Characterization and bio-activity evaluation of two new acetohydrazides synthesized from curcumin and monocarbonyl curcumin analog

JOURNAL OF SCIENCE OF HNUE DOI: 10.18173/2354-1059.2016-0050 Natural Sci. 2016, Vol. 61, No. 9, pp. 11-20 This paper is available online at 11 CHARACTERIZATION AND BIO-ACTIVITY EVALUATION OF TWO NEW ACETOHYDRAZIDES SYNTHESIZED FROM CURCUMIN AND MONOCARBONYL CURCUMIN ANALOG Duong Quoc Hoan, Nguyen Thi Thanh Xuan, Truong Minh Luong and Tran Thi Trang Faculty of Chemistry, Hanoi National University of Education Abstract. The acetohydrazides A3 containing an isoxazole ring and B3 an indazole ring, relatively were synthesized from the corresponding ester derivatives with hydrazine hydrate in good yield. Since the acetohydrazides are important starting materials in organic synthesis, the complicated structures of the acetohydrazides were characterized carefully with modern physical methods including IR, NMR and MS spectra. Almost all carbons and protons of A3 and B3 were assigned. Bio-activity testes showed that both were inactive on KB cell line and against bacteria. Keywords: Characterization, acetohydrazide, curcumin, indazole, curcumin, monocarbonyl curcumin analog. 1. Introduction Blocking the 1,3-diketone system of curcumin is to form compound A1 containing an isoxazole ring as a heterocyclic linker, and attach –CH2COOH group as a pharmacophore. (A3’) did not increase bioactivity [1]. Moreover, the modification of curcumin by using monocarbonyl linker instead of 1,3-diketone is a promising idea. For example, IC50 (µM) of ketone B1 is smaller than that of curcumin on cancer cells A549 (epithelial cancer), HepG2 (liver cancer), and MCF7 (breast cancer) [2]. However, our study show that B3’, which has an –OCH2COOH known as a pharmacophore group, did not show anti-cancer activity on KB, HepG2, Lu and MCF7 cell lines (unpublished result). Therefore, the modification of the pharmacophore groups is needed (see Figure 1). H3CO RO Pharmacophore ON OCH3 OR Pharmacophore H3CO RO OCH3 OR O ketone linkerPharmacophore Pharmacophore Heterocyclic linker R= H (A1); R= -CH2COEt (A2); R = -CH2COOH (A3') R= H (B1); R= -CH2COEt (B2); R = -CH2COOH (B3') Figure 1. Modification of curcumin and monocarbonyl curcumin analog Received May 23, 2016. Accepted November 18, 2016. Contact Duong Quoc Hoan, e-mail address: hoandq@hnue.edu.vn Duong Quoc Hoan, Nguyen Thi Thanh Xuan, Truong Minh Luong and Tran Thi Trang 12 In this paper, the modification focused on introducing a -CH2CONHNH2 group as either a new pharmacophore or starting materials for synthesizing heterocyclic derivatives [3, 4], and hydrazones, etc. Thus, their structures were characterized carefully. In addition, their bio-activities were screened on KB cancer cell line and against bacteria. 2. Content 2.1. Experiment Solvents and other chemicals were purchased from Sigma-Aldrich, Merck, and used without further purification. The 1 H NMR and 13 C NMR spectra were recorded on a Bruker Avance 500 NMR spectrometer in DMSO-d6. Chemical-shift data for each signal were reported in ppm units. IR spectra were recorded on a Mattson 4020 GALAXY Series FT-IR. Mass spectra were obtained on LC-MSD-Trap-SL spectrometer, from Mass Spectrometry Facility of the Vietnam Academy of Science and Technology. 2.2. Synthesis and bio-activity test * Synthesis of compound A1, A2, B1 and B2 These preparations followed procedures in [5]. * Synthesis of acetohydrazides Synthesis of A3: To the solution of ester A2 (0.54 g, 1.0 mmol) in ethanol (10 mL), was added 50% aqueous hydrazine hydrate (4 mL). The resulting solution was refluxed for 2 hours, then cooled down to precipitate out product. The precipitate was filtered and washed with cold ethanol to give a white solid of the compound A3 in quantitative yield, mp = 264 - 265 C Synthesis of B3: synthesis of B3 followed the procedure synthesis of A3 from B2 (1.0 mmol, 0.4 gam), 50% aqueous hydrazine hydrate (4 mL) to give a white solid of the compound B3 in quantitative yield, mp = 269 - 270 C. * Bioactivity test Bio-activity tests were followed by the Broth dilution method [6]. All tests were screened in the Laboratory of Applied Biochemistry of the Vietnam Academy of Science and Technology. 2.3. Results and discussion * Synthesis of acetohydrazides B3 The synthesis of A3 and B3 was carriedout quite simply in high yield. Hydrazine hydrate was used in excess. In the synthesis of B3, hydrazine not only substituted ethyl group but also cyclized an indazole ring (see Scheme 1). Characterization and bio-activity evaluation of two new acetohydrazides synthesized from curcumin 13 OCH3 O H3CO O N O A2 O O O O N2H4.H2O quantitative OCH3 O H3CO O N O A3 O H N NH2 H N O H2N 1 '  44' 5' 5 7' 7 1010' 1111' 14' 14 N2H4.H2O quantitative H3CO O OCH3 O NHN H3CO O OCH3 O O B2 B3O O O O O H N NH2 O H N H2N 1 55' 10' 10 11' 11 14' 14 2 3 2'3' xx' y Scheme 1. Synthesis of acetohydrazides * Characterization of acetohydride structures IR spectrum analysis: IR spectra of A3 and B3 analysis was shown in Table 1. NH2 group within their spectra showed two absorptions of N-H stretching vibrations at near 3250 cm 1 and 3340 cm -1 . Another important vibration was the absorption of C=O group. The C=O absorption of amides occurred at lower frequencies than “normal” carbonyl group, due to a resonance effect. Thus, the C=O vibrations of A3 and B3 were characterized with strong absorption at 1665 and 1677 cm -1 . The C=C and C=N stretching absorptions overlapped each other between 1450 - 1612 cm -1 , which were expectedly [7] reasonable for the designed structures. Table 1. IR and MS analysis of acetohydrides A3 and B3,  (cm -1 ) Comp. NH C-H C=O C=C, C=N MS ([M+H] + ) Calcd. Found A3 3341, 3270 3012; 2919; 2848 1677 1612; 1506; 1428 510 [C25H28N5O7] + 510 (+MS) B3 3340, 3264 3064; 2922; 2850 1665 1603; 1518; 1458 525 [C26H33N6O6] + 525 (+MS) Mass spectroscopy of A3 and B3: Mass spectra of acetohydrazide A3 and B3 showedthat molecular ion peaks matched with the expected structures. The molecular ion in compound A3 having five nitrogen atoms was found 510 aum on +MS, while B3’s having six nitrogen atoms was found 525 aum on +MS, relatively (see Table 1). NMR spectrum analysis of A3 and B3: In the 1 H NMR spectrum of A3 (Figure 3), it was easy to indentify pairs or groups of protons based on the chemical shift and spliting constant. For instance, H13 and H13’ (NH) were singlet at  9.17 ppm; While H5/H5’ were doublet at  7.36 ppm due to meta position to H9/H9’ with J = 1.5 Hz; and H2/H2’ or H3/H3’ were doublet at  7.34 ppm or 7.18 ppm because H2/H2’ or H3/H3’ were in trans-position to each other with Duong Quoc Hoan, Nguyen Thi Thanh Xuan, Truong Minh Luong and Tran Thi Trang 14 J = 16.5 Hz. Meanwhile, H9/H9’ were doublet at 7.13 or 7.14 ppm due to “ortho” position to H8/H8’ and meta position to H5/H5’ with J = 8.5 Hz and J =2.0 Hz. H8/H8’ were doublet at 6.95 ppm or 6.94 ppm with J = 8.0 Hz due to ortho position to H9/H9. H1 was a singlet at 6.90 ppm, and H11/H11’ were at 4.50 ppm, relatively. H14/H14’ (NH2) were broad peaks at 4.36 ppm. Finally, H10/H10’ were singlet at 3.85 or 3.86 ppm. However, it was impossible to distinguish precisely Hn withHn’ in the 1H NMR spectrum. Therefore, 2D NMR of A3 was recorded. Figure 2. 1 H NMR spectrum of A3 in DMSO-d6 In order to identify precisely whether Cn or Cn’ as well as Hn or Hn’, firstly, we advised to start from isoxazole ring. Cα’ (Cα’=N) at 168.17 ppm had a larger chemical shift than Cα (Cα-O), it is, at  162.09 ppm. Then, H3’ ( 7.34 (d, J 16.5 Hz, 1H)) was confirmed for having a cross peak with Cα’ on the Heteronuclear Multiple Bond Correlation (HMBC) spectrum, while C3’( 134.29 ppm) with H3’ on the Heteronuclear Single Quantum Coherence (HSQC) spectrum, relatively. H5’( 7.36 (d, J 1.5 Hz, 1H)) and H9’ ( 7.14 (dd, J 8.5, 2.0 Hz 1H)) were distingushed for their cross peaks with C3’; Definitely, C9’ ( 121.13 ppm) and C5’ ( 109.96 pmm) were also distingushed on HSQC spectrum; C7’ was at  148.49 ppm because of a cross peak with H9’ and H5’; C7’ allowed us to indentify H11’ and C11’ ( 67.20 ppm); H13’ and H14’ were not recognized since they overlapped H13 and H14; C3’ had a cross peak with H2’ ( 7.17 ppm (d, J16.0 Hz, 1H)), and C3 with H2 (7.19 (d, J 16.0 Hz, 1H), relatively. Similarly, other carbons and protons, on the other hand, could be assigned as shown in the Table 2. Characterization and bio-activity evaluation of two new acetohydrazides synthesized from curcumin 15 Figure 3. A part of HSQC and HMBC spectra in DMSO-d6 of A3 Table 2. NMR analysis for A3 1 H NMR  (ppm), J (Hz) HMBC Hn cross with Cn 13 C NMR  (ppm) HMBC Cn cross with Hn HSQC Hn cross with Cn H1 6.90 (s, 1H) C, C’ C1 98.39 - H1xC1 - - - C 162.09 H1,H3 - - - - C’ 168.17 H1’,H3’ - H2 7.19 (d, J16.0, 1H) C4, C3, C1 C2 111.66 H3 H2xC2 H2’ 7.17 (d, J16.0, 1H) C4’, C3’, C1 C2’ 114.05 H3’ H2’xC2’ H3 7.47 (d, J 16.5, 1H) C5, C9, C C3 135.98 H5, H2 H3xC3 H3’ 7.34 (d, J 16.5, 1H) C5’, C9’, C’ C3’ 134.29 H5’, H2’ H3’xC3’ - - - C4 129.64 H2 - - - - C4’ 129.27 H2’ - H5 7.35 (1H) C9, C3, C7 C5 110.12 H3, H9 H5xC5 H5’ 7.36 (1H) C9’, C3’, C7’ C5’ 109.96 H3’, H9’ H5’xC5’ - - - C6 149.33 H8 - - - - C6’ 149.33 H8’ - - - - C7 148.24 H9, H5 - Duong Quoc Hoan, Nguyen Thi Thanh Xuan, Truong Minh Luong and Tran Thi Trang 16 - - - C7’ 148.49 H9’, H5’ - H8 6.94 (d, J 8, 1H) C6, C4’ C8 113.80 - H8xC8 H8’ 6.94 (d, J 8, 1H) C6’, C4’ C8’ 113.80 - H8’xC8’ H9 7.13 (dd, J 8.5, 2.0 1H) C5, C3 C9 120.73 H5 H9xC9 H9’ 7.14 (dd, J 8.5, 2.0 1H) C5’, C3’ C9’ 121.13 H5’ H9’xC9’ H10 3.8 (s, 3H) C6 C10 55.66 - H10xC10 H10’ 3.8 (s, 3H) C6’ C10’ 55.70 - H10’xC10’ H11 4.5 (s, 2H) C7, C12 C11 67.15 - H11xC11 H11’ 4.5 (s, 2H) C7’, C12’ C11’ 67.20 - H11’xC11’ - - - C12 166.50 - - - - - C12’ 166.54 - H13 H13’ 9.17 (s, 2H) C12, C12’ - - - - H14 H14’ 4.36 (s, 4H) - - - - Note: “-“ means no data or no cross peak(s) Similar to A3, compound B3 had an asymmetric structure because the hydrazination not only did the ethoxy substitution, but also did the cyclization formed an indazole ring. It led to the difficulty of identification of all carbons and protons on its NMR spectra. In the 1 H NMR spectrum of B3, the absence of the ten protons of two ethyl group in the ester B2 supported the formation of the acetohydride B3. It was also found that protons bonded with nitrogen atoms were distinguished such as: H14 and H14’ of NH2 group at  4.32 ppm; and H13 and H13’ of NH (-CONH-, overlapped) at  9.13 ppm. In addition, there was a single peak at  7.28 ppm assigned for H3a as a doublet of NH group in the indazole ring. The proton H3 was so far from the weak field due to the hybridization from sp 2 to sp 3 and attached with NH of the indazole ring that the peak at  4.29 ppm assigned for H3; H10 and H10’ had same chemical shift at  3.79 ppm. Others protons were not indentified on the 1 H NMR spectrum. In the 13 C NMR spectrum of compound B3, there were not any peaks of carbons that belonged to the ethyl group of the ester B2. That indicated the substitution of ethyl group with hydrazine was successful. It was easy to assign the peaks at  67.6 and 67.3 ppm for C11 or C11’ (-OCH2  55.58 and 55.54 ppm for C10 or C10’ (-OCH3 groups) at  153.79 ppm for C1, relatively, since B3 had only one carbon atom. Similar to the 1 H NMR spectrum, other carbons of compound B3 were not identified due to the fact that chemical shifts were close to each other. Characterization and bio-activity evaluation of two new acetohydrazides synthesized from curcumin 17 Figure 4. 1 H NMR spectrum of B3 in DMSO-d6 Figure 5. A part of HSQC and HMBC spectra of B3 in DMSO-d6 To overcome this situation, B3 was recorded as 2D NMR spectra. The HSQC spectrum of B3 (Figure 5) showed that the protons were attached with the corresponding carbons and vice verse (see Table 3). Duong Quoc Hoan, Nguyen Thi Thanh Xuan, Truong Minh Luong and Tran Thi Trang 18 The HMBC of B3 distinguished Hn and Hn’ as well as Cn and Cn’ in the structure of B3. First of all, the C1-a bare proton carbon- was recognized at  153.7 ppm (as shown above) for having cross peaks with H3a, H2 (m, 2.64 ppm), and H3’ (s, 6.95 ppm), relatively. It was important to indentify the rest of B3 structure. For example, H3’ had a cross peak with C5’(113.57 ppm) , leading to H5’(s, 6.97 ppm) based on HSQC spectrum. While, the identification of C5’ led to the H9’ at  6.88 ppm, and C9’ at  121.88 ppm; relatively. Similarly, C6’had a cross peak with C10’ and C7’ with C11’ at  148.75 ppm, relatively. Detailed characterization was shown in Table 3. Table 3. NMR spectrum analysis for B3 1 H NMR,  (ppm) HMBC Hn crosses with Cn 13 C NMR,  (ppm) HMBC Cn crosses with Hn HSQC Hn crosses with Cn - - - C1 153.79 H3a, H3’, H2 - H2 2.64 (m, 1H) C2’, C1 C2 52.69 H(=N-NH), H3 H2xC2 - - C2’ 134.91 H9’, H2, - H3 4.28 (d, J5, 1H) Cx, C2, C5, C9 C3 72.03 - H3xC3 H3’ 6.95 (s, 1H) C1, C5’ C3’ 123.84 H5’,H9’ H3’xC3’ H3a 7.28 (d, J 5, 1H) - - - - NHxC2 - - - C4 130.47 H8 - - - - C4’ 130.26 H8’ - H5 7.07 (s, 1H) C7, C9, C6 C5 110.90 H9, H3 H5xC5 H5’ 6.97 (s, 1H) C3’, C9’, C7’, C6’ C5’ 113.57 H3’, H9’ H5’xC5’ - - - C6 149.11 H5, H8 - - C6’ 148.75 H5’, H8’ - - - C7 146.65 H5, H9 - C7’ 146.55 H5’, H9’ H8 6.92 (d, J 8.5, 1H) C4, C6 C8 113.88 - H8xC8 H8’ 6.91 (d, J 8.5, 1H) C4’, C6’ C8’ 114.28 - H9 6.89 (d, J8, 1H) C5,C6, C2 C9 119.02 H5 H9xC9’ H9’ 6.88 (d, J8, 1H) C5’,C3’, C2’,C7’ C9’ 121.88 H5’ H10 3.79 (s, 6H) C7, C7’ C10 C10’ 55.54 55.58 - H10xC10 H10’ Characterization and bio-activity evaluation of two new acetohydrazides synthesized from curcumin 19 H11 4.45 (s, 2H) C7, C12 C11 67.62 H8 H11xC11 H11’ 4.47 (s, 2H) C7’, C12’ C11’ 67.35 - - - C12 166.8 H13, H11 - C12’ 166.68 H13’, H11’ Hya 1.35 (d, J12.5, 1H) - - - - Hya x Cy Hye 1.84 (d, J11.5, 1H) - Cy 23.90 - Hye xCy Hxe 2.85 (d, J15, 1H) - - - - Hxe x Cx Hxa 1.9 (s, 1H) Cx Cx, Cx’ 27.98 - Hxa x Cx Hx’e 2.39 (t, J 13.5, 1H) Cx’ - - - Hx’e x Cx Hx’a 1.47 (d, J12, 1H) - - - - Hx’a x Cx H13 H13’ 9.15 (s, 1H) 9.13 (s, 1H) - - - - - H14 H14’ 4.32 (s, 4H) - - - - - H3a 7.28 (d, J 5, 1H) - - - - NHxC2 * Biological test Both acetohydrizeds A3 and B3 were screened. 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