Synthesis, crystal structure and b-glucuronidase inhibition activity of some new hydrazinecarboxamides and their 1,2,4-triazole derivatives

Article abstract of DOI:10.1007/s00044-011-9929-1

A new series of hydrazinecarboxamides (5a-n), bearing various methoxyphenyl and methoxyphenethyl groups, was synthesized by condensation of corresponding acid hydrazides (3a-f) with methoxyphenylisocyanates (4a-c). Dehydrocyclization of hydrazinecarboxamides (5a-n) yielded corresponding methoxyphenyl and methoxyphenethyl substituted triazoles (6a-l) by refluxing in 2 N aqueous sodium hydroxide solution. All the synthesized compounds were screened for their b-glucuronidase inhibition activity. Compounds 5a, 5e, 5h, and 5l exhibited an excellent b-glucuronidase inhibitory activity as compared to the standard inhibitor. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9929-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3885–3896
DOI 10.1007/s00044-011-9929-1
ORIGINAL RESEARCH
Synthesis, crystal structure and b-glucuronidase inhibition
activity of some new hydrazinecarboxamides and their
1,2,4-triazole derivatives
•
Muhammad Hanif Imtiaz Khan Nasim Hasan Rama
•
•
•
Shagufta Noreen Muhammad Iqbal Choudhary
•
•
Peter G. Jones Mazhar Iqbal
Received: 13 June 2011 / Accepted: 1 December 2011 / Published online: 11 December 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A new series of hydrazinecarboxamides (5a–n),
bearing various methoxyphenyl and methoxyphenethyl
groups, was synthesized by condensation of corresponding
acid hydrazides (3a–f) with methoxyphenylisocyanates
(4a–c). Dehydrocyclization of hydrazinecarboxamides
(5a–n) yielded corresponding methoxyphenyl and meth-
oxyphenethyl substituted triazoles (6a–l) by refluxing in
2 N aqueous sodium hydroxide solution. All the synthe-
sized compounds were screened for their b-glucuronidase
inhibition activity. Compounds 5a, 5e, 5h, and 5l exhibited
an excellent b-glucuronidase inhibitory activity as com-
pared to the standard inhibitor.
Introduction
Detoxification mechanism, by which a substance is bonded
with glucuronic acid via a glycosidic bond to form glucu-
ronide conjugates is known as glucuronidation. The glu-
curonide conjugation occurred pre-dominantly in liver and
the conjugates become more water soluble which can be
readily excreted via the bile in small intestine resulting to
the excretion. This phenomenon is responsible for the
excretion of different xenobiotics (drugs and pollutants),
billirubin, hormones (androgen, estrogen, mineralocorti-
coid, and glucocorticoids), fatty acid derivatives and bile
acid (Kavak et al., 2010).
Keywords Heterocycles ꢀ Cyclization ꢀ
Hydrazinecarboxamides ꢀ 1,2,4-Triazoles ꢀ
b-Glucuronidase inhibition
In reverse to glucuronidation, b-glucuronidases catalyze
hydrolysis of b-D-glucuronic acid residues from these
conjugates (McCarter and Withers, 1994). Notably, b-glu-
curonidases activities are often associated with the general
process of inflammation (Kowalczyk et al., 2011) and have
also been found responsible for the promotion of cancers
(Beaud et al., 2005). The crude extract of medicinal
mushroom Ganoderma lucidum and natural products such
as silymarin, are proved as hepatoprotective crude drug and
are also found to be the potent inhibitors of b-glucuronidase
(Kim et al., 1994, 1999). There have been several other
reports which indicates that over expression of b-glucu-
ronidase is responsible for the promotion of cancers and the
compounds which are inhibitors to b-glucuronidase have
also shown anti-cancer activities (Kowalczyk et al., 2011).
Many bacterial species such as Escherichia coli,
Klebsiela, Clostridium, Staphyllococcus, and Bacillus
possess b-glucuronidase activities (Chouiter et al., 2008;
Leung et al., 2001). The activities of bacterial b-glucu-
ronidase in the large intestine can play a major role in the
generation of toxic and carcinogenic materials (Rod and
Midtvedt, 1977). In addition, Camptothecin-11 (CPT-11), a
M. Hanif ꢀ I. Khan ꢀ N. H. Rama (&)
Department of Chemistry, Quaid-i-Azam University,
Islamabad 45320, Pakistan
e-mail: nasimhr@yahoo.com
S. Noreen ꢀ M. I. Choudhary
H.E.J. Research Institute of Chemistry, International Center
for Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
P. G. Jones
Institute for Inorganic and Analytical Chemistry,
Technical University of Braunschweig Hagenring 30, 38106
Braunschweig, Germany
M. Iqbal
National Institute for Biotechnology & Genetic Engineering
(NIBGE), Faisalabad, Pakistan
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Med Chem Res (2012) 21:3885–3896
new semi-synthetic derivative of CPT, has also been shown
to inhibit DNA topoisomerase I and a broad spectrum of
antitumor activity (Fukuoka et al., 1992; Mathijssen et al.,
2001). Hence, the bacterial b-glucuronidase can also
be considered as cancer-risk biomarker (McBain and
Macfarlane, 1998). Glucuronic acid type iminosugars in
which nitrogen atom is placed at anomeric position (Igar-
ashi et al., 1996) and glycopolymers, having pendant
acyclic mannaric group, possess excellent glucuronidase
inhibition activity (Kawaguchi et al., 2009).
of 4.46–4.16 ppm confirmed the formation of hydrazides
(3a–f).
Condensation of (3a–f) with isocyanates (4a–c) yielded
hydrazinecarboxamides (5a–n) (Zheng et al., 2003; Khan
et al., 2010), which were confirmed by IR and NMR
spectra. The disappearance of typical N=C=O stretching in
the range of 2250–2150 cm-1 and the appearance of two
peaks due to C=O groups in the range of 1716–1603 cm-1
indicated the formation of hydrazinecarboxamides (5a–n).
Typical NH stretching vibrations in (5a–n) appeared in the
1
Compounds incorporating heterocyclic ring systems
continue to attract considerable interest due to the wide
range of biological activities they possess. Among them
five membered heterocyclic compounds particularly azoles
occupy a unique place in the realm of natural and synthetic
organic chemistry (Katrizky, 1984; Saadeh et al., 2010).
1,2,4-Triazoles represent an overwhelming and rapid
developing field in modern heterocyclic chemistry. A
degree of respectability has been bestowed for 1,2,4-tria-
zole derivatives due to their wide spectrum of biological
importance (Karthikeyan et al., 2007).
range of 3423–3185 cm-1. In H NMR, three NH protons
appeared in the range of 10.01–7.89 ppm as broad signals.
Additional inference for the formation of hydrazinecarb-
oxamides was obtained by CHN analysis. These com-
pounds were the intermediates for the synthesis of
4,5-disubstituted-1,2,4-triazoles (6a–l). Dehydrocycliza-
tion (Moise et al., 2009, Shi et al., 2001) of (5a–n) in 2 N
sodium hydroxide solution yielded 4,5-disubstituted-1,
2,4-triazoles (6a–l). The structure elucidations of the newly
synthesized compounds were carried out by spectroscopic
1
techniques like IR, H NMR, and 13C NMR. Further con-
From literature, it is predictable that 1,2,4-triazoles rep-
resent important pharmacophores, and have a wide range of
therapeutic properties. They play vital role as medicinal
agents due to different biological activities and lot of work
might be carried out on this moiety for obtaining better
therapeutic molecules (Karthikeyan et al., 2007).
firmation of the synthesized compounds was carried out by
mass spectroscopy and CHN analysis.
Formation of triazoles (6a–l) was indicated by the dis-
appearance of signals due to C=O group in the IR spectra.
A slightly broad peak in the range of 3178–3153 cm-1 was
1
a typical signal for NH group. In H NMR spectra of tri-
Keeping in view the importance of these compounds, we
herein report the synthesis and b-glucuronidase inhibition
of hydrazinecarboxamides (5a–n) with different substitu-
tion pattern and their triazole derivatives (6a–l).
azoles, a deshielded, relatively broad signal, in the range of
11.68–11.54 ppm was due to NH group. NH proton exists
as proton transfer amide–imidic tautomeric form and due to
this reason, this proton is highly deshielded. Due to
unavoidable laboratory limitations and reaction conditions
only fourteen hydrazinecarboxamides (5a–n) and twelve
1,2,4-triazoles were synthesized. All the reactions were
monitored by TLC and newly synthesized compounds were
purified by column chromatography, if necessary. The
structure of one triazole derivative (6a) was unambigu-
ously deduced by single crystal X-ray diffraction analysis.
Results and discussion
Chemistry
Synthetic pathway adopted for the synthesis of hydrazin-
ecarboxamides and 4,5-disubstituted-2H-1,2,4-triazole-
3(4H)-ones is illustrated in Scheme 1. Substituted aromatic
esters (2a–f) were synthesized by the reaction of corre-
sponding substituted aromatic acids (1a–f) in the presence
of catalytic amount of sulfuric acid. The esters (2a–f) were
converted into corresponding aromatic acid hydrazides
(3a–f) by refluxing with hydrazine hydrate (80%) in
methanol (Abid et al., 2010). Synthesis of hydrazides
(3a–f) was confirmed by IR and NMR spectroscopy. In the
IR spectra, the typical sharp absorption was observed in the
range of 3338–3296 and 3204–3187 cm-1 for primary and
secondary amines, respectively.
Crystal structure determination of compound (6a)
Crystal data
C17H17N3O3, Mr = 311.34. Monoclinic, space group P21/c,
˚
a = 7.5008(3), b = 8.3750(3), c = 24.7827(8) A, b =
3
˚
97.489(4)°, V = 1543.54(10) A at 100 K, Z = 4, Dx =
1.340 Mg/m3, l = 0.094 mm-1, F(000) = 656.
Data collection
The 1H NMR exhibited a singlet in the range of
9.96–8.97 ppm for NH proton, next to the carbonyl group.
A broad singlet for two protons for NH2 group in the range
A colorless tablet 0.40 9 0.30 9 0.20 mm3 was mounted
on a glass fiber in inert oil and transferred to the cold gas
stream of the diffractometer (Oxford Diffraction Xcalibur
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Med Chem Res (2012) 21:3885–3896
3887
Scheme 1 Synthetic pathway for the synthesis of title compounds 5(a–n) and 6(a–l)
E). A total of 39910 intensities, leading to 4431 indepen-
dent reflections, were collected to 2hmax 60° using mono-
˚
chromated Mo Ka radiation (k = 0.71073 A).
Structure solution and refinement
The molecular structure (Fig. 1) was solved by routine
direct methods and refined on F2 using the program
SHELXL-97 (Sheldrick, 2008). The H atom of the NH
group was refined freely and methyl groups were refined
as idealized rigid groups allowed to rotate but not tip;
other H atoms were included using a riding model. The
final wR2 was 0.0898 for all reflections and 214 param-
eters with a conventional R1 of 0.0349; S = 1.07, max.
-3
Fig. 1 The molecular structure of compound (6a)
˚
Dq 0.302 eA
.
Crystallographic data have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication no. CCDC-800846, copies of the data can
be obtained free of charge from www.ccdc.cam.ac.
uk/data_request/cif.
The exclusive presence of the NH–C (=O) tautomer is
confirmed by the relevant molecular dimensions and by the
direct localization and free refinement of the NH hydrogen.
Ellipsoids correspond to 50% probability levels. Selected
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Med Chem Res (2012) 21:3885–3896
˚
bond lengths (A) and angles (°): N1–C5 1.2988(12),
N1–N2 1.3891(11), N2–C3 1.3503(12), C3–O1
1.2412(11), C3–N4 1.3830(11), N4–C5 1.3822(11), N4–
C11 1.4327(11); C5–N1–N2 103.96(7), C3–N2–N1
113.11(8), O1–C3–N2 128.78(8), O1–C3–N4 127.72(8),
N2–C3–N4 103.51(8), C5–N4–C3 107.67(7), C5–N4–C11
126.93(7), C3–N4–C11 125.34(7), N1–C5–N4 111.73(8),
N1–C5–C27 125.64(8), N4–C5–C27 122.60(8).
and 5l. The IC50 values indicate that compound 5h showed
the lowest IC50 = 15.5 1.29 lM among all the com-
pounds, as well as the standard. In the compound 5h, OCH3
is at ortho and para positions, of the ring A and B,
respectively when n = 2.
A slight decrease in activity was observed in compound
5e (IC50 = 16.8 1.8 lM) by switching the position of
methoxy groups at aromatic rings while n = 1. Com-
pounds in which this methoxy is present at ortho position
in both phenyl substituents, a significant lowering in IC50
was detected as in case of 5a (IC50 = 40.7 1.55 lM). In
compound 5l, with IC50 = 46.1 1.14 lM, the methoxy
in ring A is at para, while at ring B it occupies ortho
position with n = 2. If methoxy group at meta position in
A ring and ortho position in B ring as in 5i with
IC50 = 52.4 0.44 lM. These results demonstrate that
the presence of methoxy at para position on the benzene
ring when n = 2 promising for disturbance in association
with enzyme and this combination may be responsible for a
decrease in activity against b-glucuronidase enzyme.
Figure 2 reveals the packing diagram of compound (6a),
showingtheformationofmolecularchainsparalleltotheshort
x axis via classical inversion-symmetric H bonding systems
N2-H2O1 and the ‘‘weak’’ H bond C16 ꢁ H16L ꢀ ꢀ ꢀ O1.
Pharmacological studies
b-Glucuronidase inhibition assay
Two series of synthesized compounds were screened for
b-glucuronidase inhibitory activity. The first series com-
prising hydrazinecarboxamides (5a–n) showed a signifi-
cant inhibition of the enzyme while the second series
consisting of triazole derivatives (6a–l) were devoid of any
inhibition. The compounds showing more than 50% inhi-
bition were selected for IC50 evaluation. The activity
results are shown in Table 1.
Conclusion
New series of hydrazinecarboxamides (5a–n) and their tria-
zole derivatives (6a–l) were synthesized. The b-glucuroni-
dase inhibition ability of these compounds was evaluated.
Some of the compounds 5a, 5e, 5h, and 5l showed excellent
activity more than the standard drug and 5i showed significant
The hydrazinecarboxamide derivatives showed a sig-
nificant to moderate activity, mainly due to the presence of
methoxy group at different positions on both rings as side
chain. The most significant compounds were 5a, 5e, 5h, 5i,
Fig. 2 Packing diagram of
compound (6a)
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Med Chem Res (2012) 21:3885–3896
3889
General procedure for the synthesis of substituted
aromatic esters (2a–f) and aromatic acid hydrazides
(3a–f)
Table 1 Results of inhibitory activity of hydrazinecarboxamide
derivatives (5a–n) and 1,2,4-triazoles (6a–l) against b-glucuronidase
at 0.2 mM concentration
Compound
%
IC50
Compound %
inhibition
inhibition (lM) SEM
Substituted aromatic esters (2a–f) were synthesized by
refluxing corresponding substituted aromatic acids
(1a–f) (0.022 mol) in methanol (20 mL) and catalytic
amount of sulfuric acid. These esters were converted into
corresponding aromatic acid hydrazides (3a–f) by refluxing
with hydrazine hydrate (80%) in methanol following the
literature procedure (Abid et al., 2010).
5a
5b
5c
5d
5e
5f
88.8
46.5
68.3
70.8
94.5
77.6
54.6
87.9
82.9
52.0
73.3
83.7
59.3
53.6
86.2
40.7 1.55 6a
ND 6b
–
–
–
–
–
–
–
–
–
–
–
–
133.8 3.13 6c
101.4 2.97 6d
16.8 1.80 6e
67.3 1.93 6f
83.7 0.43 6g
15.5 1.29 6h
52.4 0.44 6i
178.6 3.8 6j
74.5 2.21 6k
46.1 1.14 6l
96.7 5.03
5g
5h
5i
2-(2-Methoxyphenyl) acetohydrazide (3a)
The general experimental procedure described above
afforded (3a) as white solid. Yield: 89%; m.p 121–122°C;
Rf: 0.21 (petroleum ether : ethyl acetate, 8:2); IR (Pure,
cm-1): 3312 (NH2), 3187 (NH), 1626 (C=O), 1597 (C=C),
1249 (C–O); 1H NMR (300 MHz, DMSO-d6): d (ppm)
9.26 (s, 1H, NH), 7.48–7.40 (m, 2H, Ar–H), 7.19–7.08 (m,
2H, Ar–H), 4.46 (bs, 2H, NH2), 3.96 (s, 3H, OCH3), 3.51
(s, 2H, CH2C=O); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 170.36 (C=O), {130.92, 128.64, 124.93, 120.77,
114.31, 111.31}(Ar–C), 55.81 (OCH3), 35.22 (CH2); Anal.:
Calcd. for C9H12N2O2: C, 59.99; H, 6.71; N, 15.55. Found:
C, 59.65; H, 6.50; N, 15.43.
5j
5k
5l
5m
5n
180 4.10
D-Saccharic acid
1,4-lactonea
48.4 1.25
Results are Standard error of mean (SEM) of three assays
ND Not determine; – no activity
a
Standard inhibitor
activity. Newly discovered b-glucuronidase inhibitors
required to be further assessed for their curative potential.
2-(3-Methoxyphenyl) acetohydrazide (3b)
The general experimental procedure described above
afforded (3b) as light brown solid. Yield: 82%; m.p
83–85°C; Rf: 0.20 (petroleum ether : ethyl acetate, 8:2); IR
(Pure, cm-1); 3308 (NH2), 3181 (NH), 1617 (C=O), 1593,
1491 (C=C), 1248 (C–O); 1H NMR (300 MHz, DMSO-d6):
d (ppm) 9.20 (s, 1H, NH), 7.20 (t, 1H, J = 8.1 Hz, Ar–H),
6.84–6.72 (m, 3H, Ar–H), 4.45 (bs, 2H, NH2), 3.73 (s, 3H,
OCH3), 3.31 (s, 2H, CH2C=O); 13C NMR (75 MHz,
DMSO-d6): d (ppm) 169.89 (C=O), {138.17, 129.62,
121.67, 115.16, 112.21}(Ar–C), 55.40 (OCH3), 40.98
(CH2); Anal.: Calcd. for C9H12N2O2: C, 59.99; H, 6.71; N,
15.55. Found: C, 59.68; H, 6.56; N, 15.35.
Experimental
All the common solvents were of analytical grade or dry
distilled. Melting points were measured on a Gallenkamp
melting point apparatus (MP-D) and are uncorrected. IR
Spectra were recorded on Alpha FTIR, Bruker Optics
spectrophotometer. NMR spectra have been recorded on a
Bruker Avance 300 MHz spectrometer with TMS as an
internal standard and on 75 MHz (13C) (scale in d). The
multiplicities were expressed as s = singlet, d = doublet,
t = triplet, q = quartet, dt = doublet of triplets. Mass
spectra were recorded on Mass Spec LTQXL, using ESI
(positive) probe using ethyl acetate as solvent. Samples
were diluted with LCMS grade methanol and filtered
through PTFE membrane filter (0.45 lm pore size) before
injecting them using direct syringe pump. The elemental
analysis was performed on Leco CHNO-932 Elemental
Analyzer, Leco Corporation (USA). The Rf values were
determined on pre-coated silica gel aluminum plates,
Kieslgel 60 F254 Merck (Germany), using petroleum ether:
ethyl acetate (Khan et al., 2010) as an eluent and chro-
matograms were visualized under UV at 254 and 365 nm.
2-(4-Methoxyphenyl) acetohydrazide (3c)
The general experimental procedure described above
afforded (3c) as brownish solid. Yield: 86%; m.p
126–127°C; Rf: 0.21 (petroleum ether : ethyl acetate, 8:2);
IR (Pure, cm-1); 3338 (NH2), 3205 (NH), 1610 (C=O),
1507, 1490 (C=C), 1234 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 9.16 (s, 1H, NH), 7.18 (d, 2H,
J = 8.7 Hz, Ar–H), 6.85 (d, 2H, J = 8.7, Ar–H), 4.21 (bs,
2H, NH2), 3.71 (s, 3H, OCH3), 3.27 (s, 2H, CH2C=O); 13
C
NMR (75 MHz, DMSO-d6): d (ppm) 170.38 (C=O),
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Med Chem Res (2012) 21:3885–3896
General procedure for the synthesis
{158.37, 130.82, 130.37, 128.64, 114.06}(Ar–C), 55.47
(OCH3), 40.51 (CH2); Anal.: Calcd. for C9H12N2O2: C,
59.99; H, 6.71; N, 15.55. Found: C, 59.69; H, 6.58; N,
15.49.
of hydrazinecarboxamide derivatives (5a–n)
The substituted aromatic acid hydrazide (3) (2.7 mmol)
was dissolved in methanol (30 mL) and substituted isocy-
anate (4) (2.7 mmol) was separately dissolved in methanol
(20 mL). Both of these solutions were mixed slowly with
continuous stirring. The reaction mixture was refluxed for
10–12 h and progress of the reaction was monitored by
TLC. After consumption of the starting material, the
reaction mixture was cooled to room temperature and
methanol was evaporated on a rotary evaporator to leave
behind crude corresponding hydrazinecarboxamide (5) as
oil which solidified on cooling. Crude product was purified
by recrystallization in aqueous ethanol to yield purified
hydrazinecarboxamide (5) (Zheng et al., 2003; Khan et al.,
2010).
3-(2-Methoxyphenyl) propanehydrazide (3d)
The general experimental procedure described above
afforded (3d) as light yellow crystalline solid. Yield: 85%;
m.p 82–84°C; Rf: 0.19 (petroleum ether : ethyl acetate,
8:2); IR (Pure, cm-1): 3296 (NH2), 3204 (NH), 1637
(C=O), 1525, 1491 (C=C), 1238(C–O); 1H NMR
(300 MHz, DMSO-d6): d (ppm) 9.96 (s, 1H, NH), 7.10 (d,
2H, J = 8.4 Hz, Ar–H), 6.83 (d, 2H, J = 8.4 Hz, Ar–H),
4.16 (bs, 2H, NH2), 3.70 (s, 3H, OCH3), 2.75 (t, 2H,
J = 7.5 Hz, CH2), 2.29 (t, 2H, J = 7.5 Hz, CH2); 13C
NMR (75 MHz, DMSO-d6): d (ppm) 171.33 (C=O),
{157.98, 133.54, 129.58, 114.15, 113.68}(Ar–C), 55.40
(OCH3), 35.89 (CH2), 30.63 (CH2); Anal.: Calcd. for
C10H14N2O2: C, 61.84; H, 7.27; N, 14.42. Found: C, 61.66;
H, 7.49; N, 14.31.
N-(2-Methoxyphenyl)-2{2-(2-methoxyphenyl)
acetyl}hydrazinecarboxamide (5a)
The general experimental procedure described above
afforded (5a) as white solid. Yield: 61%; m.p 177–179°C;
Rf: 0.29 (petroleum ether : ethyl acetate, 6:4); IR (Pure,
cm-1): 3341, 3185 (NH), 1663, 1619 (C=O), 1614, 1598,
1540 (C=C), 1241 (C–O); 1H NMR (300 MHz, DMSO-d6):
d (ppm) 9.95 (s, 1H, NH), 8.63 (bs, 1H, NH), 8.09 (bs, 1H,
NH), 8.04–8.02 (m, 2H, Ar–H), 7.21 (d, 2H, J = 7.2 Hz,
Ar–H), 6.98–6.86 (m, 4H, Ar–H), 3.83 (s, 3H, OCH3), 3.72
(s, 3H, OCH3), 3.41 (s, 2H, CH2); 13C NMR (75 MHz,
DMSO-d6): d (ppm) 170.81 (C=O), 158.46 (C=O),
{155.50, 148.00, 130.47, 128.86, 128.01, 122.36, 120.97,
118.57, 114.15, 111.16}(Ar–C), 56.18 (OCH3), 55.49
(OCH3), 34.62 (CH2); ESI/MS (m/z, ? ion mode): 329.91
[M ? H]?, 352.11 [M ? Na]?; Anal.: Calcd. for
C17H19N3O4: C, 62.00; H, 5.81; N, 12.76. Found: C, 61.91;
H, 5.69; N, 12.63.
3-(3-Methoxyphenyl) propanehydrazide (3e)
The general experimental procedure described above
afforded (3e) as light brown solid. Yield: 80%; m.p
88–89°C; Rf: 0.22 (petroleum ether : ethyl acetate, 8:2); IR
(Pure, cm-1): 3321 (NH2), 3197 (NH), 1635 (C=O), 1593,
1526, 1491 (C=C), 1256 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 8.98 (s, 1H, NH), 7.18 (t, 1H,
J = 7.8 Hz, Ar–H), 6.77–6.72 (m, 3H, Ar–H), 4.17 (bs, 2H,
NH2), 3.12 (s, 3H, OCH3), 2.78 (t, 2H, J = 7.5 Hz, CH2),
2.33 (t, 2H, J = 7.5 Hz, CH2); 13C NMR (75 MHz, DMSO-
d6): d (ppm) 171.25 (C=O), {159.72, 143.27, 129.73,
120.88, 114.29, 111.79}(Ar–C), 55.32 (OCH3), 35.43
(CH2), 31.49 (CH2); Anal.: Calcd. for C10H14N2O2: C,
61.84; H, 7.27; N, 14.42. Found: C, 61.75; H, 7.52; N, 14.37.
N-(3-Methoxyphenyl)-2-(2-(2-methoxyphenyl)
acetyl)hydrazinecarboxamide (5b)
3-(4-Methoxyphenyl) propanehydrazide (3f)
The general experimental procedure described above
afforded (3f) as white crystalline solid. Yield: 85%; m.p
131–133°C; Rf: 0.21 (petroleum ether : ethyl acetate, 8:2);
IR (Pure, cm-1): 3311 (NH2), 3202 (NH), 1630 (C=O),
1526, 1510 (C=C), 1244 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 8.97 (s, 1H, NH), 7.20–7.10 (m, 2H,
Ar–H), 6.93–6.85 (m, 2H, Ar–H), 4.17 (bs, 2H, NH2), 3.16
(s, 3H, OCH3), 2.79 (t, 2H, J = 7.5 Hz, CH2), 2.28 (t, 2H,
J = 7.5 Hz, CH2); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 171.57 (C=O), {157.47, 129.80, 127.80, 120.67,
110.98}(Ar–C), 55.66 (OCH3), 33.91 (CH2), 26.19 (CH2);
Anal.: Calcd. for C10H14N2O2: C, 61.84; H, 7.27; N, 14.42.
Found: C, 61.91; H, 7.52; N, 14.35.
The general experimental procedure described above
afforded (5b) as white solid. Yield.: 68%; m.p 153–155°C;
Rf: 0.28 (petroleum ether : ethyl acetate, 6:4); IR (Pure,
cm-1): 3423, 3285 (NH), 1653, 1634 (C=O), 1619, 1598,
1494 (C=C), 1241 (C–O); 1H NMR (300 MHz, DMSO-d6):
d (ppm) 9.93 (s, 1H, NH), 8.55 (bs, 1H, NH), 8.01 (bs, 1H,
NH), 8.03–8.01 (m, 2H, Ar–H), 7.21 (d, 2H, J = 7.2 Hz,
Ar–H), 6.98–6.86 (m, 4H, Ar–H), 3.82 (s, 3H, OCH3), 3.71
(s, 3H, OCH3), 3.45 (s, 2H, CH2); 13C NMR (75 MHz,
DMSO-d6): d (ppm) 170.85 (C=O), 158.61 (C=O),
{156.51, 148.06, 131.51, 128.94, 128.11, 123.42, 122.0,
118.68, 114.21, 112.27}(Ar–C), 55.75 (OCH3), 55.58
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Med Chem Res (2012) 21:3885–3896
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(OCH3), 34.25 (CH2); ESI/MS (m/z, ? ion mode): 329.89
[M ? H]?, 352.06 [M ? Na]?; Anal.: Calcd. for
C17H19N3O4: C, 62.00; H, 5.81; N, 12.76. Found: C, 61.93;
H, 5.78; N, 12.58.
NH), 7.23 (t, 1H, J = 7.8 Hz, Ar–H), 6.98–6.80 (m, 7H,
Ar–H), 3.83 (s, 3H, OCH3), 3.74 (s, 3H, OCH3), 3.47 (s,
2H, CH2); 13C NMR (75 MHz, DMSO-d6): d (ppm) 170.38
(C=O), 159.66 (C=O), {155.52, 148.63, 148.03, 137.58,
129.71, 129.29, 128.87, 122.38, 121.73, 119.60,
112.41}(Ar–C), 56.16 (OCH3), 55.40 (OCH3), 34.56
(CH2); ESI/MS (m/z, ? ion mode): 329.94 [M ? H]?,
352.08 [M ? Na]?; Anal.: Calcd. for C17H19N3O4: C,
62.00; H, 5.81; N, 12.76. Found: C, 61.93; H, 5.72; N,
12.58.
N-(2-Methoxyphenyl)-2{2-(3-methoxyphenyl)
acetyl}hydrazinecarboxamide (5c)
The general experimental procedure described above
afforded (5c) as white solid. Yield: 72%; m.p 140–142°C;
Rf: 0.31 (petroleum ether : ethyl acetate, 6:4); IR (Pure,
cm-1): 3335, 3228 (NH), 1712, 1639 (C=O), 1603, 1541
N-(2-Methoxyphenyl)-2-(3-(2-methoxyphenyl)
propanoyl)hydrazinecarboxamide (5f)
1
(C=C), 1248 (C–O); H NMR (300 MHz, DMSO-d6): d
(ppm) 9.84 (s, 1H, NH), 8.63 (bs, 1H, NH), 8.08 (bs, 1H,
NH), 7.24–6.90 (m, 8H, Ar–H), 3.83 (s, 3H, OCH3), 3.76
(s, 3H, OCH3), 3.47 (s, 2H, CH2); 13C NMR (75 MHz,
DMSO-d6): d (ppm) 170.65 (C=O), 157.61 (C=O),
{155.56, 148.03, 130.98, 128.91, 128.45, 124.21, 122.34,
120.99, 120.60, 118.64, 111.12} (Ar–C), 56.20 (OCH3),
55.85 (OCH3), 34.76 (CH2); ESI/MS (m/z, ? ion mode):
329.92 [M ? H]?, 352.10 [M ? Na]?; Anal.: Calcd. for
C17H19N3O4: C, 62.00; H, 5.81; N, 12.76. Found: C, 61.89;
H, 5.65; N, 12.61.
The general experimental procedure described above
afforded (5f) as light brown solid. Yield: 76%; m.p
154–155°C; Rf: 0.29 (petroleum ether : ethyl acetate, 6:4);
IR (Pure, cm-1): 3351, 3203 (NH), 1701, 1651 (C=O),
1598, 1542 (C=C), 1250 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 9.78 (s, 1H, NH), 8.62 (bs, 1H, NH),
8.13 (bs, 1H, NH), 7.19 (t, 1H, J = 8.1 Hz, Ar–H),
7.01–6.73 (m, 7H, Ar–H), 3.84 (s, 3H, OCH3), 3.73 (s, 3H,
OCH3), 2.85 (t, 2H, J = 7.2 Hz, CH2), 2.47 (t, 2H,
J = 7.2 Hz, CH2); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 171.84 (C=O), 159.75 (C=O), {155.56, 153.19,
148.62, 148.03, 143.04, 129.79, 129.25, 128.91, 122.36,
114.29, 111.14}(Ar–C), 56.16 (OCH3), 55.32 (OCH3),
35.18 (CH2), 31.26 (CH2); ESI/MS (m/z, ? ion mode):
343.89 [M ? H]?, 366.01 [M ? Na]?; Anal.: Calcd. for
C18H21N3O4: C, 62.96; H, 6.16; N, 12.24. Found: C, 62.85;
H, 6.05; N, 12.11.
N-(3-Methoxyphenyl)-2{2-(3-methoxyphenyl)
acetyl}hydrazinecarboxamide (5d)
The general experimental procedure described above
afforded (5d) as white solid. Yield: 68%; m.p 158–160°C;
Rf: 0.30 (petroleum ether : ethyl acetate, 6:4); IR (Pure,
cm-1); 3282, 3253 (NH), 1716, 1639 (C=O), 1618, 1594
1
(C=C), 1223 (C–O); H NMR (300 MHz, DMSO-d6): d
(ppm) 9.78 (s, 1H, NH), 8.67 (bs, 1H, NH), 8.07 (bs, 1H,
NH), 7.26–7.13 (m, 4H, Ar–H), 6.99–6.87 (m, 4H, Ar–H),
3.77 (s, 3H, OCH3), 3.71 (s, 3H, OCH3), 3.48 (s, 2H, CH2);
13C NMR (75 MHz, DMSO-d6): d (ppm) 170.70 (C=O),
160.07 (C=O), {157.61, 155.721, 154.38, 141.32, 140.83,
131.04, 130.00, 129.90, 128.43, 124.26, 120.60,
111.18}(Ar–C), 55.88 (OCH3), 55.36 (OCH3), 34.12
(CH2); ESI/MS (m/z, ? ion mode): 329.92 [M ? H]?,
352.05 [M ? Na]?; Anal.: Calcd. for C17H19N3O4: C,
62.00; H, 5.81; N, 12.76. Found: C, 61.91; H, 5.73; N,
12.55.
N-(3-Methoxyphenyl)-2-(3-(2-methoxyphenyl)
propanoyl)hydrazinecarboxamide (5g)
The general experimental procedure described above
afforded (5g) as white solid. Yield: 58%; m.p 154–155°C;
Rf: 0.31 (petroleum ether : ethyl acetate, 6:4); IR (Pure,
cm-1); 3303, 3213 (NH), 1637, 1603 (C=O), 1568, 1511
1
(C=C), 1229 (C–O); H NMR (300 MHz, DMSO-d6): d
(ppm) 9.66 (s, 1H, NH), 8.66 (bs, 1H, NH), 8.00 (bs, 1H,
NH), 7.18-7.12 (m, 4H, Ar–H), 6.97 (d, 1H, J = 8.1 Hz,
Ar–H), 6.84 (d, 2H, J = 8.4 Hz, Ar–H), 6.54 (dd, 1H,
J = 8.1, 2.1 Hz, Ar–H), 3.71 (s, 3H, OCH3), 3.36 (s, 3H,
OCH3), 2.79 (t, 2H, J = 7.2 Hz, CH2), 2.42 (t, 2H,
J = 7.2 Hz, CH2); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 171.95 (C=O), 160.05 (C=O), {158.02, 155.71,
141.31, 133.39, 129.88, 129.63, 114.19, 111.19, 107.68,
104.67}(Ar–C), 55.42 (OCH3), 55.35 (OCH3), 35.65
(CH2), 30.25 (CH2); ESI/MS (m/z, ? ion mode): 343.91
[M ? H]?, 366.05 [M ? Na]?; Anal.: Calcd. for
C18H21N3O4: C, 62.96; H, 6.16; N, 12.24. Found: C, 62.81;
H, 6.15; N, 12.13.
N-(2-Methoxyphenyl)-2-(2-(4-methoxyphenyl)
acetyl)hydrazinecarboxamide (5e)
The general experimental procedure described above
afforded (5e) as white solid. Yield: 78%; m.p 156–157°C;
Rf: 0.30 (petroleum ether : ethyl acetate, 6:4); IR (Pure,
cm-1): 3342, 3208 (NH), 1699, 1651 (C=O), 1594, 1537
1
(C=C), 1254 (C–O); H NMR (300 MHz, DMSO-d6): d
(ppm) 10.01 (s, 1H, NH), 8.67 (bs, 1H, NH), 8.12 (bs, 1H,
123

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Med Chem Res (2012) 21:3885–3896
N-(4-Methoxyphenyl)-2-(3-(2-methoxyphenyl)
propanoyl)hydrazinecarboxamide (5h)
1H, J = 8.1, 2.1 Hz, Ar–H), 3.73 (s, 3H, OCH3), 3.71 (s,
3H, OCH3), 2.84 (t, 2H, J = 7.2 Hz, CH2), 2.46 (t, 2H,
J = 7.2 Hz, CH2); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 171.85 (C=O), 160.05 (C=O), {155.69, 143.14,
141.32, 129.87, 129.79, 120.89, 114.28, 111.94, 107.68,
104.65}(Ar–C), 55.35 (OCH3), 54.36 (OCH3), 35.21
(CH2), 31.12 (CH2); ESI/MS (m/z, ? ion mode): 343.89
[M ? H]?, 366.08 [M ? Na]?; Anal.: Calcd. for
C18H21N3O4: C, 62.96; H, 6.16; N, 12.24. Found: C, 62.72;
H, 6.02; N, 12.13.
The general experimental procedure described above
afforded (5h) as white solid. Yield: 81%; m.p 140–142°C;
Rf: 0.32 (petroleum ether : ethyl acetate, 6:4); IR (Pure,
cm-1): 3321, 3196 (NH), 1651, 1638 (C=O), 1583, 1538,
1508 (C=C), 1241 (C–O); 1H NMR (300 MHz, DMSO-d6):
d (ppm) 9.63 (s, 1H, NH), 8.46 (bs, 1H, NH), 7.92 (bs, 1H,
NH), 7.36–7.30 (m, 2H, Ar–H), 7.15 (d, 2H, J = 8.4 Hz,
Ar–H), 6.87–6.82 (m, 4H, Ar–H), 3.71 (s, 3H, OCH3), 3.63
(s, 3H, OCH3), 2.79 (t, 2H, J = 7.2 Hz, CH2), 2.44 (t, 2H,
J = 7.2 Hz, CH2); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 171.97 (C=O), 158.01 (C=O), {156.07, 155.22,
154.94, 133.41, 129.62, 120.75, 120.30, 114.39,
114.28}(Ar–C), 55.59 (OCH3), 55.41 (OCH3), 35.67
(CH2), 30.25 (CH2); ESI/MS (m/z, ? ion mode): 343.91
[M ? H]?, 366.09 [M ? Na]?; Anal.: Calcd. for
C18H21N3O4: C, 62.96; H, 6.16; N, 12.24. Found: C, 62.78;
H, 6.11; N, 12.16.
N-(4-Methoxyphenyl)-2-(3-(3-methoxyphenyl)
propanoyl)hydrazinecarboxamide (5k)
The general experimental procedure described above
afforded (5k) as yellow solid. Yield: 74%; m.p 152–153°C;
Rf: 0.31 (petroleum ether : ethyl acetate, 6:4); IR (Pure,
cm-1): 3289, 3206 (NH), 1656, 1603 (C=O), 1558, 1509
1
(C=C), 1252 (C–O); H NMR (300 MHz, DMSO-d6): d
(ppm) 9.65 (s, 1H, NH), 8.47 (bs, 1H, NH), 7.93 (bs, 1H,
NH), 7.24 (d, 2H, J = 8.1 Hz, Ar–H), 7.19 (t, 1H,
J = 8.1 Hz, Ar–H), 6.87–6.74 (m, 5H, Ar–H), 3.73 (s, 3H,
OCH3), 3.70 (s, 3H, OCH3), 2.83 (t, 2H, J = 7.2 Hz, CH2),
2.45 (t, 2H, J = 7.2 Hz, CH2); 13C NMR (75 MHz,
DMSO-d6): d (ppm) 171.88 (C=O), 159.76 (C=O),
{156.06, 155.22, 154.94, 154.62, 143.17, 133.09, 132.64,
129.79, 120.89, 114.40, 114.29, 111.92}(Ar–C), 55.59
(OCH3), 55.34 (OCH3), 35.22 (CH2), 31.11 (CH2); ESI/MS
(m/z, ? ion mode): 343.83 [M ? H]?, 366.11 [M ? Na]?;
Anal.: Calcd. for C18H21N3O4: C, 62.96; H, 6.16; N, 12.24.
Found: C, 62.79; H, 6.11; N, 12.03.
N-(2-Methoxyphenyl)-2-(3-(3-methoxyphenyl)
propanoyl)hydrazinecarboxamide (5i)
The general experimental procedure described above
afforded (5i) as white solid. Yield: 73%; m.p 164–165°C;
Rf: 0.28 (petroleum ether : ethyl acetate, 6:4); IR (Pure,
cm-1): 3328, 3203 (NH), 1712, 1648 (C=O), 1609, 1512
1
(C=C), 1241 (C–O); H NMR (300 MHz, DMSO-d6,): d
(ppm) 9.76 (s, 1H, NH), 8.62 (bs, 1H, NH), 8.13 (bs, 1H,
NH), 7.16 (d, 2H, J = 8.7 Hz, Ar–H), 7.01–6.81 (m, 6H,
Ar–H), 3.86 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 2.80 (t,
2H, J = 7.2 Hz, CH2), 2.43 (t, 2H, J = 7.2 Hz, CH2); 13C
NMR (75 MHz, DMSO-d6): d (ppm) 171.9 (C=O), 158.04
(C=O), {155.57, 153.19, 148.63, 133.69, 133.32, 129.30,
122.33, 121.00, 119.60, 114.19, 111.24} (Ar–C), 56.17
(OCH3), 55.41 (OCH3), 35.64 (CH2), 30.42 (CH2); ESI/MS
(m/z, ? ion mode): 343.83 [M ? H]?, 366.12 [M ? Na]?;
Anal.: Calcd. for C18H21N3O4: C, 62.96; H, 6.16; N, 12.24.
Found: C, 62.81; H, 6.01; N, 12.09.
N-(2-Methoxyphenyl)-2-(3-(4-methoxyphenyl)
propanoyl)hydrazinecarboxamide (5l)
The general experimental procedure described above
afforded (5l) as white crystalline solid. Yield: 67%; m.p
155–157°C; Rf: 0.29 (petroleum ether : ethyl acetate, 6:4);
IR (Pure, cm-1): 3324, 3236 (NH), 1698, 1644 (C=O),
1599, 1537, 1491 (C=C), 1241(C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 9.74 (s, 1H, NH), 8.60 (bs, 1H, NH),
8.13 (bs, 1H, NH), 7.21–7.15 (m, 2H, Ar–H), 7.01–6.84
(m, 6H, Ar–H), 3.86 (s, 3H, OCH3), 3.81 (s, 3H, OCH3),
2.84 (t, 2H, J = 7.2 Hz, CH2), 2.44 (t, 2H, J = 7.2 Hz,
CH2); 13C NMR (75 MHz, DMSO-d6): d (ppm) 172.06
(C=O), 157.49 (C=O), {155.58, 153.18, 148.63, 129.86,
129.29, 127.91, 122.32, 120.89, 119.60, 118.54, 111.25,
111.02}(Ar–C), 56.17 (OCH3), 55.68 (OCH3), 33.61
(CH2), 25.99 (CH2); ESI/MS (m/z, ? ion mode): 343.88
[M ? H]?, 366.10 [M ? Na]?; Anal.: Calcd. for
C18H21N3O4: C, 62.96; H, 6.16; N, 12.24. Found: C, 62.81;
H, 6.06; N, 12.14.
N-(3-Methoxyphenyl)-2-(3-(3-methoxyphenyl)
propanoyl)hydrazinecarboxamide (5j)
The general experimental procedure described above
afforded (5j) as white crystalline solid. Yield: 75%; m.p
147–148°C; Rf: 0.29 (petroleum ether : ethyl acetate, 6:4);
IR (Pure, cm-1): 3342, 3225 (NH), 1703, 1647 (C=O),
1598, 1530 (C=C), 1261 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 9.67 (s, 1H, NH), 8.67 (bs, 1H, NH),
8.01 (bs, 1H, NH), 7.22–7.12 (m, 3H, Ar–H), 6.96 (d, 1H,
J = 8.4 Hz, Ar–H), 6.81–6.74 (m, 2H, Ar–H), 6.54 (dd,
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N-(3-Methoxyphenyl)-2-(3-(4-methoxyphenyl)
propanoyl)hydrazinecarboxamide (5m)
3-(2-Methoxybenzyl)-4-(2-methoxyphenyl)-
1H-1,2,4-triazol-5(4H)-one (6a)
The general experimental procedure described above
afforded (5m) as white solid. Yield: 69%; m.p 158–160°C;
Rf: 0.29 (petroleum ether : ethyl acetate, 6:4); IR (Pure,
cm-1): 3353, 3225 (NH), 1705, 1642 (C=O), 1596, 1530,
1491 (C=C), 1242 (C–O); 1H NMR (300 MHz, DMSO-d6):
d (ppm) 9.66 (s, 1H, NH), 8.71 (bs, 1H, NH), 8.00 (bs, 1H,
NH), 7.21–7.13 (m, 5H, Ar–H), 6.96 (t, 1H, J = 9.3 Hz,
Ar–H), 6.86 (t, 1H, J = 7.2 Hz, Ar–H), 6.55 (dd, 1H,
J = 8.1, 2.1 Hz, Ar–H), 3.79 (s, 3H, OCH3), 3.71 (s, 3H,
OCH3), 2.82 (t, 2H, J = 7.2 Hz, CH2), 2.41 (t, 2H,
J = 7.2 Hz, CH2); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 172.15 (C=O), 160.05 (C=O), {155.76, 141.34,
129.85, 129.23, 127.89, 120.73, 111.17, 111.00, 107.67,
104.64}(Ar–C), 56.52 (OCH3), 55.34 (OCH3), 33.62
(CH2), 25.84 (CH2); ESI/MS (m/z, ? ion mode): 343.94
[M ? H]?, 366.07 [M ? Na]?; Anal.: Calcd. for
C18H21N3O4: C, 62.96; H, 6.16; N, 12.24. Found: C, 62.82;
H, 6.07; N, 12.12.
The general experimental procedure described above
afforded (6a) as white crystals. Yield: 78%; m.p
139–140°C; Rf: 0.25 (petroleum ether : ethyl acetate, 5:5);
IR (Pure, cm-1): 3154 (NH), 1706 (C=O), 1578 (C=N),
1556, 1505 (C=C), 1252 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 11.60 (s, 1H, NH), 7.43 (dt, 1H,
J = 8.7, 1.8 Hz, Ar–H) 7.08 (d, 2H, J = 9.0 Hz, Ar–H),
6.97 (dt, 1H, J = 7.8, 1.2 Hz, Ar–H), 6.80–6.71 (m, 4H,
Ar–H), 3.67 (s, 3H, OCH3), 3.62 (s, 3H, OCH3), 3.36 (s,
2H, CH2); 13C NMR (75 MHz, DMSO-d6): d (ppm) 158.39
(C=O), {155.49, 154.99, 147.44, 131.23, 130.39, 127.24,
121.46, 120.92, 113.98, 112.69}(Ar–C), 55.98 (OCH3),
55.46 (OCH3), 31.58 (CH2); ESI/MS (m/z, ? ion mode):
312.13 [M ? H]?, 334.02 [M ? Na]?; Anal.: Calcd. for
C17H17N3O3: C, 65.58; H, 5.50; N, 13.50. Found: C, 65.49;
H, 5.38; N, 13.41.
3-(2-Methoxybenzyl)-4-(3-methoxyphenyl)-
1H-1,2,4-triazol-5(4H)-one (6b)
N-(4-Methoxyphenyl)-2-(3-(4-methoxyphenyl)
propanoyl)hydrazinecarboxamide (5n)
The general experimental procedure described above
afforded (6b) as white crystalline solid. Yield: 58%; m.p
150–152°C; Rf: 0.22 (petroleum ether : ethyl acetate, 5:5);
IR (Pure, cm-1): 3175 (NH), 1661 (C=O), 1582 (C=N),
1540, 1504 (C=C), 1249 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 11.63 (s, 1H, NH), 7.45 (t, 1H,
J = 8.1 Hz, Ar–H) 7.21 (dt, 1H, J = 9.3, 1.8 Hz, Ar–H),
7.01–6.86 (m, 2H, Ar–H), 6.77–6.71 (m, 4H, Ar–H), 3.78
The general experimental procedure described above affor-
ded (5n) as white solid. Yield: 61%; m.p 188–190°C; Rf: 0.33
(petroleum ether : ethyl acetate, 6:4); IR (Pure, cm-1): 3319,
3188 (NH), 1667, 1632 (C=O), 1581, 1543 (C=C), 1237
(C–O);1H NMR (300 MHz, DMSO-d6):d (ppm) 9.60(s, 1H,
NH), 8.49 (bs, 1H, NH), 7.89 (bs, 1H, NH), 7.34 (d, 2H,
J = 9.0 Hz, Ar–H), 7.21–7.14 (m, 2H, Ar–H), 6.96–6.90 (m,
2H, Ar–H), 6.88–6.83 (m, 2H, Ar–H), 3.79 (s, 3H, OCH3),
3.70 (s, 3H, OCH3), 2.80 (t, 2H, J = 7.2 Hz, CH2), 2.40 (t,
2H, J = 7.2 Hz, CH2); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 172.13 (C=O), 157.49 (C=O), {154.09, 133.12,
129.83, 127.27, 120.73, 114.29, 111.03}(Ar–C), 55.69
(OCH3), 55.60 (OCH3), 33.63 (CH2), 25.81 (CH2); ESI/MS
(m/z, ? ion mode): 343.89 [M ? H]?, 366.11 [M ? Na]?;
Anal.: Calcd. for C18H21N3O4: C, 62.96; H, 6.16; N, 12.24.
Found: C, 62.83; H, 6.05; N, 12.10.
(s, 3H, OCH3), 3.65 (s, 3H, OCH3), 3.31 (s, 2H, CH2); 13
C
NMR (75 MHz, DMSO-d6): d (ppm) 161.38 (C=O),
{157.47, 154.76, 146.93, 134.52, 130.56, 130.09, 128.32,
128.17, 120.70, 114.63, 113.63, 111.05} (Ar–C), 54.81
(OCH3), 53.58 (OCH3), 31.01(CH2); ESI/MS (m/z, ? ion
mode): 312.13 [M ? H]?, 334.06 [M ? Na]?; Anal.:
Calcd. for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50.
Found: C, 65.51; H, 5.42; N, 13.39.
3-(3-Methoxybenzyl)-4-(2-methoxyphenyl)-
1H-1,2,4-triazol-5(4H)-one (6c)
General procedure for the synthesis of 1,2,4-triazol-
5(4H)-ones (6a–l)
The general experimental procedure described above
afforded (6c) as white solid. Yield: 61%; m.p 176–178°C;
Rf: 0.23 (petroleum ether : ethyl acetate, 5:5); IR (Pure,
cm-1): 3167 (NH), 1698 (C=O), 1583 (C=N), 1571, 1526
The corresponding hydrazinecarboxamide (5), (1.5 mmol)
were refluxed in aqueous sodium hydroxide solution (2 N,
25 mL). The progress of the reaction was monitored by
TLC. After completion of reaction, the contents were
cooled to room temperature and filtered. The filtrate was
then neutralized with hydrochloric acid (2 N) to precipitate
the triazole (6) which was filtered and recrystallized from
aqueous ethanol (Moise et al., 2009; Shi et al., 2001).
1
(C=C), 1242 (C–O); H NMR (300 MHz, DMSO-d6): d
(ppm) 11.60 (s, 1H, NH), 7.43 (dt, 1H, J = 8.7, 1.8 Hz,
Ar–H) 7.08 (d, 2H, J = 9.0 Hz, Ar–H), 6.97 (dt, 1H,
J = 7.8, 1.2 Hz, Ar–H), 6.80–6.70 (m, 4H, Ar–H), 3.67 (s,
3H, OCH3), 3.62 (s, 3H, OCH3), 3.36 (s, 2H, CH2); 13C
NMR (75 MHz, DMSO-d6): d (ppm) 158.4 (C=O), {155.5,
123

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Med Chem Res (2012) 21:3885–3896
155.0, 147.4, 131.2, 130.0, 127.2, 121.5, 120.9, 114.0,
112.7}(Ar–C), 55.98, 55.50 (OCH3), 3.60 (CH2); ESI/MS
(m/z, ? ion mode): 312.09 [M ? H]?, 334.05 [M ? H]?;
Anal.: Calcd. for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50.
Found: C, 65.45; H, 5.38; N, 13.35.
IR (Pure, cm-1): 3160 (NH), 1696 (C=O), 1587 (C=N),
1543, 1497 (C=C), 1241 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 11.56 (s, 1H, NH), 7.48 (t, 1H,
J = 7.5, Ar–H) 7.23 (t, 2H, J = 7.2 Hz, Ar–H), 7.16–7.04
(m, 2H, Ar–H), 6.73 (d, 1H, J = 7.2 Hz, Ar–H), 6.62–6.60
(m, 2H, Ar–H), 3.77 (s, 3H, OCH3), 3.68 (s, 3H, OCH3),
2.70 (t, 2H, J = 7.8 Hz, CH2), 2.47 (t, 2H, J = 7.8 Hz,
CH2); 13C NMR (75 MHz, DMSO-d6): d (ppm) 159.69
(C=O), {155.46, 154.91, 147.32, 142.37, 131.32, 130.37,
129.79, 121.52, 121.24, 114.34, 112.99, 111.98}(Ar–C),
56.22 (OCH3), 55.32 (OCH3), 31.58 (CH2), 27.82 (CH2);
ESI/MS (m/z, ? ion mode): 326.11 [M ? H]?, 348.04
[M ? Na]?; Anal.: Calcd. for C18H19N3O3: C, 66.45; H,
5.89; N, 12.91. Found: C, 66.36; H, 5.78; N, 12.80.
3-(3-Methoxybenzyl)-4-(3-methoxyphenyl)-
1H-1,2,4-triazol-5(4H)-one (6d)
The general experimental procedure described above
afforded (6d) as white solid. Yield: 68%; m.p 135–137°C;
Rf: 0.27 (petroleum ether : ethyl acetate, 5:5); IR (Pure,
cm-1): 3165 (NH), 1689 (C=O), 1582 (C=N), 1556, 1492
1
(C=C), 1244 (C–O); H NMR (300 MHz, DMSO-d6): d
(ppm) 11.69 (s, 1H, NH), 7.34 (t, 1H, J = 8.1 Hz, Ar–H)
7.17 (t, 1H, J = 7.7 Hz, Ar–H), 7.01–6.80 (m, 6H, Ar–H),
3.70 (s, 3H, OCH3), 3.61 (s, 3H, OCH3), 3.44 (s, 2H, CH2);
13C NMR (75 MHz, DMSO-d6): d (ppm) 160.08 (C=O),
{157.00, 154.79, 146.35, 134.37, 130.37, 130.15, 128.69,
123.68, 120.62, 119.99, 113.44, 111.06}(Ar–C), 55.75
(OCH3), 55.69 (OCH3), 26.48 (CH2); ESI/MS (m/z, ? ion
mode): 312.05 [M ? H]?, 334.06 [M ? H]?; Anal.:
Calcd. for C17H17N3O3: C, 65.58; H, 5.50; N, 13.50.
Found: C, 65.47; H, 5.38; N, 13.38.
3-(2-Methoxyphenethyl)-4-(3-methoxyphenyl)-1H-
1,2,4-triazol-5(4H)-one (6g)
The general experimental procedure described above
afforded (6g) as light yellow solid. Yield: 63%; m.p
130–132°C; Rf: 0.24 (petroleum ether : ethyl acetate, 5:5);
IR (Pure, cm-1): 3165 (NH), 1667 (C=O), 1585 (C=N),
1540, 1510 (C=C), 1249 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 11.63 (s, 1H, NH), 7.42 (t, 1H,
J = 8.1 Hz, Ar–H) 7.15 (dt, 1H, J = 9.0, 1.8 Hz, Ar–H),
7.06–6.96 (m, 2H, Ar–H), 6.97–6.78 (m, 4H, Ar–H), 3.78
(s, 3H, OCH3), 3.65 (s, 3H, OCH3), 2.68 (t, 2H,
J = 7.8 Hz, CH2), 2.62 (t, 2H, J = 7.8 Hz, CH2); 13C
NMR (75 MHz, DMSO-d6): d (ppm) 160.28 (C=O),
{157.47, 154.76, 146.93, 134.52, 130.56, 130.09, 128.32,
128.17, 120.70, 114.63, 113.63, 111.05}(Ar–C), 55.89
(OCH3), 55.52 (OCH3), 27.07 (CH2), 26.51 (CH2); ESI/MS
(m/z, ? ion mode): 326.08 [M ? H]?, 348.11 [M ? Na]?;
Anal.: Calcd. for C18H19N3O3: C, 66.45; H, 5.89; N, 12.91.
Found: C, 66.35; H, 5.79; N, 12.78.
3-(4-Methoxybenzyl)-4-(2-methoxyphenyl)-
1H-1,2,4-triazol-5(4H)-one (6e)
The general experimental procedure described above
afforded (6e) as white crystalline solid. Yield: 67%; m.p
142–144°C; Rf: 0.26 (petroleum ether : ethyl acetate, 5:5);
IR (Pure, cm-1): 3161 (NH), 1699 (C=O), 1580 (C=N),
1505, 1497 (C=C), 1241 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 11.63 (s, 1H, NH), 7.42 (dt, 1H,
J = 8.7, 1.5 Hz, Ar–H), 7.11–7.05 (m, 3H, Ar–H), 6.97
(dt, 1H, J = 7.5, 1.6 Hz, Ar–H), 6.72 (dd, 1H, J = 8.1,
2.1 Hz, Ar–H) 6.47 (dd, 1H, J = 7.8, 1.5 Hz, Ar–H), 6.38
(m, 1H, Ar–H), 3.62 (s, 3H, OCH3), 3.61 (s, 3H, OCH3),
3.35 (s, 2H, CH2); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 159.46 (C=O), {155.49, 154.92, 147.04, 136.93,
131.24, 130.39, 129.59, 127.24, 121.45, 120.91, 114.46,
112.68, 112.65} (Ar–C), 55.95 (OCH3), 55.30 (OCH3),
32.48 (CH2); ESI/MS (m/z, ? ion mode): 312.10
[M ? H]?, 334.05 [M ? Na]?; Anal.: Calcd. for
C17H17N3O3: C, 65.58; H, 5.50; N, 13.50. Found: C, 65.48;
H, 5.41; N, 13.38.
3-(2-Methoxyphenethyl)-4-(4-methoxyphenyl)-
1H-1,2,4-triazol-5(4H)-one (6h)
The general experimental procedure described above
afforded (6h) as white solid. Yield: 69%; m.p 166–167°C;
Rf: 0.28 (petroleum ether : ethyl acetate, 5:5); IR (Pure,
cm-1): 3165 (NH), 1687 (C=O), 1585 (C=N), 1540, 1498
1
(C=C), 1249 (C–O); H NMR (300 MHz, DMSO-d6): d
(ppm) 11.55 (s, 1H, NH), 7.40 (dt, 1H, J = 8.4, 2.7 Hz,
Ar–H), 7.16–7.07 (m, 3H, Ar–H), 6.95 (dt, 2H, J = 7.8,
1.2 Hz, Ar–H), 6.81–6.76 (m, 2H, Ar–H), 3.66 (s, 3H,
OCH3), 3.58 (s, 3H, OCH3), 3.36 (s, 2H, CH2) 2.65 (t, 2H,
J = 7.8 Hz, CH2), 2.62 (t, 2H, J = 7.8 Hz, CH2); 13C
NMR (75 MHz, DMSO-d6): d (ppm) 157.03 (C=O),
{155.49, 154.94, 147.14, 131.03, 130.29, 130.15, 128.53,
123.43, 121.51, 120.85, 120.43, 112.59, 110.89} (Ar–C),
55.99 (OCH3), 55.67 (OCH3), 27.17 (CH2), 27.15 (CH2);
3-(2-Methoxyphenethyl)-4-(2-methoxyphenyl)-
1H-1,2,4-triazol-5(4H)-one (6f)
The general experimental procedure described above
afforded (6f) as white crystalline solid. Yield: 65%; m.p
175–177°C; Rf: 0.25 (petroleum ether : ethyl acetate, 5:5);
123

Med Chem Res (2012) 21:3885–3896
3895
ESI/MS (m/z, ? ion mode): 326.11 [M ? H]?, 348.13
[M ? Na]?; Anal.: Calcd. for C18H19N3O3: C, 66.45; H,
5.89; N, 12.91. Found: C, 66.35; H, 5.73; N, 12.81.
cm-1): 3165 (NH), 1698 (C=O), 1584 (C=N), 1557, 1506
1
(C=C), 1243 (C–O); H NMR (300 MHz, DMSO-d6): d
(ppm) 11.62 (s, 1H, NH), 7.47 (dt, 1H, J = 8.4, 1.5 Hz,
Ar–H), 7.22–7.11 (m, 3H, Ar–H), 7.05 (t, 1H, J = 7.5 Hz,
Ar–H), 6.95 (dd, 1H, J = 7.2, 1.2 Hz, Ar–H), 6.86 (d, 2H,
J = 8.1 Hz, Ar–H), 3.67 (s, 3H, OCH3), 3.65 (s, 3H,
OCH3), 2.76 (t, 2H, J = 7.8 Hz, CH2), 2.62 (t, 2H,
J = 7.8 Hz, CH2); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 157.42 (C=O), {155.40, 155.00, 147.88, 131.36,
130.40, 130.02, 128.27, 128.20, 121.37, 121.26, 112.87,
111.05}(Ar–C), 55.15 (OCH3), 55.49 (OCH3), 27.11
(OCH2), 26.15 (CH2); ESI/MS (m/z, ? ion mode): 326.11
[M ? H]?, 348.04 [M ? Na]?; Anal.: Calcd. for
C18H19N3O3: C, 66.45; H, 5.89; N, 12.91. Found: C, 66.33;
H, 5.80; N, 12.81.
3-(3-Methoxyphenethyl)-4-(2-methoxyphenyl)-
1H-1,2,4-triazol-5(4H)-one (6i)
The general experimental procedure described above
afforded (6i) as white crystalline solid. Yield: 71%; m.p
184–186°C; Rf: 0.25 (petroleum ether : ethyl acetate, 5:5);
IR (Pure, cm-1): 3178 (NH), 1694 (C=O), 1606 (C=N),
1582, 1507 (C=C), 1239 (C–O); 1H NMR (300 MHz,
DMSO-d6): d (ppm) 11.54 (s, 1H, NH), 7.49 (dt, 1H,
J = 8.7, 1.5 Hz, Ar–H), 7.27–7.20 (m, 2H, Ar–H), 7.07 (t,
1H, J = 7.5 Hz, Ar–H), 6.95 (d, 2H, J = 8.4 Hz, Ar–H),
6.78 (d, 2H, J = 8.4 Hz, Ar–H), 3.77 (s, 3H, OCH3), 3.68
(s, 3H, OCH3), 2.65 (t, 2H, J = 7.8 Hz, CH2), 2.46 (t, 2H,
J = 7.8 Hz, CH2); 13C NMR (75 MHz, DMSO-d6): d
(ppm) 158.08 (C=O), {155.45, 154.91, 146.36, 132.65,
130.48, 129.61, 121.52, 121.24, 114.17, 112.96}(Ar–C),
556.20 (OCH3), 55.41 (OCH3), 30.64 (CH2), 28.20 (CH2);
ESI/MS (m/z, ? ion mode): 326.11 [M ? H]?, 348.06
[M ? Na]?; Anal.: Calcd. for C18H19N3O3: C, 66.45; H,
5.89; N, 12.91. Found: C, 66.29; H, 5.69; N, 12.73.
3-(4-Methoxyphenethyl)-4-(3-methoxyphenyl)-
1H-1,2,4-triazol-5(4H)-one (6l)
The general experimental procedure described above
afforded (6l) as white solid. Yield: 65%; m.p 161–163°C;
Rf: 0.27 (petroleum ether : ethyl acetate, 5:5); IR (Pure,
cm-1): 3173 (NH), 1713 (C=O), 1601 (C=N), 1572, 1514,
1493 (C=C), 1224 (C–O); 1H NMR (300 MHz, DMSO-d6):
d (ppm) 11.68 (s, 1H, NH), 7.42 (t, 1H, J = 8.4 Hz, Ar–H),
7.05–7.01 (m, 1H, Ar–H), 6.97 (d, 2H, J = 8.7 Hz, Ar–H),
6.92–6.89 (m, 2H, Ar–H), 6.77 (d, 2H, J = 8.7 Hz, Ar–H),
3.76 (s, 3H, OCH3), 3.68 (s, 3H, OCH3), 2.66 (t, 2H,
J = 7.8 Hz, CH2), 2.51 (t, 2H, J = 7.8 Hz, CH2); 13C
NMR (75 MHz, DMSO-d6): d (ppm) 160.29 (C=O),
{158.09, 154.77, 146.75, 134.41, 132.58, 129.65, 119.83,
114.75, 114.18, 115.50}(Ar–C), 55.89 (OCH3), 55.39
(OCH3), 30.60 (OCH2), 28.45 (CH2); ESI/MS (m/z, ? ion
mode): 326.05 [M ? H]?, 348.06 [M ? Na]?; Anal.:
Calcd. for C18H19N3O3: C, 66.45; H, 5.89; N, 12.91.
Found: C, 66.29; H, 5.72; N, 12.79.
3-(3-Methoxyphenethyl)-4-(3-methoxyphenyl)-
1H-1,2,4-triazol-5(4H)-one (6j)
The general experimental procedure described above
afforded (6j) as white solid. Yield: 78%; m.p 114–115°C;
Rf: 0.26 (petroleum ether : ethyl acetate, 5:5); IR (Pure,
cm-1): 3154 (NH), 1691 (C=O), 1604 (C=N), 1578, 1490
1
(C=C), 1224 (C–O); H NMR (300 MHz, DMSO-d6): d
(ppm) 11.66 (s, 1H, NH), 7.42 (t, 1H, J = 8.1 Hz, Ar–H)
7.14 (t, 1H, J = 8.1 Hz, Ar–H), 7.05 (dd, 1H, J = 8.1,
1.8 Hz, Ar–H), 6.94–6.91 (m, 2H, Ar–H), 6.73 (dd, 2H,
J = 9.6, 2.1 Hz, Ar–H), 6.65–6.63 (m, 2H, Ar–H), 3.77 (s,
3H, OCH3), 3.68 (s, 3H, OCH3), 2.71 (OCH3), 2.49 (s, 2H,
CH2); 13C NMR (75 MHz, DMSO-d6): d (ppm) 160.31
(C=O), {159.70, 154.73, 146.63, 142.37, 134.48, 130.62,
129.80, 120.89, 119.85, 114.71, 114.36, 113.56,
112.04}(Ar–C), 55.91 (OCH3), 55.30 (OCH3), 31.52
(CH2), 28.14 (CH2); ESI/MS (m/z, ? ion mode): 326.06
[M ? H]?, 348.03 [M ? Na]?; Anal.: Calcd. for
C18H19N3O3: C, 66.45; H, 5.89; N, 12.91. Found: C, 66.36;
H, 5.77; N, 12.79.
Pharmacological assay
b-Glucuronidase assay
The inhibition efficacy of b-glucuronidase can be assessed
by using various methods (Igarashi et al., 1996; Kawaguchi
et al., 2009; Kim et al., 1994, 1999). The inhibition of
b-glucuronidase (E.C. 3.2.1.31), type-IX from E. coli by
test sample was measured by using a spectrophotometric
method (Khan et al., 2002). p-Nitrophenyl-b-D-glucuronide
was used as a substrate for the activity of enzyme. Both
enzyme and substrate were purchased from Sigma Chem-
ical Co. (USA). The total reaction volume 250 lL was
added to 96-well plate, containing 10 lL of enzyme (1 U/
well) in 185 lL of 0.1 M acetate buffer (pH 7.0), and 5 lL
3-(4-Methoxyphenethyl)-4-(2-methoxyphenyl)-1H-
1,2,4-triazol-5(4H)-one (6k)
The general experimental procedure described above
afforded (6k) as white solid. Yield: 73%; m.p 204–206°C;
Rf: 0.26 (petroleum ether : ethyl acetate, 5:5); IR (Pure,
123

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Med Chem Res (2012) 21:3885–3896
Katrizky A (1984) Comprehensive heterocyclic chemistry, vol 6.
Pergamon, Oxford
¨
Kavak DD, Altiok E, Bayraktar O, Ulku¨ S (2010) Pistacia terebinthus
(0.2 mM) test compound in triplicate dissolved in DMSO.
The solvent (DMSO, 5 lL) instead of test compound was
used as positive control. The plate was then incubated at
37°C for 30 min. After the completion of incubation per-
iod, 50 lL of substrate p-nitrophenyl-b-D-glucuronide
(0.4 mM) was added and change in absorbance was
recorded continuously for 30 min at 405 nm by using
spectrophotometer (Spectra Max M2 Molecular Devices
CA, USA). % inhibition was calculated by the given
formula;
extract: As a potential antioxidant, antimicrobial and possible b-
glucuronidase inhibitor. J Mol Cat B: Enzymes 64:167–171
Kawaguchi AW, Okawa H, Hashimoto K (2009) Synthesis of
glycopolymers bearing mannaric pendants as inhibitors on the
b-glucuronidase activity: the inhibition mechanisms of manna-
ric- and glucaric-compounds. J Polymer sci Part A: Polymer
chem 47:2032–2042
Khan KM, Shujaat S, Rahat S, Hayat S, Rahman A, Choudhary MI
(2002) b-N-Cyanoethyl acyl hydrazide derivatives: a new class
of b-glucuronidase inhibitors. Chem Pharm Bull 50:1443–1446
Khan I, Ali S, Hameed S, Rama NH, Hussain MT, Wadood A, Uddin
R, Haq ZU, Khan A, Ali S, Choudhary MI (2010) Synthesis,
antioxidant and urease inhibition activities of some new 1, 2,
4-triazole and 1, 3, 4-thiadiazole derivatives. Eur J Med Chem
45:5200–5207
% inhibition ¼ 100 ꢁ ðO:D: of S=O:D: of BÞ ꢂ 100
where B is the activity of enzyme without test compound
and S is the activity of enzyme with test compound.
Kim DH, Jin YH, Park JB, Kobashi K (1994) Silymarin and its
components are inhibitors of beta-glucuronidase. Biol Pharm
Bull 17(3):443–445
IC50 determination
Kim DH, Shim SB, Kim NJ, Jang IS (1999) Beta-glucuronidase-
inhibitory activity and hepatoprotective effect of Ganoderma
lucidum. Biol Pharm Bull 22(2):162–164
Kowalczyk MC, Spears E, Narog M, Zoltaszek R, Kowalczyk P,
Hanausek M, Yoshimi N, Saga TJ, Walaszek Z (2011) Modu-
lation of biomarkers related to tumor initiation and promotion in
mouse skin by a natural b-glucuronidase inhibitor and its
precursors. Oncol Rep 26(3):551–556
Various concentrations of test compounds were used for
the determination of IC50 values. The IC50 values represent
the amount of compound half maximal inhibitory concen-
tration with a fix concentration of enzyme. The quantitative
measurement describes the potency of compound to inhibit
a biological process. The IC50 value was calculated by
using EZ-Fit Enzyme Kinetics Program (Perrella Scientific
Inc., Amherst, MA, USA) software.
Leung JW, Liu Y, Leung PSC, Chan RCY, Inciardi JF, Cheng AF
(2001) Expression of bacterial b-glucuronidase in human bile: an
in vitro study. Gastrointest Endosc 54:346–350
Mathijssen RHJ, Alphen RJV, Verweij J, Loos WJ, Nooter K, Stoter
G, Sparreboom A (2001) Clinical pharmacokinetics and metab-
olism of Irinotecan (CPT-11). Clin Cancer Res 7:2182–2194
McBain AJ, Macfarlane GT (1998) Ecological and physiological
studies on large intestinal bacteria in relation to production of
hydrolytic and reductive enzymes involved in formation of
genotoxic metabolites. J Med Microbiol 47:407–416
Acknowledgments Authors are thankful to Dr. Basit Yameen,
Department of Chemistry, LUMS School of Science and Engineering
Lahore, Pakistan for his valuable suggestions.
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