bioorganic & medicinal...

Bioorganic & Medicinal Chemistry 23 (2015) 2435–2444

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Bioorganic & Medicinal Chemistry

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Diarylsulfonamides and their bioisosteres as dual inhibitors ofalkaline phosphatase and carbonic anhydrase: Structure activityrelationship and molecular modelling studies

http://dx.doi.org/10.1016/j.bmc.2015.03.0540968-0896/� 2015 Elsevier Ltd. All rights reserved.

Abbreviations: CA, carbonic anhydrase; CAIs, carbonic anhydrase inhibitors; AP,alkaline phosphatase; TNAP, tissue non-specific alkaline phosphatase; IAP, intesti-nal alkaline phosphatase; SAR, structure activity relationship.⇑ Corresponding authors. Tel.: +92 3324213592; fax: +92 (42) 9923 0703 (M. al-

R.); tel.: +92 992 3835916; fax: +92 992 383441 (J.I.).E-mail addresses: [email protected], [email protected].

pk (M. al-Rashida), [email protected], [email protected] (J. Iqbal).

Mariya al-Rashida a,⇑, Syeda Abida Ejaz b, Sharafat Ali a, Aisha Shaukat a, Mehwish Hamayoun b,Maqsood Ahmed c, Jamshed Iqbal b,⇑a Department of Chemistry, Forman Christian College (A Chartered University), Ferozepur Road, Lahore 54600, Pakistanb Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad, Pakistanc Department of Chemistry, The Islamia University of Bahawalpur, Pakistan

a r t i c l e i n f o

Article history:Received 6 January 2015Revised 19 March 2015Accepted 20 March 2015Available online 27 March 2015

Keywords:Alkaline phosphatase inhibitorsTissue non-specific alkaline phosphataseIntestinal alkaline phosphataseCarbonic anhydrase inhibitorsStructure activity relationship (SAR)BioisosteresHomology modeling

a b s t r a c t

The effect of bioisosteric replacement of carboxamide linking group with sulfonamide linking group, onalkaline phosphatase (AP) and carbonic anhydrase (CA) inhibition activity of aromatic benzenesulfon-amides was investigated. A series of carboxamide linked aromatic benzenesulfonamides 1a–1c, 2a–2dand their sulfonamide linked bioisosteres 3a–3d, 4a–4d was synthesized and evaluated for inhibitoryactivity against bovine tissue non-specific alkaline phosphatase (TNAP), intestinal alkaline phosphatase(IAP) and bCA II. A significant increase in CA inhibition activity was observed upon bioisostericreplacement of carboxamide linking group with a sulfonamide group. Some of these compounds wereidentified as highly potent and selective AP inhibitors. Compounds 1b, 2b, 3d, 4d 5b and 5c were foundto be selective bTNAP inhibitors, whereas compounds 1a, 1c, 2a, 2c, 2d, 3a, 3c, 4a, 4b, 4c, 5a were foundto be selective bIAP inhibitors. For most active AP inhibitor 3b, detailed kinetic studies indicated a com-petitive mode of inhibition against tissue non-specific alkaline phosphatase (TNAP) and non-competitivemode of inhibition against intestinal alkaline phosphatase (IAP). Molecular docking studies were carriedout to rationalize important binding site interactions.

� 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Alkaline phosphatase (AP, EC. 3.1.3.1) belongs to a large familyof metal containing phosphatases. APs are ubiquitously expressedin different tissues and are believed to be important in osteoblastand adipocyte differentiation, though exact mechanism throughwhich AP accomplishes this feat remains largely unknown.1,2 APis an ectoenzyme that is anchored to the plasma cell membranevia glycosylphosphatidyl inositol (GPI) linkage.3 Its catalytic pre-ferences (hydrolysis of nucleotides to nucleosides and adenosine)overlap with other members of ectonucleotidase; consequently ithas been established as a member of ectonucleotidase family.4

AP is, however, substrate nonspecific and is capable of hydrolyzinga broad range of substrates other than nucleotides such as glucose-phosphates, inorganic polyphosphates, phosphatidates, and bis(p-nitrophenyl)phosphate.4 APs are generally divided into two groups,tissue non-specific alkaline phosphatase (TNAP) and tissue specificalkaline phosphatase. The tissue specific alkaline phosphatase is offurther three types, the germ cell alkaline phosphatase (GAP),placental alkaline phosphatase (PAP) and intestinal alkaline phos-phatase (IAP).5

High concentrations of TNAP are found in mineralizing tissuesand bone. TNAP is one of the key enzymes responsible for reg-ulation of mineralization and bone formation.6 TNAP helps in boneformation in two major ways, (a) by providing an excess of phos-phate pool by virtue of its phosphatase activity (b) it is responsiblefor hydrolysis of inorganic pyrophosphatase (PPi) which is a potentinhibitor of mineralization. Ectonucleotide pyrophosphatase/phos-phodiesterase 1(NPP1 or plasma cell membrane glycoprotein-1,PC1) is responsible for maintaining enough levels of PPi. An inverseconcerted relationship of TNAP and PP1 is necessary for normal

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2436 M. al-Rashida et al. / Bioorg. Med. Chem. 23 (2015) 2435–2444

bone formation.7 Decreased level of TNAP due to gene mutationcauses hypophosphatasia, a condition characterized by severeskeletal hypomineralization.8 Over expression of TNAP causeshydroxyapatite deposition disorder (HADD) characterized byunwanted mineral deposits in periarticular soft tissues.9

Pathological aortic calcification has also been attributed toincreased concentration of TNAP. Inhibition of TNAP has been sug-gested one of the strategies for treating ectopic calcification includ-ing vascular calcification.10,11

The exact physiological role of IAP may be somewhat elusive;its role in adipognesis and regulation of lipid accumulation is cer-tainly noteworthy.12 More firmly established roles of IAP includeits protective effect against toxicity of bacterial lipopolysaccha-rides (LPS) via their dephosphorylation, regulation of bicarbonatesecretion and duodenal surface pH.13 In mammals, duodenum isthe site where gastric H+ is neutralized with secreted bicarbonate(HCO3

�) to generate CO2 and H2O. Here a consortium of IAP and car-bonic anhydrase (CA, both extracellular and cytosolic CA) isresponsible for maintaining homeostasis.14 One of the main func-tions of IAP is to maintain alkaline pH and therefore protect theduodenum. IAP has optimum functioning at alkaline pH; the activ-ity of IAP is decreased under acidic conditions causing greateraccumulation of ATP (which would otherwise be hydrolyzed byIAP). ATP, a P2Y receptor agonist, activates P2Y receptor which sig-nals enterocytes to start producing more bicarbonate. This bicar-bonate is then transported to the luminal cavity where itneutralizes the acidic environment by reacting with H+ ions andproducing H2O and CO2, this CO2 is a substrate for carbonic anhy-drase enzyme which again converts CO2 into bicarbonate ion withthe release of H+ ions.15 Inhibition of CA causes an apparentincrease in secreted bicarbonate concentration.14

ATP inhibits gut bacterial growth (gram positive bacteriaonly).16–18 Since IAP effectively hydrolyzes free ATP, luminal ATPlevels are known to increase when there is a decrease in the activ-ity of IAP (mainly due to acidic environment).15 Selective inhibitionof IAP may offer a route for increasing ATP levels which in turninhibit bacterial growth. Isozyme selective inhibitors of APs aretherefore highly sought after in order to help map out exact roleof APs in health and in disease. Sulfonamides are well known inhi-bitors of carbonic anhydrase, Price, for the first time, found someclinically used sulfonamide antibiotics to be potent inhibitors ofalkaline phosphatase19 and suggested that at least some of thepharmacological action of sulfonamides must be due to inhibitionof AP. Many compounds were identified as selective AP inhibitors.The molecular structures of synthesized compounds were eluci-dated using standard analytical techniques such as IR, C, H, N ele-mental analysis, and NMR spectroscopy. Single crystal X-raydiffraction analysis is reported for compounds 2a and 2b. Sinceno crystal structure of TNAP was available from the Protein DataBank (PDB) a homology model of bTNAP was built using hPLAPas a template and afterwards molecular docking studies werecarried out to identify structural elements necessary for efficientbinding with and to rationalize the effect of bioisosteric replacementof carboxamide linking group with sulfonamide group. Some ADMEproperties of synthesized compounds were calculated computation-ally to identify lead molecules with drug like properties.

2. Results and discussion

2.1. Chemistry

A number of benzenesulfonamide derivatives were synthesized(Scheme 1) with modulation of carefully mapped out structuralelements, in order to allow detailed structure activity relationshipstudies (SAR) to be carried out. Compounds 1a–1c and 2a–2d con-tained a carboxamide linking group between the two benzene

rings. Bioisosteric replacement of carboxamide linking group witha sulfonamide group led to the synthesis of compounds 3a–3d and4a–4d. For compounds in both series, the length of the CH2 spacergroup, between the benzene ring and the linking group, wasincreased up to two carbon atoms. All compounds had a free sul-fonamide group (–SO2NH2), the main zinc binding function (ZBF),however to test the hypothesis that terminal sulfonamide groupis a pre-requisite for AP inhibition, compound 5c that did not con-tain any free/terminal sulfonamide group was also synthesized.Single crystals suitable for analysis by X-ray diffraction analysiswere obtained for compounds 4a and 4b, their crystal data andORTEP diagrams are given in Table S1 and Figures S7–S10 inSupporting information.

Sulfonamides are well known inhibitors of carbonic anhydraseand carbonic anhydrase inhibitory activity of similar compoundshas been reported in literature.20a,b It has already beenestablished21 that a terminal sulfonamide group is essential forCA inhibition activity. We wanted to know if that was also the casefor the inhibition of another zinc containing enzyme, alkalinephosphatase, therefore two compounds 5a and 5b were designedand synthesized as analogs of compounds 4a and 3a respectively,with terminal sulfonamide group replaced by an ethoxy group(Scheme 2). To our surprise, compounds 5a and 5b were still ableto inhibit AP even without the presence of any terminal sulfon-amide group. In order to investigate further, it was decided to carryout molecular docking studies against TNAP. Since no crystal struc-ture of TNAP was available from the Protein Data Bank (PDB) ahomology model of bTNAP was built using hPLAP as a templateand afterwards molecular docking studies were carried out.

2.2. Alkaline phosphatase inhibition studies and SAR

All compounds used in this study were potent inhibitors of AP,most compounds were found to be selective IAP inhibitors,whereas some compounds were identified as selective TNAPinhibitors (Table 1). Compounds 1b, 2b, 3d, 4d, 5b and 5c werefound to be selective bTNAP inhibitors, whereas compounds 1a,1c, 2a, 2c, 2d, 3a, 3c, 4a, 4b, 4c, 5a were found to be selectivebIAP inhibitors. Detailed kinetics studies were carried out for com-pound 3b, the most active TNAP and IAP inhibitor. Compound 3bwas found to be a non-competitive inhibitor against IAP (Fig. 1A)and a competitive inhibitor against TNAP (Fig. 1B).

For carboxamide linked sulfonamides 1a–1c and 2a–2d, thesubstituent at para position of ring B had little or no effect onthe inhibition activity. Introduction of one CH2 spacer groupbetween the two aromatic rings A and B significantly increasedthe TNAP inhibition activity over IAP isozyme, making compounds1b and 2b more active TNAP inhibitors as compared to IAP. Whenthe chain length of CH2 spacer was increased further by addition ofanother CH2 group, the TNAP inhibition activity decreases, and IAPinhibition increases greatly for compound 1c but not for compound2c. The most active IAP inhibitor in this series was 1c (Ki lM,IAP = 0.186 ± 0.001; TNAP = 3.96 ± 0.99) and most active TNAPinhibitor was 2b (Ki lM, IAP = 0.603 ± 0.001; TNAP = 0.299 ± 0.13).Based on these results, it can be suggested that for the design ofTNAP selective inhibitors from this series of compounds, thereshould be a single CH2 spacer group between the two aromaticrings A and B. Further increase of CH2 chain length to (–CH2)2

spacer leads to reduced TNAP inhibition.For sulfonamide linked compounds 3a–3d and 4a–4d, the nat-

ure of substituent at para position of ring B had a marked effectin directing the selectivity between TNAP and IAP. This trend isin contrast to what was observed for the carboxamide linkedcompounds (1a–1c, 2a–2d) where the para substituent on ring Bhad little or no effect in deriving selectivity between the two iso-zymes. It was found that methyl group at para position of ring B

NH2

SO O

NH2

R

S OO

Cl

RS

O

O

NH

S

O

O

NH2+

NH2

SO O

NH2

R

O Cl

R

O

NH

S

O

ONH2

+

1a; n = 0, R = H, p-SO2NH2

1b; n = 1, R = H, p-SO2NH2

1c; n = 2, R = H, p-SO2NH2

2a; n = 0, R = F, p-SO2NH2

2b; n = 1, R = F, p-SO2NH2

2c; n = 2, R = F, p-SO2NH2

2d; n = 0, R = F, m-SO2NH2

3a; n = 0, R = CH3, p-SO2NH2

3b; n = 1, R = CH3, p-SO2NH2

3c; n = 2, R = CH3, p-SO2NH2

3d; n = 0, R = CH3, m-SO2NH2

4a; n = 0, R = F, p-SO2NH2

4b; n = 1, R = F, p-SO2NH2

4c; n = 2, R = F, p-SO2NH2

4d; n = 0, R = F, m-SO2NH2

( )n

( )n

( )n

( )n

Scheme 1. Synthesis scheme of sulfanilamide derivative containing a carboxamide linkage (1a–1c, 2a–2d), and their bioisosteres containing sulfonamide linkage (3a–3d, 4a–4d).

NH

SO

O

R1

R

NH2

R

R1

S OO

Cl

+

5a; R = OC2H5, R1 = F

5b; R = OC2H5, R1 = CH3

5c; R = H, R1 = CH3

Scheme 2. Synthesis of compounds 5a, 5b and 5c containing a sulfonamide linkagebut no terminal sulfonamide group.

M. al-Rashida et al. / Bioorg. Med. Chem. 23 (2015) 2435–2444 2437

(as in compound 3a) results in increased IAP selective inhibitionover TNAP. Replacement of this methyl group with a fluorine atom(4a), although increases IAP inhibition but at the same timereduces the selectivity between the two isozymes. By changingthe position of terminal sulfonamide group from para to meta onring A, compounds exhibited a marked preference for TNAPinhibition over IAP, thus compounds 3d and 4d, both containing

a m-SO2NH2 group were selective inhibitors of TNAP over IAP. Aninteresting trend was observed for 5a and 5b, in which terminalSO2NH2 group had been replaced by an ethoxy group. Compound5a containing a p-Me group on ring B was a selective IAP inhibitor(Ki lM, IAP = 0.37; TNAP = 39.5% inhibition) whereas its p-F isomerwas found to be a selective TNAP inhibitor (Ki lM, IAP = 12.85;TNAP = 0.12). Compound 3b had almost comparable inhibitionagainst both TNAP and IAP, similar, although less marked trendwas observed for its p-F isomer 4b. Compound 5c containing nop-substituent was still able to selectively inhibit TNAP over IAP.Thus replacement of hydrogen atom on para position of ring A witheither SO2NH2 or –OEt group leads to increased selective inhibitionof IAP over TNAP.

2.3. bCA II inhibition studies and SAR

The synthesized carboxamide containing benzenesulfonamides(1a–1c, 2a–2d) and their bioisosteres (3a–3d and 4a–4d) wereassayed against bovine cytosolic carbonic anhydrase type II(bCA II). For comparison the bCA II inhibition activity of clinicallyused standard CA inhibitor acetazolamide (AZM) was alsodetermined (Table 2). All compounds were active inhibitors ofbCA II showing excellent inhibition activity in low micro molar

Table 1Tissue non-specific alkaline phosphatase (TNAP) and intestinal alkaline phosphatase(IAP) inhibition data for the synthesized compounds

Compound TNAP IAPIC50 (lM) ± SEMor (% inhibition)aIC50 (lM) ± SEM

or (% inhibition)aKi ± SEM(lM)

1a 70.1 ± 1.52 16.61 ± 1.08 3.05 ± 0.011b 2.87 ± 1.23 0.918 ± 0.98 4.45 ± 0.551c 10.3 ± 1.21 3.96 ± 0.99 0.429 ± 0.992a 58.6 ± 1.51 25.1 ± 3.12 5.91 ± 1.212b 0.613 ± 0.99 0.299 ± 0.13 1.18 ± 0.022c 22.5 ± 0.254 4.51 ± 0.98 6.73 ± 0.012d 55.6 ± 0.032 16.2 ± 0.97 9.75 ± 1.113a 221.9 ± 3.23 91.87 ± 1.23 14.5 ± 1.133b 0.115 ± 0.02 0.05 ± 0.02 0.14 ± 0.063c 41.2 ± 2.76 17.59 ± 0.76 2.96 ± 0.043d 2.24 ± 0.26 0.95 ± 0.06 8.27 ± 1.164a 3.26 ± 0.11 1.39 ± 0.21 1.13 ± 0.114b 2.94 ± 0.15 1.25 ± 0.05 1.35 ± 0.134c 44.08%a — 11.7 ± 2.254d 0.15 ± 0.07 0.06 ± 0.01 1.27 ± 0.765a 39.55%a — 0.72 ± 0.215b 0.31 ± 0.021 0.128 ± 0.01 30.1 ± 1.515c 0.18 ± 0.03 0.07 ± 0.03 9.15 ± 0.87Levamisole 19.21 ± 0.001 1.81 ± 0.004

L-Phenyl alanine — — 80.21 ± 0.001

a The % inhibition of the enzyme activity is obtained at 0.2 mM concentration oftested compounds.

Table 2Bovine carbonic anhydrase (bCA II) inhibition data for the synthesized compounds

Compound bCA II IC50 (lM) ± SEM(or % inhibition)a

Compound bCA II IC50 (lM) ± SEM(or % inhibition)a

1a 48.4%a 3a 0.057 ± 0.0011b 0.691 ± 0.024 3b 0.134 ± 0.0041c 0.544 ± 0.014 3c 0.241 ± 0.0142a 47.3%a 3d 0.152 ± 0.0012b 0.821 ± 0.014 4a 0.1 ± 0.0032c 0.656 ± 0.017 4b 0.215 ± 0.0082d 36.4%a 4c 0.035 ± 0.0015a Inactive 4d 0.1 ± 0.0025b Inactive AZM 0.96 ± 0.18

a The % inhibition of the enzyme activity caused by 0.5 mM of the testedcompounds.


range. The most active compound in the series was 4c with IC50

value of 0.035 ± 0.001 lM. Only three carboxamide linked com-pounds 1a, 2a and 2d exhibited inhibition less than 50%, all othercompounds exhibited good inhibition activities.

The bioisosteric replacement of carboxamide linking group withsulfonamide group greatly increased the CA inhibition activity,accordingly compounds 1a–1c, 2a–2d were much less active thantheir bioisosteres 1a–1d and 2a–2d. No significant effect on CAinhibition activity was observed when the hydrogen atom on paraposition of benzene ring B was replaced with –CH3 or –F group.This was in agreement with the previously reported studies,22,23

where it was shown that fluorine substitution pattern on benzene-sulfonamides had no significant effect on CA inhibition activity. Incarboxamide linked compounds (1a–1c, 2a–2d), the introductionof one –CH2– spacer group significantly enhances the CA inhibition

Figure 1. (A) Double-reciprocal plot of the inhibition kinetics of bovine intestine alkalDouble-reciprocal plot of the inhibition kinetics of bovine tissue non-specific alkaline phoinitial velocities of the reaction were measured at different concentrations of the inhibitor3,20-(5-chlorotricyclo[3.3.1.13.7]decan])-4-yl]-1-phenyl phosphate).

activity from 48.4% (1a, containing no –CH2– spacer) to0.691 ± 0.024 lM in 1b (containing one –CH2– spacer). SimilarlyCA inhibition of 2a was increased from 47.3% to 0.821 ± 0.014 lM(in compound 2b) upon introduction of one –CH2– spacer. Whenlength of this spacer was increased up to two carbon atoms(–CH2–CH2–), there was only a slight increase in CAI activity.However, for sulfonamide linked compounds (3a–3d and 4a–4d)no such trend was observed. CA inhibition activity was found todecrease when –SO2NH2 group was changed from para to meta posi-tion on benzene ring A. Compounds 5a and 5b, although contained asulfonamide group (as linking group) but due to the absence of aterminal sulfonamide group did not inhibit CA.

2.4. Homology model building of bTNAP and molecular dockingstudies

Comparative Modelling of bovine Tissue Non Specific AlkalinePhosphatase (bTNAP) was carried out using Chimera24 andModeller25 using human placental alkaline phosphatase as a tem-plate. The overall quality of the protein was evaluated, andRamachandran plot generated using Molprobity.26 InRamachandran plot, 94.0% residues were in favored region and98.3% residues were in allowed region. Comparison of active siteresidues of bTNAP with the template are given below in Figure 2.

To rationalize the most probable binding modes of bTNAP inhi-bitors, molecular docking studies of most active inhibitors werecarried out using AutoDock 4.2 and AutoDock Tools.27 AutoDock

ine phosphatase (IAP) indicating non-competitive inhibition by compound 3b. (B)sphatase (TNAP) indicating competitive inhibition by compound 3b. Changes in the3b using substrate CDP-Star (disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-

Table 3AutoDock calculated binding free energies DG (kcal/mol).

Compound codes Binding free energies DG (kcal/mol)

1b �5.651c �5.342b �5.842c �5.073b �6.153d �5.564a �5.454b �5.304d �5.995b �5.815c �6.00

Figure 2. Comparison of active site residues of target bTNAP (gold, A), withtemplate hPALP (cyan, B).

Figure 4. Overlap of compound 3b (light brown) and 5c (blue) inside active site ofTNAP.


calculated binding free energies of bTNAP inhibitors are given inTable 3.

Detailed binding site interactions of most active inhibitor 3b(Ki = 0.05 ± 0.02 lM) are indicated in Figure 3. The terminal

Figure 3. Detailed binding site interactions of

sulfonamide group was oriented towards the two zinc metal ionssuch that a sulfonamide oxygen atom is oriented in-between thetwo zinc metal ions at a distance of 2.04 Å and 2.46 Å. This modeof orientation of sulfonamide group is in agreement with reportedbinding of sulfonamide group inside active site of TNAP.28 Theother oxygen atom of the sulfonamide group was found to beinvolved in hydrogen bond contact with Ser110 (2.19 Å), and withArg184 (2.06 Å). The nitrogen atom of sulfonamide group was alsomaking a hydrogen bond with Arg184 (2.23 Å). The benzene ring(containing 1,4-disulfonamide substituents) was found to be pi-stacked against His341.

Compound 5c is among the most active bTANP inhibitors(Ki = 0.07 ± 0.03 lM), although it did not contain any terminal sul-fonamide group. Docking studies revealed that in compound 5c,the sulfonamide linking group is involved in binding with the zincmetal ion in pretty much the same way as a terminal sulfonamidegroup in compound 3b and it binds in the same region of the activesite (Fig. 4). For compound 5c, one oxygen atom of the sulfonamidegroup is making a contact with the zinc metal atom (2.085 Å), theother oxygen atom is making hydrogen bonds with His338 (2.01 Å)and His171 (1.89 Å). Detailed binding site interactions are given inFigure 5.

Molecular docking studies of CA inhibitors is given inSupplementary information.

compound 3b inside active site of bTNAP.

Figure 5. Detailed binding site interactions of compound 5c inside active site of bTNAP.


2.5. Drug likeness evaluation and calculated ADME properties

Evaluation of ADME (Absorption, Distribution, Metabolism, andExcretion) properties of any molecule is an important step in drugdiscovery process. The ADME profile has a major impact on thelikelihood of success of a molecule as a drug. Keeping this in mind,the ADME properties of compounds were calculated usingMedChem Designer.29 The ADME properties evaluated includeddetermination of octanol–water distribution coefficients S + logPand M logP, the pH dependent octanol–water distributioncoefficient S + logD, number of hydrogen bond donors (HBDH),hydrogen bond acceptor (which is the sum of nitrogen and oxygenatoms MNO) and topological polar surface area (TPSA), a very use-ful molecular descriptor used for the prediction of drug absorption.A molecule is expected to be sufficiently bio-available if it has aTPSA value of less than 60 Å2, on the other hand, if the calculatedTPSA value exceeds 140 Å2, the molecule is generally undesirableas it is not sufficiently bioavailable.30 Typically, compounds havingmolecular weight less than 500, number of hydrogen bond accep-tor (HBDH) less than 10, number of hydrogen bond donor (MNO)less than 5 and logP value of less than 5 are considered to be orallybio-available with a favorable ADME profile.31,32 All synthesizedcompounds showed favorable ADME properties with no violationsof the Lipinski’s rule of 5. The calculated ADME properties of com-pounds are given in Table 4.

Table 4Calculated ADME properties of compounds 1a–1c, 2a–2d

Compound S + logP S + logD M logP

1a 1.935 1.932 2.0091b 1.575 1.574 2.01c 1.868 1.867 2.2532b 1.191 1.19 1.6032a 1.573 1.572 1.6082c 1.48 1.479 1.8593a 1.318 1.072 1.1683c 1.337 1.335 1.423b 1.09 1.086 1.1633d 1.365 0.832 1.1684a 1.272 0.734 1.3014b 1.137 1.126 1.3014c 1.365 1.359 1.564d 1.347 0.374 1.301

When comparing ADME properties (in particular the TPSA) oftwo types of compounds, it becomes evident that carboxamidelinked sulfonamides (1a–1c, 2a–2d), although comparatively lessinhibitory, can be expected to be much more readily bio-available,due to their decreased TPSA value (89.26), as compared to the moreactive sulfonamide linked bioisosteres (3a–3d, 4a–4d) havinggreater TPSA value (106.33).

3. Conclusions

This study reports synthesis of sulfonamide derivatives as dualinhibitors of carbonic anhydrase and alkaline phosphatase (TNAPand IAP). AP is a valuable drug target, over expression of TNAPcauses hydroxyapatite deposition disorder (HADD) and otherpathological calcification, selective TNAP inhibitors maybe devel-oped further for the treatment of pathological calcification. A seriesof carboxamide linked benzenesulfonamides (1a–1c, 2a–2d) andtheir bioisosteres (3a–3d, 4a–4d), resulting from replacement ofcarboxamide linking group with a sulfonamide linking group,was synthesized. The structures of the synthesized compoundswere ascertained using standard analytical techniques such as 1HNMR, 13C-NMR and MS spectroscopy. Single crystal X-raydiffraction analysis for 4a and 4b was carried out. All synthesizedcompounds were evaluated as inhibitors of alkaline phosphatase

MWt HBDH MNO TPSA

294.306 3 5 89.26308.333 3 5 89.26322.36 3 5 89.26290.343 3 5 89.26276.316 3 5 89.26304.37 3 5 89.26326.395 3 6 106.33354.449 3 6 106.33340.422 3 6 106.33326.395 3 6 106.33330.358 3 6 106.33344.385 3 6 106.33358.412 3 6 106.33330.358 3 6 106.33


(AP) and carbonic anhydrase (bCA II). All synthesized compoundswere able to inhibit alkaline phosphatase, both TNAP and IAP.The presence of a terminal sulfonamide group was found to be aprerequisite for CA inhibition, consequently all compounds (except5a and 5b, not containing any terminal SO2NH2 group) were able toinhibit CA. A significant increase in CA inhibition activity wasobserved upon bioisosteric replacement of carboxamide linkinggroup with a sulfonamide group, the most active CA inhibitor 4chad IC50 value of 0.035 ± 0.001 lM. Some very potent and selectiveAP inhibitors were identified. Compounds 1b, 2b, 3d, 4d 5b and 5cwere found to be selective bTNAP inhibitors, whereas compounds1a, 1c, 2a, 2c, 2d, 3a, 3c, 4a, 4b, 4c, 5a were found to be selectivebIAP inhibitors. Detailed kinetic studies revealed a competitivemode of inhibition for tissue non-specific alkaline phosphatase(TNAP) inhibitors and non-competitive mode of inhibition forintestinal alkaline phosphatase (IAP) inhibitors. Molecular dockingstudies were carried out to rationalize important binding siteinteractions of inhibitors with the enzyme. ADME properties werecalculated to estimate drug likeness of synthesized compounds. Noviolations of the Lipinski’s rule of 5 was observed, all compoundshad favorable ADME profile. While sulfonamide linked compoundswere most active inhibitors of CA, the relatively less active car-boxamide linked compounds can be expected to be more bio-avail-able due to their decreased value of TPSA.

4. Materials and methods

4.1. Materials

All chemicals were purchased from Sigma and Aldrich and wereused without further purification. Commercially available solventswere used. Ethanol was distilled and dried using standard methodsand stored over molecular sieves. Reaction progress and productpurity was checked by precoated TLC silica gel plates (0.2 mm,60 HF254, Merck). Spots were visualized under short and longwavelength UV light. Melting points were taken on a Gallenkampmelting point apparatus and are un-corrected. FTIR spectra weretaken on Perkin Elmer Spectrum BX-II. C,H,N,S elemental analysiswas carried out using LECO CHNS 630 series elemental analyzer(model 630-200-200). Mass Spectra were recorded on FinniganMAT 312 Spectrometer. 1H NMR and 13C-NMR spectra wererecorded on Bruker AM 250 and Bruker ARX 300 instruments.Chemical shift values are referenced against TMS. DMSO-d6 andMeOD were used as solvents for NMR spectroscopy.

4.2. General method of synthesis

A facile, environmentally friendly, green synthetic method, asreported by Deng and Mani 200633 was adopted with slight modi-fications. A mixture of equimolar amount of aminobenzenesulfon-amide (0.01 mol) and 4-fluoro/methyl-benzenesulfonyl chloride orappropriate benzoyl chloride (0.01 mol) were stirred together inwater containing few ml of ethanol, pH of the reaction mixturewas maintained at 8.0 by addition of sodium carbonate (0.3–0.4 g). The reaction mixture was stirred continuously for aboutthree hours. Progress of reaction was monitored using TLC. ThepH of the reaction mixture was acidified by addition of HCl to pre-cipitate out compounds which were filtered, washed, dried andfinally recrystallized from ethanol.

4.2.1. Synthesis of N-(4-sulfamoylphenyl)benzamide (1a)Yield 78%; mp 172 �C; IR (m, cm�1): 1650 (C@O), 3393 (NH2),

1100 (SO2sym), 1403 (SO2

asym); Anal. Calcd for C13H12N2O3S: C56.51, H 4.38, N 10.14; found: C 56.32, H 4.23, N 10.27. 1H NMR(500 MHz, DMSO-d6), d (ppm): 7s.99 (t, 3H, 3J = 7.7 Hz, H-30, H-50,H-40), 7.81 (d, 2H, 3J = 8.8 Hz, H-3, H-5), 7.59 (d, 2H, 3J = 7.3 Hz,

H-20, H-60), 7.54 (d, 2H, 3J = 7.6 Hz, H-2, H-6), 7.52 (s, 1H, NH),7.27 (br s, 2H, SO2NH2). 13C NMR (150.92 MHz, DMSO-d6), d(ppm): 166.09 (C@O), 142.34 (C1), 138.87 (C4), 134.62 (C10),132.21 (C40), 128.56 (C30, C50), 127.96 (C3, C5), 126.62 (C20, C60),120.01 (C2, C6).

4.2.2. Synthesis of N-[(4-sulfamoylphenyl)methyl]benzamide(1b)

Yield 83%; mp 164 �C; IR (m, cm�1): 1655 (C@O), 3390 (NH2),1149 (SO2

sym), 1315 (SO2asym); Anal. Calcd for C14H14N2O3S: C

57.92, H 4.86, N 9.65; found: C 57.87, H 4.83, N 9.57. 1H NMR(500 MHz, DMSO-d6), d (ppm): 9.12 (t, 1H, 3J = 5.8 Hz, NH), 7.96(dd, 2H, 3J = 8.8 Hz, 4J = 5.4 Hz, H-20, H-60), 7.77 (d, 2H, 3J = 8.5 Hz,H-3, H-5), 7.48 (d, 1H, 3J = 8.5 Hz, H-2, H-6.), 7.3 (t, 2H,3J = 8.8 Hz, H-30, H-50, H-40), 7.27 (br s, 2H, SO2NH2), 4.52 (d, 2H,3J = 6.0 Hz, –CH2). 13C-NMR (150.92 MHz, DMSO-d6), d (ppm):165.44 (C@O), 163.11 (C1), 143.76 (C4), 142.83 (C10), 130.79(C40), 130.03 (C30, C50), 127.86 (C3, C5), 125.85 (C20, C60), 115.32(C2, C6), 42.55 (CH2–NH).

4.2.3. Synthesis of N-[2-(4-sulfamoylphenyl)ethyl]benzamide(1c)

Yield 82%; mp 158 �C; IR (m, cm�1): 1651 (C@O), 3479 (NH2), 1156(SO2

sym), 1323 (SO2asym); Anal. Calcd for C15H16N2O3S: C, 59.19; H,

5.30; N, 9.20. Found: C, 59.21; H, 5.37; N, 9.27. 1H NMR (500 MHz,DMSO-d6), d (ppm): 8.54 (t, 1H, 3J = 5.4 Hz, H-60), 7.8 (d, 2H,3J = 6.9 Hz, H-20, H-60), 7.74 (d, 2H, 3J = 8.2 Hz, H-3, H-5), 7.5 (d, 1H,3J = 7.3 Hz, NH), 7.45 (d, 2H, 3J = 7.6 Hz, H-30, H-50), 7.42(d, 2H, 3J = 8.2 Hz, H-2, H-6), 2.92 (t, 2H, 3J = 7.3 Hz, –ArCH2), 3.51(m, 2H, –CH2–), 7.22 (br s, 2H, SO2NH2). 13C-NMR (150.92 MHz,DMSO-d6), d (ppm): 166.38 (C@O), 143.87 (C1), 142.30 (C4),134.70 (C10), 131.23 (C40), 129.25 (C30, C50), 128.40 (C20, C60),127.23 (C2, C6), 125.84 (C3, C5), 40.57 (CH2-NH), 34.91 (CH2).

4.2.4. Synthesis of 4-fluoro-N-(4-sulfamoylphenyl)benzamide(2a)


sym), 1310 (SO2asym); Anal. Calcd for C13H11N2O3SF: C,

53.05; H, 3.77; N, 9.52. Found: C, 53.14; H, 3.80; N, 9.57. 1H NMR(500 MHz, DMSO-d6), d (ppm): 7.89 (dd, 2H, 3J = 8.8 Hz,4J = 6.0 Hz, H-2, H-6), 7.44 (d, 2H, 3J = 8.8 Hz, H-3, H-5), 7.03 (t,2H, 3J = 9.0 Hz, H-30, H-50), 6.86 (s, 1H, NH), 6.58 (d, 3J = 8.8 Hz,2H, H-2, H-6), 5.78 (s, 2H, SO2NH2). 13C NMR (150.92 MHz,DMSO-d6), d (ppm): 168.86 (C40), 163.83 (C@O), 161.88 (C1),152.07 (C4), 131.20 (C10), 130.22 (C20, C60), 127.54 (C3, C5),113.75 (C2,C6), 112.58 (C30, C50).

4.2.5. Synthesis of 4-fluoro-N-[(4-sulfamoylbenzyl)methyl]benz-amide (2b)

Yield 80%; mp >200 �C; IR (m, cm�1): 1650 (C@O), 3368 (NH2), 1158(SO2

sym), 1386 (SO2asym); Anal. Calcd for C14H13N2O3SF:

C, 54.54; H, 4.25; N, 9.09. Found: C, 54.58; H, 4.30; N, 9.12. 1H NMR(500 MHz, DMSO-d6), d (ppm): 7.91 (dd, 2H, 3J = 8.5 Hz, 4J = 6.3 Hz,H-2, H-6), 7.69 (d, 2H, 3J = 8.2 Hz, H3, H-5), 7.41 (d, 2H, 3J = 7.9 Hz,H-2, H-6), 7.04 (t, 2H, 3J = 9.0 Hz, H-30, H-50), 3.76 (s, 2H, CH2).13C-NMR (150.92 MHz, DMSO-d6), d (ppm): 169.24 (C@O),165.45(C4), 136.92 (C1), 131.33 (C4), 130.12 (C10), 127.38 (C20, C60),125.62 (C2,C6), 115.30 (C3,C5), 113.82 (C30,C50), 45.26 (CH2-NH).

4.2.6. Synthesis of 4-fluoro-N-[2-(4-sulfamoylphenyl)ethyl]benz-amide (2c)



55.89; H, 4.69; N, 8.69. Found: C, 55.91; H, 4.64; N, 8.71. 1H NMR(500 MHz, DMSO-d6), d (ppm): 2.91 (t, 2H, 3J = 7.3 Hz, CH2), 3.5(2H, m, CH2NH), 8.58 (t, 1H, 3J = 5.4 Hz, NH), 7.87 (dd, 2H,


3J = 8.8 Hz, 4J = 5.7 Hz, H-20, H-60), 7.73 (d, 2H, 3J = 8.5 Hz, H-3, H-5),7.4 (d, 2H, 3J = 8.2 Hz, H-2, H-6), 7.72 (t, 2H, 3J = 9.0 Hz, H-30, H-50),7.16 (br s, 2H, SO2NH2). 13C NMR (150.92 MHz, DMSO-d6), d (ppm):165.30 (C@O), 162.94 (C1), 143.66 (C4), 131.33 (C10), 129.87 (C40),129.18 (C30, C50), 125.80 (C20, C60), 115.38 (C2, C6), 115.20 (C3, C5),40.60 (CH2–NH), 34.86 (CH2).

4.2.7. Synthesis of 4-fluoro-N-(3-sulfamoylphenyl)benzamide(2d)

Yield 78; mp >230 �C; IR (m, cm�1): 1655 (C@O), 3467 (NH2),1156 (SO2


53.05; H, 3.77; N, 9.52. Found: C, 53.09; H, 3.76; N, 9.54. 1H NMR(500 MHz, DMSO-d6), d (ppm): 7.86 (dd, 2H, 3J = 8.4 Hz,4J = 6.1 Hz, H-20, H-60), 7.03 (t, 2H, 3J = 8.8 Hz, H-30, H-50), 5.41 (s,1H, NH), 6.99 (t, 1H, 4J = 1.7 Hz, H-2), 7.14 (t, 1H, 3J = 5.0 Hz, H-5),6.92 (dt, 1H, 3J = 37.8 Hz, 4J = 0.8 Hz, H-4), 6.71 (dd, 1H,3J = 7.9 Hz, 4J = 2.2 Hz, H-6). 13C-NMR (150.92 MHz, DMSO-d6), d(ppm): 169.46 (C40), 164.36 (C@O), 162.42 (C3), 149.48 (C1),145.04 (C10), 136.98 (C5), 131.76 (C20, C60), 129.89 (C6), 114.31(C30, C50), 113.16 (C4), 110.88 (C2).

4.2.8. Synthesis of 4-methyl-N-(4-sulfamoylphenyl)benzenesul-fonamide (3a)

Yield 80%; mp 188–190 �C; IR (m, cm�1): 3380 (NH2), 1133(SO2

sym), 1346 (SO2asym); Anal. Calcd for C13H14N2O4S2: C, 47.84; H,

4.32; N, 8.58. Found: C, 47.89; H, 4.37; N, 8.54. EI-MS m/z: 326[M+], 170 [100%], 156, 91, 80, 76; 1H NMR (300 MHz, MeOD), d(ppm): 2.361 (s, 3H, CH3), 6.68 (2H, d, 3J = 8.7 Hz, H-30, H-50),7.23 (d, 2H, 3J = 9.1 Hz, H-2, H6), 7.58 (d, 2H, 3J = 9.1 Hz, H-3,H-5), 7.71 (d, 2H, 3J = 8.6 Hz, H-20, H-60).

4.2.9. Synthesis of 4-methyl-N-[(4-sulfamoylbenzyl)methyl]benzenesulfonamide (3b)



4.74; N, 8.23. Found: C, 49.37, H, 4.71; N, 8.20. EI-MS m/z: 340[M+], 325 [100%], 176, 141, 91, 77, 51; 1H NMR (300 MHz,MeOD), d (ppm): 2.418 (s, 3H, CH3), 7.01 (2H, d, 3J = 7.5 Hz, H-30,H-50), 7.17 (d, 2H, 3J = 8.1 Hz, H-2, H6), 7.33 (d, 2H, 3J = 6.2 Hz, H-3, H-5), 7.44 (d, 2H, 3J = 8.1 Hz, H-20, H-60), 3.62 (s, 2H, CH2).

4.2.10. Synthesis of 4-methyl-N-[2-(4-sulfamoylphenyl)ethyl]benzenesulfonamide (3c)



5.12; N, 7.90. Found: C, 50.85; H, 5.14; N, 7.87. EI-MS m/z: 354[M+], 198 [100%], 170, 156, 91, 80, 76; 1H NMR (300 MHz,MeOD), d (ppm): 2.41 (s, 3H, CH3), 7.22 (2H, d, 3J = 8.1 Hz, H-30,H-50), 7.39 (d, 2H, 3J = 8.1 Hz, H-2, H6), 7.78 (d, 2H, 3J = 8.1 Hz, H-3, H-5), 7.84 (d, 2H, 3J = 8.4 Hz, H-20, H-60), 3.12 (t, 2H, 3J = 6.9 Hz,CH2), 2.8 (t, 2H, 3J = 7.2 Hz, CH2).

4.2.11. Synthesis of 4-methyl-N-(3-sulfamoylphenyl)benzenes-ulfonamide (3d)



4.32; N, 8.58. Found: C, 47.80; H, 4.35; N, 8.55. EI-MS m/z: 326[M+], 171 [100%], 156, 91, 80, 76, 64, 53; 1H NMR (300 MHz,MeOD), d (ppm): 7.84 (m 2H, H-20, H-60), 7.69 (s, 1H, H-2), 7.57(d, 2H, 3J = 7.8 Hz, H-30, H-50), 7.38 (t, 1H, 3J = 8.4 Hz, H-5), 7.21–7.26 (m, 2H, H-6, H-4), 2,41 (s, 3H, CH3).

4.2.12. Synthesis of 4-fluoro-N-(4-sulfamoylphenyl)benzenesu-lfonamide (4a)

Yield 80%; mp 190–192 �C; IR (m, cm�1): 3479 (NH2), 1156 (SO2sym),

1323 (SO2asym); Anal. Calcd for C12H11N2O4S2F: C, 43.63; H, 3.36; N,

8.48. Found: C, 43.62; H, 3.35, N, 8.45. EI-MS m/z: 330 [M+], 174[100%], 156, 95, 80, 76, 16; 1H NMR (300 MHz, MeOD), d (ppm):7.84 (m, 4H, H-20, H-60, SO2NH2), 7.56 (d, 2H, 3J = 8.7 Hz, H-3, H-5),7.04 (d, 2H, 3J = 8.7 Hz, H-2, H-6), 7.13 (t, 3J = 7.8 Hz, 2H, H-30, H-50).

4.2.13. Synthesis of 4-fluoro-N-[(4-sulfamoylbenzyl)methyl]benzenesulfonamide (4b)


sym), 1326 (SO2asym); C13H13N2O4S2F: C, 45.34; H, 3.80; N, 8.14.

Found: C, 45.37, H, 3.85; N, 8.11. EI-MS m/z: 344 [M+], 185[100%], 170, 159, 155, 95, 76; 1H NMR (300 MHz, MeOD), d(ppm): 7.85 (m, 4H, H-20, H-60, SO2NH2), 7.79 (d, 2H, 3J = 8.1 Hz,H-3, H-5), 7.41 (d, 2H, 3J = 8.1 Hz, H-2, H-6), 7.25 (t, 3J = 8.7 Hz,2H, H-30, H-50), 4.15 (s, 2H, -CH2).

4.2.14. Synthesis of 4-fluoro-N-[2-(4-sulfamoylphenyl)ethyl]benzenesulfonamide (4c)


sym), 1316 (SO2asym); C14H15N2O4S2F: C, 46.92; H, 4.22; N, 7.82.

Found: C, 46.89; H, 4.25; N, 7.80. EI-MS m/z: 358 [M+], 199[100%], 159, 95, 93, 80, 76, 64, 16; 1H NMR (300 MHz, MeOD), d(ppm): 3.12 (t, 2H, 3J = 7.2 Hz, CH2), 2.48 (t, 2H, 3J = 6.9 Hz, CH2),7.83 (m, 4H, H-20, H-60, H-2, H-6), 7.33 (m, 4H, H-30, H-50, H-3, H-5).

4.2.15. Synthesis of 4-fluoro-N-(3-sulfamoylphenyl)benzenesu-lfonamide (4d)


sym), 1326 (SO2asym); Anal. Calcd for C12H11N2O4S2F: C, 43.63; H,

3.36; N, 8.48. Found: C, 43.60; H, 3.32; N, 8.46. EI-MS m/z: 330[M+], 171 [100%], 159, 155, 95, 80, 76; 1H NMR (300 MHz, MeOD),d (ppm): 7.86 (m 2H, H-20, H-60), 7.691 (s, 1H, H-2), 7.58 (d, 2H,3J = 7.8 Hz, H-30, H-50), 7.38 (t, 1H, 3J = 8.1 Hz), 7.25 (m, 2H, H-6, H-4).

4.2.16. Synthesis of N-(4-ethoxyphenyl)-4-fluorobenzenesulfon-amide (5a)


sym), 1333 (SO2asym); Anal. Calcd for C14H14NO3SF: C, 56.94; H,

4.78; N, 4.74. Found: C, 56.97; H, 4.80; N 4.76. EI-MS m/z: 295[M+], 174 [100%], 159, 136, 95, 91, 79, 76; 1H NMR (300 MHz,MeOD), d (ppm): 6.73 (d, 2H, 3J = 9.0 Hz, H-20, H-60), 6.93 (d, 2H,3J = 9.0 Hz, H-3, H-5), 7.18 (d, 2H, 3J = 8.7 Hz, H-2, H-6), 7.69 (t,3J = 8.7 Hz, 2H, H-30, H-50), 3.94 (q, 2H, 3J = 6.9 Hz, CH2), 1.34 (t,3H, 3J = 6.9 Hz, CH3).

4.2.17. Synthesis of N-(4-ethoxyphenyl)-4-methylbenzenesul-fonamide (5b)


sym), 1329 (SO2asym); Anal. Calcd for C15H17NO3S: C, 61.83; H,

5.88; N, 4.81. Found: C, 61.80; H, 5.85; N, 4.80. EI-MS m/z: 291[M+], 155 [100%], 136, 108, 91, 76, 64; 1H NMR (300 MHz,MeOD), d (ppm): 6.70 (d, 2H, 3J = 9.0 Hz, H-20, H-60), 6.72 (d, 2H,3J = 8.7 Hz, H-3, H-5), 7.25 (d, 2H, 3J = 8.1 Hz, H-2, H-6), 7.52 (t,3J = 8.1 Hz, 2H, H-30, H-50), 2.36 (s, 3H, CH3), 3.94 (q, 2H,3J = 6.9 Hz, CH2), 1.32 (t, 3H, 3J = 6.9 Hz, CH3).

4.2.18. Synthesis of 4-methyl-N-phenylbenzenesulfonamide(5c)


sym), 1329 (SO2asym); Anal. Calcd for C15H17NO3S: C, 61.83; H,

5.88; N, 4.81. Found: C, 61.80; H, 5.85; N, 4.80.

4.3. Alkaline phosphatase inhibition assay

A chemiluminescent substrate, CDP-star (disodium 2-chloro-5-(4-methoxyspiro[1,2-dioxetane-3,20-(5-chlorotricyclo[3.3.1.13.7]-decan])-4-yl]-1-phenyl phosphate), was used for the determination


of alkaline phosphatase activity of a compound against bovine kid-ney alkaline phosphatase (TNAP) enzyme and calf intestine alkalinephosphatase (IAP). The conditions for the assay were optimizedwith the slight modifications in previously used spectrophotomet-ric method.34 The assay buffer which contained 8 M DEA (pH 9.8),2.5 mM MgCl2 and 0.05 mM ZnCl2 was used. Initial screening wasperformed at a concentration of 0.2 mM of the tested compounds.The total volume of 50 lL contained 10 lL of tested compound(0.2 mM with final DMSO 1% (v/v)), followed by the addition of20 lL of TNAP (1:800 times diluted (0.8 units/mL) enzyme in assaybuffer) or 20 lL of IAP (1:800 times diluted (1 unit/mL) enzyme inassay buffer). The mixture was pre-incubated for 3–5 min at 37 �Cand luminescence was measured as pre-read using microplatereader (BioTek FLx800, Instruments, Inc. USA). Then, 20 lL ofCDP-star (final concentration of 110 lM) was added to initiate thereaction and the assay mixture was incubated again for 15 min at37 �C. The change in the luminescence was measured as after-read.The activity of each compound was compared with total activitycontrol (without any inhibitor). Levamisole (2 mM per well) and

L-phenylalanine (4 mM per well) were used as a positive controlagainst tissue-nonspecific alkaline phosphatase (TNAP) and calfintestinal alkaline phosphatase (IAP), respectively. For potentiallyactive compounds, full concentration inhibition curves were pro-duced. The compounds which exhibited over 50% inhibition ofeither the tissue-nonspecific alkaline phosphatase (TNAP) activityor calf intestinal alkaline phosphatase (IAP) activity were furtherevaluated for determination of inhibition constants (Ki values).For this purpose 6–8 serial dilutions of each compound (2 mM to20 nM) were prepared in assay buffer and their dose responsecurves were obtained by assaying each inhibitor concentrationagainst both ALPs using the above mentioned reaction conditions.All experiments were repeated three times in triplicate. TheCheng Prusoff equation was used to calculate the Ki values fromthe IC50 values, determined by the non-linear curve fitting programPRISM 5.0 (GraphPad, San Diego, California, USA).

4.4. Kinetics study

Michaelis-Menten kinetic experiments were used to determinethe enzyme inhibition interaction mode of the diarylsulfonamidesand their bioisosteres with the binding site of TNAP & IAP. For thispurpose, the initial rates of the enzyme inhibition at four differentsubstrate concentrations (55 lM, 110 lM, 165 lM & 220 lM) inthe absence and in the presence of four different concentrations(0 lM, 0.50 lM, 1.00 lM and 2.00 lM) of the selected representa-tive inhibitor 3b were measured. Michaelis-Menten kinetic parame-ters Km and Vmax of TNALP & CIALP inhibition were determined in thepresence and absence of inhibitor 3b. The maximal velocity (Vmax)

remained almost constant at different concentrations of 3b, whereasthe Michaelis constant (Km) rise with increasing inhibitor concentra-tions. The Lineweaver–Burk plots for different concentrations of 3bwere linear and intersected at the y-axis with the plot for theuninhibited enzyme. The obtained results indicate that the diaryl-sulfonamides and their bioisosteres are non-competitive inhibitorsof IAP and competitive inhibitors of TNAP.

4.5. In vitro carbonic anhydrase inhibition assay

Carbonic anhydrase inhibition was measured according to thealready published method,35 after standardization of reaction condi-tions like enzyme conc., substrate conc., buffer pH, duration ofreaction etc. The method is based on spectrophotometric determina-tion of p-nitrophenol, formed as a result of CA catalyzed hydrolysis ofp-nitrophenyl acetate. Reaction mixture consisted of 60 lL Tris–sul-fate buffer (50 mM, pH 7.6 containing 0.1 mM ZnCl2), 10 lL(0.5 mM) solution of test compound in 1%DMSO and 10 lL (50 U)

bovine enzyme per well. Contents were mixed and pre-incubated at25 �C for 10 min. Plates were pre-read at 348 nm using a 96 well platereader. Stock solution of p-nitrophenyl acetate (6 mM) was prepared(using <5% acetonitrile in buffer), 20 lL of it was added per well toachieve a final concentration of 0.6 mM per well. Total reaction vol-ume was made up to 100 lL. After 30 min incubation at 25 �C, con-tents were mixed and read at 348 nm. Suitable controls with DMSOand known inhibitor acetazolamide were included in the assay.Results reported are mean of three independent experiments(±SEM) and expressed as percent inhibition calculated by the formula,Inhibition (%) = [100� (abs of test comp/abs of control)� 100)]. IC50

values of selected compounds exhibiting >50% activity at 0.5 mMwere calculated after suitable dilutions.

4.6. Homology model building of bTNAP and molecular dockingstudies

Comparative Modelling of bovine Tissue Non Specific AlkalinePhosphatase (bTNAP) was carried out using Chimera24 andModeller.25 Sequence of target protein bTNAP (uniprot idP09487) was fetched in Chimera. Sequence similarity with targetprotein was searched using BLAST protein database,36 human pla-cental alkaline phosphatase (hPALP, PDB ID 1ZED) was identifiedamong the top five matches and was selected to be used as a tem-plate. Sequence of template protein hPALP was added to sequenceof target protein using Needleman Wunsch method embedded inChimera. Comparative modelling was run using Modeller25 viaChimera. Five models were generated out of which the best model(having 58.47% identity) was selected. The overall quality of theprotein was evaluated, and Ramachandran plot generated usingMolprobity.26 In Ramachandran plot, 94.0% residues were infavored region and 98.3% residues were in allowed region.

Molecular docking studies were carried out using AutoDock 4.2and AutoDock Tools.27 The homology built model of bTNAP wasused as receptor. For docking, the receptor was prepared usingDockPrep utility of Chimera software,24 which includes standardpreparation steps such as adding hydrogen atoms, adding gasteigercharges (using ANTECHAMBER37 utility of Chimera) and repairingincomplete side chains (if any) using Dunbrack rotamer library.Charge of +2 was added on all metal ions. Before docking the struc-tures of all inhibitors were drawn using ACD/ChemSketch38, struc-tures were 3D optimized. Gasteiger charges were added on eachligand using ANTECHAMBER37 and the energy of each moleculewas minimized through 100 steepest descents and 100 conjugategradient steps using a step size of 0.02 each using Chimera.24

Before docking, compounds were protonated (as in aqueousenvironment) using LeadIT.39 AutoDock uses AutoGrid to calculateaffinity maps, the dimensions of grid box were 70 � 70 � 70, andwas centered on the active site. Lamarckian genetic algorithm(LGA) was used for docking, the number of GA runs was set to 50and number of maximum evaluation was 5 � 106. Docked pdbqtfiles were converted into pdb files using OpenBabel.40 Dockingresults were visualized using Discover Studio Visualizer.41

Acknowledgments

This work was financially supported by German-PakistaniResearch Collaboration Program. We are grateful to Dr.Mohammad Shahid for help with docking studies andDepartment of Chemistry, Government College University,Lahore, Pakistan for providing XRD facility.

Supplementary data

Supplementary data (crystallographic data for compounds 4aand 4b reported in this paper has been deposited with the


Cambridge Crystallographic Data Center, CCDC No. 934903 and934904 respectively. Copies of the data can be obtained free ofcharge on application to The Director, CCDC, 12 Union Road,Cambridge, UK, fax: +44 1223 336033, e-mail: [email protected] or http: www.ccdc.cam.ac.uk) associated with this articlecan be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2015.03.054.

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