Identification of 6-benzyloxysalicylates as a novel class of inhibitors of 15-lipoxygenase-1

Article abstract of DOI:10.1111/cbdd.12375

Lipoxygenases metabolize polyunsaturated fatty acids into signalling molecules such as leukotrienes and lipoxins. 15-lipoxygenase-1 (15-LOX-1) is an important mammalian lipoxygenase and plays a crucial regulatory role in several respiratory diseases such as asthma, COPD and chronic bronchitis. Novel potent and selective inhibitors of 15-LOX-1 are required to explore the role of this enzyme in drug discovery. In this study we describe structure activity relationships for 6-benzyloxysalicylates as inhibitors of human 15-LOX-1. Kinetic analysis suggests competitive inhibition and the binding model of these compounds can be rationalized using molecular modelling studies. The most potent derivative 37a shows a K_i value of 1.7 μM. These structure activity relationships provide a basis to design improved inhibitors and to explore 15-LOX-1 as a drug target.

Full text of DOI:10.1111/cbdd.12375

Received Date : 12-Mar-2014
Revised Date : 24-Apr-2014
Accepted Date : 17-May-2014
Article type : Research Article
Synthesis and Evaluation of a New Series of 3,5-bis ((5-bromo-6-methyl-2-t-
aminopyrimidin-4-yl)thio)-4H-1,2,4-triazol-4-amines and their Cyclized Products
“Pyrimidinylthio pyrimidotriazolothiadiazines” as 15- Lipoxygenase Inhibitors
Running tittle: 3,5-bis( substituted pyrimidinylthio) triazolamines
Keywords: 3,5-bis((5-bromo-6-methyl-2-t-aminopyrimidin-4-yl)thio)-4H-1,2,4-triazol-4-amine,
heterocyclization, synthesis, 15-LO, pyrimidinylthio, inhibitors, docking
Tayebe Asgharia, Mehdi Bakavolia*, Mohammad Rahimizadeha, Hossein Eshghia, Sattar Saberia
Azam Karimiana, Farzin Hadizadehb*, Moreteza Ghandadic
aDepartment of Chemistry, School of Sciences, Ferdowsi University of Mashhad, 91775-1436
Mashhad, Iran
bBiotechnology Research Center, School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran
cDepartment of biotechnology, School of Pharmacy, Mashhad University of Medical Sciences,
Mashhad, Iran
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process which may lead to
differences between this version and the Version of Record. Please cite this article as an
'Accepted Article', doi: 10.1111/cbdd.12375
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*Corresponding authors: Mehdi Bakavoli, School of Sciences, Ferdowsi University of Mashhad,
Iran; email: mbakavoli@yahoo.com Tel: + 98-511- 8797022; fax: + 98-511-8796416;
Farzin Hadizadeh, School of Pharmacy, Mashhad University of Medical Sciences, Mashhad,
Iran; email: hadizadehf@mums.ac.ir; Fax: +98-511-8823251
Abstract
A series of new 3,5-bis((5-bromo-6-methyl-2-t-aminopyrimidin-4-yl)thio)-4H-1,2,4-triazol-4-
amines and their cyclized products “pyrimidinylthio pyrimidotriazolothiadiazines” were
designed, synthesized, and evaluated as potential inhibitors of 15-lipoxygenase (15-LO). Their
syntheses started by initial condensation of 2:1 equivalents of pyrimidine with triazole and
subsequent nucleophilic displacement of the chlorine atoms with secondary amines and finally
cyclocondensation in the presence of NaNH2. The compounds 4d and 4f showed the best IC50 of
15-LO inhibition (IC50 = 9 and 12 µM, respectively). Compounds 4a-g were docked into 15-LO.
We suggest that the hydrogen bonds in quaternary nitrogen of piperazine ring of compounds 4d
and 4f appears to play major role in lipoxygenase inhibition by this set of synthesized analogs
and hydrophobic nature of this protein's binding site should be considered in ongoing
investigations.
Introduction
Triazoles and their derivatives play important roles in medicinal, agricultural and industrial fields
(1-3). These compounds are known to exhibit antibacterial and antifungal (4), analgesic (5),
anticancer and antimicrobial (6), anticonvulsant (7), anti-inflammatory properties (8). Some of
the modern day drugs with triazole nucleus are Ribavirin (antiviral agent), Alprazolam
(anxiolytic agent), Fluconazole, Itraconazole (antifungal agent) and Rizatriptan (antimigrane
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agent). The commonly known systems are triazoles fused with pyridines (9), pyridazines (10),
pyrazines (11), imidazoles (12) and triazines (13).
Triazolothiadiazines are a class of heterocyclic compounds that have been described as anti-
inflammatory (14), anticandidal (15), analgesic (16), antibacterial, anticancer (17) and antiviral
agents (18). Moreover Pyrimidine-fused heterocycles often appear as the core of biologically
active compounds (19-21) such as anticancer (22), antiviral (23), anti-inflammatory (24), RET
Kinase Inhibitors (25). A literature survey reveals that there are not many examples of
triazolethiadiazine fused with pyrimidine reported. Bside more recently, 15-lipoxygenase (15-
LO) has emerged as an attractive target for therapeutic intervention (26). 15-LO has been
implicated in the progression of certain cancers (27, 28) and chronic obstructive pulmonary
disease (COPD) (27). Evidence for the inhibition of 15-LO in the treatment of vascular disease
is, however, most compelling (29). Both transgenic and knockout studies implicate a role for 15-
LO in atherogenesis (30,31). The enzyme is abundantly expressed in macrophages residing
within the atherosclerotic lesion (26). Keeping this in mind, and in continuation of our recent
studies (32, 33) on the enzyme inhibitory activity of heterocyclic compound against soybean 15-
lipoxygenase, we considered the synthesis of new derivatives of 3,5-bis((5-bromo-6-methyl-2-t-
aminopyrimidin-4-yl)thio)-4H-1,2,4-triazol-4-amines and their cyclized products pyrimidinylthio
pyrimidotriazolothiadiazines.
Method and material
Chemistry
Melting points were taken on an Electrothermal type 9100 melting point apparatus and are
uncorrected. The IR spectra were obtained in KBr disks on an Avatar 370 FT-IR Thermo-Nicolet
1
spectrometer (νmax in cm−1). The H NMR spectra were recorded on a Bruker AC at 100 MHz,
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400 MHz using tetramethylsilane (TMS) as an internal reference. Chemical shift values were
given in ppm, at room temperature using CDCl3, DMSO-d6 or CD3COCD3 as a solvent; chemical
shifts were reported in parts per million (ppm) downfield from TMS; multiplicities were
abbreviated as: s: singlet; d: doublet; q: quartet; m: multiplet; br s: broad singlet. The Mass
spectra were obtained on a Varian Mat CH-7 and Agilent 5973 instruments at 70 eV. Elemental
analysis was performed on a Thermo Finnigan Flash EA microanalyzer.
3,5-bis((5-bromo-2-chloro-6-methylpyrimidin-4-yl)thio)-4H-1,2,4-triazol-4-amine (3):
To a solution of 4-amino-1,2,4-triazolidine-3,5-dithione 2 (10 mmol, 1.48) and Et3N (26 mmol,
3.2 ml) in (40 ml) DMF was added 5-Bromo-2,4-dichloro-6-methylpyrimidine 1 (20 mmol, 4.84
gr). The solution was stirred for 2h at room temperature and then was poured into water (150
mL). The resulting precipitate was filtered off and washed with water (2 × 50 mL) and diethyl
ether (20 mL), respectively. After drying the solid, 5.50 gr of 3 were obtained as a crude product.
1
Yield: 98%, m.p. 246-247ºC; H NMR (100 MHz, DMSO-d6): δ 2.56 (s, 6H, 2CH3), 6.12 ppm
(s, 2H, NH2, D2O exchangeable); IR: ν 3332, 3252, 2958, 1523, 755cm-1; MS (m/z) 556 (M+),
558 (M++2); Anal. Calcd. for C12H8Br2Cl2N8S2: C, 25.78; H, 1.44; N, 20.04; S, 11.47. Found: C,
25.97; H, 1.48; N, 20.35; S, 11.28.
The general procedure for preparation (4a–g) compounds
The appropriate secondary amine (30 mmol) was added to a stirred mixture of compound 3 (10
mmol, 5.59 g) in acetonitrile` (100 mL), and the solution was heated under reflux for 30-60 min.
Progress of the reaction was monitored by TLC using chloroform:methanol (30:1). The solid
obtained on cooling was filtered and recrystallized from ethanol.
3,5-bis((5-bromo-6-methyl-2-morpholinopyrimidin-4-yl)thio)-4H-1,2,4-triazol-4-amine(4a):
White powder; yield: 89%; m.p. 248-249 ºC; 1H NMR (100 MHz, CDCl3): δ 2.46 (s, 6H, 2CH3),
3.48-3.57 (m, 8H, 4CH2N), 3.57-3.62 (m, 8H, 4CH2O), 5.02 ppm (s, 2H, NH2, D2O
exchangeable); IR (KBr disc): ν 3328, 3271, 2973,2916,2847, 1543, 770cm-1.; MS (m/z) 658
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(M+), 660 (M++2); Anal. Calcd. for C20H24Br2N10O2S2: C, 36.37; H, 3.66; N, 21.21; S, 9.71.
Found: C, 36.55; H, 3.62; N, 21.51; S, 9.36%.
3,5-bis((5-bromo-6-methyl-2-(piperidin-1-yl)pyrimidin-4-yl)thio)-4H-1,2,4-triazol-4-amine
(4b):
1
White powder; yield: 91%; m.p. 242 ºC; H NMR (100 MHz, CDCl3): δ 1.20-1.82 (m, 12H,
6CH2), 2.40 (S, 6H, 2CH3), 3.21-3.60 (m, 8H, 4CH2N), 5.02 ppm (s, 2H, NH2, D2O
exchangeable); IR (KBr disc): ν 3338, 3260, 2931, 2851, 1555, 768cm-1.; MS (m/z) 654 (M+),
656 (M++2); Anal. Calcd. for C22H28Br2N10S2: C, 40.25; H, 4.30; N, 21.34; S, 9.77. Found: C,
40.48; H, 4.46; N, 21.18; S, 9.91%.
3,5-bis((5-bromo-6-methyl-2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)thio)-4H-1,2,4-triazol-
4-amine (4c):
1
White powder; yield: 89%; m.p. 179 ºC; H NMR (100 MHz, CDCl3): δ 0.91 (d, J = 6 Hz, 6H,
2CH3), 1.10-1.30 (m, 2H, 2CH) 1.36-1.89 (m, 8H, 4CH2), 2.41 (s, 6H, 2CH3), 2.51-2.92 (m, 4H,
equatorial hydrogens of 2CH2N), 3.92-4.41 (m, 4H, axial hydrogens of 2CH2N), 5.03 (s, 2H,
NH2, D2O exchangeable); IR (KBr disc): ν 3329, 3256, 2947, 2922 , 1547, 1515 , 772cm-1; MS
(m/z) 682 (M+), 684 (M++2); Anal. Calcd. for C24H32Br2N10S2: C, 42.11; H, 4.71; N, 20.46; S,
9.37. Found: C, 42.53; H, 4.78; N, 20.47; S, 9.51%.
3,5-bis((5-bromo-6-methyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)thio)-4H-1,2,4-triazol-
4-amine (4d):
1
White powder; yield: 88%; m.p. 267-268ºC; H NMR (400 MHz, CDCl3): δ 2.27 (s, 6H, 2CH3)
2.32 (app. t, 8H, 4CH2N), 2.38 (s, 6H, 2CH3), 3.46 (app. t, 8H, 4CH2N), 5.13 ppm (s, 2H, NH2,
D2O exchangeable); IR (KBr disc): ν 3354, 3268, 2966, 2937, 1547, 775cm-1.; MS (m/z) 684
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(M+), 686 (M+ + 2); Anal. Calcd. for C22H30Br2N12S2: C, 38.49; H, 4.40; N, 24.48; S, 9.34 Found:
C, 38.59; H, 4.31; N, 24.44; S, 9.05%.
3,5-bis((5-bromo-6-methyl-2-(4-phenylpiperazin-1-yl)pyrimidin-4-yl)thio)-4H-1,2,4-triazol-
4-amine (4e):
White powder; yield: 87%; m.p. 199-201 ºC; 1H NMR (100 MHz, CDCl3): δ 2.50 (s, 6H, 2CH3),
3.01 (app. t, 8H, 4CH2N), 3.60 (app. t, 8H, 4CH2N), 5.10 (s, 2H, NH2, D2O exchangeable), 6.75-
7.02 (m, 6H, aromatic), 7.12-7.41 ppm (m, 4H, aromatic); IR (KBr disc): ν 3333, 3252, 2953,
2908, 1598, 1548, 758cm-1.; MS (m/z) 808 (M+), 810 (M++2); Anal. Calcd. for C32H34Br2N12S2:
C, 47.41; H, 4.23; N, 20.73; S, 7.91. Found: C, 47.63; H, 4.34; N, 20.75; S, 7.77%.
3,5-bis((5-bromo-6-methyl-2-(4-ethylpiperazin-1-yl)pyrimidin-4-yl)thio)-4H-1,2,4-triazol-4-
amine (4f):
1
White powder; yield: 91%; m.p. 220 ºC; H NMR (100 MHz, CDCl3): δ 1.16 (t, J=7Hz, 6H,
2CH3), 2.44 (s, 6H, 2CH3), 2.50-2.57 (m, 12H, 4CH2N,2CH2), 3.56 (app. t, 8H, 4CH2N), 5.12
ppm (s, 2H, NH2, D2O exchangeable); IR (KBr disc): ν 3358, 3280, 2966, 2945 , 2810 , 1545,
767cm-1.; MS (m/z) 712 (M+), 714 (M++2); Anal. Calcd. for C24H34Br2N12S2: C, 40.34; H, 4.80;
N, 23.52; S, 8.97 Found: C, 40.73; H, 4.77; N, 23.10; S, 8.37%.
3,5-bis((5-bromo-6-methyl-2-(pyrrolidin-1-yl)pyrimidin-4-yl)thio)-4H-1,2,4-triazol-4-amine
(4g):
White powder; yield: 90%; m.p. 245-246 °C; 1H NMR (400 MHz, CDCl3): δ 1.75–1.86 (m, 8H,
4CH2), 2.47 (s, 6H, 2CH3), 3.00 (m, 4H, 2CH2N), 3.47 (m, 4H, 2CH2N), 5.20 (s, 2H, NH2, D2O
exchangeable); IR (KBr disc): ν 3330, 3268, 2966, 2870, 1557, 769cm−1; MS (m/z) 626 (M+),
628 (M++2). Anal. Calcd for C20H24Br2N10S2: C, 38.23; H, 3.85; N, 22.29; S, 10.21. Found: C,
38.18; H, 3.87; N, 21.97; S, 10.01%.
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The general procedure for preparation of (5a–g) compounds:
A mixture of each of compounds 4a-g (10 mmol), NaNH2 (30mmol) in dry acetonitrile (100 mL)
was heated under reflux for 15-60 min. Progress of the reaction was monitored by TLC using
chloroform:methanol (15:1) or ethylacetate:n-hexane (15:9). 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 recrystallized
from ethanol.
4-(5-bromo-4-methyl-6-((6-methyl-8-morpholino-5H-pyrimido[5,4-e][1,2,4]triazolo[3,4-
b][1,3,4]thiadiazin-3-yl)thio)pyrimidin-2-yl)morpholine (5a):
1
White powder; yield: 75%; m.p.> 300 ºC (dec.); H NMR (100 MHz, CD3COCD3); δ 2.20 (s,
3H, CH3), 2.40 (s, 3H, CH3) 3.35-3.50 (m, 8H, 4CH2N), 3.50-3.70 (m, 8H, 4CH2O), 7.85 ppm
(br s, 1H, NH, D2O exchangeable); IR (KBr disc): ν 3260, 3166, 2958, 1611cm-1 ; MS (m/z) 578
(M+), 580 (M++2) Anal. Calcd. for C20H23BrN10O2S2: C, 41.45; H, 4.00; N, 24.17; S, 11.07
Found: C, 41.54; H, 4.10; N, 23.93; S, 10.89%.
3-((5-bromo-6-methyl-2-(piperidin-1-yl)pyrimidin-4-yl)thio)-6-methyl-8-(piperidin-1-yl)-
5H-pyrimido[5,4-e][1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (5b):
1
White powder; yield: 71%; m.p. 270 ºC (dec.); H NMR (400 MHz, CDCl3): δ 1.30-1.70 (m,
12H, 6CH2), 2.32 (s, 3H, CH3), 2.43 (s, 3H, CH3), 3.38 (app. t, 4H, 2CH2N), 3.70 (t, J=5.2Hz,
4H, 2CH2N) 7.80 ppm (br s, 1H, NH, D2O exchangeable); IR (KBr disc): ν 3260, 3170, 2931,
1631cm-1.; MS (m/z) 574 (M+), 576 (M++2); Anal. Calcd. for: C22H27BrN10S2: C, 45.91; H, 4.73;
N, 24.34; S, 11.14. Found: C, 45.33; H, 4.81; N, 24.06; S, 10.88%.
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3-((5-bromo-6-methyl-2-(4-methylpiperidin-1-yl)pyrimidin-4-yl)thio)-6-methyl-8-(4-
methylpiperidin-1-yl)-5H-pyrimido[5,4-e][1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (5c):
1
White powder; yield: 74%; m.p. 220 ºC; H NMR (100 MHz, CDCl3): δ 0.80- 1.10 (m, 6H,
2CH3), 1.31-1.85 (m, 10H, 2CH and 4CH2), 2.30 (s, 3H, CH3), 2.42 (s, 3H, CH3), 2.50-2.90 (m,
4H, equatorial hydrogens of 2CH2N), 4.10-4.70 (m, 4H, axial hydrogens of 2CH2N), 7.80 ppm
(br. s, 1H, NH, D2O exchangeable); IR (KBr disc): ν 3280, 2946, 2923, 1635cm-1.; MS (m/z) 602
(M+), 604 (M++2); Anal. Calcd. for C24H31BrN10S2: C, 47.76; H, 5.18; N, 23.21; S, 10.62.
Found: C, 47.37; H, 5.09; N, 23.04; S, 10.36%.
3-((5-bromo-6-methyl-2-(4-methylpiperazin-1-yl)pyrimidin-4-yl)thio)-6-methyl-8-(4-
methylpiperazin-1-yl)-5H-pyrimido[5,4-e][1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (5d):
1
White powder; yield: 75%; m.p.> 300 ºC (dec.); H NMR (100 MHz, CDCl3); δ 2.20-280 (m,
20H, 4CH3, 4CH2N, ), 3.40-3.90 (m, 8H, 4CH2N), 8.12 ppm (br s, 1H, NH, D2O exchangeable);
IR (KBr disc): ν 3284, 2934, 1585 cm-1.; MS (m/z) 604 (M+), 606 (M++2) Anal. Calcd. for
C22H29BrN12S2: C, 43.63; H, 4.83; N, 27.76; S, 10.59. Found: C, 43.31; H, 4.93; N, 27.45; S,
10.61%.
3-((5-bromo-6-methyl-2-(4-phenylpiperazin-1-yl)pyrimidin-4-yl)thio)-6-methyl-8-(4-
phenylpiperazin-1-yl)-5H-pyrimido[5,4-e][1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (5e):
White powder; yield: 73%; m.p. 240 ºC (dec);1H NMR (100 MHz, CDCl3): δ 2.36 (s, 3H, CH3),
2.47 (s, 3H, CH3), 2.85-3.31 (m, 8H, 4CH2N), 3.45-4.01 (m, 8H, 4CH2N), 6.69-7.08 (m, 6H,
aromatic), 7.08-7.41(m, 4H, aromatic), 8.11 ppm (br s, 1H, NH, D2O exchangeable); IR (KBr
disc): ν 3245, 2953, 2913, 1598cm-1.; MS (m/z) 728 (M+), 730 (M++2); Anal. Calcd. for
C32H33BrN12S2: C, 52.67; H, 4.56; N, 23.03; S, 8.79. Found: C, 52.39; H, 4.91; N, 23.18; S,
8.43%.
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3-((5-bromo-2-(4-ethylpiperazin-1-yl)-6-methylpyrimidin-4-yl)thio)-8-(4-ethylpiperazin-1-
yl)-6-methyl-5H-pyrimido[5,4-e][1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (5f):
White powder; yield: 70%; m.p. 260 ºC (dec); 1H NMR (100 MHz, CDCl3): δ 0.92- 1.27 (m, 6H,
2CH3), 2.06-2.69 (m, 18H, 2CH3, 4CH2N, 2CH2), 3.29-3.98 (m, 8H, 4CH2N) 8.50 ppm (br. s,
1H, NH, D2O exchangeable); IR (KBr disc): ν 3248, 2966, 1642 cm-1.; MS (m/z) 632 (M+), 634
(M++2) Anal. Calcd. for C24H33BrN12S2: C, 45.49; H, 5.25; N, 26.53; S, 10.12. Found: C, 45.23;
H, 5.40; N, 26.66; S, 10.05%.
3-((5-bromo-6-methyl-2-(pyrrolidin-1-yl)pyrimidin-4-yl)thio)-6-methyl-8-(pyrrolidin-1-yl)-
5H-pyrimido[5,4-e][1,2,4]triazolo[3,4-b][1,3,4]thiadiazine (5g):
1
White powder; yield: 77%; m.p.> 300 °C (dec.); H NMR (100 MHz, CDCl3): δ 1.68–1.92 (m,
8H, 4CH2), 2.10 (s, 3H, CH3), 2.35 (s, 3H, CH3), 2.98–3.42 (m, 8H, 4CH2N), 8.12 (s, 1H, NH,
D2O exchangeable). IR (KBr disc): ν 3268, 3190, 2968, 1615cm−1; MS (m/z) 546 (M+), 548
(M++2); Anal. Calcd for C20H23BrN10S2: C, 43.87; H, 4.23; N, 25.58; S, 11.71. Found: C, 43.52;
H, 4.26; N, 25.60; S, 11.39%.
Biology
Linoleic acid and two assay solutions (A and B) were prepared in advance. Solution A was 50
mM 3-(dimethylamino) benzoic acid (DMAB) in a 100 mM phosphate buffer (pH 7.0). Solution
B was a mixture of 10 mM 3-methyl-2-benzothiazolinone (MBTH) 3 mL, hemoglobin (5
mg/mL, 3 mL) in 50 mM phosphate buffer at pH 5.0 (25 mL). A linoleic acid solution was
prepared by mixing 5 mg of linoleic acid with 50 mg Tween 20 in 3 mL water and then diluting
with KOH 100 mM to a final volume of 5 mL. In the standard assay, the sample in ethanol (25
mL), soybean 15-lipoxygenase (SLO) (4000 units/mL in 50 mM phosphate buffer pH 7.0; 25
mL) and phosphate buffer pH 7.0 (50 mM; 900 mL) were mixed in a test tube and preincubation
was carried out for 5 min at room temperature. A control test was done with the same volume of
ethanol. After the preincubation, linoleic acid solution (50 mL) was added to start the
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peroxidation reaction, and, 7 min later, solution A (270 mL) and then solution B (130 mL) was
added to start the color formation. Further 5 min later, 200 mL of a 2% SDS solution was added
to terminate the reaction. The absorbance at 598 nm was compared with control test. SLO and
other chemicals were purchased from Sigma, Aldrich and Merck Co. respectively.
Docking
The mode of interaction between synthesized ligands and lipoxygenase was investigated by
docking. 2D structure of chemicals was drowned in ChemDraw ultera 8.0 software and 3D
structures were prepared by Hyperchem 7 software (http://www.hyper.com/), using molecular
mechanic force filed pre-optimization followed by AM1 semiempirical calculation. The X-ray
crystal structure of lipoxygenase (PDB ID: 1IK3) was downloaded from the Protein Data Bank at
the Research Collaboratory for Structural Bioinformatics (www.rcsb.org). Further preparations
such as polar hydrogen addition and water molecules elimination were done by MOE software.
Synthesized chemicals were docked into the binding site of protein by MOE software. All atoms
within a 5 Å around the co-crystallized ligand in crystal coordinates of lipoxygenase was
selected as binding site. The docking simulations were performed using Triangle matcher
placement algorithm in combination with London dG scoring function and forcefield as
refinement method. For each compound, the top-score docking poses were selected for final
ligand–target interaction analysis using LigX module in MOE Software. Validation of docking
methodology was first evaluated by docking of co-crystalized ligand into the lipoxygenase
binding site.
Results and discussion
Chemistry
The starting material 5-bromo-2,4-dichloro-6-methyl-pyrimidine 1 was prepared according to
published method (34). Treatment of compound 1 with 4-amino-1,2,4-triazolidine-3,5-dithione 2
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at room temperature afforded intermediate 3 in which the C-4 chlorine atoms in the pyrimidine
rings were replaced selectively by two sulfhydryl groups of ditiol 2 .The foregoing compound 2
was prepared according to the published procedure (35). Subsequent reaction of compound 3
with various secondary amines led to the selective replacement of the C-2 chlorine atoms in the
pyrimidine rings to produce the corresponding diheteroaryl sulfide intermediates 4a-g. The latter
compounds subsequently underwent an intramolecular SNAr reaction in the presence of NaNH2
in boiling acetonitrile to give the desired tricyclic pyrimidinylthio pyrimido[5,4-e]triazolo[5,1-
b][1,3,4]thiadiazines 5a-g. (Scheme 1)
Our attempt for replacement of Br atom of second pyrimidine ring in different solvents and
temperature was failed. Structural preference of 5a-g may be attributed to ring strain of two fused
pyrimidothiadiazine rings with triazol. The yield of compounds 4a-g were increased between 30-
40% by changing ethanol to acetonitrile as a solvent. The structure of newly synthesized
1
compounds 5a-g was confirmed by recording their IR, H NMR, elemental analysis and mass
spectra. For example, the IR spectrum of compound 4b exhibited the stretching vibration bands
at 3338 and 3260cm-1 due to symetrical and asymetrical vibrations of NH2 group while the IR
spectrum of compound 5b showed a band at 3260cm-1 for NH vibration. The 1H NMR spectrum
of the asymetrical cyclized product 5b showed two singlet at δ 2.32 and 2.44 ppm in CDCl3
1
belonging to methyl groups of the pyrimidine moieties whereas, the H NMR spectrum of the
symmetrical precursor 4b showed a singlet at δ 2.46 ppm in CDCl3 for methyl groups of the
pyrimidine moieties. The 1H NMR spectrum of the precursor 4b showed the NH2 signal at δ 5.02
1
ppm in CD3Cl which was removed on adding D2O. However, the H NMR spectrum of the
cyclized product 5b did not show this signal and instead an exchangeable broad singlet peak at δ
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7.80 ppm confirming the occurrence of heterocyclisation to 5b. The mass spectrum of 5b showed
a molecular ion peak at m/z 574 (M+) corresponding to the molecular formula C22H27BrN10S2.
Biology
The compounds 5a-g and 4a-g were evaluated in vitro to determine their activity on 15-LO
inhibition, and their inhibitory potencies were compared to 4-methyl-2-(4-methyl
piperazinyl)pyrimido[4,5-b]benzothiazine (4-MMPB) with IC50 18µM (31). Compounds 4d and
4f were the most potent inhibitors of 15-LO at a concentration of 9 and 12 µM respectively. It
was interesting to see that pyrrolidine, morpholine, piperidine, methylpiperidine, and
phenylpiperazine analogs didn’t show inhibitory activity on 15-LO (IC50 > 500 μM) (Table 1).
Also Log Dose-response 15-LO activity curves for biologically active compounds 4d, 4f and
reference 4-MMPB has been shown in Figure 1.
Docking
In order to investigate the mode of interactions between the lipoxygenase and its inhibitors in a
3D fashion, the compounds were docked into the binding site of lipoxygenase. The validity and
quality of docking procedure was evaluated by docking of co-crystalized ligand into the binding
site of lipoxygenase. The top binding pose from docking studies shows a similar orientation in
the binding pocket to the co-crystallized ligand found in crystal structure (PDB ID 1IK3). The
root mean square deviation (RMSD) between ligand docked into the binding site and co
crystallized ligand in the crystal structure for lipoxygenase was 1.4 Å indicating good ability to
reproduce the ligand binding mode observed in the experimental data (Figure 2). Figure 3 is 3D
structure of docked chemicals in protein, in this picture red compound is co-crystalysed ligand
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and docked analogs represented as line structures colored by atoms. It indicates synthetic ligands
are in suitable position between active site of protein. As mentioned in Table 1 compounds 4d
and 4f derivatives demonstrated the most potent inhibitory effects. Investigating ligand
interaction mode of docked analogs by LigX module of MOE software revealed that quaternary
nitrogen of piperazine ring in these two compounds have made appropriate site for hydrogen
binding. As shown in figure 4,5 there is a hydrogen bond between quaternary nitrogen of
piperazine ring and ser-510 in active compounds 4d, 4f. Inactive compounds 4a, b, c, g do not
have piperazine ring and quaternary nitrogen and so they cannot have hydrogen bonding to ser -
510. In inactive compound 4e which has a piperazine ring, there is a bulk, electron withdrawing
N-phenyl substituent, bigger than N-methyl (4d) and N-ethyl (4f) , which prevents quaternization
of nitrogen of piperazine. So this compound also cannot have hydrogen bond with ser-510
(Figure 6,7). It should be mentioned that presence of hydrophobic amino acids in binding site of
lipoxygenase is remarkable (green area in Figure 2 and 3 and lots of hydrophobic amino acids
colored as green in Figure 4 and 6 demonstrate this point) and can be considered as opportunity
to develop lipoxygenase inhibitors.
In conclusion hydrogen bonds in quaternary nitrogen of piperazine ring of compounds 4d and 4f
appears to play major role in lipoxygenase inhibition by this set of synthesized analogs and
hydrophobic nature of this protein's binding site should be considered in ongoing investigation.
Conclusion
A series of 3,5-bis((5-bromo-6-methyl-2-t-aminopyrimidin-4-yl)thio)-4H-1,2,4-triazol-4-amines
and their cyclized products pyrimidinylthio pyrimidotriazolothiadiazines were synthesized and
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evaluated as potential 15-LO inhibitors. Among all these synthesized compounds only two
derivatives 4d and 4f with IC50 9 and 12 µM respectively showed strong potency for 15-LO
inhibition. The mode of interactions between the lipoxygenase and its inhibitors 4a-g in a 3D
fashion showed that hydrogen bonds in quaternary nitrogen of piperazine ring of compounds 4d
and 4f appears to play a major role in lipoxygenase inhibition.
Acknowledgement
We are grateful to department of chemistry, Ferdowsi University of Mashhad, Iran for financial
Support. (Grant No 25466/3)
Conflict of Interest
The authors confirm there is no conflict of interest.
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Figures and Table Legends
Table 1: IC50 values for the 4a-g and 5a-g compounds.
Scheme 1: Synthesis of tricyclic pyrimidinylthio pyrimido[5,4-e]triazolo[5,1-b][1,3,4]thiadiazines 5a-g.
Figure 1: Log Dose-response 15-LO activity curves for biologically active compounds 4d (panel
A), 4f (panel B) and reference 4-MMPB (panel C).
Figure 2: 3D representation of docked ligand (red color) into binding site of lipoxygenase using
MOE program as well as co-crystallized one (blue color) in the crystal structure of lipoxygenase
(PDB ID: 1IK3).
Figure 3: Map surface of docked analogs in active site of enzyme. Crystallized ligand is
indicated by red and stick lines. Green: hydrophobic; violet: H bonding; blue: Mild polar.
Figure 4: The 2D representation of the interaction between compound 4d (panel A) and
compound 4f (panel B) in the crystal structure of lipoxygenase (PDB ID: 1IK3) using LigX in
MOE.
Figure 5: The 3D position of docked compound 4d (panel A) and compound 4f (panel B) in the
active site of lipoxygenase (PDB ID: 1IK3), hydrogen bonds are in purple and all hydrogens
were removed for clarification, compounds represented as stick and amino acid residues as lines
colored by atoms.
Figure 6: The 2D representation of the interaction between compound 4b (panel A), 4a (panel
B) and 4e (panel C) and lipoxygenase.
Figure 7: The 3D position of docked compound 4b (panel A), compound 4a (panel B) and
4e(panel C) in the active site of lipoxygenase (PDB ID: 1IK3), hydrogen bonds are in purple and
all hydrogens were removed for clarification, compounds represented as stick and amino acid
residues as lines colored by atoms.
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IC50 (µM)
>500
>500
>500
>500
>500
>500
9
Entry
4a
5a
4b
5b
4c
5c
4d
5d
4e
>500
>500
>500
12
N
N
5e
4f
5f
>500
>500
>500
4g
5g
Table 1: IC50 values for the 4a-g and 5a-g compounds
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.