Synthesis of Quatenary Ammonium Compounds from Eugenol

through Mannich and Methylation Reactions

and Its Antibacterial Activity

Sabarmin Perangin-angin* and Sri Wahyuni Barus

Departement of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Sumatera Utara, Medan, Indonesia

Keywords: Eugenol, Mannich Reaction, Methylation, Antibacterial,

6-[(N-iodo-N-methyl-N-methyl-N-methylamino)methyl]-4-alyl-2-methoxy phenol.

Abstract: 6-[(N-iodo-N-methyl-N-methyl-N-methylamino)methyl]-4-alyl-2-methoxyphenol was synthesized through

Mannich and methylation reaction. Mannich reaction was carried out by reacting eugenol, formaldehyde

37%, and dimethylamine 40% with ethanol at temperature of 78°C for 90 minutes and 4-allyl-6-

(dimethylamino)methyl-2-methoxyphenol was obtained with yield of 83,03%. Quartenary ammonium salt,

6-[(N-iodo-N-methyl-N-methyl-N-methylamino)methyl]-4-allyl-2-methoxyphenol, was obtained through

methylation reaction of 4-allyl-6-(dimethylamino)methyl-2-methoxyphenol with methyl iodide in ethanol.

In the FTIR spectrum, the specific peak of ammonium salt was exhibited at 948.98 and 455.20 cm-1. The

antibacterial activity of 6-[(N-iodo-N-metil-N-methyl-N-methylamino)methyl]-4-allyl-2-methoxyphenol

was performed against E. coli and S. aureus, this quaternary ammonium salt exhibited a strong activity.

1 INTRODUCTION

Eugenol is the main component contained in clove

oil (Syzygium aromaticm), it can reach 70-96%.

Therefore, the quality of clove oil is depended on the

eugenol content, the increase of eugenol content can

influence its quality and price. Eugenol has several

functional groups, i.e. hydroxyl (-OH), allyl (-CH

CH=CH

) and methoxy (-OCH

). These functional

groups can be chemically modified to be its derivate

which varies bioactivities. Eugenol has been known

as an important precursor for synthesizing a

particular compound with specific bioactivity

(Towaha, 2012).

Many researches that focused in eugenol has

been carried out, especially for synthesizing eugenol

derivatives, such as the transformation of allyl

groups in eugenol into aldehyde groups, that can be

found in vanillin which already used as food

additives. The other is the transformation of

hydroxyl groups into alkyl, acyl or acetyl, that can

be found in methyl eugenol, eugenol benzoate and

acetyl eugenol (Sastrohamidjojo, 2004).

Amine derivative compounds, such as halide

quaternary ammonium salt (R

), can be used in

wide area. Quaternary ammonium salt can act as a

phase transfer catalyst. This catalyst is widely used

for heterogeneous reactions involving ionic species

in non-polar solvents which cannot dissolve ionic

species. Ionic species are commonly found in the

liquid phase, while the compounds that will be

reacted are found in the organic phase and both

species cannot mix each other. The quaternary

ammonium salt can also act as a surfactant.

Surfactants reduce the surface tension of water by

breaking hydrogen bonds on the surface. Most

surfactants containing nitrogen bases are cationic

surfactants (Perangin-angin, 2002).

Quaternary ammonium salt can be used as an

antibacterial (Stanley, 2011). Antibacterial are

substances that can inhibit the growth of bacteria by

disrupting the microbial metabolism. Antibacterial

can only be used if they have selective toxicity, it

means they can kill bacteria that cause disease, but

they are not toxic to human. The mechanism of

antibacterial compounds can be divided into a)

inhibiting cell wall synthesis, b) disturbing the cell

wall permeability, c) inhibiting the specific enzyme,

and d) inhibiting the synthesis of nucleic acids and

proteins. Cytoplasm of all living cells are covered by

the cytoplasmic membrane that acts as a barrier with

selective permeability to carry out as active transport

Perangin-angin, S. and Wahyuni Barus, S.

Synthesis of Quatenary Ammonium Compounds from Eugenol through Mannich and Methylation Reactions and Its Antibacterial Activity.

DOI: 10.5220/0008879202230228

In Proceedings of the 1st International Conference on Chemical Science and Technology Innovation (ICOCSTI 2019), pages 223-228

ISBN: 978-989-758-415-2

223

arrangement. If the permeability of the cytoplasmic

membrane function is disrupted by substances, i.e.

surfactant, the permeability of the cell wall will

change or even become damaged (Madigan, 2005).

The Mannich reaction is a reaction to modify

enolate compounds to be dialkylamino methylene

that have active hydrogen atoms. This reaction

occurs through a condensation reaction on

functionals groups that can be enolized with

imminium ions (Mannich base), i.e. a product from

the reaction between formaldehyde and secondary

amines (Purwono and Daruningsih, 2010). In

(Karanov et al., 1995), the synthesis of 2-methoxy-

4-(1-prophenyl)-6-phenol through Mannich reaction

with eugenol, formaldehyde, and amine compound

showed an activities as plant growth control. In the

previous work, (Perangin-angin, 2002) performed a

synthesis reaction between eugenol and dimethyl

sulphate in alkaline condition, and produced 2,2-

methylene-bis-6-methoxy-4-(2-propenyl)phenol

with yield of 43%. The other study also performed a

synthesis of 6-butylaminomethyl and 6-

dibutylaminomethyl from eugenol through Mannich

reaction with yield of 50 and 78%, respectively. This

study also learn the influence of the effect of

primary and secondary amines (Hecht, 2014).

(Popovici et al., 1999) synthesized several Mannich

oxime base derivatives into quaternary ammonium

salts. For the example, 1-(2-hydroxy-5-methylpenyl)

-3-dialkylamino-1-propanone was quaternized using

methyl iodide in tetrahydrofuran (THF) and ethanol

at room temperature with yield of 93 and 65%,

respectively. (Perangin-angin, 2019) synthesized 4-

allyl-6-(hydroxymethyl)-2-methoxyphenol from

eugenol through Mannich reactions followed by

methylation and substitution reaction.

Therefore, the objective of the present research

was to synthesize quaternary ammonium salt that

based on eugenol structure through Mannich

reaction and followed by methylation reaction. The

obtained quaternary ammonium salt showed a

moderate antibacterial activity.

2 MATERIALS AND METHODS

2.1 Materials

Equipment: glassware, rotary evaporator, hotplate

with stirrer, Fourier Transform Infrared (FT-IR),

Gas Chromatography Mass Spectrometer (GC-MS),

Spectrophotometer UV-Vis.

Materials: Eugenol, Dimethylamine, Ethanol,

Formaldehyde, Methyl iodide, Na

anhydride,

Diethyl ether, Nutrient agar (NA), S. aureus, E. coli.

2.2 Synthesis of 4-Allyl-6-(Dimethyl

amino) Methyl-2-Methoxy Phenol

Compound

As much as 4.8 g of eugenol was dissolved with 28

mL of ethanol into the three neck round bottom flask

and then 3.8 g (3.48 mL ; 0.04 mol) of formaldehyde

(37 wt.%) and 5.6 g (6.3 mL ; 0.05 mol) of

dimethylamine (40 wt.%) were followed by reflux

process at 78

C for 90 minutes. The mixture was

cooled and stirred for 24 h. The excess ethanol was

then evaporated by rotary evaporator. The obtained

result was characterized by FT-IR and GC-MS.

2.3 Synthesis of 6-[(N-Iodo-N-Methyl-N

Methyl-N-Methylamino) Methyl] 4-

Allyl-2-Methoxy Phenol

As much as 4.404 g (0.2 mol) of 4-allyl-6-(dimethyl

amino)methyl-2-methoxyphenol was added into

erlenmeyer. Ethanol (35 mL) and methyl iodide (0.2

mol) were added into erlenmeyer, and tightly closed.

The mixture was stirred at room temperature with a

magnetic stirrer for 2 h and allowed to stand in the

refrigerator for one night. The precipitate formed

was filtered using filter paper and washed with

diethyl ether. The obtained product was purified by

recrystallization process using ethanol and analysed

by FT-IR.

2.4 Preparation of Nutrient Agar Slant

(NA)

About 7 g of NA was dissolved with 250 mL of

distillate water and sterilized in an autoclave at

121

C for 15 minutes.

2.5 Preparation of Medium Agar Slant

and Bacterial Culture Stock

The NA slant was prepared by adding 3 mL of NA

into test tube and placed it in the rack. Tilt the rack

onto solid surface so that the medium is slanted.

Allow the medium to harden in this position. The

culture was obtained from stock and taken with an

osse. This culture was incubated at 35

C for 18-24 h.

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224

2.6 Preparation of Mueller Hinton

Agar (MHA) Medium

MHA (19 g) was entered into erlenmeyer and

dissolved with 500 mL of distillate water sterilized

in an autoclave at 121

C for 15 minutes.

2.7 Preparation of Bacterial Inoculum

Nutrient broth (3.25 g) was dissolved with 250 mL

of distillate water and sterilized in an autoclave at

121

C for 15 minutes. Furthermore, microbial

colony was taken from culture stock using a

sterilized osse. The culture was suspended into 10

mL of sterilized nutrient broth in the test tube and

incubated at 35

C for 3 h. The optical density of

bacterial was determined using spectrophotometer

UV-Vis at 580-600 nm.

2.8 Evaluation of Antibacterial Activity

The antibacterial activity of quaternary ammonium

salt was obtained by diffusion method. Paper disk (Ǿ

6 mm) had been soaked in various concentration of

quaternary ammonium salt (10, 20, and 30%). This

paper disk then placed on the agar medium that has

been cultured with E. coli and S. aureus. The

inhibition zone was measured using calliper (mm).

3 RESULTS AND DISCUSSION

3.1 Synthesis of 4-Allyl-6-(Dimethyl

amino) Methyl-2-Methoxy Phenol

The ammonium quaternary salt, 4-Allyl-6-(dimethyl

amino)methyl-2-methoxyphenol, was obtained as

blackish brown liquid with amount of 5,51 g. The FT-

IR spectrum of 4-Allyl-6-(dimethyl amino)methyl-2-

methoxyphenol was showed in Figure 1.

Figure 1: FT-IR spectrum of 4-Allyl-6-(dimethyl

amino)methyl-2-methoxyphenol.

The spectrum showed that C-N bonding from

dimethylaminomethyl was exhibited at 1246.16 cm

. The exhibited signal at 3387 cm

-1

was identified as

vibration of OH stretching. The signal at 2970.38

and 2893.22 cm

-1

showed the vibration of aliphatic

CH, this signal was supported by the presence of

methylene signal at 1458.18 cm

-1

and methyl group

at 1303.88 cm

-1

. The presence of C=C aromatic was

shown at 1635.64 cm

-1

. The vinyl group was shown

at 987.55 cm

-1

and C-O-C from ether was shown at

1141.86 cm

-1

4-allyl-6-(dimethyl amino) methyl-2-methoxy

phenol was obtained from eugenol through the

Mannich reaction, which was reacted with iminium

ion. In the Mannich reaction of eugenol, the active

hydrogen from eugenol was replaced by the

dimethylaminomethyl. The mechanism reaction of

this quaternary ammonium salt formation was

displayed in Figure 2.

Figure 2: Mechanism reaction of the Mannich eugenol.

Synthesis of Quatenary Ammonium Compounds from Eugenol through Mannich and Methylation Reactions and Its Antibacterial Activity

225

The obtained quaternary ammonium salt of 4-

allyl-6-(dimethyl amino) methyl-2-methoxyphenol

compound using GC-MS showed peak of retention

time at 31.428 minute with purity as much 85.65%.

The mass chromatogram of the compound

synthesized by GC-MS was showed in Figure 3.

Figure 3: FT-IR spectrum of 4-Allyl-6-(dimethyl

amino)methyl-2-methoxyphenol.

The obtained result of 4-Allyl-6-

(Dimethylamino)Methyl-2-Methoxy Phenol

spectrum was corresponded in Figure 4.

Figure 4: Mass spectrum of 4-allyl-6-(dimethylamino)

methyl-2-methoxy phenol compound.

Figure 5: Mass spectrum of 4-allyl-6-(dimethylamino)

methyl-2-methoxy phenol compound.

The peak of retention time for 31.428 minute

with molecular formula C

that had relative

molecular mass was 221 g/mol. The Spectral data

showed molecular ion peaks at m/e 221 followed by

fragmentation peaks at m/e 204, 190, 176, 161, 147,

133, 117, 105, 91, 77, 58, 44, 39, and 28, which was

these values corresponded into the relative

molecular mass (Mr) of the 4-allyl-6-

(dimethylamino) methyl-2-methoxy phenol

compound synthesized. The fragmentation pattern

can be seen in Figure 5.

3.2 Synthesis of 6-[(N-Iodo-N-Methyl-

N-Methyl-N-Methylamino)

Methyl]-4-Allyl-2-Methoxy Phenol

Compound

In this study, the obtained result of 6-[(N-iodo-N-

methyl-N-methyl-N-methylamino) methyl]-4-allyl-

2-methoxy phenol was 5.71 in the form of yellow

solid and its FT-IR spectroscopy analyzed was

showed in Figure 6.

Figure 6: FT-IR spectrum of 6-[(N-iodo-N-methyl-N-

methyl-N-methylamino) methyl]-4-alyl-2-methoxy

phenol.

The FT-IR spectrum of 6-[(N-iodo-N-methyl-N-

methyl-N-methylamino) methyl)]-4-allyl-2-methoxy

phenol that can be found stretching vibration at

948.98 cm

-1

and supported by an absorption at

455,20 cm

-1

. Those Wavenumbers of 948.98 cm

-1

455.20 cm

-1

were typical of quaternary ammonium

salts that appeared simultaneously. The absorption

peak at 3379.29 cm

-1

was assigned as the O-H

vibration after that, the peak at 3008,95 cm

-1

was

corresponded as stretching vibration of C-H sp2

(=CH-) and the wavenumber at 2924,09 cm

-1

was

showed as C-H sp

on CH

. The absorption peak at

1481,33 cm

-1

indicated CH

and C=C aromatic was

showed at 1604.77 cm

-1

. The reaction of preparation

6-[(N-iodo-N-methyl-N-methyl-N-methylamino)

methyl]-4-allyl-2-methoxy phenol was showed in

Figure 7.

ICOCSTI 2019 - International Conference on Chemical Science and Technology Innovation

226

Figure 7: FT-IR spectrum of 6-[(N-iodo-N-methyl-N-

methyl-N-methylamino) methyl]-4-alyl-2-methoxy

phenol.

3.3 Antibacterial Activity

Antibacterial activity of 6-[(N-iodo-N-methyl-N-

methyl-N-methylamino) methyl]-4-alyl-2-methoxy

phenol using E. coli and S. aureus can be found in

Table 1.

Table 1: Antibacterial activity test of 6-[(N-iodo-N-

methyl-N-methyl-N-methylamino) methyl]-4-alyl-2-

methoxy phenol.

Treatment

Disc

diameter

(mm)

Clear zone diameter

(mm)

S. aureus

E. coli

10%

10.2

10.8

20%

10.8

11.4

30%

11.3

12.1

Based on antibacterial activity showed that 6-

[(N-iodo-N-methyl-N-methyl-N-methylamino)

methyl]-4-alyl-2-methoxy phenol had antibacterial

properties for both of S. aureus and E. coli. It was

caused that 6-[(N-iodo-N-methyl-N-methyl-N-

methylamino) methyl]-4-alyl-2-methoxy phenol had

a cationic charge amine group which was able to

bind food source of these bacteria that inhibited food

nutrition into bacterial cells (Nascimento et al.,

2000).

According to (Aleksandra et al., 2017) said that

antibacterial activity was classified to be 3 groups.

There were strong that produced inhibition zone

diameter at 8 mm, medium activity that produced

inhibition zone at 7-8 mm, while weak activity that

produced inhibition zone diameter less than 7 mm.

Therefore, quartenary ammonium compound has

strong antibacterial activity.

4 CONCLUSIONS

In this work, synthesis of 6-[(N-iodo-N-methyl-N-

methyl-N-methylamino) methyl]-4-allyl-2-methoxy

phenol was performed by 2 step, namely Mannich

and Methylation reactions. The obtained result of 4-

allyl-6-(dimethylamino) methyl-2-methoxy phenol

was 5.51 g with a yield of 83.03% and 6-[(N-iodo-N

-methyl-N-methyl-N-methylamino) methyl]-4-alyl-

2-methoxy phenol was 5.71 g with a yield of

78.39%. The characterization by FT-IR confirmed

the existence of 6-[(N-iodo-N-methyl-N-methyl-N-

methylamino) methyl]-4-allyl-2-methoxy phenol as

quaternary ammonium salt to the stretching

vibrations of C-N+ and the peak at 948.98 cm

-1

and

supported by absorption vibration C-N

at 455.20

-1

. Furthermore, 6-[(N-Iodo-N-methyl-N-methyl-

N-methylamino) methyl]-4-allyl-2-methoxy phenol

can be used as antibacteria which showed

antibacterial activity for both of E.coli and S. aureus.

This compound showed antibacterial activity for E.

coli was better compared to S. aureus and classified

to strong antibacterial activity.

ACKNOWLEDGEMENTS

Author would like to thank to Rector of University

of Sumatera Urara for the funding from the project

of PD-TALENTA 2019

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