Synthesis and molecular modeling studies of indole-based antitumor agents

Abstract

Indole-based compounds 30–63 were synthesized by the multi-component 1,3-dipolar cycloaddition reaction of 1-alkyl-3,5-bis(arylidene)-4-piperidones 11–25 with azomethine ylides (generated by the condensation of isatins 26–28 with sarcosine 29). The single crystal X-ray studies of 46 and 48 supported the regio- and stereoselectivity of the reaction. Most of the synthesized spiro-indoles exhibited potent antitumor properties against the HeLa (cervical cancer) cell line through in vitro sulfo-rhodamine-B bioassay, higher than that of cisplatin. Only compound 54 showed bio-potency against the HepG2 (hepatocellular cancer) cell line, comparable to that of doxorubicin hydrochloride (standard reference). 3D-Pharmacophore and 2D-QSAR studies were used to validate the observed biological data and determine the most important parameters controlling activity. The estimated bio-properties from the computational studies showed high approximations to the experimental data.

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Introduction

The indole scaffold is found in many natural and synthetic biologically active molecules.^1,2 The marketed antiviral drugs delavirdine 1, arbidol 2 and methisazone 3 are indole derivatives.³ Another indole derivative, obatoclax 4 was reported to overcome myeloid cell leukemia-1 (MCL-1) mediated resistance to apoptosis.⁴ Sunitinib 5 has been approved by the US-FDA and European Agency for the treatment of advanced renal and gastrointestinal stromal tumors.^5,6 Indibulin 6 was reported as a microtubule inhibitor (phase I/II)⁷ (Fig. 1). Many naturally occurring spiro-indoles exhibit biological activity for example; mitraphylline 7 (from Uncaria tomentosa) exhibits antiproliferative activity against brain carcinoma cell lines, neuroblastoma SKN-BE(2), and malignant glioma GAMG.⁸ Spirotryprostatins A 8 and B 9 (from the fermentation broth of Aspergillus fumigates) inhibit the G2/M progression cell division in mammalian tsFT210 cells^9,10 (Fig. 1).

Fig. 1 Biologically active indole-based compounds.

Inspired by the above reports and continuation of a research program directed towards development of novel bio-active agents with particular interest in cancer chemotherapy,^11–20 novel spiro-indoles where, the indolyl nitrogen is attached to a (cyclic-amino)methylene function were investigated in the present study. This function provides a positive ionizable fitting our candidate in the pharmacophoric active site. The synthesized spiro-indoles were screened against cervical (HeLa) and liver (HepG2) carcinoma cell lines. Cervical cancer is the fourth leading cause of cancer related death in women.^21,22 Infection with oncogenic human papillomavirus is the main cause for cervical cancer.²³ Although advances have been made in screening, diagnosis, treatment and management, the mortality due to cervical carcinoma still approaches 50%. Cervical carcinoma is treated by radiotherapy or surgery in the early stage and concurrent chemoradiation in the advanced stages.²⁴ Liver cancer is the third leading cause of cancer-related death worldwide.²⁵ Infection with hepatitis virus of either type B or C (HBV, HCV) increases the risk of liver cancer about 20 folds.²⁶ Other risk factors for hepatocellular carcinoma include alcohol abuse, diabetes, obesity, and related metabolic syndrome.²⁷ To date, the chemotherapeutic agents developed for liver cancer have shown poor efficacy with severe toxicity and side effects.²⁸ Therefore, novel chemotherapeutic agents with a higher potency and less side effects are still needed. Molecular modeling studies were undertaken to validate the observed biological data and determine the important parameters governing activity.

Results and discussion

Chemistry

The base-catalyzed condensation (KOH/EtOH) of aromatic aldehydes with 1-alkyl-4-piperidones 10 (in a 2 : 1 molar ratio) afforded the corresponding 1-alkyl-3,5-bis(arylidene)-4-piperidones 11–25 (Scheme 1). The ¹H-NMR spectra of piperidones 14 and 19 showed sharp singlet signals for the olefinic methine protons at δ_H = 7.72 and 7.76 in the case of 14 and 19, respectively confirming the E,E′-configuration.^29–31 The IR spectra of compounds 14 and 19 showed unsaturated carbonyl group at ν = 1670, 1678 cm⁻¹, respectively (Fig. S1–S6†).

Scheme 1 Synthesis of bis(arylidene)piperidones.

Multi-component reaction of 1-alkyl-3,5-bis(arylidene)-4-piperidones 11–25 with azomethine ylides (generated by the condensation of isatins 26–28 with sarcosine 29)^11–15 in refluxing ethanol afforded the corresponding dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-diones 30–63 (Scheme 2). The IR spectrum of 30 (a representative example of the family), showed the ketonic and amidic carbonyls of piperidinyl and indolyl heterocycles at ν = 1699, 1686 cm⁻¹, respectively. The ¹H-NMR spectrum of 30 showed the methylene protons of pyrrolidinyl H₂C-5′ and piperidinyl H₂C-2′′, H₂C-6′′ as diastereotopic protons. The methylene protons attached to the indolyl N-1 also exhibited diastereotopic (two doublets at δ_H = 4.16 and 4.54; J = 12.6 Hz). The methylene carbons H₂C-6′′, H₂C-5′ and H₂C-2′′ appeared in the ¹³C-NMR spectrum of 30 at δ_C = 56.9, 57.3, and 57.5, respectively. The methylene carbon attached to the indolyl N-1 (NCH₂N) appeared at δ_C = 63.3. The pyrrolidinyl methine HC-4′ exhibited at δ_C = 45.6. The signals for spiro-carbons C-3′ (C-3′′) and C-3 (C-2′) revealed at δ_C = 66.5, 75.6, respectively. Those for the carbonyl carbons C-2 and C-4′′ showed at δ_C = 176.5, 198.7, respectively (Fig. S7–S107† show the spectral data of compounds 30–63).

Scheme 2 Synthesis of indole-based compounds.

Single crystal X-ray studies

The single crystal X-ray ORTEP diagrams of compounds 46 and 48 are shown in Fig. 2 and 3, respectively. Both compounds are crystallized in the monoclinic space group P2₁/c possessing four molecules in the unit cell. The asymmetric unit in each comprises one molecule. Two spiro linkages exist in 46 and 48; at C1, where the pyrrolidine and indole heterocyclic rings are attached, and at C24, where the piperidine and pyrrolidine heterocycles are connected. In general, the bond lengths and angles of both compounds (Tables S1 and S2†) are in good agreement with each other and also with the reported structures possessing similar rings and functional groups.^31–38 The (fluorophenyl)methylidene moiety is connected to the piperidine ring at position C26 whereas, the 4-fluorophenyl ring is attached to the pyrrolidine heterocycle at C17. The (1-piperidinyl)methylene fragment is connected to the indolyl N3. The exocyclic olefinic linkage of compounds 46 and 48 has E-configuration (C26–C27). The pyrrolidine ring appears as an envelope conformation with N1 being the flap atom which lies 0.594(5) Å and 0.554(3) Å out of the plane of the remaining four atoms in 46 and 48, respectively.

Fig. 2 An ORTEP view of compound 46 showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small spheres of arbitrary radii.

Fig. 3 An ORTEP view of compound 48 showing the atom-numbering scheme. Displacement ellipsoids are drawn at the 50% probability level and H atoms are shown as small spheres of arbitrary radii.

The piperidine ring connected to C8 has a chair form with C11 and N4 deviating by 0.628 Å and 0.649 Å in 46, and by 0.544 Å and 0.652 Å in 48, respectively out of the plane of the remaining four atoms. However, the piperidine ring attached to C24 adopts a half-chair configuration where, the C35 atom lies 0.682(3) Å out of the plane of the remaining five atoms [root mean square (rms) deviation = 0.0543 Å] in 46 and lies 0.675(4) Å out of the plane of the remaining five atoms (rms deviation = 0.0426 Å) in 48. In 46, the sum of the angles around the N1 and N2 atoms are approximately 334° and 333°, respectively; and in 48 are 337° and 333°, respectively, consistent with sp³ hybridization. With respect to the piperidine ring attached to C24, the C1 occupies an axial position however, the C17 as well as the (4-fluorophenyl)methylidene residue and the N-bound alkyl substituent occupy equatorial positions.

The most significant feature of the crystal packing of 46 and 48 is the centrosymmetric eight-membered {⋯HNCO}₂ amide dimers. In the molecular packing of 46 and 48, molecules are connected by C–H⋯F and C–H⋯O interactions (listed in Tables 1 and 2) resulting in supramolecular layers. These supramolecular layers are parallel to (0 1 1) as illustrated in Fig. 4 for compound 46. However, the supramolecular layers are parallel to (1 0 0) as shown in Fig. 5 for compound 48.

Table 1 Hydrogen-bond geometry (Å, °) for compound 46^a

D–H⋯A	D–H	H⋯A	D⋯A	D–H⋯A
a Symmetry codes: (i) −1 − x, −y, −2 − z; (ii) −1 − x, −y, −2 − z; (iii) x, −1/2 − y, −1/2 + z; (iv) −1 + x, y, z.
C29–H291⋯F1^(i)	0.96	2.46(2)	3.187(6)	132(2)
C23–H231⋯F2^(ii)	0.96	2.54(3)	3.258(7)	132(3)
C3–H31⋯F2^(iii)	0.96	2.60(4)	3.240(6)	125(2)
C10–H101⋯O2^(iv)	0.96	2.64(3)	3.591(7)	166(4)

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Table 2 Hydrogen-bond geometry (Å, °) for compound 48^a

D–H⋯A	D–H	H⋯A	D⋯A	D–H⋯A
a Symmetry codes: (i) x, −1/2 − y, 1/2 + z; (ii) 2 − x, −1 − y, −1 − z; (iii) x, −3/2 − y, −1/2 + z; (iv) −1 + x, y, z.
C29–H291⋯F1^(i)	0.92	2.54(3)	3.247(6)	135(2)
C23–H231⋯F2^(ii)	0.94	2.59(3)	3.249(4)	127(3)
C3–H31⋯F2^(iii)	0.93	2.65(4)	3.312(6)	128(2)
C10–H101⋯O2^(iv)	0.96	2.71(3)	3.659(5)	169(4)

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Fig. 4 A view of the supramolecular layer parallel to (0 1 1) plane in the crystal structure of compound 46. The hydrogen-bond interactions are shown as green dashed lines.

Fig. 5 A view of the supramolecular layer parallel to (1 0 0) plane in the crystal structure of compound 48. The hydrogen-bond interactions are shown as green dashed lines.

Optimized structures

The geometric parameters of the optimized structures of compounds 46 and 48 by AM1 and PM3 computational chemistry techniques were determined and compared with the experimental X-ray observed data (Tables S1 and S2; Fig. S108–S111†). Most of the computationally determined bond lengths and angles derived from the optimized structures are correlated well with the experimentally observed ones with slight deviations. These deviations can be attributed to the fact that the experimental results are obtained for the solid phase however; the theoretical calculations are derived from the gas phase. In the solid state, the existence of the crystal field along with the intermolecular interactions connecting the molecules, are the main reasons for these differences in geometrical parameters.^31,32 The highest difference between the experimental (X-ray) and calculated bond length values for compounds 46 and 48 are 0.067, 0.075; 0.083, 0.066 Å, for AM1 and PM3 methods respectively and the root mean square errors (RMSE) are 0.0256, 0.0269; 0.0312, 0.0329 for AM1 and PM3 methods, respectively. These statistical observations explain that the bond length values obtained by any of the computational technique are strongly correlated with the experimental (X-ray) ones.

Regarding the bond angles, the highest difference between the experimental and theoretical values for compounds 46 and 48 are 6.1, 10.1; 7.4, 8.0° and the RMSE are 2.0361, 2.3048; 2.1406 and 2.2722 for AM1 and PM3 methods, respectively. Global comparisons were performed by superimposing the molecular skeletons obtained from X-ray diffraction of 46 and 48 with the corresponding ones of AM1 and PM3 techniques (Fig. S112 and S113†). Most of the rings and functional groups aligned well with each other. However, the piperidine ring attached to the methylene C8 of the theoretical optimized structures (AM1 and PM3) is aligned in different direction compared to the X-ray one. The torsion angle C7–N3–C8–N4 of compound 46 = −98.40, 55.97 and 82.18° for X-ray, AM1 and PM3, respectively support these observations. Similar words for torsion angle C7–N3–C8–N4 of compound 48 = −96.97, 55.90, 81.97° for X-ray, AM1 and PM3, respectively. The lattice form is the main factor controlling these observations in X-ray structure. However, in the conducted theoretical studies (AM1 or PM3) this effect is completely neglected, as the gas phase is adopted.

Antitumor properties

The synthesized indole-based compounds 30–63 were screened for their antitumor properties utilizing the sulfo-rhodamine-B standard method^11–20 against cervical (HeLa) and hepatocellular (HepG2) carcinoma cell lines. The results (Table 3, Fig. S114 and S115†) indicate that all the synthesized spiro-indoles (compounds 32 and 63 are exceptions, IC₅₀ = 7.75, 8.85 μM, respectively) have antitumor properties with potency higher than that of cisplatin (standard reference, IC₅₀ = 7.71 μM). Cisplatin is the clinically applicable drug either alone or in combination with topotecan for cervical carcinoma.³⁹ However, most of the synthesized spiro-alkaloids show mild antitumor properties against hepatoma cell line (HepG2), compound 54 (IC₅₀ = 7.73 μM) is the only analogue revealing bio-potency comparable to that of doxorubicin hydrochloride (IC₅₀ = 8.05 μM), the standard reference. Doxorubicin is a typical DNA-intercalating agent with broad antitumor effect against acute leukemia, lymphoma, multiple myeloma, breast cancer, osteosarcoma, soft tissue sarcoma and liver cancer.⁴⁰ Structure–activity relationship (SAR) based on the observed antitumor bioactivities against HeLa cell explains that, utilization of (4-morpholiyl)methylene function as a substituent attached to the indolyl N-1 seems more appropriate for constructing bio-active hits relative to (1-piperidinyl)methylene residue (compounds 44, 51, 53, 59 and 63 are exceptions). To explore the controlling parameters governing bio-activity and validate the observed bioactivity, computational chemistry studies including 3D-pharmacophore and 2D-quantitative structure activity relationship (2D-QSAR) were performed.

Table 3 Antitumor properties of the synthesized compounds 30–63

ID	Compd	R	R′	X	IC50^a, μg ml^−1 (μM)
					HeLa	HepG2
a IC50 is the concentration required to produce 50% inhibition of cell growth compared to the control.b Dox is doxorubicin hydrochloride (standard reference).
1	30	Ph	Me	CH2	3.62 (6.46)	12.07 (21.52)
2	31	Ph	Me	O	3.09 (5.49)	12.07 (21.45)
3	32	Ph	Me	NMe	4.46 (7.75)	11.20 (19.45)
4	33	4-ClC6H4	Me	CH2	3.80 (6.04)	11.52 (18.30)
5	34	4-ClC6H4	Me	O	3.48 (5.51)	15.76 (24.95)
6	35	4-ClC6H4	Me	NMe	4.02 (6.24)	12.17 (18.88)
7	36	4-ClC6H4	Et	CH2	3.62 (5.62)	14.57 (22.64)
8	37	4-ClC6H4	Et	O	3.37 (5.22)	14.78 (22.89)
9	38	4-ClC6H4	n-Pr	CH2	3.37 (5.12)	32.17 (48.91)
10	39	4-ClC6H4	n-Pr	O	3.26 (4.94)	33.26 (50.42)
11	40	4-ClC6H4	CH2Ph	CH2	3.62 (5.13)	22.83 (32.35)
12	41	4-ClC6H4	CH2Ph	O	3.37 (4.76)	20.87 (29.49)
13	42	2,4-Cl2C6H3	Me	O	3.15 (4.50)	20.43 (29.16)
14	43	4-FC6H4	Me	CH2	3.04 (5.09)	8.15 (13.66)
15	44	4-FC6H4	Me	O	3.15 (5.26)	21.63 (36.13)
16	45	4-FC6H4	Me	NMe	4.13 (6.75)	17.50 (28.61)
17	46	4-FC6H4	Et	CH2	3.59 (5.88)	14.13 (23.14)
18	47	4-FC6H4	Et	O	3.30 (5.39)	14.13 (23.06)
19	48	4-FC6H4	n-Pr	CH2	4.57 (7.32)	23.59 (37.76)
20	49	4-FC6H4	n-Pr	O	3.70 (5.90)	17.17 (27.40)
21	50	4-FC6H4	CH2Ph	CH2	3.30 (4.90)	16.52 (24.55)
22	51	4-FC6H4	CH2Ph	O	4.79 (7.10)	19.89 (29.48)
23	52	4-H3CC6H4	Me	CH2	3.04 (5.16)	19.89 (33.78)
24	53	4-H3CC6H4	Me	O	3.48 (5.89)	28.04 (47.46)
25	54	4-H3CC6H4	Me	NMe	3.09 (5.12)	4.67 (7.73)
26	55	4-H3COC6H4	Me	CH2	3.48 (5.61)	16.30 (26.26)
27	56	4-H3COC6H4	Me	O	3.15 (5.06)	19.78 (31.76)
28	57	4-H3COC6H4	Me	NMe	3.04 (4.78)	9.02 (14.19)
29	58	2,5-(H3CO)2C6H3	Me	CH2	3.37 (4.95)	17.39 (25.54)
30	59	2,5-(H3CO)2C6H3	Me	O	4.24 (6.21)	12.50 (18.31)
31	60	2-Thienyl	Me	CH2	4.24 (7.40)	17.61 (30.74)
32	61	2-Thienyl	Me	O	3.70 (6.44)	10.00 (17.40)
33	62	3-Pyridinyl	Me	CH2	3.91 (6.95)	47.93 (85.18)
34	63	3-Pyridinyl	Me	O	5.00 (8.85)	22.28 (39.46)
35	Dox^b	—	—	—	4.19 (7.22)	4.67 (8.05)
36	Cisplatin	—	—	—	4.19 (7.71)	3.58 (11.89)

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Molecular modeling

3D-Pharmacophore. Discovery Studio 2.5 software (Accelrys Inc., San Diego, CA, USA) was utilized for performing the 3D-pharmacophoric studies. The 3D-pharmacophore protocol was used searching for the best predictive 3D-pharmacophore hypothesis by aligning different conformations of the synthesized bio-active agents 30–63 in which the molecules are likely to bind with the receptor. The observed HYPOGEN identifies a 3D-array of five chemical features in case of HeLa (cervical carcinoma) cell line comprising two positive ionizables (PosIon-1, PosIon-2), one hydrophobic (H) and one hydrogen bonding acceptor (HBA) (Fig. S116†). Mapping of the generated pharmacophore with the synthesized bio-active agents indicates that all the compounds have the same alignment in which the nitrogen atom of the (cyclic amino)methylene fragment attached to indolyl N-1, aligned with the PosIon-1 (positive ionizable-1), and the piperidonyl nitrogen with PosIon-2 (positive ionizable-2), the indolyl carbonyl oxyen with HBA (hydrogen bonding acceptor) and the aryl group attached to the pyrrolidinyl heterocycle at C-4 with the H (hydrophobic) function (Fig. S117†). Although all the bio-active agents synthesized show the same alignment in the 3D-pharmacophore, variable estimated bio-properties and consequently variable fit values are observed (Table 4). This is due to the fact that these computed values depend on how close is the effective site of the bio-active agent to the centres of the pharmacophoric feature in addition, the weight assigned to each of the pharmacophoric feature.

Table 4 Best fit values and estimated/predicted activity values for the synthesized compounds 30–63 mapped with the generated 3D-pharmacophore model due to HeLa (cervical cancinoma) cell line

Entry	Compd	Observed IC50, μM	Estimated IC50, μM	Error^a	Fit value
a Error is the difference between the observed and estimated bio-activity values.
1	30	6.46	5.84	0.62	7.795
2	31	5.49	5.84	−0.35	7.795
3	32	7.75	6.59	1.16	7.743
4	33	6.04	5.02	1.02	7.861
5	34	5.51	5.10	0.41	7.855
6	35	6.24	6.07	0.17	7.778
7	36	5.62	5.55	0.07	7.817
8	37	5.22	5.34	−0.12	7.834
9	38	5.12	5.56	−0.44	7.817
10	39	4.94	5.14	−0.20	7.851
11	40	5.13	5.45	−0.32	7.826
12	41	4.76	5.05	−0.29	7.858
13	42	4.50	4.87	−0.37	7.875
14	43	5.09	7.21	−2.12	7.704
15	44	5.26	5.57	−0.31	7.816
16	45	6.75	6.03	0.72	7.782
17	46	5.88	6.09	−0.21	7.777
18	47	5.39	6.54	−1.15	7.746
19	48	7.32	5.58	1.74	7.815
20	49	5.90	6.36	−0.46	7.758
21	50	4.90	5.64	−0.74	7.811
22	51	7.10	5.72	1.38	7.805
23	52	5.16	5.34	−0.18	7.834
24	53	5.89	5.17	0.72	7.848
25	54	5.12	6.96	−1.84	7.719
26	55	5.61	5.13	0.48	7.852
27	56	5.06	5.38	−0.32	7.831
28	57	4.78	6.18	−1.40	7.771
29	58	4.95	4.91	0.04	7.871
30	59	6.21	5.09	1.12	7.855
31	60	7.40	6.26	1.14	7.765
32	61	6.44	5.73	0.71	7.804
33	62	6.95	6.85	0.10	7.726
34	63	8.85	7.63	1.22	7.679

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Generally, all the estimated bio-properties correlated well with the experimentally observed properties. In other words, the experimental potencies exhibited are preserved due to the computational studies. Moreover, the major structural fragments show alignment in the pharmacophoric features so; they are the main factors affecting the activity/potency. Alignment of the nitrogen atom of (cyclic amino)methylene fragment attached to indolyl N-1 with PosIon-1 is a good indicator for the importance of this residue in optimizing the bio-properties. The same words for the aryl group attached to the pyrrolidinyl heterocycle at C-4 and the piperidonyl nitrogen (aligned with H and PosIon-2, respectively) which are varied substituents. These observations support the rational design adopted for optimizing bio-effective hits.

2D-QSAR. QSAR study was undertaken utilizing CODESSA-Pro (comprehensive descriptors for structural and statistical analysis) software employing 31 synthesized indole-based analogues (compounds 30, 31, 33–40, 42–49 and 51–63; training set) exhibiting variable efficiency against HeLa (cervical carcinoma) cell line. Compounds 32, 41 and 50 (i.e. about 10% of the training set) were used as an external test set for validating the attained QSAR model, which represent high potent (compounds 41 and 50, IC₅₀ = 4.76, 4.90 μM) and mild (compound 32, IC₅₀ = 7.75 μM) effective agents. The obtained BMLR-QSAR model is statistically significant. Table 5 exhibits the QSAR's descriptors, sorted in the descending order based on student's t-criterion, which is an acceptable measure of statistical significance in multiple linear regressions. Fig. 6 shows the QSAR multi-linear model plot of correlations representing the observed versus predicted IC₅₀, μM values for the antitumor indole-based active agents. The scattered plots are uniformly distributed, covering ranges, observed 4.50–8.85, estimated/predicted 4.37–8.927 (IC₅₀, μM) values. The observed and predicted values of the training set compounds 30, 31, 33–40, 42–49 and 51–63 according to the BMLR-QSAR model are shown in Table 6.

Table 5 Descriptor of the BMLR-QSAR model for the antitumor indole-based active agents 30, 31, 33–40, 42–49 and 51–63 against HeLa (cervical cancinoma) cell line^a

Entry	ID	Coefficient	s	t	Descriptor
a IC50 (μM) = −324.653 + (0.577 × D1) + (3.069 × D2) + (0.341 × D3) − (0.455 × D4) − (0.029 × D5). N = 31, n = 5, R^2 = 0.844, R^2cvOO = 0.789, R^2cvMO = 0.975, F = 27.137, s^2 = 0.170.
1	0	−324.653	61.711	−5.261	Intercept
2	D1	0.577	0.059	9.729	Number of double bonds
3	D2	3.069	0.480	6.390	Max. n–n repulsion for bond C–C
4	D3	0.341	0.087	3.916	Number of F atoms
5	D4	−0.455	0.125	−3.654	Max. e–e repulsion for bond C–O
6	D5	−0.029	0.008	−3.663	HBSA H-bonding surface area (MOPAC PC)

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Fig. 6 BMLR-QSAR model plot of correlations representing the observed versus predicted IC₅₀, μM values for the antitumor indole-based active agents against HeLa (cervical carcinoma) cell line.

Table 6 Observed and estimated/predicted activity values for the synthesized compounds 30, 31, 33–40, 42–49 and 51–63 according to the BMLR-QSAR model due to HeLa (cervical cancinoma) cell line

Entry	Compd	Observed IC50, μM	Estimated IC50, μM	Error^a
a Error is the difference between the observed and estimated bio-activity values.
1	30	6.46	6.42	0.04
2	31	5.49	5.72	−0.23
3	33	6.04	5.63	0.41
4	34	5.51	5.04	0.47
5	35	6.24	6.00	0.24
6	36	5.62	5.49	0.13
7	37	5.22	5.65	−0.43
8	38	5.12	5.43	−0.31
9	39	4.94	5.39	−0.45
10	40	5.13	5.42	−0.29
11	42	4.50	5.25	−0.75
12	43	5.09	5.48	−0.39
13	44	5.26	5.35	−0.09
14	45	6.75	6.53	0.22
15	46	5.88	5.74	0.14
16	47	5.39	5.98	−0.59
17	48	7.32	6.76	0.56
18	49	5.90	6.07	−0.17
19	51	7.10	6.77	0.33
20	52	5.16	5.15	0.01
21	53	5.89	5.20	0.69
22	54	5.12	5.60	−0.48
23	55	5.61	5.82	−0.21
24	56	5.06	4.58	0.48
25	57	4.78	5.00	−0.22
26	58	4.95	4.37	0.58
27	59	6.21	5.94	0.27
28	60	7.40	7.07	0.33
29	61	6.44	6.65	−0.21
30	62	6.95	6.95	0.00
31	63	8.85	8.93	−0.08

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Number of double bonds, which is a constitutional descriptor, is the first descriptor controlling the obtained BMLR-QSAR model (t = 9.729). This descriptor participated positively in the model (coefficient = 0.577), meaning that the higher double bond number, the lower antitumor efficiency of the molecule. Max. n–n repulsion for bond C–C is the second descriptor controlling the 2D-QSAR model based on its t-criterion value (t = 6.390), a semi-empirical descriptor. The electron–electron repulsion between two given atoms is determined by eqn (1).⁴¹

where, A represents a given atomic species, B another atomic species, P_μνP_λσ is the density matrix elements over atomic basis {μνλσ} and 〈μν|λσ〉 is the electron repulsion integrals on atomic basis 〈μν|λσ〉. Number of F atoms (the third descriptor controlling the obtained BMLR-QSAR model, t = 3.916) is a constitutional descriptor that characterizes the atomic constitution and its inductive effect (−I) influences the aryl group electrophilicity attached at the position-4′ of pyrrolidine heterocycle. Number of F atoms in this QSAR model, serves as an indicator variable to distinguish between the only fluorine containing compound and among the rest of training set analogues. 3D-Pharmacophore shows that the aryl group attached at the pyrrolidinyl C-4 is mapped with the hydrophobic feature which may exhibit π-interaction with any of the receptor active site components. Lipophilicity in addition to −I effect of F atoms can explain not only the impotence of this descriptor for the 2D-QSAR model but also support the aforementioned observations due to 3D-pharmacophoric studies.

Max. e–e repulsion for bond C–O is a semi-empirical descriptor determined by eqn (1). HBSA H-bonding surface area (MOPAC PC) is a charge-related descriptor. This descriptor has the lowest effect on the QSAR model (coefficient = −0.029). For all the training set analogues this descriptor is zero however for compound 62 = 68.44892 (Table S3† exhibits all the QSAR descriptor values for each respective training set compound), which is a considerable value and can explain the enhanced antitumor properties of this compound relative to its structurally related analogue 63.

Validation of BMLR-QSAR model. The reliability and statistical relevance of the BMLR-QSAR model were examined by internal and external validation procedures.

Internal validation

CODESSA-Pro is capable for QSAR internal validation including, (i) Leave-One-Out (LOO) that involves developing number of models with one training set compound omitted at a time. (ii) Leave-Many-Out (LMO), which involves developing number of models with many data points (group of compounds) omitted at a time (up to 20% of the total data points). The observed correlations due to the statistical internal validation techniques are R²cvOO = 0.789, R²cvMO = 0.975. Both of them are correlated with the squared correlation coefficient of the BMLR-QSAR model (R² = 0.844). Moreover, the standard deviation of the regressions (s² = 0.170) is a measurable value for the QSAR model together with the Fisher test value (F = 27.137) that shows the ratio of the variance explained by the QSAR model and the variance due to their errors. A high value of F-test compared to the s² is a significant validation of the model.

Table 6 exhibits the experimentally observed and estimated/predicted activities of the training set. The data indicate that the error values (difference between the observed and estimated bio-properties) were in the range 0.00–0.75, thus preserving the potencies of most of the compounds tested.

External validation

Compounds 32, 41 and 50 were used as an external test set not only for validating the obtained BMLR-QSAR model but also for investigating its predicative ability. The test set analogues represent high (compounds 41 and 50) and mild (compound 32) antitumor effective agents against HeLa cell line, relative to cisplatin (standard reference). This selection provides a good idea about the capability of the obtained QSAR model for predicting analogues of wide potency range. Table 7 reveals the observed and estimated IC₅₀ values of the test set compounds. The observed data, indicate that compounds 41 and 50, which are high potent antitumor active agents reveal estimated IC₅₀ = 4.75, 5.67 μM with error value = 0.01, −0.77, respectively. Although the error value due to the estimated biological data of compound 50 is considered high, the estimated potency of this high antitumor active agent is still preserved relative to the standard reference, cisplatin (IC_50(observed) = 7.71 μM). Compound 32 which is considered a mild antitumor active analogue (IC_50(observed) = 7.75 μM), has an estimated IC₅₀ = 4.62 μM with error value = 3.13. Although, this error value is considered high, the estimated activity of this analogue is matched with its observed bio-observation which seems so close to the efficiency of the standard reference. Generally, the net results of external validation provide a good sign for the predictive capability of the attained BMLR-QSAR model particularly for optimizing high potent antitumor hits and also support the previous statement (internal validation technique) concerning its predictive power as shown by the statistical values.

Table 7 Observed, estimated and molecular descriptor values of the external test set compounds 32, 41 and 50 according to the attained BMLR-QSAR model

Entry	Compd	Observed IC50 (μM)	Estimated IC50 (μM)	Error^a	Descriptors^b
					D1	D2	D3	D4	D5
a Error is the difference between the observed and estimated bio-activity values.b D1 = number of double bonds, D2 = max. n–n repulsion for bond C–C, D3 = number of F atoms, D4 = max. e–e repulsion for bond C–O, D5 = HBSA H-bonding surface area (MOPAC PC).
1	32	7.75	4.62	3.13	3	136.5313	0	200.8148	0
2	41	4.76	4.75	0.01	3	136.4321	0	199.8628	0
3	50	4.90	5.67	−0.77	3	136.3949	2	199.0871	0

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Conclusions

From all the above it can be concluded that, indole-based compounds 30–63 were synthesized in good to excellent yields (68–86%) through the multi-component reaction of 1-alkyl-3,5-bis(arylidene)-4-piperidones 11–25 with azomethine ylides (generated through condensation of isatins 26–28 with sarcosine 29). X-ray studies of 46 and 48 provide good support for the regio- and stereoselectivity of the reaction. Many of the synthesized spiro-indoles exhibit antitumor properties against HeLa (cervical cancer) cell line through in vitro sulfo-rhodamine-B bio-assay with potencies higher than that of cisplatin (standard reference). Only compound 54 reveals bio-potency against HepG2 (hepatocellular carcinoma) cell line, comparable to that of doxorubicin hydrochloride (standard reference). HYPOGEN of the 3D-pharmacophore studies identifies a 3D-array of five chemical features in case of HeLa cell line containing two positive ionizables, one hydrophobic and one hydrogen bonding acceptor. The estimated bio-properties owing to the pharmacophic hypothesis are correlated well with the experimentally observed activities. 2D-QSAR studies led to a 5 descriptor model validated well by both internal and external validation techniques. The predicated properties obtained using the 2D-QSAR technique show high approximations to the experimentally observed data supporting the capability for optimizing high potent hits based on the attained model. The success of the computational studies although short data points were used, can be attributed to the fact that these studies utilized a homogeneous data set (non-diverse technique) of high structural resemblance (same chemical scaffold).

Experimental section

Melting points were recorded on a Stuart SMP10 melting point apparatus. IR spectra (KBr) were recorded on a Shimadzu FT-IR 8400S spectrophotometer. ¹H-NMR spectra were recorded on Varian MERCURY 300 (300 MHz), Bruker Avance (300 MHz) and Bruker Ascend 400/R (400 MHz) spectrometers. ¹³C-NMR spectra were recorded on Bruker Avance (75 MHz) and Bruker Ascend 400/R (100 MHz) spectrometers. Compounds 11–13, 15–18, 20–25 ^42–50 and 26–28 ^51,52 were prepared according to the reported procedures.

Synthesis of 3E,5E-3,5-bis[(aryl)methylidene]-1-(1-propyl)-4-piperidone (14, 19)

A mixture of 1-(1-propyl)-4-piperidone (0.75 ml, 5 mmol), and the appropriate aldehyde (10 mmol) in methanol (10 ml) containing KOH (0.56 g, 10 mmol), was stirred at room temperature (25 °C) for 3 h. The separated solid after storing the reaction mixture overnight at room temperature, was collected, washed with water, and crystallized from a suitable solvent affording the corresponding 14 and 19.

3E,5E-3,5-Bis[(4-chlorophenyl)methylidene]-1-(1-propyl)-4-piperidone (14). Pale yellow microcrystals from ethanol, mp 140–142 °C, yield 1.5 g (78%). IR ν (cm⁻¹): 1670, 1612, 1585. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 0.85 (t, J = 7.4 Hz, 3H, NCH₂CH₂CH₃), 1.45 (sextet, J = 7.4 Hz, 2H, NCH₂CH₂CH₃), 2.49 (t, J = 7.5 Hz, 2H, NCH₂CH₂CH₃), 3.76 (s, 4H, 2NCH₂), 7.30 (d, J = 8.5 Hz, 4H, arom. H), 7.38 (d, J = 8.6 Hz, 4H, arom. H), 7.72 (s, 2H, 2 olefinic CH). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 11.8, 20.5, 54.8, 59.4, 129.0, 131.7, 133.8, 133.9, 135.2, 187.1. Elemental analysis: C₂₂H₂₁Cl₂NO required C, 68.40; H, 5.48; N, 3.63, found C, 68.55; H, 5.53; N, 3.72.

3E,5E-3,5-Bis[(4-fluorophenyl)methylidene]-1-(1-propyl)-4-piperidone (19). Pale yellow microcrystals from methanol, mp 130–131 °C, yield 1.4 g (79%). IR ν (cm⁻¹): 1678, 1612, 1601. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 0.86 (t, J = 7.4 Hz, 3H, NCH₂CH₂CH₃), 1.46 (sextet, J = 7.4 Hz, 2H, NCH₂CH₂CH₃), 2.51 (t, J = 7.6 Hz, 2H, NCH₂CH₂CH₃), 3.78 (s, 4H, 2NCH₂), 7.11 (t, J = 8.7 Hz, 4H, arom. H), 7.36–7.41 (m, 4H, arom. H), 7.76 (s, 2H, 2 olefinic CH). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 11.9, 20.6, 54.9, 59.5, 115.8, 116.1, 131.6, 131.7, 132.4, 132.5, 133.27, 133.29, 135.4, 161.4, 164.7, 187.4. Elemental analysis: C₂₂H₂₁F₂NO required C, 74.77; H, 5.99; N, 3.96, found C, 74.88; H, 6.08; N, 3.81.

Synthesis of dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-diones 30–63 (general procedure)

A mixture of equimolar amounts of the appropriate 3,5-bis(arylidene)-1-alkyl-4-piperidone 11–25 (5 mmol), the corresponding isatines 26–28 and sarcosine 29 in absolute ethanol (25 ml) was boiled under reflux for the appropriate time. The separated solid while refluxing was collected and crystallized from a suitable solvent affording the corresponding 30, 34, 41, 42, 44 and 56. In case of the remaining compounds, the reaction mixture was concentrated to half of its initial volume and stored at room temperature overnight, the separated solid was collected and crystallized from a suitable solvent affording the corresponding analogues.

1′,1′′-Dimethyl-4′-phenyl-5′′-[(phenyl)methylidene]-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (30). Obtained from reaction of 11, 26 and 29. Reaction time 10 h, colorless microcrystals from ethanol, mp 155–157 °C, yield 70% (1.96 g). IR ν (cm⁻¹): 1699, 1686, 1609, 1489, 1466. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.35–1.43 (m, 2H, piperidinyl H₂C-4), 1.51–1.60 (m, 4H, piperidinyl H₂C-3/5), 1.63 (d, J = 13.2 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.99 (s, 3H, piperidinyl NCH₃), 2.17 (s, 3H, pyrrolidinyl NCH₃), 2.58 (br s, 4H, piperidinyl NCH₂-2/6), 2.86 (dd, J = 2.9, 14.6 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.27–3.36 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′, and upfield H of pyrrolidinyl H₂C-5′), 3.97 (dd, J = 8.9, 11.3 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.16 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.54 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 4.85 (dd, J = 6.9, 11.4 Hz, 1H, pyrrolidinyl HC-4′), 6.86 (d, J = 7.8 Hz, 1H, arom. H), 6.96–7.42 (m, 14H, 13 arom. H + olefinic CH). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 24.3, 26.0, 35.2, 45.3, 45.6, 52.4, 56.9, 57.3, 57.5, 63.3, 66.5, 75.6, 109.2, 122.0, 126.8, 126.9, 127.5, 128.3, 128.4, 128.7, 128.9, 129.6, 130.0, 133.7, 135.5, 137.6, 138.6, 145.2, 176.5, 198.7. Elemental analysis: C₃₆H₄₀N₄O₂ required C, 77.11; H, 7.19; N, 9.99, found C, 77.11; H, 7.27; N, 10.11.

1′,1′′-Dimethyl-1-[(4-morpholinyl)methylene]-4′-phenyl-5′′-[(phenyl)methylidene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (31). Obtained from reaction of 11, 27 and 29. Reaction time 12 h, colorless microcrystals from methanol, mp 169–170 °C, yield 72% (2.02 g). IR ν (cm⁻¹): 1694, 1612, 1601, 1491, 1468. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.62 (d, J = 13.2 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.00 (s, 3H, piperidinyl NCH₃), 2.16 (s, 3H, pyrrolidinyl NCH₃), 2.57 (t, J = 4.7 Hz, 4H, morpholinyl 2NCH₂), 2.86 (dd, J = 2.7, 14.7 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.27–3.36 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′ and upfield H of pyrrolidinyl H₂C-5′), 3.64 (t, J = 4.4 Hz, 4H, morpholinyl 2OCH₂), 3.96 (dd, J = 8.7, 11.1 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.11 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.52 (d, J = 12.3 Hz, 1H, downfield H of NCH₂N), 4.85 (dd, J = 6.8, 11.3 Hz, 1H, pyrrolidinyl HC-4′), 6.80 (d, J = 7.8 Hz, 1H, arom. H), 6.97–7.41 (m, 14H, 13 arom. H + olefinic CH). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 35.1, 45.3, 45.5, 51.3, 56.7, 57.3, 57.5, 62.6, 66.6, 66.9, 75.5, 109.0, 122.3, 126.8, 126.9, 127.6, 128.3, 128.4, 128.7, 128.9, 129.5, 130.0, 133.6, 135.3, 137.6, 138.5, 144.7, 176.5, 198.6. Elemental analysis: C₃₅H₃₈N₄O₃ required C, 74.71; H, 6.81; N, 9.96, found C, 74.77; H, 6.88; N, 10.03.

1′,1′′-Dimethyl-1-[1-(4-methylpiperazinyl)methylene]-4′-phenyl-5′′-[(phenyl)methylidene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (32). Obtained from reaction of 11, 28 and 29. Reaction time 15 h, colorless microcrystals from ethanol, mp 142–144 °C, yield 70% (2.01 g). IR ν (cm⁻¹): 1694, 1682, 1616, 1601, 1470. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.61 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.99 (s, 3H, piperidinyl NCH₃), 2.16 (s, 3H, pyrrolidinyl NCH₃), 2.25 (s, 3H, piperazinyl NCH₃), 2.38 (br s, 4H, piperazinyl 2NCH₂), 2.62 (br s, 4H, piperazinyl 2NCH₂), 2.86 (dd, J = 2.6, 15.2 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.26–3.36 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′ and upfield H of pyrrolidinyl H₂C-5′), 3.97 (dd, J = 8.7, 11.4 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.11 (d, J = 12.3 Hz, 1H, upfield H of NCH₂N), 4.56 (d, J = 12.3 Hz, 1H, downfield H of NCH₂N), 4.85 (dd, J = 6.9, 11.4 Hz, 1H, pyrrolidinyl HC-4′), 6.79 (d, J = 7.8 Hz, 1H, arom. H), 6.97–7.41 (m, 14H, 13 arom. H + olefinic CH). ¹³C-NMR (100 MHz, CDCl₃) δ (ppm): 35.0, 45.1, 45.4, 46.0, 50.7, 54.9, 56.6, 57.1, 57.3, 62.2, 66.4, 75.4, 108.9, 122.0, 126.6, 126.8, 127.3, 128.16, 128.23, 128.5, 128.7, 129.4, 129.9, 133.4, 135.2, 137.4, 138.3, 144.6, 176.2, 198.5. Elemental analysis: C₃₆H₄₁N₅O₂ required C, 75.10; H, 7.18; N, 12.16, found C, 75.18; H, 7.22; N, 12.27.

4′-(4-Chlorophenyl)-5′′-[(4-chlorophenyl)methylidene]-1′,1′′-dimethyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (33). Obtained from reaction of 12, 26 and 29. Reaction time 12 h, pale yellow microcrystals from methanol, mp 167–168 °C, yield 76% (2.40 g). IR ν (cm⁻¹): 1705, 1686, 1609, 1491, 1466. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.35–1.42 (m, 2H, piperidinyl H₂C-4), 1.48–1.56 (m, 4H, piperidinyl H₂C-3/5), 1.61 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.00 (s, 3H, piperidinyl NCH₃), 2.15 (s, 3H, pyrrolidinyl NCH₃), 2.52 (t, J = 5.1 Hz, 4H, piperidinyl NCH₂-2/6), 2.84 (dd, J = 2.7, 15.0 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.23 (d, J = 13.8 Hz, 2H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′), 3.32 (dd, J = 7.1, 8.6 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.91 (dd, J = 8.7, 11.1 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.09 (d, J = 12.3 Hz, 1H, upfield H of NCH₂N), 4.51 (d, J = 12.3 Hz, 1H, downfield H of NCH₂N), 4.79 (dd, J = 6.9, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.83 (d, J = 7.5 Hz, 1H, arom. H), 6.90–7.42 (m, 12H, 11 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 23.7, 25.2, 34.3, 44.0, 44.4, 51.4, 55.9, 56.0, 56.3, 62.2, 65.3, 74.6, 109.2, 121.3, 125.6, 126.5, 128.2, 128.3, 128.6, 128.7, 130.6, 131.4, 131.5, 132.1, 133.2, 133.5, 133.6, 135.1, 137.1, 144.7, 175.2, 197.4. Elemental analysis: C₃₆H₃₈Cl₂N₄O₂ required C, 68.67; H, 6.08; N, 8.90, found C, 68.79; H, 6.13; N, 9.04.

4′-(4-Chlorophenyl)-5′′-[(4-chlorophenyl)methylidene]-1′,1′′-dimethyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (34). Obtained from reaction of 12, 27 and 29. Reaction time 12 h, colorless microcrystals from ethanol, mp 195–197 °C, yield 79% (2.50 g). IR ν (cm⁻¹): 1705, 1682, 1614, 1491, 1466. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.60 (d, J = 12.7 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.01 (s, 3H, piperidinyl NCH₃), 2.14 (s, 3H, pyrrolidinyl NCH₃), 2.57 (t, J = 4.3 Hz, 4H, morpholinyl 2NCH₂), 2.84 (dd, J = 2.2, 14.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.21–3.25 (m, 2H, downfield H's of piperidinyl H₂C-2′′ and H₂C-6′′), 3.31 (t, J = 7.8 Hz, 1H,‏ upfield H of pyrrolidinyl H₂C-5′), 3.64 (t, J = 4.3 Hz, 4H, morpholinyl 2OCH₂), 3.89 (dd, J = 9.1, 10.7 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.09 (d, J = 12.5 Hz, 1H, upfield H of NCH₂N), 4.53 (d, J = 12.5 Hz, 1H, downfield H of NCH₂N), 4.79 (dd, J = 7.0, 11.0 Hz, 1H, pyrrolidinyl HC-4′), 6.79 (d, J = 7.8 Hz, 1H, arom. H), 6.89–7.33 (m, 12H, 11 arom. H + olefinic CH). ¹³C-NMR (100 MHz, CDCl₃) δ (ppm): 34.9, 44.7, 45.1, 51.2, 56.6, 57.0, 57.3, 62.5, 66.2, 66.7, 75.3, 108.9, 122.1, 126.4, 127.3, 128.4, 128.5, 128.9, 130.7, 131.0, 132.6, 133.4, 133.7, 134.7, 136.2, 136.8, 144.5, 176.2, 198.1. Elemental analysis: C₃₅H₃₆Cl₂N₄O₃ required C, 66.56; H, 5.75; N, 8.87, found C, 66.63; H, 5.82; N, 9.01.

4′-(4-Chlorophenyl)-5′′-[(4-chlorophenyl)methylidene]-1′,1′′-dimethyl-1-[1-(4-methylpiperazinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (35). Obtained from reaction of 12, 28 and 29. Reaction time 15 h, colorless microcrystals from methanol, mp 145–147 °C, yield 69% (2.22 g). IR ν (cm⁻¹): 1690, 1611, 1491, 1464. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.59 (d, J = 12.3 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.00 (s, 3H, piperidinyl NCH₃), 2.14 (s, 3H, pyrrolidinyl NCH₃), 2.25 (s, 3H, piperazinyl NCH₃), 2.37 (br s, 4H, piperazinyl 2NCH₂), 2.61 (br s, 4H, piperazinyl 2NCH₂), 2.83 (dd, J = 1.8, 14.4 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.21–3.34 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′ and upfield H of pyrrolidinyl H₂C-5′), 3.89 (t, J = 9.8 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.12 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.55 (d, J = 12.3 Hz, 1H, downfield H of NCH₂N), 4.78 (dd, J = 6.9, 11.1 Hz, 1H, pyrrolidinyl HC-4′), 6.80 (d, J = 7.8 Hz, 1H, arom. H), 6.89–7.34 (m, 12H, 11 arom. H + olefinic CH). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 35.0, 44.8, 45.1, 46.0, 50.8, 54.9, 56.7, 57.0, 57.3, 62.3, 66.3, 75.3, 109.1, 122.0, 126.5, 127.3, 128.4, 128.6, 128.9, 130.7, 131.1, 132.6, 133.5, 133.9, 134.6, 136.1, 136.9, 144.7, 176.2, 198.2. Elemental analysis: C₃₆H₃₉Cl₂N₅O₂ required C, 67.08; H, 6.10; N, 10.86, found C, 67.21; H, 6.14; N, 11.03.

4′-(4-Chlorophenyl)-5′′-[(4-chlorophenyl)methylidene]-1′′-ethyl-1′-methyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (36). Obtained from reaction of 13, 26 and 29. Reaction time 10 h, colorless microcrystals from ethanol, mp 160–162 °C, yield 70% (2.26 g). IR ν (cm⁻¹): 1697, 1611, 1491, 1468. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 0.72 (t, J = 7.2 Hz, 3H, NCH₂CH₃), 1.33–1.42 (m, 2H, piperidinyl H₂C-4), 1.50–1.58 (m, 4H, piperidinyl H₂C-3/5), 1.69 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.07 (sextet, J = 7.1, 12.3 Hz, 1H, upfield H of NCH₂CH₃), 2.15 (s, 3H, pyrrolidinyl NCH₃), 2.30 (sextet, J = 7.3, 12.3 Hz, 1H, downfield H of NCH₂CH₃), 2.54 (br s, 4H, piperidinyl NH₂C-2/6), 2.77 (dd, J = 2.6, 14.6 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.16 (dd, J = 1.7, 12.7 Hz, 1H, downfield H of piperidinyl H₂C-2′′), 3.27 (d, J = 14.6 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.35 (dd, J = 7.5, 8.6 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.90 (dd, J = 9.0, 10.7 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.05 (d, J = 12.7 Hz, 1H, upfield H of NCH₂N), 4.61 (d, J = 12.7 Hz, 1H, downfield H of NCH₂N), 4.79 (dd, J = 7.2, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.80 (d, J = 7.8 Hz, 1H, arom. H), 6.93–7.38 (m, 12H, 11 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 10.8, 23.6, 25.3, 34.3, 44.4, 50.7, 51.6, 52.7, 55.8, 56.3, 62.0, 64.6, 74.8, 109.1, 121.3, 125.3, 126.5, 128.1, 128.4, 128.6, 130.9, 131.3, 131.6, 133.2, 133.49, 133.5, 135.3, 137.2, 144.7, 175.0, 197.9. Elemental analysis: C₃₇H₄₀Cl₂N₄O₂ required C, 69.04; H, 6.26; N, 8.70, found C, 69.17; H, 6.29; N, 8.83.

4′-(4-Chlorophenyl)-5′′-[(4-chlorophenyl)methylidene]-1′′-ethyl-1′-methyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (37). Obtained from reaction of 13, 27 and 29. Reaction time 12 h, colorless microcrystals from ethanol, mp 188–190 °C, yield 77% (2.49 g). IR ν (cm⁻¹): 1701, 1686, 1611, 1491, 1468. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 0.71 (t, J = 7.1 Hz, 3H, NCH₂CH₃), 1.66 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.09 (sextet, J = 7.0, 12.4 Hz, 1H, upfield H of NCH₂CH₃), 2.12 (s, 3H, pyrrolidinyl NCH₃), 2.29 (sextet, J = 7.3, 12.4 Hz, 1H, downfield H of NCH₂CH₃), 2.56 (br s, 4H, morpholinyl 2NCH₂), 2.76 (dd, J = 2.7, 14.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.14 (dd, J = 1.8, 12.6 Hz, 1H, downfield H of piperidinyl H₂C-2′′), 3.26 (d, J = 14.6 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.32 (dd, J = 7.4, 8.7 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.64 (t, J = 4.5 Hz, 4H, morpholinyl 2OCH₂), 3.87 (dd, J = 8.9, 10.8 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.02 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.60 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 4.77 (dd, J = 7.3, 10.9 Hz, 1H, pyrrolidinyl HC-4′), 6.75 (d, J = 7.8 Hz, 1H, arom. H), 6.92–7.35 (m, 12H, 11 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 10.9, 34.4, 44.3, 50.7, 50.8, 52.6, 55.8, 56.3, 61.4, 64.7, 65.9, 74.9, 109.1, 121.5, 125.3, 126.6, 128.2, 128.4, 128.7, 130.9, 131.4, 131.6, 133.2, 133.4, 133.6, 135.5, 137.2, 144.5, 175.1, 197.8. Elemental analysis: C₃₆H₃₈Cl₂N₄O₃ required C, 66.97; H, 5.93; N, 8.68, found C, 67.05; H, 5.97; N, 8.81.

4′-(4-Chlorophenyl)-5′′-[(4-chlorophenyl)methylidene]-1′-methyl-1-[(1-piperidinyl)methylene]-1′′-(1-propyl)-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (38). Obtained from reaction of 14, 26 and 29. Reaction time 11 h, colorless microcrystals from methanol, mp 134–136 °C, yield 69% (2.27 g). IR ν (cm⁻¹): 1694, 1609, 1491, 1466. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 0.73 (t, J = 7.4 Hz, 3H, NCH₂CH₂CH₃), 0.96–1.21 (m, 2H, NCH₂CH₂CH₃), 1.30–1.38 (m, 2H, piperidinyl CH₂-4), 1.51–1.56 (m, 4H, piperidinyl CH₂-3/5), 1.71 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.96 (sextet, J = 5.1, 11.4 Hz, 1H, upfield H of NCH₂CH₂CH₃), 2.00–2.19 (m, 4H, downfield H of NCH₂CH₂CH₃ + pyrrolidinyl NCH₃), 2.55 (br s, 4H, piperidinyl 2NCH₂), 2.81 (dd, J = 2.6, 14.6 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.14 (dd, J = 1.3, 12.7 Hz, 1H, downfield H of piperidinyl H₂C-2′′), 3.26 (d, J = 14.7 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.36 (t, J = 8.1 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.90 (dd, J = 9.1, 10.6 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.06 (d, J = 12.8 Hz, 1H, upfield H of NCH₂N), 4.62 (d, J = 12.8 Hz, 1H, downfield H of NCH₂N), 4.78 (dd, J = 7.3, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.80 (d, J = 7.8 Hz, 1H, arom. H), 6.92–7.39 (m, 12H, 11 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 11.5, 18.7, 23.6, 25.3, 34.4, 44.6, 51.6, 53.3, 56.5, 59.1, 61.3, 62.0, 64.5, 74.9, 109.1, 121.3, 125.2, 126.6, 128.1, 128.4, 128.7, 131.0, 131.4, 131.6, 133.3, 133.6, 135.4, 137.3, 144.8, 175.1, 198.0. Elemental analysis: C₃₈H₄₂Cl₂N₄O₂ required C, 69.40; H, 6.44; N, 8.52, found C, 69.48; H, 6.48; N, 8.61.

4′-(4-Chlorophenyl)-5′′-[(4-chlorophenyl)methylidene]-1′-methyl-1-[(4-morpholinyl)methylene]-1′′-(1-propyl)-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (39). Obtained from reaction of 14, 27 and 29. Reaction time 12 h, colorless microcrystals from methanol, mp 187–189 °C, yield 73% (2.41 g). IR ν (cm⁻¹): 1699, 1684, 1611, 1489, 1466. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 0.74 (t, J = 7.3 Hz, 3H, NCH₂CH₂CH₃), 0.96–1.08 (m, 1H, upfield H of NCH₂CH₂CH₃), 1.12–1.26 (m, 1H, downfield H of NCH₂CH₂CH₃), 1.71 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.98, (sextet, J = 5.1, 11.5 Hz, 1H, upfield H of NCH₂CH₂CH₃), 2.13–2.19 (m, 4H, pyrrolidinyl NCH₃ + downfield H of NCH₂CH₂CH₃), 2.59 (br s, 4H, morpholinyl 2NCH₂), 2.82 (dd, J = 2.6, 14.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.14 (dd, J = 1.4, 12.7 Hz, 1H, downfield H of piperidinyl H₂C-2′′), 3.27 (d, J = 14.6 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.35 (t, J = 8.1 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.66 (t, J = 4.5 Hz, 4H, morpholinyl 2OCH₂), 3.88 (dd, J = 9.1, 10.7 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.05 (d, J = 12.7 Hz, 1H, upfield H of NCH₂N), 4.63 (d, J = 12.7 Hz, 1H, downfield H of NCH₂N), 4.78 (dd, J = 7.2, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.77 (d, J = 7.8 Hz, 1H, arom. H), 6.94–7.38 (m, 12H, 11 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 11.5, 18.7, 34.3, 44.4, 50.8, 53.2, 56.4, 59.1, 61.3, 64.5, 65.9, 74.9, 109.0, 121.5, 125.2, 126.6, 128.1, 128.4, 128.7, 130.9, 131.4, 131.6, 133.2, 133.45, 133.5, 135.4, 137.2, 144.5, 175.1, 197.9. Elemental analysis: C₃₇H₄₀Cl₂N₄O₃ required C, 67.37; H, 6.11; N, 8.49, found C, 67.49; H, 6.15; N, 8.54.

1′′-Benzyl-4′-(4-chlorophenyl)-5′′-[(4-chlorophenyl)methylidene]-1′-methyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (40). Obtained from reaction of 15, 26 and 29. Reaction time 10 h, pale yellow microcrystals from ethanol, mp 154–156 °C, yield 73% (2.57 g). IR ν (cm⁻¹): 1699, 1682, 1605, 1491, 1466. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.31–1.40 (m, 2H, piperidinyl H₂C-4), 1.43–1.52 (m, 4H, piperidinyl H₂C-3/5), 1.85 (d, J = 12.7 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.15 (s, 3H, pyrrolidinyl NCH₃), 2.47–2.54 (m, 4H, piperidinyl H₂CN-2/6), 2.72 (dd, J = 2.6, 14.7 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.19–3.36 (m, 3H, downfield H of piperidinyl H₂C-2′′, H₂C-6′′ + upfield H of PhCH₂), 3.40 (t, J = 8.2 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.49 (d, J = 14.0 Hz, 1H, downfield H of PhCH₂), 3.92 (dd, J = 9.2, 10.5 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.00 (d, J = 12.8 Hz, 1H, upfield H of NCH₂N), 4.50 (d, J = 12.8 Hz, 1H, downfield H of NCH₂N), 4.78 (dd, J = 7.5, 10.6 Hz, 1H, pyrrolidinyl HC-4′), 6.80–7.43 (m, 18H, 17 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 23.6, 25.3, 34.4, 45.0, 51.7, 53.0, 56.5, 56.7, 60.9, 62.3, 64.6, 75.0, 109.2, 121.6, 125.2, 126.7, 126.9, 127.9, 128.15, 128.2, 128.3, 131.0, 131.3, 131.5, 133.2, 133.5, 135.4, 135.7, 137.3, 144.7, 175.3, 197.9. Elemental analysis: C₄₂H₄₂Cl₂N₄O₂ required C, 71.48; H, 6.00; N, 7.94, found C, 71.56; H, 5.98; N, 7.98.

1′′-Benzyl-4′-(4-chlorophenyl)-5′′-[(4-chlorophenyl)methylidene]-1′-methyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (41). Obtained from reaction of 15, 27 and 29. Reaction time 11 h, pale yellow microcrystals from ethanol, mp 198–200 °C, yield 82% (2.90 g). IR ν (cm⁻¹): 1697, 1686, 1605, 1491, 1462. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.82 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.12 (s, 3H, pyrrolidinyl NCH₃), 2.48–2.58 (m, 4H, piperidinyl 2NCH₂), 2.70 (dd, J = 2.6, 14.6 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.20–3.34 (m, 3H, downfield H of piperidinyl H₂C-2′′, H₂C-6′′ + upfield H of PhCH₂), 3.38 (dd, J = 7.6, 8.7 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.48 (d, J = 14.0 Hz, 1H, downfield H of PhCH₂), 3.60 (t, J = 4.6 Hz, 4H, morpholinyl 2OCH₂), 3.89 (dd, J = 9.1, 10.5 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 3.95 (d, J = 12.8 Hz, 1H, upfield H of NCH₂N), 4.48 (d, J = 12.8 Hz, 1H, downfield H of NCH₂N), 4.76 (dd, J = 7.5, 10.7 Hz, 1H, pyrrolidinyl HC-4′), 6.77–7.40 (m, 18H, 17 arom. H + olefinic CH). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 35.2, 46.0, 51.5, 53.8, 57.3, 57.5, 61.9, 62.6, 65.6, 66.9, 76.1, 109.2, 122.5, 126.2, 127.3, 127.7, 128.3, 128.4, 128.5, 128.7, 129.1, 131.1, 131.3, 132.9, 133.5, 133.7, 134.8, 136.0, 136.6, 137.1, 144.7, 176.4, 198.8. Elemental analysis: C₄₁H₄₀Cl₂N₄O₃ required C, 69.58; H, 5.70; N, 7.92, found C, 69.69; H, 5.68; N, 8.06.

4′-(2,4-Dichlorophenyl)-5′′-[(2,4-dichlorophenyl)methylidene]-1′,1′′-dimethyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (42). Obtained from reaction of 16, 27 and 29. Reaction time 9 h, colorless microcrystals from ethanol, mp 205–207 °C, yield 80% (2.80 g). IR ν (cm⁻¹): 1701, 1611, 1483, 1466. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.73 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.90 (s, 3H, piperidinyl NCH₃), 2.09 (s, 3H, pyrrolidinyl NCH₃), 2.63 (br s, 4H, morpholinyl 2NCH₂), 2.82–2.93 (m, 2H, upfield H of piperidinyl H₂C-6′′ + downfield H of piperidinyl H₂C-2′′), 3.06 (d,‏ J = 15.0 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.49 (t, J = 8.6 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.70 (br s, 4H, morpholinyl 2OCH₂), 3.86 (t, J = 9.0 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.20 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.56 (d, J = 12.3 Hz, 1H, downfield H of NCH₂N), 5.08 (t, J = 8.3 Hz, 1H, pyrrolidinyl HC-4′), 6.83–7.47 (m, 10H, 9 arom. H + olefinic CH), 7.96 (d, J = 8.7 Hz, 1H, arom. H). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 34.2, 44.9, 50.7, 56.3, 56.9, 57.4, 57.5, 61.6, 63.1, 65.9, 75.8, 109.3, 122.0, 124.7, 126.5, 127.2, 128.4, 128.6, 129.1, 131.2, 131.3, 131.7, 132.0, 132.1, 134.1, 134.4, 134.6, 135.4, 135.7, 144.5, 175.3, 195.9. Elemental analysis: C₃₅H₃₄Cl₄N₄O₃ required C, 60.01; H, 4.89; N, 8.00, found C, 60.13; H, 4.94; N, 8.17.

1′,1′′-Dimethyl-4′-(4-fluorophenyl)-5′′-[(4-fluorophenyl)methylidene]-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (43). Obtained from reaction of 17, 26 and 29. Reaction time 12 h, pale yellow microcrystals from ethanol, mp 170–172 °C, yield 86% (2.57 g). IR ν (cm⁻¹): 1697, 1686, 1595, 1582, 1508, 1462. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.33–1.40 (m, 2H, piperidinyl H₂C-4), 1.47–1.53 (m, 4H, piperidinyl H₂C-3/5), 1.59 (d, J = 12.7 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.99 (s, 3H, piperidinyl NCH₃), 2.15 (s, 3H, pyrrolidinyl NCH₃), 2.52 (br s, 4H, piperidinyl NCH₂-2/6), 2.84 (d, J = 14.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.20–3.25 (m, 2H, downfield H's of piperidinyl H₂C-2′′ and H₂C-6′′), 3.32 (t, J = 7.9 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.90 (t, J = 9.9 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.10 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.51 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 4.80 (dd, J = 7.1, 10.9 Hz, 1H, pyrrolidinyl HC-4′), 6.82 (d, J = 7.6 Hz, 1H, arom. H), 6.96–7.14 (m, 10H, 9 arom. H + olefinic CH), 7.36 (t, J = 6.8 Hz, 2H, arom. H). ¹³C-NMR (100 MHz, CDCl₃) δ (ppm): 24.1, 25.8, 34.9, 44.7, 45.1, 52.2, 56.9, 57.1, 57.4, 63.1, 65.9, 75.5, 109.1, 114.9, 115.1, 115.3, 115.5, 121.8, 126.4, 127.2, 128.7, 130.8, 130.9, 131.2, 131.3, 131.4, 131.7, 131.8, 133.15, 133.16, 134.0, 134.1, 136.3, 145.0, 160.6, 161.4, 163.0, 163.8, 176.3, 198.4. Elemental analysis: C₃₆H₃₈F₂N₄O₂ required C, 72.46; H, 6.42; N, 9.39, found C, 72.52; H, 6.53; N, 9.48.

1′,1′′-Dimethyl-4′-(4-fluorophenyl)-5′′-[(4-fluorophenyl)methylidene]-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (44). Obtained from reaction of 17, 27 and 29. Reaction time 14 h, almost colorless microcrystals from ethanol, mp 176–178 °C, yield 73% (2.18 g). IR ν (cm⁻¹): 1705, 1684, 1612, 1508, 1489, 1464. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.60 (d, J = 12.8 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.01 (s, 3H, piperidinyl NCH₃), 2.14 (s, 3H, pyrrolidinyl NCH₃), 2.57 (br s, 4H, morpholinyl 2NCH₂), 2.84 (d, J = 14.4 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.20–3.26 (m, 2H, downfield H's of piperidinyl H₂C-2′′ and H₂C-6′′), 3.32 (t, J = 8.0 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.63 (br s, 4H, morpholinyl 2OCH₂), 3.89 (t, J = 9.9 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.11 (d, J = 12.5 Hz, 1H, upfield H of NCH₂N), 4.53 (d, J = 12.5 Hz, 1H, downfield H of NCH₂N), 4.80 (dd, J = 7.2, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.80 (d, J = 7.6 Hz, 1H, arom. H), 6.96–7.14 (m, 10H, 9 arom. H + olefinic CH), 7.35 (t, J = 6.8 Hz, 2H, arom. H). ¹³C-NMR (100 MHz, CDCl₃) δ (ppm): 34.9, 44.6, 45.1, 51.2, 56.9, 57.1, 57.3, 62.5, 66.1, 66.7, 75.4, 108.9, 115.0, 115.2, 115.3, 115.5, 122.1, 126.5, 127.4, 128.8, 130.7, 130.8, 131.15, 131.18, 131.7, 131.8, 133.06, 133.08, 133.90, 133.93, 136.4, 144.5, 160.6, 161.4, 163.1, 163.9, 176.3, 198.3. Elemental analysis: C₃₅H₃₆F₂N₄O₃ required C, 70.22; H, 6.06; N, 9.36, found C, 70.29; H, 6.17; N, 9.43.

1′,1′′-Dimethyl-4′-(4-fluorophenyl)-5′′-[(4-fluorophenyl)methylidene]-1-[1-(4-methylpiperazinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (45). Obtained from reaction of 17, 28 and 29. Reaction time 15 h, yellow microcrystals from ethanol, mp 180–182 °C, yield 82% (2.51 g). IR ν (cm⁻¹): 1686, 1605, 1585, 1508, 1462. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.59 (d, J = 12.7 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.00 (s, 3H, piperidinyl NCH₃), 2.14 (s, 3H, pyrrolidinyl NCH₃), 2.24 (s, 3H, piperazinyl NCH₃), 2.37 (br s, 4H, piperazinyl 2NCH₂), 2.62 (br s, 4H, piperazinyl 2NCH₂), 2.84 (d, J = 14.4 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.21–3.26 (m, 2H, downfield H's of piperidinyl H₂C-2′′ and H₂C-6′′), 3.32 (t, J = 7.8 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.89 (t, J = 9.9 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.12 (d, J = 12.5 Hz, 1H, upfield H of NCH₂N), 4.55 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 4.80 (dd, J = 7.0, 10.9 Hz, 1H, pyrrolidinyl HC-4′), 6.80 (d, J = 8.0 Hz, 1H, arom. H), 6.96–7.15 (m, 10H, 9 arom. H + olefinic CH), 7.35 (t, J = 6.8 Hz, 2H, arom. H). ¹³C-NMR (100 MHz, CDCl₃) δ (ppm): 35.0, 44.6, 45.1, 46.0, 50.8, 54.9, 56.9, 57.1, 57.3, 62.2, 66.1, 75.4, 109.0, 114.9, 115.1, 115.2, 115.5, 122.0, 126.5, 127.3, 128.7, 128.8, 130.75, 130.83, 131.18, 131.22, 131.7, 131.8, 133.2, 133.95, 133.98, 136.3, 144.7, 160.6, 161.3, 163.0, 163.8, 176.2, 198.3. Elemental analysis: C₃₆H₃₉F₂N₅O₂ required C, 70.68; H, 6.43; N, 11.45, found C, 70.72; H, 6.48; N, 11.53.

1′′-Ethyl-4′-(4-fluorophenyl)-5′′-[(4-fluorophenyl)methylidene]-1′-methyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (46). Obtained from reaction of 18, 26 and 29. Reaction time 10 h, colorless microcrystals from ethanol, mp 194–196 °C, yield 85% (2.59 g). IR ν (cm⁻¹): 1699, 1684, 1609, 1508, 1466. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 0.72 (t, J = 7.1 Hz, 3H, NCH₂CH₃), 1.35–1.44 (m, 2H, piperidinyl H₂C-4), 1.49–1.56 (m, 4H, piperidinyl H₂C-3/5), 1.69 (d, J = 12.7 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.08 (sextet, J = 7.0, 12.4 Hz, 1H, upfield H of piperidinyl NCH₂CH₃), 2.15 (s, 3H, pyrrolidinyl NCH₃), 2.31 (sextet, J = 7.3, 14.5 Hz, 1H, downfield H of NCH₂CH₃), 2.54 (br s, 4H, piperidinyl NH₂C-2/6), 2.80 (dd, J = 2.4, 14.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.15 (dd, J = 1.5, 12.7 Hz, 1H, downfield H of piperidinyl H₂C-2′′), 3.27 (d, J = 14.5 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.36 (t, J = 8.1 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.91 (dd, J = 9.0, 10.7 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.07 (d, J = 12.7 Hz, 1H, upfield H of NCH₂N), 4.61 (d, J = 12.7 Hz, 1H, downfield H of NCH₂N), 4.80 (dd, J = 7.3, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.81 (d, J = 7.8 Hz, 1H, arom. H), 6.93–7.14 (m, 10H, 9 arom. H + olefinic CH), 7.41 (dd, J = 5.6, 8.3 Hz, 2H, arom. H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 11.3, 24.2, 26.0, 35.1, 45.3, 51.6, 52.5, 53.4, 56.5, 57.5, 63.2, 65.4, 75.9, 109.2, 115.0, 115.2, 115.4, 115.7, 121.9, 126.3, 127.4, 128.9, 131.2, 131.3, 131.9, 132.0, 133.27, 133.29, 134.3, 134.4, 136.7, 145.2, 160.4, 161.1, 163.6, 164.4, 176.2, 199.1. Elemental analysis: C₃₇H₄₀F₂N₄O₂ required C, 72.76; H, 6.60; N, 9.17, found C, 72.84; H, 6.64; N, 9.28.

1′′-Ethyl-4′-(4-fluorophenyl)-5′′-[(4-fluorophenyl)methylidene]-1′-methyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (47). Obtained from reaction of 18, 27 and 29. Reaction time 11 h, colorless microcrystals from methanol, mp 191–193 °C, yield 73% (2.23 g). IR ν (cm⁻¹): 1707, 1686, 1607, 1508, 1468. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 0.73 (t, J = 7.2 Hz, 3H, NCH₂CH₃), 1.68 (d, J = 12.7 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.09 (sextet, J = 7.0, 12.4 Hz, upfield H of NCH₂CH₃), 2.14 (s, 3H, pyrrolidinyl NCH₃), 2.32 (sextet, J = 7.3, 12.3 Hz, 1H, downfield H of NCH₂CH₃), 2.59 (br s, 4H, morpholinyl 2NCH₂), 2.79 (dd, J = 2.6, 14.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.15 (dd, J = 1.9, 12.7 Hz, 1H, downfield H of piperidinyl H₂C-2′′), 3.28 (d, J = 14.4 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.36 (dd, J = 7.4, 8.7 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.66 (t, J = 4.5 Hz, 4H, morpholinyl 2OCH₂), 3.89 (dd, J = 8.9, 10.9 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.06 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.62 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 4.80 (dd, J = 7.3, 10.9 Hz, 1H, pyrrolidinyl HC-4′), 6.78 (d, J = 7.6 Hz, 1H, arom. H), 6.95–7.41 (m, 12H, 11 arom. H + olefinic CH). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 11.4, 35.2, 45.2, 51.5, 51.7, 53.4, 56.5, 57.5, 62.6, 65.6, 67.0, 75.9, 109.0, 115.1, 115.4, 115.5, 115.8, 122.3, 126.4, 127.6, 129.0, 131.2, 131.3, 131.5, 132.0, 132.1, 133.18, 133.2, 134.2, 134.3, 136.8, 144.8, 160.4, 161.2, 163.7, 176.2, 185.4, 199.1. Elemental analysis: C₃₆H₃₈F₂N₄O₃ required C, 70.57; H, 6.25; N, 9.14, found C, 70.71; H, 6.28; N, 9.21.

4′-(4-Fluorophenyl)-5′′-[(4-fluorophenyl)methylidene]-1′-methyl-1-[(1-piperidinyl)methylene]-1′′-(1-propyl)-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (48). Obtained from reaction of 19, 26 and 29. Reaction time 15 h, colorless microcrystals from ethanol, mp 191–193 °C, yield 87% (2.70 g). IR ν (cm⁻¹): 1697, 1682, 1609, 1508, 1466. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 0.73 (t, J = 7.4 Hz, 3H, NCH₂CH₂CH₃), 1.02–1.18 (m, 2H, NCH₂CH₂CH₃), 1.35–1.62 (m, 6H, piperidinyl 3CH₂), 1.72 (d, J = 12.9 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.98 (sextet, J = 5.1, 10.5 Hz, 1H, upfield H of NCH₂CH₂CH₃), 2.08–2.16 (m, 4H, pyrrolidinyl NCH₃ + downfield H of NCH₂CH₂CH₃), 2.58 (br s, 4H, piperidinyl 2NCH₂), 2.83 (dd, J = 2.7, 14.7 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.14 (dd, J = 2.0, 12.8 Hz, 1H, downfield H of piperidinyl H₂C-2′′), 3.27 (d, J = 14.7 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.37 (dd, J = 7.5, 9.0 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.90 (dd, J = 9.0, 10.8 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.10 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.64 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 4.80 (dd, J = 7.5, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.80–7.44 (m, 13H, 12 arom. H + olefinic CH). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 11.9, 19.6, 24.2, 26.0, 35.2, 45.4, 52.6, 54.0, 57.0, 57.6, 60.1, 63.1, 65.3, 76.0, 109.2, 115.0, 115.3, 115.5, 115.8, 122.0, 126.3, 127.4, 128.9, 131.3, 131.4, 131.5, 131.54, 132.0, 132.1, 133.3, 134.4, 136.7, 145.3, 160.4, 161.1, 163.6, 164.5, 176.2, 199.3. Elemental analysis: C₃₈H₄₂F₂N₄O₂ required C, 73.05; H, 6.78; N, 8.97, found C, 73.19; H, 6.82; N, 9.08.

4′-(4-Fluorophenyl)-5′′-[(4-fluorophenyl)methylidene]-1′-methyl-1-[(4-morpholinyl)methylene]-1′′-(1-propyl)-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (49). Obtained from 19, 27 and 29. Reaction time 16 h, colorless microcrystals from methanol, mp 186–188 °C, yield 83% (2.60 g). IR ν (cm⁻¹): 1705, 1688, 1607, 1508, 1468. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 0.71 (t, J = 7.3 Hz, 3H, NCH₂CH₂CH₃), 0.94–1.06 (m, 1H, upfield H of NCH₂CH₂CH₃), 1.10–1.20 (m, 1H, downfield H of NCH₂CH₂CH₃), 1.68 (d, J = 12.7 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.98 (sextet, J = 5.1, 11.1 Hz, 1H, upfield H of NCH₂CH₂CH₃), 2.11–2.16 (m, 4H, pyrrolidinyl NCH₃ + downfield H of NCH₂CH₂CH₃), 2.58 (br s, 4H, morpholinyl 2NCH₂), 2.80 (dd, J = 2.6, 14.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.11 (dd, J = 1.8, 12.7 Hz, 1H, downfield H of piperidinyl H₂C-2′′), 3.25 (d, J = 14.5 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.35 (dd, J = 7.4, 8.7 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.64 (t, J = 4.6 Hz, 4H, morpholinyl 2OCH₂), 3.86 (dd, J = 8.9, 10.8 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.04 (d, J = 12.7 Hz, 1H, upfield H of NCH₂N), 4.61 (d, J = 12.7 Hz, 1H, downfield H of NCH₂N), 4.77 (dd, J = 7.3, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.75 (d, J = 8.0 Hz, 1H, arom. H), 6.92–7.39 (m, 12H, 11 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 11.5, 18.7, 34.4, 44.4, 50.9, 53.3, 56.4, 56.7, 59.2, 61.4, 64.4, 66.0, 75.1, 109.1, 114.8, 115.1, 115.3, 115.7, 121.5, 125.4, 126.7, 128.7, 130.9, 132.2, 134.4, 135.7, 144.6, 175.3, 198.0. Elemental analysis: C₃₇H₄₀F₂N₄O₃ required C, 70.91; H, 6.43; N, 8.94, found C, 71.03; H, 6.41; N, 9.13.

1′′-Benzyl-4′-(4-fluorophenyl)-5′′-[(4-fluorophenyl)methylidene]-1′-methyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (50). Obtained from reaction of 20, 26 and 29. Reaction time 10 h, pale yellow microcrystals from ethanol, mp 143–144 °C, yield 77% (2.59 g). IR ν (cm⁻¹): 1699, 1680, 1601, 1508, 1466. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.32–1.40 (m, 2H, piperidinyl H₂C-4), 1.45–1.54 (m, 4H, piperidinyl H₂C-3/5), 1.86 (d, J = 12.7 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.16 (s, 3H, pyrrolidinyl NCH₃), 2.47–2.55 (m, 4H, piperidinyl NH₂C-2/6), 2.74 (dd, J = 2.4, 14.6 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.19–3.34 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′ and upfield H of PhCH₂), 3.42 (t, J = 8.2 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.49 (d, J = 13.9 Hz, 1H, downfield H of PhCH₂), 3.92 (t, J = 9.8 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.00 (d, J = 12.8 Hz, 1H, upfield H of NCH₂N), 4.50 (d, J = 12.8 Hz, 1H, downfield H of NCH₂N), 4.79 (dd, J = 7.6, 10.6 Hz, 1H, pyrrolidinyl HC-4′), 6.80–7.47 (m, 18H, 17 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 23.6, 25.3, 34.4, 45.0, 51.6, 53.0, 56.7, 60.9, 62.2, 64.6, 75.0, 109.2, 114.8, 115.0, 115.2, 115.5, 121.5, 125.3, 126.6, 126.8, 127.9, 128.2, 128.7, 130.8, 130.9, 131.0, 131.1, 131.9, 132.0, 132.7, 134.4, 135.6, 135.8, 144.7, 159.5, 160.3, 162.7, 163.6, 175.3, 198.0. Elemental analysis: C₄₂H₄₂F₂N₄O₂ required C, 74.98; H, 6.29; N, 8.33, found C, 75.12; H, 6.34; N, 8.51.

1′′-Benzyl-4′-(4-fluorophenyl)-5′′-[(4-fluorophenyl)methylidene]-1′-methyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (51). Obtained from reaction 20, 27 and 29. Reaction time 12 h, pale yellow microcrystals from methanol, mp 172–174 °C, yield 83% (2.80 g). IR ν (cm⁻¹): 1705, 1684, 1607, 1508, 1466. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.85 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.14 (s, 3H, pyrrolidinyl NCH₃), 2.53–2.57 (m, 4H, morpholinyl 2NCH₂), 2.75 (d, J = 14.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.15–3.45 (m, 4H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′, upfield H of pyrrolidinyl H₂C-5′ and upfield H of PhCH₂), 3.50 (d, J = 14.0 Hz, 1H, downfield H of PhCH₂), 3.62 (t, J = 4.5 Hz, 4H, morpholinyl 2OCH₂), 3.91 (dd, J = 9.2, 10.4 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 3.98 (d, J = 12.8 Hz, 1H, upfield H of NCH₂N), 4.51 (d, J = 12.8 Hz, 1H, downfield H of NCH₂N), 4.79 (dd, J = 7.5, 10.6 Hz, 1H, pyrrolidinyl HC-4′), 6.80–7.46 (m, 18H, 17 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 34.4, 44.9, 50.9, 53.0, 56.7, 60.9, 61.6, 64.6, 65.9, 75.1, 109.1, 114.8, 115.1, 115.2, 115.5, 121.7, 125.3, 126.7, 126.9, 127.9, 128.3, 128.8, 131.0, 131.1, 131.9, 132.0, 134.3, 134.4, 135.7, 144.5, 159.5, 160.3, 162.7, 163.6, 175.4, 197.9. Elemental analysis: C₄₁H₄₀F₂N₄O₃ required C, 72.98; H, 5.97; N, 8.30, found C, 73.13; H, 6.02; N, 8.46.

1′,1′′-Dimethyl-4′-(4-methylphenyl)-5′′-[(4-methylphenyl)methylidene]-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (52). Obtained from reaction of 21, 26 and 29. Reaction time 10 h, almost colorless microcrystals from ethanol, mp 177–178 °C, yield 71% (2.09 g). IR ν (cm⁻¹): 1701, 1686, 1609, 1512, 1487, 1466. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.41 (br s, 2H, piperidinyl H₂C-4), 1.55–1.70 (m, 5H, piperidinyl H₂C-3/5 and upfield H of piperidinyl H₂C-2′′), 2.01 (s, 3H, piperidinyl NCH₃), 2.16 (s, 3H, pyrrolidinyl NCH₃), 2.32 (s, 3H, ArCH₃), 2.33 (s, 3H, ArCH₃), 2.64 (br s, 4H, piperidinyl NH₂C-2/6), 2.87 (dd, J = 2.6, 14.3 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.23–3.33 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′ and upfield H of pyrrolidinyl H₂C-5′), 3.92 (dd, J = 8.7, 11.1 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.23 (br s, 1H, upfield H of NCH₂N), 4.58 (d, J = 13.2 Hz, 1H, downfield H of NCH₂N), 4.82 (dd, J = 7.2, 11.4 Hz, 1H, pyrrolidinyl HC-4′), 6.88–7.29 (m, 13H, 12 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 20.5, 20.8, 23.7, 25.2, 34.3, 44.2, 44.5, 51.4, 65.3, 74.7, 109.0, 121.2, 125.9, 128.6, 128.8, 128.9, 129.8, 131.6, 132.1, 135.0, 135.6, 138.6, 144.6, 175.2, 197.4. Elemental analysis: C₃₈H₄₄N₄O₂ required C, 77.52; H, 7.53; N, 9.52, found C, 77.61; H, 7.59; N, 9.59.

1′,1′′-Dimethyl-4′-(4-methylphenyl)-5′′-[(4-methylphenyl)methylidene]-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (53). Obtained from reaction of 21, 27 and 29. Reaction time 12 h, colorless microcrystals from ethanol, mp 198–200 °C (lit. mp 198–200 °C ³⁸), yield 75% (2.21 g). IR ν (cm⁻¹): 1703, 1686, 1609, 1512, 1487, 1466. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.62 (d, J = 12.8 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.98 (s, 3H, piperidinyl NCH₃), 2.12 (s, 3H, pyrrolidinyl NCH₃), 2.29 (s, 3H, ArCH₃), 2.30 (s, 3H, ArCH₃), 2.54 (t, J = 4.6 Hz, 4H, morpholinyl 2NCH₂), 2.84 (dd, J = 2.6, 14.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.23–3.30 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′ and upfield H of pyrrolidinyl H₂C-5′), 3.62 (t, J = 4.6 Hz, 4H, morpholinyl 2OCH₂), 3.90 (dd, J = 8.8, 11.2 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.07 (d, J = 12.5 Hz, 1H, upfield H of NCH₂N), 4.50 (d, J = 12.5 Hz, 1H, downfield H of NCH₂N), 4.78 (dd, J = 7.0, 11.3 Hz, 1H, pyrrolidinyl HC-4′), 6.75 (d, J = 8.0 Hz, 1H, arom. H), 6.87 (d, J = 8.0 Hz, 2H, arom. H), 6.96–7.26 (m, 10H, 9 arom. H + olefinic CH). ¹³C-NMR (100 MHz, CDCl₃) δ (ppm): 21.1, 21.4, 35.0, 45.0, 45.1, 51.2, 56.7, 57.3, 62.5, 66.3, 66.8, 75.5, 108.7, 122.1, 126.7, 127.4, 128.7, 128.9, 129.2, 130.0, 132.4, 132.6, 135.2, 136.3, 137.6, 138.9, 144.5, 176.4, 198.4. Elemental analysis: C₃₇H₄₂N₄O₃ required C, 75.23; H, 7.17; N, 9.48, found C, 75.30; H, 7.26; N, 9.61.

1′,1′′-Dimethyl-4′-(4-methylphenyl)-5′′-[(4-methylphenyl)methylidene]-1-[1-(4-methylpiperazinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (54). Obtained from reaction of 21, 28 and 29. Reaction time 15 h, colorless microcrystals from methanol, mp 201–202 °C, yield 71% (2.14 g). IR ν (cm⁻¹): 1692, 1609, 1512, 1489, 1462. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.64 (d, J = 12.9 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.01 (s, 3H, piperidinyl NCH₃), 2.15 (s, 3H, pyrrolidinyl NCH₃), 2.24 (s, 3H, piperazinyl NCH₃), 2.32 (s, 3H, ArCH₃), 2.33 (s, 3H, ArCH₃), 2.37 (br s, 4H, piperazinyl 2NCH₂), 2.62 (br s, 4H, piperazinyl 2NCH₂), 2.86 (dd, J = 2.7, 14.7 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.26–3.33 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′ and upfield H of pyrrolidinyl H₂C-5′), 3.93 (dd, J = 8.7, 11.1 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.10 (d, J = 12.3 Hz, 1H, upfield H of NCH₂N), 4.55 (d, J = 12.3 Hz, 1H, downfield H of NCH₂N), 4.81 (dd, J = 6.8, 11.3 Hz, 1H, pyrrolidinyl HC-4′), 6.77–7.29 (m, 13H, 12 arom. H + olefinic CH). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 21.3, 21.5, 35.2, 45.3, 46.2, 51.0, 55.1, 57.0, 57.5, 62.5, 66.5, 75.7, 109.1, 122.2, 127.0, 127.5, 128.8, 129.1, 129.5, 130.2, 132.7, 132.9, 135.5, 136.5, 137.7, 139.0, 144.9, 176.5, 198.6. Elemental analysis: C₃₈H₄₅N₅O₂ required C, 75.59; H, 7.51; N, 11.60, found C, 75.71; H, 7.59; N, 11.68.

1′,1′′-Dimethyl-4′-(4-methoxyphenyl)-5′′-[(4-methoxyphenyl)methylidene]-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (55). Obtained from reaction of 22, 26 and 29. Reaction time 12 h, almost colorless microcrystals from ethanol, mp 175–177 °C, yield 82% (2.55 g). IR ν (cm⁻¹): 1701, 1682, 1607, 1510, 1489, 1464. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.35–1.42 (m, 2H, piperidinyl H₂C-4), 1.50–1.59 (m, 4H, piperidinyl H₂C-3/5), 1.64 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.01 (s, 3H, piperidinyl NCH₃), 2.15 (s, 3H, pyrrolidinyl NCH₃), 2.55 (t, J = 5.0 Hz, 4H, piperidinyl NH₂C-2/6), 2.88 (dd, J = 2.7, 14.1 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.20–3.34 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′, and upfield H of pyrrolidinyl H₂C-5′), 3.79 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.91 (dd, J = 8.7, 11.4 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.12 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.54 (d, J = 12.9 Hz, 1H, downfield H of NCH₂N), 4.79 (dd, J = 7.1, 11.3 Hz, 1H, pyrrolidinyl HC-4′), 6.79–7.34 (m, 13H, 12 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 23.7, 25.3, 34.3, 43.9, 44.6, 51.5, 54.9, 55.2, 56.1, 56.7, 56.8, 62.2, 65.1, 74.8, 109.0, 113.6, 113.9, 121.2, 126.0, 126.5, 127.0, 128.4, 129.7, 130.0, 130.8, 131.8, 136.5, 144.7, 158.0, 159.7, 175.3, 197.4. Elemental analysis: C₃₈H₄₄N₄O₄ required C, 73.52; H, 7.14; N, 9.02, found C, 73.59; H, 7.22; N, 9.12.

1′,1′′-Dimethyl-4′-(4-methoxyphenyl)-5′′-[(4-methoxyphenyl)methylidene]-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (56). Obtained from reaction of 22, 27 and 29. Reaction time 15 h, colorless microcrystals from ethanol, mp 202–204 °C, yield 71% (2.21 g). IR ν (cm⁻¹): 1701, 1682, 1607, 1510, 1489, 1464. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.66 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.02 (s, 3H, piperidinyl NCH₃), 2.16 (s, 3H, pyrrolidinyl NCH₃), 2.61 (br s, 4H, morpholinyl 2NCH₂), 2.89 (dd, J = 2.4, 14.1 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.21–3.37 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′, and‏ upfield H of pyrrolidinyl H₂C-5′), 3.66 (t, J = 4.4 Hz, 4H, morpholinyl 2OCH₂), 3.79 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 3.90 (t, J = 9.9 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.14 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.55 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 4.82 (dd, J = 6.9, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.80–7.32 (m, 13H, 12 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 34.3, 43.8, 44.6, 50.7, 54.9, 55.1, 65.2, 65.9, 74.8, 99.4, 108.9, 113.6, 113.9, 121.3, 125.9, 126.5, 127.0, 128.4, 129.7, 129.9, 130.7, 131.8, 136.5, 144.4, 157.9, 159.7, 175.4, 197.3. Elemental analysis: C₃₇H₄₂N₄O₅ required C, 71.36; H, 6.80; N, 9.00, found C, 71.43; H, 6.87; N, 9.14.

1′,1′′-Dimethyl-4′-(4-methoxyphenyl)-5′′-[(4-methoxyphenyl)methylidene]-1-[1-(4-methylpiperazinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (57). Obtained from reaction of 22, 28 and 29. Reaction time 20 h, colorless microcrystals from ethanol, mp 186–188 °C, yield 69% (2.19 g). IR ν (cm⁻¹): 1703, 1680, 1607, 1510, 1464. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.63 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.00 (s, 3H, piperidinyl NCH₃), 2.14 (s, 3H, pyrrolidinyl NCH₃), 2.24 (s, 3H, piperazinyl NCH₃), 2.38 (br s, 4H, piperazinyl 2NCH₂), 2.62 (br s, 4H, piperazinyl 2NCH₂), 2.87 (dd, J = 2.3, 14.2 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.21–3.32 (m, 3H, downfield H's of piperidinyl H₂C-2′′, H₂C-6′′, and upfield H of pyrrolidinyl H₂C-5′), 3.78 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 3.90 (dd, J = 8.9, 11.0 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.11 (d, J = 12.5 Hz, 1H, upfield H of NCH₂N), 4.56 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 4.78 (dd, J = 7.0, 11.1 Hz, 1H, pyrrolidinyl HC-4′), 6.76–7.12 (m, 11H, 10 arom. H + olefinic CH), 7.31 (d. H, J = 9 Hz, 2H, arom.). ¹³C-NMR (100 MHz, CDCl₃) δ (ppm): 35.1, 44.7, 45.2, 46.0, 50.8, 54.9, 55.20, 55.24, 56.9, 57.3, 57.4, 62.2, 66.1, 75.7, 108.8, 113.6, 113.7, 121.9, 126.7, 127.3, 127.9, 128.6, 130.3, 130.4, 131.5, 131.9, 137.3, 144.6, 158.4, 159.9, 176.3, 198.4. Elemental analysis: C₃₈H₄₅N₅O₄ required C, 71.79; H, 7.13; N, 11.01, found C, 71.82; H, 7.21; N, 11.09.

4′-(2,5-Dimethoxyphenyl)-5′′-[(2,5-dimethoxyphenyl)methylidene]-1′,1′′-dimethyl-1-[(1-piperidinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (58). Obtained from reaction of 23, 26 and 29. Reaction time 16 h, yellow microcrystals from ethanol, mp 139–140 °C, yield 85% (2.89 g). IR ν (cm⁻¹): 1694, 1607, 1593, 1495, 1466. ¹H-NMR (300 Hz, CDCl₃) δ (ppm): 1.35–1.43 (m, 2H, piperidinyl H₂C-4), 1.50–1.59 (m, 4H, piperidinyl H₂C-3/5), 1.78 (d, J = 12.6 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.88 (s, 3H, piperidinyl NCH₃), 2.14 (s, 3H, pyrrolidinyl NCH₃), 2.55 (t, J = 4.8 Hz, 4H, piperidinyl NH₂C-2/6), 2.82 (dd, J = 2.7, 14.7 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 2.99 (dd, J = 2.0, 12.5 Hz, 1H, downfield H of piperidinyl H₂C-2′′), 3.15 (d, J = 14.7 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.33 (dd, J = 7.5, 8.7 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.67 (s, 3H, OCH₃), 3.71 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 4.06 (dd, J = 8.9, 10.7 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.11 (d, J = 12.3 Hz, 1H, upfield H of NCH₂N), 4.53 (d, J = 12.9 Hz, 1H, downfield H of NCH₂N), 4.88 (dd, J = 7.5, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.41 (d, J = 2.1 Hz, 1H, arom. H), 6.72–7.55 (m, 10H, 9 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 23.7, 25.3, 34.3, 45.0, 51.5, 55.2, 55.4, 55.8, 56.0, 56.7, 56.9, 62.2, 63.1, 75.7, 109.0, 111.0, 111.2, 112.1, 114.9, 115.2, 115.5, 121.4, 124.2, 125.6, 126.8, 128.1, 128.4, 131.5, 132.5, 144.8, 151.8, 151.9, 152.3, 153.0, 175.4, 195.7. Elemental analysis: C₄₀H₄₈N₄O₆ required C, 70.57; H, 7.11; N, 8.23, found C, 70.68; H, 7.31; N, 8.37.

4′-(2,5-Dimethoxyphenyl)-5′′-[(2,5-dimethoxyphenyl)methylidene]-1′,1′′-dimethyl-1-[(4-morpholinyl)methylene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (59). Obtained from reaction of 23, 27 and 29. Reaction time 15 h, pale yellow microcrystals from ethanol, mp 181–183 °C, yield 71% (2.42 g). IR ν (cm⁻¹): 1699, 1605, 1578, 1493, 1464. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.78 (d, J = 12.9 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.89 (s, 3H, piperidinyl NCH₃), 2.13 (s, 3H, pyrrolidinyl NCH₃), 2.60 (t, J = 4.5 Hz, 4H, morpholinyl 2NCH₂), 2.82 (dd, J = 2.7, 14.7 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 2.99 (dd, J = 2.1, 12.6 Hz, 1H, downfield H of piperidinyl H₂C-2′′), 3.15 (d, J = 14.4 Hz, 1H, downfield H of piperidinyl H₂C-6′′), 3.34 (dd, J = 7.5, 8.4 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.67–3.69 (m, 7H, morpholinyl 2OCH₂ + OCH₃), 3.71 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 4.05 (dd, J = 8.7, 10.8 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.12 (d, J = 12.3 Hz, 1H, upfield H of NCH₂N), 4.54 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 4.88 (dd, J = 7.4, 10.7 Hz, 1H, pyrrolidinyl HC-4′), 6.41 (d, J = 2.4 Hz, 1H, arom. H), 6.73–7.55 (m, 10H, 9 arom. H + olefinic CH). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 34.4, 45.0, 50.8, 55.2, 55.3, 55.4, 55.9, 56.8, 56.9, 61.6, 63.2, 65.9, 75.7, 108.9, 111.0, 111.2, 112.2, 114.9, 115.2, 115.4, 121.6, 124.1, 125.5, 126.9, 128.2, 128.4, 131.6, 132.4, 144.6, 151.8, 151.9, 152.3, 153.0, 175.5, 195.7. Elemental analysis: C₃₉H₄₆N₄O₇ required C, 68.60; H, 6.79; N, 8.21, found C, 68.82; H, 6.74; N, 8.35.

1′,1′′-Dimethyl-1-[(1-piperidinyl)methylene]-4′-(2-thienyl)-5′′-[(2-thienyl)methylidene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (60). Obtained from reaction of 24, 26 and 29. Reaction time 14 h, pale yellow microcrystals from methanol, mp 147–148 °C, yield 68% (1.95 g). IR ν (cm⁻¹): 1694, 1676, 1614, 1578, 1470. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.35–1.45 (m, 2H, piperidinyl H₂C-4), 1.51–1.60 (m, 4H, piperidinyl H₂C-3/5), 1.89 (d, J = 13.2 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.09 (s, 3H, piperidinyl NCH₃), 2.15 (s, 3H, pyrrolidinyl NCH₃), 2.57 (t, J = 5.1 Hz, 4H, piperidinyl NH₂C-2/6), 2.94 (dd, J = 2.6, 15.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.28–3.34 (m, 2H, downfield H's of piperidinyl H₂C-2′′ and H₂C-6′′), 3.45 (dd, J = 7.1, 8.6 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.92 (dd, J = 8.7, 11.1 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.14 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.61 (d, J = 12.3 Hz, 1H, downfield H of NCH₂N), 5.07 (dd, J = 7.5, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.82–7.54 (m, 11H, 10 arom. H + olefinic CH). ¹³C-NMR (100 MHz, CDCl₃) δ (ppm): 24.1, 25.8, 34.8, 40.9, 45.2, 52.3, 56.0, 57.1, 58.3, 63.1, 65.3, 75.8, 108.9, 121.9, 123.9, 126.0, 126.2, 126.7, 127.1, 127.8, 128.7, 129.5, 129.8, 130.4, 132.7, 138.5, 141.3, 145.1, 176.1, 197.0. Elemental analysis: C₃₂H₃₆N₄O₂S₂ required C, 67.10; H, 6.34; N, 9.78, found C, 67.36; H, 6.31; N, 9.89.

1′,1′′-Dimethyl-1-[(4-morpholinyl)methylene]-4′-(2-thienyl)-5′′-[(2-thienyl)methylidene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (61). Obtained from reaction of 24, 27 and 29. Reaction time 12 h, pale yellow microcrystals from methanol, mp 210–212 °C, yield 71% (2.04 g). IR ν (cm⁻¹): 1694, 1676, 1612, 1578, 1468. ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 1.89 (d, J = 12.9 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 2.10 (s, 3H, piperidinyl NCH₃), 2.14 (s, 3H, pyrrolidinyl NCH₃), 2.61 (t, J = 4.7 Hz, 4H, morpholinyl 2NCH₂), 2.94 (dd, J = 2.6, 15.5 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.29–3.36 (m, 2H, downfield H's of piperidinyl H₂C-2′′, and H₂C-6′′), 3.44 (dd, J = 7.2, 8.7 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.68 (t, J = 4.7 Hz, 4H, morpholinyl 2OCH₂), 3.90 (dd, J = 8.6, 11.0 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.15 (d, J = 12.9 Hz, 1H, upfield H of NCH₂N), 4.61 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 5.08 (dd, J = 7.2, 10.8 Hz, 1H, pyrrolidinyl HC-4′), 6.80–7.50 (m, 11H, 10 arom. H + olefinic CH). Elemental analysis: C₃₁H₃₄N₄O₃S₂ required C, 64.78; H, 5.96; N, 9.75, found C, 64.83; H, 6.04; N, 9.90.

1′,1′′-Dimethyl-1-[(1-piperidinyl)methylene]-4′-(3-pyridinyl)-5′′-[(3-pyridinyl)methylidene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (62). Obtained from reaction of 25, 26 and 29. Reaction time 16 h, colorless microcrystals from methanol, mp 119–120 °C, yield 72% (2.03 g). IR ν (cm⁻¹): 1701, 1686, 1611, 1481, 1466. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.32 (br s, 2H, piperidinyl H₂C-4), 1.50 (br s, 4H, piperidinyl H₂C-3/5), 1.57 (d, J = 12.7 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.92 (s, 3H, piperidinyl NCH₃), 2.09 (s, 3H, pyrrolidinyl NCH₃), 2.50 (br s, 4H, piperidinyl NH₂C-2/6), 2.81 (dd, J = 2.2, 14.6 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.16 (t, J = 12.6 Hz, 2H, downfield H's of piperidinyl H₂C-2′′ and H₂C-6′′), 3.30 (t, J = 7.8 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.85 (t, J = 9.8 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.11 (d, J = 13.1 Hz, 1H, upfield H of NCH₂N), 4.46 (d, J = 12.7 Hz, 1H, downfield H of NCH₂N), 4.74 (dd, J = 7.0, 10.7 Hz, 1H, pyrrolidinyl HC-4′), 6.82–8.18 (m, 10H, 9 arom. H + olefinic CH), 8.42 (dd, J = 4.4, 10.9 Hz, 2H, arom. H), 8.49 (s, 1H, arom. H). ¹³C-NMR (75 MHz, DMSO-d₆) δ (ppm): 23.7, 25.2, 34.3, 42.5, 44.4, 51.4, 56.0, 56.2, 57.1, 62.2, 65.2, 74.6, 109.3, 121.4, 123.3, 125.4, 126.6, 128.7, 130.2, 132.9, 133.8, 134.9, 136.3, 136.4, 144.7, 148.1, 149.2, 150.1, 150.4, 175.2, 197.3. Elemental analysis: C₃₄H₃₈N₆O₂ required C, 72.57; H, 6.81; N, 14.93, found C, 72.69; H, 6.74; N, 15.06.

1′,1′′-Dimethyl-1-[(4-morpholinyl)methylene]-4′-(3-pyridinyl)-5′′-[(3-pyridinyl)methylidene]-dispiro[3H-indole-3,2′-pyrrolidine-3′,3′′-piperidine]-2(1H),4′′-dione (63). Obtained from reaction of 25, 27 and 29. Reaction time 14 h, colorless microcrystals from methanol, mp 192–194 °C, yield 78% (2.20 g). IR ν (cm⁻¹): 1699, 1680, 1607. ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 1.58 (d, J = 12.4 Hz, 1H, upfield H of piperidinyl H₂C-2′′), 1.93 (s, 3H, piperidinyl NCH₃), 2.09 (s, 3H, pyrrolidinyl NCH₃), 2.52 (br s, 4H, morpholinyl 2NCH₂), 2.82 (dd, J = 2.3, 14.4 Hz, 1H, upfield H of piperidinyl H₂C-6′′), 3.15 (t, J = 14.4 Hz, 2H, downfield H's of piperidinyl H₂C-2′′, and H₂C-6′′), 3.32 (t, J = 7.8 Hz, 1H, upfield H of pyrrolidinyl H₂C-5′), 3.58 (t, J = 4.2 Hz, 4H, morpholinyl 2OCH₂), 3.84 (t, J = 9.7 Hz, 1H, downfield H of pyrrolidinyl H₂C-5′), 4.07 (d, J = 12.6 Hz, 1H, upfield H of NCH₂N), 4.47 (d, J = 12.6 Hz, 1H, downfield H of NCH₂N), 4.75 (dd, J = 7.0, 10.5 Hz, 1H, pyrrolidinyl HC-4′), 6.76–7.85 (m, 9H, 8 arom. H + olefinic CH), 8.16 (s, 1H, arom. H), 8.43 (dd, J = 4.2, 16.0 Hz, 2H, arom. H), 8.52 (s, 1H, arom. H). ¹³C-NMR (100 MHz, CDCl₃) δ (ppm): 34.9, 43.1, 45.1, 51.1, 56.5, 56.8, 57.5, 62.4, 66.1, 66.6, 75.3, 109.1, 122.3, 123.2, 123.6, 126.0, 127.5, 129.2, 130.8, 133.8, 134.5, 135.1, 136.5, 137.6, 144.5, 147.6, 149.4, 150.0, 150.5, 176.1, 197.6. Elemental analysis: C₃₃H₃₆N₆O₃ required C, 70.19; H, 6.43; N, 14.88, found C, 70.34; H, 6.42; N, 15.03.

Single crystal X-ray

Intensity data for 46 and 48 were collected at room temperature (298 K) on an Enraf-Nonius 590 diffractometer with a Kappa CCD detector using graphite monochromated Mo-Kα (λ = 0.71073 Å) radiation.⁵³ Data were recorded in the rotation mode using the φ and ω scan technique with 2θ_max = 34.9° and 29.5° for 46 and 48, respectively. Data were corrected for absorption effects using the multi-scan method SADABS.⁵⁴ The structures were solved by direct methods using SHELX⁵⁵ implemented in the CRYSTALS program suit.⁵⁶ The refinement was carried out by the full-matrix least-squares method with anisotropic displacement parameters for all non-hydrogen atoms based on F² using CRYSTALS. The hydrogen atoms were positioned geometrically in their idealized positions.⁵⁷ The general-purpose crystallographic tool PLATON⁵⁸ was used for the structure analysis and presentation of the results. ORTEP-3 for Windows⁵⁹ and MERCURY⁶⁰ programs were used for molecular graphics representations. Details of the data collection and refinement are given in ESI† Table S4.

Antitumor activity screening

Antitumor properties of the synthesized compounds were screened by the National Cancer Institute, Cairo University, Egypt, using the reported in vitro Sulfo-Rhodamine-B (SRB) standard method adopting HeLa (cervical) and HepG2 (liver) carcinoma cell lines.^11–20 Cells were seeded in 96 well microtiter plates at a concentration of 5 × 10⁴ to 10⁵ cell per well in a fresh medium and left for 24 h before treatment with the tested compounds to allow attachment of cells to the wall of the plate. The tested compounds were dissolved in dimethylsulfoxide (DMSO) and diluted 1000 fold in the assay. Different concentrations of the compounds under test (0, 5, 12.5, 25, and 50 μg ml⁻¹) were added to the cell monolayer. Triplicate wells were prepared for each individual dose. The monolayer cells were incubated with the tested compounds for 48 h at 37 °C, in atmosphere of 5% CO₂. After 48 h, the cells were fixed, washed and stained with sulfo-rhodamine-B stain. Excess stain was washed with acetic acid. The attached stain was recovered with Tris–EDTA buffer. Cell survival and drug activity were determined by measuring the color intensity spectrophotometrically at 564 nm using an ELISA microplate reader (Meter tech. Σ 960, USA). Data are collected as mean values for experiments that performed in three replicates for each individual dose which measured by SRB assay. Control experiments did not exhibit significant change compared to the DMSO vehicle. Doxorubicin hydrochloride and cisplatin were used as standard references during the present in vitro bioactivity screening assay. The percentage of cell survival was calculated according to eqn (2).

Surviving fraction = optical density (O.D.) of treated cells/O.D. of control cells (2)

The IC₅₀ (concentration required to produce 50% inhibition of cell growth compared to control experiment) was determined using Graph-Pad PRISM version-5 software. Statistical calculations for determination of the mean and standard error values were determined by SPSS 16 software. The observed antitumor properties were presented in Table 3 (Fig. S114 and S115†).

Computational chemistry

Structure optimization. Computational chemistry calculations using both AM1 and PM3 methods were undertaken for structural optimization. The geometry of the bio-active compounds 30–63 was optimized by the molecular mechanics force field (MM⁺), followed by either semi-empirical AM1 or PM3 methods implemented in the HyperChem 8.0 package. The structures were fully optimized without constraining any parameters, thus bringing all geometric variables to their equilibrium values. The energy minimization protocol employed the Polake–Ribiere conjugated gradient algorithm. Convergence to a local minimum was achieved when the energy gradient was ≤0.01 kcal mol⁻¹. The RHF (Restricted Hartree–Fock) method was used in the spin pairing for the two semi-empirical tools.^31,32,38,61

2D-QSAR. QSAR study was undertaken utilizing comprehensive descriptors for structural and statistical analysis (CODESSA-Pro) software. Bio-active indole-based analogues (compounds 30, 31, 33–40, 42–49 and 51–63) against HeLa (cervical carcinoma) cell line were used as a training set for constructing the QSAR model. Compounds 32, 41 and 50 (i.e. about 10% of the training set) were used as an external test set for validating the attained QSAR model. Geometry of both training and test set compounds was initially optimized by AM1 technique then, exported to CODESSA-Pro that includes MOPAC capability for the final geometry optimization. CODESSA-Pro calculated 714 molecular descriptors (constitutional, topological, geometrical, charge-related, semi-empirical, molecular-type, atomic-type and bond-type descriptors) for the exported 31 training set antitumor active agents. Different mathematical transformations [including property, 1/property, log(property) and 1/log(property)] of the experimentally observed antitumor property/activity (IC₅₀, μM) of the training set compounds were utilized searching for the best QSAR model. The best multi-linear regression (BMLR) technique was utilized which is a stepwise search for the best n-parameter regression equations (where n stands for the number of descriptors used), based on the highest R² (squared correlation coefficient), R²cvOO (squared cross-validation “leave one-out, LOO” coefficient), R²cvMO (squared cross-validation “leave many-out, LMO” coefficient), F (Fisher statistical significance criteria) values, and s² (standard deviation). The QSAR models up to 5-descriptor model describing the bio-activity of the antitumor indole-based active agents were generated (obeying the thumb rule of 6 : 1, which is the ratio between the data points and the number of QSAR descriptor).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Including spectral charts, figures of structures optimized by different computational techniques, 3D-pharmacophore mapped models, tables of geometrical parameters and tables of molecular descriptor values of 2D-QSAR model. CCDC 1455157 and 1455158. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra07061b

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