Lipase-initiated one-pot synthesis of spirooxazino derivatives: redesign of multicomponent reactions to expand substrates scope and application potential

Tetrahedron 72 (2016) 3318e3323

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Lipase-initiated one-pot synthesis of spirooxazino derivatives: redesign of multicomponent reactions to expand substrates scope and application potential Xiao-Yang Chen, Jun-Liang Wang, Xian-Fu Lin, Qi Wu * Department of Chemistry, Zhejiang University, Hangzhou 310027, People’s Republic of China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 3 January 2016 Received in revised form 20 April 2016 Accepted 22 April 2016 Available online 25 April 2016

Enzymatic multicomponent reactions (MCR) are very powerful for complex organic synthesis with environmentally friendly, highly efficient and selective characters, while sometimes having some unavoidable shortages such as narrow substrate scope and so on. Herein, this work demonstrated how to redesign one previously reported lipase-catalyzed MCR to achieve more broad substrates and more efficient synthesis. Twelve new spirooxazino derivatives with different substitutions were obtained in moderate to good yield, while all of them could not be synthesized using the previous route. Reaction conditions such as enzymes, enzyme concentration, amides and ratio of substrates were screened. Furthermore, particularly interesting is that chiral spirooxazino could also be obtained through a further developed two-enzymatic MCR process in one pot. As a domino process simultaneously constructing six new C-C/N bonds and two rings in only one step, the work will remarkably expand the application scope of enzymatic MCR for simple and green synthesis of complex compounds. Ó 2016 Elsevier Ltd. All rights reserved.

Keywords: Multicomponent reactions Spirooxazino Lipase Enzymatic reaction Candida antarctica lipase B

1. Introduction Hydrolases are known to be highly selective for the hydrolysis or transesterification of esters and amides, and recent progress in catalytic promiscuity of hydrolases has further expanded their application scope in organic synthesis.1 This promiscuous property of enzymes endows one enzyme with catalytic multi-functions, allowing multi-step reactions catalyzed by one enzyme under multicomponent reactions (MCR) manner.2 For example, the work by Yu et al. demonstrated a lipase-catalyzed three-component Mannich reaction under aqueous conditions.2a Our group developed a direct method to construct 3,4-dihydropyridin-2-ones through enzymatic condensation of aldehyde with cyanoacetamide and 1,3-dicarbonyl compounds in one-pot.2d In comparison with stepwise process, MCR are more powerful approaches for the preparation of structurally complex compounds, like spiro motifs. However, sometimes enzymatic MCR are not fully satisfactory, in some respects, such as narrow substrate scope and low reaction efficiency due to the reaction complexity and the specific structure of enzymes. Researchers often ignored less active substrates, rather than studying the internal reason and redesigning the MCR. Actually, one MCR process usually has different reaction pathways or

* Corresponding author. Tel.: þ86 571 87953001; fax: þ86 571 87952618; e-mail address: [email protected] (Q. Wu). http://dx.doi.org/10.1016/j.tet.2016.04.060 0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

routes, rational redesign or recombination of one MCR usually can bring some advantages to overcome those obstacles. There are some successful examples reported in chemical MCR, while to the best of our knowledge, similar reports in enzymatic MCR are few. For examples, standard Ugi reaction employs four components, an € lo € p4 acid, an amine, an aldehyde or ketone, and an isocyanide.3 Fu et al. reported a Ugi three-component reaction starting from alicyclic b-amino acids, for the synthesis of alicyclic b-lactams, which greatly expanded the substrate scope of Ugi reaction. Spirocycles are very important structural units widely found in natural products, synthesized pharmacological agents, agricultural products, and also some new ligands or catalysts such as spirobisoxazolines, SPINOL (1,10 -spirobiindane-7,70 -diol), SPINOLderived phosphoric acids.5 Moreover, the unique structural feature of spirocyclic compounds endows them with special fluorescent or photochromic properties, and thus important applications in the fields of fluorescent chemosensors,6a information memory and storage,6b artificial intelligent materials.6c For example, spirooxazines are the most popular class of photochromic materials because of their good performances in terms of stability and response speed.7 However, the synthesis difficulty of complicated spirocompounds usually limits their wide application in academic and industrial fields. For this reason, the development of highly efficient synthesis methodology for complicated spirocompounds, is still an enormous challenge for chemists.

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We are continuously devoting to the study of enzymatic MCR for the synthesis of complicated compounds. Recently, our group reported a novel CALB-initiated MCR for the synthesis of spirooxazino derivatives, while the substrate scope was quite narrow.8 Herein, basing on the comparative study of several synthetic routes accessing to spirooxazino derivatives, we developed a more efficient synthesis methodology for the complicated spirooxazinos derivatives than before. Twelve new spirooxazino derivatives with different substitutions were obtained in moderate to good yield, while all of them could not be synthesized using the previous route. Particularly interesting is that chiral spirooxazino could be obtained through a two-enzymatic process in one pot.

mediated MCR route (Route 2) for the synthesis of spirocompounds. A chemically prepared aldol intermediate was used as one starting molecule instead of aldehydes and cyclohexanones, and remarkable improvements of substrate scope and reaction efficiency were observed (Scheme 1).

2. Results and discussion

Scheme 1. Route 2: CALB-catalyzed MCR starting from chemo-aldol intermediate (model reaction of this work).

In our previous work, the spirocompounds were formed efficiently only for 4-nitrobenzaldehyde (3a) (Table 1).8 When other aldehydes (3bef) were used, although corresponding products could be detected, reaction yields were very low (Table 2). We found this failure was probably caused by the fact that those substituted aldehydes, as well as aliphatic aldehydes, almost could not react with cyclohexanone to form the corresponding aldol intermediate under the catalysis of Candida antarctica lipase B (CALB). After we were aware of this point, we designed another lipaseTable 1 Spirooxazinos compounds successfully obtained via Route 1 MCR8

Entry

R1

R2

Product

Yield (%)a

1 2 3 4 5 6 7 8 9 10 11 12

4-CH3O-C6H4 (1a) 4-HO-C6H4 (1b) 3-HO-C6H4 (1c) C6H5 (1d) 4-CH3-C6H4 (1e) 4-iPr-C6H4 (1f) 4-N(CH3)2-C6H4 (1g) 4-F-C6H4 (1h) C6H5 (1d) 4-F-C6H4 (1h) 4-HO-C6H4 (1b) 4-CH3O-C6H4 (1a)

H (2a) H (2a) H (2a) H (2a) H (2a) H (2a) H (2a) H (2a) p-CH3 (2b) p-CH3 (2b) p-CH3 (2b) p-CH3 (2b)

5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l

72 84 85 30 34 21 41 47 25 25 38 36

a

Data from the previous work.8

We investigated the Route 2 for the synthesis of spirooxazino derivatives using the model reaction of 4-methoxy-b-nitrostyrene (1a), cyclohexanone (2a), aldol intermediate from 4fluorobenzaldehyde (6a), and acetamide (4) (Scheme 1). After screening a lot of enzymes (Table S1, see Supplementary data), we found that only CALB catalyzed this reaction for the formation of spirocompound 5m. Control reactions did not happen under the catalysis of BSA (Bull Serum Albumin) or denatured CAL-B (Table S1, entries 3 and 4), ruling out the possibility that amino acids on the surface of CAL-B or other impurities could promote the MCR. On the other hand, these results also validated that the specific active site of CAL-B was essential in the MCR. Then the reaction conditions such as enzyme concentration, structure of amide, amount of acetamide, molar ratio of substrates and reaction time were examined further. Among the tested structurally different amides, acetamide and formamide showed the best results for this transformation, while three other amides including propanamide, benzamide and thioacetamide displayed almost no effects (Table 3). From the influence of enzyme concentration on the reaction, it was found that the use of CAL-B concentration of 60 mg/ml was enough to obtain the best yield for the tested reaction (Fig. 1). It is also noteworthy that the amount of acetamide and the molar ratio of substrates have important influence on the output of the multicomponent reaction (Figs. 2 and 3). The optimal dosage of acetamide (4) and 4-methoxy-b-nitrostyrene (1a) were 0.75 M and 1.25 M, respectively, in the tested model reaction containing 0.25 M aldol intermediate and 1 mL cyclohexanone. Moreover, the optimization of reaction time showed that 96 h was enough for this domino reaction (Fig. S1, see Supplementary data). Finally, under the optimized conditions, spirocompound 5m were obtained in the highest 67% yield (Entry 1, Table 4), showing a sharp contrast with only 7% yield in Route 1 (Entry 1, Table 2).

Table 2 The influence of structure of aldehydes on Route 1 MCRa Table 3 Amide screening for CALB-catalyzed Route 2 MCRa

Entry

R

Product

Yield (%)b

1 2 3 4 5

p-FC6H4 (3b) p-ClC6H4 (3c) p-CF3C6H4 (3d) p-CH3OC6H4 (3e) C6H5 (3f)

5m 5n 5o 5p 5q

7 <5 <5 <5 <5

a Experimental conditions: 0.25 mmol aldehyde, 1.5 mmol 4-methoxy-b-nitrostyrene, 1 mL cyclohexanone, 1.25 mmol acetamide, 70 mg CALB, 50  C, 6 days. b Yields were determined by HPLC.

Entry

Amide

Yield (%)

1 2 3 4 5

Formamide Acetamide Propanamide Benzamide Thioacetamide

27 32 6 9 0

a Experimental conditions: 0.25 M 2-((4-fluorophenyl)-(hydroxy)methyl)cyclohexanone, 0.5 M 4-methoxy-b-nitrostyrene, 1 mL cyclohexanone, 1 M amide, 60 mg CALB, 50  C, 3 days. All yields were detected by HPLC.

Then, a series of spiro-products with different substitutions at R1, R2, R3 and R4 (Table 4) were successfully obtained in moderate to good yields under the above optimized conditions. It was noteworthy that most of the products with various substitutions at R3 such as F, CF3, Cl, CH3O, H, in Table 4, could not be prepared using

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X.-Y. Chen et al. / Tetrahedron 72 (2016) 3318e3323 Table 4 CALB-catalyzed MCR starting from chemo-aldol intermediate (Route 2)a

30

Yield / %

25 20 15 10 5 0

30

40

50

60

70

80

Amount of enzyme /mg Fig. 1. The influence of enzyme concentration on Route 2 MCR. Experimental conditions: 0.25 mmol 2-((4-fluorophenyl)-(hydroxy)methyl)cyclohexanone, 0.5 mmol 4methoxy-b-nitrostyrene, 1 mL cyclohexanone, 1 mmol acetamide, 30e80 mg CAL-B, 50  C, 3 days.

40

Yield / %

30

20

0.50

0.75

1.00

1.25

1.50

Amount of acetamide / mmol Fig. 2. The influence of amide concentrations on Route 2 MCR. Experimental conditions: 0.25 mmol 2-((4-fluorophenyl)-(hydroxy)methyl)cyclohexanone, 0.5 mmol 4methoxy-b-nitrostyrene, 1 mL cyclohexanone, 0.50e1.50 mmol acetamide, 60 mg CALB, 50  C, 3 days. 60 50

Yield / %

40 30 20 10 0 0.00

R1

R2

R3

R4

Product

Yield (%)b

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

4-CH3O-C6H4 (1a) 4-CH3O-C6H4 (1a) 4-CH3O-C6H4 (1a) 4-CH3O-C6H4 (1a) 4-CH3O-C6H4 (1a) 4-F-C6H4 (1h) C6H5 (1d) 4-CH3O-C6H4 (1a) 4-CH3O-C6H4 (1a) 4-CH3O-C6H4 (1a) 3-CH3O-C6H4 (1i) 1-Naphth (1j) 4-CH3O-C6H4 (1a) C6H5 (1d) 4-F-C6H4 (1h)

H (2a) H (2a) H (2a) H (2a) H (2a) H (2a) H (2a) H (2a) 4-CH3(2b) 3-CH3(2c) H (2a) H (2a) H (2a) H (2a) H (2a)

F (6a) Cl (6b) CF3 (6c) H (6d) CH3O (6e) F (6a) F (6a) F (6f) F (6a) F (6a) F (6a) NO2 (6g) NO2 (6g) NO2 (6g) NO2 (6g)

H H H H H H H CH3 H H H H H H H

5m 5n 5o 5p 5q 5r 5s 5t 5u 5v 5w 5x 5a 5d 5h

67 33 25 43 36 32 42 20 40 35 36 77 62 (72c) 38 (30c) 36 (47c)

a Experimental conditions: 0.25 mmol aldol intermediate, 1.25 mmol nitroalkene, 1 mL monocarbonyl compound, 0.75 mmol acetamide, 60 mg CALB, 50  C, 96 h. b Yields were determined by HPLC. c Yields were from Route 1, where reaction time was 144 h.

10

0 0.25

Entry

0.25

0.50

0.75

1.00

1.25

4). This new lipase-initiated MCR provided successfully 15 complex spiro-compounds (among them, 12 are new compounds), and showed relatively wider substrate scope and generality, endowing this approach with important application potential in complex organic synthesis. Chiral spirocompounds were important in many fields, such as asymmetric catalysis and pharmacological agents. In the previous CALB-catalyzed MCR route for the synthesis of spirooxazino derivatives,8 no enantioselectivity was observed because the first step (CALB-catalyzed aldol reaction) where the stereocenters of final spiro-products were produced, failed to provide any enantioselectivity as reported in some papers from our and other groups.9 While in this redesigned CALB-catalyzed MCR (Route 2), when we used chemically prepared chiral aldol intermediate10 as substrate, corresponding chiral spirocompound could be obtained with a slightly decreased stereoselectivity (Scheme 2), because the aldol reaction was slightly reversible under the catalysis of CALB/ acetamide.

1.50

Amount of nitroalkene/mmol Fig. 3. The influence of the amount of 4-methoxy-b-nitrostyrene on the Route 2 MCR. Experimental conditions: 0.25 mmol 2-((4-fluorophenyl)-(hydroxy)methyl) cyclohexanone, 0.25e1.50 mmol 4-methoxy-b-nitrostyrene, 1 mL cyclohexanone, 0.75 mmol acetamide, 60 mg CALB, 50  C, 3 days.

the previous Route 1 method.8 While some spiro-products with nitro substitutions at R3 (5a, 5d, 5h) can be prepared via both Route 1 and Route 2 (entries 13e15 in Table 4). For the synthesis of 5a and 5h, Route 1 gave slightly higher yields than Route 2 because reaction time in Route 1 was longer than Route 2. Excitingly, the sterically demanding naphthyl group of nitroolefin 1j could be efficiently incorporated into the final spiro-compound (5x) in higher yield than other nitroolefins (entry 12 in Table 4) using Route 2, while it was also not able to be prepared via Route 1. Substituted cyclohexanone such as 3-methyl cyclohexanone (2c) could also be applied in the transformation in moderate yield (entry 10 in Table

Scheme 2. Route 2: CALB-catalyzed MCRs starting from optical pure chemo-aldol intermediate for the synthesis of chiral spirocompound.

Inspired by this stepwise process, we further developed a twoenzymatic MCR of chiral spirooxazino in one-pot (Route 3). The first step was PPL-catalyzed asymmetric aldol reaction, according to Ref. 11. Then we filtered off PPL, and added the other substrates and CALB. This process avoided to isolate the aldol intermediate, giving the product in acceptable yield and stereoselectivity (Scheme 3). At last, the above data clearly showed the advantages and disadvantages of these three routes for the synthesis of spirooxazino. For the Route 1, there are several valuable characteristics such as

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acid per 30 min) were purchased from Amano Enzyme Inc (Japan). Alkaline protease from Bacillus subtilis (BSAP) (10 U/mg, 1 U corresponds to the amount of enzyme which liberates 1 mmol folinpositive amino acids and peptides per minute at pH 7.5 and 37  C) was obtained from Wuxi Enzyme Co. Ltd. (Wuxi, PR China). All reagents used in the experiments were obtained from commercial sources and used without further purification.

4.2. Analytical methods

Scheme 3. Route 3: two-enzymatic cascade asymmetric synthesis of chiral spirooxazino in one-pot.

single enzyme-initiated MCRs process, simple and readily available starting molecules, and simultaneous construction of six new CeC/ CeN bonds and two rings in a single step, while the narrow substrate scopes limited its more application in organic synthesis. Comparatively speaking, Route 2 showed more advantages, especially wider substrate scope than Route 1, implying the success of the redesign of one enzymatic MCR to overcome some internal obstacles. Route 3, two-enzymatic cascade asymmetric synthesis of chiral spirooxazino in one-pot, displayed very attractive prospects in the complex asymmetric synthesis using green enzyme catalysts, although the stereoselectivity result was not yet fully competitive with asymmetric chemical catalysts. Directed evolution of highly selective CALB for this MCR possibly overcomes this challenge.

The 1H and 13C NMR spectra were recorded with TMS as internal standard using a Bruker AMX-400 MHz spectrometer. Chemical shifts were expressed in ppm and coupling constants (J) in Hz. Analytical HPLC was performed using an Agilent 1100 series with a reversed-phase Shim-Pack VP-ODS column (1504.6 mm), an ASH column and a UV detector. IR spectra were measured with a Nicolet Nexus FTIR 670 spectrophotometer. Melting points were determined using XT-4 apparatus and were not corrected.

4.3. General procedure of CALB-initiated multicomponent reactions

3. Conclusion

Aldol intermediate (0.25 mmol), nitroalkene (1.25 mmol), acetamide (0.75 mmol), and CALB (60 mg) in 1 mL monocarbonyl compound were stirred at 50  C for 96 h. The reaction was terminated by filtering off the enzyme. The crude residue was purified by silica gel column chromatography with an eluent consisting of petrol ether/acetone (20/1 v/v). Product-contained fractions were combined, concentrated, and dried to give 5a, 5d, 5h, 5me5x.

In conclusion, we demonstrated to redesign a lipase-catalyzed complicated MCR to expand greatly the substrate scope, thus successfully developed a more efficient and more general CALBinitiated multicomponent reaction for the one-pot synthesis of complex spirooxazino derivatives. 15 various spirooxazinos with different substitutions could be prepared in moderate to high yields, while most of them could not be prepared using the previous method. Additionally chiral spirooxazino also could be obtained using the optically pure intermediate from aldol reactions as starting material. We believe that these results can not only highlight the biocatalytic promiscuity but also afford a facile access to functionalized spirooxazino derivatives.

4.3.1. 1-(4-Fluorophenyl)-6-(4-methoxyphenyl)-7,8,9,9a-tetrahydro1 H - s p i r o [ [ 1, 3 ] o x a z i n o [ 5 , 4 , 3 - h i ] i n d o l e - 3 ,1 0 - c y c l o h e x a n e ] (5m). Orange crystalline solid, mp 211e213  C; IR (KBr) n: 2933, 2858, 1602, 1537, 1511, 1246 cm1; 1H NMR (CDCl3, d, ppm): 7.41e7.39 (m, 4H), 7.09e7.06 (m, 2H), 6.90 (s, 1H), 6.88 (s, 1H), 6.69 (s, 1H), 4.38e4.4.36 (d, J¼5.2 Hz, 1H), 3.81 (s, 3H), 2.84e2.77 (m, 2H), 2.71e2.67 (m, 1H), 2.24e2.15 (m, 2H), 2.05e1.99 (m, 2H), 1.82e1.48 (m, 9H), 1.29e1.23 (m, 2H). 13C NMR (CDCl3, d, ppm): 163.4, 161.5, 157.5, 135.7, 129.5, 128.6, 128.5, 128.4, 127.4, 123.0, 115.2, 115.1, 113.9, 112.0, 89.2, 75.1, 55.3, 41.0, 39.8, 36.0, 25.1, 24.9, 23.4, 23.0, 22.6, 22.2. HRMS (EIþ) m/z: [M]þ calcd for C28H30NO2F 431.2261; found 431.2268.

4. Experimental

4.3.2. 1-(4-Chlorophenyl)-6-(4-methoxyphenyl)-7,8,9,9a-tetrahydro1H-spiro[[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5n). Pink crystalline solid, mp 194e196  C; IR (KBr) n: 2933, 2853, 1537, 1492, 1243 cm1; 1H NMR (CDCl3, d, ppm): 7.41e7.36 (m, 6H), 6.90 (s, 1H), 6.88 (s, 1H), 6.69 (s, 1H), 4.37e4.35 (d, J¼5.2 Hz, 1H), 3.81 (s, 3H), 2.82e2.68 (m, 3H), 2.23e2.15 (m, 2H), 2.05e2.01 (m, 2H), 1.82e1.23 (m, 11H). 13C NMR (CDCl3, d, ppm): 157.4, 138.4, 133.6, 129.4, 128.5, 128.2, 127.4, 123.0, 113.9, 112.0, 112.0, 89.2, 75.1, 55.3, 41.0, 39.8, 35.9, 25.1, 24.8, 23.4, 23.0, 22.6, 22.2. HRMS (EIþ) m/z: [M]þ calcd for C28H30NO2Cl 447.1965; found 447.1969.

4.1. Materials Lipase immobilized on acrylic resin from C. antarctica (CAL-B) (10,000 U/g, recombinant, expressed in Aspergillus oryzae), Lipase from porcin pancreas type II (PPL) (30e90 U/mg protein, one unit will hydrolyze 1.0 mequiv of triacetin in 1 h at pH 7.7 at 37  C), and Amano lipase M from Mucor javanicus (Amano MJL) (10,000 U/g enzyme activity, pH 7.0, 40  C) were purchased from Sigma (Steinheim, Germany). Lipozyme immobilized from Mucor miehei (MML) (42 m/g, 1 m corresponds to the amount of enzyme which liberates 1 mol oleic acid at pH 8.0 and 40  C per minute). Lipase from hog pancreas (HPL) (2.4 U/mg, 1 U is the amount of immobilized enzyme which forms 1% octyl laurate from 0.5 mmol lauric acid and 1.0 mmol 1-octanol in 10 mL water-saturated isooctane in 1 h at 20  C) was purchased from Fluka (Switzerland). D-aminoacylase from Escherichia coli (DA) (10,000 U/mg, 1 U is defined as enzyme quantity which produces 1 mmol of D-Amino acid per 30 min) and Acylase ‘Amano’ from A. oryzae (AA) (30,000 U/g, 1 U is defined as enzyme quantity which produces 1 mmol of L-Amino

4.3.3. 6-(4-Methoxyphenyl)-1-(4-(trifluoromethyl)phenyl)-7,8,9,9atetrahydro-1H-spiro[[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5o). Pink crystalline solid, mp 189e191  C; IR (KBr) n: 2936, 2856, 1617, 1537, 1493, 1325 cm1; 1H NMR (CDCl3, d, ppm): 7.67 (s, 1H), 7.65 (s, 1H), 7.57 (s, 1H), 7.55 (s, 1H), 7.42 (s, 1H), 7.40 (s, 1H), 6.91 (s, 1H), 6.89 (s, 1H), 6.71 (s, 1H), 4.46e4.44 (d, J¼5.0 Hz, 1H), 3.82 (s, 3H), 2.84e2.82 (m, 2H), 2.79e2.77 (m, 1H), 2.24e2.18 (m, 2H), 2.06e2.02 (m, 2H), 1.84e1.32 (m, 9H), 1.29e1.25 (m, 2H). 13C NMR (CDCl3, d, ppm): 155.0, 126.8, 125.7, 124.9, 124.5, 122.7, 122.7, 120.6, 111.4, 109.6, 109.6, 86.7, 72.7, 52.7, 38.5, 37.2, 33.4, 22.5, 22.3, 20.8,

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20.4, 20.1, 19.7. HRMS (EIþ) m/z: [M]þ calcd for C29H30NO2F3 481.2229; found 481.2230. 4.3.4. 6-(4-Methoxyphenyl)-1-phenyl-7,8,9,9a-tetrahydro-1H-spiro [[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5p). Yellow crystalline solid, mp 151e153  C; IR (KBr) n: 2934, 2855, 1693, 1607, 1537, 1513, 1450, 1244 cm1; 1H NMR (CDCl3, d, ppm): 7.44e7.33 (m, 7H), 6.90 (s, 1H), 6.88 (s, 1H), 6.70 (s, 1H), 4.40e4.36 (d, J¼5.0 Hz, 1H), 3.31 (s, 3H), 2.88e2.68 (m, 3H), 2.26e2.16 (m, 2H), 2.04e1.99 (m, 2H), 1.85e1.23 (m, 12H). 13C NMR (CDCl3, d, ppm): 157.4, 139.9, 129.5, 128.9, 128.3, 127.9, 127.4, 126.9, 123.0, 113.9, 112.0, 111.9, 89.2, 75.7, 55.3, 40.9, 39.8, 36.0, 25.1, 24.9, 23.5, 23.0, 22.6, 22.2. HRMS (EIþ) m/z: [M]þ calcd for C28H31NO2 413.2355; found 413.2362. 4.3.5. 1,6-bis(4-Methoxyphenyl)-7,8,9,9a-tetrahydro-1H-spiro[[1,3] oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5q). Yellow crystalline solid, mp 182e184  C; IR (KBr) n: 2934, 2854, 1613, 1537, 1514, 1247 cm1; 1H NMR (CDCl3, d, ppm): 7.42e7.35 (m, 4H), 6.94e6.88 (m, 4H), 6.69 (s, 1H), 4.51e4.48 (d, J¼5.2 Hz, 1H), 3.83 (s, 3H), 3.81 (s, 3H), 2.88e2.68 (m, 3H), 2.35e2.32 (m, 2H), 2.09e2.01 (m, 2H), 1.87e1.22 (m, 12H). 13C NMR (CDCl3, d, ppm): 159.3, 157.3, 132.1, 129.5, 129.0, 128.1, 127.4, 122.9, 113.9, 113.7, 111.9, 111.8, 89.2, 75.3, 55.3, 55.3, 42.0, 40.7, 40.0, 36.0, 27.1, 25.1, 25.0, 24.9, 23.5, 23.0, 22.6, 22.2. HRMS (EIþ) m/z: [M]þ calcd for C29H33NO3 443.2460; found 443.2458. 4.3.6. 1,6-bis(4-Fluorophenyl)-7,8,9,9a-tetrahydro-1H-spiro[[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5r). Pink crystalline solid, mp 204e206  C; IR (KBr) n: 2932, 2853, 1601, 1533, 1510, 1228 cm1; 1H NMR (CDCl3, d, ppm): 7.44e7.38 (m, 4H), 7.10e7.00 (m, 4H), 6.72 (s, 1H), 4.38e4.36 (d, J¼4.8 Hz, 1H), 2.84e2.75 (m, 3H), 2.73e2.65 (m, 1H), 2.24e2.05 (m, 2H), 2.04e2.01 (m, 2H), 1.82e1.24 (m, 11H). 13C NMR (CDCl3, d, ppm): 163.5, 161.9, 161.5, 160.0, 135.6, 135.5, 132.8, 128.9, 128.6, 128.5, 128.4, 127.9, 127.7, 127.6, 122.4, 115.3, 115.2, 115.1, 112.4, 112.0, 89.3, 75.1, 40.9, 39.8, 36.0, 34.4, 25.1, 24.8, 23.4, 22.9, 22.8, 22.6, 22.4, 22.2. HRMS (EIþ) m/z: [M]þ calcd for C27H27NOF2 419.2061; found 419.2059. 4.3.7. 6-(4-Fluorophenyl)-1-phenyl-7,8,9,9a-tetrahydro-1H-spiro [[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5s). Yellow crystalline solid, mp 196e198  C; IR (KBr) n: 2935, 2855, 1602, 1529, 1511, 1224 cm1; 1H NMR (CDCl3, d, ppm): 7.49 (s, 1H), 7.48 (s, 1H), 7.41e7.40 (m, 2H), 7.39e7.31 (m, 2H), 7.17e7.14 (m, 1H), 7.09e7.06 (m, 2H), 6.77 (s, 1H), 4.38e4.36 (d, J¼5.0 Hz, 1H), 2.84e2.80 (m, 2H), 2.73e2.71 (m, 1H), 2.25e2.16 (m, 2H), 2.06e2.00 (m, 2H), 1.82e1.24 (m, 11H). 13C NMR (CDCl3, d, ppm): 163.4, 161.5, 136.8, 135.6, 128.6, 128.5, 128.4, 126.3, 125.1, 123.3, 115.3, 115.1, 112.7, 112.2, 89.3, 75.1, 41.0, 39.8, 36.0, 25.1, 24.9, 23.4, 23.1, 22.6, 22.2. HRMS (EIþ) m/z: [M]þ calcd for C27H28NOF 401.2155; found 401.2155. 4.3.8. 1-(4-Fluorophenyl)-6-(4-methoxyphenyl)-8-methyl-7,8,9,9atetrahydro-1H-spiro[[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5t). Yellow crystalline solid, mp 241e243  C; IR (KBr) n: 2936, 2864, 1608, 1537, 1511, 1243 cm1; 1H NMR (CDCl3, d, ppm): 7.41e7.39 (m, 4H), 7.10e7.06 (m, 2H), 6.90 (s, 1H), 6.89 (s, 1H), 6.69 (s, 1H), 4.37e4.35 (d, J¼5.0 Hz, 1H), 3.81 (s, 3H), 2.87e2.83 (m, 2H), 2.29e2.15 (m, 3H), 2.02e2.01 (m, 1H), 1.79e1.04 (m, 14H). 13C NMR (CDCl3, d, ppm): 163.4, 161.5, 157.5, 135.7, 135.6, 129.4, 128.6, 128.5, 128.4, 127.5, 122.8, 115.3, 115.1, 113.9, 112.5, 112.2, 89.3, 75.1, 55.3, 41.0, 39.8, 36.0, 33.5, 32.1, 30.6, 25.1, 22.6, 22.3. HRMS (EIþ) m/z: [M]þ calcd for C29H32NO2F 445.2417; found 445.2415. 4.3.9. 1-(4-Fluorophenyl)-6-(4-methoxyphenyl)-40 -methyl-7,8,9,9atetrahydro-1H-spiro[[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5u). Yellow crystalline solid, mp 179e181  C; IR (KBr) n: 2927, 2854, 1607, 1537, 1511, 1244 cm1; 1H NMR (CDCl3, d, ppm):

7.41e7.38 (m, 4H), 7.10e7.06 (m, 2H), 6.90 (s, 1H), 6.88 (s, 1H), 6.68 (s, 1H), 4.38e4.36 (d, J¼5.0 Hz, 1H), 3.81 (s, 3H), 2.83e2.76 (m, 3H), 2.24e2.02 (m, 4H), 1.71e1.23 (m, 11H), 0.89e0.88 (d, J¼2.8 Hz, 3H). 13 C NMR (CDCl3, d, ppm): 163.4, 161.5, 157.4, 135.7, 129.4, 128.7, 128.5, 128.4, 127.5, 127.4, 123.0, 115.2, 115.1, 113.9, 112.0, 89.1, 75.1, 55.3, 41.0, 39.6, 35.8, 31.9, 31.1, 30.7, 24.9, 23.4, 23.0, 22.0. HRMS (EIþ) m/z: [M]þ calcd for C29H32NO2F 445.2417; found 445.2419. 4.3.10. 1-(4-Fluorophenyl)-6-(4-methoxyphenyl)-30 -methyl-7,8,9,9atetrahydro-1H-spiro[[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5v). Red crystalline solid, mp 209e211  C; IR (KBr) n: 2929, 2848, 1607, 1537, 1511, 1244 cm1; 1H NMR (CDCl3, d, ppm): 7.41e7.38 (m, 4H), 7.09e7.05 (m, 2H), 6.90 (s, 1H), 6.88 (s, 1H), 6.69 (s, 1H), 4.40e4.35 (m, 1H), 3.81 (s, 3H), 2.84e2.68 (m, 3H), 2.24e2.14 (m, 2H), 2.04e1.95 (m, 2H), 1.70e0.84 (m, 12H). 13C NMR (CDCl3, d, ppm): 163.4, 161.5, 157.4, 135.7, 135.6, 129.5, 129.4, 128.6, 128.5, 128.4, 127.5, 127.4, 123.1, 123.0, 115.2, 113.9, 112.1, 112.0, 111.9, 89.8, 89.7, 75.2, 75.1, 55.3, 48.1, 44.5, 41.0, 40.9, 39.2, 35.4, 33.9, 28.9, 28.5, 24.9, 23.4, 23.0, 22.4, 22.2, 22.1, 21.9. HRMS (EIþ) m/z: [M]þ calcd for C29H32NO2F 445.2417; found 445.2421. 4.3.11. 1-(4-Fluorophenyl)-6-(3-methoxyphenyl)-7,8,9,9a-tetrahydro-1H-spiro[[1,3]oxazino[5,4,3-hi]indole-3,1 0 -cyclohexane] (5w). Dark red crystalline solid, mp 155e157  C; IR (KBr) n: 2935, 2855, 1607, 1574, 1511, 1224 cm1; 1H NMR (CDCl3, d, ppm): 7.42e7.39 (m, 2H), 7.26e7.23 (m, 1H), 7.10e7.04 (m, 4H), 6.78 (s, 1H), 6.74e6.72 (m, 1H), 4.39e4.37 (d, J¼5.0 Hz, 1H), 3.83 (s, 3H), 2.85e2.81 (m, 3H), 2.25e2.16 (m, 2H), 2.06e2.02 (m, 2H), 1.80e1.24 (m, 13H). 13C NMR (CDCl3, d, ppm): 158.2, 156.2, 154.5, 132.9, 130.4, 130.3, 124.1, 123.6, 123.2, 117.9, 113.7, 110.0, 109.8, 107.6, 107.0, 106.7, 105.3, 84.1, 69.9, 49.9, 35.7, 34.5, 30.8, 26.4, 19.8, 18.2, 17.9, 17.5, 17.4, 17.0. HRMS (EIþ) m/z: [M]þ calcd for C28H30NO2F 431.2261; found 431.2283. 4.3.12. 6-(Naphthalen-1-yl)-1-(4-nitrophenyl)-7,8,9,9a-tetrahydro1H-spiro[[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5x). Orange crystalline solid, mp 263e265  C; IR (KBr) n: 2935, 2854, 1604, 1520, 1347 cm1; 1H NMR (CDCl3, d, ppm): 8.28 (s, 1H), 8.27 (s, 1H), 8.14e8.12 (m, 1H), 7.88e7.86 (m, 1H), 7.77e7.64 (m, 3H), 7.48e7.43 (m, 4H), 6.69 (s, 1H), 4.59e4.57 (d, J¼5.2 Hz, 1H), 2.91e2.90 (m, 1H), 2.47e2.43 (m, 2H), 2.42e2.26 (m, 2H), 2.09e1.25 (m, 14H). 13C NMR (CDCl3, d, ppm): 147.6, 147.2, 134.5, 134.0, 132.3, 128.2, 127.5, 127.1, 127.0, 126.8, 126.5, 125.6, 125.5, 123.6, 122.0, 115.0, 114.4, 89.5, 75.0, 41.3, 39.7, 36.0, 25.1, 25.0, 23.2, 22.6, 22.3, 22.1. HRMS (EIþ) m/z: [M]þ calcd for C31H30N2O3 478.2256; found 478.2258. 4.3.13. 6-(4-Methoxyphenyl)-1-(4-nitrophenyl)-7,8,9,9a-tetrahydro1H-spiro[[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5a). The data of product 5a was the same as that in the literature.8 4.3.14. 1-(4-Nitrophenyl)-6-phenyl-7,8,9,9a-tetrahydro-1H-spiro [[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5d). The data of product 5d was the same as that in the literature.8 4.3.15. 6-(4-Fluorophenyl)-1-(4-nitrophenyl)-7,8,9,9a-tetrahydro1H-spiro[[1,3]oxazino[5,4,3-hi]indole-3,10 -cyclohexane] (5h). The data of product 5h was the same as that in the literature.8 4.4. Two-enzymatic cascade reaction 4-nitrobenzaldehyde (0.5 mmol), water (50 mL), PPL (20 mg) and 1 mL cyclohexanone were stirred at 37  C for 48 h. Then, PPL was filtered off, and 4-methoxy-b-nitrostyrene (2 mmol), acetamide (1 mmol) cyclohexanone (1 mL) and CALB (100 mg) were added. The whole reaction was terminated by filtering off CALB. The crude residue was purified by silica gel column chromatography with an

X.-Y. Chen et al. / Tetrahedron 72 (2016) 3318e3323

eluent consisting of petrol ether/acetone (20/1 v/v). Productcontained fractions were combined, concentrated, and dried to give chiral 5a. The dr was determined by 1H NMR, and the ee value was determined by chiral HPLC (Chiralcel AS-H column, hexane/iPrOH¼90:10, flow rate 1.0 mL/min; t (major)¼8.7 min; t (minor)¼ 11.1 min, l¼254 nm).

2.

Acknowledgements The financial support from the National Natural Science Foundation of China (No. 21272208), the Natural Science Foundation of Zhejiang Province (No. LY14B020006) and Ph.D. Programs Foundation of Ministry of Education of the People’s Republic of China (20110101110008) is gratefully acknowledged.

3. 4. 5.

Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2016.04.060. 6.

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