SYNTHESIS AND ANTIBACTERIAL ACTIVITIES OF ASPIRIN-CHALCONE DERIVATIVES
PHANG SAI FOONG 38393
Bachelor of Science with Honours Resource Chemistry
2015
Faculty of Resource Science and Technology
Synthesis and Antibacterial Activities of Aspirin-Chalcone Derivatives
Phang Sai Foong
A dissertation is submitted in partial fulfilment of the requirement for the Degree of Bachelor Science with Honours
(Resource Chemistry)
Faculty Resource Science and Technology Universiti Malaysia Sarawak
2015
ACKNOWLEDGEMENT
First of all, I would like to appreciate my supervisor, Assoc. Prof. Dr. Zainab Ngaini for guiding me along the way towards the way of completion of my research and thesis. Her critical point of view, useful advice and effective guidance had contributed greatly in the progress of my project.
Besides, I would like to address my appreciation to the lab assistants, technical staffs as well as the senior Ph. D and master students for their patient guidance since the beginning of my research. They have taught me the appropriate way to learn to use all the analytical instrument needed and gave me suitable comments in constant discussion.
In addition, I feel grateful to receive all the support from my family members and friends. They have provided substantial moral support for me whenever I encounter the difficulties in my work. Without them, I would never have such a great courage to complete my project today.
DECLARATION
I, Phang Sai Foong (38393), students from Resource Chemistry programme, Faculty of Resource Science and Technology hereby declare that the work entitled Synthesis and Antibacterial Activities of Aspirin-Chalcone Derivatives is my original work. I have not copied from any other students’ work or from any other sources except where due reference or acknowledgement is made explicitly in the text, nor has any part been written for me by another person.
_________________________
(PHANG SAI FOONG) Date :
TABLE OF CONTENT
Acknowledgement………... I
Declaration………... II
Table of Contents………. III-VI
List of Abbreviations……… VII
List of Tables, Figures and Schemes……… VIII-XI
Abstract……… 1
1.0 Introduction………..
1.1 Aspirin………..
1.2 Chalcone………...
1.3 Problem Statement………
1.4 Objectives……….
2 2 2 3 3 2.0 Literature Review………..
2.1 Aspirin………...
2.1.1 Recent study of aspirin derivatives………..
2.2 Chalcone………..
2.2.1 Antioxidant activity of chalcone
derivatives ………..
2.2.2 Anti-inflammatory activity of chalcone
derivatives ………..
2.2.3 Anticancer activity of chalcone
derivatives ………..
2.2.4 Anti-infective activity of chalcone derivatives…….
2.2.4.1 Antibacterial activity………
4 4 4 5
6
7
8 9 9
2.2.4.2 Antifungal activity………
2.2.4.3 Antiviral activity………...
2.2.4.4 Antiparasitic activity………
2.2.5 Recent studies of chalcone derivatives………
2.2.6 Recent studies of aspirin-chalcone derivatives……..
10 10 10 11 21 3.0 Materials & Methods………
3.1 Materials………
3.2 Measurements………
3.3 General procedure for alkylation of 4-hydroxybenzaldehyde, 30………..
3.3.1 Synthesis of 4-(decyloxy)benzaldehyde, 30a ……….
3.3.2 Synthesis of 4-(dodecyloxy)benzaldehyde, 30b …….
3.3.3 Synthesis of 4-(tetradecyloxy)benzaldehyde, 30c …..
3.4 General procedure for synthesis of 4-hydroxyacetophenone with benzaldehyde derivatives, 31………
3.4.1 Synthesis of (E)-3-(4-(decyloxy)phenyl)-1-(4-
hydroxyphenyl)prop-2-en-1-one, 31a ………
3.4.2 Synthesis of (E)-3-(4-(dodecyloxy)phenyl)-1-(4- hydroxyphenyl)prop-2-en-1-one, 31b ………
3.4.3 Synthesis of (E)-1-(4-hydroxyphenyl)-3-(4- (tetradecyloxy)phenyl)prop-2-en-1-one,
31c ………
3.5 General procedure for synthesis of aspirin-chalcone derivatives, 32...
23 23 23
24 24 25 26
26
27
28
29
30
3.5.1 Synthesis of (E)-4-(3-(4-
(decyloxy)phenyl)acryloyl)phenyl 2-acetoxybenzoate, 32a ……….
3.5.2 Synthesis of (E)-4-(3-(4-
(dodecyloxy)phenyl)acryloyl)phenyl 2-
acetoxybenzoate, 32b ……….
3.5.3 Synthesis of (E)-4-(3-(4-
(tetradecyloxy)phenyl)acryloyl)phenyl 2-
acetoxybenzoate, 32c ………...
3.6 Antibacterial Screening………...
31
32
33 34 4.0 Results and Discussion……….
4.1 Synthesis of 4-(decyloxy)benzaldehyde,
30a ………
4.2 Synthesis of 4-(dodecyloxy)benzaldehyde,
30b ………..
4.3 Synthesis of 4-(tetradecyloxy)benzaldehyde,
30c ………..
4.4 Synthesis of (E)-3-(4-(decyloxy)phenyl)-1-(4- hydroxyphenyl)prop-2-en-1-one,
31a ………..
4.5 Synthesis of (E)-3-(4-(dodecyloxy)phenyl)-1-(4-
hydroxyphenyl)prop-2-en-1-one, 31b………
4.6 Synthesis of (E)-1-(4-hydroxyphenyl)-3-(4-
(tetradecyloxy)phenyl)prop-2-en-1-one, 31c……….
35
35
39
43
47
51
55
4.7 Synthesis of (E)-4-(3-(4-(decyloxy)phenyl)acryloyl)phenyl 2- acetoxybenzoate, 32a………
4.8 Synthesis of (E)-4-(3-(4-(dodecyloxy)phenyl)acryloyl)phenyl 2-acetoxybenzoate,
32b………
4.9 Synthesis of (E)-4-(3-(4-
(tetradecyloxy)phenyl)acryloyl)phenyl 2-acetoxybenzoate, 32c………..
4.10 Antibacterial activity………
59
63
67 70 5.0 Conclusion and Recommendations ……… 73
6.0 References……… 74
LIST OF ABBREVIATIONS
1H NMR spectroscopy Hydrogen Nuclear Magnetic Resonance
13C NMR spectroscopy Carbon-13 Nuclear Magnetic Resonance
CDCl3 Chloroform
DMSO Dimethyl sulfoxide
FTIR Fourier Transform Infrared Spectroscopy MIC Minimum inhibitory concentrations
ppm Part per million
TLC Thin layer chromatography
LIST OF FIGURES
Figures Pages
Figure 1.1 Structure of aspirin 2
Figure 1.2 Structure of chalcone 3
Figure 2.1 Structure of Ca-ASP 5
Figure 2.2 Structure of 2’-Omethylisoliquiritigenin 6 Figure 2.3 Structure of 2’,3’,4’,6’-tetrahydroxychalcone 7 Figure 2.4 Structure of 2’,4’,6’-tris(methoxymethoxy)chalcone 7
Figure 2.5 Structure of flavokawin A 8
Figure 2.6 Structure of 2’,4’-dihydroxy-6’-methoxy-3’,5’- dimethylchalcone
8
Figure 2.7 Structure of 4-Hydroxyderricin 9
Figure 2.8 Structure of compound 10 9
Figure 2.9 Structure of isobavachalcone 10
Figure 2.10 Structure of compound 12 10
Figure 2.11 Structure of licochalcone 11
Figure 2.12 Structure of compound 20 16
Figure 2.13 Structure of 1-nonylimidazole 21
Figure 3.1 Structure of 4-(decyloxy)benzaldehyde 24 Figure 3.2 Structure of 4-(dodecyloxy)benzaldehyde 25 Figure 3.3 Structure of 4-(tetradecyloxy)benzaldehyde 26 Figure 3.4 Structure of (E)-3-(4-(decyloxy)phenyl)-1-(4-
hydroxyphenyl)prop-2-en-1-one
27 Figure 3.5 Structure of (E)-3-(4-(dodecyloxy)phenyl)-1-(4-
hydroxyphenyl)prop-2-en-1-one
28 Figure 3.6 Structure of (E)-1-(4-hydroxyphenyl)-3-(4-
(tetradecyloxy)phenyl)prop-2-en-1-one
29 Figure 3.7 Structure of (E)-4-(3-(4-(decyloxy)phenyl)acryloyl)phenyl 2-
acetoxybenzoate
31 Figure 3.8 Structure of (E)-4-(3-(4-(dodecyloxy)phenyl)acryloyl)phenyl 2-
acetoxybenzoate
32 Figure 3.9 Structure of (E)-4-(3-(4-(tetradecyloxy)phenyl)acryloyl)phenyl
2-acetoxybenzoate
33
Figure 4.1 FT-IR spectrum of 30a 36
Figure 4.2 1HNMR spectrum of 30a 37
Figure 4.3 13CNMR spectrum of 30a 38
Figure 4.4 FT-IR spectrum of 30b 40
Figure 4.5 1HNMR spectrum of 30b 41
Figure 4.6 13CNMR spectrum of 30b 42
Figure 4.7 FT-IR spectrum of 30c 44
Figure 4.8 1HNMR spectrum of 30c 45
Figure 4.9 13CNMR spectrum of 30c 46
Figure 4.10 FT-IR spectrum of 31a 48
Figure 4.11 1HNMR spectrum of 31a 49
Figure 4.12 13CNMR spectrum of 31a 50
Figure 4.13 FT-IR spectrum of 31b 52
Figure 4.14 1HNMR spectrum of 31b 53
Figure 4.15 13CNMR spectrum of 31 54
Figure 4.16 FT-IR spectrum of 31c 56
Figure 4.17 1HNMR spectrum of 31c 57
Figure 4.18 13CNMR spectrum of 31c 58
Figure 4.19 FT-IR spectrum of 32a 60
Figure 4.20 1HNMR spectrum of 32a 61
Figure 4.21 13CNMR spectrum of 32a 62
Figure 4.22 FT-IR spectrum of 32b 64
Figure 4.23 1HNMR spectrum of 32b 65
Figure 4.24 13CNMR spectrum of 32b 66
Figure 4.25 FT-IR spectrum of 32c 68
Figure 4.26 1HNMR spectrum of 32c 69
Figure 4.27 13CNMR spectrum of 32c 70
Figure 4.28 Antibacterial screening using Disc Diffusion Method 72
LIST OF SCHEMES
Schemes Pages
Scheme 1 Synthesis of aspirin 4
Scheme 2 Synthesis of chalcone 6
Scheme 3 Synthetic scheme for synthesizing the title compounds 12 Scheme 4 Synthesis of 3-[2-(2-hydroxy-4, 6-dimethoxyphenyl)-2-
oxoethyl]-2-benzofuran-1 (3H)-one
13 Scheme 5 Synthetic diagram of the chalcone derivatives 18a-h 14 Scheme 6 Synthetic diagram of the direct ester derivatives of diclofenac
19a-h
15 Scheme 7 Synthetic scheme for synthesizing the semicarbazone
compounds
16 Scheme 8 General scheme for the synthesis of compounds 21a-h 18 Scheme 9 General scheme for the synthesis of compounds 21i and 21j 18 Scheme 10 Synthesis of 4'- acetamidochalcones 19
Scheme 11 Synthesis of chlcone derivatives 20
Scheme 12 Synthesis of imidazole, 2-methylimidazole and 2-methyl-4- nitroimidazole
20
Scheme 13 The synthesis of 28a-b 22
Scheme 14 The synthesis of 29a-b 22
Scheme 15 The synthesis of 30a-c 24
Scheme 16 The synthesis of 31a-c 26
Scheme 17 The synthesis of 32a-c 30
Scheme 18 The synthesis of 30a 35
Scheme 19 The synthesis of 30b 39
Scheme 20 The synthesis of 30c 43
Scheme 21 The synthesis of 31a 47
Scheme 22 The synthesis of 31b 51
Scheme 23 The synthesis of 31c 55
Scheme 24 The synthesis of 32a 59
Scheme 25 The synthesis of 32b 63
Scheme 26 The synthesis of 32c 67
LIST OF TABLES
Tables Pages
Table 1 Antibacterial Activity of E.coli 71
Table 2 Label of compounds on the MH agar plate 72
Synthesis and antibacterial activities of aspirin-chalcone derivatives
PHANG SAI FOONG
Department of Chemistry
Faculty of Resource Science and Technology University Malaysia Sarawak
Abstract
In recent years, chalcone and its derivatives have been studied extensively in terms of their biological activities. In this study, the synthesis and characterization of aspirin-chalcone derivatives has been carried out. A series of aspirin-chalcone derivatives were successfully synthesized from the reaction of intermediates 31a-c with acetylsalicyloyl chloride to yield final product of 32a-c with overall yield of 21.8 % to 88.4 %. All the derivatives were characterized by FT-IR, 1HNMR and 13CNMR. Disc diffusion method was then used to study the antibacterial activities of the new series. All of them showed the results of poor antibacterial properties. The structure that affected the strength of antibacterial properties was discussed.
Keyword: Aspirin, chalcone, aspirin derivatives, chalcone derivatives, antibacterial activities
Abstrak
Dalam tahun-tahun kebelakangan ini, chalcone dan derivatifnya telah dikaji secara meluas dari segi aktiviti biologi mereka. Dalam kajian ini, sintesis dan pencirian derivatif aspirin - chalcone telah dijalankan. Satu siri derivatif aspirin - chalcone telah berjaya disintesis daripada reaksi 31a-c dengan klorida acetylsalicyloyl untuk menghasilkan 32a-c dengan hasil keseluruhan 21.8 % - 88.4 %. Semua derivatif telah dicirikan oleh FT- IR, 1HNMR dan
13CNMR . Kaedah cakera resapan telah digunakan untuk mengkaji aktiviti antibakteria siri baru. Semua produk menunjukkan keputusan ciri-ciri antibakteria yang lemah. Struktur yang menjejaskan kekuatan ciri-ciri antibakteria telah dibincangkan .
Kata kunci : Aspirin , chalcone , derivatif aspirin , derivatif chalcone , aktiviti anti-bakteria
1.0 Introduction 1.1 Aspirin
Aspirin 1 is known as acetylsalicylic acid. It is a salicylate drug, which is used as an analgesic to relieve pains and minor aches. In addition, aspirin is antipyretic and always being applied to reduce fever and prescribed as an anti-inflammatory medication. For now, aspirin modifications have been conducted widely and many aspirin derivatives were proven to exhibit various biological properties such as antithrombic and antiplatelet (Lechi et al., 1996), anticancer (Lechi et al., 1996; Zheng et al., 2007) and antibacterial properties (Al-Bakri et al., 2009). According to Karthikeyan et al. (2009), in the meta-analysis performed by the Antiplatelet Trialists' Collaboration, aspirin is reported to reduce the incidence of deep-vein thrombosis by 20% and that of pulmonary embolism by 69% in patients that have high risk for thromboembolic events.
1
Figure 1.1 : Structure of aspirin
1.2 Chalcone
Chalcone 2 is known as a group of compound consisting of two aromatic rings linked by an unsaturated α and β-ketone. There are various types of substituents on those two aromatic rings. Chalcone exists in most of the plants in nature. It is an intermediate precursor of
flavonoids and isoflavonoids. Chalcone has been applied widely in the field of biology and chemistry. Chalcone showed many biological properties including anticancer, antimalarial, antimicrobial, anti-inflammatory and antibacterial (Hsieh et al., 1998; Ram et al., 2000). In chemistry field, some studies showed that the photochemical and photophysical properties of chalcone enables it to be used as photoalignment and photocrosslinking unit in polymerization process, fluorescent dyes and light-emitting diodes (LEDs).
2
Figure 1.2 : Structure of chalcone
1.3 Problem Statement
Currently, there are less studies conducted on synthesizing aspirin-chalcone derivatives. So, we would like to conduct this study by synthesizing aspirin-chalcone derivatives with various substituents. Through the structure modification, it is possible to create a product which contains even stronger antibacterial properties.
1.4 Objectives
1. To synthesize hydroxyl chalcone derivatives with long alkyl chain.
2. To incorporate hydroxyl chalcone derivatives onto aspirin.
3. To characterize the synthesized compounds using FTIR, 1H and 13C NMR.
4. To study the antibacterial activities of the synthesized compounds.
2.0 Literature Review 2.1 Aspirin
Aspirin is synthesized by reacting acetic anhydride and salicylic acid through esterification reaction. Phosphoric acid is applied as catalyst to speed up the reaction. At the end of the reaction, aspirin and acetic acid will be yield as main product and by product respectively.
H3PO4
acetic anhydride salicyclic acid
aspirin
acetic acid
Scheme 1 : Synthesis of aspirin
2.1.1 Recent study of aspirin derivatives
In the study carried out by Li et al. (2014), a novel aspirin derivatives, namely Ca-ASP 3 was prepared and characterized by reacting aspirin solution with hydroxylapatite (Hap) suspension. The anti-thrombotic and gastric mucosal protection properties study was carried out by using Winstar rats. Study shown that rats that consumed 5 mmol per kg body weight of Ca-ASP showed similar anti-thrombotic activity compared to those consumed same amount of aspirin, however, they suffered gastric mucosal damage in a much lower level compared to standard aspirin.
3
Figure 2.1 : Structure of Ca-ASP
In the study carried out by Balamani and Sekar (2011), simple new aspirin Enkephalin analogues were synthesized and evaluated for its analgesic activity studies. ACOH-inducing writhing test was then carried out on mice to evaluate their analgesic effects. The mean writhing values shown that Gly-Gly-Phe-Met-NH2 sequence was important for the observed analgesic activity of compound Aspirin-Gly-Gly-Phe-Met-NH2, while the Gly-Gly-Phe- Leu-NH2 sequence was crucial for the observed analgesic activity of compound Aspirin- Gly-Gly-Phe-Leu-NH2.
2.2 Chalcone
The general procedure to synthesis chalcone is by reacting equal molar of benzaldehyde and acetophenone in 95 % ethanol as a solvent. The mixture is stirred and cooled in an ice bath.
To complete crystal the formation, it was filtered, wash in cold water and recrystallized with ethanol. The final product, chalcone is yielded.
NaOH/EtOH/H2O
benzaldehyde acetophenone
chalcone
Scheme 2 : Synthesis of chalcone
2.2.1 Antioxidant activity of chalcone derivatives
The antioxidant activity of 2’-Omethylisoliquiritigenin 4, isolated from the roots of Dalbergia odorifera, in lard was examined by Yu et al. (2007) by using oxidative stability instrument. As a result, greater antioxidant effects was obtained from this chalcone at 0.1 and 0.2 mM compared to butylated hydroxytoluene (BHT) and α-tocopherol. It was similar to α-tocopherol in inhibiting the decreasing of glutathione (GSH) level of rat lens induced by UV irradiation. The result could be useful to identify the mechanism of cataractogenesis.
4
Figure 2.2 : Structure of 2’-Omethylisoliquiritigenin
From the study conducted by Mohamad et al. (2004), the 2’,3’,4’,6’-tetrahydroxychalcone 5 isolated from dried ripe fruits of Alpinia rafflesiana exhibited more potent DPPH radical- scavenging activity (IC50 = 55 μM) compared to vitamin C (IC50 = 91 μM) and α-tocopherol (IC50 = 96 μM). From the result, chalcones were found out to be potent radical scavengers if it has catechol pattern of substitution in ring B and pyrogallol-type hydroxylated ring A.
5
Figure 2.3 : Structure of 2’,3’,4’,6’-tetrahydroxychalcone
2.2.2 Anti-inflammatory activity of chalcone derivatives
Heme oxygenase 1 (HO-1) is known as crucial anti-inflammatory enzyme induced in response to oxidative stress and cytokines. A report by Sawle et al. (2008) stated that some methoxychalcones possessed anti-inflammatory activity that correlated with their potency as HO-1 inducers. By sequentially increasing the number of methoxy substituents in the 3,4,5- and 3’,4’,5’-positions of the aryl rings, a progressive increase in HO-1 activity was obtained. Meanwhile, Jin et al. (2007) also reported that by the induction of HO-1, which was initiated by depletion of GSH, 2’,4’,6’-tris(methoxymethoxy)chalcone 6 exhibited potent anti-inflammatory activity.
6
Figure 2.4 : Structure of 2’,4’,6’-tris(methoxymethoxy)chalcone
The flavokawin A 7 isolated from Piper methysticum by Folmer et al. (2006), inhibited tumor necrosis factor (TNF)-α-induced IκBα degradation and translocation of p50 and p65 NF-κB subunits from the cytoplasm to the nucleus. Besides, important inflammation-related proteins such as p38-regulated/activated kinase, Iκβ kinase, Aurora B kinase, mitogen- activated protein kinase 3, dual-specificity tyrosine-phosphorylated and regulated kinase 1A, and also managed to be inhibited by flavokawin A.
7
Figure 2.5 : Structure of flavokawin A
2.2.3 Anticancer activity of chalcone derivatives
According to Amor et al. (2007), 2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethylchalcone 8, isolated from Syzygium samarangense showed significant differential cytotoxicity against the adenocarcinoma SKBR-3 cell lines and human mammary MCF-7 with IC50 values of 12.8 x 10-4 nM and 15 x 10-4 nM respectively. Compared to former, the positive control doxorubicin had an IC50 value of 27.6x10-4 nM against the SKBR-3 cells and an IC50 value of 2.6 x 10-4 nM against the MCF-7 cell line.
8
Figure 2.6 : Structure of 2’,4’-dihydroxy-6’-methoxy-3’,5’-dimethylchalcone
Kimura et al. (2004) isolated 4-Hydroxyderricin 9 from Angelica keiskei. Given orally at a dose of 50 mg/kg x 2/day, this compound successfully inhibited lung metastasis and prolonged the survival time in mice after the removal of subcutaneous tumors by surgical operation. This compound had additional properties by inhibiting the reduction of the numbers of lymphocytes, CD4+, CD8+ and natural killer (NK)-T cells in the spleen of tumor- removed mice (Kimura et al. 2004).
9
Figure 2.7 : Structure of 4-Hydroxyderricin
2.2.4 Anti-infective activity of chalcone derivatives 2.2.4.1 Antibacterial activity
Compound 10 identified by Bowman et al. (2007) was concluded the most active against S.
aureus, methicillin-resistant S. aureus, S. epidermidis, and B. subtilis with the MIC values 3.5, 3.0, 3.8, and 8.8 μM respectively. In comparison, the referent drug linezolid inhibited both S. aureus strains with recorded MIC values of 10.0 and 8.0 μM respectively.
10
Figure 2.8 : Structure of compound 10
2.2.4.2 Antifungal activity
Mbaveng et al. (2008) screened isobavachalcone 11 for its antifungal activity, and found out that it inhibited growth of Microsporum audorium (MIC = 0.3 μg/ml), Candida albicans (MIC = 0.3 μg/ml), C. glabrata (MIC = 0.3 μg/ml) and Trichophyton rubrum (MIC = 1.2 μg/ml).
11
Figure 2.9 : Structure of isobavachalcone
2.2.4.3 Antiviral activity
An attempt was made by Deng et al. to develop small molecules to inhibit HIV-1 integrase (IN), a crucial enzyme for viral replication. Compound 12 was synthesized successfully and in the presence of both Mn2+ and Mg2+ as cofactors, it showed potent inhibitory activity with an IC50 value of 2 μM against purified IN.
12
Figure 2.10 : Structure of compound 12
2.2.4.4 Antiparasitic activity
Licochalcone A 13, the well-known antiparasitic compound is a potent membrane-active agent that undergoes transformation of normal erythrocytes into echinocytes in parallel with the inhibition of growth of P. falciparum cultures. Ziegler et al. (2004) managed to observed the erythrocyte membrane-modifying effect transiently in mice after intravenous administration.
13
Figure 2.11 : Structure of licochalcone
2.2.5 Recent studies of chalcone derivatives
In the study conducted by Singh et al. (2010), a new pharmacophore named
‘Chalconesemicarbazone’ was designed by pharmacophore hybridization of drug design.
Singh et al. (2010) a series of novel ‘chalconylsemicarbazide’ derivatives by reacting substituted phenyl semicarbazide with various chalcone derivatives, which contained hydrogen acceptor site, hydrogen donor site and lipophilic site which may aid in receptors binding for pharmacological activities. Carrageenan-induced rat paw edema test was used to conduct in vivo determination of the anti-inflammatory activity. Meanwhile, to evaluate the analgesic activity, acetic acid-induced writhing test was used. Finally, the percentage of protection against the acetic acid-induced writhing was determined and calculated. The screening result shown that 1-(1,5-diphenylpenta-2,4-dienylidene)-4-(2-nitrophenyl) semicarbazide was most active.
