Synthesis and biological evaluation of 3,4,5-trihydroxy benzoic acid derivatives

Presented By:-

KALYAN MONDAL Regn. No. : 13-UTDPR-0117

Mrs. Saloni Kakkar Supervisor Assistant Professor (Pharm. Chemistry)

Dr. Anurag Khatkar SupervisorAssistant Professor(Pharm. Chemistry)

DEPARTMENT OF PHARMACEUTICAL SCIENCESMAHARSHI DAYANAND UNIVERSITY,

ROHTAK-124001 (HARYANA)2014

CONTENTS1. INTRODUCTION

2. OBJECTIVE

3. LITERATURE REVIEW

4. SYNTHETIC SCHEME & PROCEDURE

5. PHYSICOCHEMICAL PROPERTIES AND SPECTRAL DATA

6. BIOLOGICAL EVALUATION

7. REFERENCES

INTRODUCTION Extensive use of the antibiotics causes the development of the multidrug- resistant microbial pathogens. Rapid development of drug resistance have highlighted the need for the discovery of new antifungal agents.The unregulated production of ROS and free radicals are constantly going on in the human body by normal metabolic action, and have been identified in the pathogenesis of certain human diseases.Therapeutic approach for treating type 2 diabetes mellitus is to reduce the glucose levels. So this condition could be done by retard absorption of glucose through inhibition of α-amylase.Urease is a nickel containing metalloenzyme that rapidly catalyzes the hydrolysis of urea to the formation of ammonia and carbon dioxide.3, 4, 5-trihydroxy benzoic acid is a type of naturally occurring phenolic acid, which is generally distributed in a variety of foods and plants. 3, 4, 5-trihydroxy benzoic acid derivatives has been associated a wide range of biological activities.

OBJECTIVE

Synthesis and biological evaluation (in vitro antimicrobial, antioxidant, alpha amylase and urease inhibitory activity) of 3,4,5-trihydroxy benzoic acid derivatives.

Sr. No

3,4,5-trihydroxy benzoic acid derivatives Activity reported References

1. Octadeca-9,12-dienyl-3,4,5-hydroxybenzoate Antioxidant Aera et al., (2008)2. Novel hybrid retinoid derivatives, retinyl gallate Antioxidant Sungbum et al., (2008)

3. A novel derivative of chitosan, chitosan-gallic acid (chitosan-GA)

Antioxidant Wanvimol et al., (2007)

4. Gallic acid-grafted-chitosans (GA-g-chitosans) Antioxidant Cho et al., (2010)

5. Methyl and propyl gallate Antioxidant Maryam et al., (2014)

6. Ellagic acid Antioxidant Singh et al., (2014)

7. Ethanol extract of the peony root (Paeonia Lactiflora Pall, Paeoniaceae)

Antioxidant Seung et al., (2005)

8. The ethanolic extracts of Longan (Dimocarpus longan Lour.) seed and mango (Mangifera indica L.) kernel

Antioxidant Soong et al., (2005)

9. Gallic acid isolated from Terminalia bellerica Roxb. fruit extract lowers blood glucose with a simultaneous increase in the plasma insulin and C-peptide levels.

Antidiabetic Latha and Daisy, (2011)

Literature review of 3,4,5-trihydroxy benzoic acid derivatives

Sr. No 3,4,5-trihydroxy benzoic acid derivatives Activity reported References

10. Seabuckthorn leaf (Hippophae rhamnoides L. Elaeagnaceae) extract that increases glucose uptake in 3T3-L1 adipocytes.

Antidiabetic Prasad et al., (2009)

11. GA-g-chitosan Antidiabetic Jian et al., (2013)

12. Solanum torvum Swartz. fruit containing phenolic compounds Antidiabetic Gandhi et al., (2013)

13. 2,4,6-Trihydroxybenzaldehyde Antidiabetic Shruti et al., (2010)

14. 80 % MeOH extract of Sophora japonica leaves Antidiabetic Mohamed et al., (2014)

15. 3,4,5-trihydroxybenzoic acid, a major bioactive polyphenol present in Cyamopsis tetragonoloba, exhibited antihyperglycemic activity Antidiabetic Gandhi et al., (2014)

16. Methyl caffeate Antidiabetic Gopalsamy et al., (2011)

17. Gallic acid based steroidal phenstatin analogues Anticancer Swati et al., (2012)

18. [3-hydroxylestra-1,3,5(10)-trien,17β-acetate2yl]-(4-hydroxy,3-methoxyphenyl)-methanone Anticancer Swati et al., (2012)

19. Methyl gallate-3,5-dimethyl ether (MGDE) and isobutyl gallate-3,5-dimethyl ether (IGDE) Anticancer Saulo et al., (2009)

20. Digalloylresveratrol, a new phenolic acid derivative Anticancer Astrid et al., (2008)

Sr. No

3,4,5-trihydroxy benzoic acid derivatives Activity reported References

21. Procyanidin B2 dimer Anticancer Shen-Chieh et al., (2010)

22. Indanone derivatives Anticancer Chanda et al., (2012)

23. Mangiferin derivatives Anticancer Rivera et al., (2011)

24. [3-methoxyestra-1,3,5(10)-trien,17β-acetate2yl]-(3,4-dimethoxyphenyl)-methanone

Anticancer Kaur et al., (2009)

25. Combination of carvacrol and thymol Antimicrobial Daozong et al., (2010)

26. Laccase-mediated functionalization of chitosan by gallic acid and caffeic acid

Antimicrobial Mojca et al., (2011)

27. Chitosan-gallic acid films Antimicrobial Xiuxiu et al., (2013)

28. GA and catechin against Helicobacter pylori by an agar-well diffusion method.

Antimicrobial Roberto et al., (2013)

29. Quinolin-8-yl 3,4,5-trihydroxybenzoate Antimicrobial Khatkar et al., (2013)

30. Hexyl 3,4,5-trihydroxybenzoate Analgesic Krogh et al., (2000)

31. A bioactive derivative of chitooligosaccharides was synthesized via grafting of gallic acid onto chitooligosaccharides (G-COS)

Anti-allergic Thanh et al., (2012)

32. Gallic acid ethyl ester (GAEE) Antinociceptive Adair et al., (1999)

33. Penta-Galloyl-Glucose (PGG) Miscellaneous Jinhui et al., (2009)

34. A new hybrid derived from retinol Miscellaneous Sungbum et al., (2008)

SYNTHETIC SCHEME

OH

OH

OH

COOHSOCl2

OH

OH

OH

COCl

NOH

OH

OH

OH

CO

O

N

R-NH2

OH

OH

OH

CO-NHR

1

2-14

800 C, 4 h

Comp. R Comp. R

2. 3.

4. 5.

6. 7.

8. 9.

10. 11.

12. 13.

14

O2N

Cl

NO2

O2N

NO2Cl

F H3CO

H3COOCH3

H3C

CH3

F

H3C

H3C

General procedure for the synthesis of 3, 4, 5-trihydroxybenzoic acid derivatives

Thionyl chloride (0.6 mol) + Gallic acid (0.35 mol)

Stirred for 4 hour

800C for 30 min in water bath

3,4,5-trihydroxy benzoyl chloride + ether (15 ml)

Quinolin-8-yl 3,4,5-trihydroxybenzoate Precipitated amide

Physicochemical properties of synthesized derivatives

Comp. Mol. Formula M. Wt. m.p. ( 0C) Rf Value* % Yield

1. C16H11NO5 297.26 195-205 0.54 38.88

2. C13H11NO4 245.23 170-180 0.33 88.23

3. C13H10ClNO4 279.68 180-185 0.68 93.33

4. C13H10N2O6 290.23 140-145 0.54 82.35

5. C13H10N2O6 290.23 137-140 0.41 73.33

6. C13H9N3O8 335.23 130-135 0.60 70.00

7. C13H10ClNO4 279.68 190-193 0.46 40.00

8. C13H10FNO4 263.22 148-152 0.32 40.00

9. C14H13NO5 275.26 158-162 0.50 22.85

10. C14H13NO5 275.26 100-105 0.27 93.22

11. C14H13NO5 275.26 102-105 0.50 80.00

12. C15H15NO4 273.28 103-106 0.33 82.00

13. C13H10FNO4 263.22 180-185 0.68 91.67

14. C15H15NO4 273.28 162-167 0.67 22.22

Spectral data3,4,5-trihydroxybenzoyl chloride (Intermediates): IR (KBr pellets) cm-1: 3495 (O-H str., phenol), 3066 (C-H str., phenyl nucleus), 1541 (C=C skeletal str., phenyl), 733 (C-Cl str., aromatic); 1H NMR (DMSO-d6, δppm): 6.72 (d, 2H, Ar-H).

Quinolin-8-yl 3,4,5-trihydroxybenzoate (1): IR (KBr pellets) cm-1: 3445 (O-H str., phenol), 3098 (C-H str., aromatic), 1505 (C=C skeletal str., phenyl), 1354 (ring str., quinoline), 1225 (C-O str., ester); 1H NMR (DMSO-d6, δppm): 7.02 (d, 2H,Ar-H), 6.95-8.89 (m, 6H, qunoline)

3,4,5-trihydroxy-N-phenylbenzamide (2): IR (KBr pellets) cm-1: 3345 (O-H str., phenol), 3228 (N-H str., 2˚ amide), 3097 (C-H str., aromatic), 1583 (N-H bending, 2˚ amide), 1583 (C=C skeletal str., phenyl); 1H NMR (DMSO-d6, δppm): 6.92-7.02 (m, 7H,Ar-H), 8.83 (s, 1H, NH).

N-(2-chlorophenyl)-3,4,5-trihydroxybenzamide (3): IR (KBr pellets) cm-1: 3496 (O-H str., phenol), 3281 (N-H str., 2˚ amide), 1665 (C=O str., 2˚ amide), 1611 (N-H bending, 2˚ amide), 1539 (C=C skeletal str., phenyl), 733 (C-Cl str., aromatic); 1H NMR (DMSO-d6, δppm): 6.84-8.61 (m, 6H,Ar-H), 8.61 (s, 1H, NH)

3,4,5-trihydroxy-N-(2-nitrophenyl)benzamide (4): IR (KBr pellets) cm-1: 3493 (O-H str., phenol), 3350 (N-H str., 2˚ amide), 1616 (C=O str., 2˚ amide), 1616 (N-H bending, 2˚ amide), 1569 (C=C skeletal str., phenyl), 1385 (NO 2 sym. str., Ar-NO2); 1H NMR (DMSO-d6, δppm): 6.87-7.96 (m, 6H,Ar-H), 7.96 (s, 1H, NH).

3,4,5-trihydroxy-N-(3-nitrophenyl)benzamide (5): IR (KBr pellets) cm-1: 3495 (O-H str., phenol), 3431 (N-H str., 2˚ amide), 3073 (C-H str., aromatic ring), 1520 (C=C skeletal str., phenyl), 1618 (C=O str., 2˚ amide), 1618 (N-H bending, 2˚ amide), 1384 (NO2 sym. str., Ar-NO2); 1H NMR (DMSO-d6, δppm): 6.87-8.79 (m, 5H,Ar-H), 8.79 (s, 1H, NH).

N-(2,4-dinitrophenyl)-3,4,5-trihydroxybenzamide (6): IR (KBr pellets) cm-1: 3495 (O-H str., phenol), 3337 (N-H str., 2˚ amide), 3107 (C-H str., aromatic ring), 1584 (C=C skeletal str., phenyl), 1628 (C=O str., 2˚ amide and N-H bending, 2˚ amide), 1332 (NO2 sym. str., Ar-NO2); 1H NMR (DMSO-d6, δppm): 6.86-9.16 (m, 5H,Ar-H), 8.13 (s, 1H, NH).

ContdN-(4-chlorophenyl)-3,4,5-trihydroxybenzamide (7): IR (KBr pellets) cm-1: 3459 (O-H str., phenol), 3459 (N-H str., 2˚ amide), 3081 (C-H str., aromatic ring), 1624 (N-H bending, 2˚ amide), 1547 (C=C skeletal str., phenyl), 1653 (C=O str., 2˚ amide), 733 (C-Cl str., aromatic); 1H NMR (DMSO-d6, δppm): 6.78-6.99 (m, 6H,Ar-H), 8.56 (s, 1H, NH).

N-(2-fluorophenyl)-3,4,5-trihydroxybenzamide (8): IR (KBr pellets) cm-1: 3282 (O-H str., phenol and N-H str., 2˚ amide), 3064 (C-H str., aromatic ring), 1700 (C=O str., 2˚ amide), 1618 (N-H bending, 2˚ amide), 1539 (C=C skeletal str., phenyl), 1099 (C-F str., aromatic); 1H NMR (DMSO-d6, δppm): 6.81-7.63 (m, 6H,Ar-H), 7.95 (s, 1H, NH).

3,4,5-trihydroxy-N-(2-methoxyphenyl)benzamide (9): IR (KBr pellets) cm-1: 3495 (O-H str., phenol), 3277 (N-H str., 2˚ amide), 1608 (C= C skeletal str., phenyl and N-H bending, 2˚ amide), 1315 (C-O-C str., Ar-OCH3);

3,4,5-trihydroxy-N-(3-methoxyphenyl)benzamide (10): IR (KBr pellets) cm-1: 3463 (O-H str., phenol), 3345 (N-H str., 2˚ amide), 1597 (N-H bending, 2˚ amide), 1495 (C=C skeletal str., phenyl), 1263 (C-O-C str., Ar-OCH3); 1H NMR (DMSO-d6, δppm): 6.86-7.20 (m, 6H,Ar-H), 8.85 (s, 1H, NH), 3.75 (t, 3H, OCH3).

3,4,5-trihydroxy-N-(4-methoxyphenyl)benzamide (11): IR (KBr pellets) cm-1: 3382 (O-H str., phenol), 3382 (N-H str., 2˚ amide), 3006 (C-H str., aromatic ring), 1593 (C=C skeletal str., phenyl and N-H bending, 2˚ amide), 1300 (C-O-C str., Ar-OCH3); 1H NMR (DMSO-d6, δppm): 6.77-7.38 (m, 6H,Ar-H), 7.38 (s, 1H, NH), 3.74 (t, 3H, OCH3).

N-(2,4-dimethylphenyl)-3,4,5-trihydroxybenzamide (12): IR (KBr pellets) cm-1: 3353 (O-H str., phenol), 3227 (N-H str., 2˚ amide), 2831 (Ar-CH3), 1629 (C=O str., 2˚ amide and N-H bending, 2˚ amide), 1588 (C=C skeletal str., phenyl), 1H NMR (DMSO-d6, δppm): 6.78-7.02 (m, 5H,Ar-H), 7.96 (s, 1H, NH), 2.50 (m, 6H, CH3).

N-(4-fluorophenyl)-3,4,5-trihydroxybenzamide (13): IR (KBr pellets) cm-1: 3202 (O-H str., phenol and N-H str., 2˚ amide), 3111 (C-H str., aromatic ring), 1592 (C=C skeletal str., phenyl), 1629 (C=O str., 2˚ amide and N-H bending, 2˚ amide), 1095 (C-F str., aromatic); 1H NMR (DMSO-d6, δppm): 6.81-7.81(m, 6H,Ar-H), 7.85 (s, 1H, NH).

N-(2,6-dimethylphenyl)-3,4,5-trihydroxybenzamide (14): IR (KBr pellets) cm-1: 3495 (O-H str., phenol), 3280 (N-H str., 2˚ amide), 3064 (C-H str., aromatic ring), 1541 (C=C str., skeletal str., phenyl), 1609 (C=O str., 2˚ amide and N-H bending, 2˚ amide); 1H NMR (DMSO-d6, δppm): 6.77-6.97 (m, 5H,Ar-H), 8.60 (s, 1H, NH), 2.36 (m, 6H, CH3).

Tube dilution method (Cappucino, J.G. and Sherman, N. In Microbiology – A laboratory Manual, 4th Ed.; Addison Wesley Longman, Inc., California; 1999: pp.263)

Nutrient media Double strength nutrient broth- I.P (Antibacterial) Sabouraud Dextrose Broth – I.P

(Antifungal) (Pharmacopoeia of India, vol. I, Controller of Publications, Ministry of Health Department, Govt. of

India, New Delhi, 2007, p. 37.)

Bacterial species S. aureus, B. subtilis, E. coli

(37 ± 10C for 24h)

Fungal Species C. albicans (37 ± 10C for 48 h),

A. niger (25 ± 10C for 7 d)

Stock solution (100 µg /mL) -

Test and reference

(Norfloxacin &

Fluconazole) compounds

ANTIMICROBIAL EVALUATION

Antimicrobial activity (MIC in µM/ml)aNorfloxacin, bFluconazole, EC= E. coli, BS= B. subtilis, SA= S. aureus, CA= C. albicans,

AN= A. nigeraNorfloxacin, bFluconazole, EC= E. coli, BS= B. subtilis, SA= S. aureus, CA= C. albicans,

AN= A. nigeraNorfloxacin, bFluconazole, EC= E. coli, BS= B. subtilis, SA= S. aureus, CA= C. albicans,

AN= A. nigeraNorfloxacin, bFluconazole, EC= E. coli, BS= B. subtilis, SA= S. aureus, CA= C. albicans,

AN= A. niger

Compd. MIC(EC) MIC(BS) MIC(SA) MIC(CA) MIC(AN)

1 0.42 0.42 0.42 0.10 0.102 1.02 1.02 1.02 0.51 1.023 0.45 0.89 0.89 0.89 0.894 0.86 0.86 0.86 0.43 0.865 0.86 0.86 0.86 0.86 0.866 0.37 0.75 0.75 0.37 0.75

7 0.89 0.89 0.89 0.89 0.89

8 0.95 0.95 0.47 0.95 0.959 0.91 0.91 0.91 0.45 0.91

10 0.91 0.91 0.91 0.91 0.9111 0.91 0.91 0.91 0.91 0.9112 0.91 0.91 0.91 0.91 0.9113 0.47 0.95 0.95 0.95 0.9514 0.91 0.91 0.91 0.91 0.91Std 0.47a 0.47a 0.47a 0.50b 0.50b

aNorfloxacin, bFluconazole, EC= E. coli, BS= B. subtilis, SA= S. aureus, CA= C. albicans, AN= A. niger

Antioxidant Evaluation

Assay method used : DPPH

Reference drugs: Ascorbic acid

Antioxidant activityCompd. IC50 (μg/ml) Compd. IC50 (μg/ml)

1. 49.38 9. 82.24

2. 54.01 10. 57.78

3. 49.48 11. 51.66

4. 51.08 12. 62.08

5. 50.33 13. 57.67

6. 49.75 14. 73.80

7. 57.19Ascorbic acid* 49.11

8. 52.64

Alpha amylase inhibition assay

Assay method used :

Colorimetric method

Reference drugs:

Acarbose

Alpha amylase inhibitory activityCompound IC50 (μM/ml) Compound IC50 (μM/ml)

1. 2.00 9. 3.51

2. 4.40 10. 2.92

3. 4.51 11. 2.98

4. 2.58 12. 3.72

5. 2.34 13. 2.59

6. 2.75 14. 3.53

7. 3.92Acarbose* 3.09

8. 2.91

Urease inhibition assay

Assay method used :

Indophenol method

Reference drugs:

Thiourea*

Urease inhibitory activity

Compound IC50 (μM/ml) Compound IC50 (μM/ml)

1.0.08

9.0.16

2.0.25

10.0.23

3.0.26

11.0.12

4.0.23

12.0.17

5.0.18

13.0.07

6.0.16

14.0.17

7.0.20

Thiourea* 0.618.

0.16

O

O

HO

HO

HO

R1

HO

HO

HO

COOH

Heteroaryl group (8-hydroxy quinoline) increasesthe overall antimicrobial, antioxidant, alpha amylase inhibitory activity, and also possess good ureaseinhibitory activity.

O

NH

HO

HO

HO R

Substitution with nitro group (electron withdrawing group) increases alpha amylase inhibitory activity.

Substitution with methoxy group (electrondonating group) increases antifungal activity against C. albicans

Substitution with p-Fluro group increases urease and alpha amylase inhibitory activity

Structure activity relationship

ConclusionMost active antimicrobial

compounds:

1 (MICCA = MICAN= 0.10 µM/ml)

Most active antimicrobial compounds:

1 (IC50 =49.38 μg/ml)

Most active alpha amylase inhibitory compounds:

1 (IC50 =2.00µM/ml) Most active urease inhibitory compounds:

13 (IC50 =0.07 µM/ml)

N

O

O

OH

OH

OH

HO

HO

HO

O

HN F

Selected ReferencesAiyegoro, O. A., and Okoh, A. I. 2010. Preliminary phytochemical screening and in vitro antioxidant activities of the aqueous extract of Helichrysum longifolium DC. BMC Complem. Alterrn. M, 10(1):21.Aremu, A. O., Moyo, M., Amoo, S. O., Gruz, J., Šubrtová, M., Plíhalová, L., and Van Staden, J. 2014. Effect of a novel aromatic cytokinin derivative on phytochemical levels and antioxidant potential in greenhouse grown Merwilla plumbea. Plant Cell, Tissue Organ Cult. (PCTOC), 119(3):501-509.Arunkumar, S., Ilango, K., Manikandan, R. S., and Manikandan, N. 2009. Synthesis and anti-inflammatory activity of some novel pyrazole derivatives of gallic acid. E-J. Chem., 6:S123-S128.Božič, M., Gorgieva, S., and Kokol, V. 2012. Laccase-mediated functionalization of chitosan by caffeic and gallic acids for modulating antioxidant and antimicrobial properties. Carbohydr. Polym., 87(4):2388-2398.Cho, Y. S., Kim, S. K., Ahn, C. B., and Je, J. Y. 2011. Preparation, characterization, and antioxidant properties of gallic acid-grafted-chitosans. Carbohydr. Polym., 83(4):1617-1622.Díaz-Gómez, R., López-Solís, R., Obreque-Slier, E., and Toledo-Araya, H. 2013. Comparative antibacterial effect of gallic acid and catechin against Helicobacter pylori. LWT Food Sci. Technol., 54(2):331-335.Khatkar, A., Nanda, A., Kumar, P., and Narasimhan, B. 2013. Synthesis, antimicrobial evaluation and QSAR studies of gallic acid derivatives. Arabian J. Chem.Kumar, A., Narasimhan, B., and Kumar, D. 2007. Synthesis, antimicrobial, and QSAR studies of substituted benzamides. Bioorgan. Med. Chem., 15:4113-4124.Narasimhan, B., Judge, V., Narang, R., Ohlan, S., and Ohlan, R. 2007. Quantitative structure-activity relationship studies for prediction of antimicrobial activity of synthesized 2,4-hexadienoic acid derivatives. Bioorg. Med. Chem. Lett.,17:5836-5845.Yu, S. H., Mi, F. L., Pang, J. C., Jiang, S. C., Kuo, T. H., Wu, S. J., and Shyu, S. S. 2011. Preparation and characterization of radical and pH-responsive chitosan-gallic acid conjugate drug carriers. Carbohydr. Polym., 84(2):794-802.

Acknowledgement

Prof. B. Narasimhan, Dean & HOD, Department of Pharmaceutical Sciences, MDU,Rohtak.

Ms. Priyanka Dhiman, UGC-JRF, Department of Pharmaceutical Sciences, MDU, Rohtak.

Ms. Neelam Malik, UGC-JRF, Department of Pharmaceutical Sciences, MDU, Rohtak.

Mr. Sumit Sigroha, DST-JRF, Department of Pharmaceutical Sciences, MDU, Rohtak.