Synthesis of new derivatives of pyrimido[5,4-e][1,2,4]triazolo[3,4-b] [1,3,4]thiadiazine and their enzyme inhibitory activity assessment on soybean 15-lipoxygenase

Article abstract of DOI:10.3184/174751912X13549029453945

The synthesis of new derivatives of pyrimido[5,4-e][1,2,4]triazolo[3,4-b] [1,3,4]thiadiazine is described. These derivatives have a wide range of medicinal applications. Their inhibitory activity against the enzyme 15-lipoxygenase was also investigated.

Full text of DOI:10.3184/174751912X13549029453945

48 RESEARCH PAPER
JANUARY, 48–50
JOURNAL OF CHEMICAL RESEARCH 2013
Synthesis of new derivatives of pyrimido[5,4-e][1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazine and their enzyme inhibitory activity assessment on
soybean 15-lipoxygenase
Mehdi Bakavolia*, Seyed Mohammad Seyedia, Ali Shiria, Sattar Saberia, Mahmoud Gholamia
and Hamid Sadeghianb
aDepartment of Chemistry, School of Sciences, Ferdowsi University of Mashhad, 91775-1436 Mashhad, Iran
bDepartment of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, 91857-63788 Mashhad, Iran
The synthesis of new derivatives of pyrimido[5,4-e][1,2,4]triazolo[3,4-b][1,3,4]thiadiazine is described. These deriva-
tives have a wide range of medicinal applications. Their inhibitory activity against the enzyme 15-lipoxygenase was
also investigated.
Keywords: pyrimidotriazolothiadiazines, triazoles, annulated pyrimidines, 15-lipoxygenase inhibitor, heterocyclisation
Pyrimidine derivatives and heterocyclic annulated pyrimidines
continue to attract great interest because of their wide variety
of interesting biological activities, such as anticancer,1
antiviral,2 antitumor,3 and anti-inflammatory activities.4
Moreover, triazoles and especially fused triazoles are also an
important class of heterocyclic compounds with antifungal,5
bactericidal,5,6 anxiolytic,7,8 anticonvulsant9 and antidepressant
activities.10
Numerous methods for the synthesis of 1,2,4-triazoles have
been reported, which includes utilising toxic reagents such
as phosphorus oxychloride,11 lead tetraacetate,11,12 and bro-
mine12,13 as well as other oxidative reagents such as chlora-
mine-T,14 iodobenzene diacetate,15,16 iron(III) chloride,17 and
CuCl2.18 The synthesis of 1,2,4-triazoles by an electrochemical
method19 has also been reported. Some triazolothiadiazines
have also been reported which possess a broad spectrum of
biological activities.20–22 Keeping this in mind, and due to our
recent studies on the enzyme inhibitory activity of pyrim-
ido[5,4-e][1,2,4]triazolo[3,4-b][1,3,4]thiadiazines against 15-
lipoxygenase (15-LO), (a main group of the non-haeme,
iron-containing proteins which can catalyse hydroperoxida-
tion of polyunsaturated fatty molecules containing a cis,cis-
1,4-pentadiene structure such as arachidonic and linoleic
acid23), we considered the synthesis of pyrimidotriazolothia-
diazine compounds wherein the biologically active pyrimidine
moiety is fused to a potent triazolo[3,4-b][1,3,4]thiadiazine
ring across the 6,7-positions.
SNAr reaction in the presence of NaNH2 in boiling acetonitrile
to give the desired tricyclic 3,6-dimethyl-5H-pyrimido[5,4-e]
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines 5a–f. (Scheme 1)
The structural assignment of compounds 5a–f is based upon
1
spectroscopic and microanalytical data. For example, the H
NMR spectrum of 5d showed two singlet peaks at δ 2.44 and
2.50 ppm belonging to methyl groups of the triazole and
pyrimidine moieties, respectively. The multiplet signals in the
range of δ 3.44–3.63 ppm corresponded to the morpholine ring
proton signals. The spectrum of the precursor 4d showed the
NH2 signals at δ 4.92 ppm which was removed on adding D2O.
However, the 1H NMR spectrum of the cyclised product 5d did
not show this signal and instead an exchangeable broad singlet
peak at δ 7.72 ppm confirmed that heterocylisation to 5d had
occurred. The IR spectrum was devoid of the NH2 absorption
bands at ν 3313 and 3137 cm−1 of the precursor, but an absorp-
tion band at ν 3324 cm−1 demonstrated the existence of the NH
group in product 5d. The mass spectrum of 5d showed a
molecular ion signal at m/z 304 (M+) corresponding to the
molecular formula C12H14N7OS.
The inhibitory property of compounds 4a–f and 5a–f on
15-LO was assessed according to our previously reported pro-
cedure.23 The compounds showed very low inhibitory activity.
However, the sulfide 4c among others showed the best inhibi-
tory activity (IC50 = 499 µM). By comparing the results of
enzyme inhibitory activity of pyrimido[5,4-e][1,2,4]triazolo
[3,4-b][1,3,4]thiadiazine with the results of our previous work
on pyrimido[4,5-b][1,4]benzothiazines, we suggest that the
triazolo moiety by being an electron deficient ring, prohibits
the facile oxidation of the sulfur atom which is crucial for the
enzyme inhibitory activity. Work is currently in progress in our
laboratory with electron rich heterocycles to establish the
validity of this proposal.
We now describe the synthesis of some new derivatives of
tricyclic 3,6-dimethyl-5H-pyrimido[5,4-e][1,2,4]triazolo[3,4-b]
[1,3,4] thiadiazines 5a–f and their enzyme inhibitory activity
towards 15-LO.
Results and discussion
5-Bromo-2,4-dichloro-6-methylpyrimidine 1 was prepared
according to our previously published method.24 Treatment of
compound 1 with 1-amino-2-mercapto-5-methyl-1,2,4-triazole
2 which was prepared from the reaction of hydrazine hydrate
with CS2 followed by reaction with acetic acid according to the
published procedure25 afforded the intermediate 3. The facility
with which substitution of the C-4 chlorine atom in compound
1 occurs by nucleophilic attack of the sulfur function in the
triazole 2 had been established previously using similar condi-
tions.24 Subsequent reaction of compound 3 with various
secondary amines led to the selective replacement of the
chlorine atom at the 2-position of the pyrimidine ring and gave
the corresponding diheteroaryl sulfide intermediates 4a–f. The
latter compounds subsequently underwent an intramolecular
In summary, an interesting fused ring system containing the
pyrimido[5,4-e][1,2,4]triazolo[3,4-b][1,3,4]thiadiazine residue
was synthesised through the treatment of 5-bromo-2,4-dichloro-
6-methylpyrimidine 1 with 1-amino-2-mercapto-5-methyl-
1,2,4-triazole 2 which was subsequently reacted with secondary
amines and cyclised in presence of NaNH2 in boiling CH3CN.
The inhibitory activity of compounds 4a–f and 5a–f on 15-LO
has been assessed.
Experimental
Melting points were recorded on an Electrothermal type 9100 melting
point apparatus and are uncorrected. The IR spectra were obtained
on an Avatar 370 FT-IR Thermo-Nicolet spectrometer. The 1H NMR
(100 MHz) spectra were recorded on a Bruker AC 100 spectrometer.
The mass spectra were scanned on a Varian Mat CH-7 at 70 eV.
Elemental analysis was performed on a Thermo Finnigan Flash EA
microanalyser.
* Correspondent. E-mail: mbakavoli@um.ac.ir; mbakavoli@yahoo.com

JOURNAL OF CHEMICAL RESEARCH 2013 49
Scheme 1
3-[(5-Bromo-2-chloro-6-methylpyrimidin-4yl)thio]-5-methyl-4H-
1,2,4-triazol-4-amine (3): To a stirred solution of 5-bromo-2,4-
dichloro-6-methylpyrimidine 1 (2.44 g, 10 mmol) and Et3N (1.6 mL,
13 mmol) in CH3CN (25 mL), a solution of 1-amino-2-mercapto-5-
methyl-1,3,4-triazole 2 (1.31 g, 10 mmol) in CH3CN (30 mL) was
added dropwise over 30 min. The solution was stirred vigorously until
the white precipitate is appeared. Stirring was then continued at room
temperature for an extra 30 minutes and the resulting solid was
filtered and washed with warm water. Yield 95%, m.p. 207–208 °C,
1H NMR (DMSO-d6) δ 2.47(s, 3H, CH3), 2.62 (s, 3H, CH3), 5.73 (br s,
2H, NH2, D2O exchangeable); IR (KBr disc) ν 3247, 3149, 2998,
1524, 750 cm−1. MS (m/z) 334 (M+), 336 (M+ + 2). Anal. Calcd for
C8H8BrClN6S: C, 28.63; H, 2.40; N, 25.04; S, 9.55. Found: C, 28.53;
H, 2.37; N, 24.94; S, 9.46%.
2H, equatorial hydrogens of CH2N), 3.12–3.25 (m, 2H, axial hydro-
gens of CH2N), 4.89 (s, 2H, NH2, D2O exchangeable); IR (KBr disc)
ν 3299, 3196, 2990, 1637, 772 cm−1. MS (m/z) 397 (M+), 399 (M+ + 2).
Anal. Calcd for C14H20BrN7S: C, 42.21; H, 5.06; N, 24.61; S, 8.05.
Found: C, 42.19; H, 5.01; N, 24.57; S, 7.98%.
3-[(5-Bromo-6-methyl-2-morpholinopyrimidin-4-yl)thio]-5-methyl-
4H-1,2,4-triazol-4-amine (4d):Yield 80%, m.p. 232–234 °C, 1H NMR
(CDCl3) δ 2.44 (s, 3H, CH3), 2.50 (s, 3H, CH3), 3.44–3.49 (m, 4H,
CH2N), 3.58–3.63 (m, 4H, CH2O), 4.92 (br s, 2H, NH2, D2O exchange-
able); IR (KBr disc) ν 3313, 3137, 2986, 1597, 771 cm−1. MS (m/z)
385 (M+), 387 (M+ + 2). Anal. Calcd for C12H16BrN7OS: C, 37.31;
H, 4.18; N, 25.38; S, 8.30. Found: C, 37.27; H, 4.16; N, 25.35; S,
8.27%.
3-[(5-Bromo-6-methyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)thio]-
5-methyl-4H-1,2,4-triazol-4-amine (4e): Yield 80%, m.p. 205–207 °C,
1H NMR (CDCl3) δ 2.21 (s, 3H, CH3), 2.31 (br. t, 4H, 2CH2N), 2.42 (s,
3H, CH3), 2.59 (s, 3H, CH3), 3.51 (br. t, 4H, 2CH2N), 4.82 (s, 2H,
NH2, D2O exchangeable) IR (KBr disc) ν 3276, 3186, 2969, 1548, 771
cm−1. MS (m/z) 398 (M+), 400 (M+ + 2). Anal. Calcd for C13H19BrN8S:
C, 39.10; H, 4.80; N, 28.06; S, 8.03. Found: C, 39.05; H, 4.77; N,
28.01; S, 7.98%.
Synthesis of compounds (4a–f); general procedure
The appropriate secondary amine (12 mmol) was added to a stirred
mixture of compound 3 (3.35 g, 10 mmol) in ethanol (30 mL), and the
solution was heated under reflux for 6 h. After cooling the solution,
water (20 mL) was added and the resulting solid was filtered and
recrystallised in ethanol.
3-[(5-Bromo-6-methyl-2-(pyrrolidin-1-yl)pyrimidin-4-yl)thio]-5-
methyl-4H-1,2,4-triazol-4-amine (4a):Yield 78%, m.p. = 221–222 °C,
1H NMR (CDCl3) δ 1.88 (m, 4H, 2CH2), 2.43 (s, 3H, CH3), 2.52 (s,
3H, CH3), 3.11–3.32 (m, 4H, 2CH2–N), 5.02 (s, 2H, NH2, D2O
exchangeable). IR (KBr disc) ν 3284, 3170, 2970, 1564, 770 cm−1; MS
(m/z) 369 (M+), 371 (M+ + 2). Anal. Calcd for C12H16BrN7S: C, 38.93;
H, 4.36; N, 26.48; S, 8.66. Found: C, 38.88; H, 4.31; N, 26.42; S,
8.60%.
3-[(5-Bromo-6-methyl-2-(piperidin-1-yl)pyrimidin-4-yl)thio]-5-
methyl-4H-1,2,4-triazol-4-amine (4b): Yield 80%, m.p. 208–210 °C,
1H NMR (CDCl3) δ 1.51–1.67 (m, 6H, 3CH2), 2.41 (S, 3H, CH3), 2.5
(s, 3H, CH3), 3.41–3.58 (m, 4H, CH2N), 4.89 (s, 2H, NH2, D2O
exchangeable); IR (KBr disc) ν 3327, 3133, 2943, 1556, 770 cm−1. MS
(m/z) 383 (M+), 385 (M+ + 2). Anal. Calcd for C13H18BrN7S: C, 40.63;
H, 4.72; N, 25.51; S, 8.34. Found: C, 40.58; H, 4.70; N, 25.47; S,
8.29%.
3-[(5-Bromo-6-methyl-2-(4-phenylpiperazin-1-yl)pyrimidin-4-yl)thio]-
5-methyl-4H-1,2,4-triazol-4-amine (4f):Yield 75%, m.p. 280–282 °C,
1H NMR (CDCl3) δ 2.45 (s, 3H, CH3), 2.54 (s, 3H, CH3), 3.10 (br. t,
4H, 2CH2N), 3.59 (br. t, 4H, 2CH2N), 4.85 (s, 2H, NH2, D2O exchange-
able), 6.84–6.95 (m, 3H, aromatic), 7.26–7.31 (m, 2H, aromatic). IR
(KBr disc) ν 3276, 3219, 2949, 1583, 1546, 1503, 1445, 797 cm−1. MS
(m/z) 460 (M+), 462 (M+ + 2). Anal. Calcd for C18H21BrN8S: C, 46.86;
H, 4.59; N, 24.29; S, 6.95. Found: C, 46.83; H, 4.55; N, 24.24; S,
6.91%.
Synthesis of compounds (5a–f); general procedure
A mixture of each of compounds (4a–f) (10 mmol), NaNH2 (30 mmol)
in dry acetonitrile (50 mL) was heated under reflux for about 5h the
progress of the reaction was monitored by TLC using chloroform:
methanol (9:1). The mixture was cooled and the solvent was removed
under reduced pressure. Then, a solution of acetic acid (1 mL) in water
(20 mL) was added to the residue and the resulting precipitant was
filtered off and recrystallised from ethanol.
3-[(5-Bromo-6-methyl-2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)thio]-
5-methyl-4H-1,2,4-triazol-4-amine (4c): Yield 78%, m.p. 150–152 °C,
1H NMR (CDCl3) δ 0.95 (d, J = 9 Hz, 3H, CH3), 1.45–1.71 (m, 5H,
CH and 2CH2), 2.38 (s, 3H, CH3), 2.47 (s, 3H, CH3), 2.51–2.92 (m,
3,6-Dimethyl-8-(pyrrolidin-1-yl)-5H-pyrimido[5,4-e][1,2,4]-
triazolo[3,4-b][1,3,4]thiadiazine (5a): Yield 60%, m.p. 280–282 °C,

50 JOURNAL OF CHEMICAL RESEARCH 2013
1H NMR (CDCl3) δ 1.81–1.95 (m, 4H, 2CH2), 2.39 (s, 3H, CH3), 2.46
(s, 3H, CH3), 3.35 (t, 4H, 2CH2N), 7.22 (s, 1H, NH, D2O exchange-
able). IR (KBr disc) ν 3268, 3080, 2989, 1617 cm−1. MS (m/z) 289
(M+). Anal. Calcd for C12H15N7S: C, 49.81; H, 5.23; N, 33.88; S, 11.08.
Found: C, 49.75; H, 5.20; N, 33.85; S, 11.01%.
3,6-Dimethyl-8-(piperidin-1-yl)-5H-pyrimido[5,4-e][1,2,4]-
triazolo[3,4-b][1,3,4]thiadiazine (5b): Yield 65%, m.p. 235–237 °C,
1H NMR (CDCl3) δ 2.42–2.61 (m, 6H, 3CH2), 2.33 (s, 3H, CH3),
2.39 (s, 3H, CH3), 3.48–3.54 (m, 4H, CH2N), 7.63 (s, 1H, NH, D2O
exchangeable). IR (KBr disc) ν 3259, 2932, 1581 cm−1. MS (m/z) 303
(M+). Anal. Calcd for C13H17N7S: C, 51.47; H, 5.65; N, 32.32; S, 10.57.
Found: C, 51.43; H, 5.63; N, 32.29; S, 10.55%.
3,6-Dimethyl-8-(4-methylpiperidin-1-yl)-5H-pyrimido[5,4-e][1,2,4]-
triazolo[3,4-b][1,3,4]thiadiazine (5c): Yield 57%, m.p. 170–171 °C,
1H NMR (CDCl3) δ 0.93 (d, J = 8 Hz, 3H, CH3), 1.47–1.71 (m, 5H,
CH and 2CH2), 2.33 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.55–2.91 (m,
2H, equatorial hydrogens of CH2N), 3.23–3.41 (m, 2H, axial hydro-
gens of CH2N), 7.96 (br s, 1H, NH, D2O exchangeable); IR (KBr disc)
3335, 2949, 1579 cm−1. MS (m/z) 317 (M+).Anal. Calcd for C14H19N7S:
C, 52.98; H, 6.03; N, 30.89; S, 10.10. Found: C, 52.95; H, 6.01; N,
30.84; S, 10.04%.
Received 11 July 2012; accepted 3 December 2012
Paper 1201407 doi: 10.3184/174751912X13549029453945
Published online: 15 January 2013
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thiadiazin-8-yl)morpholine (5d): Yield 70%, m.p. 220–222 °C, H
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