Design, synthesis and in vitro study of 5,6-diaryl-1,2,4-triazine-3-ylthioacetate derivatives as COX-2 and β-amyloid aggregation inhibitors

Article abstract of DOI:10.1002/ardp.201400400

In order to find novel cyclooxygenase (COX)-2 inhibitors for treating inflammatory-based diseases such as Alzheimer's disease (AD), an ethyl carboxylate side chain was added to 5-(4-chlorophenyl)-6-(4-(methylsulfonyl)phenyl)-3-(methylthio)-1,2,4-triazine

Full text of DOI:10.1002/ardp.201400400

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Full Paper
Design, Synthesis and In Vitro Study of 5,6-Diaryl-1,2,4-triazine-3-
ylthioacetate Derivatives as COX-2 and b-Amyloid Aggregation
Inhibitors
Sakineh Dadashpour1, Tuba Tuylu Kucukkilinc2, Oya Unsal Tan3, Keriman Ozadali3, Hamid Irannejad4,
and Saeed Emami4
1
Student Research Committee, Faculty of Pharmacy, Mazandaran University of Medical Sciences, Sari,
Iran
2
Department of Biochemistry, Faculty of Pharmacy, Hacettepe University, Sihhiye, Ankara, Turkey
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hacettepe University, Ankara, Turkey
Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
3
4
Mazandaran University of Medical Sciences, Sari, Iran
In order to find novel cyclooxygenase (COX)-2 inhibitors for treating inflammatory-based diseases such as
Alzheimer’s disease (AD), an ethyl carboxylate side chain was added to 5-(4-chlorophenyl)-6-(4-
(methylsulfonyl)phenyl)-3-(methylthio)-1,2,4-triazine (lead compound II) to maintain residual inhibition
of COX-1 through interacting with Arg120. A preliminary molecular docking study on both the COX-1/
COX-2 active sites truly confirmed our hypothesis. Accordingly, a series of ethyl 5,6-diaryl-1,2,4-triazine-3-
ylthioacetate derivatives were synthesized and their chemical structures were confirmed by NMR, IR and
MS spectra. Further in vitro COX-1/COX-2 evaluations revealed that compound 6c (COX-2 IC50 ¼ 10.1 mM,
COX-1 IC50 ¼ 88.8 mM) is the most selective COX-2 inhibitor while maintaining residual inhibition of COX-
1. In order to evaluate their potential use against AD, an in vitro evaluation of b-amyloid fibril formation
was performed. The results indicated that the prototype compounds 6 are effective b-amyloid
destabilizing agents while compound 6c could inhibit 94% of the b-amyloid fibril formation after 48 h.
Finally, the in silico assessment results of their blood–brain barrier permeability were satisfactory.
Keywords: b-Amyloid aggregation / Blood–brain barrier permeability / COX-2 inhibitor / Molecular
docking / 1,2,4-Triazine-3-ylthioacetate
Received: October 26, 2014; Revised: December 23, 2014; Accepted: January 6, 2015
DOI 10.1002/ardp.201400400
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
catalyzes the first step in the conversion of arachidonic acid
into prostaglandins and thromboxanes which are important
Introduction
inflammatory mediators. COX has two isoforms, cyclooxygen-
ase-1 (COX-1) which is a housekeeping enzyme and is
constitutively expressed in normal cells and cyclooxygenase-
2 (COX-2) that is expressed and activated only in inflammatory
situations. COX is competitively inhibited by a group of drugs
named nonsteroidal anti-inflammatory drugs (NSAIDs) to
overcome inflammation and for therapeutic purposes even in
neuroinflammatory-based diseases like Alzheimer’s disease
(AD) [1]. It is well studied that accumulation of Ab peptides
promotes the pathogenesis of AD via distrupting cell
membranes and resulting cytotoxicity. Inhibition of Ab
aggregation or destabilizing preformed b-amyloid peptides
Inflammation is a pathological situation in which the immune
cells are produced in response to a necrotic agent. Cyclo-
oxygenase (COX) enzyme is an endogenous enzyme that
Correspondence: Dr. Hamid Irannejad, Department of Medicinal
Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences
Research Center, Mazandaran University of Medical Sciences,
Farahabad St., Sari 4847116547, Iran.
E-mail: irannejadhamid@gmail.com
Fax: þ98-11-33543084
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is a commonly used target for AD research. Therefore,
developing multitargeted COX inhibitors which are capable
to disaggregate Ab peptides would be a new way to challenge
AD [2].
As known, clinical administration of traditional NSAIDs
is associated with side effects such as gastric ulcers and
nephrotoxicity. Discovery and introduction of selective COX-2
inhibitors in several years later could significantly diminish the
deleterious side effects of traditional NSAIDs as well as
improving anti-inflammatory power of this group of drugs
in osteoarthritis and rheumatoid arthritis. Several selective
COX-2 inhibitors have been marketed up to now such as
celecoxib, valdecoxib and etoricoxib (Fig. 1) [3].
new selective COX-2 inhibitors with higher anti-inflammatory
effect and fewer side effects. The acetate side chain is capable
of constituting electrostatic interaction with Arg120 located
in the carboxylate site of both COX-1 and COX-2 isoforms.
However, prepared compounds were highly selective COX-2
inhibitors [6].
Regarding these facts and considering our recent introduc-
tion of 5-(4-chlorophenyl)-6-(4-(methylsulfonyl)phenyl)-3-
(methylthio)-1,2,4-triazine derivative (lead compound II)
as a potent and selective COX-2 inhibitor [7], we were
encouraged to insert a carboxylate side chain in the form of
ethyl ester on the 3-methylthio of central triazine ring to
achieve novel selective COX-2 inhibitors
6 (Fig. 4). To
It is well known that the COX-2 active site can be divided
into three parts: hydrophobic site (also present in COX-1),
which is mainly constituted by hydrophobic amino acids
including Phe518, Met522, Trp387, Leu384, Tyr385 and
Phe381. The second part is carboxylate site due to the
presence of Arg120 which is responsible for interacting with
the carboxylate side chain of non-selective COX inhibitors.
This site is also present in COX-1 and Arg120 is crucial for COX-
1 selectivity of NSAIDs. The third part of COX-2 active site is
selectivity site in which Arg513 plays an important role for
selective COX-2 inhibitors. This site is located in a secondary
hydrophilic pocket of COX-2 which is not present in COX-1.
Methylsulfonyl or sulfonamide moiety of COX-2 inhibitors
interacts through this site to infer their selectivity (Fig. 2) [4].
Unfortunately, selective COX-2 inhibitors were not devoid of
cardiovascular effects such as thrombosis and myocardial
infarction. It was shown that cardiovascular side effects arose
from suppression of COX-2 dependent prostacyclin which
mediates platelet activation and atherogenesis. Therefore,
residual inhibition of COX-1 isoform has been considered in
the design of new generation of selective COX-2 inhibitors [5].
To infer a minimal inhibition of COX-1 isoform to the
selective COX-2 inhibitors, attaching an acetate side chain
(pharmacophore of traditional NSAIDs) on the central
heterocyclic ring of a selective COX-2 inhibitor would be
logical. To achieve this goal, several compounds such as 1,5-
diarylpyrroles I (Fig. 3) have been synthesized and reported as
potentially evaluate the therapeutic effect of compounds in
neuroinflammation situations like AD, the ability of com-
pounds to disaggregate b-amyloid peptide was also assessed.
Results and discussion
Preliminary molecular docking
Preliminary in silico study was performed by molecular
docking prior to synthesis to evaluate accuracy of our
hypothesis. Compounds 6a–e were docked into the active
sites of both COX-1 and COX-2 to evaluate their selectivity
against COX isoenzymes. The best pose with the highest score
was used for comparison of results. Calculated free energy of
binding (DG, kcal/mol) for the ligand–protein complex in COX-
1 and COX-2 enzymes is shown in Table 1. As shown, all
compounds 6a–e have much higher binding energy in
complex with COX-2 enzyme than COX-1 and therefore are
considered to be selective COX-2 inhibitors. Gibbs free energy
of binding for all compounds in complex with COX-2 is in
the range of ꢀ8.66 to ꢀ9.49 kcal/mol whereas this energy in
ligand–COX-1 complex is much lower and is in the range of
ꢀ0.65 to ꢀ5.25 kcal/mol.
Mode of binding and necessary interactions of the
representative compound 6c in the active site of COX-2
enzyme is shown in Fig. 5. Docking pose showed that
methylsulfonyl moiety of compound 6c is located in the
Figure 1. Selective COX-2 inhibitors in the market.
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Figure 2. Superimposition of COX-1
(green) and COX-2 (gray) active sites
and reperesentation of the selective
COX-2 inhibitor sc-558 and its mode
of binding. The three subdivisions of
the COX-2 active site are shown.
selectivity site of COX-2 enzyme and is interacting with
Arg513, Glu192, His90, Ile517 and Phe518. Sulfur atom of this
group interacts with p-electrons of histidine ring of His90.
Backbone NH of Ile517 and Phe518 and guanidine group of
Arg513 are hydrogen bonded to sulfonyl oxygen atoms.
The p-electrons of phenyl ring (attached to sulfonyl) are in
contact with alkyl side chains of Leu352, Ser353 and Val523.
The 4-methoxyphenyl part is situated in a lipophilic space
of active site constituted by hydrophobic side chains of
Phe381, Tyr385, Trp387 and Phe518. Triazine ring has p-alkyl
interactions with Val349, Ala527 and p-cation interaction
with charged guanidine side chain of Arg120. Finally the most
important interaction that would be expected is a hydrogen
bonding with 3.1 Å distance between carbonyl oxygen atom
of compound 6c and guanidine hydrogen atom of Arg120 in
the carboxylate site of COX-2. The latter interaction also exists
between the ester part of compound 6c and Arg120 in the
COX-1 active site which leads to the concomitant inhibition of
COX-1 along with COX-2 enzyme inhibition. All compounds
showed the same binding conformation in COX-2 active site.
3 was reacted with sodium methoxide and then butyl nitrite
to produce 2-(hydroxyimino)-1-(4-(methylsulfonyl)phenyl)-2-
arylethanone derivatives 4. Triazine ring closure was per-
formed with thiosemicarbazide in acidic water to obtain
5-aryl-6-(4-methylsulfonyl)-1,2,4-triazine-3-thiols 5 [7]. At the
final step, thiol group was alkylated with ethylchloroacetate
in the presence of potassium carbonate and potassium iodide
in DMSO to give the final products 6a–e. All final compounds
Chemistry
The satisfactory results by molecular docking prompted us
to synthesize novel COX-2 inhibitors 6a–e as depicted in
Scheme 1. Accordingly, phenylacetic acid derivatives 1 were
converted to 1-(4-(methylthio)phenyl)-2-arylethanones 2 by a
fast and convenient acylation method in the presence of
trifluoroacetic acid anhydride, phosphoric acid and thioani-
sole. Then the sulfur atom of compounds 2 was oxidized to
the sulfonyl by Oxone in THF/MeOH and the resulting material
Figure 3. New selective COX-2 inhibitors with acetate side
chain.
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Figure 4. Development of our novel COX-2 inhibitors by adding ethyl carboxylate side chain.
6a–e were characterized and confirmed by 1H NMR, 13C NMR,
IR and MS spectra (Supplementary Material).
peptide concentration in CNS through b- or g-secretase
inhibition or different unknown mechanisms [9]. Once b-
amyloid plaques are formed in the brain, the major treatment
strategy is to destabilize or disaggregate the toxic b-amyloid
plaques in the brain. Therefore, developing and discovering
COX inhibitors which are capable of suppressing the initiating
neuroinflammatory process and also to destabilize the aggre-
gated amyloid peptide would be necessary [10].
In order to investigate the anti-Alzheimer potential of
compounds 6a–e, the ability of compounds to inhibit the
aggregation of b-amyloid peptides was also evaluated
in vitro. The thioflavin T fluorescence method was used to
evaluate their potential to inhibit b-amyloid fibril formation
at 100 mM concentration. Rifampicin (100 mM) was used as a
reference drug and the results are shown as percentage of
inhibition of Ab(1–40) and Ab(1–42) peptides aggregation after
24 and 48 h in Fig. 6. The percentage of inhibition of Ab(1–40)
and Ab(1–42) peptides aggregation for the reference drug
rifampicin after 24 h was 93% and 81%, respectively. All
tested compounds 6a–e had prominent effect on disaggre-
gation of both b-amyloid peptides. Their ability to inhibit the
aggregation of Ab(1–40) peptide was very similar to each other
and ranging from 85% to 89%. Compound 6b bearing the
Cyclooxygenase inhibition assay
The in vitro COX-1/COX-2 inhibition assay was performed for
the ethyl 5-aryl-6-(4-methylsulfonyl)-1,2,4-triazine-3-thioace-
tate derivatives 6a–e along with the two reference drugs, NS-
398 as a COX-2 selective and indomethacin as a nonselective
or COX-1 selective compounds. Results are summarized in
Table 2. As shown, compound 6c, with methoxy group at the
para position of phenyl ring, is the most potent and selective
COX-2 inhibitor with IC50 value of 10.1 mM (COX-2 selectivity
index ¼ 8.79). The substitution of chlorine atom at the same
place in compound 6d, decreases COX-2 selectivity index to
5.07. COX-2 and COX-1 IC50 values for compound 6d were 69.5
and 13.7 mM, respectively. In the case of fluorine atom
occupying the same place at phenyl ring (compound 6e), a
weak inhibition and an increase in the IC50 values were
observed for both COX-1 and COX-2 enzymes (COX-1
IC50 ¼ 96.8 mM, COX-2 IC50 ¼ 48.7 mM). However, substituting
methyl group could improve the inhibition of COX isoen-
zymes. In this case, COX-2 and COX-1 IC50 values were 22.7 and
56.7 mM, respectively (COX-2 SI ¼ 2.5). Compound 6a without
any substituent at phenyl ring showed a mild to moderate
inhibition of COX isoenzymes. IC50 values for COX-1 and COX-
2 were 41.2 and 34.3 mM, respectively (COX-2 SI ¼ 1.2). As
predicted and expected, all compounds were selective COX-2
inhibitors while maintaining residual inhibition of COX-1
isoenzyme. The results indicate the crucial role of ethyl
Table 1. Calculated free energy of binding for the best
pose of compounds in the active site of COX-1 and COX-2
enzymes.
acetate side chain as
modulates COX inhibition selectivity.
a new interacting motif, which
DG (kcal/mol)
DG (kcal/mol)
Compound
R
COX-1
COX-2
6a
6b
6c
6d
6e
H
Me
MeO
Cl
ꢀ3.31
ꢀ0.65
ꢀ2.65
ꢀ2.28
ꢀ5.25
ꢀ9.03
ꢀ9.49
ꢀ8.79
ꢀ8.66
ꢀ9.11
b-Amyloid aggregation assay
Administration of COX inhibitors has been effective to prevent
progression of AD in which a neuroinflammatory reaction is
normally the origin of initiation [8]. Few COX inhibitors such as
flurbiprofen have the ability to lower the toxic b-amyloid
F
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Triazine-3-thioacetates as COX-2 and Ab Formation Inhibitors
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Figure 5. Binding mode of compound 6c in
the COX-2 active site.
methyl group on the para position of phenyl ring has the most
anti-aggregation effect (89%) after 24 h among the com-
pounds tested on the Ab(1–40) peptide. Moreover, the most
selective COX-2 inhibitor 6c could inhibit the aggregation of
Ab(1–40) peptide up to 86%. But more difference was observed
among compounds tested on the Ab(1–42) peptide in which the
percentage of inhibition was in the range of 78% to 88%.
Interestingly, compound 6a showed the highest percent
inhibition after 24 h (88%) against Ab(1–42) peptide aggrega-
tion, even more than the reference drug rifampicin (81%).
Compounds 6b and 6e were in the second and third order
with the percent inhibition of 86%. The two most selective
COX-2 inhibitors 6c and 6d showed lower ability to inhibit
Ab(1–42) peptide aggregation up to 78% after 24 h. However,
Scheme 1. Reagents and conditions: (i) Thioanisole, TFAA, H3PO4, rt, stir, 5 min; (ii) Oxone1, MeOH, THF, rt, stir, 4 h; (iii) (1) MeONa,
MeOH, (2) BuONO, rt, stir; (iv) thiosemicarbazide, H2O, HCl, reflux; (v) ethylchloroacetate, K2CO3, KI, DMSO, rt, stirring.
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Table 2. In vitro cyclooxygenase inhibition assay of compounds 6a–e.
Compound
R
COX-1 IC50 (mM)a)
COX-2 IC50 (mM)a)
SIb)
6a
6b
6c
6d
H
Me
MeO
Cl
F
—
41.2
56.77
88.8
69.5
96.8
213.2
0.6
34.3
22.7
10.1
13.7
48.7
2.1
1.2
2.5
8.79
5.07
1.99
101.52
0.03
6e
NS-398
Indomethacin
—
18.5
a)The in vitro test compound concentration required to produce 50% inhibition of enzymatic activity. The result (IC50, mM) is the
mean of two determinations acquired using the COX Inhibitor Screening Assay Kit.
b)In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
much more interesting results were obtained after 48 h for
the two selective COX-2 inhibitor 6c and 6d. In the second 24 h
monitoring of fluorescence intensity, the anti-aggregation
effect of compounds 6a, 6b and 6e was decreased (fluores-
cence intensity increased) while the anti-aggregation ability
of compounds 6c and 6d was increased during the time. This
increase in disaggregation for compounds 6c and 6d led to the
maximum inhibition of 94% and 96% for Ab(1–40) and 93%
and 95% for Ab(1–42), respectively. The reference drug
rifampicin could inhibit Ab(1–40) and Ab(1–42) peptides
aggregation up to 96% and 97% after 48 h, respectively.
Compounds 6a, 6b and 6e showed much lower anti-
aggregation potential after 48 h that is ranging from 55%
to 75% for Ab(1–40) and Ab(1–42) peptides.
In silico prediction of the blood–brain barrier
permeability
Blood–brain barrier (BBB) permeability is one of the most
important challenges in the pharmacology of CNS active
drugs. The extent of CNS penetration has limited the usage of
many drugs whose target is located in the CNS. Generally,
high percentage of CNS acting drugs fails to pass the clinical
trials annually due to the CNS penetration failure. This
drawback has imposed high expenses at pharmaceutical and
drug development companies nowadays. Therefore, evaluat-
ing the capability of new compounds to pass the BBB by
in silico methods would be inevitable and helpful. Several
“rules of thumb” have been developed for prediction of
passive BBB permeability. Compounds are more likely to
penetrate the BBB if the following criteria meet. (i) Molecular
weight (MW) less than 400–500 Da. (ii) LogD (logarithm of
distribution coefficient) between ꢀ1 and 4. (iii) Number of
total hydrogen bonds (nHB) less than 10. (iv) Polar surface
area (TPSA) less than 90 Å2. Increasing the number of rules
which are not obeyed by one compound decreases the
possibility of its BBB permeability [11].
The calculated physiochemical parameters for compounds
6a–e are shown in Table 3. Also, the values of logBB
(logarithm of brain/plasma concentration) and CNS activity
were predicted for compounds 6a–e by ACD/Lab, and results
are listed in Table 3. As shown, values of MW, nHB and logD
are located in the allowed range with the exception of TPSA,
which has only a small deviation from the maximum allowed
value. Although, the values of TPSA fall a bit out of the range,
but it does not mean that these compounds have lack of CNS
activity but may reduce their extent of passage through BBB.
This fact was further confirmed by the predicted values of
logBB and CNS activity. As seen in Table 3, the predicted
Figure 6. Percent inhibition of amyloid fibril formation for
compounds 6a–e and rifampicin at 24 h (a) and 48 h (b).
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Triazine-3-thioacetates as COX-2 and Ab Formation Inhibitors
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Table 3. Calculated physicochemical parameters for compounds 6a–e and prediction of their CNS activity.
Compound
MW
logDa)
nHB
TPSAb)
Pred. logBBa)
Pred. CNS activitya)
6a
6b
6c
6d
6e
429.51
443.54
459.54
463.96
447.5
2.77
3.55
3.05
3.33
2.84
7
7
8
7
7
99.11
99.11
108.34
99.11
99.11
ꢀ0.09
ꢀ0.09
ꢀ0.09
ꢀ0.09
ꢀ0.41
Active
Active
Active
Active
Active
nHB, number of total hydrogen bonds (acceptors þ donors); logD, logarithm of distribution coefficient; logBB, logarithm of
brain/plasma concentration.
a)Calculated or predicted by ACD/Lab (www.acdlab.com).
b)Calculated by Molinspiration (www.molinspiration.com).
values of logBB for compounds 6a–d and 6e are ꢀ0.09 and
ꢀ0.41, respectively, and the favorable values of logBB for CNS
active drugs lie approximately between ꢀ1 and þ1. There-
fore, all synthesized compounds were predicted to be CNS
active.
General procedure for preparation of ethyl 5-aryl-6-(4-
methylsulfonylphenyl)-1,2,4-triazine-3-ylthioacetate
derivatives (6a–e)
The corresponding 5-aryl-6-(4-methylsulfonylphenyl)-1,2,4-
triazine-3-thiol derivatives 5a–e (1 mmol), ethyl chloroacetate
(1.5 mmol), potassium carbonate (2 mmol) and potassium
iodide (0.5 mmol) were added to DMSO (20 mL) and the
resulting mixture was stirred at room temperature for 12 h
under nitrogen atmosphere. Progress of reaction was moni-
tored by TLC. Then the reaction mixture was diluted with ethyl
acetate and washed with water several times. The organic
phase was dried over sodium sulfate, filtered and concentrat-
ed under vacuum. Final products were purified by column
chromatography (silica gel, 230–400 mesh) eluting with
chloroform/ethylacetate (5%).
Conclusion
In summary,
ylthioacetate derivatives were synthesized and their chemical
structures were characterized and confirmed by 1H NMR, 13
a series of ethyl 5,6-diaryl-1,2,4-triazine-3-
C
NMR, IR and MS spectra. Ability of compounds to inhibit COX-
1/COX-2 enzymes as well as inhibition of Ab aggregation was
assessed in vitro. All compounds could optimally inhibit both
COX isoenzymes as predicted by molecular docking analysis
which indicates the important role of the acetate side chain as
a new interacting moiety. Moreover, all compounds showed
remarkable ability for disaggregation of b-amyloid peptides.
Finally, in silico assessment revealed that the designed
compounds are BBB permeable and CNS active agents. The
obtained results indicate that the prototype compounds 6 can
be reserved as potential agents in amyloid related diseases
like AD.
Ethyl 2-(6-(4-(methylsulfonyl)phenyl)-5-phenyl-1,2,4-
triazin-3-ylthio)acetate (6a)
Yield: 30%; mp: 196°C; IR (KBr , cmꢀ1) nmax : 1731 (C O); 1312,
–
–
1151 (S O); 1H-NMR (400 MHz, CDCl3) d (ppm): 0.9 (t,
–
–
J ¼ 7.2 Hz, 3H, CH2CH3), 3.06 (s, 3H, SO2CH3), 3.80 (s, 2H,
SCH2CO), 4.21 (q, J ¼ 7.2 Hz, 2H, CH2CH3), 7.33–7.37 (m, 3H,
Ar-H), 7.50–7.53 (m, 2H, Ar-H), 7.98 (d, J ¼ 8.4 Hz, 2H, Ar-H),
8.12 (d, J ¼ 8.4 Hz, 2H, Ar-H). 13C-NMR (100 MHz, CDCl3) d
(ppm): 14.28, 32.66, 44.56, 61.12, 127.31, 128.42, 128.78,
128.83, 129.21, 133.47, 137.85, 138.74, 152.25, 155.49,
169.63, 170.48; MS (m/z, %): 429 (Mþ, 15), 399 (23), 326
(34), 299 (100), 256 (96), 220 (38), 193 (42), 165 (61). Anal.
calcd. for C20H19N3O4S2: C, 55.93; H, 4.46; N, 9.78. Found: C,
55.71; H, 4.52; N, 9.69.
Experimental
Chemistry
All commercially available reagents were purchased from
Merck or Aldrich and used without further purification.
Column chromatography was carried out on silica gel (230–
400 mesh). TLC was conducted on silica gel F254 plates. IR
spectra were taken using Perkin-Elmer FT-IR (KBr disks). Mass
spectra of the products were obtained on an Agilent 5937
mass selective detector. NMR spectra were recorded on Bruker
400 or 500 MHz instruments. The chemical shifts (d) and
coupling constants (J) are expressed in ppm and hertz (Hz),
respectively. Elemental analyses were carried out by ECS-4010
(Costech International S.p.A.) elemental analyzer. The results
of elemental analyses (C, H, N) were within ꢁ 0.4% of the
calculated values.
Ethyl 2-(6-(4-(methylsulfonyl)phenyl)-5-(p-tolyl)-1,2,4-
triazin-3-ylthio)acetate (6b)
Yield: 35%; mp: 198°C; IR (KBr , cmꢀ1) nmax: 1727 (C O); 1311,
–
–
1151 (S O); 1H-NMR (400 MHz, CDCl3) d (ppm): 1.28 (t,
–
–
J ¼ 7.2 Hz, 3H, CH2CH3), 2.34 (s, 3H, Ar-CH3), 3.06 (s, 3H,
SO2CH3), 3.80 (s, 2H, SCH2CO), 4.21 (q, J ¼ 7.21 Hz, 2H,
CH2CH3), 7.14 (d, J ¼ 8.4 Hz, 2H, Ar-H), 7.41 (d, J ¼ 8.4 Hz,
2H, Ar-H), 7.97 (d, J ¼ 8.4 Hz, 2H, Ar-H), 8.12 (d, J ¼ 8.4 Hz, 2H,
Ar-H). 13C-NMR (100 MHz, CDCl3) d (ppm): 14.23, 21.44, 33.08,
44.42, 61.06, 126.41, 127.72, 128.32, 128.40, 129.64, 138.30,
140.84, 141.79, 153.97, 156.51, 169.73, 172.51. MS (m/z, %):
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443 (Mþ, 2), 368 (4), 342 (8), 313 (85), 234 (40), 178 (55), 149
(63), 117 (100). Anal. calcd. for C21H21N3O4S2: C, 56.87; H, 4.77;
N, 9.47. Found: C, 56.71; H, 4.64; N, 9.59.
test compound causing 50% inhibition) were calculated from
the concentration-inhibition response curves (duplicate
determinations) [12].
Inhibition of Ab aggregation
Ethyl 2-(5-(4-methoxyphenyl)-6-(4-(methylsulfonyl)-
phenyl)-1,2,4-triazin-3-ylthio)acetate (6c)
Compounds 6a–e were tested for their potential to inhibit
amyloid beta peptide aggregation by thioflavin T fluores-
cence method using Ab(1–40) and Ab(1–42) peptides. Commer-
cially purchased lyophilized Ab(1–40) and Ab(1–42) peptides
(Sigma) were dissolved in 0.02% ammonia. Aliquots (10 mL)
were added to the assay medium including 120 mL 0.1 M
NaCl þ 600 mL 0.1 M KP buffer pH 7.4 þ 470 mL dH2O and
preincubated at 37°C for 24 h in the absence and presence of
inhibitors (100 mM). Preincubated Ab-peptide (100 mL) was
mixed with 50 mL of ThT (200 mM) in 50 mM glycine–NaOH
buffer (pH 8.5). Fluorescence intensities in the absence and
presence of inhibitors were measured at Exc: 448 nm,
Em: 490 nm and percentages of inhibition on aggregation
were calculated using fluorescence intensity of Ab-peptide
with no inhibitor as control. Rifampicin was tested as
reference agent at 100 mM in this study. Interaction of
Yield: 32%; mp: 150°C; IR (KBr , cmꢀ1) nmax: 1736 (C O); 1299,
–
–
1148 (S O); 1H-NMR (500 MHz, CDCl3) d (ppm): 1.28 (t,
–
–
J ¼ 7.1 Hz, 3H, CH2CH3), 3.09 (s, 3H, SO2CH3), 3.83 (s, 3H,
OCH3), 4.1 (s, 2H, SCH2CO), 4.23 (q, J ¼ 7.1 Hz, 2H, CH2CH3),
6.85 (d, J ¼ 8.75 Hz, 2H, Ar-H), 7.48 (d, J ¼ 8.75 Hz, 2H, Ar-H),
7.76 (d, J ¼ 8.25 Hz, 2H, Ar-H), 7.96 (d, J ¼ 8.25 Hz, 2H, Ar-H).
13C-NMR (100 MHz, CDCl3) d (ppm): 14.11, 32.75, 44.52, 55.69,
60.22, 115.27, 126.47, 128.45, 128.89, 129.34, 138.23, 138.74,
152.19, 157.13, 160.66, 169.86, 172.10. MS (m/z, %): 459 (Mþ,
8), 414 (4), 286 (100), 223 (15), 207 (24), 163 (18). Anal. calcd.
for C21H21N3O5S2: C, 54.89; H, 4.61; N, 9.14. Found: C, 54.96; H,
4.55; N, 9.31.
Ethyl 2-(5-(4-chlorophenyl)-6-(4-(methylsulfonyl)phenyl)-
1,2,4-triazin-3-ylthio)acetate (6d)
Yield: 43%; mp: 122°C; IR (KBr , cmꢀ1) nmax: 1735 (C O); 1307,
compounds with thioflavin T (without adding amyloid
peptide) were screened at 0, 8, 24 and 48 h and subtracted
from data as blank [13].
–
–
1152 (S O). 1H-NMR (500 MHz, CDCl3) d (ppm): 1.29 (t,
–
–
J ¼ 7.05 Hz, 3H, CH2CH3), 3.10 (s, 3H, SO2CH3), 4.11 (s, 2H,
SCH2CO), 4.23 (q, J ¼ 7.02 Hz, 2H, CH2CH3), 7.35 (d, J ¼ 8.3 Hz,
2H, Ar-H), 7.46 (d, J ¼ 8.3 Hz, 2H, Ar-H), 7.74 (d, J ¼ 8.1 Hz, 2H,
Ar-H), 7.98 (d, J ¼ 8.1 Hz, 2H, Ar-H). 13C-NMR (100 MHz,
CDCl3) d (ppm): 14.17, 33.23, 44.44, 62.11, 127.90, 129.30,
130.28, 131.18, 132.53, 138.22, 140.49, 141.46, 152.23,
154.52, 168.44, 170.51. MS (m/z, %): 464 (Mþ, 11), 418 (5),
290 (100), 292 (44), 227 (29), 176 (43). Anal. calcd. for
Molecular docking study
Crystal structures of COX-1 and COX-2 with PDB code 3N8Z
and 3NT1 were used for docking study. Docking study was
performed with AutoDock 4.2 and Discovery Studio v.4.0
(Accelrys Software, Inc.) was used to compute the ligand-
protein interactions [14].
C
20H18ClN3O4S2: C, 51.77; H, 3.91; N, 9.06. Found: C, 51.61;
H, 3.83; N, 9.19.
This research was supported by a grant from the Research
Council of Mazandaran University of Medical Sciences.
This work was related to the Pharm.D. thesis of S.D.
(Faculty of Pharmacy, Mazandaran University of Medical
Sciences).
Ethyl 2-(5-(4-fluorophenyl)-6-(4-(methylsulfonyl)phenyl)-
1,2,4-triazin-3-ylthio)acetate (6e)
Yield: 36%; mp: 153°C; IR (KBr , cmꢀ1) nmax: 1727 (C O); 1313,
–
–
1152 (S O); 1H-NMR (500 MHz, CDCl3) d (ppm): 1.10 (t,
–
–
J ¼ 7.05 Hz, 3H, CH2CH3), 3.06 (s, 3H, SO2CH3), 3.91 (s, 2H,
SCH2CO), 4.31 (dd, J ¼ 25.0 Hz, 16.65 Hz, 2H, CH2CH3), 7.03 (t,
J ¼ 8.5 Hz, 2H, Ar-H), 7.47 (dd, J ¼ 8.8 Hz, 5.35 Hz, 2H, Ar-H),
7.96 (d, J ¼ 8.6 Hz, 2H, Ar-H), 7.99 (d, J ¼ 8.6 Hz, 2H, Ar-H). 13C-
NMR (100 MHz, CDCl3) d (ppm): 14.31, 31.69, 44.51, 61.73,
117.58, 127.94, 128.38, 128.64, 128.92, 138.33, 139.41, 149.69,
156.66, 161.15, 168.65, 171.81. MS (m/z, %): 447 (Mþ, 2), 445
(20), 382 (23), 343 (51), 316 (31), 274 (100), 236 (34), 211 (44),
183 (46). Anal. calcd. for C20H18FN3O4S2: C, 53.68; H, 4.05; N,
9.39. Found: C, 53.55; H, 4.18; N, 9.29.
The authors have declared no conflict of interest.
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