Bioorganic & Medicinal Chemistry Letters 27 (2017) 4457–4461
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Synthesis of 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole derivatives and their effects on neuronal cell death Kyoji Furuta a,b,c,⇑, Yu Kawai c, Yosuke Mizuno c, Yurika Hattori c, Hiroko Koyama a,c, Yoko Hirata a,b a
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Field of Biological Molecular Sciences, United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan c Regeneration and Advanced Medical Sciences, Graduated School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan b
a r t i c l e
i n f o
Article history: Received 15 June 2017 Revised 31 July 2017 Accepted 3 August 2017 Available online 4 August 2017 Keywords: Oxindole derivative SAR Suppression of neuronal cell death Oxidative stress
a b s t r a c t Novel 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole analogs were synthesized by either of the following two pathways: (1) a sequence of Knoevenagel condensation of oxindole with (4-dimethylamino)cinnamaldehyde–hydrogenation, or (2) alkylation of oxindole dianion with [(4-dimethylamino)phenyl]alkyl halides. Subsequent alkylation at C-3 and/or N-1 of the oxindole skeleton by anion-based methods provided additional substituted derivatives for structure-activity relationship studies. Their effects on neuronal cell death induced by oxidative stress were evaluated by lactate dehydrogenase assay. Compounds with the alkyl chain length of 2–4 significantly suppressed the neuronal cell death. No significant change occurred in the activity by substitution with less-polar groups. The stereochemistry at C-3 of the oxindole core was also irrelevant for the neuroprotective effects of these compounds. Ó 2017 Elsevier Ltd. All rights reserved.
As part of our studies on the design and synthesis of small molecules with neuroprotective activities,1–9 we succeeded in elaborating a novel oxindole 1 (Fig. 1) and its analogs that significantly suppressed the oxidative stress-induced death of neuronal cells.10 Structure–activity relationship (SAR) studies clarified that substitutions with less polar functional groups on the benzene or lactam ring of the oxindole skeleton positively affect the potency, while polar groups intensely reduced the activity. In addition, the stereochemistry at C-3 of the oxindole core was not a crucial factor for the activity of the compounds. However, the effects by modifications at the methylene part, marked by the arrow in Fig. 1, have not yet been investigated. This series of compounds comprise two structural blocks of the oxindole skeleton and (dimethylamino)phenyl group. The methylene part that connects the two blocks is assumed to play an important role in regulating the spatial arrangement between them. Therefore, we planned the synthesis and validation of the neuroprotective activity of the homologs of 1 and related derivatives. Structures of the designed 3-[(4-dimethylamino)phenyl]alkyl2-oxindole derivatives with various substituents on the oxindole core are illustrated in Fig. 2. The alkyl chain length was in the range of 2–4 (n = 2–4) by considering the water solubility of the compounds. We also appended some analogs with modified or ⇑ Corresponding author at: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. E-mail address: [email protected] (K. Furuta). http://dx.doi.org/10.1016/j.bmcl.2017.08.005 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
deformed structures for further SAR studies as depicted in Fig. 3. Most of the compounds were synthesized by either one of the following two pathways: (1) a sequence of Knoevenagel condensation–hydrogenation (the upper part in Scheme 1),11–14 or (2) alkylation of oxindole dianion with halides (Scheme 2).15 Further alkylation at C-3 and/or N-1 was accomplished by the procedure described in the lower part of Scheme 1. The syntheses of the commercially unavailable starting materials are described as schemes in the Supplementary material. Knoevenagel condensation of unsubstituted or substituted oxindole and (4-dimethylamino)cinnamaldehyde afforded the intermediates A as E/Z mixtures.16 The following palladium-mediated hydrogenation of A gave compounds 3 and 10 in good yields. Compounds 15, 16 and 18 were similarly prepared by this twostep method using the corresponding aldehyde units in the total yields of 45%, 59% and 89%, respectively. The C-3-alkylated derivatives 5–9 were obtained by a consecutive five-step method; (1) Knoevenagel condensation giving A; (2) N-Boc protection of A; (3) hydrogenation of A to the saturated compound B; (4) methylation of the amide enolate generated from B; and (5) deprotection of the N-Boc group. We also applied this method for the synthesis of 17 using the appropriate aldehyde unit (five steps, 14% yield). Furthermore, the conversions of 8 to 12 and 13 were easily accomplished by N-alkylation with halides via amidate formation. For the synthesis of 11 and 14, minor modifications were made. Thus, the direct methylation of A (Y1 = H) via its amidate formation, followed
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Fig. 1. Structure of neuroprotective oxindole derivative 1.
Fig. 2. Compounds 2–14.
by hydrogenation gave 11 (two steps, 60%), while Knoevenagel condensation of 1-(4-methoxyphenyl)-2-oxindole with (4dimethylamino)cinnamaldehyde and subsequent hydrogenation produced a saturated intermediate, which in turn was methylated at C-3 via the amide enolate to produce 14. The total yield of 14 based on the starting oxindole was 63%. Compounds 2, 4, 21 and 22 were synthesized by the oxindole dianion-based method (Scheme 2) according to the reported
procedure.15 Namely, the treatment of oxindole with two equivalents of BuLi produces the dianion that preferentially reacts with an alkyl halide at the C-3 position. This method enabled the synthesis of 2 and 4, each in a single step. In a similar manner, we obtained 21 through the reaction with N-(5-iodopentyl)-N-methylaniline. Furthermore, the dianion reacted with N,N-bis(2iodoethyl)aniline to form the cyclic derivative 22. The stepwise dual-alkylation mechanism via an intermolecular anion-exchange reaction between the monoalkylated amide anion intermediates is proposed for this type of substrate.15 The dianion-based method was also applicable for the synthesis of 19 (Scheme 3). The initial attempt to alkylate the dianion generated from 25 with 1-[(4-dimethylamino)phenoxy]-2-iodoethane, by which 19 should be obtainable in a single step, was unsuccessful. However, we found that the dianion could react with 1-iodo-2(2-tetrahydro-2H-pyranoxy)ethane to afford 26. After protection of the acidic N-H in 26 with a benzyl group and deprotection of the THP ether, the resulting alcohol 28 was coupled with 4-nitrophenol under Mitsunobu conditions to produce 29. Subsequent conversion of the nitro group to dimethylamino and deprotection of the Nbenzyl group provided the desired product 19. The total yield of 19 by this scheme was 5% (yields are not optimized). The synthesis of 23 was also accomplished by an analogous procedure (Scheme 4): The dianion of 31 reacted with a long-chain alkyl halide to produce 32, which was successfully converted to the desired compound 23 by the usual method. The amide-type compound 20 was synthesized by N-acylation of triptamine followed by oxidation of the indole ring as depicted in Scheme 5.17 All the oxindole derivatives synthesized in this study are racemates. In a preceding study, we found that the stereochemistry at C-3 of compound 1 was not a crucial factor for the neuroprotective activity.10 In order to verify whether this is also the case for the homologs with an elongated carbon chain, we separated, as a representative example, the racemate 8 into two enantiomers 8a and 8b by HPLC using a chiral column (Daicel Chiralcel OD-H).10 The isomer 8a has a shorter retention time during the HPLC compared to 8b. The absolute configurations of both enantiomers were not assigned. Evaluation of the effect of the compounds on the oxidative stress-induced neuronal cell death was conducted in a similar manner as in the previous report.8,10,18 Briefly, HT22 cells were treated with each compound at 25 lM and 50 lM with 10 mM glutamate. After incubation for 24 h, the extent of cell death was determined by measuring lactate dehydrogenase (LDH), a marker of injured cells, released into the culture medium using a cytotoxicity detection kit (LDH assay). All compounds by themselves exerted no cytotoxic effects on HT22 cells up to 100 lM (data not shown). Ascorbic acid, a well-known antioxidant, prevented the glutamate-induced cell death by ca. 50% at 25 lM in this assay system.
Fig. 3. Compounds 15–23.
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Scheme 1. Synthetic route based on Knoevenagel reaction. Reagents and conditions: (i) piperidine (0.6 equiv), methanol, reflux, 4–22 h; (ii) H2, 10% Pd-C, THF, r.t., 6–24 h; (iii) Boc2O (1.5 equiv), DMAP (1.5 equiv), CH3CN, r.t., 15–30 min; (iv) NaH (1.2 equiv), DMF, 0 °C, 15 min, then CH3I (1.2 equiv), 0 °C to r.t., 20–60 min; (v) trifluoroacetic acid (7 equiv), CH2Cl2, r.t., 0.5–1 h; (vi) for 12, NaH (1.1 equiv), DMF, r.t., 15 min, then ClCH2CONMe2 (1 equiv), r.t., 1 h; (vii) for 13, NaH (1.1 equiv), DMF, r.t., 20 min, then ClCH2COOt-Bu (1 equiv), r.t., 30 min; (viii) for 13, trifluoroacetic acid/CH2Cl2 (1/1, v/v), r.t., 3 h.
Scheme 4. Synthesis of 23. Reagents and conditions: (i) BuLi (2 equiv), TMEDA (2 equiv), 78 °C, 45 min, then 1-iodo-7-(2-tetrahydro-2H-pyranoxy)heptane (1.1 equiv), 78 °C to r.t., 2 h, 35%; (ii) TsOHH2O, MeOH, r.t., 40 min; (iii) I2 (2 equiv), PPh3 (2.5 equiv), imidazole (2.5 equiv), benzene, r.t., 3 h, 75% (2 steps); (iv) Me2NHHCl (2 equiv), K2CO3 (4 equiv), MeCN, r.t., 2.5 h, 89%.
Scheme 2. Synthetic route based on alkylation of oxindole dianion. Reagents and conditions: (a) BuLi (2 equiv), TMEDA (2 equiv), THF, 78 °C, 1 h; (b) 4-(Me2N) C6H4(CH2)nI (1.2 equiv), 78 °C to r.t., 2 h; (c) PhN(Me)(CH2)5I (2 equiv), 78 to 20 °C, 30 min; (d) PhN(CH2CH2I)2 (2 equiv), 78 to 0 °C, 3 h.
The assay results are shown in Fig. 4. Overall, elongation of the carbon chain of 1 did not appreciably affect the activity. Thus, compounds 2, 3 and 4, simple homologs of 1, suppressed the neuronal cell death induced by oxidative stress. Although the activity of 2
decreased at 25 lM, compound 18, an analog of 2 with the same carbon-chain number but has methyl substituents on the chain, exhibited an activity comparable to 3 and 4. These results indicated that considerable fluctuation in the relative spatial arrangement of the two aromatic skeletons was permissible for interaction with a target. Among the derivatives, compounds 3, 6 and 7 exhibited significantly increased activities. The low-dose effects of 3, 6 and 7, together with that for 1, are summarized in Fig. 5. Evidently, these two-carbon elongated homologs exhibited increased activities
Scheme 3. Synthesis of 19. Reagents and conditions: (i) BuLi (2 equiv), TMEDA (2 equiv), THF, 78 °C, 30 min, then MeI (1.1 equiv), 78 °C, 2 h, 65%; (ii) BuLi (2 equiv), TMEDA (2 equiv), THF, 78 °C, 30 min, then ICH2CH2OTHP (2 equiv), 78 °C, 2 h, 37%; (iii) NaH (1 equiv), CH2Cl2, 0 °C, 10 min, then benzyl bromide (1.1 equiv), 0 °C, 3 h, 73%, (iv) TsOHH2O, MeOH, r.t., 30 min; (v) 4-nitrophenol (1.5 equiv), PPh3 (2 equiv), diisopropyl azodicarboxylate (2 equiv), benzene, r.t., 1 h, 63% (2 steps); (vi) H2, 10% Pd-C, THF, r.t., 16 h; (vii) (CH2O)n (10 equiv), NaBH3CN (5 equiv), AcOH, 0 °C to r.t., 5 h, 87% (2 steps); (viii) Na (7.6 equiv), liq NH3/THF (2/1, v/v), 40 °C to r.t., 5 min, 50%.
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Scheme 5. Synthesis of 20. Reagents and conditions: (i) 4-(dimethylamino)benzoyl chloride (1 equiv), Et3N (2 equiv), CH2Cl2, r.t., 16 h, 95%; (ii) DMSO/3 M HCl (1/9, v/ v), 60 °C, 21 h, 50%.
compared to 1. For compound 7, however, auxiliary involvement of the antioxidant effect of the phenolic moiety is not ruled out.19,20 The estimated IC50 values for 1 and 3 were 7.1 lM and 5.5 lM, respectively. Of interest, the observed LDH levels were somewhat lower than that of the control in some cases (compounds 5, 6, 7, 11, 15 and 16, for example). This means that the compounds concerned suppressed the cell death by over 100%. Under the experimental conditions employed, a small portion of HT22 cells dies to release LDH during incubation even in the absence of glutamate. That level of LDH was set at 100% as the control in our experiments. When HT22 cells were incubated with the compounds in the absence of glutamate, the LDH levels similarly decreased less than the control (data not shown). Therefore, we assume that the compounds also prevented such cell death together with the glutamate-mediated death, although further validation is needed. The approximate IC50 values corrected by subtracting this overlapping activity for 6 and 7 were 1.2 lM and 1.9 lM, respectively. Furthermore, the activity was only slightly affected by substituents introduced on the oxindole skeleton (Fig. 4). Consequently, the amide N–H and benzylic C–H were unnecessary to exert the effect. 10 Changes in the position and alkyl substituents of the amino moiety on the side-chain aromatic ring was also permissible (compounds 15–17). Of special interest is the retained activity of 17 possessing a large phenyl group at the amino moiety. Therefore, a considerably broad hydrophobic region around the side-chain aromatic ring is assumed to exist as the compounds’ pharamcophore. In sharp contrast, compound 13 bearing a polar carboxylic acid group completely lacked any activity, suggesting a tendency to avoid polar interactions around the oxindole skeleton. This tendency coincided very closely with those obtained for compound 1 as previously observed.10 Modifications by inserting a heteroatom into the carbon-chain also showed contrasting results. While the ether type compound
Fig. 5. Low-dose effects of 1, 3, 6, 7, 8a and 8b on glutamate-induced cell death of HT22 cells. HT22 cells were incubated with each compound (open bar, 0; rightdown oblique, 1.0 lM; right-up oblique, 2.5 lM; lattice, 5.0 lM; lateral stripes, 10 lM; dot, 25 lM) and glutamate (10 mM) for 24 h, and cell death was determined by measuring LDH released into the culture medium. The LDH level of the control cultures (c) was set at 100%.
19 exhibited a comparable activity to that of the parent analog 8, the amide derivative 20 was completely inactive. Further studies are needed to elucidate what types of factors contribute to the reduction in activity of 20. Another intriguing observation is that compound 21, whose nitrogen atom was translocated from the terminal end to the inside position of the side chain, exerted a potent suppressive effect. We designed 21 to have a nitrogen atom at the spatial position overlapping with that of 1. The analogous insidenitrogen type compound 22 with a shortened and cyclized chain resulted in a loss of activity. Therefore, the appropriate positioning of the nitrogen atom is supposed to be necessary to exert the effect. In addition, the total loss in activity of the aliphatic analog 23 indicated the indispensable requirement for the aminophenyl group located in the side chain. Finally, assay results for the enantiomers of 8 are shown in Fig. 5. Both 8a and 8b showed comparable activities, demonstrating that the stereochemistry at C-3 of the oxindole core was irrelevant for the neuroprotective effects of the compounds.10 In conclusion, we found that elongation of the carbon chain of compound 1 modestly enhanced the effect on the suppression of neuronal cell death induced by oxidative stress. The concise SAR studies proved that further modifications of the oxindole skeleton with less-polar substituents were feasible without decreasing the activities. These findings provide insights into the adequate design of compounds with desirable drug-like properties.21 Additional pharmacological features of our oxindole derivatives, including the effects on the induction of neurotrophic factors, will be described in a separate paper. Although preliminary, we observed that 8 but not 5 induced glial cell line-derived neurotrophic factor (GDNF). This finding suggests that some of the oxindole derivatives
Fig. 4. Effects of 1–23 on glutamate-induced cell death of HT22 cells. HT22 cells were incubated with each compound (open bar, 0; dot, 25 lM; check, 50 lM) and glutamate (10 mM) for 24 h, and cell death was determined by measuring LDH released into the culture medium. The LDH level of the control cultures (c) was set at 100%.
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may have distinct mechanisms of actions in their neuroprotective activity. More detailed biochemical analysis to elucidate the molecular mechanisms and in vivo studies using Parkinson’s disease model mice are currently in progress. Acknowledgment This work was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08. 005. References 1. Satoh T, Furuta K, Tomokiyo K, et al. Facilitatory roles of novel compounds designed from cyclopentenone prostaglandins on neurite outgrowthpromoting activities of nerve growth factor. J Neurochem. 2000;75:1092–1102. 2. Satoh T, Furuta K, Tomokiyo K, et al. Neurotrophic actions of novel compounds designed from cyclopentenone prostaglandins. J Neurochem. 2001;77:50–62. 3. Satoh T, Baba M, Nakatsuka D, et al. Role of heme oxygenase-1 protein in the neuroprotective effects of cyclopentenone prostaglandin derivatives under oxidative stress. Eur J Neurosci. 2003;17:2249–2255. 4. Hirata Y, Furuta K, Miyazaki S, Suzuki M, Kiuchi K. Anti-apoptotic and proapoptotic effect of NEPP11 on manganese-induced apoptosis and JNK pathway activation in PC12 cells. Brain Res. 2004;1021:241–247. 5. Satoh T, Okamoto SI, Cui J, et al. Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic phase II inducers. Proc Natl Acad Sci USA. 2006;103:768–773. 6. Furuta K, Maeda M, Hirata Y, Shibata S, Kiuchi K, Suzuki M. Synthesis of neuroprotective prostaglandin analogs: Suppression of manganese-induced apoptosis of PC12 cells. Bioorg Med Chem Lett. 2007;17:5487–5491.
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7. Shibata S, Maeda M, Furuta K, et al. Neuroprotective effects of (arylthio)cyclopentenone derivatives on manganese-induced apoptosis in PC12 cells. Brain Res. 2009;1294:218–225. 8. Shibata S, Furuta K, Maeda M, et al. (Arylthio)cyclopentenone derivatives prevent glutamate-induced HT22 cell death through a PPARc-dependent pathway. Brain Res. 2009;1296:196–202. 9. Hirata Y, Furuta K, Suzuki M, Oh-hashi K, Ueno Y, Kiuchi K. Neuroprotective cyclopentenone prostaglandins up-regulate neurotrophic factors in C6 glioma cells. Brain Res. 2012;1482:91–100. 10. Furuta K, Mizuno Y, Maeda M, Koyama H, Hirata Y. Synthesis of novel 3arylmethyl-2-oxindole derivatives and their effects on neuronal cell death. Unpublished results. 11. Walker GN. Synthesis of 5,6-dimethoxyindoles and 5,6-dimethoxyoxindoles. A new synthesis of indoles. J Am Chem Soc. 1955;77:3844–3850. 12. Walker GN, Smith RT, Weaver BN. Synthesis of new 3-(pyridylmethylene)-, 3(pyridylmethyl)-, 3-(piperidylmethyl)-, and 3-(b-alkylaminoethyl)-2indolinones. The reduction of isoindogenides, nitro compounds, and pyridines in a series of 2-indolinones. J Med Chem. 1965;8:626–637. 13. Elliot IW, Rivers P. Reduction of some oxindolyliden derivatives to 3substituted oxindoles by sodium borohydride. J Org Chem. 1964;29:2438–2440. 14. Andreani A, Rambaldi M, Locatelli A, Bossa R, Galatulas I, Ninci M. Synthesis and cardiotonic activity of 2-indolinones. Eur J Med Chem. 1990;25:187–190. 15. Kende AS, Hodges JC. Regioselective C-3 alkylations of oxindole dianion. Synth Commun. 1982;12:1–10. 16. Tacconi G, Marinone F. Preparation and characteristics of some 3-ylidene derivatives of 2-indolinone. Ricerca Sci. 1968;38:1239–1244. 17. Savige WE, Fontana A. New procedure for oxidation of 3-substituted indoles to oxindoles: modification of tryptophan residues in peptides and proteins. J Chem Soc Chem Commun. 1976;599–560. 18. Schubert D, Kimura H, Maher P. Growth factors and vitamin E modify neuronal glutamate toxicity. Proc Natl Acad Sci USA. 1992;89:8264–8267. 19. Yasuda D, Takahashi K, Ohe T, Nakamura S, Mashino T. Antioxidant activities of 5-hydroxyoxindole and its 3-hydroxy-3-phenacyl derivatives: the suppression of lipid peroxidation and intracellular oxidative stress. Bioorg Med Chem. 2013;21:7709–7714. 20. Yasuda D, Takahashi K, Kakinoki T, et al. Synthesis, radical scavenging activity and structure–activity relationship of uric acid analogs. MedChemComm. 2013;4:527–529. 21. Kerns EH, Di L. Drug-like properties: concepts, structure design and methods: from ADME to toxicity optimization. Burlington, MA: Academic Press; 2008.
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Synthesis of 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole derivatives and their effects on neuronal cell death Kyoji Furuta a,b,c,⇑, Yu Kawai c, Yosuke Mizuno c, Yurika Hattori c, Hiroko Koyama a,c, Yoko Hirata a,b a
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Field of Biological Molecular Sciences, United Graduate School of Drug Discovery and Medical Information Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan c Regeneration and Advanced Medical Sciences, Graduated School of Medicine, Gifu University, 1-1 Yanagido, Gifu 501-1194, Japan b
a r t i c l e
i n f o
Article history: Received 15 June 2017 Revised 31 July 2017 Accepted 3 August 2017 Available online 4 August 2017 Keywords: Oxindole derivative SAR Suppression of neuronal cell death Oxidative stress
a b s t r a c t Novel 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole analogs were synthesized by either of the following two pathways: (1) a sequence of Knoevenagel condensation of oxindole with (4-dimethylamino)cinnamaldehyde–hydrogenation, or (2) alkylation of oxindole dianion with [(4-dimethylamino)phenyl]alkyl halides. Subsequent alkylation at C-3 and/or N-1 of the oxindole skeleton by anion-based methods provided additional substituted derivatives for structure-activity relationship studies. Their effects on neuronal cell death induced by oxidative stress were evaluated by lactate dehydrogenase assay. Compounds with the alkyl chain length of 2–4 significantly suppressed the neuronal cell death. No significant change occurred in the activity by substitution with less-polar groups. The stereochemistry at C-3 of the oxindole core was also irrelevant for the neuroprotective effects of these compounds. Ó 2017 Elsevier Ltd. All rights reserved.
As part of our studies on the design and synthesis of small molecules with neuroprotective activities,1–9 we succeeded in elaborating a novel oxindole 1 (Fig. 1) and its analogs that significantly suppressed the oxidative stress-induced death of neuronal cells.10 Structure–activity relationship (SAR) studies clarified that substitutions with less polar functional groups on the benzene or lactam ring of the oxindole skeleton positively affect the potency, while polar groups intensely reduced the activity. In addition, the stereochemistry at C-3 of the oxindole core was not a crucial factor for the activity of the compounds. However, the effects by modifications at the methylene part, marked by the arrow in Fig. 1, have not yet been investigated. This series of compounds comprise two structural blocks of the oxindole skeleton and (dimethylamino)phenyl group. The methylene part that connects the two blocks is assumed to play an important role in regulating the spatial arrangement between them. Therefore, we planned the synthesis and validation of the neuroprotective activity of the homologs of 1 and related derivatives. Structures of the designed 3-[(4-dimethylamino)phenyl]alkyl2-oxindole derivatives with various substituents on the oxindole core are illustrated in Fig. 2. The alkyl chain length was in the range of 2–4 (n = 2–4) by considering the water solubility of the compounds. We also appended some analogs with modified or ⇑ Corresponding author at: Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan. E-mail address: [email protected] (K. Furuta). http://dx.doi.org/10.1016/j.bmcl.2017.08.005 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
deformed structures for further SAR studies as depicted in Fig. 3. Most of the compounds were synthesized by either one of the following two pathways: (1) a sequence of Knoevenagel condensation–hydrogenation (the upper part in Scheme 1),11–14 or (2) alkylation of oxindole dianion with halides (Scheme 2).15 Further alkylation at C-3 and/or N-1 was accomplished by the procedure described in the lower part of Scheme 1. The syntheses of the commercially unavailable starting materials are described as schemes in the Supplementary material. Knoevenagel condensation of unsubstituted or substituted oxindole and (4-dimethylamino)cinnamaldehyde afforded the intermediates A as E/Z mixtures.16 The following palladium-mediated hydrogenation of A gave compounds 3 and 10 in good yields. Compounds 15, 16 and 18 were similarly prepared by this twostep method using the corresponding aldehyde units in the total yields of 45%, 59% and 89%, respectively. The C-3-alkylated derivatives 5–9 were obtained by a consecutive five-step method; (1) Knoevenagel condensation giving A; (2) N-Boc protection of A; (3) hydrogenation of A to the saturated compound B; (4) methylation of the amide enolate generated from B; and (5) deprotection of the N-Boc group. We also applied this method for the synthesis of 17 using the appropriate aldehyde unit (five steps, 14% yield). Furthermore, the conversions of 8 to 12 and 13 were easily accomplished by N-alkylation with halides via amidate formation. For the synthesis of 11 and 14, minor modifications were made. Thus, the direct methylation of A (Y1 = H) via its amidate formation, followed
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Fig. 1. Structure of neuroprotective oxindole derivative 1.
Fig. 2. Compounds 2–14.
by hydrogenation gave 11 (two steps, 60%), while Knoevenagel condensation of 1-(4-methoxyphenyl)-2-oxindole with (4dimethylamino)cinnamaldehyde and subsequent hydrogenation produced a saturated intermediate, which in turn was methylated at C-3 via the amide enolate to produce 14. The total yield of 14 based on the starting oxindole was 63%. Compounds 2, 4, 21 and 22 were synthesized by the oxindole dianion-based method (Scheme 2) according to the reported
procedure.15 Namely, the treatment of oxindole with two equivalents of BuLi produces the dianion that preferentially reacts with an alkyl halide at the C-3 position. This method enabled the synthesis of 2 and 4, each in a single step. In a similar manner, we obtained 21 through the reaction with N-(5-iodopentyl)-N-methylaniline. Furthermore, the dianion reacted with N,N-bis(2iodoethyl)aniline to form the cyclic derivative 22. The stepwise dual-alkylation mechanism via an intermolecular anion-exchange reaction between the monoalkylated amide anion intermediates is proposed for this type of substrate.15 The dianion-based method was also applicable for the synthesis of 19 (Scheme 3). The initial attempt to alkylate the dianion generated from 25 with 1-[(4-dimethylamino)phenoxy]-2-iodoethane, by which 19 should be obtainable in a single step, was unsuccessful. However, we found that the dianion could react with 1-iodo-2(2-tetrahydro-2H-pyranoxy)ethane to afford 26. After protection of the acidic N-H in 26 with a benzyl group and deprotection of the THP ether, the resulting alcohol 28 was coupled with 4-nitrophenol under Mitsunobu conditions to produce 29. Subsequent conversion of the nitro group to dimethylamino and deprotection of the Nbenzyl group provided the desired product 19. The total yield of 19 by this scheme was 5% (yields are not optimized). The synthesis of 23 was also accomplished by an analogous procedure (Scheme 4): The dianion of 31 reacted with a long-chain alkyl halide to produce 32, which was successfully converted to the desired compound 23 by the usual method. The amide-type compound 20 was synthesized by N-acylation of triptamine followed by oxidation of the indole ring as depicted in Scheme 5.17 All the oxindole derivatives synthesized in this study are racemates. In a preceding study, we found that the stereochemistry at C-3 of compound 1 was not a crucial factor for the neuroprotective activity.10 In order to verify whether this is also the case for the homologs with an elongated carbon chain, we separated, as a representative example, the racemate 8 into two enantiomers 8a and 8b by HPLC using a chiral column (Daicel Chiralcel OD-H).10 The isomer 8a has a shorter retention time during the HPLC compared to 8b. The absolute configurations of both enantiomers were not assigned. Evaluation of the effect of the compounds on the oxidative stress-induced neuronal cell death was conducted in a similar manner as in the previous report.8,10,18 Briefly, HT22 cells were treated with each compound at 25 lM and 50 lM with 10 mM glutamate. After incubation for 24 h, the extent of cell death was determined by measuring lactate dehydrogenase (LDH), a marker of injured cells, released into the culture medium using a cytotoxicity detection kit (LDH assay). All compounds by themselves exerted no cytotoxic effects on HT22 cells up to 100 lM (data not shown). Ascorbic acid, a well-known antioxidant, prevented the glutamate-induced cell death by ca. 50% at 25 lM in this assay system.
Fig. 3. Compounds 15–23.
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Scheme 1. Synthetic route based on Knoevenagel reaction. Reagents and conditions: (i) piperidine (0.6 equiv), methanol, reflux, 4–22 h; (ii) H2, 10% Pd-C, THF, r.t., 6–24 h; (iii) Boc2O (1.5 equiv), DMAP (1.5 equiv), CH3CN, r.t., 15–30 min; (iv) NaH (1.2 equiv), DMF, 0 °C, 15 min, then CH3I (1.2 equiv), 0 °C to r.t., 20–60 min; (v) trifluoroacetic acid (7 equiv), CH2Cl2, r.t., 0.5–1 h; (vi) for 12, NaH (1.1 equiv), DMF, r.t., 15 min, then ClCH2CONMe2 (1 equiv), r.t., 1 h; (vii) for 13, NaH (1.1 equiv), DMF, r.t., 20 min, then ClCH2COOt-Bu (1 equiv), r.t., 30 min; (viii) for 13, trifluoroacetic acid/CH2Cl2 (1/1, v/v), r.t., 3 h.
Scheme 4. Synthesis of 23. Reagents and conditions: (i) BuLi (2 equiv), TMEDA (2 equiv), 78 °C, 45 min, then 1-iodo-7-(2-tetrahydro-2H-pyranoxy)heptane (1.1 equiv), 78 °C to r.t., 2 h, 35%; (ii) TsOHH2O, MeOH, r.t., 40 min; (iii) I2 (2 equiv), PPh3 (2.5 equiv), imidazole (2.5 equiv), benzene, r.t., 3 h, 75% (2 steps); (iv) Me2NHHCl (2 equiv), K2CO3 (4 equiv), MeCN, r.t., 2.5 h, 89%.
Scheme 2. Synthetic route based on alkylation of oxindole dianion. Reagents and conditions: (a) BuLi (2 equiv), TMEDA (2 equiv), THF, 78 °C, 1 h; (b) 4-(Me2N) C6H4(CH2)nI (1.2 equiv), 78 °C to r.t., 2 h; (c) PhN(Me)(CH2)5I (2 equiv), 78 to 20 °C, 30 min; (d) PhN(CH2CH2I)2 (2 equiv), 78 to 0 °C, 3 h.
The assay results are shown in Fig. 4. Overall, elongation of the carbon chain of 1 did not appreciably affect the activity. Thus, compounds 2, 3 and 4, simple homologs of 1, suppressed the neuronal cell death induced by oxidative stress. Although the activity of 2
decreased at 25 lM, compound 18, an analog of 2 with the same carbon-chain number but has methyl substituents on the chain, exhibited an activity comparable to 3 and 4. These results indicated that considerable fluctuation in the relative spatial arrangement of the two aromatic skeletons was permissible for interaction with a target. Among the derivatives, compounds 3, 6 and 7 exhibited significantly increased activities. The low-dose effects of 3, 6 and 7, together with that for 1, are summarized in Fig. 5. Evidently, these two-carbon elongated homologs exhibited increased activities
Scheme 3. Synthesis of 19. Reagents and conditions: (i) BuLi (2 equiv), TMEDA (2 equiv), THF, 78 °C, 30 min, then MeI (1.1 equiv), 78 °C, 2 h, 65%; (ii) BuLi (2 equiv), TMEDA (2 equiv), THF, 78 °C, 30 min, then ICH2CH2OTHP (2 equiv), 78 °C, 2 h, 37%; (iii) NaH (1 equiv), CH2Cl2, 0 °C, 10 min, then benzyl bromide (1.1 equiv), 0 °C, 3 h, 73%, (iv) TsOHH2O, MeOH, r.t., 30 min; (v) 4-nitrophenol (1.5 equiv), PPh3 (2 equiv), diisopropyl azodicarboxylate (2 equiv), benzene, r.t., 1 h, 63% (2 steps); (vi) H2, 10% Pd-C, THF, r.t., 16 h; (vii) (CH2O)n (10 equiv), NaBH3CN (5 equiv), AcOH, 0 °C to r.t., 5 h, 87% (2 steps); (viii) Na (7.6 equiv), liq NH3/THF (2/1, v/v), 40 °C to r.t., 5 min, 50%.
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Scheme 5. Synthesis of 20. Reagents and conditions: (i) 4-(dimethylamino)benzoyl chloride (1 equiv), Et3N (2 equiv), CH2Cl2, r.t., 16 h, 95%; (ii) DMSO/3 M HCl (1/9, v/ v), 60 °C, 21 h, 50%.
compared to 1. For compound 7, however, auxiliary involvement of the antioxidant effect of the phenolic moiety is not ruled out.19,20 The estimated IC50 values for 1 and 3 were 7.1 lM and 5.5 lM, respectively. Of interest, the observed LDH levels were somewhat lower than that of the control in some cases (compounds 5, 6, 7, 11, 15 and 16, for example). This means that the compounds concerned suppressed the cell death by over 100%. Under the experimental conditions employed, a small portion of HT22 cells dies to release LDH during incubation even in the absence of glutamate. That level of LDH was set at 100% as the control in our experiments. When HT22 cells were incubated with the compounds in the absence of glutamate, the LDH levels similarly decreased less than the control (data not shown). Therefore, we assume that the compounds also prevented such cell death together with the glutamate-mediated death, although further validation is needed. The approximate IC50 values corrected by subtracting this overlapping activity for 6 and 7 were 1.2 lM and 1.9 lM, respectively. Furthermore, the activity was only slightly affected by substituents introduced on the oxindole skeleton (Fig. 4). Consequently, the amide N–H and benzylic C–H were unnecessary to exert the effect. 10 Changes in the position and alkyl substituents of the amino moiety on the side-chain aromatic ring was also permissible (compounds 15–17). Of special interest is the retained activity of 17 possessing a large phenyl group at the amino moiety. Therefore, a considerably broad hydrophobic region around the side-chain aromatic ring is assumed to exist as the compounds’ pharamcophore. In sharp contrast, compound 13 bearing a polar carboxylic acid group completely lacked any activity, suggesting a tendency to avoid polar interactions around the oxindole skeleton. This tendency coincided very closely with those obtained for compound 1 as previously observed.10 Modifications by inserting a heteroatom into the carbon-chain also showed contrasting results. While the ether type compound
Fig. 5. Low-dose effects of 1, 3, 6, 7, 8a and 8b on glutamate-induced cell death of HT22 cells. HT22 cells were incubated with each compound (open bar, 0; rightdown oblique, 1.0 lM; right-up oblique, 2.5 lM; lattice, 5.0 lM; lateral stripes, 10 lM; dot, 25 lM) and glutamate (10 mM) for 24 h, and cell death was determined by measuring LDH released into the culture medium. The LDH level of the control cultures (c) was set at 100%.
19 exhibited a comparable activity to that of the parent analog 8, the amide derivative 20 was completely inactive. Further studies are needed to elucidate what types of factors contribute to the reduction in activity of 20. Another intriguing observation is that compound 21, whose nitrogen atom was translocated from the terminal end to the inside position of the side chain, exerted a potent suppressive effect. We designed 21 to have a nitrogen atom at the spatial position overlapping with that of 1. The analogous insidenitrogen type compound 22 with a shortened and cyclized chain resulted in a loss of activity. Therefore, the appropriate positioning of the nitrogen atom is supposed to be necessary to exert the effect. In addition, the total loss in activity of the aliphatic analog 23 indicated the indispensable requirement for the aminophenyl group located in the side chain. Finally, assay results for the enantiomers of 8 are shown in Fig. 5. Both 8a and 8b showed comparable activities, demonstrating that the stereochemistry at C-3 of the oxindole core was irrelevant for the neuroprotective effects of the compounds.10 In conclusion, we found that elongation of the carbon chain of compound 1 modestly enhanced the effect on the suppression of neuronal cell death induced by oxidative stress. The concise SAR studies proved that further modifications of the oxindole skeleton with less-polar substituents were feasible without decreasing the activities. These findings provide insights into the adequate design of compounds with desirable drug-like properties.21 Additional pharmacological features of our oxindole derivatives, including the effects on the induction of neurotrophic factors, will be described in a separate paper. Although preliminary, we observed that 8 but not 5 induced glial cell line-derived neurotrophic factor (GDNF). This finding suggests that some of the oxindole derivatives
Fig. 4. Effects of 1–23 on glutamate-induced cell death of HT22 cells. HT22 cells were incubated with each compound (open bar, 0; dot, 25 lM; check, 50 lM) and glutamate (10 mM) for 24 h, and cell death was determined by measuring LDH released into the culture medium. The LDH level of the control cultures (c) was set at 100%.
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may have distinct mechanisms of actions in their neuroprotective activity. More detailed biochemical analysis to elucidate the molecular mechanisms and in vivo studies using Parkinson’s disease model mice are currently in progress. Acknowledgment This work was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science. A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08. 005. References 1. Satoh T, Furuta K, Tomokiyo K, et al. Facilitatory roles of novel compounds designed from cyclopentenone prostaglandins on neurite outgrowthpromoting activities of nerve growth factor. J Neurochem. 2000;75:1092–1102. 2. Satoh T, Furuta K, Tomokiyo K, et al. Neurotrophic actions of novel compounds designed from cyclopentenone prostaglandins. J Neurochem. 2001;77:50–62. 3. Satoh T, Baba M, Nakatsuka D, et al. Role of heme oxygenase-1 protein in the neuroprotective effects of cyclopentenone prostaglandin derivatives under oxidative stress. Eur J Neurosci. 2003;17:2249–2255. 4. Hirata Y, Furuta K, Miyazaki S, Suzuki M, Kiuchi K. Anti-apoptotic and proapoptotic effect of NEPP11 on manganese-induced apoptosis and JNK pathway activation in PC12 cells. Brain Res. 2004;1021:241–247. 5. Satoh T, Okamoto SI, Cui J, et al. Activation of the Keap1/Nrf2 pathway for neuroprotection by electrophilic phase II inducers. Proc Natl Acad Sci USA. 2006;103:768–773. 6. Furuta K, Maeda M, Hirata Y, Shibata S, Kiuchi K, Suzuki M. Synthesis of neuroprotective prostaglandin analogs: Suppression of manganese-induced apoptosis of PC12 cells. Bioorg Med Chem Lett. 2007;17:5487–5491.
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