Synthesis, reactions, and biological activity of some triazine derivatives containing sulfa drug moieties

Article abstract of DOI:10.1134/S1068162015040032

Thienyl-triazine-sulphonamide conjugates were prepared from their precursor amines using triethyl orthoformate or ethyl chloroformate as cross coupling reagents. The progress of these reactions was investigated by spectral (IR, NMR, MS) and microanalytica

Full text of DOI:10.1134/S1068162015040032

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2015, Vol. 41, No. 4, pp. 437–450. © Pleiades Publishing, Ltd., 2015.
Synthesis, Reactions, and Biological Activity
of Some Triazine Derivatives Containing Sulfa Drug Moieties1
,
b
,
c
, d, 2
M. R. E. Alya , A. A. Gobouria, S. H. Abdel Hafeza , and H. A. Saada
aDepartment of Chemistry, Faculty of Science, Taif University, Taif, 21974 Kingdom of Saudi Arabia
bDepartment of Chemistry, Faculty of Applied Science, Port Said University, Port Said, Egypt
cDepartment of Chemistry, Faculty of Science, Assiut University, Assiut, Egypt
dDepartment of Chemistry, Faculty of Science, Zagazig University, Zagazig, 44511 Egypt
Received August 8, 2014; in final form, February 13, 2015
Abstract—Thienylꢀtriazineꢀsulphonamide conjugates were prepared from their precursor amines using triꢀ
ethyl orthoformate or ethyl chloroformate as cross coupling reagents. The progress of these reactions was
investigated by spectral (IR, NMR, MS) and microanalytical techniques. The synthesized compounds were
in vitro screened for antibacterial, antifungal, antioxidant, and anticancer activity. 4ꢀ[({[3ꢀMercaptoꢀ5ꢀ
oxoꢀ6ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ1,2,4ꢀtriazinꢀ4(5
out to be a powerful antibacterial agent, while all the compounds prepared were inactive against fungal speꢀ
cies tested. 4ꢀ{[({8ꢀCyanoꢀ4ꢀoxoꢀ3ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ4 ,8 ꢀ[1,2,4]triazino[3,4ꢀ ][1,3,4]thiadiazinꢀ
H
)ꢀyl]imino}methyl)amino]ꢀbenzenesulfonamide
turned
H
H
b
7ꢀyl}amino)(ethoxy)methyl]amino}benzenesulfonamide displayed in vitro promising cytotoxicity against
Ehrlich ascites carcinoma cell line with concurrent attenuation of malonodinitrile and it was unique among
other compounds being unable to increase glutathione Sꢀtransferase and reduced glutathione Sꢀtransferase
activities. This compound exhibited also good antioxidant properties that together with its cytotoxicity nomꢀ
inated it for further investigation in cancer research.
Keywords: 1,2,4ꢀtriazine, triazinothiadiazine, triazinecarbonitrile, sulfa drugs, biological activity
DOI: 10.1134/S1068162015040032
2 1
INTRODUCTION
cial Pꢀgp inhibitor coꢀadministered with anticancer
antibiotics to avert cancer resistance. Another compliꢀ
cation in cancer therapy is the oxidative stress of many
anticancer drugs. Drug candidates of dual antioxidant
potency, therefore, are highly recommended [6].
Virulence of human microbial pathogens, cancer
(as body dysfunction syndrome in consequence of
mutations), and evolution of multidrug resistant speꢀ
cies are posing serious global problems. Impairment of
drug efficacy leads to prolonged therapy and even high
mortality rates, particularly, in the case of epidemic
disieses and cancer metastasis [1]. Mutant bacteria are
Merging of specific pharmacophores in single
architecture is a promising strategy to struggle against
pathogens resistance. Sulphonamides, thiophenes,
and 1,2,4ꢀtriazines are relevant multifunctional pharꢀ
macophores. Thus, sulphonamides reported mainly as
antibacterial agents [7, 8], are also functioning as insuꢀ
lin releasing stimulants [9] and antiꢀinflammatory [10]
and cytotoxic [11] agents.
stimulated to excrete
βꢀlactamases that are able to
detoxify ꢀlactam antibiotics. Clavulanic acid (
tazobactam (II) (Scheme 1) are coꢀadministered to
neutralize these resistance vectors [2].
Intrinsic fungal resistance, primary and secondary,
is well documented in fighting mycosis. Upregulation
of drug target fungal enzymes and activation of efflux
systems are amongst the common fungal mechanisms
to bypass the drug toxicity [3, 4]. Drug efflux proteins,
for instance, Pꢀglycoproteins are resistance vectors in
several cancer species. Captopril (III) [5] is a commerꢀ
β
I) and
1,2,4ꢀTriazines documented as antiꢀHIV, antiꢀ
H5N1 virus agents [12–14] are also antiproleferative
[15, 16] agents acting on VEGFR tyrosine kinases [17]
as well as antiꢀtuberculosis [18], antiꢀanxiety, antiꢀ
inflammatory agents[19, 20], potent antidepression
and antianxiety agents [21], and antiꢀbreast cancer
candidates [22]. Finally, thiophenes are well known as
Abbreviations: BACE1, Betaꢀsecretase 1; DPPH, 2,2ꢀdiphenylꢀ
1ꢀpicrylhydrazyl; EACC, Ehrlich ascites carcinoma cell line;
GST, glutathione Sꢀtransferase; GSTꢀRd, reduced glutathione
Sꢀtransferase; HIV, human immunodeficiency virus; MDR,
multidrug resistance; Pꢀgp, Pꢀglycoproteins; SAR, structureꢀ
activity relationship; VEGFR, vascular endothelial growth facꢀ
tor receptor.
inhibitors of BACE1 preventing ꢀamyloid plaques
β
formation in Alzheimer’s disease [23, 24] and Plasmoꢀ
dium falciparum differentiation [25], antiꢀHIV [26],
antiproleferative [27, 28], antiꢀinflammatory [29–31],
antibacterial [32], and antiprotozoal [33] agents.
1
In continuation to our program on triazine chemꢀ
istry [34–38], this article describes the design and synꢀ
The article is published in the original.
Corresponding author: eꢀmail: h_saadzag@yahoo.com.
2
437

438
ALY et al.
O
O
S
COOH
H
N
O
O
S
N
O
H
N
O
N
N
O
O
CH2OH
OH
OH
H
O
(
I
)
(
II
)
(III)
Cl
NH2
N
O
HO
MeO
OH
N
NH2
OMe
(
IV)
(IV)
Scheme 1. Selected
βꢀlactamase and Pꢀgp inhibitors.
N
N
S
S
H2N
N
N
H
N
a
b
O
O
N
N
N
SH
H
SH
S
R
O
NH2
O
(
VI
)
(VII
)
(
VIIIa–e)
OEt
EtO
N
S
H
N
N
+
S
R
O
N
N
SH
O
O
NH2
(
IX)
(Xa–e)
(
VIIIa, Xa
)
(VIIIb, Xb
)
(
VIIIc, Xc
)
(
VIIId, Xd
)
(
VIIIe, Xe)
N
N
R = H
CH3CO
N
O
N
N
Scheme 2. Reagents and conditions: (a) CH(OEt) , AcOH, rfx. (92%); (b) AcOH, rfx. (64%).
3
thesis of new thienylꢀ1,2,4ꢀtriazinyl sulphonamides as ethyl orthoformate as cross coupling reagent in glacial
tripharmacophoric probes for screening their latent
antimicrobial, antioxidant, and antiproliferative
potencies—the way that might lead to discovering new
pharmacophore lead structures for application and
optimization.
acetic acid (Scheme 2). Compound (VII) showed
1H NMR signals typical for ethyl group at
1.48 ppm,
for CH3 as triplet, and at 4.52 ppm for CH2 as quarꢀ
tet. In 13C NMR spectrum, imine carbon was observed
at 150.80 ppm. The IR spectrum was free from NH2
stretching vibration bands in the range 3300–
3100 cm–1
δ
δ
δ
.
RESULTS AND DISCUSSION
Chemistry
Coupling of formamidate (VII) with available sulfa
drug species (VIIIa ) in refluxing AcOH led to
transetherification of sulfa drugs yielding sulfonaꢀ
mides (Xa ) and unique imine
IX). 1H NMR specꢀ
−
e
In earlier attempts, 4ꢀaminoꢀ3ꢀmercaptoꢀ6ꢀ[2ꢀ(2ꢀ
−
e
(
thienyl)vinyl]ꢀ1,2,4ꢀtriazinꢀ5(4H)ꢀone (VI) [13] was
activated as formamidate (VII) by refluxing with triꢀ trum of imine (IX) showed an NH2 signal at
δ
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SYNTHESIS, REACTIONS, AND BIOLOGICAL ACTIVITY
439
N
N
S
S
N
N
OEt
N
S
N
O
N
N
SH
H
O
N
SH
H+
NH2
+
–N
O
N
N
SH
H
EtO
SO2NHR
OEt
NH2
RHNO2S
NH2
(IX
)
(Xa–e)
SO2NHR
(
Xa)
(
Xb)
(
Xc)
(Xd
)
(
Xe)
N
N
R =
H
CH3CO
N
N
N
O
Scheme 3. Plausible mechanism for the formation of compound (IX).
N
S
N
O
N
N
S
a
H
(VI
)
(VIIIa
–e
)
+
N
H
N
H
R
S
O
O
(
XIa–c)
N
S
N
N
O
N
N
SH
N
S
N
H
O
N
N
H
S
H
N
N R
N
S
H
S
R
O
O
O
O
H
(
XIIa
–c
)
(XIIIa–c)
(
VIIIa, XIa
–XIIIa)(VIIIb, XIb
–XIIIb) (VIIIc, XIc
–XIIIc) (VIIId, XIId) (VIIIe, XIIe)
N
N
R =
CH3CO
H
N
O
N
N
Scheme 4. Reagents and conditions: CH(OEt) , AcOH, Ac O, rfx. (86% for (Xa);
3
2
74% for (Xb), and 68% for (Xc)).
6.46 ppm and a thiol signal at
δ
14.00 ppm. This high
Attempts to activate amines (VIIIa−e) by triethyl
downfield shift might be attributed to intramolecular
hydrogen bonding with the imine moiety in a fiveꢀ
membered ring structure. In 13C NMR spectrum, the
orthoformate followed by coupling with triazole (VI
were unsuccessful. Then, attention was given to threeꢀ
component oneꢀpot coupling of triazole (VI), amine
)
imine carbon was observed at 154.00 ppm.
δ
(
VIIIa e), and CH(OEt)3 in AcOH containing several
−
drops of Ac2O. Three target compounds (XIa c) were
successfully obtained (Scheme 4).
−
A plausible mechanism for this transetherification
is described in Scheme 3.
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440
ALY et al.
1H NMR spectra of target compounds (XIa
showed a number of NꢀH signals equivalent to those
observed in corresponding structures along with the
olefinic protons in sꢀtrans configuration according to
−c)
(
XII). The NH2 and NꢀH stretching vibration bands in
sulfonamide (XIa) were well distinguished and two
H bands were also discriminated in case of derivaꢀ
N
⎯
tives (XIb) and (XIc). The C=O stretching bands were
clearly seen in all conjugates and all these signals and
bands gave supporting evidences for modular coupling
high vicinal coupling value,
of SꢀH signal at ~14.00 ppm excludes the bicylic
J ~ 16.2 Hz. The presence
δ
structure (XIII) and the high chemical shift value supꢀ
ports structure (XI) instead of structure (XII) accordꢀ
ing to anticipated intramolecular hydrogen bond forꢀ
mation between the sulfohydryl group and the imine
nitrogen in a fiveꢀmembered ring structure, which is
of compound (VI) with sulfonamides (VIIIa
−c).
Following the same procedure, compound (XIV
[38] was cross coupled with sulfonamides (VIIIa
)
)
−c
affording conjugates (XVa−c) in excellent yields
more favorable in structure (XI) than in structure (Scheme 5).
O
O
N
O
S
CN
N
S
S
R
CN
N
N
OEt
N
N
N
H
N
NH2
N
N
H
N
S
S
O
H
(XVa
–c
)
(
XVa
)
(
XVb
)
(
XVc)
N
N
R =
H
N
N
Scheme 5. Reagents and conditions: (VIIIa
−e
), AcOH, Ac O, rfx. (77% for (XIVa),
2
82% for (XIVb), and 92% for (XIVc)).
The IR spectrum showed two NH bands at 3240
and 3150 cm–1 along with disappearance of the NH2 2200 cm–1. The CH3CH2 signals in H NMR spectra
band of precursor (XIV) reported at 3317 cm–1. The appeared typically at
.05–1.08 ppm for CH3 and
C≡
N stretching vibration band was observed around
1
δ
1
N
N
N
S
S
N
N
a
O
O
N
H
NH2
N
OEt
H
XVII
b
(
XVI
)
(
)
O
O
N
S
R
S
N
N
H
O
N
H
N
N
H
(XVIIIa–d)
(
XVIIIa) (XVIIIb
)
(
XVIIIc
)
(XVIIId)
N
N
R =
H
N
N
N
O
Scheme 6. Reagents and conditions: (a) CH(OEt) , AcOH, Ac O, rfx. (95%); (b) (VIIIa−e), AcOH,
3
2
Ac O (72% for (XVIIa), 64% for (XVIIb), 80% for (XVIIc), and 54% for (XVIId)).
2
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SYNTHESIS, REACTIONS, AND BIOLOGICAL ACTIVITY
441
3.37–3.47 ppm for CH2. The SO2N–H signal was number of NH2/NH stretching vibration bands correꢀ
observed at ) and
~10.20 ppm. The 13C NMR spectrum sponding to those in sulfonamides (XVIIIa
supported the structure of compounds (XVa XVIIIe). The C=O stretching vibration bands were
through the appearance of signals at ~59.9, 14.9, and also visible. The signals of ethyl group in H NMRꢀ
114.2 ppm arising from CH2, CH3, and
δ
−c
−c)
(
1
δ
C
≡
N
groups.
spectrum disappeared upon coupling and the N–H
signals were in good accordance with the proposed
structures; the olefinic protons in sꢀtrans configuration
were good evidences for coupling of the sulfa drug speꢀ
cies with triazole (XVII).
In a third attempt, compound (XVI) [39] was actiꢀ
vated with CH(OEt)3 affording imidoformite (XVII) in
good yield as described for the synthesis of comꢀ
pound (VII). The NH2 band at 3353 cm–1 disappeared
upon activation and the appearance of CH3CH2 sigꢀ
Unfortunately, coupling via one pot procedure was
nals in 1H NMR at
δ
1.88 and 3.88 ppm were good eviꢀ not sucsessful for sulfonamides (XIa
dences for triazole (XVII). Compound (XVII) was sucꢀ tigation, ethyl chloroformate was used as alternative cross
cessfully coupled with amines (VIIIa ) and (VIIIe coupling reagent. Thus, treatment of compound (XVI
in AcOH containing several drops of Ac2O to afford with ClCOOEt in pyridine followed by addition of the
compounds (XVIIIa ) andcompound (XVIIId) in appropriate sulfa drugs (VIIIc) and (VIIId) afforded conꢀ
−c). In the last invesꢀ
–
c
)
)
−c
very good yields (Scheme 6). IR spectra showed the jugates (XX) and (XXI) (Scheme 7).
a
(XVI)
N
S
N
O
O
O
EtO
N
N
N
H
OEt
(XIX)
b
N
O
O
O
O
O
N
N
S
N
N
S
S
S
N
O
O
N
N
N
O
O
N
N
H
H
O
N
N
O
EtO
N
H
N
H
H
COOEt
XX
EtO
(
)
(XXI)
Scheme 7. Reagents and conditions: (a) ClCOOEt, Pyr., rfx.; (b) (VIIIc) or (VIIId), rfx (83% for (XIX
)
and 89% for (XX)).
The IR spectrum of derivative (XX) showed bands for two CH3 groups, at 61.34 ppm for CH2, and at
at 3221 cm–1 for the two NH moieties and three bands
within the range 1777–1665 cm–1 for three C=O
95.06 ppm for Cꢀ4 of isoxazole, respectively.
groups. 1H NMR spectrum showed signals at
δ
1.02–
Pharmacology
1.21 and 3.42–4.12 ppm for the two C2H5 groups and
Twelve newly synthesized thienyꢀtriazinylꢀsulphoꢀ
namide tripharmacophoric probes were screened in
vitro as antimicrobial and cytotoxic agents and antioxꢀ
idants. These compounds had common thienyl and
sulphonamide pharmacophoric terminal domains and
varied only by the middle azaheteroaryl pharmacophꢀ
oric domain and the linker arm to the sulphonamide.
Therefore, they were divided into three groups.
a new signal at δ 10.13 ppm for SO2NH. On the other
hand, 13C NMR spectrum displayed signals at 16.90,
17.12, and 18.07 ppm corresponding to four CH3
groups, as well as two signals at 57.75 and 58.08 ppm
δ
for the (2CH2) moieties.
Finally, the IR spectrum of isoxazole (XXI) disꢀ
played two N–H and two C=O stretching vibration
bands within the ranges 3290–3192 cm–1 and 1742–
Group I comprized derivatives (XIa−c) and (XVIIIa−d),
where the azaheteroaryl was a 1,2,4ꢀtriazinyl moiety
and the linker originated from CH(OEt)3 as crossꢀ
1
1657 cm–1, respectively. The H NMR displayed sigꢀ
nals for CH3CH2 group at
new signal at
10.13 ppm related to SO2NH. 13C NMR
spectrum displayed signals at
δ
1.21 and 4.13 ppm and a
δ
coupler. Group II included compounds (XVa
−c),
δ
12.41 and 13.86 ppm where the azahetroaryl was presented by 1,2,4ꢀtriaziꢀ
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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442
ALY et al.
Results of antimicrobial screening against two Gramꢀnegative, two Gramꢀpositive, and two fungal species
E. coli
S. typhimurium
S. aureus
14106
1577**
B. subtilis
A. flavus
C. albicans
(
(
(
(
(
(
(
(
(
(
(
(
XIa
)
)
1742*
5
10117
3139
10126
5
16
493*
13
4
*
15
13
11
9
–
10
–
XIb
11146
*
XIc
XVa
XVb
XVc
)
*
10124
5113
*
9164
–
*
10
11
10
10
–
)
*
*
*
)
12
4
1298*
15
6
–
)
4
5
–
XVIIIa
)
)
5
5
5
–
XVIIIb
–
–
–
–
–
–
XVIIIc
XVIIId
XX
XXI
)
5
5
4
4
–
–
)
15
–
17
–
9
12
–
–
–
)
–
–
–
)
4
5
9
10
18
–
–
–
Ampicillin
2264*
22
–
1886*
–
1796*
–
Amphotericin B
–
–
20
* Inhibition zone diameters (mm) were measured at concentration of 100
values.
µ
g/mL, and the superscripts stand for some estimated MIC
90
nylꢀ[3,4ꢀb]ꢀ1,3,4ꢀthiadiazinyl, and that was the main
(XVIIId), the activity index was 0.68 or 68% of the
difference from compounds of Group I Group III control activity in the case of E.coli and 0.77 for the
.
included two compounds, (XX) and (XXI) and differed activity index, which corresponded to 77% of the conꢀ
from Group I by the urea linker originated from trol activity, in the case of S. typhimurium.
ClCOOEt as crossꢀcoupling reagent. In the following
Against Gramꢀpositive species in the case of
sections, the structureꢀactivity relationship (SAR) is
S. aureus, compound (XIb) from group I showed the
largest values of inhibition zone diameter and activity
index of 0.83 and 83% of ampicillin activity. The most
based on the classification mentioned above.
important was its low MIC90 value, that is 11% higher
than that of ampicillin. Despite the fact that the inhibiꢀ
Antimicrobial Activity
The antimicrobial screening of the newly syntheꢀ
sized probes in groups I−III was performed in vitro
tion zone diameter for the sulfonamide derivative (XVb
was lower than that of another sulfonamide derivative
XIa), the MIC90 value for compound (XVb) was higher
)
using the modified Kirby–Bauer disc diffusion method
[40]. Tested pathogenic microorganisms were Escheriꢀ
chia coli (ATCC 11775) and Salmonella typhimurium
(CAICC 31) as Gramꢀnegative bacteria, Bacillus subtiꢀ
lis and Staphylococcus aureus (ATCC 12600) as Gramꢀ
positive bacteria, and fungal species Aspergillus flavus
and Candida albicans (ATCC 26555) (table).
(
than that for compound (XIa). Thus, the MIC90 value of
sulfonamide (XVb) was 14% less than that of ampicillin,
while for sulfonamide (XIa) it was 23% less than the
MIC90 value for the control. Against B. subtilis, comꢀ
pounds (XIa) and (XVb) were the most active and had
similar activity index (0.83) corresponding to 83% of
ampicillin activity.
Against Gramꢀnegative species, compounds (XIa
)
and (XVIIId) from group I showed appreciable activiꢀ
ties. Thus, either free sulphonamide in sulfonamide
Thus, the free sulphonamide moiety in sulfonaꢀ
mide (XIa) was a common feature in high antibacterial
activity against both types of organisms, and the methꢀ
oxazole ring in sulfonamide (XVIIId) promoted the
activity against Gram negative species, while the free
pyrimidine ring in derivative (XVb) augmented the
activity against Gramꢀpositive species.
(
XIa) or the methoxazolesulphonamide in acetamide
derivative (XVIIId) were preferential over the pyrimiꢀ
dine ring in the same group or other structural variaꢀ
tions in compounds of groups II and III. The activity
index, i.e. the ratio of inhibition zone diameter to
inhibition zone diameter of the positive control [41],
was 0.77 for the sulfonamide derivative (XIa), which
In general, the compounds studied were not active
against two fungal species tested. Only compounds
corresponded to 77% of the control in case of E. coli
,
and 0.72 or 72% of the control in the case of S. typhiꢀ
(
XIb, c) showed modest activity against A. flavus and
murium. However, the MIC90 value for sulfonamide MIC90 values were too low (50% lower than that of
derivative (XIa) in the case of E. coli was 33% higher amphotericin B). The same activity was observed for
than that for ampicillin. For acetamide derivative sulfonamides (XIa, c) and (XVa−c) against C. albicans.
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SYNTHESIS, REACTIONS, AND BIOLOGICAL ACTIVITY
443
120
100
80
60
40
20
0
16
d
d
e
14
c
c
c
c
b
12
10
8
a
a
d
cd
c
6
c
b
b
ab
4
b
2
0
Fig. 1. Viability of Ehrlich ascites carcinoma cell line after
treatments. Letters a–d above the bars stand for statistical
variations at 0.001.
Fig. 2. Habig assay of glutathione Sꢀtransferase content in
EACC after treatments. Letters a–d above the bars stand
for statistical variations at 0.001.
P
<
P
<
Cytotoxicity. Cell Viability
and GSTs inhibitors are target anticancer drugs. For
instance, pyrimethamine (IV) (Scheme 1) is known
for a long time as a GST P1ꢀ1 inhibitor at a singleꢀdigit
Viability of EACC cell line in the presence of eight
compounds was separately evaluated in vitro (Fig. 1).
Results were expressed as viability percent according
to trypan blue assay [42].
micromolar concentration range (IC50 = 1
μ
M) [46].
Curcumin ( ) is also well known in this regard [47].
V
Concurrent effect of tested compounds on the GST
level in EACC cell line was investigated according to
Habig’s assay [48]. The results (Fig. 2) revealed an
augmentation in GST levels after treatment with all
compounds except for sulfonamide (XVa), which
insignificantly differed from the control. Compounds
Viability in the presence of all compounds signifiꢀ
cantly differed from that of the control assigned 100%.
Derivatives (XIc), (XVa), (XVb), and (XVIIId) (Fig. 1)
were the most activeones. Mortality of cells in their
presence fell within the range of 67–75% and insignifꢀ
icantly varied between each other. Compounds (XIa
)
(XVc) and (XVIIIc) had the highest effect with insigꢀ
and (XIb) were also effective, corresponding mortaliꢀ
ties were 59 and 60%, respectively. Mortality of cells in
the presence of sulfonamide (XVIIId) was the lowest
one (54%). Variation of derivative (XVIIIc) with comꢀ
pounds (XIa), (XIb), and (XVIIId) was insignificant,
therefore, it might be concluded that it has pseudo
variation with these compounds. Thus, free sulphonaꢀ
mide, pyrimidine, and methaoxazole sulphonamides
of compounds belonging to groups I and II could
effectively attenuate viabilities of EACC cells.
nificant variation between each other. Thus, the cytoꢀ
toxic activity of sulfonamide (XVa), which was one of
the most active derivatives in these investigations, was
not accompanied by variations in GST levels in these
cells.
In the case of assaying GSTꢀRd levels in EACC cell
line in response to treatments with test compounds
individually according to Beutler’s procedure [49], all
compounds except for (XIc) and (XVa) significantly
differed from the control and led to elevation in GSTꢀ
Rd levels. Again, only sulfonamide (XVa) had the
advantage of being cytotoxic and neutrally affecting
GSTꢀRd levels.
Oxidative and Antioxidant Parameters
in Carcinoma Cells
Malondialdehyde (MDA) is a major reactive aldeꢀ
hyde resulting from the peroxidation of membrane lipꢀ
ids in consequence of oxidative stress. MDA is
involved in carcinogensis, where increased level of
MDA is found in tumor tissues and plays a role in the
early phases of tumor growth. Therefore, MDA is conꢀ
sidered as a marker of abnormal lipid peroxidation and
cancer [50, 51].
Glutathione Sꢀtransferases (GSTs) constitute a
family of phase II isozymes that catalyze the conjugaꢀ
tion of glutathione reduced form to xenobiotic subꢀ
strates for the purpose of detoxification [43] and are
implemented in cell signaling pathways involved in
cell proliferation and cell death [44]. GSTs are
observed to be overexpressed in many carcinomas and
play an important role in cancer development and
drug resistance [45]. The high levels of these enzymes
MDA levels in EACC cell line in consequence of
in plasma from cancer patients is a marker of cancer individual treatments were determined according to
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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2015

444
ALY et al.
atom in DPPH is reduced by receiving a hydrogen
20
15
10
5
atom from the antioxidant species. As shown in Fig. 5,
compounds (XIc) and (XVa) were nearly similar in
their antioxidant activities and were the best among
other derivatives. They were 69% as active as the conꢀ
trol. As soon as cytotoxic candidates of good antioxiꢀ
dant properties are highly required in cancer therapy
to alleviate the oxidative stress of most anticancer
drugs, compound (XVa) seems to be a potential target
for further investigation in cancer related research.
d
c
c
bc
bc
c
ab ab
a
EXPERIMENTAL
Chemistry
0
All chemicals were purchased from Sigma (NY,
USA). Melting points were measured by a digital Elecꢀ
trothermal IA 9100 Series and are uncorrected. IR
spectra were recorded on an ATRꢀAlpha FTꢀIR specꢀ
Fig. 3. Beutler assay of reduced glutathione content in
EACC after treatments. Letters a–d above the bars stand
for statistical variations at 0.001.
trophotometer in the range of
4000 cm–1. 1H and 13C NMR spectra were recorded on
Br ker ACꢀ300 instrument at 300 and 75 MHz,
respectively, in DMSOꢀd6 used as a solvent. Chemical
shifts are expressed as (ppm) relative to TMS as
ν
values from 400 to
P
<
a
ü
Esterbauer and Cheesman assay [52]. The results
(Fig. 4) show that the cells treated with all compounds
significantly differed from untreated cells and disꢀ
δ
internal standard. Mass spectra were recorded on a
GCMSꢀQP 1000Ex Shimadzu spectrometer at the
Microanalytical unit, Cairo University. Elemental
analyses were performed in the Microanalytical Cenꢀ
ter, Cairo University. Pharmacological investigations
were carried out in the Biochemistry Department,
Faculty of Agriculture, Cairo University.
Ethyl[3ꢀmercaptoꢀ5ꢀoxoꢀ6ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ
1,2,4ꢀtriazinꢀ4(5H)ꢀyl]imidoformate (VII). A mixture
of the triazine derivative (VI) (0.01 mol) and triethyl
orthoformate (0.01 mol) in glacial acetic acid (25 mL)
was stirred under reflux for 1 h, cooled to room temꢀ
perature, and the precipitate formed was filtered and
crystallized from EtOH to afford formamidate (VII) as
played decreased MDA levels. Compound (XVIIIc
)
was the most active attenuant of MDA levels (40%),
while derivatives (XVa), (XVb), and (XVIIId) insignifiꢀ
cantly differed from each other and were the weakest
to reduce MDA levels (by 9–14%). Compound (XIc
significantly decreased MDA level (by 18.9%).
)
Clearly, the free terminal sulphonamide and the
central [1,2,4]triazino[3,4ꢀ ][1,3,4]thiadiazine are
essential structural motifs that evoke the activity of
derivative (XVa) from group II
b
.
Antioxidant Activity
yellow crystals. Yield 92%, mp 180
−
182
Preliminary antioxidant efficiency of synthesized trum: 1660 (C=Ostr), (C=Nstr). H NMR spectrum:
compounds was investigated by DPPH radical scavꢀ 1.48 (t, 3H, 7.2, CH3), 4.52 (q, 2H, 7.2, CH2), 6.87
enging assay [53]. The free electron on the nitrogen (d, 1H, 16.2, ThiophCH=C Triaz), 7.04 (dd, 1H, 3.60
°C. IR specꢀ
1
J
J
J
H
J
,
e
60
50
40
30
20
10
0
15
10
5
d
d
d
c
b
b
b
a
0
Fig. 4. Esterbauer and Cheesman assay of MDA formation
in EACC after treatments. Letters a–d above the bars stand
for statistical variations at 0.001.
Fig. 5. DPPH scavenging assay.
No. 4
P
<
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
2015

SYNTHESIS, REACTIONS, AND BIOLOGICAL ACTIVITY
445
4.80, Hꢀ3Thioph), 7.19 (d, 1H,
1H, 5.1, Hꢀ5Thioph), 7.59 (s, 1H, N=CH), 8.03 (d, 1H,
1H, 16.2, ThiophCH=CHTriaz), 14.00 (s, 1H, S–H); Hꢀ4Pyrim), 7.10 (dd, 1H, J
J
3.6, Hꢀ4Thioph), 7.35 (d, 1H, NꢀH.), 6.53 (d, 2H,
J
1.8, Hꢀ2Ar, Hꢀ6Ar), 6.76 (d,
Triaz), 6.98 (d, 1H, 1.8 Hz,
3.60, 4.80, Hꢀ3Thioph), 7.43
3.6 Hz, Hꢀ4Thioph), 7.62 (s, 1H, NCꢀH), 7.59
5.1, Hꢀ5Thioph), 7.64 (d, 2H, 1.8 Hz, Hꢀ
16.2, ThiophCH=C Triaz),
1.8, Hꢀ3Ar, Hꢀ5Ar), 11.24 (s, 1H, SO2N H),
J
J
J
16.2, ThiophCH=C
H
J
13C spectrum: 14.33, 61.35 (C2H5), 127.8, 128.1, (d, 1H,
J
J
128.3, 129.4, 131.2, 133.7, 141.0, 145.8, 157.8, 166.8 (d, 1H,
J
(10 C). Found: C 46.70, H, 3.86, N, 18.16%. Calcd.: 5Pyrim., Hꢀ6Pyrim), 7.96 (d, 1H,
C12H12N4O2S2 (308.38): C 46.74, H, 3.92, N, 18.17%. 8.46 (d, 2H,
J
H
−
J
14.10 (s, 1H, S–H). 13C NMR spectrum: 118.3, 122.0,
126.9, 127.0, 128.6, 129.1, 129.6, 131.4, 141.1, 141.6,
142.3, 148.1, 152.8, 155.1, 163.4, 165.4, 167.1 (20 C).
Found: C 46.83, H 3.18. N, 21.79%. Calcd.:
C20H16N8O3S3 (512.59): C 46.86, H 3.15, N 21.86%.
N'ꢀ[3ꢀMercaptoꢀ5ꢀoxoꢀ6ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ
1,2,4ꢀtriazinꢀ4(5 )ꢀyl]imidoformamide (IX). A mixꢀ
ture of compound (VII) (0.01 mol) and a derivative of
sulfa drugs (VIIIa ) (0.01 mol) in glacial AcOH
H
−e
(25 mL) was stirred under reflux for 3 h and then
cooled to room temperature. The precipitate was filtered
and crystallized from EtOH to yield compound (IX) as
Nꢀ(4,6ꢀDimethylpyrimidinꢀ2ꢀyl)ꢀ4ꢀ[({[3ꢀmercaptoꢀ
5ꢀoxoꢀ6ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ1,2,4ꢀtriazinꢀ4(5H)ꢀ
yl]imino}methyl)amino]benzenesulfonamide (XIc).
Yellow crystals. Yield 68%, mp 220 222 . IR specꢀ
trum: 3287–3190 (2 N Hstr), 1663 (C=Ostr).
1H NMR spectrum: 2.38 (s, 6H, 2 CH3), 6.75 (s, 1H,
Hstr), 6.79 (d, 2H, 1.8, Hꢀ2Ar, Hꢀ6Ar), 6.85 (d, 1H,
16.2, ThiophCH=C Triaz), 7.13 (s, 1H, H 5Pyrim), 7.45
(dd, 1H, 3.60, 4.80, Hꢀ3Thioph), 7.49 (d, 1H, 3.6
Hꢀ4Thioph), 7.63 (s, 1H, NC–H), 7.68 (d, 1H, 5.1
Hꢀ5Thioph), 7.93 (d, 1H, 16.2 Hz, ThiophC =CHTriaz),
8.02 (d, 2H, 1.8, Hꢀ3Ar, Hꢀ5Ar), 11.02 (s, 1H, SO2N H),
yellow crystals. Yield 64%, mp 243 245°C.
−
1
−
°C
IR spectrum: 3292 (NH2 str), 1660 (C=Ostr). H NMR
spectrum: 6.46 (s, 2H, NH2), 6.87 (d, 1H, 16.2,
ThiophCH=C Triaz), 7.02 (dd, 1H, 3.60, 4.80,
Hꢀ3Thioph), 7.21 (d, 1H, 3.6, Hꢀ4Thioph), 7.35 (d, 1H,
5.1, Hꢀ5Thioph), 7.60 (s, 1H, N=CH), 8.09 (d, 1H,
−
J
H
J
N
J
−
J
J
H
ꢀ
J
J
J
,
J
16.2, ThiophCH=CHTriaz), 13.98 (s, 1H, S–H).
13C NMR spectrum: 127.8, 128.1, 128.3, 129.1, 131.2,
133.6, 141.0, 145.9, 158.9, 166.7 (10 C). EIꢀMS
J
,
J
H
J
−
(m/z, %): 279.00 (1.56), 252.00 (0.32), 241.00 (0.31),
14.41 (s, 1H, S–H). 13C NMR spectrum: 15.95
(2 CH3), 118.3, 122.0, 126.9, 127.1, 128.6, 129.1,
129.6, 131.4, 141.1, 141.6, 142.3, 148.1, 152.8, 155.1,
163.4, 165.4, 167.1 (20 C). Found: C 48.84, H 3.70,
N, 20.69%. Calcd.: C22H20N8O3S3 (540.64): C 48.87,
H 3.73, N, 20.73%.
182.00 (0.37), 122 (0.32), 108 (0.50), 92 (0.31), 65
(0.47). Found: C 43.21, H 3.24, N 25.10%. Calcd.:
C10H9N5OS2 (279.34): C 43.00, H 3.25, N 25.07%.
Synthesis of Sulfonamides (XIa–c).
General Procedure
A mixture of compound (VI) (0.01 mol), glacial
AcOH (20 mL), Ac2O (2 mL), CH(OEt)3 (0.01 mol),
and a derivative of the sulfa drugs (VIIIa−e) (0.01 mol)
was stirred under reflux for 3 h and then cooled to
room temperature. The precipitate was filtered at the
pump and crystallized from EtOH.
Synthesis of Sulfonamides (XVa–c).
General Procedure
A mixture of compound (IX) (0.01 mol), glacial
AcOH (20 mL), Ac2O (2 mL), CH(OEt)3 (0.01 mol),
and a derivative of the sulfa drugs (VIIIa−c) (0.01 mol)
was stirred under reflux for 3 h and then cooled to
room temperature. The precipitate was filtered at the
pump and crystallized from chloroform/benzene.
4ꢀ[({[3ꢀMercaptoꢀ5ꢀoxoꢀ6ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ
1,2,4ꢀtriazinꢀ4(5H)ꢀyl]imino}methyl)amino]ꢀbenzeneꢀ
sulfonamide (XIa). Yellow crystals. Yield 86%,
mp 240 242 . IR spectrum: 3292 (NH2str), 3198
(NHstr), 1663 (C=Ostr). H NMR spectrum: 6.75 (s,
1H, NꢀH), 6.79 (d, 2H, 1.8, Hꢀ2Ar, Hꢀ6Ar), 6.84 (d,
1H, 16.2, ThiophCH=C Triaz), 7.13 (dd, 1H, 3.60
4.80, Hꢀ3Thioph), 7.45 (d, 1H, 3.6, Hꢀ4Thioph), 7.64 (s,
1H, N=CH), 7.93 (d, 1H, 5.1, Hꢀ5Thioph), 7.99 (d,
1H, 16.2, ThiophC =CHTriaz), 8.02 (d, 2H, 1.8 Hz,
Hꢀ3Ar, Hꢀ5Ar), 11.02 (s, 2H, NH2), 14.41 (s, 1H, SꢀH).
13C NMR spectrum: 119.2, 119.8, 126.7, 127.1, 127.3,
128.0, 128.1, 129.1, 129.4, 131.1, 141.2, 141.4, 147.9,
167.1 (16 C). Found: C 44.19, H 3.24, N 19.35%.
Calcd.: C16H14N6O3S3 (434.52): C 44.23, H 3.25;
N 19.34%.
4ꢀ{[({8ꢀCyanoꢀ4ꢀoxoꢀ3ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ4
[1,2,4]triazino[3,4ꢀ
(ethoxy)methyl]amino}benzenesulfonamide
Reddish crystals. Yield 77%, mp 229 231
trum: 3350 (NH2str), 3176 (2 N
1654 (C=Ostr). 1H NMR spectrum: 1.05 (t, 3H,
CH3), 2.77 (s, 1H, SC–H), 3.47 (q, 2H, 7.2, CH2),
4.93 (br. s, 1H, NꢀH), 6.90 (s, 2H, SO2NH2), 7.13 (d,
1H, 14.4, ThiophCH=C Triaz), 7.15 (dd, 1H, 1.2
2.4, Hꢀ3Thioph), 7.24 (s, 1H, EtOC ), 7.46 (d, 1H, 1.8
Hꢀ4Thioph), 7.63 (s, 1H, ArN ), 7.75 (d, 2H, Hꢀ2Ar, Hꢀ
6Ar), 7.82 (d, 2H, Hꢀ3Ar, Hꢀ5Ar), 7.84 (d, 1H, 5.4, Hꢀ
5Thioph), 8.17 (d, 1H, 16.2, ThiophC =CHTriaz).
13C NMR spectrum: 13.95 (CH3), 28.07 (CN
H),
HOEt), 114.3 (CN), 118.3, 119.2,
126.7, 127.1, 127.4, 128.0, 128.4, 129.4, 129.5, 131.1,
midinꢀ2ꢀylbenzenesulfonamide (XIb). Yellow crystals. 141.2, 141.5, 147.9, 167.1 (16 C). Found: C 46.29,
Yield 74%, mp 228 230 . IR specrtum: 3293–3195 H 3.72, N 20.60%. Calcd.: C21H20N8O4S3 (544.63):
(2N
Hstr), 1663 (C=Ostr). 1H NMR spectrum: 6.00 (s, C 46.31, H 3.70, N 20.57%.
H,8Hꢀ
−
°C
b
][1,3,4]thiadiazinꢀ7ꢀyl}amino)
1
(XVa).
. IR specꢀ
J
H
−
°
C
J
J
,
−
Hstr), 2202 (C
≡
Nstr),
7.2,
J
J
J
J
J
H
J
J
H
J
J
,
,
H
−
H
J
J
H
C
4ꢀ[({[3ꢀmercaptoꢀ5ꢀoxoꢀ6ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ 56.79 (CH2), 98.38 (
C
1,2,4ꢀtriazinꢀ4(5 )ꢀyl]imino}methyl)amino]ꢀ ꢀpyriꢀ
H
N
−
°C
−
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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446
ALY et al.
4ꢀ{[({8ꢀCyanoꢀ4ꢀoxoꢀ3ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ4
[1,2,4]triazino[3,4ꢀ ][1,3,4]thiadiazinꢀ7ꢀyl}amino)
(ethoxy)methyl]amino}ꢀ ꢀpyrimidinꢀ2ꢀylbenzenesulfonaꢀ
mide (XVb). Reddish crystals. Yield 82%, mp 216
218 . IR spectrum: 3190 (2N Hstr), 2200 (C Nstr),
1670 (C=Ostr). 1H NMR spectrum: 1.08 (t, 3H,
7.2,
CH3), 2.75 (s, 1H, CHCN), 3.37 (q, 2H, 7.2, CH2),
4.91 (b, 1H, NꢀH), 7.11 (d, 1H, 16.2,
ThiophCH=C Triaz), 7.13 (dd, 1H, 1.2, 2.4, Hꢀ3Thioph),
7.15 (s, 1H, EtOC ), 7.17 (s, 1H, PhN ), 7.46 (d,
1H, 1.8, Hꢀ4Thioph), 7.63 (t, 1H, H 5Pyrim), 7.71 (d,
2H, Hꢀ2Ar, Hꢀ6Ar), 7.88 (d, 2H, Hꢀ3Ar, Hꢀ5Ar), 7.91
(d, 1H, 5.4, Hꢀ5Thioph), 8.17 (d, 1H, 16.2,
Thioph.CH=C Triaz), 8.48 (d, 2H, Hꢀ4Pyrim., Hꢀ6Pyrim),
10.39 (s, 1H, SO2N
(CH3), 27.98 (S HCN), 57.53 (CH2), 97.97 (
113.1 (C N), 118.3, 120.1, 126.6, 126.8, 127.5, 128.1,
H
,8
H
ꢀ
Preparation of Compounds (XVIIIa–d).
b
General Procedure
N
A solution of compound (XVII) (0.01mol) in a
mixture of glacial AcOH (20 mL) and Ac2O (2 mL)
was treated under stirring with CH(OEt)3 (0.01 mol)
−
°
C
−
≡
J
followed by the appropriate (VIIIa−e) derivative (0.01
J
mol). After being refluxed for 3 h, it was cooled to
ambient temperature, the precipitate formed was filꢀ
tered in vacuo and crystallized from benzene to afford
J
H
J
H
−H
compounds (XVIIIa−d) as yellow crystals.
J
−
4ꢀ{[({5ꢀOxoꢀ6ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ4,5ꢀdihydroꢀ
1,2,4ꢀtriazinꢀ3ꢀyl}imino)methyl]amino}benzeneꢀsulꢀ
J
J
fonamide (XVIIIa). Yield 72%, mp over 300
spectrum: 3348–3090 (NH2 str, 2 N Hstr)
(C=Ostr). 1H NMR spectrum: 6.72 (d, 1H,
16.2 Hz,
ThiophCH=CHTriaz), 6.85 (2s, 3H, SO2NH2 and
N=CH), 7.07 (d, 2H, 2.7, Hꢀ2Ar, Hꢀ6Ar), 7.08 (dd,
1H, 3.60, 4.80, Hꢀ3Thioph), 7.09 (d, 2H, 2.7, Hꢀ3Ar
Hꢀ5Ar), 7.12 (s, 1H, N HAr), 7.29 (d, 1H, 3.6
Hꢀ4Thioph), 7.53 (d, 1H, 5.1 Hz, Hꢀ5Thioph), 8.00 (d,
1H, 16.2, Thioph =CHTriaz), 12.26 (s, 1H, H 4Triaz).
°
,
C
1662
. IR
H
13
−
−
H). C NMR spectrum: 14.37
J
C
C
HOEt),
≡
J
128.6, 129.3, 129.7, 131.4, 132.7, 136.9, 139.8, 140.8,
141.4, 141.5, 141.6, 147.9, 167.3 (20 C). Found:
C 48.17, H 3.55, N 22.42%. Calcd.: C25H22N10O4S3
(622.70): C 48.22, H 3.56, N 22.49%.
J
J
,
,
−
J
J
J
CH
ꢀ
4ꢀ{[({8ꢀCyanoꢀ4ꢀoxoꢀ3ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ4
[1,2,4]triazino[3,4ꢀ ][1,3,4]thiadiazinꢀ7ꢀyl}amino)
(ethoxy)methyl]amino}ꢀ ꢀ(4,6ꢀdimethylpyrimidinꢀ2ꢀ
yl)benzenesulfonamide (XVc). Reddish crystals. Yield
92%, mp 220 222 . IR spectrum: 3222 (3 N Hstr),
2207 (C Nstr), 1668 (C=Ostr). H NMR spectrum:
1.08 (t, 3H, 7.2, CH3), 2.05 (s, 6H, 2CH3), 2.75 (s,
1H, CH), 3.42 (q, 2H, 7.2, CH2), 3.78 (b, 1H, Nꢀ
H), 7.10 (d, 1H, 16.2, ThiophCH=C Triaz), 7.13 (dd,
1H, 1.2, 2.4, Hꢀ3Thioph), 7.15 (s, 1H, EtOC ), 7.19 (s,
1H, N HAr), 7.46 (d, 1H, 1.8, Hꢀ4Thioph), 7.64 (t, 1H,
5Pyrim.), 7.70 (d, 2H, Hꢀ2Ar, Hꢀ6Ar), 7.86 (d, 2H, Hꢀ
3Ar, Hꢀ5Ar), 7.89 (d, 1H, 5.4, Hꢀ5Thioph), 8.17 (d, 1H,
16.2 Hz, ThiophC =CHTriaz), 10.20 (s, 1H, SO2NH).
13C NMR spectrum: 13.90, 14.65 (3CH3), 28.07
(SCH), 56.79 (CH2), 98.16 ( HOEt), 113.10 (C N),
H,8Hꢀ
13C NMR spectrum: 118.3, 122.0, 126.9, 127.0, 128.0,
128.6, 129.6, 131.4, 141.4, 142.3, 148.1, 152.7, 155.1,
167.1 (16 C). Found: C 47.70, H 3.46, N 20.90%.
Calcd.: C16H14N6O3S2 (402.45): C 47.75, H 3.51,
N 20.88%.
b
N
−
°C
−
1
≡
Nꢀ(4,6ꢀDimethylpyrimidinꢀ2ꢀyl)ꢀ4ꢀ{[({5ꢀoxoꢀ6ꢀ
[2ꢀ(2ꢀthienyl)vinyl]ꢀ4,5ꢀdihydroꢀ1,2,4ꢀtriazinꢀ3ꢀyl}
imino)methyl]amino}benzenesulfonamide (XVIIIb).
J
J
J
H
Yield 64%, mp over 300
(3 N Hstr), 1665 (C=Ostr). H NMR spectrum: 6.74
(d, 1H, 16.2, ThiophCH=C Triaz), 6.82 (s, 1H,
N=CH), 7.00 (t, 1H, H 5Pyrim), 7.09 (dd, 1H, 3.60
4.80, Hꢀ3Thioph), 7.12 (s, 1H, N HAr), 7.40 (d, 1H, 3.6
Hꢀ4Thioph), 7.59 (d, 1H, 5.1, Hꢀ5Thioph), 7.70 (d, 2H,
1.8, Hꢀ2Ar, Hꢀ6Ar), 7.86 (d, 2H, 1.8, Hꢀ3Ar, Hꢀ5Ar),
8.11 (d, 1H, 16.2, ThiophC =CHTriaz), 8.48 (d, 2H,
Hꢀ4Pyrim, Hꢀ6Pyrim), 10.29 (s, 1H, H 4Triaz), 11.70 (s, 1H,
°C. IR spectrum: 3348–3101
J
H
1
−
−
J
J
H
H−
−
J
,
,
J
−
J
J
H
J
J
J
C
≡
J
H
118.30, 119.20, 126.60, 126.80, 127.50, 128.10,
128.60, 129.30, 129.60, 131.40, 132.70, 136.90,
139.50, 140.80, 141.30, 141.4, 141.60, 147.80, 167.30
(20 C). Found: C 49.80, H 4.08, N 21.57%. Calcd.:
C27H26N10O4S3 (650.75): C 49.83, H 4.03, N 21.52%.
Ethyl{5ꢀoxoꢀ6ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ4,5ꢀdihydroꢀ
1,2,4ꢀtriazinꢀ3ꢀyl}imidoformate (XVII). A mixture of
compound (XVI) (0.01 mol), glacial AcOH (20 mL),
ꢀ
13
SO2NH). C NMR spectrum: 117.7, 118.5, 118.9,
126.4, 126.6, 127.5, 127.8, 127.9, 128.2, 128.8, 129.9,
130.1, 139.9, 141.5, 141.6, 152.7, 162.3 (20 C).
Found: C 50.05, H 3.38, N, 23.24%. Calcd.:
C20H16N8O3S2 (480.52): C 49.99, H 3.36, N 23.32%.
Nꢀ(4,6ꢀDimethylpyrimidinꢀ2ꢀyl)ꢀ4ꢀ{[({5ꢀoxoꢀ6ꢀ
Ac2O (2 mL), and CH(OEt)3 (0.01 mol) was stirred [2ꢀ(2ꢀthienyl)vinyl]ꢀ4,5ꢀdihydroꢀ1,2,4ꢀtriazinꢀ3ꢀyl}imino)
under reflux for 3 h and then cooled to room temperꢀ methyl]amino}benzenesulfonamide (XVIIIc). Yield
ature. The precipitate was filtered at the pump and 80%, mp over 300
crystallized from EtOH to afford imidoformate (XVII
Hstr), 1666 (C=Ostr). 1H NMR spectrum: 1.70 (s, 6H,
as pale yellow crystals. Yield 95%, mp over 300 . IR 2 CH3), 6.79 (d, 1H, 16.2, ThiophCH=C Triaz), 6.85
Hstr), 1664 (C=Ostr). H NMR (s, 1H, N=CH), 7.03 (t, 1H, H 5Pyrim), 7.11 (dd, 1H,
spectrum: 1.88 (t, 3H, 7.2, CH3), 3.88 (q, 2H, 7.2, 3.60, 4.80, Hꢀ3Thioph), 7.12 (s, 1H, N HAr), 7.38 (d,
CH2), 6.87 (d, 1H, 16.2, ThiophCH=C Triaz), 7.00 (dd, 1H, 3.6, Hꢀ4Thioph), 7.59 (d, 1H, 5.1, Hꢀ5Thioph), 7.71
1H, 3.60, 4.80, Hꢀ3Thioph), 7.02 (d, 1H, 3.6 (d, 2H, 1.8, Hꢀ2Ar, Hꢀ6Ar), 7.89 (d, 2H, 1.8, Hꢀ3Ar
Hꢀ4Thioph), 7.53 (d, 1H, 5.1, Hꢀ5Thioph), 7.57 (s, 1H, Hꢀ5Ar), 8.15 (d, 1H, 16.2, Thioph =CHTriaz), 10.28 (s,
NC–H), 8.00 (d, 1H, 16.2, ThiophCH=C Triaz), 12.20 1H, H
4Triaz), 11.77 (s, 1H, SO2NH). 13C NMR specꢀ
°C
. IR spectrum: 3348–3101 (2N
−
)
°
C
J
H
1
spectrum: 3176 (N
−
−
J
J
J
−
J
H
J
J
J
J
,
J
J
,
J
J
CH
J
H
−
(s, 1H, triazine CONH). Found: C 52.10, H 4.37, trum: 13.04, 13.05 (2CH3), 118.4, 118.9, 126.4, 126.7,
N, 20.25%. Calcd.: C12H12N4O2S (276.31): C 52.16, 127.4, 127.7, 127.9, 128.0, 128.4, 128.7, 129.7, 130.1,
H 4.38, N 20.28%.
139.7, 141.0, 141.5, 152.4, 167.3 (20 C). Found:
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SYNTHESIS, REACTIONS, AND BIOLOGICAL ACTIVITY
447
C 51.95, H 3.98, N 22.10%. Calcd.: C22H20N8O3S2
(508.58): C 51.96, H 3.96, N 22.03%.
J
J
7.2,C
16.2, ThiophCH=C
Hꢀ3Thioph), 7.29 (s, 1H, H
Hꢀ2Ar, Hꢀ6Ar), 7.56 (d, 1H,
2H, 1.8, Hꢀ3Ar, Hꢀ5Ar), 7.74 (d, 1H,
H
2CH3), 6.11 (s, 1H, N
−
H
Triaz), 7.08 (dd, 1H,
4Isoxaz), 7.33 (d, 2H,
3.6, Hꢀ4Thioph), 7.61 (d,
5.1 Hz,
=CHTriaz),
−
Nꢀ[(4ꢀ{[({5ꢀOxoꢀ6ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ4,5ꢀdihyꢀ
droꢀ1,2,4ꢀtriazinꢀ3ꢀyl}imino)methyl]amino}ꢀphenyl)
sulfonyl]acetamide (XVIIId). Yield 54%, mp over
J
J
Hꢀ5Thioph), 7.99 (d, 1H,
J 16.2, Thioph.CH
300
(C=Ostr). 1H NMR spectrum: 1.90 (s, 3H, CH3), 6.72
(d, 1H, 16.2, ThiophCH=C Triaz), 6.85 (s, 1H,
N=CH), 7.07 (d, 2H, 2.7, Hꢀ2Ar, Hꢀ6Ar), 7.08 (dd,
1H, 3.60, 4.80, Hꢀ3Thioph), 7.09 (d, 2H, 2.7, Hꢀ3Ar
Hꢀ5Ar), 7.1 (s, 1H, N HAr), 7.29 (d, 1H, 3.6, Hꢀ
4Thioph), 7.53 (d, 1H, 5.1, Hꢀ5Thioph), 8.00 (d, 1H,
16.2, Thioph =CHTriaz), 11.96 (s, 1H, H 4Triaz),
°C
. IR spectrum: 3348–3096 (2 N Hstr), 1661
−
8.82 (s, 1H, NꢀH), 10.13 (s, 1H, SO2NH). 13C NMR
spectrum: 12.41, 13.86 (2 CH3), 61.34 (CH2), 95.06
J
H
(C− 5Isoxazole), 118.0, 122.6, 127.1, 128.0, 128.1, 128.4,
J
129.5, 129.7, 138.5, 140.2, 141.0, 141.1, 141.7, 145.3,
147.2, 152.8, 162.4 (19 C). Found: C 48.39, H 3.75,
N, 17.11%. Calcd.: C23H21N7O7S2 (571.59): C, 48.33,
H 3.70, N 17.15%.
J
J
,
−
J
J
J
CH
−
13
12.26 (s, 1H, SO2NH). C NMR spectrum: 13.05
(CH3), 116.9, 118.3, 122.1, 126.4, 126.6, 127.0, 127.1,
128.0, 128.6, 129.7, 131.4, 141.5, 142.3, 148.1, 152.7,
167.2 (17 C). Found: C 48.63, H 3.60, N 18.87%.
Calcd.: C18H16N6O4S2 (444.49): C 48.64, H 3.63,
N 18.91%.
Pharmacology Antimicrobial Assay
Briefly, 100 µL of the tested bacteria and/or fungi
were grown in 10 mL of fresh media until they reached
approximate count of 108 cells/mL for bacteria and
µ
105 cells/mL for fungi [54]. 100
L of microbial susꢀ
pension was spread onto agar plates corresponding to
the broth in which they were maintained. Isolated colꢀ
onies of each organism that might be playing a pathoꢀ
genic role were selected from primary agar plates and
tested for susceptibility by disc diffusion method
[64, 65]. Disc diffusion method used for filamentous
fungi testing was the approved standard method (M38ꢀ
A) developed by National Committee for Clinical
Laboratory Standards (NCCLS) [55] for evaluation of
the susceptibilities of filamentous fungi to antifungal
agents. Yeasts were evaluated according to the M44ꢀP
disc diffusion method.
Synthesis of Compounds (XX) and (XXI).
General Procedure
CH(OEt)3 (0.01 mol) was added to a solution of
compound (XVI) (0.01 mol) in pyridine (20 mL) and
the mixture was refluxed for 10 min, then a derivative
of (XVIIIa−e) (0.01 mol) was added and reflux was
continued for additional 2 h. The mixture was evapoꢀ
rated in vacuo and the residue was crystallized from
benzene.
Ethyl3ꢀ[{[(4ꢀ{[(4,6ꢀdimethylpyrimidinꢀ2ꢀyl)amino]
sulfonyl}phenyl)amino]carbonyl}ꢀ(ethoxycarbonyl)
amino]ꢀ5ꢀoxoꢀ6ꢀ[2ꢀ(2ꢀthienyl)vinyl]ꢀ1,2,4ꢀtriazineꢀ
Plates were inoculated with filamentous fungi
Aspergillus flavus, Gramꢀpostive bacteria Staphylococꢀ
cus aureus and Bacillus subtilis, Gramꢀnegative bacteꢀ
ria Escherichia coli and Salmonella typhimurium, and
yeast Candida albicans. Discs of sterilized whatman
No. 1 filter paper (8.0 mm diameter) were saturated
4(5
mp 218
1785 (2 C=Ostr Carbam), 1665 (C=Ost Urea). H NMR
spectrum: 1.02 (t, 3H, 7.2, CH2C 3), 1.21 (t, 3H,
7.2, CH2C 3), 2.24 (s, 6H, 2 CH3), 3.42 (q, 2H,
7.2, C 2CH3), 4.12 (q, 2H, 7.2, C 2CH3), 6.73 (d,
1H, 16.2, ThiophCH=C Triaz), 7.08 (dd, 1H, 3.60
4.80, Hꢀ3Thioph), 7.27 (s, 1H, H 5Pyrim), 7.29 (d, 2H,
3.6, Hꢀ2Ar, Hꢀ6Ar), 7.54 (d, 1H, 3.6, Hꢀ4Thioph),
7.60 (d, 2H, 1.8, Hꢀ3Ar, Hꢀ5Ar), 7.73 (d, 1H, 5.1
Hꢀ5Thioph), 7.99 (d, 1H, 16.2 Hz, ThiophC =CHTriaz),
H
)ꢀcarboxylate (XX). Orange crystals. Yield 83%,
−
220 . IR spectrum: 3221 (2 N Hstr), 1777
°
C
−
−
1
J
H
J
J
H
with 10.0
compound in DMSO and left to dry. Discs were
inserted in the assay plates and then incubated at 25
for 48 h for Aspergillus flavus, at 35 37 for 24–48 h
for Gramꢀpositive and Gramꢀnegative bacteria, and at
30 for 24–48 h for Candida albicans. After elapse of
µ
L
containing 100
µ
g/mL from each test
H
J
H
J
H
J
,
°C
−
−
°C
J
J
J
J
,
°
C
J
H
8.75 (s, 1H, NꢀH), 10.13 (s, 1H, SO2NH). 13C NMR
spectrum: 16.90, 17.12, 18.07 (3 CH3), 57.75, 58.08
(2 CH2), 117.3, 118.5, 126.3, 126.6, 127.4, 127.8,
128.0, 128.4, 129.7, 130.2, 138.5, 139.8, 141.0, 141.6,
145.0, 147.1, 152.3, 152.6, 158.2 (23 C). Found:
C 50.33, H 4.26, N 16.71%. Calcd.: C28H28N8O8S2
(668.70): C 50.29, H 4.22, N, 16.76%.
the incubation period, the inhibition zones surroundꢀ
ing the discs were measured with silipping calipers
according to the NCCLS [56]. Antimicrobial activiꢀ
ties are expressed as inhibition diameter zones (mm).
Experiments were carried out in triplicates and the
average zone of inhibition was calculated. Ampicillin
and amphotericin B were used as positive control for
antibacterial and antifungal comparisons respectively,
while DMSO was used as negative control.
Ethyl3ꢀ({[(4ꢀ{[(5ꢀmethylisoxazolꢀ3ꢀyl)amino]sulꢀ
fonyl}phenyl)amino]carbonyl}amino)ꢀ5ꢀoxoꢀ6ꢀ[2ꢀ
(2ꢀthienyl)vinyl]ꢀ1,2,4ꢀtriazineꢀ4(5
Orange crystals. Yield 89%, mp 214
trum: 3290–3192 (3 N Hstr), 1742 (C=Ostr Carbam),
1657 (C=Ostr Urea). H NMR spectrum: 1.21 (t, 3H,
7.2, CH3isoxazole), 2.28 (t, 3H, CH2CH3), 4.13 (q, 2H, nificant antibacterial and/or antifungal activities were
H
)ꢀcarboxylate (XXI).
−
216 . IR specꢀ
°C
MIC90 Evaluation
−
1
The MIC90 values of compounds that showed sigꢀ
J
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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2015

448
ALY et al.
determined by agar dilution method [55]. Positive Determination of GSH is based on the reaction of
controls were evaluated under the same conditions.
DTNB (5,5ꢀdithiobis(2ꢀnitrobenzoic acid) with GSH
to yield a yellow colored chromophore with a maximum
absorbance at 412 nm. The amount of GSH present in
the carcinoma cells calculated as mg/g protein.
In vitro Cytotoxicity
and Related Metabolic Disorders Assays
Experimental animals. Female Swiss albino mice
weighing 25–30 g were obtained from the Toxicology
Department at the Central Agricultural Pesticide Labꢀ
oratory, Agriculture Research Center, and were
Lipid Peroxidation (LPO) Level
Lipid peroxidation process is determined in carciꢀ
noma cells by the thiobarbituric acid (TBA) method,
which estimates the malondialdehyde formation
(MDA) according to Esterbauer and Cheesman.
housed at constant temperature (24 2°C) with alterꢀ
nating 12 h light and dark cycles and fed with standard
laboratory food along with water ad libitum. Animal
care and handling was according to the guidelines set
by the World Health Organization, Geneva, Switzerꢀ
land, and was approved by the committee for animal
care at Toxicology Department, Central Agricultural
Pesticide Laboratory, Agriculture Research Center.
A suspension of the carcinoma cells (250 L) was
μ
added to 1.5 mL of 1% phosphoric acid (pH 2.0) and
1 mL of 0.6% of TBA in airꢀlight tubes and placed in a
boiling water bath for 25 min. After incubation, the samꢀ
ple was cooled to room temperature and MDAꢀTBA
were extracted with 2.5 mL of butanol. The organic
Tumor cells. EACC cell line purchased from the
National Cancer Institute (NCI), Cairo University
was injected in female Swiss albino mice through serial
intraperitoneal inoculation at 7 or 8 days intervals in
our laboratory in an ascites form.
phase was separated by centrifugation at 2000
5 min and absorption was measured at 532 nm. The
concentration of MDA calculated using the absorꢀ
bance coefficient of MDAꢀTBA complex (1.56 ×
105 M–1 cm–1). Lipid peroxidation is expressed as nM
g for
MDA/mg protein.
In vitro Cytotoxicity Assay
Tissue Protein Assay
In vitro cytotoxicity of the newly synthesized comꢀ
pounds was tested against EACC cells by trypan blue
exclusion assay. EACC cell were obtained by needle
aspiration of the ascitic fluid from preinoculated mice
under aseptic conditions [57]. Tumor cell suspensions
The total protein level of carcinoma cells was deterꢀ
mined according to Bradford method [58] using cooꢀ
massie brilliant blue Gꢀ250 reagent with bovine serum
albumin as standard.
(2
×
106 cells per mL) were prepared in RPMIꢀ1640
media supplemented with 10% fetal bovine serum and
Lꢀglutamine. Test compounds at 0.18, 0.36, and
0.56 mM concentrations in DMSO were incubated
with 2 mL of cell suspension under CO2 (5%) at 37°C
overnight. The percentage of cell viabilities was evaluꢀ
ated by the trypan blue exclusion affinity.
DPPH Assay for Antioxidant Activity Screening
The antioxidant activity of the synthesized comꢀ
pounds was determined by the DPPH free radical
scavenging assay. One hundred microliter of each test
compound at 100 mM in DMSO was mixed with
900
µ
L
of 60
μ
M
solution of DPPH in MeOH to reach
. The mixture was
Cell viability (%)
= (Number of viable cells/total count of cells) 100%.
final concentrations of 18
μ
M
×
shaken vigorously and left in the dark for 60 min until
stable absorption values at 517 nm were obtained. The
radical scavenging activities (RSA) were calculated as
a percentage of DPPH discoloration using the equaꢀ
tion:
Determination of Oxidative
and Antioxidant Parameters in Carcinoma Cells
Glutathione SꢀTransferase (GST) Activity
GST activity in carcinoma cells was measured specꢀ
trophotometrically according to Habig assay using
1ꢀchloroꢀ2,4ꢀdinitrobenzene as electrophilic substrate
that binds to glutathione (GSH) with the participation
of the enzyme and forms a colored GSH complex,
which absorbs at 340 nm. The activity of GST was
expressed in terms of mmol/min/mg protein.
%RSA = (ADPPH – AS)/ADPPH × 100%,
where ADPPH is the absorption of pure DPPH and AS is
the absorption after adding the sample.
Butylated hydroxyanisole (BHA) was used as posiꢀ
tive control. Measurements were taken in triplicate
and the results were averaged.
CONCLUSIONS
Reduced Glutathione Content
Reduced glutathione content (GSHꢀRd) in carciꢀ
In this work, threeꢀpharmacophoric probes arising
noma cells was evaluated according to Beutler method from conjugation of thienyltriazines with sulphonaꢀ
using commercial reduced glutathione kits (Biodiagꢀ mides via triethyl orthoformate and ethyl chloroforꢀ
nostic for diagnostic reagents: Dokki, Giza, Egypt). mate as cross coupling reagents were synthesized and
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 41
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SYNTHESIS, REACTIONS, AND BIOLOGICAL ACTIVITY
449
17. Sharma, N.K., Kumar, Y., and Priyanka, S.S., Int. J.
Pharm. Pharm. Sci., 2010, vol. 2, pp. 118–121.
their potentials as antimicrobial, cytotoxic, and antiꢀ
oxidants were screened in vitro. Compound (XIa
)
seems to be a powerful antibacterial agent, while the
whole series was not active against considered fungal
species. Derivative (XVa) showed interesting cytotoxic
18. Gill, C., Jadhav, G., Shaikh, M., Kale, R., Ghawalkar, A.,
Nagargoje, D., and Shiradkar, M., Bioorg. Med. Chem.
Lett., 2008, vol. 18, pp. 6244–6247.
and antioxidant properties, which nominate it to be 19. Mullick, P., Khan, S.A., Begum, T., Verma, S.,
Kaushik, D., and Alam, O., Acta Pol. Pharm. Drug Res.
2009, vol. 66, pp. 379–385.
,
further investigated in cancer research.
20. Hynes, J., Kanner, S.B., Yang, X., Tokarski, J.S.,
Schieven, G.L., Dyckman, A.J., Lonial, H., Zhang, R.,
Sack, J.S., Lin, S., et al., J. Med. Chem., 2008, vol. 51,
pp. 4–16.
21. Schmitz, W.D., Brenner, A.B., Bronson, J.J., Ditta, J.L.,
Li, Y., Lodge, N.J., Molski, T.F., Olson, R.E., Grifin, C.R.,
Zhuo, X., et al., Bioorg. Med. Chem. Lett., 2010, vol. 20,
pp. 3579–3583.
ACKNOWLEDGMENTS
The financial support of Taif University,
grant number 1ꢀ434ꢀ2245, and the help of
Prof. Dr. Mohamed I. Kobesy in performing the pharꢀ
macological investigations are highly acknowledged.
22. Rankovic, Z., Cai, J., Fradera, X., Dempster, M., Misꢀ
try, A., Long, C., Hamilton, E., King, A., Boucharens, S.,
Jamieson, C., et al., Bioorg. Med. Chem. Lett., 2010,
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23. Giordanetto, F., Karlsson, O., Lindberg, J., Larsson, L.O.,
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