111

Synthesis & Antibacterial Evaluation of Juglone

Derivatives MSc candidate: Daniel Tan Teoh Chuan (P-

KM0010/06(R))Main supervisor: Assoc. Prof. Dr. Hasnah Osman

Co-Supervisor: Prof. Azlina Harun @ Kamaruddin

222

Introduction- Juglone (1)

A naturally-occuring naphthoquinone.1

Mainly found in the black walnut tree (Juglans Nigra).1

IUPAC name: 5-Hydroxy-1,4-naphthalenedione.

Molecular formula: C10H6O3 (MW: 174.15 g mol-1).

Dark orange-brown solid. Sparingly soluble in water. Applications: natural dye and

folkloric treatment for skin infections.

O

OOH

(1)

1

2

3

45

6

7

89

10

1. Thomson, R. H. (1971). Naturally Occurring Quinones. London, Academic Press.

ww

w.academ

yfloor.com/index.php%

3...pter%3D

0

http://www.morethanalive.com/Black-Walnut-hull-powder

3

Introduction- Reported Synthesis of Juglone (1)Year Authors Oxidant [O] Yield

(%)

1887 Bernthsen & Semper

Chromic acid -

1984 Wakamatsu et. al.

O2 + salcomine catalyst (3)

71

1990 Barret & Daudon

Hypervalent iodine oxidants [eg. (4)]

91

2006 Oelgemöller et. al.

O2 + sunlight + rose bengal sensitiser

79

OH

OH

[O]

O

OOH(1)(2)

N N

O OCo

(3)

IOCOCF3

OCOCF3(4)

F3CF2CF2CF2CF2CF2C

4

Introduction- The Use of Juglone (1) & 2-Bromo Acetyl Juglone (7) as Organic Reactants Diels-Alder Reactions.2,3

OMe

O

OOH

O

OOH(1)

(i) B(OAc)3 (1equivalent), CH2Cl2, 20oC

(5) rac-(ochromycinone)

+i

O O

75%

2. Guingant, A. & Barreto, M. M. (1987). Tetrahedron Letters 28 (27), 3107.

3. Krohn, K. et. al. (2004). Tetrahedron Asymmetry 15, 713.

O

OOAc

Br+

(7) (8)

O

OOH

O

OOH

O

(+)-(19)

(+)-(ochromycinone)

i

ii

iii(i) Toluene, 80-100oC, 14 h;(ii) K2CO3, MeOH; (iii) h, O2

44%

31%

5

Introduction- The Use of Juglone (1) as an Organic Reactant Direct amination.4

O

OOH

O

OOH

NH3 (g)CH3OH, rt, 24 h

NH2

(1) (9)20%

4. Arnone, A. et. al. (2007). Synthetic Communications 37, 2569.

6

Introduction- Synthetic Juglone Derivatives from Non-juglone Reactants

OAc

OAc

O

OAc O

BrNBS (4.0 eq), AcOH

60oC, 45 min,

(10) (7)>90%

i. S8, H2S2O7ii. H2O

(11)

NH2

OOH

O

(12)

NO2

NO2

5. Heinzman, S. W. & Grunwell, J. R. (1980). Tetrahedron Letters 21 (45), 4305. 6. Fariña, F. et. al. (1985). Synthesis 1985 (8), 781.

2-Bromo acetyl juglone (7) 8-Aminojuglone (12)

77

Introduction- Reported Biological Activities of Juglone Allelopathic activity against potato, tomato and alfalfa.1, 7

Antimicrobial activity against pathogenic fungus8 and bacteria.9

Antitumour activity against intestinal10 and melanoma11 tumours. Antihypertensive activity.12, 13

Larvicidal & molluscicdal activities.14

1. Thomson, R. H. (1971). Naturally Occurring Quinones. London, Academic Press.7. Rietveld, W. J. (1983). Journal of Chemical Ecology 9 (2), 295.8. Clark, A. M. et. al. (1990). Phytotherapy Research 4 (1), 11.9. Cai, L. N. et. al. (2000). Journal of Agricultural and Food Chemistry 48 (3), 909.10. Sugie, S. et. al. (1998). Cancer Letters 127 (1-2), 177.11. Aithal, B. K. et. al. (2009). Cell Biology International 33 (10), 1039.12. Neuhauscarlisle, K. et. al. (1997). Phytomedicine 4 (1), 67.13. Bhosale, S. H. et. al. (1999). Indian Journal of Pharmacology 31 (3), 222.14. Ribeiro, K. A. L. et. al. (2009). Acta Tropica 111, 44.

888

Research Objectives

1. To synthesise juglone derivatives from juglone. 2. To synthesise juglone derivatives from non-juglone

starting materials.3. To characterise all synthetic compounds.4. To evaluate the antibacterial activities of all synthetic

compounds.

9

Research Methodology- Synthesis from Juglone Reactant The Diels-Alder synthesis of ochromycinone precursor (19).

O

OOH

O

OOH

(1)

(i) Dry CHCl3, Ar, rt, 3 h (ii) DBU (2 eq.), air, rt, 13 h

(50)

+i

OSiMe3

ii

(19)

10

Research Methodology- Synthetic Preparation of Siloxy diene (50)

O EtOC HOH2C OHC

i ii iii iv

(i) NaH, (EtO)2POCH2COOEt, dry THF, rt, 5 h; (ii) LiAlH4, dry toluene, ref lux (120oC), 3 h; (iii)NMO, TPAP, 4 Å molecular sieves, dry CH2Cl2, 3 h; (iv) Dry ZnCl2 (liq), dry Et3N, dry TMSCl,dry toluene, Ar

OSiMe3(15) (47) (48) (49) (50)

O

15. Krohn, K. et. al. (1995). Liebigs Annalen 1995 (8), 1529.16. Hasnah, O. (2005). Synthetic Approaches to the Angucycline Antibiotics, Dunedin, University of Otago, PhD.

11

Research Methodology- Synthesis from Juglone Reactant Direct nitration

17. Anuradha, V. et. al. (2006). Tetrahedron Letters 47, 4933.

OH

O

OOH

O

O

Ni(NO3)2.6H2O, p -TsOH

acetone, ref lux, >24 h

(1) (51)

NO2

12

Research Methodology- Synthesis from Non-juglone Reactants Friedel-Crafts Acylation

OH

+ O

O

O

O

OOH

X R

(26) Cl Me

(27) Br Me

(65) Br H

(66) Cl H

(67) Cl Et

(26, 27, 65, 66, 67) (68) (30, 31, 52, 53, 54)

XR

XR

i

(i) AlCl3 (liq), NaCl (liq), 180-185oC, 2 mins. (ii) H3O

ii

X R

(30) Cl Me

(31) Br Me

(52) Br H

(53) Cl H

(54) Cl Et

18. Mahapatra, et. al. (2007). Bioorganic & Medicinal Chemistry 15, 7638.19. Musgrave, O. C. & Skoyles, D. (2001). Journal of the Chemical Society, Perkin Transaction 1, 1318.

13

Research Methodology- Separation and Purification of Synthetic Compounds

Silica gel column chromatography. Single-solvent recrystallisation.

14

Research Methodology- Characterisation of Synthetic Compounds

Melting point. High Resolution Electron Spray Ionisation Mass

Spectrometry (HRESIMS). Gas Chromtography-Mass Spectrometry (GC-MS). Ultraviolet spectroscopy (UV). Infrared spectroscopy (IR). Nuclear magnetic resonance spectroscopy (NMR). X-ray crystallography.

15

Research Methodology- Antibacterial Assay Broth microdilution method. Conducted in triplicates. 96-Well tissue culture plate. Sample: DMSO: water (1:9). Initial concentration: 250 g ml-1. Reference: juglone. Negative control: antibiotic. Positive control: Müller-Hinton

broth, MHB. Colour indicator: MTT [3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] www.opticsplanet.net/bd-falcon-u...lid.html

16

Results & Discussion- Preparation of (±)-(E)-Trimethyl(2-(5-methylcyclohex-1-enyl)vinyloxy) (50)

O EtOC HOH2C OHC

i ii iii iv

(i) NaH, (EtO)2POCH2COOEt, dry THF, rt, 5 h; (ii) LiAlH4, dry toluene, ref lux (120oC), 3 h; (iii)NMO, TPAP, 4 Å molecular sieves, dry CH2Cl2, 3 h; (iv) Dry ZnCl2 (liq), dry Et3N, dry TMSCl,dry toluene, Ar

OSiMe3

(15) (47) (48) (49) (50)

O

79% 56% 71% 75%

Overall yield 24%

17

Results & Discussion-Synthesis of (±)-8-Hydroxy-3-methyl-1,2,3,4-tetrahydrotetraphene-7,12-dione (19)

Non-Lewis acid promoted. Mild reaction condition. DBU base: elimination &

further aromatisation.

O

OOH

O

OOH

(1)

(i) Dry CHCl3, Ar, rt, 3 h (ii) DBU (2 eq.), air, rt, 13 h

(50)

+i

OSiMe3

ii

(19)

47%

18

Results & Discussion- Synthesis of Ochromycinone Precursor (19)

O

OOH

(1) (50)

+

OSiMe3

O

OOH

H

HO

O

OOH

(55)

(60)

H

B

O

OOH

H H

B = DBU

+ BH

SiMe3

+ SiMe3O

H

tautomerism

O O

OH

OOH

OO O

OOH

O

OH

H

B

O

OOH

(19)

+ BH +HO2

20. Kim, K. et. al. (1992). Journal of Organic Chemistry 57 (21), 5557.

19

Results & Discussion- Synthesis of 5-Hydroxy-8-nitro-1,4-naphthalenedione (51)

OH

O

OOH

O

O

Ni(NO3)2.6H2O, p -TsOH

acetone, ref lux, >24 h

(1) (51)

NO2

11%

The para-nitro substituted juglone is the sole isomeric product. Contrary to Anuradha et. al., 2006.

O

O

H

H

H

H

O

N

H

O O(51)

Hydrogen bond

202020

Results & Discussion- Syntheses of Halogen Substituted Juglone Derivatives

Compound Melting Point (oC) Yield (%)

158-160 (lit. 160-161) 8

148-150 (lit. 154) 10

206-209 24

202-204 (lit. 201-202) 10

108-111 14

O

OOH(53)

Cl

O

OOH

Br

(31)

O

OOH

Cl

(54)

O

OOH(30)

Cl

O

OOH(52)

Br

2121

Results & Discussion- Syntheses of Halogen Substituted Juglone Derivatives

OH

+ O

O

O

O

OOH(26) (68) (30)

Cl Cl

i

(i) AlCl3 (liq), NaCl (liq), 180-185oC, 2 mins. (ii) H3O

ii+

O

OOH

OH

OH(69)

8% 1%

18. Musgrave, O. C. & Skoyles, D. (2001). Journal of the Chemical Society, Perkin Transaction 1, 1318.

(69): Helminthosporin, 1,5,8-trihydroxy-3-methyl-9,10-anthracenedione

22

Results & Discussion- Syntheses of Halogen Substituted Juglone Derivatives

OH

+ O

O

O

O

OOH(26) (68) (30)

Cl Cl OH

OHOH

Cl

[H]

O

O

O

(68)

OH

OHOH

O

O

O

OOH

OH

OH

ClCltautomerism

O

OOH

OH

OH(69)

1. [H], H

2. -HCl

18. Musgrave, O. C. & Skoyles, D. (2001). Journal of the Chemical Society, Perkin Transaction 1, 1318.

232323

Results & Discussion- Antibacterial AssayCompound MIC (mM)

B. cereus S. aureus E. Coli K. Pneumoniae

0.36 0.72 0.36 0.36

0.21 0.43 0.43 0.43

0.29 0.14 0.57 0.57

0.56 1.1 0.56 0.56

O

OOH(1)

O

OOH(19)

O

OOH

NO2

(51)

O

OOH

Cl

(30)

242424

Results & Discussion- Antibacterial AssayCompound MIC (mM)

B. cereus S. aureus E. Coli K. Pneumoniae

0.47 0.94 0.47 0.47

0.50 0.99 0.50 0.50

0.23 0.46 0.23 0.23

0.53 1.1 0.53 0.53

O

OOH(31)

Br

O

OOH(69)

OH

OH

O

OOH

Br

(52)

O

OOH

Cl

(54)

252525

Results & Discussion- Antibacterial Assay

Compound MIC (mM)

B. cereus S. aureus E. Coli K. Pneumoniae

0.60 0.60 0.60 0.60

Gentamicin - - 0.027 0.014

Tetracycline 0.016 <0.004 - -

O

OOH(53)

Cl

26

Conclusion

The non-Lewis acid promoted, mild Diels-Alder reaction between juglone (1) & siloxydiene (50) afforded the ochromycinone precursor (19) in 47% yield.

8-Nitrojuglone (51) was obtained as the sole para isomeric product in 11% yield from the direct nickel(II) nitrate nitration of juglone (1).

The Friedel-Crafts acylation gave five 8-halojuglone derivatives (30, 31, 52, 53 & 54) in 8-24% yields. Two of which (52 & 54) are new derivatives.

A helminthosporin anthraquinone, (69) was obtained as the minor product of one of the Friedel-Crafts acylation reactions.

27

Conclusion

8-Nitrojuglone (51) exhibited the most significant activity against S. aureus (MIC= 0.14 mM).

Helminthosporin (69) displayed high activities against the Gram-negative bacteria tested.

All of the five 8-halojuglone derivatives showed lower antibacterial activities as compared to juglone (1).

2828

Acknowledgements

Assoc. Prof. Dr. Hasnah Osman (Main supervisor). Prof. Azlina Harun @ Kamaruddin (Co-supervisor). Madam Suriyati Mohamad. Ms. Anis Safirah. USM School of Chemical Sciences. USM School of Biological Sciences. USM Fellowship Scheme. PURGS grant: 1001/PKIMIA/832017. RU grant: 1001/PKIMIA/811016.

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