Probing the stereoselectivity of P-glycoprotein ... · Probing the stereoselectivity of...

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Probing the stereoselectivity of P-glycoprotein – synthesis, biological activity and ligand docking studies of a set of enantiopure

benzopyrano[3,4b][1,4]oxazines Ishrat Jabeen,a Penpun Wetwitayaklung,a,b Freya Klepsch,a Zahida Parveen,c Peter Chibac and

Gerhard F. Ecker*a

aDepartment of Medicinal Chemistry, University of Vienna, Althanstraße 14, 1090, Vienna, Austria

bDepartment of Pharmacognosy, Faculty of Pharmacy, Silpakorn University, Nakhon Pathom, Thailand

cMedical University of Vienna, Institute of Medical Chemistry Waehringerstraße 10, 1090, Vienna, Austria E-mail: [email protected]

Supporting Information

1. Table1 2

2. Figure1 2

3. Figure2 3

4. Homology Modeling and Docking 3

5. Biological Assay 3

6. General procedure and spectroscopic data of enantiomeric pure (S,S), and (R,R)-epioxides (4) 4

7. General procedure and spectroscopic data of L-amino acid-tert-Butyl Esters (5-7) 4

8. General procedure for cyclisation and spectral data of target compounds (11-13) 5

9. 1H- and 13C-NMR spectras of target compounds (11a-13b) 7

1. Table1 . Number of clusters obtained in common scaffold clustering in one run, in separate runs and the interacting

amino acid residues

# Compounds

(# of clusters) Clustering in one run

(# of clusters) clustering in separate runs

Interacting amino acid residue

5a; 6a; 7a 5a; 6a; 7a 5b; 6b; 7b 5b; 6b; 7b 5a,b; 6a,b; 7b 11a; 12a; 13a 11b; 12b; 13b 11b; 12b; 13b

2 1 4 1 1 4 7 1

4 1 3 1 1 7 7 1

Try307,Tyr310,Phe343, Phe336, Gln347 Phe951, Ser952, Cys956, Met69 Try307,Tyr310,Phe343, Phe336, Gln347 Tyr117, Ser952, Phe72, Met69 Try307,Tyr310,Phe343, Phe336, Gln347 Tyr307, Phe343, Ala342, Phe303 Tyr307, Phe343, Ala342, Phe303 Ala985, Ile765, Leu724

2. Figure 1. Ligand protein interaction of the selected docking poses in different positions.

Supplementary Material (ESI) for Chemical CommunicationsThis journal is (c) The Royal Society of Chemistry 2011

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7a 6b

13b (Near entry gate) 13b (Deeper inside the membrane)


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6a 3. Figure 2 Comparison of the main positioning of the benzopyrano[3,4-b][1,4]oxazines with those of the both

stereoisomers of cocrystallised tetrapeptides.

4. Homology Modeling and Docking

The homology model was generated with the modeling program Modeller 9v71 based on the sequence alignment suggested by Aller et al2. Using the automodel procedure 100 different homology models were created and refined. For a slight correction of the distorted TM helix 12 a secondary structure constraint was put on residues 885 – 918. The final model was selected on basis of the smallest number of outliers and a high DOPE score and was evaluated with the program PROCHECK3. The Ramachandran plot showed that 84.6 % of the residues lie in most favored, 12.5 % in additional allowed, 2.1 % in generously allowed regions and 0.8 % in disallowed regions. The 2.9 % of the residues that are in generously allowed or disallowed regions are located in the nucleotide binding domains (NBD) or extracellular loops (ECL) and are therefore not involved in drug binding. Compounds were docked into the homology model of human P-gp by using the software package GOLD, creating 100 poses per ligand. The binding site was defined as covering the complete transmembrane region, which leads to distribution of poses in a large area. Ligand protein complexes were minimized by the LigX graphical interface implemented in MOE by using the MMFF94 force field.

5. Biological Assay. The human T-lymphoblast cell line CCRF-CEM and the multidrug resistant CEM/vcr1000 cell line were provided by V. Gekeler (Byk Gulden, Konstanz, Germany). The resistant CEM/vcr1000 line was obtained by stepwise selection in vincristine containing medium. Cells were kept under standard culture conditions (RPMI1640 medium supplemented with 10% fetal calf serum). The P-gp-expressing resistant cell line was cultured in presence of 1000ng/ml vincristine. One week prior to the experiments cells were transferred into medium without selective agents or antibiotics. Briefly, cells were pelleted, the supernatant was removed by aspiration and cells were resuspended at a density of 1 x 106/ml in PRMI1640 medium containing 3µmol/l daunomycin. Cell suspensions were incubated at 37°C for 30min. After this time a steady state of daunorubicin accumulation was reached. Tubes were chilled on ice and cells were pelleted at 500 x g. Cells were washed once in RPMI1640 medium to remove extracellular daunorubicin. Subsequently, cells were


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resuspended in medium prewarmed to 37°C, containing either no modulator or chemosensitizer at various concentrations ranging from 3nM to 500 µM, depending on solubility and expected potency of the modifier. Generally, 8 serial dilutions were tested for each modulator. After 1, 2, 3 and 4 min aliquots of the incubation mixture were drawn and pipetted into 4 volumes of ice cold stop solution (RPMI1640 medium containing verapamil at a final concentration of 100µM). Parental CCRF-CEM cells were used to correct for simple membrane diffusion, which was less than 3% of the efflux rates observed in resistant cells. Samples drawn at the respective time points were kept in an ice water bath and measured within one hour on a Becton Dickinson FACSCalibur (Becton Dickinson, Heidelberg, Germany) flow cytometer as described. Dose response curves were fitted to the data points using non-linear least squares and EC50 values were calculated as described1. EC50 values of individual compounds are the average of at least triplicate determinations. A cv of below 20% was obtained in all determinations.

6. General procedure for the enantiomerically pure (S,S) (4a) and (R,R)- epoxide (4b). Commercial household bleach (DanKlorix) was buffered to pH 11.3 with 0.05 N Na2HPO4 and 1N NaOH and then cooled to 0°C. To 1000 mL of this solution a solution of 3 (75.58 mmol) and Mn(III) Salen catalyst (2.74x10-3 mmol) in 76 mL of CH2Cl2 was added, stirred at 0°C for 5 hr and then at room temperature overnight. The mixture was filtered through Celite and the organic phase was separated, brined once, dried (Na2SO4) and brought to dryness. Purification by flash chromatography (petroleum ether-ethylacetate; 8:2) yield 76.9% of (S,S)-4a and 78.9% of (R,R)-4b and as colourless crystals; mp 133-135oC; IR (KBr): 2227 (CN) cm-1, 1280 (epoxide) cm-1; δH (200MHz; CDCl3) 1.28 (s, 3H, CH3), 1.57(s, 3H, CH3), 1.57 (d, 1H, J = 4.52 Hz, 3-H/ 4-H), 3.89 (d, 1H, J = 4.52 Hz, 3-H/ 4-H), 6.84 (d, 1H, J = 8.53 Hz, 8-H), 7.51 (dd, 1H, J = 2.00 Hz, J = 8.41 Hz, 7-H), 7.63 (d, 1H, J = 2.01 Hz, 5-H); δC (CDCl3) 22.99(CH3), 25.46 (CH3), 49.34 (3-C), 62.27 (4-C), 74.64 (2-C), 104.27 (6-C), 118.70 (CN), 119.00 (8-C), 121.67 (4a-C), 133.77, 134.38 (5-C, 7-C), 156.45 (8a-C).

7. General procedure for L-amino acid-tert-Butyl Ester (5–7). A solution of enantiomeric pure epoxide 4a or 4b (4.97

mmol) and corresponding L-amino acid-tert-butyl ester (5.47 mmol) in 50 mL 96% ethanol was stirred at 80oC for 5 days, then evaporated in vacuo. Purification by flash chromatography (petroleum ether/ethylacetate = 8/2) yield respective L- amino acid t-butyl ester (5-7).

(3S,4R)-N-(6-cyano-3-hydroxy-2,2-dimethyl-2H-3,4-dihydro-1-benzopyran-4yl)-L-alanine-tert-butyl-Ester (5a). (S,S)-epoxide 4a and L-alanine-tert-Butyl ester gave 5a yield 67% as yellowish oil; mp 145-147 °C; IR (KBr): 2225 (CN) cm-1, 1724 (COOR) cm-1; δH (200MHz; CDCl3) 1.20 (s, 3H, 2-CH3), 1.37 (d, 3H, J = 7.03 Hz, CHCH3), 1.41 (s, 9H, C(CH3)3), 1.43 (s, 3H, 2-CH3), 1.85 (br, 1H, NH), 3.37 (d, 1H, J = 10.04 Hz, 3-H), 3.44 (d, 1H, J = 10.29 Hz, 4-H), 3.58 (q, 1H, J = 7.03 Hz, N-CH-COO), 4.60 (s, 1H, OH), 6.72 (d, 1H, J = 8.53 Hz, 8-H), 7.31 (dd, 1H, J = 2.01 Hz, J = 8.53 Hz, 7-H), 7.54 ( d, 1H, J = 1.75 Hz, 5-H) ; δC (CDCl3) 18.80, 20.31 (2x2-CH3), 26.72 (CHCH3), 27.82 (C(CH3)3), 56.75 (N-CH-CO, 4-C), 73.83 (3-C), 79.86 (C(CH3)3), 82.20 (2-C), 103.41 (6-C), 117.93 (8-C), 119.22 (CN), 126.31 (4a-C), 131.80,132.38 (5-C, 7-C), 156.49 (8a-C), 176.66 (C=O); Ms m/z 347 (0.32, M+), 160 (29.7); [α]D

20 +5.32 (c 0.141, in CH2Cl2); (Found: C, 65.69; H, 7.34; N, 8.20. C19H26N2O4 requires C, 65.88; H, 7.56; N, 8.09). (3R,4S)-N-(6-cyano-3-hydroxy-2,2-dimethyl-2H-3,4-dihydro-1-benzopyran-4yl)-L-alanine-tert-butyl-Ester (5b). (R,R)-epoxide 4b and L-alanine-tert-Butyl ester gave 5b yield 56% as yellowish oil; mp 101-102 °C; IR (KBr) 2226 (CN) cm-1, 1722 (COOR) cm-1; δH (200MHz; CDCl3) 1.18 (s, 3H, 2-CH3), 1.28 (d, 3H, J = 7.38 Hz, CHCH3 ), 1.43 (s, 9H, C(CH3)3), 1.46 (s, 3H, 2-CH3) 2.98 (q, 1H, J = 7.28 Hz, CHCH3), 3.13 (dd, 1H, J = 3.51 Hz, J = 9.92 Hz, 3-H), 3.78 (d, 1H, J = 9.79 Hz, 4-H), 4.03 (d, 1H, J = 3.51 Hz, OH), 6.81 (d, 1H, J = 8.54 Hz, 8-H), 7.40 (dd, 1H, J = 1.88 Hz, J = 8.47 Hz, 7-H), 7.87 (d, 1H, J = 1.88 Hz, 5-H)¸ δC (CDCl3) 19.11 (2-CH3), 21.25 (2-CH3), 27.19 (CHCH3), 27.78 (C(CH3)3), 51.18 (N-CH-CO), 56.42 (4-C), 70.86 (3-C), 79.68 (C(CH3)3), 82.35 (2-C), 103.78 (6-C), 118.21 (8-C), 119.48 (CN), 123.90 (4a-C), 132.38, 132.52 (5-C,7-C), 157.77 (8a-C), 178.79 (C=O); MS m/z 347 (0.12, M+), 160 (24.7); [α]D

20 -188.03 (c 0.117, in CH2Cl2; (Found: C, 66.13; H, 7.78; N, 7.88. C19H26N2O3 requires C, 88; H, 7.56; N, 8.09) (3S,4R)-N-(6-cyano-3-hydroxy-2,2-dimethyl-2H-3,4-dihydro-1-benzopyran-4yl)-L-valine-tert-butyl-Ester (6a). (S,S)-epoxide 4a and L-valine-tert-Butyl ester gave 6a yield 65% yellowish oil; mp 128-130 °C; IR (KBr): 2226 (CN) cm-1, 1721 (CO), 3478 (OH) cm-1; δH (200MHz; CDCl3) 0.91, 1.05 (each d, each 3H, each J= 6.77 Hz, CH(CH3)2), 1.13 (s, 3H, 2-CH3), 1.43 (s, 12H, CH3, C(CH3)3), 1.85 (br, 1H, NH), 2.01-2.15 (m, 1H, CH(CH3)2), 3.36 (d, 1H, J = 4.27 Hz, N-CH-CO), 3.42 (br, 2H, 3-H, 4-H), 4.16 (s, 1H, OH), 6.74 (d, 1H, J= 8.53 Hz, 8-H), 7.33 (dd, 1H, J = 8.53/1.88 Hz, 7-H), 7.69 (d, 1H, J = 1.88 Hz, 5-H); δC (CDCl3) 17.71 (CH-CH3), 18.82 (2-CH3), 19.51 (CH-CH3), 26.72 (2-CH3), 27.93 (C(CH3)3), 32.58 (CH(CH3)2), 56.72 (4-C), 66.58 (N-CH-CO), 74.28 (3-C), 79.74 (C(CH3)3), 82.16 (2-C), 103.38 (6-C), 117.92 (8-C), 119.28(CN), 126.40 (4a-C), 132.31 (5-C, 7-C), 156.49(8a-C) 176.02(C=O); Ms m/z 375 (0.79, M+), 273 (57.1%) 160 (40.9%); [α]D

20 -30.00 (c 0.113, in CH2Cl2); (Found: C, 67.33; H, 7.98; N, 7.26. C21H30N2O4 requires C, 67.35; H, 8.07; N, 7.48)


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(3R,4S)-N-(6-cyano-3-hydroxy-2,2-dimethyl-2H-3,4-dihydro-1-benzopyran-4yl)-L-valine-tert-butyl-Ester (6b). (R,R)-epoxide 4b and L-valine-tert-Butyl ester gave 6b yield 60% yellowish oil; mp 79-81°C; IR (KBr): 2226 (CN) cm-1, 1703 (C=O), 3493 (OH) cm-1; δH (200MHz; CDCl3) 0.98, 0.99 (each d, each 3H, each J= 6.77 Hz, CH(CH3)2), 1.17 (s, 3H, 2-CH3), 1.44 (s, 12H, CH3, C(CH3)3), 1.86-2.01 (m, 1H, CH(CH3)2), 2.38 (br, 1H, NH), 2.76 (d, 1H, J = 4.26 Hz, N-CH-CO), 3.15 (dd, 1H, J = 9.54/3.76 Hz, 3-H), 3.78 (d, 1H, J= 9.54 Hz, 4-H), 3.83 (d, 1H, J = 3.76 Hz, OH), 6.80 (d, 1H, J = 8.28 Hz, 8-H), 7.39 (dd, 1H, J= 8.28/1.75 Hz, 7-H), 7.94 (d, 1H, J= 1.75Hz, 5-H ); δC (CDCl3) 17.66 (CH-CH3), 19.06 (2-CH3), 19.76 (CH-CH3), 27.02 (2-CH3), 27.82 (C(CH3)3), 32.63 (CH(CH3)2), 56.54 (4-C), 60.55 (N-CH-CO), 71.01 (3-C), 79.58 (C(CH3)3), 82.31 (2-C), 103.58 (6-C), 118.18 (8-C), 119.32(CN), 123.82 (4a-C), 132.40, 133.39 (5-C, 7-C), 157.75(8a-C) 177.94(C=O); Ms m/z 375 (0.41%, M+), 273 (16%) 72 (100%); [α]D

20 -193.80 (c 0.129, in CH2Cl2); (Found: C, 67.58; H, 8.01; N, 7.25. C21H30N2O4 requires C, 67.35; H, 8.07; N, 7.48) (3S,4R)-N-(6-cyano-3-hydroxy-2,2-dimethyl-2H-3,4-dihydro-1-benzopyran-4yl)-L-phenylalanine-tert-butyl-Ester (7a). (S,S)-epoxide 4a and L-phenylalanine-tert-Butyl ester gave 7a yield 59% yellowish oil; mp 99-101oC; IR (KBr): 2225 (CN) cm-1, 1722 (C=O), 3441 (OH) cm-1; δ H (200MHz; CDCl3) 1.11 (s, 3H, 2-CH3), 1.43 (s, 3H, 2-CH3), 1.48 (s, 9H, C(CH3)3), 1.71 (br, 1H, NH), 2.74 (dd, 1H, J = 12.70/10.36 Hz, benzyl Ha), 3.22 (dd, 1H, J = 12.70/3.53 Hz, benzyl Hb), 3.31 (br, 2H, N-CH-CO, 3-H), 3.77 (dd, 1H, J= 9.79/3.41 Hz, 4-H), 4.34 (br, 1H, OH), 6.68 (d, 1H, J = 8.46 Hz, 8-H), 6.88 (s, 1H, 5-H), 7.28-7.40 (m, 6-H, 7-H, phenyl-H); δC (CDCl3) 18.76 (2-CH3), 26.74 (2-CH3), 27.89 (C (CH3)3), 40.26 (benzyl CH2), 56.90 (4-C), 63.40 (N-CH-CO), 74.09 (3-C), 79.74 (C(CH3)3), 82.53 (2-C), 103.38 (6-C), 117.79 (8-C), 118.89 (CN), 126.00 (4a-C), 127.66 (arm. CH), 129.01 (2x arom.CH), 129.06 (2x arom. CH), 131.25, 132.43 (5-C, 7-C), 137.05(arom.C), 156.39 (8a-C), 175.50 (C=O); Ms m/z 423 (1.95%, M+), 275 (47.6%), 160 (89%); [α]D

20 +0.90 (c 0.111, in CH2Cl2); (Found: C, 70.35; H, 7.09; N, 6.72. C25H30N2O4 requires C, 71.07; H, 7.16; N, 6.63) (3R,4S)-N-(6-cyano-3-hydroxy-2,2-dimethyl-2H-3,4-dihydro-1-benzopyran-4yl)-L-phenylalanine-tert-butyl-Ester (7b). (R, R)-epoxide 4b and L-phenylalanine-tert-Butyl ester gave 7b yield 53% yellowish oil; mp 94-96 oC; IR (KBr): 2229 (CN) cm-1, 1695 (C=O) cm-1; δH (200MHz; CDCl3) 1.20 (s, 3H, 2-CH3), 1.52 (s, 12H, 2- CH3, C(CH3)3), 2.40 (br, 1H, NH), 2.78 (dd, 1H, J = 13.27/8.84 Hz, benzyl Ha), 3.05 (dd, 1H, J= 13.27/4.17Hz, benzyl Hb), 3.18 (m, 2H, N-CH-CO, 3-H), 3.79 (d, 1H, J = 9.98 Hz, 4-H), 3.93 (d, 1H, J=3.41 Hz, OH), 6.84 (d, 1H, J= 8.59Hz, 8-H), 7.12(s, 1H, 5-H), 7.27(m,1H, 7-H), 7.33-7.46(m, 5H, phenyl-H); δC (CDCl3) 18.76 (2-CH3), 27.12 (2-CH3), 27.77 (C (CH3)3), 41.03 (benzyl CH2), 56.44 (4-C), 57.27 (N-CH-CO), 71.00 (3-C), 79.54 (C(CH3)3), 82.66 (2-C), 103.77 (6-C), 118.07 (8-C), 119.28 (CN), 123.32 (4a-C), 127.63 (arm. CH), 128.59 (2x arom. CH), 129.50 (2x arom.CH), 132.38, 132.63 (5-C, 7-C), 136.81(arom.C), 157.64 (8a-C), 177.66 (C=O); Ms m/z 423 (2.44%, M+), 160 (59.5%), 120 (100%); [α]D


8. General procedure for cyclisation. (4.61 mmol) of L-amino acid-tert-butyl ester (5-7) was dissolved in a small amount of

CH2Cl2 , hydrolysed by 6 mL of 70% HClO4, stirred overnight, and 4N NH4OH solution was added slowly. The precipitate was dried and used in the next reaction step without further purification. A suspension of precipitates (2.76 mmol) , 4-dimethylaminopyridine (0.69 mmol) and bis (2-oxo-3oxazolidinyl) phosphinic chloride (4.12 mmol) in CH2Cl2 (50 mL) was heated to reflux at 80°C for 10 min, then triethylamine (0.95 mL, 6.85 mmol) was added and the solution was refluxed at 70°C for 4 days. The suspension was filtered and evaporated to dryness. Purification was done by flash chromatography (petroleum ether/ethylacetate; 9:1) to yield the target compounds (11-13).

(2S,4aS,10bR)-2,5,5-trimethyl-3-oxo-1,4a,5,10b-tetrahydro-3-H[1]benzopyrano[3,4-b][1,4]oxazine-9-carbonitril (11a) yield 52% as colourless crystals; mp 157-158oC; IR (KBr): 2224 (CN) cm-1; 1751 (lactone) cm-1; δH (200MHz; CDCl3) 1.31 (s, 3H, 5-CH3), 1.52 (s, 3H, 5-CH3), 1.56 (d, 3H, J= 7.08 Hz, 2-CH3), 3.86-4.03 (m, 2H, N-CH-CO, 4a-H), 4.14 (d, 1H, J = 10.30 Hz, 10b-H), 6.87 (d, 1H , J = 8.53 Hz, 7-H), 7.46 (dd, 1H, J = 8.53/2.02 Hz, 8-H), 7.84 (d, 1H, J = 2.02 Hz, 10-H); δC (CDCl3) 18.28 (5-CH3), 19.86 (5-C), 25.88 (2-CH3), 49.63 (10b-C), 54.21 (N-CH-CO), 78.07(5-C), 83.87 (4a-C), 104.25 (9-C), 118.24 (7-C), 118.83 (CN), 121.89 (10a-C), 131.22, 133.45 (8-C, 10C), 155.59 (6a-C), 169.98 (C=O); MS m/z 272 (3.37%, M+), 185 (39.6%), 170 (100%); [α]D

20 +95.34 (c 0.118, in CH2Cl2); (Found: C, 65.94; H, 5.98; N,10.15. C15H16N2O3 requires C, 66.16; H, 5.92; N, 10.29) (2S,4aR,10bS)-2,5,5-trimethyl-3-oxo-1,4a,5,10b-tetrahydro-3-H[1]benzopyrano[3,4-b][1,4]oxazine-9-carbonitril (11b) yield 45% as colourless crystals; mp 125-127.5oC; IR (KBr) 2224 (CN) cm-1; 1737 (lactone); δH (200MHz; CDCl3) 1.31 (s, 3H, 5-CH3), 1.52 (d, 3H, J = 7.03 Hz, 2-CH3), 1.55 (s, 3H, 5-CH3), 3.90 (q, 1H, J = 7.03 Hz, N-CH-CO), 4.05 (d, 1H, J = 10.54 Hz, 4a-H), 4.21 (d, 1H, J = 10.29, 10b-H), 6.86 (d, 1H, J = 8.54 Hz, 7-H), 7.45 (dd, 1H, J = 1.88 Hz, J = 8.54 Hz, 8-H), 7.77 (d, 1H, J = 1.88, 10-H); δC (CDCl3) 18.64, 19.93 (2x2-CH3), 26.19 (2-CH3), 47.36 (10b-C), 50.88 (N-CH-CO), 77.93 (5-C), 81.08 (4a-C), 104.33 (9-C), 118.07 (7-C), 118.88 (CN), 122.62 (10a-C), 131.68, 133.25 (10-C, 8-C) 155.66 (6a-C), 171.52 (C=O); MS m/z 273 (0.7, M+), 185 (35), 170 (100); [α]D

20 -147.52 (c 0.101, in CH2Cl2); (Found: C, 66.27; H, 6.04; N, 10.05 C15H16N2O3 requires C, 66.16; H, 5.92; N, 10.29 )


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(2S,4aS,10bR)-2-isopropyl-5,5-dimethyl-3-oxo-1,4a,5,10b-tetrahydro-3-H[1]benzopyrano[3,4-b][1,4]oxazine-9-carbonitril (12a) yield 66% as yellowish solid; mp 145-146oC; IR (KBr) 2220 (CN) cm-1; 1741 (lactone); δH (200MHz; CDCl3) 1.01, 1.13 (each d, each 3H, each J= 6.95 Hz, CH(CH3)2), 1.30 (s, 3H, 5-CH3), 1.53 (s, 3H, 5-CH3), 2.49-2.64 (m, 1H, CH(CH3)2), 3.87 (d, 1H, J = 5.81Hz, N-CH-CO), 3.97 (d, 1H, J = 9.50 Hz, 4a-H), 4.06 (d, 1H, J= 9.50 Hz, 10b-H), 6.88 (d, 1H, J = 8.58 Hz, 7-H), 7.48 (dd, 1H, J = 8.58/2.02 Hz, 8-H), 7.83 (s, 1H, 10-H); δC (CDCl3) 17.46 (CH-CH3), 19.05 (5-CH3), 19.84 (CH-CH3), 25.92 (5-CH3), 31.00 (CH(CH3)2), 49.34 (10b-C), 63.77 (N-CH-CO), 78.02 (5-C), 83.16 (4a-C), 104.37 (9-C), 118.32 (7-C), 118.87 (CN), 121.35 (10a-C), 131.31, 133.45 (8-C, 10-C), 156.00 (6a-C), 169.45 (C=O); Ms m/z 300 (0.80%, M+), 170 (100%); [α]D

20 +80 (c 0.05, in CH2Cl2); (Found: C, 67.78; H, 6.67; N, 9.08. C17H20N2O3 requires C, 68.71; H, 6.71; N, 9.33) (2S,4aR,10bS)-2-isopropyl-5,5-dimethyl-3-oxo-1,4a,5,10b-tetrahydro-3-H[1]benzopyrano[3,4-b][1,4]oxazine-9-carbonitril (12b) yield 80% as white crystals; mp 120-123 oC; IR (KBr) 2224 (CN) cm-1; 1745 (lactone), 1575 (NH); δH (200MHz; CDCl3) 1.02, 1.11 (each d, each 3H, each J= 6.82 Hz, CH(CH3)2), 1.28 (s, 3H, 5-CH3), 1.53 (s, 3H, 5-CH3), 1.66 (br, 1H, NH), 2.38-2.54 (m, 1H, CH(CH3)2), 3.65 (br, 1H, 10b-H), 3.89 (t, 1H, J = 9.68 Hz, N-CH-CO), 4.15 (d, 1H, J = 9.68 Hz, 4a-H), 6.87 (d, 1H, J = 8.52 Hz, 7-H), 7.47 (dd, 1H, J=8.52/1.77 Hz, 8-H), 7.90 (br, 1H, 10-H); δC (CDCl3) 17.47 (CH-CH3), 19.62, 19.76 (CH-CH3, 5-CH3), 26.02 (5-CH3), 31.80 (CH(CH3)2), 49.30 (10b-C), 59.87 (N-CH-CO), 77.89 (5-C), 82.45 (4a-C), 104.38 (9-C), 118.22 (7-C), 118.92 (CN), 122.11 (10a-C), 131.42, 133.39 (8-C, 10-C), 155.79 (6a-C), 170.31(C=O); Ms, m/z 300 (0.66%, M+), 185 (39.7%), 170 (92%); [α]D


(2S,4aS,10bR)-2-benzyl-5,5-dimethyl-3-oxo-1,4a,5,10b-tetrahydro-3-H[1]benzopyrano[3,4-b][1,4]oxazine-9-carbonitril (13a) yield (51%) as yellowish solid; mp 140-142 °C; IR (KBr): 2226 (CN) cm-1, 1740 (lactone) cm-1; δH (200MHz; CDCl3) 1.29 (s, 3H, 5-CH3), 1.46 (s, 3H, 5-CH3), 3.23 (dd, 1H, J = 14.02/4.67 Hz, benzyl Ha), 3.47 (dd, 1H, J= 14.02/5.69 Hz, benzyl Hb), 3.69(d, 1H, J = 10.11 Hz, 4a-H), 3.97(d, 1H, J = 10.11 Hz, 10b-H), 4.21 (dd, 1H, J = 5.69/4.67 Hz, N-CH-CO), 6.84(d, 1H, J = 8.43 Hz, 7-H), 7.27-7.43 (m, 5H, phenyl-H), 7.61 (dd, 1H, J= 8.43/1.96 Hz, 8-H), 7.75 (d, 1H, J = 1.96 Hz, 10-H); δC (CDCl3) 19.78 (5-CH3), 25.75 (5-CH3), 37.65 (benzyl CH2), 49.45 (10b-C), 59.38 (N-CH-CO), 77.85 (5-C), 83.22 (4a-C) 104.26 (9-C), 118.18 (7-C), 118.80 (CN), 121.00 (10a-C), 127.47 (arom.CH), 129.07 (2x arom.CH), 129.37 (2x arom.CH), 131.13, 133.39 (8-C, 10-C), 135.99 (arom.C), 155.84 (6a-C), 169.27 (C=O); Ms m/z 348 (3.05%, M+), 257 (17%), 170 (70.8%), 91 (100%); [α]D

20 +133.33 (c 0.12, in CH2Cl2); (Found: C,72.17; H, 5.86, N, 7.96. C21H20N2O3 requires C, 72.40; H, 5.79; N, 8.04) (2S,4aR,10bS)-2-benzyl-5,5-dimethyl-3-oxo-1,4a,5,10b-tetrahydro-3-H[1]benzopyrano[3,4-b][1,4]oxazine-9-carbonitril (13b) yield (45%) as yellowish oil; mp 202-201 °C; IR (KBr): 2223 (CN) cm-1, 1744 (lactone) cm-1; δH (200MHz; CDCl3) 1.05 (s, 3H, 5-CH3), 1.48 (s, 3H, 5-CH3), 1.83 (br, 1H, NH), 3.18 (dd, 1H, J= 13.56/6.44 Hz, benzyl Ha), 3.28 (dd, 1H, J= 13.56/4.99 Hz, benzyl Hb), 3.45 (d, 1H, J= 10.11 Hz, 4a-H), 4.09 (d, 1H, J = 10.11 Hz, 10b-H), 4.11 (dd, 1H, J = 6.44/4.99 Hz, N-CH-CO), 6.81(d, 1H, J = 8.53 Hz, 7-H), 7.27-7.32 (m, 5H, phenyl-H), 7.42 (dd, 1H, J= 8.53/2.02 Hz, 8-H), 7.72 (br, 1H, 10-H); δC (CDCl3) 19.35 (5-CH3), 25.84 (5-CH3), 39.82 (benzyl CH2), 47.10 (10b-C), 56.77 (N-CH-CO), 77.75 (5-C), 82.58 (4a-C) 104.26 (9-C), 118.03 (7-C), 118.91 (CN), 121.71 (10a-C), 127.08 (arom.CH), 128.59 (2x arom.CH), 129.64 (2x arom.CH), 131.02, 133.23 (8-C, 10-C), 137.26 (arom.C), 155.70 (6a-C), 170.11 (C=O); Ms m/z 348 (2.38%, M+), 257 (20.5%), 170 (39.40%), 91 (100%); [α]D

20 -33.65 (c 0.104, in CH2Cl2); (Found: C, 72.20; H, 5.74; N, 7.87. C21H20N2O3 requires C, 72.40; H, 5.79; N, 8.04) 1. P. Chiba, G. Ecker, D. Schmid, J. Drach, B. Tell, S. Goldenberg and V. Gekeler, Mol Pharmacol, 1996, 49, 1122-

1130.

9. 1H and 13C-NMR spectras of target compounds (11a-13b)


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References 1. .A. Sali, L. Potterton, F. Yuan, H. Van Vlijmen and M. Karplus, Proteins, 1995, 23, 318-326. 2. S. G. Aller, J. Yu, A. Ward, Y. Weng, S. Chittaboina, R. Zhuo, P. M. Harrell, Y. T. Trinh, Q. Zhang, I. L. Urbatsch and G. Chang,

Science, 2009, 323, 1718-1722. 3. .R. A. Laskowski, M. W. MacArthur, D. S. Moss and J. M. Thornton, J. App. Cryst. , 1993, 26, 283-291.


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