Synthesis, enzyme inhibitory kinetics, and computational studies of novel 1-(2-(4-isobutylphenyl) propanoyl)-3-arylthioureas as Jack bean urease inhibitors

DR ZAMAN ASHRAF (Orcid ID : 0000-0002-6620-9080)

DR AAMER SAEED (Orcid ID : 0000-0002-7112-9296)

Article type : Research Article

Synthesis, enzyme inhibitory kinetics and computational studies of novel 1-(2-(4-

isobutylphenyl) propanoyl)-3-arylthioureas as Jack bean urease inhibitors

1

1,*

1

2,*

Tanzeela Abdul Fattah , Aamer Saeed , Pervaiz Ali Channar , Zaman Ashraf , Qamar

3

1

Abbas , Mubashir Hassan , Fayaz Ali Larik

1

Department of Chemistry Quaid-i-Azam University-45320, Islamabad, Pakistan.

Department of Chemistry, Allama Iqbal Open University, Islamabad 44000, Pakistan

Department of Biology, College of Natural Sciences, Kongju National University, Gongju 314-

2

3

701, Republic of Korea

*

Address for Correspondence

Aamer Saeed

Email: asaeed@qau.edu.pk, aamersaeed@yahoo.com

Contact number: +92-51-9064-2128, Fax: +92-51-9064-2241

Address for Correspondence

Zaman Ashraf,

Email: mzchem@yahoo.com

Contact number: +923215194461, Fax: +92512891471

This article has been accepted for publication and undergone full peer review but has not

been through the copyediting, typesetting, pagination and proofreading process, which may

lead to differences between this version and the Version of Record. Please cite this article as

doi: 10.1111/cbdd.13090

Abstract

In this article synthesis of a novel 1-(2-(4-isobutylphenyl)propanoyl)-3-arylthioureas (4a-j) as

jack bean urease inhibitors has been described. Freshly prepared 2-(4-isobutylphenyl)

propanoyl isothiocyanate was treated with substituted aromatic anilines in one pot using

anhydrous acetone. The compounds 4e, 4h and 4j showed IC50 values 0.0086nM, 0.0081nM

and 0.0094nM respectively. The enzyme inhibitory kinetics results showed that compound 4h

inhibit the enzyme competitively while derivatives 4e and 4j are the mixed type inhibitors.

The compound 4h reversibly binds the urease enzyme showing Ki value 0.0012nM. The Ki

values for 4e and 4j are 0.0025nM and 0.003nM respectively. The antioxidant activity results

reflected that compounds 4b, 4i and 4j showed excellent radical scavenging activity.

Moreover, the cytotoxic activity of the target compounds was evaluated using brine shrimp

assay and it was found that all of the synthesized compounds exhibited no cytotoxic effects to

brine shrimps. The computational molecular docking and molecular dynamic simulation of

title compounds was also performed and results showed that the wet lab findings are in good

agreement to the dry lab results. Based upon our results it is proposed that compound 4h may

act as a lead candidate to design the clinically useful urease inhibitors.

Keywords: Thioureas, Synthesis, Jack bean urease, Enzyme kinetics, Computaional

studies

Introduction

Ureases belongs to a family of large heteropolymeric enzymes having an active site

containing two Ni (II) atoms, largely found in Jack bean, soybeans, bacteria, fungi and other

plant seeds. They are also present in some animal tissues and intestinal micro-organisms.

Ureases catalyzes the hydrolysis of urea, forming ammonia and carbon dioxide which results

in the deficiency of nitrogen in the soil and the toxicity of excess ammonia makes soil

infertile for crops. It has been reported that ureases are responsible for dangerous factors

which include pyelonephritis, hepatic coma, peptic ulceration, stomach cancer and injection-

induced urinary stones (1-2). Besides human health ureases have a detrimental impact on the

economic impact for farmers due to excessive loss of ammonia from fertilizers. In addition,

interference of this ammonia to the atmosphere will consequently be deposited to land or

water which results in eutrophication and acidification of natural ecosystems on a regional

scale (3). Thus the development of new and effective urease inhibitors is a crucial task not

only for the treatment of human diseases but also to control the eutrophication and

acidification of natural ecosystems.

“

Ibuprofen is [2-(4-isobutylphenyl) propanoic acid], commercially available as Advil

”

or Motrin and is a well known nonsteroidal anti-inflammatory drug (NSAID) used for the

treatment of rheumatism and arthritis. The drug relieves pain and swelling by inhibiting the

formation of prostaglandins which are flat-like molecules derived from arachidonic acid.

Ibuprofen is a classical NSAID, it inhibits cyclooxygenase enzyme and it’s both isoforms

COX-1 and COX-2, involved in first step of the arachidonic acid cascade. But it produces

increased risk of side effects such as, gastrointestinal hemorrhage, ulceration, decreased renal

function, bleeding and perforation (4-7). NSAIDs side effects are believed to be involved by

two different mechanisms: a local action exerted by direct contact of drug with gastric

mucosa and generalized systemic action following absorption that is believed to be inhibition

of COX-1 (8-11). These side effects are thought to be due to free –COOH group of NSAIDs,

therefore safer and effective derivatives of NSAIDs are prepared to minimize their side

effects (12). Many derivatives of ibuprofen have been prepared such as, sugar, amide, ester

and hydrazone derivatives which show anti-inflammatory activity comparable with that of

parent drug but minimum gastrotoxicity. Thus masking of free carboxylic group is the

principal of reduced topical irritant action (13).

During the couple of decades, tremendous research focus has been devoted on the

preparation, characterization and functional studies of thiourea derivatives. 1,3-Disubstituted

thioureas are valuable building blocks for generating a variety of heterocyclic compounds.

N,N-Dialkyl-N-aroylthioureas are selective reagents for separation of platinum group metals

(14). Thiocarbonyl group containing compounds (thioureas) show higher efficiency in

inhibiting corrosion, and due to this property thiourea is an industrially significant molecule,

and has been widely used as an electroplating additive, corrosion inhibitor, rubber

accelerator, and for extracting precious metals (15). 1,3-Dibenzylthiourea (DBTU) has been

investigated as carbon steel corrosion inhibitor (16). Thioureas have found extensive

applications in the field of medicine, agriculture, analytical and coordination chemistry (17a-

17b) and are used as chelating agents (18). Various biological activities have been reported

for thioureas such as, antifungal, herbicidal, antitubercular, antifungal and insecticidal (19-

20). Thiourea derivatives of amlodipine possess antimicrobial activities (21), whereas

thiourea derivatives of bisindolylmethane act as carbonic anhydrase (II) inhibitors (22).

Zhang reported thiourea-appended 1,8-naphthalimide as chemosensor for detection of heavy

metal ions (23), and N-(4-Aminophenylethynylbenzonitrile)-N′-(1-naphthoyl)thiourea act as

chemosensor for carbon monoxide sensing (24). Ethoxy carbonyl thiourea is used as collector

for improving copper flotation recovery from copper porphyry ore (25). 1-Aroyl-3-

arylthioureas have been used in the synthesis of imidazole-2-thiones (26) and 1,3-thiazolines

(27). In this article some new ibuprofenyl-arylthioureas (4a-j) were synthesized and

evaluated for their Jack Bean Urease inhibitory potential, antioxidant and cytotoxic activities.

Experimental

Methods and materials

The chemicals, thionyl chloride, ibuprofen, 2,4-dinitroaniline, 4-bromoaniline, p-anisidine,

3,5-dinitroaniline, 3-nitroaniline, 4-chloroaniline, 4-aminobenzenesulfonamide, 2,3-

dichloroaniline, 2,4,6-trimethylaniline, benzyl amine, phenylmethanamine, all synthetic

starting materials, reagents and solvents were of analytical reagent grade or of the highest

quality commercially available and were purchased from Sigma Aldrich Chemical Co.,

Merck Germany and was used without further purification. Acetone was freshly dried over

KMnO

4 f

and distilled. R -values were determined using pre-coated silica gel aluminum plates

60F₂₅₄from Merck (Germany). Melting points were determined in an open glass capillaries

using Gallenkamp melting point apparatus (MP-D) and are uncorrected. Infrared spectra (IR)

of the compounds were recorded on a Bio-Rad-Excalibur Series Model No.FTS 300 MX

1

13

spectrophotometer as pure compounds. H and C-NMR spectra were obtained on a Bruker

00 MHz and 75.5 MHz NMR spectrometer using teteramethyl silane (TMS) as internal

3

reference standard. Elemental analyses were conducted using a LECO-183 CHNS analyser.

Procedure for the synthesis of 2-(4-isobutylphenyl)propanoyl chloride (2)

A solution of 2-(4-isobutylphenyl)propanoic acid 1 (1.0 mmol, 0.2 g) and DMF (0.05 mL) in

thionyl chloride (1.2 mmol) was refluxed for 3 h. After cooling at room temperature, the

mixture was concentrated in vacuo to afford 2-(4-isobutylphenyl)propanoyl chloride (2).

Synthesis of 1-(2-(4-isobutylphenyl) propanoyl)-3-arylthioureas (4a-j)

A solution of 2-(4-isobutylphenyl)propanoyl chloride 2 (1.0 mmol) in dry distilled acetone

(

20 mL) was added drop wise to a suspension of potassium thiocyanate (1.0 mmol ) in dry

acetone and refluxed for 1.5 h at 50ºC. After cooling on room temperature a solution of

substituted aromatic aniline (1.0 mmol) in acetone was added and the mixture was refluxed

for 7-8 h. The progress of the reaction was monitored by TLC, EtOAc : n-hexane (1:2). After

the reaction was completed it was poured onto crushed ice and the precipitates were collected

by filtration, washed, dried and recrystallized from ethanol to yield the 1-(2-(4-

isobutylphenyl)propanoyl)-3-arylthiourea derivatives (4a-j).

1-(3,5-Dinitrophenyl)-3-(2-(4-isobutylphenyl)propanoyl)thiourea (4a)

o

Light brown solid; yield: 78%; m.p: 163 C; R

f

: 0.5 (n-Hexane : Ethyl acetate 1:1); IR (KBr)

1

cm : 3371, 3223 (N-H), 3024, 2934 (Csp2-H), 2878 (Csp3–H), 1682 (C=O), 1615, 1595 (C=C

1

Ar), 1259 (C=S); H NMR (300 MHz, DMSO-d

6

): δ (ppm) 12.43 (1H, s, broad, NH), 8.69

(1H, s, broad, NH), 7.31-7.55 (d, 2H, J = 8.1 Hz, Ar-H), 7.11-7.23 (d, 2H, J = 8.1 Hz, Ar-H),

8

.55 (t, 1H, J = 7.6 Hz, Ar-H), 7.85 (d, 2H, J = 7.6 Hz, Ar-H), 4.62 (q, 1H, J = 7.10 Hz,

), 2.41 (d, 2H, J = 7.17 Hz, (CH CHCH Ar), 1.82 (m, 1H, CH(CH ), 1.40 (d, 3H,

J = 7.3 Hz, ArCHCH ), 0.86 (d, 6H, J = 6.56 Hz, CH(CH ); C NMR (75.5 MHz, DMSO-

); 178.6 (C=S), 174.5 (C=O), 149.2, 141.9, 140.5, 134.9, 133.5, 129.6, 128.2, 120.5 (Ar-

Cs), 44.5, 42.5, 31.1, 22.9, 18.2 Anal. Calcd. for C H N O S: C, 55.80; H, 5.15; N, 13.02;

CHCH

3

)

2

3 2

)

13

3

3 2

)

d

6

20

22

4

5

S, 7.45 found: C, 55.78; H, 5.13; N, 13.00; S, 7.43.

1-(4-Bromophenyl)-3-(2-(4-isobutylphenyl)propanoyl)thiourea (4b)

o

1

Brown solid; yield: 69%; m.p: 174 C; R

f

: 0.7 (n-Hexane : Ethyl acetate 1:1); IR (KBr) cm :

3288, 3211 (N-H), 3032, 2945 (Csp2-H), 2812 (Csp3–H), 1686 (C=O), 1565, 1467 (C=C Ar),

1

260 (C=S); H NMR (300 MHz, DMSO-d ): δ (ppm) 13.53 (1H, s, broad, NH), 9.76 (1H, s,

6

broad, NH), 7.21-7.39 (d, 2H, J = 8.2 Hz, Ar-H), 7.11-7.20 (d, 2H, J = 8.2 Hz, Ar-H), 7.29

d, 2H, J = 7.55 Hz, Ar-H), 6.49 (d, 2H, J = 7.55 Hz, Ar-H), 3.79 (q, 1H, J = 7.11 Hz,

(

CHCH ), 2.52 (d, 2H, J = 7.13 Hz, (CH ) CHCH Ar), 1.85 (m, 1H, CH(CH ) ), 1.56 (d, 3H,

3

3 2

2

3 2

13

3 3 2

J = 7.1 Hz, ArCHCH ), 0.91 (d, 6H, J = 6.51 Hz, CH(CH ) ); C NMR (75.5 MHz, DMSO-

6

d ); 184.3 (C=S), 176.4 (C=O), 140.5, 138.5, 133.7, 133.1, 129.1, 128.9, 127.5, 119.2 (Ar-

Cs), 46.2, 44.5, 28.7, 22.5, 18.3 Anal. Calcd. for C20

S, 3.82 found: C, 57.20; H, 5.51; N, 6.66; S, 3.80.

2

H23BrN OS: C, 57.28; H, 5.53; N, 6.68;

1-(2-(4-isobutylphenyl)propanoyl)-3-(4-methoxyphenyl)thiourea (4c)

o

1

Yellow solid; yield: 82%; m.p: 186 C; R

f

: 0.7 (n-Hexane : Ethyl acetate 2:1); IR (KBr) cm :

3

1

321, 3211 (N-H), 3076, 2945 (C -H), 2814 (C –H), 1684 (C=O), 1594, 1445 (C=C Ar),

sp2

sp3

1

6

278 (C-O-C), 1258 (C=S); H NMR (300 MHz, DMSO-d ): δ (ppm) 12.99 (1H, s, broad,

NH), 8.87 (1H, s, broad, NH), 7.33-7.45 (d, 2H, J = 8.2 Hz, Ar-H), 7.21-7.29 (d, 2H, J = 8.2

Hz, Ar-H), 6.60 (m, 2H, Ar-H), 6.49 (m, 2H, Ar-H), 3.88 (q, 1H, J = 7.10 Hz, CHCH ), 3.65

3

(

s, 3H, OCH

H, J = 7.2 Hz, ArCHCH

DMSO-d ); 180.6 (C=S), 173.5 (C=O), 155.8, 138.3, 134.1, 129.5, 129.4, 128.9, 127.1, 114.3

3

), 2.59 (d, 2H, J = 7.12 Hz, (CH

3

)

2

CHCH

2

Ar), 1.83 (m, 1H, CH(CH

3

)

2

), 1.55 (d,

1

3

), 0.95 (d, 6H, J = 6.52 Hz, CH(CH

3 2

) ); C NMR (75.5 MHz,

6

(Ar-Cs), 54.7, 45.2, 40.4, 31.5, 25.3, 18.2 Anal. Calcd. for C21

26 2 2

H N O S: C, 68.08; H, 7.07;

N, 7.56; S, 8.65 found: C, 68.06; H, 7.05; N, 7.54; S, 8.63.

1-(2,4-Dinitrophenyl)-3-(2-(4-isobutylphenyl)propanoyl)thiourea (4d)

o

Pale Yellow solid; yield: 84%; m.p: 168-169 C; R

f

: 0.64 (n-Hexane : Ethyl acetate 2:1); IR

1

(

KBr) cm : 3345, 3212 (N-H), 3023, 2932 (Csp2-H), 2838 (Csp3–H), 1690 (C=O), 1525, 1450

1

(

C=C Ar), 1265 (C=S); H NMR (300 MHz, DMSO-d

.12 (1H, s, broad, NH), 7.23-7.34 (d, 2H, J = 8.2 Hz, Ar-H), 7.12-7.20 (d, 2H, J = 8.2 Hz,

Ar-H), 8.95 (s, 1H, Ar-H), 8.35 (d, 1H, J = 8.35 Hz, Ar-H), 7.18 (d, 1H, J = 8.35 Hz, Ar-H),

.10 (q, 1H, J = 7.10 Hz, CHCH ), 2.49 (d, 2H, J = 7.13 Hz, (CH CHCH Ar), 1.86 (m, 1H,

CH(CH ) ), 1.59 (d, 3H, J = 7.3 Hz, ArCHCH ), 0.93 (d, 6H, J = 6.54 Hz, CH(CH ) ); C

6

): δ (ppm) 12.38 (1H, s, broad, NH),

9

4

3

)

2

1

3

2

3

3 2

NMR (75.5 MHz, DMSO-d

6

); 186.3 (C=S), 179.2 (C=O), 145.2, 144.3, 140.6, 140.1, 128.8,

1

5

28, 127.8, 120.1 (Ar-Cs), 44.1, 40.2, 31.7, 21.7, 18.5 Anal. Calcd. for C20 S: C,

22 4 5

H N O

5.80; H, 5.15; N, 13.02; S, 7.45 found: C, 55.78; H, 5.13; N, 13.00; S, 7.41.

1-(2-(4-isobutylphenyl)propanoyl)-3-(3-nitrophenyl)thiourea (4e)

o

Reddish brown solid; yield: 73%; m.p: 179 C; R : 0.76 (n-Hexane : Ethyl acetate 2:1); IR

f

1

(

KBr) cm : 3323, 3219 (N-H), 3123, 2935 (Csp2-H), 2874 (Csp3–H), 1680 (C=O), 1555, 1489

1

(

C=C Ar), 1255 (C=S); H NMR (300 MHz, DMSO-d

6

): δ (ppm) 13.12 (1H, s, broad, NH),

9.39 (1H, s, broad, NH), 7.30-7.45 (d, 2H, J = 8.0 Hz, Ar-H), 7.21-7.29 (d, 2H, J = 8.0 Hz,

Ar-H), 7.76 (m, 1H, Ar-H), 7.40 (s, 1H, Ar-H), 7.15 (m, 1H, Ar-H), 6.83 (m, 1H, Ar-H), 3.92

(

q, 1H, J = 7.10 Hz, CHCH ), 2.61 (d, 2H, J = 7.16 Hz, (CH ) CHCH Ar), 1.85 (m, 1H,

3

3 2

2

1

3

CH(CH

3

)

2

3 3 2

), 1.49 (d, 3H, J = 7.2 Hz, ArCHCH ), 0.88 (d, 6H, J = 6.54 Hz, CH(CH ) ); C

NMR (75.5 MHz, DMSO-d

30.6, 130.1, 128.5, 121.5, 118.5 (Ar-Cs), 43.2, 40.5, 32.5, 24.7, 17.6 Anal. Calcd. for

S: C, 62.32; H, 6.01; N, 10.90; S, 8.32 found: C, 62.30; H, 5.98; N, 10.88; S,

6

); 180.6 (C=S), 176.4 (C=O), 149.2, 140.1, 137.3, 134.2, 133.2,

1

20 23 3 3

C H N O

8.30.

1-(4-Chlorophenyl)-3-(2-(4-isobutylphenyl)propanoyl)thiourea (4f)

o

Light brown solid; yield: 56%; m.p: 175 C; R

f

: 0.56 (n-Hexane : Ethyl acetate 1:1); IR (KBr)

1

cm : 3312, 3267 (N-H), 3121, 2937 (C -H), 2865 (C –H), 1683 (C=O), 1576, 1496 (C=C

sp2

sp3

1

6

Ar), 1250 (C=S), 754 (C-Cl); H NMR (300 MHz, DMSO-d ): δ (ppm) 12.99 (1H, s, broad,

NH), 8.78 (1H, s, broad, NH), 7.55-7.60 (d, 2H, J = 8.1 Hz, Ar-H), 7.29-7.35 (d, 2H, J = 8.1

Hz, Ar-H), 7.13 (d, 2H, J = 8.4 Hz, Ar-H), 6.63 (d, 2H, J = 8.4 Hz, Ar-H), 3.99 (q, 1H, J =

7

.11 Hz, CHCH

d, 3H, J = 7.1 Hz, ArCHCH

DMSO-d ); 185.3 (C=S), 179.2 (C=O), 140.3, 136.2, 134.9, 131.2, 130.5, 130.1, 129.2, 128.8

Ar-Cs), 44.3, 41.5, 33.5, 27.1, 19.2 Anal. Calcd. for C20 OS: C, 64.07; H, 6.18; N,

3

), 2.87 (d, 2H, J = 7.15 Hz, (CH

3

)

2

CHCH

2

Ar), 1.88 (m, 1H, CH(CH

3

)

2

), 1.56

13

(

3

), 0.80 (d, 6H, J = 6.53 Hz, CH(CH

3 2

) ); C NMR (75.5 MHz,

6

(

H23ClN

2

7.47; S, 8.55 found: C, 64.05; H, 6.16; N, 7.45; S, 8.52.

N-((4-Sulfamoylphenyl) carbamothioyl-2-(4-isobutylphenyl)propanamide (4g)

o

Dark brown solid; yield: 78%; m.p: 179 C; R : 0.51 (n-Hexane : Ethyl acetate 1:1); IR (KBr)

f

1

cm : 3253, 3277 (N-H), 3150, 3012 (Csp2-H), 2950 (Csp3–H), 1663 (C=O), 1562, 1545 (C=C

1

Ar), 1263 (C=S), 1136 (C-N); H NMR (300 MHz, DMSO-d

6

): δ (ppm) 12.17 (1H, s, broad,

NH), 9.65 (1H, s, broad, NH), 7.98 (d, 2H, J = 9.2 Hz, Ar-H), 7.44 (d, 2H, J = 9.2 Hz, Ar-H),

.39-7.55 (d, 2H, J = 8.1 Hz, Ar-H), 7.27-7.32 (d, 2H, J = 8.1 Hz, Ar-H), 7.09 (s, 2H,

SO NH ) 3.76 (q, 1H, J = 7.12 Hz, CHCH ), 2.71 (d, 2H, J = 7.14 Hz, (CH CHCH Ar),

7

2

3

)

2

1

.92 (m, 1H, CH(CH ) ), 1.60 (d, 3H, J = 7.1 Hz, ArCHCH ), 0.93 (d, 6H, J = 6.54 Hz,

3

2

3

1

3

CH(CH

3

)

2

); C NMR (75.5 MHz, DMSO-d

6

); 186.2 (C=S), 179.2 (C=O), 141.5, 140.1,

139.5, 137.7, 128.2, 127.3, 126.9, 122.5 (Ar-Cs), 46.1, 43.4, 30.5, 22.3, 19.2 Anal. Calcd. for

20 25 3 3 2

C H N O S : C, 57.25; H, 6.01; N, 10.02; S, 15.29 found: C, 57.20; H, 5.98; N, 10.00; S,

15.27.

1-(2,3-Dichlorophenyl)-3-(2-(4-isobutylphenyl)propanoyl)thiourea (4h)

o

Off white solid; yield: 71%; m.p: 177 C; R

f

: 0.55 (n-Hexane : Ethyl acetate 1:1); IR (KBr)

1

cm : 3366, 3257 (N-H), 3176, 2954 (C -H), 2812 (C –H), 1681 (C=O), 1554, 1443 (C=C

sp2

sp3

1

6

Ar), 1247 (C=S), 732 (C-Cl); H NMR (300 MHz, DMSO-d ): δ (ppm) 13.21 (1H, s, broad,

NH), 9.32 (1H, s, broad, NH), 7. 21-7.34 (d, 2H, J = 8.2 Hz, Ar-H), 7.01-7.17 (d, 2H, J = 8.2

Hz, Ar-H), 6.30-6.66 (m, 3H, Ar-H), 3.96 (q, 1H, J = 7.1 Hz, CHCH ), 2.56 (d, 2H, J = 7.1

3

Hz, (CH

3

)

2

CHCH

H, J = 6.9 Hz, CH(CH

40.1, 138.6, 137.2, 134.5, 134.2, 130.6, 128.8, 128.1, 127.5, 126.3 (Ar-Cs), 46.2, 43.4, 30.3,

2

Ar), 1.95 (m, 1H, CH(CH

3

)

2

), 1.54 (d, 3H, J = 7.2 Hz, ArCHCH

3

), 0.96 (d,

13

6

1

2

5

3

)

2

); C NMR (75.5 MHz, DMSO-d ); 182.6 (C=S), 177.3 (C=O),

6

6.1, 18.3 Anal. Calcd. for C20

2 2

H22Cl N OS: C, 58.68; H, 5.42; N, 6.84; S, 7.83 found: C,

8.66; H, 5.40; N, 6.82; S, 7.81.

1-Benzyl-3-(2-(4-isobutylphenyl)propanoyl)thiourea (4i)

o

Dark brown solid; yield: 66%; m.p: 189 C; R

f

: 0.67 (n-Hexane : Ethyl acetate 1:1); IR (KBr)

1

cm : 3301, 3224 (N-H), 3112, 2945 (Csp2-H), 2813 (Csp3–H), 1680 (C=O), 1612, 1523 (C=C

1

Ar), 1240 (C=S); H NMR (300 MHz, DMSO-d

6

): δ (ppm) 12.33 (1H, s, broad, NH), 9.56

(1H, s, broad, NH), 7.12-7.35 (d, 2H, J = 8.2 Hz, Ar-H), 7.11-7.31 (d, 2H, J = 8.2 Hz, Ar-H),

7

.15-7.28 (m, 5H, Ar-H), 3.88 (q, 1H, J = 7.2 Hz, CHCH

CHCH Ar), 1.92 (m, 1H, CH(CH ), 1.56 (d, 3H, J = 7.2 Hz, ArCHCH

J = 6.8 Hz, CH(CH ) ); C NMR (75.5 MHz, DMSO-d ); 180.6 (C=S), 176.2 (C=O), 142.6,

3

), 2.61 (d, 2H, J = 7.1 Hz,

(CH

3

)

2

3

)

2

3

), 0.93 (d, 6H,

13

3

2

6

140.3, 134.6, 130.1, 128.6, 128.1, 127.9, 125.3 (Ar-Cs), 44.2, 40.3, 32.6, 27.2, 18.9 Anal.

Calcd. for C21 OS: C, 71.15; H, 7.39; N, 7.90; S, 9.04 found: C, 71.13; H, 7.37; N,

26 2

H N

7.88; S, 9.01.

1-(2-(4-isobutylphenyl)propanoyl)-3-mesitylthiourea (4j)

o

1

Yellow solid; yield: 76%; m.p: 177 C; R : 0.75 (n-Hexane : Ethyl acetate 1:1); IR (KBr) cm :

f

3388, 3260 (N-H), 3080, 2922 (Csp2-H), 2848 (Csp3–H), 1695 (C=O), 1510, 1498 (C=C Ar),

1

269 (C=S); H NMR (300 MHz, Acetone-d

6

): δ (ppm) 10.58 (1H, s, broad, NH), 8.29 (1H,

s, broad, NH), 7.39-7.41 (d, 2H, J = 8.3 Hz, Ar-H), 7.32-7.35 (d, 2H, J = 8.3 Hz, Ar-H), 7.19

3

(s, 2H, Ar-H), 4.13 (q, 1H, J = 7.11 Hz, CHCH ), 2.52 (d, 2H, J = 7.14 Hz,

(

CH ) CHCH Ar), 2.43 (s, 9H, CH Ar), 1.82 (m, 1H, CH(CH ) ), 1.53 (d, 3H, J = 7.13 Hz,

3

2

3

3 2

13

ArCHCH

3

), 0.91 (d, 6H, J = 6.54 Hz, CH(CH

3 2 6

) ); C NMR (75.5 MHz, Acetone-d ); 180.2

(C=S), 176.1 (C=O), 140.8, 140.6, 137.5, 132.5, 129.4, 128.1, 127.3, 126.9 (Ar-Cs), 46.1,

4

4.6, 30.0, 21.7, 21, 18.1 Anal. Calcd. for C H N OS: C, 72.21; H, 7.90; N, 7.32; S, 8.38

20 30 2

found: C, 72.19; H, 7.88; N, 7.30; S, 8.37.

Enzyme assay

Urease inhibition assay

The Jack bean urease activity was determined by measuring amount of ammonia produced

with indophenols method described by Weatherburn (28). The reaction mixtures, comprising

2

0 µL of enzyme (Jack bean urease, 5 U/mL) and 20 μL of compounds in 50 μL buffer (100

mM urea, 0.01 M K HPO , 1 mM EDTA and 0.01 M LiCl, pH 8.2), were incubated for 30

min at 37 ºC in 96-well plate. Briefly, 50 μL each of phenol reagents (1%, w/v phenol and

.005%, w/v sodium nitroprusside) and 50 μL of alkali reagent (0.5%, w/v NaOH and 0.1%

2

4

0

Sodium hypochloriteNaOCl) were added to each well. The absorbance at 625 nm was

measured after 10 min, using a microplate reader (OPTI Max, Tunable). All reactions were

performed in triplicate. The urease inhibition activities were calculated according to the

following formula:

Urease inhibition activity (%) = (ODcontrol – ODsample × 100) / ODcontrol

Where ODcontrol and ODsample represents the optical densities in the absence and presence of

sample, respectively. Thiourea was used as the standard inhibitor for urease.

Free radical scavenging assay

Radical scavenging activity was determined by modifying already reported method (29-30)

by 2, 2-diphenyl-1 picrylhydrazyl (DPPH) assay. The assay solution consisted of 100 µL of

DPPH (150 µM), 20 µL of increasing concentration of test compounds and the volume was

adjusted to 200 µL in each well with DMSO. The reaction mixture was then incubated for 30

minutes at room temperature. Ascorbic acid (Vitamin C) was used as a reference inhibitor.

The assay measurements were carried out by using a micro plate reader (OPTI Max, Tunable)

at 517 nm. The reaction rates were compared and the percent inhibition caused by the

presence of tested inhibitors was calculated. Each concentration was analyzed in three

independent experiments run in triplicate.

Cytotoxicity evaluation using brine shrimp assay

Culturing and harvesting of Artemia salina

Artemia salina cysts were incubated for hatching in a rectangular dish with a plastic divider

with several holes making two uneven compartments. The container was filled with 3.3%

solution of artificial sea water and dry yeast sprinkled into the larger compartment which was

darkened. The smaller compartment was illuminated with light at 28 °C. After 24 hours,

hatched A. salina cysts were transferred to fresh artificial seawater and incubated for a further

24 hours under artificial light and aeriation. The phototropic nauplii were collected by pipette

from the lighted compartment.

Brine shrimps assay

A. salina nauplii (20) were counted macroscopically using Pasteur pipette against a lighted

background and transferred into each sample vial and the solutions were made to 5mL with

test compound using serial dilutions with brine solution. A drop of dry yeast suspension was

added as food to each vial. All the vials were maintained under light. The surviving nauplii

were counted with the aid of a magnifying glass after 24 hours. The mean mortality at the

three dose levels for compound was determined and repeated thrice. Potassium dichromate

was used as reference standard. After 24 the LD50 were calculated by Probit analysis.

Kinetic study

Urease inhibition was measured by varying the concentration of urea in the presence of

different concentrations of compound 4e (0.0, 0.025, and 0.01 nM), compound 4h (0.00,

0

.0020, 0.0040 and 0.0080 nM) and compound 4j (0.00, 0.0025, 0.0050 and 0.01nM.Briefly

the urea concentration was changed from 100, 50, 25, 12.5, 6.25, 3.12 mM for urease kinetics

studies and remaining procedure was same for all kinetic studies as describes in urease

inhibition assay protocol. Maximal initial velocities were determined from initial linear

portion of absorbances up to 10 minutes after addition of enzyme at per minute’s interval.

The inhibition type on the enzyme was assayed by Lineweaver-Burk plot of inverse of

-1

velocities (1/V) versus inverse of substrate concentration 1/ [S] mM . The EI dissociation

constant Ki was determined by secondary plot of 1/V versus inhibitor concentration. The

reversible kinetics of the enzyme inhibitoe complex was also determined for different

concentration of compound 4h versus the enzyme concentration (1.25, 2.5, 5, 10, 20 and

0U/mL. Urease activity was determined by measuring ammonia production using the

3

indophenol method as reported previously (31).The results (change in absorbance per min)

were processed by using SoftMaxPro.

Computaional studies

Retrieval of target protein

The crystal structure of Jack bean urease (Canavaliaensiformis) was accessed from Protein

Data Bank (PDB) (www.rcsb.org) having PDBID 4H9M. The target protein was minimized

by employing conjugate gradient algorithm and Amber force field in UCSF Chimera 1.10.1

(32). Furthermore, the Molprobity server (33) and Protparam (34) were utilized to assess the

stereo-chemical properties of urease structure. The Ramachandran plot and values (35) were

obtained from Molprobity server. The Discovery Studio 4.1 Client (36) , a visualizing tool

employed to generate the hydrophobicity graph. The overall protein architecture and

statistical percentage values of helices, beta-sheets, coils and turn were retrieved from online

server VADAR 1.8 (37) .

Designing of synthesized ligand structures

The synthesized chemical structures were sketched in drawing ACD/ChemSketch tool and

minimized by molecular visualizing software UCSF Chimera 1.10.1. The biological

properties and drug assessment like drug-likeness values, hydrogen bond acceptors (HBA),

and hydrogen bond donors (HBD) were predicted through online tools like Molinspiration

(http://www.molinspiration.com/) and Molsoft (http://www.molsoft.com/). The number of

HBA and HBD were also confirmed by PubChem (https://pubchem.ncbi.nlm.nih.gov/).

Moreover, Lipinski’s rule of five was analyzed using Molsoft and Molinspiraion tools. The

basic biochemical properties like molar refractivity, density, surface tension and

polarizability were generated by ACD/Chemsketch.

Molecular docking simulation

Molecular docking experiment was employed on all the ligands (4a-j) against crystal

structure of urease by using PyRx docking tool (38). The grid box center values (X=10.22,

Y=24.56 and Z=46.18) were adjusted with default exhaustiveness value = 8 to maximize the

binding conformational analysis. All the compounds were docked separately against urease

with 100 numbers of runs in each docking. The docking complexes were evaluated on lowest

binding energy (Kcal/mol) values and structure activity relationship (SAR) analyses. The 3D

graphical depictions of all the docking complexes were carried out using Discovery Studio

(2.1.0).

Molecular dynamics (MD) simulations assay

The best docked energy (Kcal/mol) valued complexes (4e and 4h ligands-receptor) were

selected for MD simulation. To interpret the residual backbone flexibility of these complexes

MD simulations were carried out by Groningen Machine for Chemicals Simulations

(

GROMACS) 4.5.4 package (39) with GROMOS 96 force field (40) .The PRODRG Server

41) was employed to generate ligands topology files. Before minimization, the overall

(

system charge was neutralized by adding ions. The energy minimization (nsteps=50000) was

done by steepest descent approach (1000 ps) for each protein-ligand complex. Particle Mesh

Ewald (PME) method was employed for energy calculation and for electrostatic and Van der

Waals interactions; cut-off distance for the short-range VdW (rvdw) was set to 14 Å, where

Coulomb cut-off (r coulomb) and neighbour list (rlist) were fixed at 9 Å (42). It permits the

use of the Ewald summation at a computational cost comparable with that of a simple

truncation method of 10 Å or less, and the linear constraint solver (LINCS) (43) algorithm

was used for covalent bond constraints and the time step was set to 0.002 ps. Furthermore,

NVT (44) was performed for 100 ps to equilibrate the system with protein and ligand for

constant volume, pressure (1 atm) and temperature (300 K). Finally, a 10 ns molecular

dynamics simulation was carried out for all the complexes with nsteps 5000000. The RMSD,

RMSF, SASA and Rg analysis were carried out using Xmgrace (http://plasma-

gate.weizmann.ac.il/Grace/) and UCSF Chimera 1.10.1 software.

Results and discussion

Synthesis

The synthetic pathway used for the synthesis 1-(2-(4-isobutylphenyl)propanoyl)-3-

arylthioureas (4a-j) is indicated in scheme 1. Initially 2-(4-Isobutylphenyl)propanoyl chloride

2

was freshly prepared from ibuprofen 1 with the use of thionyl chloride this was then added

to a suspension of potassium thiocyanate in anhydrous medium of acetone to yield 2-(4-

o

Isobutylphenyl)propanoyl isothiocyanate 3 as intermediates at 50 C for 1.5 h, after cooling at

room temperature substituted aromatic anilines in acetone were added and the resulting

mixture was refluxed until completion as checked by TLC to obtain thioureas (4a-j).

-1

FT-IR spectra showed sharp absorption peaks at 3233 and 3277 cm due to N-H vibration, a

-1

peak at 3112 cm is due to aromatic stretching and a strong peak at 1680 cm corresponds to

C=O of an amide. The aromatic stretching frequency bands appear in the range 1600-1550

-1

cm . Stretching frequency band at 1258 cm confirms C=S (thione) functionality.

-1

3

Vibrational modes at 650-745 cm correspond to C-Cl stretching vibrations. The C-H (Sp )

-1

1

stretching of alkyl chains appears at around 2850 cm respectively. H-NMR, showed the

most deshieldes broad signal at 12.69 ppm for N-H embedded between C=O and C=S in

6

DMSO-d , another broad signal appears at 8.69-8.29 ppm for second N-H of the thiourea.

13

The C-NMR showed C=S and C=O signals at 180.2 ppm and 176.1 ppm respectively. The

aliphatic and aromatic carbons appear at their respective regions.

Urease inhibition assay

The synthesized target compounds 4a-j have been screened for their inhibitory effects on the

ureases. All of the target compounds exhibited good activity with IC₅₀ranging (0.0081-

0.4626 nM) while IC50 of thiourea is 18.501 nM. The excellent inhibitory potential was

shown by derivatives 4e, 4h and 4j with IC50 0.0086, 0.0081 and 0.0094 respectively as

compared to standard thiourea. In compounds 4e, 4h the presence of 3-NO and 2,3-DiCl

2

have prime importance in urease inhibitory activity. 4j with benzyl group also exhibit good

activity. Compound 4c is the least active among the series. The urease inhibition potential of

the synthesized compound is presented in Table 1.

Free radical scavenging assay

The synthesized compounds 4a-j were also evaluated for their radical scavenging activity.

Compounds 4b, 4i and 4j showed excellent radical scavenging activity as compared to the

reference vitamin C. 4i with 2,4,6-trimethyl substitution showed the higher radical

scavenging activity as shown in Table 1.

Brine shrimps assay

The cytotoxic activity of the synthesized compounds was evaluated using brine shrimps

assay. Compound 4b with 4-Br substitution showed the lowest LD50 value among all the

2 2

series is the most cytotoxic while compound 4g with 4-SO NH showed the highest LD50

value is the least active (Table 2).

Kinetic study

Based on our results we select the most potent compound like 4e, 4h and 4j to determine their

inhibition type and inhibition constant on jack bean urease. The potential of these compounds

to inhibit free enzyme and enzyme substrate complex was determined in term of EI and ESI

constants respectively. The kinetic studies of the enzyme by the Lineweaver-Burk plot of 1/V

versus 1/[S] in the presence of different compounds concentrations gave a series of straight

lines (Figures1-3). The results of 4e and 4j compounds showed that both compounds

intersected within the second quadrant (Figures 1 and 3). The analysis showed that 1/Vmax

m

increased with increasing K in the presence of increasing concentration of compounds. This

behavior of both compound (4e and 4j) indicated that it inhibits urease by two different

pathwayscompetitively forming enzymeinhibitor (EI) complex and interrupting enzyme-

substrate-inhibitor (ESI) complex in noncompetitive manner. The secondary plots of slope

versus concentration of compounds 4e and 4j showed EI dissociation constants Ki while ESI

dissociation constants Ki′ were shown by secondary plots of intercept versus concentration of

compounds. A lower value of Ki than Ki′ pointed out stronger binding between enzyme and

compounds 4e, and 4j which suggested preferred competitive over noncompetitive manners

(

Table 3). While in case of compound 4h, Lineweaver–Burk plot gave family of straight

lines, all of which intersected at the same point on the x-axis the analysis showed that 1/Vmax

remains the same to a new value while that of Km increase to new value. This behavior

indicated that compound 4h inhibits Urease competitively to form enzyme inhibitor (EI)

complex. Secondary plot of slope against concentration of 4h showed EI dissociation

constant (Ki). The results of kinetic constants and inhibition constants are summarized in

Table 3.

The inhibitory mechanism of jack bean urease by compounds 4h with concentrations (0.0,

0

.0010, 0.0020, 0.0040 and 0.0080) were investigated. The plots of the remaining enzyme

activity versus the concentration of enzyme (30, 20, 10, 5, 2.5 and 1.25 U/mL) at different

inhibitor concentrations for the catalysis of urea gave a series of parallel straight lines with

the same slopes indicating that the inhibitory effect of 4h on the urease was irreversible

(Figure 2).

Computational studies

Structural assessment of predicted protein

Jack bean urease is a class of hydrolase protein. It contains four domains with variant amino

acids numbers.Two adjustable nickel atoms are present in domain four of targeted protein.

The presence of these metals in the target protein has a significant in the downstream

signaling pathways.Six core metal binding residue (His545, His519, Kcx490, His409, His407

and Asp633) were observed in the active binding pocket of target protein. The graphical

depiction of domain architecture within target protein and active region with metal bonded

residues is mentioned in Figure 4. The VADAR statistical analysis showed the protein

architecture is entailedwith 27% helices, 31% β sheets and 41% coils. Ramachandran plots

indicated that 97.5% of residues were present in favored regions. The Ramachandran graph

values also depict the accuracy of phi (φ) and psi (ψ) angles among the coordinates of urease

protein. The predicted graphs and value showed that 96.4% residues were present in

favoredand 99.9% are part of allowed region. The predicted Ramachandran graph and

individual domains of targeted protein are mentioned in supplementary data (Fig S1and S2).

The Physiochemical properties of target protein were computed by using ProtParam as

mentioned in Table 4. The protein Mw was calculated from linear sequence by the

aggregation of average isotopic masses of residues while, theoretical pIwas measured using

pK values of amino acids (45). The literature study justified that proteins were distributed

across a wide pI range (4.31 to 11.78) (46). The predicted pI value 6.05 is comparable with

the standard values which showed the accuracy of predicted structure. Furthermore,

extinction coefficient indicates how much light a protein absorbs at a certain wavelength. The

predicted value of aliphatic and instability indexeswere also showed the stability and relative

volume occupied by aliphatic side chains. The predicted GRAVY value showed the sum of

hydropathy values of all amino acids (47). The GRAVY negative value indicates it’s more

hydrophilic and less hydrophobic behavior. The extinction coefficient, aliphatic index values

of targeted protein were calculated from the equations as mentioned in supplementary data

(

Equations i and ii). Moreover, Grand Average of Hydropathy(GRAVY) calculation principle

is also mentioned in supplementary data. All the index calculation equations are mentioned in

supplementary data.

Lipinski Rule (RO5) and drug likeness predictions

The RO5 analysis justified that most of synthesized compounds were favored the Lipinski

rule.It has been observed that hydrogenbonding affinity is considered as a significant

parameter for evaluating the drug permeability. Bakhtet al., (48) justified that exceeded

values of HBA (>10) and HBD (>5) in ligands results in poor permeation in the body. Our

chemo-informatics analyses showed that all the designed compounds possess <10 HBA and

<5 HBD which may confirm their efficacy and good penetration within the body. The

predicted results of polar surface area (PSA) and molar volume of all synthesized compounds

were also favored their efficacy. Moreover, their logP value were also comparable with

standard values <5. However, there are plenty of examples available for RO5 violation

amongst the existing drugs (49,50). The drug likeness prediction results showed that 4h was

most active drug like compounds which has greater drug score (1.74) compared to other. Our

in-silico depiction for all the compounds showed results. The predicted chemo-

informaticproperty of synthesized compounds is mentioned in Table 5.

Molecular docking and binding energy evaluation of lead structures

Molecular docking study was employedto observe the best fitted conformational position of

synthesized compounds 4a-j against target protein urease. The docked complexes were

analyzed on the basis of minimum docking energy values (Kcal/mol). Docking results

justified that compounds 4h and 4e exhibited a good binding energy value (-9.70 and -8.90

kcal/mol), respectively, as compared to others compounds. The energy values showed that

both compounds bind within the active binding region of target protein with best

conformation position. The other compounds such as 4a, 4d, 4g and 4i were also depicts their

significance by good binding energy values (-8.30, -8.40, -8.10 and -8.00 Kcal/mol),

respectively. The comparative binding energy analysis reveals that most of compounds were

binds to the target molecule with better affinity and have strong therapeutical potential

against target protein. The compounds 4his confined at good compatible position in the active

region of target protein and showed good correlation results with IC₅₀values. Both

computational docking and wet data favors that 4h predicts good therapeutic potential against

target protein. The binding energy values of all the docking complexes and IC50 values are

mentioned in Figure 5.

Binding pocket analysis of synthesized compounds against urease

The docked complexes were further analyzed on the basis of hydrogen binding interactions.

The ligands-protein binding interaction showed that all compounds were confined in the

active binding pocket of target protein with different binding poses. The best energy values

docked complexes were mentioned in Figure 6. No π-π stacking interactions were observed

between compounds and aromatic residues of urease. The 4h-receptor docked complex

reveals the best conformational state with hydrogen/hydrophobic interactions. The unique

binding pattern was absorbed in 4h docking complex. In detail, the structure activity

relationship (SAR) study showed that six hydrogen bonds were observed in 4h docking. The

chlorine molecule of benzene ring directly interacts with Ala636 with a bonding distance 2.38

Å. Two hydrogen bonds were observed between carbonyl amino (-NH ) groups moieties of

2

4h against His593 having a bonding distance 2.48Åand 2.51Å.respectively. Moreover, the

carbonyl oxygen of same compound forms hydrogen bond with His593 at a distance 2.92Å.

Similarly, carbonyl methoxy and sulfur functional groups form hydrogen bonds against

Asp494 having bonding distance 2.95Å and 2.09Å, respectively. Literature study also

justified the significance of these interacted residues in hydrogen bonding which strengthen

our docking results (51). The detail binding pattern of all the complexes is mentioned in

supplementary data (Table. S1). The comparative binding energy and SAR analysis showed

the significance of 4h compound and may consider as potent inhibitors by targeting jack bean

urease. The 3D docking depictions of all other compounds are mentioned in supplementary

data figures (Figs S3-S11).

MD simulations analysis

Based on docking energy and IC50 results, 4e and 4h docking complexes were used evaluate

the residual flexibility of receptor molecule. MD simulation study was employed at 10ns by

using Gromacs 4.5.4 tool. The Root Mean Square Deviation (RMSD) and Root Mean Square

fluctuations (RMSF) graphs were assessed to determine the protein structural behavior in

docking. Furthermore, radius of gyration (Rg) and solvent accessible surface area (SASA)

2

(

A ) were also keenly focused to analyze the compactness of target protein. Figure 7 showed

the RMSD results of both 4e and 4h complexes which interprets the protein residual

deviation and fluctuations in 10ns simulation time frame. Initially, both graph lines were

displayed an increasing trend from 0-2000 ps. The 4e complex showed higher fluctuations

than the 4h. At 2000ps time, the RMSD values of 4h and 4e were 0.25 and 0.3, respectively.

From 2000 to 4000ps the 4h complex remains stable, while 4e showed increasing trends of

fluctuations at 3000ps and after that showed decreasing fluctuations at 4000ps. Moreover

same fluctuations trends were observed from 4000ps to 8000ps. The 4e graph line was more

fluctuated than the 4h and their RMSD value also increased more than 3. Whereas, the 4h

RMSD value remain stable and showed little fluctuations which favor the stability of docking

complex. A little increasing trend was observed from 8000ps to 1000ps in 4h graph line

while 4e showed downward fluctuations. The comparative analysis justified that 4h complex

simulation graph is more stable throughout the simulation time period as compared to 4e

complex. Figure 8 showed the generated RMSF results of both 4e and 4h complexes were

showed the C and N-terminal lobes fluctuations of target protein throughout the simulation

period. The generated graph showed that N-terminal loop region is much fluctuated. Result

depicted that higher peaks in RMSF graph showed the loop conformation and its fluctuations

in the simulation time. The comparative RMSF results assured the stability of 4h against

targeted protein throughout the simulation period as compared to 4e. Both RMSD and RMSF

graphs depicts the significance of 4h over 4e complex. In MD simulations, the stable

behaviors of 4h docked complex throughout MD trajectories thus increasing the efficacy of

docking results.

The compactness of receptor protein was evaluated by Radius of gyration (Rg). The

generated results exposed that Rg value is stable at 2.05nm values throughout the simulation

time frame 0-10000ps. The Rg time graph showed that residual backbone and folding of

receptor protein was steadily stable after binding the inhibitors (Figure 9). The solvent-

2

accessible surface area (SASA) results showed that both complexes co-reside at 205 nm in

the simulations graphs (Figure 10).

Conclusions

We have described the synthesis of a novel 1-(2-(4-isobutylphenyl)propanoyl)-3-

arylthioureas (4a-j) as jack bean urease inhibitors. The title compounds were screened for

their Jack bean urease inhibition activity, compounds 4e, 4h and 4j showed IC50 values

0

.0086nM, 0.0081nM and 0.0094nM respectively. The enzyme inhibitory kinetics of the

most potent compounds was also determined and it was observed that compound 4h inhibit

the enzyme competitively while derivatives 4e and 4j are the mixed type inhibitors. The

compound 4h reversibly binds the urease enzyme showing Ki value 0.0012nM. The Ki values

calculated for compound 4e and 4j are 0.0025nM and 0.003nM respectively. The antioxidant

activity resuts showed that compounds 4b, 4i and 4j exhibited excellent radical scavenging

potential. The computational molecular docking and molecular dynamic simulation of title

compounds was also performed and results showed that the wet lab findings are in good

agreement to the dry lab results. Based upon our results it is proposed that compound 4h may

act as a lead candidate to design the clinically useful urease inhibitors.

Conflict of Interest

The authors declare no conflicts of interest in this work.

Figure legends

Figure 1: Lineweaver–Burk plots for inhibition of urease in the presence of Compound 4e. (a)

Concentrations of 4e were (0.0, 0.025, and 0.01 nM) urea concentrations were 100, 50, 25, 12.5, 6.25,

3.12 mM.

Figure 2: Lineweaver–Burk plots for inhibition of urease in the presence of Compound 4h. (a)

Concentrations of 4h were (0.00, 0.0020, 0.0040 and 0.0080 nM) urea concentrations were 100, 50,

25, 12.5, 6.25, 3.12 mM.

Figure 3: Lineweaver–Burk plots for inhibition of urease in the presence of Compound 4j. (a)

Concentrations of 4j were (0.00, 0.0025, 0.0050 and 0.01 nM) urea concentrations were 100, 50, 25,

12.5, 6.25, 3.12 mM.

Figure 4: Crystal structure of jack bean urease with active metal bonded residues.

Figure 5: The graphical depiction of docking energy and IC50values.Thebars showed the docking

energy values (Kcal/mol) while dark blue for IC50 values (nM), 4h compound depicts good results

among other.

Figure 6: Docking complex of 4h against jack bean urease. The ligand structure is mentioned in grey

color, while the functional moieties such as chlorine, amino, sulfur and oxygen are highlighted in

green, deep purple, yellow and red colors, respectively. The targeted protein residues are mentioned in

light purple color in wire format. Two nickel atoms are also present in grey color. The black dotted

lines represent the hydrogen binding while red labeling justified bond distances in angstrom (Å).

Figure 7: RMSD graphs of 4e and 4h docked complexes are mentioned in blue and maroon colors

respectively, from 0-10000ps time scale.

Figure 8: RMSF graphs of 4e and 4h docked complexes are mentioned in blue and maroon colors

respectively, from 0-10000ps time scale.

Figure 9: Radius of gyration (Rg) graphs of 4e and 4h docked complexes are mentioned in blue and

maroon colors respectively from 0-10000ps time scale.

Figure 10: Solvent Accessible Surface Area (SASA) graph of 4e and 4h from 0-10000ps.

Figure legends for Supplementary information

Figure S1: Ramachandran graph of jack bean urease.

Figure S2: Domains of jack bean urease with an architecture.

Figure S3: Docking complex of 4a against jack bean urease.

Figure S4: Docking complex of 4b against jack bean urease.

Figure S5: Docking complex of 4c against jack bean urease.

Figure S6: Docking complex of 4d against jack bean urease.

Figure S7: Docking complex of 4e against jack bean urease.

Figure S8: Docking complex of 4f against jack bean urease.

Figure S9: Docking complex of 4g against jack bean urease.

Figure S10: Docking complex of 4i against jack bean urease.

Figure S11: Docking complex of 4j against jack bean urease.

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CH₃

CH

3

OH

SOCl₂

Cl

dry acetone

CH₃

CH

3

reflux for 3 h

KSCN

1-1.5 h

O

-

SO , HCl

H C

2

H C

3

2

1

CH₃

H

N

R

CH₃

N

^C^H3

Dry acetone

N

^C^H3

C

7

-8 h reflux

O

S

H C

3

R

^N^H2

O

H C

(4a-j)

3

Compound 4a

4b

4c 4d

4e

4f

4g

4h

4i

4j

Cl

NO₂

H C

CH₃

3

R

O N

2

NO₂

NO

2

Br

OMe

NO₂

SO NH

CH

3

Cl

2

Scheme 1 Synthesis of 1-(2-(4-isobutylphenyl) propanoyl)-3-arylthioureas (4a-j)

Table 1: Urease inhibition and free radical scavenging activities of synthesized compounds

4a-j).

(

Compound

Urease (Jack bean)

IC50±SEM (nM)

0.2599±0.00825

0.2214±0.00451

0.4626±0.022

Free radical % scavenging

(100µg/mL)

30.99±1.32

69.08±4.46

14.91±0.51

20.12±0.94

29.46±1.10

39.56±1.32

45.411±2.86

1.37±0.04

4

a

4b

4

c

4d

0.1216±0.00451

0.0086±0.000167

0.0381±0.00214

0.0340±0.0020

0.0081±0.000351

0.0292±0.000981

0.0094±0.00040

18.501±0.210

4

e

4f

4

g

4h

4i

74.41±4.30

59.45±3.4

4j

Thiourea

------

Vitamin C

----

93.36±5.78

Values are expressed as mean ± SEM

SEM= Standard Error of Mean

Table 2: LD50 of the synthesized compounds (4a-j) using brine shrimp assay.

Urease (Jack bean)

Compound

IC50±SEM (nM)

4

a

210.15±15.3

209.30±12.2

215.16±16.4

210.88±15.5

234.72±20.12

218.21±18.16

235.15±20.3

225.12±22.14

212.25±18.9

222.18±20.1

0.89±0.01

4b

4

c

4d

4

e

4

f

4g

4h

4

i

4

j

Potassium dichromate

Table 3: Kinetic parameters of the jack bean urease for urea activity in the presence of

different concentration of Ibuprofen derivatives (4a-j)

Code Dose

1/Vmax

(ΔA /Min)

Km Inhibition

(mM) Type

Inhibition

Ki

(nM)

Ki′

(nM)

nM)

(

0

.00

43.846

69.230

2.040

2.631 Mixed-

4e

4h

4j

0.0025

----

0.0025

0

.0044

.01

140.769

3.030 inhibition

1.11

0.00

154.216

0.0020

4.098 Competitive Irreversible 0.0012

----

0

.0040

.0080

6.25

9.803

0.0

84.210

1.063

103.508

0.0025

2.127 Mixed-

----

0.003

0.0147

0.0050

0.01

128.070

143.859

2.941 inhibition

3.33

V max = reaction velocity, Km = Michaelis - Menten constant, Ki = EI dissociation constant,

Ki′ = ESI dissociation constant, ---- = not calculated

Table 4: Physiochemical properties of predicted structure by ProtParam.

Parameters

Molecular weight (MW)

Values

90747.7 Da

6.05

Theoretical pI

Extinction coefficient* (assuming all Cys residues are reduced)

Aliphatic index

53290

90.48

Instability index

31.75

Gran average of hydropathicity (GRAVY)

-0.152

-1

*

Extinction Coefficient units M cm at 280 nm

Table 5: Biological properties of synthesized compounds 4a-j.

Properties

4a

4b

4c

370

4.86

3

4d

430

4.22

6

4e

385

4.50

4

4f

374

5.48

2

4g

419

3.59

5

4h

408

5.96

2

4i

4j

Mw(g/mol) 430

418

354

382

LogP

HBA

HBD

4.10 5.62

4.74 5.73

6

2

4

2

PSA(A )

Mol. Vol

No. SC

108

401

1

33

371

1

40

109

400

1

71

33

83

32

34

366

1

31

412

1

381

1

375

1

366

1

396

1

381

1

Drug Score 0.08 1.23

0.99

0.97

0.51

1.61

1.08

1.74

1.09 1.56

Article Doi

Synthesis, enzyme inhibitory kinetics, and computational studies of novel 1-(2-(4-isobutylphenyl) propanoyl)-3-arylthioureas as Jack bean urease inhibitors

DOI: 10.1111/cbdd.13090

Source and publish data:

Authors:

Article abstract of DOI:10.1111/cbdd.13090

Full text of DOI:10.1111/cbdd.13090

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