Phenoxyacetohydrazide schiff bases: β-glucuronidase inhibitors

Article abstract of DOI:10.3390/molecules19078788

Phenoxyacetohydrazide Schiff base analogs 1-28 have been synthesized and their in vitro β-glucouoronidase inhibition potential studied. Compounds 1 (IC₅₀ = 9.20 ± 0.32 μM), 5 (IC₅₀= 9.47 ± 0.16 μM), 7 (IC₅₀ = 14.7 ± 0.19 μ

Full text of DOI:10.3390/molecules19078788

Molecules 2014, 19, 8788-8802; doi:10.3390/molecules19078788
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Phenoxyacetohydrazide Schiff Bases: β-Glucuronidase Inhibitors
Waqas Jamil 1,2, Shagufta Perveen 1, Syed Adnan Ali Shah 3,4, Muhammad Taha 4,5
,
Nor Hadiani Ismail 4,5, Shahnaz Perveen 6, Nida Ambreen 1, Khalid M. Khan 1,* and
Muhammad I. Choudhary 1
1
H. E. J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences,
University of Karachi, Karachi 75270, Pakistan; E-Mails: waqas143kh@yahoo.com (W.J.);
sarosh.iqbal@yahoo.com (S.P.); nida_mishi2002@yahoo.com (N.A.); hassaan2@super.net.pk (M.I.C.)
2
Institute of Advance Research Studies in Chemical Sciences, University of Sindh Jamshoro,
Hyderabad 76080, Pakistan
3
Faculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, Bandar Puncak
Alam, Selangor Darul Ehsan 42300, Malaysia; E-Mail: benzene301@yahoo.com or
syedadnan@salam.uitm.edu.my
4
Atta-ur-Rahman Institute for Natural Products Discovery (AuRIns), Level 9, FF3,
Universiti Teknologi MARA (UiTM), Puncak Alam Campus, Bandar Puncak Alam,
Selangor Darul Ehsan 42300, Malaysia; E-Mails: taha_hej@yahoo.com (M.T.);
norhadiani@puncakalam.uitm.edu.my (N.H.I.)
5
Faculty of Applied Science UiTM, Shah Alam, Selangor 40450, Malaysia
6
PCSIR Laboratories Complex, Shahrah-e-Dr. Salimuzzaman, Karachi 75280, Pakistan;
E-Mail: shamim_302@yahoo.com
* Author to whom correspondence should be addressed; E-Mail: khalid.khan@iccs.edu;
Tel.: +92-213-482-4910; Fax: +92-213-481-9018.
Received: 7 May 2014; in revised form: 6 June 2014 / Accepted: 11 June 2014 /
Published: 25 June 2014
Abstract: Phenoxyacetohydrazide Schiff base analogs 1–28 have been synthesized and their
in vitro β-glucouoronidase inhibition potential studied. Compounds 1 (IC50 = 9.20 ± 0.32 µM),
5 (IC50 = 9.47 ± 0.16 µM), 7 (IC50 = 14.7 ± 0.19 µM), 8 (IC50 = 15.4 ± 1.56 µM),
11 (IC50 = 19.6 ± 0.62 µM), 12 (IC50 = 30.7 ± 1.49 µM), 15 (IC50 = 12.0 ± 0.16 µM),
21 (IC50 = 13.7 ± 0.40 µM) and 22 (IC50 = 22.0 ± 0.14 µM) showed promising
β-glucuronidase inhibition activity, better than the standard (D-saccharic acid-1,4-lactone,
IC50 = 48.4 ± 1.25 µM).

Molecules 2014, 19
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Keywords: phenoxyacetohydrazide; Schiff bases; β-glucouoronidase; glucuronosyl-O-bonds,
D-saccharic acid-1,4-lactone
1. Introduction
A range of bioactivities are reported for hydrazide-hydrazone compounds, such as antibacterial,
anticonvulsant, antimalarial, analgesic, antiinflammatory, antiplatelets, antifungal, antituberculosis,
and anticancer activities [1–6]. A variety of semicarbazones, thiosemicarbazones and guanyl hydrazones
are found to be key compounds for drug design [7], for metal complexes [8], organocatalysis [9], and
are used for the preparation of heterocyclic rings [10]. A few pyrazole carbohydrazide hydrazone
derivatives [11] and novel 3-aryl-1-arylmethyl-1H-pyrazole-5-carbohydrazide hydrazones were found
to be proliferation inhibitors of A549 cells [12,13] Some evidence proposes a pharmacophoric
character for the hydrazone moiety present in phenylhydrazone derivatives in the inhibition of
cyclooxygenase [14]. Antioxidant [15–18], antiglycation [19–22] and antileishmanial [23] activity
have recently been reported, as well as applications in mass spectrometry [24].
The present work aimed to investigate the potential activity of a series of aryl hydrazide-hydrazones
as in vitro β-glucouoronidase inhibitors. In our designed analogues substituted phenoxy-acetohydrazides
were treated with different aromatic aldehydes to scrutinize their potential activity. The earlier reported
literature [25] showed that β-glucouoronidase is a lysosomal enzyme, present in many organs like the
spleen, kidney, lung, bile, serum and urine, etc., where its specific task is to catalyze the cleavage of
glucuronosyl-O-bonds [26–28]. It degrades glucuronic acid-containing glycosaminoglycans, like
heparan sulfate, chondroitin sulfate and dermatan sulfate [29]. An elevated level of β-glucouoronidase
was observed in various types of malignancies, such as breast, lung and gastrointestinal tract
carcinomas, and melanomas. Its high expression also observed in bronchial tumors [30]. On the other
hand, mucopolysaccharidosis type VII (MPS VII; Sly Syndrome) is caused by the deficiency of human
β-glucuronidase [31]. The circulating level of β-glucuronidase is also useful as a lysosomal enzyme in
children affected by leprosy. In borderline tuberculoid patients and lepromatous patients higher activity
of this enzyme was also observed.
2. Results and Discussion
2.1. Chemistry
Lead identification is a well defined tool in drug design and discovery. Our research group has been
involved for a decade in lead discovery programs in search of novel therapeutic agents. We have
earlier reported Schiff bases of different classes of organic compounds in the search for lead molecules
with different biological activities [32–34]. Earlier, our group reported the leishmanicidal and
β-glucurinodase inhibition potential of hydrazides derived from the corresponding esters [35–38]. In
view of the formerly reported work we synthesized hydrazide Schiff bases and screened their potential
biological activities [39–42]. Acylhydrazide Schiff base derivatives 1–28 were synthesized from an
acylhyrazide by condensing it with different aromatic aldehydes and acetophenones under reflux

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conditions in ethanol for 2–3 h (Scheme 1). The crude products (Table 1) were further recrystallized
from methanol and needle-like crystals were obtained in most of the cases. The starting acylhydrazide was
synthesized from ester of ethyl 2-(4-chloro-2-methylphenoxy) acetate by refluxing with hydrazine hydrate.
Scheme 1. Synthetic scheme for benzohydrazide followed by synthesis of Schiff bases 1–28.
O
O
O
O
NH2
O
N
N2H4 H2O
reflux, 2 h
H
Cl
Me
Cl
Me
R1 R2CO
CH3CH2OH
ref
lux, 3 h
O
NH R1
N C
O
R2
Me
Cl
Table 1. Synthesis of acylhydrazide Schiff base derivatives 1–28.
Compound
No.
Yield Compound
Yield
(%)
R1
R2
R1
R2
(%)
No.
1
H
81
15
H
88
2
H
85
16
H
92
3
4
H
H
93
87
17
18
CH3
94
89
H

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Table 1. Cont.
Compound
No.
Yield Compound
Yield
(%)
R1
R2
R1
R2
(%)
No.
5
6
H
83
19
CH3
87
H
H
86
89
20
21
H
84
7
8
9
H
H
H
94
93
91
H
H
91
94
22
23
10
11
H
H
82
88
24
25
CH3
95
91
H
4'
5'
12
13
H
H
88
81
H
H
91
86
26
27
2'
S
H
93
28
14
H
93

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2.2. β-Glucuronidase
8792
Synthetic acyl hydrazides Schiff bases 1–28 were screened for their in vitro potential as
β-glucoronidase inhibitors. The in vitro β-glucornidase inhibitory potential was evaluated by using the
literature protocol [43]. Compounds 1–28 showed diversified β-glucoronidase inhibitory activities,
with IC50 values ranging between 9.20–30.7 µM. Compounds 1, 5, 7, 8, 11, 12, 15, 21, and 22 showed
excellent β-glucoronidase inhibitory activities, with IC50 values of 9.20 ± 0.32, 9.47 ± 0.16, 14.7 ± 0.19,
15.4 ± 1.56, 19. ± 0.62, 30.7 ± 1.49, 12.0 ± 0.16, 13.7 ± 0.40, and 22.0 ± 0.14 µM, respectively, and
the remaining compounds exhibited no activity (Table 2).
Table 2. In vitro β-glucuronidase activity of compounds 1–28.
Compounds
IC50 (μM ± SEM a) Compounds
IC50 (μM ± SEM a)
12.0 ± 0.16
NA b
1
9.20 ± 0.32
NA b
15
16
17
18
19
20
21
22
23
24
25
26
27
28
-
2
3
NA b
NA b
NA b
4
NA b
5
9.47 ± 0.16
NA b
NA b
6
NA b
7
14.7 ± 0.19
15.4 ± 1.56
NA b
13.7 ± 0.40
22.0 ± 0.14
NA b
8
9
10
11
12
13
14
NA b
NA b
19.6 ± 0.62
30.7 ± 1.49
NA b
NA b
NA b
NA b
NA b
NA b
D-saccharic acid-1,4-lactone c 48.4 ± 1.25
-
SEM a is the standard error of the mean; NA b Not active; c standard inhibitor for β-glucuronidase.
It was observed that both the substituents’ nature and their position at the benzilidine part have
great importance in the β-glucoronidase inhibition activity of a compound, and apparently the acylium
part does not take part in the activity (Figure 1).
Figure 1. The two parts of molecule on which activity is based.
The best activity was shown by compound 1 (IC50 = 9.20 ± 0.32 µM, fivefold better than the
standard D-saccharic acid-1,4-lactone, IC50 value 48.4 ± 1.25 µM) which has a methoxy group at the
ortho position. Surprisingly, a marked decline in activity (to the point of being inactive) was observed

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in analog 2 which has a methoxy residue at the para position instead of the ortho position as in
compound 1. This huge difference in the activities of compounds 1 and 2 clearly indicates that a
specific group at a specific position of the benzylidine phenyl ring part plays a vital role in making a
potent β-glucornidase inhibitor in this type of compounds. The excellent activity of compound 5
(IC50 = 9.47 ± 0.16 µM) having an ortho nitro group on the phenyl ring as compared to the inactivity
of its closely related derivative 6 having a meta nitro group on phenyl ring proves our hypothesis that a
suitable group at a suitable position of the phenyl ring of benzilidine part of molecules is a prerequisite
for β-glucornidase inhibitory potential in these N-acylhydrazone Schiff bases. Comparison of activity
of chloro-containing compounds 7 (IC50 = 14.7 ± 0.19 µM), 8 (IC50 = 15.4 ± 1.56 µM), and 9
(inactive) demonstrated that the nature and location of a substitution is important for β-glucornidase
inhibitory potential. Dichloro-substituted compounds 9 and 10 were found to be completely inactive
which further proves our hypothesis. Compound 11 (IC50 = 19.6 ± 0.62 µM) having an ortho fluoro group
showed excellent activity, but a little less than analogous chloro compounds 7 and 8. We also
evaluated the effect of heterocyclic ring-containing derivatives, and it was observed that the five
membered heterocyclic thiophene ring-containing derivative 12 (IC50 30.7 ± 1.49 µM) produced
remarkable activity, while on the other hand five membered heterocyclic rings like furan and its methyl
derivatives 13 and 14 were found to be completely inactive. Almost all mono-, di- and trihydroxy
substituted compounds 16, 17, 18, 19, and 20 found to be completely inactive, but unexpectedly
compound 15 (IC50 = 12.0 ± 0.16 µM) which bears 2,3-dihydroxy substitution, was found to be very
efficient and displayed remarkable activity, better than the standard, but, compound 16, also a
3,4-dihydroxy derivative did not show any activity. N-acylhydrazones Schiff base 21 (IC50 = 13.7 ±
0.40 µM) synthesized from 1-napthaldehyde was found to be more active than 22 (IC50 = 22.0 ± 0.14 µM)
which was synthesized from 2-napthaldehyde, both without any substitution. Remaining compounds
23–28 were found to be completely inactive. This pattern of activity reveals that the substituent and its
position on the phenyl ring of benzylidine part is a driving force for β-glucornidase inhibition activity.
In conclusion, a number of potential lead molecules has been identified as β-glucuronidase
inhibitors. Compounds 1, 5, 7, 8, 11, 12, 15, 21, and 22 demonstrated excellent activity and it is
anticipated that by slight synthetic modification in these molecules, some new most active
β-glucuronidase inhibitors can be developed.
3. Experimental
3.1. General Information
1H-NMR experiments were performed on an Avance-Bruker AM 300 MHz instrument
(Wissembourg Cedex, France). A Carlo Erba Strumentazione-Mod-1106 (Milan, Italy) used to
measure CHN analysis. EI MS was performed on a Finnigan MAT-311A (Bremen, Germany). Thin
layer chromatography (TLC) was carried out on pre-coated silica gel glass plates (Kieselgel 60, 254,
E. Merck, Darmstadt, Germany). The chromatograms were visualized by UV at 254 and 365 nm or
iodine vapours.

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3.2. Biological Assays
β-Glucuronidase (E.C. 3.2.1.31 from bovine liver, G-0251) and p-nitrophenyl-β-D-glucuronide
(N-1627) were purchased From Sigma Chemical Co. (St. Louis, MO, USA). Anhydrous Na2CO3 and
all other reagents of standard grade were obtained from E. Merck. The anhydrous EtOH and CHCl3
used in the experiments were dried employing the standard methods. All other solvents and reagents
like the benzoyl chloride were of standard grade.
3.3. Assay for β-D-Glucuronidase
β-D-Glucuronidase inhibition was determined by measuring the absorbance of the p-nitrophenol
which is produce from the substrate at 405 nm. The total reaction volume was 250 µL. The reaction
mixture contains 5 µL of test compound solution, 185 µL of 0.1 M acetate buffer, and 10 µL of
enzyme, and it was incubated at 37 °C for 30 min. The plates were read on a multiplate reader at
405 nm after the addition of 50 µL of 0.4 mM p-nitrophenyl-β-D-glucuronide. All assays were
performed in triplicate.
3.4. Typical Method for the Synthesis of Compounds 1–28
To a mixture of 2-(4-chloro-2-methylphenoxyacetic acid) hydrazide (1 mmol) in methanol (25 mL)
was added a substituted aldehyde (1 mmol) and 3 drops of glacial acetic acid and the mixture was
refluxed for 3 h. After completion of the reaction (TLC analysis), it was cooled and evaporated on a
rotary evaporator. The resultant crude product was crystallized from methanol to afford 80%–90%
yields of pure product. The structures of synthetic compounds 1–28 were determined by different
spectroscopic techniques, including 1H-NMR, and EI MS spectroscopy.
2-(4-Chloro-2-methylphenoxy)-N'-[(3-(2-methoxyphenyl)-2-propenylidene]acetohydrazide (1). Yield:
1
68%; H-NMR (DMSO-d6) δ: 8.04 (d, 1H, J = 9.3 Hz, N=CH-CH), 7.63 (dd, 1H, J5',6' = 7.8 Hz,
J5',3' = 1.2 Hz, H-6'), 7.34 (dd, 1H, JCH-HC=CH = 8.1 Hz, CH-HC=CH), 7.23 (d, 1H, JHC=CH = 6 Hz,
HC=CH), 7.12–7.20 (m, 2H, H-3/5) 6.85–7.06 (m, 3H, H- 3'/4'/5'), 6.80 (d, 1H, J6,5 = 9 Hz, H-6), 5.17 (s,
2H, -OCH2), 3.84 (s, 3H, -OCH3), 2.23 (s, 3H, CH3); EI MS: m/z (%) 358 (M+, 30), 327 (100),189
(40), 175 (55), 159 (100), 125 (75.0), Anal. Calcd for C19H19ClN2O3, C = 63.60, H = 5.34, N = 7.81.
Found: C = 63.55, H = 5.31, N = 7.80.
2-(4-Chloro-2-methylphenoxy)-N'-[(3-(4-methoxyphenyl)-2-propenylidene]acetohydrazide (2). Yield:
1
70%; H-NMR (DMSO-d6) δ: 8.02 (d, 1H, J = 9.3Hz, N=CH-CH), 7.55 (d, 2H, J2',3' = J6',5' = 8.7 Hz,
H-2'/6'),7.12–7.23 (m, 2H, H-3/5),7.01 (d, 2H, J3',2' = J5',6' = 7.0 Hz, H-3'/5'), 6.76–6.87 (m, 2H, HC = CH),
6.92 (d, 1H, J5,6 = 8.4 Hz, H-6), 5.03 (s, 2H, -OCH2), 3.76 (s, 3H, OCH3), 2.21 (s, 3H, CH3); m/z (%)
358 (M+, 95), 189 (40), 175 (85), 159 (100), 125 (60). Anal. Calcd for C19H19ClN2O3, C = 63.60,
H = 5.34, N = 7.81. Found: C = 63.57, H = 5.32, N = 7.79.
2-(4-Chloro-2-methylphenoxy)-N'-[(4-ethoxyphenyl)methylidene]acetohydrazide (3). Yield: 80%; 1H-NMR
(DMSO-d6) δ: 8.20 (s, 1H, -N=CH), 7.62 (d, 2H, J2',3' = J6',5' = 8.7 Hz, H-2'/6'), 7.23–7.12 (m, 2H, H-3/5),
6.84 (d, 2H, J3',2' = J5',6' = 8.7 Hz, H-3'/5'), 6.84 (d, 1H, J6,5 = 8.7 Hz, H-6), 5.13 (s, 2H, OCH2), 4.04 (q,

Molecules 2014, 19
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2H, J = 6.9 Hz, -CH2), 2.22 (s, 3H, -CH3), 1.32 (t, 3H, J = 6.9 Hz, -CH3); EI MS: m/z (%) 347 (M+, 56),
205 (95), 155 (89), 147 (92), 119 (73). Anal. Calcd for C18H19ClN2O3, C = 62.34, H = 5.52, N = 8.08.
Found: C = 62.33, H = 5.50, N = 8.03.
2-(4-Chloro-2-methylphenoxy)-N'-[(3,4,5-trimethoxyphenyl) methylidene]acetohydrazide (4). Yield:
72%; 1H-NMR (DMSO-d6) δ: 8.43 (s, 1H, -N=CH), 7.56 (d, 1H, J2',6' = 2.7 Hz, H-2'), 7.23 (m, 1H, H-5),
7.13 (d, 1H, J3,5 = 2.7 Hz, H-3), 6.92(s, 1H, H-6'), 6.83 (d, 1H, J5,6 = 8.7 Hz H-6), 5.13 (s, 2H, -OCH2),
3.82 (s, 9H, -OCH3), 2.22 (s, 3H, -CH3); EI MS: m/z (%) 392 (M+, 62.1), 251 (15.5), 193 (100), 179
(88.9), 155 (26.8). Anal. Calcd for C19H21ClN2O5, C = 58.09, H = 5.39, N = 7.13. Found: C = 58.05,
H = 5.37, N = 7.11.
1
2-(4-Chloro-2-methylphenoxy)-N'-[(2-nitrophenyl)methylidene]acetohydrazide (5). Yield: 65%; H-NMR
(DMSO-d6) δ: 8.09(s, 1H, N=CH), 8.29 (m, 2H, H-4'/5'), 7.98 (d, 2H, J3',4' = J6',5' = 9, H-3'/6') 7.24 (m,
1H, H-5), 7.13 (d, 1H, J3,5 = 2.7 Hz, H-3), 6.89 (d, 1H, J6,5 = 8.7, H-6), 5.22 (s, 2H, -OCH2), 2.22 (s, 3H,
-CH3); EI MS: m/z (%) 347 (M+, 72), 206 (100), 155 (83.3), 125 (75.8). Anal. Calcd for C16H14ClN3O4,
C = 55.26, H = 4.06, N = 12.08. Found: C = 55.23, H = 4.02, N = 12.04.
1
2-(4-Chloro-2-methylphenoxy)-N'-[(3-nitrophenyl)methylidene]acetohydrazide (6). Yield: 70%; H-NMR
(DMSO-d6) δ: 8.40 (s, 1H, -N=CH), 8.51 (d,1H, J5',6' = 7.2 Hz H-6'), 8.12–8.25 (m, 2H, H-2'/5'), 7.76 (m,
1H, H-4'), 7.12-7.24 (m, 2H, H-3/5), 6.90 (dd, 1H, J6,5 = 6.2 Hz, H-6), 5.22 (s, 2H, OCH2), 2.23 (s, 3H,
CH3); EI MS: m/z (%) 347 (M+, 92), 206 (100), 178 (28.4), 141 (17.5), 125 (39.9). Anal. Calcd for
C19H21ClN2O5, C = 58.09, H = 5.39, N = 7.13. Found: C = 58.06, H = 5.37, N = 7.12.
2-(4-Chloro-2-methylphenoxy)-N'-[-(4-chlorophenyl)methylidene]acetohydrazide (7). Yield: 63%; 1H-NMR
(DMSO-d6) δ: 8.27 (s, 1H, -N=CH), 7.73 (d, 2H, J2',3' = J6',5' = 8.7 Hz, H-2'/6'), 7.51 (d, 2H,
J3',2' = J5',6' = 8.1 Hz, H-3'/5'), 7.13–7.23 (m, 2H, H-3/5), 6.89 (d, J6,5 = 7.8 Hz, 1H, H-6), 5.15 (s, 2H,
-OCH2), 2.19 (s, 3H, CH3); EI MS: m/z (%) 336 (M+, 20), 195 (35), 155 (55), 125 (80), 89 (100). Anal.
Calcd for C16H14Cl2N2O2, C = 56.99, H = 4.18, N = 8.31. Found: C = 56.95, H = 4.15, N = 8.28.
2-(4-Chloro-2-methylphenoxy)-N'-[-(2-chlorophenyl)methylidene]acetohydrazide (8). Yield: 66%; 1H-NMR
(DMSO-d6) δ: 8.26 (s,1H, -N=CH), 7.77 (m, 2H, H-3'/6'), 7.48 (br.t, 2H, J4',5' = J5',4' = 8.5 Hz, H-4'/5'),
7.13–7.24 (m, 1H, H-3/5), 6.89 (d, 1H, J6,5 = 8.7 Hz, H-6), 5.15 (s, 2H, -OCH2), 2.23 (s, 3H, -CH3),
2.19 (s, 3H, CH3); EI MS: m/z (%) 336 (M+, 10), 195 (30), 155 (40), 125 (75), 89 (100). Anal. Calcd for
C16H14Cl2N2O2, C = 56.99, H = 4.18, N = 8.31. Found: C = 56.96, H = 4.15, N = 8.27.
2-(4-Chloro-2-methylphenoxy)-N'-[(3,4-dichlorophenyl)methylidene]acetohydrazide (9). Yield: 70%;
1H-NMR (DMSO-d6) δ: 8.25 (s, 1H, =N-CH), 7.96 (s, 1H, H-2'), 7.68 (br. s, 2H, 5'/6'), 7.12–7.23 (m,
2H, H-3/5), 6.88 (d, 1H, J6,5 = 7.0 Hz H-6), 5.08 (s, 2H, OCH2), 2.22 (s, 3H, CH3); EI MS: m/z (%)
371 (M+, 92.2), 228 (100), 155 (87.1), 125 (89.8). Anal. Calcd for C16H13Cl3N2O2, C = 51.71, H = 3.53,
N = 7.54. Found: C = 51.68, H = 3.50, N = 7.52.
2-(4-Chloro-2-methylphenoxy)-N'-[(2, 6-dichlorophenyl)methylidene]acetohydrazide (10). Yield: 60%;
1H-NMR (DMSO-d6) δ: 8.48 (s,1H, -N=CH), 7.57 (d, 2H, J3',4' = J5',4' = 8.7 Hz, H-3'/5'), 7.44 (dd, 1H,
J4,5 = 7.2 Hz, H-4'), 7.24–7.11 (m, 2H, H-3/5), 6.78 (d, 1H, J6,5 = 8.7 Hz, H-6), 5.09 (s, 2H, -OCH2), 2.18

Molecules 2014, 19
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(s, 3H, CH3); EI MS: m/z (%) 370 (M+, 17.3), 229 (39.5), 155 (60.1), 125 (100), 89 (62). Anal. Calcd for
C16H13Cl3N2O2, C = 51.71, H = 3.53, N = 7.54. Found: C = 51.66, H = 3.51, N = 7.52.
2-(4-Chloro-2-methylphenoxy)-N'-[-(2-fluorophenyl)methylidene]acetohydrazide (11). Yield: 74%;
1H-NMR (DMSO-d6) δ: 8.52 (s, 1H, -N=CH), 7.95 (dd, 1H, J4',5' = 8.1 H-5') 7.48 (dd, 1H, J6',5' = 6.9 Hz,
H-6'), 7.25-7.31 (m, 2H, H-3'/4'), 7.12–7.21 (m, 2H, H-3/5), 6.90 (d, 1H, J6,5 = 8.7 Hz, H-6), 5.17 (s,
2H, -OCH2), 2.23 (s, 3H, CH3); EI MS: m/z (%) 320 (M+, 12), 179 (80), 125 (100), 89 (75.8). Anal. Calcd
for C16H14ClFN2O2, C = 59.91, H = 4.40, N = 8.73. Found: C = 59.89, H = 4.39, N = 8.72.
1
2-(4-Chloro-2-methylphenoxy)-N'-[3-thienylmethylidene]acetohydrazide (12). Yield: 68%; H-NMR
(DMSO-d6) δ: 8.07 (s, 1H, -N=CH), 7.12–7.24 (m, 3H, H-3, 5, 6), 6.73–9.92 (m, 3H, H-2'/4'/5'), 5.10
(s, 2H, -OCH2), 2.22 (s, 3H, CH3); EI MS: m/z (%) 308 (M+, 15), 199 (10), 155 (45), 125 (100). Anal.
Calcd for C14H13ClN2O2S, C = 54.46, H = 4.24, N = 9.07. Found: C = 54.45, H = 4.22, N = 9.04.
1
2-(4-Chloro-2-methylphenoxy)-N'-[-2-furylmethylidene]acetohydrazide (13). Yield: 70%; H-NMR
(DMSO-d6) δ: 8.17 (s, 1H, -N=CH), 7.11–7.23 (m, 2H, H-3,5), 6.90 (d, 2H, J3',4'= J5',4' = 3.3 Hz, H-3'/5'),
6.81(d, 1H, J6,5 = 8.7 Hz, 6), 6.62 (dd, 1H, J4,3 = J4,5 = 3.3 Hz, 4'), 5.07 (s, 2H, -OCH2), 2.22 (s, 3H,
-CH3); EI MS: m/z (%) 292 (M+, 35), 155 (50), 151 (80.), 125 (100). Anal. Calcd for C14H13ClN2O3,
C = 57.44, H = 4.48, N = 9.57. Found: C = 57.44, H = 4.46, N = 9.56.
2-(4-Chloro-2-methylphenoxy)-N'-[-(5-methyl-2-furyl)methylidene]acetohydrazide (14). Yield: 72%;
1H-NMR (DMSO-d6) δ: 8.07 (s, 1H, -N=CH), 7.11–7.23 (m, 2H, H-3,5), 6.88 (d, 1H, J5,6 = 8.7 Hz, H-6),
6.80 (d, 1H, J3',4' = 5.4 Hz, 3'), 6.24 (d, 1H, J4',3' = 2.7 Hz, 4'), 5.07 (s, 2H, -OCH2), 2.21 (s, 3H, -CH3)
2.19 (s, 3H, -CH3); EI MS: m/z (%) 306 (M+, 20), 65 (30), 137 (100), 125 (40). Anal. Calcd for
C15H15ClN2O3, C = 58.73, H = 4.93, N = 9.13. Found: C = 58.70, H = 4.96, N = 9.10.
2-(4-Chloro-2-methylphenoxy)-N'-[-(2,3-dihydroxyphenyl)methylidene]acetohydrazide (15). Yield: 76%;
1H-NMR (DMSO-d6) δ: 8.41 (s,1H, -N=CH), 7.24 (m, 1H, H-5), 7.13 (d, 1H, J3,5 = 2.7 Hz, H-3), 6.89
(d, 1H, J5',6' = 8.7 Hz, H-6'), 6.83 (d, 1H, J6,5 = 8.7 Hz, H-6), 6.71 (m, 2H, H-3'/4'), 5.15 (s, 2H,
-OCH2), 2.19 (s, 3H, -CH3); EI MS: m/z (%) 334 (M+, 100),300 (9), 175 (89.7), 193 (35), 179 (50),
137 (65), 125 (60). Anal. Calcd for C16H15ClN2O4, C = 57.41, H = 4.5, N = 8.37. Found: C = 57.40,
H = 3.50, N = 8.36.
2-(4-Chloro-2-methylphenoxy)-N'-[-(3,4-dihydroxyphenyl)methylidene]acetohydrazide (16). Yield: 68%;
1H-NMR (DMSO-d6) δ: 8.06 (s, 1H, -N=CH), 7.12-7.19 (m, 2H, H-3/5),7.24 (d, 1H, J2',6' = 2.7 Hz, H-2'),
6.85–6.92 (m, 2H ,H-5'/6'), 6.76 (d, J6,5 = 8.1 Hz, 1H, H-6), 5.10 (s, 2H, -OCH2), 2.22 (s, 3H, CH3); EI
MS: m/z (%) 334 (M, 36.8), 193 (23.9), 155 (71.6), 125 (88.8), 77 (100). Anal. Calcd for
C16H15ClN2O4, C = 57.41, H = 4.5, N = 8.37. Found: C = 57.39, H = 3.50, N = 8.37.
2-(4-Chloro-2-methylphenoxy)-N'-[1-(4-hydroxyphenyl)ethylidene]acetohydrazide (17). Yield: 70%;
1H-NMR (DMSO-d6) δ: 7.65 (d, 2H, J2',3' = J6',5' = 8.7 Hz , H-2'/6'), 7.23 (m, 1H, Hz H-6), 7.12 (d, 1H,
J = 2.4 Hz, H-3), 6.90 (d, J3',2'= J5',6' = 8.7 Hz, 2H, H-3'/5'), 6.77 (d, 1H, J6,5 = 8.7 Hz, H-6), 5.16 (s,
2H, -OCH2), 2.19 (s, 6H, CH3); EI MS: m/z (%) 332 (M+, 56.7), 191 (100), 177 (24.5), 149 (34.5), 134

Molecules 2014, 19
8797
(37.9). Anal. Calcd for C17H16ClN2O3, C = 61.36, H = 5.15, N = 8.42. Found: C = 61.34, H = 5.14,
N = 8.40.
2-(4-Chloro-2-methylphenoxy)-N'-[(3-hydroxyphenyl)methylidene]acetohydrazide (18). Yield: 61%;
1H-NMR (DMSO-d6) δ: 8.18 (s, 1H, -N=CH), 7.04-7.24 (m, 5H, Ar-H), 6.79–6.89 (m, 2H, H-5-6),
5.14 (s, 2H, -OCH2), 2.22 (s, 3H,-CH3); EI MS: m/z (%) 318 (M+, 97.6), 177 (100), 155 (71), 125
(65.3). Anal. Calcd for C16H15ClN2O3, C = 60.29, H = 4.74, N = 8.79. Found: C = 69.27, H = 3.49,
N = 8.34.
2-(4-Chloro-2-methylphenoxy)-N'-[1-(2,4,6-trihydroxyphenyl)ethylidene]acetohydrazide (19). Yield: 62%;
1H-NMR (DMSO-d6) δ: 8.16 (s, 1H, -N=CH), 7.21 (br.s, 2H, H-3'/5'), 7.16–7.23 (m, 2H, H-3/5), 6.86 (d,
J6,5 = 8.7 Hz, 1H, H-6), 4.98 (s, 2H, -OCH2), 2.18 (s, 3H, CH3), 1.93 (s, 3H, CH3); EI MS: m/z (%) 254
(M+, 100), 155 (91.2), 142 (75.5), 125 (100), 113 (100), 99 (86.6).
2-(4-Chloro-2-methylphenoxy)-N'-[(4-hydroxyphenyl)methylidene]acetohydrazide (20). Yield: 72%;
1H-NMR (DMSO-d6) δ: 7.88 (s, 1H, -N=CH), 7.52 (d, 2H, d, 2H, J2',3' = J6',5' = 8.7 Hz , H-2'/6'), 7.24 (d,
1H, J5,6 = 8.1 Hz, H-5), 7.13(d, 1H, J = 2.4 Hz, H-3), 6.88 (d, 2H, J3',2' = J5',6' = 8.7 Hz, H-3'/5'), 6.79
(d, 1H, J6,5 = 8.7 Hz, H-6), 5.11 (s, 2H, -OCH2), 2.22 (s, 3H, CH3); EI MS: m/z (%) 318 (M+, 9.30),
199 (10.78), 177 (27.43), 155 (44), 125 (67.50), 77 (100). Anal. Calcd for C16H15ClN2O3, C = 60.29,
H = 4.74, N = 8.79. Found: C = 69.26, H = 3.50, N = 8.34.
1
2-(4-Chloro-2-methylphenoxy)-N'-[-1-naphthylmethylidene]acetohydrazide (21). Yield: 68%; H-NMR
(DMSO-d6) δ: 8.66 (s, 1H, N=CH), 8.83 (d, J = 8.1 Hz 1H, H-8'), 8.61 (d, 1H, J4',3' = 8.4 Hz, H-4'),
8.02 (br, d, 2H, J = 7.8 Hz, H-2'/5'), 7.93 (d, 1H, J3',4' = 7.8 Hz, H-3') 7.65 (m, 2H, H-6'/7'), 7.14–7.25
(m, 2H, H-3/5), 6.95 (d, J6,5 = 8.4 Hz, 1H, H-6), 5.24 (s, 2H, -OCH2), 2.26 (s, 3H, -CH3); EI MS: m/z
(%) 352 (M+, 100), 211(65), 199. Anal. Calcd for C20H17ClN2O2, C = 68.09, H = 4.86, N = 7.94.
Found: C = 68.06, H = 4.84, N = 7.92.
1
2-(4-Chloro-2-methylphenoxy)-N'-[-2-naphthylmethylidene]acetohydrazide (22). Yield: 70%; H-NMR
(DMSO-d6) δ: 8.43 (s, 1H, N=CH), 8.16 (t, 2H, J = 8.4 Hz, H-3',6'), 7.94 (br, s, 2H, H-5/4') 7.56 (t,
2H, J5',6' = J8',7' = 9.6 Hz, H-5'/8'), 7.22 (s, 1H, H-1'), 7.17 (d, 1H, J7',8' = 8.7 Hz, H-7'), 6.92 (m, 2H,
H-5/6), 5.23 (s, 2H, OCH2), 2.23 (s, 3H, CH3); EI MS: m/z (%) 352 (M+, 72), 211 (60) 199 (35), 169
(45), 153 (100), 127 (60). Anal. Calcd for C20H17ClN2O2, C = 68.09, H = 4.86, N = 7.94. Found:
C = 68.04, H = 4.85, N = 7.91.
2-(4-Chloro-2-methylphenoxy)-N'-[(5-Chloro-2-hydroxyphenyl)methylidene]acetohydrazide (23). Yield:
1
66; H-NMR (DMSO-d6) δ: 8.23 (s,1H, -N=CH), 7.70 (d, 1H, J6',4'= 2.7 Hz H-6'),7.12–7.23 (m, 2H,
H-3/5), 7.31 (m, 1H, H-4'), 6.93 (d, 1H, J3',4' = 7.8Hz, H-3'),6.86 (d, 1H, J6,5 = 8.7 Hz, H-6), 5.17 (s,
2H, -OCH2), 2.22 (s, 3H, -CH3); EI MS: m/z (%) 352 (M+, 83.8), 210 (7.4), 197 (62.6), 155 (100), 141
(46.8). Anal. Calcd for C16H14Cl2N2O3, C = 54.41, H = 4.00, N = 7.93. Found: C = 54.39, H = 4.00,
N = 7.92.
2-(4-Chloro-2-methylphenoxy)-N'-[1(2,4-dihydroxy-5-nitrophenyl)ethylidene]acetohydrazide (24). Yield:
1
70%; H-NMR (DMSO-d6) δ: 8.23 (s, 1H, H-6'), 7.17–7.23 (m, 2H, H-3/5), 6.89 (d, 1H, J6,5 = 8.7 Hz,

Molecules 2014, 19
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H-6), 6.51 (s, 1H, H-3'), 4.82 (s, 2H, -OCH2), 2.26 (s, 3H, CH3), 2.21 (s, 3H, CH3); EI MS: m/z (%)
392 (M+, 64.4), 251 (72.1), 237 (100), 125 (89.9), 77 (68.5). Anal. Calcd for C16H14ClN3O6, C = 50.60,
H = 3.72, N = 11.07. Found: C = 50.57, H = 3.70, N = 11.05.
2-(4-Chloro-2-methylphenoxy)-N'-[(4-hydroxy-3-methoxyphenyl)methylidene]acetohydrazide (25). Yield:
1
59%; H-NMR (DMSO-d6) δ: 8.11 (s, 1H, -N=CH), 7.12–7.23 (m, 2H, H-3/5), 7.03 (s, 1H, H-2'),
6.86–6.96 (m, 2H, H-5'/6') 6.83 (d, 1H, J6,5 = 8.7 Hz, H-6), 5.12 (s, 2H, -OCH2), 3.78 (s, 3H, OCH3),
2.23 (s, 3H, CH3); EI MS: m/z (%) 348 (M+, 88.5), 207 (51), 193 (100), 155 (43), 125 (46.9). Anal.
Calcd for C17H17ClN2O4, C = 58.54, H = 4.91, N = 8.03. Found: C = 58.53, H = 4.90, N = 8.02.
2-(4-Chloro-2-methylphenoxy)-N'-{-[4-(methylsulfanyl)phenyl]methylidene}acetohydrazide (26). Yield:
63%; 1H-NMR (DMSO-d6) δ: 8.22 (s, 1H, -N=CH), 7.63 (d, 2H, J2,3 = J6,5 = 8.4 Hz, H-2'/6'), 7.31 (d, 2H,
J3,2 =J5,6 = 8.4 Hz, H-3'/5'),7.23 (m, 1H, H-5), 7.13 (d, 1H, J = 2.7 Hz, H-3), 6.89 (d, J6,5 = 8.4Hz, 1H,
H-6), 5.15 (s, 2H, -OCH2), 2.19 (s, 3H, CH3); EI MS: m/z (%) 348 (M+, 60), 207 (25), 149 (100), 125
(35), 118 (35). Anal. Calcd for C17H17ClN2O2S, C = 58.53, H = 4.91, N = 8.03. Found: C = 58.51,
H = 4.90, N = 8.02.
2-(4-Chloro-2-methylphenoxy)-N'-[(2-methylphenyl)methylidene]acetohydrazide (27). Yield: 67%;
1H-NMR (DMSO-d6) δ: 8.25 (s, 1H, -N=CH), 7.78 (d, J = 7.5 Hz, 1H, H-6'), 7.30–7.21 (m, 3H,
H-3'/4'/5'), 7.13-7.17 (m, 2H, H-3/5), 6.84 (d, 1H, J6,5 = 8.7 Hz, H-6), 5.15 (s, 2H, -OCH2), 2.23 (s,3H,
-CH3), 2.19 (s, 3H, CH3); EI MS: m/z (%) 316 (M+, 79.4), 199 (78.9), 175 (89.7), 155 (100), 141
(93.2), 118 (94.2). Anal. Calcd for C17H17ClN2O2, C = 64.46, H = 4.41, N = 8.84. Found: C = 64.45,
H = 4.40, N = 8.83.
2-(4-Chloro-2-methylphenoxy)-N'-[(4-methylphenyl)methylidene]acetohydrazide (28). Yield: 63%;
1H-NMR (DMSO-d6) δ: 8.23 (s, 1H, -N=CH), 7.59 (d, 2H, J2',3'= J6',5' = 7.8 Hz, H-2'/6'), 7.24 (d, 2H,
J3',2'= J5',6' = 8.1 Hz, H-3'/5'),7.13 (d, J = 2.7 Hz, 1H, H-3), 7.17 (m, 1H, H-5), 6.85 (d, J6,5 = 8.7 Hz,
1H, H-6), 5.15 (s, 2H, -OCH2), 2.32 (s, 3H, CH3), 2.19 (s, 3H, CH3); EI MS: m/z (%) 316 (M+, 9.61),
199 (10.80), 175 (36.26), 155 (47.70), 125 (71.62). Anal. Calcd for C17H17ClN2O2, C = 64.46,
H = 4.41, N = 8.84. Found: C = 64.44, H = 4.40, N = 8.82.
4. Conclusions
A number of potential lead molecules 1, 5, 7, 8, 11, 12, 15, 21, and 22 have been identified as
β-glucuronidase inhibitors and it is anticipated that by slight synthetic modification in these molecules,
some new most active β-glucuronidase inhibitors can be developed.
Supplementary Materials
Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/19/7/8788/s1.
Acknowledgments
Syed Adnan Ali Shah would like to acknowledge Universiti Teknologi MARA (UiTM) for the
financial support under the Principal Investigator Support Initiative (PSI) Grant Scheme with reference

Molecules 2014, 19
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number UiTM 600-RMI/DANA 5/3/PSI (251/2013) and Cumulative Impact Factor Initiative (CIFI)
Grant Scheme with reference number UiTM 600-RMI/DANA 5/3/CIFI (117/2013) and The authors
are thankful to Organization for Prohibition of Chemical Weapons (OPCW), Netherlands (Project No.
L/ICA/ICB/173681/12) and Higher Education Commission (HEC) Pakistan under “National Research
Support Program for Universities” (Project No. 1910) for financial support.
Author Contributions
All authors equally contributed.
Conflicts of Interest
The authors declare no conflict of interest.
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