A novel palladium-catalyzed cyclization of indole phytoalexin brassinin and its 1-substituted derivatives

Abstract

A novel simple synthetic approach to spiroindoline phytoalexins and their derivatives has been developed. The spirocyclization of brassinin and its 1-substituted derivatives was achieved using palladium catalyst PdCl₂(CH₃CN)₂ in DMSO at 80 °C in the presence of water, methanol or aniline.

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Introduction

In response to pathogen invasion and other types of stress (physical, biological or chemical), plants produce de novo an arsenal of chemical defenses called phytoalexins.^1,2 Indole phytoalexins are biosynthesized by economically important plants of the family Cruciferae syn. Brassicaceae (e.g., cabbage, turnip, Chinese cabbage, Japanese radish, wasabi, broccoli, rapeseed and arabidopsis). More than 40 indole phytoalexins have been isolated to date. Selected examples are shown in Fig. 1. Characteristic structural feature of cruciferous phytoalexins is indole, 1-methoxyindole, oxindole or indoline moiety with a linear side chain, 2,3-fused or 3-spiro attached heterocycle.^2,3 In addition to their antimicrobial (antifungal and antibacterial) activities,³ indole phytoalexins have been previously reported to exert cytotoxic effects against human leukemia and solid tumour cell lines in vitro^4,5 and are also interesting lead compounds for anticancer drug development. The phytoalexins brassinin (1), spirobrassinin (3) and cyclobrassinin (9) exhibited cancer chemopreventive activity in models of mammary and skin carcinogenesis.^6,7 Several indole phytoalexins have been shown to be potentiate vincristine-induced growth inhibition of U-87 MG human glioblastoma-derived cells.⁸ Brassinin (1) is also moderately active competetive inhibitor of indolamine 2,3-dioxygenase (IDO, EC 1.13.11.42), an enzyme that drives immune escape in cancer.⁹ Cruciferous phytoalexins demonstrated antitrypanosomal properties.¹⁰

Fig. 1 Structures of selected indole phytoalexins.

Stressed plants produced indole phytoalexins in small quantities and their isolation from plant tissues is unpractical. Chemical synthesis is a valuable tool to obtaining reasonable amounts of indole phytoalexins. A some methods to synthesis of indole phytoalexins and their analogs have been reported. In 2002, spirocyclization methodology toward spiroindoline phytoalexins was developed. Reaction of 1-methoxybrassinin (2) with dioxane dibromide (DDB) in dioxane in the presence of water afforded a mixture of diastereoisomers of 1-methoxyspirobrassinol (5) in 90% yield. The synthesis of a mixture of natural trans-[(±)-6a] and unnatural cis-[(±)-6b] diastereoisomer of 1-methoxyspirobrassinol methyl ether is based on a DDB-mediated cyclization of 1-methoxybrassinin (2) in the presence of methanol as a nucleophile.¹¹ Syntheses of analogs of indole phytoalexin 1-methoxyspirobrassinol methyl ether with NR₁R₂ group instead of SCH₃ were accomplished by bromine-mediated spirocyclization of 1-Boc-thioureas.¹² Racemic 1-methoxyspirobrassinin [(±)-4] was prepared by oxidation of (±)-5 using CrO₃ (40%)¹¹ or (±)-6 with pyridinium chlorochromate in 37% yield.¹³ Racemic spirobrassinin [(±)-3] was obtained by thionyl chloride or methanesulfonyl chloride mediated cyclization of dioxibrassinin.^14,15 The one-pot biomimetic syntheses of spirobrassinin [(±)-3] and 1-methoxyspirobrassinin [(±)-4] were achieved by oxidative cyclization of brassinin (1) and 1-methoxybrassinin (2) with CrO₃ or pyridinium chlorochromate.¹⁶

Catalytic cyclization approaches using palladium catalysts for formation C–S bond are extremely rare in the literature. Only a few reports describe the generation of C–S bond in which Pd catalysts have been employed.^17–20

As a continuation of our work in the indole chemistry, our present aim was to devise new route for the synthesis of spiroindoline derivatives. We decided to study the palladium-mediated intramolecular cyclization of brasssinin and its derivatives. We investigated the scope and limitation of the reaction by employing of brasssinin derivatives in the presence of water, methanol and aniline.

Results and discussion

The key intermediate of our new synthetic approach, 1-methoxybrassinin (2) was obtained by reaction of amine 10, CS₂ and CH₃I in methanol according to published procedures.²¹ All the others key intermediates, brassinin (1), 1-Boc-brassinin (14) and 1-Me-brassinin (15), were prepared by the modified procedure used previously for 1-methoxybrassinin (2). The thiourea 16 was synthesized by reaction of amine 10 with phenyl isothiocyanate in methanol in the presence of triethylamine. The thiourea 17 was obtained by the previously reported reaction of amine 11 with phenyl isothiocyanate in dioxane/chloroform (Scheme 1).¹²

Scheme 1 Synthesis of brassinin (1) and analogs 2, 14–17.

The initial palladium-catalyzed reactions were performed using 2 and 14 as substrates (Scheme 2). Substrates 2 and 14 were subjected to the conditions of the cyclization using PdCl₂(CH₃CN)₂ (10 mol%), DMSO at 80 °C in the presence of water or methanol. Application of these conditions resulted in the formation of a diastereomeric mixture of spiroindoline products 5, 6, 19 and 20. As the proposed mechanism is based in a palladacycle, studies show that high concentrations of catalyst (palladacycle) are harmful to the reaction yield.²² In another study of phytoalexin cyclization with Pd(OAc)₂/BINAP, the Pd concentration was 6–10 mol%.²³ Therefore, the amount of palladium was not optimized. For conversion was required DMSO. The use of other solvents (CH₂Cl₂, CH₃CN, MeOH, dioxane, DMF, toluene) resulted in no reaction. Only starting compound was in the reaction mixture after 24 hours. Decreasing the reaction temperature to 50 °C led to extension of reaction time from 1 h to 6 h (Table 1, entry 5). At the room temperature no reaction took place (Table 1, entry 4). From subsequent examinations of various palladium sources, PdCl₂(CH₃CN)₂ proved to be the best catalyst (Table 1, entry 1, 6, 11, 14), producing mixtures of diastereoisomers (±)-5a-(±)-5b, (±)-6a-(±)-6b, (±)-19a-(±)-19b and (±)-20a-(±)-20b in good yields (53%, 60%, 53% and 54%). The cyclization of 2 using Pd(OAc)₂ in the presence water or methanol afforded products 5 or 6 in low yields (Table 1, entry 2, 8). None of desired products 19 and 20 was obtained with palladium catalyst Pd(OAc)₂ and PdCl₂(Ph₃P)₂ (Table 1, entry 12, 13, 16). No stereoselectivity was observed in the reactions of substrates 2 and 14. The spirocyclic products (±)-6a-(±)-6b, (±)-19a-(±)-19b and (±)-20a-(±)-20b were obtained as a mixture of trans and cis-diastereoisomers in the ratio 1 : 1. Diastereoisomers of 1-methoxyspirobrassinol trans-(±)-5a and cis-(±)-5b were isolated as a mixture of diastereoisomers in the ratio 1 : 4 that easily isomerize owing to their hemiaminal structure.²⁴ Our experiments revealed that this palladium-catalyzed cyclization is efficient without reoxidant. Cyclization of 2 with 10 mol% of PdCl₂(CH₃CN)₂ and 33 mol% of CuCl in DMSO at 80 °C resulted in the formation mixture of (±)-6a-(±)-6b in 63% (Table 1, entry 7), while the reaction with the sole Pd catalyst delivered mixture of (±)-6a-(±)-6b in a 60% yield (Table 1, entry 6). Reaction of 14 with 10 mol% of PdCl₂(CH₃CN)₂ and 33 mol% of CuCl in DMSO at 80 °C led to the formation of unidentified product. Performing the cyclization reaction of 1-methoxybrassinin (2) in the presence of aniline led to the formation of a mixture of diastereoisomers of the 2-aminoderivate of phytoalexins 1-methoxyspirobrassinol methyl ether (±)-18a-(±)-18b in a good yield (Table 1, entry 10).

Scheme 2 Cyclization reactions of 1-methoxybrassinin (2) and 1-Boc-brassinin (14) using palladium catalysts.

Table 1 Reaction conditions, yields and ratios of trans- and cis-diastereoisomers produced via Scheme 2^a

Entry	Conditions	Temp. (°C)	R1	R2	Reaction time	Yield (%)	Ratio trans : cis
a Reaction conditions: 2 or 14 (0.15 mmol), Pd catalyst (10 mol%), DMSO (2.7 mL), H2O or MeOH (0.3 mL), 0.5–24 h.b 33 mol% was used.c 1.1 equiv. was used. Yield of isolated product.
1	PdCl2(CH3CN)2, H2O	80	OCH3	OH	1 h	53	22 : 78
2	Pd(OAc)2, H2O	80		OH	5 h	12	20 : 80
3	PdCl2(Ph3P)2, H2O	80		OH	3 h	43	25 : 75
4	PdCl2(CH3CN)2, MeOH	rt		OCH3	24 h	Starting compound	—
5	PdCl2(CH3CN)2, MeOH	50		OCH3	6 h	60	55 : 45
6	PdCl2(CH3CN)2, MeOH	80		OCH3	1 h	60	55 : 45
7	PdCl2(CH3CN)2, CuCl^b, MeOH	80		OCH3	1 h	63	56 : 44
8	Pd(OAc)2, MeOH	80		OCH3	4 h	28	50 : 50
9	PdCl2(Ph3P)2, MeOH	80		OCH3	1 h	43	52 : 48
10	PdCl2(CH3CN)2, PhNH2^c	80		NHPh	2 h	54	49 : 51
11	PdCl2(CH3CN)2, H2O	80	Boc	OH	1.5 h	53	57 : 43
12	Pd(OAc)2, H2O	80		OH	4 h	Trace	—
13	PdCl2(Ph3P)2, H2O	80		OH	4 h	—	—
14	PdCl2(CH3CN)2, MeOH	80		OCH3	3.5 h	54	54 : 46
15	PdCl2(CH3CN)2, CuCl^b, MeOH	80		OCH3	0.5 h	—	—
16	Pd(OAc)2, MeOH	80		OCH3	3 h	Trace	—
17	PdCl2(Ph3P)2, MeOH	80		OCH3	3.5 h	36	54 : 46

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In order to check the efficiency and limitation of this new method, we explored PdCl₂(CH₃CN)₂-catalyzed cyclization of thioureas 16 and 17. The reaction conditions [PdCl₂(CH₃CN)₂ – 10 mol%, DMSO, 80 °C] developed above were also successfully applied to the cyclization of thioureas 16 and 17 (Scheme 3). As a results, were obtained 2′-aminoanalogs of indole phytoalexins 1-methoxyspirobrassinol (±)-21 and (±)-23 and 1-methoxyspirobrassinol methyl ether (±)-22 and (±)-24. Yields and ratios of prepared diastereoisomers are reported in the Table 2. trans and cis-Diastereoisomers (±)-21a-(±)-21b, (±)-22a-(±)-22b and (±)-24a-(±)-24b arised in the ratio 1 : 1. Diastereoisomers (±)-23a and (±)-23b were isolated as a mixture of diastereoisomers in the ratio 68 : 32 owing to its unstable hemiaminal structure analogues to 1-methoxyspirobrassinol (5).

Scheme 3 Cyclization reactions of thioureas 16 and 17 catalyzed by PdCl₂(CH₃CN)₂.

Table 2 Yields and ratios of trans- and cis-diastereoisomers (±)-21a-(±)-24b produced via Scheme 3^a

Entry	Conditions	R1	R2	Reaction time	Yield (%)	Ratio trans : cis
a Reaction conditions: 16 or 17 (0.15 mmol), PdCl2(CH3CN)2 (10 mol%), DMSO (2.7 mL), H2O or MeOH (0.3 mL), 80 °C, 1.5–4 h. Yield of isolated product.
1	PdCl2(CH3CN)2, H2O	OCH3	OH	4 h	47	44 : 56
2	PdCl2(CH3CN)2, MeOH		OCH3	2 h	59	54 : 46
3	PdCl2(CH3CN)2, H2O	Boc	OH	4 h	52	68 : 32
4	PdCl2(CH3CN)2, MeOH		OCH3	1.5 h	56	53 : 47

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To test the cyclization process under palladium catalysis, brassinin (1) and 1-methylbrassinin (15) were also used (Scheme 4). In the cases of substrates 1 and 15, oxindoles (±)-3 and (±)-25 were obtained in low to moderate yields. The firs attempt to cyclize brassinin (1) and 1-methylbrassinin (15) under conditions PdCl₂(CH₃CN)₂ (10 mol%), water, DMSO at 80 °C afforded spirobrassinin [(±)-3] and 1-methylspirobrassinin [(±)-25] in a low isolated yields (13%, 17%, Table 3, entry 1 or 6). When MeOH was used instead of water, 34% or 40% conversion was observed after one hour (Table 3, entry 3 and 8). Adducts (±)-3 and (±)-25 were not observed using of a palladium catalyst PdCl₂(Ph₃P)₂ in the presence of water (Table 3, entry 2 and 7). Cyclization of 1 and 15 using PdCl₂(Ph₃P)₂ as a catalyst in the presence of methanol afforded products (±)-3 and (±)-25 in an 18% yield (Table 3, entry 5 and 10). Reactions of 1 and 15 with catalyst Pd(OAc)₂ in the presence of methanol resulted in the formation products (±)-3 and (±)-25 in only an 8% yields (Table 3, entry 4 and 9).

Scheme 4 Cyclization reactions of brassinin (1) and 1-methyl-brassinin (15) using palladium catalysts.

Table 3 Reaction conditions and yields of spirobrassinin (3) and 1-Me-spirobrasinin (25) produced via Scheme 4^a

Entry	Conditions	R	Reaction time	Yield (%)
a Reaction conditions: 1 or 15 (0.15 mmol), Pd catalyst (10 mol%), DMSO (2.7 mL), H2O or MeOH (0.3 mL), 80 °C, 1–4 h. Yield of isolated product.
1	PdCl2(CH3CN)2, H2O	H	1 h	13
2	PdCl2(Ph3P)2, H2O		2 h	—
3	PdCl2(CH3CN)2, MeOH		1 h	34
4	Pd(OAc)2, MeOH		4 h	8
5	PdCl2(Ph3P)2, MeOH		1 h	18
6	PdCl2(CH3CN)2, H2O	CH3	1 h	17
7	PdCl2(Ph3P)2, H2O		2 h	—
8	PdCl2(CH3CN)2, MeOH		1 h	40
9	Pd(OAc)2, MeOH		3 h	8
10	PdCl2(Ph3P)2, MeOH		1 h	18

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We have also studied the effect of the ligand (R)-BINAP on the stereoselectivity of PdCl₂(CH₃CN)₂-mediated cyclization of brassinin (1). The enantioselectivity of the reaction was not influenced and the enantiomers of spirobrassinin [(+)-3], [(−)-3] were obtained in a ratio 50 : 50.

We suppose that this palladium-catalyzed cyclization of brassinin (1) and its derivatives proceeds via intermediates 26 or 27. Subsequent formation of six-member palladacycle 28,^22,25,26 followed by reductive elimination provides products (±)-5, (±)-6 or (±)-3 (Scheme 5).

Scheme 5 A plausible reaction mechanism.

Conclusion

In summary, we have developed a novel synthetic method of spiroindoline derivatives through the unusual palladium-catalyzed cyclization of brassinin and its derivatives. The spirocyclization of brassinin and its derivatives proceeds using PdCl₂(CH₃CN)₂ as catalyst, DMSO as a solvent and temperature 80 °C in the presence of water, methanol or aniline. The reactions were performed using 10 mol% of PdCl₂(CH₃CN)₂ and the amount of Pd catalyst was not optimized.

Experimental

Melting points were determined on a Koffler micro melting point apparatus and are uncorrect. IR spectra were recorded on an IR-75 spectrometer (Zess Jena). ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were measured on a Varian Mercury Plus spectrometer. Chemical shifts (δ) are reported in ppm downfield from TMS as internal standard and coupling constants (J) are given in hertz. Microanalyses were performed with a Perkin-Elmer, Model 2400 analyzer. HRMS were recorded on a JEOL JMS-FAB spectrometer at ionization energy of 70 eV. The EI mass spectra were recorded on a GS-MS Trio 1000 (Fisons Instruments) spectrometer at ionization energy of 70 eV, whereas MALDI-TOF mass spectra were measured on a MALDI IV (Kratos Analytical). The samples were ionized with a N₂-laser (λ = 337 nm). The progress of chemical reactions was monitored by thin layer chromatography, using Macherey-Nagel plates Alugram® Sil G/UV254. Preparative column chromatography was performed on Kieselgel 60 Merck Type 9385 (0.040-0.063).

1.1 Synthesis of starting brassinins 1, 14, 15 and thiourea 16

1.1.1 1-(tert-butoxycarbonyl)brassinin (14). Crude product of 1-[(tert-butoxycarbonyl)indol-3-yl]methylamine (11) freshly prepared by the reduction of 1-(tert-butoxycarbonyl)indole-3-carboxaldehyde oxime (0.780 g, 3 mmol) with NaBH₄ (1.11 g, 30 mmol) in the presence NiCl₂·6H₂O (0.710 g, 3 mmol) was dissolved in methanol (24 mL).²⁷ Triethylamine (1.25 mL, 0.91 g, 9 mmol) and carbon disulfide (0.54 mL, 0.69 g, 9 mmol) were added and mixture was stirred for 5 min at room temperature. Then iodomethane (0.56 mL, 1.28 g, 9 mmol) was added and the stirring was continued for 15 min. The residue obtained after evaporation of the solvent was dissolved in dichloromethane, small amount of silica gel was added, dichloromethane evaporated and product preabsorbed on silica gel was chromatographed on silica gel (30 g, n-hexane/acetone 5 : 1). The obtained compound was further crystallized from dichloromethane/n-hexane to afford pure product 14. Yield: 0.69 g, 68%. Spectral data of the obtained 1-(tert-butoxycarbonyl)brassinin (14) were identical with those described previously.²⁷

1.1.2 Brassinin (1). Crude product of indol-3-yl-methylamine (12) freshly prepared by the reduction of indole-3-carboxaldehyde oxime (1.0 g, 6.24 mmol) with NaBH₄ (2.36 g, 62,4 mmol) in the presence NiCl₂·6H₂O (1.48 g, 6.24 mmol) was dissolved in methanol (55 mL).²⁷ Triethylamine (0.87 mL, 0.63 g, 6.24 mmol) and carbon disulfide (2.63 mL, 3.32 g, 43,7 mmol) were added and mixture was stirred for 5 min at room temperature. Then iodomethane (1.95 mL, 4.41 g, 31.2 mmol) was added and the stirring was continued for 15 min. The residue obtained after evaporation of the solvent was dissolved in dichloromethane, small amount of silica gel was added, dichloromethane evaporated and product preabsorbed on silica gel was chromatographed on silica gel (25 g, n-hexane/dichloromethane 2 : 5). The obtained compound was further crystallized from dichloromethane/n-hexane to afford pure product 1. Yield: 1.19 g, 81%. Spectral data of the obtained brassinin (1) were identical with those described previously.²⁸

1.1.3 1-Methylbrassinin (15). Crude product of (1-methylindol-3-yl)methylamine (13) freshly prepared by the reduction of 1-methylindole-3-carboxaldehyde oxime (0.87 g, 5 mmol) with NaBH₄ (1.82 g, 50 mmol) in the presence NiCl₂·6H₂O (1.30 g, 5.5 mmol) was dissolved in methanol (50 mL).²⁷ Triethylamine (0.70 mL, 0.50 g, 5 mmol) and carbon disulfide (0.60 mL, 0.76 g, 10 mmol) were added and mixture was stirred for 5 min at room temperature. Then iodomethane (0.37 mL, 0.85 g, 6 mmol) was added and the stirring was continued for 15 min. The residue obtained after evaporation of the solvent was dissolved in dichloromethane, small amount of silica gel was added, dichloromethane evaporated and product preabsorbed on silica gel was chromatographed on silica gel (40 g, n-hexane/ethyl acetate 2 : 1). Yield: 0.91 g, 73%. Spectral data of the obtained 1-methylbrassinin (15) were identical with those described previously.²⁷

1.1.4 N-(1-methoxyindol-3-yl)methyl-N′-phenylthiourea (16). Crude product of (1-methoxyindol-3-yl)methylamine (10) freshly prepared by the reduction of 1-methoxyindole-3-carboxaldehyde oxime (0.380 g, 2 mmol) with NaBH₃CN (1.26 g, 20 mmol) in the presence of TiCl₃ (5.5 mL, 16 mmol)²⁹ was dissolved in methanol (10 mL). Triethylamine (1.39 mL, 1.01 g, 10 mmol) and phenyl isothiocyanate (0.24 mL, 0.27 g, 2 mmol) were added and mixture was stirred for 25 min at room temperature. The residue obtained after evaporation of the solvent was dissolved in dichloromethane, small amount of silica gel was added, dichloromethane evaporated and product preabsorbed on silica gel was chromatographed on silica gel (15 g, n-hexane/ethyl acetate 3 : 1). The obtained compound was further crystallized from dichloromethane/n-hexane to afford pure product 16.

Yield: 0.49 g, 79%, white crystals; mp 103–104 °C (dichloromethane/n-hexane); R_f = 0.26 (n-hexane/ethyl acetate 3 : 1).ν_max (CHCl₃)/cm⁻¹: 3387, 2973, 1487, 1440, 1287, 1213, 1187, 1100, 1020, 953. δ_H (400 MHz, CDCl₃): 8.07 (bs, 1H, NH′), 7.62 (ddd, J = 0.8 Hz, 0.9 Hz, 8.0 Hz, 1H, H-4), 7.41 (ddd, J = 0.8 Hz, 0.8 Hz, 8.2 Hz, 1H, H-7), 7.32 (dd, J = 7.8 Hz, 7.8 Hz, 2H, H-3′, H-5′), 7.26 (s, 1H, H-2), 7.26 (ddd, J = 0.9 Hz, 7.2 Hz, 8.0 Hz, 1H, H-6), 7.20 (dd, J = 7.7 Hz, 7.7 Hz, 1H, H-4′), 7.14 (d, J = 7.7 Hz, 2H, H-2′, H-6′), 7.13 (ddd, J = 1.0 Hz, 7.1 Hz, 8.0 Hz, 1H, H-5), 6.18 (bs, 1H, NH), 4.98 (d, J = 4.9 Hz, 2H, CH₂), 4.05 (s, 3H, OCH₃). δ_C (100 MHz, CDCl₃): 180.3 ((C=S)), 136.3 (C-1′), 132.6 (C-7a), 130.4 (C-3′, C-5′), 127.4 (C-4′), 125.2 (C-2′, C-6′), 123.1 (C-2), 122.5 (C-6), 120.5 (C-5), 119.3 (C-4), 108.7 (C-7), 107.4 (C-3a), 66.2 (OCH₃), 41.5 (CH₂). MALDI-TOF MS, m/z (% relative int.): 334.7 [M + Na]⁺ (29), 312.7 [M + H]⁺ (14), 305.0 (16), 280.9 (21), 161.6 (100). Anal. Calc. for C₁₇H₁₇N₃OS requires: C, 65.57; H, 5.50; N, 13.49. Found: C, 65.79; H, 5.40; N, 13.71.

1.2 Typical procedure for Pd-catalyzed cyclization of brassinin and 1-substituted derivatives

A brassinin derivatives 1, 2, 14–17 (0.15 mmol) and PdCl₂(CH₃CN)₂ (0.0045 g, 0.015 mmol) in DMSO (2.7 mL) and methanol, water (0.3 mL) or aniline (0.015 mL, 0.015 g, 0.165 mmol) were heated at 80 °C for 1.5–4 h. The reaction mixture was cooled to room temperature and brine (10 mL) was added. The aqueous phase was extracted with ethyl acetate (2 × 5 mL) and the combined organic layer was dried over Na₂SO₄. The solvent was evaporated and the residue was purified by silica gel column chromatography. The glassware was washed with aqua regia (to remove residual Pd). Each reaction was performed 3 times.

1.2.1 trans and cis-(±)-1-methoxyspirobrassinol [(±)-5a and (±)-5b]. Following the general procedure, products (±)-5a and (±)-5b were obtained using 0.040 g (0.15 mmol) of 1-methoxybrassinin (2) and water (0.3 mL). Reaction mixture was stirred for 1 h. The residue obtained after evaporation of the solvent was subjected to flash chromatography on 5 g silica gel (n-hexane/ethyl acetate 2 : 1), affording (±)-5a and (±)-5b (0.022 g, 53%) as a semi-solid colourless material with all spectral data fully identical with the described natural product.²⁴

1.2.2 trans and cis-(±)-1-methoxyspirobrassinol methyl ether [(±)-6a and (±)-6b]. Following the general procedure, products (±)-6a and (±)-6b were obtained using 0.040 g (0.15 mmol) of 1-methoxybrassinin (2) and methanol (0.3 mL). Reaction mixture was stirred for 1 h. The residue obtained after evaporation of the solvent was subjected to flash chromatography on 5 g silica gel (n-hexane/diethyl ether 3 : 2), affording mixture of diastereoisomers (±)-6a and (±)-6b (0.026 g, 60%) as a colourless oil with all spectral data fully identical with the described natural²⁴ and unnatural product.¹³

1.2.3 trans and cis-(±)-1-methoxy-2-phenylamino-2′-(methylsulfanyl)spiro{indoline-3,5′-[4′,5′]-dihydrothiazol} [(±)-18a and (±)-18b]. Following the general procedure, products (±)-18a and (±)-18b were obtained using 0.040 g (0.15 mmol) of 1-methoxybrassinin (2) and aniline (0.015 mL, 0.015 g, 0.0165 mmol). Reaction mixture was stirred for 2 h. The residue obtained after evaporation of the solvent was subjected to flash chromatography on 5 g silica gel (n-hexane/ethyl acetate 2 : 1), affording mixture of diastereoisomers (±)-18a and (±)-18b (0.029 g, 54%) as a yellow oil with all spectral data fully identical with the described products.²¹

1.2.4 trans and cis-(±)-1-(tert-butoxycarbonyl)spirobrassinol [(±)-19a and (±)-19b]. Following the general procedure, products (±)-19a and (±)-19b were obtained using 0.050 g (0.15 mmol) of 1-Boc-brassinin (14) and water (0.3 mL). Reaction mixture was stirred for 1.5 h. The residue obtained after evaporation of the solvent was subjected to flash chromatography on 5 g silica gel (n-hexane/ethyl acetate 4 : 1), affording mixture of diastereoisomers (±)-19a and (±)-19b (0.028 g, 53%) as a white solid with all spectral data fully identical with the described products.¹¹

1.2.5 trans and cis-(±)-1-(tert-butoxycarbonyl)spirobrassinol methyl ether [(±)-20a and (±)-20b]. Following the general procedure, products (±)-20a and (±)-20b were obtained using 0.050 g (0.15 mmol) of 1-Boc-brassinin (14) and methanol (0.3 mL). Reaction mixture was stirred for 3.5 h. The residue obtained after evaporation of the solvent was subjected to chromatography on 5 g silica gel (dichloromethane), affording (±)-20a (0.016 g, 29%) as a colourless solid and (±)-20b (0.014 g, 25%) as a colourless oil.
trans-(±)-1-(tert-butoxycarbonyl)spirobrassinol methyl ether [(±)-20a]. Yield: 0.016 g, 29%, colourless solid; mp 68–70 °C (dichloromethane); R_f = 0.12 (dichloromethane). ν_max (CHCl₃)/cm⁻¹: 1713 (C=O), 1573 (C=N). δ_H (400 MHz, DMSO, 60 °C): 7.57 (d, J = 7.3 Hz, 1H, H-7), 7.44 (d, J = 7.5 Hz, 1H, H-4), 7.30 (dd, J = 7.8 Hz, J = 7.6 Hz, 1H, H-6), 7.07 (dd, J = 7.5 Hz, J = 7.5 Hz, 1H, H-5), 5.42 (s, 1H, H-2), 4.69 (d, J = 15.7 Hz, 1H, H_b), 4.40 (d, J = 15.7 Hz, 1H, H_a), 3.43 (s, 3H, OCH₃), 2.56 (s, 3H, SCH₃), 1.54 (s, 9H, [C(CH₃)₃]). δ_C (100 MHz, DMSO, 60 °C): 162.3 (C=N), 151.4 (C=O), 140.8 (q), 130.3 (q), 129.7, 123.7, 123.6 and 115.2 (C-arom), 98.6 (C-2), 82.0 [C(CH₃)₃], 70.6 (C-3), 65.8 (CH₂), 57.4 (OCH₃), 27.9 [C(CH₃)₃], 14.7 (SCH₃). NOESY correlations (400 MHz, DMSO): H_a/H-4, H-2/OCH₃, H_a/H_b, [C(CH₃)₃]/OCH₃, [C(CH₃)₃]/SCH₃. EIMS m/z (% relative int.): 366 [M]⁺ (19), 310 (17), 265 (17), 234 (20) 192 (14), 161 (23), 57 (100). HRMS m/z Calc. for C₁₇H₂₂N₂O₃S₂: 366.1072, found: 366.1083.
cis-(±)-1-(tert-butoxycarbonyl)spirobrassinol methyl ether [(±)-20b]. Yield: 0.014 g, 25%, colourless oil; R_f = 0.19 (dichloromethane). ν_max (CHCl₃)/cm⁻¹: 1727 (C=O), 1570 (C=N). δ_H (400 MHz, DMSO, 60 °C): 7.60 (d, J = 7.5 Hz, 1H, H-7), 7.28 (dd, J = 7.7 Hz, J = 7.8 Hz, 1H, H-6), 7.25 (d, J = 7.5 Hz, 1H, H-4), 7.05 (dd, J = 7.4 Hz, J = 7.4 Hz, 1H, H-5), 5.31 (s, 1H, H-2), 4.45 (d, J = 15.2 Hz, 1H, H_b), 3.85 (d, J = 15.2 Hz, 1H, H_a), 3.46 (s, 3H, OCH₃), 2.57 (s, 3H, SCH₃), 1.55 (s, 9H, [C(CH₃)₃]). δ_C (100 MHz, DMSO, 60 °C): 164.2 (C=N), 151.5 (C=O), 139.4 (q), 132.6 (q), 129.3, 123.4, 123.3 and 114.8 (C-arom), 95.6 (C-2), 81.8 [C(CH₃)₃)], 74.7 (CH₂), 73.3 (C-3), 57.9 (OCH₃), 27.9 [C(CH₃)₃)], 14.6 (SCH₃). NOESY correlations (400 MHz, DMSO): H_b/H-2, H-2/OCH₃, H_a/H_b, [C(CH₃)₃]/OCH₃, [C(CH₃)₃]/SCH₃. EIMS m/z (% relative int.): 366 [M]⁺ (18), 310 (17), 265 (20), 234 (21) 192 (16), 161 (30), 57 (100). HRMS m/z Calc. for C₁₇H₂₂N₂O₃S₂: 366.1072, found: 366.1087.

1.2.6 trans and cis-(±)-1-methoxy-2-hydroxy-2′-(phenylamino)spiro{indoline-3,5′-[4′,5′]-dihydrothiazol} [(±)-21a and (±)-21b]. Following the general procedure, products (±)-21a and (±)-21b were obtained using 0.047 g (0.15 mmol) of thiourea 16 and water (0.3 mL). Reaction mixture was stirred for 4 h. The residue obtained after evaporation of the solvent was subjected to flash chromatography on 5 g silica gel (n-hexane/ethyl acetate 2 : 1), affording mixture of diastereoisomers (±)-21a and (±)-21b (0.023 g, 47%) as a yellow oil.

Yield: 0.023 g, 47%, yellow oil; R_f = 0.14 (n-hexane/ethyl acetate 2 : 1). δ_H (400 MHz, CDCl₃): 7.33 (d, J = 7.4 Hz, 1H, H-4), 7.29–7.13 (m, 5H, H-3′, H-5′, H-2′, H-6′, H-6), 7.02 (dd, J = 7.4 Hz, 7.5 Hz, 2H, H-5, H-4′), 6.93 (d, J = 7.8 Hz, 1H, H-7), 5.48 (bs, 1H, OH), 5.27 (s, 1H, H-2 trans), 4.88 (s, 1H, H-2 cis), 4.47 (d, J = 12.0 Hz, 1H, H_b trans), 4.26 (d, J = 12.6 Hz, 1H, H_a cis), 4.01 (d, J = 12.6 Hz, 1H, H_b cis), 3.93 (s, 3H, OCH₃ cis), 3.88 (s, 3H, OCH₃ trans), 3.73 (d, J = 12.0 Hz, 1H, H_a trans). δ_C (100 MHz, CDCl₃): 160.8 (C=N trans), 158.5 (C=N cis), 148.5 (C-7a trans), 147.8 (C-7a cis), 145.8 (C-1′ cis), 144.5 (C-1′ trans), 130.0 (C-6 cis), 129.8 (C-6 trans), 129.0 (C-3′, C-5′), 127.1 (C-3a cis), 126.5 (C-3a trans), 124.0, 123.8, 123.8, 123.7, 123.5, 123.4 (C-4 cis, C-4 trans, C-5 cis, C-5 trans, C-4′ cis, C-4′ trans), 121.4 (C-2′, C-6′ trans), 120.9 (C-2′, C-6′ cis), 112.9 (C-7 cis), 112.8 (C-7 trans), 99.3 (C-2 trans), 98.0 (C-2 cis), 66.9 (C-3 cis), 64.4 (N–OCH₃ cis), 64.0 (C-3 trans), 63.8 (OCH₃ trans), 61.8 (CH₂ cis), 55.7 (CH₂ trans). NOESY correlations (400 MHz, CDCl₃): H_a trans/H_b trans, H_a trans/H-4, H_a cis/H_b cis, H-2 cis/H_b cis. Anal. Calc. for C₁₇H₁₇N₃O₂S requires: C, 62.36; H, 5.23; N, 12.83. Found: C, 62.11; H, 5.32; N, 12.71.

1.2.7 trans and cis-(±)-1-methoxy-2-methoxy-2′-(phenylamino)spiro{indoline-3,5′-[4′,5′]-dihydrothiazol} [(±)-22a and (±)-22b]. Following the general procedure, products (±)-22a and (±)-22b were obtained using 0.047 g (0.15 mmol) of thiourea 16 and methanol (0.3 mL). Reaction mixture was stirred for 2 h. The residue obtained after evaporation of the solvent was subjected to flash chromatography on 5 g silica gel (cyclohexane/acetone 8 : 1), affording (±)-22a (0.016 g, 32%) as a white crystals and (±)-22b (0.014 g, 27%) as a white crystals.
trans-(±)-1-methoxy-2-methoxy-2′-(phenylamino)spiro{indoline-3,5′-[4′,5′]-dihydrothiazol} [(±)-22a]. Yield: 0.016 g, 32%, white crystals; mp = 149–151 °C (dichloromethane/n-hexane); R_f = 0,11 (cyclohexane/acetone 8 : 1). ν_max (CHCl₃)/cm⁻¹ 3413, 2990, 2935, 1630, 1585, 1490, 1305, 1185, 1140, 1080, 1040, 690. δ_H (400 MHz, CDCl₃): 7.35 (dd, J = 0.5 Hz, 7.6 Hz, 1H, H-4), 7.29 (dd, J = 7.4 Hz, 8.2 Hz, 2H, H-3′, H-5′), 7.26 (ddd, J = 1.1 Hz, 7.5 Hz, 7.7 Hz, 1H, H-6), 7.18 (d, J = 8.2 Hz, 2H, H-2′, H-6′), 7.04 (dd, J = 7.4 Hz, 7.4 Hz, 1H, H-4′), 7.02 (dd, J = 7.7 Hz, 7.7 Hz, 1H, H-5), 6.95 (d, J = 7.8 Hz, 1H, H-7), 4.95 (s, 1H, H-2), 4.47 (d, J = 11.8 Hz, 1H, H_b), 3.94 (s, 3H, N–OCH₃), 3.75 (s, 3H, 2-OCH₃), 3.64 (d, J = 11.8 Hz, 1H, H_a). δ_C (100 MHz, CDCl₃): 158.7 (C=N), 148.1 (C-7a), 146.2 (C-1′), 129.8 (C-6), 129.0 (C-3′, C-5′), 127.7 (C-3a), 123.9 (C-4), 123.8 (C-5), 123.5 (C-4′), 121.0 (C-2′, C-6′), 112.9 (C-7), 108.6 (C-2), 63.8 (N–OCH₃), 63.5 (C-3), 59.9 (2-OCH₃), 57.1 (CH₂). NOESY correlations (400 MHz, CDCl₃): H_a/H_b; 2-OCH₃/H-2. Difference NOE spectra (CDCl₃): irr. at δ 4.47 (H_b) enhanced signal δ 3.64 (H_a, 24.1%), irr. at δ 4.95 (H-2) enhanced signal δ 3.75 (2-OCH₃, 7.5%). MALDI-TOF MS, m/z (% relative int.): 342.0 [M + H]⁺ (100), 312.2 (17), 200.1 (8). Anal. Calc. for C₁₈H₁₉N₃O₂S requires: C, 63.32; H, 5.61; N, 12.31. Found: C, 63.01; H, 5.72; N, 12.13.
cis-(±)-1-methoxy-2-methoxy-2′-(phenylamino)spiro{indoline-3,5′-[4′,5′]-dihydrothiazol} [(±)-22b]. Yield: 0.014 g, 27%, white crystals; mp = 166–168 °C (dichloromethane/n-hexane); R_f = 0,06 (cyclohexane/acetone 8 : 1). ν_max (CHCl₃)/cm⁻¹ 3410, 3000, 2940, 1627, 1590, 1433, 1307, 1200, 1140, 1090. δ_H (400 MHz, CDCl₃): 7.37 (dd, J = 0.5 Hz, 7.6 Hz, 1H, H-4), 7.26–7.29 (m, 5H, H-2′, H-3′, H-5′, H-6′, H-6), 7.04 (dd, J = 8.5 Hz, 8.5 Hz, 1H, H-4′), 7.03 (ddd, J = 1.0 Hz, 7.5 Hz, 7.6, 1H, H-5), 6.98 (d, J = 7.8 Hz, 1H, H-7), 4.66 (s, 1H, H-2), 4.26 (d, J = 12.6 Hz, 1H, H_a), 4.12 (d, J = 12.6 Hz, 1H, H_b), 3.95 (s, 3H, N–OCH₃), 3.74 (s, 3H, 2-OCH₃). δ_C (100 MHz, CDCl₃): 159.3 (C=N), 147.6 (C-7a), 143.3 (C-1′), 129.9 (C-6), 129.1 (C-3′, C-5′), 128.8 (C-3a), 124.0 (C-5), 123.6 (C-4′), 123.1 (C-4), 120.4 (C-2′, C-6′), 112.8 (C-7), 105.0 (C-2), 66.5 (C-3), 64.2 (CH₂), 63.8 (N–OCH₃), 59.7 (2-OCH₃). NOESY correlations (400 MHz, CDCl₃): H_a/H_b; 2-OCH₃/H-2; H_b/H-2. Difference NOE spectra (CDCl₃): irr. at δ 4.66 (H-2) enhanced signal δ 4.12 (H_b, 3.2%) and 3.74 (2-OCH₃, 7.0%), irr. at δ 4.12 (H_b) enhanced signal δ 4.66 (H-2, 8.9%) and 4.26 (H_a, 14.7%). MALDI-TOF MS, m/z (% relative int.): 342.0 [M + H]⁺ (100), 312 (14), 200.1 (5). Anal. Calc. for C₁₈H₁₉N₃O₂S requires: C, 63.32; H, 5.61; N, 12.31. Found: C, 63.59; H, 5.32; N, 12.08.

1.2.8 trans and cis-(±)-1-(tert-butoxycarbonyl)-2-hydroxy-2′-(phenylamino)spiro{indoline-3,5′-[4′,5′]-dihydrothiazol} [(±)-23a and (±)-23b]. Following the general procedure, products (±)-23a and (±)-23b were obtained using 0.058 g (0.15 mmol) of thiourea 17 and water (0.3 mL). Reaction mixture was stirred for 4 h. The residue obtained after evaporation of the solvent was subjected to flash chromatography on 5 g silica gel (n-hexane/ethyl acetate 2 : 1), affording mixture of diastereoisomers (±)-23a and (±)-23b (0.031 g, 52%) as a white solid with all spectral data fully identical with the described products.¹²

1.2.9 trans and cis-(±)-1-(tert-butoxycarbonyl)-2-methoxy-2′-(phenylamino)spiro{indoline-3,5′-[4′,5′]-dihydrothiazol} [(±)-24a and (±)-24b]. Following the general procedure, products (±)-24a and (±)-24b were obtained using 0.058 g (0.15 mmol) of thiourea 17 and methanol (0.3 mL). Reaction mixture was stirred for 1.5 h. The residue obtained after evaporation of the solvent was subjected to flash chromatography on 5 g silica gel (n-hexane/ethyl acetate 2 : 1), affording mixture of diastereoisomers (±)-24a and (±)-24b (0.035 g, 56%) as a white solid with all spectral data fully identical with the described products.¹²

1.2.10 (±)-Spirobrassinin [(±)-3]. Following the general procedure, product (±)-3 were obtained using 0.035 g (0.15 mmol) of brassinin (1) and methanol (0.3 mL). Reaction mixture was stirred for 1 h. The residue obtained after evaporation of the solvent was subjected to flash chromatography on 5 g silica gel (n-hexane/ethyl acetate 2 : 1), affording (±)-3 (0.013 g, 34%) as a colourless crystals with all spectral data fully identical with the described natural product.³⁰

1.2.11 (±)-1-Methylspirobrassinin [(±)-25]. Following the general procedure, product (±)-25 were obtained using 0.038 g (0.15 mmol) of brassinin (15) and methanol (0.3 mL). Reaction mixture was stirred for 1 h. The residue obtained after evaporation of the solvent was subjected to flash chromatography on 5 g silica gel (n-hexane/ethyl acetate 2 : 1), affording (±)-25 (0.016 g, 40%)as a white solid with all spectral data fully identical with the described product.³¹

Acknowledgements

We would like to thank the Slovak Grant Agency for Science (Grant no. 1/0954/12) for financial support of this work.

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Footnote

† Electronic supporting information (ESI) available: Copies of 1H and 13C NMR spectra for all new compounds. See DOI: 10.1039/c3ra46843g

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