European Journal of Medicinal Chemistry 74 (2014) 50e64

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Original article

Novel spirooxindoleepyrrolidine compounds: Synthesis, anticancer and molecular docking studies Y. Arun a, K. Saranraj a, C. Balachandran b, P.T. Perumal a, * a b

Organic Chemistry Division, CSIR-Central Leather Research Institute, Chennai 600020, India Division of Microbiology and Cancer Biology, Entomology Research Institute, Loyola College, Chennai 600034, India

a r t i c l e i n f o

a b s t r a c t

Article history: Received 7 November 2013 Received in revised form 17 December 2013 Accepted 22 December 2013 Available online 3 January 2014

Novel spirooxindoleepyrrolidine compounds have been synthesised through 1,3-dipolar cycloaddition of azomethine ylides generated from isatin and sarcosine or thioproline with the dipolarophile 3-(1Himidazol-2-yl)-2-(1H-indole-3-carbonyl)acrylonitrile under the optimised reaction condition. Synthesised compounds were evaluated for their anticancer activity against A549 human lung adenocarcinoma cancer cell line. Among the 29 tested compounds 4j, 6b and 6h showed very high activity 66.3%, 64.8% and 66.3% at 25 mg/mL concentration against A549 lung adenocarcinoma cancer cell line. These spirooxindoleepyrrolidine compounds can be promising therapeutic agents for A549 lung adenocarcinoma cancer cell line. Ó 2014 Elsevier Masson SAS. All rights reserved.

Keywords: Spirooxindole Pyrrolidine Multicomponent reaction Azomethine ylide cycloaddition Anticancer activity Molecular docking

1. Introduction Spirooxindole represents essential substructures for the synthesis of biologically important synthetic and natural compounds in drug discovery. Specifically, spirooxindoleepyrrolidines are found in a number of biologically active natural (Fig. 1) and synthetic products [1e8]. Interestingly, synthesised spirooxindolee pyrrolidine compounds have been identified as antimicrobial [9], anticancer [10e12], anti-inflammatory [13], antimycobacterial [14] and acetylcholinesterase (AChE) inhibitor [15,16]. The prevalence of these structures has resulted in the production of diverse libraries of small molecules for biological evaluation. Cancer is unregulated and uncontrolled cell growth and the reason for more deaths in worldwide. In particular, lung cancer is the leading cause of death from cancer and it occurs more commonly after the age of 60 years. But, some people are at a higher risk of developing lung cancer early because of their smoking habit and their life style changes. Generally, curing of cancer is difficult because of the side effect of drugs on the normal cells and makes some other abnormalities in our body and also cancer drugs have to be cheaper and more effective. Hence, developing the new therapeutic drugs for cancer treatment is more

* Corresponding author. Tel./fax: þ91 44 24913289. E-mail address: [email protected] (P.T. Perumal). 0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.ejmech.2013.12.027

important to improve the efficiency and efficacy of the drugs on the cancer cells [17]. Chemists are facing a major challenge to synthesis biologically important compounds because of multistep processes, selectivity of the desire product over to side products and tedious separation and purification steps to attain highly pure drug molecules. Conventionally, the drugs have been synthesised by multistep reaction sequences which are associated with difficult isolation of the desire product, low yields and high cost. In modern approach, the multicomponent reactions (MCRs) are useful to synthesise biologically important novel compounds. In contrast with linear-type syntheses, the MCR strategy involves the rapid combination of three or more simple molecules and produce structurally complex and diverse molecules in a single step reaction [18e20].

2. Results and discussion 2.1. Chemistry In continuation of our studies in the synthesis of novel biologically important heterocyclic compounds especially spirooxindoles [21] using multicomponent 1,3-dipolar azomethine ylide cycloaddition reaction [9,10,22], we herein report the one pot tandem reaction of 1,3-dipolar cycloaddition reaction to form the imidazole appended novel spirooxindoleepyrrolidine derivatives. In the

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51

Fig. 1. Naturally occurring spirooxindoleepyrrolidine compounds.

Scheme 1. Synthesis of spirooxindoleepyrrolidines (4aeo) from substituted isatin 1aeh, sarcosine 2 and dipolarophile 3a or 3b.

present investigation, the 1,3-dipolar cycloaddition of azomethine ylides, generated insitu via decarboxylative condensation of substituted isatins 1aeh and sarcosine 2 to 3-(1H-imidazol-2-yl)2-(1H-indole-3-carbonyl)acrylonitrile 3a or 3-(1H-imidazol-2-yl)2-(5-methoxy-1H-indole-3-carbonyl)acrylonitrile 3b in ethanol afforded novel spirooxindoleepyrrolidine 4aeo in high yields (Scheme 1). Different solvents such as methanol, ethanol, dimethyl sulfoxide and acetonitrile were used to optimise the reaction condition. Among these, ethanol is the suitable solvent to give high yields at minimum time and also purification of the product by precipitation is much easier when compared to dimethyl sulfoxide and acetonitrile. Finally, this reaction was performed by heating an equimolar mixture of substituted isatins 1aeh, sarcosine 2 and 3-(1Himidazol-2-yl)-2-(1H-indole-3-carbonyl)acrylonitrile 3a or 3-(1Himidazol-2-yl)-2-(5-methoxy-1H-indole-3-carbonyl)acrylonitrile 3b in ethanol under reflux for 1e2 h. After completion of the reaction (TLC), the reaction mixture was poured into ice-water, the resulting solid was filtered off and washed with ethanol to obtain pure spirooxindoleepyrrolidine derivatives 4aeo in 82e94% yields (Table 1). To further explore the potential of this protocol for novel spirooxindoleepyrrolidine synthesis, this reaction was further explored by heating an equimolar mixture of substituted isatin 1ae h, thioproline 5 and 3-(1H-imidazol-2-yl)-2-(1H-indole-3carbonyl)acrylonitrile 3a or 3-(1H-imidazol-2-yl)-2-(5-methoxy1H-indole-3-carbonyl)acrylonitrile 3b in ethanol under reflux for 1.5 h and obtained pure spirooxindoleepyrrolidine derivatives 6ae n in 82e95% yields (Scheme 2). The results are summarised in Table 2. The two possible regio approaches of 1,3-dipole and dipolarophile were shown in path A and B in Scheme 3. In the dipolarophile, electron density of the b-position is decreased by the carbonyl and nitrile groups. This leads to regioselective product

which cyclises only via path B rather than path A. But, two diastereomers are possible for this regioselective product which were shown in path X and Y in Scheme 3. The transition state formed by path Y stabilised by secondary orbital interaction between carbonyl groups of 1,3-dipole and dipolarophile, whereas no such secondary orbital interaction is possible in path X, hence the reaction proceeds via path Y and only leading to regio- and diastereoselective spirooxindoleepyrrolidine products. The structure of novel spirooxindoleepyrrolidine derivatives by 1,3-dipolar cycloaddition of azomethine ylide was elucidated with the help of IR, 1H NMR, 13C NMR and Mass data as illustrated for 4a. In the IR spectrum, the sharp absorption bands at 1720 and 1631 cm1 correspond to C]O stretching frequency of ketone and amide groups and the absorption band at 2243 cm1 corresponds to C^N stretching frequency. Also the absorption bands at 3394 and 3290 cm1 correspond to the NeH groups present in the product 4a. In the 1H NMR spectrum, a singlet in the region d: 10.48 ppm confirmed the presence of eNH proton of spirooxindoleepyrrolidine and the two singlets in the region d: 11.95 & 11.86 ppm confirmed the presence of two eNH proton of imidazole and indole. The peaks in the range of d: 6.65e8.15 ppm shows the 11 aromatic protons. The dd peak at d: 5.37 ppm with the J value 10.3 and 7.4 Hz for a proton shows the presence of hydrogen attached with imidazole ring. The two triplets at d: 3.91 and 3.53 ppm with the J value 7.9 and 9.5 Hz for two protons showed the presence of pyrrolidine ring eCH2 group. The signal at d: 2.10 ppm for three protons confirmed the presence of eNCH3 group. The 13C NMR, the peak at d: 76.0 ppm corresponds to the spiro carbon of spirooxindoleepyrrolidine and the peak at d: 64.9 ppm corresponds to the another spiro carbon of the compound 4a. The peaks at d: 181.0 and 174.9 ppm confirmed the presence of two carbonyl groups. In DEPT 135 and 90, the peaks at d: 54.6 & 35.0 ppm confirms the presence of eCH2 and eNCH3 groups respectively. A distinguishing peak observed at m/z: 437.17050 in

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Table 1 Synthesis of spirooxindoleepyrrolidines (4aeo) from substituted isatin 1aeh, sarcosine 2 and dipolarophile 3a or 3b. Entry

Isatin

Dipolarophile

Producta

Time (min)

1

1a

3a

90

88

90

89

90

84

90

94

90

82

90

91

110

89

110

84

4a

3a

2

Yield (%)b

1b 4b

3a

3

1c 4c

3a

4

1d 4d

3a

5

1e 4e

3a

6

1f 4f

3a

7

1g 4g

3a

8

1h 4h

Y. Arun et al. / European Journal of Medicinal Chemistry 74 (2014) 50e64

53

Table 1 (continued ) Entry

Isatin

Dipolarophile

Producta

9

Time (min)

Yield (%)b

110

82

120

90

120

83

110

86

120

82

110

85

110

85

1a 4i

3b

3b

10

1b 4j

3b

11

1c 4k

3b

12

1d 4l

3b

13

1e 4m

3b

14

1f 4n

3b

15

1g 4o a b

All the products were characterised by Mass, IR and NMR spectrum. Isolated yield.

the high resolution mass spectrum corresponds to [M þ H]þ ion of the product 4a. The structure determined from X-ray crystallographic study of the single crystal of the derivative 4a confirms the structure deduced from the spectroscopic data (Fig. 2) [23].

2.2. Pharmacology In the course of identifying various novel antitumour agents, we are particularly interested in the present work with spirooxindolee

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Y. Arun et al. / European Journal of Medicinal Chemistry 74 (2014) 50e64

Scheme 2. Synthesis of spirooxindoleepyrrolidines (6aen) from substituted isatin 1aeh, thioproline 5 and dipolarophile 3a or 3b.

pyrrolidine derivatives which have been identified as a new class of anticancer chemotherapeutic agents with significant therapeutic efficacy against A549 human lung adenocarcinoma cancer cell line.

2.2.1. Anticancer activity Anticancer activity studies have been carried out for the synthesised 29 novel spirooxindoleepyrrolidine compounds against A549 lung adenocarcinoma cancer cell line [10]. The studies

Table 2 Synthesis of spirooxindoleepyrrolidines (6aen) from substituted isatin 1aeh, thioproline 5 and dipolarophile 3a or 3b. Entry

Isatin

Dipolarophile

Producta

Time (min)

1

1a

3a

90

91

110

90

100

85

100

88

110

85

120

86

6a

3a

2

Yield (%)b

1b 6b

3a

3

1c 6c

3a

4

1d 6d

3a

5

1e 6e

3a

6

1f 6f

Table 2 (continued ) Entry

Isatin

7

Dipolarophile

Producta

3a

Time (min)

Yield (%)b

120

83

90

95

100

92

90

91

100

89

90

90

120

88

120

82

1g 6g

3a

8

1h 6h

9

1a 3b

6i

3b

10

1b 6j

3b

11

1c 6k

3b

12

1d 6l

3b

13

1e 6m

3b

14

1g 6n a b

All the products were characterised by Mass, IR and NMR spectrum. Isolated yield.

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Y. Arun et al. / European Journal of Medicinal Chemistry 74 (2014) 50e64

Scheme 3. Plausible approach of 1,3-dipole and dipolarophile for the formation of regio- and diastereoselective spirooxindoleepyrrolidines.

revealed that maximum cytotoxicity against A549 lung adenocarcinoma cancer cell line was observed at 100 to 25 mg/mL concentration. All concentrations used in the experiment could decrease the cell viability significantly (P < 0.05) in a concentrationdependent manner. All compounds showed good cytotoxicity activity against tested cell line, however some of the synthesised compounds showed prominent cytotoxic activity in vitro against A549 adenocarcinoma lung cancer cell line (Fig. 3) (Table 3). Synthesised compounds 4d, 4g, 4k, 4n, 6c and 6k showed IC50 value at 100 mg/mL but compounds 4h, 6f, 6g and 6m showed IC50 value even at 50 mg/mL. Interestingly, among all the synthesised spirooxindoleepyrrolidine compounds 4i, 4j, 6a, 6b, 6h, 6i, 6j and 6l showed very good activity with IC50 value at 25 mg/mL. In general, eOeMe substitution in the indole ring (R3) shows better activity than the unsubstituted indole products. Also, substitution of

methyl group in spirooxindole ring (R1) and substitution of eOeMe group in indole ring (R3) increases the activity of the product, particularly, compounds 4j and 6h show better activities. Interestingly, when sarcosine was replaced with thioproline, the products containing thiazolidine ring show better activity. In particular, among the tested compounds 4j, 6b and 6h showed very high activity 66.3%, 64.8% and 66.3% at 25 mg/mL concentration against A549 lung adenocarcinoma cancer cell line. These spirooxindolee pyrrolidine compounds can be promising therapeutic agents for A549 lung adenocarcinoma cancer cell line. 2.2.2. DNA fragmentation DNA purified from cells treated with 25 mg/mL of synthesised compounds 4h, 4i, 4j, 6a, 6b, 6f, 6h, 6i, 6j, 6l and 6g for 24 h was subjected to agarose gel electrophoresis to assess DNA fragmentation [24]. DNA fragmentation was observed for the synthesised compounds 4h, 6a, 6b, 6h, 6i and 6l. We have shown that the synthesised compounds inhibit the growth of human A549 lung adenocarcinoma cells and causes apoptotic DNA fragmentation (Fig. 4). 2.2.3. Apoptosis analysis of A549 cells One of the active synthesised spirooxindoleepyrrolidine compound 6b was studied for apoptosis in A549 lung adenocarcinoma cancer cells using confocal laser scanning microscope. Cancer cells were treated with or without 25 mg/mL of compound 6b for 24 h. Then apoptotic cells were identified using FITC (Fluorescein Isothiocyanate) and PI (Propidium Iodide) fluorescence under confocal laser scanning microscope. The results revealed that the compound 6b treated cells are showing apoptosis (Fig. 5). 2.3. Molecular docking studies

Fig. 2. ORTEP diagram of synthesised compound 4a.

All the synthesised novel spirooxindoleepyrrolidine compounds were subjected to molecular docking studies using the AutoDock Tools (ADT) version 1.5.6 and AutoDock version 4.2.5.1 docking program [25] to investigate the potential binding mode of inhibitors. Anaplastic Lymphoma Kinase (ALK) receptor which is tyrosine kinase receptor is an attractive oncology target for therapeutic intervention due to its critical roles of aberrant signalling in cancer [26].

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Fig. 3. Comparison of anticancer activity of synthesised spirooxindoleepyrrolidines (4aeo & 6aen) against A549 cells.

In order to verify the reproducibility of the docking calculations, the bound ligand (crizotinib) was extracted from the complexes and submitted for one-ligand run calculation. This reproduced top scoring conformation falling within root-mean-square deviation (rmsd) value of 1.64  A from bound X-ray conformation for ALK, suggesting this method is valid enough to be used for docking studies of other compounds (Fig. 6). Docking of different ligands to protein was performed using AutoDock, following the same protocol used in as that of validation study. All dockings were taken into 2.5 million energy evaluations

were performed for each of the test molecules. Docked ligand conformations were analysed in terms of energy, hydrogen bonding, and hydrophobic interaction between ligand and receptor protein ALK. Detailed analyses of the ligandereceptor interactions were carried out, and final coordinates of the ligand and receptor were saved. For display of the receptor with the ligand binding site, PyMol software was used. From the docking scores, the free energy of binding (FEB) of all compounds was calculated (Table 3). Anticancer activity studies of the synthesised compounds correlates with the calculated binding energy. Among the all

Table 3 Anticancer activity against A549 adenocarcinoma cancer cell line and calculated binding energy with ALK receptor of synthesised spirooxindoleepyrrolidines (4aeo & 6aen). Compound

4a 4b 4c 4d 4e 4f 4g 4h 4i 4j 4k 4l 4m 4n 4o 6a 6b 6c 6d 6e 6f 6g 6h 6i 6j 6k 6l 6m 6n

100 (mg/mL)

50 (mg/mL)

25 (mg/mL)

Cell inhibition

Cell inhibition

Cell inhibition

%

Mean  S.D

23.2 7.4 22.2 51.5 34.2 14.9 56.6 77.3 83.0 85.0 66.2 23.0 13.4 50.2 6.8 85.6 87.8 66.2 24.0 15.6 69.0 76.8 84.3 83.3 80.1 56.2 81.8 82.7 9.0

0.501 0.585 0.501 0.301 0.393 0.547 0.268 0.141 0.101 0.087 0.201 0.455 0.574 0.321 0.582 0.091 0.076 0.199 0.504 0.497 0.185 0.136 0.092 0.103 0.126 0.265 0.110 0.106 0.536

                            

0.007 0.0.005 0.007 0.003 0.006 0.003 0.003 0.003 0.002 0.005 0.002 0.009 0.009 0.003 0.004 0.007 0.005 0.004 0.003 0.006 0.006 0.004 0.006 0.005 0.006 0.005 0.002 0.005 0.004

%

Mean  S.D

6.3 4.3 9.0 30.9 9.8 6.8 33.4 53.5 74.4 79.2 35.6 15.2 9.9 22.2 4.3 79.3 75.1 33.5 13.4 9.0 56.3 63.4 75.8 72.1 68.5 31.1 72.1 68.4 5.1

0.611 0.605 0.536 0.429 0.522 0.599 0.411 0.289 0.152 0.121 0.383 0.501 0.536 0.501 0.572 0.131 0.154 0.391 0.574 0.536 0.261 0.215 0.142 0.172 0.198 0.421 0.170 0.194 0.559

                            

0.005 0.008 0.004 0.007 0.005 0.003 0.007 0.004 0.003 0.002 0.006 0.003 0.005 0.007 0.005 0.003 0.004 0.004 0.009 0.004 0.004 0.005 0.005 0.003 0.006 0.004 0.003 0.012 0.003

Binding energy (kcal/mol)a

%

Mean  S.D

3.1 3.3 4.5 6.9 4.4 5.9 8.8 26.7 62.8 66.3 9.9 4.7 5.1 3.7 3.0 59.9 64.8 15.5 6.0 5.1 24.5 28.0 66.3 54.4 52.9 15.7 55.2 36.2 3.3

0.633 0.611 0.615 0.578 0.602 0.605 0.563 0.456 0.221 0.196 0.536 0.563 0.559 0.620 0.635 0.254 0.218 0.497 0.623 0.559 0.451 0.421 0.198 0.281 0.296 0.515 0.273 0.391 0.611

                            

0.003 0.009 0.003 0.004 0.008 0.002 0.005 0.003 0.004 0.003 0.005 0.005 0.003 0.007 0.003 0.008 0.005 0.001 0.008 0.003 0.004 0.003 0.002 0.005 0.006 0.001 0.004 0.011 0.009

8.04 8.15 7.22 7.74 7.30 7.35 7.56 7.23 8.11 8.44 7.50 7.66 7.12 8.12 6.70 8.12 8.47 7.41 7.48 7.38 7.45 7.19 8.25 8.21 8.15 8.21 8.32 7.40 7.00

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The compound 4o, one of the least active compounds, shows very less binding energy 6.70 kcal/mol with the ALK receptor. This compound doesn’t exactly fit with the binding site of ALK receptor as crizotinib ligand and hence results in the very low binding energy. This compound interacts with only two amino acids, namely, LYS-1150 and LEU-1198 through two hydrogen bonds with ALK receptor (Fig. 9). 3. Conclusion

Fig. 4. Effect of active spirooxindoleepyrrolidines on A549 cells growth (Cells were treated with 25 mg/mL 24 h and DNA fragmentation was analysed by agarose gel electrophoresis).

compounds docked 4j, 6b, 6l and 6h show very high binding energy and also 4i, 6a, 6i and 6j show high binding energy with the ALK receptor. These results are well match with observed anticancer activity of the synthesised compounds. The results of which revealed that compound 6b, 4j, 6l and 6h as the most active with a calculated binding energy of 8.47, 8.44, 8.32 and 8.25 kcal/ mol respectively. The most active compound 6b binds very well with ALK receptor which shows very high binding energy 8.47 kcal/mol compare with all the compounds docked with ALK receptor. This compound exactly fit in the binding site of ALK receptor as like as crizotinib ligand and interacts with six amino acids, namely, LEU-1122, LEU-1198, MET-1199, ALA-1200, GLY-1202 and ASP-1203 which results seven hydrogen bonds with ALK receptor (Fig. 7). The intermediate active compound 4k binds with ALK receptor and results in binding energy of 7.50 kcal/mol. This compound interacts with five amino acids, namely, LEU-1122, LEU-1198, MET1199, GLY-1202 and ASP-1203 which results six hydrogen bonds with ALK receptor (Fig. 8).

In summary, we have reported the synthesis of novel spirooxindoleepyrrolidine compounds through 1,3-dipolar cycloaddition of an azomethine ylide generated from isatin (1aeh) and sarcosine 2 or thioproline 5 with the dipolarophile 3a or 3b, and characterisation of synthesised spirooxindoleepyrrolidine (4aeo and 6aen) compounds. These novel compounds were evaluated for their anticancer activity against A549 human lung adenocarcinoma cancer cell line. The results indicated that the synthesised spirooxindoleepyrrolidine compounds showed significant anticancer activities. Among all the compounds screened, 4j, 6b and 6h showed very high activity 66.3%, 64.8% and 66.3% at 25 mg/mL concentration against A549 lung adenocarcinoma cancer cell line. These spirooxindoleepyrrolidine compounds can be promising therapeutic agents for A549 lung adenocarcinoma cancer cell line. 4. Experimental section 4.1. Chemistry Melting points were determined in capillary tubes and are uncorrected. IR spectra were taken as KBr pellets for solids on a Perkin Elmer Spectrum RXI FT-IR. 1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were recorded in DMSO-d6 solutions with TMS as an internal standard on a Bruker instrument. Mass spectra were recorded on a Thermo Finnigan LCQ Advantage MAX 6000 ESI

Fig. 5. FITC and PI triple fluorescence staining for the detection of apoptosis in A549 cells under confocal laser scanning microscope. Cell was treated with 25 mg/mL at 24 h. (A) Control and (B) Compound 6b treated apoptotic cancer cells.

Y. Arun et al. / European Journal of Medicinal Chemistry 74 (2014) 50e64

Fig. 6. Method validation using crystallised and docked crizotinib ligand with ALK receptor.

spectrometer. Elemental analysis data were recorded using Thermo Finnigan FLASH EA 1112 CHN analyzer. 4.1.1. Experimental procedure for the synthesis of spirooxindole derivatives (4aeo) A mixture of substituted isatin 1aeh (1 mmol), sarcosine 2 (1 mmol) and 3-(1H-imidazol-2-yl)-2-(1H-indole-3-carbonyl) acrylonitrile 3a or 3-(1H-imidazol-2-yl)-2-(5-methoxy-1H-indole3-carbonyl)acrylonitrile 3b (1 mmol) were refluxed in ethanol (5 mL). After completion of the reaction as evidenced by TLC analysis, the reaction mixture was poured into ice-water, the resulting solid was filtered off and solid was washed with ethanol to afford pure spirooxindoleepyrrolidine compounds (4aeo). 4.1.1.1. 40 -(1H-Imidazol-2-yl)-30 -(1H-indole-3-carbonyl)-10 -methyl2-oxospiro[indoline-3,20 -pyrrolidine]-30 -carbonitrile (4a). White solid; mp 280  C; IR (cm1): 3394, 3290, 3133, 3059, 2976, 2864, 2243, 1720, 1631, 1518, 1439, 1243, 1161, 1101, 753; 1H NMR (400 MHz, DMSO-d6): dH: 11.95 (s, 1H), 11.86 (s, 1H), 10.48 (s, 1H), 8.14 (dd, 1H, J ¼ 4.0, 4.7 Hz), 7.80 (d, 1H, J ¼ 7.5 Hz), 7.40e7.28 (m, 2H), 7.24e7.12 (m, 3H), 6.96 (s, 2H), 6.75 (d, 1H, J ¼ 2.5 Hz), 6.66 (d, 1H, J ¼ 7.7 Hz), 5.37 (dd, 1H, J ¼ 7.4, 10.3 Hz), 3.91 (t, 1H, J ¼ 7.9 Hz), 3.53 (t, 1H, J ¼ 9.5 Hz), 2.10 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 181.0, 174.9, 144.0, 143.8, 135.8, 134.5, 131.0, 128.2, 126.4, 125.7, 124.6, 123.7, 122.8, 122.0, 121.9, 119.1, 117.0, 112.6, 112.4, 110.1, 76.0,

Fig. 7. Binding mode of the most active compound 6b with ALK receptor.

59

Fig. 8. Binding mode of intermediate active compound 4k with ALK receptor.

64.9, 54.6, 40.2, 35.0; HRMS (ESI): Mass calculated for C25H21N6O2 [M þ H]þ, 437.17260. Found [M þ H]þ, 437.17050. 4.1.1.2. 4 0 -(1H-Imidazol-2-yl)-3 0 -(1H-indole-3-carbonyl)-1,1 0 dimethyl-2-oxospiro[indoline-3,20 -pyrrolidine]-30 -carbonitrile (4b). White solid; mp 240  C; IR (cm1): 3379, 3133, 2976, 2918, 2242, 1720, 1624, 1543, 1427, 1241, 1092, 787, 708; 1H NMR (400 MHz, DMSO-d6): dH: 11.94 (s, 1H), 11.89 (s, 1H), 8.08e8.04 (m, 1H), 7.82 (d, 1H, J ¼ 7.2 Hz), 7.42e7.34 (m, 2H), 7.24e7.16 (m, 3H), 7.03 (s, 1H), 6.95 (s, 1H), 6.83 (d, 1H, J ¼ 7.8 Hz), 6.73 (s, 1H), 5.35 (dd, 1H, J ¼ 7.3, 10.5 Hz), 3.92 (dd, 1H, J ¼ 7.6, 8.4 Hz), 3.54 (dd, 1H, J ¼ 9.0, 10.2 Hz), 2.85 (s, 3H), 2.05 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 181.1, 173.2, 145.3, 143.7, 135.7, 134.4, 131.0, 128.1, 126.2, 125.2, 123.8, 123.7, 122.8, 122.5, 121.7, 119.0, 117.2, 112.5, 112.4, 108.9, 75.8, 65.3, 54.8, 40.4, 34.9, 25.9; MS m/z ¼ 451 [M þ H]þ; Anal. Calcd for C26H22N6O2: C, 69.32; H, 4.92; N, 18.66. Found: C, 69.25; H, 4.94; N, 18.59. 4.1.1.3. 5-Fluoro-40 -(1H-imidazol-2-yl)-30 -(1H-indole-3-carbonyl)10 -methyl-2-oxospiro [indoline-3,20 -pyrrolidine]-30 -carbonitrile (4c). Off white solid; mp 232  C; IR (cm1): 3381, 3123, 2976, 2918, 2241, 1718, 1631, 1520, 1439, 1243, 1161, 1101, 752; 1H NMR (400 MHz, DMSO-d6): dH: 12.06 (s, 1H), 11.95 (s, 1H), 10.59 (s, 1H), 8.14 (d, 1H, J ¼ 8.0 Hz), 7.57 (dd, 1H, J ¼ 2.0, 8.5 Hz), 7.40 (d, 1H, J ¼ 7.0 Hz), 7.25e 7.16 (m, 3H), 7.05 (s, 1H), 6.96 (s, 1H), 6.82 (s, 1H), 6.75e6.69 (m, 1H), 5.35 (dd, 1H, J ¼ 7.6, 10.1 Hz), 3.90 (t, 1H, J ¼ 8.0 Hz), 3.54 (t, 1H, J ¼ 9.8 Hz), 2.12 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.9,

Fig. 9. Binding mode of least active compound 4o with ALK receptor.

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174.9, 159.2, 156.8, 143.6, 140.3, 135.8, 134.5, 128.2, 127.1, 126.4, 126.3, 126.2, 123.8, 122.9, 121.9, 119.0, 117.5, 117.3, 117.2, 113.3, 113.0, 112.5, 111.1, 111.0, 75.9, 65.1, 54.8, 40.3, 34.9; MS m/z ¼ 455 [M þ H]þ; Anal. Calcd for C25H19FN6O2: C, 66.07; H, 4.21; N, 18.49. Found: C, 65.93; H, 4.27; N, 18.55. 4.1.1.4. 5-Chloro-40 -(1H-imidazol-2-yl)-30 -(1H-indole-3-carbonyl)10 -methyl-2-oxospiro [indoline-3,20 -pyrrolidine]-30 -carbonitrile (4d). Pale yellow solid; mp 240  C; IR (cm1): 3379, 3288, 3133, 3044, 2976, 2862, 2243, 1721, 1632, 1516, 1432, 1241, 1101, 748; 1H NMR (400 MHz, DMSO-d6): dH: 12.15 (s, 1H), 11.71 (s, 1H), 10.89 (s, 1H), 8.53 (s, 1H), 8.19 (s, 1H), 7.56 (dd, 1H, J ¼ 7.0 Hz), 7.42 (d, 1H, J ¼ 7.8 Hz), 7.31 (dd, 1H, J ¼ 1.6, 7.0 Hz), 7.26 (dd, 1H, J ¼ 1.6, 7.0 Hz), 7.23e7.18 (m, 1H), 7.17e7.14 (m, 1H), 7.07 (dd, 1H, J ¼ 2.0, 8.3 Hz), 6.54 (d, 1H, J ¼ 8.3 Hz), 5.32 (t, 1H, J ¼ 8.8 Hz), 4.07 (t, 1H, J ¼ 8.8 Hz), 3.48 (t, 1H, J ¼ 9.5 Hz), 2.04 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 174.2, 143.2, 140.6, 136.0, 134.0, 130.0, 128.1, 127.1, 126.4, 126.1, 125.5, 123.8, 122.7, 121.7, 117.9, 117.4, 113.2, 112.5, 111.5, 77.2, 63.4, 53.8, 42.0, 35.2; MS m/z ¼ 471 [M þ H]þ; Anal. Calcd for C25H19ClN6O2: C, 63.76; H, 4.07; N, 17.85. Found: C, 63.81; H, 4.01; N, 17.91. 4.1.1.5. 5-Bromo-40 -(1H-imidazol-2-yl)-30 -(1H-indole-3-carbonyl)10 -methyl-2-oxospiro [indoline-3,20 -pyrrolidine]-30 -carbonitrile (4e). Pale yellow solid; mp 188e189  C; IR (cm1): 3382, 3043, 2972, 2919, 2242, 1720, 1628, 1542, 1425, 1243, 1092, 787; 1H NMR (400 MHz, DMSO-d6): dH: 12.07 (s, 1H), 11.94 (s, 1H), 10.70 (s, 1H), 8.09 (d, 1H, J ¼ 7.9 Hz), 7.86 (s, 1H), 7.51 (d, 1H, J ¼ 9.0 Hz), 7.37 (t, 1H, J ¼ 7.6 Hz), 7.28e7.14 (m, 2H), 7.03e6.98 (m, 2H), 6.75 (s, 1H), 6.34 (d, 1H, J ¼ 8.3 Hz), 5.29 (dd, 1H, J ¼ 7.5, 9.8 Hz), 3.88 (t, 1H, J ¼ 8.0 Hz), 3.50 (t, 1H, J ¼ 9.8 Hz), 2.10 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 181.2, 174.5, 143.5, 143.4, 135.8, 134.5, 133.6, 128.3, 128.3, 127.0, 126.3, 123.8, 122.9, 121.8, 119.0, 117.2, 113.5, 112.5, 112.5, 112.1, 75.5, 65.4, 55.0, 40.5, 34.9; MS m/z ¼ 515 [M þ H]þ; Anal. Calcd for C25H19BrN6O2: C, 58.26; H, 3.72; N, 16.31. Found: C, 58.35; H, 3.69; N, 16.38. 4.1.1.6. 40 -(1H-Imidazol-2-yl)-30 -(1H-indole-3-Carbonyl)-5-iodo-10 methyl-2-oxospiro [indoline-3,20 -pyrrolidine]-30 -carbonitrile (4f). Pale yellow solid; mp 230  C; IR (cm1): 3392, 3130, 2982, 2920, 2240, 1718, 1625, 1540, 1419, 1242, 1090, 787, 712; 1H NMR (400 MHz, DMSO-d6): dH: 12.17 (s, 1H), 11.68 (s, 1H), 10.86 (s, 1H), 8.12 (d, 1H, J ¼ 3.0 Hz), 7.98e7.82 (m, 2H), 7.43 (d, 1H, J ¼ 8.1 Hz), 7.32 (d, 1H, J ¼ 8.2 Hz), 7.12 (t, 1H, J ¼ 7.5 Hz), 7.01 (m, 2H), 6.87 (s, 1H), 6.32 (d, 1H, J ¼ 8.0 Hz), 5.37 (t, 1H, J ¼ 8.8 Hz), 3.91 (t, 1H, J ¼ 7.9 Hz), 3.53 (t, 1H, J ¼ 9.5 Hz), 2.10 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 174.6, 141.8, 141.2, 139.0, 137.5, 135.9, 134.2, 127.7, 126.0, 123.9, 123.1, 122.6, 121.2, 117.9, 116.4, 112.8, 112.3, 112.2, 84.0, 76.1, 64.5, 54.7, 40.2, 35.1; MS m/z ¼ 563 [M þ H]þ; Anal. Calcd for C25H19IN6O2: C, 53.39; H, 3.41; N, 14.94. Found: C, 53.48; H, 3.37; N, 15.05. 4.1.1.7. 40 -(1H-Imidazol-2-yl)-30 -(1H-indole-3-Carbonyl)-10 -methyl5-nitro-2-oxospiro [indoline-3,20 -pyrrolidine]-30 -carbonitrile (4g). Off white solid; mp 191  C; IR (cm1): 3384, 3130, 3062, 2970, 2864, 2241, 1722, 1630, 1516, 1498, 1332, 1240, 1150, 1108, 743; 1H NMR (400 MHz, DMSO-d6): dH: 12.25 (s, 1H), 11.73 (s, 1H), 11.48 (s, 1H), 8.43 (d, 1H, J ¼ 1.8 Hz), 8.14 (d, 1H, J ¼ 3.0 Hz), 8.01 (d, 1H, J ¼ 8.8 Hz), 7.62 (d, 1H, J ¼ 8.0 Hz), 7.45 (d, 1H, J ¼ 8.1 Hz), 7.17 (t, 1H, J ¼ 7.8 Hz), 7.12e6.97 (m, 3H), 6.55 (d, 1H, J ¼ 8.8 Hz), 5.36 (t, 1H, J ¼ 8.7 Hz), 3.93 (t, 1H, J ¼ 8.0 Hz), 3.55 (t, 1H, J ¼ 9.6 Hz), 2.09 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 175.5, 148.5, 141.3, 141.0, 135.9, 134.1, 128.0, 127.6, 125.9, 100.1, 124.2, 122.7, 121.5, 121.0, 117.8, 116.0, 112.9, 112.5, 110.3, 76.0, 64.8, 54.6, 40.2, 35.1; MS m/

z ¼ 482 [M þ H]þ; Anal. Calcd for C25H19N7O4: C, 62.37; H, 3.98; N, 20.36. Found: C, 62.30; H, 3.93; N, 20.46. 4.1.1.8. 4 0 -(1H-Imidazol-2-Yl)-3 0 -(1H-indole-3-carbonyl)-1 0,5dimethyl-2-oxospiro[indoline-3,20 -pyrrolidine]-30 -carbonitrile (4h). White solid; mp 241  C; IR (cm1): 3379, 2976, 2918, 2885, 2242, 1718, 1626, 1543, 1427, 1241, 1092, 787, 708; 1H NMR (400 MHz, DMSO-d6): dH: 11.94 (s, 1H), 11.83 (s, 1H), 10.36 (s, 1H), 8.15e8.10 (m, 1H), 7.62 (s, 1H), 7.39e7.34 (m, 1H), 7.20e7.10 (m, 3H), 7.02 (s, 1H), 6.91 (s, 1H), 6.76 (s, 1H), 6.55 (d, 1H, J ¼ 7.9 Hz), 5.37 (dd, 1H, J ¼ 7.5, 10.2 Hz), 3.89 (t, 1H, J ¼ 7.9 Hz), 3.50 (t, 1H, J ¼ 9.5 Hz), 2.35 (s, 3H), 2.09 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 181.0, 174.8, 143.8, 141.5, 135.7, 134.6, 131.1, 130.8, 128.0, 126.4, 126.2, 124.6, 123.7, 122.7, 121.9, 119.0, 117.1, 112.5, 112.4, 109.9, 76.0, 64.8, 54.6, 40.2, 35.1, 21.2; MS m/z ¼ 451 [M þ H]þ; Anal. Calcd for C26H22N6O2: C, 69.32; H, 4.92; N, 18.66. Found: C, 69.21; H, 4.95; N, 18.81. 4.1.1.9. 40 -(1H-Imidazol-2-yl)-30 -(5-methoxy-1H-indole-3-carbonyl)10 -methyl-2-oxospiro [indoline-3,20 -pyrrolidine]-30 -carbonitrile (4i). White solid; mp 220  C; IR (cm1): 3384, 3160, 2851, 2924, 2243, 1714, 1622, 1439, 1269, 1212, 1092, 799, 749; 1H NMR (400 MHz, DMSO-d6): dH: 11.86 (s, 2H), 10.50 (s, 1H), 7.79 (d, 1H, J ¼ 7.5 Hz), 7.64 (s, 1H), 7.32 (t, 1H, J ¼ 7.7 Hz), 7.25 (d, 1H, J ¼ 8.8 Hz), 7.16 (t, 1H, J ¼ 7.6 Hz), 7.02 (s, 1H), 6.91 (s, 1H), 6.81 (dd, 1H, J ¼ 1.7, 8.8 Hz), 6.68e6.64 (m, 2H), 5.36 (dd, 1H, J ¼ 7.6, 9.9 Hz), 3.89 (t, 1H, J ¼ 7.9 Hz), 3.75 (s, 3H), 3.51 (t, 1H, J ¼ 9.6 Hz), 2.09 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 174.9, 156.2, 144.0, 143.8, 134.6, 130.9, 130.5, 128.1, 127.3, 125.7, 124.6, 122.0, 119.1, 117.1, 113.6, 113.2, 112.4, 110.1, 103.5, 75.9, 64.8, 55.5, 54.6, 40.2, 35.0; MS m/z ¼ 467 [M þ H]þ; Anal. Calcd for C26H22N6O3: C, 66.94; H, 4.75; N, 18.02. Found: C, 66.83; H, 4.79; N, 18.11. 4.1.1.10. 4 0 -(1H-Imidazol-2-yl)-3 0 -(5-methoxy-1H-indole-3carbonyl)-1,10 -dimethyl-2-oxo spiro[indoline-3,20 -pyrrolidine]-30 carbonitrile (4j). White solid; mp 214  C; IR (cm1): 3365, 3170, 2927, 2869, 2243, 1704, 1621, 1482, 1423, 1268, 1211, 1100, 1079, 776, 749, 708; 1H NMR (400 MHz, DMSO-d6): dH: 11.87 (s, 1H), 11.82 (s, 1H), 7.80 (d, 1H, J ¼ 7.5 Hz), 7.58e7.56 (m, 1H), 7.40 (dd, 1H, J ¼ 7.7, 7.7 Hz), 7.27e7.19 (m, 2H), 7.03 (s, 1H), 6.91 (s, 1H), 6.85e6.79 (m, 2H), 6.67 (s, 1H), 5.34 (dd, 1H, J ¼ 7.6, 10.0 Hz), 3.91 (t, 1H, J ¼ 8.0 Hz), 3.74 (s, 3H), 3.53 (t, 1H, J ¼ 9.5 Hz), 2.85 (s, 3H), 2.04 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.7, 173.2, 156.2, 145.3, 143.7, 134.4, 131.1, 130.4, 128.1, 127.2, 125.3, 123.9, 122.6, 119.0, 117.2, 113.4, 113.2, 112.5, 109.0, 103.6, 75.9, 65.1, 55.6, 54.7, 40.4, 35.0, 26.0; MS m/z ¼ 481 [M þ H]þ; Anal. Calcd for C27H24N6O3: C, 67.49; H, 5.03; N, 17.49. Found: C, 67.32; H, 5.11; N, 17.61. 4.1.1.11. 5-Fluoro-40 -(1H-imidazol-2-yl)-30 -(5-methoxy-1H-indole-3carbonyl)-10 -methyl-2-oxospiro[indoline-3,20 -pyrrolidine]-30 -carbonitrile (4k). White solid; mp 211  C; IR (cm1): 3382, 2974, 2945, 2244, 1720, 1614, 1510, 1482, 1418, 1264, 1214, 1192, 1078, 1030, 811; 1 H NMR (400 MHz, DMSO-d6): dH: 12.18 (s, 1H), 11.59 (s, 1H), 11.24 (s, 1H), 8.28 (s, 1H), 7.52 (d, 1H, J ¼ 8.6 Hz), 7.32e7.23 (m, 3H), 7.07e 7.00 (m, 1H), 6.89 (s, 1H), 6.77 (d, 1H, J ¼ 8.8 Hz), 6.52 (s, 1H), 5.12 (t, 1H, J ¼ 8.4 Hz), 3.81 (t, 1H, J ¼ 9.5 Hz), 3.69 (s, 3H), 3.53 (t, 1H, J ¼ 8.6 Hz), 2.06 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 181.8, 177.2, 159.2, 156.9, 156.2, 144.5, 140.1, 137.9, 130.5, 127.0, 126.7, 126.6, 126.5, 122.0, 118.4, 117.4, 117.2, 113.9, 113.7, 113.4, 113.3, 111.5, 111.4, 110.3, 102.7, 73.8, 65.3, 57.3, 55.4, 47.8, 33.4; MS m/z ¼ 485 [M þ H]þ; Anal. Calcd for C26H21FN6O3: C, 64.46; H, 4.37; N, 17.35. Found: C, 64.59; H, 4.37; N, 17.22. 4.1.1.12. 5-Chloro-40 -(1H-imidazol-2-yl)-30 -(5-methoxy-1H-indole3-carbonyl)-10 -methyl-2-oxospiro[indoline-3,20 -pyrrolidine]-30 -carbonitrile (4l). White solid; mp 232  C; IR (cm1): 3384, 3307, 2967,

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2934, 2237, 1719, 1636, 1550, 1516, 1475, 1240, 1428, 1240, 1199, 1097, 788; 1H NMR (400 MHz, DMSO-d6): dH: 12.04 (s, 1H), 11.68 (s, 1H), 10.86 (s, 1H), 8.09 (s, 1H), 7.46e7.44 (m, 1H), 7.31 (d, 1H, J ¼ 8.7 Hz), 7.26 (s, 1H), 7.08e7.02 (m, 2H), 6.88e6.76 (m, 2H), 6.50 (d, 1H, J ¼ 8.3 Hz), 5.27 (t, 1H, J ¼ 8.7 Hz), 4.04 (t, 1H, J ¼ 8.8 Hz), 3.72 (s, 3H), 2.01 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 174.1, 156.0, 143.2, 140.6, 133.9, 130.7, 130.0, 127.7, 127.3, 127.0, 126.1, 125.6, 117.8, 117.4, 113.5, 113.3, 113.1, 111.4, 103.2, 77.1, 63.4, 55.4, 53.9, 42.0, 35.2; MS m/z ¼ 501 [M þ H]þ; Anal. Calcd for C26H21ClN6O3: C, 62.34; H, 4.23; N, 16.78. Found: C, 62.21; H, 4.24; N, 16.71. 4.1.1.13. 5-Bromo-40 -(1H-imidazol-2-yl)-30 -(5-methoxy-1H-indole3-carbonyl)-10 -methyl-2-oxospiro[indoline-3,20 -pyrrolidine]-30 -carbonitrile (4m). Off white solid; mp 226  C; IR (cm1): 3372, 3154, 2924, 2241, 1718, 1626, 1432, 1262, 1216, 1098, 804, 742; 1H NMR (400 MHz, DMSO-d6): dH: 12.00 (s, 1H), 11.93 (s, 1H), 10.73 (s, 1H), 7.88 (s, 1H), 7.61 (s, 1H), 7.53 (d, 1H, J ¼ 8.2 Hz), 7.29 (d, 1H, J ¼ 8.8 Hz), 7.05 (s, 1H), 6.98 (s, 1H), 6.89e6.83 (m, 2H), 6.73e6.70 (m, 2H), 5.30 (dd, 1H, J ¼ 7.7, 9.9 Hz), 3.88 (t, 1H, J ¼ 8.2 Hz), 3.75 (s, 3H), 3.79 (t, 1H, J ¼ 9.6 Hz), 2.09 (s, 3H); 13C NMR (100 MHz, DMSOd6): dC: 181.0, 174.7, 156.4, 143.6, 143.5, 134.6, 133.7, 130.6, 128.4, 127.8, 127.3, 127.1, 119.1, 117.3, 113.8, 113.6, 113.5, 112.4, 112.3, 103.6, 75.6, 65.3, 55.6, 55.0, 40.6, 35.0; MS m/z ¼ 545 [M þ H]þ; Anal. Calcd for C26H21BrN6O3: C, 57.26; H, 3.88; N, 15.41. Found: C, 57.11; H, 3.72; N, 15.63. 4.1.1.14. 40 -(1H-Imidazol-2-yl)-5-iodo-30 -(5-methoxy-1H-indole-3carbonyl)-10 -methyl-2-oxo spiro[indoline-3,20 -pyrrolidine]-30 -carbonitrile (4n). Off white solid; mp 241  C; IR (cm1): 3389, 2975, 2940, 2244, 1722, 1616, 1512, 1472, 1421, 1263, 1207, 1180, 1074, 1031, 802, 731; 1H NMR (400 MHz, DMSO-d6): dH: 11.98 (s, 1H), 11.91 (s, 1H), 10.68 (s, 1H), 8.02 (s, 1H), 7.66 (d, 1H, J ¼ 8.2 Hz), 7.58 (d, 1H, J ¼ 2.0 Hz), 7.27 (d, 1H, J ¼ 8.8 Hz), 7.03 (s, 1H), 6.96 (s, 1H), 6.82 (dd, 1H, J ¼ 2.3, 8.8 Hz), 6.66 (s, 1H), 6.58 (d, 1H, J ¼ 8.2 Hz), 5.27 (dd, 1H, J ¼ 7.8, 10.0 Hz), 3.86 (t, 1H, J ¼ 8.1 Hz), 3.74 (s, 3H), 3.49 (t, 1H, J ¼ 9.3 Hz), 2.08 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 181.0, 174.4, 156.4, 143.8, 143.6, 139.4, 134.6, 134.0, 130.6, 130.5, 128.3, 127.3, 119.0, 117.2, 113.7, 113.4, 112.6, 112.4, 103.5, 84.5, 75.3, 65.3, 55.6, 55.0, 40.5, 34.9; MS m/z ¼ 593 [M þ H]þ; Anal. Calcd for C26H21IN6O3: C, 52.72; H, 3.57; N, 14.19. Found: C, 52.56; H, 3.62; N, 14.02. 4.1.1.15. 4 0 -(1H-Imidazol-2-yl)-3 0 -(5-methoxy-1H-indole-3carbonyl)-10 -methyl-5-nitro-2-oxo spiro[indoline-3,20 -pyrrolidine]30 -carbonitrile (4o). Pale yellow solid; mp 216  C; IR (cm1): 3382, 3154, 2932, 2243, 1722, 1641, 1513, 1439, 1345, 1269, 1216, 1084, 802, 756; 1H NMR (400 MHz, DMSO-d6): dH: 12.06 (s, 1H), 11.88 (s, 1H), 11.54 (s, 1H), 8.14 (s, 1H), 7.96e7.90 (m, 2H), 7.44e7.42 (m, 1H), 7.36 (d, 1H, J ¼ 8.7 Hz), 7.11 (s, 1H), 6.98e6.89 (m, 1H), 6.85 (dd, 1H, J ¼ 2.1, 8.8 Hz), 6.73 (d, 1H, J ¼ 8.4 Hz), 5.31 (t, 1H, J ¼ 8.6 Hz), 4.13 (t, 1H, J ¼ 9.3 Hz), 3.74 (s, 3H), 3.59 (t, 1H, J ¼ 8.8 Hz), 2.14 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 175.0, 156.1, 148.2, 144.1, 142.7, 142.1, 138.1, 133.2, 130.6, 127.2, 127.0, 125.4, 122.2, 117.8, 113.7, 113.5, 113.4, 110.2, 102.7, 76.6, 63.4, 55.2, 54.3, 42.3, 35.2; MS m/z ¼ 512 [M þ H]þ; Anal. Calcd for C26H21N7O5: C, 61.05; H, 4.14; N, 19.17. Found: C, 60.89; H, 4.18; N, 19.04. 4.1.2. Experimental procedure for the synthesis of spirooxindole derivatives (6aen) A mixture of substituted isatin 1aeh (1 mmol), thioproline 5 (1 mmol) and 3-(1H-imidazol-2-yl)-2-(1H-indole-3-carbonyl) acrylonitrile 3a or 3-(1H-imidazol-2-yl)-2-(5-methoxy-1H-indole3-carbonyl)acrylonitrile 3b (1 mmol) were refluxed in ethanol (5 mL). After completion of the reaction as evidenced by TLC analysis, the reaction mixture was poured into ice-water, the

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resulting solid was filtered off and solid was washed with ethanol to afford pure spirooxindoleepyrrolidine compounds (6aen). 4.1.2.1. 7 0 -(1H-Imidazol-2-yl)-6 0 -(1H-indole-3-carbonyl)-2-oxo30 ,60 ,70 ,7a0 -tetrahydro-10 H-spiro[indoline-3,50 -pyrrolo[1,2-c]thiazole]60 -carbonitrile (6a). White solid; mp 218  C; IR (cm1): 3399, 3242, 2928, 2238, 1728, 1620, 1543, 1472, 1443, 1242, 1098, 775, 747; 1H NMR (400 MHz, DMSO-d6): dH: 12.18 (s, 1H), 11.55 (s, 1H), 10.74 (s, 1H), 8.15 (s, 1H), 7.78 (d, 1H, J ¼ 7.9 Hz), 7.54 (d, 1H, J ¼ 7.6 Hz), 7.40 (d, 1H, J ¼ 8.1 Hz), 7.15 (t, 1H, J ¼ 7.6 Hz), 7.08e6.97 (m, 3H), 6.91e 6.84 (m, 2H), 6.40 (d, 1H, J ¼ 7.8 Hz), 4.89e4.82 (m, 1H), 4.58 (d, 1H, J ¼ 10.3 Hz), 3.81 (d, 1H, J ¼ 10.8 Hz), 3.29e3.20 (m, 2H), 3.08 (dd, 1H, J ¼ 1.8, 11.8 Hz); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 175.1, 142.1, 141.5, 135.9, 134.2, 130.7, 129.5, 127.8, 126.1, 123.8, 122.6, 121.4, 120.9, 120.7, 117.7, 116.7, 112.7, 112.5, 109.8, 76.0, 69.1, 64.8, 54.0, 47.9, 36.2; MS m/z ¼ 481 [M þ H]þ; Anal. Calcd for C26H20N6O2S: C, 64.98; H, 4.20; N, 17.49. Found: C, 65.11; H, 4.24; N, 17.54. 4.1.2.2. 70 -(1H-Imidazol-2-yl)-60 -(1H-indole-3-carbonyl)-1-methyl2-oxo-30 ,60 ,70 ,7a0 -tetra hydro-10 H-spiro[indoline-3,50 -pyrrolo[1,2-c] thiazole]-60 -carbonitrile (6b). Pale yellow solid; mp 217  C; IR (cm1): 3343, 3139, 3087, 2967, 2942, 2240, 1713, 1612, 1515, 1469, 1439, 1355, 1246, 1160, 1096, 1022, 81, 745, 711; 1H NMR (400 MHz, DMSO-d6): dH: 12.16 (s, 1H), 11.58 (s, 1H), 7.98 (s, 1H), 7.71 (d, 1H, J ¼ 7.9 Hz), 7.62 (d, 1H, J ¼ 7.6 Hz), 7.42 (d, 1H, J ¼ 8.1 Hz), 7.15 (t, 1H, J ¼ 7.6 Hz), 7.10e7.01 (m, 3H), 6.97 (t, 1H, J ¼ 7.6 Hz), 6.92 (s, 1H), 6.53 (d, 1H, J ¼ 7.8 Hz), 4.93e4.86 (m, 1H), 4.58 (d, 1H, J ¼ 10.3 Hz), 3.82 (d, 1H, J ¼ 10.9 Hz), 3.35e3.24 (m, 2H), 3.14 (dd, 1H, J ¼ 1.4, 11.8 Hz), 2.95 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.7, 173.3, 143.4, 141.5, 135.8, 133.7, 130.8, 129.0, 127.8, 126.0, 123.8, 122.5, 121.6, 121.2, 120.1, 117.8, 116.4, 112.6, 112.5, 108.7, 76.2, 69.1, 65.1, 54.3, 47.7, 36.4, 26.6; MS m/z ¼ 495 [M þ H]þ; Anal. Calcd for C27H22N6O2S: C, 65.57; H, 4.48; N, 16.99. Found: C, 65.42; H, 4.51; N, 17.04. 4.1.2.3. 5-Fluoro-70 -(1H-imidazol-2-yl)-60 -(1H-indole-3-carbonyl)2-oxo-30 ,60 ,70 ,7a0 -tetra hydro-10 H-spiro[indoline-3,50 -pyrrolo[1,2-c] thiazole]-60 -carbonitrile (6c). White solid; mp 214  C; IR (cm1): 3358, 3124, 2926, 2868, 2240, 1722, 1636, 1516, 1452, 1088, 1032, 936, 812, 674; 1H NMR (400 MHz, DMSO-d6): dH: 12.21 (s, 1H), 11.57 (s, 1H), 10.77 (s, 1H), 8.17 (d, 1H, J ¼ 2.6 Hz), 7.81 (d, 1H, J ¼ 7.9 Hz), 7.47e7.40 (m, 2H), 7.16 (t, 1H, J ¼ 7.5 Hz), 7.11e6.99 (m, 2H), 6.95e 6.83 (m, 2H), 6.39 (dd, 1H, J ¼ 4.5, 8.5 Hz), 4.87e4.80 (m, 1H), 4.56 (d, 1H, J ¼ 10.3 Hz), 3.83 (d, 1H, J ¼ 11.0 Hz), 3.31 (d, 1H, J ¼ 11.0 Hz), 3.27e3.19 (m, 1H), 3.14 (dd, 1H, J ¼ 1.3, 11.9 Hz); 13C NMR (100 MHz, DMSO-d6): dC: 180.7, 175.0, 158.1, 155.8, 141.4, 138.5, 136.0, 134.3, 127.8, 126.1, 123.9, 122.7, 122.4, 122.3, 121.3, 117.7, 117.5, 117.4, 117.2, 117.1, 116.4, 112.7, 112.6, 110.6, 110.5, 76.4, 69.0, 64.7, 54.1, 47.8, 36.2; MS m/z ¼ 499 [M þ H]þ; Anal. Calcd for C26H19FN6O2S: C, 62.64; H, 3.84; N, 16.86. Found: C, 62.76; H, 3.89; N, 16.72. 4.1.2.4. 5-Chloro-70 -(1H-imidazol-2-yl)-60 -(1H-indole-3-carbonyl)2-oxo-30 ,60 ,70 ,7a0 -tetra hydro-10 H-spiro[indoline-3,50 -pyrrolo[1,2-c] thiazole]-60 -carbonitrile (6d). Pale yellow solid; mp 216  C; IR (cm1): 3347, 3089, 2924, 2854, 2242, 1737, 1649, 1516, 1476, 1459, 1433, 1246, 1150, 1015, 815, 778, 756; 1H NMR (400 MHz, DMSOd6): dH: 12.22 (s, 1H), 11.65 (s, 1H), 10.87 (s, 1H), 8.14 (d, 1H, J ¼ 2.9 Hz), 7.80 (d, 1H, J ¼ 7.9 Hz), 7.59 (d, 1H, J ¼ 1.4 Hz), 7.42 (d, 1H, J ¼ 8.1 Hz), 7.16 (dd, 1H, J ¼ 7.5, 7.5 Hz), 7.11e7.03 (m, 3H), 6.91 (s, 1H), 6.39 (d, 1H, J ¼ 8.4 Hz), 4.86e4.79 (m, 1H), 4.50 (d, 1H, J ¼ 10.3 Hz), 3.82 (d, 1H, J ¼ 11.1 Hz), 3.30 (d, 1H, J ¼ 11.3 Hz), 3.26e 3.18 (m, 1H), 3.12 (d, 1H, J ¼ 11.2 Hz); 13C NMR (100 MHz, DMSO-d6): dC: 180.7, 174.7, 141.3, 141.1, 136.0, 134.2, 130.6, 129.4, 127.8, 126.0, 125.0, 123.9, 122.7, 122.6, 121.2, 117.6, 116.3, 112.8, 112.5, 111.2, 76.3, 69.0, 64.7, 54.4, 47.9, 36.2; MS m/z ¼ 515 [M þ H]þ; Anal. Calcd for

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Y. Arun et al. / European Journal of Medicinal Chemistry 74 (2014) 50e64

C26H19ClN6O2S: C, 60.64; H, 3.72; N, 16.32. Found: C, 60.55; H, 3.74; N, 16.45.

21.0; MS m/z ¼ 495 [M þ H]þ; Anal. Calcd for C27H22N6O2S: C, 65.57; H, 4.48; N, 16.99. Found: C, 65.65; H, 4.42; N, 16.81.

4.1.2.5. 5-Bromo-70 -(1H-imidazol-2-yl)-60 -(1H-indole-3-carbonyl)2-oxo-30 ,60 ,70 ,7a0 -tetra hydro-10 H-spiro[indoline-3,50 -pyrrolo[1,2-c] thiazole]-60 -carbonitrile (6e). White solid; mp 230  C; IR (cm1): 3343, 3090, 2922, 2853, 2242, 1739, 1649, 1516, 1472, 1433, 1278, 1247, 1151, 1097, 812, 716, 681; 1H NMR (400 MHz, DMSO-d6): dH: 12.21 (s, 1H), 11.67 (s, 1H), 10.88 (s, 1H), 8.14 (s, 1H), 7.80 (d, 1H, J ¼ 7.4 Hz), 7.70 (s, 1H), 7.46e7.41 (m, 1H), 7.21e7.16 (m, 2H), 7.12e 7.04 (m, 2H), 6.89 (s, 1H), 6.34 (d, 1H, J ¼ 8.1 Hz), 4.83 (t, 1H, J ¼ 7.8 Hz), 4.48 (d, 1H, J ¼ 10.1 Hz), 3.81 (d, 1H, J ¼ 11.0 Hz), 3.33e 3.18 (m, 1H), 3.12 (d, 1H, J ¼ 11.6 Hz); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 174.6, 141.5, 141.2, 135.9, 134.1, 133.4, 132.0, 129.9, 127.8, 126.0, 123.8, 123.0, 122.7, 121.2, 117.6, 116.3, 112.8, 112.5, 111.7, 76.2, 69.0, 64.6, 54.4, 47.9, 36.2; MS m/z ¼ 559 [M þ H]þ; Anal. Calcd for C26H19BrN6O2S: C, 55.82; H, 3.42; N, 15.02. Found: C, 55.67; H, 3.35; N, 15.21.

4.1.2.9. 7 0 -(1H-Imidazol-2-yl)-6 0 -(5-methoxy-1H-indole-3carbonyl)-2-oxo-30 ,60 ,70 ,7a0 -tetra hydro-10 H-spiro[indoline-3,50 -pyrrolo[1,2-c]thiazole]-60 -carbonitrile (6i). Pale yellow solid; mp 218  C; IR (cm1): 3390, 3237, 2978, 2833, 2237, 1736, 1621, 1514, 1438, 11334, 1207, 1028, 750, 685; 1H NMR (400 MHz, DMSO-d6): dH: 12.10 (d, 1H, J ¼ 2.2 Hz), 11.62 (s, 1H), 10.75 (s, 1H), 8.11 (d, 1H, J ¼ 3.2 Hz), 7.56 (d, 1H, J ¼ 7.7 Hz), 7.32 (d, 1H, J ¼ 8.8 Hz), 7.28 (d, 1H, J ¼ 2.1 Hz), 7.10e7.01 (m, 2H), 6.91 (t, 2H, J ¼ 7.6 Hz), 6.79 (dd, 1H, J ¼ 2.2, 8.8 Hz), 6.43 (d, 1H, J ¼ 7.7 Hz), 4.90e4.83 (m, 1H), 4.59 (d, 1H, J ¼ 10.3 Hz), 3.83 (d, 1H, J ¼ 10.9 Hz), 3.67 (s, 3H), 3.31e3.21 (m, 2H), 3.07 (dd, 1H, J ¼ 1.9, 11.9 Hz); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 175.0, 155.9, 142.2, 141.5, 134.2, 130.8, 130.7, 129.6, 127.8, 127.1, 120.9, 120.7, 117.7, 116.6, 113.4, 113.2, 112.7, 109.8, 103.2, 76.1, 69.1, 64.8, 55.4, 54.0, 47.9, 36.2; MS m/z ¼ 511 [M þ H]þ; Anal. Calcd for C27H22N6O3S: C, 63.52; H, 4.34; N, 16.46. Found: C, 63.66; H, 4.28; N, 16.45.

4.1.2.6. 70 -(1H-Imidazol-2-yl)-60 -(1H-indole-3-carbonyl)-5-iodo-2oxo-30 ,60 ,70 ,7a0 -tetrahydro-10 H-spiro[indoline-3,50 -pyrrolo[1,2-c]thiazole]-60 -carbonitrile (6f). White solid; mp 244  C; IR (cm1): 3337, 2920, 2854, 2242, 1739, 1650, 1614, 1516, 1433, 1307, 1276, 1231, 1154, 1023, 916, 809, 687; 1H NMR (400 MHz, DMSO-d6): dH: 12.19 (s, 1H), 11.67 (s, 1H), 10.84 (s, 1H), 8.12 (d, 1H, J ¼ 2.9 Hz), 7.82e7.78 (m, 2H), 7.41 (d, 1H, J ¼ 8.1 Hz), 7.30 (d, 1H, J ¼ 8.2 Hz), 7.16 (t, 1H, J ¼ 7.5 Hz), 7.08 (t, 1H, J ¼ 7.5 Hz), 7.04 (s, 1H), 6.87 (s, 1H), 6.21 (d, 1H, J ¼ 8.2 Hz), 4.86e4.79 (m, 1H), 4.43 (d, 1H, J ¼ 10.3 Hz), 3.80 (d, 1H, J ¼ 11.1 Hz), 3.29 (d, 1H, J ¼ 11.3 Hz), 3.26e3.20 (m, 1H), 3.11 (d, 1H, J ¼ 11.7 Hz); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 174.4, 141.9, 141.2, 139.1, 137.4, 135.9, 134.0, 127.7, 126.0, 123.8, 123.2, 122.6, 121.2, 117.7, 116.3, 112.8, 112.5, 112.1, 84.0, 76.0, 69.0, 64.6, 54.5, 47.9, 36.2; MS m/z ¼ 607 [M þ H]þ; Anal. Calcd for C26H19IN6O2S: C, 51.49; H, 3.16; N, 13.86. Found: C, 51.55; H, 3.19; N, 13.72. 4.1.2.7. 70 -(1H-Imidazol-2-yl)-60 -(1H-indole-3-carbonyl)-5-nitro-2oxo-30 ,60 ,70 ,7a0 -tetrahydro -10 H-spiro[indoline-3,50 -pyrrolo[1,2-c] thiazole]-60 -carbonitrile (6g). Off white solid; mp 234  C; IR (cm1): 3382, 3255, 2932, 2239, 1718, 1626, 1544, 1484, 1442, 1248, 1084, 785, 756; 1H NMR (400 MHz, DMSO-d6): dH: 12.27 (d, 1H, J ¼ 2.0 Hz), 11.77 (s, 1H), 11.46 (s, 1H), 8.45 (d, 1H, J ¼ 1.6 Hz), 8.16 (d, 1H, J ¼ 3.1 Hz), 7.95 (dd, 1H, J ¼ 1.9, 8.7 Hz), 7.68 (d, 1H, J ¼ 7.9 Hz), 7.44 (d, 1H, J ¼ 8.1 Hz), 7.17 (dd, 1H, J ¼ 7.6, 7.6 Hz), 7.10e6.90 (m, 3H), 6.57 (d, 1H, J ¼ 8.7 Hz), 4.92e4.86 (m, 1H), 4.53 (d, 1H, J ¼ 10.5 Hz), 3.85 (d, 1H, J ¼ 11.5 Hz), 3.45e3.41 (m, 1H), 3.32e3.24 (m, 1H), 3.21 (d, 1H, J ¼ 11.9 Hz); 13C NMR (100 MHz, DMSO-d6): dC: 180.7, 175.5, 148.7, 141.2, 141.0, 135.9, 134.0, 127.9, 127.6, 125.9, 125.0, 124.0, 122.8, 121.5, 120.9, 117.7, 116.1, 112.9, 112.6, 110.3, 76.1, 69.1, 64.7, 54.8, 48.0, 36.5; MS m/z ¼ 526 [M þ H]þ; Anal. Calcd for C26H19N7O4S: C, 59.42; H, 3.64; N, 18.66. Found: C, 59.55; H, 3.61; N, 18.52. 4.1.2.8. 70 -(1H-Imidazol-2-yl)-60 -(1H-indole-3-carbonyl)-5-methyl2-oxo-30 ,60 ,70 ,7a0 -tetra hydro-10 H-spiro[indoline-3,50 -pyrrolo[1,2-c] thiazole]-60 -carbonitrile (6h). White solid; mp 218  C; IR (cm1): 3352, 3120, 2923, 2858, 2242, 1718, 1630, 1511, 1448, 1095, 1035, 928, 809, 670, 635; 1H NMR (400 MHz, DMSO-d6): dH: 12.17 (s, 1H), 11.59 (s, 1H), 10.65 (s, 1H), 8.15 (d, 1H, J ¼ 2.1 Hz), 7.82 (d, 1H, J ¼ 7.9 Hz), 7.42 (d, 1H, J ¼ 8.1 Hz), 7.35 (s, 1H), 7.16 (t, 1H, J ¼ 7.6 Hz), 7.11e7.03 (m, 2H), 6.90 (s, 1H), 6.81 (d, 1H, J ¼ 7.9 Hz), 6.31 (d, 1H, J ¼ 7.8 Hz), 4.91e4.83 (m, 1H), 4.57 (d, 1H, J ¼ 10.3 Hz), 3.81 (d, 1H, J ¼ 10.6 Hz), 3.30e3.21 (m, 2H), 3.10 (dd, 1H, J ¼ 2.0, 11.7 Hz), 2.20 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.9, 175.0, 141.5, 139.7, 135.9, 134.1, 130.9, 129.9, 129.6, 127.8, 126.1, 123.7, 122.5, 121.4, 120.8, 117.6, 116.7, 112.8, 112.4, 109.5, 76.0, 69.0, 64.9, 53.8, 47.8, 36.1,

4.1.2.10. 7 0 -(1H-Imidazol-2-yl)-6 0 -(5-methoxy-1H-indole-3carbonyl)-1-methyl-2-oxo-30 ,60 ,70 , 7a0 -tetrahydro-10 H-spiro[indoline3,50 -pyrrolo[1,2-c]thiazole]-60 -carbonitrile (6j). Pale yellow solid; mp 225  C; IR (cm1): 3350, 3130, 3106, 2967, 2926, 2246, 1717, 1655, 1513, 1471, 1369, 1352, 1199, 1161, 1096, 1030, 776, 752, 708; 1 H NMR (400 MHz, DMSO-d6): dH: 12.05 (s, 1H), 11.62 (s, 1H), 7.92 (s, 1H), 7.63 (d, 1H, J ¼ 7.6 Hz), 7.31 (d, 1H, J ¼ 8.8 Hz), 7.17 (d, 1H, J ¼ 1.8 Hz), 7.13e7.06 (m, 2H), 6.98 (t, 1H, J ¼ 7.6 Hz), 6.92 (s, 1H), 6.78 (dd, 1H, J ¼ 1.8, 8.8 Hz), 6.52 (d, 1H, J ¼ 7.8 Hz), 4.93e4.85 (m, 1H), 4.56 (d, 1H, J ¼ 10.3 Hz), 3.81 (d, 1H, J ¼ 10.9 Hz), 3.65 (s, 3H), 3.35e3.22 (m, 2H), 3.11 (d, 1H, J ¼ 11.4 Hz), 2.93 (s, 3H); 13C NMR (100 MHz, DMSO-d6): dC: 180.8, 173.3, 155.8, 143.5, 141.5, 133.6, 130.9, 130.6, 129.0, 127.9, 127.0, 121.6, 120.2, 117.8, 116.4, 113.5, 113.3, 112.6, 108.7, 102.9, 76.3, 69.1, 65.1, 55.4, 54.3, 47.7, 36.4, 26.6; MS m/ z ¼ 525 [M þ H]þ; Anal. Calcd for C28H24N6O3S: C, 64.11; H, 4.61; N, 16.02. Found: C, 63.95; H, 4.58; N, 16.24. 4.1.2.11. 5-Fluoro-70 -(1H-imidazol-2-yl)-60 -(5-methoxy-1H-indole-3carbonyl)-2-oxo-30 ,60 ,70 , 7a0 -tetrahydro-10 H-spiro[indoline-3,50 -pyrrolo[1,2-c]thiazole]-60 -carbonitrile (6k). White solid; mp 202  C; IR (cm1): 3354, 3224, 2965, 2834, 2239, 1728, 1624, 1516, 1439, 1136, 1214, 1032, 767; 1H NMR (400 MHz, DMSO-d6): dH: 12.13 (s, 1H), 11.63 (s, 1H), 10.77 (s, 1H), 8.11 (s, 1H), 7.45 (d, 1H, J ¼ 7.8 Hz), 7.33 (d, 1H, J ¼ 8.8 Hz), 7.30 (d, 1H, J ¼ 1.9 Hz), 7.08 (s, 1H), 6.96e6.87 (m, 2H), 6.81 (dd, 1H, J ¼ 2.0, 8.9 Hz), 6.43e6.37 (m, 1H), 4.89e4.82 (m, 1H), 4.55 (d, 1H, J ¼ 10.3 Hz), 3.85 (d, 1H, J ¼ 11.0 Hz), 3.68 (s, 3H), 3.34 (d, 1H, J ¼ 11.0 Hz), 3.29e3.21 (m, 1H), 3.13 (d, 1H, J ¼ 11.4 Hz); 13 C NMR (100 MHz, DMSO-d6): dC: 180.7, 174.9, 158.1, 156.0, 155.8, 141.4, 138.5, 134.2, 130.7, 127.9, 127.1, 122.4, 122.3, 117.7, 117.5, 117.3, 117.3, 117.1, 116.4, 113.5, 113.3, 112.7, 110.6, 110.5, 103.0, 76.5, 69.0, 64.7, 55.4, 54.2, 47.7, 36.2; MS m/z ¼ 529 [M þ H]þ; Anal. Calcd for C27H21FN6O3S: C, 61.35; H, 4.00; N, 15.90. Found: C, 61.22; H, 3.97; N, 15.76. 4.1.2.12. 5-Chloro-70 -(1H-imidazol-2-yl)-60 -(5-methoxy-1H-indole3-carbonyl)-2-oxo-30 ,60 ,70 , 7a0 -tetrahydro-10 H-spiro[indoline-3,50 pyrrolo[1,2-c]thiazole]-60 -carbonitrile (6l). White solid; mp 205  C; IR (cm1): 3367, 3237, 2986, 2834, 2242, 1712, 1624, 1516, 1432, 1131, 1211, 1032, 765, 686; 1H NMR (400 MHz, DMSO-d6): dH: 12.12 (d, 1H, J ¼ 2.4 Hz), 11.75 (s, 1H), 10.86 (s, 1H), 8.09 (d, 1H, J ¼ 3.1 Hz), 7.59 (s, 1H), 7.33 (d, 1H, J ¼ 8.8 Hz), 7.26 (d, 1H, J ¼ 1.8 Hz), 7.06 (d, 2H, J ¼ 8.2 Hz), 6.98e6.89 (m, 1H), 6.80 (dd, 1H, J ¼ 2.1, 8.7 Hz), 6.38 (d, 1H, J ¼ 8.3 Hz), 4.89e4.82 (m, 1H), 4.48 (d, 1H, J ¼ 10.4 Hz), 3.84 (d, 1H, J ¼ 11.2 Hz), 3.69 (s, 3H), 3.32 (d, 1H, J ¼ 11.1 Hz), 3.28e3.19 (m, 1H), 3.11 (d, 1H, J ¼ 11.7 Hz); 13C NMR (100 MHz, DMSO-d6): dC:

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180.9, 174.7, 155.9, 141.3, 141.2, 133.9, 130.7, 129.3, 127.9, 127.0, 125.0, 122.6, 117.7, 116.2, 113.7, 113.5, 113.3, 112.8, 111.2, 102.7, 76.4, 69.1, 64.6, 55.3, 54.5, 47.9, 36.3; MS m/z ¼ 545 [M þ H]þ; Anal. Calcd for C27H21ClN6O3S: C, 59.50; H, 3.88; N, 15.42. Found: C, 59.65; H, 3.91; N, 15.35. 4.1.2.13. 5-Bromo-70 -(1H-imidazol-2-yl)-60 -(5-methoxy-1H-indole3-carbonyl)-2-oxo-30 ,60 ,70 , 7a0 -tetrahydro-10 H-spiro[indoline-3,50 pyrrolo[1,2-c]thiazole]-60 -carbonitrile (6m). Off white solid; mp 196  C; IR (cm1): 3365, 2925, 2851, 2182, 2241, 1715, 1622, 1519, 1440, 1093, 1029, 923, 803,663, 628; 1H NMR (400 MHz, DMSO-d6): dH: 12.10 (s, 2H), 10.84 (s, 1H), 8.07 (d, 1H, J ¼ 2.9 Hz), 7.69 (s, 1H), 7.32 (d, 1H, J ¼ 8.65 Hz), 7.25 (d, 1H, J ¼ 1.7 Hz), 7.18 (d, 1H, J ¼ 8.1 Hz), 7.01 (s, 2H), 6.79 (dd, 1H, J ¼ 2.0, 8.8 Hz), 6.32 (d, 1H, J ¼ 8.2 Hz), 4.83e4.87 (m, 1H), 4.47 (d, 1H, J ¼ 10.4 Hz), 3.83 (d, 1H, J ¼ 11.4 Hz), 3.71 (s, 3H), 3.34e3.22 (m, 2H), 3.10 (d, 1H, J ¼ 11.8 Hz); 13 C NMR (100 MHz, DMSO-d6): dC: 180.9, 174.6, 156.0, 141.6, 141.3, 133.9, 133.5, 132.0, 131.3, 130.7, 127.0, 123.0, 116.2, 113.8, 113.6, 113.3, 112.8, 112.8, 111.7, 102.7, 76.3, 69.0, 64.7, 55.4, 54.6, 47.9, 36.3; MS m/ z ¼ 589 [M þ H]þ; Anal. Calcd for C27H21BrN6O3S: C, 55.01; H, 3.55; N, 14.32. Found: C, 55.13; H, 3.55; N, 14.32. 4.1.2.14. 7 0 -(1H-Imidazol-2-yl)-6 0 -(5-methoxy-1H-indole-3carbonyl)-5-nitro-2-oxo-30 ,60 ,70 , 7a0 -tetrahydro-10 H-spiro[indoline3,50 -pyrrolo[1,2-c]thiazole]-60 -carbonitrile (6n). Pale yellow solid; mp 198  C; IR (cm1): 3355, 3126, 3108, 2954, 2934, 2240, 1718, 1654, 1523, 1484, 1380, 1185, 1145, 1082, 1030, 778, 714; 1H NMR (400 MHz, DMSO-d6): dH: 12.10 (s, 2H), 11.65 (s, 1H), 8.45 (s, 1H), 8.10 (d, 1H, J ¼ 3.3 Hz), 7.84e7.74 (m, 2H), 7.42 (d, 1H, J ¼ 8.1 Hz), 7.20 (d, 1H, J ¼ 7.6 Hz), 7.05e6.88 (m, 3H), 6.57 (d, 1H, J ¼ 8.8 Hz), 4.90e4.84 (m, 1H), 4.55 (d, 1H, J ¼ 10.6 Hz), 3.95 (d, 1H, J ¼ 11.0 Hz), 3.72 (s, 3H), 3.55e3.30 (m, 2H), 3.20 (d, 1H, J ¼ 11.2 Hz); 13C NMR (100 MHz, DMSO-d6): dC: 180.9, 175.0, 156.0, 148.3, 144.1, 142.7, 142.0, 138.3, 133.3, 130.5, 127.2, 126.9, 125.4, 122.3, 117.9, 113.7, 113.3, 113.4, 110.1, 102.8, 76.1, 69.1, 64.9, 55.3, 54.0, 47.9, 36.2; MS m/ z ¼ 556 [M þ H]þ; Anal. Calcd for C27H21N7O5S: C, 58.37; H, 3.81; N, 17.65. Found: C, 58.41; H, 3.86; N, 17.52. 4.2. Biological assays 4.2.1. Anticancer properties A549 human adenocarcinoma lung cancer cell line was obtained from National Institute of Cell Sciences, Pune, India. A549 lung adenocarcinoma cancer cell line was maintained in complete tissue culture medium DMEM with 10% Foetal Bovine Serum and 2 mM LGlutamine, along with antibiotics (about 100 IU/mL of penicillin, 100 mg/mL of streptomycin) with the pH adjusted to 7.2. The cytotoxicity was determined according to our previously reported method with some changes [15]. Cells (5  105) were seeded in 96 well plates containing medium with different concentrations such as 100, 50 and 25 mg/mL. The cells were cultivated at 37  C with 5% CO2 and 95% air in 100% relative humidity. After various durations of cultivation, the solution in the medium was removed. An aliquot of 100 mL of medium containing 1 mg/mL of 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was loaded to the plate. The cells were cultured for 4 h and then the solution in the medium was removed. An aliquot of 100 mL of DMSO was added to the plate, which was shaken until the crystals were dissolved. The cytotoxicity against cancer cells was determined by measuring the absorbance of the converted dye at 540 nm in an ELISA reader. Cytotoxicity of each sample was expressed as IC50 value. The IC50 value is the concentration of test sample that causes 50% inhibition of cell growth, averaged from three replicate experiments.

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4.2.2. Statistical analysis Cytotoxic properties of synthesised compounds were statistically analysed by Duncan multiple range test at P ¼ 0.05 with the help of SPSS 11.5 version software package. 4.2.3. DNA fragmentation For the DNA fragmentation assay, A549 lung adenocarcinoma cancer cells (1  106 cells) (control and treated with synthesised compound of 4h, 4i, 4j, 6a, 6b, 6f, 6h, 6i, 6j, 6l and 6g) were collected by centrifugation at 2000 rpm for 10 min and washed twice with phosphate-buffered saline (PBS, Ambion USA). The cell pellet was suspended in 100 mL of cell lysis buffer (10 mM Trise HCl buffer, pH 7.4 containing 10 mM EDTA and 0.5% triton X-100), kept at 4  C for 15 min and the cell lysate was centrifuged at 16,000 rpm for 20 min. The supernatants were incubated with proteinase K (0.4 mg/mL; SigmaeAldrich) at 60  C for 60 min, then incubated with RNase A (0.4 mg/mL; SigmaeAldrich) at 37  C for 60 min. The supernatants were mixed with 20 mL of 5 M NaCl and 120 mL of isopropyl alcohol overnight at 20  C. The supernatants were then collected by centrifugation at 16,000 rpm for 15 min. DNA samples were dissolved in TE buffer (10 mM TriseHCl, pH 7.4 and 1 mM EDTA, pH 8.0), and separated by 2% agarose gel electrophoresis. 4.2.4. Apoptosis analysis of A549 cells A549 lung adenocarcinoma cancer cells were cultured in medium supplemented with or without the synthesised compound (25 mg/mL). After 24 h of treatment, cells were washed twice with PBS (0.01 M) and suspended in binding buffer. To identify apoptotic cells, these cells were incubated with FITC and PI for 30 min at 4  C in dark room. Cells were then centrifuged and pellets were smeared. FITC (Fluorescein Isothiocyanate) and PI (Propidium Iodide) fluorescence were immediately observed under confocal laser scanning microscope (Olympus, FV1000, Japan) at 400  magn ifications. 4.3. Molecular docking studies Molecular docking studies have been done using the AutoDock Tools (ADT) version 1.5.6 and AutoDock version 4.2.5.1 docking program. 4.3.1. Protein preparation The ALK protein structure was obtained from the Protein Data Bank (PDB ID was 2XP2). The co-crystallised ligand (crizotinib) in the ALK structure was removed. Then the polar hydrogen atoms were added, lower occupancy residue structures were deleted, and any incomplete side chains were replaced using the ADT. Further ADT was used to remove crystal water, Gasteiger charges were added to each atom, and merged the non-polar hydrogen atoms to the protein structure. The distance between donor and acceptor atoms that form a hydrogen bond was defined as 1.9  A with a tolerance of 0.5  A, and the acceptore hydrogenedonor angle was not less than 120 . The structures were then saved in PDBQT file format, for further studies in ADT. 4.3.2. Ligand preparation Ligand 2D structures were drawn using ChemDraw Ultra 7.0 (ChemOffice 2002). Chem3D Ultra 7.0 was used to convert 2D structure into 3D and the energy minimised using semi-empirical AM1 method. Minimise energy to minimum RMS gradient of 0.100 was set in each iteration. All structures were saved as .pdb file format for input to ADT. All the ligand structures were then saved in PDBQT file format, to carry out docking in ADT.

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4.3.3. Grid formation A grid box with dimension of 40  40  40 Å3 with 0.375  A spacing and centred on 29.470, 47.997, 8.863 was created around the binding site of crizotinib on ALK protein using ADT. The centre of the box was set at crizotinib ligand centre and grid energy calculations were carried out. 4.3.4. Docking protocol For the AutoDock docking calculation, default parameters were used and 10 docked conformations were generated for each compound. The energy calculations were done using genetic algorithms. The outputs were exported to PyMol for visual inspection of the binding modes and interactions of the compounds with amino acid residues in the active sites. Acknowledgements One of the authors Y. Arun thanks the University Grants Commission, New Delhi, India for the research fellowship. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ejmech.2013.12.027. References [1] G.S. Singh, Z.Y. Desta, Chem. Rev. 112 (2012) 6104e6155. [2] N. Anderton, P.A. Cockrum, S.M. Cologate, J.A. Edgar, K. Flower, I. Vit, R.I. Willing, Phytochemistry 48 (1998) 437e439. [3] A. Jossang, P. Jossang, A.H. Hamid, T. Sevenet, B. Bodo, J. Org. Chem. 56 (1991) 6527e6530. [4] W.H. Wong, P.B. Lim, C.H. Chuah, Phytochemistry 41 (1996) 313e315. [5] C.B. Cui, H. Kakeya, H. Osada, Tetrahedron 52 (1996) 12651e12666.

[6] M.N.G. James, G.J.B. Williams, Can. J. Chem. 50 (1972) 2407e2412. [7] T.-H. Kang, Y. Murakami, K. Matsumoto, H. Takayama, M. Kitajima, N. Aimi, H. Watanabe, Eur. J. Pharmacol. 455 (2002) 27e34. [8] T.-H. Kang, K. Matsumoto, M. Tohda, Y. Murakami, H. Takayama, M. Kitajima, N. Aimi, H. Watanabe, Eur. J. Pharmacol. 444 (2002) 39e45. [9] G. Bhaskar, Y. Arun, C. Balachandran, C. Saikumar, P.T. Perumal, Eur. J. Med. Chem. 51 (2012) 79e91. [10] Y. Arun, G. Bhaskar, C. Balachandran, S. Ignacimuthu, P.T. Perumal, Bioorg. Med. Chem. Lett. 23 (2013) 1839e1845. [11] K. Parthasarathy, C. Praveen, C. Balachandran, P. Senthil kumar, S. Ignacimuthu, P.T. Perumal, Bioorg. Med. Chem. Lett. 23 (2013) 2708e2713. [12] A.S. Girgis, Eur. J. Med. Chem. 44 (2009) 91e100. [13] E. Rajanarendar, S. Ramakrishna, K. Govardhan Reddy, D. Nagaraju, Y.N. Reddy, Bioorg. Med. Chem. Lett. 23 (2013) 3954e3958. [14] S.M. Rajesh, S. Perumal, J.C. Menendez, P. Yogeeswari, D. Sriram, MedChemComm 2 (2011) 626e630. [15] B. Zhang, P. Feng, L.-H. Sun, Y. Cui, S. Ye, N. Jiao, Chem. Eur. J. 18 (2012) 9198e 9203. [16] M.A. Ali, R. Ismail, T.S. Choon, Y.K. Yoon, A.C. Wei, S. Pandian, R.S. Kumar, H. Osman, E. Manogaran, Bioorg. Med. Chem. Lett. 20 (2010) 7064e7066. [17] R. Siegel, D. Naishadham, A. Jemal, CA Cancer J. Clin. 63 (2013) 11e30. [18] A. Dömling, W. Wang, K. Wang, Chem. Rev. 112 (2012) 3083e3135. [19] B.B. Toure, D.G. Hall, Chem. Rev. 109 (2009) 4439e4486. [20] A. Hazra, Y.P. Bharitkar, D. Chakraborty, S.K. Mondal, N. Singal, S. Mondal, A. Maity, R. Paira, S. Banerjee, N.B. Mondal, ACS Comb. Sci. 15 (2012) 41e48. [21] N.V. Lakshmi, Y. Arun, P.T. Perumal, Tetrahedron Lett. 52 (2011) 3437e3442. [22] N.V. Lakshmi, P. Thirumurugan, P.T. Perumal, Tetrahedron Lett. 51 (2010) 1064e1068. [23] Crystallographic data for spirooxindoleepyrrolidine 1a in this letter have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication number CCDC 964149. These data can be obtained free of charge at www.ccdc.cam.ac.uk/conts/retrieving.html [or from the Cambridge Crystallographic Data Centre (CCDC),12 Union Road, Cambridge CB2 1EZ, UK; fax: þ44(0)1223 336 033; email: [email protected]]. [24] H. Akiyama, M. Endo, T. Matsui, I. Katsuda, N. Emi, Y. Kawamoto, T. Koike, H. Beppu, Biochim. Biophys. Acta Gen. Subj. 1810 (2011) 519e525. [25] http://autodock.scripps.edu/resources/references. [26] J.J. Cui, M. Tran-Dube, H. Shen, M. Nambu, P.-P. Kung, M. Pairish, L. Jia, J. Meng, L. Funk, I. Botrous, M. McTigue, N. Grodsky, K. Ryan, E. Padrique, G. Alton, S. Timofeevski, S. Yamazaki, Q. Li, H. Zou, J. Christensen, B. Mroczkowski, S. Bender, R.S. Kania, M.P. Edwards, J. Med. Chem. 54 (2011) 6342e6363.

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Novel spirooxindole-pyrrolidine compounds: synthesis, anticancer and molecular docking studies.

Novel spirooxindole-pyrrolidine compounds have been synthesised through 1,3-dipolar cycloaddition of azomethine ylides generated from isatin and sarco...
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