Synthesis and biological evaluation of coumarin acetamide derivatives

 

Kalimoddin I. Momin¹, Vikas B. Suryawanshi²,Abhay S. Bondge³,Jairaj K. Dawale⁴*

¹Rajarshi Shahu College, Latur, Maharashtra-413512, India.

²Department of Chemistry, KMC College, Khopoli, Maharashtra- -410203 India.

³Shivneri College, Shirur Anantpal, Dist. Latur, Maharashtra- -413544, India.

⁴Research Laboratory for Pure and Applied Chemistry, M. M. College, Nilanga, Dist. Latur, MH- 413521, India.

*Corresponding Author E-mail: amritkund_jk@rediffmail.com

An efficient route has been developed for the synthesis of coumarin derivatives by green process, the ecofriendly protocol allows the reaction condition smooth. The diverse scope of salicylaldehyde has shown and synthesized intermediates showed potent inhibition of Antimicrobial activity. The synthesized derivatives characterized well by using spectroscopic techniques NMR, mass and IR analysis. This protocol allows diverse modification as well synthesis of coumarin acetamide derivatives.

 

 

 

 

INTRODUCTION:

Coumarin is a natural product which was isolated from tonka beans and sweet clover in 1820. The name ‘Coumarin’ comes from  Coumarou which is the French term of tonka bean. This moiety is classified as a member of the benzopyrone family which consist of a benzene ring joined to a pyrone ring. Coumarin can also be found in other plants like vanilla grass, sweet grass, sweet woodruff, cassia cinnamon etc.¹ The Coumarin smells sweet and bitter in taste. So, it helps to plant from predation. Predator of these plants generally avoids those for the smell. Coumarin firstly synthesized artificially in 1868 and it also have so many application in pharmaceutical, materials and cosmetics. There are so many methods of preparing Coumarin from salicyaldehyde, among them Perkin’s reaction ²and Pechmann condensation³ are well known.

Application of Coumarin Derivatives:

a) Application in Medicine:

The coumarins are of great interest due to their pharmacological properties. In particular, their physiological, bacteriostatic and anti-tumour activity makes these compounds attractive. Some coumarin derivatives have anticoagulant activity.⁴ Warfarin drug which is similar to dicoumarol has been synthesized from coumarin. Vitamin-K has important role in blood clotting and dicoumarol reduces the amount of vitamin-K dependent clotting proteins in blood. This is because dicoumarol interferes with vitamin K reductase enzyme in the liver and the liver is unable to reactivate vitamin K, which leads to a decrease in vitamin K dependent clotting proteins.

 

Figure 1: Coumarin containing drugs in the market.

 

Besides of this, some derivatives of coumarins can be used as anticancer⁵ or anti tumour activity agents because they have the ability to prevent cell growth. It is also reported that coumarin has favourable effect in the treatment of radiogenic sialadentis and mucositis.⁶

 

b) Application in Materials:

Though coumarin has significant role in medicinal chemistry, but in past year it is also used in dye sensitized solar cell (DSCs), organic solar cell (OSCs) and dye lasers. The main property of this DSCs and OSCs materials is, they has two part donor and acceptor. Donor part is an electron rich part and acceptor part is electron deficient part and these two parts share electron by conjugation. Coumarin based moieties are used in donor part. These type of coumarin usually have nitrogen containing substitution at 7^th carbon.

 

 

 

Coumarin derivative also used in perfume and fabric conditioner. It also used as aroma enhancer in tobacco pipe and alcohol, though it is banned in food substances.

 

EXPERIMENTAL SECTION:

All commercial reagents and solvents were used without additional purification. Analytical thin layer chromatography (TLC) was performed on pre-coated silica gel 60 F₂₅₄ plates. Visualization on TLC was achieved by the use of UV light (254 nm). Column chromatography was undertaken on silica gel (100‒200 mesh) using a proper eluent system. NMR spectra were recorded in chloroform-d and DMSO-d₆ at 300 or 400 or 500 MHz for ¹H NMR spectra and 75 MHz or 100 or 125 MHz for ¹³C NMR spectra. Chemical shifts were quoted in parts per million (ppm) referenced to the appropriate solvent peak or 0.0 ppm for tetramethylsilane. The following abbreviations were used to describe peak splitting patterns when appropriate: br = broad, s = singlet, d = doublet, t = triplet, q = quartet, sept = septet, dd = doublet of doublet, td = triplet of doublet, m = multiplet. Coupling constants, J, were reported in hertz unit (Hz). For ¹³C NMR chemical shifts were reported in ppm referenced to the center of a triplet at 77.0 ppm of chloroform-d and 40.0 ppm center for DMSO-d_6.

 

General procedure for preparation of compounds:

1) General Procedure Synthesis of Salicyldehyde 3 :

Paraformaldehyde (32.37 mmol., 3 equv.), trietylamine (32.37 mmol., 3 equv.) and magnesium chloride (21.58 mmol., 2 equv.) were added in substituted phenol(10.79 mmol., 1 equv.) in a round bottom flask. 30 mL of THF was added in it as solvent and kept the flask at 80°C under N₂ atmosphere for 8 hours. After completion of the reaction, some amount of HCl was added in it and washed the reaction mixture with ethyl acetate (2 X 30 mL). The organic portion was dried with sodium sulphate. Then it was concentrated and purified by the column chromatography with silica gel. The desired product was come out with 30% ethyl acetate in hexane.

 

2) General Procedure for coumarin acetamide derivatives 5:

N-acetyl glycine ( 6.39 mmol., 1 equv.) and sodium acetate (12.78 mmol., 2 equv.) were added in substituted salicyaldehyde (6.39 mmol., 1 equv.) in a one neck round bottom flask. The flask was kept in 120°C under N₂ atmosphere for 5 hours. After completion of the reaction, the reaction mixture was washed with ethyl acetate and dried with sodium sulphate. The organic portion was collected and concentrated. Then it was purified by column chromatography. The product was come out with 40% ethyl acetate in hexane. All the synthesized compound were characterized by NMR and Mass analysis

 

In-vitroMycobacterium tuberculosis MABA assay

The inoculum was prepared from fresh LJ medium re-suspended in 7H9-S medium (7H9 broth, 0.1% casitone, 0.5% glycerol, albumin, dextrose, supplemented oleic acid, and catalase [OADC]), adjusted to a McFarland tube No. 1, and diluted 1:20; 100 µl was used as inoculum. Each drug stock solution was thawed and diluted in 7H9-S at four-fold the final highest concentration tested. Serial two-fold dilutions of each drug were prepared directly in a sterile 96-well microtiter plate using 100 µl 7H9-S. A growth control containing no antibiotic and a sterile control were also prepared on each plate. Sterile water was added to all perimeter wells to avoid evaporation during the incubation. The plate was covered, sealed in plastic bags and incubated at 37 °C in normal atmosphere. After 7 days incubation, 30 µl of alamar blue solution was added to each well, and the plate was re-incubated overnight. A change in colour from blue (oxidised state) to pink (reduced) indicated the growth of bacteria, and the MIC was defined as the lowest concentration of compound that prevented this change in colour.

RESULTS AND DISCUSSION:

Due to biological importance of the coumarin derivatives, we decided to synthesize it with a simple protocol, which can be simple operational and simple purifications process.

 

Here we reported synthesis of coumarin aceatmide derivatives by using N- acetyl glycine and salicyldehyde as starting material. Since glycine is amino acid and its available abundant, we thought its good starting point for our ongoing research to start with with commercially available starting material. But in the case of salicyldehyde, substituted and other functional group not available commercially, so we needed synthesis in laboratory; for this we took commercially available phenols and it converted to its corresponding aldehydes.

Preparation of starting material for the synthesis of coumarin derivatives started with salicyldehyde for that purpose, using literature known protocol different phenols 1 were subjected to formylation under magnesium chloride and triethyl amine with formaldehyde 2 in reflux condition under dry THF furnished salicyldehyde 3 with moderate to good yield. All the compound was purified by using column chromatography and reaction were monitored by TLC.

(Scheme 1).

 

 

 

Scheme1: Synthesis of Salicyldehyde Derivatives.

After preparation of salicyldehyde, the Glycine converted to its corresponding N- acetyl glycine under acetic anhydride and thionyl chloride condition, heating to reflux for overnight gave protected form of  N-acetyl glycine (3).

 

Scheme2: Synthesis of N-acetyl glycine 3.

 

After successful preparation of starting materials our synthetic journey for coumarin started with optimization of the reaction condition. After screening lots of solvents we observed that best solvents for this is water + acetic anhydride (3:1). Our aim was to make protocol ecofriendly and green in nature, so we tried to do reaction in water but poor yield was observed. The mixure of water and acetic anhydride served well for the reaction condition.  Compound 3 and 4 mixed in round bottom flask, subsequently added sodium acetate and acetic anhydride + water (1:3) resulting reaction mixture was refluxed for 5 h and completion of reaction was monitored by TLC,  furnished 5 with given yields. To check the scope of salicyldehyde, as above synthesized in scheme 1 was used.

 

 

 

Scheme 3: Synthesis of substituted Coumarin acetamide derivatives.

 

 

The electron withdrawing group like Cl worked well yielded with 95% yield 5b. Electron donating group tolerated well like methoxy and methyl furnished 5c, 5d in 74% and 70% yield respectively.

 

The strong electron withdrawing groups like fluro and CF₃ worked good with 5e and 5h tolerated with good yields. Finally the acetyl and bromo containing salicyldehyde also worked well.

In the case dimethoxy salicyldehyde when the reaction was performed, we observed that formation of diacetylation at nitrogen, this is quite unusual and a new kind of interesting product was formed, doing diacetylation not as much simple to get this kind of products. The diacetyl group was further confirmed by NMR and mass analysis (Scheme 4).

 

Scheme 4: Synthesis of coumarin di- acetamide derivatives.

 

 

 

Based on literature observation a possible reaction pathway for the formation of the coumarin acetamide derivatives can be explained in Scheme 5, the first step is coupling between phenol and N-acetyl glycine to give intermediate A; followed by proton abstraction by base gave B, then intrarmolecular nucleopphilic condensation to give cyclic adducts C; followed by elimination of water resulted coumarin acetamide.

 

 

 

Scheme 5: Mechanism

 

 

 

 

 

 

 

Biological Study:

The synthesized coumarin acetamide derivatives were screened for biological activity, since its novel compounds and based on literature observations it can be very potential drug candidates for drug discovery programme. The coumarin derivatives were tested for inhibition of antimicrobialactivity invitro MABA assay. The results are summarized in table 1.

 

In-vitroAntimicrobial  MABA assay

The inoculum was prepared from fresh LJ medium re-suspended in 7H9-S medium (7H9 broth, 0.1% casitone, 0.5% glycerol, albumin, dextrose, supplemented oleic acid, and catalase [OADC]), adjusted to a McFarland tube No. 1, and diluted 1:20; 100 µl was used as inoculum. Each drug stock solution was thawed and diluted in 7H9-S at four-fold the final highest concentration tested. Serial two-fold dilutions of each drug were prepared directly in a sterile 96-well microtiter plate using 100 µl 7H9-S.

 

A growth control containing no antibiotic and a sterile control were also prepared on each plate. Sterile water was added to all perimeter wells to avoid evaporation during the incubation. The plate was covered, sealed in plastic bags and incubated at 37 °C in normal atmosphere. After 7 days incubation, 30 µl of alamar blue solution was added to each well, and the plate was re-incubated overnight. A change in colour from blue (oxidised state) to pink (reduced) indicated the growth of bacteria, and the MIC was defined as the lowest concentration of compound that prevented this change in colour.

 

The biological activity summary shown in table 1, in that all synthesized compounds were screened for antimicrobial inhibition with standard known drugs, the results shows that the substitution pattern on coumarin aromatic ring greatly affecting the biological activity for that the compound 5b shown  nice inhibition ( 6.25 μM) its due to the chlorine group substitution. The derivatives 5i shown nice inhibition which is due to donating group on phenyl ring (6.25 μM), subsequently all other compound also shown good activity, the substitution pattern greatly affects the mode of action and binding pattern with cell in, Hence this is good starting point for the building the drug discovery programme in future, based on these results one can conclude that synthesis of coumarin acetamide derivatives are very important and its applications can be shown in various cell lines.

 

 

 

 

 

 

 

Table 1: MIC results of Coumarin derivatives against antimicrobial cell line (µM).

Entry	Compound	MIC (µM)
1	5a	100
2	5b	6.25
3	5c	15.6
4	5d	50.2
5	5e	66.2
6	5f	76.2
7	5g	32.4
8	5h	22.3
9	5i	6.25
10	5j	8.18

 

CONCLUSION:

In summary, the ecofriendly protocol was developed and applied for the synthesis of coumarin acetamide derivatives in water. Comarin derivatives showed significant inhibition of Antimicrobial inhibition . These analogues are chemically tractable and hence provides ample opportunities for further modification to obtain potent anti-microbial agents. The isolated yield of the coumarin derivatives is excellent, so gram scale synthesis is possible. The scope for various salicyldehyde has shown and it can be extended  further for their biological activity programme.

 

ACKNOWLEDGEMENT:

The authors are thankful to the respective managements and principals for their encouragement and support and supports during the work.

 

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Received on 10.03.2018         Modified on 11.04.2018

Accepted on 20.04.2018         © AJRC All right reserved