A novel class of tyrosine derivatives as dual 5-LOX and COX-2/mPGES1 inhibitors with PGE2 mediated anticancer properties

Article abstract of DOI:10.1039/c8nj04385j

Leukotriene and prostaglandin pathways are controlled by the enzymes, LOX and COX/mPGES1 respectively and are responsible for inflammatory responses. PGE₂, produced by mPGES1, leads to the progression of inflammation as well as cancer. A series of 19 novel tyrosine derivatives are synthesized, characterized and tested against 5-LOX, in vitro, and production of PGE₂, in HeLa cells. 6b-v and 6c-i, are found to possess maximum inhibitory action against 5-LOX and PGE₂ production. The compound 6b-v is found to act by disrupting the redox cycle of the 5-LOX enzyme, and its activity is comparable to that of the commercial drug, Zileuton. The activity of the other compound 6c-i is comparable to a drug in clinical trials, Licofelone, and it has been found to inhibit the mRNA expression of mPGES1 predominantly. It also arrests the HeLa cells in the S and G2/M phases of the cell cycle indicating anticancer activity. Also, compounds, 6b-iv and 6b-viii inhibit both the LT & PG pathways in the inflammation cascade. Presence of iodine in the phenyl ring appears to favour the inhibition of 5-LOX whereas chlorine favours the inhibition of PGE₂ production. These leads could be further optimized and developed as drugs against inflammation and cancer.

Full text of DOI:10.1039/c8nj04385j

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A novel class of tyrosine derivatives as dual 5-LOX
and COX-2/mPGES1 inhibitors with PGE2
mediated anticancer properties†
Cite this: New J. Chem., 2019,
43, 834
a
a
a
Ayarivan Puratchikody, * Appavoo Umamaheswari,
Navabshan Irfan,
Gayathri Ramamoorthy
bc
c
b
b
Shweta Sinha,
and Mukesh Doble
S. L. Manju,
Meera Ramanan,
b
*
Leukotriene and prostaglandin pathways are controlled by the enzymes, LOX and COX/mPGES1
respectively and are responsible for inflammatory responses. PGE , produced by mPGES1, leads to the
progression of inflammation as well as cancer. A series of 19 novel tyrosine derivatives are synthesized,
characterized and tested against 5-LOX, in vitro, and production of PGE , in HeLa cells. 6b-v and 6c-i,
are found to possess maximum inhibitory action against 5-LOX and PGE production. The compound
b-v is found to act by disrupting the redox cycle of the 5-LOX enzyme, and its activity is comparable
2
2
2
6
to that of the commercial drug, Zileuton. The activity of the other compound 6c-i is comparable to a
drug in clinical trials, Licofelone, and it has been found to inhibit the mRNA expression of mPGES1
predominantly. It also arrests the HeLa cells in the S and G2/M phases of the cell cycle indicating
anticancer activity. Also, compounds, 6b-iv and 6b-viii inhibit both the LT & PG pathways in the
inflammation cascade. Presence of iodine in the phenyl ring appears to favour the inhibition of 5-LOX
Received 28th August 2018,
Accepted 3rd December 2018
DOI: 10.1039/c8nj04385j
2
whereas chlorine favours the inhibition of PGE production. These leads could be further optimized and
rsc.li/njc
developed as drugs against inflammation and cancer.
2
converted to PGE by the membrane-bound inducible prosta-
glandin E synthase (mPGES1) enzyme. This enzyme is known to
Introduction
Inflammation is a common manifestation associated with several be up-regulated under inflammatory conditions. Earlier studies
1,2
human diseases including asthma and cancer. Inflammatory have shown that mPGES1 is coupled with COX-2 in provoking
6
response begins with the metabolism of arachidonic acid (AA) inflammatory functions. In other words, the combined action of
which is released from the lipid bilayer due to the action of the these two enzymes produce PGE
2
.
3
enzyme, PLA2, as a reaction to stress or other stimuli. Further, it is
Efforts to combat inflammation by inhibiting several crucial
channelised into two main streams namely, the leukotriene enzymes have been undertaken for the past several years.
(
(
LT) and the prostaglandin (PG) pathways by the enzymes LOX However, inhibiting one of these two pathways increases the
4
Lipoxygenase) and COX (Cycloxygenase) respectively.
amount of AA entering the other, resulting in unexpected
LTs are associated with various inflammatory diseases consequences. Hence a better strategy would be to inhibit the
1
7
including allergy and asthma. 5-LOX is the crucial enzyme which enzymes in both the pathways. Compounds including aminothia-
catalyzes AA to 5(S)-hydroperoxyeicosatetraenoic acid (5-HPETE), zole and 2-mercaptohexaenoic acid derivatives have been
5
8,9
and subsequently to LTA
4
. In the PG pathway, AA is converted to identified as dual inhibitors of 5-LOX/mPGES1. In spite of
by the action of the enzymes, COX-1/COX-2. This, in turn, is the development of several promising leads, none of them can
progress successfully towards clinical trials.
PGH
2
a
On the other hand, the role of PGE
is well proven. Excess production of PGE
2
in the etiology of cancer
is found to be associated
Drug Discovery and Development Research Group, Department of Pharmaceutical
Technology, Bharathidasan Institute of Technology, Anna University,
Tiruchirappalli, Tamil Nadu, 620024, India. E-mail: puratchipharma@gmail.com
Bioengineering and Drug Design Lab, Department of Biotechnology, Bhupat and
Jyoti Mehta School of Biosciences, Indian Institute of Technology, Madras,
Tamil Nadu, 600036, India
2
10
with various types of cancers. Several compounds including
carbazolebenzamide have been found to inhibit PGE2 and
b
11
decrease cancer progression. Similarly, Apricoxib, a drug in
c
combination with Gemcitabine-erlotinib is reported to decrease
the growth and metastasis of pancreatic cancer cell lines by
Department of Chemistry, Vellore Institute of Technology, Vellore, Tamil Nadu,
6
32014, India
1
2
†
2
Electronic supplementary information (ESI) available. See DOI: 10.1039/c8nj04385j acting through PGE .
8
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Even though, the defined role of LT in cancer is not clear yet, Similar results are observed with compounds 6b-viii (67.6%)
few papers report that 5-LOX inhibition triggers apoptosis in and 6a-ix (47.8%). Commercial drugs such as Amiodarone (for
1
3,14
prostate and pancreatic cancer cells.
Recently, a link has antiarrhythmic medication), Levothyroxine (thyroid hormone
been found between cysteinyl leukotrienes (CysLTs) and colorectal deficiency) and Idoxuridine (anti-herpesvirus and antiviral drug)
15
cancer.
possess iodine in their chemical structure. The remaining tyrosines
Surprisingly, bioactive dibromotyrosine derivatives obtained possess less than 35% inhibitory activity against the enzyme, 5-LOX.
from natural sources have been reported to exhibit antibacterial,
1
6
Inhibition of 5-LOX through the disruption of the redox cycle
anticancer and antiviral properties. They possess sterically more
complex structure and exhibit specific binding characteristics Three different mechanisms have been reported in the literature
against these enzymes when compared to their synthetic counter- for the inhibition of 5-LOX namely, redox, competitive and iron
1
7
23
2+
parts. Also it is reported that replacing the OH with hydrophobic chelation. 5-LOX contains a ‘‘non-heme’’ iron, as Fe , in the
3
+
groups including alkyl, aryl and heteroaryl in tyrosine nucleus native form and Fe in the activated state. Redox inhibitors
inhibit angiogenesis, growth and development of malignant cells inhibit the enzyme by reducing the Fe to Fe , thereby disrupting
and their migration into the surrounding tissues with negligible the redox cycle. Competitive inhibitors produce activity by binding
3+
2+
18
toxicity on the living cells indicating their anti-cancer potential.
with the active site of the enzyme. Iron chelating agents chelate the
Therefore, 19 novel substituted tyrosine scaffold were synthesized, metal and block the binding ability of the substrate, thus inhibiting
23
characterized and tested for their potency to inhibit 5-LOX in vitro the activity of the enzyme. The mechanism of action of 6b-v (the
and the production of PGE (which arises when both, the COX-2 & most active compound) is elucidated with the help of the pseudo-
2
24
mPGES1, are inhibited) in HeLa cells. This strategy may also lead peroxidase assay. This protocol determines the decrease in the
to the development of anti-cancer agents since as mentioned absorbance (at 234 nm) due to the consumption of 13(S)-hydroper-
before, excess production of PGE is implicated in several cancers. oxy octadecadienoic acid (13(S) HpODE) by the enzyme, 5-LOX, in
2
the presence of a redox inhibitor. Herein, it is observed that
absorbance decreases by 45% in 300 s when treated with compound
Results and discussion
6b-v (Fig. 1). The considerable degradation of the hydroperoxide
product suggests that 6b-v inhibits 5-LOX through the redox
mechanism. If a compound does not affect the redox cycle the
absorbance will remain constant with time but act only by binding
to the active site of this enzyme. 3-(3,4-Dihydroxyphenyl)-1H-iso-
chromen-1-one and 8-hydroxy-3-(3,4,5-trimethoxyphenyl)-1H-iso-
chromen-1-one have been reported to exhibit redox and non-
The detailed characterizations of all the 19 synthesized tyrosines
(
with the corresponding spectral details) are reported in the
ESI.† The purity of the compounds is found to be more than
5%, which is confirmed by NMR, Mass spectra and elemental
analysis. The un-optimized yield of all the compounds is above
0% and most of them are soluble in DMSO (Table 1). Except for
9
8
25
redox type of inhibition respectively. Zileuton (a reported redox
compounds (6a-ix) and (6b-i), all the compounds are solids with
molecular weights ranging from 360 to 600. Compounds, (6a-iv),
26
inhibitor ) also degrades hydroperoxide by 50% in 300 s (Fig. 1).
(
6a-vi), (6b-iii) and (6b-iv) have melting points between 100 to DPPH radical scavenging activity
1
70 1C, while others fall in the range of 220 to 500 1C.
It has been reported that 5-LOX inhibitors which possess radical
3+
scavenging activity may act by affecting the oxidation state of Fe
present in the enzyme. Antioxidant activity of compound 6b-v is
Inhibition of 5-LOX
27
The compound 6b-v exhibits the best 5-LOX enzyme inhibitory determined using a previously reported method. Zileuton and
activity (88.5% at a concentration of 50 mM) with an IC50 of 6b-v exhibit 37.8 and 35.9% radical scavenging activity at 50 mM
9
.4 ꢀ 1.2 mM (Table 1). This compound has a triazole moiety with concentration respectively indicating that they could scavenge
methoxy group at the 4th position and iodine at the 3rd and 5th the free radical, 2,2-diphenyl-1-picrylhydrazyl (DPPH). This result
positions in the phenyl ring. Zileuton, a commercial drug, which additionally proved that 6b-v inhibits 5-LOX by disrupting the
is withdrawn from the market due to its hepatotoxicity possess redox cycle of the enzyme due to its radical scavenging activity.
inhibitory activity of 95.6% at a concentration of 50 mM (with an Ascorbic acid is used as the positive control here, which exhibits
19
IC50 of 1–2 mM). Compounds 6b-iv with an oxazole (73.6%) and 80.1% antioxidant activity at the same concentration.
b-viii with a pyrimidine substitution (67.6%) exhibit good 5-LOX
inhibitory activity whereas, compound 6a-ix with bromine at the
rd and 5th positions in the phenyl ring, possesses moderate The 19 tyrosine derivatives were also tested in HeLa cells for
activity (47.8%). Literature studies indicate that electron with- their ability to inhibit the production of PGE . It is seen that
drawing groups such as F, Cl, and Br increases anti-inflammatory 6c-i, exhibits the best activity and it inhibits the production of
6
Inhibition of PGE production
2
3
2
2
0–22
activity.
An interesting activity profile has been also noted PGE by 81.6% at a concentration of 50 mM (Table 1) with an
2
that presence of halogens in the structure enhances the anti- IC50 of 9.2 ꢀ 1.5 mM. This is followed by several compounds
inflammatory activity. Substitution of iodine (being less electro- which inhibit the PGE production above 70%, namely 6b-viii
2
negative than bromine) at the 3rd and 5th positions in the phenyl (76.7%), 6b-i (76.1%), 6a-v (74.9%), 6b-vi (74.2%), 6a-x (72.2%),
ring in lieu of bromine, increases the 5-LOX inhibitory activity 6a-ii (71.5%), 6b-iii (70.9%), 6a-iii (70.6%) and 6b-vii (70.1%).
by three folds (6b-v (88.5%) and 6a-vii (29.7%) respectively). Licofelone, a drug currently in the clinical trials exhibits an IC50
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Table 1 Structures, physical properties and inhibition data at 50 mM of synthesized tyrosine derivatives
%
Inhibition
% Reduction in PGE
production ꢀ SD
2
a
b
Compound code
Chemical structure
Melting point (1C)
% Yield
87.68
of 5-LOX ꢀ SD
6
6
6
6
a-i
255–260
24.7 ꢀ 1.2
66.4 ꢀ 3.1
72.0 ꢀ 0.7
72.2 ꢀ 2.3
68.4 ꢀ 7.2
a-ii
a-iii
a-iv
4300
89.58
83.42
87.52
20.9 ꢀ 3.4
23.6 ꢀ 2.7
38.4 ꢀ 7.2
248–249
103–105
6
a-v
4300
87.63
30.8 ꢀ 0.5
74.5 ꢀ 0.5
6
6
6
a-vi
170
89.18
83.11
83.68
23.5 ꢀ 9.7
29.6 ꢀ 7.3
39.5 ꢀ 11.8
53.9 ꢀ 2.0
24.6 ꢀ 0.3
45.6 ꢀ 9.4
a-vii
a-viii
238
230–236
6
6
a-ix
a-x
Liquid
87.35
85.10
47.8 ꢀ 4.7
23.6 ꢀ 0.4
57.0 ꢀ 2.3
73.8 ꢀ 2.3
508.182
6
6
b-i
Liquid
84.12
84.55
26.3 ꢀ 4.2
25.8 ꢀ 5.0
72.1 ꢀ 5.6
21.0 ꢀ 3.4
b-ii
318–322
8
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Table 1 (continued)
%
Inhibition
% Reduction in PGE
production ꢀ SD
2
a
b
Compound code
Chemical structure
Melting point (1C)
% Yield
81.19
of 5-LOX ꢀ SD
6
b-iii
b-iv
140–142
34.6 ꢀ 1.8
71.4 ꢀ 1.2
65.8 ꢀ 3.2
6
175–177
82.72
73.6 ꢀ 6.6
6
6
6
b-v
250–254
509.17
4277
83.00
85.54
83.81
88.5 ꢀ 2.1
26.4 ꢀ 0.4
30.2 ꢀ 1.2
59.9 ꢀ 10.9
72.9 ꢀ 1.8
74.0 ꢀ 5.6
b-vi
b-vii
6
b-viii
222–224
88.66
67.6 ꢀ 1.3
34.0 ꢀ 4.1
78.4 ꢀ 2.4
81.5 ꢀ 0.1
6
c-i
277–279
84.50
a
39
b
40
In vitro cell free assay. Zilueton is used as the positive control (95.6%). In HeLa cells. Licofelone is used as the positive control (50% at 6 mM).
The combined actions of the enzymes COX-2 and mPGES1
produce PGE2 which is an important mediator of many biological
28
functions. HeLa cells express elevated levels of these enzymes
when stimulated with TNFa. During this study, reduction in the
level of PGE was observed with the compounds tested, this may
2
be attributed to the inhibition either one or of both of these
enzymes. A specific assay with pure COX-2 or mPGES1 may give
the extent of inhibition of each one of these enzymes by these
2
9
compounds.
Inhibition of mRNA expression of PG enzymes
The real time-PCR analysis was carried out to determine the
effect of 6c-i on the mRNA expression levels of COX-1, COX-2
and mPGES-1, post TNFa treatment. TNFa treatment increases
the levels of all the three enzymes by several folds. The
compound (at 50 mM concentration) decreased the mPGES-1
expression level from 6.8 (post TNFa) to 1.6 folds while Lico-
Fig. 1 Pseudoperoxidase activity plot (shown as absorbance subtracted
from control) indicating the consumption of 13(S)-HpODE by 6b-v and
Zileuton. A fall in the absorbance observed here indicates that these
compounds disrupt the redox cycle of 5-LOX.
of 6 mM and is used here as the positive control. It is noteworthy felone (6 mM) decreased it to 2.8 folds (Fig. 2). Similarly, the
to mention that compound 6c-i has comparable activity with compound decreased the COX-1 and COX-2 expression levels
this drug.
from 1.9 and 4.4 to 0.3 and 1.3 folds respectively. These results
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indicate that compound 6c-i inhibits the expression levels of all 6b-vii which exhibit more than 70% inhibitory activity have
the three enzymes but it exhibits greater activity on mPGES1 two iodine groups in the 3rd and 5th positions in the benzene
enzyme.
ring. Chlorine is more electronegative than bromine and iodine
It is reported that the presence of either sulfonamide (R–SOO– and hence may be more biologically active. A recent research
NH2) or sulfonyl (R–SOO–R) is an important criteria for COX article highlights the importance of chlorine in the place of the
inhibitors. For example, SC-57666 and NS398 are selective COX-2 hydroxyl group in enhancing the inhibition of PGE
2
and NO
3
4
inhibitors that have methylsulfonyl or methyl sulfonamide production. Further optimisation of this chlorine substituted
3
0
functional groups. Compound 6c-i has methyl sulfonamide tyrosine derivative could be attempted in an effort to develop
functional group which could be one of the reasons for its molecules with activity similar to that of the standard drug.
inhibitory action on COX-1 and COX-2.
Arylpyrrolizines (licofelone derivative) which has a combination has been attributed to the development of several cancers. It
of a free acid with substituted sulfonamide group is known to binds to the EP receptors (EP1–4) and activates several proliferative
2
Previous studies have suggested that increased PGE production
7
2
8
specifically decrease the expression level of mPGES-1 than that signalling pathways associated with tumour pathology. Com-
3
1
of COX-1, and COX-2. Compound 6c-i contained both acid and pounds including carbazolebenzamide derivative (AF3485), has
substituted sulfonamide groups which may be increasing its been found to reduce the PGE formation and hence EGFR
2
selectivity towards mPGES-1. The compound has electron with- signalling, resulting in decreased growth of A431 cells (human
9
drawing group, 3-chloro, suggesting that the presence of Cl epidermoid carcinoma). In another study, MK886 (an anti-
group at the third position of the phenyl ring increases the inflammatory compound), is observed to exhibit anti-cancer
inhibitory effect with high selectivity towards mPGES-1. It is also activity by reducing the proliferation of HL-60 cells through the
3
5
reported that chloro substituted phenyl ring exhibit better inhibition of PGE2. So compounds inhibiting PGE production
2
32
mPGES-1 inhibition both in vitro and cell-based assays. Basically, might have anti-cancer activity as well as anti-inflammatory activity.
compounds containing acidic side chain exhibits good anti- Hence all the compounds are further tested for their ability
inflammatory property, especially propanoic acid and aryl acetic to reduce the proliferation of HeLa cells at 50 mM concentration
3
3
25
acid derivatives. The latter is more lipophilic than the former.
The propanoic acid group present in compound 6c-i probably Most of the compounds do not exhibit significant cytotoxicity
makes it a potent anti-inflammatory agent. (4B90% cells are viable at this concentration after 24 h of
by MTT assay according to a previously reported method.
The current study has also identified compound 6b-v with incubation) whereas only 75.2% of HeLa cells are viable when
multiple activities namely 88.5% against 5-LOX and moderate treated with compound 6c-i indicating its ability to decrease the
inhibitory activity against PGE production (67.6%) indicating proliferation of these cells.
2
that it may be a useful lead for further investigation as a
candidate to block both the LT and PG pathways. Compounds
Cell cycle analysis
6b-iv (65.7%) and 6b-viii (78.3%) also exhibit good inhibitory Anti-cancer drugs are known to arrest the cells in different
activity against all these enzymes. Our earlier studies have observed phases of their division process thereby preventing them from
3
6
that isocoumarins exhibit good inhibitory activity against all progressing in the cell cycle. The five different phases of cell
2
5
these enzymes.
division process are G0, G1, S, G2 and M. DNA replication
Analysis of the structures of these tyrosine derivatives that happens in S phase while G phases are the resting periods or
inhibit PGE2 production indicates that they differ in their gaps between the actual cell division. The mitosis takes place
substitutions in the benzene ring and the positions of N and during the M phase. Arresting the cells in one of the phases
O in the heterocyclic ring. Compound 6c-i with 81.6 ꢀ 1.3% earlier to the mitosis stage results in cell death or decreased cell
3
7
inhibitory activity (at 50 mM) possesses chlorine in the 3rd proliferation. This process could be observed in HeLa cells by
position whereas compounds 6b-viii, 6b-i, 6b-vi, 6b-iii and Fluorescence Activated Cell Sorting (FACS) analysis.
Treatment with 50 mM of 6c-i shows the presence of a large
proportion of these cells in the S (20.6%) and G2/M (17.4%)
phases when compared to the control (without any compound
treatment) (Fig. 3a and b). S phase arrest has been a mode of
action of few anti-cancer drugs including cisplatin which disturbs
the normal DNA replication and prevents the cells to progress to
37
mitosis. Similar observation here indicates that this compound
6c-i) exhibits anti-cancer activity through this process. Likewise,
G2/M phase arrest has been a prominent mechanism of action of
(
3
8
several anticancer drugs including quinones.
Cell morphology analysis
The changes in the morphology of HeLa cells when treated with
c-i are studied using dual staining (with acridine orange and
ethidium bromide), and Nucblue live staining techniques (Fig. 4).
6
Fig. 2 mRNA expression level of COX-1/COX-2 and mPGES-1.
8
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cells (Fig. 4c and d). Also, compound treated cells exhibit central
halo indicating the absence of intact cellular DNA.
From these staining images, it is clear that the treatment of
50 mM of 6c-i reduces the number of viable cells and increases
the proportion of dead or dying cells. Fragmented nuclei and
condensation of chromatin material which are typical signs of
apoptosis are also noted (Fig. 4b). This is also reflected in the
MTT assay where a 24.8% reduction in proliferation is observed
in 6c-i treated HeLa cells. The percentage of apoptosis and
necrosis is calculated by counting the number of live cells with
normal nuclei (VN), live cells with fragmented nuclei (VA),
nonviable cells with normal nuclei (NVN) and nonviable cells
with fragmented nuclei (NVA) (formula given in the Materials &
methods section). Treatment with 6c-i shows 14.4% apoptosis
and 8.2% necrosis, and hence it appears that the cells are
Fig. 3 Cell cycle analysis of (a) untreated and (b) 50 mM of 6c-i treated
HeLa cells. (c) Proportion of cells present in different phases is shown.
*
**p o 0.0001. More 6c-i treated cells are observed in the G2M and S mainly channelised into the former rather than through the
phases than in G0/G1 phases when compared to control indicating the
anticancer activity of this compound.
latter pathway. Further experiments would be needed on apoptosis
markers to identify the exact pathway of action.
A novel set of 19 compounds, comprising of natural product-
based tyrosine scaffold, are synthesised and tested against the
enzymes in the inflammatory pathway. Compounds 6b-iv and
The former technique is used to distinguish the live and
3
6
apoptotic cells. Control cells appear healthy without cell death
or fragmentation of the nucleus (Fig. 4a). The acridine orange
stains the live cells green and the ethidium bromide stains the
dead cells orange or bright red colour (Fig. 4b, solid arrows).
HeLa cells treated with 6c-i (50 mM) shows a higher percentage
of apoptotic cells (14.4%) when compared to the untreated cells
6b-viii are found to have reasonably good anti-inflammatory
activity against 5-LOX and COX-2/mPGES1 (Fig. 5a). Inhibition
of both, LT and PG pathways by these compounds will prevent
AA from shunting on to one particular pathway. Compound
6b-v is seen here as a novel inhibitor of 5-LOX. Presence of two
iodines at the 3rd and 5th positions along with a triazole ring could
be responsible for this multiple inhibition. This compound inhibits
(3.2%) when stained with these dyes. Also compound treated
cells show more number of fragmented nuclei than the untreated
ones (Fig. 4b, dotted arrows). It is reported that combining
cisplatin and ascorbic acid exhibits similar morphological changes
5-LOX by redox mechanism. It is heartening to note that the
39
and apoptotic cells in murine ascites Dalton’s Lymphoma.
Nucblue staining is used to identify live cells which fluoresce blue
in colour. This staining shows that compound treated cells are
clumped together and puffed up when compared to the untreated
Fig. 5 (a) Inhibitory action of tyrosine derivatives against different
enzymes. 6b-v and 6c-i show maximum inhibition of 5-LOX and COX-
2/mPGES1 respectively. 6b-iv and 6b-viii are able to inhibit all the three
enzymes in both the pathways. 6b-i, which decreases PGE production
2
also reduces cell proliferation. (b) 6b-v, similar to Zileuton, acts as a redox
Fig. 4 Acridine orange/ethidium bromide stained HeLa cells (a) untreated,
(
b) 6c-i (50 mM) treated showing live (green), dead cells (red; solid arrows)
and fragmented nuclei (dotted arrows). Nucblue stained HeLa cells
c) untreated (d) 6c-i (50 mM) treated cells showing puffiness and difference
in morphology.
(
inhibitor against 5-LOX in the leukotriene pathway.
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inhibitory activity of this compound, 6b-v (88.5 ꢀ 2.3%, IC50 of
9.4 mM), against 5-LOX is comparable to that of the standard drug,
Zileuton at 50 mM (95.6 ꢀ 0.7%, IC of 1–2 mM) which is withdrawn
50
due to hepatotoxicity. It also has reasonable inhibitory activity
against the enzymes in the PG pathway (namely COX-2 and
2
mPGES1), thereby inhibiting the production of PGE .
Compound 6c-i exhibits the highest inhibition (81.6 ꢀ 1.3%,
IC50 of 9.2 mM) against the enzymes, COX-2 and mPGES1 and
its activity is comparable to that of Licofelone, a drug under
clinical trials. Hence chlorine substitution (6c-i) appears to be
advantageous in comparison to that of other halogens in
Fig. 6 Substitution of various groups in the tyrosine skeleton.
the inhibition of PGE production. Newer compounds can be
2
synthesized in the future with different chlorine substitutions
to unravel potential candidates that might compete with the
reference molecules including MK886 and Licofelone.
The best 5-LOX inhibitor, 6b-v (Fig. 5b), acts by disrupting
the redox cycle of the enzyme. This conclusion is supported by
DPPH radical scavenging and pseudoperoxidase assays (35.9%
antioxidant and B45% of pseudoperoxidase activities respectively).
PGE is well known to be involved in the etiology of cancer
2
progression through EP receptors. Compound 6c-i with an IC50 of
9
.2 ꢀ 1.5 mM also exhibits anti-cancer activity as demonstrated by
cell cycle analysis and staining studies. This compound can be
explored further to dissect its involvement in apoptotic and other
cancer related processes.
Scheme 1 Synthetic scheme of tyrosine derivatives.
The current study has identified the tyrosine moiety as a and 12 mL of 48% HX (108 mmol) was added to it. Then, 4.4 mL
promising scaffold against asthma, inflammation and cancer. of DMSO (60.7 mmol) was added, when the colourless suspension
These molecules can be further optimised in an attempt to immediately turns yellow-orange. The suspension was warmed to
develop them as potent drug candidates.
65 1C for 1.5 min at 50% microwave power (424 watts), giving a
clear light yellow solution. It was then removed from the heat and
Structural requirements for inhibiting LOX and COX/mPGES1 left at room temperature overnight. The clear yellow solution was
enzymes
then evaporated in vacuum to give a light yellow solid, which was
dissolved in 200 mL of hot water. The pH was adjusted to 5 with
solid NaHCO , and it was left at ambient temperature overnight
3
for crystallisation to complete. The suspension was filtered, the
sparkling white needles were then washed with cold water, and
left to dry, giving 2(a), 2(b) and 2(c).
Various substitutions in tyrosine skeleton shows the modified
activity according to the nature of the group and its orientation.
Substitution of triazole ring in R position increase the LOX
activity compared to the presence of oxazole and pyrimidine
ring. Electronegative substitution in benzene ring of the tyro-
sine moiety, especially, iodine increases the anti-inflammatory
4
Step 2: a solution of 2 (3.6 g) in methanolic HCl (60 mL,
2.5 N) was refluxed for 2.5 min at 50% microwave power (424 watts)
(6b-viii o 6b-I o 6a-I o 6b-vi o 6a-x) and chlorine increases
and the 60 1C the solution was concentrated in a rotary vacuum. The
residue was dissolved in ethanol (40 mL). Then the excess of
triethylamine (4.0 mL) was added (to neutralise HCl) and stirred
for 1 h at RT. The salt was filtered, and the residue was chromato-
graphed over silica gel column using chloroform–methanol (90 : 10)
as eluent to obtain 3(a), 3(b) and 3(c).
Step 3: to a mixture of 3 (1.2 g, 5.1 mmol), potassium
carbonate (1.4 g, 10.1 mmol) in acetone (50 mL) a methane
sulfonyl chloride (1.13 g, 7.6 mmol) was added and the mixture
was refluxed at 50 1C for 1 min in 50% microwave power (424 watts).
The undissolved material was filtered off, and the solvent was
evaporated from the filtrate under reduced pressure. The residue
obtained was extracted with chloroform, and the chloroform layer
was washed with brine, dried over sodium sulfate and filtered. The
residue obtained after evaporation of the solvent was chromato-
the inhibition of mRNA expression activity. On the other hand,
chlorine substituted tyrosine derivatives affect the G2/M phase
of the cell cycle thus arresting growth of cancer. Hence, it is
arrived that the molecules selected for the biological studies
possess druggability for inhibiting LOX and COX/mPGES1. This
outcome will accelerate the discovery of chemical compounds
to act against enzymes responsible for a number of cancers and
inflammatory diseases (Fig. 6).
Experimental
General procedure for the synthesis of target tyrosine
derivatives
(
The 5 step synthesis is shown in Scheme 1.)
Step 1: synthesis of R and R
L-tyrosine (5.0 g, 27.6 mmol) was suspended in 50 mL of AcOH, as eluent to afford 4(a), 4(b) and 4(c).
1
2
substituted tyrosine derivatives: graphed over silica gel column using chloroform–methanol (98 : 2)
8
40 | New J. Chem., 2019, 43, 834--846
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Step 4: solution of 4 (0.148 mmol) in dry DMF (1.0 mL) was (-NH-Str), 2890.77 (COOH str), 2109.74 (-CH
NJC
3
3
-sp -str), 1989.21
added drop-wise to cooled (5 1C) oil-free suspension of NaH (-SQO str), 1734.66 (CQO str), 1605.45 (Ar-CQC), 1499.38 (-NH-
2
(
0.148 mmol) in 1.0 mL of dry DMF. The different alkyl or aryl bend), 1417.42 (C-O), 1342.21 (Phe-C-O), 898.66 (p – sp -Ar-CH),
2
1
bromides (0.148 mmol) were then added drop-wise to the and 807.06 (O-sp -Ar-CH); H NMR (400 MHz, DMSO-d , ppm):
6
3
mixture. After the H gas evolution had ceased, the solution 1.09–1.29 (m, sp -CH), 2.64 (s, N-CH), 3.34 (s, Br-CH), 4.48–4.51
2
was warmed to room temperature and stirred overnight. The (m, Ali-CH), 4.86 (s), 7.20 (d, J = 7.0), 7.47–7.56 (m, Ar-CH), 7.73 (d,
13
solution was then poured into 1 N NaOH, and the mixture was J = 8.0, Ar-CH), 7.72 (d, J = 8.0, Ar-CH), 10.67 (s, -OH); C NMR
extracted with CHCl ): 171.7 (carboxylic-C), 154.8 (Ar-C), 134.8
(3 ꢁ 10 mL). The organic layer was then (100 MHz, DMSO-d
washed with brine (10 mL) and dried over anhydrous Na SO . The (Ar-C), 133.6 (Ar-C), 118.1 (Het-Ar-CH), 113.7 (Het-Ar-CH), 64.5
crude mixture was then purified on SilicaGel 60 using gradient (Br-C), 41.1 (S-C), 34.7 (Ali-CH ). MS (ESI) m/z calculated for
[C H Br N O S ] 512.237; found, 512.878. Elemental analysis
3
6
2
3
2
+
CHCl –MeOH or n-hexane–EtOAc systems, which afforded 5(a–c).
3
15 16
2 2 4 2
Step 5: 0.0019 mole of 5 in ethanolic KOH (5%, 20 mL) was (C, H, N %): expected C 32.7, H 2.74, N 5.45. Actual C 33.065, H
stirred at room temperature for 90 min, and the solvent was 3.603, N 6.178.
evaporated under reduced pressure. The residue was diluted
3-{3,5-Dibromo-4-[(1,3,4-oxadiazol-2-yl)methoxy]phenyl}-2-
with ice-cooled water and acidified with dil. HCl. The precipitated methanesulfonamidopropanoic acid (6(a)-iv). The title compound
solid was filtered and recrystallised from chloroform–hexane to was obtained according to the above general procedure with the
ꢂ1
obtain 6(a–c) the desired tyrosine derivatives.
yield 87.52% and m.p. 103–105 1C. IR (nmax, cm , KBr): 3386.39
3
(-NH-Str), 2921.63 (COOH str), 2674.78 (-CH
3
-sp -str), 2111.67
-SQO str), 1732.73 (CQO str), 1607.38 (Ar-CQC), 1498.42 (-NH-
-{3,5-Dibromo-4-[(1H-pyrrol-3-yl)methoxy]phenyl}-2-methane bend), 1417.42 (C-O), 1343.18 (Phe-C-O), 806.09 (p – sp -Ar-CH),
sulfonamidopropanoic acid (6(a)-i). The title compound was and 806.09 (O-sp -Ar-CH); H NMR (400 MHz, DMSO-d , ppm):
obtained according to the above general procedure with the 1.21 (t, J = 7.0, sp -CH), 2.49 (s, N-CH), 4.60 (q, J = 7.0, Ali-CH), 7.57–
yield 87.68% and m.p. 255–260 1C. IR (nmax, cm , KBr): 3386.39, 7.64 (d, J = 7.8, Ar-CH), 7.71 (t, J = 7.5), 8.52 (d, J = 8.3, Ar-CH), 11.28
Characterization
(
2
3
2
1
6
3
ꢂ1
2
13
3
2
1
181.01 (-NH-Str), 3130.87 (Ar-sp -CH), 2920.23 (COOH str), (s, -OH); C NMR (100 MHz, DMSO-d
6
): 170.5 (carboxylic-C), 163.6
-sp -str), 2541.72 (ali-CH str), 2466.51 (-SQO str), (Ar-C), 154.0 (Ar-C), 153.9 (Ar-C), 134.5 (Ar-C), 131.8 (Het-Ar-CH),
745.26 (CQO str), 1592.91 (Ar-CQC), 1507.10, 1513.85 (-NH- 118.6 (Het-Ar-CH), 70.9 (Br-C), 61.3 (S-C), 33.9 (Ali-CH ), 42.9 (Ali-
3
674.78 (-CH
3
2
3
+
bend), 1473.35 (C-H sp bend), 1415.49 (C-O), 1344.14 (Phe-C-O), CH). MS (ESI) m/z calculated for [C H Br N O S] 501.148; found,
13
15
2 3 6
2
2
1
8
35.02 (p – sp -Ar-CH), and 791.636 (O-sp -Ar-CH); H NMR 499.896. Elemental analysis (C, H, N %): expected C 31.28, H 2.63,
DMSO-d , ppm): 1.17 (t, J = 5.4, sp -CH), 2.48–2.97 (m, N-CH), N 8.42. Actual C 31.716, H 3.499, N 8.999 (carboxylic-C), (Ar-C), (Ar-
3
(
6
3
.15–3.68 (m, Br-CH), 4.21 (s, Ali-CH), 7.61–7.64 (m, Ar-CH), 8.69 CH), (Br-C), (S-C), (-CH
2
).
3-{3,5-Dibromo-4-[(5-oxo-2,5-dihydrofuran-2-yl)methoxy]phenyl}-
69.96 (carboxylic-C), 154.08 (Ar-C), 139.80 (Ar-C), 133.306 (Ar-C), 2-methanesulfonamidopropanoic acid (6(a)-v). This compound was
1
3
6
(d, J = 8.3, Ar-CH), 10.63 (s, -OH); C NMR (100 MHz, DMSO-d ):
1
1
18.4 (Ar-CH), 116.5 (Het-Ar-CH), 114.8 (Het-Ar-CH), 87.05, 55.14 prepared according to the above general procedure with the yield
ꢂ1
(Br-C), 40.124 (S-C), 38.87 (Ali-CH), 34.67 (Ali-CH
2
). MS (ESI) m/z 87.63% and m.p. 300 1C. IR (nmax, cm , KBr): 3395.07 (-NH-Str),
+
calculated for [C H Br N O S] 496.171; found, 494.921. Ele- 2713.35 (COOH str), 2111.67 (-SQO str), 1741.41 (CQO str),
mental analysis (C, H, N%): expected C 33.82, H 3.04, N 8.4. 1633.41 (Ar-CQ C), 1515.78 (-NH-bend), 818.63 (p – sp -Ar-CH),
Actual C 34.129, H 3.888, N 8.98.
-{3,5-Dibromo-4-[(1H-pyrazol-4-yl)methoxy]phenyl}-2-methane- 1.62 (t, J = 7.1, sp -CH), 2.68 (s, N-CH), 4.49 (q, J = 7.1, Ali-CH),
sulfonamidopropanoic acid (6(a)-ii). This compound was synthe- 7.58 (t, J = 7.6, Ar-CH), 7.61–7.69 (m, Ar-CH), 8.09 (d, J = 8.3), 10.89
1
5
16
2 2 5
2
2
1
and 740.53 (O-sp -Ar-CH); H NMR (400 MHz, DMSO-d , ppm):
6
3
3
13
6
sized according to the above general procedure and purified by (s, -OH); C NMR (100 MHz, DMSO-d ): 173.7 (carboxylic-C),
simple column chromatography (yield 89.58%, m.p. 300 1C). IR 154.9 (Ar-C), 150.7 (Ar-C), 133.5 (Ar-C), 131.0 (Ar-C), 122.7 (Het-Ar-
ꢂ1
(
(
n
max, cm , KBr): 3384.46 (-NH-Str), 2926.45 (COOH str), 2111.67 CH), 118.3 (Het-Ar-CH), 71.3 (Br-C), 59.9 (S-C), 43.6 (Ali-CH), 33.8
+
-SQO str), 1733.69 (CQO str), 1607.38 (Ar-CQC), 1498.42 (-NH- (Ali-CH ). MS (ESI) m/z calculated for [C H Br NO S] 513.155;
bend), 1473.35 (C-H sp bend), 1417.42 (C-O), 1343.18 (Phe-C-O), found, 511.901. Elemental analysis (C, H, N%): expected C 35.11,
98.66 (p – sp -Ar-CH), and 807.06 (O-sp -Ar-CH); H NMR (400 H 2.95, N 2.73. Actual C 35.355, H 3.802, N 3.594.
MHz, DMSO-d
2 15 15 2 7
3
2
2
1
8
3
6
, ppm): 1.26 (t, J = 7.1, sp -CH), 2.48 (q, N-CH),
3-{3,5-Dibromo-4-[(1,2-oxazol-3-yl)methoxy]phenyl}-2-methane-
2
.71–2.99 (q, S-CH), 3.31 (s, Br-CH), 7.01–7.25 (s, Ar-CH), 10.70 (s, sulfonamidopropanoic acid (6(a)-vi). The title compound was
13
-
(
OH). C NMR (100 MHz, DMSO-d
Ar-C), 134.1 (Ar-C), 132.5 (Ar-C), 118.2 (Het-Ar-CH), 117.5 (Het-Ar- yield 89.18% and mp 170 1C. IR (nmax, cm , KBr): 3660.23 (-NH-
CH), 64.8 (Br-C), 43.2 (S-C), 33.8 (Ali-CH ). MS (ESI) m/z calculated Str), 2923.56 (COOH str), 2109.74 (-SQO str), 1729.83 (CQO str),
6
): 169.7 (carboxylic-C), 154.08 synthesised according to the above general procedure with the
ꢂ1
2
+
3
for [C H Br N O S ] 495.186; found, 495.917. Elemental analysis 1589.06 (Ar-CQC), 1495.53 (C-H sp bend), 1414.53 (C-O),
15
17
2 3 4 2
2
2
(C, H, N%): expected C 33.82, H 3.04, N 8.45. Actual C 34.142, H 1346.14 (Phe-C-O), 833.09 (p – sp -Ar-CH), and 752.10 (O-p -Ar-
1
3
3.786, N 8.028.
CH); H NMR (400 MHz, DMSO-d , ppm): 1.36–1.66 (m, sp -CH),
6
3
-{3,5-Dibromo-4-[(1,3-thiazol-4-yl)methoxy]phenyl}-2-methane- 2.51 (s, N-CH), 2.95–3.13 (m), 3.45–3.72 (m, Br-CH), 4.43 (q,
1
3
sulfonamidopropanoic acid (6(a)-iii). Yield of this compound was J = 7.2, Ali-CH), 7.62–7.74 (m, Ar-CH), 10.70 (s, -OH); C NMR
obtained as 83.42% and m.p. 248 1C. IR (nmax, cm , KBr): 3181.57 (100 MHz, DMSO-d
ꢂ1
6
): 175.1 (carboxylic-C), 159.4 (Ar-C),
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Paper
1
1
54.0 (Ar-C), 150.1 (Ar-C), 133.3 (Het-Ar-C), 117.5 (Het-Ar-CH),
04.4 (Ar-CH), 77.0 (Br-C), 59.14 (S-C), 40.1 (Ali-CH), 39.7 methanesulfonamidopropanoic acid (6(a)-x). This compound
3-{3,5-Dibromo-4-[(4H-1,4-oxazin-2-yl)methoxy]phenyl}-2-
(
Ali-CH2), 34.7 (Ali-CH2). MS (ESI) m/z calculated for was synthesised according to the above general procedure with
+
ꢂ1
[C H Br N O S] 498.144; found, 496.901. Elemental analysis the yield 85.10% and m.p. 4 300 1C. IR (nmax, cm , KBr):
14 14 2 2 6
(
3
C, H, N%): expected C 33.76, H 2.83, N 5.62. Actual C 34.072, H 3699.76 (-NH-Str), 2928.38 (COOH str), 2109.74 (-SQO str),
3
.689, N 6.339.
1729.83 (CQO str), 1606.41 (Ar-CQC), 1492.63 (C-H sp bend),
2
3
-{3,5-Dibromo-4-[(1H-1,2,3-triazol-5-yl)methoxy]phenyl}-2- 1411.64 (C-O), 1343.18 (Phe-C-O), 832.13 (p – sp -Ar-CH), and
2
1
6
methanesulfonamidopropanoic acid (6(a)-vii). This compound 748.24 (O-sp -Ar-CH); H NMR (400 MHz, DMSO-d , ppm): 1.58–
was obtained according to the above general procedure with the 1.68 (m, sp -CH), 2.65–2.74 (m, N-CH), 3.50–3.65 (m Br-CH),
yield 83.11% and m.p. 238 1C. IR (nmax, cm , KBr): 3389.39, 3.72–3.85 (m Ali-CH), 7.41 (t, J = 7.5, Ar-CH), 7.99 (d, J = 8.2,
2
ꢂ1
1
3
(
(
(
(
-NH-Str), 2925.48 (COOH str), 2110.71 (-SQO str), 1733.69 Ar-CH), 11.23 (s, -OH); C NMR (100 MHz, DMSO-d ): 171.4
CQO str), 1610.27 (Ar-CQC), 1500.35 (-NH-bend), 1417.42 (carboxylic-C), 150.5 (Ar-C), 134.1 (Ar-C), 133.3 (Ar-C), 132.5
C-O), 1343.18 (Phe-C-O), 810.92 (p – sp -Ar-CH), and 741.49 (Ar-C), 118.6 (Het-Ar-CH), 102.3 (Het-Ar-HC), 75.6 (AlI-C), 60.6
O-sp -Ar-CH); H NMR (400 MHz, DMSO-d
6
2
2
1
6
, ppm): 1.45 (t, (Br-C), 49.3 (S-C), 32.5 (Ali-CH
2
). MS (ESI) m/z calculated for
S] 509.909; found, 510.916. Elemental analysis
(C, H, N%): expected C 35.18, H 3.15, N 5.47. Actual C 35.421, H
): 170.9 (carboxylic-C), 154.8 (Ar-C), 3.993, N 6.197.
3-{3,5-Diiodo-4-[(1,3-oxazol-4-yl)methoxy]phenyl}-2-methane-
5.05 (Br-C), 58.7 (S-C), 42.8 (Ali-CH), 34.5 (Ali-CH ). MS (ESI) sulfonamidopropanoic acid (6(b)-i). The title compound was
2
+
2 2 6
J = 7.1, sp -CH), 2.84–2.94 (m, N-CH), 4.48–4.59 (m, Ali-CH), [C15H16Br N O
7
NMR (100 MHz, DMSO-d
1
7
1
3
.44–7.52 (m, Ar-CH), 7.62 (t, J = 7.7, Ar-CH), 10.68 (s, -OH);
C
6
41.1 (Ar-C), 134.8 (Ar-C), 133.6 (Het-Ar-CH), 118.6 (Het-Ar-CH),
2
+
m/z calculated for [C H Br N O S] 498.147; found, 496.912. obtained according to the above general procedure with the
1
3
14
2 4 5
ꢂ3
ꢂ1
Elemental analysis (C, H, N%): expected C 31.34, H 2.83, N yield 84.12% and density 1.024 gm cm ; IR (nmax, cm , KBr):
1.25. Actual C 34.129, H 3.888, N 8.98. 3408.57 (-NH-Str), 2920.23 (COOH str), 2108.78 (-SQO str),
-{3,5-Dibromo-4-[(pyridin-4-yl)methoxy]phenyl}-2-methane- 1727.91 (CQO str), 1605.45 (Ar-CQC), 1443.46 (C-O), 1313.29
1
3
2
2
sulfonamidopropanoic acid (6(a)-viii). The title compound was (Phe-C-O), 943.06 (p – sp -Ar-CH), and 780.06 (O-sp -Ar-CH);
prepared according to the above general procedure with the
yield 83.68% and m.p. 234 1C. IR (nmax, cm , KBr): 3486.19 1.74–1.89 (m), 2.68–2.74 (m, N-CH), 3.47–3.51 (m), 3.70–3.81
1
3
6
H NMR (400 MHz, DMSO-d , ppm): 1.41–1.51 (m, sp -CH),
ꢂ1
(
(
1
7
(
3
-NH-Str), 2921.63 (COOH str), 2112.64 (-SQO str), 1733.69 (m), 4.28–4.42 (m, I-CH), 7.51-7.63 (m, Ar-CH), 8.01 (d, J = 7.9,
2
13
CQO str), 1609.31 (Ar-CQC), 1499.38 (C-H sp bend), Ar-CH), 10.88 (s, –OH); C NMR (100 MHz, DMSO-d ): 174.7
6
2
416.46 (C-O), 1343.18 (Phe-C-O), 810.92 (p – sp -Ar-CH), and (carboxylic-C), 157.2 (Ar-C), 151.1 (Ar-C), 140.0 (Ar-C), 138.9
45.35 (O-sp -Ar-CH); H NMR (100 MHz, DMSO-d , ppm): 1.28 (Ar-C), 136.3 (Ar-CH), 125.5 (Het-Ar-CH), 93.0 (Ali-CH), 70.2
6
t, J = 7.4, sp -CH), 1.34–1.57 (m), 2.41 (m), 2.97–3.15 (m, N-CH), (Ali-CH), 61.0 (Ali-CH), 44.2 (S-C), 33.7 (I-C). MS (ESI) m/z
S] 592.145; found, 592.873.
): 174.5 Elemental analysis (C, H, N%): expected C 28.4, H 2.38, N
carboxylic-C), 154.1 (Ar-C), 149.3 (Ar-C), 146.5 (Ar-C), 134.2 4.73. Actual C 28.982, H 3.261, N 5.494.
Ar-CH), 132.5 (Ar-CH), 124.3 (Het-Ar-CH), 118.2 (Het-Ar-CH), 3-{3,5-Diiodo-4-[(1,3,4-oxadiazol-2-yl)methoxy]phenyl}-2-methane-
9.8 (Br-C), 61.2 (S-C), 44.3 (Ali-CH ), 34.4 (Ali-CH ). MS (ESI) sulfonamidopropanoic acid (6(b)-ii). The title compound was
2
1
3
+
.61–3.72 (m, Br-CH), 7.45 (t, J = 7.6, Ar-CH), 7.54–7.61 (m, calculated for [C14
H I N O
14 2 2 6
1
3
Ar-CH), 11.17 (s, -OH); C NMR (100 MHz, DMSO-d
(
(
6
6
2
+
2
m/z calculated for [C H Br N O S] 508.182; found, 506.921. prepared according to the above general procedure with the
1
6
16
2 2 5
ꢂ1
Elemental analysis (C, H, N%): expected C 37.82, H 3.17, N 5.51. yield 84.55% and m.p. 318–322 1C. IR (nmax, cm , KBr): 3441.39
Actual C 37.929, H 4.012, N 6.235. (-NH-Str), 2927.41 (COOH str), 2109.74 (-SQO str), 1729.83
-{3,5-Dibromo-4-[(pyrimidin-4-yl)methoxy]phenyl}-2-methane- (CQO str), 1603.52 (Ar-CQC), 1491.67 (C-H sp bend), 1410.67
sulfonamidopropanoic acid (6(a)-ix). The title compound was (C-O), 1338.36 (Phe-C-O), 831.16 (p – sp -Ar-CH), and 749.20
synthesised according to the above general procedure with the (O-sp -Ar-CH); H NMR (400 MHz, DMSO-d
3
3
2
2
1
6
, ppm): 1.43 (t,
ꢂ3
ꢂ1
3
yield 87.35% and density 0.97 gm cm . IR (nmax, cm , KBr): J = 7.0, sp -CH), 4.45 (q, J = 7.0, I-CH), 7.26–7.54 (m, Ar-CH),
3
1
1
7
660.23 (-NH-Str), 2901.38 (COOH str), 2110.71 (-SQO str), 7.75–7.87 (d, J = 8.1, Ar-CH), 8.12–8.19 (d, J = 8.2, Ar-CH), 10.64
2
13
729.83 (CQO str), 1588.09 (Ar-CQC), 1495.53 (C-H sp bend), (s, -OH); C NMR (100 MHz, DMSO-d ): 169.9 (carboxylic-C),
6
2
417.42 (C-O), 1329.68 (Phe-C-O), 832.13 (p – sp -Ar-CH), and 157.4 (Ar-C), 153.3 (Ar-C), 140.0 (Ar-C), 139.8 (Ar-CH), 133.3
52.10 (O-sp -Ar-CH); H NMR (400 MHz, DMSO-d , ppm): 1.26 (t, (Ar-CH), 89.5 (Ali-CH), 75.4 (Ali-CH), 40.1 (S-C), 33.7 (I-C). MS
6
2
1
3
+
J = 7.1, sp -CH), 2.61–2.79 (m, N-CH), 3.17–3.30 (m), 3.75–3.89 (m, (ESI) m/z calculated for [C13
13 2 3 6
H I N O S] 593.133; found,
Br-CH), 4.35–4.43 (m, Ali-CH), 7.43 (t, J = 7.5, Ar-CH), 7.52–7.60 593.869. Elemental analysis (C, H, N%): expected C 26.33, H
1
3
(
(
(
m, Ar-CH), 8.01 (d, J = 8.2, Ar-CH), 10.67 (s, -OH); C NMR 2.21, N 7.08. Actual C 27.014, H 3.099, N 7.726.
100 MHz, DMSO-d ): 174.7 (carboxylic-C), 165.6 (Ar-C), 157.7
Ar-C), 156.6 (Ar-C), 154.8 (Ar-C), 134.3 (Ar-CH), 131.5 (Ar-CH), sulfonamidopropanoic acid (6(b)-iii). This title compound was
3-{3,5-Diiodo-4-[(1H-pyrazol-3-yl)methoxy]phenyl}-2-methane-
6
1
17.9 (Het-Ar-CH), 60.1 (Br-C), 41.3 (S-C), 32.4 (Ali-CH ). MS obtained according to the above general procedure with the yield
2
+
ꢂ1
(ESI) m/z calculated for [C15
H
15Br
2
N
3
O
5
S] 509.17; found, 81.19% and m.p. 140–142 1C. IR (nmax, cm , KBr): 3381.57 (-NH-
5
2
07.917. Elemental analysis (C, H, N%): expected C 35.38, H Str), 2926.45 (COOH str), 1990.51 (-SQO str), 1733.69 (CQO str),
3
.97, N 8.25. Actual C 35.611, H 3.822, N 8.838.
1499.38 (C-H sp bend), 1417.42 (C-O), 1273.75 (Phe-C-O),
8
42 | New J. Chem., 2019, 43, 834--846
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NJC
2
2
1
+
8
(
09.95 (p – sp -Ar-CH), and 742.46 (O-sp -Ar-CH); H NMR MS (ESI) m/z calculated for [C17
H
17
I
2
NO
, ppm): 1.66 (s, sp -CH), 2.68–2.74 (m, 601.899. Elemental analysis (C, H, N%): expected C 33.96, H
N-CH), 7.35–7.55 (m, Ar-CH), 7.67 (t, J = 7.5, Ar-CH), 7.79 (d, 2.85, N 2.33. Actual C 34.262, H 3.708, N 3.214.
5
S] 600.891; found,
3
400 MHz, DMSO-d
6
1
3
J = 8.0, Ar-CH), 8.04 (s, Ar-CH), 10.88 (s, -OH); C NMR
3-{3,5-Diiodo-4-[(pyridin-4-yl)methoxy]phenyl}-2-methane
(
100 MHz, DMSO-d ): 170.9 (carboxylic-C), 154.6 (Ar-C), 151.4 sulfonamidopropanoic acid (6(b)-vii). The title compound was
6
(
Ar-C), 140.4 (Ar-C), 139.3 (Ar-C), 134.306 (Ar-CH), 90.5 (Het-Ar- prepared according to the above general procedure with the
ꢂ1
CH), 75.4 (Ali-CH), 43.1 (S-C), 34.7 (I-C). MS (ESI) m/z calculated yield 83.81% and m.p. 277 1C. IR (nmax, cm , KBr): 3386.81
for [C14H I N O S] 591.160; found, 591.889. Elemental analysis (-NH-Str), 2921.63 (COOH str), 2113.60 (-SQO str), 1733.69
15 2 3 5
+
(
3
C, H, N%): expected C 28.44, H 2.56, N 7.11. Actual C 29.018, H (CQO str), 1609.31 (Ar-CQC), 1507.10, 1500.35 (-NH-bend),
2
.432, N 7.755.
1416.46 (C-O), 1342.21 (Phe-C-O), 810.92 (p – sp -Ar-CH), and
2
1
3-{3,5-Diiodo-4-[(1,2-oxazol-3-yl)methoxy]phenyl}-2-methane 791.636 (O-sp -Ar-CH); H NMR (100 MHz, DMSO-d , ppm): 1.12
6
3
sulfonamidopropanoic acid (6(b)-iv). This compound was (t, J = 7.3, sp -CH), 2.98 (t, J = 7.3, N-CH), 3.52 (s, I-CH), 7.26–7.37
obtained according to the above general procedure with the (m, Ar-CH), 7.45–7.55 (m, Ar-CH), 7.98–8.05 (m), 10.55 (s, -OH);
ꢂ1
13
yield 82.72% and m.p. 175–177 1C. IR (nmax, cm , KBr):
6
C NMR (100 MHz, DMSO-d ): 168.9 (carboxylic-C), 156.1
3
1
1
7
1
661.19 (-NH-Str), 2923.56 (COOH str), 2111.67 (-SQO str), (Ar-C), 149.7 (Ar-C), 145.6 (Ar-C), 139.4 (Ar-CH), 124.8 (Het-Ar-
730.80 (CQO str), 1593.88 (Ar-CQC), 1494.56 (C-H sp bend), CH), 90.7 (Het-Ar-CH), 70.2 (Ali-CH), 63.0 (Ali-CH), 42.3 (S-C),
3
2
+
410.67 (C-O), 1335.46 (Phe-C-O), 832.13 (p – sp -Ar-CH), and 33.6 (I-C). MS (ESI) m/z calculated for [C16
H I N O
16 2 2 5
S] 602.183;
, ppm): found, 602.894. Elemental analysis (C, H, N%): expected C
.40 (t, J = 7.1, sp -CH), 2.58–2.64 (m, N-CH), 4.12 (q, J = 7.1, 31.91, H 2.68, N 4.65. Actual C 32.3145, H 3.546, N 5.4175.
3-{3,5-Diiodo-4-[(pyrimidin-4-yl)methoxy]phenyl}-2-methane
m, Ar-CH), 8.08 (d, J = 8.4, Ar-CH), 10.72 (s, -OH); C NMR sulfonamidopropanoic acid (6(b)-viii). The title compound was
2
1
50.174 (O-sp -Ar-CH); H NMR (400 MHz, DMSO-d
6
3
Ali-CH), 4.49 (q, J = 7.1, I-CH), 7.39–7.50 (m, Ar-CH), 7.62–7.68
1
3
(
(
(
100 MHz, DMSO-d
6
): 174.2 (carboxylic-C), 157.7 (Ar-C), 142.0 obtained according to the above general procedure with the
ꢂ1
Ar-C), 138.9 (Ar-C), 136.5 (Ar-CH), 133.9 (Ar-CH), 121.5 (Ar-CH), yield 88.66% and m.p. 222–224 1C. IR (nmax, cm , KBr):
8
9.9 (Ar-CH), 73.6 (Ali-CH), 61.0 (Ali-CH), 41.9 (S-C), 33.5 (I-C). 3661.19 (-NH-Str), 2925.48 (COOH str), 2111.67 (-SQO str),
+
14 2 2 6
MS (ESI) m/z calculated for [C14H I N O S] 592.145; found, 1729.83 (CQO str), 1588.09 (Ar-CQC), 1507.10, 1496.49 (C-H
3
2
5
2
92.873. Elemental analysis (C, H, N%): expected C 28.42, H sp bend), 1414.53 (C-O), 1331.61 (Phe-C-O), 834.06 (p – sp -Ar-
2
1
.38, N 4.73. Actual C 28.981, H 3.261, N 5.494.
-{3,5-Diiodo-4-[(1H-1,2,3-triazol-5-yl)methoxy]phenyl}-2-methane- ppm): 1.47 (t, J = 7.0, sp -CH), 2.27–2.58 (m, N-CH) 4.43 (q,
sulfonamidopropanoic acid (6(b)-v). This compound was synthe- J = 7.0, I-CH), 5.00 (s, Ali-CH), 7.23–7.63 (m, Ar-CH), 8.06 (d,
CH), and 752.10 (O-sp -Ar-CH); H NMR (100 MHz, DMSO-d ,
6
3
3
1
3
sized according to the above general procedure with the yield J = 8.2, Ar-CH), 10.70 (s, -OH); C NMR (100 MHz, DMSO-d
6
):
ꢂ1
8
2
1
1
3.00% and m.p. 234 1C. IR (nmax, cm , KBr): 3700.73 (-NH-Str), 175.0 (carboxylic-C), 165.3 (Ar-C), 157.6 (Ar-C), 156.0 (Ar-C),
923.56 (COOH str), 2110.71 (-SQO str), 1733.69 (CQO str), 140.6 (Ar-CH), 117.6 (Het-Ar-CH), 89.9 (Ali-CH), 76.2 (Ali-CH),
608.34 (Ar-CQC), 1498.42 (C-H sp bend), 1414.53 (C-O), 60.7 (Ali-CH), 44.3 (S-C), 34.0 (I-C). MS (ESI) m/z calculated for
15 2 3 5
342.21 (Phe-C-O), 813.81 (p – sp -Ar-CH), and 747.28 (O-sp -Ar- [C15H I N O S] 603.171; found, 603.889. Elemental analysis
3
2
2
+
1
3
CH); H NMR (400 MHz, DMSO-d , ppm): 1.46 (t, J = 7.1, sp -CH), (C, H, N%): expected C 29.87, H 2.51, N 6.97. Actual C 30.377, H
6
2.20–2.33 (m, N-CH), 3.14–3.59 (t, J = 7.4, I-CH), 4.44 (q, J = 7.1, 3.385, N 7.622.
Ali-CH), 7.54-7.59 (t, J = 7.7, Ar-CH), 7.81 (d, J = 7.8, Ar-CH), 10.61
3-{3-Chloro-4-[(pyrimidin-4-yl)methoxy]phenyl}-2-methane
13
6
(s, -OH); C NMR (100 MHz, DMSO-d ): 175.2 (carboxylic-C), sulfonamidopropanoic acid (6(c)-i). The title compound was
1
54.9 (Ar-C), 141.9 (Ar-C), 139.9, 130.8 (Ar-CH), 89.8 (Ar-CH), 70.2 synthesised according to the above general procedure with the
ꢂ1
(Ali-CH), 59.2 (Ali-CH), 42.9 (S-C), 34.2 (I-C). MS (ESI) m/z yield 84.50% and m.p. 277–279 1C. IR (nmax, cm , KBr): 3660.23
+
calculated for [C13
analysis (C, H, N%): expected C 26.37, H 2.38, N 9.46. Actual C (CQO str), 1591.95 (Ar-CQC), 1493.60 (C-H sp bend), 1413.57
14 2 4 5
H I N O S] 592.148; found, 592.884. Elemental (-NH-Str), 2925.48 (COOH str), 2111.67 (-SQO str), 1729.83
3
2
27.0515, H 3.261, N 9.987.
(C-O), 1343.18 (Phe-C-O), 834.06 (p – sp -Ar-CH), and 752.102
2
1
3
-[4-(Benzyloxy)-3,5-diiodophenyl]-2-methanesulfonamido (O-sp -Ar-CH). H NMR (100 MHz, DMSO-d , ppm): 1.07–1.19
6
3
propanoic acid (6(b)-vi). The title compound was obtained (m, sp -CH), 2.92 (s, N-CH), 3.61–3.68 (m, Cl-CH), 4.49 (q, J = 7.1,
according to the above general procedure with the yield Ali-CH), 7.51–7.57 (m, Ar-CH), 7.66–7.77 (m, Ar-CH), 8.10 (d,
ꢂ1
13
8
6
5.54% and m.p. 4300 1C. IR (nmax, cm , KBr): 3387.87 J = 8.3, Ar-CH), 10.72 (s, -OH); C NMR (100 MHz, DMSO-d ):
(
(
(
(
-NH-Str), 2918.73 (COOH str), 2111.67 (-SQO str), 1734.66 172.2 (carboxylic-C), 165.3 (Ar-C), 157.3 (Ar-C), 151.5 (Ar-C),
CQO str), 1608.34 (Ar-CQC), 1500.35 (-NH-bend), 1416.46 130.6 (Ar-CH), 122.3 (Het-Ar-CH), 117.6 Het-(Ar-CH), 110.6
2
C-O), 1342.21 (Phe-C-O), 810.92 (p – sp -Ar-CH), and 742.46 (Ali-CH), 77.2 (Ali-CH), 60.7 (Cl-C), 43.3 (S-C), 34.9 (Ali-CH
2
).
2
1
+
O-sp -Ar-CH); H NMR (100 MHz, DMSO-d , ppm): 1.47 (t, MS (ESI) m/z calculated for [C H Cl N O S] 385.823; found,
6
15 16
3 5
3
J = 7.1, sp -CH), 2.35 (s, N-CH), 4.41 (q, J = 7.1, I-CH), 5.37 (s, 386.057. Elemental analysis (C, H, N%): expected C 46.70, H
Ar-CH), 7.02–7.12 (m, Ar-CH), 7.49–7.59 (m, Ar-CH), 10.99 (s, 4.18, N 10.89. Actual C 46.365, H 4.971, N 11.346.
1
3
-
1
(
OH); C NMR (100 MHz, DMSO-d
57.7 (Ar-C), 142.4 (Ar-C), 139.2 (Ar-C), 136.4 (Ar-HC), 127.9 given in the ESI.† Detailed NMR, MASS and elemental analysis
Het-Ar-CH), 90.6 (Ali-CH), 75.4 (Ali-CH), 42.2 (S-C), 38.2 (I-C). report of 19 compounds is given in the ESI.†
6
): 171.9 (carboxylic-C),
Detailed characterization of intermediate compounds is
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Biological evaluation and assays
PGE
2
estimation
, the product produced from AA by the action of the enzymes,
-LOX in a pT3 plasmid (a kind gift from Prof. Olof Rådmark, COX-2 and mPGES1, was estimated in HeLa cells. They were plated
Expression of human recombinant 5-LOX. Human recombinant PGE
2
5
Karolinska Institute, Stockholm, Sweden) was transformed into in a 24 well plate and grown to B70% confluency. Then they were
E. coli BL21 bacteria. The overnight grown culture at 18 1C was induced with TNFa for 24 h followed by treating with 50 mM of the
induced with 0.5 mM of isopropyl-D-thiogalactopyranoside compound for 3 h. The cells release PGE
containing 150 mg mL of ampicillin and then centrifuged which was added to the PGE2 EIA wells (Cayman Chemical #
2
into the supernatant
ꢂ1
(Centrifuge 5804 R, Eppendorf AG) at 5000 rpm and 4 1C for 514010 (Ann Arbor, MI)) along with 50 mL of tracer and antibody
1
5 min. The lysis of the cells was carried out in 50 mM of (provided in the kit). PGE present in the sample and that present
2
triethanolamine/HCl at a pH of 8.0, 5 mM of ethylenediamine- in the tracer competes for a limited number of antibodies. After
ꢂ1
tetraacetic acid (EDTA), 60 mg mL of trypsin inhibitor, 1 mM 18 h, the plate was developed colorimetrically using Ellman’s
ꢂ1
of phenylmethylsulphonylfluoride (PMSF) and 500 mg mL
2
reagent (provided in the PGE EIA kit) and the absorbance was
of lysozyme. The mixture was homogenised and centrifuged read at 410 nm (Enspire, Perkin Elmer, USA). This absorbance
ꢂ1
(
1
Centrifuge 5418 R, Eppendorf AG) at 19 000 ꢁ g and 4 1C for value was used to calculate the pg mL of PGE
2
produced. The
5 min. The supernatant was precipitated with ammonium percentage inhibition of PGE production by the compound was
2
sulfate (50 w/v%), centrifuged at 16 000 ꢁ g and 4 1C for 30 min calculated by normalising the value obtained from cells treated
and immediately used for activity assays.
with TNFa alone.
5-LOX activity assay
2
Percentage inhibition of PGE production = 100
The supernatant containing 5-LOX was added to 25 mM of
HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
ꢂ (100 ꢁ PGE produced by compound treated cells/
2
PGE
2
produced by TNFa induced cells)
(
pH 7.3), 0.4 M of EDTA, 10 mM of CaCl
2
, and 4 mM of
0
adenosine 5 -triphosphate (ATP). Compound at a concentration
Cell proliferation assay
of 50 mM in DMSO was added to the reaction mixture. The
enzyme reaction was initiated by the addition of 30 mM of the The compounds were tested in HeLa cells using 3-(4,5-
substrate (AA), and the activity was determined by measuring dimethylthiazol-2-yl)-3,5-diphenyltetrazolium bromide (MTT)
the formation of 5-HPETE, at 236 nm (Jasco V-550 UV-vis as per a standard protocol. The percentage of viable cells was
spectrophotometer). Zileuton was used as the positive control. calculated as,
Cell viability (%) = (OD570sample/OD570control) ꢁ 100
Pseudoperoxidase assay
The pseudoperoxidase activity of 5-LOX was determined due to
consumption of 13(S)-HPODE. Assay buffer used was 50 mM
Dual (acridine orange and EtBr) staining assay
potassium phosphate (pH 7.4), 0.3 mM CaCl
00 mM ATP, 10 mM 13-(S)-HPODE and activity was measured in of live and dead cells. HeLa cells were plated in 6-well tissue
2
, 0.1 mM EDTA, Double staining protocol was adopted to determine the number
2
terms of decrease in the absorbance with a UV-visible spectro- culture plate and allowed to grow to 60% confluence. They were
photometer (Jasco V-550 UV-vis spectrophotometer) at 234 nm. then treated with 50 mM of compound for 48 h. The cells were
ꢂ1
Reaction was initiated by the addition of 10 mM of the compound. then washed with PBS and double stained with 3 mg mL of
ꢂ1
The percent consumption of 13-(S)-HPODE was recorded in terms of acridine orange and 10 mg mL of ethidium bromide. The cells
decrease in the absorption of the product 13(S)-HPODE.
were then imaged using an inverted fluorescence microscope
(Leica Microsystems, GmbH). The green and reddish orange
DPPH assay
fluorescence emitted by acridine orange and ethidium bromide
respectively were captured and both the images were merged
using ImageJ software. The percentage of apoptotic and necrotic
cells were calculated using the following equation,
Radical scavenging activity of the compound was determined
using the stable free radical 2,2-diphenyl-1-picrylhydrazyl
(
DPPH). A solution of DPPH (0.10 mM) in methanol and test
compound (50 mM) in dmso was mixed and was incubated for
0 min at room temperature in darkness. The absorbance was
Percentage of apoptotic cells = VA + NVA/(VA + VN + NVA) ꢁ 100
3
measured at 517 nm in a Multimode Plate reader (Enspire
Perkin Elmer, version 4.10.3005.1440) and the antioxidant
activity was calculated with reference to methanol, used as
the control. Ascorbic acid was used as the positive control.
Percentage of necrotic cells = NVN/(VA + VN + NVA) ꢁ 100
where,
VN = live cells emitting bright green fluorescence
VA = live cells emitting bright green fluorescence with
fragmented (apoptotic) nuclei
NVN = dead cells emitting orange/bright red fluorescence
NVA = dead cells emitting orange/bright red fluorescence
with fragmented (apoptotic) nuclei.
ꢀ
ꢁ
A0 ꢂ As
A0
%
inhibition ¼
ꢁ 100
where A
0
= absorbance of the control (blank, without sample)
and A = absorbance of the test compounds.
s
8
44 | New J. Chem., 2019, 43, 834--846
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NJC
Table 2 Primers for RT-PCR
(81.6 ꢀ 1.3%) against COX-2 and mPGES1. It shown that the
iodine substitution more active on 5-LOX enzyme and chlorine
0 0
Forward: 5 GGAACGACATGGAGACCATCTAC-3
Reverse: 5 TCCAGGCGACAAAAGGGTTA-3
Forward: 5 GCTATTCCGGCCCCAACT-3
mPGES-1
COX-1
0
0
substitution was advantageous in the inhibition of PGE com-
2
0
0
parison to other halogens. Current medications against inflam-
mation include coxibs and NSAIDS are available but with
several side effects. So, there is a need for an alternative drug
against inflammation associated diseases. These tyrosine derivatives
could prove to be a promising scaffold against asthma, allergy,
cancer and other inflammatory diseases if further optimized.
0
0
Reverse: 5 GATGAAGGTGGCATTGACAAACT-3
Forward: 5 TATACTAGAGCCCTTCCTCCTGTGCC-3
0
0
COX-2
0
0
Reverse: 5 ACATCGCATACTCTGTTGTGTTCCC-3
Forward: 5 CTCACCATGGATGATGATATCGC-3
0
0
b actin
0
0
Reverse: 5 AGGAATCCTTCTGACCATGC-3
Nucblue staining assay
Nucblue (Hoechst stain) staining analysis was done according Conflicts of interest
to previously reported procedure. HeLa cells were plated in 6
well culture plates and allowed to grow to confluence. The cells
There are no conflicts to declare.
were treated with 50 mM compounds for 48 h. The cells were
washed with PBS and 2–3 drops of Nucblue stain was added.
The plate was incubated at 37 1C for 15 min. The cells were
Acknowledgements
visualized in fluorescence microscope (Blue filter) and documented. Shweta Sinha (SR/WOS-A/CS-126/2013) and Meera Ramanan
The microscopic field was examined for the morphology of cells. (SR/WOS-A/LS-514/2012) acknowledge the WOS-A, DST, GoI for
Live cells will appear round and uniform blue in colour whereas funding and fellowship. A. Puratchikody thanks the DST-SERB,
dead or apoptotic cells will have a central halo and shaggered edges. GoI for the financial support (SR/S1/OC-48/2011, 14-05-2013).
FACS analysis
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46 | New J. Chem., 2019, 43, 834--846
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