SYNTHESIS AND BIOLOGICAL ACTIVITY EVALUATION OF SOME DERIVATIVES SYNTHESIZED FROM CURCUMIN AND CURCUMIN ANALOG

The acetohydrazides A5 containing an isoxazole ring and B5 containing an indazole ring were synthesized from the corresponding acetohydrazide derivatives with acetic anhydride in about 80% high yield. Also, two acetohydrazones B6 and B7 were driven from acetohydrazide B4 by condensation reaction. Bioactivity testes showed that none of them were active against on bacteria except acetohydrazones  B7 showing moderate reactivity against KB cancer cell line at IC 50 = 57 mg/L.


Introduction
In our previous work, the modification of curcumin and monocarbonyl curcumin analog with heterocyclic bridges -oxazole or indazole rings and the pharmacophore groups did not give any improvement of bioactivity for these types of derivatives, Figure 1 [1,2]. Interestingly, according to literature, an acetohydrazone contains a hydrogen bonding domain (HBD); acetyl acetohydrazide contains two hydrogen bonding domains [3] that can excess DNA easily giving higher potencies for bioactivities. For example, hydrazone derivatives I, II and III worked as an anticonvulsant, antimicrobial and antimycrobacterial compounds, Figure 2 [4,5]. More examples can be found in review article of Sevim Rollas and Ş. Güniz Küçükgüzel [6]. Based on the literature, therefore, in this work, the target compound structures were designed as shown in the Figure 2. The amide group was kept to work as an HBD and the -NH2 groups were acetylated or condensed with aldehydes to improve bioactivities.

2
Experimental section

General
Solvents and other chemicals were purchased from Sigma-Aldrich, Merck and used as received, unless indicated. The 1 H NMR and 13 C NMR spectra were recorded on the Bruker Avance 500 NMR spectrometer in deuterated solvents. Chemical-shift data for each signal was reported in ppm units. IR spectra were recorded on the Mattson 4020 GALAXY Series FT-IR. Mass spectra were obtained from Mass Spectrometry Facility of Vietnam Academy of Science and Technology on LC-MSD-Trap-SL spectrometer.

Synthesis procedure
General procedure of acetylation [8]: To a solution of acetohydrazide A4 or B4 in DMF was added triethylamine and acetic anhydride. The resulting solution was stirred at room temperature for 1 h. The progress of reaction was monitored with TLC (MeOH/ DCM = 1/19). Then the mixture was diluted with water to form solid. Re-crystallization in DMSO/ water (1/2) gave pure products.

Bioactivity test
Bioactivity tests were followed by the Broth dilution method [7]. A5 and B7 were selected for bacterial test including Gram (+) (Staphylococcus aureus, Bacillus subtilis, Lactobacillus fermentum) and Gram (-) (Salmonella enteric, Escherichia coli, Pseudomonas aeruginosa) and fungal test (Candida albican). B7 was also selected for anticancer test with KB cancer cell line. All tests were screened in the Laboratory of Applied Biochemistry of Vietnam Academy of Science and Technology.

Results and discussion
Acetylation of A4 was not only occurred at -NH2 of the hydrazide groups but also at nitrogen of the indazole ring to form A5 with three acetyl amide groups. Meanwhile, the acetylation of acetohydrazide using B4 gave compound B5 with two acetyl amide groups in high yield expectedly, Fig. 3.

Fig. 3. Acetylation of acetohydrazides A4 and B4
Classic method was used to make hydrazones B6 and B7 when acetohydrazide B4 was condensed with some substituted aldehydes. Unfortunately, only substituted aldehydes which contained a nitro group drove the expected products B6 and B7. That might be explained by the increasing reactivity of these aldehydes bearing strong withdrawing electron groups as nitro group. Many other aldehydes such as p-methoxybenzaldehyde; p-hydroxylbenzaldehyde … did not give the designed products. Structures of A5, B5, B6 and B7 were first determined with IR spectroscopy method. IR spectra of all showed the vibration of N-H bond. Since each compound had at least two N-H bonds therefore, their IR spectra indicated many peaks in range of 3200-3500 cm -1 . Comparing with results published in our previous paper [1], the vibration of >C=O (CH3C=O) was larger than that of O=C-NH-NH2 group. So A5 and B5 showed two vibrations of two types of >C=O bonds. For example, IR spectrum of A5 showed vibrations at 1733 cm -1 and at 1654 cm -1 . IR spectra of B6 and B7 had one vibration of two >C=O groups. The formation of hydrazonyl groups increased wavelength number about 10 cm -1 . Other vibrations agreed with the expected structures (see experimental section). A5, B5, B6 and B7 were studied MS spectra, B5 and B6 showed the peaks of pseudo molecular ions, unfortunately, A5 and B7 did not. This method indicated molecular weight of B5 was 593 g/mol and molecular weight of B6 was 775 g/mol. 1 H NMR spectra of A5 showed all protons on its structure. In comparison to 1 H NMR spectrum of A4 whose structure was confirmed with 1D, 2D NMR and MS spectra [1], in this case, assignment of all peaks was expressed in the Figure 3. There were three signals assigned for three methyl groups of three acetyl ones. The peak at  2.24 ppm indicated the acetyl group on the nitrogen atom in the indazole ring. Next, the signals at  1.86 ppm belonged to 6 protons of H14 and H14' since they were far from unsymmetric center-indazole ring. This observation agreed with 13 C NMR spectra that showed 6 peaks in high field at  29.0, 28.4, 23. 5 Fig. 6. 1H NMR analysis of B6 1 H NMR spectra of compounds B6 and B7 gave lot of peaks than expected due to they had two isomeric forms of E and Z around the >C=N bonds with ratio about 1/2. To be simple, label from H1 to H19 used instead of accompanying with "prime number" was shown in the about 2H. An extra observation of the existence of two isomers was H11 at  5.23 ppm and at  4.73 ppm. In comparison with our previous work, other protons were assigned as shown in Figure 4. B7 was screened anticancer activity. It was against on KB cancer cell line with IC50 = 57.6 g/mL. However, neither of A5 and B7 showed positive results on bacterial tests.

Conclusion
The modification of curcumin and curcumin analog based on heterocyclic linker and pharmacophore groups gave 4 new derivatives A5, B5, B6 and B7. Acetylation gave compound A5 with three acetyl groups and B5 with two acetyl ones. B6 and B7 were a mixture of E and Z isomers. A5 and B7 did not show any activities against on bacteria or fungi, but B7 was against on KB at IC50 = 57.6 g/mL.