Synthesis, structure and cytotoxic activity against liver cancer cell (HepG2) of some n-Substituted hydrazides containing heterocycle from 5-chloro-2-hydroxybenzohydrazide or 2-hydroxy-5-iodobenzohydrazide

136 HNUE JOURNAL OF SCIENCE DOI: 10.18173/2354-1059.2018-0038 Natural Sciences 2018, Volume 63, Issue 6, pp. 136-143 This paper is available online at SYNTHESIS, STRUCTURE AND CYTOTOXIC ACTIVITY AGAINST LIVER CANCER CELL (HepG2) OF SOME N-SUBSTITUTED HYDRAZIDES CONTAINING HETEROCYCLE FROM 5-CHLORO-2-HYDROXYBENZOHYDRAZIDE OR 2-HYDROXY-5-IODOBENZOHYDRAZIDE Nguyen Tien Cong 1 , Huynh Thi Xuan Trang 1 , Le Thi Thu Huong 1 , Ho Trung Duc 1 , Tran Hoang Phuong 2 , Nguyen Thai The 2 1 Faculty of Chemistry, Ho Chi Minh City University of Education 2 Faculty of Chemistry, Ho Chi Minh City University of Science Abstract. 5-Chloro-2-hydroxybenzohydrazide and 2-hydroxy-5-iodobenzohydrazide were synthesized from 5-chloro-2-hydroxybenzoic acid and methyl 2-hydroxybenzoate (methyl salicylate), respectively. The reaction of these 5-halogeno-2-hydroxybenzohydrazides with appropriate heteryl methyl ketones gave eight new N-substituted hydrazides. The structure of the N-substituted hydrazides has been determined by IR, 1 H-NMR, 13 C-NMR, NOESY and HR-MS spectral data. The results indicated that the imine –C(CH3)=N< group in the molecule of the N-substituted hydrazides exists in Z-configuration. These compounds were also examined bioactivities. Of these compounds, 2d and 2h showed cytotoxicity against HepG2 cells, with IC5 values of 1.5 μg/mL and 1.8 μg/mL, respectively. Keywords: 5-Chloro-2-hydroxybenzohydrazide, 2-hydroxy-5-iodobenzohydrazide, N-substituted hydrazide, configuration, heterocyclic. 1. Introduction Salicylic acid and its derivatives (i.e. aspirin, methyl salicylate, salol, salicylanilide) are known primarily as an analgesic, antipyretic, antioxidant, antimicrobial, antiproliferative and cytotoxic agents [1, 2] widely used in medicine. Hydrazone derivatives or N-substituted hydrazides are molecules containing highly reactive azomethine group (-CO-NH-N=C<), these are found to possess anti-tumoral [3], anti-convulsant [4], anti-microbial [5], analgesic [6], anti-tubercular [7] and anti-inflammatory [8] activities. For example, 4-hydroxybenzoic acid [(5-nitro-2-furyl)methylene]-hydrazide (nifuroxazide) is an intestinal antiseptic [9] and 4-fluorobenzoic [(5-nitrothiophene-2-yl)methylene]hydrazide showed the highest inhibition (99%) antituberculosis activity at a constant concentration level (6.25 μg/mL) [10]. Hydrazones have been described as useful intermediate compounds for synthesizing various heterocyclic rings of different ring sizes with one or several heteroatoms [11]. As a continuation of our studies in synthesis and transformation of hydrazides of 5-halogeno-2-hydroxybenzohydrazide [12-14], herein we report on the synthesis of some N-substituted hydrazides by condensation of 5-chloro-2- hydroxybenzohydrazide or 2-hydroxy-5-iodobenzohydrazide with heteryl methyl ketones. Received June 6, 2018. Revised July 3, 2018. Accepted July 10, 2018. Contact Le Thi Thu Huong, e-mail address: huonghctn@yahoo.com Synthesis, structure and cytotoxic activity against liver cancer cell (HepG2) of some n-substituted hydrazides 137 2. Content 2.1. Experiments General procedures: All chemicals and solvents were obtained from commercial sources and used without any further purification. Melting points were determined in open capillaries and the values are uncorrected. IR spectra were recorded in KBr discs on a Shimadzu FTIR 8400S spectrophotometer. NMR spectra were recorded on a Bruker Avance spectrometer (500 MHz for 1 H-NMR and 125MHz for 13 C-NMR) using DMSO-d6 as solvent and tetramethylsilane (0.00 ppm) as an internal reference. Mass spectra were taken on a Bruker micrOTOF-Q 10187. 5-Chloro-2-hydroxybenzohydrazide (1a) was prepared from 5-cholorosalicylic acid according to the previously reported methods [15]. 2-Hydroxy-5-iodobenzohydrazide (1b) was synthesized from methyl salicylate by using method reported earlier [11]. 5-Chloro-2-hydroxybenzohydrazide (1a): white needles, mp. 174 o C, yield 64%; IR (ν, cm-1): 3403, 3318, 1636, 1582, 718; 1 H-NMR (δ, ppm): 12.47 (1H, br, OH), 10.10 (1H, br, NH), 7.87 (1H, d, 4 J=2.5, ArH), 7.42 (1H, dd, 3 J= 9.0 Hz, 4 J=2.5 Hz, ArH), 6.94 (1H, d, 3 J=9.0 Hz, ArH), 4.72 (2H, br, NH2). 2-Hydroxy-5-iodobenzohydrazide (1b): white needles, mp. 178 oC, yield 79%; IR (ν, cm-1): 3405, 3322, 1626, 1574, 529; 1 H-NMR (δ, ppm): 12.41 (1H, br, OH), 10.12 (1H, br, NH), 8.12 (1H, d, 4 J =2.0, ArH), 7.65 (1H, dd, 3 J = 9.0 Hz, 4 J=2.0 Hz, ArH), 6.75 (1H, d, 3 J =9.0 Hz, ArH), 4.80 (2H, br, NH2); 13 C-NMR: 166.1 (CO), 158.9, 141.3, 135.5, 119.9, 117.4, 80.5. Scheme 1. Synthetic pathway The transformation of the two 5-halogeno-2-hydroxybenzohydrazides into N-substituted compounds by reaction with definite heteryl methyl ketones is outlined in Scheme 1. General procedure for the synthesis of 2-hydroxy-5-iodo-N’-(1-arylethylidene) benzohydrazide compounds (2a-h): Equimolar quantity of hydrazide (1a) or hydrazide (1b) and a definite heteryl methyl ketone was refluxed in ethanol for 2 hours. The reaction mixture was cooled down to room temperature and the precipitate obtained was filtered off and crystallized from dioxane or mixture of DMF and water to give the corresponding products. The physical data and IR, MS spectral data of the prepared N-substituted hydrazide compounds are listed in Table 1, specifically the LC-HR-ESI-MS of 2h (see Figure 1). Nguyen Tien Cong, Huynh Thi Xuan Trang, Le Thi Thu Huong, Ho Trung Duc, Tran Hoang Phuong, Nguyen Thai The 138 Figure 1. The LC-HR-ESI-MS of 2h Bioassay: Cytotoxic assay was carried out as the same method described in [16, 17]. Table 1. Physical and spectral data of the N-substituted hydrazides (2a-h) Comp. (X) Solvent for Cryst. Yield (%) Mp. ( o C) IR,  (cm-1) MS found [Calc.] NH, OH C–H C=O, C=N, C=C C-Cl [C-I] 2a (Cl) DMF:H2O 72 240- 241 3451 3278 3080 2932 1641 1559 711 317.0033 (M+Na) + [317.0127] 2b (Cl) Dioxane 68 259- 260 3289 3070 1682 1597 764 290.0706 (M+H) + [290.0696] 2c (Cl) DMF:H2O 66 258- 259 3444 3278 3055 2936 1641 1554 722 290.0539 (M+H) + [290.0696] 2d (Cl) DMF:H2O 76 243- 244 3440 3299 2932 1634 1577 759 379.0309 (M+Na) + [379.0462] Synthesis, structure and cytotoxic activity against liver cancer cell (HepG2) of some n-substituted hydrazides 139 2e (I) DMF:H2O 79 234- 235 3424 3073 2930 1639 1564 [625] 408.9345 (M+Na) + [408.9484] 2f (I) Dioxane 68 240- 242 3279 3055 2909 1674 1597 [633] 382.0020 (M+H) + [382.0052] 2g (I) DMF:H2O 69 244- 245 3451 3247 3071 2884 1663 1548 [649] 403.9770 (M+Na) + [403.9872] 2h (I) DMF:H2O 78 239- 240 3417 3024 1647 1599 [628] 470.9823 (M+Na) + [470.9818] 2.2. Results and discussion Esterification of 5-chlorosalicylic acid with methanol in the presence of concentrated sulfuric acid gave methyl 5-chlorosalicylate [15]. Methyl 2-hydroxy-5-iodobenzoate was yielded in the addition of NaOCl drop-wise to the solution of methyl salicylate and KI in methanol [11]. The reaction of methyl 5-halogeno-2-hydroxybenzoate compounds including methyl 5-chloro-2-hydroxybenzoate and methyl 2-hydroxy-5-iodobenzoate in excess hydrazine hydrate gave 5-chloro-2-hydroxybenzohydrazide (1a) and 2-hydroxy-5-iodobenzohydrazide (1b), respectively. IR spectrum of 5-halogeno-2-hydroxybenzohydrazides (1a, b) showed absorption around 1630 cm -1 corresponding to C=O stretching vibration of the amide group. Two bands near 3400 and 3320 cm -1 appeared due to the presence of OH and NH2 groups, respectively. The 1 H-NMR spectrum of 5-halogeno-2-hydroxybenzohydrazides displayed a broad singlet in region of 4.80- 4.40 ppm (2H) corresponding to NH2 protons. In each of the spectra, two broad peaks appeared around 12.45 ppm and 10.10 ppm were in turn represented for OH and NHCO protons. The appearance of three signals in the aromatic area with intensity of 1H for each signals including a doublet with 4 J = 2.0-2.5 Hz; a doublet of doublet with 3 J = 9.0 Hz and 4 J=2.0 Hz and a doublet with 3 J = 9.0 Hz in the separate spectra indicated that the benzene rings must have three substituents at positions of 1, 2 and 5. The condensation of the hydrazides with different heteryl methyl ketones such as 2-acetylthiophene, 3-acetylpyridine, 4-acetylpyridine and 3-acetylcoumarin afforded eight N- substituted hydrazide compounds. The reactions occur easily and in almost of cases, an appearance of precipitates can be seen as soon as the solutions are boiling. The molecular structure of the synthesis compounds were established based on analytical and spectral data. The IR spectra of the N-substituted hydrazides showed common characteristic absorption bands at 3323-3236 cm -1 (NH/OH), 1685-1645 cm -1 (NH-C=O), which are similar spectra of the hydrazides (1a) and (1b). However, the absence of those bands due to the NH2 group and the appearance of characteristic absorption bands for the Csp 3–H bonds (in the CH3 group) below 3000 cm -1 provided the evidence for the condensation reaction between hydrazides and heterocyclic ketones. A similar pattern for the proton resonance was also observed in the 1 H-NMR spectrum of N-substituted hydrazides. In the 1 H-NMR spectra of 2a-h compounds, the OH and NH protons were observed at 11.54-10.50 ppm as singlets; the resonance signal of the CH3 protons bonding to the azomethine group appeared at 3.42-2.31 ppm as a singlet while the remaining protons appeared at the aromatic area, matching with the characteristic of the molecules. Nguyen Tien Cong, Huynh Thi Xuan Trang, Le Thi Thu Huong, Ho Trung Duc, Tran Hoang Phuong, Nguyen Thai The 140 Table 2. The 1 H-NMR spectral data (δ, ppm and J, Hz) of the N-substituted 5-halogeno-2-hydroxybenzohydrazides Comp. (X) Thiophen-2-yl Pyridin-3-yl Pyridin-4-yl Coumarin-3-yl X = Cl X = I X = Cl X = I X = Cl X = I X = Cl X = I H 2a 12.04 (s) 12.01 (s) 12.06 (s) 12.03 (s) 12.10 (s) 12.07 (s) 12.07 (s) 12.07 (s) H 3 7.06 (d, J = 8.5) 6.86 (d, J = 8.5) 7.07 (d, J = 8.0) 6.88 (d, J = 8.5) 7.07 (d, J = 9.0) 6.88 (d, J = 8.5) 7.07 (d, J = 7.0) 6.89 (d, J = 8.5) H 4 7.47 (dd, J = 8.5, J = 2.5) 7.71 (dd, J = 8.5, 2.0) 7.50 (m) 7.72 (d, J = 7.5) 7.49 (dd, J = 9.0, 2.5) 7.74 – 7.69 (m) 7.50 (m) 7.73 (d, J = 8.5) H 6 7.92 (d, J = 2.5) 8.19 (d, J = 2.0) 7.94 (d, J = 2.5) 8.21 (s) 7.93 (d, J = 2.0) 8.21 (d, J = 1.5) 7.92 (m) 8.22 (d, J = 2.0) H 8 11.24 (s) 11.19 (s) 11.40 (s) 11.35 (s) 11.45 (s) 11.41 (s) 11.39 (s) 11.39 (s) H 9a 2.38 (s) 2.36 (s) 2.39 (s) 2.37 (s) 2.35 (s) 2.33 (s) 2.30 (s) 2.29 (s) H 11 7.56 (d, J = 2.5) 7.55 (d, J = 3.0) 9.03 (d, J = 2.0) 9.01 (s) 7.79 (d, J = 5.0) 7.78 (d, J = 5.0) - - H 12 7.13 (dd, J = 4.5, J = 4.0) 7.15 – 7.08 (m) 8.63 (d, J = 3.5) 8.62 (s) 8.67 (d, J = 5.0) 8.65 (d, J = 4.5) - - H 13 7.65 (d, J = 5.0) 7.63 (d, J = 5.0) 7.50 (m) 7.48 (dd, J = 7.0, 4.8) 8.67 (d, J = 5.0) 8.65 (d, J = 4.5) 7.50 (m) 7.48 (d, J = 8.0) H 14 - - 8.22 (d, J = 8.0) 8.21 (s) 7.79 (d, J = 5.0) 7.78 (d, J = 5.0) 7.69 (dd, J = 7.0) 7.69 (t, J = 7.5) H 15 - - - - - - 7.42 (m) 7.42 (t, J = 7.5) H 16 - - - - - - 7.92 (m) 7.93 (d, J = 7.5) H 18 - - - - - - 8.30 (s) 8.30 (s) Synthesis, structure and cytotoxic activity against liver cancer cell (HepG2) of some n-substituted hydrazides 141 Table 3. 13 C-NMR spectral data (δ, ppm and J, Hz) of the N-substituted 5-halogeno-2-hydroxybenzohydrazides Comp. (X) Thiophen-2-yl Pyridin-3-yl Pyridin-4-yl Coumarin-3-yl X = Cl X = I X = Cl X = I X = Cl X = I X = Cl X = I C 1 120.1 121.0 120.2 121.0 120.2 121.0 120.1 121.0 C 2 155.8 156.8 155.9 156.8 155.8 156.8 155.7 154.0 C 3 119.4 120.1 119.4 120.1 119.4 120.2 119.6 120.2 C 4 133.3 141.8 133.4 142.0 133.5 142.1 133.5 142.0 C 5 123.8 82.2 123.8 82.2 123.9 82.2 123.9 82.2 C 6 129.7 138.8 130.1 139.0 130.2 139.1 130.2 139.1 C 7 160.9 160.8 161.3 161.2 161.3 161.2 164.2 161.1 C 9 150.2 150.1 150.7 150.6 150.7 150.6 151.5 150.5 C 9a 14.8 14.8 14.3 14.3 13.8 13.8 16.2 16.2 C 10 143.4 143.4 133.9 133.9 145.3 145.3 123.4 123.3 C 11 130.0 129.7 148.1 148.1 121.0 121.1 159.8 159.8 C 12 129.0 128.9 151.3 151.3 150.5 150.5 142.3 142.3 C 13 128.1 128.1 124.0 124.0 150.5 150.5 116.5 116.5 C 14 - - 134.3 134.3 121.0 121.1 128.9 128.3 C 15 - - - - - - 125.4 125.3 C 16 - - - - - - 129.8 129.8 C 17 - - - - - - 119.3 119.3 C 18 - - - - - - 133.0 133.0 In the 13 C-NMR spectra, signals are clearly observed for all carbon atoms. The 1 H-NMR and 13 C-NMR data for 2a-h compounds were presented in Tables 2 and 3, respectively. In order to determine the configuration of the reported compounds, the NOESY spectrum of representative N-substituted hydrazides 2h was analyzed. However, there were not any different protons made cross peaks in the NOESY spectrum of this compound. It would be explained by the fact that the two groups (NH and CH3) are on opposite side of the double bond. This is similar to Nguyen Tien Cong, Huynh Thi Xuan Trang, Le Thi Thu Huong, Ho Trung Duc, Tran Hoang Phuong, Nguyen Thai The 142 characteristic of spectra of the N’-(1-arylethylidene)-2-hydroxy-5-iodobenzohydrazide compounds [14]. Therefore, imine –C(CH3)=N< group of the N-substituted hydrazide may exist in the Z- configuration. Eight compounds 2a-h were tested for their cytotoxic activity (Table 4). Compound 2d and compound 2h show 1.5 μg/ml and 1.8 μg/ml of IC50 values against the human liver cancer HepG2 cell line respectively. Particularly, both of these compounds are derivatives of coumarin. Table 4. Cytotoxic activity of compounds 2a-h Compound HepG2 Compound HepG2 In vitro Test Cell survival (%) IC50 (µg/ml) In vitro Test Cell survival (%) IC50 (µg/ml) DMSO 100 - 2d 0 1.5 Ellipticine 1.340.8 2e 96.742.0 - 2a 86.453.0 - 2f 91.431.5 - 2b 94.472.1 - 2g 99.100.6 - 2c 89.642.8 - 2h 12.791.0 1.8 3. Conclusion Eight novel N’-(1-hetarylethylidene)-5-halogeno-2-hydroxybenzohydrazide compounds were synthesized by reaction of 5-chloro-2-hydroxybenzohydrazide or 2-hydroxy-5- iodobenzohydrazide and appropriate hetaryl methyl ketones. The structures of the synthesized N- substituted hydrazides were determined by IR, 1 H-NMR, 13 C-NMR, NOESY and HR-MS spectral data. The reported hydrazones exist in the Z-configuration. Compounds 2d and 2h showed cytotoxic activity against the human liver cancer HepG2 cell line. Acknowledgement. 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