An efficient one-pot synthesis of coumarin-amino acid derivatives as potential anti-inflammatory and antioxidant agents

Article abstract of DOI:10.1080/00397911.2020.1735442

A series of seven coumarinyl-amino acid ester conjugates have been synthesized and characterized by NMR (¹H and 13C) and mass spectra. Further, the compounds were investigated for their therapeutic applications such as anti-inflammatory and antioxidant activities. Among the synthesized compounds most of the analogs showed good efficiency compared with the standard.

Full text of DOI:10.1080/00397911.2020.1735442

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: 0039-7911 (Print) 1532-2432 (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
An efficient one-pot synthesis of coumarin-amino
acid derivatives as potential anti-inflammatory
and antioxidant agents
Mamata Devendra Naik, Yadav D. Bodke, Vijay Kumar M & Revanasiddappa
BC
To cite this article: Mamata Devendra Naik, Yadav D. Bodke, Vijay Kumar M &
Revanasiddappa BC (2020): An efficient one-pot synthesis of coumarin-amino acid derivatives
as potential anti-inflammatory and antioxidant agents, Synthetic Communications, DOI:
10.1080/00397911.2020.1735442
To link to this article: https://doi.org/10.1080/00397911.2020.1735442
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https://doi.org/10.1080/00397911.2020.1735442
An efficient one-pot synthesis of coumarin-amino acid
derivatives as potential anti-inflammatory and
antioxidant agents
Mamata Devendra Naika, Yadav D. Bodkeb, Vijay Kumar Mc, and
Revanasiddappa BCc
aDepartment of P.G. Studies and Research in Industrial Chemistry, Jnanasahyadri, Kuvempu University,
b
Shimoga, India; Department of P.G. Studies and Research in Chemistry, Jnanasahyadri, Kuvempu
c
University, Shimoga, India; Department of Pharmaceutical Chemistry, NGSM Institute of Pharmaceutical
Sciences, NITTE, Mangalore, India
ABSTRACT
ARTICLE HISTORY
Received 6 November 2019
A series of seven coumarinyl-amino acid ester conjugates have been
synthesized and characterized by NMR (1H and 13C) and mass spec-
tra. Further, the compounds were investigated for their therapeutic
applications such as anti-inflammatory and antioxidant activities.
Among the synthesized compounds most of the analogs showed
good efficiency compared with the standard.
KEYWORDS
Amino acid ester;
anti-inflammatory activity;
antioxidant activity;
conjugates; coumarin-
3-carboxylic acid
GRAPHICAL ABSTRACT
Introduction
Coumarins are some versatile heterocyclic nuclei which constitute one of the important
structural motifs of natural products. The analogs of coumarins are synthesized by a
variety of methods to enhance its bio-medicinal properties. Coumarin is found to be
CONTACT Yadav D. Bodke
ydbodke@gmail.com
Department of P.G. Studies and Research in Chemistry,
Jnanasahyadri, Kuvempu University, Shimoga, Karnataka 577451, India.
Supplemental data for this article can be accessed on the publisher’s website
ß 2020 Taylor & Francis Group, LLC

2
M. D. NAIK ET AL.
O
O
O
O
R
OH
EDC/HOBt,NMM
o
R-H.HCl
+
0-5 C, overnight
O
O
II(a-g)
III (a-g)
I
CH
O
3
O
HN
NH
NH
NH
H C
where, R =
O
3
O
O
H C
O
CH
H C
O
3
O
3
3
c
a
b
d
NH
O
NH
OH
O
H C
3
NH
O
O
O
H C O
3
f
g
OH
e
Scheme 1. The synthetic pathway involved in the synthesis of target peptides III (a–g).
one of the key molecular templates which block the inflammation by inhibiting lipoxy-
genase (LOX), cyclooxygenase (COX) which are produced in the pathways of arachi-
donic acid metabolism and also by other mechanisms. Various other contributions
made on coumarins also justify its role in the development of molecules possessing
anti-inflammatory activity. Coumarin compounds from both plant and synthetic origin
are found to possess the special capacity to scavenge reactive oxygen species (ROS) and
regulate stress-related chronic diseases.[1–2] The substitution at the third and fourth pos-
ition of coumarin nucleus have been studied more because of its great utility in the
medicinal field and drug discovery.[3–5] Coumarin-3-carboxylic acid has been used as
one of the key synthon for several natural/synthetic pharmacological agents.[6]
Acid amine coupling is a well-recognized tool in peptide synthesis. Amino acids are
the essential constitutes of most of the proteins which are building blocks of human
life. Conjugation of amino acids with other biologically active scaffolds is found to
enhance their physiological effects.[7–9] The high bioavailability, low toxicity and effect-
ive biological activities of peptide derivatives have opened up new perspectives in drug
design and the field of biomedical research.[10]
The presence of amide functionality in a large array of biological molecules signifies
their role in modern biology.[11] Activation of the carboxylic group using different
coupling reagents is found to be a more convenient method than converting carboxylic
acid to acid chlorides followed by the reaction with amine for peptide synthesis. There
are several coupling agents available in the market and one to select an appropriate cou-
pling agent based on the mechanism through which it acts on substrates. There are
many literature reports which justify that the presence of an amide bridge linker
between coumarin and other heterocycles is associated with many biological
activities.[12–14]
Keeping in view the above, the therapeutic applications of amino acids and coumarin
scaffolds in the modern drug discovery, this work pertains simple and convenient acid

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3
amine coupling reaction involving coumarin-3-carboxylic acid and different amino acid
esters as depicted in Scheme 1. Further, the synthesized scaffolds were evaluated for
in vitro antioxidant and anti-inflammatory activity.
Result and discussion
Chemistry
The desired bioactive conjugates were synthesized by the well-known acid amine cou-
pling reaction. One of the key synthon required for the synthesis of target compounds,
coumarin 3-carboxylic acid I was synthesized by reported procedure[13] and coupled
with different commercially available amino acid esters of L-configuration (glycine ethyl
ester hydrochloride, alanine methyl ester hydrochloride, valine benzyl ester hydrochlor-
ide, leucine methyl ester hydrochloride, serine methyl ester hydrochloride, phenylalanine
methyl ester hydrochloride, and tyrosine methyl ester hydrochloride) to get desired con-
jugated molecules III(a–g) as shown in Scheme 1.
Amide functionality is obtained by the condensation of carboxylic acid and amine
group. In this reaction, the elimination of water molecules is necessary. Therefore, these
reactions were performed using certain coupling agents at ambient temperature. Thus
in the present investigation, we have used EDC.HCl/HOBt as a coupling agent in the
presence of N-methyl morpholine as a base. The carboxylic group is activated by treat-
ing it with carbodiimide to convert it to a good leaving group prior to coupling with
the amino acid ester. HOBt helps in reducing the racemization by converting the
O-acyl urea to activated ester. The ester so formed reacts with amines to give the
corresponding amide.[15] Further, the synthesized compounds were well characterized
by different spectral techniques and the spectral data of the synthesized compounds
were in accordance with expected values (see Supplementary information).
The postulated structure of synthesized methyl-3-hydroxy-2-[(2-oxo-2H-1-benzo-
1
pyran-3-carbonyl) amino] propanoate (IIIe) was confirmed by FT-IR, H NMR, 13C
NMR and mass spectral data. The FT-IR spectrum of compound (IIIe) showed an
absorption band at 3321 cmꢀ1 attributed to the –NH stretching of the AMIDE I linkage.
The other stretching vibrations at 1638 and 1710 cmꢀ1 correspond to AMIDE I car-
bonyl and coumarin carbonyl, respectively. A broadband at 3388 cmꢀ1 corresponds to
–OH stretching vibration of serine methyl ester amino acid. Formation of the com-
pound (IIIe) was further confirmed by the proton NMR spectrum, wherein the forma-
tion of a new amide bond was confirmed by the absence of –COOH signal and
appearance of a doublet at d 9.59–9.57 ppm which corresponds to –NH proton. Further,
the molecular ion peak at 276.02 [M þ H] in the mass spectrum of the compound con-
firmed the proposed structure. The physicochemical data of synthesized compounds III
(a–g) has been displayed in Table S1 (given in Supplementary file).
Biological evaluation
The in vitro anti-inflammatory activity of all the newly synthesized coumarin-amino acid
conjugates was assessed by two functional models: (a) Protein denaturation by Bovine
Serum albumin and (b) Protein Denaturation Egg Albumin Denaturation Method.

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M. D. NAIK ET AL.
The antioxidant efficacy of synthesized compounds was screened by DPPH radical
scavenging and Nitric oxide assay.
Anti-inflammatory activity
Protein denaturation by bovine serum albumin method
The potency of the synthesized peptide analogs was evaluated for their anti-inflammatory
activity by protein denaturation using Bovine Serum Albumin Method and the outcome
of the screening is summarized in Table S2 in the Supplementary file.
The bio-screening profile revealed that all the newly synthesized conjugates showed
good to moderate inhibition efficacy. Among the synthesized compounds, phenylalanine
conjugate (IIIf) showed potent inhibition efficacy compared to standard and compound
IIIc exhibited good protein denaturation inhibition with minimum inhibitory concen-
tration (36.29). This may be attributed due to the presence of a hydrophobic phenyl
group. The compound with tyrosine conjugation (IIIg) showed the least activity among
the series, it may be due to the presence of phenolic group which is hydrophilic,
whereas coumarin coupled with aliphatic amino acids like serine, alanine and glycine
showed moderate activity, which may be due to the presence of polar alcoholic and a
non-polar alkyl group.
Protein denaturation by egg albumin denaturation method
The anti-inflammatory efficiency of the conjugates was also screened by protein
denaturation using Egg Albumin Denaturation Method and the result of the assay is
tabulated in Table S3 in the Supplementary file.
It was observed from the table that the highest activity was displayed by the Serine
conjugate IIIe followed by another conjugate containing leucine amino acid ester (IIId)
both are aliphatic. Peptide template in which coumarin-3-carboxylic acid is coupled
with aromatic amino acids such as phenylalanine (IIIf) and valine benzyl ester (IIIc)
displayed moderate activity. It is quite interesting that the aliphatic amino acid
conjugates which showed good activity in egg albumin denaturation method
whereas the same showed moderate activity in the bovine serum albumin method.
Similarly, the conjugates containing aromatic amino acid segment exhibited potent
activity in the BSA method but in the case of egg albumin denaturation, the same
conjugates presented weak potency. From both activity profiles, it is clear that the
conjugate containing the tyrosine skeleton exhibited poor efficiency toward inhibition of
protein denaturation.
Antioxidant activity
It is well documented that free radicals play a major role in the inflammation process
since many of the existing non-steroidal drugs were found to act by inhibiting free
radical production or radical scavengers.[16] The compounds with anti-inflammatory
properties are expected to possess antioxidant activity. Based on these facts we have
assessed the synthesized conjugates to their free radical scavenging ability with regard to
standard antioxidant agents.

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DPPH free radical scavenging activity
DPPH free radical scavenging assay is one of the most widely accepted protocols for
screening the ability of synthesized compounds to act as inhibitors/scavengers of free
radicals. This assay is based on the formation of stable DPPH free radical in the
presence of electron or hydrogen donating antioxidants. The newly synthesized couma-
rin-amino acid ester conjugates III (a–g) were evaluated for their radical scavenging
capacity by taking ascorbic acid as standard. All the compounds showed varying degrees
of inhibition efficacy and the result is expressed as IC50 value (Supplementary
Table S4). It is also observed from the table that the scavenging efficiency of each com-
pound increases with an increase in the concentration of test samples.
Among the series, compound IIIg and IIIe showed potent radical scavenging efficacy
with the least IC50 value 28.23 and 31.45 mg/mL, respectively, with comparable to the
IC50 value of standard ascorbic acid (20.53 mg/mL). This may be due to the presence of
hydrogen-donating phenolic –OH and the aliphatic hydroxyl group of tyrosine and ser-
ine amino acid fragment of target compounds respectively. Further, compound IIIa
with glycine amino acid conjugation showed poor activity. Compounds IIIc, IIId and
IIIb showed good to moderate radical scavenging property.
Nitric oxide scavenging activity
Nitric oxide radical plays a key role in many biological functions of mammalian and
also human body at low concentration. Overexpression of nitric oxide contributes to
physiological damages. NO free radical acts cellular messengers and protects some body
organs. The synthesized coumarin conjugates were evaluated for their nitric oxide
antiradical capacity and the result of the assay are given in Table S5 in the
Supplementary file.
Among the synthesized derivatives, significant scavenging activity was shown by the
compounds with phenolic and aliphatic –OH groups with the least IC50 values compar-
able with the standard. Similarly, the compound with glycine amino acid conjugation
(IIIa) showed a declining trend in the activity profile. The rest of the compounds
showed good to moderate nitric oxide scavenging property.
Conclusion
A series of coumarinyl-amino acid ester conjugates were synthesized by using
acid-amine coupling reagent and appraised for their biological efficiency. From the
anti-inflammatory result, it was clear that conjugates with aromatic amino acids showed
potent inhibition of protein denaturation of bovine serum albumin whereas aliphatic
amino acids showed compelling inhibition of egg albumin protein. The presence of
phenolic and aliphatic hydroxyl group in the side-chain functionality of coumarinyl-
amino acid ester conjugates showed a declining trend in the activity data. On the other
hand, the antioxidant activity results revealed that the presence of different functional
groups on the side-chain of amino acid fragment affects the radical scavenging activity
such as –OH group in tyrosine and serine exhibited the highest efficiency toward
DDPH radical scavenging. Similarly, the electron-donating groups presented as good
scavengers of the nitric oxide free radical. Overall, conjugation of the different amino

6
M. D. NAIK ET AL.
acid esters with coumarin-3-carboxylic acid was found to exhibit a varying degree of
biological potency.
General procedure for coupling of amino acids with 2-oxo-2H-1-benzopyran-3-
carboxylic acid
To the cooled solution of 2-oxo-2H-1-benzopyran-3-carboxylic acid I (1 mmol) in
DMF, N-methyl morpholine (1 mmol) was added and stirred for 15 min at 0–5 ꢁC.
EDC.HCl (1 mmol) was added to the above solution while maintaining the temperature
at 0–5 ꢁC followed by the addition of HOBt (1 mmol) in DMF. The resultant reaction
mixture was stirred for additional 10–15 min at the same temperature. To this homoge-
neous reaction mixture, a pre-cooled solution of esters of amino acids II (a–g) (1 mmol)
in DMF was added slowly and pH of the solution was adjusted to eight by adding
N-methyl morpholine and stirred overnight at room temperature. After completion of
the reaction, the whole mass was poured to ice-cold water and stirred for 30 min.
The precipitate formed was filtered, dried and recrystallized from ethanol to get the
desired products III (a–g).
Ethyl [(2-oxo-2H-1-benzopyran-3-carbonyl) amino] acetate (IIIa)
Yield: 88%; White solid; mp: 171–173 ꢁC; 1H NMR (400 MHz, DMSO-d6, d ppm):
1.16–1.19 (t, J¼ 12 Hz, 3H, ester-CH3), 4.09 (s, 2H, CH2), 7.41–7.48 (q, 2H, Ar–H),
7.71–7.72 (d, J¼ 4 Hz, 1H, Ar–H), 7.97–7.95 (d, J ¼ 8 Hz, 1H, Ar–H), 8.87 (s, 1H,
Ar–H), 9.03 (s, 1H, amide NH). 13C NMR (100 MHz, DMSO-d6, d ppm): 14.51 (ester-
CH3), 41.92 (amide-CH2), 61.01 (ester-CH2), 116.598, 118.533, 118.826, 125.597,
130.860, 134.772, 148.63 and 154.43 (coumarin C–Ar), 160.73, 161.82 (coumarin –C–O)
and 169.86 (glycine ester –C–O). HRMS (ESI): m/z Anal. Calcd: 275.2567; Found:
276.0385 (M þ H).
Acknowledgments
The authors thank the Chairman, Department of Industrial Chemistry, Kuvempu University,
Shankaraghatta for providing laboratory facilities.
Disclosure statement
No potential conflict of interest was reported by the author(s).
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