Synthesis of sulfadiazinyl acyl/aryl thiourea ...

Bioorganic & Medicinal Chemistry 26 (2018) 3707–3715

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Synthesis of sulfadiazinyl acyl/aryl thiourea derivatives as calf intestinal alkaline phosphatase inhibitors, pharmacokinetic properties, lead optimization, Lineweaver-Burk plot evaluation and binding analysis☆ ⁎

T

⁎

Sajid-ur-Rehmana, Aamer Saeeda, , Gufran Saddiquea, Pervaiz Ali Channara, Fayaz Ali Larika, , Qamar Abbasc, Mubashir Hassanb, Hussain Razab, Tanzeela Abdul Fattaha, Sung-Yum Seob a b c

Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan Department of Biological Sciences, College of Natural Sciences, Kongju National University, 56 Gongjudehak-Ro, Gongju, Chungnam 314-701, Republic of Korea Department of Physiology, University of Sindh, Jamshoro, Pakistan

A R T I C LE I N FO

A B S T R A C T

Keywords: Sulfadiazine drug derivatives Acyl/aryl thioureas Calf intestinal alkaline phosphatase Kinetic studies Pharmacokinetics Binding analysis

To seek the new medicinal potential of sulfadiazine drug, the free amino group of sulfadiazine was exploited to obtain acyl/aryl thioureas using simple and straightforward protocol. Acyl/aryl thioureas are well recognized bioactive pharmacophore containing moieties. A new series (4a–4j) of sulfadiazine derived acyl/aryl thioureas was synthesized and characterized through spectroscopic and elemental analysis. The synthesized derivatives 4a–4j were subjected to calf intestinal alkaline phosphatase (CIAP) activity. The derivative 4a–4j showed better inhibition potential compared to standard monopotassium phosphate (MKP). The compound 4c exhibited higher potential in the series with IC50 0.251 ± 0.012 µM (standard KH2PO4 4.317 ± 0.201 µM). Lineweaver-Burk plots revealed that most potent derivative 4c inhibition CIAP via mixed type pathway. Pharmacological investigations showed that synthesized compounds 4a–4j obey Lipinsk’s rule. ADMET parameters evaluation predicted that these molecule show significant lead like properties with minimum possible toxicity and can serve as templates in drug designing. The synthetic compounds show none mutagenic and irritant behavior. Molecular docking analysis showed that compound 4c interacts with Asp273, His317 and Arg166 amino acid residues.

1. Introduction Alkaline Phosphatases (APs, E.C. 3.1.3.1) belongs to the family of ectonucleotidase enzymes. As the name alkaline indicates the enzyme AP performs ideally in basic medium. It plays important role in dephosphorylating compounds and cleaves the phosphodiester bond.1 It assists in the transfer of phosphoryl group (PO3) from phosphate ester or anhydride. AP also helps in the hydrolysis of essential nucleopeptide adenosine monophosphatase (AMP).2 The hydrolysis of AMP results in the release of adenosine and adenosine is key signaling modulator in immunological, cardiovascular and angiogenic response. AP is found in plethora of organisms ranging from bacteria to men. AP contains three metal ions two Zn2+ and one Mg2+ and these three metals play crucial role in the active site of AP.3 Slight variation in structure of AP alters the function and gene sequencing varies in human beings.4 The gene coding distribution determine the homology and alkaline phosphatase (AP) is divided into two major classes, one is tissue specific AP (90–98% homology), which is further sub classified into three categories ☆ ⁎

placental (PLAP), intestinal (IAP) and germ cell (GCAP) and the other is tissue non-specific alkaline phosphatase (TNAP). TNAP is mainly found in the tissue of bone, kidney, liver, central nervous system and teeth.5 The complete profile of their physiological functions is still obscure. TNAP assists in the growth of bones by furnishing inorganic phosphates via phosphatase activity or by inhibiting inorganic pyrophosphate (PPi).6 Different pathogenic disorders have been ensued by alkaline phosphatase enzyme, such as, imbalance in the distribution of TNAP results in hydroxyapatite deposition disorder (HDD).7 The IAP is found in the epithelial cells of small intestine and impedes the assimilation of unwanted substance in small intestine by the absorption of lipopolysaccharide (LPS).8,9 The excess level of IAP leads to development of pathological conditions such as inflammatory bowl disease.10–12 IAP and TNAP structurally resemble with each other and few inhibitors of these are reported in literature. Homoarginine, Levamisole, tertamisole, Theophylline and L-Phenylalanine are selective inhibitors of AP reported in literature.13–16 Discovering new uses of commercial drugs has been the current

ACD/Installer, version 11.01, Advanced Chemistry Development, Inc., Toronto, ON, Canada, www.acdlabs.com, 2015. Corresponding authors. E-mail addresses: [email protected] (A. Saeed), [email protected] (F. Ali Larik).

https://doi.org/10.1016/j.bmc.2018.06.002 Received 1 May 2018; Received in revised form 31 May 2018; Accepted 1 June 2018 Available online 02 June 2018 0968-0896/ © 2018 Elsevier Ltd. All rights reserved.


Sajid-ur-Rehman et al.

chlorine atom at para position. The compound 4d showed less potency and it possess methoxy group at para position. Compounds 4j, 4g, 4h and 4i possess different carbon chain length. Among these various alkyl chain length containing derivatives, compound 4j showed minimum potential against CIAP. Compound 4h relatively showed better inhibition compared to 4g.

interest of researchers. Sulfadiazine is a sulfonamide containing marketed drug and it serves as second line of treatment for otitis media. It is taken orally and prevents the patients from rheumatic fever, chancroid, chlamydia and infections by Haemophilus influenzae. Sulfadiazine shows its therapeutic potential by inhibiting the enzyme dihydropteroate synthetase. Acyl/aryl thioureas possess diverse applications in medicinal and material chemistry. The free NeH atoms present in thiourea play pivotal role in the synthesis of wide variety of heterocycles. The carbonyl and thiocarbonyl functionalities serve as soft and hard donors for formation of metal complexes. The simple and intriguing structural features of thioureas has grabbed unprecedented upsurge in medicinal and coordination chemistry. Thioureas possess broad spectrum of biological activities including antitumor, antiviral, antimicrobial, antiparasitic, insecticidal, herbicidal, pesticidal, fungicidal, urease inhibition, acetylcholinesterase and butyrylcholinesterase inhibition and carbonic anhydrase inhibition.17–22 The synthetic routes applied for the synthesis of thioureas are simple and high yielding this prompts synthetic chemists to design conjugates of thioureas with other bioactive molecules. Along these lines, we have incorporated aryl/acyl thioureas with sulfadiazine drug to evaluate their role as potent selective inhibitors of intestinal alkaline phosphatase (IAP).

2.3. Free radical scavenging activity Newly synthesized series of sulfadiazine based acyl/aryl thioureas were checked against radical scavenging activity. From our results, the only compounds (4b and 4f) showed good radical scavenging potency, while, our other all synthesized compounds did not show significant potency as compared to the standard even at the high concentrations (100 µg/mL), the Ascorbic acid (vitamin C) was used as a standard for the radical scavenging activity (Fig. 1). 2.4. Kinetic mechanism Presently most potent compound 4c was studied for their mode of inhibition against alkaline phosphatase. The potential of the compound to inhibit the free enzyme and enzyme-substrate complex was determined in terms of EI and ESI constants respectively. The kinetic studies of the enzyme by the Lineweaver-Burk plot of 1/V versus substrate para nitrophenyl phosphate disodium salt 1/[S] in the presence of different inhibitor concentrations gave a series of straight lines as shown in Fig. 2 (A). The results of compound 4c gives series of straight lines, intersected within the second quadrant. The analysis showed that Vmax decreased with increasing Km in the presence of increasing concentrations of compound 4c. This behavior of compound 4c indicated that it inhibits alkaline phosphatase by two different pathways; competitively forming enzyme inhibitor (EI) complex and interrupting enzyme-substrate-inhibitor (ESI) complex in noncompetitive manner. The secondary plots of slope versus concentration of compound 4c showed EI dissociation constants Ki Fig. 2 (B) while ESI dissociation constants Ki′ were shown by secondary plots of intercept versus concentration of compound 4c Fig. 2 (C). A lower value of Ki than Ki′ pointed out stronger binding between enzyme and compound 4c which suggested preferred competitive over noncompetitive manners (Table 2). The results of kinetic constants and inhibition constants are presented in Table 2.

2. Results and discussions 2.1. Chemistry A new series of acyl/aryl thioureas having sulfadiazine drug linked was synthesized as outline in Scheme 1. Various acyl/aryl tagged acid chlorides were reacted with potassium thiocyanate using dry acetone as a solvent to obtain isothiocyanate a key intermediate in the synthesis of acyl/aryl thioureas. Isothiocyanate was further reacted with a commercial drug sulfadiazine to achieve sulfadiazine incorporated acyl/ aryl thioureas in good yield. The presence of three NeH groups were characterized through 1H NMR spectroscopy. The NeH groups associated with carbonyl and thiocarbonyl of thiourea moiety appeared at 12–11 ppm value while NeH linked with sulfonamide appeared 7–6 ppm value. The appearance of signals at 8–7 ppm value was assigned to aromatic protons. The aliphatic protons were assigned to signals that appeared at 3–2 ppm value. The 13C NMR spectroscopy was exploited to assign signals to distinct carbons. The carbon of thiocarbonyl appeared at 180 ppm value while carbonyl carbon appeared at 175–170 ppm value. The appearance of signals at around 140–120 ppm value were attributed to aryl carbons. The sp3 carbons appeared at around 40–20 ppm value. In FTIR, eNH of thioureas gave a broad band above 3200 cm−1 due to the intramolecular hydrogen bonding between the oxygen of the carbonyl and –NH. Just around 3000 cm−1 AreH stretch was present and carbonyl group appeared as an intense band in the region of 1700–1600 cm−1. C]S was available between 1050 cm−1 and 1250 cm−1 for all the compounds.

3. Pharmacokinetics and binding analysis 3.1. Structural assessment and physiochemical evaluation of alkaline phosphate Human placental alkaline phosphate is a class of hydrolase single chain protein which consist of 539 amino acids with embedded zinc and magnesium ions which involve in downstream signaling pathways. The VADAR analysis showed the overall protein architecture consist of 31% helices, 26% β sheets and 42% coils. Moreover, the Ramachandran plot indicated that 96.4% of residues were present in favored regions which shows the precision of phi (φ) and psi (ψ) angles among the coordinates of alkaline phosphate. The hydrophobicity and Ramachandran graphs are mentioned in Supplementary data (Fig. S1-2). The basic physiochemical properties of alkaline phosphatase such as molecular weight (Mw) and theoretical pI were generated by accumulation of average isotopic masses and pK values of residues, respectively.23 Previous research data showed that the computational predicted pI value of protein structures is ranging from 4.31 to 11.78.24 The predicted pI value 5.81 of alkaline phosphatase is comparable with the standard value which showed the accuracy and reliability of targeted protein. The predicted values of aliphatic and instability indexes is presented the relative volume engaged by aliphatic side chains of targeted protein. The GRAVY value is the sum of hydropathic values of all amino acids

2.2. Calf intestinal alkaline phosphatase inhibition assay All the synthesized compounds 4a–4j were evaluated for their potential to calf intestinal alkaline phosphatase (CIAP). The results of CIAP are summarized in Table 1. The synthesized compounds were designed by using commercial drug sulfadiazine and this drug was further exploited to incorporate acyl/aryl thioureas. Various electron withdrawing groups and one donating group were added to phenyl ring. Chlorine substituted derivative 4c showed better potential compared to fluorine substituted 4a. Dinitrosubstituted 4b showed more potency than halogen substituted derivatives (Chlorine or Fluorine atom bearing). The derivative 4e bears chlorine atom at ortho position of phenyl ring showed less potency compared to 4c which possess 3708



Scheme 1. Synthetic outline of the sulfadiazine based acyl/aryl thioureas 4a–4j. Table 1 Alkaline phosphatase inhibitory activity of sulfadiazine base acyl/ aryl thioureas 4a–4j. Compound

Alkaline phosphatase IC50 (µM)

4a 4b 4c 4d 4e 4f 4g 4h 4i 4j KH2PO4

1.547 0.896 0.251 3.291 3.458 0.344 1.636 1.704 0.492 4.256 4.317

± ± ± ± ± ± ± ± ± ± ±

0.075 0.04 0.012 0.160 0.178 0.0159 0.0797 0.092 0.032 0.301 0.201

Fig. 1. Antioxidant activity on DPPH, percentage activity of newly synthesized compounds, values were presented as mean ± SEM (Standard error of the mean). 100 µg/mL concentration of all compounds.

Values are presented as Mean ± SEM Standard error of mean.

present in the protein.25 However, more negative value indicates it’s more hydrophilic and less hydrophobic behavior [23]. The generated values are mentioned in Table 3.

our designed chemical structures. The prior research data showed that polar surface area (PSA) is significant tool to observe the drug absorption.26,27 The molecular lipophilicity and molar refractivity values of chemical structures are also significant in protein binding and bioavailability. It has been observed that the standard values for molar refractivity (40–130 cm3), PSA (< 89 Å2), and molecular weight (160–480 g/mol), respectively.28 Furthermore, the total number of atoms in any drug molecule is range from 20 to 70, having average

3.2. Ligands assessment and authentication of Lipinski Rule of Five The generated properties of synthesized ligands (Table 4) and Lipinski Rule were analyzed to validate the drug likeness performance in 3709



Fig. 2. Lineweaver-Burk plots for inhibition of Alkaline phosphatase in the presence of Compound 4c (A) Concentrations of 4c were 0.00, 0.015, and 0.3 µM. (B) The plot represent the slope (C) the vertical intercepts versus inhibitors concentrations to determine inhibition constants.

3.3. ADMET properties of synthetic ligands

Table 2 Kinetic parameters of the Alkaline phosphatase for para nitrophenylphosphate disodium salt activity in the presence of different concentrations of 4c. Concentration (µM)

Vmax (ΔA /Sec)

Km (mM)

Inhibition Type

Ki (µM)

Ki′ (µM)

0.00 0.015 0.3

1.2 × 10−5 8.0 × 10−6 4.9 × 10−5

0.833 1.333 2.00

Mixed inhibition

0.115

0.415

The good pharmacokinetic properties and better efficacy are key principle in the development of novel drugs. The generated pharmacokinetic properties assessment is evaluated to check the effectiveness of lead compounds. The pkCSM web tool was employed on all synthetic compounds (4a–4j) to judge the pharmacokinetic properties (Table 5). The water and intestinal solubility (log mol/L & % absorbed), respectively and skin permeability (logKp) predicted values showed good results of newly designed compounds. It has been observed that ligands having good in absorption property can reach the target molecule with effective potency by crossing gut barrier with passive penetration.32 Moreover, compounds showed very good water absorption and intestinal solubility results, compared to a standard value (> 30 %abs). The predicted skin permeability results of all synthetic ligands showed better values compared to standard value (−2.5 logKp), and may depicted as good lead chemical compounds. Moreover BBB and CNS permeability results values of all synthetic ligands were also showed good comparable results against standard values (> 0.3 to < −1 log BB and > −2 to < −3 logPS) respectively. Research data showed that ligand structure having > 0.3 (log BB) value have more potential to cross BBB. Whereas, ligands having < −1 value may perceive as poorly distributed in the brain. The ligands structures which contain > −2 (logPS) value are nominated as good penetrant to CNS, whereas, other having < −3 are impossible to cross in CNS. Our generated values results showed that all the synthetic ligands showed good effective behavior to cross internal membranes barriers. Moreover, their computational metabolic behavior was confirmed by positive results of CYP3A4. The toxicity and excretion generated results were also justified the good drug like potential of synthetic ligands by computationally analyzing the AMES toxicity, MTD and LD50 values. All compounds show non-mutagenic and non-toxic behavior except 4a and 4j. These predicted pharmacokinetic results values justified that our novel synthetic compounds have lead like potential for further evaluation.

Vmax = the reaction velocity; Km = Michaelis-Menten constant; Ki = EI dissociation constant; Ki′ = ESI dissociation constant. Table 3 Physiochemical properties of alkaline phosphatase by ProtParam. Sr. No.

Parameters

Values

1 2 3

Molecular weight (MW) Theoretical pI Extinction coefficient* (assuming all Cys residues are reduced) Aliphatic index Instability index Gran average of hydropathicity (GRAVY)

52299.88 Da 5.81 47,330

4 5 6

75.22 33.53 −0.368

* Extinction Coefficient units M−1 cm−1 at 280 nm.

value of 48.28 Table 2 predicted results depicted that all the compounds showed comparable justification with the standard values. However, the overall justification can predicted as that, compounds may consider as good therapeutically active compounds against target protein. The Lipinski's analysis also favor in that all compounds were comparable with standard values of RO5 (HBA: < 10, HBD: < 5, logP, < 500 g/ mol). Research data showed that exceed numbers of HBA and HBD results in poor permeation.29 Our synthetic compounds were good in Lipinski’s analysis whereas, various examples are available for RO5 violation in the present drug databases.30,31 3710



Table 4 Cheminformatics properties of ligands. Properties

4a

4b

4c

4d

4e

4f

4g

4h

4i

4j

Mol.weight (g/mol) No. HBA No. HBD Mol.LogP Mol. PSA (A2) Stereo centers Mol.Vol (A3) Molar Refractivity (cm3) Surface Tension (dyne/cm) Density (g/cm3) Polarizability (cm3) Drug Score Lipinski Rule

431.05 6 3 2.43 94.46 0 362.60 108.14 84.6 1.549 42.87 1.24 Yes

505.05 10 05 1.35 170.98 0 417.31 120.09 103.9 1.690 47.61 0.54 Yes

447.02 6 3 2.87 94.46 0 373.88 112.85 87.1 1.565 44.74 1.38 Yes

443.07 7 3 2.25 102.00 0 388.53 114.39 81.3 1.487 45.35 1.03 Yes

447.02 6 3 2.75 94.46 0 372.01 112.85 87.1 1.565 44.74 1.07 Yes

480.98 6 3 3.47 94.46 0 389.28 117.68 87.3 1.618 46.65 1.03 Yes

379.08 6 3 1.70 94.52 0 338.61 97.20 80.6 1.451 38.53 0.94 Yes

435.14 6 3 3.63 94.52 0 410.23 115.73 68.9 1.330 45.88 0.53 Yes

421.12 6 3 3.15 94.52 0 392.32 111.10 71.3 1.355 44.04 0.53 Yes

351.05 6 3 0.73 94.63 0 301.97 87.94 89.8 1.538 34.86 1.02 Yes

3.4. Lead optimization and lipophilicity values

3.5. Binding energy analysis of docking complexes

The ligand efficiency (LE), lipophilic ligand efficiency (LLE) and lipophilicity-corrected ligand efficiency (LELP) values of all compounds were predicted to confirm their drug-likeness efficacy. Previous research showed that lipophilicity is a basic parameter to enhance the lead structure efficacy33 and from lead to drug candidate.34,35 Prior research shows the lipophilicity of various chemical structures on the basis of cLogP value and predict the standard values for LE, LLE and LELP (LE > ∼0.30 kcal/mol/HA, LLE > ∼0.5 kcal/mol, LELP −10 < to < 10 and cLogP < 3).36 The generated cLogP values showed that the synthetic ligands were comparable with the standard results. The LE predicted values of compounds showed comparable results with standard value. The synthetic compounds show none mutagenic and irritant behavior, respectively Table 6.

The synthesized compounds (4a–4j) were docked against alkaline phosphatase to observe the good ligand position in the active region of receptor. The predicted docked complexes were examined on the basis of lowest energy values (kcal/mol) and hydrogen/hydrophobic interactions pattern. Molecular docking experiment results showed that compound 4c possess significant docking energy value (−8.3 kcal/mol) compared to others compounds. The other compounds also presents significance energy values against receptor molecule. However, the chemical skeleton in all ligands were similar, therefore most of synthetic ligands show good and efficient energy values having little energy difference. Moreover, the prior data also showed the standard error for energy values for Autodock is 2.5 kcal/mol. The comparative binding energy analysis reveals that 4c is most active compound and

Table 5 Pharmacokinetic assessment of synthesized compounds. ADMET Properties

4a

4b

4c

4d

4e

4f

4g

4h

4i

4j

Absorption

WS IS SP

−4.94 76.71 −3.12

−5.11 70.48 −2.75

−5.27 77.60 −3.11

−4.89 74.8 −3.09

−5.27 77.60 −3.118

−5.72 78.78 −3.04

−4.18 71.01 −3.28

−5.20 74.04 −3.02

−4.95 73.29 −3.09

−3.72 69.50 −3.36

Distribution

BBBP CNSP

−1.40 −3.11

−1.66 −2.98

−1.37 −2.49

−1.40 −3.17

−1.37 −2.493

−1.53 −2.37

−1.34 −2.94

−1.46 −2.93

−1.43 −2.93

−1.27 −2.95

Metabolism

CYP3A4 inhibitor

No

Yes

Yes

Yes

Yes

Yes

No

Yes

No

No

Excretion

TC

−0.72

−0.26

−0.70

−0.54

−0.442

−0.51

−0.454

−0.33

−0.36

−0.45

Toxicity

AMES toxicity Max. tolerat. dose ORAT (LD50) HT SS

Yes 0.82 2.58 Yes No


No 0.79 2.69 Yes No

No 0.797 2.503 Yes No

No 0.793 2.699 Yes No

No 0.72 2.79 No No

No 0.90 2.60 Yes No

No 0.97 2.58 Yes No

No 0.95 2.59 Yes No


*

Abbreviation: WS = Water solubility (log mol/L), ISa = intestinal solubility (%abs), SP = skin permeability (log Kp), BBBP = blood brain barrier permeability (Log BB), CNSP = CNS permeability (LogPS), TC = Total clearance (log ml/min/kg), ORAT = Oral Rat Acute Toxicity, HT = Hepatotoxicity, SS = Skin Sensitization.

Table 6 Ligand efficacy prediction values. Ligands

cLogP

cLogS

LE

LLE

LELP

Mutagenic

Tumorigenic

Irritant

4a 4b 4c 4d 4e 4f 4g 4h 4i 4j

2.93 3.33 1.55 3.30 0.25 2.70 2.19 2.02 2.70 0.65

−4.45 −4.72 −3.64 −5.74 −5.19 −5.01 −4.58 −4.29 −5.01 −3.10

0.41 0.40 0.48 0.38 0.39 0.40 0.41 0.39 0.40 0.54

5.61 5.09 7.24 5.17 9.34 5.86 6.46 6.66 5.86 8.53

6.99 8.42 3.22 8.52 0.64 6.66 5.36 5.09 6.66 1.18

none none none none none none none none none none

high high high high high high high high high high

none none none none none none none none none none

3711



mixed type of mechanism and possess Ki 0.115 µM. Pharmacokinetics disclosed that all the compounds 4a–4j obey Lipinsk’s rule and show promising drug score. ADMET parameters evaluation unveiled that compounds show good lead like efficiency with no mutagenic and irritant effect. The comparative binding energy analysis reveals that 4c is most active compound and confined a good compatible position within binding pocket of alkaline phosphatase. From the results of CIAP assay and computational studies (Pharmacokinetics, ADMET parameters evaluation and binding analysis) it can be concluded that compound 4c can serve structural model for medicinal chemists to design potent inhibitions of CIAP. 5. Experimental Fig. 3. The bar chart of docking energy values.

5.1. General methods and materials Melting points were recorded using a digital Gallenkamp (Tokyo, Japan) model MPD.BM 3.5 apparatus and are uncorrected. 1H NMR spectra were determined as CDCl3 solutions at 300 MHz using a Bruker AM-300 spectrophotometer using TMS as an internal reference and 13C NMR spectra were determined at 75 MHz using a Bruker 75 MHz NMR spectrometer in DMSO solution. FTIR spectra were recorded on an FTS 3000 MX spectrophotometer. Mass Spectra (EI, 70 eV) on a MAT 312 instrument, and elemental analyses were conducted using a LECO-183 CHNS analyzer.

occupied good conformational pattern inside active region of target (Fig. 3). 3.6. Binding pocket analysis of alkaline phosphatase docked complexes The docking structures were analyzed on binding interactions pattern. Docking energy and binding analysis depicted compound 4c was actively binds in the active region of target protein (Fig. 4). The structure activity relationship (SAR) study depicted that three hydrogen bonds were seen in 4c-docked complex. The amino group of benzene ring is directly interacts with Asp273 by hydrogen interaction with bond distance 2.17 Å. Furthermore, carbonyl group and carbonyl sulphur of 4c made couple of hydrogen bonds at His317 and Arg166 with appropriate bond distances 2.31 and 3. 06 Å, respectively. The comparative analysis depicted the significance of 4c and it may considered as potent inhibitor against target protein. All other docking complexes of all compounds are listed in Supplementary data (Figs. S13–S21).

5.2. General procedure for the synthesis of sulfadiazine based acyl/aryl thioureas A solution of potassium thiocyanate (5 mmol) in dry acetone was stirred and then a solution of commercially available acid chloride (5 mmol) in dry acetone (10 mL) was added drop wise to a reaction mixture. The reaction mixture was refluxed for half an hour, after cooling, a solution of sulfadiazine (5 mmol) in dry acetone (10 mL) was added and resulting mixture was stirred at 50 °C for 5–6 h.20 The progress of reaction was monitored through TLC (n-hexane: ethyl acetate 4:1). After completion the reaction mixture was poured onto crushed ice and the precipitates were collected by filtration, dried, and recrystallized from ethanol to afford the desired thiourea derivatives with excellent yield.

4. Conclusion To test the hypothesis that new uses of commercial drugs can been researched by incorporating bioactive entities. We summarize that Sulfadiazine commercial drug was linked with acyl/aryl thioureas to obtain a series of new drug derivatives 4a–4j. The structure elucidation of compounds 4a–4j was performed through spectroscopic and elemental analysis. Newly synthesized compounds 4a–4j were subjected to calf intestinal alkaline phosphatase (CIAP) and free radical scavenging activities. The preliminary structure activity relationship revealed that aryl thioureas showed better potency against CIAP than acyl thioureas. Moreover, mono chlorine atom contain derivative 4c was found to most potent derivative in the series with IC50 0.251 ± 0.012 µM. Lineweaver-Burk plot showed that most derivative 4c inhibits CIAP via

6. Experimental data 6.1. 4-Fluoro-N-((4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)carbamothioyl) benzamide (4a) Light yellow solid, Yield; 73%, m.p: 235 °C, Rf = 0.65 (n-hexane: ethyl acetate, 4:1), FTIR υ (cm−1) 3168.3 (NeH, stretching), 2926.6

Fig. 4. Putative binding mode of synthesized compounds and Figure A shows compound 4c interactions. 3712



(DMSO‑d6, 300 MHz); δ (ppm) 12.35 (s, 1H, NH), 11.33 (s, 1H, NH), 11.12 (s, 1H, NH), 8.32 (d, 2H, AreH, J = 7.3 Hz), 8.12 (d, 2H, AreH, J = 7.1 Hz), 7.63 (d, 2H, AreH), 7.46–7.23 (m, 4H, AreH), 7.12 (t, 1H, AreAreH); 13C NMR (75 MHz DMSO‑d6) δ (ppm) 179.3 (C]S), 178.4 (C]O), 171.6, 154.6, 154.3, 151.4, 144.5, 141.7, 139.6, 133.4, 131.4, 130.2, 126.8, 122.1, 121.6, 120.4, 118.6, 116.3 (AreC), Anal. Calcd. For C18H14ClN5O3S2: C, 48.16; H, 3.04; N, 15.46; S, 14.31 found: C, 48.06; H, 2.96; N, 15.27; S, 14.01.

(Aromatic CeH, stretching), 1709.8 (C]O, stretching), 1609.5 (CeC, stretching), 1447.3 (CeH, bending), 1252.4 (C]S, stretching). 1H NMR (DMSO‑d6, 300 MHz); δ (ppm) 12.35 (s, 1H, NH), 11.74 (s, 1H, NH), 11.37 (s, 1H, NH), 8.61 (d, 2H, AreH, J ] 8.2 Hz), 8.11 (d, 2H, AreH, J = 7.6 Hz), 7.87 (d, 2H, AreH), 7.76 (d, 2H, AreH), 7.53 (d, 2H, AreH), 7.06 (t, 1H, AreH); 13C NMR (75 MHz DMSO‑d6) δ (ppm) 180.4 (C]S), 178.1 (C]O), 172.5, 158.8, 157.3, 151.4, 142.1, 140.3, 137.7, 134.2, 131.7, 130.7, 128.8, 124.1, 121.6, 120.4, 118.6, 116.3 (AreC), Anal. Calcd. For C18H14FN5O3S2: C, 50.01; H, 3.08; N, 16.12; S, 14.51 found: C, 49.87; H, 2.97; N, 15.98; S, 14.30.

6.6. 2,4-Dichloro-N-((4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl) carbamothioyl)benzamide (4f)

6.2. 3,5-Dinitro-N-((4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl) carbamothioyl)benzamide (4b)

Light yellow solid, Yield; 71%, m.p: 253 °C, Rf = 0.62 (n-hexane: ethyl acetate, 4:1), FTIR υ (cm−1) 3138.6 (NeH, stretching), 2957.4 (Aromatic CeH, stretching), 1741.3 (C]O, stretching), 1624.3 (CeC, stretching), 1418.2 (CeH, bending), 1234.6 (C]S, stretching). 1H NMR (DMSO‑d6, 300 MHz); δ (ppm) 12.36 (s, 1H, NH), 11.34 (s, 1H, NH), 11.22 (s, 1H, NH), 8.43 (d, 2H, AreH, J = 7.2 Hz), 8.23 (d, 2H, AreH, J = 6.5 Hz), 7.53 (d, 2H, AreH), 7.43–7.22 (m, 3H, AreH), 7.13 (t, 1H, AreH); 13C NMR (75 MHz DMSO‑d6) δ (ppm) 179.7 (C]S), 177.9 (C] O), 171.5, 153.7, 154.1, 152.3, 145.2, 142.4, 138.6, 134.8, 132.7, 131.2, 127.3, 123.2, 121.9, 120.4, 118.5, 116.8 (AreC), Anal. Calcd. For C18H13Cl2N5O3S2: C, 44.61; H, 2.54; N, 15.42; S, 13.18 found: C, 44.46; H, 2.45; N, 15.27; S, 13.08.

Red solid, Yield; 77%, m.p: 225 °C, Rf = 0.52 (n-hexane: ethyl acetate, 4:1), FTIR υ (cm−1) 3172.3 (NeH, stretching), 2934.2 (Aromatic CeH, stretching), 1711.5 (C]O, stretching), 1633.6 (CeC, stretching), 1430.3 (CeH, bending), 1263.4 (C]S, stretching). 1H NMR (DMSO‑d6, 300 MHz); δ (ppm) 12.15 (s, 1H, NH), 11.63 (s, 1H, NH), 11.33 (s, 1H, NH), 8.31 (d, 2H, AreH, J = 7.2 Hz), 8.12 (d, 2H, AreH, J = 7.3 Hz), 7.97 (d, 2H, AreH), 7.66 (d, 2H, AreH), 7.43 (s, 1H, AreH), 7.03 (t, 1H, AreH); 13C NMR (75 MHz DMSO‑d6) δ (ppm) 180.6 (C]S), 177.1 (C]O), 171.3, 168.3, 156.3, 150.4, 141.1, 139.3, 135.9, 132.1, 130.7, 129.9, 128.4, 125.3, 121.3, 120.2, 119.4, 116.6 (AreC), Anal. Calcd. For C18H13N7O7S2: C, 42.81; H, 2.48; N, 19.32; S, 12.54 found: C, 42.70; H, 2.37; N, 19.08; S, 12.30.

6.7. N-((4-(N-(Pyrimidin-2-yl)sulfamoyl)phenyl)carbamothioyl) butyramide (4g)

6.3. 4-Chloro-N-((4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)carbamothioyl) benzamide (4c)

Light brown solid, Yield; 67%, m.p: 176 °C, Rf = 0.57 (n-hexane: ethyl acetate, 4:1), FTIR υ (cm−1) 3038.6 (NeH, stretching), 2956.6 (Aromatic CeH, stretching), 1696.4 (C]O, stretching), 1573.5 (CeC, stretching), 1412.3 (CeH, bending), 1163.2 (C]S, stretching). 1H NMR (DMSO‑d6, 300 MHz); δ (ppm) 12.75 (s, 1H, NH), 11.84 (s, 1H, NH), 11.57 (s, 1H, NH), 8.51 (d, 2H, AreH, J = 6.5 Hz), 8.01 (d, 2H, AreH, J = 3.9 Hz), 7.75 (d, 2H, AreH), 7.07 (t, 1H, AreH), 3.36 (t, 2H), 2.51 (quint, 2H), 2.43 (quint, 2H), 1.57 (quint, 2H),1.55 (quint, 2H), 1.24 (sex, 2H), 0.85 (t, 3H); 13C NMR (75 MHz DMSO‑d6) δ (ppm) 179.4 (C] S), 176.1 (C]O), 172.5, 158.8, 157.3, 142.1, 137.7, 130.7, 128.8, 124.1, 118.6, 116.3 (AreC), 40.51, 39.7, 36.2, 29.1, 25.4, 22.6, 14.4, Anal. Calcd. For C19H25N5O3S2: C, 52.19; H, 5.48; N, 15.94; S, 14.41 found: C, 51.07; H, 5.37; N, 15.73; S, 14.30.

Brown crystalline solid, Yield; 90%, m.p: 240 °C, Rf = 0.61 (nhexane: ethyl acetate, 4:1), FTIR υ (cm−1) 3176.3 (NeH, stretching), 2956.3 (Aromatic CeH, stretching), 1719.8 (C]O, stretching), 1613.6 (CeC, stretching), 1427.2 (CeH, bending), 1232.6 (C]S, stretching). 1 H NMR (DMSO‑d6, 300 MHz); δ (ppm) 12.55 (s, 1H, NH), 11.63 (s, 1H, NH), 11.33 (s, 1H, NH), 8.31 (d, 2H, AreH, J = 7.2 Hz), 8.21 (d, 2H, AreAreH, J = 7.6 Hz), 7.67 (d, 2H, AreH), 7.56 (d, 2H, AreH), 7.33 (d, 2H, AreH), 7.13 (t, 1H, AreH); 13C NMR (75 MHz DMSO‑d6) δ (ppm) 179.4 (C]S), 177.1 (C]O), 171.4, 156.8, 155.3, 150.4, 143.1, 140.8, 138.7, 134.7, 132.7, 130.6, 127.8, 123.1, 121.9, 120.1, 119.6, 117.3 (AreC), Anal. Calcd. For C18H14ClN5O3S2: C, 48.17; H, 3.05; N, 15.46; S, 14.21 found: C, 48.07; H, 2.97; N, 15.28; S, 14.01.

6.8. N-((4-(N-(Pyrimidin-2-yl)sulfamoyl)phenyl)carbamothioyl) octanamide (4h)

6.4. 4-Methoxy-N-((4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl) carbamothioyl)benzamide (4d) Light yellow solid, Yield; 69%, m.p: 254 °C, Rf = 0.58 (n-hexane: ethyl acetate, 4:1), FTIR υ (cm−1) 3186.1 (NeH, stretching), 2998.3 (Aromatic CeH, stretching), 1689.8 (C]O, stretching), 1617.6 (CeC, stretching), 1420.2 (CeH, bending), 1289.6 (C]S, stretching). 1H NMR (DMSO‑d6, 300 MHz); δ (ppm) 12.11 (s, 1H, NH), 11.63 (s, 1H, NH), 11.23 (s, 1H, NH), 8.11 (d, 2H, AreH, J = 7.5 Hz), 8.01 (d, 2H, AreH, J = 7.1 Hz), 7.63 (d, 2H, AreH), 7.36 (d, 2H, AreH), 7.34 (d, 2H, AreH), 7.11 (t, 1H, AreH), 2.3 (s, 3H); 13C NMR (75 MHz DMSO‑d6) δ (ppm) 182.4 (C]S), 179.1 (C]O), 173.2, 156.3, 153.3, 151.4, 145.1, 142.8, 139.5, 136.8, 133.6, 130.3, 128.7, 124.1, 122.6, 120.3, 119.2, 117.6 (AreC), 55 (CH3), Anal. Calcd. For C19H17N5O4S2: C, 51.37; H, 3.65; N, 15.49; S, 14.21 found: C, 51.07; H, 3.47; N, 15.28; S, 14.01.

White solid, Yield; 80%, m.p: 215 °C, Rf = 0.61 (n-hexane: ethyl acetate, 4:1), FTIR υ (cm−1) 3078.7 (NeH, stretching), 2926.6 (Aromatic CeH, stretching), 1699.8 (C]O, stretching), 1579.9 (CeC, stretching), 1407.3 (CeH, bending), 1152.4 (C]S, stretching). 1H NMR (DMSO‑d6, 300 MHz); δ (ppm) 12.75 (s, 1H, NH), 11.84 (s, 1H, NH), 11.57 (s, 1H, NH), 8.51 (d, 2H, AreH, J = 6.5 Hz), 8.01 (d, 2H, AreH, J = 3.9 Hz), 7.75 (d, 2H, AreH), 7.07 (t, 1H, AreH), 3.36 (t, 2H), 2.51 (quint, 2H), 2.43 (quint, 2H), 1.57 (quint, 2H),1.55 (quint, 2H), 1.24 (sex, 2H), 0.85 (t, 3H); 13C NMR (75 MHz DMSO‑d6) δ (ppm) 179.4 (C] S), 176.1 (C]O), 172.5, 158.8, 157.3, 142.1, 137.7, 130.7, 128.8, 124.1, 118.6, 116.3 (AreC), 40.51, 39.7, 36.2, 29.1, 25.4, 22.6, 14.4, Anal. Calcd. For C19H25N5O3S2: C, 52.19; H, 5.48; N, 15.94; S, 14.41 found: C, 51.07; H, 5.37; N, 15.73; S, 14.30.

6.5. 2-Chloro-N-((4-(N-(pyrimidin-2-yl)sulfamoyl)phenyl)carbamothioyl) benzamide (4e)

6.9. N-((4-(N-(Pyrimidin-2-yl)sulfamoyl)phenyl)carbamothioyl) heptanamide (4i)

White solid, Yield; 76%, m.p: 243 °C, Rf = 0.59 (n-hexane: ethyl acetate, 4:1), FTIR υ (cm−1) 3136.3 (NeH, stretching), 2956.3 (Aromatic CeH, stretching), 1739.8 (C]O, stretching), 1623.6 (CeC, stretching), 1417.2 (CeH, bending), 1233.6 (C]S, stretching). 1H NMR

Yellow solid, Yield; 75%, m.p: 210 °C, Rf = 0.58 (n-hexane: ethyl acetate, 4:1), FTIR υ (cm−1) 3108.3 (NeH, stretching), 2928.6 (Aromatic CeH, stretching), 1676.8 (C]O, stretching), 1517.5 (CeC, stretching), 1421.4 (CeH, bending), 1276.9 (C]S, stretching). 1H NMR 3713



(DMSO‑d6, 300 MHz); δ (ppm) 12.64 (s, 1H, NH), 11.74 (s, 1H, NH), 11.43 (s, 1H, NH), 8.32 (d, 2H, AreH, J = 6.3 Hz), 8.0 (d, 2H, AreH, J = 3.2 Hz), 7.36 (d, 2H, AreH), 7.06 (t, 1H, AreH), 3.46 (t, 2H), 2.63 (quint, 2H), 2.33 (quint, 2H), 1.53 (quint, 2H), 1.31 (sex, 2H), 0.76 (t, 3H); 13C NMR (75 MHz DMSO‑d6) δ (ppm) 178.5 (C]S), 175.3 (C]O), 171.8, 157.3, 156.4, 141.1, 136.4, 131.7, 127.2, 123.1, 119.6, 117.1 (AreC), 40.42, 39.4, 36.1, 28.1, 24.4, 15.4, Anal. Calcd. For C18H23N5O3S2: C, 50.92; H, 5.43; N, 16.42; S, 14.95 found: C, 50.67; H, 5.27; N, 15.93; S, 14.46.

stock solution of DPPH with the concentration of (150 µM), from the stock solution we used 100 µL in assay reaction, 20 µL of increasing concentration of compounds and volume was adjusted up to 200 µL in each well with methyl alcohol (CH3OH). After adjusted the final volume of assay mixture, the assay reaction mixture was then incubated at room temperature for 30 min. In this study, as a reference inhibitor Ascorbic acid (Vitamin C) was used. The measurement of the assay was observed at 517 nm, using a microplate reader (OPTI MAX, Tunable USA). The rate of reaction was compared and the percent inhibition caused by the presence of tested inhibitors were calculated. In a triplicate form, all our compounds were repeated three times.

6.10. N-((4-(N-(Pyrimidin-2-yl)sulfamoyl)phenyl)carbamothioyl) acetamide (4j)

6.14. In-silico experiment Light yellow solid, Yield; 70%, m.p: 204 °C, Rf = 0.54 (n-hexane: ethyl acetate, 4:1), FTIR υ (cm−1) 3095.4 (NeH, stretching), 2934.6 (Aromatic CeH, stretching), 1685.6 (C]O, stretching), 1508.6 (CeC, stretching), 1437.2 (CeH, bending), 1262.3 (C]S, stretching). 1H NMR (DMSO‑d6, 300 MHz); δ (ppm) 12.43 (s, 1H, NH), 11.64 (s, 1H, NH), 11.36 (s, 1H, NH), 8.12 (d, 2H, AreH, J = 6.3 Hz), 8.03 (d, 2H, AreH, J = 3.2 Hz), 7.26 (d, 2H, AreH), 7.02 (t, 1H, AreH), 3.36 (s, 3H); 13C NMR (75 MHz DMSO‑d6) δ (ppm) 178.3 (C]S), 176.2 (C]O), 172.6, 151.3, 146.4, 131.1, 130.4, 129.7, 126.2, 123.7, 118.7, 116.1 (AreC), 30.42, Anal. Calcd. For C13H13N5O3S2: C, 44.21; H, 3.47; N, 19.62; S, 17.95 found: C, 43.97; H, 3.27; N, 19.23; S, 17.56.

6.14.1. Retrieval of alkaline phosphate structure from PDB The human placental alkaline phosphate crystal structure having 1.8 Å resolution was accessed from Protein Data Bank (PDB) having PDBID 1EW2.39 The retrieved alkaline phosphate structure was minimized by UCSF Chimera 1.16 tool.40 Protparam was used to predict stereo-chemical properties of alkaline phosphate41 while hydrophobicity and Ramachandran plots were accessed from Discovery Studio 4.1 Client of target alkaline phosphate.42 The proteins helices, beta-sheets, coils and turn were generated from VADAR 1.8 server.43 6.14.2. In-silico designing and chemo-informatics analysis of ligands The synthesized chemical structures (4a–4j) were drawn in ACD/ ChemSketch tool (https://www.acdlabs.com/; ACD/Installer, version 11.01, 2015) and further minimized by UCSF Chimera 1.16 tool. The basic chemo-informatics properties such as molecular weight (g/mol), hydrogen bond acceptors and hydrogen bond donors (HBA/D), logP, molecular volume (A3), molar refractivity, density, polarizability and drug likeness score were evaluated using ChemSketch and Molsoft tool (http://www.molsoft.com/), respectively. Furthermore, the ADMET assessment was performed by using online pkCSM tool.44 Data Warrior tool was used to predicts the ligand and lipophilic ligand efficiency (LE, LLE), and lipophilicity-corrected ligand efficiency (LELP) values.45

6.11. Alkaline phosphatase inhibition activity The Newly synthesized compounds were evaluated against alkaline phosphatase (CIALP) to screen putative inhibitors, the activity of calf intestinal alkaline phosphatase (CIALP) was measured by the spectrophotometric assay as previously described by J. Iqbal.37 The reaction mixture of assay comprised of Buffer solution 50 mM Tris-HCl, containing (5 mM MgCl2, 0.1 mM ZnCl2 pH 9.5), the compound (0.1 mM with the final concentration of DMSO 1% (V/V), and 5 µL Enzyme (CIALP 0.025 U/mL), after adding enzyme the reaction mixture was pre-incubated for 10 min. After incubation time the 10 µL of substrate (0.5 mM p-NPP (para nitrophenylphosphate disodium salt) was added to each well and mix very well to initiate the reaction and reaction mixture was incubated again at 37 °C for 30 min. After the Incubation period of 30 min, the change in absorbance of released p-nitrophenolate was measured at 405 nm, using a 96-well microplate reader (OPTIMAX, Tunable USA). In Triplicate form, all the experiments were repeated three times. In this study as a reference inhibitor of calf intestinal alkaline phosphatase (CIALP), the KH2PO4 was used in this assay.

6.15. Molecular docking PyRx docking tool was used to perform molecular docking experiment for all the ligands against alkaline phosphate.46 The grid box center values of (center_X = 43.3, center_Y=23.1612and center_ Z = 9.1269) and size Values were specified as (X = 65.56, Y = 71.79, and Z = 64.64) for better conformational pattern in the active binding site of target receptor. All the synthesized ligands were docked separately against alkaline phosphate with default exhaustiveness value = 8. The docked complexes were analyzed on the basis of lowest binding energy (kcal/mol) values and binding interaction pattern between the ligands and receptor. The graphical depictions of all docking complexes were accomplished by Discovery Studio (2.1.0) and UCSF Chimera 1.10.1 tool.

6.12. Kinetic analysis The kinetic inhibition analysis was followed by the same method as already described in (Alkaline Phosphatase Inhibition Activity) section, to determine the mechanism of enzyme inhibition, based upon IC50 results we have select the most potent inhibitor 4c to determine the mechanism of enzyme inhibition. In this analysis the concentrations of inhibitor (0.0, 0.015, and 0.3 µM), and the substrate (p-NPP) concentrations (10, 5, 2.5, 1.25 and 0.625 mM) were used. Maximal initial velocities were determined from the initial linear portion of absorbance up to 10 min after addition of enzyme at per minute’s interval. The inhibition type on the enzyme was assayed by Lineweaver-Burk plot of the inverse of velocities (1/V) versus the inverse of substrate concentration 1/[S] mM−1. The EI dissociation constant Ki was determined by the secondary plot of 1/V versus inhibitor concentration.

Conflict of interest Authors declare no any conflict of interest A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmc.2018.06.002. References

6.13. Free radical scavenging activity Through some modifications, already reported method of Larik et al.38, the radical scavenging activity was determined by 2, 2-diphenyl-1 picrylhydrazyl (DPPH) assay. In this assay we have prepared

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