Horseradish peroxidase catalyzed oxidative cross-coupling reactions: the synthesis of ‘unnatural’ dihydrobenzofuran lignans

Tetrahedron Letters 52 (2011) 3856–3860

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Horseradish peroxidase catalyzed oxidative cross-coupling reactions: the synthesis of ‘unnatural’ dihydrobenzofuran lignans Francesco Saliu ⇑, Eeva-Liisa Tolppa, Luca Zoia, Marco Orlandi Department of Environmental Science, University of Milano-Bicocca, Piazza della Scienza 1, 20126 Milano, Italy

a r t i c l e

i n f o

Article history: Received 11 January 2011 Revised 12 May 2011 Accepted 16 May 2011 Available online 23 May 2011 Keywords: Dihydrobenzofuran lignans Radical cross-coupling reaction Horseradish peroxidase

a b s t r a c t The possibility to afford by a biomimetic reaction ‘unnatural’ products, which could offer a better bioactivity profile than natural analogues, is outlined and the first applications to the synthesis of lignans and related compounds have been reported. Here we describe the synthesis of new heterodimers, having a phenylcoumaran skeleton, by horseradish peroxidase catalyzed cross-coupling reactions of methyl esters of substituted hydroxycinnamic acids. Ó 2011 Elsevier Ltd. All rights reserved.

Due to their wide bioactivity as well as their structural variety, lignans, neolignans and related compounds are attractive targets in synthetic chemistry.1,2 The most cited example of bioactive lignan is podophyllotoxin, a dimeric phenylpropanoid known as a constituent of Podophyllum peltatum, which is the pharmacological precursor of the important anticancer drug etoposide.3,4 The other well studied molecule is the dihydrobenzofuran lignan 30 ,4-di-Omethylcedrusin which is one of the active principles of ‘Sangre de Drago’ (or ‘dragon’s blood’), the latex produced by some Croton spp (trees from the Euphorbiacae family, growing in the Andes) and employed for centuries in the traditional medicine of South America as a haemostatic and wound healing agent.5 With the aim of identifying new potential antitumour agents which could act as analogues of natural products, an extensive study has been carried out on synthetic dihydrobenzofuran lignans and related benzofurans obtained by dehydrogenative coupling from methyl esters of hydroxycinnamic acids. As an example, methyl ferulate, methyl coumarate and methyl caffeate dimers showed significant activities towards the leukaemia and breast cancer cell lines.6,7 In particular, the 2R,3R dimerization product of methyl caffeate was twice as active as an inhibitor of tubulin polymerization compared with the racemic mixture, while the 2S,3S-enantiomer had minimal activity. Methyl caffeate and methyl ferulate dimers were also tested for antiangiogenic activity.8 Recently, a study on lipophilic esters of caffeic acid showed a high activity against chloroquine-resistant Plasmodium falciparum and indicated a dihydrobenzofuran derivative of n-butyl caffeate as a promising antileishmanial lead compound.9 ⇑ Corresponding author. Tel.: +39 02 64482813; fax: +39 02 64482839. E-mail address: [email protected] (F. Saliu). 0040-4039/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tetlet.2011.05.072

The standard oxidative phenol coupling protocols employ metals such as Ag2O, MnO2 or enzymes. Different from the biosynthetic pathway, which provides enantiopure lignans, these methods normally give rise to racemic compounds and are combined with preparative-scale chiral HPLC for the resolution of the products. Cross-coupling reactions are widely studied due to their implications for the understanding of the lignification process in vascular plants and they seem to be governed by a combination of factors such as oxidation potential and radical reactivity.10,11 Recently, equimolecular mixtures of pairs of substituted hydroxycinnamic acid suberin precursors were submitted to HRP catalyzed dehydrogenative oxidation.12 Here we report the synthesis of new dihydrobenzofuran lignans obtained from cross-coupling reactions between methyl esters of hydroxycinnamic acids. In addition, we test a cross coupling reaction starting from the R-methylbenzylamides of sinapic acid and ferulic acid. All the HRP catalyzed oxidative dehydrogenation reactions were performed in dioxane-aqueous buffer pH 3.5. MnO2 was used in dichloromethane, as described by Daquino et al.13 N,N-bis (salicylidene)ethylenediaminocobalt(II) complex was used in dichloromethane at room temperature with air as the oxidant. Preliminary homocoupling experiments were carried out using the methyl esters and the methyl benzyl amides of substituted hydroxycinnamic acids as substrates (Fig. 1, 1–5). Dimerization products were purified from crude reaction mixtures by silica gel chromatography and identified by 1H NMR using published data.14–18 The isolated homodimers were used as standard to HPLC–MS analysis19 in cross-coupling reaction experiments.

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F. Saliu et al. / Tetrahedron Letters 52 (2011) 3856–3860

Figure 1. Substrates in the dehydrogenative oxidation reactions.

Table 1 Dimerization products in cross-coupling reactions of methyl ester of hydroxycinnamic acids Entry

Mixture of

Catalyst

Conversion

Homocoupling products detected

Isolated yield in the cross-coupling products

Unidentified oligomers (%)

1 2 3 4 5

1+2 2+3 2+3 1+3 1+3

HRP HRP MnO2 HRP MnO2

1 2 2 1 1

6 (32%), b–b products from 1 6 (10%), 10 (17%) 6 (4%), 10 (16%) 10 (16%), b–b products from 1 10 (17%), b–b products from 1

Trace 7 (24%) 7 (19%)a 9 (14%), 11 (9%) 9 (12%)

13 15 32 14 27

(57%), (65%), (66%), (48%), (55%),

2 3 3 3 3

(68%) (69%) (82%) (74%) (83%)

Weight conversion. Reaction conditions are described in the text. a A compound having m/z 445 was detected in the HPLC-APCI-MS chromatogram of the crude reaction mixture but it was not possible to isolate this compound by flash chromatography.

Figure 2. Chemical structures of the major products isolated from the dehydrogenative oxidation of methyl ester of substituted para-hydroxycinnamic acids.

Cross-coupling reactions20 were carried out using equimolecular mixtures of two methyl esters of substituted para-hydroxycinnamic acids. Heterodimers were easily identified as major products by HPLC–MS analysis of the crude reaction mixtures (Table 1). Only in the case of the reaction between methyl ferulate and methyl caffeate (Table 1, entry 1) were the corresponding heterodimers observed in trace amounts and the major product was a b-5 homodimer from methyl ferulate (Fig. 2, 6). The same chromatographic peaks were observed in both HRP and MnO2 mediated oxidation reactions, but the latter led to the major formation of oligomers and a major consumption of starting products. For quantitative analysis, the products were separated by flash chromatography and weighted. The HPLC-APCI-MS analysis of the crude reaction mixtures from the HRP mediated oxidation of methyl sinapate (3) and methyl ferulate (2) (Table 1, entry 2) showed as major peaks the b–b homodimer from methyl sinapate (10), the b-5 homodimer from methyl ferulate (6) and two cross-coupling products at m/z 445 [M+H]+.

Purification and isolation of one of these heterodimers were achieved by flash chromatography on silica gel. The 1H NMR spectrum of this compound is consistent with structure 7 proposed in Figure 2. In particular the presence of an A2 and an AB aromatic system implicates a substitution at position 8–50 . The scalar coupling constants supported the presence of two trans olefinic protons (70 H and 80 H at d 7.64 and 6.32 ppm, respectively; J = 15.10 Hz), and two methine protons (7H and 8H at d 6.11 and d 4.32 ppm, respectively, J = 7.08 Hz). The presence of only one chemical shift in the 60–90 ppm region suggests a carbon atom bonding to an oxygen atom (87.9 ppm) and confirms the b-5 benzofuran structure. The cross-coupling reactions between methyl sinapate (2) and methyl caffeate (1) led to the formation of two cross-coupling products having an APCI-MS molecular ion at m/z 431 [M+H]+, and the b–b homodimer from methyl sinapate. The products deriving from methyl caffeate (1) dimerization were formed only in small amounts. NMR analysis of the major cross-coupling products

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Table 2 1 H NMR data for the cross-coupling products Position

(7) H NMR, d (ppm), J (Hz)

(9) 1 H NMR, d (ppm), J (Hz)

(11) 1 H NMR, d (ppm), J (Hz)

6.63, 6.11, 4.32, 6.98, — 7.08, 7.64, 6.32,

6.62, 6.10, 4.34, 6.89, — 7.11, 7.62, 6.31,

6.60, 5.18, 4.86, 7.10, 7.20, 7.05, 7.65, 6.32,

1

2–6 7 8 20 50 60 70 80

2H, 1H, 1H, 1H,

s d, 7.08 d, 7.08 s

1H, s 1H, d, 15.10 1H, d, 15.10

2H, 1H, 1H, 1H,

s d, 7.10 d, 7.10 s

1H, s 1H, d, 15.08 1H, d, 15.08

2H, s 1H, d, 2.30 1H, d, 2.30 1H,d, 1.81 1H,d, 8.33 1H,dd, 1.81, 8.33 1H, d, 15.11 1H, d, 15.11

at m/z 431 allowed assigning also in this case the b-5 structure. Compound 9 displayed the same aromatic system observed in the 1H NMR analysis of compound 7 (Table 2), characterized by two trans olefinic protons and two methine protons. Also in this case the 13C NMR analysis showed only one chemical shift in the 60–90 ppm region. The minor product at m/z 431 displayed an A2 and an AMX aromatic system and two trans olefinic protons. The similar chemical shifts of the methine protons at 5.18 and 4.86 ppm (7H and 8H) suggested that the hydroxyl groups of methyl caffeate (1) are bound to the carbons situated on the aliphatic chain of the methyl sinapate (3) in positions 7 and 8. This result is supported by the 13C NMR spectrum, in which signals were found in the 60–80 ppm region. These signals are attributable to two carbon atoms bearing an oxygen atom. These observations indicated a 1–4 benzodioxane ring structure 11. Noteworthy, when HRP was replaced with MnO2 as the oxidant, this compound was identified only in trace amount in the crude reaction mixture (Fig. 3). Both in the methyl sinapate (3)–methyl caffeate (1) and methyl sinapate (3)–methyl ferulate (2) cross-coupling crude reaction mixtures the related chromatograms showed some additional peaks having a large mass than can be correlated to trimer or higher oligomeric products. By monitoring the peaks corresponding to the homocoupling products, we observed that they started to be

3 25 3.25

Figure 4. Compounds 7 and 9 have a dihydrobenzofuran structure resembling the natural compound 2R,3S 30 ,4-di-O-methylcedrusin 12, the synthetic derivative Benfur6 14 and the promising antileishmanial7 13.

formed at the beginning of the reaction and a small part of them was consumed to form oligomers. These observations suggest that the dimers are intermediates in oxidative coupling reactions that may form subsequently higher oligomers which may remain undetected because they are sparingly soluble. Mass balance after silica gel column chromatography confirms that 14–32% of crude reaction material is not eluted and can be related with height weighted oligomers. In addition, we noted that the peak corresponding to methyl sinapate decreased faster than the peaks corresponding to the other substrates. The dihydrobenzofuran structure of the cross-coupling products 7 and 9 is similar to that of the natural compound 2R,3S methylcedrusine, which has been extensively studied with its congeners ( Fig. 4) as an antitumoural, antiangiogenic and antileshmania

mAU(x100) Extract-239nm,4nm , ((1.00))

3.00 2.75 2.50 2.25 2.00 1.75

10

1.50 1.25 1.00 0.75

11

9

0.50

3

1

0.25 0.00 0.25 -0.25 50 0.50 -0 00 0.0

1 1.0 0

2.0 20

3.0 30

4.0 40

5.0 50

6.0 60

7.0 70

8.0 80

9.0 90

10.0 10 0

11.0 11 0

12.0 12 0

13.0 13 0

14.0 14 0

Figure 3. Chromatogram from the MnO2 oxidation of an equimolecular mixture of methyl sinapate and methyl caffeate.

min

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F. Saliu et al. / Tetrahedron Letters 52 (2011) 3856–3860 Table 3 Dimerization products in cross-coupling reactions of chiral amides of substituted para-hydroxycinnamic acids Entry

Mixture of

Catalyst

Conversion

Homocoupling products

Cross-coupling products

Unidentified oligomers

1

4+5

HRP

4 (60%), 5 (67%)

15 (6%), 16 (15%)

b-5 (27%), 17

14%

Inten.(x100,000) 6.0 5.5 593

5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5

203

0.0 200

247

290

250

317

300

351 368 385

350

472

418 436

400

Inten.(x1,000,000)

513

500

698 681

616

565

550

600

650

m/z

Inten.(x1,000,000) 4.5

4.0 3.5

4.0

653

623

3.5

3.0

3.0

2.5

2.5

2.0

2.0

1.5

1.5

1.0 0.5

489

450

1.0 225 257

0.0 219 200 250

298

300

328

352 368

350

402

400

428 445

450

476

506

500

532 550 564

550

593

600

623

668

650

699

m/z

0.5 0.0 209 200

254

250

297

300

323 338 358

350

401

400

430 448

450

476

502

500

519 547 569

594

550

600

651 669

650

697

m/z

Figure 5. APCI mass spectrum of the products of the cross-coupling reaction between the sinapic acid methyl benzyl amide and ferulic acid methyl benzyl amide. Experimental mass were 15 593.2 ± 0.1, 16 654.3 ± 0.1 and 17 623.3 ± 0.1 uma.

agent. As expected, our compounds did not show optical activity and should be described as racemic mixtures. The literature data21 showed pronounced difference in the activity when racemic and both enantiomers of dihydrobenzofuran lignans were evaluated separately. In this light, the enantioselective synthesis of compounds 7 and 9 may be a crucial issue for further preparative development. Stereocontrol in enzymatic oxidation of substituted parahydroxycinnamic acid has been already reported in homocoupling reactions22 by the use of aminoacids as the chiral auxiliary. A pair of diasteroisomers was obtained starting from ethyl S-alaninate amide of ferulic acid. After the cleavage of the chiral auxiliary from the major diateroisomers with LiOOH, an optically pure homodimer of ferulic acid was isolated. To the best of our knowledge this strategy has not been tested yet in a cross-coupling reaction and is valuable in order to avoid expensive chiral chromatography. As a preliminary attempt, we performed an HRP mediated oxidation of an equimolecular mixture of amides of ferulic acid (4) and sinapic acid (5) having an R-methyl benzyl amine group as chiral auxiliary.23 These substrates were prepared according to the procedure described by Bolzacchini et al .24 The HPLC-APCI-MS chromatogram of the crude reaction mixture showed a major peak having m/z 623, corresponding to a cross-coupling product (17).

The other detected products (Table 3) were the b-5 benzofuran (15) from the dimerization of ferulic methyl benzyl amide (4) and the b–b aryltetralin (16) from the dimerization of sinapic methyl benzyl amide (5). In Figure 5 the APCI mass spectra of these compounds are reported. Thus, compound 17 was isolated by flash column chromatography and submitted to RP-HPLC. Two peaks corresponding to a pair of diasteroisomers having a trans b-5 benzofuran structure were detected. The diasteroisomeric excess was evaluated to be 38%.25 In conclusion, by carrying out HRP catalyzed oxidative crosscoupling reactions of methyl esters of hydroxycinnamic acids, we have synthesized in good yield new ‘unnatural’ products having the dihydrobenzofuran structure of the natural analogue 30 ,4-diO-methylcedrusin. In addition, a diasteroisomeric excess in a new dihydrobenzofuran bis R-methylbenzylamide (17) was obtained. Studies are needed to improve the stereoselection in the final dihydrobenzofuran structures with the use of easily removable auxiliaries. The preferred regioselectivity in all these cross-coupling reactions seemed to be 8–50 . The mechanistic reasons for this behaviour have to be further elucidated. HRP oxidation is beneficial for mild reaction conditions and is environmentally friendly since the reaction occurs in water and

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the wastes do not contain metals. The bioactivity profiles of the products described in this Letter are currently under evaluation in our laboratory. Acknowledgements We warmly thank University of Milan-Bicocca FAR 2010 for the financial support and our students Giovanni Di Gennaro, Andrea Bosisio and Lorenzo Beretta. References and notes 1. Haworth, R. D. J. Chem. Soc. 1942, 448–456. 2. Spatafora, C.; Tringali, C. In Targets in Heterocyclic System Chemistry and Properties; Spinelli, D., Attanasi, O. A., Eds.; Società Chimica Italiana: Rome, Italy, 2007; Vol. 11, pp 284–312. 3. Ionkova, I. Pharmacognosy Rew. 2007, 1, 57–58. 4. Bruschi, M.; Orlandi, M.; Rindone, M.; Rindone, B.; Saliu, F.; Suarez-Bertoa, R.; Tolppa, E.L.; Zoia, L. Biomimetics Learning from Nature. InTech, March 2010. 5. Chen, Z. P.; Cai, Y.; Phillipson, J. D. Planta Med. 1994, 60, 541–545. 6. Manna, S. K.; Bose, J. S.; Gangan, V.; Raviprakash, N.; Navaneetha, T.; Raghavendra, P. B.; Babajan, B.; Kumar, C. S.; Jain, S. K. J. Bio. Chem. 2010, 285(29), 22318–22327. 7. Pieters, L.; Van Dyck, S.; Gao, M.; Bai, R.; Hamel, E.; Vlietinck, A.; Lemiere, G. J. Med. Chem. 1999, 26, 5475–5481. 8. Apers, S.; Dietrich, P.; Burgermeister, J.; Baronikova, S.; Van Dyck, S.; Lemiere, G.; Arnold Vlietinck, A.; Pieters, L. J. Nat. Prod. 2002, 65, 718–720. 9. Van Miert, S.; Van Dyck, S.; Schmidt, T. J.; Brun, R.; Vlietinck, A.; Lemiere, G.; Pieters, L. Bioorg. Med. Chem. 2005, 13, 661–669. 10. Fournand, D.; Cathalab, B.; Lapierre, C. Phytochemistry 2003, 62, 139–146. 11. Syrjänen, K.; Brunow, G. J. Chem. Soc., Perkin Trans. 1 1998, 3425–3429. 12. Arrieta-Baez, D.; Stark, R. E. Phytochemistry 2006, 67, 743–753. 13. Daquino, C.; Rescifina, A.; Spatafora, C.; Tringali, C. Eur. J. Org. Chem. 2009, 6289–6300. 14. Setala, H.; Pajunen, A.; Kilpelainen, I.; Brunow, G. J. Chem. Soc., Perkin Trans. 1 1994, 1163–1165. 15. Ahmed, R.; Lehrer, M.; Stevenson, R. Tetrahedron Lett. 1973, 10, 747–750. 16. Bruschi, M.; Orlandi, M.; Rindone, B.; Rummakko, P.; Zoia, L. J. Phys. Org. Chem. 2006, 19, 592–596. 17. Wallis, A. F. Tetrahedron Lett. 1969, 5287–5288. 18. Wallis, A. F. Aust. J. Chem. 1973, 26, 1571–1576. 19. The extracted crude reaction mixtures were previously analyzed by HPLCAPCI-MS in order to have a wide indication of all the products present. The instrument was settled on the positive ionization mode with a flow rate of 2.5 mL min1 of molecular nitrogen as the carrier gas. The APCI ion source temperature was fixed at 250 °C and the detector voltage set to +1.5 kV. The mass acquisition range was comprised within 100 and 600 amu with a 100 amu s1 scan speed. Chromatographic separation was achieved by using an Ascentis C18 15 cm  4.6 mm  5 lm (SUPELCO, Bellefonte, USA) column at 25 °C and gradient elution with solvent A (water) and solvent B (methanol) using the following elution profile: 0–10 min linear gradient from 90% A: 10% B to 65% A: 45% B: linear gradient from 10 to 20 min to 30% A: 70% B and continuing isocratic at 40% A: 70% B for eight additional minutes. Flow rate: 0.6 mL min1. Injection volume: 60 lL. 20. Substrates (1, 2, and 3) were prepared by simple Fischer esterification reactions of the substituted para-hydroxycinnamic acids, using methanol as the solvent and sulphuric acid as the catalyst. Cross-coupling reactions were performed by preparing a solution with equimolar amounts of two substituted parahydroxycinnamic acid methyl esters at a concentration of 1.0 mmol in dioxane (14 mL) and 0.02 M phosphate/citric acid buffer pH 3.5 (4.0 mL). This solution was added of a 0.86 M aqueous hydrogen peroxide solution (0.60 mL, 0.5 mmol) and aqueous HRP (0.93 mL, 837 U) at 0 °C in small portions over 15 min. The mixture was then stirred at 0 °C for 30 min and then allowed to reach room temperature. The extents of the enzymatic reactions were determined through the decrease in TLC analysis for both the substrates. After 4 h a saturated aqueous NaCl solution (20 mL) was added. The dioxane was then evaporated under reduced pressure and the resulting solution was

21. 22. 23.

24. 25.

extracted with ethyl acetate (4  20 mL). The combined organic extracts were washed with 10% aqueous NaHCO3 (25 mL), then with water saturated with NaCl (25 mL), and dried over Na2SO4. The solvent was evaporated under reduced pressure, and the residue was chromatographed on a silica gel flash column (Merck silica gel 60, 0.040–0.063 mm, 230–400 mesh ASTM) with hexane–ethyl acetate (gradient mode from 4:1 to 1:1). Compound 7: Methyl(E-3-[2-(4-hydroxy-3,5-dimethoxy-phenyl)-7-methoxy-3methoxycarbonyl-2,3-dihydro-1-benzofuran-5-yl]propen-2-enoate. Amorphous yellow–brown powder. UV kmax (MeOH) nm: 206, 329. 1H NMR (CDCl3, dH): 7.64 (1H, d, J = 15.10 Hz); 7.08 (1H, s); 6.98 (1H, s); 6.63 (2H, s); 6.32(1H, d, J = 15.10 Hz); 6.11(1H, d, J = 7.08 Hz); 5.61 (1H, s,); 4.32 (1H, d, J = 7.10 Hz); 3.91 (6H, s); 3.87 (3H,s) 3.81 (3H, s); 3.69 (3H, s).13C NMR: 172.8, 168.1, 145.3, 143.2, 139.8, 132.5, 127.6, 119.5, 118.1, 114.9, 110.4, 104.1, 87.9, 56.8, 56.5, 56.2, 53.6, 52.4. APCI-MS m/z 445.1 ± 0.1 [M+H]+. Compound 9: Methyl(E-3-[2-(4-hydroxy-3,5-dimethoxy-phenyl)-7-hydroxy-3methoxycarbonyl-2,3-dihydro-1-benzofuran-5-yl]propen-2-enoate. Amorphous yellow–brown powder. UV kmax (MeOH) nm: 210, 325. 1H NMR (CDCl3, dH): 7.62 (1H, d, J = 15.08 Hz); 7.11 (1H, s); 6.89 (1H, s); 6.62 (2H, s,); 6.31 (1H, d, J = 15.08 Hz); 6.10 (1H, d, J = 7.10 Hz); 5.60 (1H, s); 5.30 (1H, s); 4.34 (1H, d, J = 7.10 Hz); 3.91 (6H, s); 3.80 (3H, s); 3.70 (3H, s).13C NMR: 172.5, 168.2, 145.1, 143.0, 132.6, 127.4, 119.4, 118.2, 117.9, 110.8, 104.2 88.2, 56.8, 56.2, 53.6, 52.5 APCI-MS m/z 431.1 ± 0.1 [M+H]+. Compound 11: Methyl(E-3-[2-(4-hydroxy-3,5-dimethoxy-phenyl-3methoxycarbonyl-2,3-dihydro-1-4-benzodioxan-7-yl]propen-2-enoate. Amorphous red powder. UV kmax (MeOH) nm: 298, 239. 1H NMR (CDCl3, dH): 7.65 (1H, d, J = 15.11 Hz); 7.20 (1H, d, J = 8.3 Hz); 7.10 (1H, d, J = 1.8 Hz); 7.05 (1H, dd, J = 1.8 Hz, J = 8.3 Hz); 6.60 (2H, s); 6.32 (1H, d, J = 15.11 Hz); 5.63 (1H, s); 5.18 (1H, d, J = 2.30 Hz); 4.86 (1H, d, J = 2.30 Hz); 3.94 (6H, s); 3.82 (3H, s); 3.68 (3H, s). 13C NMR: 172.3, 167.9, 146.0, 143.8, 132.9, 128.4, 127.6, 117.4, 115.8, 104.1, 74.1, 62.6, 56.7, 53.5, 52.3. APCI-MS m/z 431.1 ± 0.1 [M+H]+. Lemiere, G.; Gao, M.; De Groot, A.; Dommisse, R.; Lepoivre, J.; Pieters, L.; Buss, V. J. Chem. Soc., Perkin Trans. 1 1995, 1775–1779. Zoia, L.; Bruschi, M.; Orlandi, M.; Tolppa, E. L.; Rindone, B. Molecules 2008, 13, 129–148. The HRP catalyzed oxidative cross-coupling reaction of ferulic acid amide (4) and sinapic acid amide (5) having R-methyl benzyl amine as chiral auxiliary was carried out with the same method described for the cross-coupling reactions of p-hydroxycinnamic acid methyl esters. The preparation of substrates 4 and 5 was achieved as follow: to a solution of sinapic acid (3, 4.46 mmol) or ferulic acid (2, 4.46 mmol) in anhydrous THF (40 mL), TEA (0.62 mL, 5.1 mmol), DCC (1 g, 4.84 mmol) and (R)-methyl benzyl amine (4.46 mmol) were added. The mixture was stirred at reflux for 4 h. The extent of the reaction was monitored by TLC (hexane/ethyl acetate 1:1). Some drops of acetic acid were added to remove the excess of DCC. After filtration the solvent was evaporated under reduced pressure and the residue diluted in ethyl acetate (100 mL). The resulting solution was washed with a pH 4 solution of aqueous HCl (60 mL), with a 10% aqueous NaHCO3 solution (60 mL), with saturated brine (60 mL) and finally dried over Na2SO4. The solvent was evaporated under reduced pressure, and the residue was purified by silica gel flash chromatography, (eluent ethyl acetate and petroleum ether 7:3). The yield in 5 and 6 ranged from 30% to 35%. Bolzacchini, E.; Brunow, G.; Meinardi, S.; Orlandi, M.; Rindone, B.; Rummakko, P. Tetrahedron Lett. 1998, 39, 3291–3294. The fraction containing 17 collected by flash column chromatography (Merck silica gel 60, 0.040–0.063 mm, 230–400 mesh ASTM) was successively analyzed by RP-HPLC-DAD using a Gemini 5u C18 15 cm  3.0 mm  5 lm (PHENOMENEX, USA) column at 25 °C and gradient elution with solvent A (water) and solvent B (methanol) using the following elution profile: 0–6 min isocratic B = 25%, from 6–20 min linear gradient from 25% to 70% of B and continuing isocratic 70% B for eight additional minutes. Flow rate: 0.8 mL min1. Injection volume: 40 lL. The diasteroisomeric excess (%) was evaluated on the basis of the ratio of the integral of the two peaks detected at 298 nm in the diode array extracted chromatogram. The value of [diasteroisomer1 + diasteroisomer2] is the yield of 17 reported in Table 3 (27%), the ratio of the two peak integral was 2.2. Compound 17 1H NMR (CDCl3, dH): 7.64 (1H, d, 15.07); 7.34–7.11 (10H aromatic) 7.03 (1H, s); 6.98 (1H, s); 6.63 (2H, s); 6.58 (1H, d, J = 15.09 Hz); 6.16 (1H, d, J = 7.07 Hz); 5.61 (1H, s,); 5.53–5.39 (2H, m), 5.20 (broad), 4.44 (broad), 4.32 (1H, t, J = 7.10); 3.92 (6H, s); 3.88 (3H, s) 1.42–1.48 (6H,m). APCI-MS m/z 623.3 ± 0.1 [M+H]+.