Synthesis and antioxidant activity of thymol and carvacrol based Schiff bases

Article abstract of DOI:10.1016/j.bmcl.2012.12.001

Thymol and carvacrol are well known antioxidants found in the extract of the plants of thyme species. The Schiff bases of 2-iso-propyl-5-methyl-phenol (thymol/1a), 2-tert-butyl-5-methyl-phenol (1b) and 5-iso-propyl-2-methyl-phenol (carvacrol/1c) exhibited much better antioxidant activity than thymol and carvacrol in DPPH assay. Ten compounds (4k, 4l, 4r, 5k, 5l, 5q, 5r, 6k, 6l and 6r) showed better or similar activity as compared to the reference compound ascorbic acid. Twenty-four most active compounds were also screened by ABTS method and showed 60-90% inhibition at 5 μg/mL concentration.

Full text of DOI:10.1016/j.bmcl.2012.12.001

Bioorganic & Medicinal Chemistry Letters 23 (2013) 641–645
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antioxidant activity of thymol and carvacrol based
Schiff bases
⇑
Beena, Deepak Kumar, Diwan S. Rawat
Department of Chemistry, University of Delhi, Delhi 110007, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Thymol and carvacrol are well known antioxidants found in the extract of the plants of thyme species.
The Schiff bases of 2-iso-propyl-5-methyl-phenol (thymol/1a), 2-tert-butyl-5-methyl-phenol (1b) and
5-iso-propyl-2-methyl-phenol (carvacrol/1c) exhibited much better antioxidant activity than thymol
and carvacrol in DPPH assay. Ten compounds (4k, 4l, 4r, 5k, 5l, 5q, 5r, 6k, 6l and 6r) showed better or
similar activity as compared to the reference compound ascorbic acid. Twenty-four most active com-
Received 6 August 2012
Revised 27 November 2012
Accepted 1 December 2012
Available online 8 December 2012
pounds were also screened by ABTS method and showed 60–90% inhibition at 5 lg/mL concentration.
Keywords:
Antioxidant
Thymol
Ó 2012 Elsevier Ltd. All rights reserved.
Carvacrol
Schiff base
DPPH
ABTS
Reactive oxygen species (ROS) such as hydroxyl radicals, super-
oxide radicals, singlet oxygen, hydrogen peroxide radical are con-
stantly formed as a result of normal organ functions or excessive
oxidative stress.1,2 Their presence in the biological system is very
harmful as these species are responsible for the damage of biomol-
ecules such as nucleic acid, proteins, lipids, DNA and carbohy-
drates3 and this may cause many diseases such as cancer,
atherosclerosis, aging, hair loss, inflammation, immunosupress-
sion, diabetes and neurodegenerated disorders (such as Alzhei-
mer’s and Parkinson’s diseases).4–7 The balance between the
production and elimination of ROS is normally controlled by the
body defence mechanism through the use of different enzymes
such as superoxide dismutase (SOD), catalase and glutathione per-
oxidase. However when it is disturbed oxidative stress is generated
leading to oxidative damage to biomolecules. Antioxidants are
compounds which slow down or prevent the oxidation of other
molecules.8,9 They interact with free radicals and prevent the dam-
age by ROS. Thus the treatment with antioxidants is potentially a
way to overcome the oxidative stress. Because of this, there is a
great interest in the discovery of natural and synthetic antioxi-
dants that can serve as protective agents against these diseases.
Phenolic compounds are known for their antimicrobial and antiox-
idant properties. They act as free radical scavengers and their anti-
oxidant potential depends on the substituent present and the
extent of structure conjugation.10
Extracts of the plants of thyme species possess a wide range of
biological and pharmacological properties.11–13 The rich essential
oils present in thyme species are used for the treatment of several
diseases as well as for food preservatives.14 The most frequently
occurring constituents of these species are thymol (2-iso-propyl-
5-methylphenol, 1a) and its isomer carvacrol (5-iso-propyl-2-
methyl-phenol, 1c). They exhibit antibacterial, antifungal, antiviral,
antitumor and anti-inflammatory activities.15–22 They also act as
antioxidant,23 free radical scavenger24 and anti-lipidperoxidative
agents.25 Thymol and carvacrol act as biocidal agent by causing
disruption of the bacterial membrane. Thymol and carvacrol are
used as antiseptic, in medical practice, agriculture, cosmetics and
food industry. Thymol has antimicrobial properties and has the
ability to reduce bacterial resistance to drugs such as penicillins
(synergistic) and it is an active ingredient of Listerine (mouthwash)
and Vicks. Encouraged by these observations and as a part of our
ongoing programme towards the synthesis of medicinal relevant
molecules26–35 we became interested to modify thymol and carva-
crol, and study their antioxidant activity.
The synthesis of the compounds was carried out according to
the procedure outlined in Schemes 1 and 2. Firstly, 2-iso-propyl-
5-methyl-phenol (thymol/1a) or 2-tert-butyl-5-methyl-phenol
(1b) or 5-iso-propyl-2-methyl-phenol (carvacrol/1c) were con-
verted to compound with 4-nitroso substituent (2a/2b/2c) by
treating ethanolic solution of these phenols (1a/1b/1c) with concd
hydrochloric acid and sodium nitrite at 0 °C.36,37 The nitroso com-
pounds (2a–2c) were reduced in an ammonical solution by passing
H2S gas (Scheme 1). The substituted 4-aminophenols (3a–3c) ob-
tained are highly unstable and subject to oxidation over a period
⇑
Corresponding author. Tel.: +91 11 27662683; fax: +91 11 27667501.
E-mail address: dsrawat@chemistry.du.ac.in (D.S. Rawat).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.12.001

642
Beena et al. / Bioorg. Med. Chem. Lett. 23 (2013) 641–645
R
NO
R1
R
R
NH2
R1
(i)
(ii)
HO
R1
HO
HO
1a, R = i-Pr, R1 = Me
1b, R = t-Bu, R1 = Me
1c, R = Me, R1 = i-Pr
2a-2c
3a-3c
Scheme 1. Reagents and conditions: (i) NaNO2, concd HCl, EtOH, 0 °C; (ii) H2S gas, NH4OH, 30–40 min.
CHO
R2
R2
R
N
R
NH2
R1
(i)
HO
R1
HO
3a, R = i-Pr, R1 = Me
3b, R = t-Bu, R1 = Me
3c, R = Me, R1 = i-Pr
(4a-4u)
(5a-5u)
(6a-6u)
o, R2 = 3,5-OMe
a, R2 = H
h, R2 = 4-Cl
p, R2 = 3,4,5-OMe
q, R2 = 3,5-Br, 2-OH
r, R2 = 3,5-Br, 4-OH
s, R2 = 2,3-Methylenedioxy
t, R2 = 2-Pyridyl
b, R2 = 4-Me i, R2 = 4-Br
c, R2 = 4-Et
j, R2 = 4-NO2
d, R2 = 4-n-Pr k, R2 = 2-OH
e, R2 = 4-i-Pr l, R2 = 4-OH
f, R2 = 4-t-Bu m, R2 = 4-OMe
u, R2 = 3-Pyridyl
n, R2 = 3,4-OMe
g, R2 = 4-F
Scheme 2. Reagents and conditions: (i) dry MeOH, 30–35 °C, N2 atm, 3–4 h.
100
of time. The 4-aminophenols (3a–3c) prepared according to
Scheme 1 were condensed with different benzaldehydes as de-
picted in Scheme 2. This reaction was carried out at room temper-
ature (30–35 °C) using dry MeOH as solvent and maintaining inert
condition using nitrogen.
In the present study, the antioxidant activity of the synthesized
compounds was assessed in vitro by the 1,1-diphenyl-2-pic-
rylhydrazyl (DPPH) radical scavenging assay.38–41 This method is
based on measuring the continual absorbance decrease of the eth-
anolic solution of the DPPH at 517 nm, in the presence of antioxi-
dant compound. The DPPH has an odd electron so it can accept an
electron or hydrogen free radical. In the presence of antioxidant,
this odd electron becomes paired due to H transfer from antioxi-
dant and hence DPPH absorbance decreases.
90
80
70
60
50
40
30
20
10
0
5a, R = H
5b, R = 4-Me
5k, R = 2-OH
5l, R = 4-OH
5m, R = 4-OMe
5n, R = 3,4-OMe
5o, R = 3,5-OMe
5p, R = 3,4,5-OMe
5q, R = 3,5-Br,2-OH
5r, R = 3,5-Br,4-OH
5s, R = 3,4-OCH2O
5t, R = 2-Pyridyl
5u, R = 3-Pyridyl
1b
AscAcid
Figures 1–4 depict the percentage antioxidant activity at vari-
ous concentrations and IC50 of the synthesized compounds. Com-
pound 1c (carvacrol) showed better antioxidant activity than 1a
(thymol) and 1b. Compound 1a and 1b exhibited almost same level
0
20
40
60
80
100
Concentration (μg/mL)
Figure 2. Antioxidant activity of compounds 5a–5u by DPPH method (EtOH).
of activity (IC50 = 167.57 and 173.93 lg/mL). To study the struc-
ture–activity relationship (SAR) of antioxidant activity, Schiff bases
of 1a (thymol), 1b and 1c (carvacrol) containing strong and weak
100
100
90
90
6a, R = H
4a, R = H
80
6b, R = 4-Me
80
70
60
50
40
30
20
10
0
4b, R = 4-Me
6k, R = 2-OH
70
4k, R = 2-OH
6l, R = 4-OH
4l, R = 4-OH
6m, R = 4-OMe
6n, R = 3,4-OMe
6o, R = 3,5-OMe
6p, R = 3,4,5-OMe
6q, R = 3,5-Br,2-OH
6r, R = 3,5-Br,4-OH
6s, R = 3,4-OCH2O
6t, R = 2-Pyridyl
6u, R = 3-Pyridyl
1c
60
50
40
30
20
10
0
4m, R = 4-OMe
4n, R = 3,4-OMe
4o, R = 3,5-OMe
4p, R = 3,4,5-OMe
4q, R = 3,5-Br,2-OH
4r, R = 3,5-Br,4-OH
4s, R = 3,4-OCH2O
4t, R = 2-Pyridyl
4u, R = 3-Pyridyl
1a
AscAcid
AscAcid
0
20
40
60
80
100
0
20
40
60
80
100
Concentration (μg/mL)
Concentration (μg/mL)
Figure 3. Antioxidant activity of compounds 6a–6u by DPPH method (EtOH).
Figure 1. Antioxidant activity of compounds 4a–4u by DPPH method (EtOH).

Beena et al. / Bioorg. Med. Chem. Lett. 23 (2013) 641–645
643
100
90
180
160
140
120
100
80
4k, 2-OH
4l, 4-OH
4m, 4-OMe
4n, 3,4-OMe
4o, 3,5-OMe
4p, 3,4,5-OMe
4q, 3,5-Br-2-OH
4r, 3,5-Br-4-OH
5k, 2-OH
80
70
4a-4u
5a-5u
6a-6u
60
60
5l, 4-OH
5m, 4-OMe
40
50
5n, 3,4-OMe
5o, 3,5-OMe
5p, 3,4,5-OMe
5q, 3,5-Br-2-OH
5r, 3,5-Br-4-OH
6k, 2-OH
20
40
30
20
10
0
6l, 4-OH
6m, 4-OMe
6n, 3,4-OMe
6o, 3,5-OMe
6p, 3,4,5-OMe
6q, 3,5-Br-2-OH
6r, 3,5-Br-4-OH
Figure 4. IC50 values of the Schiff bases by DPPH method (EtOH).
0
0
20
40
60
80
100
Concentration (µg/mL)
electron donating or withdrawing substituents were synthesized
(4a–4u, 5a–5u and 6a–6u). Figures 1–3 depict the percentage anti-
oxidant activity of the compounds at various concentrations. Most
of the compounds exhibited potent antioxidant activity
(IC50 <50 lg/mL) as shown in Figure 4, even better than their par-
ent compounds (1a–1c). Compounds having OH group at para po-
Figure 6. DPPH antioxidant activity in chloroform.
6m) showed good level of activity (IC50 = 12–14 lg/mL). Introduc-
ing, another OMe group at 3-position (4n, 5n, 6n) makes the com-
pounds slight less active. Again compounds with 3,5-OMe (4o, 5o,
6o) and 3,4,5-OMe (4p, 5p, 6p) were found to be less active than 4-
OMe. Compounds having 2-pyridyl (4t, 5t, 6t) moiety was more ac-
tive than 3-pyridyl (4u, 5u, 6u). Compounds having halogens at
para position of the benzene ring showed mild activity due to their
ꢀI effect, which destabilizes the free radical (VI/Fig. 5). Whereas al-
kyl group containing compounds showed mild activity but better
than the halogen containing compounds due to their +I effect, they
stabilizes the radical to some extent which cause increase in anti-
oxidant activity in comparison to halogen derivatives.
sition (4l, 4r, 5l, 5r, 6l, 6r) were found to be the most potent
compounds with IC50 values ranging between 7.55 and 9.76
mL, better than the reference compound ascorbic acid
(IC50 = 11.66 g/mL). These compounds can be considered as the
lg/
l
best compounds in terms of antioxidant activity as IC50 values low-
er than 10 mg/mL are usually considered to be effective activities
in antioxidant properties.42,43 Compounds with OH at ortho posi-
tion (4k, 5k, 6k) also showed potent activity compared to ascorbic
acid. Compounds with methoxy substituent exhibited slightly low-
er activity than the hydroxyl group containing compounds. For
example compound having OMe group at para position (4m, 5m,
To find the exact mechanism of radical scavenging activity of
these compounds, the antioxidant activity of 24 most active
DPPH2
H
H
DPPH
DPPH
ArOH
ArO
SPLET Mechanism
ArO
R2
R
N
DPPH2
ArOH =
HO
DPPH
R1
ArOH
HAT Mechanism
ArO
R2
O2N
R2
O2N
R
O
N
R
N
H
NO2
NO2
N
N
N
N
R1
HO
R1
O2N
O2N
DPPH
(4-6)
I
R2
R2
R2
R
N
R
O
N
R
O
N
R1
R1
R1
O
III
I
II
R2
R2
R2
R
O
N
R
O
N
R
O
N
R1
R1
R1
VI
IV
V
Figure 5. Proposed mechanism of reaction of Schiff bases with DPPH.

644
Beena et al. / Bioorg. Med. Chem. Lett. 23 (2013) 641–645
120
100
80
60
40
20
0
pathways namely SPLET and HAT mechanisms in ethanol and chlo-
roform, respectively, in DPPH assay. While in ABTS method, the
reaction between ABTSÅ+ and Schiff bases involves electron transfer
followed by proton transfer (ET–PT) mechanism.
4k-4r
5k-5r
6k-6r
Acknowledgments
k
l
m
n
o
p
q
r
Asc
D.S.R. thanks Council of Scientific and Industrial Research (CSIR)
[No. 02(0049)/12/EMR-II] New Delhi, India for financial support.
Beena and D.K. are thankful to CSIR for the award of senior re-
search fellowship. Authors are thankful to CIF-USIC, University of
Delhi, Delhi for spectrophotometer and NMR spectral data, RSIC,
CDRI, Lucknow for mass.
acid
Figure 7. Percentage inhibition by ABTS radical cation method.
compounds (4k–4r, 5k–5r and 6k–6r) was also carried out in non-
polar solvent chloroform (Fig. 6). From the activity results, a similar
structure activity relationship was observed in both the solvents
and the IC50 values of the compounds measured in chloroform
were also comparable to those in ethanol (slightly better in case
of ethanol). In ethanol, ionization of phenolic compounds may take
place due to high dielectric constant of ethanol (ca. 24.55). The cor-
responding phenoxide ion, is much strong electron donor than
PhOH and can transfer one electron to the DPPH free radical rap-
idly. Thus the partial dissociation of phenols in ethanol and rela-
tively high reduction potential of DPPH free radical suggests the
SPLET (sequential proton loss electron transfer) mechanism
(Fig. 5). But on the other side, chloroform having much lower
dielectric constant (ca. 4.80) has less ability to ionize phenols,
therefore interaction between phenol and DPPH free radical occur
primarily by HAT (hydrogen atom transfer) mechanism (Fig. 5).
Slight better activity in ethanol than in chloroform is the conse-
quences of different pathways by which compounds interact with
DPPH radicals. SPLET or HAT mechanism both ultimately results in
the formation of same phenoxyl radical PhOÅ, therefore the stabil-
ization of this free radical finally decides the effect of different sub-
stitution on the antioxidant activity. Electron donating groups on
the ortho or para position of the benzene ring enhance the activity
by stabilization of the free radical, while electron withdrawing
groups decrease the antioxidant activity. The most active com-
Supplementary data
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.bmcl.2012.12.001.
References and notes
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In conclusion we have synthesized sixty three Schiff bases of
different substituted amino phenols (3a, 3b, 3c) and evaluated
them for their in vitro antioxidant activity by DPPH method. All
the compounds exhibited better activity than the parent com-
pounds (1a, 1b, 1c). Eight compounds showed excellent activity
even better than the reference (ascorbic acid) and compound 5r
was found to be the most potent with IC50 7.55 lg/mL. Antioxidant
activity data of selected compounds by DPPH and ABTS assays con-
firms the antioxidant potential of these compounds. The Schiff base
derivatives showed antioxidant property by two different

Beena et al. / Bioorg. Med. Chem. Lett. 23 (2013) 641–645
645
32. Kumar, D.; Joshi, S.; Rohilla, R. K.; Roy, N.; Rawat, D. S. Bioorg. Med. Chem. Lett.
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38. Assay for DPPH scavenging activity: The absorbance values were taken at 5
different concentrations (5, 10, 25, 50 and 100 g/mL) of the tested compounds
l
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36. (a)Synthesis of 2-iso-propyl-5-methyl-4-nitrosophenol (2a): To a stirred solution
of 2-iso-propyl-5-methylphenol (1a; 5 g, 0.03 mol) in ethanol (25 mL), concd
HCl (25 mL) was added. The reaction mixture was cooled to 0 °C and sodium
nitrite (3.44 g, 0.04 mol) was added slowly to this with vigorous stirring. The
solid separated was filtered and recrystallized with ethanol. Yield 75%; lit. mp
158 °C; 1H NMR (400 MHz, CDCl3): d 1.14 (d, J = 6.6 Hz, 6H, CH(CH3)2), 2.19 (s,
3H, CH3), 3.09 (septet, J = 6.6 Hz, 1H, CH(CH3)2), 3.51 (s, 1H, OH), 6.34 (d,
J = 1.5 Hz, 1H, ArH), 7.54 (s, 1H, ArH).
with DPPH at 517 nm. The ethanolic solution of DPPH was added to the
solution of the synthesized compounds in ethanol. After 30 min incubation in
dark the absorbance was measured against ethanol as blank. Ascorbic acid was
used as reference.
The antioxidant activity was calculated as follows: %Activity = [(Ao ꢀ Ai)/
A0] ꢁ 100 where, Ao is the absorbance in the absence and Ai in the presence
of tested compounds. The IC50 values were also determined for all the
compounds by linear regression method.
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(b)Synthesis of 4-amino-2-iso-propyl-5-methylphenol (3a): To 2-iso-propyl-5-
methyl-4-nitrosophenol (2a; 3.75 g) 30% aq ammonia (300 mL) was added. The
brown solution was filtered and to the clear filtrate hydrogen sulfide gas was
passed for 30–40 min. The white solid obtained was filtered and recrystallised
from ethanol to get amino-2-isopropyl-5-methylphenol (3a). Yield 80%; mp
177 °C.
44. Assay for ABTSÅ+ scavenging activity: A 7 mM ABTS and 2.45 mM potassium
persulfate (K2S2O8) solution was made in double distilled water and stored in
the dark at room temperature for 12–16 h before use, to generate the radical
cation. The ABTSÅ+ solution was diluted in ethanol to obtain an absorbance
value of 0.7–0.9 at 734 nm at 30 °C. Then, a 1 mL solution of ABTSÅ+ was added
(c)Typical procedure for the synthesis of 4-(benzylideneamino)-2-iso-propyl-5-
methylphenol (4a) and related compounds (4–6): To a stirred solution of 4-
amino-2-iso-propyl-5-methylphenol (3a; 200 mg, 0.0012 mol) in dry MeOH
(15 mL), benzaldehyde (128.5 mg, 0.0012 mol) was added and reaction
mixture was stirred at 30–35 °C for 3–4 h under nitrogen atmosphere. The
completion of reaction was confirmed by TLC. The solvent was removed under
reduced pressure and the residue thus obtained was washed with hexane. The
crude product was crystallized with MeOH to get compound 4a.
37. Vashi, B. S.; Mehta, D. S.; Shah, V. H. Indian J. Chem. 1995, 34B, 802.
to 1 mL ethanolic solution of the compound. The absorbance of 5 lg/mL
solution was recorded after 1 min and then calculated the percentage
inhibition.
45. Belkheiri, N.; Bouguerne, B.; Bedos-Belval, F.; Duran, H.; Bernis, C.; Salvayre, R.;
Negre-Salvayre, A.; Baltas, M. Eur. J. Med. Chem. 2010, 45, 3019.
46. Menezes, J. C. J. M. D. S.; Kamat, S. P.; Cavaleiro, J. A. S.; Gaspar, A.; Garrido, J.;
Borges, F. Eur. J. Med. Chem. 2011, 46, 773.
47. Shang, Y. J.; Qian, Y. P.; Liu, X. D.; Dai, F.; Shang, X. L.; Jia, W. Q.; Liu, Q.; Fang, J.
G.; Zhou, B. J. Org. Chem. 2009, 74, 5025.