Synthesis and Preliminary In Vitro Anti-inflammatory Evaluation of Mannich Bases Derivatives of 4’-Methoxy-substituted of Asymmetrical Cyclovalone Analogs

Two of Mannich bases derivatives of 4’-methoxy-substituted of asymmetrical cyclovalone analog (ACA) ( 2a and 2b ) were synthesized. The synthesized compounds and the other two Mannich bases derivatives of 4'-methoxy-substituted ACA ( 2c and 2d ) were evaluated for their in-vitro antiinflammatory activity preliminary by protein denaturation inhibition method using a final concentration of 1.57 μM. The study found that all the Mannich bases exhibited anti-inflammatory potential with inhibition ranging from 33.17- 42.47%. The activity of 2b (42,47%) and 2d (41.90%) was higher than that of diclofenac sodium (35.27%) and the parent compound 1 (38.16%). As a conclusion, 2b and 2d have a prospect as a potential candidate for an anti-inflammatory agent. Further study should be done using more specific methods.


INTRODUCTION
Curcumin is well-documented to have antiinflammatory activity with low toxicity. However, clinical usage of the compound is limited due to its stability and bioavailability (Prasad et al. 2014;Anand et al., 2008;Wang et al., 1997). The monocarbonyl analogs of curcumin (MACs), such as cyclovalone, showed a more stable chemical structure and better pharmacokinetic profile. Several of them were more active as antiinflammatory agents than curcumin. (Liang et al., 2008;Zhao et al., 2013;Lamperti et al., 2014;Zhao et al., 2015)..

Synthesis of Mannich Bases derivatives of 4'methoxy ACA (2a and 2b)
The compounds 2a and 2b were synthesized using the method for preparation of 2c and 2d reported previously (Prasetyaningrum et al., 2018, Putri et al., 2018. A cooled solution in ethanol of 1 (2 mmol) was stirred and added a solution in ethanol of dimethylamine (for 2a) or 1methylpiperazine (for 2b) (6 mmol) and formaldehyde solution (6 mmol). The mixture was further stirred for 30 minutes at r.t., then refluxed for 3 h (for 2a) and 6 h (for 2b) until the reaction was completed (TLC monitoring). After that, the products were isolated and then purified using a column chromatography technique to get 2a and b.

Preliminary
in-vitro anti-inflammatory Evaluation.
The evaluation was done using inhibition of albumin denaturation technique, as reported previously with minor modification (Putri et al., 2018). Mixture of 0.5mL of standard or test compounds solution in methanol (15.72 uM) or solvent (for control) and 4.5mL Bovine Saline Albumin (BSA) 0,5% in tris-buffer saline pH 6.3 was incubated at 37°C for 15min, then heated at 70°C in a water bath for 5min, and cooled to reach room temperature. The turbidity was measured at 660nm. The inhibition (%) of the denaturation was calculated with the formula:

% inhibition = [Ac -
As ] x 100% Ac where Ac was absorbance of control; As was absorbance with sample addition.

Chemistry
Scheme of the synthesis of compound 2a-b (Figure 2). Figure 2. Scheme of the synthesis of Mannich bases derivatives of 4'-methoxy-substituted ACA Infrared, 1 proton, and 13 carbon NMR and mass spectral data obtained were presented in Table II. The infrared spectra 2a and 2b (Figures 3) showed the bands of C-O-C and C-N at 1,246-1.023cm -1 ; C-H aliphatic at 2.935-2.829, while the a,ß-unsaturated carbonyl groups, the C=C aromatic or ethylenic and alkyl were observed as strong band peaks at 1.658-1.507 and 1.459cm -1 , respectively. The 1 proton NMR (Figures 4), the protons of methylene connecting N of dimethylamine or N-methylpiperazine to aromatic ring appeared as a singlet at 3.71 ppm. Six protons from dimethylamine group in 2a appeared as singlet peak at 2.34 ppm, eight protons from piperazine group in 2b appeared as multiplet peak at 2.99ppm, and three protons of N-methylpinerazine group in 2b appeared as a singlet at 2.31ppm. While, the two protons from the ethenyl chain appeared as two singlets at 7.61-7.67ppm (1H, respectively) confirming the structures were asymmetric (Silverstein et al., 2005). The structures elucidation was supported further by 13 carbon NMR ( Figures 5 and mass spectra (Figures 6). All data confirmed full agreement with the structures expected.

In-vitro Anti-inflammatory activity
Protein denaturation in vivo is one of the causes of inflammation. One of the mechanisms of antirheumatic activity of NSAIDs is by inhibiting this denaturation (Umapathy et al. 2010;Gunathilake et al., 2018). Denaturation of protein can be induced by UV or heat. The denaturation is making protein aggregation, which can lead to the production of superoxide and nitric oxide by macrophages, which stimulates inflammation (Guzik et al., 2003). Compounds inhibiting the heatinduced protein denaturation are considered to have potential anti-inflammatory activity (Chandra et al. 2012;Jagtap et al., 2011).  The preliminary activity of the synthesized compounds (Table II and Figure 7). At a final concentration of 1.57μM indicated that all the Mannich bases inhibited the heat-induced protein denaturation ranging from 33.17-42.47%. This study found that the activity of 2b methyl-4'-methoxy ACA] (42.47%) and 2d (5-morpholinomethyl-4'methoxy ACA) (41.90%) was higher than that of diclofenac sodium (35.27%), the parent compound 1 (38.16%), and cyclovalone (19.64%). It was in line with the result of the introduction of the Mannich bases to dehydrozingerone reported earlier . Therefore, the compounds have a prospect as a potential candidate for an anti-inflammatory agent. However, to ensure their biological activities, further study should be done using more specific methods.

CONCLUSION
Two Mannich base derivatives of asymmetrical cyclovalone analogs (ACA) were synthesized successfully. Their preliminary in-vitro anti-inflammatory evaluation indicated that the compound 2b [5-(N-methylpiperazino)methyl-4'methoxy ACA] and 2d (5-morpholinomethyl-4'methoxy ACA) exhibited the highest activity. Their biological activities were higher than diclofenac sodium. However, further study should be done using more specific methods.