Isonicotinohydrazones as inhibitors of alkaline phosphatase and ecto-5′-nucleotidase

Article abstract of DOI:10.1111/cbdd.12861

A series of isonicotinohydrazide derivatives was synthesized and tested against recombinant human and rat ecto-5′-nucleotidases (h-e5′NT and r-e5′NT) and alkaline phosphatase isozymes including both bovine tissue-non-specific alkaline phosphatase (b-TNAP)

Full text of DOI:10.1111/cbdd.12861

Received Date : 18-Apr-2016
Revised Date : 16-Jun-2016
Accepted Date : 13-Aug-2016
Article type : Research Article
Isonicotinohydrazones as Inhibitors of Alkaline Phosphatase and Ecto-5'-
nucleotidase
Pervaiz Ali Channar,‡a Syed Jawad Ali Shah,‡b Sidra Hassan,b Zaib un Nisa,b Joanna Lecka,d,e
Jean Sévigny,d,e Jürgen Bajorath,f Aamer Saeed,*a Jamshed Iqbal*b
a Department of Chemistry, Quaid-I-Azam University, 45320, Islamabad, Pakistan
b
Centre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad, Pakistan
c
Medicinal/Pharmaceutical Chemistry Laboratory, Korea Research Institute of Chemical
Technology141-Gajeongro, Yuseong, Daejeon 305-600, South Korea.
d Département de microbiologie-infectiologie et d'immunologie, Faculté de Médecine, Université
Laval, Québec, QC, Canada
e Centre de Recherche du CHU de Québec – Université Laval, Québec, QC, Canada
f Department of Life Science Informatics, B-IT, LIMES Program Unit Chemical Biology and
Medicinal Chemistry, Rheinische Friedrich-Wilhelms-Universität, Dahlmann Str. 2, D-53113
Bonn, Germany
‡These authors contributed equally.
*Corresponding authors.
aamersaeed@yahoo.com (A. Saeed); drjamshed@ciit.net.pk (J. Iqbal)
Address correspondence to:
Prof. Dr. Jamshed Iqbal
Centre for Advanced Drug Research,
COMSATS Institute of Information Technology,
This article has been accepted for publication and undergone full peer review but has not been
through the copyediting, typesetting, pagination and proofreading process, which may lead to
differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.12861
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Abbottabad, Postal Code 22060, Pakistan
Telephone: +92-992-383591-96
Fax: +92-992-383441
E-Mail: drjamshed@ciit.net.pk
Running Title:
Isonicotinohydrazones as TNAP and e5'NT Inhibitors.
Abstract:
A series of isonicotinohydrazides derivatives were synthesized and tested against recombinant
human and rat ecto-5'-nucleotidases (h-e5'NT & r-e5'NT) and alkaline phosphatase isozymes
including both bovine tissue-nonspecific alkaline phosphatase (b-TNAP) and tissue specific calf
intestinal alkaline phosphatase (c-IAP). These enzymes are implicated in vascular calcifications,
hypophosphatasia, solid tumors and cancers, such as colon, lung, breast, pancreas and ovary. All
tested compounds were active against both enzymes. The most potent inhibitor of h-e5’NT was
derivative
(E)-N'-(1-(3-(4-fluorophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-
(3j) whereas, derivative (E)-N'-(4-hydroxy-3-
yl)ethylidene)isonicotinohydrazide
methoxybenzylidene)isonicotinohydrazide (3g) exhibited significant inhibitory activity against r-
e5'NT. In addition, the derivative (E)-N'-(4′-Chlorobenzylidene)isonicotinohydrazide (3a) was
most potent inhibitor against calf Intestinal alkaline phosphatase and the derivative (E)-N'-(4-
hydroxy-3-methoxybenzylidene)isonicotinohydrazide (3g) was found to be most potent inhibitor
of bovine tissue non-specific alkaline phosphatase. Furthermore, after assay binding modes of
the most compounds against e5’NT (human and rat e5’NT) and AP (including b-TNAP and c-
IAP) was determined computationally.
Introduction:
All cells have the ability to release ATP, an extracellular nucleotide, either intrinsically or under
a pathological condition. These molecules have an important role in regulation of a variety of
physiological functions, such as inflammation, blood clotting, pain perception, immune
reactions, smooth muscle contraction, cell proliferation, and cancer (1, 2). Ecto-nucleotidases are
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surface-located enzymes, particularly regulating the availability of the important signaling
molecules (nucleotides) in extracellular environment and are thus vital for maintaining normal
cell functioning (3).
Ecto-nucleotidases enzymes are categorized into four main groups: the alkaline phosphatases
(APs), the ecto-5′-nucleotidases (e5′NT), the ecto-nucleoside-5′-triphosphate
diphosphohydrolases (NTPDases) and the ecto-nucleotide pyrophosphatases
(NPPs)(4). Ecto-5’-nucleotidase (e5′NT, EC 3.1.3.5) is widely distributed in the body, i.e.
brain, liver, intestine and lymphocyte membrane (5). The enzyme attached to the extracellular
surface membrane by glycosyl phosphatidyl inositol linkage with its catalytic site containing
divalent cation i.e. Zn faces the extracellular surface (6). Eukaryotic ecto-5’-nucleotidase consist
of two structural domain, i.e. C–terminal and N-terminal. Both terminals are connected with
small hinge region that allows them to display greater domain movement and change their
conformation i.e. open or closed (7). Ecto-5'-nucleotidase catalytic activity involves the
dephosphorylation of nucleoside monophosphate into its respective nucleoside.
Previous studies described the non-enzymatic function of e5'NT (CD73) in the body such as
cell–cell, cell–matrix interactions (8-11), in drug resistance (12) and tumor-promoting functions
(13). The expression of CD73 was associated with tumor metastasis, invasion and
neovascularization. CD73 exhibited broad substrate specificity, but adenosine 5-monophosphate
is highly preferable. There is a positive correlation of higher adenosine level with tumor
progression and invasion (9). ADP and ATP are the physiological antagonist of ecto-5’-
nucleotidase with Ki values in the low micro molar range, even though these nucleotides shows
instability as they can be easily hydrolyzed by other ectonucleotidases such as NTPDases, APs
and NPPs (7). The structural analog of ADP has been synthesized. The potential role of e5'NT in
various cancers has been elaborated in previous studies, therefore, e5'NT inhibitors are
important pharmaceutical targets cancer and inflammatory diseases (14, 15) Until now, only few
inhibitors have been discovered such as sulfonamides (16), anthraquinones (17) and
polyoxometalates (POMs) (18) and various polyphenols (19). Sulphonamides and
anthraquinones inhibit the catalytic site of enzyme by showing the competitive mechanism,
whereas POMs and the polyphenol display noncompetitive mechanism of inhibition. Due to
upregulation of e5'NTin various carcinomas, there is a need to synthesize novel inhibitors of
e5'NT having less toxicity, more selectivity and potency towards its target.
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Alkaline phosphatase (AP; EC 3.1.3.1), a metal containing enzyme, is involved in catalyses of
phosphoryl transfer reaction. There is only one alkaline phosphatase found in Escherichia coli
(ECAP), but four types of alkaline phosphatase isoenzymes have been isolated from humans. In
humans, three isozymes are classified as tissue-specific: first one is placental (PLAP), second is
germ cell (GCAP), and the third is present in intestine (IAP) (3). The fourth one tissue-
nonspecific (TNAP), 50% alike to the other three isozymes, is present in bone, liver, and kidney
(20-22). TNAP gene is present on chromosome number 1p34–36 (23) and mutations in the
TNAP gene resulted in hypophosphatasia, a hereditary disorder, characterized by defective bone
mineralization (24). Evidences showed that the major role of TNAP in bones is hydrolysis of
PPi, a potent inhibitor of mineralization, thus ensuring normal bone growth (25, 26).
Different pathological conditions have been ensued due to high levels of APs (3). Consequently,
it necessitates to design small, potent inhibitors of APs having ability to look at the causative
mechanisms, or cure the diseases. Up till now, very few selective inhibitors of APs have been
described due to high structural similarity between two isozymes; tissue-specific IAP and TNAP.
AP is inhibited by various compounds on the basis of mechanisms of inhibition. L-
phenylalanine, imidazole, histamine and theophylline are uncompetitive inhibitors of alkaline
phosphatase while phosphate, phosphoethanolamine and phenylphosphonate inhibit alkaline
phosphatase competitively (27-29). Levamisole derivatives have also been found selective
inhibitors of TNAP (30) while selective inhibitors of IAP such as ML260 (N-(2,5-
dimethylphenyl)-2-(2-oxo-2,3-dihydro-1,3-benzoxazole-6-sulphonamide acetamide) have also
been reported (31).
To determine the accurate mechanisms and the related consequences due to increased levels of
nucleotides it is requisite for the screening of ecto-5’-nucleotidase inhibitors for the management
of cancer, central nervous system and cardiovascular disorders. (3, 32). Previously
isonicotinohydrazide derivatives have been synthesized and screened for In vitro potential of the
compounds against Mycobacterium tuberculosis (33). Herein, we synthesized different
isonicotinohydrazides derivatives and evaluated their AP and ecto-5’-nucleotidase inhibitory
potential for the possible management of the ailments discussed above.
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Results and Discussion
Chemistry
The synthetic layout of the aimed isonicotinohydrazones (3a-3j) is presented in the Scheme 1.
Isoniazid 1 was condensed with a range of substituted aromatic and heteroaromatic aldehydes in
dry ethanol, catalyzed by acetic acid to afford the desired hydrazones (3a-3j) in good yields
(Scheme 1, Table 1). The structures of hydrazones (3a-3j) were established with 1H NMR
spectroscopy. A broad singlet belongs to –NH- group of synthesized hydrazones was observed in
the range of 12.26 to 11.87 (δ in ppm). Two sets of protons (H2′H6′ and H3′H5′) of 4-substituted
pyridine ring appeared as two pseudoquartets or multiplets at δ 8.75 and 7.74 ppm. The
characteristic singlet for –N=CH appeared between 7.1-7.7 ppm. The compounds were obtained
in 65-90% yields depend upon the nature of substituents on aromatic ring and type of aldehyde
(Table 1).
O
O
O
N
Ar
H
Ar
N
H
NH2
(2)
N
H
N
3a-3j
Isonicotinohydrazones
N
Dry EtOH / HOAc
(1)
Isonicotinohydrazide
2'
3'
1'
4'
R
Ar =
R = Substituents
6' 5'
3a-g
Ar =
F
N
N
O
N
N
3h
CH3
3i
(3j)
Scheme 1: Synthetic layout of isonicotinohydrazones (3a-3j)
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Table 1: Synthesized isonicotinohydrazones (3a-3j)
Compound
Ar
Yield (%)
3a
3b
3c
3d
3e
3f
4′-Chlorophenyl
4′-bromophenyl
4′-Fluorophenyl
4′-Methoxyphenyl
2′-hydroxyphenyl
3′-Nitrophenyl
4′-Hydroxy-3′-
methoxyphenyl
4′-pyridyl
80
85
78
88
70
79
90
3g
68
65
3h
3i
3′-pyridyl
70
3j
Alkaline Phosphatase and Ecto-5’-nucleotidase Activity and SAR:
A series of N-ethylidene isonicotinohydrazide derivatives were synthesized and their biological
potential was evaluated by performing in-vitro assays on both ecto-5'-nucleotidase (human and
rat) and alkaline phosphatase isoenzymes (b-TNAP and c-IAP). The derivatives were tested at
concentration of 200 µM for APs and 100 µM for e5′NT. These derivatives exhibited more
inhibition for e5′NT as compared to alkaline phosphatase even in lower micro molar range. All
the derivatives exhibited initibion of h-e5′NT wth IC50 SEM in the range of 0.21 0.05 to
13.8 0.12 µM and of r-e5′NT IC50 SEM = 0.21 0.05 to 7.13 0.63 µM. The most potent
inhibitor of h- e5′NT was 3j having IC50 SEM = 0.19 0.02 exhibiting ≈1.9 fold selectivity over
r- e5′NT. This compound also exhibited ≈222 fold improvement in inhibition as compared to
reference standard i.e sulfamic acid (IC50 SEM = 42.1 7.8 µM). The detailed structural
comparison of this derivative with other compounds suggested that the activity of this compound
might be due to the presence of pyrazol ring. The most potent inhibitor of r-e5′NT was 3g having
inhibitory value of IC50 SEM = 0.14 0.001 exhibiting ≈4.8 fold selectivity over h-e5′NT. This
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compound also exhibited ≈552 fold improvement in inhibition as comared to reference standard
i.e. sulfamic acid (IC50 SEM = 77.3 7.0 µM). The comprehensive study of its structure in
comparison with the other derivatives of the series suggested that the activity of this compound
might be due to the presence of electron donating group on the phenyl ring i.e MeO at 4 position
making the ring stable by electronic cloud. This compound exhibited ≈3.2 fold improvement in
inhibition as compared to compound 3e (IC50 SEM = 0.45 0.03 µM) in which the phenyl ring
was un-substituted. Similarly, ≈2.2 fold maximum inhibition was seen in 3g, its inhibitory
potential was compared with 3d (IC50 SEM = 0.31 0.01 µM) in which the MeO was substituted
to phenyl ring.
When the activity of other derivatives was observed it can be suggested that the presence of
electronegative substititution exhibited maximum inhibition of r-e5′NT as compared to h- e5′NT.
The effect was interestingly observed in case of compounds 3a, 3b and 3c. Among these three,
compound 3c exhibited maximum inhibitory value i.e IC50 SEM = 0.44 0.02 µM and
compound 3b the least inhibition i.e IC50 SEM = 7.13 0.63 µM, having F and Br substitution
respectively. The reverse effect was observed for h-e5′NT i.e. the compounds having less
electronegative substitution exhibited more inhibition of h-e5′NT e.g compound 3b exhibited
~31 fold selectivity over r-e5′NT.
As for as alkaline phosphatase activity is concerned, the compounds were screened against two
isozymes of APs i.e. b-TNAP, c-IAP. All synthetic compounds containing isonicotinohydrazide
nucleus, showed potent inhibition against c- IAP, though, some were found to be potent
inhibitors of b-TNAP comparable to their respective reference standards used in the study.
It is evident from Table-2 that compounds 3b, 3c, 3d, 3f, 3g, 3i and 3j showed potent b-TNAP
inhibition with the inhibitory values (IC50) in the range of 0.35 0.01 to 4.13 0.91 µM as
compared to standard inhibitor i.e. Levamisole with IC50 values 19.21 0.001 µM. The compound
3g exhibited maximum inhibition potential against TNAP with IC50 values 0.35 0.01 µM. While
the whole series (10 compounds) was found to be active against c-IAP with the inhibitory values
of IC50 0.67 0.02 to 17.5 1.01 µM and showed more potency than the familiar reference
standard used in this study i.e. L-Phenylalanine with IC50 values 80.21 0.001µM. Maximum
inhibition activity against c-IAP with IC50 values 0.67 0.02 µM was exhibited by the compound
3a.
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Whereas the derivatives 3b, 3c, 3d, 3f, 3g, 3i and 3j showed dual inhibition against both the
isoenzymes. All these compounds showed inhibitory values in the range of 0.35 0.01 to
4.13 0.91 µM and 0.67 0.02 to 17.5 1.01 µM, against b-TNAP & c-IAP, respectively.
Similarly the derivatives 3a, 3e, and 3h showed selective and potent inhibition of c-IAP with
IC50 values in the range of 0.67 0.02 to 2.46 0.87 µM. Through a comprehensive study of the
structure of the active compound it is clear that potent activity against b-TNAP was due to the
substitution at C-3 and C-4 of the benzene ring of hydrazide derivative with electron donating
groups (-OH and –OCH3). The compound 3g showed potent inhibition against b-TNAP due to
the presence of hydroxyl and methoxy groups on the benzene ring, thus promoting its activity.
While the presence of electrophilic substitution (bromine) at the para position of the same
nucleus (3b) reduces its activity against b-TNAP as compared to compound 3g. Electron
donating groups (-OH and -OCH3) present at C-4 and C-3 of the benzene ring, enhancing its
nucleophilic character (more responsive towards electrophiles) thus, making it more reactive. It
resulted in carbocation formation due to stabilization of the transition state of the benzene ring
system, leading to faster reaction rate of electrophilic attack. This phenomenon bettered
solubility of the compound in polar solvent (water) because anions are extremely solvated by H-
bonding.
While the isonicotinohydrazide derivative (3a) showed potent inhibition of c-IAP with IC50
values 0.67 0.02 µM, due to substitution on the benzene ring with chlorine, an electron
withdrawing group. Since the electron withdrawing group (chlorine) is present at para position of
the benzene ring, hence boosting its reaction rate and lessening nucleophilic property of the
compound (less reactive towards electrophiles). Due to electronegativity, chlorine withdraws
electron density from the benzene ring system, deactivates it weakly, develops an inductive
withdrawal effect, the consequence of which is a slower rate of reaction. By donating electrons
to the benzene ring system, chlorine also displays the resonance effect, but this effect is
suppressed by inductive effect. Due to this attribute, 3a showed increased inhibition potential
against c-IAP.
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Table 2. Ecto-5′-nucleotidase and Alkaline Phosphatase inhibition activity by the synthesized compounds
Ecto-5-nucleotidase Alkaline Phosphatase
Compound
h- e5'NT
r- e5'NT
b-TNAP
c-IAP
IC50 (µM)
IC50 (µM) SEM IC50 (µM) SEM
IC50 (µM) SEM
SEM
1.67 0.58
0.23 0.02
6.76 0.16
13.78 0.12
1.34 0.26
1.90 0.01
0.68 0.01
2.12 0.15
0.21 0.05
0.19 0.02
42.1 7.8
1.08 0.24
7.13 0.63
0.44 0.02
0.31 0.01
0.45 0.03
0.21 0.05
0.14 0.001
2.89 0.41
3.01 0.12
0.36 0.01
77.3 7.0
48.95%
4.13 0.91
2.26 0.56
0.56 0.06
45.14%
0.67 0.02
9.72 1.51
17.5 1.01
1.74 0.16
1.44 0.05
7.34 0.93
1.92 0.03
2.46 0.87
1.46 0.06
3.98 0.96
3a
3b
3c
3d
3e
1.82 0.63
0.35 0.01
44.74%
3f
3g
3h
3i
2.43 0.86
1.06 0.26
3j
Sulfamic acid
Levamisole
L-Phenylalanine
19.2 .001
-
-
80.2 0.001
The IC50 value is the concentration at which 50% of the enzymatic activity is inhibited. Ecto-5-nucleotidase (human
and rat) activity was performed at final concentration of 100 µM while alkaline phosphatase (b-TNAP, c-IAP)
activity was done at the final concentration of 200 µM.
Homology Modeling
From the Protein Data Bank, the X-ray structure of the closed form (PDB ID 4H2I) of human
e5'NT was taken as structural templates for modeling of rat e5'NT (89 % sequence identity).
Homology modeling was carried out using Molecular Operating Environment (MOE) 2014, 09
(34). The active site region of the human and rat enzyme is highly conserved, with only one
residue replacement, Phe500 (human) vs. Tyr502 (rat). The superposition of the human template
structure and rat model of the closed (active) form yielded a Cα RMSD values of less than 0.5 Å.
Ramachandran diagrams confirming high stereochemical quality of the models are provided as
Supplementary Information.
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Molecular Docking
To model a putative binding mode of synthesized compounds, docking studies were carried
using the crystal structure of the closed (active) form human e5’NT and the corresponding rat
e5’NT homology model as well as previously reported models of bovine tissue non-specific and
intestinal alkaline phosphatase..
Molecular docking of the most potent compound 3j was performed using the human e5'NT
crystal structure, while docking of compound 3g was carried out using the closed form of the rat
e5'NT homology model. Compound 3j in the docking model with the human e5'NT closed form
is involved in hydrogen bonding interactions with residue Asn390 and Asp506. These
interactions are similar to those formed by an adenosine moiety (35). In the model, the dihydro
pyrazole ring of the compound 3j was sandwiched between residue Phe417 and Phe500. The
fluorobenzene ring of compound 3j also formed putative hydrogen bonding interactions with
Thr420 and Asp524. Docking of compound 3g into the rat e5'NT model resulted in putative
hydrogen bonding interactions with residues Arg356, Asn247 and His245. Such interactions
have also been reported in case of adenosine (35) as well as sulfonic acid derivatives (36). The
pyridine ring of the compound formed stacking interaction with residue Phe419 (corresponding
to residue Phe417 of human e5'NT) and Tyr502 (Phe500 residue of human e5'NT). The modeled
binding modes of compound 3j and compound 3g are shown in Figure 1.
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Fig. 1 Putative binding mode of compound 3j (colored green) in the human e5'NT crystal structure (brown) and
compound 3g (colored magenta) in the rat e5'NT homology model (cyan). Hydrogen bonding interactions are shown
as dashed green lines, pi-pi interactions as solid yellow lines, and interaction with fluorine interactions as dashed
blue lines.
In the docking model with bovine tissue non-specific alkaline phosphatase, the most potent
compound 3g formed hydrogen bonding interactions with the catalytic Ser110 and with residue
His338. Residue Arg184 and His451 also formed hydrogen bonding interactions with the
inhibitor, consistent with previously observed interactions (37). Residue His451 interacted with
the pyridine ring of the compound. In the case of calf intestinal alkaline, the most potent
compound 3a formed putative hydrogen bonding interactions with residue Ser111 and Arg185
and additional interactions were observed with His172. Figure 2 shows the putative binding
mode of compound 3g and 3a in the models of bovine tissue non-specific and intestine specific
alkaline phosphatase, respectively.
Fig. 2 Putative binding mode of compound 3g (colored magenta) in the model of bovine tissue non-specific alkaline
phosphatase (brown) and compound 3a (colored cyan) in calf intestinal alkaline phosphatase (green). Putative
hydrogen bonding interactions are shown as green lines, stacking interactions as solid yellow lines, and pi-pi t-
shaped interactions as dashed purple dashed lines.
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Conclusion
The bioassay results of the synthesized series and their modelling and docking studies suggested
that the derivatives of Isonicotinohydrazones possess significant activity against APs and e5’NT.
The compound 3j was found to be most active against human e5’NT with an IC50 value of
0.19 0.02. Compound 3a was determined to be most potent against c-IAP with an IC50 value of
0.67 0.02 and compound 3g was found to be most active against rat e5’NT with an IC50 value of
0.14 0.00, as well as potent against b-TNAP with an IC50 value of 0.35 0.01. These newly
synthesized class of compounds can be used to develop novel drug candidates against defective
bone mineralization, solid tumors and cancers of different organs.
Acknowledgements
J. Iqbal is thankful to the Organization for the Prohibition of Chemical Weapons (OPCW), The
Hague, The Netherlands and Higher Education Commission of Pakistan for the financial support
through Project No. 20-3733/NRPU/R&D/ 14/520 for the financial support. JS received support
from the Canadian Institutes of Health Research (CIHR) and was also a recipient of a
“Chercheur National” research award from the Fonds de recherche du Québec – Santé (FRQS).
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