Design, Synthesis, and Biological Evaluation of New Peptide Analogues as Selective COX-2 Inhibitors

Article abstract of DOI:10.1002/ardp.201700158

A new class of peptide derivatives possessing SO₂Me and N₃ pharmacophores at the para position of a phenyl ring bound to different aromatic amino acids were synthesized based on solid-phase synthesis methodology, and evaluated as selective cyclooxygenase-2 (COX-2) inhibitors. One of the analogues, i.e., compound 2a as the representative of this series, was recognized as the highest selective COX-2 inhibitor with a COX-2 selectivity index of >500. The structure–activity relationships (SARs) acquired indicated that compound 2a containing a 4-(methylsulfonyl)benzoyl group as a pharmacophore and tyrosine as a ring bearing amino acid in the second position and glutamic acid as the C-terminal amino acid can give the essential geometry to provide selective COX-2 inhibitory activity. Antiproliferative activity of the synthesized peptides (1a–7b) was also determined against four different human cancer cell lines, including MCF-7, HepG2, A549, and HeLa. According to our results, A549, HepG2, and MCF7 seemed to be more sensitive cell lines than HeLa cells encountering these compounds, which gave inhibitory action with IC₅₀ values from 4.8 to 64.4 μM. In this regard, compounds 3a and 2b displayed the best inhibitory activity against the cell lines. Moreover, a good correlation was observed between the antiproliferative potency and the COX-2 inhibitory activity of compounds 1a, 2a, 2b, and 5b. Such findings suggest that one of the mechanism of anticancer activity of these peptides may be through the COX-2 inhibitory action.

Full text of DOI:10.1002/ardp.201700158

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Arch. Pharm. Chem. Life Sci. 2017, 350, e1700158
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Full Paper
Design, Synthesis, and Biological Evaluation of New Peptide
Analogues as Selective COX-2 Inhibitors
Mohammad A. Ahmaditaba1, Soraya Shahosseini1, Bahram Daraei2, Afshin Zarghi1
,
ꢀ
1
and Mohammad H. Houshdar Tehrani
1
Department of Medicinal Chemistry, School of Pharmacy, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
Department of Toxicology, School of Medical Sciences, Tarbiat Modares University, Tehran, Iran
2
A new class of peptide derivatives possessing SO2Me and N3 pharmacophores at the para position of
a phenyl ring bound to different aromatic amino acids were synthesized based on solid-phase
synthesis methodology, and evaluated as selective cyclooxygenase-2 (COX-2) inhibitors. One of the
analogues, i.e., compound 2a as the representative of this series, was recognized as the highest
selective COX-2 inhibitor with a COX-2 selectivity index of >500. The structure–activity relationships
(SARs) acquired indicated that compound 2a containing a 4-(methylsulfonyl)benzoyl group as a
pharmacophore and tyrosine as a ring bearing amino acid in the second position and glutamic acid
as the C-terminal amino acid can give the essential geometry to provide selective COX-2 inhibitory
activity. Antiproliferative activity of the synthesized peptides (1a–7b) was also determined against
four different human cancer cell lines, including MCF-7, HepG2, A549, and HeLa. According to our
results, A549, HepG2, and MCF7 seemed to be more sensitive cell lines than HeLa cells encountering
these compounds, which gave inhibitory action with IC50 values from 4.8 to 64.4 mM. In this regard,
compounds 3a and 2b displayed the best inhibitory activity against the cell lines. Moreover, a good
correlation was observed between the antiproliferative potency and the COX-2 inhibitory activity of
compounds 1a, 2a, 2b, and 5b. Such findings suggest that one of the mechanism of anticancer
activity of these peptides may be through the COX-2 inhibitory action.
Keywords: Anticancer / Antiproliferative activity / COX-2 inhibitor / Peptide derivatives / Solid-phase
synthesis
Received: May 16, 2017; Revised: August 2, 2017; Accepted: August 7, 2017
DOI 10.1002/ardp.201700158
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
COX-2, and COX-3 enzymes. COX-1, exists naturally, is
responsible for many physiological activities such as gastroin-
Introduction
Cyclooxygenase (COX) is an essential enzyme involved in
conversion of arachidonic acid to prostaglandins [1]. Three
isoforms of this enzyme identified so far are named as COX-1,
testinal, homeostasis, and renal functions. In contrast, COX-2
is provoked in response to pathogenic conditions and triggers
proinflammatory status [2]. COX-1 and COX-2 enzymes have
an essential role in the biosynthesis of different prostanoids
and consequently these enzymes take part in pain and fever
mechanism [3]. COX-3, a lately discovered enzyme, does not
Correspondence: Prof. Mohammad H. Houshdar Tehrani,
Department of Medicinal Chemistry, School of Pharmacy, Shahid
Beheshti University of Medical Sciences, Tehran 14155-6153, Iran.
E-mail: m_houshdar@sbmu.ac.ir
ꢀAdditional correspondence: Prof. Afshin Zarghi,
E-mail: zarghi@sbmu.ac.ir
Fax: þ98-21-88665341
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show any prostaglandin-involved COX activity [4]. Traditional
non-steroidal anti-inflammatory drugs (NSAIDs) are not
selective inhibitors of COX enzymes and therefore show
many side effects including gastrointestinal and kidney
dysfunctions. Because COX-2 is specifically involved in
producing inflamed conditions, it was assumed that the
application of selective COX-2 inhibitors by blocking prosta-
glandin and thromboxane synthesis alleviate inflammation
and pain in patient along with a reduced gastric irritation and
risk of peptic ulceration. For this reason, many selective COX-2
inhibitors with various structures have been synthesized and
introduced to the market as safe and secure agents for
treatment of inflammation with minimum related side
effects. However, some of COX-2 inhibitors such as rofecoxib
and valdecoxib showed cardiovascular side effects and so
have been withdrawn from the market [5].
Accordingly, many researchers are interested in introducing
novel templates of COX-2 inhibitors with higher potency and
lower side effects. Recent researches for introducing thera-
peutic peptides and increasing attractions in this field have
resulted in the production of around 140 therapeutic peptides
which are now being evaluated in clinical trials [6]. Mean-
while, in a previous study, a new series of tripeptides was
reported as COX inhibitors [7]. The peptides were checked
experimentally by utilizing surface plasmon resonance (SPR)
method. The authors identified a tripeptide which could be a
possible lead for another class of COX inhibitors. In another
study, a series of fluorobenzoylated di- and tripeptides were
described as COX-2 inhibitors which displayed noticeable
potency and selectivity compared with celecoxib [8].
Structure–activity relationship (SAR) surveys of COX-2 inhib-
itors have demonstrated that the substitution of N3 (azide)
moiety or SO2Me (or SO2NH2) at the para position of one of
the phenyl rings frequently makes high selectivity in COX-2
inhibitory activity [9–11]. Therefore, it is attractive to develop
novel selective COX-2 inhibitors with peptide structure
containing necessary pharmacophoric groups which show
anti-inflammatory effects. COX-2 enzyme also seems to be
expressed at high levels in many types of cancerous cells such
as colon cancer [12]. It has been shown that PGE2 is raised in
colon cancer where COX-2 overexpression leads to increasing
metastatic potential of tumors [13–15]. These explorations
were confirmed by anticancer effect of celecoxib as a potent
and selective COX-2 inhibitor [16, 17]. Consequently, it would
be attractive to develop novel selective COX-2 inhibitors with
peptide structure which show anti-inflammatory effects as
well as anticancer activity.
view of containing any COX-2 inhibitory as well as anti-
proliferative activities.
Results and discussion
Chemistry
The synthesis of peptides (1a–7b) was carried out according to
the solid-phase peptide synthesis approach using Fmoc
methodology in a manual reaction vessel. This solid-phase
synthesis could give products with high purity and good yield.
To introduce a SO2Me group at N-terminal position, at first 4-
methylthiobenzaldehyde was treated with oxone. This
reagent oxidized methyl as well as aldehyde groups,
simultaneously, to give 4-(methylsulfonyl)benzoic acid. This
latter compound was used to link to the peptide-resin, like as
a last amino acid. For preparing peptides carrying an azide
group, 4-aminobenzoic acid was first converted to azidoben-
zoic acid by NaNO2 and NaN3 reagents. The azido compound,
thus prepared, was used as a last amino acid for attaching to
the peptide-resin. Cleaving final peptide from resin could give
a peptide product containing carboxyl groups at one end and
SO2 Me or N3 group at the other end (Scheme 1).
Molecular modeling
By docking of compound 2a into the COX-2 binding
site (Fig. 2), it shows two hydrogen bonds with His90
513
̊
̊
(distance ¼ 3.03 A) and Arg
(distance ¼ 4.13 A). Further-
more, the acidic functional group of docked compound shows
one hydrogen bond with Trp387 (distance ¼ 4.98 A). In
̊
addition, docking shows the hydrophobic pocket made by
the side chains of residues Leu531, Leu359, and Val349 surrounds
the phenyl group. Figure 3 shows the superimposition of
celecoxib and the synthesized peptides while docked into the
COX-2 binding site and shows that all the compounds are well
integrated in the selective COX-2 active site pocket. The
docking scores of synthesized compounds and celecoxib are
mentioned in Table 1.
Biological activity
The data of COX-1/COX-2 inhibition assay are mentioned in
Table 2. SAR data (IC50 values) acquired by the calculation of
the in vitro potency of the heading compounds to inhibit the
COX-1 and COX-2 isozymes displayed that the most peptides
(1a–7b) were acceptable inhibitors of the COX-2 isozyme with
IC50 values in the 0.06–0.44 mM range, and COX-2 selectivity
indices in the 84.3–>500 range [18]. Our results showed that
the COX inhibition from the points of potency and selectivity
was affected by the nature of amino acid substituents and
SO2Me or N3 pharmacophore at the para position of the end
phenyl ring. Based upon, compounds (4a, 5a, 4b) and (6a, 1b)
possessing histidine and tryptophan, respectively, as the ring
bearing amino acids showed low selectivity index which may
In the present work, we report the design and synthesis of a
new series of peptide derivatives as COX-2 inhibitors (Fig. 1).
The rational design is based on the use of pharmacophoric
parts of COX-2 inhibitors containing SO2Me or N3 (azide)
pharmacophore at the para position of a phenyl ring on one
end with aromatic or cyclic amino acids in the middle and
acidic amino acids on the other end of the peptide molecules
to imitate the structure of COX-2 inhibitors. The synthesized
peptides are then employed for evaluating their properties in
be explained by
a weak interaction of histidine and
tryptophan with COX-2 active site. Subsequently, compounds
containing glutamic acid as the C-terminal amino acid (such as
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Figure 1. Chemical structures of some
known selective COX-2 inhibitors (2a, 5b),
two of our designed scaffolds.
1a, 2a, 2b, and 5b) were generally more selective COX-2
inhibitors compared with the other analogues having aspartic
acid. This finding could be described as a result of difference
in the side chain of glutamic acid being longer than aspartic
4.8 to 64.4 mM. Nevertheless, compounds 4a, 5a, 1b, and 7b
did not show any cytotoxic activity for HepG2 cell line at
100 mM concentration. These results also showed that the
synthesized peptides did not have noticeable cytotoxic
activity against HeLa cell line except peptides 1a, 7a, 2b,
4b, 5b, and 7b which had a moderate cytotoxic activity. These
results inferred that the compounds constructed with
tryptophan and histidine as the ring bearing amino acids
may generally decrease the cytotoxic activity. Whereas,
compounds containing phenylalanine, tyrosine, and proline
as the central core amino acids may relatively improve the
cytotoxic activity. These results also indicated that there is not
a meaningful difference in cytotoxic activity among the
peptides containing azide or methylsulfonyl group at the para
position of their phenyl rings. Based on the obtained results,
in some cases such as for compounds 1a, 2a, 2b, and 5b there
was a good correlation between antiproliferative activity and
COX-2 selective inhibitory activity whereas for some other
compounds, i.e., 3a, 7a, and 7b, this correlation was not
detected. Therefore, it can be assumed that one of the
mechanisms of cytotoxic activity of these synthesized peptides
might be through the COX-2 inhibitory action. The cytotoxic-
ity outcomes for human fibroblasts showed no significant
effects resulted by compounds 1a–7b.
acid, which could make better hydrogen binding with Trp387
.
These results also marked that the peptide derivatives
carrying azide group (compounds 2b, 3b, 5b, and 6b)
displayed higher potency for COX-2 inhibitory activity
compared with methyl sulfonyl analogues. This may be
defined as the result of hydrogen binding between nitrogen
atoms of azide group with the COX-2 active site for better
interaction. Our results indicated that 2a, 2b, and 5b showed
the highest COX-2 selectivity (SI ¼ >500, 439.2, and 463.6,
respectively) among the synthesized compounds.
Antiproliferative activity of the synthesized peptides
(1a–7b) was determined against four different human cancer
cell lines, including MCF-7 (breast cancer cells), HepG2 (liver
hepatocellular cells), A549 (adenocarcinoma human alveolar
basal epithelial cells), and HeLa (human cervix cancer cells).
The results are shown in Table 3. In accordance with our
results, A549, MCF-7, and HepG2 were more sensitive cell lines
than HeLa cells exposed to the synthesized peptides, i.e.,
compounds 2a–6a. The most peptides had very good activity
against A549, MCF-7, and HepG2 with mean IC50 values from
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Scheme 1. Synthesis route. Reagents and conditions: (a) DIC, HOBT, DMAP, Fmoc-Asp or Fmoc-Glu, shaking 2h; (b) piperazine 10% in
DMSO, 30 min; (c) DIC, HOBT, Fmoc-XAA, shaking 3 h; (d) piperazine 10% in DMSO, 30 min; (e) DIC, HOBT, 4-(methylsulfonyl)benzoic
acid or para-azidobenzoic acid, shaking 3 h; (f) trifluoroacetic acid/DCM/anisole/triisopropyl-silane, 2 h.
higher than celecoxib, a known COX-2 inhibitor drug. The
results also marked that the peptide derivatives containing
Conclusions
With the aim of discovery of new and potent COX-2 inhibitors,
we designed new peptide scaffolds which could show activity
as COX-2 inhibitors. The synthesis of peptide analogues was
based on solid-phase peptide synthesis using Wang resin. The
outcome of in vitro COX-1/COX-2 inhibition assay showed that
the most of the designed peptides had an acceptable
inhibitory activity and in some cases, such as for compounds
2a, 2b, and 5b, with a selectivity index of COX-2 inhibition
azide moiety display higher potency than analogues with
methyl sulfonyl group for COX-2 inhibitory activity. Anti-
proliferative activity of the synthesized peptides showed that
compounds containing phenylalanine, tyrosine, and proline
as the central core amino acids may relatively improve the
cytotoxic activity. Based on the obtained results, for some
cases such as compounds 1a, 2a, 2b, 5b there was a good
correlation between antiproliferative activity and COX-2
Figure 2. 3D representations of the binding
of compound 2a (colored according to the
atom type) in the active site of COX-2 (COX-2
structure with PDB code 6cox was used for
docking). (A) The red-dotted lines show the
hydrogen bonds in protein–ligand complex.
(B) The protein is represented by colored
ribbon and shows superimposition of cele-
coxib (pink and stick) and 2a (cyan and stick)
with COX-2 by the essential amino acid
residues at the active site. All pictures were
prepared with PyMOL.
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(Bachem, Switzerland and Sigma–Aldrich, UK). Peptide
synthesis solvents, reagents were analytical grade and
provided from commercial sources (Merck, Germany) and
used without further purification unless otherwise noted.
Infrared spectra were acquired on a Perkin-Elmer 1420 ratio
recording spectrometer. A Bruker FT-500 MHz instrument
(Brucker Biosciences, USA) was used to acquire 1H NMR
spectra; DMSO-d6 was used as solvent. Coupling constant (J)
values were estimated in hertz (Hz) and spin multiples were
given as
s
(singlet),
d
(double),
t
(triplet),
q
(quartet), m (multiplet), and br (broad). The mass spectral
measurements were performed on a 6410 Agilent LCMS triple
quadruple mass spectrometer (LCMS) with an electrospray
ionization (ESI) interface.
The InChl codes with biological activity data for the
synthetic peptides are given in the Supporting Information.
Figure 3. Three-dimensional superimposed representations of
celecoxib and the synthesized compounds with COX-2 using
PyMOL program with the essential amino acid residues at the
active site.
General procedure for attaching the first amino acid
The first amino acid, Fmoc-Xaa-OH, was linked to the Wang
resin (100–200 mesh, 1% DVB, 1 mmol/g) using HOBt (2 eq)
and DIC (1 eq) as activating agents and a catalytic amount of
DMAP. The Fmoc protecting group was removed by treating
the protected amino acid-resin with a 10% solution of
piperazine in DMF (30 min). The resin was then washed with
DMF (5ꢁ).
inhibitory action, whereas, for some other compounds this
was not the case. As a conclusion, these data indicate that one
of the mechanisms of anticancer activity of the designed
peptides could be mediated through COX-2 inhibition.
Experimental
General procedure for the preparation of peptides 1a–7b
The following reactant materials: Fmoc-amino acid,
4-(methylsulfonyl)benzoic acid or 4-azidobenzoic acid (2.0
eq), DIC (1 eq), and HOBt (2 eq), were dissolved in DMF or
DCM, added to the resin and shaken slowly. The coupling time
Chemistry
General
Na-Fmoc-protected amino acids, Wang resin, HOBt, DIC,
piperazine, and trifluoroacetic acid were purchased from
Table 1. Docking scores for compounds 1a–7b.
Docking score
(kcal/mol)
Compounds
Y
First amino acid
Second amino acid
1a
2a
3a
4a
5a
6a
7a
1b
2b
3b
4b
5b
6b
7b
Celecoxib
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
SO2Me
N3
N3
N3
N3
N3
Phe
Tyr
Tyr
His
Glu
Glu
Asp
Glu
Asp
Asp
Asp
Asp
Glu
Asp
Asp
Glu
Asp
Asp
ꢂ6.1
ꢂ7.4
ꢂ5.3
ꢂ7.4
ꢂ7.8
ꢂ8.1
ꢂ8.3
ꢂ8.4
ꢂ7.6
ꢂ8.3
ꢂ7.3
ꢂ8.2
ꢂ8.1
ꢂ7.3
ꢂ11.5
His
Trp
Pro
Trp
Pro
Tyr
His
Phe
Phe
Pro
N3
N3
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Table 2. In vitro COX-1 and COX-2 enzyme inhibition assay data for compounds 1a–7b.
COX-1
IC50 ꢃ SD (mM)
COX-2
a)
a)
Peptide
Compounds
IC50 ꢃ SD (mM)
Selectivity indexb) (SI)
1a
2a
3a
4a
5a
6a
7a
1b
2b
3b
4b
5b
6b
7b
Celecoxib
p-MeSO2Bz-Phe-Glu
p-MeSO2Bz-Tyr-Glu
p-MeSO2Bz-Tyr-Asp
p-MeSO2Bz-His-Glu
p-MeSO2Bz-His-Asp
p-MeSO2Bz-Trp-Asp
p-MeSO2Bz-Pro-Asp
p-N3Bz-Trp-Asp
p-N3Bz-Pro-Glu
p-N3Bz-Tyr-Asp
p-N3Bz-His-Asp
p-N3Bz-Phe-Glu
55.7
>100
33.9
ND
0.19
0.2
0.17
ND
293.1
>500
199.4
ND
23.6
25.2
50.2
45.8
52.1
28.9
31.2
64.9
12.4
47.6
24.3
0.28
0.19
0.44
>100
0.12
0.14
0.22
0.14
0.06
0.24
0.06
84.3
132.6
114.1
ND
439.2
206.4
141.8
463.6
206.6
198.3
405
p-N3Bz-Phe-Asp
p-N3Bz-Pro-Asp
a) Values are means of two determinations acquired using an ovine COX-1/COX-2 assay kit and the deviation from the mean is
<10% of the mean value.
b) In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
was 3 h. The peptide-resin was washed with DMF (3ꢁ), DCM
(3ꢁ), and methanol (3ꢁ). Peptides were deprotected and
cleaved from the solid support with trifluoroacetic acid/DCM/
anisole/triisopropylsilane (50:45:2.5:2.5) for 2 h. The resin was
filtered off and the crude peptide was precipitated by adding
dropwise to a cold diethyl ether. The product was filtered and
lyophilized.
50:50) was added. The mixture was stirred at room tempera-
ture for 24 h. After evaporation of THF, the residue was
extracted with chloroform, washed with 10% aqueous
sodium bicarbonate, and dried with anhydrous sodium sulfate
and then the solvent was evaporated. In many cases, off-white
to pale yellow solid was formed. Yield: 70–94%.
General procedure for the preparation of 4-azidobenzoic
acid
4-Aminobenzoic acid (1.64 g, 12 mmol) was dissolved in water
(10 mL), and HCl (6 mL, 12 N) was added. The mixture
was stirred at room temperature for 1 h, then cooled down
General procedure for the preparation of 4-(methylsulfonyl)-
benzoic acid
4-Methylthiobenzaldehyde (3 mL) was dissolved in THF
(10 mL), and to which oxone (10 g) in THF/water (30 mL,
Table 3. The in vitro antiproliferative activities of peptides 1a–7b derivatives.
No.
Compounds
MCF-7 (IC50 mM)
HEP-G2 (IC50 mM)
A549 (IC50 mM)
HELA (IC50 mM)
Celecoxib
19.3 ꢃ 0.04
64.4 ꢃ 0.03
16.0 ꢃ 0.13
17.5 ꢃ 0.04
44.1 ꢃ 0.02
>100
16.0 ꢃ 0.03
5.7 ꢃ 0.04
31.1 ꢃ 0.03
4.8 ꢃ 0.12
>100
16.0 ꢃ 0.02
13.9 ꢃ 0.03
20.6 ꢃ 0.03
5.7 ꢃ 0.04
>100
12.9 ꢃ 0.23
61.6 ꢃ 0.18
23.8 ꢃ 0.03
>100
19.9 ꢃ 0.03
5.8 ꢃ 0.02
28.4 ꢃ 0.02
>100
27.9 ꢃ 0.04
21.4 ꢃ 0.02
>100
>100
>100
>100
>100
1a
2a
3a
4a
5a
6a
7a
1b
2b
3b
4b
5b
6b
7b
p-MeSO2Bz-Phe-Glu
p-MeSO2Bz-Tyr-Glu
p-MeSO2Bz-Tyr-Asp
p-MeSO2Bz-His-Glu
p-MeSO2Bz-His-Asp
p-MeSO2Bz-Trp-Asp
p-MeSO2Bz-Pro-Asp
p-N3Bz-Trp-Asp
p-N3Bz-Pro-Glu
p-N3Bz-Tyr-Asp
p-N3Bz-His-Asp
p-N3Bz-Phe-Glu
>100
18.1 ꢃ 0.01
64.3 ꢃ 0.01
23.0 ꢃ 0.05
>100
19.3 ꢃ 0.02
62.3 ꢃ 0.02
63.2 ꢃ 0.03
18.7 ꢃ 0.04
61.5 ꢃ 023
>100
>100
24.4 ꢃ 0.03
5.7 ꢃ 0.03
12.1 ꢃ 0.23
>100
62.2 ꢃ 0.02
19.7 ꢃ 0.06
18.4 ꢃ 0.12
19.1 ꢃ 0.04
>100
17.7 ꢃ 0.04
>100
19.9 ꢃ 0.02
20.6 ꢃ 0.04
>100
p-N3Bz-Phe-Asp
p-N3Bz-Pro-Asp
18.6 ꢃ 0.04
21.8 ꢃ 0.03
17.9 ꢃ 0.02
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Peptide Analogues as Selective COX-2 Inhibitors
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to 0–5°C in an ice bath, and to which, an aqueous solution of
NaNO2 (0.89 g in 10 mL, 13 mmol) was added dropwise. After
5 min, a solution of sodium azide (0.0.84 g in 10 mL water,
13 mmol) was added to the mixture and stirred for 5 min. The
precipitate was filtered, extracted with ethanol (20 mL), and
dried by vacuum to give a light yellow solid (1.52 g, yield
75%). The crude solid was used for the next step of synthesis.
Synthesis of p-MeSO2Bz-His-Asp (5a)
Yield:78%;whitesolid;IR(KBr):n(cmꢂ1)1305,1141(SO2),1400–
1
–
1600(aromatic),1736,1704(C O); HNMR(DMSO):dppm2.63–
–
2.74 (m, 2H, CH2, aspartic acid), 2.63–2.74 (m, 2H, CH2,
imidazole), 3.1 (s, 3H, SO2CH3), 4.26–4.27 (d, 1H, CH), 4.77–
4.79 (d, 1H, CH), 7.20–7.22 (d, 2H, J ¼ 10Hz, 4-methylsulfonyl-
phenyl H2 and H6), 7.32 (s, 1H, CH, imidazole), 7.87–7.89 (d,
J ¼ 10Hz, 2H, 4-methylsulfonyl phenyl H3 and H5), 8.38–8.39 (d,
1H, NH), 8.77–8.78 (d, 1H, NH), 8.93 (s, 1H, CH, imidazole), 12.5
(br, 2H, COOH), 13.79 (s, 1H, NH, imidazole); LC-MS (ESI) m/z:
451.2 (Mꢂ1).
Synthesis of p-MeSO2Bz-Phe-Glu (1a): p-(methylsulfonyl)-
benzoyl-phenylalanine-glutamic acid
Yield: 72%; white solid; IR (KBr): n (cmꢂ1) 1324, 1154 (SO2),
1400–1600 (aromatic), 1731, 1740 (C O); 1H NMR (DMSO): d
–
–
ppm 1.84–2.03 (m, 2H, CH2, glutamic acid), 2.34–2.50 (t, 2H,
CH2, glutamic acid), 2.98–3.17 (m, 2H, CH2, benzyl), 3.1 (s, 3H,
SO2CH3), 4.26–4.27 (d, 1H, CH), 4.77–4.79 (d, 1H, CH), 7.15–7.18
(t, J ¼ 9 Hz, 1H, phenyl H4), 7.34–7.36 (d, 2H, J ¼ 9.5 Hz, phenyl
H2 and H6), 7.79–7.99 (m, 4H, 4-methylsulfonyl phenyl
H2–H6), 8.38–8.39 (d, 1H, NH), 8.77–8.78 (d, 1H, NH), 12.6
(br, 2H, COOH); LC-MS (ESI) m/z: 474.7 (Mꢂ1).
Synthesis of p-MeSO2Bz-Trp-Asp (6a)
Yield: 57%; white solid; IR (KBr): n (cmꢂ1) 1305, 1144 (SO2),
1400–1600 (aromatic), 1738, 1727 (C O); 1H NMR (DMSO): d
–
–
ppm 2.63–2.74 (m, 2H, CH2, aspartic acid), 2.83–3.05 (m, 2H,
CH2, indole), 3.1 (s, 3H, SO2CH3), 4.26–4.27 (d, 1H, CH), 4.77–
4.79 (d, 1H, CH), 6.62–6.64 (d, 2H, J ¼10 Hz, 4-methylsulfonyl
phenyl H3 and H5), 7.14–7.16 (d, J ¼ 10 Hz, 2H, 4-methylsul-
fonyl phenyl H2 and H6), 7.69 (s, 1H, CH, indole), 8.002–8.005
(m, 4H, indole), 8.38–8.39 (d, 1H, NH), 8.77–8.78 (d, 1H, NH),
12.4 (br, 2H, COOH); LC-MS (ESI) m/z: 499.7 (Mꢂ1).
Synthesis of p-MeSO2Bz-Tyr-Glu (2a)
Yield: 81%; white solid; IR (KBr): n (cmꢂ1) 1305, 1145 (SO2),
1400–1600 (aromatic), 1737, 1727 (C O); 1H NMR (DMSO): d
–
–
ppm 1.84–2.03 (m, 2H, CH2, glutamic acid), 2.34–2.50 (m, 2H,
CH2, glutamic acid), 2.98–3.17 (m, 2H, CH2, benzyl), 3.1 (s, 3H,
SO2CH3), 4.26–4.27 (d, 1H, CH), 4.77–4.79 (d, 1H, CH), 4.9 (s, 1H,
phenol), 6.60–6.62 (d, J¼ 8 Hz, 2H, phenol H3 and H5),
7.08–7.10 (d, 2H, J¼ 8 Hz, phenol H2 and H6), 7.17–7.19
(d, 2H, J¼ 10 Hz, 4-methylsulfonyl phenyl H2 and H6), 7.83–7.85
(d, J¼ 10 Hz, 2H, 4-methylsulfonyl phenyl H3 and H5), 8.38–8.39
(d, 1H, NH), 8.77–8.78 (d, 1H, NH), 12.3 (br, 2H, COOH); LC-MS (ESI)
m/z: 490.7 (Mꢂ1).
Synthesis of p-MeSO2Bz-Pro-Asp (7a)
Yield: 52%; white solid; IR (KBr): n (cmꢂ1) 1307, 1136 (SO2),
1400–1600 (aromatic), 1741 (C O); 1H NMR (DMSO): d ppm
–
–
1.80–1.82 (m, 2H, CH2, pyrrolidine), 2.10–2.32 (m, 2H, CH2,
pyrrolidine), 2.57 (s, 3H, SO2Me), 2.63–2.74 (m, 2H, CH2,
aspartic acid), 2.99 (t, 2H, CH2), 4.26–4.27 (d, 1H, CH), 4.68–4.69
(d, 1H, CH), 7.75–7.77 (d, J ¼ 8 Hz, 2H, 4-methylsulfonyl phenyl
H3 and H5), 8.00–8.017 (d, 2H, J ¼ 8 Hz, 4-methylsulfonyl
phenyl H2 and H6), 8.2 (d, 1H, NH), 12.4 (br, 2H, COOH); LC-MS
(ESI) m/z: 411 (Mꢂ1).
Synthesis of p-MeSO2Bz-Tyr-Asp (3a)
Yield: 55%; white solid; IR (KBr): n (cmꢂ1) 1305, 1161 (SO2),
Synthesis of p-N3Bz-Trp-Asp (1b): p-azidobenzoyl-
1400–1600 (aromatic), 1737, 1732 (C O); 1H NMR (DMSO): d
tryptophan-aspartic acid
–
–
ppm 2.63–2.74 (m, 2H, CH2, aspartic acid), 2.98–3.17 (m, 2H,
CH2, benzyl), 3.1 (s, 3H, SO2CH3), 4.26–4.27 (d, 1H, CH),
4.77–4.79 (d, 1H, CH), 4.9 (s, 1H, phenol), 6.60–6.62 (d, J ¼ 8 Hz,
2H, phenol H3 and H5), 7.08–7.10 (d, 2H, J ¼ 8 Hz, phenol H2
and H6), 7.17–7.19 (d, 2H, J ¼ 10 Hz, 4-methylsulfonyl phenyl
H2 and H6), 7.83–7.85 (d, J ¼ 10 Hz, 2H, 4-methylsulfonyl
phenyl H3 and H5), 8.38–8.39 (d, 1H, NH), 8.77–8.78 (d, 1H, NH),
12.4 (br, 2H, COOH); LC-MS (ESI) m/z: 477.0 (Mꢂ1).
Yield: 61%; white solid; IR (KBr): n ) 1400–1600
(cmꢂ1
(aromatic), 1727 (C O), 2134 (N ); 1H NMR (DMSO): d ppm
–
–
3
2.63–2.74 (m, 2H, CH2, aspartic acid), 2.83–3.05 (m, 2H, CH2,
indole), 4.26–4.27 (d, 1H, CH), 4.77–4.79 (d, 1H, CH), 6.62–6.64
(d, 2H, J ¼ 10 Hz, 4-azidophenyl H3 and H5), 7.14–7.16 (d,
J ¼ 10 Hz, 2H, 4-azidophenyl H2 and H6), 7.69 (s, 1H, CH,
indole), 8.002–8.005 (m, 4H, indole), 8.38–8.39 (d, 1H, NH),
8.77–8.78 (d, 1H, NH), 12.7 (br, 2H, COOH); LC-MS (ESI) m/z:
462.8 (Mꢂ1).
Synthesis of p-MeSO2Bz-His-Glu (4a)
Yield:75%;whitesolid;IR(KBr):n(cmꢂ1)1320,1178(SO2),1400–
Synthesis of p-N3Bz-Pro-Glu (2b)
1
1600 (aromatic), 1742 (C O); H NMR (DMSO): d ppm 1.84–2.03
Yield: 54%; white solid; IR (KBr): n ) 1400–1600
(cmꢂ1
–
–
(aromatic), 1741 (C O); 2153 (N ); 1H NMR (DMSO): d ppm
–
–
(m, 2H, CH2, glutamic acid), 2.34–2.50 (m, 2H, CH2, glutamic
acid), 2.63–2.74 (m, 2H, CH2, imidazole), 3.1 (s, 3H, SO2CH3),
4.26–4.27 (d, 1H, CH), 4.77–4.79 (d, 1H, CH), 7.20–7.22 (d, 2H,
J ¼ 10Hz, 4-methylsulfonyl phenyl H2 and H6), 7.32 (s, 1H, CH,
imidazole), 7.87–7.89 (d, J ¼ 10 Hz, 2H, 4-methylsulfonyl phenyl
H3 and H5), 8.38–8.39 (d, 1H, NH), 8.77–8.78 (d, 1H, NH), 8.93 (s,
1H, CH, imidazole), 12.7 (br, 2H, COOH), 13.4 (s, 1H, NH,
imidazole); LC-MS (ESI) m/z: 467.0 (Mꢂ1).
3
1.80–1.82 (m, 2H, CH2, pyrrolidine), 1.84–2.03 (m, 2H, CH2,
glutamic acid), 2.10–2.32 (2H, CH2, pyrrolidine), 2.34–2.50
(m, 2H, CH2, glutamic acid), 2.99 (t, 2H, CH2), 4.26–4.27 (d, 1H,
CH), 4.68–4.69 (d, 1H, CH), 7.75–7.77 (d, J ¼ 8 Hz, 2H,
4-azidophenyl H3 and H5), 8.00–8.017 (d, 2H, J ¼ 8 Hz,
4-azidophenyl H2 and H6), 8.2 (d, 1H, NH), 12.4 (br, 2H,
COOH); LC-MS (ESI) m/z: 388.1 (Mꢂ1).
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Arch. Pharm. Chem. Life Sci. 2017, 350, e1700158
M. A. Ahmaditaba et al.
Archiv der Pharmazie
Synthesis of p-N3Bz-Tyr-Asp (3b)
6COX). All docking computations were done with the
AutoDock Vina program [19].
Yield: 78%; white solid; IR (KBr):
n ) 1400–1600
(cmꢂ1
(aromatic), 1737, 1725 (C O), 2132 (N ); 1H NMR (DMSO): d
–
–
3
Biological assays
ppm 2.63–2.74 (m, 2H, CH2, aspartic acid), 2.98–3.17 (m, 2H,
CH2, benzyl), 4.26–4.27 (d, 1H, CH), 4.77–4.79 (d, 1H, CH), 4.9 (s,
1H, phenol), 6.60–6.62 (d, J ¼ 8 Hz, 2H, phenol H3 and H5),
7.08–7.10 (d, 2H, J ¼ 8 Hz, phenol H2 and H6), 7.17–7.19 (d, 2H,
J ¼ 10 Hz, 4-azidophenyl H2 and H6), 7.83–7.85 (d, 2H, J ¼ 10
Hz, 4-azidophenyl H3 and H5), 8.38–8.39 (d, 1H, NH), 8.77–8.78
(d, 1H, NH), 12.5 (br, 2H, COOH); LC-MS (ESI) m/z: 454.1 (Mꢂ1).
In vitro COX inhibition assays
The ability of the synthesized compounds to inhibit ovine
COX-1 and COX-2 (IC50 value, mM) was determined using
chemiluminescent enzyme assays kit (Cayman Chemical, Ann
Arbor, MI, USA) according to the previously reported
method [18].
Synthesis of p-N3Bz-His-Asp (4b)
General procedure of the MTT assay
Yield: 81%; white solid; IR (KBr):
n
(cmꢂ1
)
1400–1600
The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazo-
lium bromide) based assay was carried out by seeding 10 ꢁ 103
cancer cells per 200 mL RPMI complete culture medium in each
well of 96-well culture plates. The day after seeding, culture
medium was replaced with medium containing celecoxib (as
the standard sample) as well as different concentrations of
newly synthesized peptides and RPMI culture medium (as the
control sample with no drug in it). The cells were then
incubated at 37°C in 5% CO2 incubator for 48 h. Then MTT
solution (10 mL, 4 mg/mL) was added to each well and further
incubation was carried out at 37°C for 3 h. At the end of
incubation, formazan crystals were dissolved in 100 mL of
DMSO and the plates were read in a plate reader (Synergy H4,
USA) at 570 nm. This experiment was performed in triplicate
determination each time [20].
(aromatic), 1758 (C O), 2140 (N ); 1H NMR (DMSO): d ppm
–
–
3
2.63–2.74 (m, 2H, CH2, aspartic acid), 2.63–2.74 (m, 2H, CH2,
imidazole), 4.26–4.27 (d, 1H, CH), 4.77–4.79 (d, 1H, CH), 7.20–
7.22 (d, 2H, J ¼ 10 Hz, 4-azidophenyl H2 and H6), 7.32 (s, 1H,
CH, imidazole), 7.87–7.89 (d, J ¼ 10 Hz, 2H, 4-azidophenyl H3
and H5), 8.38–8.39 (d, 1H, NH), 8.77–8.78 (d, 1H, NH), 8.93 (s,
1H, CH, imidazole), 12.5 (br, 2H, COOH), 13.56 (s, 1H, NH,
imidazole); LC-MS (ESI) m/z: 413.7 (Mꢂ1).
Synthesis of p-N3Bz-Phe-Glu (5b)
Yield: 65%; white solid; IR (KBr):
n ) 1400–1600
(cmꢂ1
(aromatic), 1785, 1773 (C O), 2148 (N ); 1H NMR (DMSO): d
–
–
3
ppm 1.84–2.03 (m, 2H, CH2, glutamic acid), 2.34–2.50 (m, 2H,
CH2, glutamic acid), 2.98–3.17 (m, 2H, CH2, benzyl), 4.26–4.27
(d, 1H, CH), 4.77–4.79 (d, 1H, CH), 7.15–7.18 (t, J ¼ 9 Hz, 1H,
phenyl H4), 7.34–7.36 (d, 2H, J ¼ 9.5 Hz, phenyl H2 and H6),
7.81–7.99 (m, 4H, 4-azidophenyl H2–H6), 8.38–8.39 (d, 1H, NH),
8.77–8.78 (d, 1H, NH), 12.6 (br, 2H, COOH); LC-MS (ESI) m/z:
437.8 (Mꢂ1).
The authors would like to thank the Research Deputy of
Shahid Beheshti University of Medical Sciences and the
Research Office for providing financial support of this project
with the grant number 1191.
Synthesis of p-N3Bz-Phe-Asp (6b)
The authors have declared no conflict of interest.
Yield: 61%; white solid; IR (KBr):
n ) 1400–1600
(cmꢂ1
(aromatic), 1744, 1726 (C O), 2149 (N ); 1H NMR (DMSO): d
–
–
3
ppm 2.63–2.74 (m, 2H, CH2, aspartic acid), 2.99 (t, 2H, CH2,
benzyl), 4.26–4.27 (d, 1H, CH), 4.68–4.69 (d, 1H, CH), 7.15–7.18
(t, J ¼ 9 Hz, 1H, phenyl H4), 7.34–7.36 (d, 2H, J ¼ 9.5 Hz, phenyl
H2 and H6), 7.82–7.99 (m, 4H, 4-azidophenyl H2–H6),
8.38–8.39 (d, 1H, NH), 8.77–8.78 (d, 1H, NH), 12.2 (br, 2H,
COOH); LC-MS (ESI) m/z: 425.1 (Mꢂ1).
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Synthesis of p-N3Bz-Pro-Asp (7b)
Yield: 52%; white solid; IR (KBr):
n
(cmꢂ1
)
1400–1600
(aromatic), 1741 (C O); 2153 (N ); 1H NMR (DMSO): d ppm
–
–
3
1.80–1.82 (m, 2H, CH2, pyrrolidine), 2.10–2.32 (m, 2H, CH2,
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CH2), 4.26–4.27 (d, 1H, CH), 4.68–4.69 (d, 1H, CH), 7.75–7.77 (d,
J ¼ 8 Hz, 2H, 4-azidophenyl H3 and H5), 8.00–8.017 (d, 2H,
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www.archpharm.com
(9 of 9) e1700158