Influence of the diversified structural variations at the imine functionality of 4-bromophenylacetic acid derived hydrazones on alkaline phosphatase inhibition: Synthesis and molecular modelling studies

Article abstract of DOI:10.1039/c5ra14836g

Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to humans with an ability to dephosphorylate and transphosphorylate a wide range of substrates. In humans, four AP isozymes have been identified such as tissue-nonspecific (TNAP), intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of the activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. To identify potent inhibitors of APs, a diverse range of 4-bromophenylacetic acid derived hydrazone derivatives has been synthesized and characterized by spectro-analytical methods and, in the case of 4i and 4q, by single crystal X-ray diffraction analysis. Among the tested series, several compounds were identified as lead candidates showing IC₅₀ values from micro to nanomolar ranges. Compound 4k displayed exceptional activity with an IC₅₀ value of 10 nM against h-IAP. This inhibitory effect is ～10000-fold more potent than the standard drug l-phenylalanine. Compounds 4p, 4g and 4e were potent inhibitors of TNAP, PLAP and GCAP, respectively. Molecular docking studies of the respective potent inhibitors have been carried out to rationalize the important binding modes of the most active inhibitors.

Full text of DOI:10.1039/c5ra14836g

View Article Online
View Journal
RSC Advances
This article can be cited before page numbers have been issued, to do this please use: J. Iqbal, I. Khan, A.
Ibrar, S. Ejaz, S. Khan, S. Shah, S. Hameed, J. Simpson, J. Lecka and J. sevigny, RSC Adv., 2015, DOI:
10.1039/C5RA14836G.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. This Accepted Manuscript will be replaced by the edited,
formatted and paginated article as soon as this is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/advances

Page 1 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
Influence of the diversified structural variations at the imine functionality of 4-
bromophenylacetic acid derived hydrazones on alkaline phosphatase inhibition: Synthesis
and molecular modelling studies
Imtiaz Khana, Aliya Ibrara, Syeda Abida Ejazb, Shafi Ullah Khanb, Syed Jawad Ali Shahb Shahid
,
Hameeda,*, Jim Simpsonc, Joanna Leckad,e, Jean Sévignyd,e and Jamshed Iqbalb,*
aDepartment of Chemistry, Quaid-i-Azam University, Islamabad-45320, Pakistan
bCentre for Advanced Drug Research, COMSATS Institute of Information Technology,
Abbottabad-22060, Pakistan
cDepartment of Chemistry, University of Otago, P.O. Box 56, Dunedin, 9054, New Zealand
dDepartment of Microbiology-Infectiology and Immunology, Faculty of Medicine, Centre de
Recherche du CHU de Québec – Université Laval, Québec, QC, G1V 4G2, Canada
eCentre de Recherche du CHU de Québec – Université Laval, Québec, QC, Canada
_________________________________________________________________________
The present study is aimed at developing novel small molecule inhibitors of alkaline
phosphatases based on bromophenylacetic acid derived hydrazones.
1

RSC Advances
Page 2 of 36
View Article Online
DOI: 10.1039/C5RA14836G
Putative binding mode of 4g inside the active pocket of h-PLAP
________________________________________________________________________
*Corresponding authors. Tel.: +92-51-9064-2133; Fax: +92-51-9064-2241; E-mail:
shameed@qau.edu.pk
(S.
Hameed);
+92-992-383591/96;
Fax:
+92-992-383441.
drjamshed@ciit.net.pk (J. Iqbal)
2

Page 3 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
Abstract
Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man
with an ability to dephosphorylate and transphosphorylate a wide range of substrates. In humans,
four AP isozymes have been identified such as tissue-nonspecific (TNAP), intestinal (IAP),
placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP
isozymes may have therapeutic implications in distinct diseases and cellular processes. To
identify potent inhibitors of APs, a diverse range of 4-bromophenylacetic acid derived hydrazone
derivatives has been synthesized and characterized by spectro-analytical methods and, in case of
4i and 4q, by single crystal X-ray diffraction analysis. Among the tested series, several
compounds were identified as lead candidates showing IC50 value from micro to nanomolar
range. Compound 4k displayed an exceptional activity with an IC50 value of 10 nM against h-
IAP. This inhibitory effect is ~10,000-fold more potent than the standard drug L-phenylalanine.
Compounds 4p, 4g and 4e were potent inhibitors of TNAP, PLAP and GCAP, respectively.
Molecular docking studies of the respective potent inhibitors have been carried out to rationalize
the important binding modes of the most active inhibitors.
Introduction
Alkaline phosphatases (APs, EC 3.1.3.1) are ubiquitous ectoenzymes extensively distributed in
nature from bacteria to humans, with the ability to perform important physiological processes.
Their main functions comprised of catalyzing dephosphorylation and transphosphorylation
reactions on a wide spectrum of physiological and non-physiological substrates.1–3 In humans,
four AP isozymes have been identified with differential tissue expression and encoded by
distinct genes. These are mainly categorized into two groups: the tissue-specific alkaline
phosphatases which include the intestinal (IAP), placental (PLAP), and germ cell (GCAP)
3

RSC Advances
Page 4 of 36
View Article Online
DOI: 10.1039/C5RA14836G
isozymes and the tissue-nonspecific alkaline phosphatase (TNAP), and can be discriminated by
their amino acid sequences and their inhibition by specific inhibitors.4
TNAP is highly expressed in the developing neural tube, liver and kidney.5 Recent and
contemporary studies have documented persuasive proof indicating the major role of TNAP in
bone tissue hydrolyzing the extra cellular inorganic pyrophosphate, PPi, to avoid accumulation of
this mineralization inhibitor, thereby ensuring normal bone mineralization. Thus, small molecule
inhibitors of TNAP have the potential to probe the causative mechanism, or treat disorders of
pyrophosphate metabolism such as in generalized arterial calcification of infancy and related rare
genetic diseases as well as in chronic kidney disease.6
On the other hand, placental alkaline phosphatase (PLAP) is mainly expressed in significant
amounts in the syncytiotrophoblast cells of the placenta from about the eighth week of gestation
throughout pregnancy.7 PLAP was one of the first enzymes recognized as an oncofetal protein
expressed in a variety of cancers.8 The germ cells alkaline phosphatase (GCAP) is also expressed
in the placenta, although at 1/50 the level of PLAP,9 but it is expressed in testicular germ cell
tumors of the testis, particularly seminomas.10 PLAP and GCAP are often co-expressed in
ovarian cancers,11 and it has been suggested that transformation from normal to malignant
trophoblast might be associated with a switch from PLAP to GCAP expression.12 Moreover,
intestinal alkaline phosphatase (IAP) appears to have a role in fat absorption in the
gastrointestinal tract,13 and as a gut mucosal defense factor,14 where its expression coincides with
the stage of colonization by commensal bacterial flora.15 Several isozyme-selective inhibitors of
APs have been reported (Fig. 1), including L-Phe, L-Trp, L-Leu,16 and L-homoarginine.17 Also,
levamisole and theophylline are used as TNAP inhibitors.6 However, in the recent years, several
research groups have identified and optimized small molecule inhibitors of AP isozymes.18
4

Page 5 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
Hydrazones constitute an important class of biologically active drug molecules which has
attracted attention of medicinal chemists due to their wide range of pharmacological properties.
These compounds are reported to possess a notable profile of biological actions like
antimicrobial, anticonvulsant, antihypertensive, antituberculosis, analgesic, anti-inflammatory,
antitumoral, antiglycation, antiproliferative and antimalarial activities.19 Hydrazones have also
been developed as novel inhibitors of IMP-1, a metallo-β-lactamase, antioxidant, and
antileishmanial agents.20 Moreover, many industrial and biologically active compounds utilize
hydrazones as key intermediates for cycloaddition and ring closure reactions.21 The attachment of
hydrazones has been employed both in the design of ligands for further complexation with
transition metals,22 and during the processes of hit-to-lead conversion.23
We have recently developed chromone-based sulfonamides, diaryl sulfonamides and quinoline-
carboxylic acids as potential inhibitors of alkaline phosphatases.24 With this interest, we
continued our studies towards the identification of a new class of potent inhibitors of APs, and
designed a small molecules library of hydrazone derivatives and showed their successful
screening against human recombinant APs. This study identified several potent inhibitory
scaffolds targeting various isoforms of APs. To identify the important binding interactions of the
most active inhibitors, molecular docking studies were performed.
5

RSC Advances
Page 6 of 36
View Article Online
DOI: 10.1039/C5RA14836G
Fig. 1 Examples of AP inhibitors.
Results and discussion
Synthesis of hydrazones
The synthetic route applied for the synthesis of a variety of hydrazone analogues is depicted in
Scheme 1. Starting from 4-bromophenylacetic acid (1), which was first converted to methyl 2-(4-
bromophenyl)acetate (2) by heating at reflux in methanol under acidic conditions followed by
reaction with hydrazine hydrate to afford 2-(4-bromophenyl)acetohydrazide (3).25 Compound 3
was condensed with a range of aldehydes to provide corresponding hydrazones in good yields.26
The scope and generality of this reaction was validated by using a range of aliphatic, aromatic
and heteroaromatic aldehydes. A diverse variation of electron‒rich (‒OH, ‒OMe, ‒OEt) and
electron‒poor (‒NO2) substituents was tolerated on aryl ring of aldehydes producing the desired
hydrazones in good yield. However, the aldehyde bearing para-NO2 group gave complex
mixture of unidentifiable products. In addition, aldehydes with hydroxyl‒protected groups were
also used in this condensation reaction affording Schiff bases in excellent yields. Apart from
aromatic groups, aldehyde with long chain alkyl group (4s) was also tested. Heteroaromatic
aldehyde like furfural was also found to be a good coupling partner affording compound 4r in
6

Page 7 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
85% yield. The reaction involving aldehyde with more sterically demanding biphenyl ring (4q)
was also taken into account.
Entry
4a
R
Entry
4b
R
Entry
4c
R
4d
4g
4e
4h
4f
4i
4j
4k
4n
4l
4m
4o
7

RSC Advances
Page 8 of 36
View Article Online
DOI: 10.1039/C5RA14836G
4p
4s
4q
4r
Scheme 1 Synthesis of hydrazones (4a‒s).
Characterization of hydrazones
The formation of compounds (4a‒s) was indicated by their FTIR spectral data where
characteristic absorptions were observed in the range of 3218‒3171 cm‒1 attributed to the N‒H
1
group in addition to those of C=N in the range of 1650‒1582 cm‒1. In H NMR spectra, N‒H
protons resonated in the range of 11.57‒11.18 ppm along with additional aromatic protons of the
aldehyde moiety. The disappearance of signals for ‒NH2 and ‒CHO also confirmed the
13
formation of hydrazones. C NMR spectra also aided in the confirmation of compounds (4a‒s)
where distinctive signals for imine carbon were observed in the range of 168.9‒152.2 ppm. The
additional resonances for carbons related to aldehyde coupling partner were also observed in
their respective regions with appropriate chemical shift values. 13C NMR spectra were also
devoid of aldehyde carbon signals ~190 ppm which confirmed the condensation reaction
between hydrazide and aldehydes towards the formation of Schiff bases. The purity of the
synthesized compounds was ascertained by elemental analysis. For a full structural elucidation,
the X-ray diffraction measurements were carried out for the synthesized compounds 4i and 4q
(see ESI for experimental details). The molecular structures of compounds 4i and 4q are
illustrated in Fig. 2.
8

Page 9 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
(a)
(b)
Fig. 2 ORTEP of (a) 4i and (b) 4q drawn with 50% ellipsoidal probability along with the atom
numbering scheme.
Enzyme inhibition studies
Alkaline phosphatase inhibition
The inhibitory activity potential of the synthesized hydrazones (4a-s) against human recombinant
APs including TNAP, IAP, PLAP and GCAP was measured using spectrophotometric method.27
The IC50 values were determined for compounds which exhibited over 50% inhibition of either
of the above mentioned enzymes and the results are presented in Table 1. Levamisole was used
as a positive control for both TNAP and PLAP with an IC50 value of 25 2 and 120 3 µM,
respectively. Whereas, L-phenylalanine served as a positive control for both IAP and GCAP with
an IC50 value of 100 3 and 300 5 µM, respectively. The preliminary data generated from this
study led to the identification of 4p, 4k and 4g as the most potent inhibitors of TNAP, IAP and
PLAP with IC50 values of 0.026 0.001, 0.01 0.001 and 0.048 0.009 µM, respectively.
Whereas, only compound 4e inhibited the GCAP with an IC50 value of 0.74 0.06 µM.
Structure-activity relationship analysis
9

RSC Advances
Page 10 of 36
View Article Online
DOI: 10.1039/C5RA14836G
In the light of preliminary data presented in Table 1, a general SAR strategy was developed
around the scaffold 4 focusing on the variation of substituents with different nature and number
on the aryl ring attached to the imine carbon. In general, among the evaluated hydrazone
derivatives, several compounds were identified as potent inhibitors of APs with IC50 value
ranging from 5 µM to 10 nM. The incorporation of hydroxyl groups on the phenyl ring generally
increased the inhibitory potency up to 27-fold as compared to the standard drug levamisole when
tested against h-TNAP (4a; IC50 = 0.94 0.11 µM). The presence of an ortho-hydroxyl group
along with the para-benzylated hydroxyl group in compound 4c showed a significant increase in
the inhibitory potential which is ~309-fold strong inhibition (IC50 = 0.081 0.001 µM). The
compounds with hydroxyl groups capped with alkyl or acetyl groups led to a marked reduction
in activity (4d; 41% inhibition). Para-substituent seems essential for activity towards h-TNAP as
the data indicates lack of anything in para-position shows a large drop in activity (compounds
4e, 4g and 4i).
These compounds also possess benzylated, acetylated and allaylated hydroxyl groups at ortho-
and/ormeta-position of the phenyl ring. On moving the substituent to para-position, a marked
increase in inhibition potential was noticed. For example, compounds 4h and 4k showed ~79-
and ~192-fold strong inhibition as compared to the levamisole with IC50 values of 0.32 0.002
and 0.13 0.001 µM, respectively. Notably, the compounds with para-isopropyl (4l) and ethoxy
(4m) group also exhibited potent inhibition of TNAP with IC50 values of 0.051 0.006 and 0.23
0.04 µM, respectively. This inhibitory potential is ~490- and ~109-fold higher as compared to
the standard drug. The introduction of a polarized nitro group on the phenyl ring at ortho- and
meta-position completely eliminates the activity. The most potent compound of this series
against TNAP was obtained by introducing an α-branched substituent at the imine carbon. This
10

Page 11 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
derivative (4p) displayed ~961-fold increased inhibitory potential with an IC50 value of 0.026
0.001 µM. Extending the steric bulk by introducing a biphenyl ring or changing the aromatic
nature of the substituent afforded compounds 4q and 4r with reduced activity. However, the
introduction of an alkyl substituent (n-Bu) maintained the good inhibition profile with an IC50
value of 0.47 0.02 µM which is ~53-fold strong inhibitory efficacy as compared to positive
control, levamisole (IC50 = 25
2 µM). Overall, from the presented results it could be
anticipated that the substituent nature and position on the phenyl ring impart a large impact on
the TNAP inhibitory potency.
The synthesized hydrazone analogues were also evaluated against h-IAP using L-phenylalanine
as a positive control (IC50 = 100 3 µM). On the initial examination of the results it could be
noticed that several screened compounds displayed remarkable inhibitory activity ranging from
2.09 µM to 10 nm. In the series, the dihydroxylated compound (4a) showed strong inhibitory
activity with an IC50 value of 0.12 0.001 µM which is ~833-fold higher potential as compared
to L-phenylalanine. The variation of the substituent position and nature of hydroxyl protecting
groups decreased the potency but still these compounds showed a beneficial effect by inhibiting
h-IAP with a better ability as compared to the standard drug. These compounds (4c and 4d) were
potent inhibitors with ~48- and ~625-fold higher potency. The compounds with ortho-benzylated
(4e), para-acetylated (4h), and ortho-allylated (4i) hydroxyl groups showed less than 50%
inhibition. However, the compound (4g) with a meta-acetylated hydroxyl group displayed ~312-
fold strong inhibition strength with an IC50 = 0.32 0.04 µM, whereas, 4k bearing a para-
allylated hydroxyl substituent emerged as the most potent analogue showing an exceptional
activity with an IC50value of 10 nM. This inhibitory effect is ~10,000-fold stronger as compared
to the standard drug L-phenylalanine. Also, this compound is the lead member of the evaluated
11

RSC Advances
Page 12 of 36
View Article Online
DOI: 10.1039/C5RA14836G
series. Steric bulk was unfavorable for activity in addition to the electron-withdrawing groups on
the aryl ring. Also, the compounds with hetero-aromatic (4r) and aliphatic (4s) substituents were
not suitable candidates for h-IAP inhibition. However, compound 4m incorporating an electron-
donating (-OEt) group inhibited the IAP with ~170-fold enhanced efficacy.
Similarly, the synthesized hydrazone derivatives were also tested against h-PLAP, another
isozyme of the alkaline phosphatase family. Several compounds showed an excellent inhibition
results. The activity data is summarized in Table 1. The compound 4a with free hydroxyl groups
inhibited the PLAP enzyme with ~222-fold better efficacy and displayed an IC50 value of 0.54
0.03 µM. Also, the compounds 4c and 4d with free or protected hydroxyl groups at variable
positions showed a good inhibition profile with an IC50 value of 0.36 0.01 and 0.26 0.01 µM,
respectively. These results demonstrated the strong inhibitory action of compounds 4a and 4d
against PLAP which is ~333- and ~461-fold, respectively. Similar to the previous results, ortho-
benzylated product could not inhibit the PLAP with better efficiency. However, the compound
with a meta-acetylated hydroxyl group (4g) demonstrated the highest inhibitory potential against
PLAP among the tested compounds. This compound showed an IC50 value of 0.048 0.009 µM
which is ~2500-fold strong inhibition potential. On moving the substituent from meta- to para-
position afforded compound 4h with reduced activity but still this inhibition is ~107-fold higher
as compared to levamisole. Compounds with ortho- and para-allylated hydroxyl (4i and 4k)
groups were also potent inhibitors where 4k inhibited PLAP more strongly (~4-fold) as
compared to 4i and ~2033-fold as compared to positive control. The compounds with para-
substituents like 4l and 4m maintained a similar trend of inhibition by inhibiting PLAP with
~293-fold (IC50 = 0.41 0.01 µM) and ~1428-fold (IC50 = 0.084 0.001 µM), respectively. In
contrast to the previous results, the compounds (4n and 4o) incorporating electron-deficient (-
12

Page 13 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
NO2) group at ortho- and meta-positions of the phenyl ring attached to the imine carbon
exhibited remarkable inhibitory potential against PLAP with an IC50 value of 0.066 0.003 and
0.23 0.06 µM, respectively. In this case, the ortho-substituted derivative (4n) was 3- and 1818-
fold superior as compared to 4o and levamisole, respectively, in terms of inhibition results. The
incorporation of an additional carbon unit spacer between the phenyl ring and the imine moiety
produced compound 4p which exhibited ~24-fold strong inhibition (IC50 = 5.04 0.94 µM). The
introduction of a heterocyclic moiety such as furanyl (4r) resulted in a significant inhibition of
PLAP with an IC50 value of 0.14 0.02 µM. This potential is ~857-fold higher as compared to
the levamisole and is in contrast with the inhibition data of this compound against TNAP and
IAP where less than 50% inhibition has been observed. Compound 4s with an alkyl group also
demonstrated excellent inhibition potential with ~222-fold better efficacy (IC50 = 0.54 0.01
µM).
The synthesized compounds were also tested for their inhibitory potential against h-GCAP where
only 4e incorporating ortho-benzylated hydroxyl group emerged as a significantly potent and
lead member with an IC50 value of 0.74 0.06 µM. This inhibition is ~405-fold strong as
compared to the L-phenylalanine (IC50 = 300 5 µM) and is in contrast with the previous results
obtained for this compound against other isozymes of alkaline phosphatase such as TNAP, IAP
and PLAP. The rest of the compounds showed less than 50% inhibition.
To sum up, these data clearly demonstrate that the nature of the substituent and the position on
the phenyl ring are the main and critical factors determining the potency (and selectivity) for the
inhibition of the various isoforms of alkaline phosphatase.
13

RSC Advances
Page 14 of 36
View Article Online
DOI: 10.1039/C5RA14836G
Table 1. ALP inhibition data for hydrazones (4a-s)
Alkaline phosphatase inhibition
h-IAP h-PLAP
IC50 SEM (µM) / %inhibition
120
Compounds
Substituent (R)
h-TNAP
h-GCAP
—
—
25
2
—
3
—
Levamisole
—
100
3
—
300
5
L-Phenylalanine
0.94 0.11
0.12 0.001
0.54 0.03
28.8%
—
4a
4b
—
—
—
0.081 0.001
41.2%
2.09 0.78
0.16 0.02
42.8%
0.36 0.01
0.26 0.01
44.8%
11.2%
7.28%
4c
4d
4e
43.8%
0.74 0.06
—
—
—
—
4f
4g
4h
42.1%
0.32 0.04 0.048 0.009
36.7%
33.2%
0.32 0.002
46.1%
1.12 0.013
14

Page 15 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
38.2%
33.2%
—
0.21 0.02
41.9%
4i
—
—
—
4j
0.13 0.001
0.01 0.001 0.059 0.003
45.7%
4k
0.051 0.006
0.23 0.04
37.5%
0.41 0.01
21.5%
38.7%
4l
0.59 0.03 0.084 0.001
4m
38.9%
43.6%
36.8%
29.7%
0.066 0.003
0.23 0.06
44.9%
23.6%
4n
4o
0.026 0.001
37.1%
47.2%
44.1%
5.04 0.94
36.3%
33.5%
34.8%
4p
4q
41.4%
41.2%
31.2%
0.14 0.002
0.54 0.01
24.6%
23.8%
4r
4s
0.47 0.02
The IC50 is the concentration at which 50% of the enzyme activity is inhibited. The % inhibition is the value that
compound inhibits specific isozyme at 200 µM concentration. The values are reported with standard error of
mean (IC50 SEM). The experiments were performed in triplicate, n = 3 (See method for detail).
15

RSC Advances
Page 16 of 36
View Article Online
DOI: 10.1039/C5RA14836G
Homology modelling and molecular docking studies
Homology models of human derived tissue non-specific and intestinal alkaline phosphatase was
performed previously24 and is used for molecular docking of our compounds. However,
homology modelling for h-GCAP was carried out using Modeller v9.1428 via Chimera v1.10.1.29
Basic Local Alignment Search Tool (BLAST) of NCBI protein database identified crystal
structure of human derived placental alkaline phosphatase as top ranking templates and PDB ID
1ZED was thus used for modelling because of its high percentage identity with human germ cell
alkaline phosphatase (i.e. 97.7% identity). Five homology models were generated. The
Ramachandran plot of the homology model shows good stereochemical quality of the model
with 97.72% residues in favored region and only 0.21% as Ramachandran outliers (see ESI for
details).
The active site analysis of the human derived germ cells alkaline phosphatase with its template
protein structure revealed only a single amino acid residue change from amino acid Glu429 (of
h-PLAP) to amino acid Gly429 (inside h-GCAP). All other amino acids of the active pocket are
highly conserved (see ESI for details).
Molecular docking studies were performed to rationalize the most plausible binding modes of
APs inhibitors. Docking studies were carried out for the most active inhibitors against specific
AP isoenzymes. These compounds oriented themselves in a similar pattern inside the APs active
site. However, for each active compound inside their respective isozyme the individual
interactions in some cases were observed, while in other cases some interaction differs. This
difference in individual interaction is either due to the presence or absence of specific functional
groups present in individual active compound or due to the difference in the amino acid makeup
of the individual isozyme.
16

Page 17 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
The compound 4g inside the h-PLAP forms characteristic hydrogen bonding interaction with
Arg166, Thr431 and with Glu429 and is in compliance with previously reported interactions.30
Substitution of phenyl acetate in case of compound 4g was proved to be more favorable inside
the active pocket of h-PLAP by providing as extra carbonyl functional group that can form
additional hydrogen bonding interactions with amino acid Arg166 and possible interaction with
Ser92.
Similar to compound 4g, almost similar binding mode was determined for compound 4k inside
h-IAP i.e., hydrogen bonding interaction was observed with amino acid Arg166 and Thr431.
Unlike compound 4g hydrogen bonding interaction occurs with Ser429 (specific amino acid of
h-IAP instead of amino acid residue Glu429 of h-PLAP). p-Allyloxybenzene substitution in case
of compound 4k was found to be oriented deep in the active site groove of h-IAP and oxygen
moiety of this substitution is responsible for the interaction of compound with amino acid
Arg166, however the catalytic amino acid Ser92 was found to be little farther away to form any
interaction with compound 4k.
Molecular docking of ethylbenzene substituted compound 4p inside the h-TNAP yields
interactions similar to that of compound 4g, i.e. it forms hydrogen bonding interactions with
amino acid residue Thr437. Unlike compound 4g, where the h-PLAP specific amino acid Glu429
is involved in hydrogen bonding interaction with protonated nitrogen of compound 4g, no such
interaction was observed because of h-TNAP basic amino acid residue His434. However, amino
acid His434 was observed to form a Pi-Pi stacking interaction with bromobenzene of compound
4p. Other interaction such as interaction with amino acid His437 and Glu435 was also observed.
Except interaction with amino acid Arg167 all these interaction are similar to interactions
previously reported.24 Ethylbenzene substituted group in case of compound 4p was oriented in
17

RSC Advances
Page 18 of 36
View Article Online
DOI: 10.1039/C5RA14836G
direction opposite to that of earlier substitution of 4g or 4k due to hydrophobic nature of the
substitution and thus no interaction with amino acid Arg167 was observed.
Molecular docking of compound 4e also revealed poses similar to other compounds (4g, 4k and
4p). Similar to the compound 4g, a hydrogen bonding interaction of amino acid Thr431 with
carbonyl moiety of the compound was observed. The benzyloxybenzene substitution of the
compound 4e aligned itself in such a way that its oxygen moiety forms polar interaction with
amino acid His432. Additionally, imidazole ring of amino acid His432 forms a Pi-Pi stacking
interaction with the benzene of benzyloxy substitution of compound 4e and thus, stabilized the
pose. The molecular docking interactions of the most active compounds 4e, 4g, 4k and 4p inside
h-GCAP, h-PLAP, h-IAP and h-TNAP respectively, can be seen in Fig. 3.
18

Page 19 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
Fig. 3 Putative binding mode of compound 4p (colored green) inside h-TNAP model (amino
acid colored brown), compound 4g (colored light blue) inside h-PLAP (amino acid colored
green), compound 4k (colored magenta) inside h-IAP (amino acid colored cyan) and compound
4e (colored yellow) inside h-GCAP (colored blue).
19

RSC Advances
Page 20 of 36
View Article Online
DOI: 10.1039/C5RA14836G
Conclusions
In summary, a series of hydrazone derivatives (4a-s) based on 4-bromophenyl acetic acid was
designed and synthesized with a diverse range of substituents at the imine functionality. The
resulting compounds were established on the basis of spectro-analytical data and, in case of 4i
and 4q, by X-ray crystallography. The synthesized compounds displayed alkaline phosphatase
inhibition in low nanomolar to micromolar range and several compounds were identified as lead
candidates. Compound 4k displayed an exceptional activity with an IC50value of 10 nM against
h-IAP with a strong inhibitory effect of ~10,000-fold as compared to the standard drug L-
phenylalanine. Compounds 4p, 4g and 4e were potent inhibitors of human TNAP, PLAP and
GCAP, respectively. Molecular docking studies results revealed the important interactions
between the most active inhibitors and specific APs isoenzymes. The presented SAR and the
identified inhibitors provide an excellent platform for further optimization of AP inhibitors.
Experimental
Materials and methods
Unless specified otherwise, all commercially available reagents and solvents were used as
received. Thin layer chromatography (TLC) was performed on Merck DF-Alufoilien 60F254 0.2
mm precoated plates. Compounds were visualized by exposure to UV light at 254 and 365 nm.
Melting points were recorded on a Stuart melting point apparatus (SMP3) and are
uncorrected.Infra-red (IR) spectra were recorded on FTS 3000 MX, Bio-Rad Merlin (Excalibur
model) spectrophotometer. NMR spectra were acquired on Bruker AV300 spectrometer at room
1
temperature unless otherwise stated. H and 13C NMR spectra were referenced to external
tetramethylsilane via the residual protonated solvent (1H) or the solvent itself (13C). All chemical
shifts are reported in parts per million (ppm). For (CD3)2SO, the shifts are referenced to 2.50
20

Page 21 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
ppm for 1H NMR spectroscopy and 39.52 ppm for 13C NMR spectroscopy.Abbreviations used in
the description of resonances are: s (singlet), br s (broad singlet), d (doublet), dd (doublet of
doublet), t (triplet), q (quartet), m (multiplet), Ar (aromatic). The elemental analysis was
performed on Leco CHNS-932 Elemental Analyzer, Leco Corporation (USA).
Preparation of hydrazones (4a-s)
Preparation of 2-(4-bromophenyl)acetohydrazide (3)
The synthesis of 2-(4-bromophenyl)acetohydrazide (3) was accomplished according to a
literature reported procedure.25 The data was found to be consistent with those observed
previously.31
General procedure for the preparation of hydrazone derivatives (4a-s)
To a stirred solution of 2-(4-bromophenyl)acetohydrazide (3) (1.0 mmol) in methanol was added
appropriate aldehyde (1.2 mmol) and the reaction mixture was heated at reflux for 7 h. After
completion of the reactions (monitored by TLC analysis), the reaction mixture was cooled to
room temperature.The excess solvent was removed under reduced pressure and the purification
of the residue by recrystallization (30% acetone/hexane) afforded the desired hydrazone
derivatives(4a-s).26
2-(4-Bromophenyl)-N'-(3,4-dihydroxybenzylidene)acetohydrazide (4a)
The general experimental procedure described above afforded 4a as a light brown solid (81%,
289 mg). m.p 217-218 °C; Rf: 0.76 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3444 (OH), 3243
(NH), 3063 (Ar-H), 1645 (C=O), 1594 (C=N), 1547, 1520 (C=C); 1H NMR (300 MHz, DMSO-
d6): δ 11.18 (s, 1H, NH), 9.15 (s, 1H, OH), 8.01 (s, 2H, OH, C-H), 7.52-7.48 (m, 3H, Ar-H),
7.28-7.17 (m, 2H, Ar-H), 6.82 (dd, 2H, J = 8.1,7.2, Ar-H), 3.93 (s, 2H, CH2); 13C NMR (75
MHz, DMSO-d6): δ 171.9, 166.2, 148.4, 148.2, 135.8, 132.2, 131.8, 131.6, 131.5, 126.1, 121.0,
21

RSC Advances
Page 22 of 36
View Article Online
DOI: 10.1039/C5RA14836G
120.2, 44.2. Analysis Calcd. for C15H13BrN2O3 (348.01): C, 51.60; H, 3.75; N, 8.02. Found: C,
51.42; H, 3.69; N, 7.91.
2-(4-Bromophenyl)-N'-(2,4-dihydroxybenzylidene)acetohydrazide (4b)
The general experimental procedure described above afforded 4b as a brown solid (70%, 250
mg). m.p 201-202 °C; Rf: 0.73 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3424 (OH), 3198 (NH),
1
3053 (Ar-H), 1637 (C=O), 1580 (C=N), 1574, 1523 (C=C); H NMR (300 MHz, DMSO-d6): δ
11.07 (s, 1H, NH), 9.28 (s, 1H, OH), 7.76 (s, 2H, OH, C-H), 7.56-7.40 (m, 3H, Ar-H), 7.37-7.21
13
(m, 2H, Ar-H), 6.87-6.73 (m, 2H, Ar-H), 3.90 (s, 2H, CH2); C NMR (75 MHz, DMSO-d6): δ
170.8, 165.0, 146.1, 139.6, 134.9, 131.5, 130.7, 130.1, 129.5, 127.0, 122.3, 120.4, 45.6. Analysis
Calcd. for C15H13BrN2O3 (348.01): C, 51.60; H, 3.75; N, 8.02. Found: C, 51.48; H, 3.67; N, 7.85.
N'-(4-(Benzyloxy)-2-hydroxybenzylidene)-2-(4-bromophenyl)acetohydrazide (4c)
The general experimental procedure described above afforded 4c as a white solid (79%, 346 mg).
m.p 200-201 °C; Rf: 0.73 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3440 (OH), 3187 (NH), 3065
1
(Ar-H), 1662 (C=O), 1627 (C=N), 1605, 1566 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.40
(s, 1H, NH), 10.23 (s, 1H, OH), 8.32 (s, 1H, C-H), 8.20 (s, 1H, Ar-H), 7.60-7.23 (m, 8H, Ar-H),
13
6.59-6.53 (m, 3H, Ar-H), 5.11 (s, 2H, OCH2), 3.91 (s, 2H, CH2); C NMR (75 MHz, DMSO-
d6): δ 171.6, 166.2, 158.3, 148.1, 141.9, 137.2, 135.4, 132.1, 131.9, 131.6, 131.5, 128.9, 128.4,
128.2, 120.3, 113.7, 69.8, 44.3. Analysis Calcd. for C22H19BrN2O3 (438.06): C, 60.15; H, 4.36;
N, 6.38. Found: C, 59.98; H, 4.47; N, 6.24.
N'-(3-(Acetoxy)-4-methoxybenzylidene)-2-(4-bromophenyl)acetohydrazide (4d)
The general experimental procedure described above afforded 4d as a light yellow solid (83%,
335 mg). m.p 198-199 °C; Rf: 0.73 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3195 (NH), 3068 (Ar-
1
H), 1667 (C=O), 1650 (C=N), 1591, 1488 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.51 (s,
22

Page 23 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
1H, NH), 8.20 (s, 1H, C-H), 7.97 (s, 1H, Ar-H), 7.53-7.48 (m, 2H, Ar-H), 7.28 (d, 2H, J = 8.4
Hz, Ar-H), 7.17-7.13 (m, 2H, Ar-H), 3.99 (s, 3H, OCH3), 3.55 (s, 2H, CH2), 2.27 (s, 3H, CH3);
13C NMR (75 MHz, DMSO-d6): δ 172.5, 168.9, 166.7, 151.6, 146.5, 140.9, 133.6, 131.8, 131.6,
131.5, 123.7, 120.8, 120.3, 56.3, 40.9, 20.9. Analysis Calcd. for C18H17BrN2O4 (404.04): C,
53.35; H, 4.23; N, 6.91. Found: C, 53.12; H, 4.47; N, 6.79.
N'-(2-(Benzyloxy)benzylidene)-2-(4-bromophenyl)acetohydrazide (4e)
The general experimental procedure described above afforded 4e as a white solid (78%, 329 mg).
m.p 170-171 °C; Rf: 0.75 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3187 (NH), 3069 (Ar-H), 1661
1
(C=O), 1597 (C=N), 1541, 1487 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.48 (s, 1H, NH),
8.49 (s, 1H, C-H), 7.91-7.83 (m, 2H, Ar-H), 7.52-7.46 (m, 2H, Ar-H), 7.45-7.31 (m, 2H, Ar-H),
7.29-7.26 (m, 2H, Ar-H), 7.18 (d, 2H, J = 8.4 Hz, Ar-H), 7.08-6.97 (m,3H, Ar-H), 5.18 (s, 2H,
13
OCH2), 3.97 (s, 2H, CH2); C NMR (75 MHz, DMSO-d6): δ 172.2, 166.4, 157.1, 142.2, 139.3,
137.2, 136.9, 135.7, 135.6, 132.2, 131.8, 131.6, 131.5, 129.0, 120.3, 120.3, 70.1, 44.6. Analysis
Calcd. for C22H19BrN2O2 (422.06): C, 62.42; H, 4.52; N, 6.62. Found: C, 62.31; H, 4.39; N, 6.71.
2-((2-(2-(4-Bromophenyl)acetyl)hydrazono)methyl)phenyl acetate (4f)
The general experimental procedure described above afforded 4f as an off-white solid (71%, 265
mg). m.p 181-182 °C; Rf: 0.69 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3172 (NH), 3057 (Ar-H),
1750, 1639 (C=O), 1600 (C=N), 1560, 1490 (C=C); 1H NMR (300 MHz, DMSO-d6): δ 11.17 (s,
1H, NH), 8.57 (s, 1H, C-H), 7.80-7.77 (m, 4H, Ar-H), 7.63-7.56 (m, 4H, Ar-H), 3.72 (s, 2H,
13
CH2), 2.33 (s, 3H, CH3); C NMR (75 MHz, DMSO-d6): δ 171.4, 167.2, 153.2, 144.0, 139.1,
134.8, 131.8, 131.4, 131.1, 130.4, 129.0, 127.6, 126.9, 45.7, 25.1. Analysis Calcd. for
C17H15BrN2O3 (374.03): C, 54.42; H, 4.03; N, 7.47. Found: C, 54.05; H, 3.91; N, 7.35.
3-((2-(2-(4-Bromophenyl)acetyl)hydrazono)methyl)phenyl acetate (4g)
23

RSC Advances
Page 24 of 36
View Article Online
DOI: 10.1039/C5RA14836G
The general experimental procedure described above afforded 4g as an off-white solid (75%, 280
mg). m.p 197-198 °C; Rf: 0.76 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3192 (NH), 3065 (Ar-H),
1755, 1645 (C=O), 1607 (C=N), 1578, 1485 (C=C); 1H NMR (300 MHz, DMSO-d6): δ 11.29 (s,
1H, NH), 8.61 (s, 1H, C-H), 7.99-7.90 (m, 3H, Ar-H), 7.84-7.77 (m, 3H, Ar-H), 7.66-7.59 (m,
2H, Ar-H), 3.87 (s, 2H, CH2), 2.30 (s, 3H, CH3); 13C NMR (75 MHz, DMSO-d6): δ 171.0, 166.8,
151.6, 140.5, 138.5, 132.1, 131.9, 131.7, 131.6, 130.3, 128.7, 127.9, 127.6, 43.5, 24.0. Analysis
Calcd. for C17H15BrN2O3 (374.03): C, 54.42; H, 4.03; N, 7.47. Found: C, 54.25; H, 4.27; N, 7.37.
4-((2-(2-(4-Bromophenyl)acetyl)hydrazono)methyl)phenyl acetate (4h)
The general experimental procedure described above afforded 4h as a yellow solid (77%, 288
mg). m.p 207-208°C; Rf: 0.68 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3218 (NH), 3068 (Ar-H),
1726, 1661 (C=O), 1594 (C=N), 1557, 1513 (C=C); 1H NMR (300 MHz, DMSO-d6): δ 11.40 (s,
1H, NH), 8.34 (s, 1H, C-H), 7.80-7.71 (m, 2H, Ar-H), 7.62-7.54 (m, 2H, Ar-H), 7.45-7.33 (m,
13
2H, Ar-H), 7.19-7.14 (m, 2H, Ar-H), 3.92 (s, 2H, CH2), 2.30 (s, 3H, CH3); C NMR (75 MHz,
DMSO-d6): δ 171.4, 166.7, 159.4, 140.2, 138.2, 132.7, 132.2, 131.7, 131.6, 127.1, 125.9, 44.5,
28.5. Analysis Calcd. for C17H15BrN2O3 (374.03): C, 54.42; H, 4.03; N, 7.47. Found: C, 54.21;
H, 4.32; N, 7.29.
N'-(2-(Allyloxy)benzylidene)-2-(4-bromophenyl)acetohydrazide (4i)
The general experimental procedure described above afforded 4i as a light brown solid (77%,
286 mg). m.p 176-177 °C; Rf: 0.72 (10% MeOH/CHCl3); IR (ATR, cm-1): 3171 (NH), 3074 (Ar-
1
H), 1667 (C=O), 1609 (C=N), 1573, 1519 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.45 (s,
1H, NH), 8.59 (s, 1H, C-H), 7.88-7.79 (m, 1H, Ar-H), 7.52-7.47 (m, 2H, Ar-H), 7.39-7.24 (m,
3H, Ar-H), 7.15-6.96 (m, 2H, Ar-H), 6.11-6.00 (m, 1H,C-H), 5.50-5.44 (m, 1H, C-H), 5.32-5.26
(m, 1H, C-H), 4.63 (br s, 2H, OCH2), 3.97 (s, 2H, CH2); 13C NMR (75 MHz, DMSO-d6): δ
24

Page 25 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
158.2, 157.0, 156.9, 142.4, 139.3, 135.7, 133.7, 132.2, 131.6, 131.5, 126.0, 125.8, 121.4, 117.6,
69.0, 38.8. Analysis Calcd. for C18H17BrN2O2 (372.05): C, 57.92; H, 4.59; N, 7.51. Found: C,
57.88; H, 4.68; N, 7.46.
N'-(3-(Allyloxy)benzylidene)-2-(4-bromophenyl)acetohydrazide (4j)
The general experimental procedure described above afforded 4j as a brown solid (70%, 260
mg). m.p 165-166 °C; Rf: 0.68 (10% MeOH/CHCl3); IR (ATR, cm-1): 3155 (NH), 3067 (Ar-H),
1
1660 (C=O), 1622 (C=N), 1569, 1524 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.40 (s, 1H,
NH), 8.48 (s, 1H, C-H), 7.80-7.72 (m, 1H, Ar-H), 7.50-7.44 (m, 2H, Ar-H), 7.37-7.28 (m, 3H,
Ar-H), 7.21-6.99 (m, 2H, Ar-H), 6.18-6.08 (m, 1H,C-H), 5.54-5.48 (m, 1H, C-H), 5.30-5.26 (m,
13
1H, C-H), 4.69 (br s, 2H, OCH2), 3.92 (s, 2H, CH2); C NMR (75 MHz, DMSO-d6): δ 159.1,
157.8, 155.7, 144.1, 140.2, 136.4, 133.7, 132.6, 131.9, 131.3, 128.4, 126.2, 123.8, 119.5, 68.7,
39.7. Analysis Calcd. for C18H17BrN2O2 (372.05): C, 57.92; H, 4.59; N, 7.51. Found: C, 57.79;
H, 4.50; N, 7.38.
(E)-N'-(4-(Allyloxy)benzylidene)-2-(4-bromophenyl)acetohydrazide (4k)
The general experimental procedure described above afforded 4k as a yellow solid (74%, 265
mg). m.p 140-141 °C; Rf: 0.74 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3218 (NH), 3071 (Ar-H),
1666 (C=O), 1622 (C=N), 1600, 1573, 1505 (C=C); 1H NMR (300 MHz, DMSO-d6): δ 11.54 (s,
1H, NH), 8.43 (s, 1H, C-H), 7.97-7.82 (m, 2H, Ar-H), 7.78-7.62 (m, 2H, Ar-H), 7.54-7.33 (m,
4H, Ar-H), 6.95-6.79 (m, 1H, C-H), 6.14-5.97 (m, 2H, CH2), 5.56 (m, 2H, OCH2), 3.89 (s, 2H,
13
CH2); C NMR (75 MHz, DMSO-d6): δ 172.2, 158.2, 141.6, 139.2, 132.7, 132.2, 131.7, 131.4,
130.6, 127.9, 126.1, 125.3, 65.7, 45.3. Analysis Calcd. for C18H17BrN2O2 (358.07): C, 57.92; H,
4.59; N, 7.51. Found: C, 57.74; H, 4.35; N, 7.37.
2-(4-Bromophenyl)-N'-(4-isopropylbenzylidene)acetohydrazide (4l)
25

RSC Advances
Page 26 of 36
View Article Online
DOI: 10.1039/C5RA14836G
The general experimental procedure described above afforded 4l as a yellow solid (73%, 261
mg). m.p 149-150 °C; Rf: 0.70 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3194 (NH), 3033 (Ar-H),
1
1652 (C=O), 1607 (C=N), 1572, 1504 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.31 (s, 1H,
NH), 8.37 (s, 1H, C-H), 7.90-7.86 (m, 2H, Ar-H), 7.78-7.64 (m, 2H, Ar-H), 7.59-7.46 (m, 2H,
Ar-H), 6.99-6.84 (m, 2H, Ar-H), 2.86-2.78 (m, 1H, C-H), 2.26 (d, 6H, J = 6.9 Hz, CH3), 3.83 (s,
13
2H, CH2); C NMR (75 MHz, DMSO-d6): δ 171.3, 156.7, 140.2, 132.8, 132.1, 131.6, 131.4,
130.6, 126.4, 124.9, 46.7, 44.2, 29.7. Analysis Calcd. for C18H19BrN2O (358.07): C, 60.18; H,
5.33; N, 7.80. Found: C, 60.22; H, 5.17; N, 7.66.
2-(4-Bromophenyl)-N'-(4-ethoxybenzylidene)acetohydrazide (4m)
The general experimental procedure described above afforded 4m as an off-white solid (83%,
299 mg). m.p 160-161 °C; Rf: 0.72 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3181 (NH), 3085 (Ar-
1
H), 1661 (C=O), 1598 (C=N), 1547, 1518 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.23 (s,
1H, NH), 8.57 (s, 1H, C-H), 8.05-8.02 (m, 2H, Ar-H),7.53-7.47 (m, 2H, Ar-H), 7.40-7.33 (m,
2H, Ar-H), 7.30-7.25 (m, 2H, Ar-H), 5.12 (q, 2H, J = 6.9 Hz, OCH2), 3.87 (s, 2H, CH2), 1.34 (t,
3H, J = 6.6 Hz, CH3); 13C NMR (75 MHz, DMSO-d6): δ 170.3, 158.6, 139.8, 134.6, 132.4,
131.8, 131.5, 131.4, 130.1, 126.4, 55.9, 39.7, 28.4. Analysis Calcd. for C17H17BrN2O2 (360.05):
C, 57.92; H, 4.59; N, 7.51. Found: C, 57.86; H, 4.38; N, 7.42.
N'-(2-Nitrobenzylidene)-2-(4-bromophenyl)acetohydrazide (4n)
The general experimental procedure described above afforded 4n as an off-white solid (75%, 271
mg). m.p 239-240 °C; Rf: 0.78 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3181 (NH), 3075 (Ar-H),
1
1665 (C=O), 1582 (C=N), 1529, 1514 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.57 (s, 1H,
NH), 8.09 (s, 1H, C-H), 7.89-7.82 (m, 2H, Ar-H), 7.53-7.47 (m, 2H, Ar-H), 7.27-7.23 (m, 2H,
Ar-H), 6.88 (d, 1H, J = 4.9 Hz, Ar-H), 6.63-6.60 (m, 1H, Ar-H), 3.90 (s, 2H, CH2); 13C NMR (75
26

Page 27 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
MHz, DMSO-d6): δ 172.2, 159.7, 148.7, 145.6, 145.4, 133.6, 132.3, 131.9, 131.6, 114.0, 113.6,
112.6, 38.5. Analysis. Calcd. for C15H12BrN3O3 (361.01): C, 49.74; H, 3.34; N, 11.60. Found: C,
49.61; H, 3.27; N, 11.39.
N'-(3-Nitrobenzylidene)-2-(4-bromophenyl)acetohydrazide (4o)
The general experimental procedure described above afforded 4o as a light brown solid (84%,
303 mg). m.p 210-211°C; Rf: 0.78 (10% MeOH/CHCl3); IR (ATR, cm-1): 3191 (NH), 3057 (Ar-
1
H), 1666 (C=O), 1610 (C=N), 1552, 1525 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.38 (s,
1H, NH), 8.24-8.19 (m, 2H, Ar-H), 8.15 (s, 1H, C-H), 8.05-8.01 (m, 2H, Ar-H), 7.88-7.71 (m,
4H, Ar-H), 3.92 (s, 2H, CH2); 13C NMR (75 MHz, DMSO-d6): δ 171.2, 152.2, 148.7, 142.5,
138.8, 135.1, 132.3, 131.8, 131.4, 131.5, 128.5, 128.3, 37.6.Analysis Calcd. for C15H12BrN3O3
(361.01): C, 49.74; H, 3.34; N, 11.60. Found: C, 49.58; H, 3.39; N, 11.42.
2-(4-Bromophenyl)-N'-(2-phenylpropylidene)acetohydrazide (4p)
The general experimental procedure described above afforded 4p as a white solid (78%, 268
mg). m.p 160-161 °C; Rf: 0.75 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3201 (NH), 3045 (Ar-H),
1657 (C=O), 1608 (C=N), 1629, 1587, 1556 (C=C); 1H NMR (300 MHz, DMSO-d6): δ 11.25 (s,
1H, NH), 7.61 (s, 1H, C-H), 7.51-7.47 (m, 2H, Ar-H), 7.40-7.31 (m, 2H, Ar-H), 7.26-7.19 (m,
5H, Ar-H), 3.83 (s, 2H, CH2), 3.71-3.67 (m, 1H, C-H), 1.38 (d, 3H, J = 6.6 Hz, CH3); 13C NMR
(75 MHz, DMSO-d6): δ 172.0, 166.3, 153.6, 143.0, 132.1, 131.8, 131.6, 129.2, 127.9, 127.2,
42.5, 19.1, 18.9. Analysis Calcd. for C17H17BrN2O (344.05): C, 59.14; H, 4.96; N, 8.11. Found:
C, 59.02; H, 4.85; N, 8.24.
N'-(Biphenyl-4-ylmethylene)-2-(4-bromophenyl)acetohydrazide (4q)
The general experimental procedure described above afforded 4q as an off-white solid (79%, 310
mg). m.p 213-214 °C; Rf: 0.75 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3179 (NH), 3022 (Ar-H),
27

RSC Advances
Page 28 of 36
View Article Online
DOI: 10.1039/C5RA14836G
1
1667 (C=O), 1639 (C=N), 1591, 1551 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.70 (s, 1H,
NH), 8.25 (s, 1H, C-H), 7.80-7.70 (m, 4H, Ar-H), 7.54-7.45 (m, 4H, Ar-H), 7.41-7.36 (m, 2H,
13
Ar-H), 7.31-7.27 (m, 3H, Ar-H), 4.00 (s, 2H, CH2); C NMR (75 MHz, DMSO-d6): δ 172.4,
166.6, 146.7, 143.1, 139.8, 135.7, 132.2, 131.9, 131.6, 131.5, 129.5, 128.3, 127.8, 127.1, 38.8.
Analysis Calcd. for C18H17BrN2O2 (392.05): C, 57.92; H, 4.59; N, 7.51. Found: C, 57.86; H,
4.54; N, 7.29.
2-(4-Bromophenyl)-N'-(furan-2-ylmethylene)acetohydrazide (4r)
The general experimental procedure described above afforded 4r as a white solid (85%, 260 mg).
m.p 185-186 °C; Rf: 0.71 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3173 (NH), 3054 (Ar-H), 1659
1
(C=O), 1587 (C=N), 1538, 1514 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.34 (s, 1H, NH),
8.45 (s, 1H, C-H), 7.98-7.92 (m, 3H, Ar-H), 7.47-7.38 (m, 2H, Ar-H), 7.10-7.04 (m, 2H, Ar-H),
13
3.83 (s, 2H, CH2); C NMR (75 MHz, DMSO-d6): δ 171.2, 153.3, 146.6, 142.8, 139.9, 134.6,
132.5, 131.7, 131.5, 131.4, 38.6. Analysis Calcd. for C13H11BrN2O2 (306.00): C, 50.84; H, 3.61;
N, 9.12. Found: C, 50.78; H, 3.49; N, 9.04.
2-(4-Bromophenyl)-N'-pentylideneacetohydrazide (4s)
The general experimental procedure described above afforded 4s as a light yellow solid (74%,
219 mg). m.p 119-120 °C; Rf: 0.74 (10% MeOH/CHCl3); IR (ATR, cm‒1): 3211 (NH), 3068 (Ar-
1
H), 1669 (C=O), 1626 (C=N), 1589, 1552 (C=C); H NMR (300 MHz, DMSO-d6): δ 11.28 (s,
1H, NH), 7.98 (s, 1H, C-H), 7.78-7.75 (m, 2H, Ar-H), 7.40-7.31 (m, 2H, Ar-H), 3.84 (s, 2H,
CH2), 1.98-1.95 (m, 2H, CH2), 1.33-1.29 (m, 4H, CH2), 0.98 (t, 3H, J = 6.9 Hz, CH3); 13C NMR
(75 MHz, DMSO-d6): δ 172.6, 153.6, 132.5, 131.8, 131.4, 131.1, 44.4, 33.2, 30.5, 28.9, 20.8.
Analysis Calcd. for C13H17BrN2O (296.05): C, 52.54; H, 5.77; N, 9.43. Found: C, 52.63; H, 5.56;
N, 9.32.
28

Page 29 of 36
RSC Advances
View Article Online
DOI: 10.1039/C5RA14836G
Biological protocol
Cell transfection with human alkaline phosphatases and protein sample preparation
COS-7 cells were transfected with plasmids expressing human APs (TNAP, PLAP, IAP, and
GCAP)32 in 10-cm plates, by using Lipofectamine. The confluent cells were then incubated for 5
h at 37 °C in DMEM/F-12 in the absence of fetal bovine serum and with 6 µg of plasmid DNA
and 24 µL of Lipofectamine reagent. The same volume of DMEM/F-12 containing 20% FBS
was added to stop the transfection and the cells were harvested 48–72 h later.
The transfected cells were washed three times at 4 °C, with Tris–saline buffer, collected by
scraping in the harvesting buffer (95 mM NaCl, 0.1 mM PMSF, and 45 mM Tris buffer, pH 7.5)
and washed twice by centrifugation at 300g for 5 min at 4 °C.33 Subsequently, cells were allowed
to resuspend in the harvesting buffer containing 10 µg/mL aprotinin and sonicated. Cellular and
nuclear debris were discarded by 10 min centrifugation (300×g at 4 °C). Glycerol was added
(final concentration of 7.5%) to the resulting supernatant and the samples were kept at ‒80 °C
until used. Protein concentration was determined by the Bradford microplate method and bovine
serum albumin was used as a reference standard.32
Alkaline phosphatase inhibition assay
A chemiluminescent substrate, CDP-star with or without tested inhibitors, was used for the
determination of AP activity. For this the recombinant form of the following enzymes expressed
32,33
in COS-7 cells as before
were prepared: TNAP, IAP, PLAP and GCAP.6,18 The conditions
for the assay were optimized with the slight modifications in previously used spectrophotometric
method.27 The assay buffer was composed of 2.5 mM MgCl2,0.05 mM ZnCl2 and 8 M DEA (pH
9.8). The compounds were tested initially at the final concentration of 200 µM. The total volume
of 50 µL contained 10 µL of each tested compound (final 200 µM with DMSO 1% (v/v)),
29