Anti-Microbial activities of two Platinum(II) complexes bearing ethyl eugenoxyacetate and synthesis, structure of [PtCl(ethyl eugenoxyacetate-1H)(morpholine)]

Abstract: Two organo-metallic complexes of platinum(II), [PtCl(Eteug-1H)(C9H7N)] (P1), [Pt(Eteug-1H)(OC9H6N)] (P2), have been experimented on anti-microbial activity for the first time. P1 has activity against L.fermentum and B.subtilis bacterias with the IC50 values of 165.05, 220.58 µg/mL, respectively, P2 exhibits higher cytotoxicity on B.subtilis, S.aureus bacteria and C.albicans fungi with the IC50 values from 37.75  63.06 µg/mL. A new complex - [PtCl(Eteug-1H)(morpholine)] (P3)- has been synthesized and determined structure by the weight and thermal analysis method on ESI-MS, IR, 1H NMR, 13C NMR, HMQC, NOESY spectro-scopies. In P3, Eteug co-ordinates with Pt(II) through ethylenic double bond of the allyl group and C5 of benzene ring, while morpholine co-ordinates with Pt(II) via the N atom with Pt-N bond of e-type, then occupies cis-position in comparison with the allyl group.

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JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2016-0057 Natural Sci. 2016, Vol. 61, No. 9, pp. 60-67 This paper is available online at 60 ANTI-MICROBIAL ACTIVITIES OF TWO PLATINUM(II) COMPLEXES BEARING ETHYL EUGENOXYACETATE AND SYNTHESIS, STRUCTURE OF [PtCl(ETHYL EUGENOXYACETATE-1H)(MORPHOLINE)] Nguyen Thi Thanh Chi 1 , Nguyen Thi My Hoa 2 and Truong Thi Cam Mai 3 1 Faculty of Chemistry, Hanoi National University of Education 2 Faculty of Chemistry, Hanoi Pedagogical University No. 2 3 Faculty of Chemistry, Quy Nhon University Abstract: Two organo-metallic complexes of platinum(II), [PtCl(Eteug-1H)(C9H7N)] (P1), [Pt(Eteug-1H)(OC9H6N)] (P2), have been experimented on anti-microbial activity for the first time. P1 has activity against L.fermentum and B.subtilis bacterias with the IC50 values of 165.05, 220.58 µg/mL, respectively, P2 exhibits higher cyto- toxicity on B.subtilis, S.aureus bacteria and C.albicans fungi with the IC50 values from 37.75  63.06 µg/mL. A new complex - [PtCl(Eteug-1H)(morpholine)] (P3)- has been synthesized and determined structure by the weight and thermal analysis method on ESI-MS, IR, 1 H NMR, 13 C NMR, HMQC, NOESY spectro-scopies. In P3, Eteug co-ordinates with Pt(II) through ethylenic double bond of the allyl group and C5 of benzene ring, while morpholine co-ordinates with Pt(II) via the N atom with Pt-N bond of e-type, then occupies cis-position in comparison with the allyl group. Keywords: Ethyl eugenoxyacetate, platinum(II) complexes, quinoline derivaties, morpholine, anti-microbial activities. 1. Introduction Since FDA approval of Cisplatin for the treatment of metastatic ovarian and testicular cancers was granted in 1978, it has urged researchers to develop new platinum complexes for medical applications. Although over thousand complexes have been prepared and tested thus far, only three platinum drugs including Cisplatin, Carboplatin and Oxaliplatin have been approved for clinical use worldwide. Nevertheless, they all have toxic side-effects and are not universally effective in all cancer types [1, 2]. Recently, a research group at Hanoi National University of Education has made complexes of K[PtCl3(Eteug)] and [PtCl(Eteug-1H)]2 of which Eteug is ethyl eugenoxyacetate - a derivative of eugenol (the main component of clove basil oil) [3]. The inter-action between these complexes Received November 8, 2016. Accepted November 30, 2016. Contact Nguyen Thi Thanh Chi, e-mail address: chintt@hnue.edu.vn Anti-microbial activities of two platinum(II) complexes bearing ethyl eugenoxyacetate and synthesis 61 and different amines formed new complexes with promising anti-cancer activities, especially those containing hetero-cyclic amine ligands [4, 5]. However, these resulting complexes have not been tested for microbial activities. In addition, platinum(II) complex containing Eteug and morpholine has not been studied yet. In this paper, we describe the anti-microbial activity of two platinum (II) complexes, [PtCl(Eteug-1H)(quinoline)] and [Pt(Eteug-1H)(8-oxiquinoline)]. The synthesis and structure of complex [PtCl(Eteug-1H)(morpholine)] were also reported. 2. Content 2.1. Experiment 2.1.1. Synthesis of complexes * Synthesis of [PtCl(Eteug-1H)(C9H7N)] (P1) and [Pt(Eteug-1H)(OC9H6N)] (P2) P1 and P2 were synthesized by the reaction between [PtCl(Eteug-1H)]2 and quinoline or 8- hidro-xiquinolin in acetone solvent according to the procedures of Tran Thi Da et al. [5]. * Synthesis of [PtCl(Eteug-1H)(C4H8ONH)] (P3) [PtCl(Eteug-1H)(C4H8ONH)] was prepared as follows: A mixture of 480 mg (1 mmol) [PtCl(Eteug-1H)]2 (prepared according to the synthetic protocol of Da et al. [3]) and 10 mL of acetone was added gradually to 0.1 mL of morpholine (1.16 mmol). The reaction mixture was stirred at room temperature. The starting compound - [PtCl(Eteug-1H)]2 - was dissolved completely after a few minutes to form a clear solution which later appeared with white-blue precipitates A part of the solvent was evaporated from the starting mixture which was later cooled for 1 hour. The solid compound obtained was isolated by filtration, next washed with water andether, then re-crystallized by volume 1 : 1 from acetone/ethanol to afford blue-white prismatic crystals which are insoluble in water, but slightly soluble in ethanol and acetone, and highly soluble in chloroform, finally yielded 78%. 2.1.2. Apparatus and methods Pt was analyzed according to the weight method [3] at the Faculty of Chemistry of Hanoi National University of Education. Thermal analysis curve of the complex was recorded in Ar atmosphere on DTG-60H instrument, at temperatures ranging from 25 to 800 o C (with temperature rate of 10 o C per minute). The ESI-MS spectrum of P3 was recorded on a 1100 Series LC-MSD-Trap-SL; while the IR spectra were recorded on IMPACT-410 NICOLET spectro-meter in KBr discs in the range 400 - 4000 cm -1 ; the NMR spectra were recorded on a Brucker AVANCE 500 MHz; all were at 298 - 300 K in CDCl3 with TMS as the internal standard at the Institute of Chemistry, Vietnam Academy of Science and Technology. The anti-microbial activities of P1, P2 were tested on gram (+) bacteria including lactobacillus fermentum (L.fermentum), bacillus subtilis (B.subtilis), staphylococcus aureus (S.aureus); and gram (-) bacteria including salmonella enterica (S.enterica), escherichia coli (E.coli), pseudomonas aeruginosa (P.aeruginosa), and Candida albicans (C.albicans) fungi at the Institute of Chemistry of Natural Compounds, Vietnam Academy of Science and Technology. Nguyen Thi Thanh Chi, Nguyen Thi My Hoa and Truong Thi Cam Mai 62 2.2. Results and discussion 2.2.1. Anti-microbial activities survey of P1, P2 Basing on the protocol of Tran Thi Da et al. [5], we synthesized P1, P2 with a high yield. The reaction equations are described in schemes (1) and (2). The IR and 1 H NMR spectra of P1 and P2 are the same as those published in [5]. The structures of P1 and P2 have been determined by using elemental analysis IR, 1D & 2D NMR spectra [5]. P1, P2 were experimented in vitro activity against microbes including L. fermentum, B. subtilis, S. aureus, S. enterica, E. coli, P.aeruginosa and C. albicans. The results are shown in Table 1. Table 1. The result of anti-microbial activity of P1 and P2 Comp. The minimum and half-maximal inhibitory concentration (MIC and IC50: µg/mL) Gram(+) Gram (-) Fungi L.fermentum B.subtilis S.aureus S.enterica E.coli P.aeruginosa C.albicans MIC IC50 MIC IC50 MIC IC50 MIC IC50 MIC IC50 MIC IC50 MIC IC50 P1 256 165.05 - 220.58 - - - - - - - - - - P2 - - 256 37.75 256 63.06 - - - - - - - 47.09 Anti-microbial activities of two platinum(II) complexes bearing ethyl eugenoxyacetate and synthesis 63 The data in Table 1 show that P1 and P2 have activities against Gram (+) bacteria and fungi. Specifically, P1 exhibits cyto-toxic activity against L. fermentum and B. subtilis bacteria with the IC50 value of 165.05, 220.58 µg/mL, respectively. While P2 exhibits higher cyto-toxicity against B. subtilis, S. aureus bacteria, and C. albicans fungi. The IC50 values are 37.75, 63.06 and 47.09 µg/mL, respectively. 2.2.2. Determination the structure of [PtCl(Eteug-1H)(morpholine)] (P3) According to [5, 6], the interaction between a dinuclear chelate ring complex of [PtCl(olefin-1H)]2 with an amine formed the complex [PtCl(olefin-1H)(amine)]. Based on the method described in [5, 6], P3 was synthesized by the reaction of [PtCl(Eteug-1H)]2 and morpholine with the yield of 78%. The reaction equation is described as shown in scheme (3). The numeration of the examined compound in scheme (3) is only used for the analysis of its NMR spectra. To determine the structure of P3, we used the weight and thermal analysis method; EDX, ESI-MS, IR, 1 H NMR, 13 C NMR, HMQC and NOESY spectro-scopies. Atomic ratio of Pt:Cl shows a good agreement (1.00:1.00) between the theoretical value for the expected formula of P3 in scheme (3), and the actual value (1.01:1.00) determined from the EDX spectra. Percentage of Pt determined by weight method is 34% in accordance with the data (34.42%) calculated from the formula [PtCl(Eteug-1H)(morpholine)]. This is further confirmed by thermal analysis method, of which the result is shown in Figure 1. Figure 1. Thermal analysis diagram of P3 Nguyen Thi Thanh Chi, Nguyen Thi My Hoa and Truong Thi Cam Mai 64 In the differential thermal analysis curve (DTA) appears a weakly endothermic effect at about 185 o C. However, no effect in the thermo-gravimetric analysis curve (TGA) reaches 200 o C (Figure 1). This indicates that there is no crystallized and co-ordinated water in P3, and the melting of the compound might occur at 185 o C. The DTA curve shows a weak exothermic peak at about 205 o C corresponding to a 46.251% mass loss in the TGA curve, and two others from 280  450 oC to a 19.078%, similarly. This is due to the 3-step de-composition of the compound which produces Pt and other gaseous compounds with the total mass loss of 65.329%, leaving a residue mass of 34.671% for Pt. This value is in good agreement (34.42%) with that calculated from the formula [PtCl(Eteug-1H)(morpholine)]. Additionally, at temperature higher than 450 o C, there is no effect in both the DTA and TGA curves. This means that the complex is pure and de- composed completely at below 450 o C. The process can be displayed as following: [PtCl(Eteug-1H)(C4H8ONH)]  Pt + gaseous compounds Figure 2 shows the partial +MS and –MS spectra of P3. In the positive-mode ESI-MS, there is a peak at m/z 529.2 au with a relative intensity of 100% (base peak), in consistence with pseudo-molecular ion [P3-Cl] + i.e. [Pt(Eteug-1H)(C4H8ONH)] + ; In the negative-mode ESI-MS there is peak at m/z 478.4 au also with relative intensity of 100% in line with pseudo-molecular ion [P3-morpholine-1H] - i.e. [PtCl(Eteug-2H)] - . Figure 2. Partial positive mode (a) and negative mode (b) ESI-MS spectra of P3 Table 2. Main bands in the IR spectrum of P3, cm -1 Compound νNH νCH aromatic νCH aliphatic νC=O ν(C=C, C=N) ν(Pt-C5, Pt-N) νPt-C=C P3 3243 3057 2972 2858 1741 1571 1479 649 581 442 Anti-microbial activities of two platinum(II) complexes bearing ethyl eugenoxyacetate and synthesis 65 The IR spectrum of P3 shows characteristic bands for the presence of Eteug and morpholine (Table 2). For instance, the spectrum displays an intense band at 1741 cm -1 corresponding to the νC=O band for Eteug. Concurrently, the absence of a band at 1640 cm -1 from the C=C double bond of allyl group in non-coordinated Eteug indicates that Eteug has coordinated with Pt(II) through this bond. This co-ordination results in a decrease in wave number of C=Canken, and an appearance of a new band for Pt-(C=C) at 442 cm -1 . The band for N-H stretching vibration in P3 is observed at 3243 cm -1 , and around 3450-3300 cm -1 in non-coordinated morpholine [7]. This indicates that morpholine has coordinated with Pt(II) via N atom. The assignment of 1 H NMR signals of P3 is based on the chemical shift (δ), intensity, spin – spin splitting patterns (shape), value of splitting constant (J) and the NOESY spectrum. The results are listed in Table 3. Table 3. 1H NMR signals of Eteug and morpholine in P3, δ (ppm), J (Hz) Compound H3 H6 H7a H7b H8a H8b H9 H10cis H10tran s H1 1 H12 Eteug Free 6.72 6.77 4.76 3.79 3.33 5.94 5.06 5.09 4.25 1.22 P3 6.57 s 6.91 s 4.65 s 3.80 s 2.59 d 3 J 16.5 3.81 ov 4.62 ov 3.79 ov 3.77 ov 4.28 m 1.23 t 3 J 7.0 Morpholine 1 eH 1 eH 2 aH 2 eH 2 aH NH NH O   e e a a Fre e 2.90 3.60 1.90 P3 3.07 d 2 Jae 13.0 2.97 d 2 Jae 13.0 2.88 qd 2 Jae 13.0; 3 Jaa 13.0 3 Jaa(N) 13.0; 3 Jae 3.0 3.87 ov 3.53 td 2 Jae 13.0 3 Jaa 13.0 3 Jae 3.0 3.12 br Table 3 shows the resonances for all protons of Eteug and morpholine in P3, except for H5. They are all different from those in free Eteug and morpholine ligands. The resonances of the ethylenic protons (H9, H10cis and H10trans) are upfield in comparison with those of non- coordinated Eteug with   1.3 ppm, and the H5 signal is absent showing that Eteug has coordinated with Pt(II) not only through the C=Callyl but also the C5 of benzene ring [3, 5, 6]. Upon co-ordination to Pt(II), six protons of morpholine (except for NH) give a rise to the five resonances, and only two resonances in free morpholine (Table 3). Moreover, the resonance of H-(N) shifts downfield compared to itself in free morpholine and the 3 Jaa(N) value calculated from the resonance of aH in P3 within 13.0 Hz. These elucidate morpholine coordinates with Pt(II) via the N atom with Pt-N bond being of e-type as discribled in scheme (3). The 13 C NMR (Table 4) further confirms the structure of P3. The assignment of the 13 C NMR signals is based on its chemical shift with HMQC, experienced rules on 13 C NMR spectra of analogous complexes [3, 5, 6]. For example, in the partial HMQC spectrum (Figure 3), the cross Nguyen Thi Thanh Chi, Nguyen Thi My Hoa and Truong Thi Cam Mai 66 peaks a, b of eH and aH show that two signals at 67.84 and 68.08 ppm belong to 2Cβ. Similarly, cross peaks f, g of aH and eH indicate two signals of 2Cα at 47.84 and 48.22 ppm. The cross peaks c, d, e point out signals of C10, C7b, C8, respectively. The results of the 13 C NMR assignment are listed in Table 4. (a) (b) Figure 3. Partial HMQC spectrum (a) and NOESY spectrum (b) of P3 Table 4. 13C NMR signals of Eteug and morpholine in P3, δ (ppm) Comp. C1 C2 C3 C4 C5 C6 C7a C7b C8 P3 143.58 147.87 108.81 141.89 122.36 119.43 66.43 56.10 38.45 C9 C10 C=O C11 C12 Cα Cβ 80.16 56.32 169.40 61.18 14.63 47.84 / 48.22 67.84/ 68.08 Table 4 shows resonances for the presence of Eteug and morpholine in P3. For example, the characteristic signal for the carbonyl group, C=O, of Eteug appears at 169.40 ppm (Table 4). In the 13 C NMR spectrum, all the signals are changed with respect to their signals in the free ligands [5, 8] due to the co-ordination of the ligands. Particularly, the resonances of the ethylenic carbons (C9, C10) are upfield. This indicates that Eteug co-ordinates with Pt(II) at ethylenic double bond of the allyl group resulting in re-hybridization from sp 2 to some sp 3 character of the carbon atoms [4, 8]. In addition, the co-ordination of Eteug with Pt(II) via the C5 of the aromatic ring makes the intensity of C5 lower than that in the free ligand. Based on the analyzed results above, it is indicated that in P3, Eteug co-ordinates with Pt(II) through the C=Callyl and C5, while morpholine coordinates with Pt(II) via the N atom with Pt-N- bond being of e-type. However, the question poses whether the morpholin is in cis position to the C=Callyl or C5. To answer it, we recorded the NOESY spectrum of P3 (Figure 3b), In the spectrum, the cross peaks a, b and c between H-(N), eH and aH with H10cis indicate that these Anti-microbial activities of two platinum(II) complexes bearing ethyl eugenoxyacetate and synthesis 67 protons are close together H-(N). In other words, the morpholine occupies the cis-position in comparison to the allyl group, as shown in scheme (3). 3. Conclusion In this paper, we present the evaluation for activity against microbial of two complexes [PtCl(Eteug-1H)(C9H7N)] (P1), [Pt(Eteug-1H)(OC9H6N)] (P2) and synthetic, structural studies of complex [PtCl(Eteug-1H)(C4H8ONH)] (P3). The result of microbial resistance test indicates that P1 has activity against L. fermentum and B. subtilis bacterias with the IC50 value of 165.05 and 220.58 µg/mL, respectively, while P2 exhibits higher cytotoxicity on B. subtilis, S. aureus bacteria and C. albicans fungi with the IC50 values of 37.75, 63.06 and 47.09 µg/mL, respectively. P3 was characterized by the weight and thermal analyses on ESI-MS, IR, 1 H NMR, 13 C NMR, HMQC, NOESY spectroscopies. The results reveal that Eteug coordinates with Pt(II) at ethylenic double bond of the allyl group, morpholine coordinates with Pt(II) via the N atom with Pt -N bond of e-type. 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