Synthesis of novel xanthene based analogues: Their optical properties, jack bean urease inhibition and molecular modelling studies

Article abstract of DOI:10.1016/j.saa.2020.118667

In this work, a series of the rhodamine 6G based derivatives 5a-5g, were synthesized. The structural framework of the synthesized compounds was established by using ¹H NMR, ¹³C NMR, FT-IR, and LC-MS analytical methods. The spectroscopic properties of the target compounds were determined by using absorption and fluorescence study in four different solvents. Furthermore, the synthesized derivatives were assessed for in-vitro screening against jack bean urease inhibition and in-silico molecular docking study. The result reveals that all the compounds exhibit good urease inhibitory activity against this enzyme but among the series, the compound 5a & 5c with an IC₅₀ values of 0.1108 ± 0.0038 μM and 0.1136 ± 0.0295 μM shows to be most auspicious inhibitory activity compared to a standard drug (Thiourea) having IC₅₀ value 4.7201 ± 0.0546 μM. Subsequently, the molecular docking experiment was analysed to distinguish the enzyme-inhibitor binding interaction.

Full text of DOI:10.1016/j.saa.2020.118667

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Synthesis of the Novel Xanthene Based Analogues: their Optical
Properties, Jack Bean Urease Inhibition and Molecular Modelling
Studies
Balasaheb D. Vanjare, Prasad G. Mahajan, Nilam C. Dige,
Hussain Raza, Mubashir Hassan, Sung-Yum Seo, Ki Hwan Lee
PII:
S1386-1425(20)30646-6
https://doi.org/10.1016/j.saa.2020.118667
SAA 118667
DOI:
Reference:
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
To appear in:
Received date: 4 April 2020
Revised date:
17 June 2020
27 June 2020
Accepted
date:
Please cite this article as: B.D. Vanjare, P.G. Mahajan, N.C. Dige, et al., Synthesis
of the Novel Xanthene Based Analogues: their Optical Properties, Jack Bean Urease
Inhibition and Molecular Modelling Studies, Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy (2020), https://doi.org/10.1016/j.saa.2020.118667
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Synthesis of the Novel Xanthene Based Analogues: their Optical Properties, Jack Bean
Urease Inhibition and Molecular Modelling Studies
Balasaheb D. Vanjare1 · Prasad G. Mahajan1 · Nilam C. Dige2 · Hussain Raza2 · Mubashir
Hassan3 · Sung-Yum Seo2 · Ki Hwan Lee*1
1 Department of Chemistry, Kongju National University, Gongju, Chungnam 32588, Republic of
Korea.
2 Department of Biological Sciences, Kongju National University, Gongju, Chungnam 32588,
Republic of Korea.
3 Institute of Molecular Biology and Biotechnology, The University of Lahore-54590, Pakistan
Corresponding author: Ki Hwan Lee
✉ khlee@kongju.ac.kr
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Abstract
In this work, a series of the rhodamine 6G based derivatives 5a-5g, were synthesized. The
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structural framework of the synthesized compounds was established by using H NMR, C
NMR, FT-IR, and LC-MS analytical methods. The spectroscopic properties of the target
compounds were determined by using absorption and fluorescence study in four different
solvents. Furthermore, the synthesized derivatives were assessed for in-vitro screening against
jack bean urease inhibition and in-silico molecular docking study. The result reveals that all the
compounds exhibit good urease inhibitory activity against this enzyme but among the series, the
compound 5a & 5c with an IC50 values of 0.1108±0.0038 µM and 0.1136±0.0295 µM shows to
be most auspicious inhibitory activity compared to a standard drug (Thiourea) having IC50 value
4.7201±0.0546 µM. Subsequently, the molecular docking experiment was analysed to
distinguish the enzyme-inhibitor binding interaction.
Keywords: Rhodamine derivatives; Photophysical properties; Urease inhibition; Radical
scavenging activity; Molecular Modelling.
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1. Introduction
Nowadays, most of the scientist are engrossed in modifying or synthesizing an organic
heterocyclic compound which has importance in various field such as, pharmaceutical,
biotechnology and fluorescence spectroscopy [1-4]. Rhodamine and its accompanying products
are a family of fluorone dye, which are one of the highly crucial heterocyclic compounds and
widely used in many scientific fields [5-8]. Xanthene is one of the crucial heterocyclic scaffolds
that appears in the rhodamine or its derivatives making them more active. Therefore, it has been
noticed, xanthene and its derivatives have gained more admiration because of their bioactive
nature [9-10]. For instance, a significant amount of xanthene core holding framework has been
introduced in many therapeutically important drugs (Scheme.1) candidate containing anti-
inflammatory activities, antibacterial activities, photodynamic therapy, antiviral effects,
antifungal activities, and enzyme inhibition. Also, there are several reports accessible on natural
products which comprise a xanthene structural frame [9,11,12].
Apart from biological applications, xanthene core containing compounds demonstrates
substantial spectroscopic assets such as stokes shift, quantum yield, high molar extinction
coefficient, absorption and fluorescence emission, fluorescence lifetime etc [13-15]. Moreover,
to qualify the criteria of the delicate fluorescence, the compound/dye must exhibit the finest
fluorescence response when exposed in the visible scale. As per the literature survey, it has been
observed that the framework containing a conjugated π-system and presence of electron-
donating/withdrawing substituents in the relevant functionality, exhibit decent fluorogenic
response [16]. Thus, we have integrated a rhodamine-based derivative since it comprises the
xanthene structure as well as the conjugated system. In practice, rhodamine and its derivatives
are rated as a versatile dye and which have been broadly utilized because of the excellent
spectroscopic outcomes. Moreover, these compounds exit in two forms, one is spirocyclic close
ring form (non-fluorescent) and the other is spirocyclic open ring form (fluorescent) [17].
Currently, compounds dealing with excellent spectroscopic assets have gained more
consideration in the field of chemosensor, optoelectronics, cell imaging study and organic light-
emitting diode (OLED) [18-20]. In this work, our major objectives were to synthesize of the
novel compounds, with good yield, that are easy to replicate and target compounds that could be
biologically active in contrast to different activities.
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Enzyme inhibition is a standout amongst the highly fortunate and obligatory zones in the
pharmaceutical field [21-23]. The organic chemist and pharmacist are inclined to contribute to
the development of incipient medicines in the areas of biotechnology and pharmacology, due to
which many valuable medicines are discovered to remedy numerous diseases [24-26]. In enzyme
inhibition mechanism, the certain inhibitor binds with enzyme and blocks its activity. By
blocking the action of an enzyme, it can correct the metabolic imbalance. There are many drugs
which act as an enzyme inhibitor; thus, many organic chemists are concentrating on the synthesis
of the bioactive compounds [27-31].
In 1926, the scientist James Summer scrutinized that urease is a protein from its crystalline
nature, that can function as an enzyme. After 70 years, the precise structure of the urease was
first explained by Karplus in 1995 as Urease (E.C 3.5.1.5) which belongs to a supergroup of
amidohydrolases and phosphodiesterase [32-35]. Furthermore, the first organic compound
synthesized in the laboratory was urea and the first enzyme crystallised in history was urease
[36-39]. It was isolated from Jack bean (JB) plant and recognized as metalloenzyme as it
possesses an active site of two Nickel (II) atoms, which are involved in the hydrolysis of urea to
produce volatile ammonia and carbamate. Variety of urease has expansively originated in nature
amongst organisms, plants, microbes, green growth, and invertebrates [40-44]. Bacteria
produced from urease have an adverse effect on human health. Helicobacter pylori is a bacteria
produced from the urease which can cause infections of the urinary tract and gastrointestinal [45-
47]. Some other diseases related with urease are hepatic coma, urinary catheter encrustation,
hepatic encephalopathy, pyelonephritis, urolithiasis, etc. Urease inhibitors have a crucial function
in the inhibition of harmful consequence caused by urease enzyme and significantly help to
recuperate human health [48-50a].
Herein, we have constructed and synthesized numerous rhodamine 6G centered derivatives by
the reaction of the rhodamine 6G and different hydrazide compounds in basic media. The
structural interpretation of the synthesized compounds was confirmed by using different
analytical techniques, for instance, LCMS, 1H NMR, 13C NMR & FT-IR spectroscopy skills. The
spectroscopic properties of the synthesized target compounds have been evaluated in distinct
solvents including non-polar, polar-aprotic, and polar protic solvents. Moreover, from the
biomedical applications point of view, the synthesized target compounds were screened against
urease inhibitory activity. From the in-vitro study, we observed that distinct substituent appears
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in nucleophile and significantly influences the urease inhibitory activity. Additionally, the
bioactivity outcomes were endorsed by the free radical scavenging survey and the molecular
modelling studies.
Scheme 1. Biologically crucial structure of xanthene derivatives.
2. MATERIALS AND METHODS
2.1 Chemistry
All the reagents were procured from Sigma Aldrich and solvents required in synthesis,
purification technique and for photophysical study were purchased from Samchun chemicals
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(Daejeon, South Korea), which were used without purification. H NMR and C NMR for the
synthesized target compounds were undertaken at 400 MHz spectrophotometers in DMSO-d6
solvent, by using a Bruker Avance (Germany). The chemical shift and coupling constant values
mentioned in ppm and Hz, respectively. The mass analysis (LC-MS) were recorded on
2795/ZQ2000 (waters) spectrometer. The FT-IR spectra were recorded on Fourier IR
spectrophotometer (Perkin Elmer, USA). The reaction progress was tracked by a thin layer
chromatography (TLC) method. The melting points of the compounds (5a-5g) were determined
by using Fisher Scientific (USA) melting point instrument and are uncorrected. The UV-Visible
absorption and fluorescence emission spectra of the target compounds (5a-5g) were computed on
a Shimadzu Spectrophotometer and FS-2 fluorescence spectrophotometer (Scinco, Korea). The
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UV-Visible absorption and emission spectra of the targets compound were measured in various
solvents like chloroform (CHCl3), acetonitrile (ACN), dimethyl formamide (DMF) and methanol
(MeOH) at 100 µM (1 × 10-4 mole/L). Path length of the cells used for absorption and emission
studied was 1 cm. The decay curves were obtained using a Time Correlated single photon
counting (TCSPC) spectrophotometer (HORIBAiHR320) employing a nanosecond diode laser
with time correlation of 0.0548 ns per channel and operated at its respective wavelength (530-
560 nm). The fluorescence quantum yield of the target compounds (5a-5g) in different solvents
were determined by using equation 1.
(1)
Where, A represents the area under the fluorescence spectra and OD is optical density of
the compound at the excitation wavelength, ƞ is the refractive index of the solvent used.
Quinine sulphate in 0.1 M sulphuric acid was taken as the reference (Φf) = 0.546) [2].
2.1.1 General method for the synthesis of compounds (2a-2g), (3a-3g) and (5a-5g).
2.1.1a General procedure for the Synthesis of (2/3/4) un/substituted ethyl benzoate (2a-2g)
As presented in Scheme 2, the unsubstituted/substituted aromatic acid compound (1a-1g ,1
mmol) were dissolved in ethanol (15 mL) and stirred for 10 min. Concentrated H2SO4 was added
to it and followed by refluxing for 5-6 hrs. The formation of the product was checked by using
TLC in hexane / Ethyl acetate (4:6) [50a]. After completion of the reaction, the reaction mass
was gradually cooled to ambient temperature and concentrated by rotary evaporator. Water was
added to the residue and extracted by using ethyl acetate. Further, ethyl acetate part (organic
layer) was washed by a saturated solution of sodium bicarbonate (to remove the excess of acid)
and followed by water and brine washing. The ethyl acetate layer was dried over anhydrous
magnesium sulphate, filtered, and distilled to afford a (2a-2g) with 70-80% yield and used for
the next step.
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Scheme 2. Synthesis route, reagents, and conditions: i) ethanol, conc. H2SO4 ii) hydrazine
hydrate, ethanol, reflux. iii) potassium tert-butoxide (K-tOBu), DMF, RT
2.1.1b General procedure for the synthesis of (2/3/4) un/substituted-benzhydrazides (3a-3g)
As displayed in Scheme 2, the un/substituted ester derivative (2a-2g, 1.0 mmol), hydrazine
hydrate (1.2 mmol) were taken in ethyl alcohol (10 mL) and the reaction mixture was heated to
reflux temperature and maintained until the reaction was completed (Checked by TLC) [50a].
After achievement of the reaction, the reaction was progressively cooled to room temperature.
Slowly, solid precipitated out in the reaction mass, was filtered, washed with cold ethanol, and
dried to afford a product (2/3/4) un/substituted-Benz hydrazides with 80-87% yield (3a-3g).
2.1.1c General procedure for the synthesis of N-(3', 6’-bis (ethyl amino)-2', 7’-dimethyl-3-
oxospiro [isoindoline-1, 9’-xanthene]-2-yl) (2/3/4-un/substituted-benzamides) compounds (5a-
5g)
As shown in Scheme 2, rhodamine 6G (1.0 mmol) and compound (3a-3g) (1.0 mmol) were
taken in DMF (8 mL) and cooled to 0-5 °C. Potassium tert-butoxide was added slowly and the
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reaction was stirred at 0-5 °C for 1 h then the temperature was gradually augmented to 25-30 ° C
and stirred until the reaction gets completed (6-7 hrs / monitored by TLC) [50b]. The reaction
mixture was quenched by gradual addition of the ice-cold water, the solid gets suspended in the
reaction mass which was finally filtered and dried. The impure solid was purified by column
chromatography, using dichloromethane/ methanol (9.5:0.5) as an eluent to afford a pink
coloured target compound with 80-86% yield (5a-5g).
N-(3', 6’-bis (ethyl amino)-2', 7’-dimethyl-3-oxospiro [isoindoline-1, 9’-xanthene]-2-yl)
benzamide (5a):
1
Pink solid; Melting point : >260°C; Yield 80 %; Fig. ES1: H NMR (400 MHz, DMSO-d6) δ
10.09 (s, 1H, -NH), 7.86 (d, J = 6.8 Hz, 1H, Ar-H), 7.66 – 7.32 (m, 7H, Ar-H), 7.02 (d, J = 6.9
Hz, 1H, Ar-H), 6.32 (s, 2H, Ar-H), 6.20 (s, 2H, Ar-H ), 4.98 (s, 2H, [-NH-CH2-CH3]2), 3.21 –
3.01 (m, 4H, [-CH2-CH3]2), 1.86 (s, 6H, Ar-CH3), 1.19 (t, J = 7.1 Hz, 6H, [-CH2-CH3]2). Fig.
ES2: 13C NMR (101 MHz, DMSO-d6) δ 170.64, 166.11, 162.46, 152.33, 147.82, 132.82, 128.85,
122.67, 117.87, 104.87, 96.13, 55.49, 37.93, 17.66, 14.83. Fig. ES3: IR (KBr, cm-1): 3401.41 (-
NH-COAr), 3355.10 (Ar-NH-CH2-CH3), 3239.17, 3022.15 (aromatic alkene stretching -C=C-
H), 2969.87 (aliphatic -NH-C-H2-CH3), 2868.22 (aliphatic -NH-CH2-C-H3), 1680.79
(Secondary amide -NH-CO-Ar), 1202.68 (aromatic amine C-N); Fig. ES4: LC-MS: 533.6 m/z.
N-(3', 6’-bis (ethyl amino)-2', 7’-dimethyl-3-oxospiro [isoindoline-1, 9’-xanthene]-2-yl)-2-
hydroxybenzamide (5b):
Pink solid; Melting point: 147°C; Yield 82 %. Fig. ES5:1H NMR (400 MHz, DMSO) δ 11.39 (s,
1H, -OH), 9.93 (s, 1H, -NH), 7.90 – 7.77 (m, 1H, Ar-H), 7.68 (dd, J = 8.1, 1.6 Hz, 1H, Ar-H),
7.62 – 7.52 (m, 2H, Ar-H), 7.40 – 7.33 (m, 1H, Ar-H), 7.01 (dd, J = 7.9, 4.2 Hz, 1H, Ar-H), 6.87
– 6.81 (m, 2H, Ar-H), 6.36 (m, 2H, Ar-H), 6.20 (m, 2H, Ar-H), 5.03 (s, 2H, [-NH-CH2-CH3]2),
3.16 – 3.02 (m, 4H, , [-NH-CH2-CH3]2), 1.88 (s, 6H, Ar-CH3), 1.19 (t, J = 7.1 Hz, 6H, [-NH-
CH2-CH3]2). Fig. ES6: 13C NMR (101 MHz, DMSO) δ 163.85, 160.76, 152.02, 148.04, 129.98,
128.84, 117.86, 117.04, 116.13, 105.14, 104.01, 95.57, 37.94, 17.61, 14.53. Fig. ES7: IR (KBr,
cm-1) : 3324.86 (Ar-NH-CH2-CH3), 2929.31 (aliphatic -NH-C-H2-CH3), 2851.22 (aliphatic -NH-
CH2-C-H3), 1623.69 (Secondary amide -NH-CO-Ar), 1513.04 ( -C=C-), 1218.28 (aromatic
amine C-N); Fig. ES8: LC-MS: 549 m/z.
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N-(3', 6’-bis (ethyl amino)-2', 7’-dimethyl-3-oxospiro [isoindoline-1, 9’-xanthene]-2-yl)-3-
hydroxybenzamide (5c)
Pink solid; Melting point: 120°C; Yield 80 %. Fig. ES9:1H NMR (400 MHz, DMSO) δ 10.04 (s,
1H, -NH), 7.96 – 7.16 (m, 5H, Ar-H), 7.15 – 6.96 (m, 3H, Ar-H), 6.31 (s, 2H, Ar-H), 6.19 (s, 2H,
Ar-H), 4.98 (s, 2H, [-NH-CH2-CH3]2), 3.16 – 3.05 (m, 4H, [-NH-CH2-CH3]2), 1.86 (s, 6H, Ar-
13
CH3), 1.19 (t, J = 7.1 Hz, 6H, [-NH--CH2-CH3]2 ). Fig. ES10: C NMR (101 MHz, CdCl3) δ
152.38, 147.82, 129.44, 128.83, 123.61, 117.32, 104.62, 95.83, 61.25, 50.79, 38.27, 30.74, 16.59,
14.47; Fig. ES11: IR (KBr, cm-1) : 3323.83 (secondary amine Ar-NH-CH2CH3), 2929.26
(aliphatic -NH-C-H2-CH3), 2851.29 (aliphatic -NH-CH2-C-H3), 1705.24 ( carbonyl), 1621.96
(Secondary amide -NH-CO-Ar), 1217.82 (aromatic amine C-N), 903.67 (monosubstituted); Fig.
ES12: LC-MS: 549.3 m/z.
N-(3', 6’-bis (ethyl amino)-2', 7’-dimethyl-3-oxospiro [isoindoline-1, 9’-xanthene]-2-yl)-3-
methoxybenzamide (5d)
1
Pink solid; Melting point: 252°C; Yield 83 %, Fig. ES13: H NMR (400 MHz, DMSO) δ 9.98
(s, 1H, -NH), 7.84 (d, J = 7.2 Hz, 1H, Ar-H), 7.62 – 7.51 (m, 2H, Ar-H), 7.18 – 7.12 (m, 1H, Ar-
H), 7.06 – 6.91 (m, 3H, Ar-H), 6.86 (dd, J = 8.1, 2.5 Hz, 1H, Ar-H), 6.32 (s, 2H, Ar-H), 4.97 (s,
2H, Ar-H), 3.82 (s, 3H, -OCH3), 4.97 (s, 2H, [-NH-CH2-CH3]2), 3.20 – 3.01 (m, 4H, [-NH-CH2-
CH3]2), 1.86 (s, 6H, AR-CH3), 1.19 (t, J = 7.1 Hz, 6H, [-NH-CH2-CH3]2). Fig. ES14:13C NMR
(101 MHz, CdCl3) δ 152.44, 147.76, 133.96, 133.38, 129.41, 129.11, 129.06, 128.51, 123.56,
119.39, 118.26, 115.37, 96.35, 50.68, 38.40, 30.92, 29.76, 16.72, 14.69; Fig. ES15: IR (KBr, cm-
1) : 3400.03 (aliphatic -NH-CH2-C-H3), 3246.00, 3022.15 (aromatic alkene stretching -C=C-H),
2970.22 (aliphatic -NH-C-H2-CH3), 2870.26 (aliphatic -NH-CH2-C-H3), 1680.32 (Secondary
amide -NH-CO-Ar), 1272.95 (aromatic amine C-N), 967.31 (monosubstituted);; Fig. ES16: LC-
MS: 563.4 m/z.
N-(3', 6’-bis (ethyl amino)-2', 7’-dimethyl-3-oxospiro [isoindoline-1, 9’-xanthene]-2-yl)-4-
methoxybenzamide (5e)
Pink solid; Melting point: 170°C; Yield 87 % , Fig. ES17:1H NMR (400 MHz, DMSO) δ 10.01
(s, 1H, -NH), 8.00 – 7.73 (m, 1H, Ar-H ), 7.62 – 7.45 (m, 4H, Ar-H), 7.29 – 7.11 (m, 1H, Ar-H),
7.02 (dd, J = 26.0, 7.4 Hz, 1H, Ar-H), 6.92 -6.91 (s, 1H, Ar-H), 6.30 (s, 2H, Ar-H), 6.21 (s, 2H,
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Ar-H), 4.97 (s, 2H, [-NH-CH2-CH3]2), 3.76 (s, 3H, -OCH3), 3.14 – 3.07 (m, 4H, [-NH-CH2-
CH3]2), 1.86 (s, 6H, Ar-CH3 ), 1.19 (t, J = 7.1 Hz, 6H, [-NH-CH2-CH3]2). Fig. ES18: 13C NMR
(101 MHz, DMSO) δ 165.16, 164.16, 162.64, 152.26, 148.27, 130.26, 124.86, 124.58, 123.18,
117.55, 113.91, 104.58, 95.53, 66.16, 55.99, 37.67, 17.91, 14.53. Fig.ES19: IR (KBr, cm-1)
:
3402.92 (-NH-COAr), 3241.52 (Ar-NH-CH2-CH3), 3022.11 (aromatic alkene stretching -C=C-
H), 2970.10 (aliphatic -NH-C-H2-CH3), 2870.05 (aliphatic -NH-CH2-C-H3), 1682.71
(Secondary amide -NH-CO-Ar), 1273.49 (aromatic amine C-N), 967.30; Fig. ES20: LC-MS:
563.6 m/z.
N-(3', 6’-bis (ethyl amino)-2', 7’-dimethyl-3-oxospiro [isoindoline-1, 9’-xanthene]-2-yl)-2-
fluorobenzamide (5f)
Pink solid; Melting point: >260°C; Yield 80 % Fig. ES21: 1H NMR (400 MHz, DMSO) δ 10.01
(s, 1H, -NH), 7.86 (dd, J = 6.5, 1.5 Hz, 1H, Ar-H), 7.62 – 7.55 (m, 2H, Ar-H), 7.52 – 7.41 (m,
1H, Ar-H), 7.37 – 7.13 (m, 3H, Ar-H), 7.04 (d, J = 6.7 Hz, 1H, Ar-H), 6.27 (s, 2H, Ar-H), 6.23
(s, 2H, Ar-H), 4.99 (s, 2H, [-NH-CH2-CH3]2 ), 3.18 – 3.04 (m, 4H, [-NH-CH2-CH3]2), 1.84 (s,
6H, Ar-CH3), 1.19 (t, J = 7.1 Hz, 6H, [-NH-CH2-CH3]2); Fig. ES22:13C NMR (101 MHz,
DMSO) δ 163.89, 162.99, 161.53, 155.90, 152.19, 152.01, 148.03, 129.46, 128.95, 124.49,
123.10, 117.76, 104.52, 95.82, 66.16, 55.38, 37.94, 17.62, 14.68. Fig. ES23:IR (KBr, cm-1)
:
3403.29 (-NH-COAr), 3250.36 (Ar-NH-CH2-CH3), 3022.16 (aromatic alkene stretching -C=C-
H), 2969.98 (aliphatic -NH-C-H2-CH3), 2868.97 (aliphatic -NH-CH2-C-H3), 1682.66
(Secondary amide -NH-CO-Ar), 1273.52 (aromatic amine C-N), 967.34; Fig. ES24: LC-MS:
551.6 m/z.
N-(3', 6’-bis (ethyl amino)-2', 7’-dimethyl-3-oxospiro [isoindoline-1, 9’-xanthene]-2-yl)-4-
fluorobenzamide (5g)
1
Pink solid; Melting point : >260°C; Yield 85 %, Fig. ES25: H NMR (400 MHz, DMSO) δ
10.01 (s, 1H, -NH), 7.86 (dd, J = 6.4, 1.5 Hz, 1H, Ar-H), 7.58 (m, 2H, Ar-H), 7.47 (m, 1H, Ar-
H), 7.31 – 7.13 (m, 3H, Ar-H), 7.04 (dd, J = 6.5, 1.3 Hz, 1H, Ar-H), 6.27 (s, 2H, Ar-H), 6.23 (s,
2H, Ar-H), 5.00 (s, 2H, [-NH-CH2-CH3]2), 3.15 – 3.03 (m, 4H, [-NH-CH2-CH3]2), 1.84 (s, 6H,
Ar-CH3 ), 1.19 (t, J = 7.1 Hz, 6H, [-NH-CH2-CH3]2). Fig. ES26: 13C NMR (101 MHz, CdCl3) δ
152.28, 151.75, 147.74, 133.81, 132.16, 128.87, 128.46, 123.55, 117.88, 116.08, 115.84, 104.54,
96.44, 50.67, 38.41, 30.91, 16.72, 14.68; Fig. ES27:IR (KBr, cm-1) : 3401.67 (-NH-COAr),
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3240.23 (Ar-NH-CH2-CH3), 3022.18 (aromatic alkene stretching -C=C-H), 2970.22 (aliphatic -
NH-C-H2-CH3), 2870.72 (aliphatic -NH-CH2-C-H3), 1683.20 (Secondary amide -NH-CO-Ar),
1274.04 (aromatic amine C-N), 967.31; Fig. ES28: LC-MS: 551.6 m/z.
2.2 Biological evaluation
2.2.1 In-Vitro urease Inhibition Activity
For the synthesized molecules, the in-vitro studies were performed by evaluating them against
the jack bean urease enzyme. The enzyme (Jack bean urease) activity was determined by
evaluating the absorbance and the amount of ammonia generated using the already reported
indophenol technique [51-54]. The reaction mixture contained enzyme (20 µL) (Jack bean
urease, 5 U/mL) and 20 μL respective compounds (5a-5g) in 50 μL potassium phosphate buffer
(100 mM urea, 10 mM K2HPO4, 1 mM EDTA and 10 mM LiCl, pH 8.2), were incubated at 37
ºC in 96-well plate for 30 minutes. Briefly, each well was supplemented with 50 μL each of
phenol reagents (1%, w/v phenol and 0.005%, w/v sodium nitroprusside) and 50 μL of alkali
reagent (0.5%, w/v NaOH and 0.1% Sodium hypochlorite (NaOCl). Absorbance was measured
after 10 minutes by using a microplate reader (OPTI Max, Tunable USA) at 625 nm. All the
measurements were conducted in triplicate. The urease inhibition activities were calculated by
using the equation 2.
Urease Inhibition Activity (%) = [ODcontrol- ODsample / ODcontrol] x 100
(2)
Where, ODcontrol and ODsample indicate the optical densities in the absence and presence of the
sample, respectively. Thiourea has been used as positive control for the activity of urease.
2.2.2 Kinetic analysis:
The mode of inhibition was determined by kinetic analysis study. Based on the most powerful
IC50 values, compound 5a was chosen. Kinetics have been achieved by varying the urea
concentration in the vicinity of distinct concentrations of compound 5a (0.00, 0.06, 0.12 and 0.24
µM). In urease kinetics study, the concentration of the urea was ranged from 100, 50, 25, 12.5,
6.25 and 3.12 mM, respectively, but the rest of the procedure was same for all kinetic study as
described in urease inhibition assay protocol. Maximum initial velocities were determined from
the original absorbance part, up to 10 minutes later the enzyme was added at the interval of each
minute. The type of the inhibition on the enzyme was scrutinized by plotting a Lineweaver-Burk
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graph of inverse of velocities (1/V) against the inverse of substrate concentration 1/[S] mM-1. In
addition, the EI dissociation constant (Ki) was calculated by plotting a secondary graph of 1/V
against inhibitor concentration. Urease activity was measured using the indophenol method as
earlier reported by evaluating ammonia quantification. Urease activity was determined by
measuring ammonia production using the indophenol method as reported previously [55-57].
The results (change in absorbance per min) were processed by using SoftMaxPro.
2.2.3 Free radical scavenging assay:
Radical scavenging activity was not easily affected by altering a previously reported technique
[58-59] by 2, 2-diphenyl-1 picrylhydrazyl (DPPH) assay. The test solution consisted of 100 µL
of DPPH (150 µM), 20 µL of increasing concentration of test compounds and the volume was
adapted to 200 µL with methanol in each well. Afterward, the assay reaction mixture was
incubated at ambient temperature for 30 minutes. Furthermore, Ascorbic acid (Vitamin C) was
used as a reference inhibitor. Measurements of the assay were achieved by using a microplate
reader (OPTI Max, Tunable) at 517 nm. In this study, we compared the reaction rates, and
calculated the percent inhibition induced by the presence of tested inhibitors. All experiments
were repeated thrice.
2.3. In-silico methodology
2.3.1. Retrieval of Jack bean urease in protein preparation wizard
The structure of the jack bean urease was gained from Protein Data Bank (PDB) (www.rcsb.org)
having PDBID 4H9M in protein preparation wizard. The preferred urease protein structure has
been pre-processed, improved, and minimized using Maestro interface default parameters [60].
2.3.2. Grid generation and molecular docking
The improved Jack bean urease structure was organized using the "Protein Preparation Wizard"
workflow in Schrödinger Suite before molecular docking. Bond orders were assigned, and
hydrogen atoms was supplemented with hydrogen atoms. The structure was then minimised with
the OPLS_2005 force field to achieve a converged root mean square deviation (RMSD) of 0.30
Å. The active site of the enzyme is defined from the co-crystallized ligands from Protein Data
Bank and literature data [61, 62]. Furthermore, the docking experiment was performed against all
synthesized ligands (5a-5g) sketched in 2D sketcher and target protein by using the Glide
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docking protocol [63]. By using Glide experiment, the anticipated binding energies (docking
ratings) and conformational positions of the ligands within the active protein section were also
performed. Throughout the docking simulations, both partial flexibility and full flexibility
around the active site residues are performed by Glide/SP/XP and induced fit docking (IFD)
approaches [64].
3. Results and discussion
3.1. Chemistry
3.1.1 Synthesis
The synthetic path for rhodamine derivatives (5a-5g) is presented in Scheme 2. The various
aromatic ester derivatives (2a-2g) were prepared by reacting the acid compounds (1a-1g) in
ethanol, refluxed under acidic condition. Further, the un/substituted ester derivatives were
reacted at reflux temperature with hydrazine hydrate in ethanol. This replacement reaction was
performed to insert a hydrazide functionality (3a-3g) into the various acid derivatives, these
un/substituted hydrazide derivatives were further used for the synthesis of the target compounds
(5a-5g). Finally, the equivalent rhodamine 6G derivatives were synthesized by the reaction of the
different un/substituted aromatic benzhydrazide (3a-3g) with rhodamine 6G in presence of
anhydrous potassium tert-butoxide under nitrogen atmosphere at ambient temperature. However,
the progress of the reaction was checked by TLC technique. The synthesized target compounds
were purified by using column chromatography method, in which the crude solid of target
compounds (5a-5g), initially adsorbed on silica (60-120 mesh), and the column was packed
using 100-200 mesh size silica, in which dichloromethane (MDC) and methanol used as an
eluting solvent. Afterwards, a single spot on TLC was segregated individually for all the target
compounds (5a-5g). Finally, the structural clarification of the target compounds (5a-5g) were
done by using FT-IR, LC-MS, 1H NMR and 13C NMR analytical methods.
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3.1.2 Photophysical Study
The absorption and emission spectra of the target compounds were assessed in variety of
solvents including non-polar (chloroform), polar aprotic (ACN and DMF) and polar protic
(methanol) solvents at 100 µM (1 × 10-4 M). The absorption and emission graphs were used to
evaluate the spectroscopic parameters for instance absorption and emission wavelength, Stokes
shift, molar extinction coefficient, fluorescence quantum yield, and fluorescence lifetime values
of the respective compounds in a variety of solvents. Figs. 1A, B, C, and D relates to absorption
spectra of the target compounds (5a-5g) in four distinct solvents such as chloroform, ACN,
DMF, and methanol. Fig. 1A demonstrates maximum absorption band within the spectral region
500-530 nm for the compounds 5a-5g in chloroform. Similarly, Figs. 1B, 1C and D exhibit the
absorption maxima ranges from 520-535 nm, 530-540 nm, and 520-530 nm in ACN, DMF and
methanol solvent, respectively. The bands showed in the absorption spectra were attributed to the
π→π* transitions of the conjugated skeleton on the xanthene ring. Therefore, from the absorption
spectrum can be stated that, as the polarity of the solvent enhances from non-polar to polar
aprotic solvent, the absorption maxima slightly changes towards red shift. A small spectral shift
was observed in chloroform, ACN and DMF solvents but in case of polar protic solvent
(methanol), it switched towards blue zone. Besides, if the electron donating substituents (like -F,
-OH or -OCH3) are present at ortho position or in the absence of substituents with respect to
carbonyl group, under such scenario the compounds (5a, 5b and 5f) exhibits an increased peak
broadening along with reduced absorbance values. The variation of the results might be arisen
because of the ortho substituted (5b & 5f) / unsubstituted (5a) xanthene compounds, can
influence to the conjugated π-electrons system causes to restriction the transition from bonding
molecular orbital to the antibonding molecular orbital. This sort of behaviour has been detected
in chloroform, ACN and DMF solvents but not in case of methanol. In details, rhodamine and its
derivative consist of a highly strained five membered spirocyclic ring. In the presence of the
polar protic solvent such as methanol, xanthene associated amino group gets protonated causing
delocalization of the π electrons and results into the opening of the spirocyclic ring [65]. As a
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result, the absorbance intensity of the synthesized target compounds rises in polar protic solvent
but the same is not valid for non-polar and polar-aprotic solvent.
Fig. 1. UV-Vis absorption spectra of the target compounds (5a-5g) at 1 × 10-4 mole/L in different
solvents such as A) chloroform B) ACN C) DMF and D) methanol.
Fluorescence emission features assets of the target compounds 5a-5g were investigated in
chloroform, ACN, DMF and methanol solvents at ambient temperature as an expressive means
of covering examination of electronic relations of the substituted/unsubstituted rhodamine core
in the excited state. The fluorescence emission spectra of the target compounds 5a-5g in
chloroform, ACN, DMF and methanol solvents (As displayed in Figs. 2A, B, C & D)
demonstrates a major specific broad peak varying from 510-650 nm, since the transition takes
place from higher energy level (excited state) to lower energy level (ground state) transitions i.e.
n*→π and π*→π. The rhodamine derivatives 5c, 5d, 5e and 5g exhibit strong fluorescence
emission intensity in all solvents nevertheless compounds 5a, 5b and 5f shows less intensity
expect in methanol. Also, the relative quantum yield and a fluorescence lifetime values for
compounds 5a, 5b and 5f manifest electronic impact of the unsubstituted phenyl ring and ortho-
substituted phenyl group (-OH and -F) present in the respective compounds i.e. 5a, 5b & 5c
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respectively (Table. 1 & Fig. 3). This variation might be possibly caused because of the
moderately and strongly activating groups (-F, -OCH3, and -OH) present in respective
compounds. In practice, when these substituents are present at ortho/ meta/ para position or
without substituents with respect to the amide functionality, experiences of different electronic
effects on the phenyl moiety triggers to deliver variation in the photophysical properties of the
respective compounds in various solvents.
Fluorescence spectra of the synthesized derivatives guide us to evaluate the distinct
photophysical factors such as, stokes shift, fluorescence quantum yield for each compound in a
variety of solvents. In fact, the excellent fluorescent properties or the changeover in
spectroscopic properties owing to change in the various polarity of the solvents are the key
hypothesis of this experiment. The calculated photophysical parameters, for all the synthesized
derivatives in variety of solvents are organized in Tables. 1, 2, 3 & 4 separately. The estimated
values of the quantum yield suggest that the quantum yield value decreases as the polarity of the
solvent increases (except for methanol) owing to the charge transfer phenomenon. In fact, the
fluorescence quantum yield values for compound 5a, 5b, 5c, & 5d increases in polar protic
solvent (methanol) because of the negative solvatokinetic effect. Whereas compound 5e, 5f, and
5g had lower quantum yield values in a highly polar solvent (methanol) equated with the other
solvents acquiring less polarity (CHCl3 and ACN) due to the positive solvatokinetic effect [16].
In addition, the present -NH unit transfer an electron towards the xanthene platform leads to an
intramolecular charge transfer (ICT) effect to take place [16, 66]. Because of the ICT effect, the
impact of the solvent polarity upon the emission band was more remarkable compared with the
absorption spectra. Furthermore, the dipole moment concurrently increases to solvent molecule
in the excited state via relaxation process. The spectral shift is usually dependent upon the
changeover in dipole moment and the disparity in dielectric constants of the respective solvents
which triggers alteration of fluorescence lifetime values. The lifetime value decrease implies, the
decrease in the radiative pathways from the excited state. Hence, the fluorescence lifetime values
are also dependent on the dielectric constant, dipole moment and the polarity of the solvents
which are used for spectroscopic parameter studies [66-67]. The transformations detected for all
the synthesized compounds in various solvents are slight but nevertheless performs a significant
function in an excited state phenomenon of the compounds and changes in their spectral assets.
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Overall, emission spectra of all the synthesized derivatives are affected by changing the polarity
of the solvent. After comparing all the target compounds 5a-5g and their fluorescence emission
response in respect to nonpolar (chloroform), polar aprotic (ACN & DMF), and polar protic
(methanol) solvent reveals that, the compound 5c shows maximum fluorescence emission
response along with good photophysical properties equated with the other derivatives (5a, 5b &
5d-5g).
Fig. 2. Fluorescence emission spectra of compounds (5a-5g) at 1 × 10-4 mole/L in different
solvents such as A) chloroform B) ACN C) DMF and D) methanol.
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Table 1: Photophysical properties presented by compounds 5a-5g in chloroform, ACN, DMF
and methanol solvent.
Compound
λabs
(nm)
λem
(nm)
Stokes Shift
(nm)
Ɛ
ɸf
τ
(ns)
(M-1 cm-1)
Chloroform (CHCl3)
5a
5b
5c
5d
5e
5f
507
523
532
529
530
524
530
535
536
540
538
543
538
536
28
13
8
110
260
1280
660
790
210
640
0.094
0.035
0.083
0.080
0.099
0.086
0.041
3.17
3.07
3.55
3.26
3.27
3.44
3.42
9
13
14
6
5g
Acetonitrile (ACN)
5a
5b
5c
5d
5e
5f
531
545
550
546
547
549
548
545
14
28
24
22
24
22
20
90
0.062
0.022
0.132
0.095
0.127
0.079
0.072
2.68
2.35
3.43
3.27
3.38
3.16
3.32
522
522
525
525
526
525
320
940
670
800
190
630
5g
Dimethylformamide (DMF)
5a
5b
5c
5d
5e
5f
538
537
531
535
535
537
535
555
557
557
560
560
557
560
17
20
26
25
25
20
25
100
210
1000
430
690
150
550
0.081
0.027
0.090
0.074
0.087
0.066
0.036
3.19
2.10
3.43
3.06
3.26
3.16
3.55
5g
Methanol (MeOH)
5a
5b
5c
5d
5e
525
548
552
545
550
551
23
25
21
23
21
460
820
1600
1170
1270
0.219
0.040
0.091
0.084
0.078
3.19
2.05
3.13
2.59
2.65
527
524
527
530
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5f
5g
530
529
550
550
20
21
850
990
0.054
0.039
3.05
3.33
λabs= Maximum absorption wavelength; λem =fluorescence emission wavelength; Ɛ= molar
extinction coefficient; ɸf= fluorescence quantum yield and τ= fluorescence lifetime.
3.2 In Vitro urease activity and structural activity relationship (SAR):
A series of the rhodamine-based derivatives (5a-5g) were synthesized systematically as
presented in Scheme 2. The objective behind the new compounds was to synthesize the
therapeutically active compounds which can be used in future for the development of a new
drug. The synthesized target compounds (5a-5g) were employed for their urease inhibitory
activity, as a result fluctuating inhibitory activity was detected with modifying the various
substituents and its position on the benzamide functionality within the target compounds. The
inhibitory activity ranged from 0.1108±0.0038 to 0.8644±0.0828 µM, all the derivatives display
respectable outcome for the urease inhibitory activity but among them compound 5c (IC50 value
0.1108±0.0038 µM) shows most potent activity compared with standard inhibitor thiourea (IC50
value 4.7201±0.0546 µM). The results are given in Table 2.
The activity which is observed by the in-vitro study for the compounds (5a-5g), is all because of
the involvement of the whole molecule. Therefore, to scrutinize the importance of the attached
different substituent (Such as -OH, -OCH3 or –F) on the activity limited structural activity
relationship (SAR) study was employed. Figs. 3-6 display the structure of the different target
compounds.
As shown in Fig. 3, among the two hydroxy (-OH) regio-isomers i.e. 5b and 5c, the compound
containing the hydroxy substituent at meta position i.e. 5c (IC50 = 0.1108±0.0038 µM) displays a
decent inhibition than 5b (IC50 = 0.1432±0.0612 µM), in which ortho position occupied by the
hydroxy group. Thus, it can be deduced from the in-vitro study that, the inhibition rate decreases
when the electron-donating substituents are present at the ortho position. It means the interaction
of the meta substituted ligand (5c) was more efficient than ortho-substituted ligand (5b).
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Table 2: In-vitro IC50 value for urease inhibition of compounds 5a-5g.
(5a-5g)
Sr. No
Compounds
5a
Urease activity
IC50 ± SEM (µM)
R1
-H
R2
-H
R3
-H
1
0.1108±0.0038
2
3
4
5
6
7
8
-OH
-H
-H
-OH
-OCH3
-H
-H
-H
0.1432±0.0612
0.1136±0.0295
0.1456±0.0194
0.8644±0.0828
0.1525±0.0201
0.1489±0.0343
4.7201±0.0546
5b
5c
5d
5e
5f
-H
-H
-H
-OCH3
-H
-F
-H
-H
-H
-F
5g
Thiourea
SEM= Standard error of the mean; values are expressed in mean ± SEM.
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Fig. 3. Structural activity relationship of 5a, 5b, and 5c.
Besides, as displayed in Fig. 4, the hydroxy group (-OH) was superseded by an electron-
donating group such as methoxy (-OCH3) at meta and para-position, respectively. The outcomes
exhibit that, the meta substituted derivative 5d (IC50 = 0.1456±0.0194 µM) demonstrate higher
inhibition than para-substituted derivative 5e (IC50 = 0.8644±0.0828 µM). Yet again, it confirmed
that when an electron-donating (-OCH3) substituent was present at meta-position (5d) the
interaction with the enzyme was more powerful than the para-substituted derivative (5e), which
cause a decline in the urease inhibitory activity at para-position associated with meta substituted
derivatives (Fig. 4).
Fig. 4. Structural activity relationship of 5d and 5e.
Likewise, the ortho and para position of the benzamide functionality in the target compounds 5f
(IC50 = 0.1525±0.0201 µM) and 5g (IC50 = 0.1489±0.0343 µM) has been replaced by the fluorine
(electron donating) group, as presented in Fig. 5. Herein, fluorine donates the lone pair of
electrons through resonance which activates the phenyl ring of the benzamide functionality and
leads to increases the electron density at ortho and para positions. Therefore, both the target
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compounds (5f and 5g) appears virtually the same interaction with the enzyme. But among them,
the para substituted compound proves decent inhibitory activity as compared to the para-
substituted compound. By equating both compounds, the para-substituted compound displays
fairly urease inhibitory activity than ortho-substituted due to the fluorine substituents present at
ortho position, which is more closer to the carbonyl functionality and have further prospects to
generate a steric interaction between them than para substituted fluorine compound 5g.
Fig. 5. Structural activity relationship of 5f and 5g.
Furthermore, the authentication of the impact of the electron-donating substituents on the
benzamide functionality present in the target compounds have been demonstrated in the SAR
study. Wherein, we have equated numerous substituted target compounds (5b, 5c, 5d, 5e, 5f and
5g) with the unsubstituted derivative 5a (IC50= 0.1108±0.0038 µM). At this time, it has been
remarked that the unsubstituted and meta-substituted compound i.e. 5a & 5c, reveal an excellent
role as compared not only with the ortho and para substituted target compounds but also with the
standard drug thiourea (IC50 = 4.7201±0.0546 µM) as indicated in Fig. 6 and Table. 2.
In summary, while comparing all the derivatives 5a-5g, along with their activity and
attachment of the different substituents. The findings have shown that, when an electron-
donating substituent like –OH, -OCH3 or –F are present at ortho or para-position, the interaction
of the enzyme was not efficient compared with the meta substituted derivatives. It is well
understood that the electron-donating are ortho/para directing in aromatic benzene ring.
Therefore, it suggests that the substituents that appear at ortho/para position could be more
sensible to enhance the conjugation along with the benzamide carbonyl moiety, which causes to
decrease the interaction with an enzyme, as a result, decrease the urease inhibitory activity.
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Whereas, in the case of meta-substitution, the conjugation phenomenon along with carbonyl
functionality is absent and causes to increases the rate of the inhibition compared with the ortho
and para-substituted derivatives. Likewise, there will be no more potential in the benzamide
functional group to enhance the electron density of the phenyl ring in the absence of the
substituents. Hence, this may lead to increase in the effectual interaction between the energetic
site of the enzyme and the unsubstituted ligand 5a causes it to increase the urease inhibitory
activity.
Fig. 6. Structure activity relationship of 5a-5g.
Therefore, it was determined from structural activity relationships that, if the electron-donating
groups were present at ortho or para-position, the interaction with enzyme was not as effective as
with the meta-substituted derivatives, but it was more operative in absence of the electron-
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donating groups. The xanthene derivatives 5a-5g can be arranged as per trend observed in in-
vitro study: 5a>5c>5b>5d>5g>5f>5e.
3.3 In Vitro analysis:
3.3.1 Kinetic analysis:
To estimate the sort of inhibition and inhibition constant on jack bean urease, compound 5a was
chosen for further study as per our obtained result. In terms of EI (Enzyme inhibitor) and ESI
(Enzyme Substrate Inhibitor) constants, the potential of the synthesized compounds to inhibit
free enzyme and enzyme-substrate complexes was determined. The Lineweaver-Burk graph of
1/V against 1/ [S] plotted (where, ‘V’ is the velocity of reaction and ‘S’ is the reaction of
substrate) in the existence of discrete compounds in enzyme kinetic experiments was employed.
This shows a series of straight lines (Fig. 7A). From the outcome it was observed that within the
second quadrant the compound 5a was intersected. Besides, the analysis displays the value of
Vmax decreases with increase in the inhibitor doses but on the other hand, the Km remains
constant. This behaviour indicates that compound 5a inhibits the urease non-competitively to
form the enzyme inhibitor complex. The Secondary plot of slope against the concentration of
inhibitors displayed enzyme inhibitor dissociation constant (Ki) (Fig.7B). The kinetic results are
presented in Table 3.
Table 3. Kinetic parameters of the jack bean urease for urea activity in the presence of different
Concentrations of 5a.
Concentration
(µM)
Vmax
Km
Inhibition Type
Ki
(ΔA /Min)
0.001049091
0.000408182
0.000222481
(mM)
(µM)
0.00
2
2
2
Non-Competitive
0.04
0.06
0.12
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0.24
0.000139091
2
Where, Vmax = velocity of the reaction, Km = Michaelis-Menten constant, Ki = EI dissociation
constant.
Fig. 7. Lineweaver–Burk plots for inhibition of Urease in the presence of compound 5a. (A)
Concentrations of 5a were 0.00, 0.06, 0.12 and 0.24 µM. (B) The insets represent the plot of the
slope or the vertical versus inhibitor 5a concentrations to determine inhibition constants. The
lines were drawn using linear least squares fit.
3.3.2 Free radical scavenging activity:
All the synthesized compounds (5a-5g) were evaluated for DPPH free radical scavenging ability.
In the comparison of the standard, the compound 5b showed an excellent activity other than all
the synthesized compounds which did not show significant activity, even at high concentration
(100 µg/ mL). Vitamin C was used as a reference drug. The results are presented in Fig.8.
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Fig. 8. Free radical % scavenging activity of synthetic compounds values were represented as
mean ± SEM (Standard error of the mean). All compounds concentrations were 100 µg/mL.
3.4 In Silico analysis
3.4.1. Structural and physicochemical evaluation of Jack bean urease
Jack bean urease is a class of metal-containing hydrolase protein consisting of four domains with
distinct residue numbers. The VADAR assessment exhibited that the structure of the protein
contained 27% helices, 31% β sheets and 41% coils in the target protein. In addition,
Ramachandran plots stated that 97.5 % of the residues were present in preferred areas, showing
the accuracy of the corners of phi (φ), and psi (ψ) angles among the jack bean urease coordinates.
The overall protein structure is mentioned in Fig 9.
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Fig. 9. The overall protein structure of jack bean urease.
3.4.2 Binding energy evaluation of the synthesized compounds
To forecast the best-fitted conformational site of the synthesized compounds (5a-5g), the
compounds were docked against jack bean urease. The generated docked complexes were
examined based on glide docking energy values (kcal/mol) and bonding interaction
(hydrogen/hydrophobic) pattern. The binding energy value depicts the conformational position
within the active region of the target protein. The docking results show that 5c showed higher
energy value (-4.78 kcal/mol) compared to other ligands. The binding difference was not much
fluctuated among all the docking energy score as the basic ligand skeleton was similar in all
synthetic compounds. The other compounds possessed different energy values such as -3.41, -
4.08, -3.35, -3.48, -3.08 and -4.03 kcal/mol (Fig. 10) [26, 56].
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Fig. 10. Docking energy values of ligands.
3.4.3 Ligand-Binding analysis of urease docked complexes
Molecular docking approach is one of the most significant approach to understand and visualize
the binding interaction outline of designed ligands against target protein structure [68]. Based on
in-vitro results (IC50) 5a was selected for close binding interaction pattern. In detailed binding
analysis, single π-π interaction was observed in 5a docking complex. The benzene ring structure
of 5a forms π-π interaction with Arg639. The other residues which are in close contact with the
ligand structure are Ile411, Leu415, Glu418, Arg439, Gln635, Met635, Gly638, Val640, Pro573
and Met588. These amino acids are core binding pocket residues and prior docking research data
ensured the importance of these residues in bonding with other urease inhibitors, which
strengthen our docking results [52, 69]. The comparative SAR analysis showed the significance
of 5a might be considered as potent inhibitor by targeting jack bean urease. The three-
dimensional (3D) docking graphical depiction for 5a is mentioned in Fig 11. and all other
ligands 2D docking images are justified in Fig 12.
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Fig. 11. Binding pocket of urease protein.
Fig. 12. Docking complexes of ligands 5a-5g.
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