Identification of novel chromone based sulfonamides as highly potent and selective inhibitors of alkaline phosphatases

Article abstract of DOI:10.1016/j.ejmech.2013.06.015

A new series of structurally diverse chromone containing sulfonamides has been developed. Crystal structures of three representative compounds (2a, 3a and 4a) in the series are reported. All compounds were screened for their inhibitory potential against alkaline phosphatases (ALPs). Two main classes of ALP isozymes were selected for this study, the tissue non-specific alkaline phosphatase (TNALP) from bovine and porcine source and the tissue-specific intestinal alkaline phosphatases (IALPs) from bovine source. All sulfonamide compounds had a marked preference for IALP (K_i, up to 0.01 ± 0.001 μM) over TNALPs. Kinetics studies of the compounds showed competitive mode of inhibition. Molecular docking studies were carried out in order to characterize the selective inhibition of the compounds. An additional interesting aspect of these chromone sulfonamides is their inhibitory activity against ecto-5′-nucleotidase enzyme.

Full text of DOI:10.1016/j.ejmech.2013.06.015

European Journal of Medicinal Chemistry 66 (2013) 438e449
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Identification of novel chromone based sulfonamides as highly potent
and selective inhibitors of alkaline phosphatases
,
b
c
b
Mariya al-Rashida a , Rabia Raza , Ghulam Abbas , Muhammad Shakil Shah ,
**
George E. Kostakis d, Joanna Lecka e f, Jean Sévigny e f, Muhammad Muddassar g,
,
,
Constantina Papatriantafyllopoulou h, Jamshed Iqbal b
,
*
a Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur Road, 54600 Lahore, Pakistan
b Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
c Interdisciplinary Research Centre in Biomedical Materials (IRCBM), COMSATS Institute of Information Technology, Lahore 54590, Pakistan
d Institute of Nanotechnology, Karlsruhe Institute of Technology, Hermann-von-Helmholtz Platz 1, 76344 Eggenstein-Leopoldshafen, Germany
e Centre de Recherche en Rhumatologie et Immunologie, CHU de Québec, Québec, QC, Canada
f Département de Microbiologie-Infectiologie et d’Immunologie, Faculté de Médecine, Université Laval, Québec, QC, Canada
g Zhang Initiative Research Unit, Advanced Science Institute, RIKEN, 2-1 Hirosawa, Saitama, Wako 351-0198, Japan
h Department of Chemistry, University of Cyprus, CY-1678 Nicosia, Cyprus
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of structurally diverse chromone containing sulfonamides has been developed. Crystal
structures of three representative compounds (2a, 3a and 4a) in the series are reported. All compounds
were screened for their inhibitory potential against alkaline phosphatases (ALPs). Two main classes of
ALP isozymes were selected for this study, the tissue non-specific alkaline phosphatase (TNALP) from
bovine and porcine source and the tissue-specific intestinal alkaline phosphatases (IALPs) from bovine
Received 14 April 2013
Received in revised form
7 June 2013
Accepted 10 June 2013
Available online 19 June 2013
source. All sulfonamide compounds had a marked preference for IALP (Ki, up to 0.01 ꢀ 0.001
mM) over
TNALPs. Kinetics studies of the compounds showed competitive mode of inhibition. Molecular docking
studies were carried out in order to characterize the selective inhibition of the compounds. An additional
interesting aspect of these chromone sulfonamides is their inhibitory activity against ecto-50-nucleo-
tidase enzyme.
Keywords:
Alkaline phosphatase inhibitors
Ecto-50-nucleotidase inhibitors
Chromones
Ó 2013 Elsevier Masson SAS. All rights reserved.
Sulfonamides
Molecular docking
Structure activity relationships (SARs)
1. Introduction
regulatory role of ALPand plasma cell membrane glycoprotein-1 (PC-
1) in bone mineralization via modulation of extracellular PPi levels.
Alkaline phosphatases (ALPs, EC 3.1.3.1) are dimeric enzymes that
catalyze hydrolysis of phosphomonoesters at an alkaline pH [1].
Different isozymes of alkaline phosphatases are grouped under two
categories; the tissue specific alkaline phosphatases (placental PALP,
germ cell GALP, intestinal IALP) and tissue non-specific alkaline
phosphatase (TNALP). The exact physiological function of these en-
zymes is obscure [2]. Avital role of ALP in biomineralization has been
known for a long time by virtue of its high concentrations in
mineralizing tissues. Hessle et al. [3] have provided evidence for the
The concerted relation of TNALP and PC-1 is an inverse one, TNALP
hydrolyzes PPi (a potent inhibitor of mineralization) and PC-1 is
responsible for maintaining sufficient levels of extracellular PPi.
Together TNALP and PC-1 are important for balanced bone forma-
tion. Deficiency of TNALP due to gene mutation is responsible for the
severe disorder of bones and elevated levels of extracellular PPi [4].
On the other hand unwanted deposition of hydroxyapatite (HA)
along with other forms of calcium phosphate in soft tissues is termed
hydroxyapatite deposition disorder (HADD) [5]. It has been sug-
gested that the pool of Pi required for HA deposition is provided by
TNALP via hydrolysis of PPi and nucleoside triphosphates. Another
major source of phosphate supply is through intestinal absorption
where IALP may play a role. IALP has also been suggested to be
involved in lipid absorption as a parallel increase has been observed
in triacylglycerol concentration and IAP activity, during fat absorp-
tion in thoracic duct lymph [6,7].
Abbreviations: ALP, alkaline phosphatase; IALP, intestinal alkaline phosphatase;
TNALP, tissue non specific alkaline phosphatase; ecto-50-NT, ecto-50-nucleotidase.
* Corresponding author. Tel.: þ92 992 3835916; fax: þ92 992 383441.
** Corresponding author. Tel.: þ92 3432106432; fax: þ92 (42) 9923 0703.
E-mail addresses: maria_al_rashida@hotmail.com (M. al-Rashida), jamshediqb@
gmail.com, drjamshed@ciit.net.pk (J. Iqbal).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.06.015

M. al-Rashida et al. / European Journal of Medicinal Chemistry 66 (2013) 438e449
439
TNALP is an important therapeutic target for modulation of
biomineralization. Inhibition of TNALP may have advantages in
treatment of HADD, tumor calcification and vascular mineralization
[8]. IALP was found to be over-expressed in mucosal biopsies in
patients with inflammatory bowel disease specifically Crohn’s
disease [9]. Here the role of IALP selective inhibitors as potential
therapeutic agents cannot be over looked. Because of high homol-
ogy between tissue specific IALP and TNALP, a very few selective
inhibitors of IALP have been reported; moreover inhibition con-
stants of these inhibitors are relatively high and poorly selective
[10e12]. Selective sulfonamide inhibitors of IALP are still lacking in
literature, the only other aryl sulfonamides known as selective in-
hibitors are those of TNALP described by Dahl et al. [13]. This
prompted us toward the development of potent and selective
chromone based sulfonamide inhibitors of TNALP and IALP.
heteroaryl substituted sulfonamides and had the general formula e
RSO2NHR0.
Owing to the presence of three electron deficient centers (C2,
C3 and C4) in the molecule of 3-formylchromone (3-FC), the re-
actions of 3-FC with primary amines do not always yield Schiff
base products. Ethanol is sufficiently nucleophilic and adds into
the C2eC3 olefinic bond of chromone without causing ring
cleavage. Chromone containing sulfonamide enamines were
therefore obtained. Compounds 1ae1e and 2ae2e, formed via
reaction of 3-FC with 4- and 3-ABS respectively, have already been
reported by us [14e16]. When 2-ABS was reacted with 3-
formylchromone (3a), 6-fluoro-3-formylchromone (3b) and 6-
bromo-3-formylchromone (3c),
a benzothiadiazine ring was
formed. However, when 6,8-dibromo-3-formylchromone (3d) and
6-ethyl-3-formylchromone (3e) were reacted with 2-ABS, no
ringed product was obtained and the structures were essentially
similar to those obtained via reaction of 4- and 3-ABS. Single
crystals suitable for analysis by X-ray diffraction were obtained for
compounds 2a, 3a and 4a, their crystal structures are reported
here (Table 1) along with molecular diagrams (Figs. 1e6).
2. Results and discussion
2.1. Chemistry
Depending on the sulfonamide nitrogen substitution, two types
of compounds have beenprepared. Compounds of type A, containing
N-un-substituted sulfonamide moiety (eRSO2NH2, Scheme 1; 1ae
1e, 2ae2e, 3ae3e). The compounds of type B (Scheme 2; 4ae4f)
were similarly prepared by reacting 3-formylchromones with N-
2.2. Enzyme inhibition studies (ALP, ecto-50-NT) and SAR
The synthesized compounds were tested for their inhibitory
potencies against two major groups of alkaline phosphatases i.e.
NH2
2
R
O
O
O
CH3
SO2NH2
4-ABS
1
R
NH2
HN
R2
(1a-1e)
O
2
SO2NH2
SO2NH2
R
SO2NH2
3-ABS
O
O
CH3
R1
O
O
1
NH2
R
SO2NH2
1a, 2a, 3a; R1 = H; R2 = H
1b, 2b, 3b; R1 = F; R2 = H
1c, 2c, 3c; R1 = Br; R2 = H
1d, 2d, 3d; R1 = Br; R2 = Br
1e, 2e, 3e; R1 = Et; R2 = H
O
HN
(2a-2e)
2
R
2-ABS
O
H
N
1
R
O
HN
S
(3a-3c)
O
O
2
R
O
O
CH3
1
R
SO2NH2
O
HN
(3d, 3e)
Scheme 1. Synthesis of compounds 1ae1e, 2ae2e and 3ae3e.

440
M. al-Rashida et al. / European Journal of Medicinal Chemistry 66 (2013) 438e449
CH3
O
O
H2N
O
O
p-TsOH
EtOH
O
+
NH
R
S
R
NHR'
O
O
O
O
S
NHR'
O
(4a-4f)
CH3
O
CH3
S
N
N
4a; R = F; R' =
4b; R = H; R' =
HN
N
4e; R = H; R' =
N
HN
CH3
O
N
N
4c; R = Br; R' =
4d; R = H; R' =
HN
O
4f; R = H; R' =
HN
N
CH3
Scheme 2. Synthesis of compounds 4ae4f.
tissue specific and tissue non specific alkaline phosphatase. Bovine
intestinal ALP was used as tissue specific ALP while two sources for
TNALP were used that are porcine kidney and bovine kidney. The
results showed that all compounds exhibited almost comparable
inhibition on both TNALPs and were generally much more potent
and selective toward IALP. With the only exception of few com-
pounds like 3c, 2a, 2b and 4b (for which there was almost com-
parable inhibition against both IALP and TNALPs), all other
compounds have shown a marked preference for IALP over both
TNALPs (Table 2). A graphical representation emphasizing on se-
lective inhibition of bovine IALP over TNALP inhibition for com-
pounds of type A (Fig. 7) and type B (Fig. 8) is given. Among
compounds of type A, IALP inhibition activities of both m- and p-
sulfonamide compounds were increased many fold upon substi-
tution of chromone ring. Consequently 1a and 2a were weaker IALP
inhibitors as compared to other compounds containing substituted
chromone rings. The effect of halogenation of chromone ring on
ALP activity was significant.
Table 1
Summary of data collection and refinement parameters for compounds 2a, 3a and
4a.
Compound
2a
3a
4a
Formula
C18H17N2O5S
C16H10N2O4S
326.32
C23H19FN4O6S2
530.54
Formula weight
Crystal size
Crystal system
Space group
373.40
0.14 ꢁ 0.10 ꢁ 0.07 0.17 ꢁ 0.10 ꢁ 0.04 0.34 ꢁ 0.25 ꢁ 0.18
Monoclinic
C2/c
Monoclinic
P21/c
Monoclinic
P21/n
Unit cell
dimensions
ꢀ
a [A]
25.8629 (19)
8.4035 (6)
15.7334 (10)
15.0557 (16)
7.0036 (5)
13.9873 (15)
4.9031 (2)
31.9121 (14)
16.4915 (8)
ꢀ
b [A]
ꢀ
c [A]
b
[deg]
98.879 (7)ꢂ
3378.5 (4)
8
113.760 (13)ꢂ
1349.9 (2)
4
93.888 (3)ꢂ
2574.5 (2)
4
3
ꢀ
V [A ]
Z
Density
1.468
1.606
1.369
(Dx Mg mꢃ3
)
F(000)
T [K]
1560
100
0.23
2.8e28.9
ꢃ30 / 28
ꢃ9 / 9
ꢃ18 / 18
Multi-scan
672
100
0.26
2.9e29.1
ꢃ17 / 17
ꢃ6 / 8
ꢃ16 / 11
Multi-scan
1096
296
0.259
2.6e28.4
ꢃ6 / 6
ꢃ42 / 41
ꢃ20 / 21
Multi-scan
m
q
h
k
l
(Mo-K
range [ꢂ]
a
) [mmꢃ1
]
Absorption
correction
Measured
reflections
Independent
reflections
Rint
8321
2965
4843
2385
20,520
6408
0.056
251
0.91/ꢃ0.36
0.065
216
2.35/ꢃ0.72
0.0899
290
0.42/ꢃ0.28
Parameters
Max. difference
peak/hole
ꢀꢃ3
[e A
]
S
1.07
1.26
0.92
Reflections with
2525
1914
2456
I > 2
s
(I)
R[F2 > 2
wR(F2)
s
(F2)]
0.047
0.123
0.109
0.317
0.055
0.168
Fig. 1. ORTEP plot of 2a showing atom labeling. Thermal ellipsoids are drawn at 50%
probability level.

M. al-Rashida et al. / European Journal of Medicinal Chemistry 66 (2013) 438e449
441
Fig. 4. Molecular packing diagram of 3a.
Fig. 2. Molecular packing diagram of 2a.
decrease in bovine TNALP inhibition. For these compounds 6-
substitution at the chromone ring not only caused a decrease in
bovine TNALP inhibition but at the same time also caused a sig-
nificant increase in IALP inhibition (Table 2). This behavior is epit-
omized by comparing 1a (potent TNALP inhibitor; weak IALP
inhibitor) and its 6-fluoro analog 1b (potent IALP inhibitor; weak
TNALP inhibitor) (Fig. 10). This important SAR consideration can be
a prolific lead in design of isozyme selective ALP inhibitors from
this class of compounds. For the rest of the series 2ae2e, 3ae3e and
4ae4f, 6-substitution at chromone ring did cause an average
decrease in bovine TNALP inhibition, but the trend is not unani-
mous for all compounds, for example compounds 2b, 2d, 2e, 3c and
4b did not show this behavior.
In order to characterize the mechanism of inhibition, double
reciprocal plots of substrate concentration (mMꢃ1) and initial ve-
locity were plotted for most potent inhibitor (3c) against CIALP.
Enzyme kinetics was determined by evaluating the effect of 0, 100
and 300 nm of 3c. A direct linear relationship in the Km value and
inhibitor concentration was observed i.e. Km value increased with
increasing inhibitor concentration. However Vmax value remained
unaffected in the absence and presence of inhibitors. Thus, it was
inferred that both the compounds showed competitive inhibition.
Double-reciprocal plot of the inhibition kinetics of 3c is shown in
Fig. 11.
When SO2NH2 group was at meta position, 6,8-di-substitution
on chromone ring was preferred, hence compound 2d was most
potent IALP inhibitor among m-SO2NH2 containing compounds.
The IALP inhibition tendency decreased significantly when bulky
groups at 6-position of chromone ring were substituted by rela-
tively compact groups, hence activity decreased in the order
Et > Br > F [ H. Similar trends were observed for p-SO2NH2
containing compounds. In general bulky substituents at 6-position
of chromone ring were preferred. Hence bromine containing 1c
was more active than fluorine containing 1b. However, in contrast
to m-series, in p-SO2NH2 containing compounds simultaneous
substitution at 6 and 8 position of chromone ring resulted in
decreased IALP inhibition. The least active compound in this series
was 1a which contained no substitution at chromone ring. Among
compounds derived from 2-ABS (3ae3e), compound 3c was most
active IALP inhibitor followed by 3a. Compounds in this series were
also found to selectively inhibit bovine IALP over bovine TNALP. A
comparison of concentration response curve of 3c against bovine
IALP, bovine TNALP and porcine TNALP is represented in Fig. 9.
Compounds of type B (4ae4f) with N-substituted sulfonamide
group were also found be selective inhibitors of IALP over TNALP.
Fluorine containing compound 4a was most active. However when
the fluorine was replaced by a hydrogen atom in 4b, a decrease in
IALP inhibition was observed. Compounds 4e and 4f inhibited IALP
at a similar degree, but had different level of TNALP inhibition.
An interesting inference, regarding selective inhibition can be
drawn from the structure activity relationship studies of p-SO2NH2
containing compounds (1ae1e). For all compounds in this series,
substituents at 6-position of chromone ring caused a significant
Since the compounds prepared by us showed excellent potential
to inhibit metal containing ALP which hydrolyzes phosphomo-
noesters, it was hypothesized that these compounds could also act
Fig. 3. ORTEP plot of 3a showing atom labeling. Thermal ellipsoids are drawn at 50%
Fig. 5. ORTEP plot of 3a showing atom labeling. Thermal ellipsoids are drawn at 30%
probability level.
probability level.

442
M. al-Rashida et al. / European Journal of Medicinal Chemistry 66 (2013) 438e449
Fig. 6. Molecular packing diagram of 4a.
as inhibitors of yet another metallophosphoesterase enzyme ecto-
50-NT (EC 3.1.3.5, lymphocyte differentiation antigen CD73) [17] for
which a few inhibitors are known [18e20]. Ecto-50-NT is anchored
to the cell membrane via GPI linkages and is responsible for
dephosphorylation of ribo- and deoxyribonucleotide-50-mono-
phosphates to corresponding nucleosides, with AMP as the
preferred substrate [21]. It is over expressed in tumors and is
implicated in promoting metastatic activity of tumors by increasing
extracellular concentrations of adenosine [22,18]. Keeping this in
mind all compounds were assayed against ecto-50-NT (Table 3). All
compounds derived from 4-ABS (1ae1e) inhibited ecto-50-NT
modestly, up to 52% of inhibition at 0.1 mM concentration. It has
been found that substitution of chromone ring increases ecto-50-NT
inhibition. Among compounds derived from 3-ABS, the 6,8-
dibromo compound 2d and 6-ethyl substituted 2e showed inhibi-
tion. Compounds derived from 2-ABS, 3ae3c were devoid of any
ecto-50-NT inhibitory activity, whereas, compounds 3d and 3e
exhibited some inhibition. The 6,8-dibromo compound 3d showed
notable 63% inhibition, whereas, the ethyl substituted 3e did not
have any effect on the ecto-50-NT activity. Among compounds of
type B, all were inactive except 4b which exhibited a weak unim-
pressive 26% inhibition.
An additional relationship can be derived from comparison of
SAR of three structural isomers 1d, 2d and 3d. Among all com-
pounds analyzed in this series, 3d is an active inhibitor of ecto-50-
NT (63% inhibition). As the position of sulfonamide group is
changed from o- to m- as in 2d, ecto-50-NT activity decreases to 54%.
When sulfonamide group is at para position (1d), the activity de-
creases even further and is only 43%. Although the decrease in in-
hibition is not sharp, but given the lack of ecto-50-NT inhibitors in
literature, this SAR may be exploited in hopes of optimizing in-
hibitors from this class of compounds. Interestingly, for the same
structural isomers, the IALP inhibition activity also decreases in the
same manner (Fig. 12). In our opinion it is important to assay in-
hibitors of ALP against ecto-50-NT as well, since both these enzymes
are widely distributed in living systems, both are AMP hydrolyzing
ectoenzymes. Although the inhibition shown by these compounds
is only modest, nevertheless, this information is noteworthy
especially when one considers the scarcity of known ecto-50-NT
inhibitors in the available literature.
Table 2
Inhibition activities of chromone based sulfonamide derivatives 1ae1e, 2ae2e, 3ae
3d and 4ae4f against calf intestine alkaline phosphatase, porcine kidney and bovine
kidney tissue non-specific alkaline phosphatase.
Compound
Ki ꢀ SEM (
m
M)a
IALPb
TNALPb
(porcine kidney)
TNALPb
(bovine kidney)
(bovine intestine)
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
90.6 ꢀ 8.9
1.43 ꢀ 0.4
0.41 ꢀ 0.002
5.1 ꢀ 0.2
10.9 ꢀ 1.8
66.8 ꢀ 3.0
44.6 ꢀ 2.9
55.3 ꢀ 2.5
68.8 ꢀ 3.0
41.7 ꢀ 8.3
19.9 ꢀ 0.1
51.3 ꢀ 1.4
67.9 ꢀ 12
20.1 ꢀ 1.2
21.3 ꢀ 6.3
2.8 ꢀ 0.4
6.8 ꢀ 1.0
220 ꢀ 37
48 ꢀ 13
63 ꢀ 16
2.6 ꢀ 0.03
33.5 ꢀ 8.0
17.1 ꢀ 0.02
8.7 ꢀ 0.09
0.8 ꢀ 0.001
2.1 ꢀ 0.08
0.049 ꢀ 0.007
0.78 ꢀ 0.13
0.021 ꢀ 0.007
0.2 ꢀ 0.01
0.4 ꢀ 0.001
0.01 ꢀ 0.001
0.085 ꢀ 0.01
9.3 ꢀ 0.01
0.41 ꢀ 0.06
7.8 ꢀ 0.02
7.8 ꢀ 0.03
60 ꢀ 6.2
41 ꢀ 8.3
18 ꢀ 2.2
44 ꢀ 6.7
26 ꢀ 9.9
16.8 ꢀ 9.3
27 ꢀ 5.8
43 ꢀ 7.4
0.13 ꢀ 0.01
29 ꢀ 4.1
29 ꢀ 3.0
21 ꢀ 7.8
0.25 ꢀ 0.07
74 ꢀ 14
0.021 ꢀ 0.002
11.7 ꢀ 1.2
18.0 ꢀ 1.1
31.8 ꢀ 7.6
0.079 ꢀ 0.01
73.6 ꢀ 6
2.3. Molecular docking studies
Molecular docking studies were carried out to determine the
probable binding modes of these compounds in homology modeled
active sites of both the enzymes. Fig. 13 shows plausible binding
modes for three representative compounds 2a, 3a and 4a (XRD data
for these compounds is also reported here). 3D generated models
structural analyses showed that both isozymes contain similar
residues in active site except with the difference of Arg-127 and Ser
447 in IALP with Gly-126 and Arg 450 in TNALP. The top ranked best
21.2 ꢀ 2.1
52.5 ꢀ 3.5
35.4 ꢀ 1.3
22 ꢀ 5.9
38 ꢀ 11
23 ꢀ 11
a
Values are the mean ꢀ SEM of three experiments.
b
TNALP, tissue-non specific alkaline phosphatase; IALP, calf intestinal alkaline
phosphatase.

M. al-Rashida et al. / European Journal of Medicinal Chemistry 66 (2013) 438e449
443
Br
O
O
O
HN
(1a)
O
O
O
O
O
O
O
O
O
HN
(1d)
O
O
O
F
Et
Br
Br
HN
HN
HN
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
(1c)
(1b)
1.43
220
(1e)
0.41
48
5.1
63
IALP (Ki
µ
M)
2.6
60
0.002
13
0.2
16
90.6
6.8
0.4
37
0.03
6.2
8.9
1.0
TNALP ( Ki µM)
Br
O
O
O
O
O
O
O
O
O
O
O
O
O
O
F
Et
Br
Br
O
HN
HN
HN
HN
HN
SO2NH2
SO2NH2
SO2NH2
SO2NH2
SO2NH2
(2a)
(2c)
(2b)
(2e)
(2d)
33.5 8.0
41 8.3
8.7 0.09
44 6.7
0.8 0.001
26 9.9
17.1 0.02
18 2.2
2.1 0.08
16.8 9.3
Br
O
O
O
O
O
O
O
O
O
H
N
H
N
H
N
O
O
O
Br
Et
S
O
Br
S
O
F
S
O
HN
HN
O
HN
O
HN
O
HN
H2NO2S
H2NO2S
(3d)
(3b)
(3c)
(3a)
(3e)
0.2 0.01
29 4.1
0.021 0.007
0.13 0.01
0.78 0.13
43 7.4
0.4 0.001
29 3.0
0.049 0.007
27.3 5.8
Fig. 7. Comparison of bovine IALP and bovine TNALP inhibition activities (Ki, mM) of compounds 1ae1e, 2ae2e and 3ae3e.
probable binding mode of the most active IALP inhibitor (4a) is
shown in Fig. 14. The sulfone group of 4a makes strong ionic
interaction with the Zn ion in active site and thiadiazole group
formulates H-bonding with the Arg-127, whereas carbonyl oxygen
of chromen makes H-bonding with the Arg169 present at the edge
of binding site. In our modeling studies it has been observed that in
IALP, most of the compounds have similar binding interaction
pattern. Whereas in TNALP, the binding modes and interaction
patterns are not consistent like those in IALP. This might be due to
the absence of non conserved residue Arg-127 in TNALP, which is
responsible for H-bonding interactions of these compounds in IALP.
This difference might be the explanation for the relative selectivity
of these compounds series for IALP over TNALP enzyme.
study the selective inhibition showed by these compounds. Com-
M) were
found to be most active IALP inhibitors. This is the first study
describing compounds based on chromone scaffold that exhibit not
only potent but also highly selective IALP inhibition activities as
compared to TNALP.
pounds 3c (Ki, 0.021 ꢀ 0.007
mM) and 4a (Ki, 0.01 ꢀ 0.001
m
4. Experimental
All reagents were purchased from either Sigma or Aldrich and
were used as such. Commercially available solvents were used.
Ethanol was distilled and dried using standard methods and stored
over molecular sieves. Reaction progress and product purity was
monitored by TLC silica gel plates (0.2 mm, 60 HF254, Merck). TLC
spots were analyzed under short and long wavelength UV radia-
tion. Melting points were taken on a Gallenkamp melting point
apparatus and were uncorrected. Purity of compounds was estab-
lished by using HPLC (Shimadzu Liquid Chromatograph LC-10 AS
with UV Spectrophotometric Detector, Shimadzu SPD-6A) on C18
3. Conclusions
Series of novel chromone containing sulfonamides was syn-
thesized and evaluated for their alkaline phosphatase inhibitory
activity. Two major classes of ALP, i.e., tissue non specific TNALP
(from bovine and porcine source) and intestinal IALP (from bovine
source) were selected for enzyme assay. All compounds showed
excellent and selective IALP inhibition over TNALP with Ki value up
column (M2 Analytical column 250 ꢁ 4.6 mm; 5
mm). All the
chromatograms obtained were very similar due to chemical simi-
larity of the compounds. IR spectra were recorded on Perkin Elmer
Spectrum BX-II. 1H NMR and 13C NMR spectra were recorded on
to 0.01 ꢀ 0.001
mM. Molecular docking studies were carried out to

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M. al-Rashida et al. / European Journal of Medicinal Chemistry 66 (2013) 438e449
O
O
O
O
O
O
O
NH
NH
NH
Br
F
O
S
S
O
O
O
O
N
N
S
S
S
NH
N
N
N
NH
NH
O
O
O
(4b)
(4c)
(4a)
0.085 0.01
0.25 0.07
9.3 0.01
74 14
K
IALP (
µ
M)
0.01 0.001
21 7.8
i
K
µ
TNALP (
M)
i
O
O
O
O
O
O
O
O
N
NH
NH
NH
O
N
O
O
O
O
O
S
S
N
S
NH
O
NH
N
NH
N
O
O
O
(4f)
(4d)
(4e)
IALP (K
µ
M)
M)
i
0.41 0.06
7.8 0.03
23 11
7.8 0.02
38 11
TNALP (K
µ
i
22 5.9
Fig. 8. Comparison of bovine IALP and bovine TNALP inhibition activities (Ki, mM) of compounds 4ae4f.
Bruker (300, 400 and 500 MHz) AMX Spectrometer. Chemical shift
values were referenced against TMS. DMSO-d6 was used as solvent
for NMR spectroscopy. Mass Spectra were recorded on Finnigan
MAT 312 Spectrometer. Calf intestine alkaline phosphatase IALP
(1000 U), porcine kidney alkaline phosphatase TNALP (100 U), Tris
HCl, MgCl2, ZnCl2 and phenyl phosphate disodium salt were pur-
chased from Sigma, Germany. For the spectrophotometric deter-
mination of p-nitrophenol which was product of ALP, a 96 well
plate microplate reader from Bio-Tek ELx 800Ô, Instruments, Inc.
USA was used.
4.1.2. 3-(1,1-Dioxido-3,4-dihydro-2H-1,2,4-benzothiadiazin-3-yl)-
6-fluoro-4H-chromen-4-one (3b)
Yield 81% as white crystalline needles, m.p. 198e200 ꢂC; IR (
n,
cmꢃ1): 1645 (C]O), 1603 (C]C), 1288 (CeO), 3429 (NH), 1154
(SOs2ym), 1376 (SOa2sym). EIeMS m/z (rel. int. %) 344.0 (7), 281 (76),
190.1 (44), 164 (13), 139.1 (22), 92.1 (29), 65.1 (62). 1H NMR (DMSO-
d6, 500 MHz)
d
: 6.02 (1H, d, J ¼ 9.5 Hz, H-8), 6.84 (1H, d, J ¼ 8.3 Hz,
H-60), 6.80 (1H, t, J ¼ 7.7 Hz, H-40), 7.30 (1H, s, H-11), 7.353 (1H, t,
J ¼ 7.8 Hz, H-50), 7.54 (1H, d, J ¼ 7.8 Hz, H-30), 7.77e7.83 (3H, m, NH,
H-7, eSO2NH), 7.87 (1H, dd, J ¼ 4.2 Hz, J ¼ 9.1 Hz, H-5), 8.65 (1H, s,
H-2). 13C NMR (DMSO-d6, 400 MHz)
d: 173.88 (C-4), 160.46 (C-6),
158.02 (C-9), 152.12 (C-20), 143.57 (C-10), 124.47 (C-10), 119.84 (C-3),
4.1. General method for the synthesis of compounds 3ae3e and
4ae4f
A solution of 0.001 M 6-(un)substituted 3-formylchromone in
10 mL ethanol was stirred with heating until dissolved. Catalytic
amount of p-toluenesulfonic acid (p-TsOH) was added (for com-
pounds 3ae3e) followed by the addition of equimolar solution of
appropriate sulfonamide in 10 mL of ethanol. The reaction mixture
was refluxed for 3.5 h and kept overnight. Solid product was ob-
tained by filtration and purified by recrystallization from a mixture
of hot ethanol and acetone (1:1).
4.1.1. 3-(1,1-Dioxido-3,4-dihydro-2H-1,2,4-benzothiadiazin-3-yl)-
4H-chromen-4-one (3a)
Yield 65% as yellow crystalline solid, m.p. 188e190 ꢂC; IR (
n,
cmꢃ1): 1648 (C]O), 1557 (C]C), 1286 (CeO), 3343 (NH), 1154
(SO2sym), 1376 (SO2as). EIeMS m/z (rel. int. %) 326.0 (14), 263.2
(73.2), 172.0 (73.5), 121.0 (35.8), 92.1 (100), 65.0 (87.2). 1H NMR
(DMSO-d6, 300 MHz)
d
: 6.06 (1H, d, J ¼ 12.0 Hz, H-8), 6.81 (1H, t,
J ¼ 7.7 Hz, H-40), 6.85 (1H, d, J ¼ 8.3 Hz, H-60), 7.31 (1H, s, H-11), 7.34
(1H, ddd, J ¼ 1.2 Hz, J ¼ 7.9 Hz, H-50), 7.52e7.90 (5H, m, ArH
(chromone), eSO2NH, NH), 8.15 (1H, dd, J ¼ 1.2 Hz, J ¼ 7.9 Hz, H-30),
1H, 8.63 (s, H-2). 13C NMR (DMSO-d6, 400 MHz)
d: 174.49 (C-4),
155.63 (C-9),143.64 (C-20),123.81 (C-10),121.48 (C-10),120.40 (C-3),
156.82 (C-2), 134.84 (C-7), 133.06 (C-50), 126.10 (C-5), 125.28 (C-30),
123.81 (C-6), 118.60 (C-8), 117.11 (C-40), 116.58 (C-60), 60.27 (C-11).
Fig. 9. Concentration-response curves of 3c against bovine TNALP, bovine IALP, and
porcine TNALP.

M. al-Rashida et al. / European Journal of Medicinal Chemistry 66 (2013) 438e449
445
Fig. 10. 6-Substitution at chromone ring causes a decrease in bovine TNALP inhibition activity and an increase in bovine IALP inhibition.
157.10 (C-2), 133.09 (C-50), 123.80 (C-30), 123.17 (C-7), 121.50 (C-8),
117.17 (C-40), 116.32 (C-5), 110.04 (C-60), 60.18 (C-11).
4.1.4. 2-{[(6,8-Dibromo-2-ethoxy-4-oxo-2H-chromen-3(4H)-
ylidene)methyl]amino} benzenesulfonamide (3d)
Yield 72% as yellow needles, m.p. 166e168 ꢂC; IR ( , cmꢃ1): 1640
n
4.1.3. 6-Bromo-3-(1,1-dioxido-3,4-dihydro-2H-1,2,4-
(C]O), 1592 (C]C), 1280 (CeO), 3345 (NH2), 1153 (SOs2ym), 1336
(SOa2sym). EIeMS m/z (rel. int. %) 420.1 (7), 247.0 (100), 172.0 (31),
benzothiadiazin-3-yl)-4H-chromen-4-one (3c)
Yield 77% as colorless needles, m.p. 224e226 ꢂC; IR (
n
, cmꢃ1):
146.0 (9), 121.1 (17), 76.1 (28). 1H NMR (DMSO-d6, 400 MHz)
d: 1.11
1645 (C]O), 1602 (C]C), 1287 (CeO), 3355 (NH), 1157 (SO2sym),
1316 (SO2asym). EIeMS m/z (rel. int. %) 406.0 (15), 343.1 (31), 252.0
(27), 172.1 (25), 155.0 (40), 92.1 (100), 65.1 (75). 1H NMR (DMSO-d6,
(3H, t, J ¼ 7.2 Hz, CH3), 3.77 (2H, q, J ¼ 7.2 Hz, CH2), 12.24 (1H, d,
J ¼ 12.4 Hz, NH), 8.14 (1H, d, J ¼ 12.4 Hz, CH enamine), 7.90 (1H, s,
H-7), 8.07 (1H, s, H-5), 6.14 (1H, s, H-2), 7.86 (1H, d, J ¼ 7.6 Hz, H-60),
7.77 (4H, m, eSO2NH2, ArH benzene), 7.33 (1H, m, H-40). 13C NMR
400 MHz)
d
: 6.02 (1H, br s, NH), 6.81 (1H, t, J ¼ 7.6 Hz, H-40), 6.84
(1H, d, J ¼ 8.4 Hz, H-60), 7.31 (1H, s, H-11), 7.35 (1H, t, J ¼ 7.8 Hz, H-
50), 7.54 (1H, d, J ¼ 7.6 Hz, H-8), 7.76 (1H, d, J ¼ 7.8 Hz, H-30), 7.84
(1H, br s, eSO2NH), 8.05 (1H, dd, J ¼ 2.4 Hz, J ¼ 7.8 Hz, H-7), 8.20
(1H, d, J ¼ 2.4 Hz, H-5), 8.65 (1H, s, H-2). 13C NMR (DMSO-d6,
(DMSO-d6, 300 MHz) d
: 177.86 (C-4), 151.45 (C-9), 124.97 (C-20),
113.87 (C-10), 139.42 (C-10), 113.01 (C-6), 142.14 (C-8), 103.58 (C-3),
100.63 (C-2), 145.71 (C-7), 138.75 (C-5), 116.96 (C-50), 116.74 (C-40),
127.86 (C-30), 127.44 (C-60), 138.87 (C-11), 63.57 (CH2 ethoxy), 14.75
(CH3 ethoxy).
400 MHz) d
: 173.31 (C-4), 154.61 (C-9), 143.55 (C-20), 127.33 (C-10),
124.72 (C-10), 120.58 (C-6), 118.54 (C-3), 157.19 (C-2), 137.49 (C-7),
133.09 (C-5), 123.80 (C-50), 121.51 (C-30), 121.51 (C-8), 117.19 (C-4),
116.33 (C-60), 60.16 (C-11).
4.1.5. 2-{[(6-Ethyl-2-ethoxy-4-oxo-2H-chromen-3(4H)-ylidene)
methyl]amino} benzenesulfonamide (3e)
Yield 72% as yellow needles, m.p. 166e168 ꢂC; IR ( , cmꢃ1): 1640
n
(C]O), 1592 (C]C), 1280 (CeO), 3345 (NH2), 1153 (SOs2ym), 1336
(SOa2sym). EIeMS m/z (rel. int. %) 420.1 (7), 247.0 (100), 172.0 (31),
146.0 (9), 121.1 (17), 76.1 (28). 1H NMR (DMSO-d6, 400 MHz)
d: 1.01
(3H, t, J ¼ 7.2 Hz, CH3 ethyl), 1.20 (3H, t, J ¼ 7.5 Hz, CH3 ethoxy), 3.68
(2H, q, J ¼ 7.2 Hz, CH2 ethoxy), 2.65 (2H, q, J ¼ 7.5 Hz, CH2 ethyl),
11.81 (1H, d, J ¼ 12.3 Hz, NH), 8.15 (1H, d, J ¼ 12.6 Hz, H11), 7.40 (1H,
dd, J ¼ 2.4 Hz, J ¼ 8.7 Hz, H-7), 7.53 (2H, d, J ¼ 8.7 Hz, H-40, H-50),
Table 3
Percent inhibition values against human ecto-50-NT at 0.1 mM concentration.
Compound
% inhibition
Ecto-50-NT
Compound
% inhibition
Ecto-50-NT
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
5.7
16
52
43
13
0
0
0
54
30
0
3b
3c
3d
3e
4a
4b
4c
4d
4e
4f
0
0
63
4
0
26
0
0
0
0
Fig. 11. Double-reciprocal plots of the inhibition kinetics of bovine intestine alkaline
phosphatase by compound 3c. Changes in the initial velocities of the reaction were
measured at different concentrations of the inhibitor 3c using substrate (4-NPP)
concentrations of 0.01e1 mM.

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M. al-Rashida et al. / European Journal of Medicinal Chemistry 66 (2013) 438e449
Br
Br
Br
O
O
O
O
O
O
NH2
S
Br
O
O
Br
Br
O
NH2
O
HN
O
HN
O
HN
S
O
O
S
3d
NH2
2d
1d
O
>
IALP (Ki µM)
5.1
>
0.2
0.8 0.001
0.2 0.01
Ecto-5'-NT (% inhib)
43
63
54
Fig. 12. IALP and Ecto-50-NT inhibition activities for 3 structural isomers 1d, 2d and 3d showing a decrease in inhibition activity as the sulfonamide group is shifted from o- through
m- to p-position of the benzene ring.
7.01 (1H, d, J ¼ 8.4 Hz, H-8), 7.80 (2H, d, J ¼ 8.7 Hz, H-30, H-60), 7.29
(2H, br s, eSO2NH2), 7.64 (1H, s, H-5), 5.91 (1H, s, H-2). 13C NMR
CH2), 5.44 (1H, s, H-2), 11.76 (1H, d, J ¼ 12.4 Hz, NH), 8.18 (1H, d,
J ¼ 12.4 Hz, H-11), 7.53 (1H, d, J ¼ 8.8 Hz, H-30, H-50), 7.77 (2H, d,
J ¼ 8.8 Hz, H-20, H-60), 7.15 (1H, dd, J ¼ 4.4 Hz, J ¼ 8.8 Hz, H-8), 7.49
(1H, d, J ¼ 8.7 Hz, H-5), 7.43 (2H, m, H-7, NH). 13C NMR (DMSO-d6,
(DMSO-d6, 300 MHz) d
: 180.78 (C-4), 153.87 (C-9), 138.73 (C-20),
122.09 (C-10), 142.54 (C-10), 137.22 (C-6), 117.99 (C-8), 105.00 (C-3),
99.84 (C-2), 134.50 (C-7), 124.18 (C-5), 63.01 (CH2 ethoxy), 27.30
(CH2 ethyl), 15.56 (CH3 ethoxy), 14.95 (CH3 ethyl), 127.57 (C-60),
127.44 (C-30), 116.33 (C-40), 116.17 (C-50), 143.91 (C-11).
400 MHz)
d: 63.17 (CH2), 16.05 (CH3), 18.53 (CH3 thiadiazole),
179.47 (C-4), 167.80 (C-12), 158.25 (C-13), 155.88 (C-9), 154.52 (C-
6), 151.89 (C-10), 142.93 (C-40), 136.67 (C-10), 104.59 (C-3), 144.64
(C-11), 127.55 (C-30, C-50), 116.83 (C-20, C-60), 99.93 (C-2), 121.77 (C-
7), 123.01 (C-8), 110.93 (C-5).
4.1.6. 4-{[(6-Fluoro-2-ethoxy-4-oxo-2H-chromen-3(4H)ylidene)
methyl]amino}-N-(5-methyl-1,3,4-thiadiazol-2-yl)
benzenesulfonamide (4a)
4.1.7. 4-{[(2-Ethoxy-4-oxo-2H-chromen-3(4H)ylidene)methyl]
amino}-N-(5-methyl-1,3,4-thiadiazol-2-yl)benzenesulfonamide
(4b)
Yield 62% as yellow crystalline solid, m.p. 162e164 ꢂC; IR (
n,
cmꢃ1): 1651 (C]O), 1563 (C]C), 1261 (CeO), 3338 (NH), 1136
(SOs2ym), 1382 (SOa2sym). EIeMS m/z (rel. int. %) 444.0 (33), 380.1
(48), 282.1 (78), 255.0 (30), 190.1 (58), 164.0 (37), 139.0 (46), 92.0
Yield 81% as yellow crystalline solid, m.p. 172e174 ꢂC; IR (
n,
cmꢃ1): 1650 (C]O), 1570 (C]C), 1287 (CeO), 3429 (NH), 1154
(SOs2ym), 1376 (SOa2sym). EIeMS m/z (rel. int. %) 426.2 (8), 362.1 (29),
172.1 (37), 92.1 (40), 76.0 (53), 65.1 (52), 59.0 (100). 1H NMR
(37), 59.0 (100). 1H NMR (DMSO-d6, 400 MHz)
d: 1.08 (3H, t,
J ¼ 6.8 Hz, CH3), 1.06 (3H, s, CH3 thiadiazole), 3.72 (2H, q, J ¼ 6.8 Hz,
(DMSO-d6, 300 MHz)
d
: 1.09 (3H, t, J ¼ 7.2 Hz, CH3), 1.60 (3H, s, CH3
thiadiazole), 3.73 (2H, q, J ¼ 7.3 Hz, CH2), 5.94 (1H, s, H-2),11.79 (1H,
d, J ¼ 12.3 Hz, NH), 8.14 (1H, d, J ¼ 12.3 Hz, H-11), 7.77 (2H, d,
J ¼ 8.7 Hz, H-30, H-50), 7.52 (2H, d, J ¼ 8.8 Hz, H-20, H-60), 7.08 (1H, d,
J ¼ 8.1 Hz, H-8), 7.154 (1H, t, J ¼ 7.5 Hz, H-6), 7.84 (1H, dd, J ¼ 1.5,
J ¼ 7.8 Hz, H-5), 7.56 (1H, ddd, J ¼ 1.8, J ¼ 7.8, H-7). 13C NMR (DMSO-
d6, 300 MHz) d: 63.08 (CH2), 16.03 (CH3), 18.53 (CH3 thiadiazole),
180.61 (C-4), 167.76 (C-12), 155.72 (C-13), 154.50 (C-9), 121.96 (C-6),
Fig. 14. Superimposed model structures of IALP (magenta) and TNALP (yellow) en-
zymes. Backbones are represented with cartoon and non conserved amino acids are
represented with the sticks and dotted spheres. Zinc ions are shown in orange color
(left). Predicted binding mode of the most active compound (4a) in the active site of
IALP enzyme. Yellow dotted lines are showing the H-bond and ionic interactions.
Distances are measured in angstroms (right). (For interpretation of the references to
color in this figure legend, the reader is referred to the web version of this article.)
Fig. 13. Plausible binding modes for compounds 2a, 3a and 4a (2a ¼ cyan, 3a ¼ green,
4a ¼ white). (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of this article.)

M. al-Rashida et al. / European Journal of Medicinal Chemistry 66 (2013) 438e449
447
148.96 (C-10), 143.34 (C-11), 143.09 (C-40), 136.38 (C-10), 105.04 (C-
3), 135.21 (C-11), 127.57 (C-30, C-50), 116.60 (C-20, C-60), 99.90 (C-2),
121.96 (C-7), 125.30 (C-8), 112.81 (C-5).
NMR (DMSO-d6, 400 MHz)
d
: 1.09 (3H, t, J ¼ 7.2 Hz, CH3), 3.28 (6H, s,
eOCH3), 3.79 (2H, q, J ¼ 7.2 Hz, CH2), 5.95 (1H, s, ArH pyrimidine),
5.93 (1H, s, H-2), 11.75 (1H, d, J ¼ 12.4 Hz, NH), 8.13 (1H, d,
J ¼ 12.4 Hz, H-11), 7.91 (2H, d, J ¼ 8.8 Hz, H-30, H-50), 7.55 (2H, d,
J ¼ 8.8 Hz, H-20, H-60), 7.08 (1H, d, J ¼ 8.0 Hz, H-8), 7.15 (1H, t,
J ¼ 7.6 Hz, H-6), 7.84 (1H, dd, J ¼ 1.6, J ¼ 7.6 Hz, H-5), 7.52e7.55 (1H,
4.1.8. 4-{[(6-Bromo-2-ethoxy-4-oxo-2H-chromen-3(4H)ylidene)
methyl]amino}-N-(pyridin-2-yl)benzenesulfonamide (4c)
Yield 70% as yellow crystalline solid, m.p. 158e160 ꢂC; IR (
n
,
m, H-7), 11.48 (1H, br s, SO2NH). 13C NMR (DMSO-d6, 300 MHz)
d:
cmꢃ1): 1650 (C]O), 1554 (C]C), 1265 (CeO), 3352 (NH), 1158
(SO2sym), 1347 (SO2asym). EIeMS m/z (rel. int. %) 340.2 (100),
341.1 (51), 220.2 (31), 172.0 (30), 121.0 (37), 92.1 (26), 77.0 (38), 55.0
18.43 (CH3), 55.89 (CH2), 188.27 (C-4), 157.69 (C-9), 144.41 (C-10),
138.78 (C-40), 124.56 (C-10), 104.84 (C-3), 163.37 (C-11), 129.22 (C-
30, C-50), 118.82 (C-20, C-60), 112.34 (C-2), 135.12 (C-7), 116.33 (C-8),
126.64 (C-5), 125.21 (C-6), 84.13 (C-13), 54.29 (eOCH3), 53.59 (e
OCH3), 174.80 (C-14), 171.61 (C-15).
(52). 1H NMR (DMSO-d6, 400 MHz)
d
: 1.08 (3H, t, J ¼ 7.2 Hz, CH3),
3.72 (2H, q, J ¼ 7.3 Hz, CH2), 5.97 (1H, s, H-2), 11.61 (1H, d,
J ¼ 12.4 Hz, NH), 8.01 (1H, br s, SO2NH), 8.25 (1H, d, J ¼ 12.4 Hz, H-
11), 7.85 (2H, d, J ¼ 8.8 Hz, H-30, H-50), 7.54 (2H, d, J ¼ 8.8 Hz, H-20,
H-60), 7.07 (1H, d, J ¼ 8.0 Hz, H-8), 6.88 (1H, t, J ¼ 7.5 Hz, H-6), 7.75
4.2. Biochemical assays
(3H, m), 7.15 (2H, m). 13C NMR (DMSO-d6, 300 MHz)
d
: 62.76 (CH2),
4.2.1. Alkaline phosphatase activity
15.84 (CH3), 179.52 (C-4), 155.66 (C-12), 99.83 (C-13), 113.61 (C-15),
138.39 (C14), 142.85 (C-9), 135.84 (C-6), 142.96 (C-10), 133.11 (C-40),
122.31 (C-10), 101.95 (C-3), 143.87 (C16), 134.69 (C-11), 128.24 (C-30,
C-50), 116.13 (C-20, C-60), 96.26 (C-2), 123.72 (C-7), 125.60 (C-8),
110.31 (C-5).
Initial screening was performed at a concentration of 0.1 mM of
the tested compounds. For potentially active compounds, full
concentrationeinhibition curves were produced. To screen putative
inhibitors, activities of calf intestinal alkaline phosphatase (IALP)
and tissue-non specific alkaline phosphatase (TNALP) were
measured by spectrophotometric assay as previously described
4.1.9. 4-{[(2-Ethoxy-4-oxo-2H-chromen-3(4H)ylidene)methyl]
[23]. The reaction mixture comprised of 75
HCl, 5 mM MgCl2, 0.1 mM ZnCl2, pH 9.5) and 10
compound (0.1 mM with final DMSO 1% (v/v)). This mixture was
incubated for 10 min after adding 5 L of IALP (0.025 U/mL) or 5
of TNALP (0.5 U/mL). Then, 10 L of substrate (0.5 mM p-NPP) was
m
L buffer (50 mM Trise
amino}-N-(pyridin-2-yl)benzenesulfonamide (4d)
m
L of the tested
Yield 78% as yellow crystalline solid, m.p. 150e152 ꢂC; IR (
n,
cmꢃ1): 1644 (C]O), 1599 (C]C), 1276 (CeO), 3345 (NH), 1157
(SO2sym), 1342 (SO2asym). EIeMS m/z (rel. int. %) 340.2 (100),
341.1 (51), 220.2 (31), 172.0 (30), 121.0 (37), 92.1 (26), 77.0 (38), 55.0
m
mL
m
added to initiate the reaction and the assay mixture was incubated
again for 30 min at 37 ꢂC. The change in absorbance of released p-
nitrophenolate was monitored at 405 nm, using a 96-well micro-
plate reader (Bio-Tek ELx 800Ô, Instruments, Inc. USA). The effect
on the activity of each sample containing the inhibitor was
compared with the control sample (without inhibitor). The com-
pounds which exhibited over 50% inhibition of either the calf in-
testinal alkaline phosphatase (IALP) or tissue-non specific alkaline
phosphatase (TNALP) activity were further evaluated for determi-
nation of inhibition constants (Ki values). For this purpose 8e10
serial dilutions of each compound spanning three orders of
magnitude (1 mM to 10 nM) were prepared in assay buffer and
their dose response curves were obtained by assaying each inhib-
itor concentration against all the ALPs using the above mentioned
reaction conditions. All experiments were repeated three times in
triplicate. The ChengePrusoff equation was used to calculate the Ki
values from the IC50 values, determined by the non-linear curve
fitting program PRISM 5.0 (GraphPad, San Diego, California, USA).
(52). 1H NMR (DMSO-d6, 400 MHz)
d
: 1.08 (3H, t, J ¼ 7.2 Hz, CH3),
3.71 (2H, q, J ¼ 7.3 Hz, CH2), 5.92 (1H, s, H-2), 11.76 (1H, d,
J ¼ 12.4 Hz, NH), 8.01 (1H, br s, SO2NH), 8.11 (1H, d, J ¼ 12.4 Hz, H-
11), 7.85 (2H, d, J ¼ 8.8 Hz, H-30, H-50), 7.49 (2H, d, J ¼ 8.8 Hz, H-20,
H-60), 7.07 (1H, d, J ¼ 8.0 Hz, H-8), 6.88 (1H, t, J ¼ 7.5 Hz, H-6), 7.72
(1H, dd, J ¼ 1.5, J ¼ 7.8 Hz, H-5), 7.55 (1H, ddd, J ¼ 1.6, J ¼ 7.6, H-7).
13C NMR (DMSO-d6, 300 MHz)
d: 62.96 (CH2), 14.84 (CH3), 180.52
(C-4), 155.66 (C-12), 99.83 (C-13), 113.61 (C-15), 138.39 (C14),
142.85 (C-9), 121.84 (C-6), 142.96 (C-10),134.09 (C-40), 122.31 (C-10),
104.95 (C-3), 143.87 (C16), 134.69 (C-11), 128.34 (C-30, C-50), 116.33
(C-20, C-60), 96.26 (C-2), 121.72 (C-7), 125.60 (C-8), 112.31 (C-5).
4.1.10. 4-{[(2-Ethoxy-4-oxo-2H-chromen-3(4H)ylidene)methyl]
amino}-N-(6-methoxypyridazin-3-yl)benzenesulfonamide (4e)
Yield 66% as yellow crystalline solid, m.p. 174e176 ꢂC; IR (
n,
cmꢃ1): 1640 (C]O), 1592 (C]C), 1284 (CeO), 3345 (NH), 1153
(SOs2ym), 1336 (SOa2sym). EIeMS m/z (rel. int. %) 436.2 (4), 371.2 (100),
172.0 (28), 146.1 (17), 138.1 (48), 121.1 (34), 92.0 (26), 77.0 (25), 65.1
(27). 1H NMR (DMSO-d6, 300 MHz)
d
: 1.09 (3H, t, J ¼ 7.2 Hz, CH3),
4.2.2. Cell transfection with ecto-50-nucleotidase
3.37 (3H, s, eOCH3), 3.71 (2H, q, J ¼ 7.2 Hz, CH2), 5.93 (1H, s, H-2),
11.79 (1H, d, J ¼ 12.3 Hz, NH), 8.14 (1H, d, J ¼ 12.3 Hz, H-11), 7.82
(2H, d, J ¼ 8.4 Hz, H-30, H-50), 7.51 (2H, d, J ¼ 8.7 Hz, H-20, H-60), 7.08
(1H, d, J ¼ 8.4 Hz, H-8), 7.14 (1H, t, J ¼ 7.2 Hz, H-6), 7.83e7.80 (1H, m,
H-5), 7.55e7.51 (1H, m, H-7), 7.387e7.34 51 (2H, m, ArH pyr-
COS-7 cells were transfected in 10-cm plates by using Lip-
ofectamine, as reported previously [24], with plasmids expressing
ecto-50-NT (plasmid sequence deposited to Gene Bank: DQ186653).
In short, confluent cells were incubated for 5 h at 37 ꢂC in DMEM/F-
12 in the absence of fetal bovine serum and with 6
mg of plasmid
idazine), 8.11 (1H, br s, SO2NH). 13C NMR (DMSO-d6, 300 MHz)
d:
DNA and 24 L of Lipofectamine reagent. The transfection was
m
63.07 (CH2), 14.93 (CH3), 15.15 (eOCH3), 180.98 (C-4), 167.80 (C-12),
122.06 (C-13), 155.1 (C-15), 155.71 (C-9), 121.94 (C-6), 155.55 (C-10),
142.93 (C-40), 122.46 (C-10), 104.94 (C-3), 144.00 (C-11), 127.98 (C-
30, C-50), 116.51 (C-20, C-60), 99.92 (C-2), 134.77 (C-7), 118.42 (C-8),
125.67 (C-5).
stopped by adding the same volume of DMEM/F-12 containing 20%
FBS and the cells were harvested 48e72 h later. Alkaline phos-
phatase (EC 3.1.3.1 from calf intestine), tissue non-specific alkaline
phosphatase (TNALP) from bovine, and the tissue-specific intestinal
alkaline phosphatases (IALPs) from bovine, tissue-non specific
alkaline phosphatase from porcine kidney were obtained from
SigmaeAldrich, Steinheim, Germany.
4.1.11. 4-{[(2-Ethoxy-4-oxo-2H-chromen-3(4H)ylidene)methyl]
amino}-N-(2,6-dimethoxypyrimidin-4-yl)benzenesulfonamide (4f)
Yield 75% as yellow crystalline solid, m.p. 181e183 ꢂC; IR (
n
,
4.2.3. Preparation of membrane fractions
cmꢃ1): 1639 (C]O), 1568 (C]C), 1282 (CeO), 3444 (NH), 1156
(SOs2ym), 1337 (SOa2s). EIeMS m/z (rel. int. %) 246.1 (14), 172.0 (12),
120.1 (26), 92.1 (31), 82.9 (100), 77.0 (12), 69.1 (17), 65.0 (37). 1H
Preparation of protein extracts was done as before [24]. Briefly,
transfected cells were washed three times at 4 ꢂC, with Trisesaline
buffer, collected by scraping in the harvesting buffer (95 mM NaCl,

448
M. al-Rashida et al. / European Journal of Medicinal Chemistry 66 (2013) 438e449
0.1 mM PMSF, and 45 mM Tris buffer, pH 7.5) and washed twice by
4.3.2. Molecular docking simulations
centrifugation at 300 g for 5 min at 4 ꢂC. Subsequently, cells were
The molecular docking of the compounds in the generated ho-
mology models was performed using the GOLD (Genetic Optimi-
zation for Ligand Docking) program. GOLD [30] uses the genetic
algorithm (GA) to search the full length of conformational flexibility
of the ligands inside the protein binding site. Before docking all
compounds were sketched and minimized using MMFF-94 force
field implemented in MOE. Hydrogen atoms were added to both
the model structures and ionization states of metals ions were
resuspended in the harvesting buffer containing 10 mg/mL aproti-
nin and sonicated. Nuclear and cellular debris were discarded by
10 min centrifugation at 4 ꢂC. Glycerol was added to the resulting
supernatant at a final concentration of 7.5%. Samples were kept at
ꢃ80 ꢂC until used. Protein concentration was estimated using
Bradford microplate assay and bovine serum albumin was used as a
standard [25].
ꢀ
fixed. For docking simulations 12 A spherical binding site was used
4.2.4. CE instrumentation
across the zinc ions available in the binding pocket of both the
enzymes. During docking simulations the protein residues
remained rigid, except Ser, Thr and Tyr hydroxyl groups, in order to
optimize hydrogen bonding interactions with the docked com-
pounds. For each solution 10 GA operations were run and best
ranked solution based on the chemscore was selected for each
molecule, all other parameters with their default values were used.
A P/ACE MDQ CE system (Beckman Instruments, Fullerton, USA)
was used for CE separations of substrate and product of ecto-50-NT.
UV detection system of the instrument was coupled with a diode
array detector (DAD). All instrumental operations like perfor-
mance, data collection and peak area analysis were performed
using P/ACE MDQ software 32 KARAT (Beckman Instruments,
Fullerton, USA). The temperature of sample storing unit and
capillary were kept at 25 ꢂC. An eCAPÔ capillary [40 cm (30 cm
Acknowledgment
effective length) ꢁ internal diameter (i.d.) 75
m
m ꢁ outside
diameter (o.d.) 375 m], from Beckman Instruments, Fullerton,
m
PhD scholarship (HEC Indigenous 5000 PhD Fellowship Program,
Batch-III) given to Mariya-al-Rashida funded this research project.
This work was also financially supported by COMSTECHeTWAS and
German-Pakistani Research Collaboration Programme to JI. J.S.’s
laboratory was supported by the Canadian Institutes of Health
Research.
USA, was used to perform electrophoretic separations. The sepa-
ration was performed using an applied constant voltage of 15 kV
and data acquisition rate of 8 Hz. Analytes were detected at
260 nm. The capillary was conditioned by pressure washing with
NaOH and water for 2 min and subsequently with buffer (25 mM
sodium tetra borate buffer, pH 6.5) for 1 min.
Appendix A. Supplementary material
4.2.5. Ecto-50-nucleotidase activity assays
Ecto-50-Nucleotidase assays were performed as reported previ-
ously [26]. Stock solutions of 10 mM of each inhibitor were pre-
Supplementary material associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.ejmech.
2013.06.015.
pared in DMSO. A sample of 10 mL of each tested compound and
10 m mg/mL) protein extract was pre-
L of human ecto-50-NT (6.94
incubated at 37 ꢂC for 10 min in the presence of assay buffer (final
concentration 10 mM Hepes, 2 mM MgCl2, 1 mM CaCl2, pH 7.4).
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the substrate at a final concentration of 500 M and was allowed to
proceed for 10 min at 37 ꢂC. Enzymatic reaction was stopped by
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L of
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into the CE instrument. Under the applied conditions less than 10%
of the substrate was converted into product allowing the enzyme to
work at Vmax for the time of the enzymatic assay. Adenosine con-
centration was determined by calculating the area under its
absorbance peak at 260 nm.
mL of AMP as
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