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Photoinduced isomerization and hepatoxicities semaxanib, sunitinib and related 3 substituted indolin 2 ones

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  • Photoinduced isomerization and hepatoxicities semaxanib, sunitinib and related 3 substituted indolin 2 ones

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DOI: 10.1002/cmdc.201500475 Full Papers Photoinduced Isomerization and Hepatoxicities of Semaxanib, Sunitinib and Related 3-Substituted Indolin-2ones Mun Hong Ngai,[a] Choon Leng So,[a] Michael B Sullivan,[b] Han Kiat Ho,[a] and Christina L L Chai*[a] tion ranged from 0.009 to 0.048 hÀ1 Selected compounds were tested for cytotoxicity in the TAMH liver cell line E/Z mixtures of four compounds were also assessed for toxicity in the TAMH and HepG2 cell lines In some cases, the stereochemically pure drug was more toxic than the E/Z mixtures, but a general statement cannot be made Our studies show that each stereoisomer could contribute differently to toxicity, suggesting that stereochemical purity issues that could arise from isomerization cannot be ignored 3-Substituted indolin-2-ones are an important class of compounds that display a wide range of biological activities Sunitinib is an orally available multiple tyrosine kinase inhibitor that has been approved by the US Food and Drug Administration (FDA) for the treatment of renal cell cancer Sunitinib and a related compound, semaxanib, exist as thermodynamically stable Z isomers, which photoisomerize to E isomers in solution In this study, 17 3-substituted indolin-2-ones were synthesized, and the kinetics of their photoisomerization were studied by H NMR spectroscopy The rate constants for photoisomeriza- Introduction and negative results in phase II/III studies.[5–7] Subsequent studies focused on structural modifications of semaxanib, leading to the discovery of sunitinib (2), a new multitargeted receptor tyrosine kinase inhibitor.[8] Sunitinib has been approved by the US Food and Drug Administration (FDA) for treatment of advanced renal cell cancer and imatinib-resistant or imatinib-intolerant gastrointestinal stromal tumors (GISTs).[9, 10] Both semaxanib and sunitinib have Z stereochemistry at the double bond, and both can undergo photoisomerization to the E isomer The Z isomer is the thermodynamically stable form, due to the presence of intramolecular hydrogen bonding between the C2 carbonyl group of the oxindole and the NH group of the pyrrole ring.[4] Thermal reversion of the less stable E isomer to the Z isomer is also reported to occur Some pharmaceutical implications of this isomerization process, for example, the stabilities of the administered drug, have been recognized for both semaxanib and sunitinib, leading to the development of analytical methods for detection of the isomers.[11–14] However, not much has been reported with regard to the photoisomerization process of semaxanib,[11–13] sunitinib,[15] and related 3-pyrrolylmethylidene oxindoles, or whether the stereoisomers display differential biological activities In the latter context, we were specifically interested in determining whether the E and Z isomers display different toxicities that may lead to undesired side effects later in the drug development process This is especially relevant in view of the toxicity effects observed with semaxanib in clinical trials In this study, we report the photoisomerization studies of semaxanib and 3-substituted indolin-2-one analogues and report the toxicities of the compounds and their isomers, first against the TAMH cell line as a metabolically competent model The 3-substituted indolin-2-ones (oxindoles) are an important class of compounds that have been well-explored for their biological activities and continue to attract much interest due to their promise in drug development.[1] Of these oxindoles, the 3-pyrrolylmethylidene oxindoles are particularly exciting, as they have been shown to inhibit receptor tyrosine kinases (RTKs) The selectivities of these oxindoles against particular RTKs are dependent on the substituents, especially at the C3 position Semaxanib (1) is an example of this class of compounds and shows potent activity against vascular endothelial growth factor (VEGF).[2–4] Semaxanib proceeded to clinical trials, but its development was halted due to toxicity issues [a] Dr M H Ngai,+ C L So,+ Prof Dr H K Ho, Prof Dr C L L Chai Department of Pharmacy, National University of Singapore 18 Science Drive 4, Singapore 117543 (Singapore) E-mail: phacllc@nus.edu.sg [b] Dr M B Sullivan Institute of High-Performance Computing Agency for Science Technology and Research, Singapore Fusionopolis Way, #16-16 Connexis, Singapore 138632 (Singapore) [+] These authors contributed equally to this work ChemMedChem 2016, 11, 72 – 80 72  2016 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Full Papers to recapitulate in vivo bioactivation events, and in another liver cell line, HepG2, to validate our findings Table Yield and Z/E isomer ratios of 3-substituted indolin-2-ones Results and Discussion Synthesis The 3-substituted indolin-2-ones were prepared following reported procedures using a Knoevenagel condensation between substituted indoline-2-ones and aldehydes in the presence of piperidine in ethanol (Scheme 1).[4] Oxindole, 5-fluoro-, Compd R1 R2 R3 5a 5b 5c 5d 5e 5f 5g 5h 5i 6a 6b 6c 7a 7b 7c H H H H H H H H CH3 CH3 H H H CH3 CH3 CH3 – H F Ac NO2 OMe NHAc OH NH2 H F H H H H H H – H H H H H H H H H H Ac CH3 Boc Ac CH3 Boc – Yield [%] Z/E isomer ratio 62 58 53 74 73 71 57 34 46 15 78 53 90 58 85 90 66 100:0 100:0 100:0 100:0 100:0 100:0 100:0 100:0 0:100 3:97 100:0 100:0 100:0 16:84 11:89 14:86 100:0 doline-2-one ring The same acylation and alkylation method was applied to the synthesis of N-methylpyrrole compounds a–c, and a mixture of E and Z isomers were obtained, despite starting with the pure E isomer of h (Scheme 3) The relative Scheme Synthesis of 3-substituted indolin-2-ones Reagents and conditions: a) piperidine, EtOH, 75 8C and 5-nitrooxindoles were commercially available 5-Acetyloxindole was prepared via Friedel–Crafts acetylation of oxindole.[16] Oxidation of oxindole with phenyliodine(III)-bis(trifluoroacetate) (PIFA) gave 5-hydroxyoxindole, which was subsequently methylated to give 5-methoxyoxindole.[17] 5-Acetamidoxindole was synthesized from 5-nitrooxindole in two steps, according to a literature procedure.[18] For all of the 3-substituted indoline-2-ones with R1 = H (1, a–f), only the Z isomer was obtained This was verified with NOE experiments in which an NOE effect was observed between the vinylic proton and the C4 aromatic proton Compounds with R1 = CH3 (5 h–i) were isolated exclusively as the E isomer (i.e., h) or as mixtures of both the Z and E isomers (i.e., i; Table 1) The 5-amino indolinone g was synthesized by reduction of 5-nitro indolinone c (Scheme 2).[19] N1-substituted compounds a–c were synthesized by acylation or alkylation of semaxanib Acylation and alkylation of semaxanib occurred exclusively at the nitrogen atom of the in- Scheme Synthesis of N1-substituted indolin-2-one derivatives a–c and N1-substituted N1’-methylindolin-2-one derivatives a–c Reagents and conditions: a) R3 = Ac: Ac2O, Et3N, DMAP, CH2Cl2, RT; R3 = CH3 : NaH, MeI, DMF (6 b) or THF (7 b), RT; R3 = Boc: (Boc)2O, DMAP, CH2Cl2, RT ratios of the stereoisomers for a–c were determined by H NMR spectroscopy (Table 1) It was observed that the vinyl hydrogen for the Z isomer resonated downfield to the E isomer by ~ 0.2–0.3 ppm Compound was synthesized by condensing indoline-2-one with pyrrole-2-carboxaldehyde under standard Knoevenagel conditions.[4] Scheme Synthesis of 5-amino indolinone g Reagents and conditions: a) Pd/C (10 %), H2, EtOH, RT, 16 h, 34 % ChemMedChem 2016, 11, 72 – 80 www.chemmedchem.org 73  2016 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Full Papers Photoinduced isomerization studies In order to assess the effect of substituents on the ease of the photoisomerization process, isomerization studies were carried out The isomerization was monitored by 1H NMR spectroscopy, as this method can be carried out in real-time, is rapid and quantitative, and can be carried out in a neutral solvent ([D6]DMSO) In a typical experiment, the indolin-2-ones in [D6]DMSO were exposed to fluorescent light, and their 1H NMR spectra were acquired at various time intervals (0.25, 0.5, 1, 2, 3, 4, 5, 6, 12, and 24 h) Table shows the ratio of Z/E isomers Figure Kinetics of Z!E photoisomerization of semaxanib (1) in [D6]DMSO Data were determined at various time points by 1H NMR spectroscopy Table Indolinone Z/E isomer ratios before (t0) and after 24 h light exposure (t24), and chemical shifts (d) of the vinyl protons Compd R1 R2 R3 5a 5b 5c 5d 5e 5f 5g 5h 5i 6a 6b 6c 7a 7b 7c H H H H H H H H CH3 CH3 H H H CH3 CH3 CH3 – - H F Ac NO2 OMe NHAc OH NH2 H F H H H H H H – – H H H H H H H H H H Ac CH3 Boc Ac CH3 Boc – – t0 Z/E ratio t24 100:0 100:0 100:0 100:0 100:0 100:0 100:0 100:0 0:100 3:97 100:0 100:0 100:0 16:84 11:89 14:86 100:0 100:0 64:36 70:30 89:11 94:6 69:31 88:12 100:0 100:0 18:82 28:72 74:26 83:17 72:28 23:77 20:80 25:75 65:35 55:45 Table Rate constants of Z!E isomerization in light and reversion in dark of semaxanib and 3-substituted indolin-2-one analogues in [D6]DMSO d [ppm] Z isomer E isomer 7.55 7.63 7.77 7.95 7.57 7.77 7.40 7.29 7.59 7.67 7.66 7.60 7.63 7.81 7.66 7.75 7.73 7.74 7.33 7.38 7.41 7.48 7.32 7.49 NA NA 7.42 7.50 7.51 7.41 7.46 7.65 7.51 7.58 7.40 7.51 www.chemmedchem.org R1 R2 R3 5a 5b 5c 5d 5e 5f 5g 5h 6a H H H H H H H H CH3 H H F Ac NO2 OMe NHAc OH NH2 H H H H H H H H H H H Ac Rate constant [hÀ1][a] Isomerization Reversion 0.048 0.048 0.043 0.009 0.019 0.021 None None 0.090[b] 0.041 0.002 0.003 0.018 0.006 0.061 0.001 – – None 0.033 [a] Concentration of the samples was 10 mm in [D6]DMSO [b] Isomerization from E to Z room temperature, and 1H NMR spectra were recorded at different time intervals to study the E-to-Z isomer conversion The E isomer of semaxanib reverted back to the Z isomer in the dark with a slower observed rate constant of 0.002 hÀ1 In a similar manner, the rate constants of Z!E or E!Z photoisomerization and reversion of semaxanib and related 3-substituted indolin-2-one derivatives were measured as shown in Table In general, the rate constants for isomerization ranged from (no isomerization for compounds f and g) to 0.09 hÀ1 With the exception of h, the rate constants measure Z!E photoisomerization For those with measurable reversion, the rate constants ranged from 0.001 to 0.061 hÀ1 Thus there is little discernible correlation between the rate constants for isomerization and reversion with the nature of the substituents of the indolin-2-one before exposure to light and the Z/E ratio after exposure to light for 24 h With the exception of 5-hydroxy compound f and 5-amino compound g, the Z/E ratio of all other indolinones changed upon exposure to fluorescent light, in the range of ~ 6–36 % The kinetics of the photoisomerization of selected indolin-2ones were investigated Figure shows the formation of the E isomer of semaxanib in solution after exposure to light at different time intervals Prior to light exposure, only (Z)-semaxanib was present, and the formation of the E isomer reached equilibrium at 36 % after 24 h The formation of the E isomer followed first-order kinetics, and the rate constant for isomerization of (Z)-semaxanib to (E)-semaxanib was determined to be 0.048 hÀ1 (Table 3) The E isomer of semaxanib was not stable in solution and reverted back to the Z isomer when left in the dark To determine the reversion kinetics of semaxanib in the dark, the compound was exposed to fluorescent light for 24 h, following which, the sample was kept in the dark at ChemMedChem 2016, 11, 72 – 80 Compd Theoretical studies We attempted to rationalize the observed ease of photoisomerization by assessing the HOMO and LUMO energies of compounds a–h and a We used a similar approach to that taken by Tomasi et al.,[20] using B3LYP/6-311 + + G(d,p)//B3LYP/ 6-311G(d,p) with IEF-PCM solvation in DMSO, as implemented in Gaussian 09.[21] A summary of the calculated UV/Vis results is shown in Table 74  2016 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Full Papers Table Calculated electronic spectra for selected indolinones Table HOMO and LUMO energies of selected (Z)-indolinones Compd l [nm] Oscillator strength Compd l [nm] Oscillator strength Compd (Z)-1 422.3 361.1 305.3 423.9 367.2 307.6 421.6 375.2 348.2 525.1 415.5 356.3 439.0 395.7 304.6 424.9 373.9 307.4 439.6 390.4 305.2 476.4 401.0 303.8 427.5 365.0 325.3 430.2 346.4 315.1 0.679 0.230 0.025 0.625 0.309 0.026 0.749 0.009 0.309 0.022 0.864 0.353 0.232 0.699 0.025 0.606 0.343 0.023 0.297 0.623 0.025 0.087 0.825 0.021 0.613 0.191 0.028 0.717 0.086 0.109 (E)-1 415.1 358.1 295.6 421.3 365.0 298.5 415.4 376.4 348.5 526.5 412.5 359.8 437.8 385.6 295.0 420.9 369.8 300.9 438.3 382.2 295.6 473.8 390.2 297.7 419.3 362.1 317.6 419.2 346.8 324.0 0.604 0.182 0.026 0.543 0.241 0.028 0.645 0.010 0.159 0.030 0.667 0.237 0.228 0.542 0.026 0.510 0.263 0.052 0.285 0.486 0.027 0.122 0.634 0.061 0.570 0.085 0.026 0.704 0.105 0.039 5a 5b 5c 5d 5e 5f 5g 5h 6a (Z)-5 a (Z)-5 b (Z)-5 c (Z)-5 d (Z)-5 e (Z)-5 f (Z)-5 g (Z)-5 h (Z)-6 a (E)-5 a (E)-5 b (E)-5 c (E)-5 d (E)-5 e (E)-5 f (E)-5 g (E)-5 h (E)-6 a www.chemmedchem.org Z isomer HOMO À2.27 À2.34 À2.39 À2.89 À2.25 À2.33 À2.27 À2.22 À2.22 À2.48 À5.47 À5.55 À5.57 À5.70 À5.46 À5.53 À5.45 À5.31 À5.45 À5.59 HOMO-1 À6.33 À6.31 À6.54 À6.82 À5.86 À6.23 À5.93 À5.66 À6.27 À6.69 dolinone Consistent with experimental observations, calculations show that the Z isomer is the more stable isomer for compounds 1, a–g, and a, while the E isomer is the more stable isomer for h Cytotoxicity studies The cytotoxicities of the compounds were first determined using the MTT cell proliferation assay against the TAMH cell line, selected for its ability to metabolize xenobiotics and to reproduce the toxicity observed with classical hepatotoxicants, such as acetaminophen.[22] The 50 % inhibitory concentration value (IC50) was estimated using GraphPad Prism (Table 6) Table Cytotoxicity and Z/E isomer ratios of 3-substituted indolin-2ones An examination of the oscillator strengths (f) of each pair of isomers in the UV region of ~ 400 nm shows that compounds 1, a–c, e, h, and a have high f values, in the range of ~ 415 nm to 430 nm, as compared with compounds d, f, and g With the exception of d and h, these are reflective of the ease of Z!E isomerization, such that those with large f values for the Z isomers are easier to isomerize and vice versa With h, E!Z isomerization occurs readily, as is reflected by the high f value for the E isomer Based solely on our results, the isomerization of d does not fit the trend of the others The UV/Vis results for d and f are very similar, but the rates of their photoisomerization are different If one considers that photoisomerization is a consequence of S0 to S1 transitions, then the calculated HOMO and LUMO energies of the Z isomers of 1, a–h, and a may provide further insight into their ease of isomerization As shown in Table 5, the FMO energies are broadly similar, and the energy difference between the HOMO and LUMO does not explain the observed isomerization rates From these calculations, the dihedral angle of C=CÀCÀN for the Z isomers is roughly zero, indicating planarity, while the same dihedral angle is ~ 108 out of plane for the E isomers The exception is h, where both the E and Z isomers have a dihedral angle of ~ 308, which is the consequence of N-methylation at the pyrrole ring, which in turn, does not allow for intramolecular hydrogen bonding with the carbonyl group on the inChemMedChem 2016, 11, 72 – 80 LUMO Compd R1 R2 R3 5a 5h 5i 6a 6b 6c 7a 7b 7c H H CH3 CH3 H H H CH3 CH3 CH3 – – H F H F H H H H H H – – H H H H Ac CH3 Boc Ac CH3 Boc – – Z/E ratio IC50 [mm][a] 100:0 100:0 0:100 3:97 100:0 100:0 100:0 16:84 11:89 14:86 100:0 100:0 6.28 Ỉ 1.24 2.17 Ỉ 0.47 > 50 > 50 9.77 Ỉ 0.67 3.76 Ỉ 0.61 0.69 Ỉ 0.12 > 50 > 50 11.94 Ỉ 0.08 11.28 Ỉ 0.65 21.53 Ỉ 2.40 [a] Values are the mean Ỉ SD of three independent experiments When comparing the compounds with a hydrogen atom on the N1’ of the pyrrole ring (i.e., 1, 2, a, a–c, 8), (Z)-8 was found to be the least toxic to TAMH cells (IC50 = 21.53 mm) The IC50 value of (Z)-2 was 11.28 mm, which is higher than for indolinones 1, a, and a–c Compounds substituted at the N1 posi75  2016 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Full Papers tion of the oxindole ring, such as (Z)-6 b (R3 = CH3) and (Z)-6 c (R3 = Boc), were more toxic (IC50 values of 3.76 and 0.69 mm, respectively) (Z)-6 a (R3 = Ac) was less toxic (IC50 = 9.77 mm) than the unsubstituted (Z)-1 (R3 = H; IC50 = 6.28 mm) Fluorine substitution on the oxindole core (compound a; R2 = F) had increased toxicity relative to compound (Z)-1 In contrast, compounds with substitution at the nitrogen atom of the pyrrole ring were generally less toxic to the TAMH cell line With the exception of c (IC50 = 11.94 mm), all compounds in this series showed IC50 values greater than 50 mm Overall, the toxicities of the compounds varied Boc substitution at the N1 position of the indolin-2-ones resulted in c and c (R3 = Boc), and these were the most toxic compounds within their series (R1 = H or Me) Moreover, it could be inferred that methylation at the N1’ position and/or the E isomeric forms could decrease the toxicity of the 3-[(substituted pyrrol2-yl)methylidenyl]indolin-2-one series To determine if the E isomers were indeed less toxic, the E/Z mixtures of 1, 2, a, and were also tested against the TAMH and HepG2 cell lines (Z)-8 is the parent compound, while (Z)1 (semaxanib) and (Z)-2 (sunitinib) are potent anticancer agents (Z)-5 a was also selected, because it is similar to (Z)1 but has an isosteric replacement (R2 = F) The stability of 100 mm drug stocks of these compounds in glass vials was independently determined They were exposed to fluorescent lighting, and after 24 h, the E/Z ratios were determined using NMR spectroscopy by diluting 50 mL of the stocks solution to 500 mL in [D6]DMSO In all of the cases above, isomerization was observed The observed E/Z ratios were only slightly lower than those listed in Table 2, with the exception of a, for which the percentage of (E)-5 a was significantly lower (Table 7) For and a, the E/Z mixtures were more toxic than (Z)1 and (Z)-5 a, while the reverse was true for and in the TAMH cell line The differences between the IC50 values between the pure Z isomers and the E/Z mixtures were not large for a and (2.17 mm vs 1.59 mm and 11.28 mm vs 16.66 mm, respectively) The IC50 value was 6.28 mm for (Z)-1 but was 2.99 mm for the E/Z mixture, which contained 31 % E isomer and 69 % Z isomer For (Z)-8, the IC50 value was 21.53 mm, while its E/Z mixture was less toxic to the TAMH cell line (IC50 > 50 mm) For HepG2 cell line, Z isomers of 1, a, and were more toxic than their respective E/Z mixtures, while the E/Z mixture of was not significantly more toxic than (Z)-8 (2.38 and 2.17 mm, respectively) It was observed that the E/Z mixture of was the most toxic compound in the HepG2 cell line Thus, E/Z isomerism can affect the toxicity of the compounds Whether the E/Z mixtures or the isomerically pure samples were more toxic could not be generalized, as this was dependent on the structures of the compounds and the differential handling of the compounds by the host cell line (Table 7) Conclusions A total of 16 compounds that were structurally related to semaxanib (1) and sunitinib (2) were synthesized in yields of 15 % to 90 % For a–g, a–c, and 8, only the Z isomers were obtained In contrast, only the E isomer of h was obtained, while i and a–c were obtained as E/Z mixtures but with the E form predominating Overall, a majority of the compounds tested were able to undergo isomerization The rate of isomerization and the amount of the less stable isomer varied The stabilities of the E/Z mixtures were also examined, and it was found that without continual exposure to the light source, some mixtures converted back to the more stable form, with variable rates of reversion All compounds were tested in the TAMH cell line Generally, compounds with N1’ substitutions that had the E isomers as the predominant form were less toxic to the TAMH cell line, while compounds that were in the Z forms decreased cell viability to a greater extent The E/Z mixtures of the four selected compounds that were of clinical importance (1, 2, a, and 8) were tested for toxicity in the TAMH and HepG2 cell lines With respect to TAMH cell line toxicity, the E/Z mixture of was more toxic to the cells than (Z)-1, while the reverse was true for and However, it should be noted that the differences in the IC50 values between the pure Z isomers and the E/Z mixtures for a and were not large For the HepG2 cell line, Z isomers of 1, a, and were more toxic than the E/Z mixtures The differences in the observed trends between the two liver cells lines are indicative of the sensitivities of the responses of the cell lines to the stereoisomers We note that HepG2 is a liver cancer cell line, and the effects of these RTK inhibitors on HepG2 cannot be dissociated from the toxicity The significance of our study should be realized Such findings could prompt the appropriate handling of clinically available drugs (e.g., sunitinib) and demonstrate that protection of the drugs from light could be vital For future lead compounds that contain exocyclic double bonds, it may be advantageous to separately investigate the contributions of their more stable isomers, less stable isomers, and/or their E/Z mixtures to activities and toxicities Removing such unsaturation and/or design- Table Z/E isomer ratios of the indolinones and their corresponding IC50 values against TAMH and HepG2 cell lines Compd 5a Z/E ratio 100:0 100:0 100:0 100:0 TAMH IC50 [mm][a] 6.28 Æ 1.24 11.28 Æ 0.65 2.17 Æ 0.47 21.53 Æ 2.40 Z/E ratio HepG2 8.17 Ỉ 0.39 5.85 Ỉ 1.20 12.11 Ỉ 0.26 2.38 Ỉ 1.67 69:31 69:31 83:17 56:44 TAMH IC50 [mm][a] 2.99 Ỉ 0.36 16.66 Ỉ 1.52 1.59 Æ 0.29 > 50 HepG2 37.55 Æ 10.03 10.36 Æ 4.43 21.57 Ỉ 2.74 2.17 Ỉ 1.13 [a] Values are the mean Ỉ SD of three independent experiments ChemMedChem 2016, 11, 72 – 80 www.chemmedchem.org 76  2016 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Full Papers ing stereochemically stable compounds may be feasible, provided that the activities are not compromised This would avoid the issue of E/Z isomerization matography (CH2Cl2) afforded (Z)-5 a as an orange solid in 58 % yield (60.9 mg): mp: 275–277 8C (lit.[24] 271–273 8C); 1H NMR (400 MHz, CDCl3): d = 13.14 (br s, H), 7.65 (br s, H), 7.33 (s, H), 7.17 (m, H), 6.81 (m, H), 6.00 (s, H), 2.38 (s, H), 2.33 ppm (s, H); 13C NMR (100 MHz, CDCl3): d = 169.9, 168.9, 160.2, 157.9, 137.9, 133.7, 132.7, 128.1, 128.0, 127.2, 124.4, 113.1, 111.8, 111.6, 109.6, 109.5, 104.7, 104.4, 14.0, 11.7 ppm (number of carbon signals was greater than expected due to F coupling); HRMS (ESI-TOF): (m/ z) calcd for C15H13FN2O [M + H] + 257.1090, found 257.1090 Experimental Section General methods: (Z)-2 (Sunitinib malate) was used as purchased from LC laboratories All other reagents and solvents were purchased from Sigma–Aldrich or Alfa Aesar and were used without further purification unless otherwise specified Reactions involving air- or moisture-sensitive reagents were performed with dried glassware under a nitrogen atmosphere Thin layer chromatography (TLC) was performed on Merck precoated silica gel plates Visualization was accomplished with UV light or by staining with KMnO4 solution Compounds were purified by flash chromatography on columns using Merck silica gel 60 (230–400 mesh) unless otherwise specified The purity of the compounds were determined using analytical HPLC with a Phenomenex Kinetex 2.6 mm C18 100 Š (150 ” 4.60 mm) column at 254 nm All compounds were > 95 % pure Mass spectra were recorded on an Applied Biosystems MDS SCIEX API 2000 mass spectrometer High-resolution mass spectra (HRMS) were recorded on an Agilent mass spectrometer using electrospray ionization–time of flight (ESI-TOF) Analytical LC–MS was performed on a Phenomenex Luna mm C18 100 Š (50 ” 3.0 mm) column Melting points (mp) were recorded on a Gallenkamp melting point apparatus and are uncorrected NMR spectra were recorded at 400 MHz for 1H and at 100 MHz for 13C on a Bruker spectrometer with CDCl3 or [D6]DMSO as solvent The chemical shifts are given in ppm, using the proton solvent residue signal (CDCl3 : d = 7.26; [D6]DMSO: d = 2.50) as a reference in the H NMR spectrum The deuterium coupled signal of the solvent was used as reference in 13C NMR (CDCl3 : d = 77.0; [D6]DMSO: d = 39.5) The following abbreviations were used to describe the signals: s = singlets, d = doublet, t = triplet, m = multiplet, br = broad signal (Z)-5-Acetyl-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2one (5 b): Following the general procedure, a solution of 5-acetyl2-oxindole (100 mg, 0.57 mmol) and 3,5-dimethyl-2-carboxaldehyde (4 a; 84 mg, 0.68 mmol), and piperidine (3 drops) in EtOH (7 mL) was stirred at 75 8C for h to give 5-acetyl derivative b as a brown solid (84.8 mg, 53 %): mp: 297–298 8C (decomposed); H NMR (400 MHz, [D6]DMSO): d = 13.33 (s, H), 11.14 (br s, H), 8.35 (d, J = 1.6 Hz, H), 7.75–7.77 (m, H), 6.96 (d, J = 8.4 Hz, H), 6.05 (d, J = Hz, H), 2.59 (s, H), 2.36 (s, H), 2.34 ppm (s, H); 13 C NMR (100 MHz, [D6]DMSO): d = 196.9, 169.8, 141.8, 136.7, 133.0, 130.5, 126.9, 126.6, 125.9, 124.7, 118.5, 113.0, 111.3, 108.8, 26.6, 13.5, 11.4 ppm; HRMS (ESI-TOF): (m/z) calcd for C17H16N2O2 [M + H] + 281.1290, found 281.1287 (Z)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-5-nitroindolin-2one (5 c): Following the general procedure, a solution of 5-nitro-2oxindole (96 mg, 0.54 mmol) and 3,5-dimethyl-2-carboxaldehyde (4 a; 80 mg, 0.65 mmol), and piperidine (3 drops) in EtOH (8 mL) was stirred at 75 8C for h The resulting precipitate was filtered and washed with cold EtOH to give 5-nitro c as a brown solid (114.2 mg, 75 %): mp: > 300 8C (lit.[25] > 280 8C); 1H NMR (400 MHz, [D6]DMSO): d = 13.35 (s, H), 11.43 (s, H), 8.78 (d, J = 2.4 Hz, H), 8.03 (dd, J = 8.4, 2.4 Hz, H), 7.95 (s, H), 7.04 (d, J = 8.4 Hz, H), 6.10 (d, J = 2.0 Hz, H), 2.38 (s, H), 2.36 ppm (s, H); 13C NMR (100 MHz, [D6]DMSO): d = 169.8, 142.9, 142.0, 138.3, 134.9, 127.2, 126.8, 126.4, 121.5, 113.8, 113.6, 109.8, 108.9, 13.6, 11.4 ppm; HRMS (ESI-TOF): (m/z) calcd for C15H13N3O3 [M + H] + 284.1035, found 284.1039 General procedure for Knoevenagel condensation: Piperidine (3 drops) was added to a solution of 5-substitiuted-2-oxindole (1.0 equiv) and 3,5-dimethyl-2-carboxaldehyde (1.2 equiv) in EtOH (8 mL) The reaction mixture was heated at 75 8C for h The reaction mixture was then cooled to room temperature, and the resulting precipitate was filtered and washed with cold EtOH to give the 5-substituted-2-oxindole compound (Z)-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-5-methoxyindolin2-one (5 d): Following the general procedure, a solution of 5-methoxy-2-oxindole (75 mg, 0.46 mmol) and 3,5-dimethyl-2-carboxaldehyde (4 a; 68 mg, 0.55 mmol), and piperidine (3 drops) in EtOH (7 mL) was stirred at 75 8C for h The residue was purified by column chromatography (CH2Cl2/MeOH, 99:1) to give 5-methoxy derivative d as a brown solid (90.1 mg, 73 %): mp: 256–257 8C (decomposed); 1H NMR (400 MHz, [D6]DMSO): d = 13.44 (s, H), 10.56 (s, H), 7.57 (s, H), 7.39 (d, J = 2.4 Hz, H), 6.75 (d, J = 8.4 Hz, H), 6.67 (dd, J = 8.4, 2.4 Hz, H), 6.00 (d, J = 2.0 Hz, H), 3.77 ppm (s, H); 13C NMR (100 MHz, [D6]DMSO): d = 169.5, 154.7, 135.5, 132.0, 131.6, 126.8, 126.6, 123.6, 113.2, 112.4, 111.9, 109.6, 104.1, 55.6, 13.5, 11.3 ppm HRMS (ESI-TOF): (m/z) calcd for C16H16N2O2 [M + H] + 269.1290, found 269.1286 (3Z)-3-[(3,5-Dimethyl-1H-pyrrol-2-yl)methylidene]-1,3-dihydro2H-indol-2-one ((Z)-1): Following the general procedure, a solution of 3,5-dimethyl-2-carboxaldehyde (4 a; 111 mg, 0.90 mmol), indolin2-one (100 mg, 0.75 mmol), and piperidine (3 drops) in EtOH (2 mL) was stirred at 90 8C for h The resulting precipitate was filtered, washed with cold EtOH, and dried to give (Z)-1 as an orange solid in 62 % yield (111 mg): mp: 232–234 8C (lit.[23] 220–222 8C); 1H NMR (400 MHz, [D6]DMSO): d = 13.35 (br s, H), 10.77 (br s, H), 7.71 (d, J = 7.2 Hz, H), 7.55 (s, H), 7.09 (m, H), 6.97 (m, H), 6.87 (d, J = 7.6 Hz, H), 6.00 (d, J = 2.4 Hz, H), 2.32 (s, H), 2.30 ppm (s, H); 13 C NMR (100 MHz, [D6]DMSO): d = 169.3, 138.0, 135.5, 131.4, 126.5, 125.7, 125.6, 123.3, 120.7, 117.9, 112.6, 112.4, 109.1, 13.4, 11.2 ppm; HRMS (ESI-TOF): (m/z) calcd for C15H14N2O [M + H] + 239.1184, found 239.1182 (Z)-N-(3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-2-oxoindolin-5yl)acetamide (5 e): Following the general procedure, a solution of 5-acetamide-2-oxindole (64 mg, 0.34 mmol) and 3,5-dimethyl-2-carboxaldehyde (4 a; 50 mg, 0.41 mmol), and piperidine (3 drops) in EtOH (5 mL) was stirred at 75 8C for h The residue was purified by column chromatography (CH2Cl2/MeOH, 99:1) to give 5-acetamide derivative e as a yellow solid (70.8 mg, 71 %): mp: > 350 8C (decomposed); 1H NMR (400 MHz, [D6]DMSO): d = 13.36 (s, H), 10.70 (s, H), 9.75 (s, H), 7.77 (d, J = 2.0 Hz, H), 7.36 (s, H), 7.22 (dd, J = 8.4, 2.0 Hz, H), 6.79 (d, J = 8.4 Hz, H), 6.01 (d, J = 2.0 Hz, H), 2.32 (s, H), 2.28 (s, H), 2.02 ppm (s, H); 13C NMR (100 MHz, (Z)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-5-fluoroindolin-2one ((Z)-5 a): Following the general procedure, a solution of 3,5-dimethyl-2-carboxaldehyde (4 a; 50 mg, 0.41 mmol), 5-fluoroindolin2-one (61 mg, 0.41 mmol), and piperidine (3 drops) in EtOH (3 mL) was stirred at 75 8C for 12 h Purification by silica gel column chroChemMedChem 2016, 11, 72 – 80 www.chemmedchem.org 77  2016 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Full Papers [D6]DMSO): d = 169.5, 167.7, 135.7, 134.2, 133.1, 131.5, 126.4, 125.7, 122.8, 118.0, 112.8, 112.5, 110.1, 109.1, 23.7, 13.5, 11.2 ppm; HRMS (ESI-TOF): (m/z) calcd for C17H17N3O2 [M + H] + 296.1399, found 296.1404 (Z)-3-((1H-pyrrol-2-yl)methylene)indolin-2-one ((Z)-8): Following the general procedure, a solution of 1H-pyrrole-2-carbaldehyde (86 mg, 0.9 mmol), indolin-2-one (100 mg, 0.75 mmol), and piperidine (3 drops) in EtOH (2 mL) was stirred at 90 8C for h The resulting precipitate was filtered, washed with cold EtOH, and dried to give (Z)-8 as an orange solid in 66 % yield (104 mg): mp: 234– 236 8C (lit.[26] 210–213 8C) ; 1H NMR (400 MHz, [D6]DMSO): d = 13.34 (br s, H), 10.88 (br s, H), 7.74 (s, H), 7.63 (d, J = 7.2 Hz, H), 7.35 (br s, H), 7.14 (td, J = 7.6, 1.2 Hz, H), 7.00 (m, H), 6.88 (d, J = 7.6 Hz, H), 6.83 (m, H), 6.36–6.34 ppm (m, H); 13C NMR (100 MHz, [D6]DMSO): d = 169.1, 138.9, 129.5, 126.8, 126.2, 125.5, 125.1, 121.1, 120.1, 118.4, 116.7, 111.3, 109.4 ppm; HRMS (ESI-TOF): (m/z) calcd for C13H10N2O [M + H] + 211.0871, found 211.0873 (Z)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-5-hydroxyindolin2-one (5 f): Following the general procedure, a solution of 5-hydroxy-2-oxindole (34 mg, 0.23 mmol) and 3,5-dimethyl-2-carboxaldehyde (4 a; 33 mg, 0.27 mmol), and piperidine (3 drops) in EtOH (5 mL) was stirred at 75 8C for 16 h The residue was purified by column chromatography (CH2Cl2/MeOH, 97:3 to 95:5) to give 5-hydroxy derivative f as an orange solid (33.2 mg, 57 %): 1H NMR (400 MHz, [D6]DMSO) d13.41 (s, H), 10.46 (s, H), 7.40 (s, H), 7.09 (d, J = 2.4 Hz, H), 6.65 (d, J = 8.4 Hz, H), 6.53 (dd, J = 8.4, 2.4 Hz, H), 5.98 (d, J = 2.4 Hz, H), 2.30 (s, H), 2.28 ppm (s, H); 13C NMR (100 MHz, [D6]DMSO): d = 169.4, 152.2, 135.2, 131.1, 130.9, 126.8, 126.4, 122.9, 113.5, 112.7, 112.3, 109.6, 105.3, 13.5, 11.2 ppm; HRMS (ESI-TOF): (m/z) calcd for C15H14N2O2 [M + H] + 255.1134, found 255.1132 General procedure for the acylation reaction for the synthesis of a, a, c, and c: A mixture of (Z)-1 or (E)-5 h (1.0 equiv), DMAP (0.15 equiv), and (Boc)2O or Ac2O (1.2 equiv) with triethylamine (1.2 equiv) in CH2Cl2 was stirred under nitrogen at room temperature The reaction was monitored using TLC until no (Z)-1 or (E)-5 h could be detected The solvent was evaporated, and the mixture was purified using column chromatography (petroleum ether/ EtOAc, 4:1), unless otherwise specified If a mixture of E and Z isomers was obtained, the analytical data reported correspond to the major isomer The minor isomer is not reported (Z)-5-Amino-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2one (5 g): 10 % Pd/C (18 mg, 0.017 mmol Pd) was added to a suspension of 5-nitro compound c (60 mg, 0.21 mmol) in EtOH (2 mL) The reaction mixture was stirred under hydrogen atmosphere overnight The reaction mixture was then filtered through a pad of Celite The residue was purified by column chromatography (CH2Cl2/MeOH, 1:0 to 99:1) to give 5-amino derivative g as an orange solid (17.8 mg, 34 %): mp: 249–251 8C (decomposed); H NMR (400 MHz, [D6]DMSO): d = 13.39 (s, H), 10.34 (s, H), 7.29 (s, H), 6.89 (s, H), 6.56 (d, J = 8.0 Hz, H), 6.38 (dd, J = 8.0, 1.6 Hz, H), 5.96 (s, H), 4.59 (br s, H), 2.30 (s, H), 2.26 ppm (s, H); 13 C NMR (100 MHz, [D6]DMSO): d = 169.2, 143.1, 134.7, 130.3, 129.4, 126.3, 122.0, 114.1, 112.3, 112.1, 109.6, 104.3, 13.4, 11.2 ppm; HRMS (ESI-TOF): (m/z) calcd for C15H15N3O [M + H] + 254.1293, found 254.1292 (Z)-1-Acetyl-3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)indolin-2one ((Z)-6 a): Following the general procedure, a mixture of (Z)1 (200 mg, 0.84 mmol), DMAP (15 mg, 0.13 mmol), triethylamine (102 mg, 1.01 mmol), and Ac2O (121 mg, 1.01 mmol) in CH2Cl2 (6 mL) was stirred under nitrogen at room temperature Purification by silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded (Z)-6 a as an orange solid in 78 % yield (184 mg): mp: 196–198 8C ;1H NMR (400 MHz, CDCl3): d = 12.61 (br s, H), 8.25 (m, H), 7.49 (m, H), 7.40 (s, H), 7.20 (m, H), 6.04 (s, H), 2.80 (s, H), 2.42 (s, H), 2.35 ppm (s, H); 13C NMR (100 MHz, CDCl3): d = 171.4, 168.8, 138.1, 136.2, 134.5, 127.3, 126.4, 126.1, 124.4, 124.0, 116.3, 116.3, 113.5, 110.1, 27.1, 14.1, 11.7 ppm; HRMS (ESI-TOF): (m/ z) calcd for C17H16N2O2 [M + H] + 281.1290, found 281.1293 (E)-3-((1,3,5-Trimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one ((E)-5 h): Following the general procedure, a solution of b (200 mg, 1.46 mmol), indolin-2-one (162 mg, 1.21 mmol), and piperidine (3 drops) in EtOH (5 mL) was stirred at 75 8C for 24 h Purification by silica gel column chromatography (petroleum ether/ EtOAc, 4:1!100% EtOAc) afforded (E)-5 h as an orange solid in 46 % yield (140 mg): mp: 170–172 8C; 1H NMR (400 MHz, CDCl3): d = 7.67 (s, H), 7.64 (s, H), 7.17 (m, H), 6.95 (dt, J = 4.0, 8.0 Hz, H), 6.87 (d, J = 8.0 Hz, H), 5.94 (s, H), 3.48 (s, H), 2.29 (s, H), 1.97 ppm (s, H); 13C NMR (100 MHz, CDCl3): d = 170.1, 140.2, 135.3, 128.1, 126.8, 125.5, 125.3, 124.7, 123.0, 122.4, 121.7, 111.0, 109.3, 31.8, 13.8, 12.6 ppm; HRMS (ESI-TOF): (m/z) calcd for C16H16N2O [M + H] + 253.1341, found 253.1342 (Z)-tert-Butyl 3-((3,5-dimethyl-1H-pyrrol-2-yl)methylene)-2-oxoindoline-1 carboxylate ((Z)-6c): Following the general procedure, a mixture of (Z)-1 (50 mg, 0.21 mmol), DMAP (4 mg, 0.03 mmol), and (Boc)2O (37 mg, 0.17 mmol) in CH2Cl2 (4 mL) was stirred under nitrogen at room temperature Purification by silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded (Z)-6 c as an orange solid in 90 % yield (51.8 mg): 1H NMR (400 MHz, CDCl3): d = 12.82 (br s, H), 7.73 (m, H), 7.49 (m, H), 7.39 (s, H), 7.17 (m, H), 6.02 (s, H), 2.37 (s, H), 2.34 (s, H), 1.70 ppm (s, H); 13 C NMR (100 MHz, CDCl3): d = 167.9, 149.4, 138.1, 135.6, 134.1, 127.3, 126.0, 125.7, 123.7, 123.6, 116.6, 114.7, 113.3, 110.0, 84.2, 28.2, 14.0, 11.7 ppm; HRMS (ESI-TOF): (m/z) calcd for C20H22N2O3 [M + Na] + 361.1528, found 361.1523 (E)-5-Fluoro-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methylene)indolin2-one ((E)-5i): Following the general procedure, a solution of b (157 mg, 1.14 mmol), 5-fluoroindolin-2-one (208 mg, 1.37 mmol), and piperidine (3 drops) in EtOH (5 mL) was stirred at 75 8C for 12 h Purification by silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded i as an E/Z mixture ((E)-5 i:(Z)-5 i = 97:3) as an orange solid in 15 % (46.2 mg) combined yield: 1H NMR E isomer (400 MHz, CDCl3): d = 8.52 (s, H), 7.68 (s, H), 6.86 (m, H), 5.97 (s, H), 3.49 (s, H), 2.29 (s, H), 1.98 ppm (s, H); 13 C NMR E isomer (100 MHz, CDCl3): d = 170.2, 159.9, 157.6, 136.2, 136.1, 126.7, 126.7, 126.2, 126.2, 114.3, 114.0, 110.2, 109.9, 109.5, 109.4, 31.8, 13.9, 12.7 ppm (number of carbon signals was greater than expected due to F coupling); HRMS (ESI-TOF): (m/z) calcd for C16H15FN2O [M + H] + 271.1247, found 271.1239 ChemMedChem 2016, 11, 72 – 80 www.chemmedchem.org (E)-1-Acetyl-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methylene)indolin2-one ((E)-7 a): Following the general procedure, a mixture of (E)5 h (100 mg, 0.40 mmol), DMAP (7 mg, 0.06 mmol), triethylamine (61 mg, 0.60 mmol), and Ac2O (72 mg, 0.60 mmol) in CH2Cl2 (6 mL) was stirred under nitrogen at room temperature Purification by silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded a as an E/Z mixture ((E)-7 a:(Z)-7 a = 84:16) as an orange solid in 58 % (68.2 mg) combined yield: 1H NMR E isomer (400 MHz, [D6]DMSO): d = 8.18 (d, J = Hz, H), 7.65 (s, H), 7.31 (m, H), 7.17 (m, H), 6.00 (s, H), 3.48 (s, H), 2.66 (s, H), 2.28 (s, H), 1.87 ppm (s, H); 13C NMR E isomer (100 MHz, [D6]DMSO): d = 170.5, 167.7, 138.5, 137.1, 128.1, 126.4, 126.2, 125.7, 124.3, 122.9, 78  2016 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Full Papers 121.3, 118.8, 115.4, 111.5, 31.6, 26.4, 13.9, 12.3 ppm; HRMS (ESITOF): (m/z) calcd for C18H18N2O2 [M + H] + 295.1447, found 295.1439 Isomerization study: Photoisomerization experiments were performed with 10 mm [D6]DMSO solutions of 3-substituted indoline2-one derivatives in NMR tubes The NMR tubes were then exposed to fluorescent light (Philips Tornado 5W ES 6500 K cool daylight, 285 lumens) at ambient temperature (22–24 8C), with a light intensity of ~ 1700 lux (measured with a Gossen Luna-Pro F light meter) The samples were analyzed by 1H NMR spectroscopy at various time points (0.25, 0.5, 1, 2, 3, 4, 5, 6, 12, 24 h) For the analysis of isomerization in the dark, after the samples were exposed to light for 24 h, the samples were then protected from light At various time points (0.5, 1, 2, 3, 4, 5, 6, 12, 24, 48, 72 h), samples were analyzed by 1H NMR spectroscopy (E)-tert-Butyl 2-oxo-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methylene)indoline-1-carboxylate ((E)-7 c): Following the general procedure, a mixture of (E)-4 a (50 mg, 0.20 mmol), DMAP (4 mg, 0.03 mmol), and (Boc)2O (52 mg, 0.24 mmol) in CH2Cl2 (5 mL) was stirred under nitrogen at room temperature Purification by silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded c as an E/Z mixture ((E)-7 c:(Z)-7 c = 86:14) as an orange oil in 90 % (63.4 mg) combined yield: 1H NMR E isomer (400 MHz, CDCl3): d = 7.89 (m, H), 7.68 (s, H), 7.24 (m, H), 7.06 (m, H, H-7), 5.97 (s, H), 3.46 (s, H), 2.28 (s, H), 1.94 (s, H), 1.67 ppm (s, H); 13 C NMR E isomer (100 MHz, [D6]DMSO): d = 165.5, 148.8, 138.0, 136.6, 128.2, 126.3, 125.8, 125.3, 123.6, 122.3, 121.5, 119.0, 114.0, 111.3, 83.3, 31.5, 27.7, 13.9, 12.3 ppm; HRMS (ESI-TOF): (m/z) calcd for C21H24N2O3 [M + Na] + 375.1685, found 375.1678 Isomerization of 100 mm compound stocks: The 100 mm drug stocks of (Z)-1, (Z)-5 a, (Z)-2, and (Z)-10 were exposed to fluorescent lighting At the end of 24 h, the E/Z ratios were determined using NMR spectroscopy by diluting 50 mL of the stock solutions to 500 mL using [D6]DMSO General procedure for the alkylation reaction for the synthesis of b and b: (Z)-1 (1.0 equiv) in DMF or THF (2 mL) was added to a stirring suspension of NaH (1.0 equiv for the synthesis of b; 2.2 equiv for b) under nitrogen After h, iodomethane (1.0 equiv for the synthesis of b; 2.2 equiv for b) was added The reaction was monitored using TLC until no (Z)-1 could be detected Upon completion, 0.5 mL of saturated ammonium chloride was added The mixture was extracted with EtOAc (3 ” 10 mL) The combined organic layers were washed with brine and dried with Na2SO4 The solvent was evaporated, and the mixture was purified using column chromatography (petroleum ether/EtOAc, 4:1), unless otherwise specified If a mixture of E and Z isomers was obtained, the analytical data correspond to the major isomer The minor isomer is not reported Biology Reagents and general procedures: Gentamicin and the soybean trypsin inhibitor were from Invitrogen Phosphate-buffered saline (PBS) was purchased from Bio-Rad, and 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide (MTT) dye was purchased from Duchefa Drug stocks (10 mm and 100 mm) were prepared in DMSO and were stored at À20 8C TAMH cells were maintained in a T75 flask with Dulbecco’s modified Eagle’s medium (DMEM):Nutrient Mixture F-12 (DMEM/F-12) supplemented with ITS (5 mg mLÀ1 insulin, mg mLÀ1 transferrin, ng mLÀ1 selenium), 10 mm nicotinamide, 100 nm dexamethasone, and 10 mg mLÀ1 gentamicin, while HepG2 was maintained in minimal essential medium in the presence of 10 % fetal bovine serum Both lines were incubated at 37 8C in 95 % air and % CO2 (Z)-3-((3,5-Dimethyl-1H-pyrrol-2-yl)methylene)-1-methylindolin2-one ((Z)-6b): Following the general procedure, (Z)-1 (20 mg, 0.08 mmol) in DMF (2 mL) was added to a stirring suspension of NaH (3.5 mg, 0.09 mmol) under nitrogen After h, iodomethane (13 mg, 0.09 mmol) was added The mixture was quenched and extracted Purification by silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded (Z)-6 b as an orange solid in 53 % yield (10.7 mg): mp: 162–164 8C; 1H NMR (400 MHz, CDCl3): d = 13.25 (br s, H), 7.51 (d, J = 8.0 Hz, H), 7.40 (s, H), 7.19 (dt, J = 1.2, 8.0 Hz, H), 7.08 (dt, J = 1.2, 8.0 Hz, H), 6.88 (d, J = 8.0 H, H), 5.97 (s, H), 3.38 (s, H), 2.38 (s, H), 2.33 ppm (s, H); 13C NMR (100 MHz, CDCl3): d = 168.4, 139.7, 136.5, 132.1, 127.0, 125.6, 125.5, 123.1, 121.6, 117.0, 112.5, 111.9, 107.8, 26.1, 13.9, 11.6 ppm; HRMS (ESI-TOF): (m/z) calcd for C16H16N2O [M + H] + 253.1341, found 253.1343 MTT cell proliferation assay: The TAMH cells were trypsinized to produce a single-cell suspension and were resuspended using 0.5 mg mLÀ1 of soybean trypsin inhibitor After centrifugation, the cell pellet was resuspended using the DMEM/F-12 media After counting the cells using a hemocytometer, the cell suspension was diluted to provide the desired density of 15 000 cells per well and then seeded into 96-well plates, where the cells were allowed to attach for 24 h The spent media was removed Drug stocks were diluted appropriately using DMEM/F-12 medium immediately before each assay Stocks (200 mL) were added to each well and were incubated for 24 h Cell viability was determined by reduction in MTT by viable cell dehydrogenases MTT was added to give a final concentration of 400 mg mLÀ1 in each well, and the plates were incubated at 37 8C for h before aspirating the supernatant and solubilizing the insoluble formazan product using 100 mL DMSO Absorbance at 570 nm was measured using an Infinite 200 microplate reader (Tecan) Cell viability in percentage was plotted against the concentrations of the drug IC50 values were determined using GraphPad Prism Software (E)-1-methyl-3-((1,3,5-trimethyl-1H-pyrrol-2-yl)methylene)indolin-2-one ((E)-7b): Following the general procedure, (Z)-1 (50 mg, 0.21 mmol) in THF (2 mL) was added to a stirring suspension of NaH (20 mg, 0.48 mmol) under nitrogen After h, iodomethane (69 mg, 0.48 mmol) was added The mixture was quenched and extracted Purification by silica gel column chromatography (petroleum ether/EtOAc, 4:1) afforded b as an E/Z mixture ((E)-7 b:(Z)7 b = 89:11) as an orange oil in 85 % (47.5 mg) combined yield: H NMR (E)-isomer (400 MHz, CDCl3): d = 7.65 (s, H), 7.21 (m, H), 6.97 (m, H), 6.83 (d, J = 8.0 Hz, H), 5.93 (s, H), 3.47 (s, H), 3.31 (s, H), 2.28 (s, H), 1.96 ppm (s, H); 13C NMR (E)-isomer (100 MHz, CDCl3): d = 168.9, 143.2, 134.9, 128.1, 126.8, 125.2, 124.8, 122.7, 122.2, 121.6, 110.8, 107.5, 31.7, 26.1, 13.8, 12.6 ppm; HRMS (ESITOF): (m/z) calcd for C17H18N2O [M + H] + 267.1497, found 267.1496 ChemMedChem 2016, 11, 72 – 80 www.chemmedchem.org Acknowledgements The authors thank Ms David P Sheela (National University of Singapore) for her assistance with some of the biological assays This work was supported by a National University of Singapore (NUS) start-up grant to C.L.L.C (R148000146133) and the A*STAR 79  2016 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim Full Papers Computational Resource Centre (for M.B.S.) for the use of its high-performance computing facilities Keywords: indolin-2-ones · computational cytotoxicity · kinetics · photoisomerization chemistry [16] T C Crawford (Pfizer Inc., New York), US Pat No US4652658, 1987 [17] J Caballero, C Munoz, J H Alzate-Morales, S Cunha, L Gano, R Bergmann, J Steinbach, T Kniess, Eur J Med Chem 2012, 58, 272 – 280 [18] M H Kim, A L Tsuhako, E W Co, D T Aftab, F Bentzien, J Chen, W Cheng, S Engst, L Goon, R R Klein, D T Le, M Mac, J J Parks, 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Kirkpatrick, Nat Rev Drug Discovery 2006, 5, 279 – 280 [10] S Faivre, G Demetri, W Sargent, E Raymond, Nat Rev Drug Discovery 2007, 6, 734 – 745 [11] A Sistla, N Shenoy, Drug Dev Ind Pharm 2005, 31, 1001 – 1007 [12] a) Y Zhao, J Sukbuntherng, J Pharm Biomed Anal 2005, 38, 479 – 486; b) Y Zhao, J Sukbuntherng, L Antonian, J Pharm Biomed Anal 2004, 35, 513 – 522 [13] A Sistla, W L Yang, N Shenoy, J Chromatogr A 2006, 1110, 73 – 80 [14] M C Etienne-Grimaldi, N Renee, H Izzedine, G Milano, J Chromatogr B 2009, 877, 3757 – 3761 [15] a) M Maafi, L Y Lee, J Pharm Biomed Anal 2015, 110, 34 – 41; b) S K Dewan, S Sangwan, R Thapar, M Prasad, J Appl Chem 2014, 3, 1055 – 1058 ChemMedChem 2016, 11, 72 – 80 www.chemmedchem.org Received: October 14, 2015 Published online on November 23, 2015 80  2016 Wiley-VCH Verlag GmbH & Co KGaA, Weinheim ... 35 6 .3 439 .0 39 5.7 30 4.6 424 .9 37 3.9 30 7.4 439 .6 39 0.4 30 5 .2 476.4 401.0 30 3.8 427 .5 36 5.0 32 5 .3 430 .2 34 6.4 31 5.1 0.679 0. 23 0 0. 025 0. 625 0 .30 9 0. 026 0.749 0.009 0 .30 9 0. 022 0.864 0 .35 3 0. 23 2 0.699... 36 9.8 30 0.9 438 .3 3 82. 2 29 5.6 4 73. 8 39 0 .2 297.7 419 .3 3 62. 1 31 7.6 419 .2 34 6.8 32 4.0 0.604 0.1 82 0. 026 0.5 43 0 .24 1 0. 028 0.645 0.010 0.159 0. 030 0.667 0. 23 7 0 .22 8 0.5 42 0. 026 0.510 0 .2 63 0.0 52. .. isomer HOMO ? ?2. 27 ? ?2 .34 ? ?2 .39 ? ?2. 89 ? ?2. 25 ? ?2 .33 ? ?2. 27 ? ?2. 22 ? ?2. 22 ? ?2. 48 À5.47 À5.55 À5.57 À5.70 À5.46 À5. 53 À5.45 À5 .31 À5.45 À5.59 HOMO-1 À6 .33 À6 .31 À6.54 À6. 82 À5.86 À6. 23 À5. 93 À5.66 À6 .27 À6.69
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Xem thêm: Photoinduced isomerization and hepatoxicities semaxanib, sunitinib and related 3 substituted indolin 2 ones, Photoinduced isomerization and hepatoxicities semaxanib, sunitinib and related 3 substituted indolin 2 ones