Synthesis of triazole Schiff bases: Novel inhibitors of nucleotide pyrophosphatase/phosphodiesterase-1

Article abstract of DOI:10.1016/j.bmc.2014.08.032

A series of Schiff base triazoles 1-25 was synthesized and evaluated for their nucleotide pyrophosphatase/phosphodiesterase-1 inhibitory activities. Among twenty-five compounds, three compounds 10 (IC₅₀ = 132.20 ± 2.89 μM), 13 (IC₅₀

Full text of DOI:10.1016/j.bmc.2014.08.032

Bioorganic & Medicinal Chemistry 22 (2014) 6509–6514
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of triazole Schiff bases: Novel inhibitors of nucleotide
pyrophosphatase/phosphodiesterase-1
a
a
a,b
Khalid Mohammed Khan a, , Salman Siddiqui , Muhammad Saleem , Muhammad Taha
,
⇑
Syed Muhammad Saad a, Shahnaz Perveen c, M. Iqbal Choudhary a,d
a H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University Karachi, Karachi 75270, Pakistan
b Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia
c PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280, Pakistan
d Department of Biochemistry, Faculty of Science, King Abdulaziz University, Jeddah 21412, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 10 July 2014
Revised 22 August 2014
Accepted 26 August 2014
Available online 16 September 2014
A series of Schiff base triazoles 1–25 was synthesized and evaluated for their nucleotide pyrophospha-
tase/phosphodiesterase-1 inhibitory activities. Among twenty-five compounds, three compounds 10
(IC50 = 132.20 2.89
l
M), 13 (IC50 = 152.83 2.39
l
M), and 22 (IC50 = 251.0 6.64
lM) were identified
as potent inhibitors with superior activities than the standard EDTA (IC50 = 277.69 2.52
l
M). The newly
identified inhibitors may open a new avenue for the development of treatment of phosphodiesterase-I
related disorders. These compounds were also evaluated for carbonic anhydrase, acetylcholinesterase
and butyrylcholinesterase inhibitory potential and were found to be inactive. The compounds showed
non-toxic effect towards PC3 cell lines.
Keywords:
Nucleotide pyrophosphatase/
phosphodiesterase-1
Schiff bases of triazoles
PC3 cell lines
Ó 2014 Elsevier Ltd. All rights reserved.
Chondrocalcinosis
1. Introduction
phosphodiesterase-I.39 Due to close structural resemblance of
1,2,4-triazoles with above-mentioned compounds, we synthesized
Triazoles are five membered organic heterocyclic compounds
having a di-unsaturated ring comprising of three nitrogen and
two carbon atoms. 1,2,4-Triazoles are cyclic hydrazones with H-
atom (or substitution) on either hydrazide N-atom or an amide
N-atom. According to the literature, 1H-1,2,4-triazoles and their
analogues have diverse biological activities, such as antiasthamat-
ic,1,2 antiviral,3 antifungal,4 antibacterial,5 hypnotic,6 pesticidal,7
breast cancer preventive,8,9 anticonvulsant, anticancer, antiinflam-
matory,10–19 CNS depressant,20 and antihypertensive21,22 activities.
1,2,4-Triazole derivatives have been used as mimics and isoster-
es.23–30 Vasodilatory,31,32 psychotropic,33,34 cyclooxygenase and
5-lipoxygenase inhibition,35 hypoglycemic,36 antitumor activi-
ties,37 plant growth regulating and anticoagulant properties are
also reported for this class of compounds.38
1,2,4-triazoles as potential inhibitors of nucleotide pyrophospha-
tase/phosphodiesterase-1. Enzyme inhibition studies on 1,2,4-tria-
zoles proved our hypothesis.
Nucleoside-50-monophosphates are released by the catalytic
action of nucleotide pyrophosphatase/phosphodiesterase-1 (NPP/
PDEs) from a variety of nucleotides and their derivatives.40 These
PDEs/NPPs occur in both forms as soluble proteins in the body or
membrane proteins with active site outside the body. These
enzymes are widely distributed throughout the body cells, as well
as in a variety of organs, such as liver cells, serum, mammalian
intestinal mucosa, and snake venom.41–43 Plasma cell membrane
glycoprotein (NPP1/PC-1) is the major agent for bone calcification
and related tissues. The hyperactivity of PDE1 results in
chondrocalcirosis,44 whereas down regulation of enzymes severes
calcification in mice.45,46 Human calcification syndrome is found
to be associated with the down regulation of PDEs.
Easily attainable heterocyclic compounds of synthetic origin are
ideal candidates for drug discovery and development against defined
biological targets. Based on this hypothesis, we recently reported
1,3,4-oxadiazole-2(3H)-thiones and 1,3,4-thiadiazole-2(3H)-thiones
(Fig. 1), as potent inhibitors of nucleotide pyrophosphatase/
Only a few studies have been conducted on the inhibition of
nucleotide pyrophosphatases/phosphodiesterases by natural or
synthetic compounds.47–53 Progress in the discovery and
development of inhibitors slowed largely due to the unavailability
of the three-dimensional structures of the target enzymes.54
⇑
Corresponding author. Tel.: +92 21 34824910; fax: +92 21 34819018.
E-mail addresses: hassaan2@super.net.pk, khalid.khan@iccs.edu (K.M. Khan).
http://dx.doi.org/10.1016/j.bmc.2014.08.032
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

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K.M. Khan et al. / Bioorg. Med. Chem. 22 (2014) 6509–6514
R2
Table 1
Phosphodiesterase-I inhibition by Schiff bases of triazole 1–25
N
N
Compounds
Structures
IC50 SEMa
(lM)
R1
X
OH
X = O or S
H
N
1
NAb
Figure 1. 1,3,4-Oxadiazole-2(3H)-thiones and 1,3,4-thiadiazole-2(3H)-thiones.
N
N
N
MeO
OH
During the current study, triazole Schiff bases 1–25 were syn-
thesized by using very simple and one step synthetic procedure
keeping in mind the easy adoptability by pharma industries and
cheap commercialization. All the synthetic Schiff bases 1–25 were
screened for the inhibition of the snake venom nucleotide pyro-
phosphatase/phosphodiesterase-1. According to literature survey,
these compounds are novel inhibitors of PDE1. This is the first
report on the inhibitory potential of Schiff base of triazoles against
PDE1/NPP1.
H
2
3
NAb
NAb
N
N
N
N
OH
H
N
N
N
N
Me
H
MeO
N
N
4
5
6
7
8
NAb
NAb
NAb
NAb
NAb
N
N
N
2. Results and discussion
2.1. Chemistry
MeO
H
N
Cl
N
N
N
Cl
Triazole Schiff bases 1–25 were synthesized by refluxing differ-
ent substituted benzaldehydes and amino triazole in equimolar
proportion in ethanol as a solvent with a catalytic amount of
H2SO4 for 4–10 h. Progress of reaction was periodically monitored
by TLC. After completion of reaction, solvent was evaporated under
reduced pressure. The solid mass was re-crystallized with ethanol,
which gave pure compounds (Scheme 1). These resulting com-
pounds were characterized by 1H NMR and EI mass spectrometry.
Elemental analyses of all the compounds were found satisfactory.
Compounds 3, 4, 6, 7, 8, 13, and 25 were synthesized for the first
time.
H
N
O2N
EtO
N
N
H
N
N
N
N
OH
H
MeS
Cl
N
N
N
N
In vitro snake venom NPP1 inhibitory activity of all derivatives
of triazole Schiff bases 1–25 was carried out, whereas EDTA was
used as the positive control. Compounds 10, 13, and 22 were found
to be most potent inhibitors against PDE1/NPP1 enzymes with IC50
H
N
9
NAb
N
N
N
Cl
Me
OH
OH
values 132.20 2.89, 152.83 2.39, and 251.0 6.64
tively, better than the standard EDTA (IC50 = 277.69 2.52
Compounds 23, 21, and 20 were found to be weakly active with
IC50 values between 404 and 1164 M. Nonetheless, all other
l
M, respec-
H
N
N
N
lM).
10
11
132.2 2.89
Me
Br
N
N
l
derivatives were found to be inactive. The IC50 values of all the
inhibitors against snake venom PDE1/NPP1 are shown in Table 1.
EDTA is used for the treatment of rheumatoid arthritis, osteoar-
thritis, diabetes, Parkinson’s disease, and multiple sclerosis. Skin
conditions, including scleroderma and psoriasis are also being trea-
ted with EDTA. As the PDEs hyperactivity is involved in a number
of diseases such as arthritis, hypertension, and cardiovascular dis-
eases,55 therefore, it is usually used as standard for PDE1/NPP1
enzymes.
H
N
NAb
NAb
N
N
N
Br
OH
N
N
N
N
12
13
N
N
N
N
N
152.83 2.39
HO
HO
OH
2.2. Structure–activity relationship
N
N
N
14
15
NAb
NAb
Structure–activity relationship (SAR) revealed that activity is
mainly due to substitutions on benzene ring. Compound 10 having
N
N
N
N
HO
N
O
N
N
N
N
N
EtOH, H2SO4
N
N
+
Δ
NAb
H
R
N
R
N
H
NH2
16
N
H
MeO
OH
1 25
( -
)
Scheme 1. Synthesis of triazole Schiff bases 1–25.

K.M. Khan et al. / Bioorg. Med. Chem. 22 (2014) 6509–6514
6511
M)
Table 2
Table 1 (continued)
Cytotoxcity of Schiff bases of triazole 1-25 against PC3 cell lines
Compounds
Structures
IC50 SEMa
(lM)
Compounds
IC50 SEM (
lM)
Compounds
IC50 SEM (l
N
N
N
N
1
2
3
4
5
6
7
8
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
14
15
16
17
18
19
20
21
22
23
24
25
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
17
NAb
EtO
OH
N
N
N
N
18
19
NAb
NAb
MeO
N
N
9
N
N
10
11
12
13
EtO
HO
OH
N
N
Doxorubicin Std.
0.912 0.12
N
N
20
21
22
1164 100.45
823.1 31.56
251.0 6.64
OH
2.3. Cytotoxicity studies
N
N
N
N
Br
Cl
All the compounds 1–25 were found to be inactive against the
anticancer activity (PC3 Cell Line) (Table 2).
N
N
N
N
N
3. Conclusion
OH
OH
Three compounds 10, 13, and 22 were identified as potential
lead compounds for nucleotide pyrophosphatase/phosphodiester-
ase-1 inhibition. These compounds were also evaluated for car-
bonic anhydrase and found to be inactive. The result illustrates
that these synthetic compounds are selective towards nucleotide
pyrophosphatase/phosphodiesterase-1 and may be useful for the
treatment of disorders related to PDEs hyperactivity after further
mechanistic studies.
Br
N
N
N
23
24
404.56 10.74
N
N
N
N
NAb
MeO
MeO
N
N
4. Experimental
N
N
25
NAb
MeS
EDTA (Std.)
4.1. Material and methods
277.69 2.52
a
Standard error of mean.
NA = Not active.
The phosphodiesterase-1 (Broth atrox crude dried snake
venom) was purchased from Sigma–Aldrich, USA. The tris buffer
was purchased from Scharlau, Spain Cat #TR0423, magnesium
acetate was purchased from Sigma–Aldrich, USA Cat #M-0631,
bis(p-nitrophenyl) phosphate was used as a substrate (Sigma–
Aldrich, USA N-3002). 3-Amino-4(H)-1,2,4-triazoles, 4-amino-
4(H)-1,2,4-triazoles, and different benzaldehydes of analytical
grades were purchased from TCI, Japan. Ethanol was dried using
magnesium turnings. 1H NMR experiments were run on Avance
Bruker AM 300 MHz instrument. Electron impact mass spectrome-
try (EI-MS) analyses were performed on a Finnigan MAT-311A
(Germany) mass spectrometer. CHN analyses were performed on
a Carlo Erba Strumentazione-Mod-1106, Milano, Italy. Thin layer
chromatography (TLC) was monitored on precoated silica gel alu-
minum plates (Kieselgel 60, 254, E. Merck, Germany). TLC Chro-
matograms were visualized under UV at wavelengths of 254 and
365 nm or by using iodine vapours spray.
b
N,N0-dimethyl substituent exhibited
IC50 = 132.2 2.89
in compound 13 with ortho hydroxyl groups,
activity (IC50 = 152.83 2.39 M) was observed. Compound 22
(IC50 = 251.0 6.64 M) was found to be an excellent inhibitor
with ortho hydroxyl and meta chloro groups. However, the pres-
ence of bromo group, as in compound 23 (IC50 = 404.56 10.74 -
M) decreased the activity. It may be due to larger size of bromo
substituent than a chloro group which developed steric and
electronic hindrances for interaction with the active site residues
of PDE1. Furthermore, the presence of chloro and hydroxyl
groups in compound 22 enhanced the activity, as compared to
the compound 23 which may be due to more electronegativity
of chlorine than bromine. Compounds 21 (IC50 = 823.1 31.56
a
potent activity with
lM, as compared to other analogues. Similarly
a
comparable
l
l
l
lM), and 20 (IC50 = 1164 100.45 lM) were found to be weak
inhibitors with different substituents. Weak inhibitor 20 indicated
that the presence of three hydroxyl groups caused a marked
decrease in activity which may be due to steric hindrance making
4.2. Nucleotide pyrophosphatases/phosphodiesterases-1
inhibition assay
p
-electrons non-available to chelate with the active site amino
The activity of snake venom PDE1/NPP1 was carried out by the
standard procedure as reported in literature56 with some modifica-
tions, such as 33 mM tris–HCl as buffer at pH of 8.8, magnesium
acid residues of the enzyme. In compound 21, the presence of
bromo residue enhanced the activity as compared to compound
20 which possessed three hydroxyl groups contributing in the
steric hindrance.
In brief, this study has identified compounds 10, 13, and 22 as
leads for further research towards the treatment of phosphodies-
terase-I related disorders.
acetate at
a concentration of 30 mM as a co-factor, and
7.42 ꢀ 10ꢁ4 units of PDE1 enzyme dissolved in cold de-ionized
water. After 30 min of incubation at 37 °C, the reaction was moni-
tored by the addition of substrate (bis(p-nitrophenyl)phosphate) at
0.33 mM of total concentration. The rate of p-nitrophenol release

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K.M. Khan et al. / Bioorg. Med. Chem. 22 (2014) 6509–6514
0
0
0
0
0
0
was monitored at 410 nm in 96-well plate by spectrophotometer
(Molecular Devices, CA, USA).
All the reactions were performed in triplicate, and the initial
rates were measured as the rate of changes in the Vmax/min (maxi-
mal velocity/min) and used in subsequent calculations.
The formula for % inhibition is:
J6 ,5 = 8.4 Hz, H-60), 7.59 (dd, 1H, J5 ,6 = 8.4 Hz, J5 ,3 = 1.8 Hz, H-50);
EI-MS m/z (% rel. abund.): 242 [M+2]+ (6), 240 [M]+ (10), 207
(37), 205 (100); Anal. Calcd for C9H6Cl2N4, C = 44.84; H = 2.51;
Cl = 29.41; N = 23.24. Found: C = 44.82; H = 2.52; N = 23.23.
4.3.6. N-[(40-Nitrophenyl)methylidene]-4H-1,2,4-triazol-3-
amine (6)
%Inhibition ¼ 100 ꢁ ðVmax of test=Vmax of controlÞ ꢀ 100
IC50 Values were calculated by using the EZ-fit enzyme kinetics
software (USA).
Yield: 62%; 1H NMR (400 MHz, DMSO-d6): d 9.35 (s, N@CHAAr),
8.55 (s, 1H, H-5), 8.36 (d, 2H, J3 ,2 = J5 ,6 = 8.8 Hz, H-30, H-50), 7.82
0
0
0
0
(d, 2H, J2 ,3 = J6 ,5 = 8.8 Hz, H-20, H-60); EI-MS m/z (% rel. abund.):
217 [M]+ (74), 76 (13); Anal. Calcd for C9H7N5O2, C = 49.77;
H = 3.25; N = 32.25. Found: C = 49.76; H = 3.24; N = 32.26.
0
0
0
0
4.3. General procedure for the synthesis of compounds 1–25
In a typical procedure, 3-arylimino-1,2,4-triazoles and 4-aryli-
mino-1,2,4-triazoles 1–25 were synthesized by mixing 3-amino-
1,2,4-triazole or 4-amino-1,2,4-triazole (2 mmol), substituted
benzaldehydes (2 mmol) and H2SO4 (2 mL) in ethanol (15 mL).
The mixtures were refluxed for 4–10 h, while progress of the reac-
tion was monitored through thin layer chromatography. When
reaction was completed, solvent was evaporated on a rotary evap-
orator under reduced pressure and residue was washed with hot
hexane. Resulting compounds were crystallized by ethanol to give
title compounds in moderate to good yields.
4.3.7. N-[(40-Ethoxy-20-hydroxyphenyl)methylidene]-4H-1,2,4-
triazol-3-amine (7)
Yield: 77%; 1H NMR (400 MHz, DMSO-d6): d 9.39 (s, N@CHAAr),
8.43 (s, 1H, H-5), 7.35 (d, 1H, J6 ,5 = 8.0 Hz, H-60), 7.15 (d, 1H,
0
0
J5 ,6 = 8.0 Hz, H-50), 6.90 (m, 1H, H-30), 4.08 (q, 2H, OCH2), 1.35 (t,
0
0
3H, CH3); EI-MS m/z (% rel. abund.): 232 [M]+ (94), 215 (100),
175 (40); Anal. Calcd for
C11H12N4O2, C = 56.89; H = 5.21;
N = 24.12; O = 13.78. Found: C = 56.87; H = 5.20; N = 24.11.
4.3.8. N-[40-(Methylsulfanyl)phenyl]methylidene-4H-1,2,4-
triazol-3-amine (8)
4.3.1. N-[(30-Hydroxyphenyl)methylidene]-4H-1,2,4-triazol-3-
amine (1)
Yield: 57%; 1H NMR (400 MHz, DMSO-d6): d 13.99 (s, 1H, H-4),
Yield: 74%; 1H NMR (300 MHz, DMSO-d6): d 9.41 (s, 1H,
0
0
0
0
9.15 (s, N@CHAAr), 8.22 (s, 1H, H-5), 7.91 (d, 2H, J2 ,3 = J6 ,5 = 8.4
Hz, H-20, H-60), 7.38 (d, 2H, J3 ,2 = J5 ,6 = 8.4 Hz, H-30, H-50), 3.32 (s,
N@CHAAr), 8.41 (s, 1H, H-5), 7.79 (d, 1H, J6 ,5 = 7.6 Hz, H-60),
0
0
0
0
0
0
3H, SCH3); EI-MS m/z (% rel. abund.): 218 [M]+ (95), 217 (100),
0
0
0
0
0
0
7.44 (t, 1H, J4 ,3 ,5 = 7.6 Hz, H-4 ), 6.98 (m, 2H, H-5 , H-3 ); EI-MS
m/z (% rel. abund.): 188 [M]+ (48), 171 (100), 84 (31); Anal. Calcd
for C9H8N4O, C = 57.44; H = 4.28; N = 29.77; O = 8.50. Found:
C = 57.42; H = 4.27; N = 29.78.
149 (77); Anal. Calcd for
C10H10N4S, C = 55.02; H = 4.62;
N = 25.67; S = 14.69. Found: C = 55.03; H = 4.61; N = 25.66.
4.3.9. N-[(30,5-Dichloro-20-hydroxyphenyl)methylidene]-4H-
1,2,4-triazol-3-amine (9)
4.3.2. N-[(20-Hydroxy-30-methoxyphenyl)methylidene]-4H-
1,2,4-triazol-3-amine (2)
Yield: 40%; 1H NMR (500 MHz, DMSO-d6): d 14.27 (s, 1H, H-4),
13.80 (s, 1H, OH), 9.41 (s, N@CHAAr), 8.63 (s, 1H, H-5), 7.94 (d, 1H,
Yield: 95%; 1H NMR (300 MHz, DMSO-d6): d 9.40 (s, N@CHAAr),
J4 ,6 = 1.1 Hz, H-40), 7.78 (d, 1H, J6 ,4 = 1.1 Hz, H-60); EI-MS m/z (%
rel. abund.): 258 [M+2]+ (41), 256 [M]+ (62), 243 (12), 241 (66),
239 (100); Anal. Calcd for C9H6Cl2N4O, C = 42.05; H = 2.35;
Cl = 27.58; N = 21.79; O = 6.22. Found: C = 42.04; H = 2.34;
N = 21.78.
8.42 (s, 1H, H-5), 7.38 (dd, 1H, J6 ,5 = 7.8 Hz, J6 ,4 = 1.3 Hz, H-60),
0
0
0
0
0
0
0
0
7.35 (dd, 1H, J4 ,5 = 7.6 Hz, J4 ,6 = 1.2 Hz, H-40), 6.91 (t, 1H,
J = 7.8 Hz, H-50), 3.82 (s, 3H, OCH3); EI-MS m/z (% rel. abund.):
218 [M]+ (47), 201 (100), 92 (24); Anal. Calcd for C10H10N4O2,
C = 55.04; H = 4.62; N = 25.68. Found: C = 55.02; H = 4.61;
N = 25.67.
0
0
0
0
4.3.10. N-[40-(Dimethylamino)phenyl]methylidene-4H-1,2,4-
triazol-3-amine (10)
4.3.3. N-[(20-Hydroxy-50-methylphenyl)methylidene]-4H-1,2,4-
triazol-3-amine (3)
Yield: 51%; 1H NMR (500 MHz, DMSO-d6): d 13.75 (s, 1H, H-4),
Yield: 65%; 1H NMR (400 MHz, DMSO-d6): d 9.35 (s, N@CHAAr),
0
0
0
0
8.99 (s, N@CHAAr), 8.04 (s, 1H, H-5), 7.78 (d, 2H, J2 ,3 = J6 ,5 = 8.5
Hz, H-20, H-60), 7.38 (d, 2H, J3 ,2 = J5 ,6 = 8.5 Hz, H-30, H-50), 3.03 (s,
6H, 2 ꢀ NCH3); EI-MS m/z (% rel. abund.): 215 [M]+ (100), 214
(100), 146 (85); Anal. Calcd for C11H13N5, C = 61.38; H = 6.09;
N = 32.54. Found: C = 61.37; H = 6.10; N = 32.53.
8.39 (s, 1H, H-5), 7.58 (s, 1H, H-60), 7.26 (d, 1H, J4 ,3 = 8.0 Hz, H-40),
0
0
0
0
0
0
6.88 (d, 1H, J3 ,4 = 8.0 Hz, H-30), 4.01 (s, 1H, OH), 3.16 (s, 3H, CH3),
2.26 (s, 3H, OCH3); EI-MS m/z (% rel. abund.): 202 [M]+ (72), 185
(100), 84 (86); Anal. Calcd for C10H10N4O, C = 59.40; H = 4.98;
N = 27.71; O = 7.91. Found: C = 59.41; H = 4.97; N = 27.70.
0
0
4.3.11. N-[(30,50-Dibromo-20-hydroxyphenyl)methylidene]-4H-
1,2,4-triazol-3-amine (11)
4.3.4. N-[(30,40-Dimethoxyphenyl)methylidene]-4H-1,2,4-
triazol-3-amine (4)
Yield: 51%; 1H NMR (300 MHz, DMSO-d6): d 14.24 (bs, 2H, H-4,
Yield: 69%; 1H NMR (400 MHz, DMSO-d6): d 13.92 (s, 1H, H-4),
9.11 (s, N@CHAAr), 8.22 (s, 1H, H-5), 7.58 (s, 1H, H-20), 7.53 (d, 1H,
0
0
OH), 9.38 (s, N@CHAAr), 8.61 (s, 1H, H-5), 8.07 (d, 1H, J4 ,6 = 2.4 Hz,
H-40), 7.96 (d, 1H, J6 ,4 = 2.4 Hz, H-60); EI-MS m/z (% rel. abund.):
348 [M++4] (51), 346 [M+2]+ (100), 344 [M]+ (56), 225 (26); Anal.
Calcd for C9H6Br2N4O, C = 31.24; H = 1.75; Br = 46.19; N = 16.19;
O = 4.62. Found: C = 31.22; H = 1.74; N = 16.17.
0
0
J6 ,5 = 8.4 Hz, H-60), 7.11 (d, 1H, J5 ,6 = 8.4 Hz, H-40), 3.84 (s, 3H,
OCH3), 3.83 (s, 3H, OCH3); EI-MS m/z (% rel. abund.): 232 [M]+
(99), 231 (100), 84 (9); Anal. Calcd for C11H12N4O2, C = 56.89;
H = 5.21; N = 24.12; O = 13.78. Found: C = 56.87; H = 5.20;
N = 24.11.
0
0
0
0
4.3.12. N-[(20-Hydroxyphenyl)methylidene]-4H-1,2,4-triazol-4-
amine (12)
Yield: 64%; 1H NMR (300 MHz, DMSO-d6): d 9.15 (s, 3H, H-3,
4.3.5. N-[(20,40-Dichlorophenyl)methylidene]-4H-1,2,4-triazol-3-
amine (5)
H-5, N@CHAAr), 7.77 (d, 1H, J6 ,5 = 7.8 Hz, H-60), 7.40 (t, 1H,
0
0
J4 ,3 ,5 = 8.4 Hz, H-40), 7.0 (d, 1H, J3 ,4 = 8.4 Hz, H-30), 6.92 (t, 1H,
Yield: 56%; 1H NMR (300 MHz, DMSO-d6): d 9.50 (s, N@CHAAr),
0
0
0
0
0
+
J5 ,4 ,6 = 7.5 Hz, H-50); EI-MS m/z (% rel. abund.): 188 [M] (99),
8.42 (s, 1H, H-5), 7.82 (d, 1H, J3 ,5 = 1.8 Hz, H-30), 7.20 (d, 1H,
0
0
0
0
0

K.M. Khan et al. / Bioorg. Med. Chem. 22 (2014) 6509–6514
6513
119 (44), 84 (100); Anal. Calcd for C9H8N4O, C = 57.44; H = 4.28;
N = 29.77, O = 8.50. Found: C = 57.42; H = 4.27; N = 29.78.
(s, 3H, CH3); EI-MS m/z (% rel. abund.): 216 [M]+ (100), 188 (41),
106 (84); Anal. Calcd for C11H12N4O, C = 61.10; H = 5.59;
N = 25.91; O = 7.40. Found: C = 61.11; H = 5.57; N = 25.92.
4.3.13. N-[(20,30-Dihydroxyphenyl)methylidene]-4H-1,2,4-triaz-
ol-4-amine (13)
4.3.20. N-[(20,40,60-Trihydroxyphenyl)methylidene]-4H-1,2,4-
triazol-4-amine (20)
Yield: 72%; 1H NMR (300 MHz, DMSO-d6): d 9.71 (s, 2H,
2 ꢀ OH), 9.15 (s, 3H, H-3, H-5, N@CHAAr), 7.22 (dd, 1H, J6 ,5 = 7.8
Yield: 42%; 1H NMR (500 MHz, DMSO-d6): d 10.62 (s, 2H, H-3,
H-5), 10.26 (s, N@CHAAr), 9.10 (s, 2H, 2xOH), 9.02 (s, 1H, OH),
5.89 (s, 2H, H-30, H-50); EI-MS m/z (% rel. abund.): 220 [M]+ (2),
151 (41), 44 (100); Anal. Calcd for C9H8N4O3, C = 49.09; H = 3.66;
N = 25.45; O = 21.80. Found: C = 49.08; H = 3.67; N = 25.43.
0
0
Hz, J6 ,4 = 1.5 Hz, H-60), 6.98 (dd, 1H, J4 ,5 = 7.8, J4 ,6 = 1.5 Hz, H-40),
0
0
0
0
0
0
6.76 (t, 1H, J5 ,6 ,4 = 7.8 Hz, H-50); EI-MS m/z (% rel. abund.): 204
[M]+ (100), 135 (84), 107 (51); Anal. Calcd for C9H8N4O2,
C = 52.94; H = 3.95; N = 27.44; O = 15.67. Found: C = 52.93;
H = 3.93; N = 27.43.
0
0
0
4.3.21. N-[(40-Bromophenyl)methylidene]-4H-1,2,4-triazol-4-
amine (21)
4.3.14. N-[(30-Hydroxyphenyl)methylidene]-4H-1,2,4-triazol-4-
amine (14)
Yield: 45%; 1H NMR (500 MHz, DMSO-d6): d 9.12 (s, 2H, H-3, H-
5), 9.08 (s, N@CHAAr), 7.78 (s, 4H, H-20, H-30, H-50, H-6); EI-MS m/z
(% rel. abund.): 252 [M+2]+ (94), 250 [M]+ (100), 89 (88); Anal.
Calcd for C9H7BrN4, C = 43.05; H = 2.81; Br = 31.82; N = 22.31.
Found: C = 43.04; H = 2.80; N = 22.30.
Yield: 46%; 1H NMR (300 MHz, DMSO-d6): d 9.86 (s, 1H, OH),
9.12 (s, 2H, H-3, H-5), 8.99 (s, N@CHAAr), 7.35 (m, 1H, H-50),
7.35 (m, 1H, H-60), 7.25 (m, 2H, H-40, H-20); EI-MS m/z (% rel.
abund.): 188 [M]+ (100), 84 (93); Anal. Calcd for C9H8N4O,
C = 57.44; H = 4.28; N = 29.77; O = 8.50. Found: C = 57.42;
H = 4.27; N = 29.78.
4.3.22. N-[(50-Chloro-20-hydroxyphenyl)methylidene]-4H-1,2,4-
triazol-4-amine (22)
Yield: 48%; 1H NMR (300 MHz, DMSO-d6): d 9.17 (s, 2H, H-3, H-
4.3.15. N-[(40-Hydroxyphenyl)methylidene]-4H-1,2,4-triazol-4-
amine (15)
5), 9.09 (s, N@CHAAr), 7.75 (d, 1H, J6 ,4 = 2.7 Hz, H-60), 7.44 (dd, 1H,
0
0
J4 ,3 = 9.0, J4 ,6 = 3.0 Hz, H-40), 7.00 (d, 1H, J3 ,4 = 9.0 Hz, H-30); EI-MS
m/z (% rel. abund.): 224 [M+2]+ (21), 222 [M]+ (60), 153 (100); Anal.
Calcd for C9H7ClN4O, C = 48.55; H = 3.17; Cl = 15.92; N = 25.17;
O = 7.19. Found: C = 48.54; H = 3.16; N = 25.16.
Yield: 76%; 1H NMR (400 MHz, DMSO-d6): d 10.34 (s, 1H, OH),
0
0
0
0
0
0
0
0
9.05 (s, 2H, H-3, H-5), 8.91 (s, N@CHAAr), 7.69 (d, 2H, J2 ,3
=
J6 ,5 = 8.8 Hz, H-20, H-60), 6.95 (d, 2H, J3 ,2 = J5 ,6 = 8.8 Hz, H-30, H-
50); EI-MS m/z (% rel. abund.): 188 [M]+ 100, 106 (95); Anal. Calcd
for C9H8N4O, C = 57.44; H = 4.28; N = 29.77; O = 8.50. Found:
C = 57.42; H = 4.27; N = 29.78.
0
0
0
0
0
0
4.3.23. N-[(50-Bromo-20-hydroxyphenyl)methylidene]-4H-1,2,4-
triazol-4-amine (23)
Yield: 60%; 1H NMR (300 MHz, DMSO-d6): d 10.80 (s, 1H, OH),
4.3.16. N-[(20-Hydroxy-30-methoxyphenyl)methylidene]-4H-1,2,
4-triazol-4-amine (16)
0
0
9.17 (s, 2H, H-3, H-5), 9.08 (s, N@CHAAr), 7.89 (d, 1H, J6 ,4 = 2.4 Hz,
H-60), 7.55 (dd, 1H, J4 ,3 = 8.7 Hz, J4 ,6 = 2.4 Hz, H-40), 6.95 (d, 1H,
Yield: 61%; 1H NMR (300 MHz, DMSO-d6): d 9.86 (s, 1H, OH),
0
0
0
0
+
J3 ,4 = 8.7 Hz, H-30); EI-MS m/z (% rel. abund.): 268 [M+2] (5),
266 [M]+ (5), 199 (100); Anal. Calcd for C9H7BrN4O, C = 40.47;
H = 2.64; Br = 29.92; N = 20.98; O = 5.99. Found: C = 40.48;
H = 2.63; N = 20.97.
0
0
0
0
0
0
9.16 (s, 3H, H-3, H-5, N@CHAAr), 7.37 (dd, 1H, J6 ,5 = 7.8, J6 ,4 = 1.2
Hz, H-60), 7.14 (dd, 1H, J4 ,5 = 7.8, J4 ,6 = 1.2 Hz, H-40), 6.89 (t, 1H,
0
0
0
0
J5 ,6 ,4 = 7.8 Hz, H-50), 3.85 (s, 3H, OCH3); EI-MS m/z (% rel. abund.):
218 [M]+ (100), 149 (100), 106 (83); Anal. Calcd for C10H10N4O2,
C = 55.04; H = 4.62; N = 25.68; O = 14.66. Found: C = 55.02;
H = 4.61; N = 25.67.
0
0
0
4.3.24. N-[(30,40-Dimethoxyphenyl)methylidene]-4H-1,2,4-triaz-
ol-4-amine (24)
Yield: 73%; 1H NMR (300 MHz, DMSO-d6): d 9.08 (s, 3H, H-3, H-
4.3.17. N-[(30-Ethoxy-20-hydroxyphenyl)methylidene]-4H-1,2,4-
triazol-4-amine (17)
5), 9.97 (s, N@CHAAr), 7.42 (d, 1H, J2 ,6 = 1.8 Hz, H-20), 7.37 (dd, 1H,
0
0
J6 ,5 = 8.4 Hz, J6 ,2 = 1.8 Hz, H-60), 7.13 (d, 1H, J5 ,6 = 9.0 Hz, H-50),
3.84 (s, 1H, OCH3), 3.82 (s, 1H, OCH3); EI-MS m/z (% rel. abund.):
232 [M]+ (100), 166 (27), 135 (40); Anal. Calcd for C11H12N4O2,
C = 56.89; H = 5.21; N = 24.12; O = 13.78. Found: C = 56.87;
H = 5.20; N = 24.11.
Yield: 48%; 1H NMR (300 MHz, DMSO-d6): d 9.85 (s, 1H, OH),
0
0
0
0
0
0
0
0
9.16 (s, 3H, H-3, H-5, N@CHAAr), 7.35 (dd, 1H, J6 ,5 = 8.1 Hz,
J6 ,4 = 1.2 Hz, H-60), 7.14 (dd, 1H, J4 ,5 = 8.1 Hz, J4 ,6 = 1.2 Hz, H-40),
0
0
0
0
0
0
6.88 (t, 1H, J5 ,6 ,4 = 7.8 Hz, H-50), 4.09 (s, 2H, OCH2), 1.36 (s, 3H,
CH3); EI-MS m/z (% rel. abund.): 232 [M]+ (100), 164 (36), 135
(100); Anal. Calcd for C11H12N4O2, C = 56.89; H = 5.21; N = 24.12;
O = 13.78. Found: C = 56.87; H = 5.23; N = 24.10.
0
0
0
4.3.25. N-[40-(Methylthio)phenyl]methylidene-4H-1,2,4-triazol-
4-amine (25)
Yield: 76%; 1H NMR (300 MHz, DMSO-d6): d 9.10 (s, 2H, H-3, H-
4.3.18. N-[(40-Methoxyphenyl)methylidene]-4H-1,2,4-triazol-4-
amine (18)
5), 9.07 (s, N@CHAAr), 7.76 (d, 1H, J2 ,3 = 8.4 Hz, H-20, H-60), 7.40 (d,
0
0
1H, J3 ,2 = 8.4, H-30, H-50), 1.13 (s, 3H, CH3); EI-MS m/z (% rel.
abund.): 218 [M]+ (91), 149 (100), 122 (70), 84 (56); Anal. Calcd
for C10H10N4S, C = 55.02; H = 4.62; N = 25.67; S = 14.69. Found:
C = 55.03; H = 4.61; N = 25.66.
Yield: 49%; 1H NMR (300 MHz, DMSO-d6): d 9.08 (s, 2H, H-3, H-
0
0
5), 8.98 (s, N@CHAAr), 7.79 (d, 2H, J2 ,3 = J6 ,5 = 8.7 Hz, H-60, H-20),
0
0
0
0
7.11 (d, 2H, J3 ,2 = J5 ,6 = 8.7 Hz, H-50, H-30), 3.84 (s, 3H, OCH3); EI-
0
0
0
0
MS m/z (% rel. abund.): 202 [M]+ (100), 120 (73), 105 (25); Anal.
Calcd for
Found: C = 59.41; H = 4.97; N = 27.70.
C10H10N4O, C = 59.40; H = 4.98; N = 27.71; O = 7.91.
Acknowledgments
4.3.19. N-[(40-Ethoxyphenyl)methylidene]-4H-1,2,4-triazol-4-
amine (19)
The authors are grateful to the Higher Education Commission
(HEC), Pakistan, for providing financial support through Project
20-1910 under the ‘National Research Program for Universities’,
and for award of indigenous fellowship through HEC 5000 indige-
nous fellowship program to one of the students.
Yield: 70%; 1H NMR (300 MHz, DMSO-d6): d 9.07 (s, 2H, H-3, H-
5), 8.98 (s, N@CHAAr), 7.78 (d, 2H, J2 ,3 = J6 ,5 = 9.0 Hz, H-60, H-20),
0
0
0
0
7.11(d, 2H, J3 ,2 = J5 ,6 = 9.0 Hz, H-50, H-30) 4.11 (s, 2H, OCH2), 1.35
0
0
0
0

6514
K.M. Khan et al. / Bioorg. Med. Chem. 22 (2014) 6509–6514
30. Boyd, S. A.; Fung, A. K. L.; Baker, W. R.; Mantei, R. A.; Stein, H. H.; Cohen, J.;
References and notes
Barlow, J. L.; Klinghofer, V.; Wessale, J. L.; Vreburg, K. M.; Polakowski, J. S.;
Adler, A. L.; Calzadilla, S. V.; Kovar, P.; Yao, Z.; Hutchins, C. W.; Denissen, J. F.;
Grabowski, B. A.; Cepa, S.; Hoffman, D. J.; Garren, K. W.; Kleinert, H. D. J. Med.
Chem. 1994, 37, 2991.
1. Al-Masoudi, N. A.; Al-Soud, Y. A.; Ali, I. A. Nucleos. Nucleot. Nucl. 2007, 26, 37.
2. Naito, Y.; Akahoshi, F.; Takeda, S.; Takada, T.; Kajii, M.; Nishimura, H.; Sugiura,
M.; Fukaya, C.; Kagitani, Y. J. Med. Chem. 1996, 39, 3019.
31. Hovsepian, T. R.; Dilanian, E. R.; Engoyan, A. P.; Melik-Ohanjanian, R. G. Chem.
Heterocycl. Compd. 2004, 40, 1194.
32. Tenor, E.; Ludwig, R. Pharmazie 1971, 26, 534.
33. Baiocchi, L.; Chiari, A.; Frigerio, A.; Ridolfi, P. Arzneim.-Forsch. 1973, 23, 400.
34. Hökfelt, B.; Jöhnsson, A. J. Med. Chem. 1962, 5, 247.
35. Boschelli, D. H.; Connor, D. T.; Bornemeier, D. A.; Dyer, R. D.; Kennedy, J. A.;
Kuipers, P. J.; Okonkwo, G. C.; Schrier, D. J.; Wright, C. D. J. Med. Chem. 1802,
1993, 36.
36. Iqbal, A. K. M.; Khan, A. Y.; Kalashetti, M. B.; Belavagi, N. S.; Gong, Y. D.; Khazi, I.
A. Eur. J. Med. Chem. 2012, 53, 308.
37. Al-Soud, Y. A.; Al-Dweri, M. N.; Al-Masoudi, N. A. IL Farmaco 2004, 59, 775.
38. Eliott, R.; Sunley, R. L.; Griffin, D. A. U.K. Pat. Appl. GB. 1986, 2, 175301.
39. Khan, K. M.; Fatima, N.; Rasheed, M.; Jalil, S.; Ambreen, N.; Perveen, S.;
Choudhary, M. I. Bioorg. Med. Chem. 2009, 17, 7816.
40. Razzell, W. E. Methods Enzymol. 1963, 6, 236.
41. Bollen, M.; Gijsbers, R.; Ceulemans, H.; Stalmans, W.; Stefan, C. Crit. Rev.
Biochem. Mol. Biol. 2000, 35, 393.
42. Goding, J. W.; Grobben, B.; Slegers, H. Biochim. Biophys. Acta 2003, 1638, 1.
43. Johnson, K.; Hashimoto, S.; Lotz, M.; Pritzker, K.; Goding, J.; Terkeltaub, R.
Arthritis Rheum. 2001, 44, 1071.
44. Okawa, A.; Nakamura, I.; Oto, S.; Moriya, H.; Nakamura, Y.; Ikegawa, S. Nat.
Genet. 1998, 19, 271.
45. Ahmed, V. U.; Abbasi, M. A.; Hussain, H.; Akhtar, M.; Farooq, U.; Fatima, N.;
Choudhary, M. I. Phytochemistry 2003, 63, 217.
46. Iqbal, K.; Anis, I.; Muhktar, N.; Malik, A.; Fatima, N.; Choudhary, M. I.
Heterocycles 2003, 60, 151.
47. Ahmed, V. U.; Abbasi, M. A.; Zubair, M.; Fatima, N.; Farooq, U.; Choudhary, M. I.
Helv. Chim. Acta 2004, 87, 67.
48. Choudhary, M. I.; Fatima, N.; Abbasi, A.; Jalil, S.; Ahmad, V. U.; Atta-ur-Rahman
Bioorg. Med. Chem. 2004, 12, 5793.
49. Abbasi, M. A.; Ahmed, V. U.; Zubair, M.; Fatima, N.; Farooq, U.; Hussain, S.;
Lodhi, M. A.; Choudhary, M. I. Planta Med. 2004, 70, 1189.
50. Atta-ur-Rahman; Naz, H.; Fadimatou, M. T.; Yasin, A.; Fatima, N.; Ngounou, F.
N.; Kimbu, S. F.; Sondengam, B. L.; Choudhary, M. I. J. Nat. Prod. 2005, 68, 189.
51. Ahmed, V. U.; Zubair, M.; Abbasi, M. A.; Kousar, F.; Fatima, N.; Choudhary, Z.
Naturforsch 2005, 60B, 1101.
3. De Clercq, E. J. Clin. Virol. 2004, 30, 115.
4. Collin, X.; Sauleau, A.; Coulon, J. Bioorg. Med. Chem. Lett. 2003, 13, 2601.
5. Sui, Z.; Guan, J.; Hlasta, D. J.; Macielag, M. J.; Foleno, B. D.; Goldschmidt, R. M.;
Loeloff, M. J.; Webb, G. C.; Barrett, J. F. Bioorg. Med. Chem. Lett. 1929, 1998, 8.
6. Hester, J. B.; Rudzik, A. D.; Kamdar, B. V. J. Med. Chem. 1971, 14, 1078.
7. Tanaka, G. Japan Kokai, 74, 95973. Chem. Abstr. 1975, 82, 156320h.
8. Lambert, P. A.; Somers, K. D.; Kohn, E. C.; Perry, R. R. Surgery 1997, 122, 372.
9. Akçay, H. T.; Bayrak, R. Spectrochim. Acta A: Mol. Biomol. Spectrosc. 2014, 122,
142.
10. Clemons, M.; Coleman, R. E.; Verma, S. Cancer Treat. Rev. 2004, 30, 325.
11. Bekircan, O.; Bektas, H. Molecules 2006, 11, 469.
12. Kane, J. M.; Baron, B. M.; Dudley, M. W.; Sorenson, S. M.; Staeger, M. A.; Miller,
F. P. J. Med. Chem. 1990, 33, 2772.
13. Küçükgüzel, I.; Küçükgüzel, S. G.; Rollas, S.; Ötük-Sanißs, G.; Özdemir, O.;
Bayrak, I.; Altug˘, T.; Stables, J. P. IL Farmaco 2004, 59, 893.
14. Gilbert, B. E.; Knight, V. Antimicrob. Agents Chemother. 1986, 30, 201.
15. Holla, B. S.; Veerendra, B.; Shivananda, M. K.; Poojary, B. Eur. J. Med. Chem.
2003, 38, 759.
16. Turan-Zitouni, G.; Sivaci, M. F.; Kiliç, S.; Erol, K. Eur. J. Med. Chem. 2001, 36, 685.
17. Bekircan, O.; Kucuk, M.; Kahveci, B.; Kolayli, S. Arch. Pharm. 2005, 338, 365.
18. Wade, P. C.; Vogt, B. R.; Kissick, T. P.; Simpkins, L. M.; Palmer, D. M.; Millonig, R.
C. J. Med. Chem. 1982, 25, 331.
19. Gruta, A. K.; Bhargava, K. P. Pharmazie 1978, 33, 430.
20. Modzelewska, B.; Kalabun, J. Pharmazie 1999, 54, 503.
21. Meyer, W. E.; Tomcufcik, A. S.; Chan, P. S.; Haug, M. J. Med. Chem. 1989, 32, 593.
22. Siddiqui, A. A.; Mishra, R.; Shaharyar, M.; Husain, A.; Rashid, M.; Pal, P. Bioorg.
Med. Chem. Lett. 2011, 21, 1023.
23. Lagoja, I. M.; Pannecouque, C.; Musumeci, L.; Froeyen, M.; Aerschot, A. V.;
Balzarini, J.; Herdewijn, P.; Clercq, E. D. Helv. Chim. Acta 1883, 2002, 85.
24. Boeglin, D.; Cantel, S.; Heitz, A.; Martinez, J.; Fehrentz, J. A. Org. Lett. 2003, 5,
4465.
25. Wadsworth, H. J.; Jenkins, S. M.; Orlek, B. S.; Cassidy, F.; Clark, M. S. G.; Brown,
F.; Riley, G. J.; Graves, D.; Hawlins, J.; Naylor, C. B. J. Med. Chem. 1992, 35, 1280.
26. Jenkins, S. M.; Wadsworth, H. J.; Bromidge, S.; Orlek, B. S.; Wyman, P. A.; Riley,
G. J.; Hawkins, J. J. Med. Chem. 1992, 35, 2392.
27. Burrell, G.; Evans, J. M.; Hadley, M. S.; Hicks, F.; Stemp, G. Bioorg. Med. Chem.
Lett. 1994, 4, 1285.
28. Tully, W. R.; Gardner, C. R.; Gillepsie, R. J.; Westwood, R. J. Med. Chem. 1991, 34,
2060.
29. Thompson, S. K.; Eppley, A. M.; Frazee, J. S.; Darcy, M. G.; Lum, R. T.; Tomaszeck,
T. A.; Ivanoff, L. A.; Morris, J. F.; Sternberg, E. J.; Lambert, D. M.; Fernandez, A.
V.; Petteway, S. R.; Meek, T. D.; Metclaff, B. W.; Gleason, J. G. Bioorg. Med. Chem.
Lett. 1994, 4, 2441.
52. Choudhary, M. I.; Fatima, N.; Khan, K. M.; Jalil, S.; Iqbal, S.; Atta-ur-Rahman
Bioorg. Med. Chem. 2006, 14, 8066.
53. Mostafa, M.; Nahar, N.; Mosihuzzaman, M.; Sokeng, S.; Fatima, N.; Atta-ur-
Rahman, ; Choudhary, M. I. Nat. Prod. Res. 2006, 20, 686.
54. Abby, L. P.; Daniel, L. B. Expert Opin. Ther. Pat. 2010, 20, 1619.
55. Olszewer, E.; Carter, J. P. Med. Hypotheses 1988, 27, 41.
56. Razzell, W. E.; Khorana, H. G. J. Biol. Chem. 1959, 234, 2114.