Synthesis of novel 7-fluoro-3-substituted-1,2,4-triazolo[3,4-b]benzothiazoles (FTBs) as potent antifungal agents: molecular docking and in silico evaluation

Article abstract of DOI:10.1007/s11164-016-2599-3

A novel series of fluorinated 1,2,4-triazolo[3,4-b]benzothiazoles was synthesized by fusion of proven antifungal lead 1,2,4-triazole with flourinated benzothiazole nucleus exploiting lead hybridization strategy. Their in vitro antifungal assay against various phytopathogenic fungi revealed that a 2- or 3-chlorinated aryl group at the 3-position of the fused system yielded outstanding and remarkable results of fungitoxicity. Compounds 3b and 3c were found to inflict the best fungitoxicity against most of the test fungi (EC₅₀ value as low as 0.24 mmoles/L) with results better than or comparable to standards. In silico molecular docking and Lipinskii indices were in agreement with the observed trend of antifungal activity. Moreover, the toxicity analysis showed that the compounds belong to class III of toxicity which is the same as that of the recommended standards used.

Full text of DOI:10.1007/s11164-016-2599-3

Res Chem Intermed
DOI 10.1007/s11164-016-2599-3
Synthesis of novel 7-fluoro-3-substituted-1,2,4-
triazolo[3,4-b]benzothiazoles (FTBs) as potent
antifungal agents: molecular docking and in silico
evaluation
Sharu Kukreja1 Anjali Sidhu1 Vineet K. Sharma2
•
•
Received: 28 December 2015 / Accepted: 29 May 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract A novel series of fluorinated 1,2,4-triazolo[3,4-b]benzothiazoles was
synthesized by fusion of proven antifungal lead 1,2,4-triazole with flourinated
benzothiazole nucleus exploiting lead hybridization strategy. Their in vitro anti-
fungal assay against various phytopathogenic fungi revealed that a 2- or 3-chlori-
nated aryl group at the 3-position of the fused system yielded outstanding and
remarkable results of fungitoxicity. Compounds 3b and 3c were found to inflict the
best fungitoxicity against most of the test fungi (EC50 value as low as 0.24 mmoles/
L) with results better than or comparable to standards. In silico molecular docking
and Lipinskii indices were in agreement with the observed trend of antifungal
activity. Moreover, the toxicity analysis showed that the compounds belong to class
III of toxicity which is the same as that of the recommended standards used.
Keywords 7-Fluoro-3-substituted-1,2,4-triazolo[3,4-b]benzothiazoles Á Toxtree
analysis Á Molecular docking Á Lipinski filtration Á Antifungal evaluation
Introduction
Lead hybridization-based synthesis [1] of combinational heterocycles is a matter of
immense interest and an important source for the discovery of new bioactive
molecules which are likely to have diverse and augmented biological activities. The
novel molecules thus formed inflict multisite modes of action, lower doses of usage,
effects of the same molecules at different stages of growth, high bioavailability and
low or zero resistance.
&
Anjali Sidhu
anjalisidhu@pau.edu
1
2
Department of Chemistry, Punjab Agricultural University, Ludhiana, Punjab, India
Department of Plant Pathology, Punjab Agricultural University, Ludhiana, Punjab, India
123

S. Kukreja et al.
Triazole and their analogues, considered as bioisosteres of imadazole [2], belongs
to the most rapidly expanding group as fungicides [3–8]. They have well-known
inhibitiory effects on the cytochrome P450-dependent lanosterol 14a-demethylase
(CYP51) which affects the sterol biosynthesis of fungal cell membranes [8]. They
have additional advantages of low toxicity [9], high bioavailability [10] and a broad
spectrum of activity [11] against several fungi. So far, more than 20 triazole
fungicides have been commercialized in the agricultural field. On the other hand,
1,3-benzothiazoles have already been extensively reported for their antifungal
[12, 13], antibacterial [14], antiparasitic [15], antimicrobial [16], anticancer [17],
antitubercular [18], analgesic [19], antitumor [20], antihelmintic [21], anti-
inflammatory [22], and anti-oxidant [23] activities, and are also part of commer-
cially available fungicides [24], herbicides [25], plant desiccants and defoliant [26]
compounds. The combination of 1,2,4-triazole and benzothiazol have shown
synergestic effects in combination with each other [1]. So, there is overwhelming
attention on their mutual derivatization with each other for synergistic and
augmented fungi-toxicological profiles of the molecules.
Most ofthe organic moleculeshavefailed to reach commercialization due to lowtissue
uptake and, thus, low bioavalability [27–29]. The presence of fluorine in the molecules is
known to induce improved pharmokinetic profiles of the bioactive molecule,s improving
lipophilicity and suppressing the metabolic detoxification by increasing the in vivo
lifetime of the molecules and, thus, increasing the tissue bioavailability. So, the share of
commercial fluorinated compounds is on the increase nowadays.
The vitality of pharmokinetic profiles and biofunctionality of 1,2,4-triazoles and
1,3-benzothiazoles along with the importance of the introduction of fluorine in
biologically active compounds, impelled us to take up the synthesis of series of
novel 7-fluoro-3-substituted-1,2,4-triazolo[3,4-b]benzothiazoles (FTBs) to check
the effect of fusion of these leads on the antifungal potential of the compounds. Due
to the development of resistance to 1,2,4-triazole fungicides (reduced risk
fungicides), various phytopathogenic fungi have become the optimal candidates
for the potential evaluation of 1,2,4-triazolo analogues as resistance measures. In
continuity with our work, with remarkable results of the combination of 1,2,4-
triazole and 1,3-benzothiazole with spacers between them [1], this paper reports the
synthesis of novel 7-fluoro-3-substituted-1,2,4-triazolo[3,4-b]benzothiazoles having
these moieties in the form of fused heterocycles and their antifungal evaluation
against various phytopathogenic fungi. In silico Toxtree analysis, docking studies in
the active site of lanosterol 14a-demethylase [30] and Lipinski characters [31] have
been further performed to rationalize the trends of the observed bioactivity.
Experimental
General information
All chemicals were of analytical grade and used directly. Melting points were
determined in scientific melting point apparatus and were uncorrected. The purity of
the compounds were checked by TLC using Merck silica gel 60 F254 and visualized
123

Synthesis of novel 7-fluoro-3-substituted-1,2,4-triazolo[3…
by exposure to iodine vapors or UV light. IR spectra were recorded on a Perkin
Elmer RX1 FTIR spectrophotometer, using potassium bromide pellets, and the
frequencies are expressed in cm-1. The 1H NMR and 13C NMR spectra were
recorded with a Bruker Avance II 400 NMR spectrometer, using tetramethylsilane
as the internal reference, with CDCl3 and DMSO-d6 as solvents. The chemical shifts
are reported in parts per million (d ppm). Elemental analyses were performed on a
Perkin Elmer 2400 Series II CHNS/O elemental analyzer. The mass spectra were
recorded on Perkin Elmer Clarus 500 Mass Spectrometer. All spectral data were
consistent with the proposed structure.
General procedure for the synthesis of 6-flourbenzothiazol-2-amines (1)
4-fluoroaniline (0.1 mol) and potassium thiocyanate (0.4 mol) were dissolved in
glacial acetic acid (40 ml) and cooled. Bromine (0.04 mol) mixed with glacial
acetic acid (24 ml) was added from a dropping funnel at such a rate that temperature
did not rise above 5–6 °C. After all the bromine had been added, the solution was
stirred with a glass rod for an additional 2 h at low temperature. The mixture was
allowed to stand overnight, during which period orange–yellow precipitates settled
at the bottom. Water was added and the slurry formed was heated at 85 °C on a
steam bath for 20 min and filtered hot. The filtrate was cooled and neutralized with
ammonia solution to pH 6. Off-white precipitates obtained were collected, washed
with water and recrystallized from benzene to get pure crystals. IR (KBr) mmax: 3452
(N–H str), 3080 and 813 (C–H aromatic str), 3010 (=C–H str), 1680 (C=C str), 1350
1
(C=N str). The HNMR (DMSO-d6) spectrum of this product showed signals: d
6.5–7.4 (3H, m, Ar C–H) and at 5.4 (2H, br, NH2) ppm. The peaks in its 13CNMR
(DMSO-d6) d: 108.0, 113.9, 117.8, 131.6, 148.8, 158.5 and 166.3 ppm.
Synthesis of 6-flourohydrazinylbenzothiazol (2)
Concentrated hydrochloric acid (0.067 mol) was added dropwise with stirring to
hydrazine hydrate (0.12 mol) at 5–6 °C followed by ethylene glycol (30 mL);
thereafter, 6-fluorobenzo[d]thiazol-2-amine (1) (20 mmol) was added in portions
and the resultant mixture was refluxed for 2–3 h and cooled at room temperature.
The reaction progress was monitored by TLC using toluene:ethylacetate (75:25) as
mobile phase. The reaction mixture was filtered and the resulting precipitates were
washed with distilled water. The resulting crude was recrystallized from ethanol. IR
(KBr) mmax: 3010 (=C–H str), 1645 (C=C str), 1398 (C=N str). The 1HNMR
(DMSO-d6) spectrum of this product showed signals: d 7.2–7.4 (3H, m, Ar C–H), at
4.0 (1H, br, NH) and 4.75 (2H, br, NH2) ppm. The peaks in its 13CNMR (DMSO-d6)
d: 106.1, 112.4, 115.5, 129.7, 145.5, 157.5, 171.2 ppm.
General procedure for the synthesis of 7-flouro-3-phenyl-1,2,4-triazolo[3,4-
b]benzothiazoles (3a–3t)
The mixture of 6-fluoro-2-hydrazinylbenzo[d]thiazole (2) (0.01 mol) and benzalde-
hyde/substituted benzaldehyde (0.01 mol) was refluxed in ethanol (15 ml) at
123

S. Kukreja et al.
70–80 °C for 3 h. The separated product obtained was filtered off, washed with
distilled water and recrystallized from methanol to give the corresponding
hydrazone. The product obtained was further dissolved in acetic acid (20 ml) at
room temperature followed by the addition of sodium acetate (0.5 g). Bromine
(2 mmol) in acetic acid (10 ml) was added dropwise to the refluxing reaction
mixture. After 1 h, the mixture was poured onto crushed ice (100 g). The precipitate
obtained was filtered off and crystallized from ethanol-dimethylformamide (1:1) to
give crystals of (3a–3t).
7-flouro-3-phenyl-1,2,4-triazolo[3,4-b]benzothiazole (3a) IR (KBr) mmax: 1600
([C=N str), 1522 (C=C str), 1245 (C–F str) and 852 (C–S str) cm-1. The
1HNMR (CDCl3) spectrum of this product showed signals: d 7.0–7.6 (3H, m, fused
benzothiazole ring) and 7.7–8.0 (5H, m, phenyl ring) ppm. The peaks in its
13CNMR (DMSO-d6) d: 106.1, 112.4, 115.5, 129.7, 145.5, 157.5, 171.2 ppm.
3-(20Chlorophenyl)-7-flouro-1,2,4-triazolo[3,4-b]benzothiazole (3b) IR (KBr) mmax
:
2853 (C–H str), 1610 ([C=N str), 1531(C=C str), 1356 (C–F str), 825 (C–Cl str) and
714 (C–S str) cm-1. The 1HNMR (CDCl3) spectrum of this product showed signals:
d 7.9–8.3 (3H, m, fused benzothiazole ring) and 7.3–7.5 (4H, m, phenyl ring) ppm.
The peaks in its 13CNMR (DMSO-d6) d: 107.8, 114.4, 123.4, 127.5, 128.0, 129.4,
130.4, 131.7, 132.3, 136.7, 138.1, 151.2, 158.6, 159.9.
3-(30-Chlorophenyl)-7-flouro-1,2,4-triazolo[3,4-b]benzothiazole (3c) IR (KBr) mmax
:
2853 (C–H str), 1623 ([C=N str), 1531 (C=C str), 1398 (C–F str), 755 (C–Cl str)
1
and 601 (C–S str) cm-1. The HNMR (CDCl3) spectrum of this product showed
signals: d 7.2–8.1 (3H, m, fused benzo ring) and 7.4–8.1 (4H, m, chlorophenyl ring)
ppm. The peaks in its 13CNMR (DMSO-d6) d: 106.4, 113.4, 124.3, 127.5, 128.0,
129.4, 130.4, 131.5, 133.3, 137.7, 139.1, 153.2, 159.3, 161.2.
3-(40-Chlorophenyl)-7-flouro-1,2,4-triazolo[3,4-b]benzothiazole (3d) IR (KBr) mmax
:
2956 (C–H str), 1595 ([C=N str), 1563 (C=C str), 1265 (C–F str), 985 (C–Cl str)
1
and 814 (C–S str) cm-1. The HNMR (CDCl3) spectrum of this product showed
signals: d 7.2–7.5 (3H, m, fused benzo ring) and 7.9–8.4 (4H, m, chlorophenyl ring)
ppm. The peaks in its 13CNMR (DMSO-d6) d: 105.2, 116.3, 125.1, 127.8, 129.5,
129.9, 130.8, 132.6, 134.1, 136.3, 140.1, 151.3, 156.6, 157.3.
7-Flouro-3-(40-flourophenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3e) IR (KBr) mmax
:
2893 (C–H str), 1600 ([C=N str), 1516 (C=C str), 1322 (C–F str) and 714 (C–S str)
1
cm-1. The HNMR (CDCl3) spectrum of this product showed signals: d 7.6–7.9
(3H, m, fused benzo ring) and 7.1–7.4 (4H, m, fluorophenyl ring) ppm. The peaks in
its 13CNMR (DMSO-d6) d: 108.4, 113.2, 116.7, 116.6, 123.3, 128.7, 128.9, 130.3,
132.2, 136.5, 150.4, 157.9, 162.3, 163.5.
7-Flouro-3-(20-hydroxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3f) IR (KBr)
m
max: 3301 (O–H str), 2913 (C–H str), 1576 ([C=N str), 1501 (C=C str), 1321
(C–F str), 1176 ([C–OH str) and 714 (C–S str) cm-1. The 1HNMR (CDCl3)
spectrum of this product showed signals: d 7.6 (3H, m, fused benzothiazole ring),
8.8 (1H, br, phenolic proton) and between 7.0 and 7.2 (4H, m, substituted phenyl
123

Synthesis of novel 7-fluoro-3-substituted-1,2,4-triazolo[3…
ring) ppm. The peaks in its 13CNMR (DMSO-d6) d: 108.8, 113.4, 117.9, 118.9,
121.3, 123.6, 130.9, 132.3, 133.4, 136.5, 151.3, 154.3, 158.8, 161.2.
7-Flouro-3-(30-hydroxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3g) IR (KBr)
m
max: 3313 (O–H str), 2799 (C–H str), 1583 ([C=N str), 1512 (C=C str), 1287
(C–F str), 1216 ([C–OH str) and 797 (C–S str) cm-1. The 1HNMR (CDCl3)
spectrum of this product showed signals: d 7.5–7.6 (3H, m, fused benzothiazole
ring), 7.2–7.4 (4H, m, substituted pheyl group) and 9.30 (1H, br, phenolic proton)
ppm. The peaks in its 13CNMR (DMSO-d6) d: 107.5, 112.5, 116.4, 118.5, 120.4,
122.4, 131.9, 132.4, 134.5, 138.9, 152.4, 155.4, 158.6, 164.6.
7-Flouro-3-(40-hydroxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3h) IR (KBr)
m
max: 3312 (O–H str), 2879 (C–H str), 1644 ([C=N str), 1531 (C=C str), 1343
1
(C–F str), 1299 ([C–OH str) and 723 (C–S str) cm-1. The H NMR (CDCl3)
spectrum of the product showed signals: d 7.4–7.7 (3H, m, fused benzothiazole
ring), 7.0–7.2 (4H, m, substituted phenyl ring) and 9.33 (1H, br, phenolic proton)
ppm. The peaks in its 13CNMR (DMSO-d6) d: 107.2, 111.9, 115.4, 117.4, 121.4,
122.4, 131.9, 132.4, 134.5, 140.1, 153.3, 154.4, 158.6, 166.9.
7-Flouro-3-(40-methoxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3i) IR (KBr)
m
max: 2875 (C–H str), 1676 (O–C str), 1564 ([C=N str), 1502 (C=C str), 1376 (C–F
1
str), 1096 (C–O–C str) and 713 (C–S str) cm-1. The HNMR (CDCl3) spectrum of
the product showed signals: d 7.2–7.6 (3H, m, fused benzothiazole ring), 7.9–8.1
(4H, m, substituted phenyl ring) and 3.13 (3H, s, –OCH3) ppm. The peaks in its
13CNMR (DMSO-d6) d: 57.6, 108.8, 113.4, 114.6, 115.5, 123.7, 126.0, 130.1, 130.1,
132.9, 137.9, 150.3, 159.8, 161.7, 163.4.
7-Flouro-3-(20-nitrophenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3j) IR (KBr) mmax
:
2875 (C–H str), 1564 ([C=N str), 1528 (C=C str), 1501 (NO2 str), 1386 (C–F str)
and 845 (C–S str) cm-1.The 1H NMR (CDCl3) spectrum of product showed signals:
d 7.0–7.2 (3H, m, Ar C–H) and 7.9–8.4 (4H, m, nitrophenyl group) ppm. The peaks
in its 13CNMR (DMSO-d6) d: 105.7, 112.4, 113.4, 118.0, 124.5, 126.7, 131.1, 131.1,
135.3, 139.5, 154.3, 159.8, 160.1, 161.4.
7-Flouro-3-(30-nitrophenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3k) IR (KBr) mmax
:
2793 (C–H str), 1614 ([C=N str), 1578 (C=C str), 1494 (NO2 str), 1345 (C–F str)
and 898 (C–S str) cm-1. The 1H NMR (CDCl3) spectrum of product showed signals:
d 7.3–7.5 (3H, m, Ar C–H) and 7.7–8.3 (4H, m, nitrophenyl group) ppm. The peaks
in its 13CNMR (DMSO-d6) d: 109.2, 111.1, 114.3, 116.3, 123.0, 126.7, 133.4, 133.4,
137.1, 142.5, 154.3, 159.8, 164.2, 169.4.
7-Flouro-3-(40-nitrophenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3l) IR (KBr) mmax
:
2793 (C–H str), 1614 ([C=N str), 1588 (C=C str), 1523 (NO2 str), 1335 (C–F str)
and 816 (C–S str) cm-1. The 1H NMR (CDCl3) spectrum of product showed signals:
d 7.7–8.0 (3H, m, fused benzothiazole ring) and 8.1–8.3 (4H, m, nitrophenyl ring)
ppm. The peaks in its 13CNMR (DMSO-d6) d: 102.3, 117.1, 119.3, 121.3, 125.0,
129.7, 135.4, 135.4, 139.1, 149.5, 158.3, 162.8, 168.2, 169.4.
123

S. Kukreja et al.
7-Flouro-3-(30,40-dimethoxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3m) IR (KBr)
m
max: 2832 (C–H str), 1614 ([C=N str), 1594 (C=C str), 1376 (C–F str), 1105 (C–
1
O–C str) and 915 (C–S str) cm-1. The H NMR (CDCl3) spectrum of product
showed signals: d 7.0–7.2 (3H, m, benzothiazole ring), 6.9–8.3 (3H, m, phenyl
group) and d 3.3 (6H, s, OCH3 groups) ppm. The peaks in its 13CNMR (DMSO-d6)
d: 59.4, 59.4, 105.7, 112.4, 113.4, 118.0, 124.5, 126.7, 131.1, 131.1, 135.3, 139.5,
154.3, 159.8, 160.1, 161.4.
7-Flouro-3-(40-hyroxy-30-methoxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3n) IR
(KBr) mmax: 3306 (O–H str), 2912 (C–H str), 1667 ([C=N str), 1611 (C=C str), 1322
1
(C–F str), 1087 (C–O–C str) and 878 (C–S str) cm-1. The H NMR (CDCl3)
spectrum of product showed signals: d 7.7–8.0 (3H, m, benzothiazole), 6.9–7.1 (3H,
m, phenyl group), 3.8 (3H, s, CH3 group) and 9.83 (1H, br, phenolic proton) ppm.
The peaks in its 13CNMR (DMSO-d6) d: 57.9, 108.3, 113.3, 119.6, 123.4, 125.9,
135.3, 135.3, 139.7, 142.2, 148.8, 151.6, 156.8, 160.0, 163.4.
7-Flouro-3-(40-ethoxy-30-methoxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3o) IR
(KBr) mmax: 3243 (O–H str), 2832 (C–H str), 1614 ([C=N str), 1599 (C=C str),
1324 (C–F str), 1176 ([C–OH str), 996 (C–O–C str) and 754 (C–S str) cm-1. The
1H NMR (CDCl3) spectrum of product showed signals: d 7.6–8.0 (3H, m,
benzothiazole ring), 6.9–7.2 (3H, m, substituted phenyl group), 4.0 (2H, m, CH2),
1.32 (3H, m, CH3) and 9.83 (1H, br, OH group) ppm. The peaks in its 13CNMR
(DMSO-d6) d: 17.8, 67.9, 107.2, 119.7, 121.6, 124.4, 129.6, 138.5, 138.5, 140.1,
144.6, 149.2, 153.6, 157.0, 161.4, 164.0.
3-(40-dimetylaminophenyl)-7-Flouro-1,2,4-triazolo[3,4-b]benzothiazole (3p) IR (KBr)
m
max: 2874 (C–H str), 1601 ([C=N str), 1531(C=C str), 1354 (C–F str) and 899 (C–
1
S str) cm-1. The H NMR (CDCl3) spectrum of product showed signals: d 7.3–7.5
(3H, m, benzothiazole ring), 6.7–7.0 (4H, m, substituted phenyl group) and 3.1 (6H,
m, CH3) ppm. The peaks in its 13CNMR (DMSO-d6) d: 43.7, 43.7, 105.7, 112.4,
112.4, 115.9, 123.5, 125.7, 128.9, 128.9, 132.9, 138.7, 150.7, 156.3, 159.8, 161.7.
7-Flouro-3-(30,40,50-trimethoxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3q) IR
(KBr) mmax: 2753 (C–H str), 1645 ([C=N str), 1603 (C=C str), 1354 (C–F str), 1123
1
(C–O–C str) and 769 (C–S str) cm-1. The H NMR (CDCl3) spectrum of product
showed signals: d 7.0–7.2 (3H, m, fused benzo ring), 3.0–3.3 (9H, s, CH3) and 6.9
(2H, m, substituted phenyl group) ppm. The peaks in its 13CNMR (DMSO-d6) d:
56.7, 58.1, 62.4, 108.1, 113.4, 113.4, 119.4, 121.5, 123.7, 128.4, 128.4, 132.9,
138.7, 152.3, 157.7, 160.1, 164.5.
7-Flouro-3-(40hydroxy-30,50-dimethoxyphenyl)-1,2,4-triazolo[3,4-b]benzothiazole (3r) IR
(KBr) mmax: 3218 (O–H str), 2873 (C–H str), 1598 ([C=N str), 1531 (C=C str), 1395
1
(C–F str), 1054 (C–O–C str) and 715 (C–S str) cm-1. The H NMR (CDCl3)
spectrum of product showed signals: d 7.8–8.1 (3H, m, fused benzo ring), 6.7 (2H, s,
substituted phenyl ring), 8.7 (1H, br, OH) and 3.8 (6H, s, CH3) ppm. The peaks in its
13CNMR (DMSO-d6) d: 56.7, 56.7, 103.8, 117.5, 119.5, 120.4, 124.4, 126.7, 129.2,
129.2, 135.3, 138.7, 151.3, 159.7, 165.1, 169.5.
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Synthesis of novel 7-fluoro-3-substituted-1,2,4-triazolo[3…
7-Flouro-3-furanyl-1,2,4-triazolo[3,4-b]benzothiazole (3s) IR (KBr) mmax: 2897
(C–H str), 1612 ([C=N str), 1601 (C=C str), 1376 (C–F str) and 829 (C–S str)
cm-1. The 1H NMR (CDCl3) spectrum of product showed signals: d 7.2–7.5 (3H, m,
fused benzo ring) and 7.7–8.3 (3H, s, furan ring) ppm. The peaks in its 13CNMR
(DMSO-d6) d: 106.7, 109.3, 112.2, 114.8, 123.9, 134.6, 138.9, 145.4, 149.8, 154.4,
159.9, 163.0.
7-Flouro-3-styrenyl-1,2,4-triazolo[3,4-b]benzothiazole (3t) IR (KBr) mmax: 2793
(C–H str), 1602 ([C=N str),1589 (C=C str), 1357 (C–F str) and 763 (C–S str) cm-1
.
1
The H NMR (CDCl3) spectrum of product showed signals: d 7.8–8.0 (3H, m,
benzothiazole ring), 7.3–7.6 (5H, s, phenyl ring) and 6.5 (2H, s, CH) ppm. The
peaks in its 13CNMR (DMSO-d6) d: 108.9, 114.5, 123.4, 127.9, 128.9, 128.9, 130.1,
130.1, 133.4, 134.6, 137.9, 138.7, 142.2, 148.3, 158.8, 162.3.
In silico analysis
Toxtree analysis
Different derivatives were screened for different physico-chemical properties by
using different software. Toxtree v.2.6.6 is an open-source software application that
places chemicals into categories and predicts various kinds of toxic effects by
applying various decision-tree approaches. Toxtree was developed by IdeaConsult
(Sofia, Bulgaria) under the terms of an ECB contract. The software is made freely
available by ECB as a service to scientific researchers and anyone with an interest in
the application of computer-based estimation methods in the assessment of chemical
toxicity. The new module with the revised list of SAS also includes quantitative
structure–activity relationship (QSAR) models that enable the toxicity evaluations
for a number of chemical classes to be fine-tuned.
In order to find out the toxic hazards of all the synthesized compounds, two-
dimensional models of the compounds were first converted into its simplified
molecular-input line-entry system (SMILES format). Then, simply putting the
SMILES code into the chemical identifier row available in the Toxtree software, we
can easily obtain the toxic characters.
Docking procedure
The compounds were built using the builder tool kit of the software package
ArgusLab 4.0.1 and the geometry was optimized using the semi-empirical quantum
mechanical method, PM3. Due to the non-availability of fungal lanosterol
demethylase, the crystal coordinates of human lanosterol 14a-demethylase (PDB
ID 2 VKU) were downloaded from the protein data bank (www.rcsb.org) and the
active site of the enzyme was located, carrying a 4,40-dihydroxybenzophenone
(DHBP) as a ligand in the active site. The molecule to be docked in the active site of
the protein was pasted in the work space carrying the structure of the enzyme. The
docking programme implements an efficient grid-based docking algorithm which
approximates an exhaustive search within the free volume of the binding site cavity.
123

S. Kukreja et al.
The conformational space was explored by the geometry optimization of the flexible
ligand (rings were treated as rigid) in combination with the incremental construction
of the ligand torsions. Thus, docking occurs between the flexible ligand parts of the
compound and enzyme. The final positions of the compounds were ranked by lowest
interaction energy values (docking score in Kcal/mol). H-bond interactions between
the compound and enzyme were explored.
Lipinski filtration
Fungitoxicity profiles were further rationalized for their potential bioactivity as
antifungals or pro-antifungals by theoretically calculating the Lipinski parameters of
all the synthesized molecules using Molinspiration which is a web-based software.
It is already referred to as the Pfizer’s rule of five or simply the rule of five (RO5).
This rule was formulated by Christopher A. Lipinski in 1997. According to
Lipinski’s rule of five, molecules should have log P values of B5 in order to be
readily bioavailable. Lipinski’s rule says that the molecular weight should be below
500, the number of hydrogen bond acceptors should not be more than 5 and also the
number of hydrogen bond donors should not be more than 5. These numbers are the
upper limits for the biomolecules to be able to penetrate through biomembranes. In
order to calculate the molecular properties, first we have either to draw the molecule
online (www.molinspiration.com) or simply to input the SMILES code, then all the
Lipinski characters along with the bioactivity and 3D structure of the molecule are
generated.
Antifungal assays
The in vitro antifungal activity of all the title compounds were evaluated against five
phytopathogenic fungi viz. Puccinia triticina, Ustilago tritici, Erysiphe graminis,
Puccinia striiformis and Alternaria alternata which are often encountered, and
compared with the standard fungicides Tilt (1-[[2-(2,4-dichlorophenyl)-4-propyl-
1,3-dioxolan-2-yl]methyl]-1H-1,2,4-triazole) and Vitavax (5,6-dihydro-2-methyl-
1,4-oxathiin-3-carboxamide). The isolates of phytopathogenic fungi were provided
by the Plant Pathology Department of the Punjab Agricultural University and the
standards Tilt and Vitavax, serving as the positive control, were obtained from their
respective manufacturers.
Five micromoles per litre of each of the test compounds and standards used were
made by dissolving the appropriate amount of the compound in 1.0 ml of dimethyl
sulfoxide (DMSO) and making the final volume 10 ml. These were capped and
refrigerated to be used as stock solutions. Serial dilution of the stock solutions up to
0.1 mmoles per litre was done as and when required.
The in vitro effective concentrations of the compounds were determined by the
spore germination inhibition technique [35]. The EC50 refers to the concentration
which induces a response halfway. For assays, 0.02 ml of the title compounds to be
tested and 0.02 ml of spore suspension were seeded in the cavity of a cavity slide
and incubated at 15 2 °C. Growth was determined at 24 h for all the
123

Synthesis of novel 7-fluoro-3-substituted-1,2,4-triazolo[3…
phytopathogenic fungi. The results of assays in terms of EC50 are summarized in
Table 2. The data points are the means of triplicates.
Results and discussion
Synthetic chemistry
Scheme 1 shows the schematic representation of synthetic route for the preparation
of the target compounds. Compound 1 was synthesized by the reaction of aldehydes
with 4-flouroaniline followed by bromocyclization.
The reaction of compound 1 with hydrazine hydrate in ethylene glycol yielded
6-fluoro-2-hydrazinylbenzo[d]thiazole 2. Further, 7-fluoro-3-substituted-1,2,4-tria-
zolo[3,4-b]benzothiazoles 3a–3t were prepared by refluxing the mixture of
compound 2 and a suitable aromatic aldehyde in ethanol. The substituted
hydrazones obtained were further bromocyclised to give corresponding FTBs,
1
3a–3t. The structures of the compounds were confirmed by IR, H NMR and 13C
NMR and elemental analysis (Table 1) which were correlated with the proposed
structures, confirming the formation of the compounds.
In silico analysis
Toxtree analysis
Estimation of toxic hazards by Cramer rules was carried out using Toxtree v.2.6.6
software [32], which showed that the compounds belong to the class III level of
toxicity, which was same as that of the toxicity level standard fungicides used.
O
Ethylene glycol
NH2NH2.H2O
F
F
F
S
N
S
N
S
N
Ar
C
H
Ar
NHN C
NH2
NHNH2
EtOH
Conc. HCl
H
(1)
(2)
Br2/AcOH
Sodium Acetate
(3a-3t)
Ar
(3k)= 3'NO2-C6H4
(3l)= 4'NO2-C6H4
(3m)= 3'OCH3, 4'OCH3-C6H3
(3n)= 3'OCH3, 4'OH-C6H3
(3o)= 3'OCH2CH3, 4'OH-C6H3
(3p)= 4'N(CH3)2-C6H4
(3q)= 3'OCH3, 4'OCH3, 5'OCH3-C6H2
(3r)= 3'OCH3, 4'OH, 5'OCH3- C6H2
(3s)= C4H3O
(3a)= C6H5
F
S
(3b)= 2'Cl-C6H4
(3c)= 3'Cl-C6H4
(3d)= 4'Cl-C6H4
(3e)= 4'F-C6H4
(3f )= 2'OH-C6H4
(3g)= 3'OH-C6H4
(3h)= 4'OH-C6H4
(3i)= 4'OCH3-C6H4
(3j)= 2'NO2-C6H4
N
N
N
Ar
(3a-3t)
(3t)= -CH=CH-C6H5
Scheme 1 General synthesis of 7-flouro-3-phenyl-1,2,4-triazolo[3,4-b]benzothiazoles
123

S. Kukreja et al.
Table 1 Physical characteristics and elemental analysis of all the synthesized compounds
Compds
Yield (%)
M.P (°C)
Mol. formula
Elemental analysis (calculated %/found %)
C
H
N
1
89
84
75
63
61
66
54
62
67
63
69
54
51
54
64
63
64
51
53
62
52
49
195
223
235
239
252
247
267
234
247
243
268
285
292
287
247
241
246
249
257
252
265
261
C7H5FN2S
C7H6FN3S
C14H8FN3S
(48.33/49.99)
(44.79/45.89)
(61.77/62.44)
(54.57/55.36)
(54.36/55.34)
(54.43/55.34)
(57.32/58.34)
(58.24/58.94)
(57.44/58.94)
(57.94/58.94)
(59.77/60.19)
(52.76/53.50)
(52.34/53.50)
(52.16/53.50)
(57.33/58.35)
(57.22/57.54)
(57.39/58.35)
(60.33/61.52)
(56.12/56.82)
(55.22/55.65)
(55.21/55.59)
(64.23/65.02)
(2.12/3.00)
(2.98/3.30)
(2.12/2.99)
(1.78/2.32)
(2.99/2.32)
(2.23/2.32)
(2.39/2.86)
(2.12/2.83)
(2.09/2.83)
(2.42/2.83)
(3.22/3.37)
(2.74/2.24)
(2.22/2.24)
(2.44/2.24)
(3.24/3.67)
(2.98/3.20)
(3.22/3.67)
(3.87/3.19)
(3.22/3.93)
(3.21/3.50)
(2.12/2.33)
(3.12/3.41)
(15.67/16.66)
(21.97/22.94)
(14.87/15.60)
(13.21/13.83)
(13.23/13.83)
(13.00/13.83)
(14.42/14.83)
(13.97/14.73)
(13.74/14.73)
(13.87/14.73)
(13.87/14.04)
(17.67/17.83)
(16.99/17.83)
(17.23/17.83)
(12.33/12.76)
(12.89/13.33)
(12.34/12.76)
(17.22/17.94)
(11.22/11.69)
(11.8/12.17)
(16.27/16.21)
(14.19/14.23)
2
3a
3b
3c
3d
3e
3f
C
C
C
C
C
C
C
C
C
C
C
C
C
14H7ClFN3S
14H7ClFN3S
14H7ClFN3S
14H7F2N3S
14H8FN3OS
14H8FN3OS
14H8FN3OS
15H10FN3OS
14H7FN4O2S
14H7FN4O2S
14H7FN4O2S
16H12FN3O2S
15H10FN3O2S
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
C16H12FN3O2S
C
C
C
C
C
16H13FN4S
17H14FN3O3S
16H12FN3O3S
12H6FN3OS
16H10FN3S
Docking studies
Molecular docking scores of all the synthesized compounds were performed in the
active site of the enzyme lanosterol 14a-demethylase (PDB ID 2VKU), using
ArgusLab 4.0.1. All the compounds get concealed in the interior of the enzyme and
none of these compounds exhibited H-bond interactions with active sites of
lanosterol 14a-demethylase because of no labile hydrogens (–NH, –OH or HF)
present in the molecule, but they still have high favorable free energies of binding
because of the close strong interactions with amino acid residues, viz. glycine,
alanine, phenylalanine, cystein and threonine (Fig. 1). Compounds 3b, 3c, 3g and 3t
had the most favourable free energies of binding, viz. -11.12, -11.76, -10.99 and
-10.74 kcal/mol, respectively. Significant differences in their respective docking
scores were observed which was probably due to hydrophobic and other dipole–
dipole interactions [33, 34] of the test compound with amino acid residues in the
active site. Docking scores of most of the synthesized compounds were better than
the compounds having these two leads separated by spacers [1], and their bioactive
assay, therefore, followed the trend.
123

Synthesis of novel 7-fluoro-3-substituted-1,2,4-triazolo[3…
Fig. 1 Docking poses of the
compounds 3b, 3c, 3g and 3t
(shown in green), respectively,
in the active site (shown in red)
of cytochrome P450 lanosterol
14a-demethylase
123

S. Kukreja et al.
Lipinski filtration
Lipinski parameters of the synthesized compounds are presented in Table 2. All the
compounds had molecular weights less than 360. Compounds 3q and 3r had H-bond
acceptors more than 5 but all of them had the number of hydrogen bond donors (i.e.
the sum of OH and NH groups) less than 5. The log p value was unfavorable (more
than 5) in the case of compounds 3j, 3k, 3l and 3t. Combining all the factors,
compounds 3j, 3k, 3l, 3q, 3r and 3t were expected to be non-relevant for
bioactivity. But for real results, all the compounds were evaluated for fungitoxicity.
Antifungal assay
Fungicidal evaluation of the series of title compounds was performed against
various phytopathogenic fungi by a spore germination inhibition technique
representing their results in terms of EC50 values. The five fungi used in the
Table 2 The docking scores and Lipinski characters
Compds
Final docked
energy (kcal/mol)
Log p
Molecular weight
n ON
n OHNH
1
-8.62
-9.19
-10.46
-11.12
-11.76
-10.73
-10.15
-9.51
-10.99
-9.97
-9.76
-9.54
-9.43
-9.68
-9.59
-9.02
-9.99
-8.95
-8.14
-8.67
-8.45
-10.74
2.59
2.10
4.37
4.93
4.93
4.93
4.53
3.99
3.99
3.99
4.29
5.16
5.16
5.16
4.12
3.86
4.20
4.66
4.00
3.73
2.96
5.16
168
183
269
303
303
303
287
285
285
285
295
314
314
314
329
315
329
312
359
345
259
295
2
3
3
3
3
3
3
4
4
4
4
5
5
5
5
5
5
4
6
6
4
3
1
2
0
0
0
0
0
1
1
1
0
0
0
0
0
1
1
0
0
1
0
0
2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
Log p logarithm of the octanol–water partition coefficient; n ON number of hydrogen bond acceptors
(sum of O and N atoms); n OHNH number of hydrogen bond donors (sum of OH and NH groups)
123

Synthesis of novel 7-fluoro-3-substituted-1,2,4-triazolo[3…
fungicidal bioassay were Puccinia triticina, Ustilago tritici, Erysiphe graminis,
Puccinia striiformis and Alternaria alternata. Commercial agricultural fungicide
Vitavax (5,6-dihydro-2-methyl-1,4-oxathiin-3-carboxamide) against Ustilago tritici
and Tilt (1-[[2-(2,4-dichlorophenyl)-4-propyl-1,3-dioxolan-2-yl]methyl]-1H-1,2,4-
triazole) against the rest of the fungi were used as standards. For the sake of
comparison of fungitoxicity of the molecules with standard fungicides, the results
were appropriately expressed in terms of mmoles/L.
Investigations of antifungal screening listed in Table 3 clearly revealed that most
of the synthesized fluorinated heterocylics showed significant inhibition of
germination against all the test fungi. This series were found to be most active
against A. alternata. Ten out of 20 compounds had antifungal potential better than
the standard compound Tilt (ED50 0.69 mmoles/L) with compound 3c the best with
ED50 0.39 m moles/L which was much more favorable than the standard. Against E.
Table 3 Antifungal potential of the synthesized compounds against various phytopathogenic fungi in
terms of EC50 (mmoles/L)
Compounds
P. triticina
U. tritici
E. graminis
P. striiformis
A. alternata
EC50 (m moles/L)
1
4.64
2.19
0.92
0.49
0.47
0.82
1.74
1.22
0.46
1.75
1.69
1.59
1.53
2.31
1.31
0.79
1.51
1.60
1.32
2.11
1.93
0.83
0.65
–
2.08
0.55
0.65
0.33
0.60
0.79
0.31
0.25
0.81
0.87
0.33
1.47
1.27
0.76
0.30
0.63
0.60
0.80
0.69
0.72
1.54
0.73
–
4.37
0.59
0.92
0.27
0.24
0.37
1.74
0.84
0.27
0.70
0.84
0.40
0.59
0.89
0.75
1.12
0.40
1.60
1.81
1.76
0.96
0.41
0.40
–
3.86
3.01
1.67
0.47
0.36
1.81
2.26
1.57
0.87
1.92
1.11
0.66
1.12
2.26
1.20
1.09
1.36
1.12
1.25
1.30
1.73
0.64
0.50
–
2.85
1.21
0.46
0.54
0.39
1.32
1.21
0.86
0.82
0.70
0.42
0.41
0.54
0.61
0.45
0.66
0.75
0.80
0.52
1.43
1.92
0.62
0.69
–
2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
Tilta
Vitavaxb
a
0.31
Standard fungicide against A.alternata, E. graminis, P. triticina and P.striiformis
b
Standard fungicide against U. tritici
123

S. Kukreja et al.
graminis, compounds 3b, 3c and 3d have antifungal potential with EC50 values of
0.27, 0.24 and 0.27 mmoles/L, respectively, which were again lower than the
standard fungicide Tilt (EC50 = 0.40 mmoles/L). Similar trends were repeated
against P. triticina, where compounds 3b, 3c and 3g showing promising results
(EC50 values 0.49, 0.47 and 0.46 mmoles/L, respectively) better than the standard
(Tilt EC50 values 0.60 mmoles/L). Against P. striiformis, compounds 3b and 3c
showed EC50 values of 0.47 and 0.36 m moles/L, lower than the Tilt (EC50 values
0.50 m moles/L). In the case of U.tritici, compounds 3e and 3f were the most
effective compounds with EC50 values of 0.31 and 0.25, respectively, which were
comparable to the standard fungicide Vitavax (EC50 values 0.31 mmoles/L).
All the 7-fluoro-3-substituted-1,2,4-triazolo[3,4-b]benzothiazoles exhibited anti-
fungal activity very much better than the precursors 1 and 2. The presence of aryl
groups at the 3-position was important for favorable fungitoxicity as the compounds
3a has lower fungitoxicity than the rest of the series. Compounds 3b, 3c and 3d
exhibited appreciable fungitoxicity followed by compounds 3f and 3t, which
evidently endorsed that the presence of chloro, flouro and vinyl phenyl groups at the
3-position was important for appreciable results of antifungal potential. Compounds
3b and 3c were the best against all the fungi with significant inhibition of
germination in comparison to the standards, indicating the importance of the chloro
group in the phenyl ring at position 3. These results also corresponded well to the
best docking scores in these molecules. All the compounds mentioned also
performed best in silico docking scores. Their other drug-like indices along with log
p values were in the approved range of the Lipinskii rule. The results of antifungal
potential of the fused 1,2,4-triazole with benzothiazole were better than the non-
fused analogues separated by spacers [1], endorsing the ring fusion of the leads than
their presence in the same molecule separated by the spacers.
Conclusion
A novel series of 7-fluoro-3-substituted-1,2,4-triazolo[3,4-b]benzothiazoles synthe-
sized on the basis of the lead hybridization strategy gave synergistic effects of
heterocyclic fusion on their antifungal potential against various test fungi. Their
EC50 values in some cases were lower than the standard fungicides used. Favorable
in silico and in vitro antifungal evaluation studies provides an impetus to the further
exploration of compounds 3b, 3c, 3d, 3g, and 3f as potential leads for in vivo trials,
in place of existing 1,2,4-triazoles, as reduced risk fungicides. Further designing,
synthesis and structural optimization are in progress.
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