Biology-oriented drug synthesis (BIODS), in vitro urease inhibitory activity, and in silico studies on ibuprofen derivatives

Article abstract of DOI:10.1007/s11030-019-10032-x

Abstract: Novel ibuprofen derivatives 1–19 including ibuprofen hydrazide 1, and substituted thiourea derivatives 2–19 were synthesized and characterized by EI-MS, FAB-MS, HREI-MS, HRFAB-MS, ¹H-, and ¹³C-NMR spectroscopic techniques.

Full text of DOI:10.1007/s11030-019-10032-x

Molecular Diversity
https://doi.org/10.1007/s11030-019-10032-x
ORIGINAL ARTICLE
Biology‑oriented drug synthesis (BIODS), in vitro urease inhibitory
activity, and in silico studies on ibuprofen derivatives
Faiza Seraj1 · Kanwal1,6 · Khalid Mohammed Khan1,2 · Ajmal Khan3 · Muhammad Ali3 · Ruqaiya Khalil4 ·
Zaheer Ul‑Haq4 · Shehryar Hameed1 · Muhammad Taha2 · Uzma Salar4 · Shahnaz Perveen5
Received: 15 October 2019 / Accepted: 30 December 2019
© Springer Nature Switzerland AG 2020
Abstract
Novel ibuprofen derivatives 1–19 including ibuprofen hydrazide 1, and substituted thiourea derivatives 2–19 were synthe-
1
sized and characterized by EI-MS, FAB-MS, HREI-MS, HRFAB-MS, H-, and 13C-NMR spectroscopic techniques. The
synthetic molecules 1–19 were examined for their in vitro urease inhibition and were found to display a diversifed degree of
inhibitory potential in the range of IC50 =2.96–178 μM as compared to the standard thiourea (IC50 =21.32 0.22 μM). Out
of nineteen, thirteen derivatives 2–4, 6, 7, 9, 11–15, 17, and 18 demonstrated remarkable inhibitory activity with IC50 values
of 2.96 1.11 to 16.1 1.07 μM, compound 5 exhibited moderate inhibition with IC50 value of 37.3 0.41 μM, whereas,
compounds 1, 8, and 10 demonstrated weak inhibition against urease enzyme. Almost all structural features are participating
in the activity; however, limited structure–activity relationship was discussed on the basis of diferent structural features,
i.e., diferent functional groups and their positions at aryl part. In addition, molecular docking study was performed in order
to understand the ligands binding interactions with the active site of urease enzyme.
Electronic supplementary material The online version of this
3
article (https://doi.org/10.1007/s11030-019-10032-x) contains
supplementary material, which is available to authorized users.
UoN Chair of Oman’s Medicinal Plants and Marine
Natural Products, University of Nizwa, P.O. Box 33,
616 Birkat al Mauz, Nizwa, Oman
* Khalid Mohammed Khan
4
5
6
Dr. Panjwani Center for Molecular Medicine and Drug
Research, International Center for Chemical and Biological
Sciences, University of Karachi, Karachi 75270, Pakistan
khalid.khan@iccs.edu; drkhalidhej@gmail.com
1
H. E. J. Research Institute of Chemistry, International Center
PCSIR Laboratories Complex, Karachi,
Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280,
Pakistan
for Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
2
Department of Clinical Pharmacy, Institute for Research
Institute of Marine Biotechnology, Universiti Malaysia
and Medical Consultations (IRMC), Imam Abdulrahman Bin
Faisal University, P.O. Box 31441, Dammam, Saudi Arabia
Terengannu, 21300 Kuala Terengannu, Terengannu, Malaysia
Vol.:(0123456789)
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Molecular Diversity
Graphic abstract
Scheme-1: Synthesis of ibuprofen derivatives
Keywords Ibuprofen · Biology-oriented drug synthesis (BIODS) · Urease inhibition · Thiourea derivatives · In silico
studies
is increased due to excess formation of ammonia leading
to the super-saturation of calcium phosphate resulting in
stone formation [16]. Urease enzyme promotes the growth
of Gram-negative bacterium, Helicobacter pylori (HP),
which is involved in many pathological conditions like pep-
tic ulcer, pyelonephritis, hepatic coma, and formation of
urinary stones [17, 18]. So, it is essential to introduce new,
selective, and potent inhibitors for urease having signifcant
stability, bioavailability, and less toxicity [19–21].
Introduction
Ibuprofen (IBP) is the frst member of propionic acid deriva-
tives which is most signifcant nonsteroidal anti-infamma-
tory drugs (NSAIDs) [1, 2]. It is extensively used for curing
osteoarthritis, rheumatoid arthritis, and musculoskeletal pain
[3–5]. It was introduced for the frst time in 1969 in UK after
the approval of FDA in 1947 and then it became available
worldwide [6, 7]. In addition to analgesic and anti-infamma-
tory activities, antimalarial, antimicrobial, anticonvulsant,
and antitubercular activities are also reported for S-ibuprofen
derivatives [8–10]. Only the (S) enantiomer of ibuprofen
potentially inhibits cyclooxygenase enzyme [11, 12].
We used biology-oriented drug synthesis (BIODS)
approach for the synthesis of library of ibuprofen deriva-
tives in order to evaluate their diversifed and unknown bio-
logical activities. Previously, using BIODS approach, our
research group has reported many lead compounds, such as
metronidazole derivatives for their antidiabetic potential by
inhibiting α-amylase and β-glucuronidase enzymes, piroxi-
cam derived compounds for their anti-nociceptive activity,
furbiprofen-based compounds for their α-amylase inhibitory
activity, and S-naproxen-based analogs as urease inhibitors
(Fig. 1) [22–26].
Urease is nickel-based metalloenzyme which belongs
to the family of phosphotriestreases and amidohydrolases,
it is responsible for the production of carbon dioxide and
ammonia from the hydrolysis of urea. Urease enzyme of
jack bean was crystallized for the frst time from plant source
Canavalia ensiformis in 1926 and mainly found in algae,
fungi, bacteria, and plants [13–15]. Each human being is
responsible to produce about 10 kg of urea per year due to
diferent metabolic processes. Thus, the alkalinity of urine
Since our research group has already reported the diverse
biological activities of compounds derived from NSAIDs,
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Molecular Diversity
Fig. 1 Lead candidates derived from commercially available drugs
therefore, in continuation of our previous work, we had
selected ibuprofen for chemical modifcations in order to
explore biological potential. It is worth noting that our
research group has reported various lead compounds as
potential inhibitors of urease enzyme and recently S-nap-
roxen derivatives were reported as urease inhibitors. These
results encourage us to explore the urease inhibitory activity
of newly synthesized derivative of ibuprofen. To the best of
our knowledge, the synthetic molecules (1–19) are reported
for the frst time as urease inhibitors.
was synthesized via one-pot reaction of ibuprofen, hydra-
zine hydrate, and 1,1-carbonyl diimidazole (CDI) in THF.
Further reaction of hydrazide with diferent substituted
phenyl isothiocyanates in dichloromethane with continu-
ous stirring at room temperature aforded compounds 2–19
(Scheme 1). The reaction progress was examined by TLC
(hexane:ethyl acetate). Upon completion of reaction, the
solvent was evaporated and residue was washed with hot
hexane and excess of distilled water to aford pure prod-
ucts. Solid product was crystallized from ethanol. All
synthetic compounds 1–19 were characterized by EI-MS,
FAB-MS, HREI-MS, HRFAB-MS, 1H- and 13C-NMR. Of
nineteen molecules, fve compounds 1, 2, 5, 13, and 18 are
structurally known [4, 27, 28].
Results and discussion
Chemistry
Novel derivatives (1–19) of commercially available drug
ibuprofen were synthesized. First, ibuprofen hydrazide
Scheme 1 Synthesis of ibupro-
fen derivatives
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Molecular Diversity
Compound 8 (IC50 =178 0.71 μM) having bromo sub-
stituent at meta-position showed weak inhibitory potential.
Interestingly, compound 9 (IC50 = 10.3 1.05 μM) with
fouro group at ortho-position showed excellent inhibi-
tory activity, however, addition of another fuoro group
at para-position leads to decreased activity in compound
10 (IC50 = 77.7 0.77 μM) might be due to increased
inductive efect of two fouro groups or increased steric
hindrance which results in less interaction within active
site of enzyme. In general, it was observed that halogen
substituted at ortho-position of aryl ring exhibited good
inhibitory potential, addition of halogens or changing their
position resulted in loss of activity (Fig. 5).
In vitro urease inhibitory activity
Ibuprofen hydrazide 1 and synthetic compounds 2–19 were
screened them for their in vitro urease inhibition. The results
listed in Table 1 showed that all compounds were found to
be potent with IC50 values of 2.96 1.11 to 16.1 1.07 μM,
while, compound 5 displayed moderate inhibition with IC50
value of 37.3 0.41 μM in comparison with the standard
thiourea (IC50 = 21.32 0.22 μM). Compounds 16 and 19
were found to be inactive. The general structure of synthetic
molecules have been shown in Fig. 2.
Interestingly, nitro-substituted compounds
showed different pattern, among them compound 12
(IC50 = 6.98 1.15 μM) bearing nitro group at para-posi-
tion displayed an increased inhibition in comparison with
meta-substituted compound 11 (IC50 = 8.72 0.52 μM).
Both of these compounds were potentially active in com-
parison with the standard thiourea might be due to the
chelation of oxygen atoms with enzyme active site (Fig. 6).
Compounds with mono- and dimethyl substitutions
showed varying degree of inhibitory activity. Among
them, compound 13 (IC50 = 5.26 1.35 μM) with an
ortho-methyl substitution showed an increased inhibi-
tory potential in comparison with the standard thiourea
(IC50 = 21.32 0.22 μM) which indicated that methyl
group efciently interacted within the active site of enzyme
might be due to hyper-conjugation efect. Nevertheless,
dimethyl-substituted derivative 14 (IC50 =10.5 5.39 μM)
was found to be less active in comparison with compound
13 might be the inclusion of another methyl substituent
at ortho-position resulted in increased steric hindrance
thus leads to decreased activity. However, both of these
compounds were more active as compared to the standard
(Fig. 7).
Structure–activity relationship (SAR)
The parent molecule ibuprofen displayed no inhibition
against urease enzyme, whereas, hydrazide of ibuprofen 1
(IC50 = 202 1.23 μM) was weakly active as compared to
the standard thiourea (IC50 =21.32 0.22 μM) might be due
to the weak binding interactions of hydrazide functional-
ity. Surprisingly, compound 2 (IC50 =16.1 1.07 μM) with
unsubstituted benzene ring and the thiourea functionality
was found to be potentially active in comparison to the
standard might be due to incorporation of thiourea func-
tionality. Compounds 3–19 with substituted benzene ring
also exhibited good inhibition against urease. It showed that
substitution on benzene ring and incorporation of thiourea
moiety is playing signifcant role in the urease inhibitory
activity of synthetic compounds (Fig. 3).
Among chloro-substituted derivatives, compound 3
(IC50 = 5.36 1.27 μM) bearing ortho-chloro substituent
showed potential inhibitory activity as compared to the stand-
ard thiourea (IC50 =21.32 0.22 μM). The change in position
of chloro group to meta and para, respectively, in compounds 4
(IC50 =15.2 0.98 μM) and 5 (IC50 =37.3 0.41 μM) resulted
in decreased activity as compared to compound 3. It showed
that ortho-position is contributing actively might be due to
the better binding interactions with the active site of enzyme.
The activity was decreased threefold in case of meta-substitu-
tion, nonetheless, it was more active as compared to standard,
however, para-substituted compound was least active. The
dichloro-substituted derivative 6 (IC50 =6.16 1.68 μM) hav-
ing chlorine atoms para to each other was found to be good
inhibitor as compared to compound 4 might be due to the pres-
ence of chloro group at ortho-position, whereas, it was less
active in comparison with compound 3 due to meta-substitu-
tion. Compound 7 (IC50 =2.96 1.11 μM) showed sevenfold
more activity in comparison with the standard thiourea and
was most active compound of the series. Compound 7 having
chloro and a nitro groups meta to each other showed good
activity, might be the nitro group having two oxygen atoms
resulted in the enhanced activity of this compound due to bet-
ter binding interactions (Fig. 4).
Compounds 15–17 were positional isomers of each
other bearing trifuoromethyl group at diferent positions
of aryl ring, and all these derivatives were found to be
potentially active than the standard thiourea. Among them,
compound 15 (IC50 = 9.85 1.16 μM) with ortho-trifuo-
romethyl was found to possess excellent inhibitory activ-
ity. Nevertheless, compound 17 (IC50 = 8.53 0.64 μM)
bearing para trifuoromethyl group was more active as
compared to its ortho-substituted analog 15 while meta-
substituted isomer 16 was completely inactive. Com-
pound 18 (IC50 = 3.5 3.26 μM) bearing methoxy group
at ortho-position demonstrated potent inhibitory activity
and was the second most active derivative. The activity
of this group might be due to better binding interactions
of methoxy group with the active site of enzyme (Fig. 8).
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Molecular Diversity
Table 1 In vitro urease
Compound No.
1
R
IC50 SEMa (µM)
inhibition of compounds (1–19)
202 1.23
Ibuprofen Hydrazide Derivatives
R
16.1 1.07
5.36 1.27
2
3
15.2 0.98
37.3 0.41
4
5
6.16 1.68
6
2.96 1.11
178 0.71
10.3 1.05
77.7 0.77
7
8
9
10
8.72 0.52
6.98 1.15
5.26 1.35
11
12
13
10.5 5.39
9.85 1.16
14
15
NAb
16
17
18
19
8.53 0.64
3.5 3.26
NAb
Thioureac
21.32 0.22
ICa (mean standard error of mean); NAb (not active); Standardc (inhibitor for urease
50
activity)
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Molecular Diversity
Fig. 2 General structure of synthetic compounds
Fig. 3 SAR of compounds 1–2
Among the halogen-containing compounds, compound 3
(IC50 =5.36 1.27 μM) showed the highest inhibitory activ-
ity. The simulated binding pose of the compound 3 is pre-
sented in Fig. 9c (S=−8.25). As evident from the Fig. 9, the
chloro-benzene moiety of the ligand is involved in aromatic
interaction with Ala636 (green dashed lines), while the
ligand displays hydrogen bonds with His593 and Cme593.
As mentioned earlier in SAR, the combina-
tion of other groups with chloro such as compound 7
(IC50 =2.96 1.11 μM) with nitro and chloro groups meta to
each other was found to be most potent and sevenfold more
active as compared to the standard thiourea. Figure 10a pre-
sents the binding mode of the compound 7. The compound
7 exhibited hydrogen bonding interaction with the His593
and Cme593. The benzene ring can be seen stacked against
the His593, disrupting the catalytic process.
In silico studies
Molecular docking studies were conducted to understand
the plausible ligand binding mechanism. The docking stud-
ies were carried out by using the crystal structure of Jack
Bean urease (PDB 4GY7). Ibuprofen along with the nineteen
newly synthesized derivatives was docked. The interaction
patterns were understood by visualization of resulting poses.
The top-ranked docked pose of the ibuprofen is presented
in Fig. 9a. As evident from the fgure the benzene ring of the
ibuprofen is stacked against the His593. While the oxygen
atom exhibited bi-dentate interaction with one of the nickel
atoms. The strategic position of the carboxylic functional
group in the binding site of the urease (near the catalytic
center) suggests that the modifcations in this area might
increase the inhibitory potential of the ibuprofen. In this con-
nection, ibuprofen was modifed to obtain compound 1. The
compound exhibited additional contacts within the binding
site with the Met637. The benzene ring of the compound 1
was stacked against the His593 (Fig. 9b).
Among the compounds bearing nitro substitutions,
compounds 12 with nitro group at the para-position dis-
played better activity (IC50 = 6.98 1.15 μM). The top-
ranked simulated pose of the compound 12 suggested that
the compound exhibit π-stacking interaction with His539
(Fig. 10b). The contact between His539 and the ligand is
The compound 1 was further modified to yield a
series of the ibuprofen hydrazide derivatives (2–19).
1 3

Molecular Diversity
Fig. 4 SAR of compounds 3–7
Fig. 5 SAR of compounds 8–10
1 3

Molecular Diversity
Fig. 6 SAR of compounds 11–12
Fig. 7 SAR of compounds 13–14
further stabilized by the hydrogen bond. Another hydrogen
bond is observed between the ligand and Cme592. The
para-substituted nitro group exhibits hydrogen bonding
interaction with the Ni901 at the catalytic center.
3-urease complex. As evident from the fgure, there is no
signifcant diference in the coordinates of the active site
residues and the system presented over all stability.
Compound 13 (IC50 =5.26 1.35 μM) bearing an ortho-
methyl substitution demonstrated an increased inhibitory
activity which could be attributed to the hyper-conjugation
efect. Figure 10c depicted the simulated binding mode
of the compound 13 which indicates that methyl group
interacted more efciently with the active site of enzyme.
The methyl-benzene ring of the ligand exhibits π-cation
interactions with His409 and His539 which rationalize the
observed higher inhibitory activity of 13.
Conclusion
Novel derivatives of ibuprofen 1–19 were synthesized and
screened for their in vitro inhibition against urease enzyme.
The synthetic compounds displayed good to moderate inhibi-
tion against urease enzyme in comparison with the standard
thiourea. All compounds were found to be excellent inhibitors
except 16 and 19 which were inactive. Limited structure–activ-
ity relationship (SAR) proposed that the variation in the activ-
ity of diferent molecules might be due to the position and
nature of the substituted groups. Moreover, the docking study
revealed that the synthetic molecules (ligands) have extensive
binding interactions with the active site of enzyme. This study
identifed many lead candidates derived from ibuprofen and
further exploration of these molecules for future research to
get novel urease inhibitors.
The binding mode of the compound 18
(IC50 = 3.5 3.26 μM) with methoxy substitution is pre-
sented in Fig. 10d. The methoxy group of the ligand inter-
acts with the Trp356. Moreover, the ligand exhibits bi-
dentate hydrogen bonds with the Ala436.
The molecular dynamics simulation of the compound 3
was carried out to understand the dynamics of the observed
protein–ligand interaction. Figure 11 presents the super-
imposed coordinates of the active site of the compound
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Molecular Diversity
Fig. 8 SAR of compounds 15–19
Fig. 9 The binding mode of the ibuprofen a ibuprofen hydrazide; compound 1 b ibuprofen hydrazide derivative; compound 3 c hydrogen bonds
are presented as blue dashed lines. Green dashes depict the hydrophobic and aromatic interactions. (Color fgure online)
1 3

Molecular Diversity
Fig. 10 The simulated binding
mode of the compounds 7 (a),
12 (b), 13 (c), and 18 (d). The
hydrogen bonds are presented as
blue dashed lines. Green dashes
depict the hydrophobic and
aromatic interactions. (Color
fgure online)
were used. Spots were visualized with a dual wavelength
of 254 and 366 nm under ultraviolet light or iodine vapors.
Electron impact mass spectra were recorded on MAT 113D
Experimental
Materials and methods
1
and MAT 312 mass spectrometers. The H-NMR spectra
were recorded on Bruker AM machines, functioning at 300
and 400 MHz and 13C-NMR were recorded on 75, 100,
and 125 MHz. Chemical shift (δ) values, relative to tetra-
methylsilane (TMS) as an internal standard, are presented
in ppm, and the coupling constant (J) values are presented
in Hz. Melting point of the compounds were determined
on a Stuart® SMP10 melting point apparatus.
All analytical grade reagents were purchased from Sigma-
Aldrich, USA. For thin layer chromatography (TLC), silica
gel-coated aluminum plates GF-254 (Merck, Germany)
Synthesis of ibuprofen hydrazide (1)
Ibuprofen (1 mmol) and CDI (1.2 mmol) were taken in a
round-bottomed fask (100 mL) and dissolved in minimum
volume of tetrahydrofuran (THF) in the presence of trieth-
ylamine (TEA) to make a saturated solution. The reaction
mixture was then stirred for 15 min, followed by the addi-
tion of hydrazine hydrate (0.5 mL) and THF (5 mL) and
it was further refuxed for 3 h at 60 °C with constant stir-
ring. Reaction progress was monitored by the TLC (7:3;
hexane:ethyl acetate) and then after completion of reaction,
Fig. 11 The superimposed coordinates of the active site residues,
before (gray) and after simulation (pink). The nickel atoms are pre-
sented as green balls. (Color fgure online)
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Molecular Diversity
reaction mixture was poured onto crushed ice. The precipi-
tates were formed immediately which were fltered, and to
aford pure product, precipitates were washed with excess
of distilled water, dried in vacuum and crystallized from
ethanol. Structure of the compound 1 was elucidated by the
N‑(2‑Chlorophenyl)‑2‑(2‑(4‑isobutylphenyl)propanoyl)
hydrazine‑1‑carbothioamide (3)
Yield: 75%; M.P.: 219–221 °C; 1H-NMR (400 MHz, DMSO-
d6): δH 10.16 (s, 1H, NH–CO), 9.76 (s, 1H, NH–CS), 9.14
(s, 1H, NH), 7.60 (s, 1H, H-6ʹ), 7.46 (dd, 1H, J3ʹ,4ʹ =7.8 Hz,
J3ʹ,5ʹ = 1.0 Hz, H-3ʹ), 7.32 (dt, 1H, J5ʹ,4ʹ/5ʹ,6ʹ = 7.7 Hz,
J5ʹ,3ʹ =1.0 Hz, H-5ʹ), 7.24 (m, 3H, H-2, H-6, H-4ʹ), 7.08 (d,
2H, J3,2/5,6 =8.0 Hz, H-3, H-5), 3.66 (d, 1H, J2b,3b =5.2 Hz,
H-2b), 2.39 (d, 2H, J1a,2a = 7.2 Hz, H-1a), 1.78 (m, 1H,
H-2a), 1.38 (d, 3H, J3b,2b = 7.2 Hz, H-3b), 0.84 (d, 6H,
J3a,2a/4a,2a = 6.4 Hz, H-3a, H-4a); 13C-NMR (125 MHz,
DMSO-d6): δc 181.2 (C=S), 173.3 (C=O), 139.4 (C-1),
138.5 (C-4), 136.2 (C-1ʹ), 129.6 (C-2ʹ), 129.1 (C-6ʹ), 128.7
(C-2, -6), 127.4 (C-3, -5, -4ʹ), 127.2 (C-3ʹ), 126.9 (C-5ʹ),
44.1 (C-1a), 42.8 (C-2b), 29.5 (C-2a), 22.1 (C-3a, -4a), 18.9
(C-3b); FAB+-MS m/z: 390 (M++1); HRFAB+-MS m/z:
Calcd for C20H25ON3ClS [390.1411], Found [390.1407].
1
EI-MS, FAB-MS, HREI-MS, HRFAB-MS, and H-NMR
spectroscopic techniques.
2‑(4‑Isobutylphenyl)propanehydrazide (1) [4]
Yield: 72%; M.P.: 60–62 °C; 1H-NMR (400 MHz, DMSO-
d6): δH 9.10 (s, 1H, NH), 7.20 (d, 2H, J2,3/6,5 =8.0 Hz, H-2,
H-6), 7.05 (d, 2H, J3,2/5,6 =8.0 Hz, H-3, H-5), 4.15 (s, 2H,
NH2), 3.46 (q, 1H, H-2b), 2.39 (d, 2H, J1a,2a =7.2 Hz, H-1a),
1.78 (m, 1H, H-2a), 1.30 (d, 3H, J3b,2b =6.8 Hz, H-3b), 0.84
(d, 6H, J3a,2a/4a,2a =6.8 Hz, H-3a, H-4a); EI-MS m/z (% rel
abund.): 220 (M+, 65), 188 (57), 161 (100).
General procedure for the synthesis of substituted
thiourea of ibuprofen (2–19)
N‑(3‑Chlorophenyl)‑2‑(2‑(4‑isobutylphenyl)propanoyl)
hydrazine‑1‑carbothioamide (4)
Ibuprofen hydrazide 1 (0.5 mmol) and corresponding sub-
stituted phenyl isothiocyanates (0.5 mmol) were taken into
a round-bottomed fask (100 mL) and dissolved in dichlo-
romethane (DCM) (5 mL). Reaction mixture was stirred
at room temperature. Reaction progress was monitored by
TLC (6:4; hexane:ethyl acetate) analysis. After completion
of reaction, the solvent was evaporated in vacuo and the
residue obtained was washed with distilled water followed
by washing with hot hexane. Pure products were obtained by
crystallization from ethanol. All compounds were character-
ized by EI-MS, FAB-MS, HREI-MS, HRFAB-MS, 1H-, and
13C-NMR spectroscopic techniques. Spectroscopic data of
all known compound were found to be in agreement with
the reported data.
Yield: 88%; M.P.: 195–197 °C; 1H-NMR (400 MHz, DMSO-
d6): δH 10.08 (s, 1H, NH–CO), 9.74 (s, 1H, NH–CS), 9.48
(s, 1H, NH), 7.61 (s, 1H, H-2ʹ), 7.39 (m, 1H, H-6ʹ), 7.35 (t,
1H, J5ʹ,4ʹ/5ʹ,6ʹ = 7.8 Hz, H-5ʹ), 7.26 (d, 2H, J2,3/6,5 = 8.0 Hz,
H-2, H-6), 7.20 (d, 1H, J4ʹ,5ʹ = 7.2 Hz, H-4ʹ), 7.09 (d, 2H,
J3,2/5,6 = 8.0 Hz, H-3, H-5), 3.67 (d, 1H, J2b,3b = 6.0 Hz,
H-2b), 2.40 (d, 2H, J1a,2a = 7.2 Hz, H-1a), 1.78 (m, 1H,
H-2a), 1.38 (d, 3H, J3b,2b = 6.8 Hz, H-3b), 0.84 (d, 6H,
J3a,2a/4a,2a = 6.4 Hz, H-3a, H-4a); 13C-NMR (100 MHz,
DMSO-d6): δc 221.8 (C=S), 170.6 (C=O), 139.4 (C-1),
138.5 (C-4), 129.6 (C-3ʹ), 128.7 (C-2, -6, -5ʹ), 127.1 (C-3, -5,
-2ʹ, -4ʹ),124.49 (C-6ʹ), 44.1 (C-1a), 42.8 (C-2b), 29.5 (C-2a),
22.1 (C-3a, -4a), 18.2 (C-3b); EI-MS m/z (% rel abund.): 389
(M+,2), 355 (27), 340 (9), 312 (8), 220 (26), 188 (26), 161
(100); HREI-MS m/z: Calcd for C20H24ON3ClS [389.1327],
Found [389.1329].
2‑(2‑(4‑Isobutylphenyl)
propanoyl)‑N‑phenylhydrazine‑1‑carbothioamide (2) [4]
1
Yield: 80%; M.P.: 170–172 °C; H-NMR (400 MHz,
N‑(4‑Chlorophenyl)‑2‑(2‑(4‑isobutylphenyl)propanoyl)
hydrazine‑1‑carbothioamide (5) [27]
DMSO-d6): δH 10.05 (s, 1H, NH–CO), 9.56 (s, 1H, NH–CS),
9.37 (s, 1H, NH), 7.41 (d, 2H, J2ʹ,3ʹ/6ʹ,5ʹ =7.2 Hz, H-2ʹ, H-6ʹ),
7.32 (t, 2H, J3ʹ,2ʹ/3ʹ,4ʹ/5ʹ,6ʹ/5ʹ,4ʹ = 7.8 Hz, H-3ʹ, H-5ʹ), 7.26 (d,
2H, J2,3/6,5 =7.6 Hz, H-2, H-6), 7.15 (t, 1H, J4ʹ,3ʹ/4ʹ,5ʹ =7.2 Hz,
H-4ʹ), 7.09 (d, 2H, J3,2/5,6 =8.0 Hz, H-3, H-5), 3.67 (q, 1H,
H-2b), 2.40 (d, 2H, J1a,2a = 6.8 Hz, H-1a), 1.78 (m, 1H,
H-2a), 1.38 (d, 3H, J3b,2b = 6.8 Hz, H-3b), 0.84 (d, 6H,
J3a,2a/4a,2a =6.8 Hz, H-3a, H-4a); EI-MS m/z (% rel abund.):
355 (M+, 1), 337 (2), 321 (43), 306 (13), 278 (7), 262 (5),
220 (23), 188 (23), 161 (100).
1
Yield: 79%; M.P.: 150–152 °C; H-NMR (400 MHz,
DMSO-d6): δH 10.06 (s, 1H, NH–CO), 9.66 (s, 1H, NH–CS),
9.47 (s, 1H, NH), 7.46 (d, 2H, J2ʹ,3ʹ/6ʹ,5ʹ =7.6 Hz, H-2ʹ, H-6ʹ),
7.37 (d, 2H, J3ʹ,2ʹ/5ʹ,6ʹ = 8.4 Hz, H-3ʹ, H-5ʹ), 7.26 (d, 2H,
J2,3/6,5 = 8.0 Hz, H-2, H-6), 7.08 (d, 2H, J3,2/5,6 = 8.0 Hz,
H-3, H-5), 3.66 (d, 1H, J2b,3b =6.8 Hz, H-2b), 2.40 (d, 2H,
J1a,2a = 7.2 Hz, H-1a), 1.78 (m, 1H, H-2a), 1.38 (d, 3H,
J3b,2b =6.8 Hz, H-3b), 0.84 (d, 6H, J3a,2a/4a,2a =6.8 Hz, H-3a,
H-4a); EI-MS m/z (% rel abund.): 389 (M+, 1), 355 (21), 262
(5), 220 (28), 188 (26), 161 (100).
1 3

Molecular Diversity
N‑(2,5‑Dichlorophenyl)‑2‑(2‑(4‑isobutylphenyl)propanoyl)
hydrazine‑1‑carbothioamide (6)
N‑(2‑Fluorophenyl)‑2‑(2‑(4‑isobutylphenyl)propanoyl)
hydrazine‑1‑carbothioamide (9)
Yield: 82%; M.P.: 180–182 °C; 1H-NMR (400 MHz, DMSO-
d6): δH 10.20 (s, 1H, NH–CO), 9.92 (s, 1H, NH–CS), 9.19
(s, 1H, NH), 7.71 (s, 1H, H-6ʹ),7.52 (d, 1H, J3ʹ,4ʹ =8.8 Hz,
H-3ʹ), 7.33 (d, 1H, J4ʹ,3ʹ = 6.8 Hz, H-4ʹ), 7.26 (d, 2H,
J2,3/6,5 =8.0 Hz, H-2, H-6), 7.08 (d, 2H, J3,2/5,6 =8.0 Hz, H-3,
H-5), 3.65 (s, 1H, H-2b), 2.40 (d, 2H, J1a,2a =7.2 Hz, H-1a),
1.78 (m, 1H, H-2a), 1.38 (d, 3H, J3b,2b =7.2 Hz, H-3b), 0.84
(d, 6H, J3a,2a/4a,2a = 6.4 Hz, H-3a, H-4a); FAB+-MS m/z:
424 (M++1); 13C-NMR (75 MHz, DMSO-d6): δc 139.4
(C-1), 138.4 (C-4, 1ʹ), 137.4 (C-5ʹ), 130.8 (C-2ʹ), 130.5
(C-4ʹ), 128.7 (C-2, -6, -3ʹ), 127.1 (C-3, -5, -6ʹ), 44.1 (C-1a),
42.8 (C-2b), 29.5 (C-2a), 22.1 (C-3a, -4a), 18.3 (C-3b);
HRFAB+-MS m/z: Calcd for C20H24ON3Cl2S [424.0999],
Found [424.1017].
Yield: 68%; M.P.: 195–197 °C; 1H-NMR (400 MHz, DMSO-
d6): δH 10.12 (s, 1H, NH–CO), 9.73 (s, 1H, NH–CS), 9.22 (s,
1H, NH), 7.39 (s, 1H, H-3ʹ), 7.26 (d, 3H, J2,3/6,5/6ʹ,5ʹ =7.6 Hz,
H-2, H-6, H-6ʹ), 7.18 (m, 2H, H-4ʹ, H-5ʹ), 7.08 (d, 2H,
J3,2/5,6 = 8.0 Hz, H-3, H-5), 3.64 (s, 1H, H-2b), 2.40 (d,
2H, J1a,2a = 7.2 Hz, H-1a), 1.78 (m, 1H, H-2a), 1.38 (d,
3H, J3b,2b =6.8 Hz, H-3b), 0.84 (d, 6H, J3a,2a/4a,2a =6.8 Hz,
H-3a, H-4a); 13C-NMR (125 MHz, DMSO-d6): δc 139.4
(C-4), 128.7 (C-2,6), 127.2 (C-3, -5), 126.9 (C-5ʹ), 123.9
(C-4ʹ), 115.7 (C-6ʹ), 115.5 (C-3ʹ), 44.2 (C-1a), 42.8 (C-2b),
29.6 (C-2a), 22.1 (C-3a, -4a), 18.3 (C-3b); FAB+-MS m/z:
374 (M++1); HRFAB+-MS m/z: Calcd for C20H25ON3FS
[374.1708], Found [374.1702].
N‑(2,4‑Difuorophenyl)‑2‑(2‑(4‑isobutylphenyl)propanoyl)
hydrazine‑1‑carbothioamide (10)
N‑(2‑Chloro‑4‑nitrophenyl)‑2‑(2‑(4‑isobutylphenyl)
propanoyl)hydrazine‑1‑carbothioamide (7)
Yield: 71%; M.P.: 230–232 °C; 1H-NMR (400 MHz, DMSO-
d6): δH 10.11 (s, 1H, NH–CO), 9.75 (s, 1H, NH–CS), 9.24
(s, 1H, NH), 7.34 (s, 1H, H-3ʹ), 7.28 (dd, 1H, J5ʹ,6ʹ =9.8 Hz,
J5ʹ,3ʹ =2.2 Hz, H-5ʹ), 7.25 (d, 2H, J2,3/6,5 =7.6 Hz, H-2, H-6),
7.08 (ovp. m, 3H, H-3, H-5, H-6ʹ), 3.62 (s, 1H, H-2b), 2.40
(d, 2H, J1a,2a =7.2 Hz, H-1a), 1.78 (m, 1H, H-2a), 1.37 (d,
3H, J3b,2b =7.2 Hz, H-3b), 0.84 (d, 6H, J3a,2a/4a,2a =6.4 Hz,
H-3a, H-4a); 13C-NMR (75 MHz, DMSO-d6): δc 173.0,
(C=S), 164.0 (C=O), 162.7 (C-2ʹ), 160.4 (C-4ʹ), 139.3
(C-1), 138.5 (C-4), 128.7 (C-2, -6), 127.2 (C-3, -5), 126.9
(C-6ʹ), 125.4 (C-1ʹ), 104.1 (C-5ʹ), 103.7 (C-3ʹ), 44.1 (C-1a),
42.8 (C-2b), 29.5 (C-2a), 22.1 (C-3a, -4a), 18.3 (C-3b);
EI-MS m/z (% rel abund.): 391 (M+, 1), 357 (42), 342 (10),
314 (13), 220 (21), 188 (23), 161 (100); HREI-MS m/z:
Calcd for C20H23ON3F2S [391.15453], Found [391.1530].
1
Yield: 83%; M.P.: 166–168 °C; H-NMR (400 MHz,
DMSO-d6): δH 10.29 (s, 1H, NH–CO), 10.14 (s, 1H,
NH–CS), 9.30 (s, 1H, NH), 8.33 (s, 1H, H-3ʹ), 8.18 (broad
s, 2H, H-5ʹ,6ʹ), 7.27 (d, 2H, J2,3/6,5 =8.0 Hz, H-2, H-6), 7.09
(d, 2H, J3,2/5,6 =8.0 Hz, H-3, H-5), 3.67 (s, 1H, H-2b), 2.40
(d, 2H, J1a,2a =7.2 Hz, H-1a), 1.78 (m, 1H, H-2a), 1.39 (d,
3H, J3b,2b =6.8 Hz, H-3b), 0.84 (d, 6H, J3a,2a/4a,2a =6.4 Hz,
H-3a, H-4a); 13C-NMR (75 MHz, DMSO-d6): δc 180.5
(C=S), 170.1 (C=O), 142.2 (C-4ʹ), 139.5 (C-1), 138.3 (C-4),
128.8(C-2, -6), 128.3 (C-2ʹ), 127.1 (C-3, -5, -6ʹ), 124.4
(C-3ʹ), 122.3 (C-5ʹ), 44.1 (C-1a), 42.9 (C-2b), 29.5 (C-2a),
22.1 (C-3a, -4a), 18.3 (C-3b); FAB+-MS m/z: 435 (M++1);
HRFAB+-MS m/z: Calcd for C20H24O3N4ClS [435.1262],
Found [435.1258].
N‑(3‑Bromophenyl)‑2‑(2‑(4‑isobutylphenyl)propanoyl)
hydrazine‑1‑carbothioamide (8)
2‑(2‑(4‑Isobutylphenyl)propanoyl)‑N‑(3‑nitrophenyl)
hydrazine‑1‑carbothioamide (11)
1
Yield: 92%; M.P.: 215–217 °C; 1H-NMR (400 MHz, DMSO-
d6): δH 10.07 (s, 1H, NH–CO), 9.73 (s, 1H, NH–CS), 9.46
(s, 1H, NH), 7.73 (s, 1H, H-2ʹ), 7.45 (d, 1H, J4ʹ,5ʹ =8.0 Hz,
H-4ʹ), 7.29 (ovp. m, 4H, H-5ʹ, H-6ʹ, H-2, H-6), 7.09 (d,
2H, J3,2/5,6 =8.0 Hz, H-3, H-5), 3.66 (d, 1H, J2b,3b =6.4 Hz,
H-2b), 2.40 (d, 2H, J1a,2a = 7.2 Hz, H-1a), 1.79 (m, 1H,
H-2a), 1.38 (d, 3H, J3b,2b = 7.2 Hz, H-3b), 0.84 (d, 6H,
J3a,2a/4a,2a = 6.4 Hz, H-3a, H-4a); 13C-NMR (100 MHz,
DMSO-d6): δc 173.0, (C=S), 165.0 (C=O), 140.7 (C-1),
139.4 (C-4), 138.5 (C-1ʹ), 129.9 (C-3ʹ), 128.7 (C-2, -6,
-5ʹ), 127.3 (C-4ʹ), 127.1 (C-3, -5, -2ʹ, -6ʹ), 44.1 (C-1a),
42.8 (C-2b), 29.5 (C-2a), 22.1 (C-3a, -4a), 18.2 (C-3b);
FAB+-MS m/z: 434 (M++1); HRFAB+-MS m/z: Calcd for
C20H25ON3BrS [434.0911], Found [434.0902].
Yield: 74%; M.P.: 160–162 °C; H-NMR (400 MHz,
DMSO-d6): δH 10.12 (s, 1H, NH–CO), 9.89 (s, 1H,
NH–CS), 9.81 (s, 1H, NH), 8.47 (s, 1H, H-2ʹ), 7.99 (d, 1H,
J4ʹ,5ʹ =6.4 Hz, H-4ʹ), 7.94 (d, 1H, J6ʹ,5ʹ =7.2 Hz, H-6ʹ), 7.62
(t, 1H, J5ʹ,4ʹ/5ʹ,6ʹ =8.2 Hz, H-5ʹ), 7.27 (d, 2H, J2,3/6,5 =8.0 Hz,
H-2, H-6), 7.09 (d, 2H, J3,2/5,6 =8.0 Hz, H-3, H-5), 3.68 (d,
1H, J2b,3b =6.4 Hz, H-2b), 2.40 (d, 2H, J1a,2a =6.8 Hz, H-1a),
1.78 (m, 1H, H-2a), 1.39 (d, 3H, J3b,2b = 7.2 Hz, H-3b),
0.84 (d, 6H, J3a,2a/4a,2a = 6.8 Hz, H-3a, H-4a); 13C-NMR
(125 MHz, DMSO-d6): δc 178.0, (C=S), 164.0 (C=O),
145.7 (C-3ʹ), 140.4 (C-4), 139.4 (C-1), 138.5 (C-1ʹ), 129.2
(C-6ʹ), 128.8 (C-2, -6), 127.2 (C-3, -5, -5ʹ), 119.3 (C-4ʹ, -2ʹ),
44.1 (C-1a), 42.9 (C-2b), 29.6 (C-2a), 22.1 (C-3a, -4a), 18.3
1 3

Molecular Diversity
(C-3b); FAB+-MS m/z: 401 (M++1); HRFAB+-MS m/z:
2‑(2‑(4‑Isobutylphenyl)propanoyl)‑N‑(2‑(trifuoromethyl)
phenyl)hydrazine‑1‑carbothioamide (15)
Calcd for C20H25O3N4S [401.1641], Found [401.1647].
1
2‑(2‑(4‑Isobutylphenyl)propanoyl)‑N‑(4‑nitrophenyl)
hydrazine‑1‑carbothioamide (12)
Yield: 85%; M.P.: 185–187 °C; H-NMR (400 MHz,
DMSO-d6): δH 10.12 (s, 1H, NH–CO), 9.76 (s, 1H, NH–CS),
9.05 (s, 1H, NH), 7.67 (m, 2H, H-3ʹ, H-4´), 7.47 (m, 2H,
H-5ʹ, H-6ʹ), 7.26 (d, 2H, J2,3/6,5 =8.0 Hz, H-2, H-6), 7.07 (d,
2H, J3,2/5,6 =8.0 Hz, H-3, H-5), 3.64 (s, 1H, H-2b), 2.39 (d,
2H, J1a,2a =6.8 Hz, H-1a), 1.77 (m, 1H, H-2a), 1.37 (d, 3H,
J3b,2b =6.8 Hz, H-3b), 0.83 (d, 6H, J3a,2a/4a,2a =6.8 Hz, H-3a,
H-4a); 13C-NMR (75 MHz, DMSO-d6): δc 139.3 (C-1),
138.5 (C-4), 132.4 (C-1ʹ), 132.1 (C-5ʹ), 128.7 (C-2, -6),
127.1 (C-3, -5, 3ʹ), 126.0 (C-4ʹ, -6ʹ), 125.9 (C-2ʹ, C-CF3),
44.1 (C-1a), 42.7 (C-2b), 29.5 (C-2a), 22.0 (C-3a, -4a), 18.2
(C-3b); FAB+-MS m/z: 424 (M++1); HRFAB+-MS m/z:
Calcd for C21H25ON3F3S [424.1671], Found [424.1670].
Yield: 85%; M.P.: 170–172 °C; 1H-NMR (400 MHz, DMSO-
d6): δH 10.31 (s, 1H, NH–CO), 10.02 (s, 1H, NH–CS), 9.78
(s, 1H, NH), 8.19 (d, 2H, J3ʹ,2ʹ/5ʹ,6ʹ = 9.2 Hz, H-3ʹ, H-5ʹ),
7.86 (d, 2H, J2ʹ,3ʹ/6ʹ,5ʹ = 9.2 Hz, H-2ʹ, H-6ʹ), 7.26 (d, 2H,
J2,3/6,5 =8.0 Hz, H-2, H-6), 7.09 (d, 2H, J3,2/5,6 =8.0 Hz, H-3,
H-5), 3.68 (s, 1H, H-2b), 2.40 (d, 2H, J1a,2a =7.2 Hz, H-1a),
1.79 (m, 1H, H-2a), 1.39 (d, 3H, J3b,2b =6.8 Hz, H-3b), 0.84
(d, 6H, J3a,2a/4a,2a =6.8 Hz, H-3a, H-4a); FAB+-MS m/z: 401
(M++1); 13C-NMR (75 MHz, DMSO-d6): δc 180.7 (C=S),
173.0 (C=O), 145.7 (C-4ʹ), 139.4 (C-1), 139.2 (C-4), 138.4
(C-1ʹ), 128.7 (C-2, -6), 127.1 (C-3, -5, -2ʹ, -6ʹ), 124.7 (C-3ʹ,
-5ʹ), 44.1 (C-1a), 42.7 (C-2b), 29.5 (C-2a), 22.1 (C-3a, -4a),
18.3 (C-3b); HRFAB+-MS m/z: Calcd for C20H25O3N4S
[401.1646], Found [401.1647].
2‑(2‑(4‑Isobutylphenyl)propanoyl)‑N‑(3‑(trifuoromethyl)
phenyl)hydrazine‑1‑carbothioamide (16)
1
Yield: 59%; M.P.: 248–250 °C; H-NMR (400 MHz,
DMSO-d6): δH 10.08 (s, 1H, NH–CO), 9.79 (s, 1H,
NH–CS), 9.65 (s, 1H, NH), 7.86 (s, 1H, H-2ʹ), 7.77 (d, 1H,
J4ʹ,5ʹ = 8.4 Hz, H-4ʹ), 7.55 (t, 1H, J5ʹ,4ʹ/5ʹ,6ʹ = 7.8 Hz, H-5ʹ),
7.49 (m, 1H, H-6ʹ), 7.27 (d, 2H, J2,3/6,5 = 8.0 Hz, H-2,
H-6), 7.09 (d, 2H, J3,2/5,6 =8.0 Hz, H-3, H-5), 3.67 (d, 1H,
J2b,3b = 6.4 Hz, H-2b), 2.40 (d, 2H, J1a,2a = 7.2 Hz, H-1a),
1.78 (m, 1H, H-2a), 1.38 (d, 3H, J3b,2b =6.8 Hz, H-3b), 0.84
(d, 6H, J3a,2a/4a,2a =6.8 Hz, H-3a, H-4a); 13C-NMR (75 MHz,
DMSO-d6): δc 188.3 (C=S), 172.8 (C=O), 139.9 (C-1),
139.4 (C-4), 138.5 (C-1ʹ), 129.2 (C-3ʹ), 128.7 (C-2, -6, -2ʹ,
-5ʹ), 127.2 (C-3, -5, -4ʹ, -6ʹ), 110.0 (C-CF3), 44.1 (C-1a),
42.8 (C-2b), 29.5 (C-2a), 22.1 (C-3a, -4a), 18.3 (C-3b);
EI-MS m/z (% rel abund.): 423 (M+, 2), 389 (72), 374 (27),
346 (35), 262 (24), 220 (45), 203 (94), 188 (63), 161 (100);
HREI-MS m/z: Calcd for C21H24ON3F3S [423.1619], Found
[423.1592].
2‑(2‑(4‑Isobutylphenyl)propanoyl)‑N‑(o‑tolyl)
hydrazine‑1‑carbothioamide (13) [27]
Yield: 82%; M.P.: 195–197 °C; 1H-NMR (400 MHz, DMSO-
d6): δH 10.04 (s, 1H, NH–CO), 9.44 (s, 1H, NH–CS), 9.16
(s, 1H, NH), 7.26 (d, 2H, J2,3/6,5 =8.0 Hz, H-2, H-6), 7.19
(m, 4H, H-3ʹ, H-4ʹ, H-5ʹ, H-6ʹ), 7.07 (d, 2H, J3,2/5,6 =8.0 Hz,
H-3, H-5), 3.64 (d, 1H, J2b,3b =6.8 Hz, H-2b), 2.39 (d, 2H,
J1a,2a = 6.8 Hz, H-1a), 2.11 (s, 3H, CH3-2ʹ), 1.77 (m, 1H,
H-2a), 1.37 (d, 3H, J3b,2b = 6.8 Hz, H-3b), 0.84 (d, 6H,
J3a,2a/4a,2a =6.8 Hz, H-3a, H-4a); EI-MS m/z (% rel abund.):
369 (M+, 1), 335 (30), 262 (5), 220 (26), 188 (25), 174 (72),
161 (100).
N‑(2,6‑Dimethylphenyl)‑2‑(2‑(4‑isobutylphenyl)propanoyl)
hydrazine‑1‑carbothioamide (14)
2‑(2‑(4‑Isobutylphenyl)propanoyl)‑N‑(4‑(trifuoromethyl)
phenyl)hydrazine‑1‑carbothioamide (17)
Yield: 72%; M.P.: 180–182 °C; 1H-NMR (400 MHz, DMSO-
d6): δH 10.06 (s, 1H, NH–CO), 9.39 (s, 1H, NH–CS), 9.01 (s,
1H, NH), 7.26 (d, 2H, J2,3/6,5 =8.0 Hz, H-2, H-6), 7.04 (ovp.
m, 5H, H-3, H-5, H-3ʹ, H-4ʹ, H-5ʹ), 3.63 (q, 1H, H-2b), 2.39
(d, 2H, J1a,2a =7.2 Hz, H-1a), 2.10 (s, 6H, CH3-2ʹ, CH3-6ʹ),
1.77 (m, 1H, H-2a), 1.38 (d, 3H, J3b,2b =6.8 Hz, H-3b), 0.83
(d, 6H, J3a,2a/4a,2a =6.8 Hz, H-3a, H-4a); 13C-NMR (75 MHz,
DMSO-d6): δc 181.4 (C=S), 173.1 (C=O), 139.3 (C-1),
138.7 (C-4), 136.8 (C-1ʹ), 136.6 (C-2ʹ), 136.4 (C-6ʹ), 128.7
(C-2, -6), 127.4 (C-3ʹ, -5ʹ), 127.1 (C-3, -5), 126.7 (C-4ʹ),
44.1 (C-1a), 42.8 (C-2b), 29.5 (C-2a), 22.1 (C-3a, -4a), 18.3
(C-3b), 17.8 (C-2ʹ, -6ʹ, CH3); EI-MS m/z (% rel abund.): 383
(M+, 2), 349 (49), 220 (9), 188 (100); HREI-MS m/z: Calcd
for C22H29ON3S [383.2041], Found [383.2031].
1
Yield: 81%; M.P.: 170–172 °C; H-NMR (400 MHz,
DMSO-d6): δH 10.09 (s, 1H, NH–CO), 9.82 (s, 1H, NH–CS),
9.59 (s, 1H, NH), 7.71 (d, 4H, J=16.8 Hz, H-2ʹ, H-3ʹ, H-5ʹ,
H-6ʹ), 7.26 (d, 2H, J2,3/6,5 =7.6 Hz, H-2, H-6), 7.09 (d, 2H,
J3,2/5,6 =8.0 Hz, H-3, H-5), 3.67 (s, 1H, H-2b), 2.40 (d, 2H,
J1a,2a = 7.2 Hz, H-1a), 1.78 (m, 1H, H-2a), 1.38 (d, 3H,
J3b,2b =6.8 Hz, H-3b), 0.84 (d, 6H, J3a,2a/4a,2a =6.8 Hz, H-3a,
H-4a); 13C-NMR (125 MHz, DMSO-d6): δc 142.9 (C-4),
139.4 (C-1, -4ʹ), 138.5 (C-1ʹ), 128.8 (C-2, -6, -2ʹ, -6ʹ), 127.2
(C-3, -5, -3ʹ, -5ʹ), 125.1 (C-CF3), 44.1 (C-1a), 42.8 (C-2b),
29.6 (C-2a), 22.1 (C-3a, -4a), 18.3 (C-3b); FAB+-MS m/z:
1 3

Molecular Diversity
(
)
424 (M++1); HRFAB+-MS m/z: Calcd for C21H25ON3F3S
% Inhibition = 100 − ODtestwell∕ODcontrol × 100
[424.1677], Found [424.1670].
2‑(2‑(4‑Isobutylphenyl)propanoyl)‑N‑(2‑methoxyphenyl)
hydrazine‑1‑carbothioamide (18) [27]
Molecular docking assay
Molecular docking studies were carried out to predict the
molecular basis of the observed urease inhibition demon-
strated by the newly synthesized ibuprofen hydrazide deriva-
tives. In this connection, the crystal structure of the Jack
Bean urease under the accession code 4GY7 was retrieved
from RCSB-PDB [30]. The protein preparation module in
MOE v2018.0101 was implemented to fll in the missing
loops and atoms, assign bond orders, and for the treatment
of formal charges using AMBER 10:EHT force feld. The
prepared structure was further used to establish the bind-
ing mode of the newly synthesized derivatives. The global
search coordinates were generated using the centroids of
the residues Kcx-490, His-492, His-519, His-545, Asp-
633, Ni-901, and Ni-902. The primary placement method
was Triangle Matcher, and the London dG was selected as
placement scoring method. The Rigid Receptor protocol was
used for refnement of the initially generated 30 poses. The
top-ranked fve poses scored by the GBVI/WSA dG meth-
ods were retrieved for subsequent analysis. The top-ranked
binding pose of each compound was analyzed visually using
the Protein–Ligand Interaction Profler (PLIP) web server
[31]. All the visuals were rendered using Chimera and MOE.
The compounds were sketched using the builder module
in MOE. The molecules were charged and minimized at neu-
tral pH using the MMFFF 94x force feld [32].
1
Yield: 88%; M.P.: 110–112 °C; H-NMR (400 MHz,
DMSO-d6): δH 10.20 (s, 1H, NH–CO), 9.72 (s, 1H,
NH–CS), 8.12 (s, 1H, NH), 7.27 (d, 2H, J2,3/6,5 = 8.0 Hz,
H-2, H-6), 7.10 (m, 4H, H-3, H-5, H-5ʹ, H-6ʹ), 7.02 (d, 1H,
ʹʹ
3,4
ʹʹ ʹ ʹ
4,5/4,3
J =8.0 Hz, H-3ʹ), 6.91 (t, 1H, J
=7.8 Hz, H-4ʹ), 3.73
(s, 3H, OCH3), 3.69 (d, 1H, J2b,3b =6.8 Hz, H-2b), 2.40 (d,
2H, J1a,2a =6.8 Hz, H-1a), 1.78 (m, 1H, H-2a), 1.39 (d, 3H,
J3b,2b =6.8 Hz, H-3b), 0.83 (d, 6H, J3a,2a/4a,2a =6.8 Hz, H-3a,
H-4a); FAB+-MS m/z: 386 (M++1).
N‑(4‑((4‑(dimethylamino)phenyl)diazenyl)
phenyl)‑2‑(2‑(4‑isobutylphenyl)propanoyl)
hydrazine‑1‑carbothioamide (19) [28]
1
Yield: 90%; M.P.: 215–217 °C; H-NMR (400 MHz,
DMSO-d6): δH 10.10 (s, 1H, NH–CO), 9.70 (s, 1H, NH–CS),
7.74 (m, 4H, H-2ʹ, H-3ʹ, H-5ʹ, H-6ʹ), 7.64 (m, 2H, H-2″,
H-6″), 7.27 (d, 2H, J2,3/6,5 =8.0 Hz, H-2, H-6), 7.09 (d, 2H,
″″ ″ ″
3,2/5,6
J3,2/5,6 = 7.6 Hz, H-3, H-5), 6.83 (d, 2H, J
= 8.8 Hz,
H-3″, H-5″), 3.69 (s, 1H, H-2b),3.04 (s, 6H, N-(CH3)2, 2.41
(d, 2H, J1a,2a =6.8 Hz, H-1a), 1.79 (pent, 1H, H-2a), 1.39 (d,
3H, J3b,2b =6.8 Hz, H-3b), 0.85 (d, 6H, J3a,2a/4a,2a =6.4 Hz,
H-3a, H-4a); ESI+-MS m/z: 503 (M++1); HRESI+-MS m/z:
Calcd for C28H35SON6 [503.2583], Found [503.2588].
A short production run of 500 ps was carried out in cases
of compounds 3 and 7 to optimize the docking poses gener-
ated by MOE. The system was initially solvated using TIP3P
water model under periodic boundary conditions. The cut-
of for the non-bonded interactions was set as 8 Å. The sys-
tem was initially minimized to remove the steric clashes
followed by the gradual increase in the temperature up to
300 K. The pressure was set to 1 atm. The trajectories were
saved 2 fs.
In vitro urease inhibitory assay
Urease inhibitory potentials of ibuprofen derivatives 1–19
were performed by reported method [29]. Reaction mixture
comprising enzyme solution (25 μL) and bufer solution
(55 μL) containing urea (100 mM) were incubated with test
compounds (5 μL and 1 mM concentration) at 30 °C for
15 min in 96-well plate. By using the indophenols method,
ammonia production was measured to determine the urease
activity. Alkali reagent (70 μL; 0.5% w/v NaOH and 0.1%
active chloride NaOCl) and phenol reagent (45 μL; 1% w/v
phenol and 0.005% w/v sodium nitroprusside) were added
to each well. After 50 min, the absorbance at 630 nm was
measured using a microplate reader (Molecular Device,
USA). All steps were performed thrice in a fnal volume
of 200 μL. By using SoftMax Pro software (Molecular
Device, USA), the results (change in absorbance per min)
were calculated. Assays were performed at pH 8.2 (0.01 M
K2HPO4·3H2O, 1 mM EDTA and 0.01 M LiCl). Thiourea
was used as standard inhibitor of urease. From the following
formula, percentage inhibitions were calculated:
Acknowledgements The authors are thankful to Pakistan Academy of
Sciences for providing fnancial support to Project No. (5-9/PAS/440).
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