Synthesis and antibacterial activity of 4-Piperidone curcumin analogues against Gram-positive and Gram-negative bacteria

 

Prashinta Nita Damayanti¹, Ritmaleni²*, Erna Prawita Setyowati³

¹Student of Postgraduate Program, Master Program of Pharmaceutical Sciences and Technology,

Faculty of Pharmacy, Gadjah Mada University, Sekip Utara, Yogyakarta 55281, Indonesia

²Departement Of Pharmaceutical Chemistry, Faculty of Pharmacy,

Gadjah Mada University, Sekip Utara, Yogyakarta 55281, Indonesia

³Departement Of Pharmaceutical Biology, Faculty of Pharmacy,

Gadjah Mada University, Sekip Utara, Yogyakarta 55281, Indonesia

 

ABSTRACT:

The rapid development of antibiotics resistance makes antibiotics become less effective for the treatment of bacterial infections. Accordingly, it is so important to discovery and development of new antibacterial agents. Three 4-piperidone curcumin analogues (3,5-bis-(2-chlorobenzylidene)-4-piperidone (1), 3,5-bis-(4-chlorobenzylidene)-4-piperidone (2) and 3,5-bis-(2,4-dichlorobenzylidene)-4-piperidone (3) were synthesized from 4-piperidone monohydrate hydrochloride with 2-chlorobenzaldehyde, 4-chlorobenzaldehyde and 2,4-dichlorobenzaldehyde. The Claisen-Schmidt condensation reaction was used in acid condition. All the compounds showed light yellow crystal with percentage of yield 39, 41 and 34% respectively. All the structure compounds were confirmed by using IR, ¹H-NMR, ¹³C-NMR, and MS. The evaluation of antibacterial activity conducted by Agar diffusion method against Gram-positive bacteria such as Bacillus subtilis (ATCC 6633) and Enterococcus faecalis (ATCC 29212), and Gram-negative bacteria such as Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853). The result showed that all the compounds showed significant activity but they were more effective against Gram-positive bacteria as compared to their Gram-negative counterparts. Analogues curcumin compound showed that there was antibacterial activity up to concentration 62,5 μg/mL on Gram positive bacteria and 125 μg/mL on Gram negative bacteria. The compound 3,5-bis-(2,4-dichlorobenzylidene)-4-piperidone (3) containing two substituent -Cl on ortho and para position in the benzene ring has best activity than compound 1 and 2.

 

 

 

INTRODUCTION:

Antibiotics resistance is a global challenge for human health. Recent studies showed that most of bacteria are resistant to various antibiotics¹. The abuse of antibiotics in medical treatment and animal husbandry makes bacteria resistant to antibiotics. The rapid development of antibiotics resistance makes antibiotics become less effective for the treatment of bacterial infections².

 

Every year, there are million of people all over the world die because bacterial infections. Accordingly, it is so important to discovery and development of new antibacterial agents.

 

Natural products and/or their semisynthetic derivatives are most valuable source of novel bioactive molecules in antibacterial drug discovery³. Previous studied, reported curcumin showed antibacterial activity against several microorganisms such as Escherichia coli, Bacillus subtilis, Helicobacter pylori, dan Mycobacterium tuberculosis (M. tuberculosis)^4,5,6. However, pure curcumin was highly unstable to light and chemical degradation and have poor solubility in water^7,8.

 

Sardjiman, 1997⁹ have modificated structure diketone of curcumin to be monoketone of curcumin analogue through Claisen-Schmidt condensation reaction. HGV-6 is a curcumin analogue have substituent -Cl and -OH on the benzene ring. At antibacterial activity test, HGV-6 requires high concentration to inhibit Gram positive bacteria at concentration ≥1000 μg/mL and did not inhibit Gram negative bacteria.

 

Heterocyclic compounds are interesting synthetic targets because many important biochemical compounds and drugs contain heterocyclic ring structures¹⁰. Among the family of heterocyclic compounds, nitrogen containing heterocycles especially 4-piperidone is such important molecules which often exhibiting wide range of biological activities such as antimicrobial, antitumor, antimalarial and antiviral^11,12. In this study, we synthesized some new 4-piperidone curcumin analogue which have substituent –Cl as substituent of HGV-6 and evaluated the structure-activity relationship and evaluated as antibacterial agents against Gram-positive and Gram-negative bacteria.

 

Figure 1. Chemical structure of 3,5-bis-(2’-chlorobenzyliden)-4-piperidone (1), 3,5-bis-(4’-chlorobenzyliden)-4-piperidone (2) and 3,5-bis-(2’,4’-dichlorobenzyliden)-4-piperidone (3)

 

MATERIAL AND METHODS:

Chemicals:

The following chemicals and materials were used : 2-chlorobenzaldehyde (Sigma Aldrich), 4-chlorobenzaldehyde (Sigma Aldrich), 2,4-dichlorobenzaldehyde (Sigma Aldrich), 4-piperidone monohydrate hydrochloride (Matrix Scientific), glacial acetic acid (Merck), chloric acid (Merck), methanol (Merck), ethyl acetate (Merck), hexane (Merck), chloroform (Merck), aquadest. All other ingredients used were of analytical grade.

 

Culture media:

Mueller Hilton Agar (MHA), Brain Heart Infusion (BHI) broth, the test bacteria used in this study were a clinical isolate of Bacillus subtilis (ATCC 6633), Enterococcus faecalis (ATCC 29212), Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853) supplied by the Laboratory Microbiology Faculty of Pharmacy, Gadjah Mada University.

 

General procedure for synthesis:

Synthesis of 3,5-bis-(2’-chlorobenzyliden)-4-piperidone (1), 3,5-bis-(4’-chlorobenzyliden)-4-piperidone (2) and 3,5-bis-(2’,4’-dichlorobenzyliden)-4-piperidone (3) was carried out using Claisen-Schmidt condensation reaction. 1 mmol of 4-piperidone monohydrate hydrochloride and 2 mmol 2-chlorobenzaldehyde (1), 4-chlorobenzaldehyde (2), and 2,4-dichlorobenzaldehyde (3) was stirred in glacial acetic acid at temperature 65^oC until clear solution was obtained. Then, add HCl (37%) to the mixture and left at temperature 65^oC for 96 h. The compound were filtered on a Buchner funnel, washed with aquadest, crystallized from methanol and dried at 45°C for 24 h. All compounds were characterized using spectroscopy IR, ¹H-NMR, ¹³C-NMR and MS.

Spectral Studies:

The reaction were followed and checked by TLC (silica gel F254) for completion using EtOAc:Hex (1:1) and EtOAc: Hex (1:2). The spots were examined under UV lamp; whereas the identity and purity of the products were checked using EtOAc: CHCl₃ (1:20) and the Rf-value has been reported. The IR spectra of the products were recorded on a 300 MHz Shimadzu FTIR spectrometer using KBr pellets. ¹H-NMR and ¹³C-NMR spectra were recorded in DMSO on 500 Mhz Shimadzu FT-NMR relative to TMS as internal standard and mass spectra were recorded on a QP-2010S mass spectrometer at 70eV.

 

3,5-bis-(2’-chlorobenzyliden)-4-piperidone (1):

Yield 39 %; yellow solid; mp 211-213 ^o C (MeOH); Rf= 0,5 (EtOAc:CHCl₃ 1:20); IR (KBr) 3448,72 (NH); 3062,96 (CH=CH); 2893,22 (CH-CH) ; 1681,93 (C=O); 1627,92 (CH=CH); ¹H-NMR (500 MHz, DMSO) δ 4,351 (4H, s, -CH₂); 7,476 (6H, m, Ar-H); 7,630 ( 2H, dd, J=9; 2,5 Hz, Ar-H) ; 7,982 (2H, s, C=CH); 10,057 (1H, br, N-H); ¹³C-NMR (DMSO): δ 43,657; 127,559; 129,814; 129,997 ; 130,813; 131,619; 131,734; 134,105; 135,785; 182,319; EI-MS (m/z) 343.05 C₁₉H₁₅Cl₂NO

 

3,5-bis-(4’-chlorobenzyliden)-4-piperidone (2):

Yield 41 %; yellow crystal; mp 255-257 ^o C (MeOH); Rf= 0,5 (EtOAc:CHCl₃ 1:20); IR (KBr) 3448,72 (NH); 3062,96 (CH=CH); 2893,22 (CH-CH) ; 1674,21 (C=O); 1627,92 (CH=CH); ¹H-NMR (500 MHz, DMSO) δ 4,456 (4H, s, -CH₂); 7,558 (4H, d, J=8,5 Hz, Ar-H); 7,594 ( 4H, dd, J=9; 2 Hz, Ar-H) ; 7,855 (2H, s, C=CH); 9,936 (1H, br, N-H); ¹³C-NMR (DMSO): δ 43,753; 128,471; 129,018; 132,329 ; 132,579; 134,825; 137,916; 182,271; EI-MS (m/z) 343.05 C₁₉H₁₅Cl₂NO

 

3,5-bis-(4’-chlorobenzyliden)-4-piperidone (3):

Yield 34 %; yellow crystal; mp 180-181 ^o C (MeOH); Rf= 0,6 (EtOAc:CHCl₃ 1:20); IR (KBr) 3443,29 (NH); 3070,68 (CH=CH); 2931,80 (CH-CH) ; 1674,21 (C=O); 1627,92 (CH=CH); ¹H-NMR (500 MHz, DMSO) δ 3,856 (4H, s, -CH₂); 7,446 (2H, d, J=8,5 Hz, Ar-H); 7,510 ( 2H, dd, J=11;2,5 Hz, Ar-H) ; 7,660 (2H, s, C=CH); ¹³C-NMR (DMSO): δ 47,122; 127,463; 129,219; 129,325 ; 131,705; 132,272; 134,355; 134,950; 137,820; 187,195 EI-MS (m/z) 411 C₁₉H₁₃Cl₄NO

 

Anti-bacterial activity:

All the three compounds (1-3) were screened for their antibacterial activity against Gram positive bacteria: Bacillus subtilis (ATCC 6633), Enterococcus faecalis (ATCC 29212), Gram negative bacteria: Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853) bacterial strains. Bacteria were grown overnight at 37^oC in Brain Heart Infusion medium. After incubation, the optical density (absorbance) at 600nm (OD600) was recorded using Spectrometer UV-Vis and the density of bacteria is 1-2 x 10⁸ cfu/mL ¹³. The bacteria were streaked on the surface of the agar (Mueller Hinton Agar) using sterile swab stick, using different plates and different swab sticks for different organisms. Then, six series concentration (1000 μg/mL-31,25 μg/mL) of compounds that solved in dimethyl sulfoxide (DMSO) were added in paper disk (5 mm) on agar. DMSO were used as negative control and Amoxicillin were used as positive control. Petri dishes were incubated for 24 h at 37°C. The results of antibacterial activity were evaluated as diameter of the zones of inhibition in milimetres¹⁴.

 

RESULT AND DISCUSSION:

Synthesis of 4-piperidone curcumin analogues:

The aim of this research is to synthesize the curcumin analog compound 3,5-bis-(2'-chlorobenzyliden)-4-piperidone (1), 3,5-bis- (4'-chlorobenzyliden) -4-piperidone (2), and 3,5-bis- (2',4'-dichlorobenzyliden)-4-piperidone (3). Each curcumin analog compound is substituted with chlorine group in various positions. Halogen groups such as Cl have good antibacterial activity¹⁵. The synthesis of 1, 2, and 3 compounds was prepared based on target molecules by methods developed by Stuart Warren (2008)¹⁶ (fig 2).

 

Figure 2. α, β disconnect analysis

 

Synthesis was carried out by Claisen-Schmidt condensation method using 37% concentrated HCI catalyst and glacial acetic acid solvent (fig 3). With the aid of an acid catalyst, the ketone group forms an enol compound as nucleophile, the anion is reversibly convertible to the carbonyl group of the aldehyde molecule. This aldehyde molecule previously undergoes a protonation of its carbonyl group, producing a β-hydroxy carbonyl compound. Nucleophile attack on carbon atoms from carbon oxygen double bonds (carbonyl group) of the aldehyde molecule, it occurs because carbon has positive charge. The reaction takes place, followed by dehydration process and produces a α, β unsaturated carbonyl¹⁷.

 

Figure 3: The synthesis of 3,5-bis-(2’-chlorobenzyliden)-4-piperidone (1), 3,5-bis-(4’-chlorobenzyliden)-4-piperidone (2) and 3,5-bis-(2’,4’-dichlorobenzyliden)-4-piperidone (3)

 

The formed product from the synthesis of analogue curcumin is a single compound with yield of 39%, 41%, and 34%, respectively. IR analysis shows a typical functional group, in the frequency 3300-3500 cm^-1 is a functional group of amines and in the frequency 1600-1700 cm^-1 appears the peak of the C = O carbonyl group. The ketone carbonyl peak generally appears at a wavelength of 1715 cm^-1, but the resonant effect (C = O and α, β- unsaturated) causes the vibrational frequency to decrease and shift toward lower wave numbers¹⁸. The C=C of α, β- unsaturated peak appears at wavelength of 1627,92 cm^-1. The ¹H-NMR spectrum of these compound were simple in appearance. The proton of α, β- unsaturated appeared at 7,60-7,90 ppm. This is due to the anisotropic effect of the carbonyl group of the curcumin analog resulting in the downfield of α, β- unsaturated protons¹⁹. The -NH peak of compound 3 didn’t appear of the ¹H-NMR spectra because proton of -NH peak may exchange with deuterium solvent.

 

Antibacterial activity:

The antibacterial study was carried out for all compounds as the procedure mentioned, each test was performed in triplicate and the recorded zone of inhibitions are given in table 1, 2 and 3.

Table 1: Antibacterial activity of compound 1

Concentration (µg/mL)	Bacillus subtilis (ATCC 6633)	Enterococcus faecalis (ATCC 29212)	Escherichia coli (ATCC 25922)	Pseudomonas aeruginosa (ATCC 27853)
1000	10, 9, 8	13, 14, 11	11, 10, 10	10, 10, 10
500	8, 8, 8	10, 9, 10	9, 8, 8	10, 9, 9
250	8, 9, 8	8, 10, 9	-	9, 8, 7
125	7, 8, 8	8, -, -	-	-
62,5	-	-	-	-
31,25	-	-	-	-
Amoxicillin	6, 9, 8 (31,25 µg/mL)	7, 8, 7 (31,25 µg/mL)	25, 25, 25 (62,5 µg/mL)	22, 21, 22 (62,5 µg/mL)

 

Table 2: Antibacterial activity of compound 2

Concentration (µg/mL)	Bacillus subtilis (ATCC 6633)	Enterococcus faecalis (ATCC 29212)	Escherichia coli (ATCC 25922)	Pseudomonas aeruginosa (ATCC 27853)
1000	6, 6, 6	10, 9, 7	11, 10, 10	7, 6, 6
500	6, 6, 6	8, 9, 7	9, 8, 8	6, 6, 6
250	-	8, 6, 6	-	-
125	-	8, 6, 6	-	-
62,5	-	-	-	-
31,25	-	-	-	-
Amoxicillin	8, 8, 8 (31,25 µg/mL)	11, 8, 7 (31,25 µg/mL)	25, 25, 25 (62,5 µg/mL)	19, 18, 18 (62,5 µg/mL)

 

Table 3: Antibacterial activity of compound 3

Concentration (µg/mL)	Bacillus subtilis (ATCC 6633)	Enterococcus faecalis (ATCC 29212)	Escherichia coli (ATCC 25922)	Pseudomonas aeruginosa (ATCC 27853)
1000	11, 10, 9	13, 12, 12	9, 9, 8	10, 9, 8
500	9, 9, 9	9, 9, 9	9, 8, 8	9, 8, 8
250	9, 8, 8	7, 8, 7	8, 8, 7	8, 7, 7
125	8, 7, 7	7, -, -	7, 6, 6	6, 6, -
62,5	6, 7, 6	-	-	-
31,25	-	-	-	-
Amoxicillin	19, 21, 20 (62,5 µg/mL)	7, 7, 7 (31,25 µg/mL)	23, 22, 23 (62,5 µg/mL)	21, 20, 25 (62,5 µg/mL)

 

Inhibitory diameter was seen through visual observation after the petri dish was incubated for 18-24 hours. If there is a clear zone around the paper disk then there is antimicrobial activity or microbial growth inhibition occurs. Interestingly, all of them were found positive for their activity against Gram-positive bacteria such as Bacillus subtilis (ATCC 6633) and Enterococcus faecalis (ATCC 29212), and Gram-negative bacteria such as Escherichia coli (ATCC 25922), and Pseudomonas aeruginosa (ATCC 27853). The activity of antibacterial agents is depend on concentration of the curcumin analogues. The higher concentration of curcumin analog compound showed the higher antibacterial activity²⁰. All the 4-piperidone curcumin analog used in this study indicated broad antibacterial spectrum because of its show antibacterial activity against Gram-positive and negative bacteria. Structure-activity analysis of the results demonstrated that compound with electron-withdrawing substituent -Cl active as antibacterial agent^21,22,23, but the position of substituent -Cl in the benzene ring is important. Generally, ortho-substituted derivates compounds showed better antibacterial activity than others²⁴. The compound 1 and 2 imply that ortho-substitution with electron withdrawing groups more active as antibacterial agents than para-substitution. The compound 3 containing 2 substituent -Cl on ortho and para position in the benzene ring has best activity. Compound 3 can inhibit Gram positive bacteria up to concentration of 62.5 µg / mL and Gram negative up to concentration of 125 μg / mL. The compounds were found to be more effective against Gram positive bacteria than against Gram negative bacteria. These compounds probably act on the synthesis peptidoglycan from the cell wall, which explains by the antibacterial effects of some compounds that higher on Gram-positive bacteria instead of Gram-negative ones. The cell wall of Gram-positive bacteria is more simple than Gram negative bacteria. The major part of the cell walls of Gram-positive bacteria is peptidoglycan, so lyophilic molecules are able to penetrate the cell²⁵. On the contrary, the cell wall of Gram-negative bacteria is more complex which limits access of the antimicrobial agents to their targets in the bacterial cells²⁶. It has peptidoglycan layer thinner but the peptidoglycan linked to lipopolysaccharide layer. This is the reason that the cell wall of Gram-negative bacteria stronger than Gram-positive bacteria and relatively resistant to lyophilic compound²⁷.

 

CONCLUSION:

Three compounds of 4-piperidone curcumin analogues have been synthesized by using Claisen-Schmidt condensation reaction with acid catalyst and evaluated for antibacterial activity against Gram-positive and Gram-negative bacteria. The result showed that all the compounds showed significant activity but they were more effective against Gram-positive bacteria as compared to their Gram-negative counterparts.

 

ACKNOWLEDGEMENT:

The authors would like to thank to Faculty of Pharmacy, Gadjah Mada University, Yogyakarta.

 

CONFLICT OF INTEREST:

The authors have declared “no conflict of interest with respect to the research, authorship, and/or publication of this article”.

 

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Received on 14.11.2019          Modified on 31.12.2019

Accepted on 15.02.2020         © RJPT All right reserved