An evaluation of spirooxindoles as blocking agents of SARS-CoV-2 spike/ACE2 fusion and M pro inhibitory agents: Synthesis, biological evaluation and computational analysis

An evaluation of spirooxindoles as blocking agents of SARS-CoV-2 spike/ACE2 fusion and M pro inhibitory agents: Synthesis, biological evaluation and computational analysis | 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 Article An evaluation of spirooxindoles as blocking agents of SARS-CoV-2 spike/ACE2 fusion and M pro inhibitory agents: Synthesis, biological evaluation and computational analysis Albert Enama Ehinak, Maloba M. M. Lobe, Conrad V. Simoben, Ian Tietjen, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4535655/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 Both tetrahydroisoquinolines (THIQs) and oxindoles (OXs) display a broad range of biological activities, including antiviral activity. They are, therefore, recognized as privileged scaffolds in drug discovery. Here, we describe the synthesis of spirofused tetrahydroisoquinoline–oxindole hybrids (spirooxindoles) and their evaluation as potential blocking agents of both SARS-CoV-2 spike/ACE fusion and inhibitors of the main protease (M pro ). The most active synthesized compound showed a 50% inhibitory concentration (IC 50 ) of 3.6 µM against SARS-CoV-2 spike/ACE fusion. None of the tested compounds was shown to be active against M pro . The most active compound possesses a bulky naphthyl group, which addresses voluminous hydrophobic regions of the ACE2 binding site and interacts with the hydrophobic residues of the target; this finding agrees with previous studies revealing that bulky compounds block spike/ACE2 fusion, e.g., the natural product hopeaphenol. Therefore, spirooxindoles may provide useful leads in the search for SARS-CoV-2 spike/ACE fusion blocking agents. Biological sciences/Drug discovery/Drug screening Biological sciences/Drug discovery/Medicinal chemistry Biological sciences/Drug discovery/Pharmacology Mpro SARS-CoV-2 spike/ACE2 spirooxindoles tetrahydroisoquinolines Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent of coronavirus disease 19 (COVID-19), has emerged as a very important public health concern 1 . Since its outbreak in December 2019, in Wuhan city in Hubei province of China, the disease has resulted in significant global morbidity and mortality with over 680 million confirmed cases and 6,805,012 deaths 2,3 . Recent reports have identified four structural proteins of SARS-CoV-2 as potential targets for drug development. These structural proteins include the spike, envelope, nucleocapsid, and membrane proteins 4 . Based on established knowledge about the virus, research groups have focused their efforts on two viral proteins: i) the spike (s)-glycoprotein 5 , to disrupt its recognition of the membrane-bound angiotensin-converting enzyme 2 (ACE2), thereby hindering the interaction of the receptor binding domain (RBD) located at the S1 subunit of the spike protein and the angiotensin-converting enzyme II (ACE2) which is responsible for cellular entry 6 ; and ii) the main viral protease (M pro , 3CL pro ), which is one of two viral proteases responsible for cleaving viral polypeptides to generate mature proteins, to inhibit viral replication 7 . SARS-CoV-2 main protease is a critical protein that plays a vital role in the replication cycle of the SARS-CoV-2 virus, and its inhibition is a promising strategy for the development of antiviral therapies 8 . One promising approach to drug discovery is that which takes advantage of privileged scaffolds. According to Evans et al. , a privileged scaffold is a molecular framework that has high affinity for a diverse array of receptors 9 . Thus, privileged scaffolds are often used as a starting point in drug discovery; the combination of two or more of such privileged scaffolds, called molecular hybridization, has been highly exploited in drug discovery 10 . Spirocyclic compounds, especially spirooxindoles, have been widely studied in recent years because of their unique three-dimensional structures and broad range of biological activities. Both synthetic and naturally occurring spirooxindoles (Figure 1) display diverse pharmacological properties such as anticancer 11 , antimicrobial 12 , anti-inflammatory 12 , antimalarial 13 , antiviral 14 , antidiabetic 15 , and antioxidant activity 16 . Spirotryprostatin A, for example, is an indole alkaloid of the 2,5-diketopiperazine class of natural products that was first identified by Cui et al . from the fungal species Aspergillus fumigatus as a mammalian cell cycle inhibitor 17 . When tested in an anti- Trypanosoma assay, Spirotryprostatin A was inactive, although its semi-synthetic analogues of the diketopiperazine class showed antitrypanosomal activity 18 . It was later found that the potential anticancer activity of Spirotryprostatin A was due to its antimitotic properties 19 . Isorhynchophylline is the main alkaloid in Uncaria species which is widely used in Traditional Chinese medicine. Plants from this genus are known for their therapeutic value in the treatment of cardiovascular and central nervous system (CNS) related diseases 20 . Recent studies have shown that isorhynchophylline has potent antihypertensive and neuroprotective activities, and several other activities, including cardiovascular and CNS , e.g. bradycardia, arrhythmia, and sedation, vascular dementia, and amnesia 21-23 . Amongst the spiro compounds, spirobrassinin is a sulfur-containing phytoalexin originally isolated from the daikon Rhaphanus sativus L. var. Hortensis (Cruciferae) 24 . The total synthesis and anticancer properties of this compound have been described 25 . Mitraphylline is an oxindole derivative originally isolated from the leaves of Mitragyna speciosa 26 . The compound was also isolated from the bark of Uncaria tomentosa (Cat's Claw) along with several isomeric alkaloids 27 . Current investigation on this alkaloid focuses on its antiproliferative and cytotoxic effects and its ability to induce apoptosis in human breast cancer, sarcoma as well as lymphoblastic leukaemia cell lines in vivo 28-30 . Elacomine is a hemiterpene spirooxindole alkaloid first isolated from Elaeagnus commutate 31 . The total synthesis was done via a five-step synthetic route from 6-methoxytryptamine, and the synthetic sequence was shown to yield a racemic mixture of isoelacomine and elacomine 32 . A reinvestigation of the alkaloid content of the roots of E. commutata revealed that both elacomine and isoelacomine occur naturally in racemic form 33 . The spirobacillenes are unusual spiro-cyclopentenones identified from the species Lysinibacillus fusiformis KMC003 34 . Park et al. also demonstrated that spirobacillene A shows inhibitory activity against the production of nitric oxide (NO) and reactive oxygen species (ROS) 34 . As for cipargamin (KAE609), the compound recently showed efficacy in Phase II clinical trials in Sub-Saharan Africa in patients presenting with uncomplicated Plasmodium falciparum malaria 35 . The present study focuses on the investigation of the recently described 3’,4’-dihydro-2’H-spiro[indoline-3,1’-isoquinolin]-2-ones (DSIIQs), which were designed as molecular hybrids of two privileged scaffolds, tetrahydroisoquinoline (THIQ) and oxindole (OX), as M pro and spike/ACE2 fusion inhibitors. This investigation aims at the discovery of novel spirooxindoles that target two SARS-CoV-2 viral proteins; the spike/ACE2 fusion that could potentially prevent transmission and those that target the main protease (M pro ), i.e. can prevent viral replication. A compound that hits both targets may present a higher genetic barrier to viral resistance as the two proteins involved would need to simultaneously undergo mutation to produce a drug-resistant strain. Besides, a compound that inhibits multiple aspects of viral replication could have synergistic activities and thus be especially potent and promising to be taken further down the drug discovery pipeline. Materials and methods Test compounds Synthesis of the tested compounds (Fig. 2 ) has been previously described 36–38 . The synthesis of additional spirooxindoles ( 12a, 13l, 14g-14i and 17a-17d) is described below in the Experimental section. General experimental procedure Chemicals were purchased from Sigma-Aldrich Chemicals Company and were used as supplied. All solvents were reagent grade. Where necessary, solvents and starting materials were purified using standard procedures. Solvent removal was carried out under reduced pressure using a Buchi rotary evaporator at temperatures not greater than 60°C. Melting points were measured using a Mel-Temp II apparatus with the use of open capillaries and were uncorrected. The progress of all reactions was monitored by thin layer chromatography (TLC) on aluminum-backed silica gel 60 F254 plates obtained from Sigma-Aldrich; visualization was by UV light at 254 nm or by staining with iodine. Compounds were purified by medium-pressure liquid chromatography over silica gel 60-to-400 mesh, using solvent mixtures that are specified below. Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Avance III spectrometer operating at 600 MHz (H1) and 150 MHz (13C). Spectra were recorded in deuterated solvents and referenced to residual solvent signals. Chemical shifts (δ) were measured in parts per million. Hydrogen and carbon assignments were done using gradient correlation spectroscopy (gCOSY), gradient heteronuclear single quantum correlation (gHSQC) spectroscopy, and heteronuclear multiple bond correlation (gHMBC) techniques. Multiplicities are reported as singlet (s), doublet (d), doublet of doublets (dd), doublet of triplets (dt), triplet (t), triplet of doublets (td) and multiplet (m). Coupling constants (J) are reported in Hertz. For biological evaluation, all compounds were converted to the corresponding hydrochlorides by treatment of the free bases with methanolic HCl. All compounds are greater than 95% pure by high-performance liquid chromatography (HPLC) analysis. Synthesis of compounds Synthesis of 5,7-dibromo-6',7'-dihydroxy-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one (12c). Method A The compound was synthesized via the phenolic Pictet–Spengler reaction reported by Ngo Hanna et al . [ 41 ]. To a solution of 5,7-dibromoisatin (1.5 g, 5.1 mmol) in absolute ethanol (10 ml) was added dopamine (1 g, 5.1 mmol) and triethylamine (1 ml). The reaction mixture was stirred and heated under reflux for 7–10 h, and subsequently concentrated under reduced pressure to remove the solvent. Distilled water was added to the resulting viscous mass and the product which precipitated out was extracted into ethyl acetate (3 × 30 ml). The combined organic extracts were dried over anhydrous sodium sulphate and concentrated to minimum volume. The crude product was further purified by column chromatography (hexane: ethyl acetate—60 : 40). The final product was recrystallized from absolute ethanol. Yield, 1.7 g, 76% (brown solid). M.p. 256–258 o C (HCl salt). 1 H NMR (CD 3 OD, 700 MHz): δ ppm 2.74 (dt, J = 16.1, 4.6 Hz, 1H, H4’a), 2.90 (ddd, J = 15.9, 5.4 Hz, 1H, H4’b), 3.10–3.15 (m, H3’a), 3.71–3.77 (m, H3’b), 5.91 (s, 1H, H8’), 6.62 (s, 1H, H5’), 7.27 (d, J = 1.8 Hz, 1H, H4), 7.64 (d, J = 1.8 Hz, 1H, H6). 13 C NMR (CD 3 OD, 175 MHz): δ ppm 27.2 (C4’), 38.4 (C3’), 64.7 (C3/C1’), 102.8 (C7), 111.9 (C8’), 114.9 (C5), 115.4 (C5’), 123.5 (C8’a), 126.7 (C4), 127.3 (C4’a), 133.6 (C6), 138.7 (C3a), 140.9 (C7a), 143.8 (C7’), 145.0 (C6’), 180.1 (C2). FTMS + cESI : m/z 440.92 M + . General method for the synthesis of 6-methoxy- & 6’,7’-dimethoxy-3’,4’-dihydro-2’ H -spiro[indoline-3,1’-isoquinolin]-2-ones (13l &14g - i). Method B. A mixture of the appropriate isatin (1 equiv), methoxyphenethylamine (1.2 equiv) and polyphosphoric acid (2 g) was heated in an oil bath (bath temperature at 100°C), while stirring mechanically for 5 hours. Upon completion of the reaction, as revealed by TLC, the reaction mixture was allowed to cool to about 50°C and quenched by slow addition of water. To this mixture was added a saturated solution of sodium carbonate to adjust the pH to 11. The floating product obtained was extracted into ethyl acetate (3 × 30 ml). The combined organic extracts were dried over anhydrous sodium sulphate, and concentrated under reduced pressure to obtain the crude product. The latter was purified by flash chromatography on silica gel using suitable solvent systems. Yields ranged between 60 and 98%. 6'-methoxy-5-methyl-3',4'-dihydro-2' H -spiro[indoline-3,1'-isoquinolin]-2-one (13l) Method B. Prepared from 5-methylisatin (2.8 g, 17 mmol), 3-dimethoxyphenethylamine (2.6 g 17 mmol) and polyphosphoric acid (3 g). The crude product was purified by flash chromatography (hexane: ethyl acetate—80: 20). Yield, 4.6 g, 92% (brown solid), M.p. 208–209 o C. 1 H NMR (DMSO-d 6 , 600 MHz): δ ppm 1.46 (s, 3H, 5-C H 3 ), 2.07–2.13 (m, 1H, H4’a), 2.23 (ddd, J = 16.5, 8.7, 5.3 Hz, 1H, H4’b), 2.39 (dt, J = 12.8, 5.2 Hz, 1H, H3’a), 2.96 (d, J = 5.1 Hz, 4H, H3’b, m, 4H, 7’-OC H 3 ), 5.64 (d, J = 8.6 Hz, 1H, H8’), 5.80 (dd, J = 8.6, 2.7 Hz, 1H, H7’), 5.95 (d, J = 2.7 Hz, 1H, H5’), 6.07 (d, J = 7.87 Hz, 1H, H7), 6.14–6.17 (m, 1H, H4), 6.29 (ddd, J = 7.9, 1.7, 0.8 Hz, 1H, H6). 13 C NMR (DMSO-d 6 , 150 MHz): δ ppm 18.9 (5- C H 3 ), 27.6 (C4’), 37.5 (C3’), 53.4 (6’- O C H 3 ), 62.9 (C3/C1’), 108.7 (C7), 111.8 (C7’), 112.6 (C5’), 124.3 (C4), 125.4 (C8’a), 126.4 (C8’), 128.3 (C6), 131.5 (C3a), 134.5 (C7a), 136.4 (C4’a), 138.4 (C5), 158.0 (C6’), 180.3 (C2). FTMS + cESI : m/z 295.14 [M + 1] + . 5,7-dibromo-6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2' H -spiro[indoline-3,1'-isoquinolin]-2-one (14g) Method B. Prepared from 5,7-dibromo-1-(4-methylbenzyl)indoline-2,3-dione (1.8 g, 4.4 mmol), 3,4-dimethoxyphenethylamine (0.8 g, 4.4 mmol) and polyphosphoric acid (2 g). The crude product was purified by flash chromatography (hexane : ethyl acetate—60 : 40). Yield, 1.8 g, 71% (yellow oil), M.p. 239–240 o C (HCl salt). 1 H NMR (DMSO-d 6 , 600 MHz): δ ppm 1.93 (dt, J = 16.2, 4.3 Hz, 1H, H4’a), 2.11 (ddt, J = 15.3, 9.3, 4.9 Hz, 1H, H4’b), 2.27 (ddd, J = 12.7, 5.4, 4.0 Hz, 1H, H3’a), 2.58 (s, 3H, 7’-OC H 3 ), 2.85–2.89 (m, 1H, H3’b), 2.92 (s, 3H, 6’-OC H 3 ), 4.39 (d, J = 16.1 Hz, 1H, C H 2 -Ar ), 4.51 (d, J = 16.3 Hz, 1H, C H 2 -Ar ), 5.00 (s, 1H, H8’), 5.91 (s, 1H, H5’), 6.23 (d, J = 7.8 Hz, 2H, H3”, H5”), 6.30 (m, 2H, H2”, H6”), 6.40 (d, J = 2.0 Hz, 1H, H4), 6.77 (d, J = 1.9 Hz, 1H, H6). 13 C NMR (DMSO-d 6 , 150 MHz): δ ppm 18.8 (4”- C H 3 ), 28.6 (C4’), 37.5 (C3’), 43.0 ( C H 2 -Ar ), 54.2 (7’-O C H 3 ), 54.2 (6’-O C H 3 ), 62.0 (C3/C1’), 101.9 (C7), 107.9 (C8’), 111.6 (C5’), 115.0 (C5), 123.8 (C8’a), 125.5 (C2”, C6”), 126.3 (C4), 128.1 (C3”, C5”), 128.3 (C4’a), 133.5 (C1”), 135.8 (C6), 136.0 (C3a), 138.5 (C4”), 139.0 (C7a), 147.1 (C7’), 148.3 (C6’), 178.3 (C2). FTMS + cESI : m/z 573.02 [M + 1] + . 1-(4-fluorobenzyl)-6',7'-dimethoxy-5-methyl-3',4'-dihydro-2' H -spiro[indoline-3,1'-isoquinolin]-2-one (14h) Method B. Prepared from 5-methyl-1-(4-fluorobenzyl)indoline-2,3-dione (1 g, 3.7 mmol), 3,4 - dimethoxyphenethylamine (0.8 g, 4.4 mmol) and polyphosphoric acid (3 g). The crude product was purified by flash chromatography (hexane: ethyl acetate—60 : 40). Yield, 1.4 g, 90% (brown solid), M.p. 99–101 o C 1 H NMR (DMSO-d 6 , 600 MHz): δ ppm 2.19 (s, 3H, 5-C H 3 ), 2.71 (dt, J = 15.9, 4.1 Hz, 1H, H4’a), 2.88 (ddd, J = 15.1, 9.3, 5.4 Hz, 1H, H4’b), 3.05 (ddd, J = 12.5, 5.4, 4.1 Hz, 1H, H3’a), 3.29(s, 3H, 7’-OC H 3 ), 3.65 (ddd, J = 12.4, 9.3, 4.3 Hz, 1H, H3’b), 3.74 (s, 3H, 6’-OC H 3 ), 4.76 (d, J = 15.6 Hz, 1H, C H 2 -Ar ), 4.96 (d, J = 15.6 Hz, 1H, C H 2 -Ar ), 5.72 (s, 1H, H8’), 6.76 (s, 1H, H5’), 6.92 (dd, J = 4.8, 3.1 Hz, 2H, H4, H7), 7.05 (ddd, J = 8.0, 1.8, 0.9 Hz, 1H, H6), 7.16–7.18 (m, H3”, H5”), 7.42 (dd, J = 8.6, 5.5 Hz, 2H, H2”, H6”). 13 C NMR (DMSO-d 6 , 150 MHz): δ ppm 21.0 (5- C H 3 ), 28.7 (C4’), 38.9 (C3’), 42.2 ( C H 2 -Ar ), 55.8 (7’-O C H 3 ), 56.0 (6’-O C H 3 ), 63.6 (C3/C1’), 109.2 (C7), 109.4 (C8’), 113.0 (C5’), 115.8 (C3”, C5”), 125.5 (C4), 127.2 (C8’a), 129.3 (C6), 129.5 (C4’a), 129.9 (C2”, C6”), 132.2 (C3a), 133.5 (C1”), 135.5 (C5), 140.4 (C7a), 148.5 (C7’), 148.5 (C6’), 161.2 (C4”), 178.8 (C2). FTMS + cESI : m/z 433.19 [M + 1] + . 6',7'-dimethoxy-5-methyl-1-(4-methylbenzyl)-3',4'-dihydro-2' H -spiro[indoline-3,1'-isoquinolin]-2-one (14i) Method B. Prepared from 5-methyl-1-(4-methylbenzyl)indoline-2,3-dione (1 g, 3.8 mmol), 3,4 - dimethoxyphenethylamine (0.82 g, 4.5 mmol) and polyphosphoric acid (2 g). The crude product was purified by flash chromatography (hexane: ethyl acetate—60 : 40). Yield, 0.9 g, 56% (brown solid), M.p. 111–112 o C 1 H NMR (DMSO-d 6 , 600 MHz): δ ppm 2.18 (s, 3H, 5-C H 3 ), 2.26 (s, 3H, 4”-C H 3 ), 2.70 (dt, J = 15.9, 4.2 Hz, 1H, H4’a), 2.87 (ddd, J = 15.2, 9.3, 5.4 Hz, 1H, H4’b), 3.04 (dt, J = 12.5, 4.9 Hz, 1H, H3’a), 3.29(s, 3H, 7’-OC H 3 ), 3.64 (m, 1H, H3’b), 3.73 (s, 3H, 6’-OC H 3 ), 4.69 (d, J = 15.5 Hz, 1H, C H 2 -Ar ), 4.95 (d, J = 15.5 Hz, 1H, C H 2 -Ar ), 5.73 (s, 1H, H8’), 6.75 (s, 1H, H5’), 6.86 (d, J = 8.0 Hz, 1H, H7), 6.90 (d, J = 1.8 Hz, 1H, H4), 7.03 (ddd, J = 8.0, 1.8, 0.9 Hz, 1H, H6), 7.13 (d, J = 7.7 Hz, 2H, H3”, H5”), 7.23–7.27 (m, 2H, H2”, H6”). 13 C NMR (DMSO-d 6 , 150 MHz): δ ppm 21.0 (5- C H 3 ), 21.1 (4”- C H 3 ), 28.7 (C4’), 38.9 (C3’), 42.8 ( C H 2 -Ar ), 55.7 (7’-O C H 3 ), 55.9 (6’-O C H 3 ), 63.5 (C3/C1’), 109.3 (C7), 109.4 (C8’), 112.9 (C5’), 125.4 (C4), 127.3 (C8’a), 127.9 (C2”, C6”), 129.3 (C6), 129.5 (C4’a), 129.6 (C3”, C5”), 132.1 (C3a), 134.2 (C1”), 135.6 (C5), 137.1 (C4”), 140.6 (C7a), 147.5 (C7’), 148.4 (C6’), 178.7 (C2). FTMS + cESI : m/z 429.21 [M + 1] + . General method for the synthesis of 2'-N-arylalkyl-6',7'-dimethoxy-1-(4- substituted benzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one (17a,b). Method C The compounds were prepared from previously the described 36 6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one ( 14e ) ( 1 equiv) and the corresponding 4-substituted benzyl halides (1.4 equiv). An acetonitrile (10 mL) solution of 14e and K 2 CO 3 (2 equiv) was stirred at room temperature for 1 hour. Thereafter, 4-substituted benzyl halide (1.4 equiv) and KI (0.2 equiv) were added and the reaction mixture heated at 80 o C for 2 hours. Upon completion of the reaction, the solvent was removed under reduced pressure, and the resulting viscous mass adjusted to pH 10 by the addition of an aqueous solution of Na 2 CO 3 . The product was extracted into dichloromethane (30 mL x 3), and the combined organic extracts dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash chromatography (hexane:ethyl acetate—70:30). Yields ranged between 65 and 75%. 2'-N-(4-fluorobenzyl)-6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one (17a) Method C. The compound was prepared from 6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one ( 14e) (1 g, 2.4 mmol) and 4-fluorobenzylchloride (0.51 g, 3.5 mmol). The crude product was purified by flash chromatography (hexane : ethyl acetate—70 : 30). Yield, 1 g, 75% (yellow oil). 1 H NMR (DMSO-d 6 , 700 MHz): δ ppm 2.26 (s, 3H, 4”-C H 3 ), 2.71 (dt, J = 15.7, 3.3 Hz, 1H, H4’a), 2.75 (ddd, J = 11.9, 5.4, 2.9 Hz, 1H, H3’a), 2.88 (ddd, J = 16.0, 10.7 5.5, Hz, 1H, H4’b), 3.22 (m, 4H, N1’-C H 2 , 7’-OC H 3 ), 3.29 (d, J = 4.0 Hz, 1H, N1’-C H 2 ), 3.54 (td, J = 11.1, 3.8 Hz, 1H, H3’b), 3.73 (s, 3H, 6’-OC H 3 ), 4.79 (d, J = 15.2 Hz, 1H, N1-C H 2 ), 5.01 (d, J = 15.2 Hz, 1H, N1-C H 2 ), 5.69 (s, 1H, H8’), 6.77 (s, 1H, H5’), 7.05 (td, J = 7.52, 1.0 Hz, 1H, H5), 7.09–7.12 (m, 3H, H7, H3”’, H5”’), 7.19 (dd, J = 7.4, 1.3 Hz, 1H, H3”, H5”), 7.29 (ddd, J = 8.5, 3.9, 2.6 Hz, 3H, H6, H2”’, H6”’), 7.30–7.32 (m, 2H, H2”, H6”). 13 C NMR (DMSO-d 6 , 175 MHz): δ ppm 21.0 (4”- C H 3 ), 28.9 (C4’), 42.7 (C3’), 42.9 (N1- C H 2 ), 54.0 (N1’- C H 2 ), 55.6 (7’-O C H 3 ), 55.9 (6’-O C H 3 ), 68.9 (C3/C1’), 109.7 (C8’), 109.8 (C7), 112.4 (C5’), 115.4 (2C, C3”’, C5”’), 123.8 (C5), 124.7 (C4), 126.7 (C8’a), 128.2 (2C, C2”, C6”), 128.5 (C4’a), 129.6 (C6), 129.7 (2C, C3”, C5”), 130.2 (2C, C2”’, C6”’), 133.5 (C3a), 134.2 (C1”), 135.5 (C1”’), 137.4 (C4”), 143.7 (C7a), 147.5 (C7’), 148.5 (C6’), 161.1 (C4”’), 177.2 (C2). FTMS + cESI : m/z 523.24 [M + 1] + . 6',7'-dimethoxy-1,2'-bis(4-methylbenzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one (17b) Method C. The target compound was prepared from 6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one 14e ( 1 g, 2.4 mmol) and 4-methylbenzylchloride (0.51 g, 3.6 mmol). The crude product was purified by flash chromatography (hexane: ethyl acetate—70: 30). Yield, 0.8 g, 65% (yellow oil). 1 H NMR (DMSO-d 6 , 700 MHz): δ ppm 2.25 (s, 6H, 4”-C H 3 , 4”’-C H3 ), 2.69(dt, J = 15.8, 3.4 Hz, 1H, H4’a), 2.75 (dt, J = 5.7, 3.0 Hz, 1H, H3’a), 2.86 (ddd, J = 16.0, 10.7 5.6, Hz, 1H, H4’b), 3.21 (m, 5H, N2’-C H 2 , 7’-OC H 3 ), 3.51–3.55 (m, 1H, H3’b), 3.72 (s, 3H, 6’-OC H 3 ), 4.79 (d, J = 15.2 Hz, 1H, N1-C H 2 ), 5.01 (d, J = 15.3 Hz, 1H, N1-C H 2 ), 5.70 (s, 1H, H8’), 6.76 (s, 1H, H5’), 7.05 (td, J = 7.5, 1.0 Hz, 1H, H5), 7.09 (t, J = 8.0 Hz, 3H, H7, H3”’, H5”’), 7.11–7.15 (m, 4H, H3”, H5”, H2”’, H6”’), 7.18–7.20 (m, 1H, H4), 7.29 (td, J = 7.7, 1.3 Hz, 1H, H6), 7.30–7.33 (m, 2H, H2”’, H6”’). 13 C NMR (DMSO-d 6 , 175 MHz): δ ppm 21.0 (2C, 4”- C H 3, 4”’- C H 3, ), 28.9 (C4’), 42.5 (C3’), 42.9 (N1- C H 2 ), 54.5 (N2’- C H 2 ), 55.6 (7’-O C H 3 ), 55.9 (6’-O C H 3 ), 68.9 (C3/C1’), 109.7 (C8’), 109.8 (C7), 112.4 (C5’), 123.8 (C5), 124.7 (C4), 126.7 (C8’a), 127.0 (C4’a), 128.2 (2C, C2”, C6”), 128.4 (2C, C3”’, C5”’), 129.1 (C6), 129.3 (2C, C2”’, C6”’), 129.6 (C4”’), 129.7 (2C, C3”, C5”), 133.6 (C3a), 134.2 (C1”), 136.6 (C1”’), 137.4 (C4”), 143.7 (C7a), 147.5 (C7’), 148.5 (C6’), 177.2 (C2). FTMS + cESI : m/z 519.26 [M + 1] + . Synthesis of N -ethyl-6',7'-dimethoxy-1-(4-methylbenzyl)-2-oxo-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinoline]-2'-carboxamide (17c) (Method D) The target compound was prepared from the previously described 6’,7’-dimethoxy-1-(4-methylbenzyl)-3’,4’-dihydro-2’H-spiro[indoline-3,1’-isoquinolin]-2-one 14e ( 1 g, 2.4 mmol) and ethylisocyanate (0.21 g, 0.23 mL, 2.9 mmol, 1.2 eq). An acetonitrile solution of 14e and ethyl isocyanate was heated to 60 o C for 2 hours. Upon completion of the reaction, the mixture was allowed to cool to room temperature, made basic by slow addition of aqueous sodium bicarbonate to pH 10. The product was extracted into ethyl acetate (30 mL x 2), and the combined organic extracts dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by flash chromatography (hexane: ethyl acetate—70:30). Yield, 0.6 g, 50% (white solid). M.p. 193–194 o C. 1 H NMR (DMSO-d 6 , 700 MHz): δ ppm 0.96 (t, J = 7.17 Hz, 3H, N1”’-CH 2 C H 3 ), 2.27 (s, 3H, 4”-C H 3 ), 2.90 (ddd, J = 15.4, 4.8, 3.4 Hz, 1H, H4’a), 2.92–3.01 (m, 3H, 1H, H4’b, N1”’-C H 2 CH 3 ), 3.11 (s, 3H, 7’-OC H 3 ), 3.55–3.60 (m, 1H, H3’a), 3.72 (s, 3H, 6’-OC H 3 ), 3.97 (td, J = 12.2, 4.6 Hz, 1H, H3’b), 4.62 (d, J = 15.5 Hz, 1H, N1-C H 2 ), 4.96 (d, J = 15.5 Hz, 1H, N1-C H 2 ), 5.76 (s, 1H, H8’), 6.84 (s, 1H, H5’), 6.89 (td, J = 7.5, 1.0 Hz, 1H, H5), 6.93 (dt, J = 7.9, 0.7 Hz, 1H, H7), 7.03 (dd, J = 7.3, 1.25 Hz, 1H, H4), 7.10–7.13 (d, J = 7.8 Hz, 2H, H3”, H5”), 7.17 (td, J = 7.7, 1.3 Hz, 1H, H6), 7.31–7.34 (m, 2H, H2”, H6”). 13 C NMR (DMSO-d 6 , 175 MHz): δ ppm 15.8 (N1”’-CH 2 C H 3 ), 21.1 (4”- C H 3 ), 30.0 (C4’), 35.4 (N1”’- C H 2 CH 3 ), 42.2 (C3’), 43.4 (N1- C H 2 ), 55.4 (7’-O C H 3 ), 55.9 (6’-O C H 3 ), 65.5 (C3/C1’), 108.9 (C7), 109.1 (C8’), 112.3 (C5’), 122.3 (C4), 122.7 (C5), 126.5 (C8’a), 128.2 (C4’a), 128.3 (2C, C2”, C6”), 128.9 (C6), 129.5 (2C, C3”, C5”), 134.5 (C1”), 135.7 (C3a)137.0 (C4”), 143.5 (C7a), 147.7 (C7’), 148.3 (C6’), 156.7 (C2”’), 177.3 (C2). FTMS + cESI : m/z 486.24 [M + 1] + . Synthesis 6',7'-dimethoxy-2'-methyl-1-(4-methylbenzyl)-3',4'-dihydro-2' H -spiro[indoline-3,1'-isoquinolin]-2-one (17d) (Method E) This compound was prepared from previously synthesized 6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2' H -spiro[indoline-3,1'-isoquinolin]-2-one ( 14e ) 36 ( 1 g, 2.4 mmol) and formaldehyde (0.3 mL of 37% formalin, 3.6 mmol, 1.5 eq). To a formic acid solution of 14e formaldehyde was added dropwise. The resulting mixture was heated at 60 oC for 3 hours, allowed to cool to room temperature, and made basic by slowly adding 2 M aqueous sodium hydroxide. The product was extracted into ethyl acetate (30 mL x 3), and the combined organic extracts dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by flash chromatography (hexane: ethyl acetate—50: 50). Yield, 0.8g, 78% (yellow oil). 1 H NMR (DMSO-d 6 , 700 MHz): δ ppm 2.06 (s, 3H, N2’-C H 3 ), 2.26 (s, 3H, 4”-C H 3 ), 2.78 (dt, J = 15.8, 3.6 Hz, 1H, H4’a), 2.88 (ddd, J = 11.3, 5.7, 2.9 Hz, 1H, H3’a), 3.03 (ddd, J = 16.0, 10.5, 5.6 Hz, 1H, 1H, H4’b), 3.22 (s, 3H, 7’-OC H 3 ), 3.63 (td, J = 10.9, 4.1 Hz, 1H, H3’b), 3.73 (s, 3H, 6’-OC H 3 ), 4.77 (d, J = 15.4 Hz, 1H, N1-C H 2 ), 4.97 (d, J = 15.5 Hz, 1H, N1-C H 2 ), 5.65 (s, 1H, H8’), 6.77 (s, 1H, H5’), 6.99–7.01 (m, 2H, H4, H5), 7.06 (d, J = 7.9 Hz, 1H, H7), 7.14 (d, J = 7.8 Hz, 2H, H3”, H5”), 7.26–7.29 (m, 3H, H6, H2”, H6”). 13 C NMR (DMSO-d 6 , 175 MHz): δ ppm 21.0 (4”- C H 3 ), 28.7 (C4’), 39.6 (N2’- C H 3 ), 42.7 (N1- C H 2 ), 46.9 (C3’), 55.6 (7’-O C H 3 ), 55.9 (6’-O C H 3 ), 69.0 (C3/C1’), 109.6 (C8’), 109.6 (C7), 112.4 (C5’), 123.5 (C5), 124.8 (C4), 126.7 (C8’a), 128.0 (C4’a), 128.1 (2C, C2”, C6”), 129.4 (2C, C3”, C5”), 129.7 (C6), 133.2 (C3a), 134.2 (C1”), 137.3 (C4”), 143.5 (C7a), 147.4 (C7’), 148.5 (C6’), 177.3 (C2). FTMS + cESI : m/z 429.21 [M + 1] + . Description of biological screening procedures AlphaScreen assay SARS-CoV-2 spike-RBD binding to ACE2 was determined using AlphaScreen technology-based assay as described previously 42 . For RBD-ACE2 assays, 2 nM of ACE2-Fc (Sino Biological, Chesterbrook, PA, USA) was incubated with 5 nM HIS-tagged SARS-CoV-2 Spike-RBDs representing the parental USA-WA/2020 (“Wild-type” (WT)) sequence (SinoBiological) in the presence of 5 µg/mL nickel chelate donor bead in a total of 10 µL of 20 mM Tris (pH 7.4), 150 mM KCl, and 0.05% CHAPS. Test compounds were diluted to 100x final concentration in DMSO. 5 µL of ACE2-Fc/Protein A acceptor bead was first added to the reaction, followed by 100 nL test compound and then 5 µL of RBD-HIS/Nickel chelates donor beads. All conditions were performed in duplicate. Following incubation at room temperature for 2 hours, luminescence signals were measured using a ClarioStar plate reader (BMC Labtech, Cary, NC, USA). Data were then normalised to percent inhibition, where 100% equalled the AlphaScreen signal in the absence of RBD-HIS, and 0% denoted AlphaScreen signal in the presence of both protein and DMSO vehicle control. To measure PD1/PD-L1 binding, 0.5 nM of human PD-L1-Fc (Sino Biological) was incubated with 5 nM HIS-tagged human PD1 (Sino Biological) in the presence of 5 µg/mL protein A and 5 µg/mL nickel chelate donor beads in a total volume of 10 µL of 20 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% Tween-20. Proteins and test agents were then added, incubated, and analysed as described above. M pro inhibition assay The M pro inhibition assay was done using the fluorogenic assay as described previously 42 . Firstly, 5 µl of 25 nM M pro diluted in assay buffer (25 mM HEPES [pH 7.4]), 150 mM NaCl, 5 mM DTT, and 0.005% Tween) was dispensed into black, low-volume, 384-well plates. Test compounds were serially diluted into 100% DMSO, and 0.1 ml was added to the assay using a Janus MDT Nanohead tool (PerkinElmer). Assays were initiated by addition of 5 µl of 5 µM fluorogenic substrate, and fluorescence at 355 nm excitation and 460 nm emission was monitored every 5 min for 50 min using an Envision plate reader (PerkinElmer). The rate of substrate cleavage was determined using linear regression of the raw data values obtained during the time course. The slopes of these progress curves were then normalized to percentage inhibition, where 100% equaled the rate in the absence of M pro (which was typically 0), and 0% equaled the rate of cleavage in the presence of M pro and 0.1% DMSO. Molecular modeling procedures Target proteins for docking Molecular modeling protocols were performed as previously reported 43–47 . The protein structures (ID: 6M0J) for SARS-CoV-2 spike/ACE2 and (PDB ID: 6W63) for M pro , respectively, corresponding to the Wuhan strain were retrieved from the Protein Data Bank (PDB) 48–50 and used for the entire study. Protein preparation All water molecules were deleted using the Molecular Operating Environment (MOE) software 51 . The Protein Preparation Wizard integrated in the Schrödinger package software 52–53 was used to prepare the proteins by adding the missing hydrogen bonds, assigning bond orders and filling the missing side chains using PRIME. After this the protein structures were energy minimized to reduce atomic clashes and optimized their interactions with the ligands during docking. From the Schrödinger software, the commercialized maestro package’s Epik-tool was used to predict the protonation states at a pH of 7.0 54 . Finally a restrained energy minimization step was carried out using the Optimized Potentials for Liquid Simulations 2005 (OPLS2005) forcefield 55 on both proteins. During the protein optimization step, the root mean square deviation (RMSD) of the displacement of the atoms was set to end with the minimization at 0.3 Å. Ligand preparation The MOE 51 builder module was used to generate the 3D models of the library of synthesized spirooxindoles. For consistency, only the R stereoisomers were prepared for docking, as these addressed the voluminous hydrophobic regions in the ACE2 site more appropriately during trial docking. The generated 3D structures were then energy minimized using the MMFF94 force field 56–60 . The ligands were further prepared for docking using the LigPrep tool to generate all the plausible tautomers of each ligand as implemented in Schrödinger’s Maestro software package 54 . Using the incorporated OPLS2005 force field 55 , the spirooxindole 3D structure library was further energy minimized. The ConfGen tool (implemented in the Schrödinger package) was then used to compute 60 conformers per ligand in the 3D library, by setting all other options to default except for the minimization of the output 61 . Docking and scoring Docking was carried out using the Glide program incorporated in the Maestro package distributed by Schrödinger 52–53 as shown in our recent publications 43–47 , with some modifications. Docking validation results on this protein have already been reported in our previously reported studies 45–47 . After the protein preparation phase, a docking grid box was generated for the spike/ACE2 complex to investigate how the ligands will bind around the following amino acid residues; Asp597, Thr598, Lys516, Val321, Gln121, Lys578, Ala283, Ser91, Asn746, Gln68, Pro744, Glu518 and Thr610. The co-crystalized ligand (X77) was used as the centroid to generate the docking grid box for M pro as seen in our recently published work 62 . The ligand size for each of these grid boxes, which is the area where all the generated 3D structures were docked, was set to a maximum ligand size of 36 Å. While writing 10 poses per ligand conformer, 20 poses were included for each ligand conformer and taking into consideration the input of ring conformation, all other settings were allowed to default. The outputs were scored using standard precision (SP) GlideScore as the scoring function 63 . Selection of binding modes After the extraction of the results and the computation of carefully selected descriptors, the specific area ligands bound with the protein in the receptor binding domain (RBD) of both the Spike/ACE2 and M pro , the binding modes and the residues taking part in the interaction during binding were observed using MOE 51 . Browsing through the docking results and establishing the ligand interactions of each docked protein-ligand complex made it possible to establish structure-activity relationships (SAR) in the RBD in both cases and to identify some ligand moieties important for activity and selectivity. The ligands in both protein RBD were then superimposed to highlight their preferred binding modes. Results and discussion Activities in the AlphaScreen assay and M pro inhibitory assay For the synthesized compounds, the 50% inhibitory concentrations (IC 50 values) for spike/ACE2 binding (AlphaScreen) and inhibition of M pro are shown in Table 1 , alongside the best docking score for each ligand. The cut-off concentrations to distinguish between active, moderately active, and inactive ligands for SARS-CoV-2 enzymatic assays were adopted from recent literature 64 and are summarized in Table 2 . Table 1 Biological assay results and docking results for spike/ACE2 and M pro . Compound ID spike/ACE2 M pro IC 50 (µM) Glide SP Score IC 50 (µM) Glide SP Score 10d 20.4 -6.75 > 100 -7.72 10f 9.7 -6.42 23.3 -6.89 10g 14.7 -6.52 69.0 -7.79 10h 9.8 -6.73 > 100 -8.15 10j 7.4 -7.12 > 100 -6.91 10k 35.2 -5.89 > 100 -7.40 10l 6.1 -6.15 > 100 -7.38 11a 71.3 -6.58 > 100 -6.90 11b > 100 -6.10 > 100 -7.16 11c > 100 -5.36 > 100 -6.21 11d 70.8 -6.68 > 100 -7.69 11e 15.7 -6.56 > 100 -6.93 11f 28.6 -6.38 > 100 -6.88 11g 10.2 -6.42 > 100 -7.05 11h 21.6 -6.72 > 100 -7.03 11i 20.5 -6.96 > 100 -7.54 11j 3.6 -7.07 > 100 -7.31 11k 20.6 -6.25 > 100 -7.31 11l 8.2 -6.72 > 100 -7.43 11m 7.9 -5.93 > 100 -7.45 12a 10.3 -6.61 > 100 -7.19 12b 8.7 -6.06 24.9 -6.89 12c 9.0 -5.39 35.1 -6.75 13a > 100 -6.18 > 100 -6.71 13b > 100 -5.86 > 100 -6.88 13c 35.8 -5.30 58.8 -6.76 13d 33.9 -6.07 > 100 -7.68 13e 53.97 -6.64 > 100 -7.00 13k > 100 -6.59 > 100 -7.78 13l 7.9 -5.69 > 100 -6.92 14a > 100 -5.80 > 100 -6.77 14b > 100 -5.79 > 100 -6.89 14c > 100 -5.08 > 100 -6.70 14d 22.4 -6.53 > 100 -7.35 14e 45.4 -6.53 > 100 -7.06 14f 9.4 -5.27 > 100 -6.66 14g 13.1 -6.43 > 100 -7.37 14h 5.6 -5.90 > 100 -7.48 14i 14.8 -6.33 > 100 -7.11 14j 6.7 -6.74 30.5 -7.39 14k 18.5 -5.17 66.7 -6.22 14m 17.8 -5.46 29.9 -6.86 14n 44.7 -5.53 > 100 -7.06 15a > 100 -5.00 > 100 -6.73 15b 12.4 -4.78 > 100 -7.06 15c 8.4 -5.09 > 100 -6.89 15d 44.7 -5.12 > 100 -7.06 15e 49.6 -5.37 > 100 -6.99 16a > 100 -5.16 > 100 -6.95 16b 9.9 -4.74 > 100 -6.28 17a 39.5 -5.51 > 100 -8.00 17b 63.0 -5.42 > 100 -7.65 17c > 100 -5.44 > 100 -7.44 17d 50.3 -5.51 > 100 -7.20 17e 17.4 -5.61 > 100 -6.64 18a > 100 -5.31 > 100 -6.52 18b 12.6 -6.21 > 100 -7.06 19a > 100 -4.78 > 100 -6.34 19b 10.6 -5.64 > 100 -7.12 GC-376 (control) 0.0031 -11.61 Table 2 Standard acceptable cut-off activity values for both SARS-CoV-2 enzymes as defined in the literature 64 . Enzymatic assays (spike/ACE, M pro ) Active Moderately active Inactive Cut-off concentration IC 50 < 10 µM 10 µM < IC 50 20 µM On this basis, the ligands were classified into categories A (active, with IC 50 < 10 µM), B with (moderately active, with 10 µM < IC 50 20 µM) for the spike/ACE2 assay. All tested compounds were inactive in the M pro assay. The classification of the ligands into categories A to C is shown in Table S1 (Supplementary Data). Of the 60 tested spirooxindoles, 15 fell under category A including 10f , 10h , 10j , 10l , 11j , 11l , 11m , 12b , 12c , 14f , 14h , 14j , 15c , 18c , and 18d , the most active compound being 11j (IC 50 = 3.6 µM). There were 11 compounds in category B, which include 10g , 10k , 11e , 11g , 12a , 14g , 14i , 15b , 17a , 17f , 18a , and 18e . The remaining compounds were inactive (category C). We could further identify a subset of non-selective compounds in categories A and B (referred to as A’ and B’, respectively), which we could define as active compounds and moderately active compounds against spike/ACE2, which could contain some pharmacophore features required for binding to M pro . These are compounds that could be slightly modified to derive dual inhibitors of spike/ACE2 and M pro . Category A’ includes compounds 10f , 12b , 12c , and 14j , while category B’ includes compounds 10g , 18a , and 18e . Our discussion of the structure-activity relationships will focus on the common features of compounds in categories A, A’, B, and B’ which are absent from category C and vice versa. Although there was no correlation between the activities of the compounds and their docking scores towards the spike/ACE2 site, the orientations of the top-scoring poses could carefully explain the structure-activity relations. It was observed that the most active compound ( 11j , IC 50 = 3.60 µM) interacted with Arg375 via the N-H of the isoquinoline moiety (Fig. 4 ), while the naphthyl group makes several arene-H interactions with Asp332. Although these amino acids make similar interactions with almost all the actives, compound 11j distinguishes itself by the strong hydrophobic interactions resulting from the interaction field produced by the naphthyl moiety. This matches with the strong hydrophobic patch created by the amino acids Phe22, Ser26, Leu333, and Ile361 (shown in Figs. 4 B and 4 C), which is an indication that the activity of this compound could be driven by the strong hydrophobic interactions between the naphthyl moiety and this patch. This suggests that more active compounds could be designed and synthesized by introducing other hydrophobic groups around the naphthyl (F, CH 3 , Cl, CF 3 , Br, etc.) moiety, a feature which is conspicuously absent from the moderately active and inactive compounds. The 8-hydroxy isomer of the most active compound ( 11j ), i.e. compound 10j was shown to be about twofold less active (IC 50 = 7.4 µM). A superposition of the two isomers has been shown in the spike/ACE2 pocket in Fig. 5 . While the 6-hydroxy group in compound 11j is free to make H-bond interactions with the protein backbone, this possibility is hindered in compound 10j , which rather forms intramolecular H-bonding with the carbonyl of the oxindole moiety. This could explain the observed activity of compound 11j compared with compound 10j . The top-scoring poses of the rest of the two molecules show almost perfect superposition (Fig. 5 ). Conclusion To the best of our knowledge, this is the first report that shows that spirooxindoles have activity against SARS-CoV-2 spike/ACE2 binding. Nine newly reported and fifty already published 36–38 spirooxindoles, synthesized by Pictet-Spengler cyclodehydration, were screened against both spike/ACE2 binding and M pro inhibition. While all IC 50 values against M pro were shown to be > 20 µM, it was shown that 15 compounds had IC 50 < 10 µM in the spike/ACE2 assay, 11 compounds were shown to be moderately active, while the rest were inactive. Molecular docking and evaluation of the structure-activity relationship showed that H-bonding between the isoquinoline moiety and the Arg375/Asn376 pair was required for activity. Besides, the presence of a bulky hydrophobic moiety attached to the oxindole is important for activity by potentially forming π-π stacking with Trp331, arene-H interactions with Asp332, and the strong hydrophobic interactions with the patch created by the amino acids Phe22, Ser26, Leu333, and Ile361. It would be necessary to further design new naphthyl-based analogues with hydrophobic substituents that address this region of the binding pocket to improve the activity against SARS-CoV-2 spike/ACE2 binding. Declarations Funding We acknowledge financial support from the Bill & Melinda Gates Foundation through the Calestous Juma Science Leadership Fellowship awarded to Fidele Ntie-Kang (grant award number: INV-036848 to University of Buea). FNK also acknowledges joint funding from the Bill & Melinda Gates Foundation and LifeArc (award number: INV-055897 and Grant ID: 10646) under the African Drug Discovery Accelerator program. FNK acknowledges further funding from the Alexander von Humboldt Foundation for a Research Group Linkage project. Acknowledgment We acknowledge the technical support of Mr. Cyril T. Namba-Nzanguim. CRediT authorship contribution statement Albert Enama Ehinak: Conceptualization, Methodology, Data curation, Formal analysis, Investigation, Writing – original draft. Maloba M.M. Lobe: Conceptualization, Methodology, Data curation, Formal analysis, Investigation, Writing – original draft. Conrad V. Simoben: Conceptualization, Formal analysis, Writing – review & editing. Ian Tietjen: Conceptualization, Funding acquisition, Investigation, Methodology, Writing – review & editing. Joel Cassel: Conceptualization, Investigation, Methodology, Writing – review & editing. Joseph M. Salvino: Funding acquisition, Investigation, Methodology, Supervision, Writing – review & editing. Luis J. Montaner: Funding acquisition, Investigation, Methodology, Supervision, Writing – review & editing. Wolfgang Sippl: Conceptualization, Formal analysis, Supervision, Writing – review & editing. Simon M. N. Efange: Conceptualization, Formal analysis, Supervision, Writing – review & editing. Fidele Ntie-Kang: Funding acquisition, Investigation, Methodology, Supervision, Writing – original draft, Writing – review & editing. Declaration of competing interest The authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Data availability All data generated or analysed during this study are included in this published article and its supplementary information files. References Pinto, G. P. et al. Screening of world approved drugs against highly dynamical spike glycoprotein of SARS-CoV-2 using CaverDock and machine learning. Comput. Struct. Biotechnol. J. 19, 3187–3197 (2021). https://doi.org/10.1016/j.csbj.2021.05.043 Wrobel, A. G. et al. 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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-4535655","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":317040716,"identity":"662aead5-7b45-455a-81dc-357db4a26242","order_by":0,"name":"Albert Enama Ehinak","email":"","orcid":"","institution":"University of Buea","correspondingAuthor":false,"prefix":"","firstName":"Albert","middleName":"Enama","lastName":"Ehinak","suffix":""},{"id":317040717,"identity":"9847c7ec-392d-4e48-b696-76661583ed4c","order_by":1,"name":"Maloba M. M. 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The active compounds are depicted in stick representation, while amino acid residues are shown in line representation. H-bonds are shown as grey broken lines and atoms take their usual colours (e.g. grey for C, red for O, blue for N, etc). H-atoms are omitted for the sake of clarity and the figure is generated using MOE (Chemical Computing Group, 2016).\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-4535655/v1/ca9d47fe7f037295af4b268a.png"},{"id":58829282,"identity":"feecfd5b-6c08-4015-90a1-53e08d60273b","added_by":"auto","created_at":"2024-06-21 17:34:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":881464,"visible":true,"origin":"","legend":"\u003cp\u003eProtein-ligand interactions of the most active of the synthesized siprooxindoles \u003cstrong\u003e11j\u003c/strong\u003e; (A) a 2D representation showing the H-bonding with Arg375 and Arene-H interactions with Asp332, (B) a 3D representation cast against the background of the molecular surface showing hydrophobic regions in grey, polar regions in blue and mildly polar regions in cyan, (C) a 3D representation cast against the background of the van der Waals surface highlighting the amino acid residues that form the hydrophobic patch.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-4535655/v1/81888f477b445d17c103e846.png"},{"id":58829610,"identity":"b1e08bab-4d84-4d85-8abb-f3d198bf58ae","added_by":"auto","created_at":"2024-06-21 17:35:09","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1340735,"visible":true,"origin":"","legend":"\u003cp\u003eA superposition of the isomers (compounds \u003cstrong\u003e10j\u003c/strong\u003e and \u003cstrong\u003e11j\u003c/strong\u003e) in the spike/ACE2 pocket, showing protein-ligand interactions with Arg375 and Asp332, with Asn376 in the background of the molecular surface. The colour coding of the molecular surface is as in Fig. 4B, while inter-molecular H-bonding is shown in grey broken lines and intra-molecular H-binding is shown in yellow broken lines. Both ligands are shown in stick representation and C-atoms of compound \u003cstrong\u003e10j\u003c/strong\u003e are shown in green, while those of compound \u003cstrong\u003e11j \u003c/strong\u003eare shown in grey.\u003c/p\u003e","description":"","filename":"Fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-4535655/v1/620deebd61f8736db856f168.png"},{"id":59400537,"identity":"45afac97-be6b-4660-bd0f-423cbbda99ce","added_by":"auto","created_at":"2024-07-01 10:08:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4900310,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4535655/v1/1ae4f35a-1f52-4b56-937e-37072354a2aa.pdf"},{"id":58829716,"identity":"9d26890d-0955-44f5-a9a6-731d73bed418","added_by":"auto","created_at":"2024-06-21 17:35:20","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":3101526,"visible":true,"origin":"","legend":"","description":"","filename":"SpirooxindoleSuppl.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4535655/v1/0c343171080c31e5b2097e87.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"An evaluation of spirooxindoles as blocking agents of SARS-CoV-2 spike/ACE2 fusion and M pro inhibitory agents: Synthesis, biological evaluation and computational analysis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent of coronavirus disease 19 (COVID-19), has emerged as a very important public health concern\u003csup\u003e1\u003c/sup\u003e. Since its outbreak in December 2019, in Wuhan city in Hubei province of China, the disease has resulted in significant global morbidity and mortality with over 680 million confirmed cases and 6,805,012 deaths\u003csup\u003e2,3\u003c/sup\u003e. Recent reports have identified four structural proteins of SARS-CoV-2 as potential targets for drug development. These structural proteins include the spike, envelope, nucleocapsid, and membrane proteins\u003csup\u003e4\u003c/sup\u003e. Based on established knowledge about the virus, research groups have focused their efforts on two viral proteins: i) the spike (s)-glycoprotein\u003csup\u003e5\u003c/sup\u003e, to disrupt its recognition of the membrane-bound angiotensin-converting enzyme 2 (ACE2), thereby hindering the interaction of the receptor binding domain (RBD) located at the S1 subunit of the spike protein and the angiotensin-converting enzyme II (ACE2) which is responsible for cellular entry\u003csup\u003e6\u003c/sup\u003e; and ii) the main viral protease (M\u003csup\u003epro\u003c/sup\u003e, 3CL\u003csup\u003epro\u003c/sup\u003e), which is one of two viral proteases responsible for cleaving viral polypeptides to generate mature proteins, to inhibit viral replication\u003csup\u003e7\u003c/sup\u003e. \u0026nbsp;SARS-CoV-2 main protease is a critical protein that plays a vital role in the replication cycle of the SARS-CoV-2 virus, and its inhibition is a promising strategy for the development of antiviral therapies\u003csup\u003e8\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eOne promising approach to drug discovery is that which takes advantage of privileged scaffolds. According to Evans \u003cem\u003eet al.\u003c/em\u003e, a privileged scaffold is a molecular framework that has high affinity for a diverse array of receptors\u003csup\u003e9\u003c/sup\u003e. Thus, privileged scaffolds are often used as a starting point in drug discovery; the combination of two or more of such privileged scaffolds, called molecular hybridization, has been highly exploited in drug discovery\u003csup\u003e10\u003c/sup\u003e. Spirocyclic compounds, especially spirooxindoles, have been widely studied in recent years because of their unique three-dimensional structures and broad range of biological activities. Both synthetic and naturally occurring spirooxindoles (Figure 1) display diverse pharmacological properties such as anticancer\u003csup\u003e11\u003c/sup\u003e, antimicrobial\u003csup\u003e12\u003c/sup\u003e, anti-inflammatory\u003csup\u003e12\u003c/sup\u003e, antimalarial\u003csup\u003e13\u003c/sup\u003e, antiviral\u003csup\u003e14\u003c/sup\u003e, antidiabetic\u003csup\u003e15\u003c/sup\u003e, and antioxidant activity\u003csup\u003e16\u003c/sup\u003e. Spirotryprostatin A, for example, is an indole alkaloid of the 2,5-diketopiperazine class of natural products that was first identified by Cui \u003cem\u003eet al\u003c/em\u003e. from the fungal species \u003cem\u003eAspergillus fumigatus\u003c/em\u003e as a mammalian cell cycle inhibitor\u003csup\u003e17\u003c/sup\u003e. When tested in an anti-\u003cem\u003eTrypanosoma\u003c/em\u003e assay, Spirotryprostatin A was inactive, although its semi-synthetic analogues of the diketopiperazine class showed antitrypanosomal activity\u003csup\u003e18\u003c/sup\u003e. It was later found that the potential anticancer activity of Spirotryprostatin A was due to its antimitotic properties\u003csup\u003e19\u003c/sup\u003e. Isorhynchophylline is the main alkaloid in \u003cem\u003eUncaria\u003c/em\u003e species which is widely used in Traditional Chinese medicine. Plants from this genus are known for their therapeutic value in the treatment of cardiovascular and central nervous system (CNS) related diseases\u003csup\u003e20\u003c/sup\u003e. Recent studies have shown that isorhynchophylline has potent antihypertensive and neuroprotective activities, and several other activities, including cardiovascular and CNS , e.g. bradycardia, arrhythmia, and sedation, vascular dementia, and amnesia\u003csup\u003e21-23\u003c/sup\u003e. Amongst the spiro compounds, spirobrassinin is a sulfur-containing phytoalexin originally isolated from the daikon \u003cem\u003eRhaphanus sativus\u003c/em\u003e L. var. Hortensis (Cruciferae)\u003csup\u003e24\u003c/sup\u003e. The total synthesis and anticancer properties of this compound have been described\u003csup\u003e25\u003c/sup\u003e. Mitraphylline is an oxindole derivative originally isolated from the leaves of \u0026nbsp;\u003cem\u003eMitragyna speciosa\u003c/em\u003e\u003csup\u003e26\u003c/sup\u003e. \u0026nbsp;The compound was also isolated from the bark of \u003cem\u003eUncaria tomentosa\u003c/em\u003e (Cat\u0026apos;s Claw) along with several isomeric alkaloids\u003csup\u003e27\u003c/sup\u003e. Current investigation on this alkaloid focuses on its antiproliferative and cytotoxic effects and its ability to induce apoptosis in human breast cancer, sarcoma as well as lymphoblastic leukaemia cell lines \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e28-30\u003c/sup\u003e. Elacomine is a hemiterpene spirooxindole alkaloid first isolated from \u003cem\u003eElaeagnus commutate\u003c/em\u003e\u003csup\u003e31\u003c/sup\u003e. The total synthesis was done \u003cem\u003evia\u003c/em\u003e a five-step synthetic route from 6-methoxytryptamine, and the synthetic sequence was shown to yield a racemic mixture of isoelacomine and elacomine\u003csup\u003e32\u003c/sup\u003e. A reinvestigation of the alkaloid content of the roots of \u003cem\u003eE. commutata\u003c/em\u003e revealed that both elacomine and isoelacomine occur naturally in racemic form\u003csup\u003e33\u003c/sup\u003e. The spirobacillenes are unusual spiro-cyclopentenones identified from the species \u003cem\u003eLysinibacillus fusiformis\u0026nbsp;\u003c/em\u003eKMC003\u003csup\u003e34\u003c/sup\u003e. Park \u003cem\u003eet al.\u003c/em\u003e also demonstrated that spirobacillene A shows inhibitory activity against the production of nitric oxide (NO) and reactive oxygen species (ROS)\u003csup\u003e34\u003c/sup\u003e. As for cipargamin (KAE609), the compound recently showed efficacy in Phase II clinical trials in Sub-Saharan Africa in patients presenting with uncomplicated \u003cem\u003ePlasmodium falciparum\u003c/em\u003e malaria\u003csup\u003e35\u003c/sup\u003e.\u003c/p\u003e\n\u003cp\u003eThe present study focuses on the investigation of the recently described 3\u0026rsquo;,4\u0026rsquo;-dihydro-2\u0026rsquo;H-spiro[indoline-3,1\u0026rsquo;-isoquinolin]-2-ones (DSIIQs), which were designed as molecular hybrids of two privileged scaffolds, tetrahydroisoquinoline (THIQ) and oxindole (OX), as M\u003csup\u003epro\u003c/sup\u003e and spike/ACE2 fusion inhibitors. This investigation aims at the discovery of novel spirooxindoles that target two SARS-CoV-2 viral proteins; the spike/ACE2 fusion that could potentially prevent transmission and those that target the main protease (M\u003csup\u003epro\u003c/sup\u003e), i.e. can prevent viral replication. A compound that hits both targets may present a higher genetic barrier to viral resistance as the two proteins involved would need to simultaneously undergo mutation to produce a drug-resistant strain. Besides, a compound that inhibits multiple aspects of viral replication could have synergistic activities and thus be especially potent and promising to be taken further down the drug discovery pipeline.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003eTest compounds\u003c/h2\u003e \u003cp\u003eSynthesis of the tested compounds (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) has been previously described\u003csup\u003e36\u0026ndash;38\u003c/sup\u003e. The synthesis of additional spirooxindoles (\u003cb\u003e12a, 13l, 14g-14i\u003c/b\u003e and \u003cb\u003e17a-17d)\u003c/b\u003e is described below in the Experimental section.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eGeneral experimental procedure\u003c/h2\u003e \u003cp\u003eChemicals were purchased from Sigma-Aldrich Chemicals Company and were used as supplied. All solvents were reagent grade. Where necessary, solvents and starting materials were purified using standard procedures. Solvent removal was carried out under reduced pressure using a Buchi rotary evaporator at temperatures not greater than 60\u0026deg;C. Melting points were measured using a Mel-Temp II apparatus with the use of open capillaries and were uncorrected. The progress of all reactions was monitored by thin layer chromatography (TLC) on aluminum-backed silica gel 60 F254 plates obtained from Sigma-Aldrich; visualization was by UV light at 254 nm or by staining with iodine. Compounds were purified by medium-pressure liquid chromatography over silica gel 60-to-400 mesh, using solvent mixtures that are specified below. Nuclear magnetic resonance (NMR) spectra were obtained using a Bruker Avance III spectrometer operating at 600 MHz (H1) and 150 MHz (13C). Spectra were recorded in deuterated solvents and referenced to residual solvent signals. Chemical shifts (δ) were measured in parts per million. Hydrogen and carbon assignments were done using gradient correlation spectroscopy (gCOSY), gradient heteronuclear single quantum correlation (gHSQC) spectroscopy, and heteronuclear multiple bond correlation (gHMBC) techniques. Multiplicities are reported as singlet (s), doublet (d), doublet of doublets (dd), doublet of triplets (dt), triplet (t), triplet of doublets (td) and multiplet (m). Coupling constants (J) are reported in Hertz. For biological evaluation, all compounds were converted to the corresponding hydrochlorides by treatment of the free bases with methanolic HCl. All compounds are greater than 95% pure by high-performance liquid chromatography (HPLC) analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis of compounds\u003c/h2\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eSynthesis of 5,7-dibromo-6',7'-dihydroxy-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one (12c). Method A\u003c/h2\u003e \u003cp\u003eThe compound was synthesized via the phenolic Pictet\u0026ndash;Spengler reaction reported by Ngo Hanna \u003cem\u003eet al\u003c/em\u003e. [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. To a solution of 5,7-dibromoisatin (1.5 g, 5.1 mmol) in absolute ethanol (10 ml) was added dopamine (1 g, 5.1 mmol) and triethylamine (1 ml). The reaction mixture was stirred and heated under reflux for 7\u0026ndash;10 h, and subsequently concentrated under reduced pressure to remove the solvent. Distilled water was added to the resulting viscous mass and the product which precipitated out was extracted into ethyl acetate (3 \u0026times; 30 ml). The combined organic extracts were dried over anhydrous sodium sulphate and concentrated to minimum volume. The crude product was further purified by column chromatography (hexane: ethyl acetate\u0026mdash;60 : 40). The final product was recrystallized from absolute ethanol. Yield, 1.7 g, 76% (brown solid). M.p. 256\u0026ndash;258 \u003csup\u003eo\u003c/sup\u003e C (HCl salt).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eH NMR\u003c/b\u003e (CD\u003csub\u003e3\u003c/sub\u003eOD, 700 MHz): δ ppm 2.74 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.1, 4.6 Hz, 1H, H4\u0026rsquo;a), 2.90 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9, 5.4 Hz, 1H, H4\u0026rsquo;b), 3.10\u0026ndash;3.15 (m, H3\u0026rsquo;a), 3.71\u0026ndash;3.77 (m, H3\u0026rsquo;b), 5.91 (s, 1H, H8\u0026rsquo;), 6.62 (s, 1H, H5\u0026rsquo;), 7.27 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8 Hz, 1H, H4), 7.64 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8 Hz, 1H, H6). \u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC NMR\u003c/b\u003e (CD\u003csub\u003e3\u003c/sub\u003eOD, 175 MHz): δ ppm 27.2 (C4\u0026rsquo;), 38.4 (C3\u0026rsquo;), 64.7 (C3/C1\u0026rsquo;), 102.8 (C7), 111.9 (C8\u0026rsquo;), 114.9 (C5), 115.4 (C5\u0026rsquo;), 123.5 (C8\u0026rsquo;a), 126.7 (C4), 127.3 (C4\u0026rsquo;a), 133.6 (C6), 138.7 (C3a), 140.9 (C7a), 143.8 (C7\u0026rsquo;), 145.0 (C6\u0026rsquo;), 180.1 (C2). \u003cb\u003eFTMS\u0026thinsp;+\u0026thinsp;cESI\u003c/b\u003e: m/z 440.92 M\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGeneral method for the synthesis of 6-methoxy- \u0026amp; 6\u0026rsquo;,7\u0026rsquo;-dimethoxy-3\u0026rsquo;,4\u0026rsquo;-dihydro-2\u0026rsquo;\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-spiro[indoline-3,1\u0026rsquo;-isoquinolin]-2-ones (13l \u0026amp;14g - i). Method B.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA mixture of the appropriate isatin (1 equiv), methoxyphenethylamine (1.2 equiv) and polyphosphoric acid (2 g) was heated in an oil bath (bath temperature at 100\u0026deg;C), while stirring mechanically for 5 hours. Upon completion of the reaction, as revealed by TLC, the reaction mixture was allowed to cool to about 50\u0026deg;C and quenched by slow addition of water. To this mixture was added a saturated solution of sodium carbonate to adjust the pH to 11. The floating product obtained was extracted into ethyl acetate (3 \u0026times; 30 ml). The combined organic extracts were dried over anhydrous sodium sulphate, and concentrated under reduced pressure to obtain the crude product. The latter was purified by flash chromatography on silica gel using suitable solvent systems. Yields ranged between 60 and 98%.\u003c/p\u003e \u003cp\u003e \u003cb\u003e6'-methoxy-5-methyl-3',4'-dihydro-2'\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-spiro[indoline-3,1'-isoquinolin]-2-one (13l)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMethod B. Prepared from 5-methylisatin (2.8 g, 17 mmol), 3-dimethoxyphenethylamine (2.6 g 17 mmol) and polyphosphoric acid (3 g). The crude product was purified by flash chromatography (hexane: ethyl acetate\u0026mdash;80: 20). Yield, 4.6 g, 92% (brown solid), M.p. 208\u0026ndash;209 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eH NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 600 MHz): δ ppm 1.46 (s, 3H, 5-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.07\u0026ndash;2.13 (m, 1H, H4\u0026rsquo;a), 2.23 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.5, 8.7, 5.3 Hz, 1H, H4\u0026rsquo;b), 2.39 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.8, 5.2 Hz, 1H, H3\u0026rsquo;a), 2.96 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.1 Hz, 4H, H3\u0026rsquo;b, m, 4H, 7\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 5.64 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6 Hz, 1H, H8\u0026rsquo;), 5.80 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6, 2.7 Hz, 1H, H7\u0026rsquo;), 5.95 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.7 Hz, 1H, H5\u0026rsquo;), 6.07 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.87 Hz, 1H, H7), 6.14\u0026ndash;6.17 (m, 1H, H4), 6.29 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 1.7, 0.8 Hz, 1H, H6). \u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 150 MHz): δ ppm 18.9 (5-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 27.6 (C4\u0026rsquo;), 37.5 (C3\u0026rsquo;), 53.4 (6\u0026rsquo;- O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 62.9 (C3/C1\u0026rsquo;), 108.7 (C7), 111.8 (C7\u0026rsquo;), 112.6 (C5\u0026rsquo;), 124.3 (C4), 125.4 (C8\u0026rsquo;a), 126.4 (C8\u0026rsquo;), 128.3 (C6), 131.5 (C3a), 134.5 (C7a), 136.4 (C4\u0026rsquo;a), 138.4 (C5), 158.0 (C6\u0026rsquo;), 180.3 (C2). \u003cb\u003eFTMS\u0026thinsp;+\u0026thinsp;cESI\u003c/b\u003e: m/z 295.14 [M\u0026thinsp;+\u0026thinsp;1]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e5,7-dibromo-6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2'\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-spiro[indoline-3,1'-isoquinolin]-2-one (14g)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMethod B. Prepared from 5,7-dibromo-1-(4-methylbenzyl)indoline-2,3-dione (1.8 g, 4.4 mmol), 3,4-dimethoxyphenethylamine (0.8 g, 4.4 mmol) and polyphosphoric acid (2 g). The crude product was purified by flash chromatography (hexane : ethyl acetate\u0026mdash;60 : 40). Yield, 1.8 g, 71% (yellow oil), M.p. 239\u0026ndash;240 \u003csup\u003eo\u003c/sup\u003eC (HCl salt).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eH NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 600 MHz): δ ppm 1.93 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.2, 4.3 Hz, 1H, H4\u0026rsquo;a), 2.11 (ddt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.3, 9.3, 4.9 Hz, 1H, H4\u0026rsquo;b), 2.27 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.7, 5.4, 4.0 Hz, 1H, H3\u0026rsquo;a), 2.58 (s, 3H, 7\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.85\u0026ndash;2.89 (m, 1H, H3\u0026rsquo;b), 2.92 (s, 3H, 6\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 4.39 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.1 Hz, 1H, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-Ar ), 4.51 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.3 Hz, 1H, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-Ar ), 5.00 (s, 1H, H8\u0026rsquo;), 5.91 (s, 1H, H5\u0026rsquo;), 6.23 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 2H, H3\u0026rdquo;, H5\u0026rdquo;), 6.30 (m, 2H, H2\u0026rdquo;, H6\u0026rdquo;), 6.40 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.0 Hz, 1H, H4), 6.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.9 Hz, 1H, H6). \u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 150 MHz): δ ppm 18.8 (4\u0026rdquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 28.6 (C4\u0026rsquo;), 37.5 (C3\u0026rsquo;), 43.0 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e-Ar ), 54.2 (7\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 54.2 (6\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 62.0 (C3/C1\u0026rsquo;), 101.9 (C7), 107.9 (C8\u0026rsquo;), 111.6 (C5\u0026rsquo;), 115.0 (C5), 123.8 (C8\u0026rsquo;a), 125.5 (C2\u0026rdquo;, C6\u0026rdquo;), 126.3 (C4), 128.1 (C3\u0026rdquo;, C5\u0026rdquo;), 128.3 (C4\u0026rsquo;a), 133.5 (C1\u0026rdquo;), 135.8 (C6), 136.0 (C3a), 138.5 (C4\u0026rdquo;), 139.0 (C7a), 147.1 (C7\u0026rsquo;), 148.3 (C6\u0026rsquo;), 178.3 (C2). \u003cb\u003eFTMS\u0026thinsp;+\u0026thinsp;cESI\u003c/b\u003e: m/z 573.02 [M\u0026thinsp;+\u0026thinsp;1]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e1-(4-fluorobenzyl)-6',7'-dimethoxy-5-methyl-3',4'-dihydro-2'\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-spiro[indoline-3,1'-isoquinolin]-2-one (14h)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMethod B. Prepared from 5-methyl-1-(4-fluorobenzyl)indoline-2,3-dione (1 g, 3.7 mmol), 3,4 - dimethoxyphenethylamine (0.8 g, 4.4 mmol) and polyphosphoric acid (3 g). The crude product was purified by flash chromatography (hexane: ethyl acetate\u0026mdash;60 : 40). Yield, 1.4 g, 90% (brown solid), M.p. 99\u0026ndash;101 \u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eH NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 600 MHz): δ ppm 2.19 (s, 3H, 5-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.71 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9, 4.1 Hz, 1H, H4\u0026rsquo;a), 2.88 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.1, 9.3, 5.4 Hz, 1H, H4\u0026rsquo;b), 3.05 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.5, 5.4, 4.1 Hz, 1H, H3\u0026rsquo;a), 3.29(s, 3H, 7\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 3.65 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.4, 9.3, 4.3 Hz, 1H, H3\u0026rsquo;b), 3.74 (s, 3H, 6\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 4.76 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.6 Hz, 1H, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-Ar ), 4.96 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.6 Hz, 1H, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-Ar ), 5.72 (s, 1H, H8\u0026rsquo;), 6.76 (s, 1H, H5\u0026rsquo;), 6.92 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.8, 3.1 Hz, 2H, H4, H7), 7.05 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0, 1.8, 0.9 Hz, 1H, H6), 7.16\u0026ndash;7.18 (m, H3\u0026rdquo;, H5\u0026rdquo;), 7.42 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.6, 5.5 Hz, 2H, H2\u0026rdquo;, H6\u0026rdquo;). \u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 150 MHz): δ ppm 21.0 (5-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 28.7 (C4\u0026rsquo;), 38.9 (C3\u0026rsquo;), 42.2 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e-Ar ), 55.8 (7\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 56.0 (6\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 63.6 (C3/C1\u0026rsquo;), 109.2 (C7), 109.4 (C8\u0026rsquo;), 113.0 (C5\u0026rsquo;), 115.8 (C3\u0026rdquo;, C5\u0026rdquo;), 125.5 (C4), 127.2 (C8\u0026rsquo;a), 129.3 (C6), 129.5 (C4\u0026rsquo;a), 129.9 (C2\u0026rdquo;, C6\u0026rdquo;), 132.2 (C3a), 133.5 (C1\u0026rdquo;), 135.5 (C5), 140.4 (C7a), 148.5 (C7\u0026rsquo;), 148.5 (C6\u0026rsquo;), 161.2 (C4\u0026rdquo;), 178.8 (C2). \u003cb\u003eFTMS\u0026thinsp;+\u0026thinsp;cESI\u003c/b\u003e: m/z 433.19 [M\u0026thinsp;+\u0026thinsp;1]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e6',7'-dimethoxy-5-methyl-1-(4-methylbenzyl)-3',4'-dihydro-2'\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-spiro[indoline-3,1'-isoquinolin]-2-one (14i)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eMethod B. Prepared from 5-methyl-1-(4-methylbenzyl)indoline-2,3-dione (1 g, 3.8 mmol), 3,4 - dimethoxyphenethylamine (0.82 g, 4.5 mmol) and polyphosphoric acid (2 g). The crude product was purified by flash chromatography (hexane: ethyl acetate\u0026mdash;60 : 40). Yield, 0.9 g, 56% (brown solid), M.p. 111\u0026ndash;112 \u003csup\u003eo\u003c/sup\u003eC\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eH NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 600 MHz): δ ppm 2.18 (s, 3H, 5-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.26 (s, 3H, 4\u0026rdquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.70 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9, 4.2 Hz, 1H, H4\u0026rsquo;a), 2.87 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.2, 9.3, 5.4 Hz, 1H, H4\u0026rsquo;b), 3.04 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.5, 4.9 Hz, 1H, H3\u0026rsquo;a), 3.29(s, 3H, 7\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 3.64 (m, 1H, H3\u0026rsquo;b), 3.73 (s, 3H, 6\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 4.69 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.5 Hz, 1H, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-Ar ), 4.95 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.5 Hz, 1H, C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e-Ar ), 5.73 (s, 1H, H8\u0026rsquo;), 6.75 (s, 1H, H5\u0026rsquo;), 6.86 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 1H, H7), 6.90 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.8 Hz, 1H, H4), 7.03 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0, 1.8, 0.9 Hz, 1H, H6), 7.13 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7 Hz, 2H, H3\u0026rdquo;, H5\u0026rdquo;), 7.23\u0026ndash;7.27 (m, 2H, H2\u0026rdquo;, H6\u0026rdquo;). \u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 150 MHz): δ ppm 21.0 (5-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 21.1 (4\u0026rdquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 28.7 (C4\u0026rsquo;), 38.9 (C3\u0026rsquo;), 42.8 (\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e-Ar ), 55.7 (7\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 55.9 (6\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 63.5 (C3/C1\u0026rsquo;), 109.3 (C7), 109.4 (C8\u0026rsquo;), 112.9 (C5\u0026rsquo;), 125.4 (C4), 127.3 (C8\u0026rsquo;a), 127.9 (C2\u0026rdquo;, C6\u0026rdquo;), 129.3 (C6), 129.5 (C4\u0026rsquo;a), 129.6 (C3\u0026rdquo;, C5\u0026rdquo;), 132.1 (C3a), 134.2 (C1\u0026rdquo;), 135.6 (C5), 137.1 (C4\u0026rdquo;), 140.6 (C7a), 147.5 (C7\u0026rsquo;), 148.4 (C6\u0026rsquo;), 178.7 (C2). \u003cb\u003eFTMS\u0026thinsp;+\u0026thinsp;cESI\u003c/b\u003e: m/z 429.21 [M\u0026thinsp;+\u0026thinsp;1]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eGeneral method for the synthesis of 2'-N-arylalkyl-6',7'-dimethoxy-1-(4- substituted benzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one (17a,b). Method C\u003c/h2\u003e \u003cp\u003eThe compounds were prepared from previously the described\u003csup\u003e36\u003c/sup\u003e 6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one (\u003cb\u003e14e\u003c/b\u003e) \u003cb\u003e(\u003c/b\u003e1 equiv) and the corresponding 4-substituted benzyl halides (1.4 equiv). An acetonitrile (10 mL) solution of \u003cb\u003e14e\u003c/b\u003e and K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e (2 equiv) was stirred at room temperature for 1 hour. Thereafter, 4-substituted benzyl halide (1.4 equiv) and KI (0.2 equiv) were added and the reaction mixture heated at 80\u003csup\u003eo\u003c/sup\u003eC for 2 hours. Upon completion of the reaction, the solvent was removed under reduced pressure, and the resulting viscous mass adjusted to pH 10 by the addition of an aqueous solution of Na\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e. The product was extracted into dichloromethane (30 mL x 3), and the combined organic extracts dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by flash chromatography (hexane:ethyl acetate\u0026mdash;70:30). Yields ranged between 65 and 75%.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2'-N-(4-fluorobenzyl)-6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one (17a)\u003c/h2\u003e \u003cp\u003eMethod C. The compound was prepared from 6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one (\u003cb\u003e14e)\u003c/b\u003e (1 g, 2.4 mmol) and 4-fluorobenzylchloride (0.51 g, 3.5 mmol). The crude product was purified by flash chromatography (hexane : ethyl acetate\u0026mdash;70 : 30). Yield, 1 g, 75% (yellow oil).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eH NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 700 MHz): δ ppm 2.26 (s, 3H, 4\u0026rdquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.71 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.7, 3.3 Hz, 1H, H4\u0026rsquo;a), 2.75 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.9, 5.4, 2.9 Hz, 1H, H3\u0026rsquo;a), 2.88 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.0, 10.7 5.5, Hz, 1H, H4\u0026rsquo;b), 3.22 (m, 4H, N1\u0026rsquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e, 7\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 3.29 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;4.0 Hz, 1H, N1\u0026rsquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e), 3.54 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.1, 3.8 Hz, 1H, H3\u0026rsquo;b), 3.73 (s, 3H, 6\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 4.79 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.2 Hz, 1H, N1-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e), 5.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.2 Hz, 1H, N1-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e), 5.69 (s, 1H, H8\u0026rsquo;), 6.77 (s, 1H, H5\u0026rsquo;), 7.05 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.52, 1.0 Hz, 1H, H5), 7.09\u0026ndash;7.12 (m, 3H, H7, H3\u0026rdquo;\u0026rsquo;, H5\u0026rdquo;\u0026rsquo;), 7.19 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.4, 1.3 Hz, 1H, H3\u0026rdquo;, H5\u0026rdquo;), 7.29 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.5, 3.9, 2.6 Hz, 3H, H6, H2\u0026rdquo;\u0026rsquo;, H6\u0026rdquo;\u0026rsquo;), 7.30\u0026ndash;7.32 (m, 2H, H2\u0026rdquo;, H6\u0026rdquo;). \u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 175 MHz): δ ppm 21.0 (4\u0026rdquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 28.9 (C4\u0026rsquo;), 42.7 (C3\u0026rsquo;), 42.9 (N1-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 54.0 (N1\u0026rsquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 55.6 (7\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 55.9 (6\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 68.9 (C3/C1\u0026rsquo;), 109.7 (C8\u0026rsquo;), 109.8 (C7), 112.4 (C5\u0026rsquo;), 115.4 (2C, C3\u0026rdquo;\u0026rsquo;, C5\u0026rdquo;\u0026rsquo;), 123.8 (C5), 124.7 (C4), 126.7 (C8\u0026rsquo;a), 128.2 (2C, C2\u0026rdquo;, C6\u0026rdquo;), 128.5 (C4\u0026rsquo;a), 129.6 (C6), 129.7 (2C, C3\u0026rdquo;, C5\u0026rdquo;), 130.2 (2C, C2\u0026rdquo;\u0026rsquo;, C6\u0026rdquo;\u0026rsquo;), 133.5 (C3a), 134.2 (C1\u0026rdquo;), 135.5 (C1\u0026rdquo;\u0026rsquo;), 137.4 (C4\u0026rdquo;), 143.7 (C7a), 147.5 (C7\u0026rsquo;), 148.5 (C6\u0026rsquo;), 161.1 (C4\u0026rdquo;\u0026rsquo;), 177.2 (C2). \u003cb\u003eFTMS\u0026thinsp;+\u0026thinsp;cESI\u003c/b\u003e: m/z 523.24 [M\u0026thinsp;+\u0026thinsp;1]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e6',7'-dimethoxy-1,2'-bis(4-methylbenzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one (17b)\u003c/h2\u003e \u003cp\u003eMethod C. The target compound was prepared from 6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinolin]-2-one \u003cb\u003e14e (\u003c/b\u003e1 g, 2.4 mmol) and 4-methylbenzylchloride (0.51 g, 3.6 mmol). The crude product was purified by flash chromatography (hexane: ethyl acetate\u0026mdash;70: 30). Yield, 0.8 g, 65% (yellow oil).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eH NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 700 MHz): δ ppm 2.25 (s, 6H, 4\u0026rdquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e,\u003c/sub\u003e 4\u0026rdquo;\u0026rsquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH3\u003c/span\u003e), 2.69(dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8, 3.4 Hz, 1H, H4\u0026rsquo;a), 2.75 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.7, 3.0 Hz, 1H, H3\u0026rsquo;a), 2.86 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.0, 10.7 5.6, Hz, 1H, H4\u0026rsquo;b), 3.21 (m, 5H, N2\u0026rsquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e, 7\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 3.51\u0026ndash;3.55 (m, 1H, H3\u0026rsquo;b), 3.72 (s, 3H, 6\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 4.79 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.2 Hz, 1H, N1-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e), 5.01 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.3 Hz, 1H, N1-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e), 5.70 (s, 1H, H8\u0026rsquo;), 6.76 (s, 1H, H5\u0026rsquo;), 7.05 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5, 1.0 Hz, 1H, H5), 7.09 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;8.0 Hz, 3H, H7, H3\u0026rdquo;\u0026rsquo;, H5\u0026rdquo;\u0026rsquo;), 7.11\u0026ndash;7.15 (m, 4H, H3\u0026rdquo;, H5\u0026rdquo;, H2\u0026rdquo;\u0026rsquo;, H6\u0026rdquo;\u0026rsquo;), 7.18\u0026ndash;7.20 (m, 1H, H4), 7.29 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 1.3 Hz, 1H, H6), 7.30\u0026ndash;7.33 (m, 2H, H2\u0026rdquo;\u0026rsquo;, H6\u0026rdquo;\u0026rsquo;). \u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 175 MHz): δ ppm 21.0 (2C, 4\u0026rdquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3,\u003c/sub\u003e 4\u0026rdquo;\u0026rsquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3,\u003c/sub\u003e), 28.9 (C4\u0026rsquo;), 42.5 (C3\u0026rsquo;), 42.9 (N1-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 54.5 (N2\u0026rsquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 55.6 (7\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 55.9 (6\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 68.9 (C3/C1\u0026rsquo;), 109.7 (C8\u0026rsquo;), 109.8 (C7), 112.4 (C5\u0026rsquo;), 123.8 (C5), 124.7 (C4), 126.7 (C8\u0026rsquo;a), 127.0 (C4\u0026rsquo;a), 128.2 (2C, C2\u0026rdquo;, C6\u0026rdquo;), 128.4 (2C, C3\u0026rdquo;\u0026rsquo;, C5\u0026rdquo;\u0026rsquo;), 129.1 (C6), 129.3 (2C, C2\u0026rdquo;\u0026rsquo;, C6\u0026rdquo;\u0026rsquo;), 129.6 (C4\u0026rdquo;\u0026rsquo;), 129.7 (2C, C3\u0026rdquo;, C5\u0026rdquo;), 133.6 (C3a), 134.2 (C1\u0026rdquo;), 136.6 (C1\u0026rdquo;\u0026rsquo;), 137.4 (C4\u0026rdquo;), 143.7 (C7a), 147.5 (C7\u0026rsquo;), 148.5 (C6\u0026rsquo;), 177.2 (C2). \u003cb\u003eFTMS\u0026thinsp;+\u0026thinsp;cESI\u003c/b\u003e: m/z 519.26 [M\u0026thinsp;+\u0026thinsp;1]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis of\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e\u003cb\u003e-ethyl-6',7'-dimethoxy-1-(4-methylbenzyl)-2-oxo-3',4'-dihydro-2'H-spiro[indoline-3,1'-isoquinoline]-2'-carboxamide (17c) (Method D)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe target compound was prepared from the previously described 6\u0026rsquo;,7\u0026rsquo;-dimethoxy-1-(4-methylbenzyl)-3\u0026rsquo;,4\u0026rsquo;-dihydro-2\u0026rsquo;H-spiro[indoline-3,1\u0026rsquo;-isoquinolin]-2-one \u003cb\u003e14e (\u003c/b\u003e1 g, 2.4 mmol) and ethylisocyanate (0.21 g, 0.23 mL, 2.9 mmol, 1.2 eq). An acetonitrile solution of \u003cb\u003e14e\u003c/b\u003e and ethyl isocyanate was heated to 60 \u003csup\u003eo\u003c/sup\u003e C for 2 hours. Upon completion of the reaction, the mixture was allowed to cool to room temperature, made basic by slow addition of aqueous sodium bicarbonate to pH 10. The product was extracted into ethyl acetate (30 mL x 2), and the combined organic extracts dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by flash chromatography (hexane: ethyl acetate\u0026mdash;70:30). Yield, 0.6 g, 50% (white solid). M.p. 193\u0026ndash;194 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eH NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 700 MHz): δ ppm 0.96 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.17 Hz, 3H, N1\u0026rdquo;\u0026rsquo;-CH\u003csub\u003e2\u003c/sub\u003eC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.27 (s, 3H, 4\u0026rdquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.90 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.4, 4.8, 3.4 Hz, 1H, H4\u0026rsquo;a), 2.92\u0026ndash;3.01 (m, 3H, 1H, H4\u0026rsquo;b, N1\u0026rdquo;\u0026rsquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 3.11 (s, 3H, 7\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 3.55\u0026ndash;3.60 (m, 1H, H3\u0026rsquo;a), 3.72 (s, 3H, 6\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 3.97 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;12.2, 4.6 Hz, 1H, H3\u0026rsquo;b), 4.62 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.5 Hz, 1H, N1-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e), 4.96 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.5 Hz, 1H, N1-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e), 5.76 (s, 1H, H8\u0026rsquo;), 6.84 (s, 1H, H5\u0026rsquo;), 6.89 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.5, 1.0 Hz, 1H, H5), 6.93 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9, 0.7 Hz, 1H, H7), 7.03 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.3, 1.25 Hz, 1H, H4), 7.10\u0026ndash;7.13 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 2H, H3\u0026rdquo;, H5\u0026rdquo;), 7.17 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.7, 1.3 Hz, 1H, H6), 7.31\u0026ndash;7.34 (m, 2H, H2\u0026rdquo;, H6\u0026rdquo;). \u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 175 MHz): δ ppm 15.8 (N1\u0026rdquo;\u0026rsquo;-CH\u003csub\u003e2\u003c/sub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 21.1 (4\u0026rdquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 30.0 (C4\u0026rsquo;), 35.4 (N1\u0026rdquo;\u0026rsquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003eCH\u003csub\u003e3\u003c/sub\u003e), 42.2 (C3\u0026rsquo;), 43.4 (N1-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 55.4 (7\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 55.9 (6\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 65.5 (C3/C1\u0026rsquo;), 108.9 (C7), 109.1 (C8\u0026rsquo;), 112.3 (C5\u0026rsquo;), 122.3 (C4), 122.7 (C5), 126.5 (C8\u0026rsquo;a), 128.2 (C4\u0026rsquo;a), 128.3 (2C, C2\u0026rdquo;, C6\u0026rdquo;), 128.9 (C6), 129.5 (2C, C3\u0026rdquo;, C5\u0026rdquo;), 134.5 (C1\u0026rdquo;), 135.7 (C3a)137.0 (C4\u0026rdquo;), 143.5 (C7a), 147.7 (C7\u0026rsquo;), 148.3 (C6\u0026rsquo;), 156.7 (C2\u0026rdquo;\u0026rsquo;), 177.3 (C2). \u003cb\u003eFTMS\u0026thinsp;+\u0026thinsp;cESI\u003c/b\u003e: m/z 486.24 [M\u0026thinsp;+\u0026thinsp;1]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSynthesis 6',7'-dimethoxy-2'-methyl-1-(4-methylbenzyl)-3',4'-dihydro-2'\u003c/b\u003e \u003cb\u003eH\u003c/b\u003e \u003cb\u003e-spiro[indoline-3,1'-isoquinolin]-2-one (17d) (Method E)\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThis compound was prepared from previously synthesized 6',7'-dimethoxy-1-(4-methylbenzyl)-3',4'-dihydro-2'\u003cem\u003eH\u003c/em\u003e-spiro[indoline-3,1'-isoquinolin]-2-one (\u003cb\u003e14e\u003c/b\u003e)\u003csup\u003e36\u003c/sup\u003e \u003cb\u003e(\u003c/b\u003e1 g, 2.4 mmol) and formaldehyde (0.3 mL of 37% formalin, 3.6 mmol, 1.5 eq). To a formic acid solution of \u003cb\u003e14e\u003c/b\u003e formaldehyde was added dropwise. The resulting mixture was heated at 60 oC for 3 hours, allowed to cool to room temperature, and made basic by slowly adding 2 M aqueous sodium hydroxide. The product was extracted into ethyl acetate (30 mL x 3), and the combined organic extracts dried over anhydrous sodium sulphate and concentrated under reduced pressure. The crude product was purified by flash chromatography (hexane: ethyl acetate\u0026mdash;50: 50). Yield, 0.8g, 78% (yellow oil).\u003c/p\u003e \u003cp\u003e \u003csup\u003e \u003cb\u003e1\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eH NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 700 MHz): δ ppm 2.06 (s, 3H, N2\u0026rsquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e3\u003c/sub\u003e), 2.26 (s, 3H, 4\u0026rdquo;-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 2.78 (dt, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8, 3.6 Hz, 1H, H4\u0026rsquo;a), 2.88 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;11.3, 5.7, 2.9 Hz, 1H, H3\u0026rsquo;a), 3.03 (ddd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.0, 10.5, 5.6 Hz, 1H, 1H, H4\u0026rsquo;b), 3.22 (s, 3H, 7\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 3.63 (td, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.9, 4.1 Hz, 1H, H3\u0026rsquo;b), 3.73 (s, 3H, 6\u0026rsquo;-OC\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e3\u003c/span\u003e\u003c/sub\u003e), 4.77 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.4 Hz, 1H, N1-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e), 4.97 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.5 Hz, 1H, N1-C\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eH\u003c/span\u003e\u003csub\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003e2\u003c/span\u003e\u003c/sub\u003e), 5.65 (s, 1H, H8\u0026rsquo;), 6.77 (s, 1H, H5\u0026rsquo;), 6.99\u0026ndash;7.01 (m, 2H, H4, H5), 7.06 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.9 Hz, 1H, H7), 7.14 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 2H, H3\u0026rdquo;, H5\u0026rdquo;), 7.26\u0026ndash;7.29 (m, 3H, H6, H2\u0026rdquo;, H6\u0026rdquo;). \u003csup\u003e\u003cb\u003e13\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eC NMR\u003c/b\u003e (DMSO-d\u003csub\u003e6\u003c/sub\u003e, 175 MHz): δ ppm 21.0 (4\u0026rdquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 28.7 (C4\u0026rsquo;), 39.6 (N2\u0026rsquo;-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 42.7 (N1-\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e2\u003c/sub\u003e), 46.9 (C3\u0026rsquo;), 55.6 (7\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 55.9 (6\u0026rsquo;-O\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eC\u003c/span\u003eH\u003csub\u003e3\u003c/sub\u003e), 69.0 (C3/C1\u0026rsquo;), 109.6 (C8\u0026rsquo;), 109.6 (C7), 112.4 (C5\u0026rsquo;), 123.5 (C5), 124.8 (C4), 126.7 (C8\u0026rsquo;a), 128.0 (C4\u0026rsquo;a), 128.1 (2C, C2\u0026rdquo;, C6\u0026rdquo;), 129.4 (2C, C3\u0026rdquo;, C5\u0026rdquo;), 129.7 (C6), 133.2 (C3a), 134.2 (C1\u0026rdquo;), 137.3 (C4\u0026rdquo;), 143.5 (C7a), 147.4 (C7\u0026rsquo;), 148.5 (C6\u0026rsquo;), 177.3 (C2). \u003cb\u003eFTMS\u0026thinsp;+\u0026thinsp;cESI\u003c/b\u003e: m/z 429.21 [M\u0026thinsp;+\u0026thinsp;1]\u003csup\u003e+\u003c/sup\u003e.\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eDescription of biological screening procedures\u003c/h2\u003e \u003cdiv id=\"Sec10\" class=\"Section4\"\u003e \u003ch2\u003eAlphaScreen assay\u003c/h2\u003e \u003cp\u003eSARS-CoV-2 spike-RBD binding to ACE2 was determined using AlphaScreen technology-based assay as described previously\u003csup\u003e42\u003c/sup\u003e. For RBD-ACE2 assays, 2 nM of ACE2-Fc (Sino Biological, Chesterbrook, PA, USA) was incubated with 5 nM HIS-tagged SARS-CoV-2 Spike-RBDs representing the parental USA-WA/2020 (\u0026ldquo;Wild-type\u0026rdquo; (WT)) sequence (SinoBiological) in the presence of 5 \u0026micro;g/mL nickel chelate donor bead in a total of 10 \u0026micro;L of 20 mM Tris (pH 7.4), 150 mM KCl, and 0.05% CHAPS. Test compounds were diluted to 100x final concentration in DMSO. 5 \u0026micro;L of ACE2-Fc/Protein A acceptor bead was first added to the reaction, followed by 100 nL test compound and then 5 \u0026micro;L of RBD-HIS/Nickel chelates donor beads. All conditions were performed in duplicate. Following incubation at room temperature for 2 hours, luminescence signals were measured using a ClarioStar plate reader (BMC Labtech, Cary, NC, USA). Data were then normalised to percent inhibition, where 100% equalled the AlphaScreen signal in the absence of RBD-HIS, and 0% denoted AlphaScreen signal in the presence of both protein and DMSO vehicle control. To measure PD1/PD-L1 binding, 0.5 nM of human PD-L1-Fc (Sino Biological) was incubated with 5 nM HIS-tagged human PD1 (Sino Biological) in the presence of 5 \u0026micro;g/mL protein A and 5 \u0026micro;g/mL nickel chelate donor beads in a total volume of 10 \u0026micro;L of 20 mM HEPES (pH 7.4), 150 mM NaCl, and 0.005% Tween-20. Proteins and test agents were then added, incubated, and analysed as described above.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eM\u003csup\u003epro\u003c/sup\u003e inhibition assay\u003c/h2\u003e \u003cp\u003eThe M\u003csup\u003epro\u003c/sup\u003e inhibition assay was done using the fluorogenic assay as described previously\u003csup\u003e42\u003c/sup\u003e. Firstly, 5 \u0026micro;l of 25 nM M\u003csup\u003epro\u003c/sup\u003e diluted in assay buffer (25 mM HEPES [pH 7.4]), 150 mM NaCl, 5 mM DTT, and 0.005% Tween) was dispensed into black, low-volume, 384-well plates. Test compounds were serially diluted into 100% DMSO, and 0.1 ml was added to the assay using a Janus MDT Nanohead tool (PerkinElmer). Assays were initiated by addition of 5 \u0026micro;l of 5 \u0026micro;M fluorogenic substrate, and fluorescence at 355 nm excitation and 460 nm emission was monitored every 5 min for 50 min using an Envision plate reader (PerkinElmer). The rate of substrate cleavage was determined using linear regression of the raw data values obtained during the time course. The slopes of these progress curves were then normalized to percentage inhibition, where 100% equaled the rate in the absence of M\u003csup\u003epro\u003c/sup\u003e (which was typically 0), and 0% equaled the rate of cleavage in the presence of M\u003csup\u003epro\u003c/sup\u003e and 0.1% DMSO.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eMolecular modeling procedures\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section3\"\u003e \u003ch2\u003eTarget proteins for docking\u003c/h2\u003e \u003cp\u003eMolecular modeling protocols were performed as previously reported\u003csup\u003e43\u0026ndash;47\u003c/sup\u003e. The protein structures (ID: 6M0J) for SARS-CoV-2 spike/ACE2 and (PDB ID: 6W63) for M\u003csup\u003epro\u003c/sup\u003e, respectively, corresponding to the Wuhan strain were retrieved from the Protein Data Bank (PDB)\u003csup\u003e48\u0026ndash;50\u003c/sup\u003e and used for the entire study.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProtein preparation\u003c/h2\u003e \u003cp\u003eAll water molecules were deleted using the Molecular Operating Environment (MOE) software\u003csup\u003e51\u003c/sup\u003e. The Protein Preparation Wizard integrated in the Schr\u0026ouml;dinger package software\u003csup\u003e52\u0026ndash;53\u003c/sup\u003e was used to prepare the proteins by adding the missing hydrogen bonds, assigning bond orders and filling the missing side chains using PRIME. After this the protein structures were energy minimized to reduce atomic clashes and optimized their interactions with the ligands during docking. From the Schr\u0026ouml;dinger software, the commercialized maestro package\u0026rsquo;s Epik-tool was used to predict the protonation states at a pH of 7.0\u003csup\u003e54\u003c/sup\u003e. Finally a restrained energy minimization step was carried out using the Optimized Potentials for Liquid Simulations 2005 (OPLS2005) forcefield\u003csup\u003e55\u003c/sup\u003e on both proteins. During the protein optimization step, the root mean square deviation (RMSD) of the displacement of the atoms was set to end with the minimization at 0.3 \u0026Aring;.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLigand preparation\u003c/h2\u003e \u003cp\u003eThe MOE\u003csup\u003e51\u003c/sup\u003e builder module was used to generate the 3D models of the library of synthesized spirooxindoles. For consistency, only the R stereoisomers were prepared for docking, as these addressed the voluminous hydrophobic regions in the ACE2 site more appropriately during trial docking. The generated 3D structures were then energy minimized using the MMFF94 force field\u003csup\u003e56\u0026ndash;60\u003c/sup\u003e. The ligands were further prepared for docking using the LigPrep tool to generate all the plausible tautomers of each ligand as implemented in Schr\u0026ouml;dinger\u0026rsquo;s Maestro software package\u003csup\u003e54\u003c/sup\u003e. Using the incorporated OPLS2005 force field\u003csup\u003e55\u003c/sup\u003e, the spirooxindole 3D structure library was further energy minimized. The ConfGen tool (implemented in the Schr\u0026ouml;dinger package) was then used to compute 60 conformers per ligand in the 3D library, by setting all other options to default except for the minimization of the output\u003csup\u003e61\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDocking and scoring\u003c/h2\u003e \u003cp\u003eDocking was carried out using the Glide program incorporated in the Maestro package distributed by Schr\u0026ouml;dinger\u003csup\u003e52\u0026ndash;53\u003c/sup\u003e as shown in our recent publications\u003csup\u003e43\u0026ndash;47\u003c/sup\u003e, with some modifications. Docking validation results on this protein have already been reported in our previously reported studies\u003csup\u003e45\u0026ndash;47\u003c/sup\u003e. After the protein preparation phase, a docking grid box was generated for the spike/ACE2 complex to investigate how the ligands will bind around the following amino acid residues; Asp597, Thr598, Lys516, Val321, Gln121, Lys578, Ala283, Ser91, Asn746, Gln68, Pro744, Glu518 and Thr610. The co-crystalized ligand (X77) was used as the centroid to generate the docking grid box for M\u003csup\u003epro\u003c/sup\u003e as seen in our recently published work\u003csup\u003e62\u003c/sup\u003e. The ligand size for each of these grid boxes, which is the area where all the generated 3D structures were docked, was set to a maximum ligand size of 36 \u0026Aring;. While writing 10 poses per ligand conformer, 20 poses were included for each ligand conformer and taking into consideration the input of ring conformation, all other settings were allowed to default. The outputs were scored using standard precision (SP) GlideScore as the scoring function\u003csup\u003e63\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSelection of binding modes\u003c/h2\u003e \u003cp\u003eAfter the extraction of the results and the computation of carefully selected descriptors, the specific area ligands bound with the protein in the receptor binding domain (RBD) of both the Spike/ACE2 and M\u003csup\u003epro\u003c/sup\u003e, the binding modes and the residues taking part in the interaction during binding were observed using MOE\u003csup\u003e51\u003c/sup\u003e. Browsing through the docking results and establishing the ligand interactions of each docked protein-ligand complex made it possible to establish structure-activity relationships (SAR) in the RBD in both cases and to identify some ligand moieties important for activity and selectivity. The ligands in both protein RBD were then superimposed to highlight their preferred binding modes.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results and discussion","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eActivities in the AlphaScreen assay and M\u003csup\u003epro\u003c/sup\u003e inhibitory assay\u003c/h2\u003e \u003cp\u003eFor the synthesized compounds, the 50% inhibitory concentrations (IC\u003csub\u003e50\u003c/sub\u003e values) for spike/ACE2 binding (AlphaScreen) and inhibition of M\u003csup\u003epro\u003c/sup\u003e are shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, alongside the best docking score for each ligand. The cut-off concentrations to distinguish between active, moderately active, and inactive ligands for SARS-CoV-2 enzymatic assays were adopted from recent literature\u003csup\u003e64\u003c/sup\u003e and are summarized in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.\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\u003eBiological assay results and docking results for spike/ACE2 and M\u003csup\u003epro\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompound ID\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c3\" namest=\"c2\"\u003e \u003cp\u003espike/ACE2\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eM\u003csup\u003epro\u003c/sup\u003e\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(\u0026micro;M)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGlide SP Score\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eIC\u003c/b\u003e\u003csub\u003e\u003cb\u003e50\u003c/b\u003e\u003c/sub\u003e \u003cb\u003e(\u0026micro;M)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGlide SP Score\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e10d\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e20.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-6.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.72\u003c/p\u003e \u003c/td\u003e 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colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11d\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e70.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-6.68\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.69\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e11e\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e15.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-6.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e 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align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.48\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14i\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-6.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14j\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-6.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e30.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.39\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14k\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.17\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e66.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14m\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.46\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e29.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e14n\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.53\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e15a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.00\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.73\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e15b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-4.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e15c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e8.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.09\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.89\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e15d\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e44.7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e15e\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e49.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.37\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.99\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e16a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.95\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e16b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-4.74\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e17a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e39.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-8.00\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e17b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e63.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.42\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.65\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e17c\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.44\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e17d\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e50.3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.51\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e17e\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e17.4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.61\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e18a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.52\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e18b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-6.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.06\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e19a\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-4.78\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-6.34\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003e19b\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e10.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-5.64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;100\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-7.12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGC-376 (control)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0031\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-11.61\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 \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\u003eStandard acceptable cut-off activity values for both SARS-CoV-2 enzymes as defined in the literature\u003csup\u003e64\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eEnzymatic assays (spike/ACE, M\u003csup\u003epro\u003c/sup\u003e)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eActive\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eModerately active\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eInactive\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCut-off concentration\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;10 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 \u0026micro;M\u0026thinsp;\u0026lt;\u0026thinsp;IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;20 \u0026micro;M\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e \u0026gt; 20 \u0026micro;M\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\u003eOn this basis, the ligands were classified into categories A (active, with IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;10 \u0026micro;M), B with (moderately active, with 10 \u0026micro;M\u0026thinsp;\u0026lt;\u0026thinsp;IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;20 \u0026micro;M) or C (inactive, with IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026gt;\u0026thinsp;20 \u0026micro;M) for the spike/ACE2 assay. All tested compounds were inactive in the M\u003csup\u003epro\u003c/sup\u003e assay. The classification of the ligands into categories A to C is shown in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e (Supplementary Data). Of the 60 tested spirooxindoles, 15 fell under category A including \u003cb\u003e10f\u003c/b\u003e, \u003cb\u003e10h\u003c/b\u003e, \u003cb\u003e10j\u003c/b\u003e, \u003cb\u003e10l\u003c/b\u003e, \u003cb\u003e11j\u003c/b\u003e, \u003cb\u003e11l\u003c/b\u003e, \u003cb\u003e11m\u003c/b\u003e, \u003cb\u003e12b\u003c/b\u003e, \u003cb\u003e12c\u003c/b\u003e, \u003cb\u003e14f\u003c/b\u003e, \u003cb\u003e14h\u003c/b\u003e, \u003cb\u003e14j\u003c/b\u003e, \u003cb\u003e15c\u003c/b\u003e, \u003cb\u003e18c\u003c/b\u003e, and \u003cb\u003e18d\u003c/b\u003e, the most active compound being \u003cb\u003e11j\u003c/b\u003e (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.6 \u0026micro;M). There were 11 compounds in category B, which include \u003cb\u003e10g\u003c/b\u003e, \u003cb\u003e10k\u003c/b\u003e, \u003cb\u003e11e\u003c/b\u003e, \u003cb\u003e11g\u003c/b\u003e, \u003cb\u003e12a\u003c/b\u003e, \u003cb\u003e14g\u003c/b\u003e, \u003cb\u003e14i\u003c/b\u003e, \u003cb\u003e15b\u003c/b\u003e, \u003cb\u003e17a\u003c/b\u003e, \u003cb\u003e17f\u003c/b\u003e, \u003cb\u003e18a\u003c/b\u003e, and \u003cb\u003e18e\u003c/b\u003e. The remaining compounds were inactive (category C). We could further identify a subset of non-selective compounds in categories A and B (referred to as A\u0026rsquo; and B\u0026rsquo;, respectively), which we could define as active compounds and moderately active compounds against spike/ACE2, which could contain some pharmacophore features required for binding to M\u003csup\u003epro\u003c/sup\u003e. These are compounds that could be slightly modified to derive dual inhibitors of spike/ACE2 and M\u003csup\u003epro\u003c/sup\u003e. Category A\u0026rsquo; includes compounds \u003cb\u003e10f\u003c/b\u003e, \u003cb\u003e12b\u003c/b\u003e, \u003cb\u003e12c\u003c/b\u003e, and \u003cb\u003e14j\u003c/b\u003e, while category B\u0026rsquo; includes compounds \u003cb\u003e10g\u003c/b\u003e, \u003cb\u003e18a\u003c/b\u003e, and \u003cb\u003e18e\u003c/b\u003e. Our discussion of the structure-activity relationships will focus on the common features of compounds in categories A, A\u0026rsquo;, B, and B\u0026rsquo; which are absent from category C and vice versa. Although there was no correlation between the activities of the compounds and their docking scores towards the spike/ACE2 site, the orientations of the top-scoring poses could carefully explain the structure-activity relations.\u003c/p\u003e \u003cp\u003eIt was observed that the most active compound (\u003cb\u003e11j\u003c/b\u003e, IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.60 \u0026micro;M) interacted with Arg375 \u003cem\u003evia\u003c/em\u003e the N-H of the isoquinoline moiety (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), while the naphthyl group makes several arene-H interactions with Asp332. Although these amino acids make similar interactions with almost all the actives, compound \u003cb\u003e11j\u003c/b\u003e distinguishes itself by the strong hydrophobic interactions resulting from the interaction field produced by the naphthyl moiety. This matches with the strong hydrophobic patch created by the amino acids Phe22, Ser26, Leu333, and Ile361 (shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), which is an indication that the activity of this compound could be driven by the strong hydrophobic interactions between the naphthyl moiety and this patch. This suggests that more active compounds could be designed and synthesized by introducing other hydrophobic groups around the naphthyl (F, CH\u003csub\u003e3\u003c/sub\u003e, Cl, CF\u003csub\u003e3\u003c/sub\u003e, Br, etc.) moiety, a feature which is conspicuously absent from the moderately active and inactive compounds.\u003c/p\u003e \u003cp\u003eThe 8-hydroxy isomer of the most active compound (\u003cb\u003e11j\u003c/b\u003e), i.e. compound \u003cb\u003e10j\u003c/b\u003e was shown to be about twofold less active (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.4 \u0026micro;M). A superposition of the two isomers has been shown in the spike/ACE2 pocket in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. While the 6-hydroxy group in compound \u003cb\u003e11j\u003c/b\u003e is free to make H-bond interactions with the protein backbone, this possibility is hindered in compound \u003cb\u003e10j\u003c/b\u003e, which rather forms intramolecular H-bonding with the carbonyl of the oxindole moiety. This could explain the observed activity of compound \u003cb\u003e11j\u003c/b\u003e compared with compound \u003cb\u003e10j\u003c/b\u003e. The top-scoring poses of the rest of the two molecules show almost perfect superposition (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eTo the best of our knowledge, this is the first report that shows that spirooxindoles have activity against SARS-CoV-2 spike/ACE2 binding. Nine newly reported and fifty already published\u003csup\u003e36\u0026ndash;38\u003c/sup\u003e spirooxindoles, synthesized by Pictet-Spengler cyclodehydration, were screened against both spike/ACE2 binding and M\u003csup\u003epro\u003c/sup\u003e inhibition. While all IC\u003csub\u003e50\u003c/sub\u003e values against M\u003csup\u003epro\u003c/sup\u003e were shown to be \u0026gt;\u0026thinsp;20 \u0026micro;M, it was shown that 15 compounds had IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;\u0026lt;\u0026thinsp;10 \u0026micro;M in the spike/ACE2 assay, 11 compounds were shown to be moderately active, while the rest were inactive. Molecular docking and evaluation of the structure-activity relationship showed that H-bonding between the isoquinoline moiety and the Arg375/Asn376 pair was required for activity. Besides, the presence of a bulky hydrophobic moiety attached to the oxindole is important for activity by potentially forming π-π stacking with Trp331, arene-H interactions with Asp332, and the strong hydrophobic interactions with the patch created by the amino acids Phe22, Ser26, Leu333, and Ile361. It would be necessary to further design new naphthyl-based analogues with hydrophobic substituents that address this region of the binding pocket to improve the activity against SARS-CoV-2 spike/ACE2 binding.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge financial support from the Bill \u0026amp; Melinda Gates Foundation through the Calestous Juma Science Leadership Fellowship awarded to Fidele Ntie-Kang (grant award number: INV-036848 to University of Buea). FNK also acknowledges joint funding from the Bill \u0026amp; Melinda Gates Foundation and LifeArc (award number: INV-055897 and Grant ID: 10646) under the African Drug Discovery Accelerator program. FNK acknowledges further funding from the Alexander von Humboldt Foundation for a Research Group Linkage project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe acknowledge the technical support of Mr. Cyril T. Namba-Nzanguim.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAlbert Enama Ehinak:\u0026nbsp;\u003c/strong\u003eConceptualization, Methodology, Data curation, Formal analysis, Investigation, Writing \u0026ndash; original draft. \u003cstrong\u003eMaloba M.M. Lobe:\u003c/strong\u003e Conceptualization, Methodology, Data curation, Formal analysis, Investigation, Writing \u0026ndash; original draft. \u003cstrong\u003eConrad V. Simoben:\u0026nbsp;\u003c/strong\u003eConceptualization, Formal analysis, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eIan Tietjen:\u0026nbsp;\u003c/strong\u003eConceptualization, Funding acquisition, Investigation, Methodology, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eJoel Cassel:\u003c/strong\u003e Conceptualization, Investigation, Methodology, Writing \u0026ndash; review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Joseph M. Salvino:\u0026nbsp;\u003c/strong\u003eFunding acquisition, Investigation, Methodology, Supervision, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eLuis J. Montaner:\u0026nbsp;\u003c/strong\u003eFunding acquisition, Investigation, Methodology, Supervision, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eWolfgang Sippl: \u0026nbsp;\u003c/strong\u003eConceptualization, Formal analysis, Supervision, Writing \u0026ndash; review \u0026amp; editing. \u003cstrong\u003eSimon M. N. Efange: \u0026nbsp;\u003c/strong\u003eConceptualization, Formal analysis, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003cstrong\u003e\u0026nbsp;Fidele Ntie-Kang:\u0026nbsp;\u003c/strong\u003eFunding acquisition, Investigation, Methodology, Supervision, Writing \u0026ndash; original draft, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analysed during this study are included in this published article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePinto, G. 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Pharmacol. 14, 1193282 (2023). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fphar.2023.1193282\u003c/span\u003e\u003cspan address=\"10.3389/fphar.2023.1193282\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Schemes","content":"\u003cp\u003eScheme 1 and 2 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":"Mpro, SARS-CoV-2, spike/ACE2, spirooxindoles, tetrahydroisoquinolines","lastPublishedDoi":"10.21203/rs.3.rs-4535655/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4535655/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBoth tetrahydroisoquinolines (THIQs) and oxindoles (OXs) display a broad range of biological activities, including antiviral activity. They are, therefore, recognized as privileged scaffolds in drug discovery. Here, we describe the synthesis of spirofused tetrahydroisoquinoline\u0026ndash;oxindole hybrids (spirooxindoles) and their evaluation as potential blocking agents of both SARS-CoV-2 spike/ACE fusion and inhibitors of the main protease (M\u003csup\u003epro\u003c/sup\u003e). The most active synthesized compound showed a 50% inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) of 3.6 \u0026micro;M against SARS-CoV-2 spike/ACE fusion. None of the tested compounds was shown to be active against M\u003csup\u003epro\u003c/sup\u003e. The most active compound possesses a bulky naphthyl group, which addresses voluminous hydrophobic regions of the ACE2 binding site and interacts with the hydrophobic residues of the target; this finding agrees with previous studies revealing that bulky compounds block spike/ACE2 fusion, e.g., the natural product hopeaphenol. Therefore, spirooxindoles may provide useful leads in the search for SARS-CoV-2 spike/ACE fusion blocking agents.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e","manuscriptTitle":"An evaluation of spirooxindoles as blocking agents of SARS-CoV-2 spike/ACE2 fusion and M pro inhibitory agents: Synthesis, biological evaluation and computational analysis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-21 16:46:12","doi":"10.21203/rs.3.rs-4535655/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":"bb9e631e-e6c6-4bc2-b202-e908eb38b214","owner":[],"postedDate":"June 21st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":33526205,"name":"Biological sciences/Drug discovery/Drug screening"},{"id":33526206,"name":"Biological sciences/Drug discovery/Medicinal chemistry"},{"id":33526207,"name":"Biological sciences/Drug discovery/Pharmacology"}],"tags":[],"updatedAt":"2024-07-01T10:00:49+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-21 16:46:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4535655","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4535655","identity":"rs-4535655","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}