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Electronic Supplementary Information Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late Stage Diels-Alder Reaction Alan R. Healy and Nicholas J. Westwood* Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry. This journal is © The Royal Society of Chemistry 2015

Stage Diels-Alder Reaction Synthesis of the Bioactive ... fileElectronic Supplementary Information Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late Stage Diels-Alder Reaction

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Page 1: Stage Diels-Alder Reaction Synthesis of the Bioactive ... fileElectronic Supplementary Information Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late Stage Diels-Alder Reaction

Electronic Supplementary Information

Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late

Stage Diels-Alder Reaction

Alan R. Healy and Nicholas J. Westwood*

Electronic Supplementary Material (ESI) for Organic & Biomolecular Chemistry.This journal is © The Royal Society of Chemistry 2015

Page 2: Stage Diels-Alder Reaction Synthesis of the Bioactive ... fileElectronic Supplementary Information Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late Stage Diels-Alder Reaction

Table of Contents1. Late-stage DA cyclisations in the biosynthesis of tetramic acid derived natural products.........3

2. UPLC Analysis..............................................................................................................................5

3. Experimental procedures ...........................................................................................................6

4. NMR spectra of novel compounds .............................................................................................9

5. Bibliography..............................................................................................................................17

Page 3: Stage Diels-Alder Reaction Synthesis of the Bioactive ... fileElectronic Supplementary Information Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late Stage Diels-Alder Reaction

1. Late-stage DA cyclisations in the biosynthesis of tetramic acid derived natural products.

NH

O

HO

O

OH

OH

PyrE3

NH

O

HO

O

OH

OH

H

H

Pyrl4NH

OO

OH

H

H

HO

HO

NH

OO

O

H

H

CO2H

HO

HO

OOOOO

NH2O

O

NH

NH

R

OH

R = H: Pyrroindomycin AR = Cl: Pyrroindomycin B

HNO

OHO CO2H

OH

HN

O

O

H

H

HO HO CO2HcghA

HN

O O

HN

O O

HN

H

HN

O O

HNHN

O O

HN

H

O OH

O

HN

O O

H

HN

OO

H

O

O

OHO

O

chaetoglobosin A

cytochalasin E

Sch210972(3)

(A)

(B)

(C)

Scheme S1 (A) Dedicated cyclases “Diels-Alderases” PyrE3 and Pyrl4 have very recently been show to act in tandem to catalyse the formation of two cyclohexene rings in the biosynthesis of Pyrroindomycins A and B.1 These enzyme-catalysed [4+2] cycloadditions occur after the formation of the tetramic acid ring core. (B) Proposed Sch210972 (3) biosynthetic pathway involving a cghA catalysed late-stage Diels-Alder cycloaddition.2 (C) An enzymatic IMDA cycloaddition is proposed in the biosynthesis of chaetoglobosin A and cytochalasin E involving a partially reduced tetramic acid ring system.2–4a

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O

PO

EtOEtO

N

O

OO

CO2Et

NHO CO2Et

O

O

HO

O

PO

EtOEtO

N

O

OO

CO2EtN

CO2EtO

OHO

PO

EtOEtO O

NCO2EtO

OHO

PO

EtOEtO HO

O

9

5

(a)

10

N

O

O

HO

HO CO2Et

Scheme S2. One-pot domino cyclisation-HWE olefination. Reagents and conditions: (a) (i) tBuOK, THF, 0 C, 1 hour. (ii) 10, THF, 0 C r.t., 16 h, 85%. For a very similar mechanism for a related transformation see reference 4b

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2. UPLC Analysis

Figure S1. The UPLC traces were obtained on an ACQUITY UPLC BEH C18 column (0.6 ml/min; solvent: 55 % MeCN (0.1% TFA)). (A) UPLC trace of the BF3.OEt2 catalysed IMDA cycloaddition (Table 1, entry 3). (B) UPLC trace of the Mg(II)-bis(oxazoline) complex 12 catalysed IMD cycloaddition (Table 1, entry 4).

3a 3b (A)

(B)

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3. Experimental procedures

Isopropyl (2S,4S)-4-(4-(diethoxyphosphoryl)-N-methyl-3-oxobutanamido)-2-isopropyl-5-

oxotetrahydrofuran-2-carboxylate (9)

4N 1'

2'3'

4'P5O

2 3

O

1''O

2'''

O

3''

4''

1''' 2'''

O O O

OO

7'

8'

7' 8'O2

3

4O

N

1''

OO

3'''

4'''

2''

SO

O2

3

4O

NH

1''

OO

3'''

4'''

2''

76 9To a solution of 6 (205 mg, 0.62 mmol, 1.0 eq.) in THF (1.5 mL) at 0 C was added HCl (0.62 mL, 4.0

eq., 4 N in dioxane). The reaction was stirred for 10 minutes at this temperature before the addition

of a solution of NaBH3CN (117 mg, 1.86 mmol, 3.0 eq.) in MeOH (3 mL). The reaction was stirred for a

further 1 hour at 0 C before being concentrated in vacuo. The residue was partitioned between a

saturated aqueous solution of NaHCO3 (5 mL) and EtOAc (5 mL). The aqueous layer was extracted with

EtOAc (3 5 mL) and the combined organic extracts were washed with brine (10 mL), dried over

Na2SO4, filtered and concentrated in vacuo to yield the crude free amine 7. 1H NMR (500 MHz,

Chloroform-d) δ 4.25 (qd, J = 7.1, 2.2 Hz, 2H, C3’’’-H2), 3.54 (dd, J = 11.5, 8.4 Hz, 1H, C4-H), 2.74 (dd, J

= 12.8, 8.4 Hz, 1H, C3-H2), 2.46 (s, 3H, NHCH3), 2.25 (hept, J = 6.9 Hz, 1H, C1’’-H), 2.05 (dd, J = 12.8,

11.5 Hz, 1H, C3-H2), 1.78 (br s, 1H, NHCH3), 1.30 (t, J = 7.1 Hz, 3H, C4’’’-H3), 1.02 (d, J = 6.9 Hz, 3H, C2’-

H3), 0.99 (d, J = 6.9 Hz, 1H, C2’-H3); 13C NMR (126 MHz, Chloroform-d) δ 175.9 (C5), 171.2 (C1’’’), 87.3

(C2), 62.2 (C3’’’), 58.1 (C4), 35.9 (C3), 34.4 (NCH3), 34.3 (C1’’), 17.0 (C2’), 16.4 (C2’), 14.3 (C4’’’). To a

solution of 7 in anhydrous MeCN (6 mL) was added a solution of 8 in anhydrous MeCN (3 mL), and the

reaction was heated at reflux for 2.5 hours. The reaction was concentrated in vacuo and purified via

the Biotage SP4 (silica-packed SNAP column 12 g; 0-8% MeOH/DCM) to give the title product 9 as an

orange oil (220 mg, 79%). In CDCl3 at room temperature the title compound 9 exists as a (7 : 3) enol :

keto mixture. The NMR signals are reported for the major keto tautomer. 1H and 13C spectra are

complicated by 31P splitting. IR (thin film) ν max: 2978, 2936, 1784 (C=O), 1734 (C=O), 1636 (C=O), 1236,

1184, 1022; 1H NMR (500 MHz, Chloroform-d) δ 4.83 – 4.71 (m, 1H, C4-H), 4.33 – 4.21 (m, 2H, C3’’-H2),

4.21 – 4.07 (m, 4H, (C7’-H2)2), 3.81 (s, 2H, C2’-H2), 3.25 (dd, J = 22.6, 4.4 Hz, 2H, C4’-H2), 3.01 (s, 3H,

NCH3), 2.68 (dd, J = 12.8, 9.1 Hz, 1H, C3-H2), 2.47 (dd, J = 12.8, 11.4 Hz, 1H, C3-H2), 2.38 – 2.29 (m, 1H,

C1’’’-H), 1.37 – 1.28 (m, 9H, C4’’-H3, (C8’-H3)2), 1.05 (d, J = 6.9 Hz, 3H, C2’’’-H3), 1.02 (d, J = 6.9 Hz, 3H,

C2’’’-H3); 13C NMR (126 MHz, Chloroform-d) δ 195.7 (C3’), 172.1 (C5), 171.0 (C1’’), 167.3 (C1’), 87.2

(C2), 63.0 (d, J = 6.5 Hz, C7’), 62.4 (C3’’), 56.7 (C4), 49.7 (C2’), 42.5 (d, J = 125.9 Hz, C4’), 35.3 (NCH3),

34.2 (C1’’’), 31.5 (C3), 17.0 (C2’’’), 16.5 (C2’’’), 16.4 (C8’), 14.3 (C4’’). 31P NMR (202 MHz, Chloroform-d)

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δ 19.2. m/z (ES+) 472.17 ([M+Na]+, 100 %); HRMS (ES+) Calcd for C19H32O9NPNa [M+Na]+: 472.1707,

found 472.1696; = -9.2 (c 1.0, MeOH).[α]20D

Ethyl (S)-2-hydroxy-2-(((S,E)-4-((2E,8E,10E)-1-hydroxydodeca-2,8,10-trien-1-ylidene)-1-methyl-3,5-

dioxopyrrolidin-2-yl)methyl)-3-methylbutanoate (5)

4N 1'

2'3'

4'P5O

2 3

O

1''O

2'''

O

3''

4''

1''' 2'''

O O O

OO

7'

8'

7' 8'

5 N23

4

1'' 2''3''

4''

4''

HO1'''

O

O

3'''

4'''O

O

1'

2'3'

4'5'

6'7'

8'

HO

9'

10'11'

12'

9 5

To a solution of 9 (100 mg, 0.22 mmol, 1.0 eq.) in THF (2.5 mL) at 0 C was added tBuOK (0.24 mL, 1.1

eq., 1M in THF) and the reaction was stirred for 40 minutes. To the mixture was added a solution of

105 (102 mg, 0.67 mmol, 3.0 eq.; 85% E,E-diene geometry) in THF (1 mL) and the reaction was stirred

for a further 15 minutes before being warmed to room temperature and stirred for 16 hours. The

reaction was quenched by the addition of an aqueous solution of HCl (3 mL, 1N) and extracted with

DCM (3 10 mL). The organic extracts were combined, washed with brine (10 mL), dried over Na2SO4,

filtered, concentrated in vacuo and purified via the Biotage SP4 (Reverse-phase silica-packed SNAP

column 4 g; 20-100% H2O/(MeOH:MeCN)) to give the title product 5 as an yellow oil (83 mg, 85%). In

CDCl3 at room temperature the 1H and 13C NMR spectra of 5 are complicated by the presence of

keto/enol tautomers and a minor E,Z,E isomer. The NMR signals are reported for the major tautomer.

IR (thin film) ν max: 2964, 2931, 1717 (C=O), 1690 (C=O), 1645 (C=O), 1586, 1449, 1242, 989; 1H NMR

(500 MHz, Chloroform-d) δ 7.24 – 7.13 (m, 1H, C3’-H), 7.08 – 7.03 (m, 1H, C2’-H), 6.04 – 5.90 (m, 2H,

C9’-H, C10’-H), 5.62 – 5.45 (m, 2H, C8’-H, C11’-H), 4.24 (q, J = 7.1 Hz, 2H, C3’’’-H2), 3.67 (dd, J = 9.6, 2.2

Hz, 1H, C2-H), 2.97 (s, 3H, NCH3), 2.37 – 2.29 (m, 2H, C4’-H2), 2.30 – 2.28 (m, 1H, C1’’-H2), 2.10 – 2.00

(m, 3H, C3’’-H, C7’-H2), 1.90 (dd, J = 14.5, 9.6 Hz, 1H, C1’’-H2), 1.71 (d, J = 6.4 Hz, 3H, C12’-H3), 1.57 –

1.45 (m, 2H, C5’-H2), 1.46 – 1.37 (m, 2H, C6’-H2), 1.31 (t, J = 7.1 Hz, 3H, C4’’’-H3), 0.94 (d, J = 7.3 Hz, 3H,

C4’’-H3), 0.93 (d, 7.3 Hz, 3H, C4’’-H3); 13C NMR (126 MHz, Chloroform-d) δ 196.3 (C3), 175.5 (C1’’’),

175.1 (C1’), 173.4 (C5), 151.8 (C3’), 131.6 (C10’), 131.4 (C8’), 130.8 (C9’), 127.2 (C11’), 121.6 (C2’), 98.9

(C4), 78.8 (C2’’), 64.5 (C2), 61.6 (C3’’’), 36.8 (C3’’), 34.9 (C1’’), 33.3 (C4’), 32.4 (C7’), 29.1 (C6’), 27.7

(C5’), 26.8 (NCH3), 18.1 (C12’), 17.5 (C4’’), 16.4 (C4’’), 14.5 (C4’’’); m/z (ES-) 446.25 ([M-H]-, 100 %);

HRMS (ES-) Calcd for C25H36O6N [M-H]-: 446.2548, found 446.2549.

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JBIR-22 (3a/b)

9

87

611

10

54

32

1HO 3' 2'

N5'4'

6' 7'

8'9'

12'

O

10'OH

OH 11'

12O

O

H

H

9

87

611

10

54

32

1HO 3' 2'

N5'4'

6' 7'

8'9'

12'

O

10'OH

OH 11'

12O

O

H

H

3a 3b

The 1:1 mixture of JBIR-22 (3a/b) was synthesised as reported in Ref[8]. 3a/b was obtained as an

amorphous white solid (73 mg). Spectroscopic data was obtained for JBIR-22 3a/b-Et2NH salt. = [α]23D

+22.0 (c 0.7, MeOH); 1H NMR (500 MHz, Acetone-d6) δ 5.57 – 5.48 (m, 2H, 2 C4-H), 5.33 – 5.27 (m,

2H, 2 C5-H), 3.93 (dd, J = 11.1, 5.5 Hz, 1H, C2-H (3b)), 3.90 (dd, J = 11.1, 5.5 Hz, 1H, C2-H (3a)), 3.37

(dd, J = 10.1, 1.3 Hz, 2H, 2 C5’-H), 2.72 (s, 6H, 2 C10’-H3), 2.67 – 2.56 (m, 2H, 2 C3-H), 2.31 (dt, J

= 13.9, 1.5 Hz, 2H, 2 C6’-H2), 2.04 – 1.94 (m, 4H, 2 C8’-H, 2 x 1 of C10-H2), 1.76 – 1.66 (m, 8H, 2 1

of C7-H2, 2 C6-H, 2 1 of C8-H2, 2 1 of C9-H2), 1.63 (dd, J = 13.8, 10.1 Hz, 2H, 2 1 of C6’-H2), 1.51

– 1.41 (m, 2H, 2 C11-H), 1.30 – 1.28 (m, 4H, 2 1 of C8-H2, 2 1 of C9-H2), 1.09 – 0.98 (m, 2H, 2 1

of C7-H2), 0.91 (d, J = 6.7 Hz, 6H, 2 C9’-H3), 0.77 (d, J = 7.2 Hz, 3H, C12-H3 (3a)), 0.75 (d, J = 7.2 Hz, 3H,

C12-H3 (3b)), 0.73 – 0.67 (m, 2H, 2 1 of C10-H2); 13C NMR (126 MHz, Acetone) δ 196.40 (C1), 196.36

(C1), 196.0 (C4’), 195.8 (C4’), 180.5 (C11’), 180.4 (C11’), 174.8 (C2’), 174.7 (C2’), 133.6 (C4), 133.5 (C4),

131.2 (C5), 131.1 (C5), 101.5 (2 C3’), 80.0 (C7’), 79.9 (C7’), 63.3 (C5’), 63.2 (C5’), 51.07 (C2), 50.98

(C2), 43.4 (C6), 43.3 (C6), 37.4 (C11), 37.3 (C11), 37.2 (C6’), 37.1 (C6’), 36.62 (C8’), 36.60 (C8’), 34.4

(C7), 34.3 (C7), 32.3 (C3), 32.2 (C3), 31.1 (C10), 31.0 (C10), 27.7 (C9/C8), 27.6 (C9/C8), 18.7 (C9’), 18.7

(C9’), 18.34 (C12), 18.33 (C12), 16.8 (2 C12’); m/z (ES-) 418.23 ([M-H]-, 100 %); HRMS (ES-) Calcd for

C23H32O6N [M-H]-: 418.2235, found 418.2225. Spectroscopic data are in agreement with that reported

previously by us for the single diastereomers JBIR-22 3a and 3b.5 See Section 6 for the NMR spectra of

the 1:1 mixture of 3a/b.

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4. NMR spectra of novel compounds

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)

0

500

1000

1500

2000

2500

3000

3500

4000

6.11

12.5

0

1.29

1.38

1.11

2.03

2.01

5.33

2.94

1.00

NP

O

O

O

O

CH 3

C H 3CH 3

C H 3

O O O

O

O

C H 3

C H 3

9, CDCl3, 500 MHz

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102030405060708090100110120130140150160170180190200210220f1 (ppm)

-100

0

100

200

300

400

500

600

700

800

900

1000

1100

1200

1300

14.2

816

.44

16.4

816

.96

31.5

034

.23

35.2

7

41.9

842

.98

49.7

2

56.6

762

.42

62.9

663

.01

87.1

9

167.

3017

0.95

172.

09

195.

66

NP

O

O

O

O

CH 3

C H 3CH 3

C H 3

O O O

O

O

C H 3

C H 3

9, CDCl3, 500 MHz

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0.81.01.21.41.61.82.02.22.42.62.83.03.23.43.63.84.04.24.44.64.85.05.25.45.65.8f2 (ppm)

0

10

20

30

40

50

60

70

80

90

f1 (

ppm

)

9 (HSQC), CDCl3, 500 MHz

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0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5f1 (ppm)

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

6.77

3.61

2.44

2.42

3.45

1.01

3.01

3.55

3.08

1.08

2.41

2.04

2.00

1.09

1.01

N C H 3

C H 3

OH

O

O

CH 3

C H 3O

OOH

CH 3

5, CDCl3, 500 MHz

Page 13: Stage Diels-Alder Reaction Synthesis of the Bioactive ... fileElectronic Supplementary Information Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late Stage Diels-Alder Reaction

102030405060708090100110120130140150160170180190200210220f1 (ppm)

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

12000

13000

14000

14.4

916

.37

17.4

718

.13

26.7

927

.70

29.0

932

.35

33.3

134

.87

36.7

5

61.5

664

.48

78.7

8

98.9

0

121.

56

127.

2013

0.81

131.

3713

1.63

151.

75

173.

4017

5.11

175.

47

196.

28

N C H 3

C H 3

OH

O

O

CH 3

C H 3O

OOH

CH 3

5, CDCl3, 500 MHz

Page 14: Stage Diels-Alder Reaction Synthesis of the Bioactive ... fileElectronic Supplementary Information Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late Stage Diels-Alder Reaction

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0f2 (ppm)

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

f1 (

ppm

)

5 (HSQC), CDCl3, 500 MHz

Page 15: Stage Diels-Alder Reaction Synthesis of the Bioactive ... fileElectronic Supplementary Information Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late Stage Diels-Alder Reaction

0.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5f1 (ppm)

-1000

0

1000

2000

3000

4000

5000

6000

7000

8000

9000

10000

11000

0.93

2.98

5.97

1.39

1.18

5.05

2.05

0.95

1.16

2.72

0.84

1.00

1.11

1.06

OHN

CH 3C H 3

O

C H 3

O HO H

C H 3

O

O

H

H

OHN

CH 3C H 3

O

C H 3

O HO H

C H 3

O

O

H

H

3a/b, Acetone-d6, 500 MHz

Page 16: Stage Diels-Alder Reaction Synthesis of the Bioactive ... fileElectronic Supplementary Information Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late Stage Diels-Alder Reaction

0102030405060708090100110120130140150160170180190200210220f1 (ppm)

-400

-200

0

200

400

600

800

1000

1200

1400

1600

1800

2000

2200

2400

2600

2800

3000

3200

3400

3600

3800

400016.8

418

.33

18.3

418

.68

18.7

326

.78

27.6

731

.00

31.0

532

.30

34.3

434

.39

36.6

036

.62

37.2

737

.39

43.3

143

.40

50.9

851

.07

63.2

263

.27

79.9

379

.95

101.

49

131.

1013

1.20

133.

4513

3.60

174.

7117

4.76

180.

3718

0.52

195.

8419

6.00

196.

3619

6.40

OHN

CH 3C H 3

O

C H 3

O HO H

C H 3

O

O

H

H

OHN

CH 3C H 3

O

C H 3

O HO H

C H 3

O

O

H

H

3a/b, Acetone-d6, 500 MHz

Page 17: Stage Diels-Alder Reaction Synthesis of the Bioactive ... fileElectronic Supplementary Information Synthesis of the Bioactive Tetramic Acid JBIR-22 using a Late Stage Diels-Alder Reaction

5. Bibliography

1 Z. Tian, P. Sun, Y. Yan, Z. Wu, Q. Zheng, S. Zhou, H. Zhang, F. Yu, X. Jia, D. Chen, A. Mándi, T. Kurtán and W. Liu, Nat. Chem. Biol., 2015, 1–10.

2 K. Watanabe, Chem. Pharm. Bull., 2014, 62, 1153–1165.

3 W. L. Kelly, Org. Biomol. Chem., 2008, 6, 4483–93.

4 G. Li, S. Kusari and M. Spiteller, Nat. Prod. Rep., 2014, 31, 1175–1201.

5 A. R. Healy, M. Izumikawa, A. M. Z. Slawin, K. Shin-ya and N. J. Westwood, Angew. Chemie Int. Ed., 2015, 54, 4046–4050.