Brine Shrimp Lethality Assay of Ethyl Cinnamate Derivatives

Synthesized by Microwave Irradiation of Cinnamic Acids with

Ethyl Acetate

Rose Malina Annuur

, Nisa Urribah

, Lia Meilawati

, Faris Hermawan

Salahuddin Salahuddin

, Megawati Megawati

and Zetryana Puteri Tachrim

1e*

Research Center for Pharmaceutical Ingredient and Traditional Medicine, National Research and Innovation Agency,

Kawasan Sains Teknologi (KST) BJ Habibie, South Tangerang, Banten, 15314, Indonesia

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Negeri Semarang,

Semarang, Central Java, 50229, Indonesia

Keywords: Microwave Irradiation, Ethyl Cinnamates, Brine Shrimp Lethality, Cinnamic Acids.

Abstract: The simple and low-cost bioassay of brine shrimp lethality assay can be used to examine the toxicity effect

of potential drugs. Ethyl p-methoxycinnamate is a major compound that can be isolated from the rhizome of

Kaempferia galanga which was suggested that its ethyl ester is the most important functional group

contributing to its bioactivity. In general, the synthesis of this typical ethyl ester of cinnamic acids is derived

from the reaction of ethanol and cinnamic acid under reflux with the contribution of acid as a catalyst. In this

study, cinnamic acid and selected para-substituted cinnamic acid derivatives are subjected to microwave

irradiation-guided ethyl esterification by using ethyl acetate and sulfuric acid. This alternative method for

ethyl esterification yielded 19–39% products of ethyl cinnamate derivatives and ethyl p-chloro cinnamate

showed the highest toxicity level among the tested ethyl cinnamate derivatives with LC

of 1.29 µg/mL.

1 INTRODUCTION

The simple and low-cost bioassay of brine shrimp

lethality assay can be used to examine the toxicity

effect of potential drugs (Michael, et al., 1956; Dash,

et al., 2014). Ethyl p-methoxycinnamate is a major

compound derived from Kaempferia galanga which

is a main constituent as an anti-inflammatory agent

(Elshamy, et al., 2019; Umar, et al. 2014). It is

suggested by the study on the structure-activity

relationship that the ethyl ester form of this cinnamic

acid derivative is the most important functional group

contributing to the anti-inflammatory activity

(Komala, et al., 2018). In general, the synthesis of this

typical ethyl ester of cinnamic acids is derived from

the reaction of ethanol and cinnamic acid under reflux

with the contribution of sulfuric acid (Anthony, et al.,

https://orcid.org/0009-0002-7107-507X

https://orcid.org/0009-0006-6923-4050

https://orcid.org/0000-0003-0580-0579

https://orcid.org/0000-0002-6698-0668

https://orcid.org/0000-0001-6637-541X

2022). Ethyl acetate under sulfuric acid can be

hydrolyzed (Jaques, 1971; Vinnik and Librovich,

1975) for presenting ethanol and can be donated as an

ethyl ester group candidate for direct

transesterification with cinnamic acid. Microwave

irradiation is believed to increase the efficiency

during organic synthesis by offering rapid

temperature increments (Hoz, et al., 2005). To the

best knowledge, no reported study has synthesized

ethyl cinnamate by microwave irradiation of ethyl

acetate and sulfuric acid. Since several cinnamic acid

derivatives are relatively less soluble in the ethanol,

thus by taking advantage of ethyl acetate hydrolysis

under a high concentration of sulfuric acid this study

then directly conducts the ethyl esterification of

selected cinnamic acids with sulfuric acid as a

catalyst under microwave irradiation. The

representative ethyl ester of cinnamic acid derivatives

Annur, R. M., Urribah, N., Meilawati, L., Hermawan, F., Salahuddin, S., Megawati, M. and Tachrim, Z. P.

Brine Shrimp Lethality Assay of Ethyl Cinnamate Derivatives Synthesized by Microwave Irradiation of Cinnamic Acids with Ethyl Acetate.

DOI: 10.5220/0013471800004612

Paper published under CC license (CC BY-NC-ND 4.0)

In Proceedings of BRIN’s 2nd International Conference for Health Research (ICHR 2024), pages 5-8

ISBN: 978-989-758-755-9

then aims to understand its toxicity by utilizing a

brine shrimp lethality assay.

2 MATERIALS AND METHODS

2.1 Generals

Hexane and ethyl acetate were distillate before use,

the distilled ethyl acetate was further dried with

MgSO

before reaction, conc. H

(96%, Merck),

and reverse osmosis (RO) water were produced on-

site. Non-modified domestic microwave oven 900-

Watt (Moderna MG 2516) was utilized for the

synthesis and the reaction mixture was covered with

heat resistance glass before irradiation. Seawater was

taken from Anyer, Banten, Indonesia, stored at room

temperature, and filtered before use. NMR spectra

(

H and

C) were collected using AVANCE NEO

700MHz NMR (BRIN, Serpong, Indonesia). GC-MS

data were collected using GC-MS Agilent 7890B GC

and 5977A MSD (BRIN, Serpong, Indonesia).

2.2 Synthesis of Ethyl Cinnamate

Derivatives via Microwave

Irradiation of Cinnamic Acids with

Ethyl Acetate

Cinnamic acid derivatives (1–3, 100 mg, 0.51–0.69

mmol) are dissolved in 20 mL ethyl acetate. 1 mL

conc. H

is added to the cinnamic acid derivatives

solution in the ice bath. This solution was then

microwaves irradiated for 60 seconds x 12 times and

intervals with cooling for 3 minutes. After the

reaction, the mixture was cooled to room temperature

and added 20 mL of ethyl acetate. Then, RO water

was added, and the pH was adjusted to 9–10 with 5%

NaOH

(aq)

. Separation of ethyl acetate fraction and

water fraction was conducted. The ethyl acetate

fraction was washed with brine, dried with MgSO

and evaporated to result in ethyl cinnamate

derivatives (5–7).

Ethyl cinnamate (5). Colourless liquid.

H NMR

(700 MHz, Chloroform-d) δ 7.68 (d, J = 15.9 Hz, 1H),

7.51 (dd, J = 6.7, 3.1 Hz, 2H), 7.39 – 7.35 (m, 3H),

6.43 (d, J = 16.0 Hz, 1H), 4.26 (q, J = 7.2 Hz, 2H),

1.33 (t, J = 7.2 Hz, 3H) ppm.

C NMR (176 MHz,

CDCl

) δ 167.0, 144.6, 134.4, 130.2, 128.9, 128.0,

118.3, 60.5, 14.3 ppm. C

calculated m/z 131.05

and found m/z 131.10 (100%).

Ethyl 4-chlorocinnamate (6). Colourless liquid.

H NMR (700 MHz, Chloroform-d) δ 7.62 (d, J =

16.0 Hz, 1H), 7.44 (d, J = 8.4 Hz, 2H), 7.35 (d, J =

8.4 Hz, 2H), 6.40 (d, J = 16.1 Hz, 1H), 4.26 (q, J =

7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H) ppm.

C NMR

(176 MHz, CDCl

) δ 166.8, 143.2, 136.2, 133.1,

129.3, 129.3, 119.0, 60.7, 14.4 ppm. C

ClO

calculated m/z 165.10 and found m/z 165.00 (100%).

Ethyl 4-nitrocinnamate (7). Colourless crystal.

NMR (700 MHz, Chloroform-d) δ 8.25 (d, J = 8.8 Hz,

1H), 7.71 (d, J = 16.2 Hz, 2H), 7.68 (d, J = 8.7 Hz,

2H), 6.56 (d, J = 16.1 Hz, 1H), 4.30 (q, J = 7.0 Hz,

2H), 1.36 (t, J = 7.2 Hz, 3H) ppm.

C NMR (176

MHz, CDCl

) δ 166.2, 148.6, 141.7, 140.7, 128.7,

124.3, 122.7, 61.1, 14.4 ppm. C

calculated

m/z 176.03 and found m/z 176.10 (100%).

2.3 Isolation of

Ethyl 4-Metoxycinnamate (9)

Isolation of ethyl 4-metoxycinnamate (9) was

conducted according to literature (Tachrim, et al.,

2022).

2.4 Brine Shrimp Lethality Assay

The procedure for BSLT followed the literature

(Primahana, et al, 2015). Brine shrimps were hatched

using brine shrimp eggs (Artemia salina) in a conical-

shaped vessel, filled with filtered seawater. The

number of dead and surviving nauplii in each tube

was counted and recorded. LD

values were

determined from the best-fit line plotted

concentration versus percentage lethality.

3 RESULT AND DISCUSSION

The reaction of cinnamic acid derivatives (1–4) with

ethyl acetate and H

via microwave irradiation

can be conducted without a reflux system within 60

seconds of irradiation time with 3 minutes cooling

process. This interval is utilized for the prevention of

further ethyl acetate evaporation due to the rapid

high-temperature condition offered by microwave

irradiation. The resulted ethyl cinnamate derivatives

(5–7) resulted in moderate yield from the

correspondent cinnamic acid (1), p-chloro (2), and p-

nitro (3), respectively (Scheme 1). As for p-hydroxy

(4, Scheme 1), after quenching the reaction mixture,

no ethyl 4-hydroxycinnamate (8, Scheme 1) is

isolated. Compound 4 might need a longer reaction

time to conduct with the ethyl acetate system, since

this compound has low solubility towards ethyl

acetate and found up until 10 minutes of irradiation it

cannot be dissolved completely.

ICHR 2024 - BRIN’s International Conference for Health Research (ICHR)

The conventional method of refluxing cinnamic

acid derivatives with ethanol for the synthesis of ethyl

ester derivatives (5–7) was also tested and showed a

higher yield (60–80%) with 5 hour reaction time. In

comparing these results with the current method,

microwave irradiation is relatively more efficient due

to its faster reaction time. Herein, the ethyl acetate

utilization in sulfuric acid can offer esterification of

semi-polar compounds of cinnamic acid derivatives

(1–3) into its ethyl ester derivatives (5–7). This

method can be used as an alternative to direct ethanol

utilization (Fisher esterification) in which ethanol can

be formed from the hydrolysis of ethyl acetate

(Jaques, 1971; Vinnik and Librovich, 1975) under

high temperature by microwave irradiation.

Scheme 1: Synthesis of ethyl cinnamate derivatives via

microwave irradiation of cinnamic acids with ethyl acetate.

The bracket showed an isolated yield.

No ethyl 4-

hydroxycinnamate is isolated.

Ethyl 4-methoxycinnamate

is isolated from rhizomes of K. galanga according to

literature (Tachrim, et al., 2022).

The result of brine shrimp assay lethality assay of

synthesized ethyl cinnamate derivatives 5–7 and ethyl

4-methoxycinnamate 9 isolated from the rhizome of

K. galanga are shown in Table 1 and Fig. 1. Based on

these results, the toxicity level of all tested compound

is 6 > 9 > 5 > 7. The synthesized ethyl cinnamate 5

and p-nitro 7 showed moderate toxicity compared

with the ethyl 4-methoxycinnamate 9 isolated from

the rhizome of K. galanga. p-Chloro 6 showed high

toxicity among all tested compounds with LC

1.29 µg/mL and has highest % mortality on 10 ppm.

For comparison, the reported brine shrimp lethality

assay on crude CH

extract of K. galanga with the

major compound of ethyl cinnamate and ethyl 4-

methoxycinnamate showed almost 100% mortality

can be achieved at a concentration of 10 ppm

(Othman, et al., 2006). The previous brine shrimp

lethality assay of methyl cinnamate also showed a

moderate LC

of ~120 µg/mL (Primahana, et al.,

2015). Despite further study is needed to understand

the structure correlation with the toxicity, the high

toxicity of p-chloro 6 is due to the introduction of

chloro as a substituent on the aromatic moiety.

Moreover, this compound has the potential to be used

for the study of its bioactivity in the future.

Table 1: Brine shrimp lethality assay of synthesized ethyl

cinnamate derivatives.

Mortalit

(%)

(ppm)

ppm

100

ppm

500

ppm

1000

ppm

5 (R = H) 0 6 100 100 234.42

6 (R = Cl) 61 98 100 100 1.29

7 (R = NO

) 4 17 74 93 301.99

9 (R = OMe) 5 17 52 82 173.78

Figure 1: Effect of synthesized ethyl cinnamate derivatives

5–7 and 9 on brine shrimp nauplii.

4 CONCLUSIONS

The reaction of cinnamic acid derivatives (1–3) with

ethyl acetate and H

via microwave irradiation

can be used as an alternate method for the synthesis

of their representative ethyl esters (5–7). Among the

synthesized ethyl cinnamate derivatives 5–7 and ethyl

4-methoxycinnamate 9 isolated from the rhizome of

K. galanga, ethyl p-chloro cinnamate 6 showed the

highest toxicity level with LC

of 1.29 µg/mL. The

brine shrimp lethality assay tested in this study can

provide the toxicity data comparison of several ethyl

esters of cinnamic acid derivatives before in vivo

study and contribute to the preliminary study of drug

development.

ACKNOWLEDGEMENTS

RMA was supported by the Postdoctoral Fellowship

National Research and Innovation Agency,

Indonesia. NU thanks to Research Assistant

Fellowship National Research and Innovation

Agency, Indonesia.

100

0.5 1.0 1.5 2.0 2.5 3.0 3.5

Percent of

mortality (%)

Log conc.

5 6 7 9 Linear (5) Linear (6) Linear (7) Linear (9)

Brine Shrimp Lethality Assay of Ethyl Cinnamate Derivatives Synthesized by Microwave Irradiation of Cinnamic Acids with Ethyl Acetate

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