Synthesis and biological evaluation of spirofused pyrazole and pyrrolidine hybrids from tryptanthrin

Synthesis and biological evaluation of spirofused pyrazole and pyrrolidine hybrids from tryptanthrin | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis and biological evaluation of spirofused pyrazole and pyrrolidine hybrids from tryptanthrin Devika Krishnan, Sruthi Sudheendran Leena, Ashkar Ali B., E. G. Jayasree, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9343418/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Twenty-two spirofused heterocycles containing tryptanthrin - pyrazole/pyrrolidine hybrids were synthesized via classical (3 + 2) cycloaddition reaction of ethyl ( E )-2-(12-oxoindolo[2,1- b ]quinazolin-6(12 H )-ylidene)acetate with nitrile imine / azomethine ylide. The compounds were screened in vitro for antibacterial activity against the ESKAPE pathogen panel, from which spiropyrazoles 3d and 3f (MIC = 8 µg m/L) were found to be most potent against S. aureus ATCC 29213. Anticancer evaluation of the molecules against HCT-116 human colon cancer cell line by MTT assay indicated dispiropyrrolidines 6a and 6c exhibited highest activity with LC₅₀ values of 23.57 µg/mL and 32.99 µg/mL respectively. Molecular docking studies demonstrated the potential of 6a and 6c as promising candidates for modulating the activity of the IDO1 protein. These findings further support the experimental results obtained from in vitro biological studies and highlight the prospective utility of pyrazole/pyrrolidine derivatives for future therapeutic applications. Pyrazoles pyrrolidines tryptanthrin anti- bacterial and anticancer activity Figures Figure 1 Figure 2 Figure 3 1. Introduction The rise of antimicrobial resistance (AMR) has intensified the global risk associated with infectious diseases [ 1 ]. This challenge is especially evident with Staphylococcus aureus , a bacterium capable of developing multidrug resistance (MDR), which can result in severe complications due to its complex nature and wide range of virulence factors [ 2 ]. Methicillin-resistant Staphylococcus aureus (MRSA) poses a serious clinical threat, consistently associated with high rates of illness and death [ 3 ]. Glycopeptide antibiotics like vancomycin are commonly used to treat MRSA infections; however, the emergence of vancomycin-resistant S. aureus (VRSA), recognized by the World Health Organization as a high-priority antibiotic-resistant pathogen, highlights the critical need for the discovery and development of novel antimicrobial agents effective against the MDR pathogens [ 4 ]. Parallelly, cancer is a leading cause of death worldwide; 20 million new cancer cases were recorded in 2022 and it is estimated to increase to 35 million new cases by 2050 [ 5 ]. Cancer cells also develop multiple and complex mechanisms to evade drug-induced cytotoxicity, making resistance to drugs a significant obstacle to successful cancer therapy [ 6 ]. In this context, searching new antibacterial and anticancer agents which can be effective against MDR pathways is very crucial in modern times. The work embodied in the current manuscript discloses the synthesis and promising preliminary results about the antibacterial and anticancer activities of certain tryptanthrin incorporated heterocycles. Tryptanthrin (indolo[2,1- b ]quinazoline-6,12-dione), a bioactive indoloquinazoline alkaloid isolated from several medicinal plants, including Strobilanthes cusia (Assam indigo), Isatis tinctoria (Chinese woad), and Polygonum tinctorium (Japanese indigo) [ 7 , 8 ] has gained considerable scientific interest due to its diverse pharmacological activities such as antibacterial, anti-inflammatory, anticancer, antifungal, and antiviral properties [ 9 , 10 ]. Owing to its significant therapeutic potential, numerous tryptanthrin hybrid molecules have been chemically synthesized and evaluated for their efficacy against diverse diseases and infections [ 9 , 11 – 13 ]. Investigations by our research group have identified tryptanthrin derived spiro compounds as promising candidates for antibacterial activity, particularly against Staphylococcus aureus . A series of spiro-fused tryptanthrin-thiopyrano[2,3- b ]indole hybrid molecules targeting drug-resistant S. aureus , led us to the identification of nitro-substituted hybrid molecule with potent activity against S. aureus ATCC 29213, with a good selectivity index [ 14 ]. Later, a series of tryptanthrin appended dispiropyrrolidine oxindoles [ 15 ] and 4-spiropiperidines [ 16 ] were also reported and among them bromo substituted derivatives exhibited lowest MIC values of 0.125 µg/mL and 4 µg/mL respectively. Figure 1 depicts the structures of tryptanthrin hybrid molecules active against S. aureus made previously in our lab. The anticancer activities of various tryptanthrin derived heterocyclic compounds have also been well recognized [ 17 ]. In line with the latter, our group has also reported the synthesis of tryptanthrin-1,2,3-triazole hybrids that exhibited potent anticancer activity against breast and colon cancer cell lines [ 18 ]. Owing to our continued interest in developing effective antibacterial/anticancer agents from tryptanthrin, we now report the synthesis of two distinct classes of spiro compounds through 1,3-dipolar cycloaddition reactions, using ethyl ( E )-2-(12-oxoindolo[2,1- b ]quinazolin-6(12 H )-ylidene)acetate as dipolarophile and nitrile imine / azomethine ylide as dipoles. The cycloaddition of nitrile imine [ 19 ] to the above dipolarophile resulted in a pyrazole ring; a promising scaffold in drug development with a broad spectrum of biological activities such as antibacterial, anti-inflammatory, antiviral, anticancer and antimicrobial properties [ 20 – 22 ]. Meanwhile, the use of azomethine ylide as the dipole resulted in the formation of spiropyrrolidines another significant scaffold extensively explored in the synthesis of novel bioactive compounds [ 23 , 24 ]. This study thus reports the synthesis of tryptanthrin appended spiropyrazoles and dispiropyrrolidines and evaluates their in vitro antibacterial activity against ESKAPE pathogens, and in vitro anticancer activity against human colon cancer cell lines which is further supported by molecular docking study. 2. Results and Discussion 2.1 Synthesis of tryptanthrin appended spiropyrazoles and dispiropyrrolidines In separate reactions, ethyl ( E )-2-(12-oxoindolo[2,1- b ]quinazolin-6(12 H )-ylidene)acetate 1a was reacted with (i) nitrile imine and (ii) azomethine ylide which led to the formation of tryptanthrin appended spiropyrazole 3a and dispiropyrrolidine 6a respectively. As a pilot reaction, compound 1a was treated with the nitrile imine, (formed in situ by the dehydrochlorination of the corresponding hydrazonyl chloride 2a ), in acetonitrile at room temperature and after completion of the reaction as observed from TLC, the product 3a was isolated by silica gel column chromatography using 80:20 hexane-ethyl acetate in 92% yield. In a discrete reaction, azomethine ylide was generated by refluxing isatin 4 , and sarcosine 5a in ethanol and the cycloaddition with 1a was proceeded by refluxing at 85℃ until complete consumption of isatin was observed from TLC. The product 6a was isolated by silica gel column chromatography using 70:30 hexane-ethyl acetate in 91% yield (Scheme 1 ). The structure of the compounds 3a and 6a were characterized by IR, 1 H NMR, 13 C NMR and CHN analysis. The FTIR spectrum of 3a showed sharp peaks at 1672 cm − 1 and 1713 cm − 1 corresponding to the amide and ester carbonyls respectively. In the 1 H NMR spectrum of 3a , the singlet at δ 5.24 ppm corresponded to -CH proton and the multiplets ranging from δ 6.74–8.66 ppm corresponds to 18 aromatic protons. In the 13 C NMR spectrum the spiro carbon was observed at δ 65.5 ppm. The amide carbonyl carbon was seen at δ 166.5 ppm while the imine carbons were seen at δ 159.7 and 159.0 ppm. In the FTIR spectrum of 6a three characteristic stretchings at 1695 cm − 1 , 1705 cm −1 and 1722 cm − 1 can be attributed to amide, indole and ester carbonyl groups. In the 1 H NMR spectrum, a singlet at δ 2.22 ppm corresponds to N-CH 3 protons, the two multiplets in the range δ 3.69–3.61 and δ 3.50–3.44 ppm corresponded to N-CH 2 protons and a triplet at δ 3.78 corresponds to -CH proton of the pyrrolidine ring. The NH proton of oxindole was observed as a singlet at δ 10.46 ppm. In the 13 C NMR spectrum the spiro carbons were observed at δ 60.6 and 78.6 ppm. The amide carbonyl groups were observed at δ 159.5 and δ 169.8 ppm while ester carbonyl was seen at δ 176.4 ppm. The elemental analysis and purity of 3a and 6a were ascertained by CHN analysis. In order to optimize the conditions, the reaction was carried out by varying the solvent, temperature and time (Table 1 ). For formation of 3a , use of polar aprotic solvent anhydrous CH 3 CN was found to be beneficial leading to 92% yield (entry 1), while use of polar protic solvent EtOH was found to be advantageous for formation of 6a (obtained in 88% yield, entry 5) which was subsequently improved to 91% by adding toluene at 85 ºC (Entry 7). Table 1 Optimization of reaction condition. Entry Solvent T (⁰C) Time (h) Yield (%) b 3a-j 6a-j 1 CH 3 CN rt 4 92 40 2 CH 3 CN 80 4 92 45 3 MeOH rt 24 65 70 4 EtOH rt 24 68 80 5 EtOH 85 12 70 88 6 Toluene 110 12 75 65 7 EtOH-Toluene 85 12 75 91 a The reactions were performed with 1a (1 mmol), 2a (1 mmol), and base (1 mmol) in the solvent (5.0 mL) for formation of 3a - j The reaction was performed with 1a (1 mmol), 4 (1 mmol), and 5a (2 mmol) in the solvent (5.0 mL) for formation of 6a - j b Isolated yield. Employing the optimized reaction conditions, we investigated the scope of the reaction by varying the substituents on both the dipolarophile and the 1,3- dipoles (Scheme 2 ). When electron-withdrawing groups such as halogens were introduced at the C 8 position, the desired compounds ( 3b–d & 6b–d) were obtained in good yields. However, introducing an electron-donating methoxy group at the same position led to a slight decrease in the yield of compounds ( 3e & 6e) . Additionally, substituting C 2 /C 8 /both positions with halogens ( 3f & 6f-h ) resulted in moderate yields. Varying the substituents on 1,3- dipoles by changing the substituents at phenyl ring of nitrile imine ( 3g-j ) and changing the amino acid part of azomethine ylide to proline and thioproline ( 6i-j ) resulted in products with moderate yield. Substrate scope is further extended by varying the dipolorophile to 2-(12-oxoindolo[2,1-b]quinazolin-6(12H)-ylidene)malononitrile and the corresponding spiropyrazole 3k and dispiropyrrolidine 6k were obtained in good yields as shown in Scheme 3 . 2.2. In vitro biological studies 2.2.1. In vitro antimicrobial activity against bacterial pathogen panel All compounds 3a-k and 6a-k were evaluated for their antibacterial activity against the bacterial pathogen panel to determine the minimum inhibitory concentration (MIC). Levofloxacin was used as the positive control and results were depicted in Table 2 . As can be seen, among the synthesized compounds, 3b - 3f, 3h, 3j-k, 6c and 6j-k exhibited antibacterial activity against S. aureus ATCC 29213 of which the 8-fluoro substituted spiropyrazole ( 3d ) and 2- chloro, 8-bromo substituted spiropyrazole ( 3f ) exhibited a low MIC of 8 µg/mL. Table 2 MIC ( µ g/mL) of 3a-k and 6a-k against bacterial pathogen panel along with comparator antibiotic Sl. No. Compound name E. coli ATCC 25922 S.aureus ATCC 29213 K.pneumoniae BAA 1705 A.baumannii BAA 1605 P.aeruginosa ATCC 27853 E. coli IMP 4213 1 3a > 64 > 64 > 64 > 64 > 64 > 64 2 3b > 64 32 > 64 > 64 > 64 > 64 3 3c > 64 16 > 64 > 64 > 64 > 64 4 3d > 64 8 > 64 > 64 > 64 > 64 5 3e > 64 16 > 64 > 64 > 64 > 64 6 3f > 64 8 > 64 > 64 > 64 > 64 7 3g > 64 > 64 > 64 > 64 > 64 > 64 8 3h > 64 16 > 64 > 64 > 64 > 64 9 3i > 64 > 64 > 64 > 64 > 64 > 64 10 3j > 64 16 > 64 > 64 > 64 > 64 11 3k > 64 64 > 64 > 64 > 64 > 64 12 6a > 64 > 64 > 64 > 64 > 64 > 64 13 6b > 64 > 64 > 64 > 64 > 64 > 64 14 6c > 64 16 > 64 > 64 > 64 > 64 15 6d > 64 > 64 > 64 > 64 > 64 > 64 16 6e > 64 > 64 > 64 > 64 > 64 > 64 17 6f > 64 > 64 > 64 > 64 > 64 > 64 18 6g > 64 > 64 > 64 > 64 > 64 > 64 19 6h > 64 > 64 > 64 > 64 > 64 > 64 20 6i > 64 > 64 > 64 > 64 > 64 > 64 21 6j > 64 64 > 64 > 64 > 64 > 64 22 6k > 64 64 > 64 > 64 > 64 > 64 23 Levofloxacin 0.0156 0.25 64 4 1 0.0078 2.2.2. In vitro anticancer studies by MTT assay Recent reports on the potential anticancer effects of several tryptanthrin derived heterocyclic compounds [ 17 ] highlight the importance of investigating the anticancer activity of the synthesized pyrazole and pyrrolidine hybrids of tryptanthrin. Since colorectal cancer ranks as the third most prevalent malignant disease worldwide, compounds 3d , 3f , 6b and 6c were subjected to in vitro evaluation against the HCT-116 colon cancer cell line using the MTT assay (Fig. 2 ) and the results are presented in Table 3 . It was found that chloro substituted dispiropyrrolidine 6b and bromo substituted dispiropyrrolidine 6c exhibited significant anticancer properties against HCT-116 colon cancer cell line with LC 50 32.99 µ g/mL and 23.57 µ g/mL respectively. By varying concentrations of both 6b and 6c (6.25, 12.5, 25, 50 and 100 µ g/mL), percentage viability decreased appreciably. The concentration dependent cell death and low LC 50 value observed for the compounds highlights its potential efficacy as potent anticancer agents. Table 3 LC 50 value calculated at the end of 24 h incubation Sl. No. Compound name LC 50 ( µ g/mL) 1 3d 198.8 2 3f 187.6 3 4 6b 6c 32.99 23.57 2.3. Molecular docking studies Furthermore, molecular docking studies were carried out to support and validate the in vitro findings of the anticancer study. Indoleamine 2,3-dioxygenase (IDO1), an intracellular enzyme that is highly expressed in a variety of tumors including colorectal cancer, breast cancer and cervical squamous cell carcinoma was considered as a classical target of tryptanthrin and its derivatives [ 25 , 26 ]. Structure activity relationship (SAR) analysis revealed that the presence of an electron withdrawing group at the C-8 position of tryptanthrin significantly contributes to IDO1 inhibition [ 27 ]. Studies on the inhibitory effects of tryptanthrin on colorectal cancer have shown that the underlying mechanisms are significantly associated with the inhibition of Topo I and IDO1 proteins [ 28 ]. Therefore, molecular docking studies were performed to evaluate the binding affinity and interaction profile of the selected ligands 6b and 6c against the IDO1 protein as the target. The three-dimensional crystal structure of IDO1 protein (PDB ID: 2D0T, 4-phenylimidazole bound form of human indoleamine 2,3-dioxygenase) was retrieved from the Protein Data Bank. Protein preparation was carried out using PyMol Software [ 29 ]. Ligand structures were energy minimized employing Gaussian program and B3LYP/6-311 + G(d) level of theory prior to docking. Docking simulations were performed using PyRx Software [ 30 ]. Binding affinities were recorded in kcal/mol, and the best-ranked poses were selected based on binding energy and interaction consistency. Protein–ligand interactions, including van der waal, π–π stacking and π–alkyl interactions were analyzed using ChimeraX Software [ 31 ]. Both 2D and 3D interaction maps were generated for visualization. The docking outcomes, presented in Table 4, reveal the binding affinities between the ligands and the protein. Both ligands demonstrated strong binding affinity (-9.4 kcal/mol) towards the 2D0T protein exhibiting similar interactions. Figures 3 (a) & (b) illustrate the 2D and 3D representations of these interactions. Interactions between the ligands 6b and 6c against 2D0T protein, emphasizing various types of non-covalent interactions that contribute to binding affinity and stability. The diagram highlights π-interactions, including π-Alkyl (light pink lines), and π-π stacking (pink lines) interactions that are observed with residues such as ARG B:231, LEU B:234, PHE B:291 and LEU B:384, indicating significant aromatic and electrostatic contributions. These diverse interactions underscore the ligand's strong binding affinity and its potential effectiveness as a modulator of the 2D0T protein which supports the experimental results of in vitro anticancer studies. 3. Conclusion A series of twenty-two tryptanthrin based spiro compounds containing pyrazole and pyrrolidine frameworks were synthesized through (3 + 2) cycloaddition reactions of tryptanthrin ylidene with nitrile imines and azomethine ylides. All the synthesized compounds were tested for antibacterial activity against ESKAPE pathogens of which 3b - 3f, 3h, 3j-k, 6c & 6j-k exhibited antibacterial activity against S. aureus ATCC 29213 while 3d and 3f exhibited a low MIC of 8 µg/mL. Compounds 3d , 3f , 6b & 6c were evaluated for anticancer activity against HCT-116 cell line by MTT assay and the compound 6b & 6c were found to be active with LC 50 32.99 µ g/mL and 23.57 µ g/mL respectively. The molecular docking studies reveal valuable insights into the ligand's binding mechanism and establish its potential as a promising candidate for modulating the 2D0T protein's activity, which further supports the experimental results of in vitro anticancer study. All these results position tryptanthrin-pyrazole/pyrrolidine hybrids as promising molecules for further antibacterial/anticancer studies in future. 4. Experimental section 4.1 Materials and methods All reagents and solvents were procured from commercial suppliers (Spectrochem Pvt. Ltd., SIGMA-ALDRICH Chemicals Pvt. Ltd., and Merck Specialities Pvt. Ltd.) and were used without further purification. The reaction progress was monitored by thin-layer chromatography (TLC) performed on silica gel 60 F254 pre-coated aluminium sheets. TLC was visualized by a 254 nm UV lamp and iodine staining. NMR spectra were recorded on a 400MHz Bruker Avance FTNMR spectrometer using CDCl 3 and DMSO as solvents. 4.2.1. General procedure for the synthesis of tryptanthrins (1a-h) Tryptanthrin derivatives were synthesized from the corresponding isatins and isatoic anhydrides according to a previously reported procedure [7]. Isatin (1 mmol) was taken in a round bottom flask, followed by the addition of isatoic anhydride (1 mmol) and triethylamine (5 mmol). A minimal amount of dry toluene was added as solvent and the reaction mixture was refluxed at 110 ℃ for three hours. The precipitated product was filtered, washed using methanol and dried. The product was recrystallized from ethanol. 4.2.2 General procedure for the synthesis of Spiropyrazole compounds (3a-k) Hydrazonyl chloride (0.25 mmol) was stirred in acetonitrile in a 50 ml round-bottom flask at room temperature followed by the addition of triethylamine (0.25 mmol) as base for the in-situ generation of nitrile imine. It is then added with tryptanthrin ylidene (0.25 mmol and allowed to stir for 3–4 hours. The completion of reaction was observed from TLC, and the product was isolated by silica gel column chromatography using 80:20 hexane-ethyl acetate in 78–92% yield. 4.2.2.1 Ethyl-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylat e, C 32 H 24 N 4 O 3 , (3a) : Light yellow powder; Yield: 92%; mp: 278–280 ℃; FTIR (cm − 1 ): 3030, 2917, 1713, 1672, 1639, 1590, 1337, 1315. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.66 (d, J = 8 Hz, 1H), 8.48 (d, J = 8 Hz, 1H), 7.78–7.74 (m, 4H), 7.61–7.55 (m, 2H), 7.47–7.42 (m, 4H), 7.28–7.24 (m, 1H), 7.02 (t, J = 8 Hz, 2H), 6.77–6.74 (m, 3H), 5.24 (s, 1H), 3.78 (q, J = 10.1 Hz, 2H), 0.81 (t, J = 7.2 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 166.5, 163.8, 159.7, 159.0, 147.1, 144.1, 142.6, 139.3, 134.7, 131.6, 131.2, 129.0, 128.9, 128.6, 128.4, 128.3, 127.8, 127.5, 126.9, 126.8, 126.7, 126.1, 125.9, 121.7, 121.3, 117.2, 115.5, 115.3, 65.5, 61.7, 29.7, 13.5. Elemental Analysis: Analytically calculated (%): C, 74.99; H, 4.72; N, 10.93; O, 9.36 Found %: C, 7.98; H, 4.73; N, 10.94 4.2.2.2 Ethyl-8-chloro-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C 32 H 23 ClN 4 O 3 , (3b) : Light yellow powder; Yield: 88%; mp: 273–275 ℃; FTIR (cm − 1 ): 3032, 2919, 1717, 1679, 1632, 1593, 1339, 1317. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.52 (d, J = 8.4 Hz, 1H), 8.37–8.35 (dd, J = 8 Hz, J = 0.8 Hz, 1H), 7.69–7.64 (m, 4H), 7.52–7.47 (m, 1H), 7.43–7.40 (m, 1H), 7.37–7.31 (m, 4H), 7.02 (t, J = 8 Hz, 2H), 6.95 (t, J = 8 Hz, 2H), 6.72–6.66 (m, 3H), 5.1 (s, 1H), 3.78 (q, J = 7.2 Hz, 2H), 0.81 (t, J = 7.2 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 166.4, 163.4, 160.8, 159.5, 147.0, 144.3, 142.4, 140.2, 134.8, 132.5, 131.3, 130.4, 129.7, 129.2, 129.1, 128.6, 128.5, 128.3, 128.0, 127.5, 127.0, 126.1, 125.74, 123.5, 122.7, 121.6, 118.2, 115.3, 65.7, 62.0, 29.5, 13.5. Elemental Analysis: Analytically calculated %: C,70.26; H, 4.24; Cl, 6.48; N, 10.24; O, 8.77 Found %: C, 70.27; H, 4.25; N, 10.25. 4.2.2.3 Ethyl-8-bromo-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C 32 H 23 BrN 4 O 3 , (3c) : Light yellow powder; Yield: 85%; mp: 276–278 ℃; FTIR (cm − 1 ): 3034, 2919, 1715, 1675, 1636, 1598, 1335, 1318. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.56 (d, J = 8.4 Hz, 1H) 8.45 (d, J = 8 Hz, 1H), 7.79–7.74 (m, 4H), 7.69–7.66 (dd, J = 8.8 Hz, J = 2 Hz, 1H) 7.61–7.57 (dt, J = 7.3 Hz, J = 4.3 Hz, 2H), 7.47–7.41 (m, 3H), 7.05 (t, J = 8 Hz, 2H), 6.82–6.75 (m, 3H), 5.22 (s, 1H), 3.88 (q, J = 9.2 Hz, 2H), 0.92 (t, J = 7.2 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 170.6, 166.4, 159.5, 159.4, 146.5, 145.6, 142.9, 140.3, 140.2, 138.2, 134.8, 134.2, 133.9, 131.4, 129.8, 129.3, 129.1, 128.6, 128.5, 126.4, 126.1, 125.7, 123.5, 122.0, 121.6, 118.4, 115.8, 115.3, 65.8, 62.0, 29.7, 13.6. Elemental Analysis: Analytically calculated %: C, 64.50; H, 3.29; N, 10.75. Found %: C, 64.52; H, 3.31; N, 10.77. 4.2.2.4 Ethyl-8-fluoro-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C 32 H 23 FN 4 O 3 (3d) : Yellow powder; Yield: 87%; mp: 280–282 ℃; FTIR (cm -1 ): 3171, 3051, 1680, 1628, 1546, 1456, 1337, 1248, 1156, 1061. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.53 (q, J = 4.4 Hz, 1H), 8.31 (d, J = 1.6 Hz, 2H), 7.66–7.60 (m, 4H), 7.36–7.32 (m, 3H), 7.14–7.08 (m, 3H), 6.95 (t, J = 8 Hz, 2H), 6.72–6.63 (m, 2H), 5.12 (s, 1H), 3.78 (q, J = 4.6 Hz, 2H), 0.80 (t, J = 7 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 165.3, 158.7, 158.0, 157.2, 144.4, 143.4, 141.4, 134.2, 134.1, 133.0, 130.3, 128.7, 128.2, 128.0, 127.6, 125.3, 125.1, 121.7, 120.8, 117.7, 117.6, 117.2, 116.9, 114.4, 112.8, 112.5, 64.5, 61.0, 28.6, 12.6. Elemental Analysis: Analytical calculated (%): C, 72.44; H, 4.37; N, 10.56. Found (%): 72.40; H, 4.35; N, 10.52. 4.2.2.5 Ethyl-8-methoxy-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C 33 H 26 N 4 O 4 (3e) : Yellow powder; Yield: 78%; mp: 286–288 ℃; FTIR (cm -1 ): 3170, 3059, 1672, 1630, 1540, 1450, 1330, 1250, 1158, 1065. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.61 (d, J = 8.8 Hz, 1H) 8.45 (t, J = 6.2 Hz, 1H), 7.84 (d, J = 8 Hz, 1H), 7.81–7.74 (m, 2H) 7.69–7.58 (m, 2H), 7.53–7.41 (m, 3H), 7.07–6.96 (m, 2H), 6.94–6.85 (m, 3H), 6.78 (q, J = 8.4 Hz, 2H), 5.22 (s, 1H), 3.87 (q, J = 7.2 Hz, 2H), 3.76 (s, 3H), 0.80 (t, J = 7.28 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 166.3, 166.1, 158.5, 158.3, 145.5, 145.1, 144.4, 142.4, 137.9, 136.8, 135.2, 134.3, 134.1, 131.3, 129.8, 129.7, 129.3, 129.1, 129.0, 128.7, 126.4, 126.2, 126.1, 122.7, 121.8, 120.3, 118.7, 115.4, 65.7, 62.1, 57.1, 29.3, 13.6. Elemental Analysis: Analytical calculated (%): C, 73.05; H, 4.83; N, 10.33. Found (%): C, 73.01; H, 4.85; N, 10.30. 4.2.2.6 Ethyl 8-bromo-2-chloro-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C 32 H 22 BrClN 4 O 3 , (3f) : Yellow powder; Yield: 83%; mp: 288–290 ℃; FTIR (cm -1 ): 3032, 2914, 1717, 1677, 1637, 1599, 1334, 1312. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.44 (d, J = 8.8 Hz, 1H), 8.30 (d, J = 2.4 Hz, 1H), 7.66–7.51 (m, 5H), 7.51 (d, J = 2.4 Hz, 1H), 7.38–7.32 (m, 3H), 6.95 (2H, t, J = 8Hz), 6.73–6.64 (3H, m), 5.09 (s, 1H), 3.80 (q, J = 2.9 Hz, 2H), 0.83 (t, J = 7.2 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 168.6, 166.3, 158.5, 158.3, 145.5, 144.4, 142.4, 137.9, 135.2, 134.3, 134.1, 131.3, 129.8, 129.3, 129.1, 129.0, 128.7, 126.4, 126.1, 122.7, 121.8, 120.3, 119.0, 118.6, 115.4, 115.4, 65.7, 62.1, 29.7, 13.6. Elemental Analysis: Analytical calculated (%): C, 61.41; H, 3.54; N, 8.95. Found (%): C, 61.40; H, 3.55; N, 8.92. 4.2.2.7 Ethyl-8-bromo-2'-(4-fluorophenyl)-12-oxo-5'-phenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C 32 H 22 BrFN 4 O 3 (3g) : Yellow powder; Yield: 82%; mp: 290–292 ℃; FTIR (cm -1 ): 3178, 3045, 1685, 1630, 1545, 1450, 1335, 1240, 1160, 1070. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.62 (d, J = 2 Hz, 1H), 8.38 (d, J = 7.6 Hz, 1H), 7.82–7.77 (m, 1H), 7.72–7.69 (m, 1H), 7.67–7.60 (m, 2H), 7.52 (t, J = 7.4 Hz, 1H), 7.05 (t, J = 8.6 Hz, 3H), 6.95 (t, J =8Hz, 3H), 6.71 (q, J = 7.2 Hz, 2H), 6.64 (d, J = 7.6 Hz, 1H), 5.11 (s, 1H), 3.73 (q, J = 3.3 Hz, 2H), 0.78 (t, J = 8.6 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 166.3, 162.8, 159.8, 159.4, 146.8, 142.4, 140.0, 137.1, 135.2, 135.0, 130.2, 129.9, 129.0, 128.3, 128.1, 128.0, 127.9, 127.1, 126.9, 126.7, 125.0, 121.8, 117.7, 115.9, 115.7, 115.5, 65.4, 62.0, 29.7, 14.1. Elemental Analysis: Analytical calculated (%): C, 63.06; H, 3.64; N, 9.19; Found (%): C, 63.05; H, 3.66; N, 9.18. 4.2.2.8 Ethyl-8-bromo-2-chloro-2'-(4-fluorophenyl)-12-oxo-5'-phenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C 32 H 21 BrClFN 4 O 3 (3h) : Yellow powder; Yield: 80%; mp: 296–298 ℃; FTIR (cm -1 ): 3172, 3051, 1682, 1630, 1552, 1455, 1335, 1250, 1158, 1065. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.44 (d, J = 8.4 Hz, 1H), 8.31 (d, J = 2.4 Hz, 1H), 7.65–7.58 (m, 5H), 7.49 (d, J = 2 Hz, 1H), 7.05 (t, J = 8.6 Hz, 2H), 6.98–6.92 (m, 2H), 6.72 (t, J = 7.2 Hz, 1H), 6.63 (d, J = 7.6 Hz, 2H), 5.07 (s, 1H), 3.79 (q, J = 2.6 Hz, 2H), 0.83 (t, J = 7.2 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 166.2, 160.3, 158.4, 158.3, 145.4, 143.4, 142.4, 137.9, 135.2, 134.3, 134.2, 129.8, 129.1, 128.9, 128.8, 128.0, 127.9, 126.4, 122.7, 121.9, 120.3, 118.7, 115.9, 115.7, 115.4, 65.6, 62.2, 29.7, 13.6. Elemental Analysis: Analytical calculated (%): C, 59.69; H, 3.29; N, 8.70; Found (%): C, 59.66; H, 3.28; N, 8.69 4.2.2.9 Ethyl (4'S,6S)-8-bromo-12-oxo-5'-phenyl-2'-(4-(trifluoromethyl)phenyl)-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C 33 H 22 BrF 3 N 4 O 3 (3i) : Yellow powder; Yield: 87%; mp: 300–302 ℃; FTIR (cm -1 ): 3175, 3051, 1680, 1630, 1544, 1450, 1335, 1240, 1150, 1060. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.38 (d, J = 1.6 Hz, 1H), 8.36 (d, J = 1.2 Hz, 1H), 7.99–7.91 (m, 1H), 7.75–7.67 (m, 3H), 7.62–7.58 (m, 4H), 7.54–7.51 (m, 1H), 7.47–7.45 (m, 1H), 6.98 (t, J = 8 Hz, 2H), 6.75–6.67 (m, 2H), 5.13 (s, 1H), 3.79 (q, J = 7.2 Hz, 2H), 0.82 (t, J = 6 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 167.2, 166.1, 159.5, 158.4, 146.3, 142.0, 140.8, 138.2, 134.9, 134.4, 130.9, 129.5, 129.2, 128.8, 128.3, 128.2, 127.0, 126.4, 126.2, 125.6, 124.6, 123.7, 123.2, 122.2, 120.1, 118.6, 115.8, 115.5, 65.3, 62.3, 29.7, 14.1. Elemental Analysis: Analytical calculated (%): C, 60.10; H, 3.36; N, 8.50; Found (%): C, 60.12; H, 3.34; N, 8.51. 4.2.2.10 Ethyl(4'S,6S)-8-bromo-2-chloro-12-oxo-5'-phenyl-2'-(4-(trifluoromethyl)phenyl)-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C 33 H 21 BrClF 3 N 4 O 3 (3j) : Yellow powder; Yield: 85%; mp: 295–297 ℃; FTIR (cm -1 ): 3168, 3050, 1685, 1636, 1550, 1459, 1340, 1250, 1158, 1065. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.44 (d, J = 8.8 Hz, 1H), 8.31 (d, J = 2.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 2H), 7.65–7.56 (m, 5H), 7.48 (d, J = 1.6 Hz, 1H), 6.98 (t, J = 8 Hz, 2H), 6.75 (t, J = 7.6 Hz, 1H), 6.70–6.64 (m, 2H), 5.10 (s, 1H), 3.80 (q, J = 7 Hz, 2H), 0.83 (t, J = 7 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 166.0, 160.0, 158.2, 158.2, 145.4, 142.8, 141.9, 137.9, 135.3, 134.7, 134.5, 134.3, 129.8, 129.2, 128.8, 128.6, 126.5, 126.3, 126.1, 125.6, 122.7, 122.6, 122.3, 120.4, 119.1, 118.7, 115.6, 65.2, 62.3, 29.7, 13.6. Elemental Analysis: Analytical calculated (%): C, 57.12; H, 3.05; N, 8.07; Found (%): C, 57.13; H, 3.02; N, 8.05. 4.2.2.11 (R)-12-oxo-2',5'-diphenyl-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4',4'(2'H)-dicarbonitrile; C 31 H 18 N 6 O (3k) : Yellow powder; Yield: 93%; mp: 298–300 ℃; FTIR (cm -1 ): 2105, 1676, 1594, 1486, 1456, 1352, 1229, 1095. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 8.80 (d, J = 7.6 Hz, 1H), 8.71 (d, J = 8 Hz, 1H), 8.51 (d, J = 7.6 Hz, 1H), 8.41–8.39 (dd, J = 0.8 Hz, J = 8 Hz, 1H), 8.10–8.06 (m, 2H), 8.02-8.00 (dd, J = 0.6 Hz, J = 8.4 Hz, 1H), 7.86–7.83 (m, 1H), 7.79–7.74 (m, 3H), 7.69–7.65 (m, 1H), 7.59–7.55 (m, 2H), 7.08 (t, J = 8 Hz, 2H), 6.95–6.85 (m, 2H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 162.9, 159.3, 158.9, 146.2, 145.8, 142.0, 141.4, 134.9, 133.5, 133.0, 129.8, 129.1, 128.5, 128.4, 127.1, 126.6, 126.4, 126.1, 124.5, 122.0, 119.8, 118.6, 118.0, 116.8, 116.5, 116.2, 109.9, 109.4, 68.9, 53.2. Elemental Analysis: Analytical calculated (%): C, 75.91; H, 3.70; N, 17.13; Found (%): C, 75.93; H, 3.71; N, 17.10 4.2.3 General procedure for the synthesis of dispiropyrrolidine compounds (6a-k) The dispiropyrrolidine compounds were synthesized by a three-component reaction of isatin (1 mmol), sarcosine (2 mmol) and tryptanthrin ylidene (1 mmol) by refluxing in ethanol- toluene mixture for 85℃ until complete consumption of isatin was observed from TLC. The product was isolated by silica gel column chromatography using 70:30 hexane-ethyl acetate in 70–91% yield. 4.2.3.1 Ethyl-1'-methyl-2,12''-dioxo-12'' H -dispiro[indoline-3,2'-pyrrolidine-3',6''- indolo[2,1- b ]quinazoline]-4'-carboxylate; C 29 H 24 N 4 O 4 (6a) : Yellow solid; Yield: 85%; mp: 250–252 ℃; FTIR (cm -1 ): 1722, 1705, 1695, 1514, 750. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 10.46 (s, 1H), 8.32–8.27 (m, 2H), 8.19 (q, J = 7.8, 1H), 8.00 (s, 1H), 7.58–7.49 (m, 3H), 7.36 (t, J = 7.4 Hz, 1H), 6.95 (t, J = 7.7 Hz, 1H), 6.53 (t, J = 7.6 Hz, 2H), 6.12 (d, J = 7.5 Hz, 1H), 4.95 (q, J = 5.8 Hz, 2H), 3.78 (t, J = 10 Hz, 1H), 3.69–3.61 (m, 1H), 3.50–3.44 (m, 1H), 2.22 (s, 3H), 0.50 (t, J = 7.1 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 176.4, 169.8, 159.5, 155.6, 146.9, 141.7, 139.2, 134.3, 132.7, 132.2, 130.1, 129.7, 127.9, 127.3, 126.7, 125.8, 122.6, 122.3, 121.1, 119.1, 117.8, 109.8, 78.6, 60.6, 51.6, 49.1, 35.0, 29.7, 13.3. Elemental Analysis: Analytical calculated (%): C, 60.96; H, 4.06; N, 9.80; Found (%): C, 60.94; H, 4.06; N, 9.81. 4.2.3.2 Ethyl-8''-chloro-1'-methyl-2,12''-dioxo-12'' H -dispiro[indoline-3,2'-pyrrolidine- 3',6''-indolo[2,1- b ]quinazoline]-4'-carboxylate; C 29 H 23 ClN 4 O 4 (6b) : Yellow solid; Yield: 91%; mp: 214–216 ℃; FTIR (cm -1 ): 1721, 1718,1685, 1575, 690. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 10.89 (s, 1H). 8.32 (d, J = 8.8 Hz, 1H), 8.23–8.21 (m, 1H), 8.13–8.12 (m, 1H), 7.66–7.65 (m, 1H), 7.59–7.57 (m, 1H), 7.44–7.40 (m, 2H), 7.32–7.30 (m, 1H), 6.57–6.53 (m, 1H), 6.45 (d, J = 8 Hz, 1H), 6.34 (d, J = 7.2 Hz, 1H), 4.06 (q, J = 8.8 Hz, 2H), 2.56–2.54 (m, 1H), 2.30 (t, J = 7.6 Hz, 2H), 1.96 (s, 3H), 0.68 (t, J = 6.8 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 175.6, 165.1, 157.2, 140.7, 139.0, 137.2, 135.5, 134.7, 131.7, 130.0, 129.1, 128.4, 128.1, 127.3, 125.0, 121.0, 119.9, 119.7, 118.1, 116.4, 113.2, 110.2, 86.2, 61.5, 52.7, 46.7, 29.7, 22.7, 14.1. Elemental Analysis: Analytical calculated (%): C, 70.72; H, 4.91; N, 11.38; Found (%): C, 70.70; H, 4.92; N, 11.36. 4.2.3.3 Ethyl-8''-bromo-1'-methyl-2,12''-dioxo-12'' H -dispiro[indoline-3,2'-pyrrolidine- 3',6''-indolo[2,1- b ]quinazoline]-4'-carboxylate; C 29 H 23 BrN 4 O 4 (6c) : Yellow solid; Yield: 88%; mp: 225–227 ℃; FTIR (cm − 1 ): 1724, 1719, 1691, 1595, 1450. 1 H NMR (δ ppm, 400 MHz, DMSO -d6 ): 10.71 (s, 1H), 8.10 (d, J = 7.7, 1H), 8.02 (q, J = 5, 1H), 7.84 (d, J = 7.8, 1H), 7.79–7.71 (m, 1H), 7.59 (d, J = 8.2 Hz, 1H), 7.48–7.41 (m, 1H), 7.26–7.19 (m, 1H), 7.14 (d, J = 8.2 Hz, 1H), 6.96 (dd, J = 10.4 J = 2.4 Hz, 1H), 6.45 (d, J = 2.4Hz, 1H), 6.04–5.99 (m, 1H), 4.67 (d, J = 11 Hz, 2H), 4.25 (d, J = 11.7 Hz, 1H), 4.20–4.16 (m, 1H), 4.01–3.94 (m, 1H), 2.99 (s, 3H), 0.85 (t, J = 6.3 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, DMSO- d6 ): 182.0, 171.2, 159.4, 144.0, 141.0, 139.4, 137.6, 135.8, 135.6, 132.9, 129.3, 128.3, 127.9, 125.9, 124.3, 121.2, 120.0, 117.3, 116.2, 116.0, 112.1, 83.0, 67.6, 60.1, 46.2, 33.3, 23.2, 15.1, 9.1. Elemental Analysis: Analytical calculated (%): C, 66.10; H, 4.40; N, 10.63; Found (%): C, 66.10; H, 4.42; N, 10.62. 4.2.3.4 Ethyl-8''-fluoro-1'-methyl-2,12''-dioxo-12'' H -dispiro[indoline-3,2'-pyrrolidine- 3',6''-indolo[2,1- b ]quinazoline]-4'-carboxylate; C 29 H 23 FN 4 O 4 (6d) : Yellow solid; Yield: 80%; mp: 252–254 ℃; FTIR (cm -1 ): 1723, 1714, 1683, 1584, 720. 1 H NMR (δ ppm, 400 MHz, DMSO -d6 ): 10.2 (s, 1H), 8.02 (q, J = 5.1 Hz, 1H), 7.84 (d, J = 7.8 Hz, 1H), 7.78–7.71 (m, 3H), 7.59 (d, J = 8.2 Hz, 1H), 7.48–7.41 (m, 2H), 6.96 (d, J = 11 Hz, 1H), 6.48–6.42 (m, 1H), 6.04–5.99 (m, 1H), 4.67 (d, J = 11.6 Hz, 2H), 4.20–4.16 (m, 1H), 4.01–3.94 (m, 2H), 2.92 (s, 3H), 1.13 (t, J = 7 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, DMSO- d6 ): 174.4, 165.3, 156.5, 137.4, 137.2, 136.6, 130.6, 128.8, 128.0, 127.7, 127.2, 124.6, 124.4, 123.3, 121.4, 120.8, 120.2, 120.0, 118.9, 114.2, 112.3, 107.6, 73.3, 62.4, 55.3, 42.5, 29.4, 22.5, 14.4. Elemental Analysis: Analytical calculated (%): C, 68.23; H, 4.54; N, 10.97; Found (%): C, 68.24; H, 4.55; N, 10.96. 4.2.3.5 Ethyl-8''-methoxy-1'-methyl-2,12''-dioxo-12'' H -dispiro[indoline-3,2'- pyrrolidine-3',6''-indolo[2,1- b ]quinazoline]-4'-carboxylate; C 30 H 26 N 4 O 5 (6e) : Yellow solid; Yield: 74%; mp: 260–262 ℃; FTIR (cm -1 ): 1725, 1718, 1690, 1591, 735. 1 H NMR (δ ppm, 400 MHz, DMSO -d6 ): 11.67 (s, 1H), 8.49 (d, J = 7.6 Hz, 2H), 8.33 (d, J = 8 Hz, 2H), 7.96 (d, J = 3.6 Hz, 2H), 7.90–7.86 (m, 2H), 7.73–7.77 (m, 1H), 7.49 (t, J = 7.6 Hz, 2H), 4.34 (q, J = 3.6 Hz, 2H), 3.73 (s. 3H), 2.68–2.66 (m, 1H), 2.33–2.32 (m, 2H), 1.97 (s, 3H), 0.69 (t, J = 9.2 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, DMSO- d6 ): 181.8, 170.8, 158.1, 146.8, 145.4, 144.9, 137.3, 135.9, 135.8, 131.6, 130.5, 130.4, 127.4, 124.7, 124.4, 123.6, 121.4, 119.6, 119.0, 114.6, 110.7, 82.7, 60.8, 56.4, 46.3, 32.2, 25.7, 21.5, 12.8. Elemental Analysis: Analytical calculated (%): C, 68.95; H, 5.02; N, 10.72; Found (%): C, 68.94; H, 5.01; N, 10.73. 4.2.3.6 Ethyl-2''-chloro-1'-methyl-2,12''-dioxo-12'' H -dispiro[indoline-3,2'-pyrrolidine- 3',6''-indolo[2,1- b ]quinazoline]-4'-carboxylate; C 29 H 23 ClN 4 O 4 (6f) : Yellow solid; Yield: 71%; mp: 243–245 ℃; FTIR (cm -1 ): 1720, 1715, 1697, 1514, 819. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 10.33 (s, 1H), 8.56 (d, J = 8 Hz, 1H), 8.39–8.36 (m, 1H), 7.97 (d, J = 8 Hz, 1H), 7.85 (d, J = 7.6 Hz, 1H), 7.81–7.76 (m, 2H), 7.74–7.70 (m, 1H), 7.63–7.59 (m, 1H), 7.47–7.38 (m, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.07–7.04 (m, 1H), 3.91 (q, J = 3.6 Hz, 2H), 2.90 (t, J = 10.4 Hz, 1H), 2.72 (d, J = 7.6 Hz, 1H), 2.27 (t, J = 7.6 Hz, 1H), 1.96 (s, 3H), 0.48 (t, J = 7.2 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 177.0, 169.4, 152.8, 142.5, 134.3, 133.1, 131.8, 130.0, 129.2, 128.2, 127.2, 126.5, 124.9, 123.3, 121.8, 116.7, 114.8, 112.8, 111.2, 110.0, 72.4, 65.3, 52.9, 42.2, 29.7, 20.5, 11.5. Elemental Analysis: Analytical calculated (%): C, 66.10; H, 4.40; N, 10.63; Found (%): C, 66.11; H, 4.40; N, 10.62. 4.2.3.7 Ethyl-2''-bromo-1'-methyl-2,12''-dioxo-12'' H -dispiro[indoline-3,2'-pyrrolidine- 3',6''-indolo[2,1- b ]quinazoline]-4'-carboxylate; C 29 H 23 BrN 4 O 4 (6g) : Yellow solid; Yield: 73%; mp: 236–238 ℃; FTIR (cm -1 ): 1722, 1714, 1685, 1592, 724. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 9.99 (s, 1H), 8.33–8.27 (m, 2H), 8.19 (d, J = 6.6 Hz, 1H), 8.00 (d, J = 5.5 Hz, 1H), 7.59–7.56 (m, 2H), 7.53–7.49 (m, 1H), 7.38–7.34 (m, 1H), 6.97–6.9 (m, 1H), 6.52 (t, J = 8.3 Hz, 2H), 4.96 (q, J = 12.5 Hz, 2H), 4.01–3.94 (m, 1H), 3.78 (t, J = 10.2 Hz, 1H), 3.69–3.62 (m, 1H), 2.25 (s, 3H), 0.63 (t, J = 7.1 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 182.5, 170.3, 158.1, 146.6, 146.3, 144.3, 138.2, 135.1, 133.0, 131.9, 130.7, 130.3, 130.2, 127.5, 127.2, 125.4, 124.1, 123.7, 121.9, 118.0, 101.9, 72.6, 62.7, 44.1, 31.9, 29.6, 22.6, 14.1. Elemental Analysis: Analytical calculated (%): C, 60.96; H, 4.06; N, 9.80; Found (%): C, 60.96; H, 4.05; N, 9.81. 4.2.3.8 Ethyl-2''-bromo-8''-chloro-1'-methyl-2,12''-dioxo-12'' H -dispiro[indoline-3,2'- pyrrolidine-3',6''-indolo[2,1- b ]quinazoline]-4'-carboxylate; C 29 H 22 BrClN 4 O 4 (6h) : Yellow solid; Yield: 70%; mp: 248–250 ℃; FTIR (cm -1 ): 1721, 1717, 1682, 1595, 1414. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 10.34 (s, 1H), 8.45 (d, J = 8.8 Hz, 1H), 8.32 (d, J = 2.4 Hz, 1H), 7.96 (d, J = 2.2 Hz, 1H), 7.85–7.82 (m, 1H), 7.75–7.72 (m, 1H), 7.46 (d, J = 9.2 Hz, 2H), 7.28 (t, J = 2.4 Hz, 1H), 7.07–7.04 (m, 2H), 3.91 (q, J = 2.4 Hz, 2H), 2.75–2.71 (m, 1H), 2.55–2.51 (m, 1H), 2.10 (t, J = 4 Hz, 1H), 1.96 (s, 3H), 0.80 (t, J = 6.4 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 182.5, 169.4, 158.1, 147.5, 146.6, 146.3, 144.3, 138.3, 135.1, 130.7, 130.2, 127.5, 127.2, 125.4, 123.7, 121.9, 117.9, 107.0, 106.0, 104.1, 101.9, 81.4, 60.5, 46.9, 41.8, 29.7, 22.7, 14.1. Elemental Analysis: Analytical calculated (%): C, 57.49; H, 3.66; N, 9.25; Found (%): C, 57.49; H, 3.65; N, 9.26. 4.2.3.9 Ethyl-8''-bromo-2,12''-dioxo-5',6',7',7a'-tetrahydro-1' H , 12'' H -dispiro[indoline- 3,3'-pyrrolizine-2',6''-indolo[2,1- b ]quinazoline]-1'-carboxylate; C 31 H 25 BrN 4 O 4 (6i) : Yellow solid; Yeild: 80%; mp: 225–227 ℃; FTIR (cm -1 ): 1720, 1713, 1681. 1592. 1418. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 9.08 (s, 1H), 8.60–8.53 (m, 2H), 8.47 (d, J = 8.4 Hz, 1H), 8.41 (d, J = 8 Hz, 1H), 8.35 (t, J = 5.6 Hz, 2H), 7.74–7.73 (m, 2H), 7.68–7.45 (m, 1H), 7.39 (t, J = 4 Hz, 2H), 3.69 (q, J = 7.6 Hz, 2H), 2.75–2.71 (m, 1H), 2.55–2.51 (m, 1H), 2.28 (t, J = 7.2 Hz, 3H), 1.37–1.33 (m, 2H), 1.31–1.30 (m, 1H), 0.69 (t, J = 8 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 177.0, 170.0, 158.3, 145.7, 141.7, 139.9, 137.1, 130.0, 129.8, 129.3, 129.2, 126.9, 126.2, 126.0, 122.8, 122.6, 122.2, 120.8, 116.6, 109.7, 78.8, 61.8, 60.5, 51.7, 49.2, 35.0, 29.7, 22.7, 13.2. Elemental Analysis: Analytical calculated (%): C, 62.32; H, 4.22; N, 9.38; Found (%): C, 62.33; H, 4.23; N, 9.36. 4.2.3.10 Ethyl-8''-bromo-2,12''-dioxo-7',7a'-dihydro-1' H ,3' H ,12'' H -dispiro[indoline- 3,5'-pyrrolo[1,2- c ]thiazole-6',6''-indolo[2,1- b ]quinazoline]-7'-carboxylate; C 30 H 23 BrN 4 O 4 S (6j): Yellow solid; Yield: 89%; mp: 249–251 ℃; FTIR (cm -1 ): 1721, 1719, 1687, 1538, 1425, 918. 1 H NMR (δ ppm, 400 MHz, CDCl 3 ): 9.85 (s, 1H), 8.32 (d, J = 8.8 Hz, 1H), 8.23–8.21 (m, 1H), 8.13–8.12 (m, 1H), 7.81–7.65 (m, 2H), 7.59–7.57 (m, 2H), 6.96–6.92 (m, 1H), 6.57–6.53 (m, 1H), 6.45 (d, J = 7.6 Hz, 1H), 6.34 (d, J = 7.6 Hz, 1H), 4.72 (q, J = 6 Hz, 2H), 3.89 (d, J = 8 Hz, 1H), 3.74–3.67 (m, 1H), 3.62 (d, J =8Hz, 1H), 3.56–3.52 (m, 2H), 3.23 (q, J = 4 Hz, 2H), 0.58 (t, J = 10.8 Hz, 3H). 13 C NMR (δ ppm, 100 MHz, CDCl 3 ): 177.3, 168.8, 159.4, 154.1, 146.7, 140.8, 139.2, 134.5, 133.1, 131.2, 130.3, 129.3, 127.9, 127.5, 126.7, 125.9, 123.5, 122.5, 121.1, 119.3, 118.1, 109.9, 79.4, 69.0, 66.4, 60.9, 51.2, 36.3, 29.7, 13.4. Elemental Analysis: Analytical calculated (%): C, 58.54; H, 3.77; N, 9.10; Found (%): C, 58.54; H, 3.76; N, 9.10. 4.2.3.11 (3R,3'S)-1'-methyl-2,12''-dioxo-12''H-dispiro[indoline-3,2'-pyrrolidine-3',6''-indolo[2,1-b]quinazoline]-4',4'-dicarbonitrile; C 28 H 18 N 6 O 2 (6k) : Yellow solid: Yield: 90%; mp: 260–262 ℃; FTIR (cm -1 ): 2149,1680, 1546, 1456, 765. 1 H NMR (δ ppm, 400 MHz, DMSO- d6 ): 11.05 (s, 1H), 8.43 (d, J = 7.6 Hz, 1H), 8.30–8.28 (dd, J = 1.2 Hz, J = 8 Hz, 1H), 7.91–7.86 (m, 1H), 7.82 (d, J = 7.2 Hz, 1H), 7.62–7.58 (m, 2H), 7.52–7.48 (m, 1H), 7.45–7.41 (m, 1H), 7.28–7.23 (m, 1H), 7.18–7.13 (m, 1H), 6.86 (s, 2H), 3.89 (d, J = 10 Hz, 1H). 3.64 (d, J = 10 Hz, 1H). 2.86 (s, 3H). 13 C NMR (δ ppm, 100 MHz, DMSO- d6 ): 164.4, 163.8, 159.5, 147.8, 138.6, 137.8, 136.2, 135.1, 129.3, 128.6, 127.8, 127.5, 126.8, 125.7, 124.6, 121.4, 121.3, 116.4, 64.2, 56.0, 55.5, 33.5, 21.5. Elemental Analysis: Analytical calculated (%): C, 71.48; H, 3.86; N, 17.86; Found (%): C, 71.44; H, 3.83; N, 17.85. 4.3 Biological studies 4.3.1 In vitro antibacterial study The compounds were screened against a bacterial panel comprising ESKAPE pathogens, including Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 29213, Klebsiella pneumoniae BAA 1705, Acinetobacter baumannii BAA 1605, Pseudomonas aeruginosa ATCC 27853 and Escherichia coli IMP 4213. The strains were obtained from the Biodefense and Emerging Infections Research Resources Repository / Network on Antimicrobial Resistance in Staphylococcus aureus / American Type Culture Collection (BEI/NARSA/ATCC, USA) and routinely cultured in Mueller–Hinton agar (MHA) and Mueller–Hinton broth II (MHBII). Prior to the experiment, a single colony from an MHA plate was inoculated into MHBII and incubated overnight at 37°C with shaking for 18–24 h to obtain the starter culture. 4.3.2 In vitro anticancer study Fifteen milligrams of MTT (Sigma, M-5655) was dissolved in 3 ml of PBS until fully solubilized and then sterilized through filter sterilization. Following a 24-hour incubation period, the contents in the wells were discarded, and 30 µl of the prepared MTT solution was added to each test and cell control well. The plate was gently shaken to ensure proper mixing and then incubated at 37°C in a humidified incubator containing 5% CO₂ for 4 hours. After incubation, the supernatant was carefully removed, and 100 µl of MTT solubilization solution (dimethyl sulfoxide, DMSO; Sigma Aldrich, USA) was added and the wells were mixed gently by pipetting up and down to dissolve the formazan crystals. Finally, the absorbance was measured at 540 nm using a microplate reader. 4.4 Molecular docking The three-dimensional crystal structure of the protein was retrieved from the Protein Data Bank (PDB ID: 2D0T). Protein preparation was carried out using PyMol Software. Ligand structures were energy minimized using Gaussian program and B3LYP/6-311 + G(d) level of theory prior to docking. Docking simulations were carried out using PyRx Software. Binding affinities were recorded in kcal/mol, and the best ranked poses were selected based on binding energy and interaction consistency. Protein–ligand interactions, including van der waal, π–π stacking and π–alkyl interactions were analyzed using ChimeraX Software. Declarations Conflict of interest The authors declare no competing interests. Author Contribution DK wrote the original draftDK and SLS did the data collection and formal analysisAAB and JEG contributed in the docking studyAS, AL and SC did the biological evaluation AD did the conceptualization, validation and final draft Acknowledgements The corresponding author thank ANRF for funding in terms of POWER research grant (SPG/2023/000179). DK thank CSIR and SSL thank University of Kerala for the Junior Research Fellowships respectively. All authors acknowledge CLIF, University of Kerala, for providing instrumental facilities. References Davies J, Davies D (2010) Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74:417–433. https://doi.org/10.1128/MMBR.00016-10 Kumar S, Mahato RP, Ch S, Soniya Kumbham (2025) Current strategies against multidrug-resistant Staphylococcus aureus and advances toward future therapy. Microbe 6:100281. https://doi.org/10.1016/j.microb.2025.100281 Turner NA, Sharma-Kuinkel BK, Maskarinec SA, Eichenberger EM, Shah PP, Carugati M, Holland TL, Fowler VG Jr (2019) Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat Rev Microbiol 17:203–218. https://doi.org/10.1038/s41579-018-0147-4 Belete MA, Gedefie A, Alemayehu E, Debash H, Mohammed O, Gebretsadik D, Tilahun M (2023) The prevalence of vancomycin-resistant Staphylococcus aureus in Ethiopia: a systematic review and meta-analysis. Antimicrob resist infect contro 12:86. https://doi.org/10.1186/s13756-023-01291-3 Casolino R, Tatah L, Charnaud S, Santero M, Ilbawi A, Ross AL (2025) The WHO global landscape of cancer clinical trials. Nat Med 31:2901–2912. https://doi.org/10.1038/s41591-025-03926-x Vasan N, Baselga J, Hyman DM (2019) A view on drug resistance in cancer. Nature 575:299–309. https://doi.org/10.1038/s41586-019-1730-1 Tucker AM, Grundt P (2012) The chemistry of tryptanthrin and its derivatives. Arkivoc 1:546–569 Jahng Y (2013) Progress in the studies on tryptanthrin, an alkaloid of history. Arch Pharm Res 36:517–535. https://doi.org/10.1007/s12272-013-0091-9 Brandão P, Pineiro M, Burke AJ (2022) Tryptanthrin and its derivatives in drug discovery: synthetic insights. Synthesis 54:4235–4245. https://doi.org/10.1055/s-0040-1719901 Kaur R, Manjal SK, Rawal RK, Kumar K (2017) Recent synthetic and medicinal perspectives of tryptanthrin. Bioorg Med Chem 25:4533–4552. https://doi.org/10.1016/j.bmc.2017.07.003 Alam K, Hossain MS, Zhao Y, Zhang Z, Xu S, Hao J, Li A (2024) Tryptanthrins as multi-bioactive agents: discovery, diversity distribution and synthesis. Bioorg Chem 154:108071. https://doi.org/10.1016/j.bioorg.2024.108071 Catanzaro E, Betari N, Arencibia JM, Montanari S, Sissi C, De Simone A, Milelli A (2020) Targeting topoisomerase II with trypthantrin derivatives: Discovery of 7-((2-(dimethylamino) ethyl) amino) indolo [2, 1-b] quinazoline-6, 12-dione as an antiproliferative agent and to treat cancer. Eur J Med Chem 202:112504. https://doi.org/10.1016/j.ejmech.2020.112504 Li MH, Wang Z, Liang Y, Lu S, Hou BL (2025) Synthesis, characterization, crystal structure, and anti-inflammatory evaluation of thienyl-substituted tryptanthrins. J Mol Struct 1342:142651. https://doi.org/10.1016/j.molstruc.2025.142651 Leena SS, Kaul G, Akhir A, Saxena D, Chopra S, Deepthi A (2022) Green synthesis and antibacterial evaluation of spiro fused tryptanthrin-thiopyrano [2, 3-b] indole hybrids targeting drug-resistant S. aureus . Bioorg Chem 128:106046. https://doi.org/10.1016/j.bioorg.2022.106046 Leena SS, Akhir A, Saxena D, Maitra R, Chopra S, Deepthi A (2023) Synthesis of tryptanthrin appended dispiropyrrolidine oxindoles and their antibacterial evaluation. RSC Med Chem 14:1165–1171. https://doi.org/10.1039/D3MD00017F Al-Sharifi HK, Leena SS, Saxena D, Maitra R, Chopra S, Deepthi A (2024) Green synthesis of tryptanthrin appended 4-spiropiperidines and their anti-bacterial evaluation. Tetrahedron 151:133786. https://doi.org/10.1016/j.tet.2023.133786 Zhou X (2024) Recent advances of tryptanthrin and its derivatives as potential anticancer agents. RSC Med Chem 15:1127–1147. https://doi.org/10.1039/D3MD00698K Meenakshy CB, Sruthi SL, Jayasree EG, Sandhya KS, Deepthi A (2024) Synthesis and Anticancer Evaluation of Tryptanthrin-1, 2, 3‐Triazole Hybrids. J Heterocycl Chem 61:1891–1898. https://doi.org/10.1002/jhet.4916 Deepthi A, Acharjee N, Sruthi SL, Meenakshy CB (2022) An overview of nitrile imine based [3 + 2] cycloadditions over half a decade. Tetrahedron 116:132812. https://doi.org/10.1016/j.tet.2022.132812 Viveka S, Dinesha, Shama P, Nagaraja GK, Ballav S, Kerkar S (2015) Design and Synthesis of Some New Pyrazolyl-Pyrazolines as Potential Anti-Inflammatory, Analgesic and Antibacterial Agents. Eur J Med Chem 101:442–451. https://doi.org/10.1016/j.ejmech.2015.07.002 Mantzanidou M, Pontiki E, Hadjipavlou-Litina D (2021) Pyrazoles and Pyrazolines as Anti-InflammatoryAgents. Molecules 26:3439. https://doi.org/10.3390/molecules26113439 Kotnala M, Singh G, Singh K, Nath R, Panda KC, Kumar A, Dutta S (2024) Exploring the Synthetic Strategies and Biological Activities of Pyrazole Derivatives. Nat Campano 28:3238 Li Petri G, Raimondi MV, Spanò V, Holl R, Barraja P, Montalbano A (2021) Pyrrolidine in drug discovery: a versatile scaffold for novel biologically active compounds. Top Curr Chem 379:34. https://doi.org/10.1007/s41061-021-00347-5 Poyraz S, Döndaş HA, Döndaş NY, Sansano JM (2023) Recent insights about pyrrolidine core skeletons in pharmacology. Front Pharmacol 14:1239658. https://doi.org/10.3389/fphar.2023.1239658 Yang S, Li X, Hu F, Li Y, Yang Y, Yan J, Kuang C, Yang Q (2023) Discovery of tryptanthrin derivatives as potent inhibitors of indoleamine 2,3-dioxygenase with therapeutic activity in Lewis lung cancer (LLC) tumor-bearing mice. J Med Chem 56:8321–8331. https://doi.org/10.1021/jm401195n Tang K, Wu YH, Song Y, Yu B (2021) Indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors in clinical trials for cancer immunotherapy. J Hematol Oncol 14:68. https://doi.org/10.1186/s13045-021-01080-8 Qian S, Zhang M, Chen Q, He Y, Wang W, Wang Z (2016) IDO as a drug target for cancer immunotherapy: recent developments in IDO inhibitors discovery. RSC Adv 6:7575–7581. https://doi.org/10.1039/C5RA25046C Lu S, Hou BL, Wang T, Ma K, Huang A, Wu X, Liang YN, Wang Z (2025) Antitumor effects of tryptanthrin on colorectal cancer by regulating the mitogen-activated protein kinase signaling pathway and targeting topo I and IDO1. ACS omega 10:3206–3221. https://doi.org/10.1021/acsomega.4c11189 Yuan S, Stephen Chan HC, Hu Z (2017) Using PyMOL as a platform for computational drug design. Wiley Interdiscip Rev Comput Mol Sci 7:e1298. https://doi.org/10.1002/wcms.1298 Sargis D, Olson AJ (2015) Small-molecule library screening by docking with PyRx. Methods Mol Biol 243–250. https://doi.org/10.1007/978-1-4939-2269-7_19 Meng, Elaine C (2023) UCSF ChimeraX: Tools for structure building and analysis. Protein Sci 32:e4792. https://doi.org/10.1002/pro.4792 Schemes Schemes 1 to 3 are available in the Supplementary Files section Additional Declarations No competing interests reported. Supplementary Files Supportinginformation.docx SC1.png Scheme 1. (3 + 2) Cycloaddition of 1a with nitrile imine and azomethine ylide for the synthesis of spiropyrazole and dispiropyrrolidine. SC2.png Scheme 2. Synthesis of spiropyrazoles 3a-j and dispiropyrrolidines 6a-j SC3.png Scheme 3. Synthesis of spiropyrazole 3k and dispiropyrrolidine 6k Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9343418","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":623281274,"identity":"6b0a11e8-2c65-4bb1-9a88-bf3235b1835f","order_by":0,"name":"Devika Krishnan","email":"","orcid":"","institution":"University of Kerala","correspondingAuthor":false,"prefix":"","firstName":"Devika","middleName":"","lastName":"Krishnan","suffix":""},{"id":623281278,"identity":"ae50cb95-8c69-4527-894c-cdfe506e04e0","order_by":1,"name":"Sruthi Sudheendran Leena","email":"","orcid":"","institution":"University of Kerala","correspondingAuthor":false,"prefix":"","firstName":"Sruthi","middleName":"Sudheendran","lastName":"Leena","suffix":""},{"id":623281279,"identity":"ad0eaca2-6d00-4817-b45f-f6b466392fb9","order_by":2,"name":"Ashkar Ali B.","email":"","orcid":"","institution":"Cochin University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Ashkar","middleName":"Ali","lastName":"B.","suffix":""},{"id":623281281,"identity":"de78cf86-6b8c-4586-b238-ff337389b0da","order_by":3,"name":"E. G. Jayasree","email":"","orcid":"","institution":"Cochin University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"E.","middleName":"G.","lastName":"Jayasree","suffix":""},{"id":623281287,"identity":"f6c219e8-31bb-4559-929b-177f04607337","order_by":4,"name":"Aparna Sahoo","email":"","orcid":"","institution":"CSIR-Central Drug Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Aparna","middleName":"","lastName":"Sahoo","suffix":""},{"id":623281290,"identity":"b694d1c3-eb54-4e32-8938-153d8bf0f0c3","order_by":5,"name":"Ankita Lama","email":"","orcid":"","institution":"CSIR-Central Drug Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Ankita","middleName":"","lastName":"Lama","suffix":""},{"id":623281291,"identity":"5f0cff12-bb5f-4eb7-bd52-9e5c47eaf363","order_by":6,"name":"Sidharth Chopra","email":"","orcid":"","institution":"CSIR-Central Drug Research Institute","correspondingAuthor":false,"prefix":"","firstName":"Sidharth","middleName":"","lastName":"Chopra","suffix":""},{"id":623281292,"identity":"90405399-51cc-4c50-9fd7-cbb8c37c1a03","order_by":7,"name":"Ani Deepthi","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYFACxgYJEGXAzHwASEnIEK1FwoCZLQFE8xBlD0QLA48BiENYC//s5sYbHyru1Jmz83x+daPGgoeB/fDRDXhtuHOw2XLGmWcSls2826xzjgEdxpOWdgOvNTcS26R52w5LGBzm3WacwwbUIsFjhleLPEjL338gLTzPjHP+EaHFAKSFsQGshflxbhsRWgxvJDZb9hx7JrnhMJsZc26fBA8bIb/I3Uh/eONHzR1+g/OHH3/O+VYnx89++Bh+70PAARDBBk4GbEQoh2th/kCk6lEwCkbBKBhhAAAF9Un+stlm0gAAAABJRU5ErkJggg==","orcid":"","institution":"University of Kerala","correspondingAuthor":true,"prefix":"","firstName":"Ani","middleName":"","lastName":"Deepthi","suffix":""}],"badges":[],"createdAt":"2026-04-07 10:25:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9343418/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9343418/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":107067597,"identity":"3d2a2adf-c532-4711-aacd-6192a4475dbe","added_by":"auto","created_at":"2026-04-16 11:26:12","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":121138,"visible":true,"origin":"","legend":"\u003cp\u003eTryptanthrin hybrid molecules active against \u003cem\u003eS. aureus\u003c/em\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9343418/v1/abe12a3bca759b63226d0a7c.png"},{"id":107067521,"identity":"ed061461-7579-45ff-bf87-b2a3f94423d7","added_by":"auto","created_at":"2026-04-16 11:25:59","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1150675,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical representation of anticancer effect of the compounds on HCT-116 cell line by MTT assay\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9343418/v1/ea768c221cf8a9546841dafd.png"},{"id":107067342,"identity":"aa66db06-375f-42db-8434-243bc86fd376","added_by":"auto","created_at":"2026-04-16 11:25:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":667410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e(a) \u003c/strong\u003e2D and 3D interactions of \u003cstrong\u003e6b\u003c/strong\u003e with 2DOT \u003cstrong\u003e(b)\u003c/strong\u003e 2D and 3D interactions of \u003cstrong\u003e6c\u003c/strong\u003e with 2DOT\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9343418/v1/6569cdcffb7ff4752d41df78.png"},{"id":107485954,"identity":"653f099e-6873-4c93-873e-c4f71393a5a2","added_by":"auto","created_at":"2026-04-22 02:37:00","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3525112,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9343418/v1/effa113c-d7fa-4c97-bf64-a2ca3effed73.pdf"},{"id":107067563,"identity":"2121af40-f606-426b-aa85-b6d33ccd8c4a","added_by":"auto","created_at":"2026-04-16 11:26:05","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5474178,"visible":true,"origin":"","legend":"","description":"","filename":"Supportinginformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-9343418/v1/e95509de366492c98e6a3386.docx"},{"id":107067594,"identity":"f5cb86d6-0cb0-4826-976b-bbab5c6b6c57","added_by":"auto","created_at":"2026-04-16 11:26:11","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":127754,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e. (3 + 2) Cycloaddition of \u003cstrong\u003e1a\u003c/strong\u003e with nitrile imine and azomethine ylide for the synthesis of spiropyrazole and dispiropyrrolidine.\u003c/p\u003e","description":"","filename":"SC1.png","url":"https://assets-eu.researchsquare.com/files/rs-9343418/v1/31a8eb4044bd2c670351785c.png"},{"id":107067602,"identity":"883cf3ba-1707-4a05-a8a5-bd47ee85e773","added_by":"auto","created_at":"2026-04-16 11:26:15","extension":"png","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":968732,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 2\u003c/strong\u003e. Synthesis of spiropyrazoles \u003cstrong\u003e3a-j \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003edispiropyrrolidines \u003cstrong\u003e6a-j\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SC2.png","url":"https://assets-eu.researchsquare.com/files/rs-9343418/v1/153deb99695fda1e9d3ccf7f.png"},{"id":107067569,"identity":"e7f68184-9e18-4188-8d05-16e22a9f01fe","added_by":"auto","created_at":"2026-04-16 11:26:10","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":126900,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 3. \u003c/strong\u003eSynthesis of spiropyrazole \u003cstrong\u003e3k \u003c/strong\u003eand\u003cstrong\u003e \u003c/strong\u003edispiropyrrolidine \u003cstrong\u003e6k\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"SC3.png","url":"https://assets-eu.researchsquare.com/files/rs-9343418/v1/31b44fcadd77964ed0833ace.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synthesis and biological evaluation of spirofused pyrazole and pyrrolidine hybrids from tryptanthrin","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe rise of antimicrobial resistance (AMR) has intensified the global risk associated with infectious diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. This challenge is especially evident with \u003cem\u003eStaphylococcus aureus\u003c/em\u003e, a bacterium capable of developing multidrug resistance (MDR), which can result in severe complications due to its complex nature and wide range of virulence factors [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) poses a serious clinical threat, consistently associated with high rates of illness and death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Glycopeptide antibiotics like vancomycin are commonly used to treat MRSA infections; however, the emergence of vancomycin-resistant \u003cem\u003eS. aureus\u003c/em\u003e (VRSA), recognized by the World Health Organization as a high-priority antibiotic-resistant pathogen, highlights the critical need for the discovery and development of novel antimicrobial agents effective against the MDR pathogens [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Parallelly, cancer is a leading cause of death worldwide; 20\u0026nbsp;million new cancer cases were recorded in 2022 and it is estimated to increase to 35\u0026nbsp;million new cases by 2050 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Cancer cells also develop multiple and complex mechanisms to evade drug-induced cytotoxicity, making resistance to drugs a significant obstacle to successful cancer therapy [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In this context, searching new antibacterial and anticancer agents which can be effective against MDR pathways is very crucial in modern times. The work embodied in the current manuscript discloses the synthesis and promising preliminary results about the antibacterial and anticancer activities of certain tryptanthrin incorporated heterocycles.\u003c/p\u003e \u003cp\u003eTryptanthrin (indolo[2,1-\u003cem\u003eb\u003c/em\u003e]quinazoline-6,12-dione), a bioactive indoloquinazoline alkaloid isolated from several medicinal plants, including \u003cem\u003eStrobilanthes cusia\u003c/em\u003e (Assam indigo), \u003cem\u003eIsatis tinctoria\u003c/em\u003e (Chinese woad), and \u003cem\u003ePolygonum tinctorium\u003c/em\u003e (Japanese indigo) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] has gained considerable scientific interest due to its diverse pharmacological activities such as antibacterial, anti-inflammatory, anticancer, antifungal, and antiviral properties [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Owing to its significant therapeutic potential, numerous tryptanthrin hybrid molecules have been chemically synthesized and evaluated for their efficacy against diverse diseases and infections [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Investigations by our research group have identified tryptanthrin derived spiro compounds as promising candidates for antibacterial activity, particularly against \u003cem\u003eStaphylococcus aureus\u003c/em\u003e. A series of spiro-fused tryptanthrin-thiopyrano[2,3-\u003cem\u003eb\u003c/em\u003e]indole hybrid molecules targeting drug-resistant \u003cem\u003eS. aureus\u003c/em\u003e, led us to the identification of nitro-substituted hybrid molecule with potent activity against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213, with a good selectivity index [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Later, a series of tryptanthrin appended dispiropyrrolidine oxindoles [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e] and 4-spiropiperidines [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] were also reported and among them bromo substituted derivatives exhibited lowest MIC values of 0.125 \u0026micro;g/mL and 4 \u0026micro;g/mL respectively. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e depicts the structures of tryptanthrin hybrid molecules active against \u003cem\u003eS. aureus\u003c/em\u003e made previously in our lab. The anticancer activities of various tryptanthrin derived heterocyclic compounds have also been well recognized [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In line with the latter, our group has also reported the synthesis of tryptanthrin-1,2,3-triazole hybrids that exhibited potent anticancer activity against breast and colon cancer cell lines [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOwing to our continued interest in developing effective antibacterial/anticancer agents from tryptanthrin, we now report the synthesis of two distinct classes of spiro compounds through 1,3-dipolar cycloaddition reactions, using ethyl (\u003cem\u003eE\u003c/em\u003e)-2-(12-oxoindolo[2,1-\u003cem\u003eb\u003c/em\u003e]quinazolin-6(12\u003cem\u003eH\u003c/em\u003e)-ylidene)acetate as dipolarophile and nitrile imine / azomethine ylide as dipoles. The cycloaddition of nitrile imine [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] to the above dipolarophile resulted in a pyrazole ring; a promising scaffold in drug development with a broad spectrum of biological activities such as antibacterial, anti-inflammatory, antiviral, anticancer and antimicrobial properties [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Meanwhile, the use of azomethine ylide as the dipole resulted in the formation of spiropyrrolidines another significant scaffold extensively explored in the synthesis of novel bioactive compounds [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. This study thus reports the synthesis of tryptanthrin appended spiropyrazoles and dispiropyrrolidines and evaluates their \u003cem\u003ein vitro\u003c/em\u003e antibacterial activity against ESKAPE pathogens, and \u003cem\u003ein vitro\u003c/em\u003e anticancer activity against human colon cancer cell lines which is further supported by molecular docking study.\u003c/p\u003e"},{"header":"2. Results and Discussion","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Synthesis of tryptanthrin appended spiropyrazoles and dispiropyrrolidines\u003c/h2\u003e \u003cp\u003eIn separate reactions, ethyl (\u003cem\u003eE\u003c/em\u003e)-2-(12-oxoindolo[2,1-\u003cem\u003eb\u003c/em\u003e]quinazolin-6(12\u003cem\u003eH\u003c/em\u003e)-ylidene)acetate \u003cb\u003e1a\u003c/b\u003e was reacted with (i) nitrile imine and (ii) azomethine ylide which led to the formation of tryptanthrin appended spiropyrazole \u003cb\u003e3a\u003c/b\u003e and dispiropyrrolidine \u003cb\u003e6a\u003c/b\u003e respectively. As a pilot reaction, compound \u003cb\u003e1a\u003c/b\u003e was treated with the nitrile imine, (formed \u003cem\u003ein situ\u003c/em\u003e by the dehydrochlorination of the corresponding hydrazonyl chloride \u003cb\u003e2a\u003c/b\u003e), in acetonitrile at room temperature and after completion of the reaction as observed from TLC, the product \u003cb\u003e3a\u003c/b\u003e was isolated by silica gel column chromatography using 80:20 hexane-ethyl acetate in 92% yield. In a discrete reaction, azomethine ylide was generated by refluxing isatin \u003cb\u003e4\u003c/b\u003e, and sarcosine \u003cb\u003e5a\u003c/b\u003e in ethanol and the cycloaddition with \u003cb\u003e1a\u003c/b\u003e was proceeded by refluxing at 85℃ until complete consumption of isatin was observed from TLC. The product \u003cb\u003e6a\u003c/b\u003e was isolated by silica gel column chromatography using 70:30 hexane-ethyl acetate in 91% yield (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe structure of the compounds \u003cb\u003e3a\u003c/b\u003e and \u003cb\u003e6a\u003c/b\u003e were characterized by IR, \u003csup\u003e1\u003c/sup\u003eH NMR, \u003csup\u003e13\u003c/sup\u003eC NMR and CHN analysis. The FTIR spectrum of \u003cb\u003e3a\u003c/b\u003e showed sharp peaks at 1672 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1713 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e corresponding to the amide and ester carbonyls respectively. In the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum of \u003cb\u003e3a\u003c/b\u003e, the singlet at δ 5.24 ppm corresponded to -CH proton and the multiplets ranging from δ 6.74\u0026ndash;8.66 ppm corresponds to 18 aromatic protons. In the \u003csup\u003e13\u003c/sup\u003eC NMR spectrum the spiro carbon was observed at δ 65.5 ppm. The amide carbonyl carbon was seen at δ 166.5 ppm while the imine carbons were seen at δ 159.7 and 159.0 ppm. In the FTIR spectrum of \u003cb\u003e6a\u003c/b\u003e three characteristic stretchings at 1695 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1705 cm\u003csup\u003e\u0026minus;1\u003c/sup\u003eand 1722 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e can be attributed to amide, indole and ester carbonyl groups. In the \u003csup\u003e1\u003c/sup\u003eH NMR spectrum, a singlet at δ 2.22 ppm corresponds to N-CH\u003csub\u003e3\u003c/sub\u003e protons, the two multiplets in the range δ 3.69\u0026ndash;3.61 and δ 3.50\u0026ndash;3.44 ppm corresponded to N-CH\u003csub\u003e2\u003c/sub\u003e protons and a triplet at δ 3.78 corresponds to -CH proton of the pyrrolidine ring. The NH proton of oxindole was observed as a singlet at δ 10.46 ppm. In the \u003csup\u003e13\u003c/sup\u003eC NMR spectrum the spiro carbons were observed at δ 60.6 and 78.6 ppm. The amide carbonyl groups were observed at δ 159.5 and δ 169.8 ppm while ester carbonyl was seen at δ 176.4 ppm. The elemental analysis and purity of \u003cb\u003e3a\u003c/b\u003e and \u003cb\u003e6a\u003c/b\u003e were ascertained by CHN analysis.\u003c/p\u003e \u003cp\u003eIn order to optimize the conditions, the reaction was carried out by varying the solvent, temperature and time (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). For formation of \u003cb\u003e3a\u003c/b\u003e, use of polar aprotic solvent anhydrous CH\u003csub\u003e3\u003c/sub\u003eCN was found to be beneficial leading to 92% yield (entry 1), while use of polar protic solvent EtOH was found to be advantageous for formation of \u003cb\u003e6a\u003c/b\u003e (obtained in 88% yield, entry 5) which was subsequently improved to 91% by adding toluene at 85 \u0026ordm;C (Entry 7).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eOptimization of reaction condition.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEntry\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eSolvent\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eT (⁰C)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" morerows=\"1\" nameend=\"c5\" namest=\"c4\" rowspan=\"2\"\u003e \u003cp\u003eTime (h)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eYield (%)\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e3a-j\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e6a-j\u003c/b\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eCH\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003cb\u003eCN\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003ert\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e92\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCH\u003csub\u003e3\u003c/sub\u003eCN\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e92\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e45\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMeOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ert\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEtOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003ert\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eEtOH\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e85\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e70\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eToluene\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e110\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eEtOH-Toluene\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e85\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e91\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003csup\u003ea\u003c/sup\u003eThe reactions were performed with \u003cb\u003e1a\u003c/b\u003e (1 mmol), \u003cb\u003e2a\u003c/b\u003e (1 mmol), and base (1 mmol) in the solvent (5.0 mL) for formation of \u003cb\u003e3a\u003c/b\u003e-\u003cb\u003ej\u003c/b\u003e The reaction was performed with \u003cb\u003e1a\u003c/b\u003e (1 mmol), \u003cb\u003e4\u003c/b\u003e (1 mmol), and \u003cb\u003e5a\u003c/b\u003e (2 mmol) in the solvent (5.0 mL) for formation of \u003cb\u003e6a\u003c/b\u003e-\u003cb\u003ej\u003c/b\u003e \u003csup\u003eb\u003c/sup\u003eIsolated yield.\u003c/p\u003e \u003cp\u003eEmploying the optimized reaction conditions, we investigated the scope of the reaction by varying the substituents on both the dipolarophile and the 1,3- dipoles (Scheme \u003cspan refid=\"Sch2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). When electron-withdrawing groups such as halogens were introduced at the C\u003csup\u003e8\u003c/sup\u003e position, the desired compounds (\u003cb\u003e3b\u0026ndash;d \u0026amp; 6b\u0026ndash;d)\u003c/b\u003e were obtained in good yields. However, introducing an electron-donating methoxy group at the same position led to a slight decrease in the yield of compounds (\u003cb\u003e3e \u0026amp; 6e)\u003c/b\u003e. Additionally, substituting C\u003csup\u003e2\u003c/sup\u003e/C\u003csup\u003e8\u003c/sup\u003e/both positions with halogens (\u003cb\u003e3f\u003c/b\u003e \u0026amp; \u003cb\u003e6f-h\u003c/b\u003e) resulted in moderate yields. Varying the substituents on 1,3- dipoles by changing the substituents at phenyl ring of nitrile imine (\u003cb\u003e3g-j\u003c/b\u003e) and changing the amino acid part of azomethine ylide to proline and thioproline (\u003cb\u003e6i-j\u003c/b\u003e) resulted in products with moderate yield.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubstrate scope is further extended by varying the dipolorophile to 2-(12-oxoindolo[2,1-b]quinazolin-6(12H)-ylidene)malononitrile and the corresponding spiropyrazole \u003cb\u003e3k\u003c/b\u003e and dispiropyrrolidine \u003cb\u003e6k\u003c/b\u003e were obtained in good yields as shown in Scheme \u003cspan refid=\"Sch3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. \u003cem\u003eIn vitro\u003c/em\u003e biological studies\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1. \u003cem\u003eIn vitro\u003c/em\u003e antimicrobial activity against bacterial pathogen panel\u003c/h2\u003e \u003cp\u003eAll compounds \u003cb\u003e3a-k\u003c/b\u003e and \u003cb\u003e6a-k\u003c/b\u003e were evaluated for their antibacterial activity against the bacterial pathogen panel to determine the minimum inhibitory concentration (MIC). Levofloxacin was used as the positive control and results were depicted in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. As can be seen, among the synthesized compounds, \u003cb\u003e3b\u003c/b\u003e- \u003cb\u003e3f, 3h, 3j-k, 6c\u003c/b\u003e and \u003cb\u003e6j-k\u003c/b\u003e exhibited antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 of which the 8-fluoro substituted spiropyrazole (\u003cb\u003e3d\u003c/b\u003e) and 2- chloro, 8-bromo substituted spiropyrazole (\u003cb\u003e3f\u003c/b\u003e) exhibited a low MIC of 8 \u0026micro;g/mL.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eMIC (\u003cem\u003e\u0026micro;\u003c/em\u003eg/mL) of \u003cb\u003e3a-k\u003c/b\u003e and \u003cb\u003e6a-k\u003c/b\u003e against bacterial pathogen panel along with comparator antibiotic\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSl. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompound name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e ATCC 25922\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eS.aureus\u003c/em\u003e ATCC 29213\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eK.pneumoniae\u003c/em\u003e BAA 1705\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eA.baumannii\u003c/em\u003e BAA 1605\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eP.aeruginosa\u003c/em\u003e ATCC 27853\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e IMP 4213\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e1\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e2\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3d\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3e\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e6\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3f\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e7\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3g\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3h\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e9\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3i\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3j\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e3k\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e12\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e13\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e15\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6d\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e16\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6e\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e17\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6f\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e18\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6g\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e19\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6h\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e20\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6i\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e21\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6j\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e22\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6k\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e23\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eLevofloxacin\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0.0156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e0.0078\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2. \u003cem\u003eIn vitro\u003c/em\u003e anticancer studies by MTT assay\u003c/h2\u003e \u003cp\u003eRecent reports on the potential anticancer effects of several tryptanthrin derived heterocyclic compounds [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e] highlight the importance of investigating the anticancer activity of the synthesized pyrazole and pyrrolidine hybrids of tryptanthrin. Since colorectal cancer ranks as the third most prevalent malignant disease worldwide, compounds \u003cb\u003e3d\u003c/b\u003e, \u003cb\u003e3f\u003c/b\u003e, \u003cb\u003e6b\u003c/b\u003e and \u003cb\u003e6c\u003c/b\u003e were subjected to \u003cem\u003ein vitro\u003c/em\u003e evaluation against the HCT-116 colon cancer cell line using the MTT assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) and the results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. It was found that chloro substituted dispiropyrrolidine \u003cb\u003e6b\u003c/b\u003e and bromo substituted dispiropyrrolidine \u003cb\u003e6c\u003c/b\u003e exhibited significant anticancer properties against HCT-116 colon cancer cell line with LC\u003csub\u003e50\u003c/sub\u003e 32.99 \u003cem\u003e\u0026micro;\u003c/em\u003eg/mL and 23.57 \u003cem\u003e\u0026micro;\u003c/em\u003eg/mL respectively. By varying concentrations of both \u003cb\u003e6b\u003c/b\u003e and \u003cb\u003e6c\u003c/b\u003e (6.25, 12.5, 25, 50 and 100 \u003cem\u003e\u0026micro;\u003c/em\u003eg/mL), percentage viability decreased appreciably. The concentration dependent cell death and low LC\u003csub\u003e50\u003c/sub\u003e value observed for the compounds highlights its potential efficacy as potent anticancer agents.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e value calculated at the end of 24 h incubation\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSl. No.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCompound name\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLC\u003csub\u003e50\u003c/sub\u003e (\u003cem\u003e\u0026micro;\u003c/em\u003eg/mL)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3d\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e198.8\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3f\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e187.6\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e3\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e4\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003e6b\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e6c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cb\u003e32.99\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e23.57\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Molecular docking studies\u003c/h2\u003e \u003cp\u003eFurthermore, molecular docking studies were carried out to support and validate the \u003cem\u003ein vitro\u003c/em\u003e findings of the anticancer study. Indoleamine 2,3-dioxygenase (IDO1), an intracellular enzyme that is highly expressed in a variety of tumors including colorectal cancer, breast cancer and cervical squamous cell carcinoma was considered as a classical target of tryptanthrin and its derivatives [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Structure activity relationship (SAR) analysis revealed that the presence of an electron withdrawing group at the C-8 position of tryptanthrin significantly contributes to IDO1 inhibition [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Studies on the inhibitory effects of tryptanthrin on colorectal cancer have shown that the underlying mechanisms are significantly associated with the inhibition of Topo I and IDO1 proteins [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Therefore, molecular docking studies were performed to evaluate the binding affinity and interaction profile of the selected ligands \u003cb\u003e6b\u003c/b\u003e and \u003cb\u003e6c\u003c/b\u003e against the IDO1 protein as the target. The three-dimensional crystal structure of IDO1 protein (PDB ID: 2D0T, 4-phenylimidazole bound form of human indoleamine 2,3-dioxygenase) was retrieved from the Protein Data Bank. Protein preparation was carried out using PyMol Software [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Ligand structures were energy minimized employing Gaussian program and B3LYP/6-311\u0026thinsp;+\u0026thinsp;G(d) level of theory prior to docking. Docking simulations were performed using PyRx Software [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Binding affinities were recorded in kcal/mol, and the best-ranked poses were selected based on binding energy and interaction consistency. Protein\u0026ndash;ligand interactions, including van der waal, π\u0026ndash;π stacking and π\u0026ndash;alkyl interactions were analyzed using ChimeraX Software [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Both 2D and 3D interaction maps were generated for visualization. The docking outcomes, presented in Table\u0026nbsp;4, reveal the binding affinities between the ligands and the protein.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eBoth ligands demonstrated strong binding affinity (-9.4 kcal/mol) towards the 2D0T protein exhibiting similar interactions. Figures\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e(a) \u0026amp; (b) illustrate the 2D and 3D representations of these interactions. Interactions between the ligands \u003cb\u003e6b\u003c/b\u003e and \u003cb\u003e6c\u003c/b\u003e against 2D0T protein, emphasizing various types of non-covalent interactions that contribute to binding affinity and stability. The diagram highlights π-interactions, including π-Alkyl (light pink lines), and π-π stacking (pink lines) interactions that are observed with residues such as ARG B:231, LEU B:234, PHE B:291 and LEU B:384, indicating significant aromatic and electrostatic contributions. These diverse interactions underscore the ligand's strong binding affinity and its potential effectiveness as a modulator of the 2D0T protein which supports the experimental results of \u003cem\u003ein vitro\u003c/em\u003e anticancer studies.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Conclusion","content":"\u003cp\u003eA series of twenty-two tryptanthrin based spiro compounds containing pyrazole and pyrrolidine frameworks were synthesized through (3\u0026thinsp;+\u0026thinsp;2) cycloaddition reactions of tryptanthrin ylidene with nitrile imines and azomethine ylides. All the synthesized compounds were tested for antibacterial activity against ESKAPE pathogens of which \u003cb\u003e3b\u003c/b\u003e- \u003cb\u003e3f, 3h, 3j-k, 6c \u0026amp; 6j-k\u003c/b\u003e exhibited antibacterial activity against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213 while \u003cb\u003e3d\u003c/b\u003e and \u003cb\u003e3f\u003c/b\u003e exhibited a low MIC of 8 \u0026micro;g/mL. Compounds \u003cb\u003e3d\u003c/b\u003e, \u003cb\u003e3f\u003c/b\u003e, \u003cb\u003e6b\u003c/b\u003e \u0026amp; \u003cb\u003e6c\u003c/b\u003e were evaluated for anticancer activity against HCT-116 cell line by MTT assay and the compound \u003cb\u003e6b\u003c/b\u003e \u0026amp; \u003cb\u003e6c\u003c/b\u003e were found to be active with LC\u003csub\u003e50\u003c/sub\u003e 32.99 \u003cem\u003e\u0026micro;\u003c/em\u003eg/mL and 23.57 \u003cem\u003e\u0026micro;\u003c/em\u003eg/mL respectively. The molecular docking studies reveal valuable insights into the ligand's binding mechanism and establish its potential as a promising candidate for modulating the 2D0T protein's activity, which further supports the experimental results of \u003cem\u003ein vitro\u003c/em\u003e anticancer study. All these results position tryptanthrin-pyrazole/pyrrolidine hybrids as promising molecules for further antibacterial/anticancer studies in future.\u003c/p\u003e"},{"header":"4. Experimental section","content":"\u003cdiv\u003e\n\u003ch2\u003e4.1 Materials and methods\u003c/h2\u003e\n\u003cp\u003eAll reagents and solvents were procured from commercial suppliers (Spectrochem Pvt. Ltd., SIGMA-ALDRICH Chemicals Pvt. Ltd., and Merck Specialities Pvt. Ltd.) and were used without further purification. The reaction progress was monitored by thin-layer chromatography (TLC) performed on silica gel 60 F254 pre-coated aluminium sheets. TLC was visualized by a 254 nm UV lamp and iodine staining. NMR spectra were recorded on a 400MHz Bruker Avance FTNMR spectrometer using CDCl\u003csub\u003e3\u003c/sub\u003e and DMSO as solvents.\u003c/p\u003e\n\u003cdiv\u003e\n\u003ch2\u003e4.2.1. General procedure for the synthesis of tryptanthrins (1a-h)\u003c/h2\u003e\n\u003cp\u003eTryptanthrin derivatives were synthesized from the corresponding isatins and isatoic anhydrides according to a previously reported procedure [7]. Isatin (1 mmol) was taken in a round bottom flask, followed by the addition of isatoic anhydride (1 mmol) and triethylamine (5 mmol). A minimal amount of dry toluene was added as solvent and the reaction mixture was refluxed at 110 ℃ for three hours. The precipitated product was filtered, washed using methanol and dried. The product was recrystallized from ethanol.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003e4.2.2 General procedure for the synthesis of Spiropyrazole compounds (3a-k)\u003c/h2\u003e\n\u003cp\u003eHydrazonyl chloride (0.25 mmol) was stirred in acetonitrile in a 50 ml round-bottom flask at room temperature followed by the addition of triethylamine (0.25 mmol) as base for the \u003cem\u003ein-situ\u003c/em\u003e generation of nitrile imine. It is then added with tryptanthrin ylidene (0.25 mmol and allowed to stir for 3\u0026ndash;4 hours. The completion of reaction was observed from TLC, and the product was isolated by silica gel column chromatography using 80:20 hexane-ethyl acetate in 78\u0026ndash;92% yield.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.1 Ethyl-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylat\u003c/strong\u003ee, \u003cstrong\u003eC\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e24\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e,\u003c/sub\u003e\u003cstrong\u003e(3a)\u003c/strong\u003e: Light yellow powder; Yield: 92%; mp: 278\u0026ndash;280 ℃; FTIR (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3030, 2917, 1713, 1672, 1639, 1590, 1337, 1315. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.66 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 8.48 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 7.78\u0026ndash;7.74 (m, 4H), 7.61\u0026ndash;7.55 (m, 2H), 7.47\u0026ndash;7.42 (m, 4H), 7.28\u0026ndash;7.24 (m, 1H), 7.02 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H), 6.77\u0026ndash;6.74 (m, 3H), 5.24 (s, 1H), 3.78 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.1 Hz, 2H), 0.81 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 166.5, 163.8, 159.7, 159.0, 147.1, 144.1, 142.6, 139.3, 134.7, 131.6, 131.2, 129.0, 128.9, 128.6, 128.4, 128.3, 127.8, 127.5, 126.9, 126.8, 126.7, 126.1, 125.9, 121.7, 121.3, 117.2, 115.5, 115.3, 65.5, 61.7, 29.7, 13.5. Elemental Analysis: Analytically calculated (%): C, 74.99; H, 4.72; N, 10.93; O, 9.36 Found %: C, 7.98; H, 4.73; N, 10.94\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.2 Ethyl-8-chloro-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eClN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e, \u003cstrong\u003e(3b)\u003c/strong\u003e: Light yellow powder; Yield: 88%; mp: 273\u0026ndash;275 ℃; FTIR (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3032, 2919, 1717, 1679, 1632, 1593, 1339, 1317. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.52 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 1H), 8.37\u0026ndash;8.35 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8 Hz, 1H), 7.69\u0026ndash;7.64 (m, 4H), 7.52\u0026ndash;7.47 (m, 1H), 7.43\u0026ndash;7.40 (m, 1H), 7.37\u0026ndash;7.31 (m, 4H), 7.02 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H), 6.95 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H), 6.72\u0026ndash;6.66 (m, 3H), 5.1 (s, 1H), 3.78 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 2H), 0.81 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 166.4, 163.4, 160.8, 159.5, 147.0, 144.3, 142.4, 140.2, 134.8, 132.5, 131.3, 130.4, 129.7, 129.2, 129.1, 128.6, 128.5, 128.3, 128.0, 127.5, 127.0, 126.1, 125.74, 123.5, 122.7, 121.6, 118.2, 115.3, 65.7, 62.0, 29.5, 13.5. Elemental Analysis: Analytically calculated %: C,70.26; H, 4.24; Cl, 6.48; N, 10.24; O, 8.77 Found %: C, 70.27; H, 4.25; N, 10.25.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.3 Ethyl-8-bromo-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eBrN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e, \u003cstrong\u003e(3c)\u003c/strong\u003e: Light yellow powder; Yield: 85%; mp: 276\u0026ndash;278 ℃; FTIR (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 3034, 2919, 1715, 1675, 1636, 1598, 1335, 1318. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.56 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 1H) 8.45 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 7.79\u0026ndash;7.74 (m, 4H), 7.69\u0026ndash;7.66 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2 Hz, 1H) 7.61\u0026ndash;7.57 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3 Hz, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.3 Hz, 2H), 7.47\u0026ndash;7.41 (m, 3H), 7.05 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H), 6.82\u0026ndash;6.75 (m, 3H), 5.22 (s, 1H), 3.88 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.2 Hz, 2H), 0.92 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 3H).\u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 170.6, 166.4, 159.5, 159.4, 146.5, 145.6, 142.9, 140.3, 140.2, 138.2, 134.8, 134.2, 133.9, 131.4, 129.8, 129.3, 129.1, 128.6, 128.5, 126.4, 126.1, 125.7, 123.5, 122.0, 121.6, 118.4, 115.8, 115.3, 65.8, 62.0, 29.7, 13.6. Elemental Analysis: Analytically calculated %: C, 64.50; H, 3.29; N, 10.75. Found %: C, 64.52; H, 3.31; N, 10.77.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.4 Ethyl-8-fluoro-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eFN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(3d)\u003c/strong\u003e: Yellow powder; Yield: 87%; mp: 280\u0026ndash;282 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 3171, 3051, 1680, 1628, 1546, 1456, 1337, 1248, 1156, 1061. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.53 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.4 Hz, 1H), 8.31 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.6 Hz, 2H), 7.66\u0026ndash;7.60 (m, 4H), 7.36\u0026ndash;7.32 (m, 3H), 7.14\u0026ndash;7.08 (m, 3H), 6.95 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H), 6.72\u0026ndash;6.63 (m, 2H), 5.12 (s, 1H), 3.78 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.6 Hz, 2H), 0.80 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 165.3, 158.7, 158.0, 157.2, 144.4, 143.4, 141.4, 134.2, 134.1, 133.0, 130.3, 128.7, 128.2, 128.0, 127.6, 125.3, 125.1, 121.7, 120.8, 117.7, 117.6, 117.2, 116.9, 114.4, 112.8, 112.5, 64.5, 61.0, 28.6, 12.6. Elemental Analysis: Analytical calculated (%): C, 72.44; H, 4.37; N, 10.56. Found (%): 72.40; H, 4.35; N, 10.52.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.5 Ethyl-8-methoxy-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e33\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e26\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(3e)\u003c/strong\u003e: Yellow powder; Yield: 78%; mp: 286\u0026ndash;288 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 3170, 3059, 1672, 1630, 1540, 1450, 1330, 1250, 1158, 1065. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.61 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H) 8.45 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.2 Hz, 1H), 7.84 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 7.81\u0026ndash;7.74 (m, 2H) 7.69\u0026ndash;7.58 (m, 2H), 7.53\u0026ndash;7.41 (m, 3H), 7.07\u0026ndash;6.96 (m, 2H), 6.94\u0026ndash;6.85 (m, 3H), 6.78 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 5.22 (s, 1H), 3.87 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 2H), 3.76 (s, 3H), 0.80 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.28 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 166.3, 166.1, 158.5, 158.3, 145.5, 145.1, 144.4, 142.4, 137.9, 136.8, 135.2, 134.3, 134.1, 131.3, 129.8, 129.7, 129.3, 129.1, 129.0, 128.7, 126.4, 126.2, 126.1, 122.7, 121.8, 120.3, 118.7, 115.4, 65.7, 62.1, 57.1, 29.3, 13.6. Elemental Analysis: Analytical calculated (%): C, 73.05; H, 4.83; N, 10.33. Found (%): C, 73.01; H, 4.85; N, 10.30.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.6 Ethyl 8-bromo-2-chloro-12-oxo-2',5'-diphenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e22\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eBrClN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e, \u003cstrong\u003e(3f)\u003c/strong\u003e: Yellow powder; Yield: 83%; mp: 288\u0026ndash;290 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 3032, 2914, 1717, 1677, 1637, 1599, 1334, 1312. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H), 8.30 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 7.66\u0026ndash;7.51 (m, 5H), 7.51 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 7.38\u0026ndash;7.32 (m, 3H), 6.95 (2H, t, \u003cem\u003eJ\u003c/em\u003e= 8Hz), 6.73\u0026ndash;6.64 (3H, m), 5.09 (s, 1H), 3.80 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.9 Hz, 2H), 0.83 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 3H).\u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 168.6, 166.3, 158.5, 158.3, 145.5, 144.4, 142.4, 137.9, 135.2, 134.3, 134.1, 131.3, 129.8, 129.3, 129.1, 129.0, 128.7, 126.4, 126.1, 122.7, 121.8, 120.3, 119.0, 118.6, 115.4, 115.4, 65.7, 62.1, 29.7, 13.6. Elemental Analysis: Analytical calculated (%): C, 61.41; H, 3.54; N, 8.95. Found (%): C, 61.40; H, 3.55; N, 8.92.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.7 Ethyl-8-bromo-2'-(4-fluorophenyl)-12-oxo-5'-phenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e22\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eBrFN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(3g)\u003c/strong\u003e: Yellow powder; Yield: 82%; mp: 290\u0026ndash;292 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 3178, 3045, 1685, 1630, 1545, 1450, 1335, 1240, 1160, 1070. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.62 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2 Hz, 1H), 8.38 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 7.82\u0026ndash;7.77 (m, 1H), 7.72\u0026ndash;7.69 (m, 1H), 7.67\u0026ndash;7.60 (m, 2H), 7.52 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 1H), 7.05 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6 Hz, 3H), 6.95 (t, \u003cem\u003eJ\u003c/em\u003e=8Hz, 3H), 6.71 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 2H), 6.64 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 5.11 (s, 1H), 3.73 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.3 Hz, 2H), 0.78 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 166.3, 162.8, 159.8, 159.4, 146.8, 142.4, 140.0, 137.1, 135.2, 135.0, 130.2, 129.9, 129.0, 128.3, 128.1, 128.0, 127.9, 127.1, 126.9, 126.7, 125.0, 121.8, 117.7, 115.9, 115.7, 115.5, 65.4, 62.0, 29.7, 14.1. Elemental Analysis: Analytical calculated (%): C, 63.06; H, 3.64; N, 9.19; Found (%): C, 63.05; H, 3.66; N, 9.18.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.8 Ethyl-8-bromo-2-chloro-2'-(4-fluorophenyl)-12-oxo-5'-phenyl-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e32\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e21\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eBrClFN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(3h)\u003c/strong\u003e: Yellow powder; Yield: 80%; mp: 296\u0026ndash;298 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 3172, 3051, 1682, 1630, 1552, 1455, 1335, 1250, 1158, 1065. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 1H), 8.31 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 7.65\u0026ndash;7.58 (m, 5H), 7.49 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2 Hz, 1H), 7.05 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6 Hz, 2H), 6.98\u0026ndash;6.92 (m, 2H), 6.72 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 1H), 6.63 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 2H), 5.07 (s, 1H), 3.79 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.6 Hz, 2H), 0.83 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 166.2, 160.3, 158.4, 158.3, 145.4, 143.4, 142.4, 137.9, 135.2, 134.3, 134.2, 129.8, 129.1, 128.9, 128.8, 128.0, 127.9, 126.4, 122.7, 121.9, 120.3, 118.7, 115.9, 115.7, 115.4, 65.6, 62.2, 29.7, 13.6. Elemental Analysis: Analytical calculated (%): C, 59.69; H, 3.29; N, 8.70; Found (%): C, 59.66; H, 3.28; N, 8.69\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.9 Ethyl (4'S,6S)-8-bromo-12-oxo-5'-phenyl-2'-(4-(trifluoromethyl)phenyl)-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e33\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e22\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eBrF\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(3i)\u003c/strong\u003e: Yellow powder; Yield: 87%; mp: 300\u0026ndash;302 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 3175, 3051, 1680, 1630, 1544, 1450, 1335, 1240, 1150, 1060. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.38 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.6 Hz, 1H), 8.36 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.2 Hz, 1H), 7.99\u0026ndash;7.91 (m, 1H), 7.75\u0026ndash;7.67 (m, 3H), 7.62\u0026ndash;7.58 (m, 4H), 7.54\u0026ndash;7.51 (m, 1H), 7.47\u0026ndash;7.45 (m, 1H), 6.98 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H), 6.75\u0026ndash;6.67 (m, 2H), 5.13 (s, 1H), 3.79 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 2H), 0.82 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 167.2, 166.1, 159.5, 158.4, 146.3, 142.0, 140.8, 138.2, 134.9, 134.4, 130.9, 129.5, 129.2, 128.8, 128.3, 128.2, 127.0, 126.4, 126.2, 125.6, 124.6, 123.7, 123.2, 122.2, 120.1, 118.6, 115.8, 115.5, 65.3, 62.3, 29.7, 14.1. Elemental Analysis: Analytical calculated (%): C, 60.10; H, 3.36; N, 8.50; Found (%): C, 60.12; H, 3.34; N, 8.51.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.10 Ethyl(4'S,6S)-8-bromo-2-chloro-12-oxo-5'-phenyl-2'-(4-(trifluoromethyl)phenyl)-2',4'-dihydro-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e33\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e21\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eBrClF\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e3\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(3j)\u003c/strong\u003e: Yellow powder; Yield: 85%; mp: 295\u0026ndash;297 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 3168, 3050, 1685, 1636, 1550, 1459, 1340, 1250, 1158, 1065. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.44 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H), 8.31 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 7.74 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 2H), 7.65\u0026ndash;7.56 (m, 5H), 7.48 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.6 Hz, 1H), 6.98 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H), 6.75 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 6.70\u0026ndash;6.64 (m, 2H), 5.10 (s, 1H), 3.80 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7 Hz, 2H), 0.83 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 166.0, 160.0, 158.2, 158.2, 145.4, 142.8, 141.9, 137.9, 135.3, 134.7, 134.5, 134.3, 129.8, 129.2, 128.8, 128.6, 126.5, 126.3, 126.1, 125.6, 122.7, 122.6, 122.3, 120.4, 119.1, 118.7, 115.6, 65.2, 62.3, 29.7, 13.6. Elemental Analysis: Analytical calculated (%): C, 57.12; H, 3.05; N, 8.07; Found (%): C, 57.13; H, 3.02; N, 8.05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.2.11 (R)-12-oxo-2',5'-diphenyl-12H-spiro[indolo[2,1-b]quinazoline-6,3'-pyrazole]-4',4'(2'H)-dicarbonitrile; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO (3k)\u003c/strong\u003e: Yellow powder; Yield: 93%; mp: 298\u0026ndash;300 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 2105, 1676, 1594, 1486, 1456, 1352, 1229, 1095. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 8.80 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 8.71 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 8.51 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 8.41\u0026ndash;8.39 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.8 Hz, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 8.10\u0026ndash;8.06 (m, 2H), 8.02-8.00 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.6 Hz, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 1H), 7.86\u0026ndash;7.83 (m, 1H), 7.79\u0026ndash;7.74 (m, 3H), 7.69\u0026ndash;7.65 (m, 1H), 7.59\u0026ndash;7.55 (m, 2H), 7.08 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H), 6.95\u0026ndash;6.85 (m, 2H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 162.9, 159.3, 158.9, 146.2, 145.8, 142.0, 141.4, 134.9, 133.5, 133.0, 129.8, 129.1, 128.5, 128.4, 127.1, 126.6, 126.4, 126.1, 124.5, 122.0, 119.8, 118.6, 118.0, 116.8, 116.5, 116.2, 109.9, 109.4, 68.9, 53.2. Elemental Analysis: Analytical calculated (%): C, 75.91; H, 3.70; N, 17.13; Found (%): C, 75.93; H, 3.71; N, 17.10\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003e4.2.3 General procedure for the synthesis of dispiropyrrolidine compounds (6a-k)\u003c/h2\u003e\n\u003cp\u003eThe dispiropyrrolidine compounds were synthesized by a three-component reaction of isatin (1 mmol), sarcosine (2 mmol) and tryptanthrin ylidene (1 mmol) by refluxing in ethanol- toluene mixture for 85℃ until complete consumption of isatin was observed from TLC. The product was isolated by silica gel column chromatography using 70:30 hexane-ethyl acetate in 70\u0026ndash;91% yield.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3.1 Ethyl-1'-methyl-2,12''-dioxo-12''\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003e-dispiro[indoline-3,2'-pyrrolidine-3',6''- indolo[2,1-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e]quinazoline]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e24\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(6a)\u003c/strong\u003e: Yellow solid; Yield: 85%; mp: 250\u0026ndash;252 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 1722, 1705, 1695, 1514, 750. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 10.46 (s, 1H), 8.32\u0026ndash;8.27 (m, 2H), 8.19 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1H), 8.00 (s, 1H), 7.58\u0026ndash;7.49 (m, 3H), 7.36 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4 Hz, 1H), 6.95 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 1H), 6.53 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 2H), 6.12 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5 Hz, 1H), 4.95 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 2H), 3.78 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10 Hz, 1H), 3.69\u0026ndash;3.61 (m, 1H), 3.50\u0026ndash;3.44 (m, 1H), 2.22 (s, 3H), 0.50 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 176.4, 169.8, 159.5, 155.6, 146.9, 141.7, 139.2, 134.3, 132.7, 132.2, 130.1, 129.7, 127.9, 127.3, 126.7, 125.8, 122.6, 122.3, 121.1, 119.1, 117.8, 109.8, 78.6, 60.6, 51.6, 49.1, 35.0, 29.7, 13.3. Elemental Analysis: Analytical calculated (%): C, 60.96; H, 4.06; N, 9.80; Found (%): C, 60.94; H, 4.06; N, 9.81.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3.2 Ethyl-8''-chloro-1'-methyl-2,12''-dioxo-12''\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003e-dispiro[indoline-3,2'-pyrrolidine- 3',6''-indolo[2,1-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e]quinazoline]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eClN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(6b)\u003c/strong\u003e: Yellow solid; Yield: 91%; mp: 214\u0026ndash;216 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 1721, 1718,1685, 1575, 690. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 10.89 (s, 1H). 8.32 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H), 8.23\u0026ndash;8.21 (m, 1H), 8.13\u0026ndash;8.12 (m, 1H), 7.66\u0026ndash;7.65 (m, 1H), 7.59\u0026ndash;7.57 (m, 1H), 7.44\u0026ndash;7.40 (m, 2H), 7.32\u0026ndash;7.30 (m, 1H), 6.57\u0026ndash;6.53 (m, 1H), 6.45 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 6.34 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 1H), 4.06 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 2H), 2.56\u0026ndash;2.54 (m, 1H), 2.30 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 2H), 1.96 (s, 3H), 0.68 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.8 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 175.6, 165.1, 157.2, 140.7, 139.0, 137.2, 135.5, 134.7, 131.7, 130.0, 129.1, 128.4, 128.1, 127.3, 125.0, 121.0, 119.9, 119.7, 118.1, 116.4, 113.2, 110.2, 86.2, 61.5, 52.7, 46.7, 29.7, 22.7, 14.1. Elemental Analysis: Analytical calculated (%): C, 70.72; H, 4.91; N, 11.38; Found (%): C, 70.70; H, 4.92; N, 11.36.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3.3 Ethyl-8''-bromo-1'-methyl-2,12''-dioxo-12''\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003e-dispiro[indoline-3,2'-pyrrolidine- 3',6''-indolo[2,1-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e]quinazoline]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eBrN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(6c)\u003c/strong\u003e: Yellow solid; Yield: 88%; mp: 225\u0026ndash;227 ℃; FTIR (cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e): 1724, 1719, 1691, 1595, 1450. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, DMSO\u003cem\u003e-d6\u003c/em\u003e): 10.71 (s, 1H), 8.10 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 1H), 8.02 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5, 1H), 7.84 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8, 1H), 7.79\u0026ndash;7.71 (m, 1H), 7.59 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 7.48\u0026ndash;7.41 (m, 1H), 7.26\u0026ndash;7.19 (m, 1H), 7.14 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 6.96 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.4 \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 6.45 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4Hz, 1H), 6.04\u0026ndash;5.99 (m, 1H), 4.67 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11 Hz, 2H), 4.25 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.7 Hz, 1H), 4.20\u0026ndash;4.16 (m, 1H), 4.01\u0026ndash;3.94 (m, 1H), 2.99 (s, 3H), 0.85 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.3 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e): 182.0, 171.2, 159.4, 144.0, 141.0, 139.4, 137.6, 135.8, 135.6, 132.9, 129.3, 128.3, 127.9, 125.9, 124.3, 121.2, 120.0, 117.3, 116.2, 116.0, 112.1, 83.0, 67.6, 60.1, 46.2, 33.3, 23.2, 15.1, 9.1. Elemental Analysis: Analytical calculated (%): C, 66.10; H, 4.40; N, 10.63; Found (%): C, 66.10; H, 4.42; N, 10.62.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3.4 Ethyl-8''-fluoro-1'-methyl-2,12''-dioxo-12''\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003e-dispiro[indoline-3,2'-pyrrolidine- 3',6''-indolo[2,1-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e]quinazoline]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eFN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(6d)\u003c/strong\u003e: Yellow solid; Yield: 80%; mp: 252\u0026ndash;254 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 1723, 1714, 1683, 1584, 720. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, DMSO\u003cem\u003e-d6\u003c/em\u003e): 10.2 (s, 1H), 8.02 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.1 Hz, 1H), 7.84 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 1H), 7.78\u0026ndash;7.71 (m, 3H), 7.59 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.2 Hz, 1H), 7.48\u0026ndash;7.41 (m, 2H), 6.96 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11 Hz, 1H), 6.48\u0026ndash;6.42 (m, 1H), 6.04\u0026ndash;5.99 (m, 1H), 4.67 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.6 Hz, 2H), 4.20\u0026ndash;4.16 (m, 1H), 4.01\u0026ndash;3.94 (m, 2H), 2.92 (s, 3H), 1.13 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e): 174.4, 165.3, 156.5, 137.4, 137.2, 136.6, 130.6, 128.8, 128.0, 127.7, 127.2, 124.6, 124.4, 123.3, 121.4, 120.8, 120.2, 120.0, 118.9, 114.2, 112.3, 107.6, 73.3, 62.4, 55.3, 42.5, 29.4, 22.5, 14.4. Elemental Analysis: Analytical calculated (%): C, 68.23; H, 4.54; N, 10.97; Found (%): C, 68.24; H, 4.55; N, 10.96.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3.5 Ethyl-8''-methoxy-1'-methyl-2,12''-dioxo-12''\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003e-dispiro[indoline-3,2'- pyrrolidine-3',6''-indolo[2,1-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e]quinazoline]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e30\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e26\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e5\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(6e)\u003c/strong\u003e: Yellow solid; Yield: 74%; mp: 260\u0026ndash;262 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 1725, 1718, 1690, 1591, 735. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, DMSO\u003cem\u003e-d6\u003c/em\u003e): 11.67 (s, 1H), 8.49 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 2H), 8.33 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 2H), 7.96 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.6 Hz, 2H), 7.90\u0026ndash;7.86 (m, 2H), 7.73\u0026ndash;7.77 (m, 1H), 7.49 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 2H), 4.34 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.6 Hz, 2H), 3.73 (s. 3H), 2.68\u0026ndash;2.66 (m, 1H), 2.33\u0026ndash;2.32 (m, 2H), 1.97 (s, 3H), 0.69 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.2 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e): 181.8, 170.8, 158.1, 146.8, 145.4, 144.9, 137.3, 135.9, 135.8, 131.6, 130.5, 130.4, 127.4, 124.7, 124.4, 123.6, 121.4, 119.6, 119.0, 114.6, 110.7, 82.7, 60.8, 56.4, 46.3, 32.2, 25.7, 21.5, 12.8. Elemental Analysis: Analytical calculated (%): C, 68.95; H, 5.02; N, 10.72; Found (%): C, 68.94; H, 5.01; N, 10.73.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3.6 Ethyl-2''-chloro-1'-methyl-2,12''-dioxo-12''\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003e-dispiro[indoline-3,2'-pyrrolidine- 3',6''-indolo[2,1-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e]quinazoline]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eClN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(6f)\u003c/strong\u003e: Yellow solid; Yield: 71%; mp: 243\u0026ndash;245 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 1720, 1715, 1697, 1514, 819. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 10.33 (s, 1H), 8.56 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 8.39\u0026ndash;8.36 (m, 1H), 7.97 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 7.85 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 7.81\u0026ndash;7.76 (m, 2H), 7.74\u0026ndash;7.70 (m, 1H), 7.63\u0026ndash;7.59 (m, 1H), 7.47\u0026ndash;7.38 (m, 1H), 7.36 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 7.07\u0026ndash;7.04 (m, 1H), 3.91 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;3.6 Hz, 2H), 2.90 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.4 Hz, 1H), 2.72 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 2.27 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 1.96 (s, 3H), 0.48 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 177.0, 169.4, 152.8, 142.5, 134.3, 133.1, 131.8, 130.0, 129.2, 128.2, 127.2, 126.5, 124.9, 123.3, 121.8, 116.7, 114.8, 112.8, 111.2, 110.0, 72.4, 65.3, 52.9, 42.2, 29.7, 20.5, 11.5. Elemental Analysis: Analytical calculated (%): C, 66.10; H, 4.40; N, 10.63; Found (%): C, 66.11; H, 4.40; N, 10.62.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3.7 Ethyl-2''-bromo-1'-methyl-2,12''-dioxo-12''\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003e-dispiro[indoline-3,2'-pyrrolidine- 3',6''-indolo[2,1-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e]quinazoline]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e23\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eBrN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(6g)\u003c/strong\u003e: Yellow solid; Yield: 73%; mp: 236\u0026ndash;238 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 1722, 1714, 1685, 1592, 724. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 9.99 (s, 1H), 8.33\u0026ndash;8.27 (m, 2H), 8.19 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.6 Hz, 1H), 8.00 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.5 Hz, 1H), 7.59\u0026ndash;7.56 (m, 2H), 7.53\u0026ndash;7.49 (m, 1H), 7.38\u0026ndash;7.34 (m, 1H), 6.97\u0026ndash;6.9 (m, 1H), 6.52 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.3 Hz, 2H), 4.96 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.5 Hz, 2H), 4.01\u0026ndash;3.94 (m, 1H), 3.78 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.2 Hz, 1H), 3.69\u0026ndash;3.62 (m, 1H), 2.25 (s, 3H), 0.63 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 182.5, 170.3, 158.1, 146.6, 146.3, 144.3, 138.2, 135.1, 133.0, 131.9, 130.7, 130.3, 130.2, 127.5, 127.2, 125.4, 124.1, 123.7, 121.9, 118.0, 101.9, 72.6, 62.7, 44.1, 31.9, 29.6, 22.6, 14.1. Elemental Analysis: Analytical calculated (%): C, 60.96; H, 4.06; N, 9.80; Found (%): C, 60.96; H, 4.05; N, 9.81.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3.8 Ethyl-2''-bromo-8''-chloro-1'-methyl-2,12''-dioxo-12''\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003e-dispiro[indoline-3,2'- pyrrolidine-3',6''-indolo[2,1-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e]quinazoline]-4'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e29\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e22\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eBrClN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(6h)\u003c/strong\u003e: Yellow solid; Yield: 70%; mp: 248\u0026ndash;250 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 1721, 1717, 1682, 1595, 1414. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 10.34 (s, 1H), 8.45 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H), 8.32 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 7.96 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.2 Hz, 1H), 7.85\u0026ndash;7.82 (m, 1H), 7.75\u0026ndash;7.72 (m, 1H), 7.46 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.2 Hz, 2H), 7.28 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 1H), 7.07\u0026ndash;7.04 (m, 2H), 3.91 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.4 Hz, 2H), 2.75\u0026ndash;2.71 (m, 1H), 2.55\u0026ndash;2.51 (m, 1H), 2.10 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 Hz, 1H), 1.96 (s, 3H), 0.80 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.4 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 182.5, 169.4, 158.1, 147.5, 146.6, 146.3, 144.3, 138.3, 135.1, 130.7, 130.2, 127.5, 127.2, 125.4, 123.7, 121.9, 117.9, 107.0, 106.0, 104.1, 101.9, 81.4, 60.5, 46.9, 41.8, 29.7, 22.7, 14.1. Elemental Analysis: Analytical calculated (%): C, 57.49; H, 3.66; N, 9.25; Found (%): C, 57.49; H, 3.65; N, 9.26.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3.9 Ethyl-8''-bromo-2,12''-dioxo-5',6',7',7a'-tetrahydro-1'\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e,\u003cstrong\u003e12''\u003c/strong\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003cstrong\u003e-dispiro[indoline- 3,3'-pyrrolizine-2',6''-indolo[2,1-\u003c/strong\u003e\u003cstrong\u003eb\u003c/strong\u003e\u003cstrong\u003e]quinazoline]-1'-carboxylate; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e31\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e25\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eBrN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e4\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(6i)\u003c/strong\u003e: Yellow solid; Yeild: 80%; mp: 225\u0026ndash;227 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 1720, 1713, 1681. 1592. 1418. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 9.08 (s, 1H), 8.60\u0026ndash;8.53 (m, 2H), 8.47 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.4 Hz, 1H), 8.41 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 8.35 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.6 Hz, 2H), 7.74\u0026ndash;7.73 (m, 2H), 7.68\u0026ndash;7.45 (m, 1H), 7.39 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 Hz, 2H), 3.69 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 2H), 2.75\u0026ndash;2.71 (m, 1H), 2.55\u0026ndash;2.51 (m, 1H), 2.28 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 3H), 1.37\u0026ndash;1.33 (m, 2H), 1.31\u0026ndash;1.30 (m, 1H), 0.69 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 177.0, 170.0, 158.3, 145.7, 141.7, 139.9, 137.1, 130.0, 129.8, 129.3, 129.2, 126.9, 126.2, 126.0, 122.8, 122.6, 122.2, 120.8, 116.6, 109.7, 78.8, 61.8, 60.5, 51.7, 49.2, 35.0, 29.7, 22.7, 13.2. Elemental Analysis: Analytical calculated (%): C, 62.32; H, 4.22; N, 9.38; Found (%): C, 62.33; H, 4.23; N, 9.36.\u003c/p\u003e\n\u003cdiv\u003e\n\u003ch2\u003e4.2.3.10 Ethyl-8''-bromo-2,12''-dioxo-7',7a'-dihydro-1'\u003cem\u003eH\u003c/em\u003e,3'\u003cem\u003eH\u003c/em\u003e,12''\u003cem\u003eH\u003c/em\u003e-dispiro[indoline- 3,5'-pyrrolo[1,2-\u003cem\u003ec\u003c/em\u003e]thiazole-6',6''-indolo[2,1-\u003cem\u003eb\u003c/em\u003e]quinazoline]-7'-carboxylate; C\u003csub\u003e30\u003c/sub\u003eH\u003csub\u003e23\u003c/sub\u003eBrN\u003csub\u003e4\u003c/sub\u003eO\u003csub\u003e4\u003c/sub\u003eS (6j):\u003c/h2\u003e\n\u003cp\u003eYellow solid; Yield: 89%; mp: 249\u0026ndash;251 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 1721, 1719, 1687, 1538, 1425, 918. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 9.85 (s, 1H), 8.32 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.8 Hz, 1H), 8.23\u0026ndash;8.21 (m, 1H), 8.13\u0026ndash;8.12 (m, 1H), 7.81\u0026ndash;7.65 (m, 2H), 7.59\u0026ndash;7.57 (m, 2H), 6.96\u0026ndash;6.92 (m, 1H), 6.57\u0026ndash;6.53 (m, 1H), 6.45 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 6.34 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 4.72 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6 Hz, 2H), 3.89 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 3.74\u0026ndash;3.67 (m, 1H), 3.62 (d, \u003cem\u003eJ\u003c/em\u003e=8Hz, 1H), 3.56\u0026ndash;3.52 (m, 2H), 3.23 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4 Hz, 2H), 0.58 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.8 Hz, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e): 177.3, 168.8, 159.4, 154.1, 146.7, 140.8, 139.2, 134.5, 133.1, 131.2, 130.3, 129.3, 127.9, 127.5, 126.7, 125.9, 123.5, 122.5, 121.1, 119.3, 118.1, 109.9, 79.4, 69.0, 66.4, 60.9, 51.2, 36.3, 29.7, 13.4. Elemental Analysis: Analytical calculated (%): C, 58.54; H, 3.77; N, 9.10; Found (%): C, 58.54; H, 3.76; N, 9.10.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e4.2.3.11 (3R,3'S)-1'-methyl-2,12''-dioxo-12''H-dispiro[indoline-3,2'-pyrrolidine-3',6''-indolo[2,1-b]quinazoline]-4',4'-dicarbonitrile; C\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e28\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eH\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e18\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eN\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003eO\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003e2\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e(6k)\u003c/strong\u003e: Yellow solid: Yield: 90%; mp: 260\u0026ndash;262 ℃; FTIR (cm\u003csup\u003e-1\u003c/sup\u003e): 2149,1680, 1546, 1456, 765. \u003csup\u003e1\u003c/sup\u003eH NMR (\u0026delta; ppm, 400 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e): 11.05 (s, 1H), 8.43 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.6 Hz, 1H), 8.30\u0026ndash;8.28 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.2 Hz, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8 Hz, 1H), 7.91\u0026ndash;7.86 (m, 1H), 7.82 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.2 Hz, 1H), 7.62\u0026ndash;7.58 (m, 2H), 7.52\u0026ndash;7.48 (m, 1H), 7.45\u0026ndash;7.41 (m, 1H), 7.28\u0026ndash;7.23 (m, 1H), 7.18\u0026ndash;7.13 (m, 1H), 6.86 (s, 2H), 3.89 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10 Hz, 1H). 3.64 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10 Hz, 1H). 2.86 (s, 3H). \u003csup\u003e13\u003c/sup\u003eC NMR (\u0026delta; ppm, 100 MHz, DMSO-\u003cem\u003ed6\u003c/em\u003e): 164.4, 163.8, 159.5, 147.8, 138.6, 137.8, 136.2, 135.1, 129.3, 128.6, 127.8, 127.5, 126.8, 125.7, 124.6, 121.4, 121.3, 116.4, 64.2, 56.0, 55.5, 33.5, 21.5. Elemental Analysis: Analytical calculated (%): C, 71.48; H, 3.86; N, 17.86; Found (%): C, 71.44; H, 3.83; N, 17.85.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003e4.3 Biological studies\u003c/h2\u003e\n\u003cdiv\u003e\n\u003ch2\u003e4.3.1 \u003cem\u003eIn vitro\u003c/em\u003e antibacterial study\u003c/h2\u003e\n\u003cp\u003eThe compounds were screened against a bacterial panel comprising ESKAPE pathogens, including \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 29213, \u003cem\u003eKlebsiella pneumoniae\u003c/em\u003e BAA 1705, \u003cem\u003eAcinetobacter baumannii\u003c/em\u003e BAA 1605, \u003cem\u003ePseudomonas aeruginosa\u003c/em\u003e ATCC 27853 and \u003cem\u003eEscherichia coli\u003c/em\u003e IMP 4213. The strains were obtained from the Biodefense and Emerging Infections Research Resources Repository / Network on Antimicrobial Resistance in Staphylococcus aureus / American Type Culture Collection (BEI/NARSA/ATCC, USA) and routinely cultured in Mueller\u0026ndash;Hinton agar (MHA) and Mueller\u0026ndash;Hinton broth II (MHBII). Prior to the experiment, a single colony from an MHA plate was inoculated into MHBII and incubated overnight at 37\u0026deg;C with shaking for 18\u0026ndash;24 h to obtain the starter culture.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003e4.3.2 \u003cem\u003eIn vitro\u003c/em\u003e anticancer study\u003c/h2\u003e\n\u003cp\u003eFifteen milligrams of MTT (Sigma, M-5655) was dissolved in 3 ml of PBS until fully solubilized and then sterilized through filter sterilization. Following a 24-hour incubation period, the contents in the wells were discarded, and 30 \u0026micro;l of the prepared MTT solution was added to each test and cell control well. The plate was gently shaken to ensure proper mixing and then incubated at 37\u0026deg;C in a humidified incubator containing 5% CO₂ for 4 hours. After incubation, the supernatant was carefully removed, and 100 \u0026micro;l of MTT solubilization solution (dimethyl sulfoxide, DMSO; Sigma Aldrich, USA) was added and the wells were mixed gently by pipetting up and down to dissolve the formazan crystals. Finally, the absorbance was measured at 540 nm using a microplate reader.\u003c/p\u003e\n\u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv\u003e\n\u003ch2\u003e4.4 Molecular docking\u003c/h2\u003e\n\u003cp\u003eThe three-dimensional crystal structure of the protein was retrieved from the Protein Data Bank (PDB ID: 2D0T). Protein preparation was carried out using PyMol Software. Ligand structures were energy minimized using Gaussian program and B3LYP/6-311\u0026thinsp;+\u0026thinsp;G(d) level of theory prior to docking. Docking simulations were carried out using PyRx Software. Binding affinities were recorded in kcal/mol, and the best ranked poses were selected based on binding energy and interaction consistency. Protein\u0026ndash;ligand interactions, including van der waal, \u0026pi;\u0026ndash;\u0026pi; stacking and \u0026pi;\u0026ndash;alkyl interactions were analyzed using ChimeraX Software.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003ch2\u003eConflict of interest\u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDK wrote the original draftDK and SLS did the data collection and formal analysisAAB and JEG contributed in the docking studyAS, AL and SC did the biological evaluation AD did the conceptualization, validation and final draft\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eThe corresponding author thank ANRF for funding in terms of POWER research grant (SPG/2023/000179). DK thank CSIR and SSL thank University of Kerala for the Junior Research Fellowships respectively. All authors acknowledge CLIF, University of Kerala, for providing instrumental facilities.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDavies J, Davies D (2010) Origins and evolution of antibiotic resistance. Microbiol Mol Biol Rev 74:417\u0026ndash;433. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1128/MMBR.00016-10\u003c/span\u003e\u003cspan address=\"10.1128/MMBR.00016-10\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKumar S, Mahato RP, Ch S, Soniya Kumbham (2025) Current strategies against multidrug-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and advances toward future therapy. Microbe 6:100281. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.microb.2025.100281\u003c/span\u003e\u003cspan address=\"10.1016/j.microb.2025.100281\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTurner NA, Sharma-Kuinkel BK, Maskarinec SA, Eichenberger EM, Shah PP, Carugati M, Holland TL, Fowler VG Jr (2019) Methicillin-resistant Staphylococcus aureus: an overview of basic and clinical research. Nat Rev Microbiol 17:203\u0026ndash;218. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41579-018-0147-4\u003c/span\u003e\u003cspan address=\"10.1038/s41579-018-0147-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelete MA, Gedefie A, Alemayehu E, Debash H, Mohammed O, Gebretsadik D, Tilahun M (2023) The prevalence of vancomycin-resistant Staphylococcus aureus in Ethiopia: a systematic review and meta-analysis. Antimicrob resist infect contro 12:86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13756-023-01291-3\u003c/span\u003e\u003cspan address=\"10.1186/s13756-023-01291-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCasolino R, Tatah L, Charnaud S, Santero M, Ilbawi A, Ross AL (2025) The WHO global landscape of cancer clinical trials. Nat Med 31:2901\u0026ndash;2912. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41591-025-03926-x\u003c/span\u003e\u003cspan address=\"10.1038/s41591-025-03926-x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVasan N, Baselga J, Hyman DM (2019) A view on drug resistance in cancer. Nature 575:299\u0026ndash;309. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41586-019-1730-1\u003c/span\u003e\u003cspan address=\"10.1038/s41586-019-1730-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTucker AM, Grundt P (2012) The chemistry of tryptanthrin and its derivatives. Arkivoc 1:546\u0026ndash;569\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJahng Y (2013) Progress in the studies on tryptanthrin, an alkaloid of history. Arch Pharm Res 36:517\u0026ndash;535. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s12272-013-0091-9\u003c/span\u003e\u003cspan address=\"10.1007/s12272-013-0091-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrand\u0026atilde;o P, Pineiro M, Burke AJ (2022) Tryptanthrin and its derivatives in drug discovery: synthetic insights. Synthesis 54:4235\u0026ndash;4245. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1055/s-0040-1719901\u003c/span\u003e\u003cspan address=\"10.1055/s-0040-1719901\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKaur R, Manjal SK, Rawal RK, Kumar K (2017) Recent synthetic and medicinal perspectives of tryptanthrin. Bioorg Med Chem 25:4533\u0026ndash;4552. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bmc.2017.07.003\u003c/span\u003e\u003cspan address=\"10.1016/j.bmc.2017.07.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAlam K, Hossain MS, Zhao Y, Zhang Z, Xu S, Hao J, Li A (2024) Tryptanthrins as multi-bioactive agents: discovery, diversity distribution and synthesis. Bioorg Chem 154:108071. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bioorg.2024.108071\u003c/span\u003e\u003cspan address=\"10.1016/j.bioorg.2024.108071\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCatanzaro E, Betari N, Arencibia JM, Montanari S, Sissi C, De Simone A, Milelli A (2020) Targeting topoisomerase II with trypthantrin derivatives: Discovery of 7-((2-(dimethylamino) ethyl) amino) indolo [2, 1-b] quinazoline-6, 12-dione as an antiproliferative agent and to treat cancer. Eur J Med Chem 202:112504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejmech.2020.112504\u003c/span\u003e\u003cspan address=\"10.1016/j.ejmech.2020.112504\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi MH, Wang Z, Liang Y, Lu S, Hou BL (2025) Synthesis, characterization, crystal structure, and anti-inflammatory evaluation of thienyl-substituted tryptanthrins. J Mol Struct 1342:142651. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.molstruc.2025.142651\u003c/span\u003e\u003cspan address=\"10.1016/j.molstruc.2025.142651\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeena SS, Kaul G, Akhir A, Saxena D, Chopra S, Deepthi A (2022) Green synthesis and antibacterial evaluation of spiro fused tryptanthrin-thiopyrano [2, 3-b] indole hybrids targeting drug-resistant \u003cem\u003eS. aureus\u003c/em\u003e. Bioorg Chem 128:106046. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bioorg.2022.106046\u003c/span\u003e\u003cspan address=\"10.1016/j.bioorg.2022.106046\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeena SS, Akhir A, Saxena D, Maitra R, Chopra S, Deepthi A (2023) Synthesis of tryptanthrin appended dispiropyrrolidine oxindoles and their antibacterial evaluation. RSC Med Chem 14:1165\u0026ndash;1171. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D3MD00017F\u003c/span\u003e\u003cspan address=\"10.1039/D3MD00017F\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Sharifi HK, Leena SS, Saxena D, Maitra R, Chopra S, Deepthi A (2024) Green synthesis of tryptanthrin appended 4-spiropiperidines and their anti-bacterial evaluation. Tetrahedron 151:133786. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tet.2023.133786\u003c/span\u003e\u003cspan address=\"10.1016/j.tet.2023.133786\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou X (2024) Recent advances of tryptanthrin and its derivatives as potential anticancer agents. RSC Med Chem 15:1127\u0026ndash;1147. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/D3MD00698K\u003c/span\u003e\u003cspan address=\"10.1039/D3MD00698K\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeenakshy CB, Sruthi SL, Jayasree EG, Sandhya KS, Deepthi A (2024) Synthesis and Anticancer Evaluation of Tryptanthrin-1, 2, 3‐Triazole Hybrids. J Heterocycl Chem 61:1891\u0026ndash;1898. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jhet.4916\u003c/span\u003e\u003cspan address=\"10.1002/jhet.4916\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDeepthi A, Acharjee N, Sruthi SL, Meenakshy CB (2022) An overview of nitrile imine based [3\u0026thinsp;+\u0026thinsp;2] cycloadditions over half a decade. Tetrahedron 116:132812. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tet.2022.132812\u003c/span\u003e\u003cspan address=\"10.1016/j.tet.2022.132812\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eViveka S, Dinesha, Shama P, Nagaraja GK, Ballav S, Kerkar S (2015) Design and Synthesis of Some New Pyrazolyl-Pyrazolines as Potential Anti-Inflammatory, Analgesic and Antibacterial Agents. Eur J Med Chem 101:442\u0026ndash;451. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.ejmech.2015.07.002\u003c/span\u003e\u003cspan address=\"10.1016/j.ejmech.2015.07.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMantzanidou M, Pontiki E, Hadjipavlou-Litina D (2021) Pyrazoles and Pyrazolines as Anti-InflammatoryAgents. Molecules 26:3439. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/molecules26113439\u003c/span\u003e\u003cspan address=\"10.3390/molecules26113439\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKotnala M, Singh G, Singh K, Nath R, Panda KC, Kumar A, Dutta S (2024) Exploring the Synthetic Strategies and Biological Activities of Pyrazole Derivatives. Nat Campano 28:3238\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi Petri G, Raimondi MV, Span\u0026ograve; V, Holl R, Barraja P, Montalbano A (2021) Pyrrolidine in drug discovery: a versatile scaffold for novel biologically active compounds. Top Curr Chem 379:34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s41061-021-00347-5\u003c/span\u003e\u003cspan address=\"10.1007/s41061-021-00347-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePoyraz S, D\u0026ouml;ndaş HA, D\u0026ouml;ndaş NY, Sansano JM (2023) Recent insights about pyrrolidine core skeletons in pharmacology. Front Pharmacol 14:1239658. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2023.1239658\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2023.1239658\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang S, Li X, Hu F, Li Y, Yang Y, Yan J, Kuang C, Yang Q (2023) Discovery of tryptanthrin derivatives as potent inhibitors of indoleamine 2,3-dioxygenase with therapeutic activity in Lewis lung cancer (LLC) tumor-bearing mice. J Med Chem 56:8321\u0026ndash;8331. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/jm401195n\u003c/span\u003e\u003cspan address=\"10.1021/jm401195n\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang K, Wu YH, Song Y, Yu B (2021) Indoleamine 2, 3-dioxygenase 1 (IDO1) inhibitors in clinical trials for cancer immunotherapy. J Hematol Oncol 14:68. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13045-021-01080-8\u003c/span\u003e\u003cspan address=\"10.1186/s13045-021-01080-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQian S, Zhang M, Chen Q, He Y, Wang W, Wang Z (2016) IDO as a drug target for cancer immunotherapy: recent developments in IDO inhibitors discovery. RSC Adv 6:7575\u0026ndash;7581. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1039/C5RA25046C\u003c/span\u003e\u003cspan address=\"10.1039/C5RA25046C\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLu S, Hou BL, Wang T, Ma K, Huang A, Wu X, Liang YN, Wang Z (2025) Antitumor effects of tryptanthrin on colorectal cancer by regulating the mitogen-activated protein kinase signaling pathway and targeting topo I and IDO1. ACS omega 10:3206\u0026ndash;3221. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1021/acsomega.4c11189\u003c/span\u003e\u003cspan address=\"10.1021/acsomega.4c11189\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan S, Stephen Chan HC, Hu Z (2017) Using PyMOL as a platform for computational drug design. Wiley Interdiscip Rev Comput Mol Sci 7:e1298. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/wcms.1298\u003c/span\u003e\u003cspan address=\"10.1002/wcms.1298\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSargis D, Olson AJ (2015) Small-molecule library screening by docking with PyRx. Methods Mol Biol 243\u0026ndash;250. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-1-4939-2269-7_19\u003c/span\u003e\u003cspan address=\"10.1007/978-1-4939-2269-7_19\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeng, Elaine C (2023) UCSF ChimeraX: Tools for structure building and analysis. Protein Sci 32:e4792. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/pro.4792\u003c/span\u003e\u003cspan address=\"10.1002/pro.4792\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eSchemes 1 to 3 are available in the Supplementary Files section\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Pyrazoles, pyrrolidines, tryptanthrin, anti- bacterial and anticancer activity","lastPublishedDoi":"10.21203/rs.3.rs-9343418/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9343418/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTwenty-two spirofused heterocycles containing tryptanthrin - pyrazole/pyrrolidine hybrids were synthesized \u003cem\u003evia\u003c/em\u003e classical (3\u0026thinsp;+\u0026thinsp;2) cycloaddition reaction of ethyl (\u003cem\u003eE\u003c/em\u003e)-2-(12-oxoindolo[2,1-\u003cem\u003eb\u003c/em\u003e]quinazolin-6(12\u003cem\u003eH\u003c/em\u003e)-ylidene)acetate with nitrile imine / azomethine ylide. The compounds were screened \u003cem\u003ein vitro\u003c/em\u003e for antibacterial activity against the ESKAPE pathogen panel, from which spiropyrazoles \u003cb\u003e3d\u003c/b\u003e and \u003cb\u003e3f\u003c/b\u003e (MIC\u0026thinsp;=\u0026thinsp;8 \u0026micro;g m/L) were found to be most potent against \u003cem\u003eS. aureus\u003c/em\u003e ATCC 29213. Anticancer evaluation of the molecules against HCT-116 human colon cancer cell line by MTT assay indicated dispiropyrrolidines \u003cb\u003e6a\u003c/b\u003e and \u003cb\u003e6c\u003c/b\u003e exhibited highest activity with LC₅₀ values of 23.57 \u0026micro;g/mL and 32.99 \u0026micro;g/mL respectively. Molecular docking studies demonstrated the potential of \u003cb\u003e6a\u003c/b\u003e and \u003cb\u003e6c\u003c/b\u003e as promising candidates for modulating the activity of the IDO1 protein. These findings further support the experimental results obtained from \u003cem\u003ein vitro\u003c/em\u003e biological studies and highlight the prospective utility of pyrazole/pyrrolidine derivatives for future therapeutic applications.\u003c/p\u003e","manuscriptTitle":"Synthesis and biological evaluation of spirofused pyrazole and pyrrolidine hybrids from tryptanthrin","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-16 11:24:17","doi":"10.21203/rs.3.rs-9343418/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9c2d3c85-9934-456b-88d8-e396f7ac8848","owner":[],"postedDate":"April 16th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-04-19T16:24:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-16 11:24:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9343418","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9343418","identity":"rs-9343418","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}