Open Access

Synthesis and antibacterial activity against ralstonia solanacearum for novel hydrazone derivatives containing a pyridine moiety

Contributed equally
Chemistry Central Journal20126:28

DOI: 10.1186/1752-153X-6-28

Received: 21 January 2012

Accepted: 7 April 2012

Published: 7 April 2012

Abstract

Background

Ralstonia solanacearum, one of the most important bacterial diseases on plants, is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range. In order to discover new bioactive molecules and pesticides acting on tobacco bacterial wilt, we sought to combine the active structure of hydrazone and pyridine together to design and synthesize a series of novel hydrazone derivatives containing a pyridine moiety.

Results

A series of hydrazone derivatives containing a pyridine moiety were synthesized. Their structures were characterized by 1 H-NMR, 13 C-NMR, IR, and elemental analysis. The preliminary biological activity tests showed that compound 3e and 3g exhibited more than 80% activity against Ralstonia solanacearum at 500 mg/L, especially compound 3g displayed relatively good activity to reach 57.0% at 200 mg/L.

Conclusion

A practical synthetic route to hydrazone derivatives containing a pyridine moiety by the reaction of intermediates 2 with different aldehydes in ethanol at room temperature using 2-chloronicotinic acid and 2-amino-5-chloro-3-methylbenzoic acid as start materials is presented. This study suggests that the hydrazone derivatives containing a substituted pyridine ring could inhibit the growth of Ralstonia solanacearum.

Background

Ralstonia solanacearum (R. solanacearum), one of the most important bacterial diseases on plants, is a devastating, soil-borne plant pathogen with a global distribution and an unusually wide host range, which belongs to the β-proteobacteria and is considered as a "species complex". It causes a wilt disease with deadly effects in many important crops, such as in potato, tomato and eggplant firstly described by Smith in 1896 and subsequently in tobacco in 1908 [13]. Once the plants infected with bacterial wilt, the main symptoms of plants were rapid death, but stems and leaves remained green. Tobacco bacterial wilt, caused by R. solanacearum, is one of the main plant diseases in tobacco production and occurred in the most common and serious form. The high incidence of plant mortality and the lack of effective control methods make R. solanacearum become one of the world's most destructive plant pathogens [4, 5].

Pyridine is an important class of heterocyclic ring, which have been attracted more and more attention in the pesticides areas due to their broad-activities, such as fungicidal activity [68], insecticidal activity [9, 10] and herbicidal activity [11, 12]. In fungicidal activity regard, some pyridine derivatives can prevent Erysiphe graminis, Botrytis cinerea [13], Pyricularia oryzae [14] and Phytophthora infestans [15]. Currently, some pyridine compounds have been developed and commercialized, for example, fluopicolide [16], boscalid [17] and picoxystrobin [18] (Figure 1). In the recent literature, hydrazones demonstrated significant antimicrobial activity [19], antitubercular activity [20] and antitumoral activity [21] in medicinal areas. On the other hand, hydrazones also exhibit good fungicidal activity against Phytophthora infestans [22], Cladosporium cucumerinum and Colletotrichum orbiculare [23] in the pesticides areas, some of them containing hydrazone structure have been commercialized, such as benquinox and ferimzone [24] (Figure 1).
Figure 1

Figure.

Controlling of plant bacterial diseases has long been a challenging mission in the agricultural sector. The application of traditional pesticides has not proved much effective and at the same time high residue level or negative impact on the environment were caused. Copper formulation, a commercial bactericide can enhance resistance in host tobacco plant. Despite being useful in the treatment of plants affected by tobacco bacterial wilt, the use of copper formulation for field trial is largely limited due to its phytotoxicity, strong alkali and low mobility. Therefore, the search for new antibacterial agent still remains a daunting task in pesticide science [25]. In order to discover new molecules and pesticide acting on tobacco bacterial wilt, we sought to combine the active structure of hydrazone and pyridine together to design and synthesize a class of novel hydrazone derivatives containing a pyridine moiety. Thus, 2-chloronicotinic acid and 2-amino-5-chloro-3-methylbenzoic acid were used as start materials, 11 novel analogues of hydrazones containing pyridine were synthesized. All the compounds were unequivocally characterized by IR, NMR and elemental analysis. The biological activities on R. solanacearum were tested, the results showed that most of the synthesized compounds exhibited antibacterial activity against R. solanacearum to a certain extent, compounds 3e and 3g showed good antibacterial activity at 200 mg/L. especially compounds 3g displayed excellent antibacterial activity (57.0%) at 200 mg/L. To the best of our knowledge, this is the first report on the antibacterial activity of hydrazone derivatives containing a pyridine moiety.

Results and discussion

Synthesis

The synthetic route to the title compounds is demonstrated in Additional file 1. 6- chloro-2-(2-chloropyridin-3-yl)-8-methyl-4H-benzo[d] [1, 3]oxazin-4-one 1 were prepared by treatment of 2-chloronicotinic acid with 2-amino-5-chloro-3-methylbenzoic acid using readily available starting materials and a simple procedure as describing in the literature [26]. The further reaction of intermediates 1 with 80% hydrazine hydrate could proceed readily at room temperature to give intermediates 2 [27]. Subsequent treatment of intermediates 2 with different aldehydes in ethanol at room temperature afforded the desired compounds (3a to 3k). The synthetic route in Scheme 1 had several advantages, which included short steps, short reaction times and excellent yields (e.g. the yields for 3b, 3d, 3e and 3j were 93.3%, 96.6%, 96.6% and 93.6%, respectively), especially the mild conditions (room temperature). Additional file shows the structures, yields and elemental analysis data for title compounds in more detail [see Additional file 2].

All the synthesized compounds (3a-3k) were characterized on the basis of their spectroscopic data. The IR absorption bands near 3260-3180 cm-1, 3100-2910 cm-1, 1666-1610 cm-1, 1600-1575 cm-1 and 1367-1352 cm-1 confirmed the presence of two N-H, Ar-H, amide [28], - C = N- [29] and -CH3 functional groups, respectively. In the 1H-NMR spectra of the title compounds, the -CONHAr proton appeared as a broad singlet at 12.17-10.53; the -CONHN proton appeared as a broad singlet at 10.34-10.17; the -C = NH proton mainly appeared as a broad singlet at 8.73-8.20; the pyridine ring of 4 and 6 positions protons were occurred double-double peaks near 8.52-8.51 and 8.01-7.94; the methyl (Ar-CH3) proton signals were observed as a singlet near 2.34-2.30. Meanwhile, we observed the titles compounds possess E and Z configuration in the 1H-NMR spectra, but the E configuration was mainly forms by the spectra analysis, such as E configuration of -CONHAr proton appeared at 12.11, but Z proton was occurred at 12.01 in 3e, and the E/Z is approximately equal to 3.60.

Biological activity and structure-activity relationship

The antibacterial activity of compounds 3a-3k against R. solanacearum was assayed by the reported method [30]. Kocide, one of the proven commercial agents for controlling R. solanacearum, was used as the reference of bactericides. The results provided in Table 1 indicate that most of the prepared compounds have weak to good antibacterial activity against R. solanacearum at 500 mg/L. Compounds 3e and 3g displayed higher activities than compounds 3a-3d, 3f to 3h-3k at 500 mg/L. For example, inhibition of 3e (R2 = 3-trifluoromethylphenyl) and 3g (R2 = N, N-dimethylamino) on tobacco bacterial wilt was 80.9% and 100.0% at 500 mg/L, whereas 3a (R2 = Et), 3b (R2 = 2-chlorophenyl) and 3f(R = 4-tert-butylphenyl) was only 5.0%, 6.0% and 39.0% at the same condition. Primary structure- activity relationships revealed that when the CONHN = CHR2 was simultaneously substituted by N, N-dimethylamino (at the R2 group) and 3-trifluoromethylphenyl (at the R2 group), the activity on R. solanacearum increased. However, the introduction of CH2CH3, 2-chlorophenyl, 4-fluorophenyl, 2,6-dichlorophenyl, 4-tert-butylphenyl, 3, 4-dichlorophenyl, 2, 3-dimethoxyphenyl, 2, 4-dimethoxyphenyl and 2, 5-dimethoxy- phenyl groups into R2 groups decreased the activity of the compound. Furthermore, the mono- substituent at the different positions on phenyl of the R2 group also affected the bioactivity of the compound. For instance, the compound with trifluromethyl group at 3-position on phenyl ring (3e) displayed good activity, while the compounds with tert-butyl at 4-position on phenyl ring (3f) exhibited moderate activity and the compound with chlorine at 2-position on phenyl ring (3b) and the compound with fluorine at 4-position on phenyl ring (3c) showed no antibacterial activities on R. solanacearum. Compared with the same substituents on phenyl, the substituents at the 2, 3-positions, the corresponding molecules always had higher inhibition rates for tobacco bacterial wilt. For example, the inhibition rates of 3i (R2 = 2, 3-dimethoxyphenyl) and 3k (R2 = 2,4-dimethoxyphenyl) was 40.3%, 36.1% and the inhibition rates of 3j (R2 = 2,5-dimethoxyphenyl) on tobacco bacterial wilt was 25.2%.
Table 1

Antibacterial activity of compounds 3a to 3 k, Kocide against R. solanacearum

 

inhibition rates (%)

Compounds

500 mg/L

200 mg/L

3a

5.0

0.0

3b

6.0

0.0

3c

5.0

1.1

3d

18.2

15.2

3e

80.9

33.3

3f

39.0

18.2

3g

100.0

57.1

3h

35.0

12.4

3i

40.3

0.0

3j

25.2

1.9

3k

36.1

0.0

Kocide 3000

100.0

100.0

Cu(OH)2

  

Experimental

Chemistry

Unless otherwise stated, all the reagents and reactants were purchased from commercial suppliers; melting points were uncorrected and determined on a XT-4 binocular microscope (Beijing Tech Instrument Co., China). The 1H-NMR and 13 C-NMR spectra were recorded on a JEOL ECX 500 NMR spectrometer (JEOL Ltd., Japan) at room temperature operating at 500 MHz for 1H-NMR and 125 MHz for 13 C-NMR by using CDCl3 or DMSO as solvents and TMS as an internal standard; infrared spectra were recorded in KBr on a IR Pristige-21 spectrometer (Shimadzu corporation, Japan); elemental analysis was performed on an Elemental Vario-III CHN analyzer (Elementar, German). The course of the reactions was monitored by TLC; analytical TLC was performed on silica gel GF 254.

Intermediate 1 and 2 were prepared according to the reported methods [26, 27] and used without further purifications [Additional file 3].

Antibacterial biological assay

Antibacterial activities of some title compounds against R. solanacearum were evaluated by the turbidimeter test [30], whereas Kocide® 3000 was the positive control. The compounds were dissolved in 30 μL DMSO and diluted with water containing TWEEN-20 (0.1 mg/L) to generate a final concentration of 500 mg/L and 200 mg/L, which were added to the toxic nutrient broth (NB) liquid medium in 5 mL tubes, respectively. To the above tubes, 40 μL NB liquid medium containing R. solanacearum pathogen was individually added, then shaken at 30°C and 180 r.p.m. for 48 h, the relative inhibition rate of the circle mycelium compared to blank assay was calculated via the following equation.

Relative inhibition rate % = A 0 - A 1 A 0 × 100 %

A0: Corrected OD values of the control medium of bacilli;

A1: Corrected OD values of the medium of toxic.

Conclusion

In conclusion, a series of the novel hydrazone derivatives containing a substituted pyridine ring were designed and synthesized. The reaction of intermediates 2 with different aldehydes in ethanol at room temperature used 2-chloronicotinic acid and 2-amino-5-chloro-3-methylbenzoic acid as start materials provides a ready access to a series of the novel hydrazone derivatives containing pyridine moiety. The antibacterial tests indicated that some of the synthesized compounds possessed of moderately high activity against R. solanacearum. The title compounds 3e and 3g exhibited favourable activity against tobacco bacterial wilts in vitro compared to the commercial bactericides Kocide 3000. The antibacterial tests showed that when the R2 group of CONHN = CHR2 was 3-trifluoromethylphenyl or N, N-dimethylamino, the corresponding compounds presented good antibacterial activities. The structure of the target products needs to be optimized to enhance their antibacterial activity. Future structural modification and biological evaluation should be carried out to explore the full potential of this novel class of antibacterial molecules.

Notes

Declarations

Acknowledgements

This work was supported the National Key Program for Basic Research (No. 2010CB 126105), Key Technologies R&D Program (No.2011BAE06B05-6) and National Natural Science Foundation of China (No.21162004), the Special Foundation of Governor for Outstanding talents in Guizhou (No.2011-38).

Authors’ Affiliations

(1)
State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University
(2)
Research and Development Center for Fine Chemicals, Guizhou University

References

  1. Salanoubat M, Genin S, Artiguenave F, Gouzy J, Mangenot S, Arlat M, Billaultk A, Brottier P, Camus JC, Cattolico L, Chandler M, Choisne N, Claudel-Renard C, Cunnac S, Demange N, Gaspin C, Lavie M, Moisan A, Robert C, Saurin W, Schiex T, Siguier P, Thébault P, Whalen M, Wincker P, Levy M, Weissenbach J, Boucher CA: Genome sequence of the plant pathogen Ralstonia solanacearum. Nature. 2002, 415: 497-502. 10.1038/415497a.View ArticleGoogle Scholar
  2. Álvarezi B, Biosca EG, López MM: On the life of Ralstonia solanacearum, a destructive bacterial plant pathogen. Current research, technology and education topics in applied microbiology and microbial biotechnology. Edited by: Mendez-Vilas A. 2010, Spain: Formatex, Bada-joz, 1: 267-279.Google Scholar
  3. Li ZF, Wu SL, Bai XF, Liu Y, Lu JF, Liu Y, Xiao BG, Lu XP, Fan LJ: Genome Sequence of the Tobacco Bacterial Wilt Pathogen Ralstonia solanacearum. J Bacteriol. 2011, 193: 6088-6089. 10.1128/JB.06009-11.View ArticleGoogle Scholar
  4. Turner M, Jauneau A, Genin S, Tavella MJ, Vailleau F, Gentzbittel L, Jardinaud MF: Dissection of Bacterial Wilt on Medicago truncatula Revealed Two Type III Secretion System Effectors Acting on Root Infection Process and Disease Development. Plant Physiol. 2009, 150: 1713-1722. 10.1104/pp.109.141523.View ArticleGoogle Scholar
  5. Mukaihara T, Tamura N: Identification of novel Ralstonia solanacearum type III effector proteins through translocation analysis of hr B-regulated gene products. Microbiology. 2009, 155: 2235-2244. 10.1099/mic.0.027763-0.View ArticleGoogle Scholar
  6. Minakata S, Hamada T, Komatsu M, Tsubo H, Kikuta H, Ohshiro Y: Synthesis and Biological Activity of 1H-Pyrrolo [2, 3-b] pyridine Derivatives: Correlation between Inhibitory Activity against the Fungus Causing Rice Blast and Ionization Potential. J Agric Food Chem. 1997, 45: 2345-2348. 10.1021/jf9607730.View ArticleGoogle Scholar
  7. Bis SJ, Canada EJ, Cooper DH, Galka CS, Kirby N, Ouimette DG, Podhorez DE, Pieczko M, Rezac R, Rieder B, Swayze JK, Hegde VB, Sampson GL: 2-Methoxyimino-2-(pyridinyl oxymethyl)phenyl acetamides with carboxylic acid derivatives on the pyridine ring as fungicides. WO 9833772, 1998[Chem Abstr 1998, 129:161497]
  8. Foor SR, Walker MP: Synergistic fungicide compositions containing at least one N'- (2-pyridinyl) methyl-3-pyridinecarboxamide derivative and one or more further fungicides useful for controlling fungal plant diseases. WO 2003079787. 2003[Chem Absr 2003, 140:141086]
  9. Wakita T, Kinoshita K, Yasui N, Yamada E, Kawahara N, Kodaka K: Synthesis and Structure-activity Relationships of Dinotefuran Derivatives: Modification in the Nitroguanidine Part. J Pestic Sci. 2004, 29: 348-355. 10.1584/jpestics.29.348.View ArticleGoogle Scholar
  10. Shao XS, Xu ZP, Zhao XF, Xu XY, Tao LM, Li Z, Qian XH: Synthesis, Crystal Structure, and Insecticidal Activities of Highly Congested Hexahydroimidazo[1,2-a] pyridine Derivatives: Effect of Conformation on Activities. J Agric Food Chem. 2010, 58: 2690-2695. 10.1021/jf902513t.View ArticleGoogle Scholar
  11. Hegde SG, Mahoney MD: Synthesis and Herbicidal Activity of 5-(Haloalkyl)-Substituted Thiazole [4,5-b] pyridine-3(2H)-acetic Acid Derivatives. J Agric Food Chem. 1993, 41: 2131-2134. 10.1021/jf00035a058.View ArticleGoogle Scholar
  12. Hanagan MA, Selby TP, Sharpe PL, Sheth RB, Stevenson TM: Herbicidal Amides. WO 2005070889[Chem Abstr 2005, 143:193910]
  13. Mansfield DJ, Rieck H, Greul JN, Coqueron PY, Genix P, Grosjean-Cournoyer MC, Perez J, Villier A: Preparation of heteroarylcarboxamides as fungicides. EP 1449841, 2004 [Chem Abstr 2004, 141:207064]
  14. Watanabe T, Araki N: Pyridylmethyl Derivatives of 2,6-Dichloroisonicotinic Acid, Process for Production of the Same, and Disease Controllers for Agricultural and Horticultural Use. WO 2005068430, 2005[Chem Abstr 2005, 143:172764]
  15. Lee SF, Gliedt M: Substituted Isoxazoles as Fungicides. WO, 2006031631[P]. 2006.
  16. Mansfield DJ, Cooke T, Thomas PS, Coqueron PY, Vors JP, Briggs GG, Lachaise H, Rieck H, Desbordes P, Grosjean-Cournoyer MC: Novel 2-Pyridylethylbenzamide Derivative. WO, 2004016088[P]. 2004. [Chem Abstr 2004, 140:176744]
  17. Elcken K, Goetz N, Harreus A, Eberhard A, Gisela L, Harald R: Anilide Derivatives and Their Use to Combat Botrytis. EP 0545099, 1993[Chem Abstr 1993, 119:160132]
  18. Clough JM, Godfrey CRA, Hutchings MG, Aathony VM: Methyl 3-methoxy-2-phenylpropenoate derivatives useful as plant fungicides and plant growth regulators, their fungicidal compositions, and processes and intermediates for their preparation. EP 278595, 1988[Chem Abstr 1988, 109:270725]
  19. Küçükgüzei ŞG, Oruç EE, Rollas S, Sahin F, Ozbek A: Synthesis, Characterization and biological activity of novel 4- thiazolidinones, 1,3,4-oxadiazoles and some related compounds. Eur J Med Chem. 2002, 37: 197-206. 10.1016/S0223-5234(01)01326-5.View ArticleGoogle Scholar
  20. Rollas S, Gülerman N, Erdeniz H: Synthesis and antimicrobial activity of some new hydrazones of 4-fluorobenzoic acid hydrazide and 3-acetyl-2,5-disubstituted-1,3,4-oxadiazo- lines. Farmaco. 2002, 57: 171-174. 10.1016/S0014-827X(01)01192-2.View ArticleGoogle Scholar
  21. Savini L, Chiasserini L, Travagli V, Pellerano C, Novellino E, Cosentino S, Pisano MB: New α-heterocyclichydrazones: evaluation of anticancer, anti-HIV and antimicrobial activity. Eur J Med Chem. 2004, 39: 113-122. 10.1016/j.ejmech.2003.09.012.View ArticleGoogle Scholar
  22. Young D, Shaber S, Avila-adame C, Breaux N, Ruiz J, Siddall T, Webster J: Fungicidal compositions including hydrazone derivatives and copper useful for controlling growth of fungi and their preparation. WO 2010083319, 2010[Chem Abstr 2010, 153:204054]
  23. Yang XD: Synthesis and biological activity of hydrazone derivatives containing pyrazole. J Chem Res. 2008, 9: 489-491.View ArticleGoogle Scholar
  24. Okuno T, Furusawa I, Matsuura K, Shishiyama J: Mode of action of ferimzone, a novel systemic fungicide for rice disease: Biological properties against Pyricularia oryzae in vitro. Phytopathology. 1989, 79: 827-832. 10.1094/Phyto-79-827.View ArticleGoogle Scholar
  25. Hayward A: C: Biology and epidemiology of bacterial wilt caused by Pseudomonas solanacearum. Annu Rev Phytopathol. 1991, 29: 65-87. 10.1146/annurev.py.29.090191.000433.View ArticleGoogle Scholar
  26. Lahm GP: Preparation of novel anthranilamides useful for controlling invertebrate pests. WO 2006023783, 2006[Chem Abstr 2010, 144:274265]
  27. Wu J, Song BA, Hu DY, Yue M, Yang S: Design, synthesis and insecticidal activities of novel pyrazole amides containing hydrazone substructures. Pest Manag Sci. 2011, DOI 10.1002/ps.2329Google Scholar
  28. Wu J, Yang S, Song BA, Bhadury PS, Hu DY, Zeng S, Xie HP: Synthesis and insecticidal activities of novel neonicotinoid analogs bearing an amide moiety. J Heterocyclic Chem. 2011, 48: 901-906. 10.1002/jhet.663.View ArticleGoogle Scholar
  29. Kandile NG, Mohamed MI, Zaky H, Mohamed HM: Novel pyridazine derivatives: Synthesis and antimicrobial activity evaluation. Eur J Med Chem. 2009, 44: 1989-1996. 10.1016/j.ejmech.2008.09.047.View ArticleGoogle Scholar
  30. Lee JY, Moon SS, Hwang BK: Isolation and in vitro and in vivo activity against Phytophthora capsici and Colletotrichum orbiculare of phenazine-1-carboxylic acid from Pseudomonas aeruginosa strain GC-B26. Pest Manag Sci. 2003, 59: 872-882. 10.1002/ps.688.View ArticleGoogle Scholar

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© Wu et al 2012