Structure–Activity Relationship Study of 3-Amino-2-indolyllactam Derivatives: Development of Inhibitors of Oxidative Stress-Induced Necrosis

Kosuke Dodo; Kenji Hayamizu; Tadashi Shimizu; Mikiko Sodeoka

doi:10.1248/cpb.c16-00259

Abstract

Modification of our previously reported selective inhibitor of oxidative stress-induced necrosis, 2-(1H-indol-3-yl)-3-pentylamino-maleimide (IM-54) by regioselective reduction of the C-4 carbonyl group afforded a 3-amino-2-indolyllactam (IL-1) with more potent activity. To examine the structure–activity relationship of IL derivatives, we developed new synthetic routes with flexibility to incorporate a range of substituents at a late stage. The synthesized IL derivatives were evaluated for activity to inhibit necrotic cell death induced by hydrogen peroxide. Among them, IL-12 showed the most potent activity (IC₅₀=49 nM) among the IL and indolylmaleimide (IM) derivatives examined.

Classically, cell death has been classified into two major categories, apoptosis and necrosis, depending on the morphological phenotypes.¹⁾ Apoptosis is associated with characteristic changes, such as membrane blebbing, cellular shrinkage, nuclear condensation, and formation of apoptotic bodies. In contrast, necrosis results in cellular swelling and rupture of the cell membrane, which might be induced by accidental physical injury. Therefore, only apoptosis was believed to be regulated, while necrosis was considered as uncontrolled cell death. As a result, cell death research initially focused mainly on apoptosis, and caspases were identified as key proteases mediating apoptotic signal transduction.^2,3) Since then, the main signaling pathways of apoptosis have been identified, often with the aid of caspase inhibitors.⁴⁾ However, recent studies have revealed the existence of a number of new types of regulated non-apoptotic cell death.^5,6) Among them, necroptosis was defined as regulated necrosis induced by a physiological death ligand, such as Fas ligand or tumor necrosis factor-alpha (TNF-α).^7,8) Other types of regulated cell death involving necrotic morphology have also been reported.⁹⁾ Thus, novel cell death inhibitors, such as necrostatin-1,⁷⁾ are required to study these novel necrotic cell death processes. We have reported an indolylmaleimide (IM) derivative, 2-(1H-indol-3-yl)-3-pentylamino-maleimide (IM-54; Fig. 1), as a novel inhibitor of necrotic cell death induced by oxidative stress, such as H₂O₂.^10–12) IM-54 did not inhibit apoptotic cell death induced by etoposide, and moreover, it inhibited necrotic cell death at much lower concentration than that of H₂O₂ used, and did not react with H₂O₂ directly, indicating that it is not a simple suicide-type antioxidant. Thus, we considered that IM-54 might have novel mechanism of action, and could be a useful molecule for cell death research. Furthermore, since oxidative stress and necrosis are known to be involved in ischemia–reperfusion (I/R) injuries,^13,14) IM-54 could be a lead compound for development of therapeutics for these diseases. Therefore, we performed further structural development of IM-54 to find more potent derivatives. Here, we report that some indolyllactam (IL) derivatives show greatly increased activity as inhibitors of oxidative stress-induced necrosis.

Fig. 1. Structure of IM-54

Results and Discussion

Structural Development from IM-54 to IL Derivative

In our previous work,¹¹⁾ IM derivatives having various alkyl chain at C-3 position were synthesized and evaluated for cell death–inhibitory activity. We found that alkylamino and alkyloxy chains afforded much stronger activity than alkyl chains (Fig. 2). This suggested that delocalization of lone-pair electrons from nitrogen or oxygen to the maleimide ring serves to enhance the activity.¹¹⁾

Fig. 2. Structure–Activity Relationship of Alkyl Chain of IM Derivatives

However, two types of electron delocalization can occur in IM-54, i.e., between indole and the C-4 carbonyl group and between aminoalkyl and the C-1 carbonyl group (Fig. 3). We hypothesized that removal of the C-4 carbonyl group would enhance delocalization of the lone-pair electrons of nitrogen to the C-1 carbonyl group, resulting in more potent activity.

Fig. 3. Design of IL Derivatives

Since the C-3 nitrogen atom would render the C-1 carbonyl group more resistant to reduction by hydride,¹⁵⁾ the C-4 carbonyl group is expected to be selectively reduced by hydride. As expected, treatment of IM-54 with an excess of lithium aluminum hydride gave the desired hydroxylactam 1 in good yield without reduction at the C-1 carbonyl group (Chart 1). The structure of compound 1 was confirmed by heteronuclear multiple bond correlation (HMBC) analysis. Removal of the hydroxyl group via thioether 2 afforded the indolyllactam derivative IL-1¹⁶⁾ (Chart 1). Namely, in the presence of a catalytic amount of 10-camphorsulfonic acid (CSA), hydroxylactam 1 was treated with 1-propanethiol to afford 2, and desulfurization of 2 with Raney Ni in EtOH proceeded smoothly to give the desired IL-1 in good yield.

Chart 1. Synthesis of IL-1 from IM-54

The inhibitory activity of IL-1 against H₂O₂-induced necrotic cell death was examined by means of lactate dehydrogenase (LDH) assay. In this assay, rupture of cellular membrane, a hallmark of necrosis, is quantified based on the activity of released cytosolic LDH.¹⁷⁾ The IC₅₀ value (concentration needed for 50% inhibition of LDH release induced by H₂O₂ treatment) of IL-1 was 92 nM, indicating that it is twice more potent than IM-54 (IC₅₀=210 nM).

Structure–Activity Relationship (SAR) Study of IL Derivatives

Encouraged by the above result, we next planned to examine the SAR of IL derivatives. For this purpose, we set out to develop a flexible new synthetic route, in which various substituents, including an alkyl group at the lactam C-4 position, could be introduced at a late stage. The planned route is illustrated in Fig. 4.

Fig. 4. New Synthetic Plan for IL Derivatives

To introduce the various amine substituents (R³) at the final stage by simple condensation reaction, we selected the tetramic acid as a key intermediate. This should be accessible by Rh-catalyzed coupling reaction of diazolactam with indole, as reported by Wood et al.¹⁸⁾ The diazolactam could be synthesized from amino acid via Dieckmann condensation, followed by Regitz diazo transfer reaction and alkylation with alkyl halide. By changing the starting amino acid, various C-4 substituents (R²) could be introduced, and the substituent at lactam nitrogen (R¹) could be also varied by changing the alkyl halide.

To establish the new synthetic route, we firstly examined the synthesis of IL-1 from glycine ester (Chart 2). According to the reported method,¹⁸⁾ we synthesized diazolactam 6 from glycine ethyl ester 3. Amide bond formation between 3 and monoethyl malonic acid afforded 4, which was refluxed with NaOEt in benzene to afford lactam 5 via Dieckmann condensation. This in turn was converted to diazolactam 6 through Regitz diazo transfer reaction with MsN₃.¹⁹⁾ After methylation at lactam nitrogen with methyl iodide (MeI), we carried out C–C bond-forming reaction between N-methyl indole and diazolactam 7 with Rh₂(OAc)₄ as a catalyst.^18,20) Rhodium carbenoid derived from diazolactam 7 efficiently reacted with methyl indole to provide tetramic acid 8. Finally, IL-1 was acquired by reaction with propylamine in acetic acid under reflux conditions.²¹⁾ IL-2–IL-4 having different C-3 side chains were similarly synthesized (Chart 2).

Chart 2. Synthesis of IL Derivatives Having Various Aminoalkyl Groups

To investigate the effects of C-4 substitution on the cell death–inhibitory activity, we also synthesized C-4-methyl derivative IL-5 from DL-alanine 9 in 6 steps according to the same procedure (Chart 3).

Chart 3. Synthesis of C-4-Methyl IL Derivative IL-5

The newly synthesized IL derivatives were examined for activity to inhibit H₂O₂-induced necrotic cell death by means of LDH assay (Table 1). Compared with IL-1, none of the IL derivatives having different C-3 side chains showed improved activity. Substitution of the pentyl group with a benzyl (IL-2) or cyclopentyl (IL-3) group caused a clear decrease of the activity, while pyrrolidine substitution (IL-4) decreased the activity by more than one order of magnitude. The C-4 methyl derivative IL-5 also showed weaker activity than IL-1, suggesting that substitution at C-4 is not a promising option for enhancing the activity. Finally, IL-1 was the most potent inhibitor among the synthesized IL derivatives.

Table 1. Cell Death–Inhibitory Activities of IL Derivatives

Compound		R¹	IC₅₀ (nM)
IM-54			210
	IL-1	H	92
	IL-2	H	110
	IL-3	H	250
	IL-4	H	870
	IL-5	CH₃	110

Development of N-Acyl IL Derivatives

The above IL derivatives were found to decompose partially during storage even at −20°C. In the case of IM derivatives, conjugation of the indole ring to the electron-withdrawing C-4 carbonyl group is thought to be important for stabilization (Fig. 5). In contrast, the electron density on the indole ring of IL-1 would be high, and this may be the reason for the greater instability of IL-1 and its derivatives. Therefore, we speculated that introduction of an electron-withdrawing group (EWG) at indolyl nitrogen would improve the stability of the IL derivatives, and so we set out to synthesize N-acyl IL derivatives.

Fig. 5. Hypothesis for Stabilization of IL Derivatives

To introduce an acyl group at indolyl nitrogen, we first protected the hydroxyl group of compound 15, which was prepared from diazolactam 7 and indole. As shown in Chart 4, compound 15 was treated with acetyl chloride and Et₃N to afford compound 16. The acyl group was then introduced at indolyl nitrogen by using acid anhydride, N,N-dimethyl-4-aminopyridine (DMAP) and Et₃N. Under these reaction conditions, the acetyl group was also replaced with the same acyl group. But, these 3-acyloxy derivatives were successfully converted to the desired N-acyl IL derivatives IL-6–IL-16 by reaction with pentylamine in acetic acid (Chart 4). To reduce the hydrophobicity of N-acyl IL compounds, we also introduced an aminopropyl group (Chart 4, IL-17–IL-27), which showed the comparable activity to an aminopentyl group in SAR study of IM derivatives.

Chart 4. Synthesis of N-Acyl IL Derivatives

Moreover, N-acyl IL derivatives IL-28 and IL-29 with different alkylamino groups at the C-3 position were synthesized by reaction with the corresponding amine (Chart 5). As expected, the N-acyl IL derivatives were found to be stable during storage.

Chart 5. Modification of the Aminoalkyl Group of N-Acyl IL Derivatives

With these N-acyl IL derivatives in hand, we examined their cell death–inhibitory activities by means of LDH assay (Table 2). A simple change of the substituent on indole nitrogen from methyl (IL-1) to acetyl (IL-6) caused a significant decrease of cell death–inhibitory activity, implying the importance of the electron density of the indole ring or the substituent at indolyl nitrogen. Interestingly, however, the activity varied dramatically depending upon the nature of the N-acyl group. IL-12 having a n-heptanoyl group showed the most potent activity (IC₅₀=49 nM) among the synthesized IL derivatives, being more potent than IL-1 (IC₅₀=92 nM). Among IL derivatives having an aminopentyl chain (IL-6–IL-16), those with a shorter or longer acyl group showed weaker activity than IL-12. The IL derivatives having an aminopropyl chain (IL-17–IL-27) showed the same trend. IL-23 having a n-heptanoyl group showed the strongest activity. Moreover, the n-heptanoyl group also increased the activity of the N-cyclopentyl derivative IL-29. IL-29 (IC₅₀=98 nM) showed greater cell death–inhibitory activity than IL-3 (IC₅₀=250 nM). Therefore, the n-heptanoyl group increases both the cell death–inhibitory activity and the stability of IL derivatives. The developed synthetic routes should also be useful for further optimization of various substituents.

Table 2. Cell Death–Inhibitory Activities of N-Acyl IL Derivatives

	R¹	IC₅₀ (nM)	Compound	R¹	IC₅₀ (nM)
IL-1		92	IM-54		210
IL-6	CH₃	740	IL-17	CH₃	2200
IL-7	CH₂CH₃	380	IL-18	CH₂CH₃	1300
IL-8	(CH₂)₂CH₃	360	IL-19	(CH₂)₂CH₃	810
IL-9	CH(CH₃)₂	360	IL-20	CH(CH₃)₂	1200
IL-10	(CH₂)₃CH₃	170	IL-21	(CH₂)₃CH₃	280
IL-11	(CH₂)₄CH₃	60	IL-22	(CH₂)₄CH₃	95
IL-12	(CH₂)₅CH₃	49	IL-23	(CH₂)₅CH₃	69
IL-13	(CH₂)₆CH₃	67	IL-24	(CH₂)₆CH₃	97
IL-14	(CH₂)₁₀CH₃	120	IL-25	(CH₂)₁₀CH₃	89
IL-15	(CH₂)₁₄CH₃	640	IL-26	(CH₂)₁₄CH₃	180
IL-16	(CH₂)₂C₆H₅	66	IL-27	(CH₂)₂C₆H₅	97
IL-28	(CH₂)₅CH₃	170	IL-29	(CH₂)₅CH₃	98

Conclusion

In this study, we designed, synthesized and evaluated IL derivatives based on IM-54, which we previously reported as a novel inhibitor of necrotic cell death induced by oxidative stress. Reduction of the C-4 carbonyl group on the maleimide ring of IM-54 proceeded regioselectively, and the resulting derivative, IL-1, showed increased activity. IL derivatives having various C-3 substituents and indole N-acyl groups were also synthesized using newly developed synthetic routes. Acylation of indolyl nitrogen improved both the chemical stability and necrosis-inhibitory activity of IL derivatives. Finally, among the IM and IL derivatives obtained, IL-12 showed the most potent activity (IC₅₀=49 nM). Further SAR studies aiming at developing pharmaceutical candidates are in progress.

Experimental

Synthesis of IL Derivatives

General

NMR spectra were obtained on a JEOL JNM-ECS400 or JEOL JNM-AL300 spectrometer, operating at 400 MHz and 300 MHz for ¹H-NMR, and at 100.4 MHz and 75.5 MHz for ¹³C-NMR. Chemical shifts (δ) for ¹H-NMR and ¹³C-NMR are given in parts per million (ppm) relative to CDCl₃, CD₃OD or DMSO-d₆ as a reference, with coupling constants in Hz. The data are presented in the following order: chemical shift, signal area, multiplicity (s=singlet, d=doublet, t=triplet, q=quartet, m=multiplet and/or multiple resonances, and br s=broad singlet), integration in natural numbers. Electrospray ionization (ESI)-MS was measured on a Bruker microTOF-QII-RSL. Matrix assisted laser desorption/ionization-time-of-flight (MALDI-TOF)/MS was conducted on Bruker Daltonics microflex with matrix dimer and angiotensin I as internal standards. Column chromatography was performed with silica gel 60N (40–50 µm) purchased from Kanto Chemical Co., Inc. (Japan). Routine thin layer chromatography was performed on silica gel 60 F₂₅₄ plates (Merck, Germany). All reactions were carried out under a nitrogen atmosphere with dry solvents under anhydrous conditions, unless otherwise noted, and were monitored by thin layer chromatography.

Compound 1

To a stirred solution of IM-54 (62 mg, 0.19 mmol) in tetrahydrofuran (THF) (2.0 mL) at 0°C was added LiAlH₄ (72 mg, 1.9 mmol). The resulting mixture was stirred at room temperature for 5 min, then quenched with an aqueous solution of potassium sodium tartrate. The aqueous layer was extracted with AcOEt, and the organic layer was dried over Na₂SO₄, and concentrated under reduced pressure. The residue was collected by filtration and rinsed with AcOEt to give 1 (55 mg, 88%) as a white powder. ¹H-NMR (300 MHz, CD₃OD) δ: 7.45 (d, J=7.6 Hz, 1H), 7.35 (d, J=7.8 Hz, 1H), 7.16 (dd, J=7.8, 7.3 Hz, 1H), 7.07 (s, 1H), 7.03 (dd, J=7.6, 7.3 Hz, 1H), 5.91 (br s, 1H), 5.24 (s, 1H), 4.62 (br s, 1H), 3.80 (s, 3H), 3.10 (t, J=7.0 Hz, 2H), 2.93 (s, 3H), 1.50–1.21 (m, 2H), 1.20–0.89 (m, 4H), 0.75 (t, J=7.3 Hz, 3H); ¹³C-NMR (75.5 MHz, CD₃OD) δ: 175.6, 160.1, 138.2, 130.2, 130.0, 122.5, 121.1, 120.0, 110.2, 106.5, 92.5, 83.0, 44.8, 32.9, 31.4, 29.9, 26.5, 23.2, 14.3; high resolution (HR)-ESI-MS: m/z 350.1842 (Calcd for C₁₉H₂₅N₃O₂ 350.1839 [M+Na]⁺).

Compound 2

To a stirred solution of 1 (62 mg, 0.16 mmol) in CH₂Cl₂ (0.75 mL) were added CSA (2.0 mg, 0.0084 mmol) and 1-propanethiol (21 µL, 0.24 mmol). The resulting mixture was stirred at room temperature for 1 h, then quenched with an aqueous solution of NaHCO₃. The aqueous layer was extracted with AcOEt, and the organic layer was dried over Na₂SO₄. The residue was purified by flash column chromatography (SiO₂, eluent; AcOEt–hexane=2 : 1) to give 2 (56 mg, 92%) as a colorless oil. ¹H-NMR (300 MHz, CDCl₃) δ: 7.46 (dd, J=7.5, 1.1 Hz, 1H), 7.32 (dd, J=7.5, 1.1 Hz, 1H), 7.22 (s, 1H), 7.21 (ddd, J=7.5, 7.0, 1.1 Hz, 1H), 7.10 (ddd, J=7.5, 7.0, 1.1 Hz, 1H), 4.89 (s, 1H), 4.50 (t, J=6.3 Hz, 1H), 3.80 (s, 3H), 3.34 (dtd, J=13.0, 7.0, 6.3 Hz, 1H), 3.12 (dtd, J=13.0, 7.0, 6.3 Hz, 1H), 3.02 (s, 3H), 2.40 (td, J=12.1, 7.3 Hz, 1H), 2.31 (td, J=12.1, 7.3 Hz, 1H), 1.60 (tq, J=7.3, 7.3 Hz, 2H), 1.43 (tt, J=7.0, 7.0 Hz, 2H), 1.28–1.10 (m, 4H), 0.97 (t, J=7.3 Hz, 3H), 0.83 (t, J=7.0 Hz, 3H); ¹³C-NMR (75.5 MHz, CDCl₃) δ: 171.7, 154.5, 136.6, 128.7, 127.6, 121.3, 120.0, 119.1, 109.4, 105.1, 97.3, 64.1, 44.2, 32.8, 30.2, 28.7, 27.8, 26.0, 22.6, 22.2, 13.8, 13.7; HR-ESI-MS m/z: 408.2084 (Calcd for C₂₂H₃₁N₃OS 408.2080 [M+Na]⁺).

IL-1

To a stirred solution of 2 (36 mg, 0.093 mmol) in EtOH (0.93 mL) was added Raney Ni (50% slurry in water). The resulting mixture was stirred at room temperature for 1 h under a hydrogen atmosphere, and then the solid was collected by filtration and rinsed with EtOH. The filtrate was washed with water, and the organic layer was dried over Na₂SO₄ and concentrated under reduced pressure. The residue was purified by flash column chromatography (SiO₂, eluent; AcOEt–hexane=1 : 2–2 : 1) to give IL-1 (25 mg, 87%) as a colorless amorphous solid. ¹H-NMR (300 MHz, CDCl₃) δ: 7.49 (d, J=7.8 Hz, 1H), 7.33 (d, J=8.1 Hz, 1H), 7.26 (s, 1H), 7.22 (dd, J=8.1, 7.0 Hz, 1H), 7.10 (dd, J=7.8, 7.0 Hz, 1H), 4.51 (t, J=6.0 Hz, 1H), 3.98 (s, 2H), 3.80 (s, 3H), 3.09 (dt, J=6.0, 7.3 Hz, 2H), 3.06 (s, 3H), 1.63–1.45 (m, 2H), 1.42–1.18 (m, 4H), 0.90 (t, J=7.0 Hz, 3H); ¹³C-NMR (75.5 MHz, CDCl₃) δ: 173.3, 154.5, 136.7, 128.2, 126.4, 121.3, 120.0, 118.9, 109.5, 105.3, 96.3, 50.2, 44.2, 32.8, 30.2, 29.3, 28.9, 22.3, 13.9; HR-ESI-MS m/z: 334.1896 (Calcd for C₁₉H₂₅N₃O 334.1890 [M+Na]⁺).

Compound 4¹⁸⁾

To a stirred solution of glycine ethyl ester hydrochloride 3 (3.0 g, 22 mmol), ethyl hydrogen malonate (2.8 g, 22 mmol) and triethylamine (2.3 g, 23 mmol) in CH₂Cl₂ (56 mL) were added N,N′-dicyclohexylcarbodiimide (DCC) (4.9 g, 24 mmol) and DMAP (0.13 g, 1.1 mmol) at 0°C. The reaction mixture was stirred at 0°C for 30 min and then allowed to warm to room temperature, while being stirred for an additional 6 h. The solid urea by-product was removed by filtration. The filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO₂, eluent; CHCl₃) to give 4 (5.0 g, >99%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 7.60 (s, 1H), 4.21 (q, J=7.1 Hz, 2H), 4.20 (q, J=7.2 Hz, 2H), 4.05 (d, J=7.1 Hz, 2H), 3.34 (s, 2H), 1.28 (t, J=7.1 Hz, 3H), 1.27 (t, J=7.2 Hz, 3H).

Compound 5¹⁸⁾

A solution of sodium ethoxide in ethanol prepared from sodium metal (0.43 g, 19 mmol) and absolute EtOH (12 mL) was added to a solution of the crude diester 4 (4.1 g, 19 mmol) in benzene (23 mL). The resulting mixture was brought to reflux for 4 h, and then quenched with water after cooling to room temperature. The biphasic mixture was separated, and the aqueous layer was acidified to pH 1 with 1 N HCl at 0°C and then extracted with CHCl₃. The organic layer was dried over MgSO₄ and concentrated in vacuo to provide 5 (2.3 g, 96%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 7.08 (br s, 1H), 4.42 (q, J=7.1 Hz, 2H), 4.06 (s, 2H), 1.39 (t, J=7.1 Hz, 3H).

Compound 6¹⁸⁾

A solution of ester 5 (1.2 g, 7.4 mmol) and H₂O (0.22 mL) in CH₃CN (0.33 L) was heated to reflux for 2 h. The volume of CH₃CN was reduced to approximately 30% of the original volume in vacuo. The remaining solution was cooled to 0°C and treated sequentially with triethylamine (1.5 g, 15 mmol) and methanesulfonyl azide (1.8 g, 15 mmol) in CH₃CN (40 mL) via an additional funnel. The reaction mixture was stirred for 20 min at 0°C. The resulting dark red solution was allowed to warm to room temperature, stirred for an additional 2 h, and then concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO₂, eluent; MeOH–CHCl₃=1 : 100) to give diazo lactam 6 (0.57 g, 64%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 5.70 (s, 1H), 3.93 (s, 2H).

Compound 7

To a stirred solution of diazolactam 6 (0.20 g, 1.6 mmol) and methyl iodide (6.8 g, 48 mmol) in N,N-dimethylformamide (DMF) (3.0 mL) was added sodium hydride (77 mg, 1.7 mmol, 55% in paraffin) at 0°C. The resulting mixture was stirred for 1 h at 0°C, and then quenched with an aqueous solution of NH₄Cl. The aqueous layer was extracted with CHCl₃. The organic layer was dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO₂, eluent; CHCl₃) to give N-methyl diazolactam 7 (0.12 g, 54%) as a yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ: 3.79 (s, 2H), 2.96 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 185.9, 161.8, 65.8, 56.5, 29.8.

Compound 8

A mixture of N-methylindole (93 mg, 0.71 mmol), N-methyl diazolactam 7 (33 mg, 0.24 mmol) and Rh₂(OAc)₄ (2.1 mg, 0.0050 mmol) in benzene (3.0 mL) was heated to reflux for 1.5 h. The reaction mixture was cooled to room temperature and concentrated in vacuo. The resulting residue was dissolved in AcOEt, and the solution was extracted with 1 N NaOH solution. The aqueous layer was then acidified to pH 1 with 1 N HCl and extracted with AcOEt. The combined organic layer was dried over MgSO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO₂, eluent; MeOH–CH₂Cl₂=1 : 50–1 : 20) to give tetramic acid 8 (44 mg, 88%) as a yellow powder. ¹H-NMR (400 MHz, CDCl₃) δ: 7.81 (d, J=8.3 Hz, 1H), 7.40 (s, 1H), 7.34 (d, J=8.3 Hz, 1H), 7.16 (dd, J=8.3, 7.6 Hz, 1H), 7.02 (dd, J=8.3, 7.6 Hz, 1H), 4.02 (s, 2H), 3.81 (s, 3H), 3.02 (s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 171.7, 162.0, 136.2, 128.0, 126.4, 122.3, 120.9, 118.3, 109.3, 105.5, 100.7, 51.2, 32.4, 28.5; HR-ESI-MS m/z: 265.0958 (Calcd for C₁₄H₁₄N₂O₂ 265.0947 [M+Na]⁺).

General Procedure for the Introduction of an Aminoalkyl Group (General Procedure A)

Typical Example for IL-1

To a stirred solution of 8 (13 mg, 0.060 mmol) in AcOH (0.14 mL) was added pentylamine (27 mg, 0.30 mmol). The reaction mixture was stirred for 1 h at 100°C, and then poured into an aqueous solution of NaHCO₃. The aqueous layer was extracted with AcOEt. The combined organic layer was dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO₂, eluent; MeOH–CH₂Cl₂=1 : 50) and Gel Permeation Chromatography (column; YMC-GPC-T4000, eluent; CHCl₃) to give IL-1 (9.1 mg, 49%) as a pale brown powder. The spectral data agreed with the data of IL-1 obtained in Experimental.

IL-2

Reaction of 8 (30 mg, 0.12 mmol) with benzylamine (66 mg, 0.62 mmol) according to General Procedure A afforded IL-2 (36 mg, 88%) as a white powder. ¹H-NMR (300 MHz, CDCl₃) δ: 7.47 (d, J=7.8 Hz, 1H), 7.35–7.15 (m, 8H), 7.07 (ddd, J=7.8, 7.1, 1.1 Hz, 1H), 4.97–4.94 (m, 1H), 4.34 (d, J=6 Hz, 2H), 3.97 (s, 2H), 3.81 (s, 3H), 3.04 (s, 3H); ¹³C-NMR (75.5 MHz, CDCl₃) δ: 172.9, 154.0, 138.1, 136.7, 128.8 (2C), 128.3, 127.6, 126.8 (2C), 126.4, 121.3, 120.0, 119.0, 109.5, 105.0, 97.7, 50.3, 48.1, 32.9, 29.4; HR-ESI-MS m/z: 354.1581 (Calcd for C₂₁H₂₁N₃O 354.1577 [M+Na]⁺).

IL-3

Reaction of 8 (30 mg, 0.12 mmol) with cyclopentylamine (53 mg, 0.62 mmol) according to General Procedure A afforded IL-3 (29 mg, 76%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 7.47 (d, J=8.0 Hz, 1H), 7.34 (d, J=8.0 Hz, 1H), 7.28 (s, 1H), 7.22 (dd, J=8.0, 7.3 Hz, 1H), 7.11 (dd, J=8.0, 7.3 Hz, 1H), 4.56 (br s, 1H), 4.05 (s, 2H), 3.81 (s, 3H), 3.69 (dtt, J=6.2, 6.2, 6.2 Hz, 1H), 3.10 (s, 3H), 1.26–2.04 (m, 8H); ¹³C-NMR (100 MHz, CDCl₃) δ: 173.3, 154.1, 136.9, 128.4, 126.5, 121.4, 120.1, 119.1, 109.7, 105.5, 55.8, 50.6, 34.4 (2C), 33.0, 29.5, 23.9 (2C); HR-ESI-MS m/z: 332.1717 (Calcd for C₁₉H₂₃N₃O 332.1733 [M+Na]⁺).

IL-4

Reaction of 8 (17 mg, 0.060 mmol) with pyrrolidine (22 mg, 0.31 mmol) according to General Procedure A afforded IL-4 (13 mg, 64%) as a yellow powder. ¹H-NMR (400 MHz, CDCl₃) δ: 7.46 (d, J=8.0 Hz, 1H), 7.28 (d, J=8.3 Hz, 1H), 7.23 (s, 1H), 7.17 (dd, J=8.3, 7.2 Hz, 1H), 7.05 (dd, J=8.0, 7.2 Hz, 1H), 3.98 (s, 2H), 3.78 (s, 3H), 3.19–3.16 (m, 4H), 3.06 (s, 3H), 1.78–1.75 (m, 4H); ¹³C-NMR (100 MHz, CDCl₃) δ: 174.4, 155.0, 136.3, 130.6, 129.4, 121.0, 120.3, 118.8 (2C), 108.8, 107.2, 52.3, 48.6, 32.8 (2C), 29.4, 25.2 (2C); HR-ESI-MS m/z: 318.1581 (Calcd for C₁₈H₂₁N₃O 318.1577 [M+Na]⁺).

Compound 10

To a stirred solution of 9 (5.0 g, 33 mmol) in CH₂Cl₂ (85 mL) at 0°C was added a solution of ethyl hydrogen malonate (4.5 g, 34 mmol) and triethylamine (3.3 g, 33 mmol), followed by DCC (7.4 g, 36 mmol) and DMAP (0.20 g, 1.6 mmol). The mixture was stirred at 0°C for 1.5 h and allowed to warm to room temperature while being stirred for an additional 1 h. The solid urea by-product was removed by filtration, and the filtrate was concentrated in vacuo to afford a yellow oil. Further purification was carried out by flash column chromatography (SiO₂, eluent; MeOH–CH₂Cl₂=1 : 49) to give 10 (8.2 g, >99%) as a yellow oil. ¹H-NMR (400 MHz, CDCl₃) δ: 7.62 (br s, 1H), 4.59 (q, J=7.2 Hz, 1H), 4.22 (q, J=7.1 Hz, 2H), 4.21 (q, J=7.1 Hz, 2H), 3.33 (s, 2H), 1.44 (d, J=7.2 Hz, 3H), 1.30 (t, J=7.1 Hz, 3H), 1.29 (t, J=7.1 Hz, 3H); MALDI-TOF-MS (CHCA) m/z: 254 (Calcd for C₁₀H₁₇NO₅ 254.1 [M+Na]⁺).

Compound 11

To a stirred solution of sodium ethoxide in ethanol prepared from sodium metal (0.82 g, 33 mmol) and absolute EtOH (21 mL) was added a solution of the crude diester 10 (7.5 g, 33 mmol) in benzene (39 mL). The resulting mixture was brought to reflux for 3.5 h. The reaction mixture was allowed to cool to room temperature and then diluted with H₂O. The layers were separated and the aqueous layer was acidified to pH 1 with 1 N HCl at 0°C, and then extracted with CHCl₃. The organic layer was dried over MgSO₄, and concentrated in vacuo to provide 11 (4.9 g, 76%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 6.47 (br s, 1H), 4.41 (q, J=7.0 Hz, 2H), 4.26–4.14 (m, 1H), 1.44 (d, J=6.8 Hz, 3H), 1.40 (t, J=7.0 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 189.7, 169.5, 167.7, 77.5, 61.8, 52.6, 17.7, 14.6; MALDI-TOF-MS (CHCA) m/z: 208 (Calcd for C₈H₁₁NO₄ 208.06 [M+Na]⁺).

Compound 12

A solution of ester 11 (2.2 g, 11 mmol) and H₂O (0.33 mL) in CH₃CN (0.50 L) was heated to reflux for 4 h. The volume of CH₃CN was reduced to approximately 30% of the original volume in vacuo. The solution was cooled to 0°C and treated with triethylamine (2.3 g, 22 mmol) followed by methanesulfonyl azide (2.7 g, 22 mmol) in CH₃CN (30 mL) via an additional funnel. The reaction mixture was stirred for 20 min at 0°C. The resulting dark red solution was allowed to warm to room temperature, and stirred for an additional 3 h. The resulting mixture was concentrated under reduced pressure, and purified by flash column chromatography (SiO₂, eluent; MeOH–CHCl₃=1 : 50) to give 12 (1.3 g, 82%) as a yellow powder. ¹H-NMR (400 MHz, CDCl₃) δ: 6.61 (br s, 1H), 4.05 (qd, J=6.9, 1.5 Hz, 1H), 1.42 (d, J=6.9 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 190.2, 163.6, 64.9, 57.5, 17.8.

Compound 13

To a stirred solution of 12 (50 mg, 0.36 mmol) and methyl iodide (1.5 g, 11 mmol) in DMF (0.79 mL) was added sodium hydride (17 mg, 0.38 mmol, 55% in paraffin) at 0°C. The mixture was stirred for 1 h, and then the solvent was removed under reduced pressure. The residue was taken up in CHCl₃ and the solution was washed with water and brine. The organic layer was dried over MgSO₄, and concentrated in vacuo. The crude product was purified by column chromatography (SiO₂, eluent; MeOH–CHCl₃=1 : 19), giving 13 as a yellow oil (58 mg, >99%). ¹H-NMR (400 MHz, CDCl₃) δ: 3.84 (q, J=6.8 Hz, 1H), 2.98 (s, 3H), 1.42 (d, J=6.8 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 190.0, 161.4, 62.5, 28.0, 15.6.

Compound 14

A mixture of N-methylindole (0.72 g, 5.5 mmol), 13 (0.28 g, 1.8 mmol) and Rh₂(OAc)₄ (16 mg, 0.040 mmol) in benzene (30 mL) was heated to reflux for 2 h, then cooled to room temperature and concentrated in vacuo to afford a brown residue, which was dissolved in AcOEt. This solution was extracted with 1 N NaOH solution. The aqueous layer was acidified to pH 1 with I N HCl and extracted with AcOEt. The combined organic layers were washed with H₂O and brine, and then dried over MgSO₄. The organic layer was concentrated in vacuo to provide a crude mixture, which was purified by column chromatography (SiO₂, eluent; MeOH–CH₂Cl₂=1 : 19), giving 14 as a white powder (0.41 g, 87%). ¹H-NMR (400 MHz, DMSO-d₆) δ: 7.89 (d, J=7.6 Hz, 1H), 7.57 (s, 1H), 7.38 (d, J=7.6 Hz, 1H), 7.12 (dd, J=7.6, 7.6 Hz, 1H), 6.99 (dd, J=7.6, 7.6 Hz, 1H), 3.97 (q, J=6.8 Hz, 1H), 3.78 (s, 3H), 2.85 (s, 3H), 1.36 (d, J=6.8 Hz, 3H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 170.4, 166.3, 136.0, 128.2, 126.3, 122.0, 120.7, 118.1, 109.2, 105.0, 99.9, 55.9, 32.4, 26.2, 16.3; HR-ESI-MS m/z: 279.1109 (Calcd for C₁₅H₁₆N₂O₂ 279.1104 [M+Na]⁺).

IL-5

Reaction of 14 (20 mg, 0.080 mmol) with pentylamine (68 mg, 0.8 mmol) according to General Procedure A afforded IL-5 (14 mg, 54%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 7.44 (d, J=7.6 Hz, 1H), 7.33 (d, J=8.3 Hz, 1H), 7.20–7.40 (m, 2H), 7.12 (dd, J=8.3, 7.6 Hz, 1H), 4.30 (br s, 1H), 4.11–4.06 (m, 1H), 3.81 (s, 3H), 3.13 (dt, J=7.1, 7.1 Hz, 2H), 3.04 (s, 3H), 1.60–1.20 (m, 9H), 0.86 (t, J=6.7 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.1, 160.2, 137.0, 128.9, 127.2, 121.5, 120.2, 119.2, 109.7, 105.5, 97.3, 56.0, 45.4, 33.2, 31.0, 29.2, 27.2, 22.7, 18.1, 14.3; HR-ESI-MS m/z: 348.2046 (Calcd for C₂₀H₂₇N₃O 348.2046 [M+Na]⁺).

Compound 15

A mixture of indole (1.1 g, 9.5 mmol), N-methyl diazolactam 7 (0.44 g, 3.1 mmol) and Rh₂(OAc)₄ (28 mg, 0.060 mmol) in benzene (39 mL) was heated to reflux for 2 h. The reaction mixture was cooled to room temperature and concentrated in vacuo to afford a brown residue, which was dissolved in AcOEt. This solution was extracted with 1 N NaOH solution. The aqueous layer was acidified to pH 1 with 1 N HCl, and extracted with AcOEt. The combined organic layer was dried over Na₂SO₄, and concentrated under reduced pressure. The residue was recrystallized from AcOEt–hexane to afford 15 (0.50 g, 63%) as a white powder. ¹H-NMR (400 MHz, DMSO-d₆) δ: 11.0 (s, 1H), 10.8 (s, 1H), 7.92 (d, J=8.0 Hz, 1H), 7.60 (s, 1H), 7.33 (d, J=8.0 Hz, 1H), 7.04 (dd, J=8.0, 7.7 Hz, 1H), 6.93 (dd, J=8.0, 7.7 Hz, 1H), 3.94 (s, 2H), 2.90 (s, 3H); ¹³C-NMR (100 MHz, DMSO-d₆) δ: 171.6, 161.8, 135.6, 130.8, 125.9, 123.6, 121.8, 120.6, 118.0, 110.9, 106.1, 51.1, 28.5; HR-ESI-MS m/z: 251.0796 (Calcd for C₁₃H₁₂N₂O₂ 251.0791 [M+Na]⁺).

Compound 16

To a stirred solution of 15 (78 mg, 0.44 mmol) and triethylamine (67 mg, 0.66 mmol) in CH₂Cl₂ (8.8 mL) at −15°C was added dropwise acetyl chloride (38 mg, 0.48 mmol), via a syringe. The mixture was stirred at −15°C for 1.5 h, and quenched with an aqueous solution of NH₄Cl. The aqueous layer was extracted with AcOEt, and washed with brine. The organic layer was dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO₂, eluent; MeOH–CH₂Cl₂=1 : 50) to give 16 (80 mg, 87%) as a white powder. ¹H-NMR (400 MHz, CD₃OD) δ: 7.70 (d, J=8.0 Hz, 1H), 7.67 (s, 1H), 7.40 (d, J=8.0 Hz, 1H), 7.14 (dd, J=8.0, 7.5 Hz, 1H), 7.07 (dd, J=8.0, 7.5 Hz, 1H), 4.40 (s, 2H), 3.12 (s, 3H), 2.21 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 169.9, 167.4, 152.2, 136.1, 126.1, 125.9, 122.3, 121.0, 120.1, 115.0, 111.6, 105.5, 51.8, 29.3, 21.2; HR-ESI-MS m/z: 293.0892 (Calcd for C₁₅H₁₄N₂O₃ 293.0897 [M+Na]⁺).

General Procedure for the Acylation of Indolyl Nitrogen (General Procedure B)

Typical Example for 17a

To a stirred solution of 16 (10 mg, 0.040 mmol) and triethylamine (13 mg, 0.55 mmol) in CH₂Cl₂ (0.40 mL) at room temperature was added dropwise acetyl anhydride (9.8 mg, 0.10 mmol), via a syringe, followed by DMAP (0.45 mg, 0.0040 mmol). The mixture was stirred at room temperature for 8.5 h and then poured into an aqueous solution of NH₄Cl. The aqueous layer was separated and extracted with AcOEt. The organic solution was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO₂, eluent: AcOEt–hexane=3 : 10–7 : 10) to give 17a as a white powder (9.7 mg, 84%). ¹H-NMR (400 MHz, CDCl₃) δ: 8.37 (d, J=8.0 Hz, 1H), 8.07 (s, 1H), 7.71 (d, J=7.3 Hz, 1H), 7.39–7.29 (m, 2H), 4.45 (s, 2H), 3.01 (s, 3H), 2.68 (s, 3H), 2.24 (s, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 169.3, 166.8, 156.5, 145.7, 134.7, 128.1, 126.4, 124.8, 123.3 (2C), 121.1, 115.8, 110.2, 51.3, 28.6, 23.9, 20.9.

Compound 17b

Reaction of 16 (20 mg, 0.074 mmol) with propionic anhydride (25 mg, 0.19 mmol) according to General Procedure B afforded 17b (15 mg, 59%) as a pale brown powder. ¹H-NMR (400 MHz, CD₃OD) δ: 8.46 (d, J=8.0 Hz, 1H), 7.80 (s, 1H), 7.67 (d, J=6.9 Hz, 1H), 7.35 (dd, J=8.0, 7.1 Hz, 1H), 7.28 (dd, J=7.1, 6.9 Hz, 1H), 4.51 (s, 2H), 3.12 (s, 3H), 3.07 (q, J=7.2 Hz, 2H), 2.55 (q, J=7.5 Hz, 2H), 1.31 (t, J=7.2 Hz, 3H), 1.11 (t, J=7.5 Hz, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ: 174.4, 171.8, 171.2, 158.9, 137.9, 137.0, 132.7, 130.0, 126.7, 126.3, 124.6, 122.2, 117.6, 53.4, 30.1, 29.5, 28.6, 9.22, 9.17.

Compound 17c

Reaction of 16 (0.20 g, 0.74 mmol) with n-butyric anhydride (0.46 g, 2.9 mmol) according to General Procedure B afforded 17c (0.15 g, 56%) as a brown powder. ¹H-NMR (400 MHz, CD₃OD) δ: 8.46 (d, J=8.0 Hz, 1H), 8.00 (s, 1H), 7.70 (d, J=7.8 Hz, 1H), 7.35 (dd, J=7.8, 7.2 Hz, 1H), 7.28 (dd, J=8.0, 7.2 Hz, 1H), 4.50 (s, 2H), 3.12 (s, 3H), 3.01 (t, J=7.3 Hz, 2H), 2.50 (t, J=7.3 Hz, 2H), 1.85 (tq, J=7.3, 7.3 Hz, 2H), 1.62 (tq, J=7.3, 7.3 Hz, 2H), 1.08 (t, J=7.3 Hz, 3H), 0.90 (t, J=7.3 Hz, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ: 173.6, 170.9 (2C), 158.9, 137.0, 130.0, 126.8, 126.3 (2C), 124.6, 122.2, 117.6, 113.5, 53.4, 38.6, 36.9, 29.5, 19.4, 19.2, 14.2, 13.9.

Compound 17d

Reaction of 16 (0.20 g, 0.74 mmol) with isobutyric anhydride (0.46 g, 2.9 mmol) according to General Procedure B afforded 17d (0.11 g, 40%) as a yellow oil. ¹H-NMR (400 MHz, CD₃OD) δ: 8.47 (d, J=8.0 Hz, 1H), 8.04 (s, 1H), 7.65 (d, J=7.6 Hz, 1H), 7.36 (dd, J=8.0, 7.6 Hz, 1H), 7.29 (t, J=8.0, 7.6 Hz, 1H), 4.53 (s, 2H), 3.52 (qq, J=6.8, 6.8 Hz, 1H), 3.14 (s, 3H), 2.76 (qq, J=6.9, 6.9 Hz, 1H), 1.35 (d, J=6.8 Hz, 6H), 1.17 (d, J=6.9 Hz, 6H); ¹³C-NMR (100 MHz, CD₃OD) δ: 177.9, 174.5, 171.3, 159.3, 137.2, 130.1, 126.7, 126.4, 124.7, 122.2, 117.8, 113.4, 112.2, 53.3, 35.5, 35.0, 29.5, 20.0 (2C), 19.1 (2C).

Compound 17e

Reaction of 16 (0.20 g, 0.74 mmol) with pentanoic anhydride (0.54 g, 2.9 mmol) according to General Procedure B afforded 17e (0.11 g, 39%) as a pale brown powder. ¹H-NMR (400 MHz, CD₃OD) δ: 8.45 (d, J=8.0 Hz, 1H), 8.00 (s, 1H), 7.66 (d, J=8.0 Hz, 1H), 7.35 (dd, J=8.0, 7.6 Hz, 1H), 7.28 (dd, J=8.0, 7.6 Hz, 1H), 4.50 (s, 2H), 3.12 (s, 3H), 3.04 (t, J=7.3 Hz, 2H), 2.52 (t, J=7.3 Hz, 2H), 1.81 (tt, J=7.6, 7.3 Hz, 2H), 1.57 (tt, J=7.5, 7.3 Hz, 2H), 1.50 (tq, J=7.6, 7.3 Hz, 2H), 1.29 (tq, J=7.5, 7.4 Hz, 2H), 1.01 (t, J=7.3 Hz, 3H), 0.86 (t, J=7.4 Hz, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ: 177.9, 174.5, 171.3, 159.3, 137.2, 130.1, 126.7, 126.4, 124.7, 122.2, 117.8, 113.4, 112.2, 53.3, 35.5 (2C), 35.0 (2C), 29.5, 20.0 (2C), 19.1 (2C).

Compound 17f

Reaction of 16 (0.20 g, 0.74 mmol) with hexanoic anhydride (0.62 g, 2.9 mmol) according to General Procedure B afforded 17f (0.14 g, 44%) as a brown powder. ¹H-NMR (400 MHz, CD₃OD) δ: 8.46 (d, J=8.3 Hz, 1H), 8.00 (s, 1H), 7.66 (d, J=7.8 Hz, 1H), 7.35 (dd, J=7.8, 7.3 Hz, 1H), 7.28 (dd, J=8.3, 7.3 Hz, 1H), 4.50 (s, 2H), 3.13 (s, 3H), 3.04 (t, J=7.3 Hz, 2H), 2.52 (t, J=7.3 Hz, 2H), 1.83 (tt, J=7.3, 7.3 Hz, 2H), 1.58 (tt, J=7.3, 7.3 Hz, 2H), 1.45–1.43 (m, 4H), 1.24–1.22 (m, 4H), 0.96 (t, J=6.9 Hz, 3H), 0.83 (t, J=6.8 Hz, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ: 173.5, 171.0, 170.8, 158.8, 139.8, 136.8, 129.8, 126.5, 126.1, 124.5, 122.0, 117.5, 113.3, 111.9, 53.2, 36.5, 34.9, 32.5, 32.2, 29.3, 25.3, 23.6, 23.3, 14.4, 14.2.

Compound 17g

Reaction of 16 (0.20 g, 0.74 mmol) with heptanoic anhydride (0.70 g, 2.9 mmol) according to General Procedure B afforded 17g (0.16 g, 48%) as a pale brown powder. ¹H-NMR (400 MHz, CD₃OD) δ: 8.45 (d, J=8.3 Hz, 1H), 7.99 (s, 1H), 7.66 (d, J=8.1 Hz, 1H), 7.35 (dd, J=8.1, 7.6 Hz, 1H), 7.27 (dd, J=8.3, 7.6 Hz, 1H), 4.48 (s, 2H), 3.12 (s, 3H), 3.02 (t, J=7.4 Hz, 2H), 2.51 (t, J=7.2 Hz, 2H), 1.81 (tt, J=7.4 Hz, 2H), 1.57 (tt, J=7.2 Hz, 2H), 1.47–1.20 (m, 12H), 0.93 (t, J=7.3 Hz, 3H), 0.84 (t, J=6.7 Hz, 3H); ¹³C-NMR (100 MHz, CD₃OD) δ: 173.5, 170.8 (2C), 158.8, 136.8, 129.8, 126.5, 126.1, 124.4, 122.0, 117.5, 111.9, 53.2, 36.6, 34.9, 32.8, 32.5, 30.0 (2C), 29.6, 29.3, 25.7, 25.5, 23.7, 23.5, 14.4, 14.3.

General Procedure for the Acylation of Indolyl Nitrogen (General Procedure C)

Typical Example for 17h

To a stirred solution of caprylic acid (0.21 g, 1.5 mmol) and triethylamine (0.44 g, 3.1 mmol) in CH₂Cl₂ (4.0 mL) at 0°C was added dropwise thionyl chloride (86 mg, 0.72 mmol), via a syringe. The mixture was allowed to warm to room temperature and stirred for 1 h. Compound 16 (0.10 g, 0.37 mmol) and DMAP (4.5 mg, 0.037 mmol) were added, and stirring was continued for another 57 h. The reaction mixture was quenched with an aqueous solution of NH₄Cl. The aqueous layer was separated and extracted with CH₂Cl₂. The organic layer was washed with brine, dried over Na₂SO₄, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (SiO₂, eluent; AcOEt–hexane=1 : 10–3 : 10) to give 17h as a white powder (40 mg, 23%). ¹H-NMR (400 MHz, CDCl₃) δ: 8.54 (d, J=8.3 Hz, 1H), 8.09 (s, 1H), 7.65 (d, J=7.3 Hz, 1H), 7.36 (dd, J=7.4, 7.3 Hz, 1H), 7.27 (dd, J=8.3, 7.4 Hz, 1H), 4.39 (s, 2H), 3.14 (s, 3H), 2.96 (t, J=7.5 Hz, 2H), 2.49 (t, J=7.6 Hz, 2H), 1.83 (tt, J=7.5, 7.5 Hz, 2H), 1.64–1.25 (m, 18H), 0.89 (t, J=6.8 Hz, 3H), 0.87 (t, J=7.0 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.1, 170.1, 169.2, 155.4, 136.0, 128.6, 125.6, 125.5, 123.6, 121.0, 117.1, 113.2, 111.2, 52.2, 36.2, 34.6, 32.0, 31.9, 29.5 (2C), 29.4, 29.2, 29.1, 24.9, 24.8, 23.0, 22.9, 14.5, 14.4.

Compound 17i

Reaction of 16 (0.10 g, 0.37 mmol) with lauric acid (0.30 g, 1.5 mmol) according to General Procedure C afforded 17i (49 mg, 22%) as a yellow powder. ¹H-NMR (300 MHz, CDCl₃) δ: 8.54 (d, J=8.1 Hz, 1H), 8.09 (s, 1H), 7.65 (d, J=7.7 Hz, 1H), 7.36 (dd, J=7.7, 7.6 Hz, 1H), 7.27 (dd, J=8.1, 7.6 Hz, 1H), 4.39 (s, 2H), 3.13 (s, 3H), 2.95 (t, J=7.2 Hz, 2H), 2.49 (t, J=6.9 Hz, 2H), 1.82 (tt, J=7.2, 7.2 Hz, 2H), 1.62 (tt, J=6.9, 6.9 Hz, 2H), 1.40–1.03 (m, 32H), 0.90–0.86 (m, 6H); ¹³C-NMR (75.5 MHz, CDCl₃) δ: 171.9, 169.9, 169.0, 155.2, 135.7, 128.4, 125.4, 125.2, 123.3, 120.8, 116.8, 112.9, 110.9, 51.9, 35.9, 34.2, 31.9 (2C), 29.6 (4C), 29.5 (3C), 29.4, 29.3, 29.1 (4C), 28.9, 24.5, 24.4, 22.7, 14.1 (2C).

Compound 17j

Reaction of 16 (0.10 g, 0.37 mmol) with palmitic acid (0.38 g, 1.5 mmol) according to General Procedure C afforded 17j (85 mg, 33%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.54 (d, J=8.3 Hz, 1H), 8.09 (s, 1H), 7.66 (d, J=8.1 Hz, 1H), 7.36 (dd, J=8.1, 7.7 Hz, 1H), 7.29–7.25 (m, 1H), 4.39 (s, 2H), 3.14 (s, 3H), 2.96 (t, J=7.3 Hz, 2H), 2.50 (t, J=7.2 Hz, 2H), 1.83 (tt, J=7.3, 7.3 Hz, 2H), 1.62 (tt, J=7.2, 7.2 Hz, 2H), 1.41–1.26 (m, 48H), 0.90–0.87 (m, 6H); ¹³C-NMR (75.5 MHz, CDCl₃) δ: 169.9, 168.9 (2C), 155.0, 135.7, 128.3, 125.4, 125.2, 123.3, 120.7, 116.8, 112.7, 110.9, 51.9, 35.9, 34.2, 31.9 (2C), 29.7 (14C), 29.6, 29.5, 29.4 (2C), 29.2, 29.1, 29.0, 24.6, 24.5, 22.8 (2C), 14.2 (2C).

Compound 17k

Reaction of 16 (0.20 g, 0.74 mmol) with hydrocinnamic anhydride (0.90 g, 3.2 mmol) according to General Procedure B afforded 17k (0.23 g, 64%) as a light brown powder. ¹H-NMR (400 MHz, CD₃OD) δ: 8.55 (d, J=8.3 Hz, 1H), 8.07 (s, 1H), 7.61 (d, J=8.0 Hz, 1H), 7.37 (dd, J=8.0, 7.1 Hz, 1H), 7.32–7.12 (m, 11H), 4.33 (s, 2H), 3.30 (t, J=7.4 Hz, 2H), 3.16 (t, J=7.7 Hz, 2H), 3.11 (s, 3H), 2.94 (t, J=7.4 Hz, 2H), 2.83 (t, J=7.7 Hz, 2H); ¹³C-NMR (100 MHz, CD₃OD) δ: 170.6, 168.8, 168.7, 154.9, 140.1, 139.2, 135.6, 128.6 (2C), 128.6, 128.4 (3C), 128.1 (2C), 126.6 (2C), 126.3, 125.3, 125.1, 123.5, 120.6, 116.8, 112.9, 111.1, 51.8, 37.6, 35.9, 30.5, 30.3, 29.1.

IL-6

Reaction of 17a (16 mg, 0.051 mmol) with pentylamine (45 mg, 0.51 mmol) according to General Procedure A afforded IL-6 (10 mg, 58%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.49 (d, J=8.3 Hz, 1H), 7.58 (s, 1H), 7.47 (d, J=7.8 Hz, 1H), 7.35 (dd, J=8.3, 7.6 Hz, 1H), 7.26 (dd, J=7.8, 7.6 Hz, 1H), 4.59 (br s, 1H), 4.01 (s, 2H), 3.12 (dt, J=6.7, 6.7 Hz, 2H), 3.06 (s, 3H), 2.63 (s, 3H), 1.57 (tt, J=7.2, 7.2 Hz, 2H), 1.35–1.31 (m, 4H), 0.91 (t, J=6.6 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.7, 169.0, 156.4, 136.0, 129.4, 125.3, 124.2, 123.7, 120.2, 117.3, 113.6, 94.6, 50.7, 44.7, 30.5, 29.6, 29.3, 24.4, 22.7, 14.3; HR-ESI-MS m/z: 362.1842 (Calcd for C₂₀H₂₅N₃O₂ 362.1839 [M+Na]⁺).

IL-7

Reaction of 17b (9.0 mg, 0.026 mmol) with pentylamine (23 mg, 0.26 mmol) according to General Procedure A afforded IL-7 (4.0 mg, 42%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.3 Hz, 1H), 7.64 (s, 1H), 7.47 (d, J=7.6 Hz, 1H), 7.35 (dd, J=7.6, 7.5 Hz, 1H), 7.26 (dd, J=8.3, 7.5 Hz, 1H), 4.57 (br s, 1H), 4.02 (s, 2H), 3.13 (dt, J=7.0, 7.0 Hz, 2H), 3.07 (s, 3H), 2.97 (q, J=6.8 Hz, 2H), 1.57 (tq, J=7.0, 6.5 Hz, 2H), 1.28–1.25 (m, 7H), 0.91 (t, J=6.5 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.7, 172.5, 156.3, 136.1, 129.2, 125.3, 123.6, 120.2, 117.4, 113.6, 113.4, 94.7, 50.7, 44.7, 30.5, 29.6 (2C), 29.3, 22.7, 14.3, 11.4; HR-ESI-MS m/z: 376.1991 (Calcd for C₂₁H₂₇N₃O₂ 376.1995 [M+Na]⁺).

IL-8

Reaction of 17c (20 mg, 0.054 mmol) with pentylamine (47 mg, 0.54 mmol) according to General Procedure A afforded IL-8 (8.8 mg, 44%) as a pale brown powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.52 (d, J=8.4 Hz, 1H), 7.64 (s, 1H), 7.47 (d, J=7.6 Hz, 1H), 7.35 (dd, J=7.7, 7.6 Hz, 1H), 7.26 (dd, J=8.4, 7.7 Hz, 1H), 4.60 (br s, 1H), 4.01 (s, 2H), 3.13 (dt, J=7.1, 7.1 Hz, 2H), 3.07 (s, 3H), 2.90 (t, J=7.5 Hz, 2H), 1.85 (tq, J=7.5, 7.4 Hz, 2H), 1.57 (tt, J=7.5, 7.1 Hz, 2H), 1.35–1.33 (m, 4H), 1.05 (t, J=7.4 Hz, 3H), 0.91 (t, J=6.7 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.6, 171.7, 156.2, 136.1, 129.3, 125.2, 123.7, 123.5, 120.1, 117.4, 113.4, 94.7, 50.6, 44.6, 38.1, 30.5, 29.5, 29.3, 22.7, 18.5, 14.3, 14.1; HR-ESI-MS m/z: 390.2149 (Calcd for C₂₂H₂₉N₃O₂ 390.2152 [M+Na]⁺).

IL-9

Reaction of 17d (23 mg, 0.064 mmol) with pentylamine (55 mg, 0.64 mmol) according to General Procedure A afforded IL-9 (11 mg, 45%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.56 (d, J=8.3 Hz, 1H), 7.70 (s, 1H), 7.47 (d, J=7.8 Hz, 1H), 7.35 (dd, J=7.8, 7.4 Hz, 1H), 7.26 (dd, J=8.3, 7.4 Hz, 1H), 4.57 (br s, 1H), 4.01 (s, 2H), 3.38 (qq, J=6.8, 6.8 Hz, 1H), 3.13 (dt, J=6.9, 6.9 Hz, 2H), 3.07 (s, 3H), 1.57 (tt, J=6.9, 6.9 Hz, 2H), 1.34–1.33 (m, 4H), 1.34 (d, J=6.8 Hz 6H), 0.91 (t, J=6.8 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 176.1, 172.7, 156.2 147.5, 136.2, 129.3, 125.3, 123.6 (2C), 120.1, 113.4, 94.7, 50.6, 44.6, 34.1, 30.5, 29.5, 29.3, 22.7, 19.8 (2C), 14.3; HR-ESI-MS m/z: 390.2153 (Calcd for C₂₂H₂₉N₃O₂ 390.2152 [M+Na]⁺).

IL-10

Reaction of 17e (20 mg, 0.052 mmol) with pentylamine (44 mg, 0.52 mmol) according to General Procedure A afforded IL-10 (12 mg, 61%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.52 (d, J=8.3 Hz, 1H), 7.64 (s, 1H), 7.46 (d, J=7.8 Hz, 1H), 7.34 (dd, J=7.8, 7.4 Hz, 1H), 7.26 (dd, J=8.3, 7.4 Hz, 1H), 4.59 (br s, 1H), 4.01 (s, 2H), 3.12 (dt, J=6.9, 6.9 Hz, 2H), 3.07 (s, 3H), 2.93 (t, J=7.6 Hz, 2H), 1.81 (tt, J=7.6, 7.5 Hz, 2H), 1.57 (tt, J=7.3, 6.9 Hz, 2H), 1.45 (tq, J=7.5, 6.8 Hz, 2H), 1.35–1.33 (m, 4H), 0.97 (t, J=7.3 Hz, 3H), 0.91 (t, J=6.8 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 171.6, 156.0, 135.8, 128.9, 124.9 (2C), 123.4, 123.2, 119.8, 117.1, 113.0, 94.4, 50.3, 44.3, 35.7, 30.2, 29.2, 29.0, 26.7, 22.4 (2C), 14.0, 13.9; HR-ESI-MS m/z: 404.2304 (Calcd for C₂₃H₃₁N₃O₂ 404.2308 [M+Na]⁺).

IL-11

Reaction of 17f (20 mg, 0.047 mmol) with pentylamine (41 mg, 0.47 mmol) according to General Procedure A afforded IL-11 (12 mg, 37%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.0 Hz, 1H), 7.65 (s, 1H), 7.46 (d, J=7.8 Hz, 1H), 7.35 (dd, J=7.8, 7.1 Hz, 1H), 7.26 (dd, J=8.0, 7.1 Hz, 1H), 4.57 (br s, 1H), 4.02 (s, 2H), 3.13 (q, J=7.0 Hz, 2H), 3.07 (s, 3H), 2.93 (t, J=7.5 Hz, 2H), 1.83 (tt, J=7.5, 7.5 Hz, 2H), 1.57 (tt, J=7.0, 7.0 Hz, 2H), 1.40–1.33 (m, 8H), 0.99–0.89 (m, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.7, 172.0, 156.3, 136.1, 129.2, 125.3, 123.7, 123.6, 120.1, 117.4, 113.3, 94.7, 50.7, 44.6, 36.2, 31.7, 30.5, 29.6, 29.3, 24.7, 22.8, 22.7, 14.3 (2C); HR-ESI-MS m/z: 418.2468 (Calcd for C₂₄H₃₃N₃O₂ 418.2465 [M+Na]⁺).

IL-12

Reaction of 17g (20 mg, 0.044 mmol) with pentylamine (39 mg, 0.44 mmol) according to General Procedure A afforded IL-12 (11 mg, 61%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.52 (d, J=8.3 Hz, 1H), 7.64 (s, 1H), 7.46 (d, J=7.8 Hz, 1H), 7.34 (dd, J=7.8, 7.7 Hz, 1H), 7.26 (dd, J=8.3, 7.7 Hz, 1H), 4.59 (br s, 1H), 4.02 (s, 2H), 3.13 (dt, J=6.9, 6.9 Hz, 2H), 3.07 (s, 3H), 2.92 (t, J=7.2 Hz, 2H), 1.81 (tt, J=7.2, 6.7 Hz, 2H), 1.57 (tt, J=6.9, 6.9 Hz, 2H), 1.42 (tt, J=6.7, 6.7 Hz, 2H), 1.38–1.33 (m, 8H), 0.93–0.89 (m, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.5, 171.8, 156.1, 135.9, 129.0, 125.0, 123.4, 123.3, 119.9, 117.1, 113.1, 94.4, 50.3, 44.3, 35.9, 31.5, 30.1, 29.2, 28.9, 28.8, 24.5, 22.5, 22.3, 14.0, 13.9; HR-ESI-MS m/z: 432.2624 (Calcd for C₂₅H₃₅N₃O₂ 432.2621 [M+Na]⁺).

IL-13

Reaction of 17h (21 mg, 0.042 mmol) with pentylamine (33 mg, 0.42 mmol) according to General Procedure A afforded IL-13 (6.5 mg, 37%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.1 Hz, 1H), 7.65 (s, 1H), 7.46 (d, J=7.8 Hz, 1H), 7.35 (dd, J=7.8, 7.1 Hz, 1H), 7.26 (dd, J=8.1, 7.2 Hz, 1H), 4.58 (br s, 1H), 4.02 (s, 2H), 3.13 (dt, J=7.0, 7.0 Hz, 2H), 3.07 (s, 3H), 2.93 (t, J=7.4 Hz, 2H), 1.82 (tt, J=7.4, 7.4 Hz, 2H), 1.57 (tt, J=7.0, 7.0 Hz, 2H), 1.43–1.30 (m, 12H), 0.92–0.87 (m, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ: 171.6, 166.0, 156.0, 135.8, 128.9, 125.0, 123.4, 123.2, 119.8, 117.1, 113.0, 94.4, 50.3, 44.3, 35.9, 31.7, 30.2, 29.3, 29.2, 29.1, 29.0, 24.7, 22.7, 22.4, 14.1, 14.0; HR-ESI-MS m/z: 446.2777 (Calcd for C₂₆H₃₇N₃O₂ 446.2778 [M+Na]⁺).

IL-14

Reaction of 17i (20 mg, 0.034 mmol) with pentylamine (29 mg, 0.34 mmol) according to General Procedure A afforded IL-14 (4.2 mg, 26%) as a pale brown powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.0 Hz, 1H), 7.65 (s, 1H), 7.46 (d, J=7.6 Hz, 1H), 7.35 (dd, J=7.6, 7.1 Hz, 1H), 7.26 (dd, J=8.0, 7.1 Hz, 1H), 4.56 (br s, 1H), 4.03 (s, 2H), 3.14 (dt, J=6.7, 6.7 Hz, 2H), 3.08 (s, 3H), 2.93 (t, J=7.5 Hz, 2H), 1.82 (tt, J=7.5, 7.5 Hz, 2H), 1.57–1.26 (m, 22H), 0.93–0.86 (m, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.4, 171.7, 156.0, 135.8, 128.9, 125.0, 123.4 (2C), 123.3, 119.7, 117.1, 113.0, 50.3, 44.3, 36.0, 31.9, 30.2, 29.7 (3C), 29.5 (2C), 29.4, 29.3, 29.0, 24.7, 22.8, 22.4, 14.2, 14.0; HR-ESI-MS m/z: 502.3408 (Calcd for C₃₀H₄₅N₃O₂ 502.3404 [M+Na]⁺).

IL-15

Reaction of 17j (20 mg, 0.028 mmol) with pentylamine (25 mg, 0.28 mmol) according to General Procedure A afforded IL-15 (5.9 mg, 39%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.0 Hz, 1H), 7.64 (s, 1H), 7.46 (d, J=7.6 Hz, 1H), 7.35 (dd, J=7.6, 7.4 Hz, 1H), 7.26 (t, J=8.0, 7.4 Hz, 1H), 4.56 (br s, 1H), 4.02 (s, 2H), 3.13 (dt, J=6.7, 6.7 Hz, 2H), 3.08 (s, 3H), 2.93 (t, J=7.6 Hz, 2H), 1.82 (tt, J=7.6, 7.6 Hz, 2H), 1.57–1.26 (m, 30H), 0.93–0.86 (m, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.4, 171.7, 156.0, 135.8, 128.9, 125.0, 123.4, 123.2, 119.8, 117.1, 113.0, 94.4, 50.3, 44.3, 36.0, 32.0, 30.2, 29.8, 29.7 (5C), 29.5 (2C), 29.4, 29.3 (2C), 29.0, 24.7, 22.8, 22.4, 14.2, 14.0; HR-ESI-MS m/z: 558.4030 (Calcd for C₃₄H₅₃N₃O₂ 558.4030[M+Na]⁺).

IL-16

Reaction of 17k (20 mg, 0.041 mmol) with pentylamine (35 mg, 0.41 mmol) according to General Procedure A afforded IL-16 (13 mg, 72%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.0 Hz, 1H), 7.63 (s, 1H), 7.47 (d, J=7.6 Hz, 1H), 7.38–7.21 (m, 7H), 4.56 (br s, 1H), 4.01 (s, 2H), 3.27 (t, J=7.4 Hz, 2H), 3.17–3.10 (m, 4H), 3.06 (s, 3H), 1.56 (tt, J=7.1, 7.1 Hz, 2H), 1.34–1.33 (m, 4H), 0.91 (t, J=7.1 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.6, 170.8, 156.3, 140.6, 136.1, 129.3, 128.8 (2C), 128.7 (2C), 126.6, 125.4, 123.7, 123.4, 120.2, 117.4, 113.7, 94.6, 50.7, 44.7, 37.9, 30.7, 30.5, 29.6, 29.3, 22.7, 14.3; HR-ESI-MS m/z: 452.2312 (Calcd for C₂₇H₃₁N₃O₂ 452.2308 [M+Na]⁺).

IL-17

Reaction of 17a (20 mg, 0.064 mmol) with propylamine (38 mg, 0.64 mmol) according to General Procedure A afforded IL-17 (5.5 mg, 28%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.50 (d, J=8.0 Hz, 1H), 7.59 (s, 1H), 7.47 (d, J=7.6 Hz, 1H), 7.35 (dd, J=7.6, 7.4 Hz, 1H), 7.27 (dd, J=8.0, 7.4 Hz, 1H), 4.60 (br s, 1H), 4.02 (s, 2H), 3.10 (dt, J=6.9, 6.9 Hz, 2H), 3.06 (s, 3H), 2.63 (s, 3H), 1.59 (tq, J=7.4, 6.9 Hz, 2H), 0.96 (t, J=7.4 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.3, 168.7, 156.0, 135.7, 129.1, 125.0, 123.9, 123.4, 119.9, 117.0, 113.3, 94.3, 50.3, 46.1, 29.2, 24.1, 23.8, 11.4; HR-ESI-MS m/z: 334.1527 (Calcd for C₁₈H₂₁N₃O₂ 334.1526 [M+Na]⁺).

IL-18

Reaction of 17b (20 mg, 0.059 mmol) with propylamine (35 mg, 0.59 mmol) according to General Procedure A afforded IL-18 (15 mg, 77%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.51 (d, J=8.3 Hz, 1H), 7.63 (s, 1H), 7.45 (d, J=7.6 Hz, 1H), 7.34 (dd, J=7.6, 7.2 Hz, 1H), 7.26 (dd, J=8.3, 7.2 Hz, 1H), 4.63 (br s, 1H), 4.01 (s, 2H), 3.10 (dt, J=7.1, 7.1 Hz, 2H), 3.06 (s, 3H), 2.96 (q, J=7.3 Hz, 2H), 1.60 (tq, J=7.5, 7.1 Hz, 2H), 1.32 (t, J=7.3 Hz, 3H), 0.97 (t, J=7.5 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.3, 172.2, 156.0, 135.8, 128.9, 124.9, 123.2 (2C), 119.8, 117.0, 113.1, 94.3, 50.3, 46.1, 29.2 (2C), 23.8, 11.4, 8.82; HR-ESI-MS m/z: 348.1681 (Calcd for C₁₉H₂₃N₃O₂ 348.1682 [M+Na]⁺).

IL-19

Reaction of 17c (20 mg, 0.054 mmol) with propylamine (32 mg, 0.54 mmol) according to General Procedure A afforded IL-19 (11 mg, 60%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.52 (d, J=8.0 Hz, 1H), 7.64 (s, 1H), 7.47 (d, J=7.6 Hz, 1H), 7.35 (dd, J=7.6, 7.3 Hz, 1H), 7.26 (dd, J=8.0, 7.3 Hz, 1H), 4.61 (br s, 1H), 4.01 (s, 2H), 3.10 (dt, J=7.0, 7.0 Hz, 2H), 3.07 (s, 3H), 2.90 (t, J=7.4 Hz, 2H), 1.85 (tq, J=7.4, 7.4 Hz, 2H), 1.60 (tq, J=7.0, 7.0 Hz, 2H), 1.05 (t, J=7.4 Hz, 3H), 0.97 (t, J=7.0 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.3, 171.4, 155.9 135.8, 129.0, 124.9, 123.4, 123.2, 119.8, 117.1, 113.1, 94.4, 50.3, 46.1, 37.8, 29.2, 23.8, 18.2, 13.8, 11.4; HR-ESI-MS m/z: 362.1833 (Calcd for C₂₀H₂₅N₃O₂ 362.1839 [M+Na]⁺).

IL-20

Reaction of 17d (23 mg, 0.063 mmol) with propylamine (37 mg, 0.63 mmol) according to General Procedure A afforded IL-20 (9.0 mg, 42%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.60 (d, J=8.0 Hz, 1H), 7.70 (s, 1H), 7.47 (d, J=7.8 Hz, 1H), 7.34 (dd, J=8.0, 7.5 Hz, 1H), 7.26 (dd, J=7.8, 7.5 Hz, 1H), 4.59 (br s, 1H), 4.02 (s, 2H), 3.38 (qq, J=6.8, 6.8 Hz, 1H), 3.11 (dt, J=7.1 Hz, 2H), 3.07 (s, 3H), 1.60 (tq, J=7.1, 7.1 Hz, 2H), 1.35 (d, J=6.8 Hz, 6H), 0.97 (t, J=7.1 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 176.1, 172.7, 156.2, 136.2, 129.3, 125.3, 123.6 (2C), 120.0, 117.6, 113.4, 94.8, 50.6, 46.4, 34.1, 29.5, 24.1, 19.8 (2C), 11.7; HR-ESI-MS m/z: 362.1842 (Calcd for C₂₀H₂₅N₃O₂ 362.1839 [M+Na]⁺).

IL-21

Reaction of 17e (20 mg, 0.052 mmol) with propylamine (30 mg, 0.52 mmol) according to General Procedure A afforded IL-21 (12 mg, 65%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.52 (d, J=8.1 Hz, 1H), 7.64 (s, 1H), 7.46 (d, J=7.6 Hz, 1H), 7.34 (dd, J=7.6, 7.2 Hz, 1H), 7.26 (t, J=8.1, 7.2 Hz, 1H), 4.62 (br s, 1H), 4.01 (s, 2H), 3.10 (dt, J=7.0, 7.0 Hz, 2H), 3.06 (s, 3H), 2.92 (t, J=7.6 Hz, 2H), 1.80 (tt, J=7.6, 7.6 Hz, 2H), 1.60 (tq, J=7.0, 7.0 Hz, 2H), 1.45 (tq, J=7.6, 7.6 Hz, 2H), 0.99–0.95 (m, 6H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.7, 171.9, 156.3, 136.1, 129.3, 125.2, 123.7, 123.6, 120.1, 117.4, 113.3, 94.7, 50.6, 46.4, 36.0, 29.5, 27.1, 24.1, 22.7, 14.2, 11.7; HR-ESI-MS m/z: 376.1995 (Calcd for C₂₁H₂₇N₃O₂ 376.1998 [M+Na]⁺).

IL-22

Reaction of 17f (20 mg, 0.047 mmol) with propylamine (28 mg, 0.47 mmol) according to General Procedure A afforded IL-22 (8.8 mg, 51%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.0 Hz, 1H), 7.65 (s, 1H), 7.46 (d, J=7.8 Hz, 1H), 7.35 (dd, J=7.8, 7.1 Hz, 1H), 7.26 (dd, J=8.0, 7.1 Hz, 1H), 4.60 (br s, 1H), 4.02 (s, 2H), 3.11 (dt, J=7.0, 7.0 Hz, 2H), 3.07 (s, 3H), 2.92 (t, J=7.6 Hz, 2H), 1.83 (tt, J=7.6, 7.6 Hz, 2H), 1.60 (tq, J=7.0, 7.0 Hz, 2H), 1.41–1.39 (m, 4H), 0.97 (t, J=7.0 Hz, 3H), 0.92 (t, J=7.1 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.4, 171.6, 156.0, 135.8, 128.9, 124.9, 123.4, 123.3, 119.8, 117.1, 113.0, 94.4, 50.3, 46.1, 35.9, 31.4, 29.3, 24.4, 23.8, 22.5, 14.0, 14.3; HR-ESI-MS m/z: 390.2161 (Calcd for C₂₂H₂₉N₃O₂ 390.2152 [M+Na]⁺).

IL-23

Reaction of 17g (20 mg, 0.044 mmol) with propylamine (26 mg, 0.44 mmol) according to General Procedure A afforded IL-23 (9.0 mg, 53%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.3 Hz, 1H), 7.65 (s, 1H), 7.46 (d, J=7.6 Hz, 1H), 7.35 (dd, J=7.6, 7.5 Hz, 1H), 7.26 (dd, J=8.3, 7.5 Hz, 1H), 4.60 (br s, 1H), 4.02 (s, 2H), 3.11 (dt, J=7.0, 7.0 Hz, 2H), 3.07 (s, 3H), 2.93 (t, J=7.4 Hz, 2H), 1.82 (tt, J=7.4, 7.4 Hz, 2H), 1.60 (tq, J=7.0, 7.0 Hz, 2H), 1.42–1.33 (m, 6H), 0.97 (t, J=7.0 Hz, 3H), 0.90 (t, J=7.2 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.7, 172.0, 156.3, 136.1, 129.2, 125.3, 123.7, 123.6, 120.1, 117.4, 113.3, 50.7, 46.4, 36.3, 31.9, 30.1, 29.6, 29.2, 24.9, 24.1, 22.9, 14.4, 11.7; HR-ESI-MS m/z: 404.2312 (Calcd for C₂₃H₃₁N₃O₂ 404.2308 [M+Na]⁺).

IL-24

Reaction of 17h (21 mg, 0.042 mmol) with propylamine (25 mg, 0.42 mmol) according to General Procedure A afforded IL-24 (7.5 mg, 46%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.0 Hz, 1H), 7.65 (s, 1H), 7.46 (d, J=7.8 Hz, 1H), 7.35 (dd, J=7.8, 7.6 Hz, 1H), 7.26 (dd, J=8.0, 7.6 Hz, 1H), 4.59 (br s, 1H), 4.02 (s, 2H), 3.12 (dt, J=7.0, 7.0 Hz, 2H), 3.07 (s, 3H), 2.93 (t, J=7.5 Hz, 2H), 1.82 (tt, J=7.5, 7.3 Hz, 2H), 1.60 (tq, J=7.3, 7.0 Hz, 2H), 1.42–1.30 (m, 8H), 0.97 (t, J=7.3 Hz, 3H), 0.97 (t, J=7.0 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.5, 171.8, 156.1, 136.0, 129.1, 125.1, 123.6, 123.4, 120.0, 117.3, 113.2, 94.6, 50.5, 46.2, 37.8, 36.1, 31.9, 29.4, 29.3, 24.8, 23.9, 22.8, 14.3, 11.6; HR-ESI-MS m/z: 418.2475 (Calcd for C₂₄H₃₃N₃O₂ 418.2470 [M+Na]⁺).

IL-25

Reaction of 17i (20 mg, 0.034 mmol) with propylamine (20 mg, 0.34 mmol) according to General Procedure A afforded IL-25 (9.8 mg, 60%) as a pale brown powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.0 Hz, 1H), 7.65 (s, 1H), 7.46 (d, J=7.8 Hz, 1H), 7.35 (dd, J=7.8, 7.7 Hz, 1H), 7.26 (dd, J=8.0, 7.7 Hz, 1H), 4.60 (br s, 1H), 4.02 (s, 2H), 3.11 (dt, J=7.0, 7.0 Hz, 2H), 3.07 (s, 3H), 2.92 (t, J=7.5 Hz, 2H), 1.82 (tt, J=7.5, 7.5 Hz, 2H), 1.60 (tq, J=7.0, 7.0 Hz, 2H), 1.26–1.44 (m, 16H), 0.97 (t, J=7.0 Hz, 3H), 0.88 (t, J=6.8 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.4, 171.6, 156.0, 135.8, 128.9, 124.9, 123.4, 123.2, 119.8, 117.1, 113.0, 94.5, 50.3, 46.1, 36.0, 31.9, 29.6, 29.5 (2C), 29.4, 29.3 (3C), 24.7, 23.8, 22.7, 14.2, 11.4; HR-ESI-MS m/z: 474.3109 (Calcd for C₂₈H₄₁N₃O₂ 474.3091 [M+Na]⁺).

IL-26

Reaction of 17j (20 mg, 0.028 mmol) with propylamine (17 mg, 0.28 mmol) according to General Procedure A afforded IL-26 (8.6 mg, 60%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.3 Hz, 1H), 7.65 (s, 1H), 7.47 (d, J=7.8 Hz, 1H), 7.35 (dd, J=7.8, 7.6 Hz, 1H), 7.26 (dd, J=8.3, 7.6 Hz, 1H), 4.58 (br s, 1H), 4.02 (s, 2H), 3.11 (dt, J=7.0, 7.0 Hz, 2H), 3.07 (s, 3H), 2.93 (t, J=7.5 Hz, 2H), 1.82 (tt, J=7.5, 7.5 Hz, 2H), 1.60 (tq, J=7.0, 7.0 Hz, 2H), 1.42–1.26 (m, 24H), 0.97 (t, J=7.0 Hz, 3H), 0.88 (t, J=6.8 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.2, 171.5, 155.9, 135.6, 128.8, 124.8, 123.2, 123.1 (2C), 119.6, 117.0, 112.8, 50.2, 45.9, 35.8, 31.8, 29.6 (6C), 29.4, 29.3 (2C), 29.1, 24.5 (2C), 23.6, 22.6, 14.0, 11.3; HR-ESI-MS m/z: 530.3686 (Calcd for C₃₂H₄₉N₃O₂ 530.3717 [M+Na]⁺).

IL-27

Reaction of 17k (20 mg, 0.041 mmol) with propylamine (24 mg, 0.41 mmol) according to General Procedure A afforded IL-27 (14 mg, 76%) as a white powder. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.3 Hz, 1H), 7.62 (s, 1H), 7.47 (d, J=7.8 Hz, 1H), 7.37–7.19 (m, 7H), 4.59 (br s, 1H), 4.01 (s, 2H), 3.27 (t, J=7.5 Hz, 2H), 3.17–3.09 (m, 4H), 3.06 (s, 3H), 1.59 (tq, J=7.3, 7.3 Hz, 2H), 0.96 (t, J=7.3 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.3, 170.5, 156.0, 140.3, 135.8, 129.0, 128.5 (2C), 128.3 (2C), 126.2, 125.0, 123.4, 123.1, 119.9, 117.1, 113.3, 94.3, 50.3, 46.1, 37.6, 30.4, 29.2, 23.8, 11.4; HR-ESI-MS m/z: 424.2000 (Calcd for C₂₅H₂₇N₃O₂ 424.1995 [M+Na]⁺).

IL-28

Reaction of 17g (20 mg, 0.044 mmol) with benzylamine (47 mg, 0.44 mmol) according to General Procedure A afforded IL-28 (11 mg, 59%) as a green oil. ¹H-NMR (400 MHz, CDCl₃) δ: 8.51 (d, J=8.1 Hz, 1H), 7.65 (s, 1H), 7.49 (d, J=7.8 Hz, 1H), 7.22–7.39 (m, 7H), 5.04 (br s, 1H), 4.36 (d, J=6.1 Hz, 2H), 4.00 (s, 2H), 3.04 (s, 3H), 2.92 (t, J=7.6 Hz, 2H), 1.81 (tt, J=7.6, 7.1 Hz, 2H), 1.42 (tt, J=7.1, 7.1 Hz, 2H), 1.34–1.33 (m, 4H), 0.90 (t, J=6.7 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.2, 171.6, 155.6, 137.6, 135.8, 129.0 (2C), 127.9 (2C), 126.9 (2C), 125.0, 123.6, 123.3, 119.8, 117.1, 112.8, 95.7, 50.4, 48.2, 35.9, 31.6, 29.2, 28.9, 24.6, 22.6, 14.1; HR-ESI-MS m/z: 452.2314 (Calcd for C₂₇H₃₁N₃O₂ 452.2308 [M+Na]⁺).

IL-29

Reaction of 17g (20 mg, 0.044 mmol) with cyclopentylamine (38 mg, 0.44 mmol) according to General Procedure A afforded IL-29 (11 mg, 60%) as a red oil. ¹H-NMR (400 MHz, CDCl₃) δ: 8.53 (d, J=8.3 Hz, 1H), 7.65 (s, 1H), 7.44 (d, J=7.6 Hz, 1H), 7.34 (dd, J=7.6, 7.1 Hz, 1H), 7.26 (dd, J=8.3, 7.1 Hz, 1H), 4.59 (br s, 1H), 4.06 (s, 2H), 3.70 (dtt, J=6.9 Hz, 1H), 3.07 (s, 3H), 2.92 (t, J=7.6 Hz, 2H), 2.03–1.97 (m, 2H), 1.85–1.32 (m, 14H), 0.90 (t, J=7.1 Hz, 3H); ¹³C-NMR (100 MHz, CDCl₃) δ: 172.6, 172.0, 155.7, 136.1, 129.1, 125.2, 123.7, 123.5, 120.0, 117.5, 113.3, 94.8, 56.1, 50.9, 36.2, 34.6 (2C), 31.9, 29.6, 29.2, 24.9 (2C), 24.1, 22.9, 14.4; HR-ESI-MS m/z: 430.2472 (Calcd for C₂₅H₃₃N₃O₂ 430.2465 [M+Na]⁺).

Evaluation of Cell Death–Inhibitory Activity against Necrosis Induced by H₂O₂

Cell Culture

HL-60 cells were maintained in RPMI 1640 medium supplemented with 100 U/mL penicillin, 100 µg/mL streptomycin, and 5% heat-inactivated fetal bovine serum (FBS). Cells were grown in a humidified incubator at 37°C under 5% CO₂–95% air.

Lactate Dehydrogenase (LDH) Assay

Necrosis was measured in terms of LDH leakage into the culture medium with a Cytotoxicity detection kit^PLUS (Roche Applied Science). HL-60 cells (4×10⁴ cells/well) were suspended in fresh medium in a 96-well plate. After 2 h incubation, the cells were treated with test compounds (DMSO solution) for 0.5 h and then H₂O₂ (100 µM at final concentration) was added (final volume 100 µL). The final DMSO concentration was 0.5% in all experiments. After 3 h, released LDH was quantified according to the manufacturer’s protocol. The change in absorbance at 490 nm was measured to quantify released LDH. The IC₅₀ values were calculated as the concentration causing 50% inhibition of LDH release induced by H₂O₂ treatment.

Acknowledgments

We thank Dr. Go Hirai (RIKEN) and Dr. Toshiaki Teruya (University of Ryukyus) for helpful discussions. This work was supported in part by a Grant-in-Aid for Scientific Research on Innovative Areas (Homeostatic regulation by various types of cell death) (26110004) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. T.S. was supported by a Research Fellowship for Young Scientists from the Japan Society for the Promotion of Science.

Conflict of Interest

The authors declare no conflict of interest.

References

1) Kerr J. F., Wyllie A. H., Currie A. R., Br. J. Cancer, 26, 239–257 (1972).
2) Ellis H. M., Horvitz H. R., Cell, 44, 817–829 (1986).
3) Thornberry N. A., Lazebnik Y., Science, 281, 1312–1316 (1998).
4) Van Noorden C. J., Acta Histochem., 103, 241–251 (2001).
5) Degterev A., Yuan J., Nat. Rev. Mol. Cell Biol., 9, 378–390 (2008).
6) Galluzzi L., Vitale I., Abrams J. M., Alnemri E. S., Baehrecke E. H., Blagosklonny M. V., Dawson T. M., Dawson V. L., El-Deiry W. S., Fulda S., Gottlieb E., Green D. R., Hengartner M. O., Kepp O., Knight R. A., Kumar S., Lipton S. A., Lu X., Madeo F., Malorni W., Mehlen P., Nuñez G., Peter M. E., Piacentini M., Rubinsztein D. C., Shi Y., Simon H.-U., Vandenabeele P., White E., Yuan J., Zhivotovsky B., Melino G., Kroemer G., Cell Death Differ., 19, 107–120 (2012).
7) Degterev A., Huang Z., Boyce M., Li Y., Jagtap P., Mizushima N., Cuny G. D., Mitchison T. J., Moskowitz M. A., Yuan J., Nat. Chem. Biol., 1, 112–119 (2005).
8) Galluzzi L., Vanden Berghe T., Vanlangenakker N., Buettner S., Eisenberg T., Vandenabeele P., Madeo F., Kroemer G., Int. Rev. Cell Mol. Biol., 289, 1–35 (2011).
9) Vanden Berghe T., Linkermann A., Jouan-Lanhouet S., Walczak H., Vandenabeele P., Nat. Rev. Mol. Cell Biol., 15, 135–147 (2014).
10) Katoh M., Dodo K., Fujita M., Sodeoka M., Bioorg. Med. Chem. Lett., 15, 3109–3113 (2005).
11) Dodo K., Katoh M., Shimizu T., Takahashi M., Sodeoka M., Bioorg. Med. Chem. Lett., 15, 3114–3118 (2005).
12) Sodeoka M., Dodo K., Chem. Rec., 10, 308–314 (2010).
13) Zweier J. L., Talukder M. A. H., Cardiovasc. Res., 70, 181–190 (2006).
14) Sanderson T. H., Reynolds C. A., Kumar R., Przyklenk K., Hüttemann M., Mol. Neurobiol., 47, 9–23 (2013).
15) Regioselective reduction of bisindolylmaleimide was reported: Link J. T., Danishefsky S. J., Tetrahedron Lett., 35, 9135–9138 (1994).
16) Luzzio F. A., Zacherl D. P., Figg W. D., Tetrahedron Lett., 40, 2087–2090 (1999).
17) Chan F. K.-M., Moriwaki K., De Rosa M. J., Methods Mol. Biol., 979, 65–70 (2013).
18) Wood J. L., Stoltz B. M., Dietrich H.-J., Pflum D. A., Petsch D. T., J. Am. Chem. Soc., 119, 9641–9651 (1997).
19) Danheiser R. L., Miller R. F., Brisbois R. G., Park S. Z., J. Org. Chem., 55, 1959–1964 (1990).
20) Pirrung M. C., Zhang S. J., Lackey K., Sternbach D. D., Brown F., J. Org. Chem., 60, 2112–2124 (1995).
21) Tanaka M., Sagawa S., Hoshi J., Shimoma F., Yasue K., Ubukata M., Ikemoto T., Hase Y., Takahashi M., Sasase T., Ueda N., Matsushita M., Inaba T., Bioorg. Med. Chem., 14, 5781–5794 (2006).

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