Design, synthesis and biological evaluation of glutamic acid derivatives as anti-oxidant and anti-inflammatory agents

Article abstract of DOI:10.1016/j.bmcl.2017.11.012

A series of glutamic acid derivatives was synthesized and evaluated for their antioxidant activity and stability. We found several potent and stable glutamic acid derivatives. Among them, compound 12b exhibited good in vitro activity, chemical stability and cytotoxicity. A prototype compound 12b showed an anti-inflammatory effect in LPS-stimulated RAW 264.7 cell lines and in a zebrafish model.

Full text of DOI:10.1016/j.bmcl.2017.11.012

Accepted Manuscript
Design, synthesis and biological evaluation of glutamic acid derivatives as anti-
oxidant and anti-inflammatory agents
Suvarna H. Pagire, Eunhye Lee, Haushabhau S. Pagire, Eun Jung Bae, Soo Jung
Ryu, Dahye Lee, Min Hee Kim, Geum Ran Kim, Kyu-Seok Hwang, Sukyung
Ahn, Jin Hee Maeng, Jin Sook Song, Myung Ae Bae, Don Hang Lee, Jin Hee
Ahn
PII:
S0960-894X(17)31087-9
https://doi.org/10.1016/j.bmcl.2017.11.012
BMCL 25418
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
1 October 2017
5 November 2017
6 November 2017
Please cite this article as: Pagire, S.H., Lee, E., Pagire, H.S., Jung Bae, E., Jung Ryu, S., Lee, D., Hee Kim, M., Ran
Kim, G., Hwang, K-S., Ahn, S., Hee Maeng, J., Sook Song, J., Ae Bae, M., Hang Lee, D., Hee Ahn, J., Design,
synthesis and biological evaluation of glutamic acid derivatives as anti-oxidant and anti-inflammatory agents,
Bioorganic & Medicinal Chemistry Letters (2017), doi: https://doi.org/10.1016/j.bmcl.2017.11.012
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Bioorganic & Medicinal Chemistry Letters
Design, synthesis and biological evaluation of glutamic acid derivatives as anti-
oxidant and anti-inflammatory agents
Suvarna H. Pagirea,†, Eunhye Leeb,†, Haushabhau S. Pagirea, Eun Jung Baea, Soo Jung Ryua, Dahye Leea,
Min Hee Kima, Geum Ran Kimc,Kyu-Seok Hwangc, Sukyung Ahnb, Jin Hee Maengb, Jin Sook Songc,
Myung Ae Baec, Don Hang Leeb,*, Jin Hee Ahna,*
a Department of Chemistry, Gwangju Institute of Science and Technology, Gwangju 61005, Republic of Korea
bUtah-Inha Drug Delivery Systems (DDS) and Advanced Therapeutics Research Center, Incheon 21988, Republic of Korea
c Drug Discovery Division, Korea research Institute of Chemical Technology, Daejeon, 305-600, Republic of Korea
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A series of glutamic acid derivatives was synthesized and evaluated for their
antioxidant activity and stability. We found several potent and stable glutamic acid
derivatives. Among them, compound 12b exhibited good in vitro activity, chemical
stability and cytotoxicity. A prototype compound 12b showed an anti-inflammatory
effect in LPS-stimulated RAW 264.7 cell lines and in a zebrafish model.
2017 Elsevier Ltd. All rights reserved.
Keywords:
Glutamic acid derivatives
Ramalin
Antioxidant
Reactive oxygen species (ROS) are produced in living
Despite these properties, the use of Ramalin is limited
because it is unstable, and spontaneously decomposes at room
temperature.8 To overcome its instability, a method for stabilizing
Ramalin using vitamin C has been reported, however, the
instability associated with the parent structure unsolved. This
situation prompted us to structurally modify Ramalin to improve
its stability as well as its potency. In the present study, we report
the synthesis, structure activity relationships (SAR) and
biological evaluation of glutamic acid derivatives as anti-oxidant
and anti-inflammatory agents.
organisms either due to normal cell metabolisms in situ or due to
environmental factors. When ROS (free radicals) such as
superoxide anions, hydroxyl radicals, hydrogen peroxide, and
hypochlorous acid cannot be effectively eliminated, their
accumulation in the body generates oxidative stress. This can
cause chronic and degenerative illnesses such as inflammation,
cardiovascular diseases, cancer, aging-related disorders,
metabolic disorders, and atherosclerosis.1 ROS attack unsaturated
fatty acids resulting in membrane lipid peroxidation, reduced
membrane fluidity, loss of enzymes and receptor activities and
damage to membrane proteins, ultimately leading to cell
inactivation.2 Despite the existence of natural defense
mechanisms, excessive production of ROS over the lifetime of a
cell causes progressive oxidative damage and ultimately cell
death.3
The Ramalin structure can be divided based on three
functional units such as an ortho hydroxy phenyl group,
hydrazine group and L-glutamic acid as shown in Figure 2. We
tried to modify and optimize Ramalin to identify more stable and
potent derivatives.
Many antioxidant compounds are extracted from plants
and tested as the main ingredients of cosmetics. Ramalin, isolated
from the Antarctic lichen, Ramalina terebrata (Ramalinaceae)
has anti-oxidant and anti-inflammatory activities.4-6 It has also
been shown in vivo to inhibit melanogenesis.7
Figure 1. Structure of Ramalin.
Figure 2. Structure of Ramalin divided into three functional units.

The synthesis of glutamic acid derivatives 4(a-l), 6a, 8a,
and 12a,b is depicted in Scheme 1. A commercially available
aniline derivative 1(a-l) was reacted with sodium nitrite under
acidic conditions to obtain the phenyl hydrazine hydrochloride
2(a-l). Compound 2(a-l) reacted with 1-benzyl N-
carbobenzoxy-L-glutamate 13 and O-(benzotriazol-1-yl)-
N,N,N',N'-tetramethyluronium tetrafluoroborate to give Cbz
protected glutamic acid derivatives 3(a-l). Benzyloxy
carbamate and benzyl groups were deprotected with hydrogen
gas and palladium on charcoal to obtain 4(a-l). 2-Hydroxy
aniline 1a coupled with 1-benzyl N-carbobenzoxy-L-
glutamate 13 followed by hydrogenation to give 6a.
(2-(Benzyloxy)phenyl)hydrazine 2a was coupled with 5-
(benzyloxy)-2-(((benzyloxy)carbonyl)amino)-5-oxopentanoic
acid 14 to afford 7a followed by hydrogenation to give 8a. 1-
Fluoro-4-(methylsulfonyl)benzene 9a and 1,2-difluoro-4-
(methylsulfonyl)benzene 9b were treated with hydrazine
monohydrate afforded the hydrazine hydrochlorides 10a,b.
Subsequent condensation with 1-benzyl N-carbobenzoxy-L-
glutamate 13 followed by hydrogenation was carried out to
obtain 12a,b.
The in vitro antioxidant activity of the synthesized glutamic acid
derivatives was evaluated using a DPPH assay. The
assay
Sixteen
different types of glutamic acid derivatives were screened by
using the DPPH assay. Ascorbic acid was used as a reference
compound.
Initially, the SARs of glutamic acid derivatives were
investigated and the results are summarized in Table 1. Ramalin
exhibited antioxidant activity with an IC50 value of 8.67 g/ml,
which is 2 fold higher than that of ascorbic acid. Replacement of
hydrazine in Ramalin with an amide (6a) caused loss of in vitro
activity. In addition, opposite glutamic acid (8a) and protected
glutamic acid derivatives (3a) showed decreased antioxidant
activities. On the basis of these data, we retained the hydrazine
and glutamic acid moieties and further optimized the
hydroxyphenyl moiety.
Diverse mono-substituted phenylhydrazine glutamic acid
derivatives were synthesized and evaluated for their activity. As
can be seen in Table 2, the o-methyl derivative (4b) showed
better in vitro activity than Ramalin (4a) with an IC50 value of
6.24 g/ml. The o-ethyl derivative (4c) exhibited decreased
activity. Although several meta substituted compounds were
synthesized and evaluated, however the activities were not
effective than Ramalin. Para methyl (4h) and methysulfonyl (12a)
derivatives were found to possess single digit IC50 value of 9.99
and 9.57 g/ml, respectively. Based on these data, we further
optimized the phenylhydrazine moiety with more substituents.
Several disubstituted phenylhydrazine glutamic acid
derivatives were synthesized and evaluated. As shown in Table 3,
2,4-di-substituted glutamic acid derivative 4j showed better in
vitro activity than 3,5-di-substituted 4l. Therefore, more 2,5-di-
substitued compounds were evaluated. Compound 12b showed
good in vitro activity with an IC50 value of 8.72 g/ml.
Scheme 1. Reagents and conditions: (a) 1) Sodium nitrite, 6M HCl, H2O,
0 oC; 2) Tin chloride, 6M HCl, 0 oC, 75% (over two steps); (b) 1-Benzyl N-
carbobenzoxy-L-glutamate 13 or 5-(benzyloxy)-2-(((benzyloxy)carbonyl)-
amino)-5-oxopentanoic acid 14, o-(benzotriazol-1-yl)-N,N,N',N'-tetramethy-
luroniumtetrafluoroborate, DIPEA, DMF, room temperature, 85%; (c) H2,
Pd/C, methanol, room temperature, 80-85%; (d) Hydrazine monohydrate,
ethanol, reflux, 70%.
Table 1. Anti-oxidant activity of Ramalin derivatives
compound
Structure
IC50 (g/ml)
4a(Ramalin)
8.67

6a
8a
162.42
37.19
3a
44.98
17.38
Ascorbic acid
Table 2. Anti-oxidant activity of glutamic acid derivatives with monosubstitutents
compound
Structure
IC50 (g/ml)
4a(Ramalin)
8.67
6.24
4b
4c
17.14
10.86
10.67
4d
4e
4f
10.1
11.44
9.99
4g
4h
13.77
9.57
4i
12a

Table 3. Anti-oxidant activity of glutamic acid derivatives with disubstituents
compound
Structure
IC50 (g/ml)
28.01
4j
316.86
15.75
8.72
4l
4k
12b
Table 4. Chemical stability of Ramalin and glutamic acid derivatives
compound
initial
1day(45 ℃)
7day(45 ℃)
14day(45 ℃) 21day(45 ℃) 28day(45 ℃)
100%*
45.71%
-
-
-
-
4a(Ramalin)
100%
100%
100%
100%
82.33%
85.1%
100.2%
99.4%
73.78%
82.0%
94.6%
95.0%
56.74%
72.2%
85.5%
87.0%
4b
4h
58.1%
79.2%
88.8%
50.4%
70.7%
87.2%
12a
12b
Conditions: Compound stability was analyzed by HPLC after storage at 45 oC; 1 mg of each sample was dissolved in 1mL DW.
*Calculated HPLC AUC of day 0 as 100%
On the basis of the DPPH assay results, we chose 4b, 4h, 12a
and 12b for chemical stability studies. Ramalin was unstable at
To evaluate cell viability (compound toxicity) in vitro, a
modified WST-1 assay was performed.10 WST-1 (4-[3-(4-
iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate) assay has been broadly used to measure toxin effects
on mammalian cells.11-13 As shown in Figure 3, glutamic acid
derivatives 4b, 4h and 12b were less cytotoxic than Ramalin 4a
at 100 µg/mL.
o
45 C (Table 4) as well as room temperature.8 Compounds12a
and 12b showed improved stability. Over ~ 95% of parent
compound remained after 7 days at 45 oC.
1 2 0
1 0 0
8 0
On the basis of in vitro stability and toxicity test results,
compound 12b was selected as a prototype. Compound 12b was
evaluated for inhibition of nitric oxide (NO) production in LPS-
stimulate RAW264.7 mouse macrophage cell lines.14 NO is an
important molecule for host defense immune system against
various pathogens such as bacteria, fungi, viruses and parasites.15
Over-expression of NO induces tissue damage related with acute
and chronic inflammations.16 To accomplish this study, cells were
simultaneously treated with 0.3 μg/mL LPS and different
concentrations of compound 12b. The RAW 264.7 cells were
activated and increased NO production by LPS, and NO
production was measured as nitrite concentration in the culture
medium after 24 h incubation. Compound 12b significantly
inhibited (p<0.01) the nitrite accumulation in LPS-activated
RAW 264.7 cells in a concentration dependent manner.
6 0
*
4 0
2 0
0
C o n tro l
4 a
4 b
4 h
1 2 b
Figure 3. Effects of glutamic acid derivatives on cell viability in Raw 264.7
mouse macrophages. Cells were treated with glutamic acid derivatives (100
μg/mL) for 24h. Relative cell viability was evaluated using a modified WST-
1 assay. All results are representative of 3 independent experiments. (means ±
SD) *P<0.01 compared with the control group.

In conclusion, we investigated the antioxidant activity of a
series of glutamic acid derivatives. Several glutamic acid
derivatives with ortho and para substituents were found to be
potent anti-oxidant agents. Among the synthesized glutamic acid
derivatives, compounds 4b, 12a, and 12b showed better in vitro
activity, chemical stability and cytotoxicity than Ramalin 4a.
Compound 12b, which is a prototype, showed anti-inflammatory
effects in LPS-stimulated RAW 264.7 cell lines and in a
zebrafish model.
Acknowledgments
This work was supported by grants from the Ministry of Trade,
Industry and Energy, Korea (2017-10063396), the Ministry of
Science, ICT & Future Planning(MSIP)/National Research
Foundation of Korea(NRF) (2016M3A9B6902868) and the
Korea Health Industry Development Institute (KHIDI), funded
by the Ministry of Health & Welfare (A111345).
Figure 4. Inhibition of NO production in LPS-stimulated RAW 264.7 cell
lines. NO production was measured using the Griess reagent. All results are
representative of 3 independent experiments. Values are means ± SD.
**P<0.01 compared with the LPS group.
†These authors contributed equally to this work
To investigate the anti-inflammatory effects of 12b in vivo, we
used a zebrafish model. The Tg(mpx:GFP) zebrafish line is an in
vivo model used for the analysis of inflammatory response, which
expresses GFP under the control of the neutrophil-specific
myeloperoxidase promoter.17 At 3 dpf (3 days post fertilization),
Tg(mpx:GFP) larvae were anesthetized with tricaine and
amputated tail fin. After amputation of tail fin, GFP-expressing
neutrophils migrated to the wounded region. At 4 hours post
amputation, the embryonic medium containing wounded larvae
was treated with 12b for 6 hours. 12b decreased neutrophil
recruitment to wounded tail fin. However, un-amputated larvae
did not recruit neutrophils recruitment in response to 12b
treatment. Dexamethasone was used as a positive control. This
result confirms that 12b suppresses neutrophil-mediated wound
inflammation.
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2 0 0
1 5 0
1 0 0
**
**
5 0
Supplementary Material
**
**
0
(  g / ml
)
5 0
1 0
Supplementary data associated with this article can be found,
in the online version at doi:
D E X
0 .1 % D M S O
1 2 b
Figure 5. In vivo anti-inflammatory activity of compound 12b.The compound
reduced neutrophil numbers in wound sites in response to injury in Tg
(mpx:GFP) zebrafish. **P<0.01

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Suvarna H. Pagire, Eunhye Lee, Haushabhau S. Pagire, Eun Jung Bae, Soo Jung Ryu, Min Hee Kim, Dahye Lee, Geum
Ran Kim, Kyu-Seok Hwang, Sukyung Ahn, Jin Hee Maeng, Jin Sook Song, Myung Ae Bae, Don Hang Lee, Jin Hee Ahn