Synthesis , Antioxidant , and Anti-inflammatory Activity of Morpholine Mannich base of AMACs ( ( 2 E , 6 E )-2-( { 4-hydroxy-3-[ morpholin-4-yl-) methyl ] phenyl } methylidene )-6-( phenylmethylidene ) cyclohexan-1-one ) and Its Analogs

© 2018 Titah Nidya Putri et al. This is an open access article distributed under the terms of the Creative Commons Attribution License -NonCommercialShareAlikeUnported License (http://creativecommons.org/licenses/by-nc-sa/3.0/). *Corresponding Author dan Hayun Hayun, Faculty of Pharmacy, Universitas Indonesia, Depok 16424, West Java, Indonesia. E-mail: hayun.ms @ ui.ac.id Synthesis, Antioxidant, and Anti-inflammatory Activity of Morpholine Mannich base of AMACs ((2E, 6E)-2-({4-hydroxy-3[morpholin-4-yl-)methyl]phenyl}methylidene)-6-(phenylmethylidene) cyclohexan-1-one) and Its Analogs


INTRODUCTION
Curcumin, ((1E, 6E)-1,7-bis(4-hydroxy-3methoxyphenyl)hepta-1,6-diene-3,5-dione), is the active principle of Curcuma sp.The compound is well-documented to exhibit good biological activities with low toxicity as well as antioxidant, anti-inflammatory, and others.However, due to its low stability and bioavailability, its clinical application is limited (Anand et al., 2008;Wang et al., 1997;Rosemond et al., 2004;Grogan, 2005).Many curcumin analogs have been synthesized and investigated, such as mono-carbonyl analogs of curcumin (MACs), to improve the bioactivities, stability, and bioavailability.The compound and its analogs containing a cyclohexanone or cyclopentanone linker between the two phenyl rings exhibited greater antiinflammatory and antioxidant activity and a higher stability and a better pharmacokinetic profile than that of curcumin (Zhao et al., 2013;Lamperti et al., 2014).The structures of the above MACs are generally symmetric.Nowadays, some asymmetrical monocarbonyl of curcumin (AMACs) have been synthesized and among of the AMACs exhibited potent anti-inflammatory (Zhang et al., 2014a;Zhang et al., 2014b;Aluwi et al., 2016), antioxidant and antitumor (Li et al., 2015).The reports suggest that the AMACs are good lead compounds to be developed for discovering antiinflammatory and antioxidant agents.
Introduction of aminoalkyl substituent via Mannich base reactions are known to increase the biological activity of compounds.The Mannich base serves as an important pharmacophore moiety to potentiate the biological activity of the drug (Bala et al., 2015).Ibuprofen-containing morpholine Mannich base substituent exhibited higher anti-inflammatory activity than that of diclofenac.The larger structure of the compound making it easier to interact with cyclooxygenase-2 having a larger active site (Sujith et al., 2009).Meanwhile, quercetin containing morpholine Mannich base substituent showed the highest antioxidant activity compared to quercetin, and quercetin containing piperazine Mannich base, and quercetin containing dopamine Mannich base (Joshi et al., 2013).However, the study of the Mannich bases of AMACs as an antioxidant and anti-inflammatory agent has never been reported.Therefore, herein we report the synthesis, antioxidant and anti-inflammatory activity of morpholine Mannich base of AMACs.

EXPERIMENTALS Materials and instruments
All chemicals were reagent-grade and purchased commercially and used without purification.The purity of the synthesized compounds was tested by TLC method on silica gel 60 F254 plates (Merck).Melting points were determined in the capillary tube using melting point apparatus (Analogue Model SMP11, Stuart Scientific) and are uncorrected.The IR spectra were recorded on an FT-IR spectrophotometer (8400S, Shimadzu).The NMR spectra were recorded on NMR spectrometer (Agilent) at 500 MHz for 1 H and 125 MHz for 13 C using TMS as an internal standard.High-resolution mass spectra (HR-MS) were recorded on a Waters LCT Premier XE (ESI-TOF) system in negative mode.
A solution of the AMACs (3a-f) (2 mmol) in ethanol (5 ml) was cooled in the ice bath, added morpholine (5-7 mmol) and formaldehyde solution 37% (5-7 mmol) and stirred for 30 min at room temperature.Subsequently, the mixture was refluxed for 3-7 h until the starting material disappeared, which was monitored by TLC.The solvent of the mixture was evaporated and methanol (40 ml) was added and subsequently evaporated.A colored precipitate obtained was filtered off, washed with cold methanol and dried at room temperature and purified by column chromatography to afford compound 4a-f.

Anti-inflammatory Activity
The title compounds (4a-f) and the parent compound AMACs (3a-f) were screened for anti-inflammatory activity using inhibition of albumin denaturation technique reported previously (Saso et al., 2001), with slight modification.Bovine Saline Albumin (BSA) solution 0.5% (w/v) was prepared in Tris-buffer saline and adjusted pH to 6.3 using glacial acetic acid.The reaction mixtures (5 ml) consisted of 0.5 ml of varying concentrations of standard diclofenac sodium or test compounds in methanol and 4.5 ml of the BSA solution were heated for 10 minutes at 70°C ± 2 in a test tube placed in water bath, then cooled and its turbidity measured at 660 nm and absorbance read using UV-Vis Spectrophotometer in 1 ml cuvettes in three replicates.The control was prepared as above and a similar volume of methanol was used instead of the test compounds.The percentage inhibition was calculated by using following formula: The capacity of the compound to inhibit the denaturation was calculated by plotting percentage inhibition to control against concentration and expressed as an IC 50 value.
The synthesized compounds (4a-f) were characterized by IR, 1 H-NMR, 13 C-NMR dan mass spectra.The IR spectra of compounds 4a-f showed the bands of C-H aromatic at 3,044-3,080 cm −1 and C-H aliphatic at 2913-2980.The α,β-unsaturated carbonyl groups, the C=C aromatic or ethylenic and C-O-C and C-N of the compounds are observed as strong bands at 1,732-1,734 cm −1 , 1,447-1,665 and 1,140-1,265 cm −1 , respectively.In the 1 H-NMR spectra, the two protons of the ethenyl chain of the compounds are observed as two singlets at a range of 7.67-7.78ppm ( 1 H, respectively) indicating the asymmetrical of the structure.The protons of methylene connecting N of morpholine to phenyl ring (Ar-CH 2 -N-) are observed as a singlet at 3.74-3.77ppm, while the four protons of CH 2 connected to N (CH2-CH 2 -N-) and the four protons of CH 2 connected to O (CH2-CH 2 -O-) in the morpholine skeleton are observed as triplet at 2.58-2.61ppm and 3.75-3.77ppm, respectively (Silverstein et al., 2005).The structures were further supported by 13 C-NMR and HR-ESI-MS spectra of the compounds which showed the complete agreement with the desired molecular structures.

Antioxidant Activity
The antioxidant activity of the title compounds (4a-f) and the parent compound AMACs (3a-f) were evaluated using 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenger method.The choice of the method because it is fast, easy, sensitive, reliable and does not require a special reaction and device (Soare et al., 1977;Blois, 1958).The antioxidant compound reacts with DPPH radical through the hydrogen atom donation mechanism and producing the alteration of DPPH's color from violet to yellow measured by visible spectrophotometer (Aksoy et al., 2013;Blois, 1958).The result of the antioxidant activity of the synthesized compounds (3a-f and 4a-f) are shown in Table 1 and Figure  1.The data exhibited that the AMACs containing morpholine Mannich base group showed lower antioxidant activity than that of the parent compounds, AMACs, except for compound 4e and 4f.The result is in line with the effect of substitution of morpholine Mannich base to the antioxidant activity of cyclovalone reported previously (Hayun et al., 2017).All the synthesized compounds showed lower antioxidant activity than that of the symmetrical MAC, cyclovalone, and quercetin.Compound 4f, the AMAC without methoxy group at the ortho position of the hydroxyl group, showed the lowest antioxidant activity.These results show the important role of the hydroxyl and the methoxy groups for antioxidant activity as earlier reported (Li et al., 2015).However, compound 3d, the AMACs having a p-fluor group, showed antioxidant activity, which is comparable to cyclovalone (IC 50 68.9µM).

Anti-inflammatory Activity
The anti-inflammatory activity of the title compounds (4a-f) and the parent compound AMACs (3a-f) were evaluated using inhibition of heat-induced albumin denaturation method.
Protein denaturation in vivo (occurred in certain rheumatic diseases) stimulates the production of autoantigens stimulating inflammation (Umapathy et al., 2010;Grant et al., 1970).Several anti-inflammatory drugs have shown the ability to inhibit heatinduced albumin denaturation (Grant et al., 1970).A fluorinated phenyl styryl ketone inhibited of heat-induced bovine serum albumin (BSA) denaturation and possess anti-inflammatory (Nargund et al., 1992).Although there was no complete correlation, several compounds of acetamido[(phenyl-4'-yl)oxymethyl)]-2-(p-substituted phenylamino)-1,2,4-triazoles and -1,3,4-thiadiazoles which showed good inhibition of denaturation also showed significant anti-inflammatory activity by carrageenaninduced edema in the rat paw (Nargund et al., 1993).The results of the anti-inflammatory activity evaluation of the synthesized compound were presented in Table 2 and Figure  All the AMACs containing morpholine Mannich base substituent showed inhibition of BSA denaturation activity higher than that of the parent compounds, AMACs; indicating that substitution of a morpholine Mannich base increased the activity of the parent compounds.In addition, the presence of methoxy at the ortho position of the hydroxyl group of the compound, revealed significant contribution towards anti-inflammatory activity as shown in compounds 3f and 4f compared to 3a and 4a, respectively.

CONCLUSION
In summary, we successfully synthesized a series of asymmetrical mono-carbonyl analogs of curcumin (AMACs) containing morpholine Mannich base ((2E, 6E)-2-({4-hydroxy-3-[morpholin-4-yl-)methyl]phenyl}methylidene)-6-(phenylmethylidene)cyclohexan-1-one).All the compounds and the parent compounds, AMACs, exhibited antioxidant and antiinflammatory activity.The presence of morpholine Mannich base substituent in the AMACs enhanced the inflammatory activity of the compounds but generally lowered their antioxidant activity.The results suggest that newly synthesized compounds are potential agents to be used in the treatment of inflammatory diseases.However, further studies are required to investigate the mechanism of the biological activity of the compound as an antiinflammatory agent.

2 .
The data revealed that all the synthesized compound exhibited inhibition of BSA denaturation with the range of IC 50 = 25.3-80.1 µM.All the synthesized compounds showed lower activity than that of symmetrical MAC, cyclovalone.However, the compounds 4c and 4d, the AMACs having morpholine Mannich base substituent on the phenolic moiety and methoxy or fluoro substituent at para position on another phenyl ring of the compounds, exhibited a potent anti-inflammatory activity (IC 50 = 25.3 µM and 26.3 µM, respectively), which almost comparable to cyclovalone (IC 50 = 22.4 µM) and the standard diclofenac sodium (IC 50 = 20.3µM).

Table 1 :
The free radical scavenging activity (IC 50 ) of the title compounds (4a-f) and the parent compounds (AMACs,

Table 2 :
The inhibition of heat-induced BSA denaturation activity (IC 50 ) of the title compounds (4a-f) and the parent compounds (AMACs,