New amino acid clubbed Schiff bases inhibit carbonic anhydrase II, α-glucosidase, and urease enzymes: in silico and in vitro

Article abstract of DOI:10.1007/s00044-020-02696-0

Combating pathological conditions related to hyperactivity of enzymes remains a formidable challenge for health. Small molecules therapy constitutes one of the means to circumvent the medical disorders resulting from enzyme hyperactivity. In this regard, we have synthesized structurally diverse amino acid hybrid Schiff bases (5a–5l and 10a–10k) and evaluated them for carbonic anhydrase II, α-glucosidase, and urease inhibitory potential. These new chemical scaffolds showed variable efficacies against the selected enzymes. The results indicated that compounds 5b (11.8 ± 1.33 μM), 10i (83.3 ± 1.13 μM), and 10f (88.2 ± 2.27 μM) are the most active scaffolds against carbonic anhydrase II, α-glucosidase, and urease, respectively. A structure–activity relationship revealed the most structural features contributing to the overall activities. Molecular docking suggested that these compounds possess excellent binding interactions with the active site residues of the targets by interacting through hydrogen bonding, π–π, and π–cation interactions.

Full text of DOI:10.1007/s00044-020-02696-0

MEDICINAL
CHEMISTRY
RESEARCH
Medicinal Chemistry Research (2021) 30:712–728
https://doi.org/10.1007/s00044-020-02696-0
ORIGINAL RESEARCH
New amino acid clubbed Schiff bases inhibit carbonic anhydrase II,
α-glucosidase, and urease enzymes: in silico and in vitro
Kashif Rafiq1,2 Majid Khan
1,3 ●
Niaz Muhammed Ajmal Khan Najeeb Ur Rehman
2 ●
1 ●
1 ●
●
1 ●
1 ●
1 ●
4 ●
5 ●
Balqees Essa Mohammad Al-Yahyaei Mohammed Khiat Sobia Ahsan Halim Zarbad Shah Rene Csuk
Ahmed Al-Harrasi
1
Received: 1 July 2020 / Accepted: 16 December 2020 / Published online: 9 January 2021
©
The Author(s), under exclusive licence to Springer Science+Business Media, LLC part of Springer Nature 2021
Abstract
Combating pathological conditions related to hyperactivity of enzymes remains a formidable challenge for health. Small
molecules therapy constitutes one of the means to circumvent the medical disorders resulting from enzyme hyperactivity. In
this regard, we have synthesized structurally diverse amino acid hybrid Schiff bases (5a–5l and 10a–10k) and evaluated
them for carbonic anhydrase II, α-glucosidase, and urease inhibitory potential. These new chemical scaffolds showed
variable efficacies against the selected enzymes. The results indicated that compounds 5b (11.8 ± 1.33 µM), 10i (83.3 ±
1
.13 µM), and 10f (88.2 ± 2.27 µM) are the most active scaffolds against carbonic anhydrase II, α-glucosidase, and urease,
respectively. A structure–activity relationship revealed the most structural features contributing to the overall activities.
Molecular docking suggested that these compounds possess excellent binding interactions with the active site residues of the
targets by interacting through hydrogen bonding, π–π, and π–cation interactions.
●
●
●
●
●
Keywords Amino acid hybrid Schiff bases Carbonic anhydrase II α-Glucosidase Urease Structure–activity relationship
Molecular docking
Introduction
Amino acids play an important role for the establishment of
innovative drugs using nonproteogenic and unnatural amino
acids [1–8]. They are “building blocks of life” and have
important biochemical and medicinal applications [9].
Proteogenic amino acid showed physical properties are
governed by the structure and nature of their side chains:
polar (hydrophilic) vs. nonpolar (hydrophobic); neutral,
acidic, or basic; aromatic vs. nonaromatic; essential vs.
nonessential; and achiral vs. chiral. For the construction of
all peptides and proteins in living system, 20 amino acids
are used [10].
Supplementary information The online version of this article (https://
doi.org/10.1007/s00044-020-02696-0) contains supplementary
material, which is available to authorized users.
*
*
Najeeb Ur Rehman
najeeb@unizwa.edu.om
Ahmed Al-Harrasi
aharrasi@unizwa.edu.om
1
Natural and Medical Sciences Research Center, University of
Nizwa, P.O. Box 33 Birkat Al Mauz, PC 616 Nizwa, Sultanate of
Oman
L-β-phenylalanine and L-leucine are the amino acids,
which have capability to make different Schiff bases. Schiff
bases are mostly known as imines or azomethines and act as
ligands in various metal complexes. Hugo Schiff, a German
chemist, was the first to synthesize Schiff bases in 1864 by
condensing primary amines with carbonyl compounds [11].
These biologically dynamic Schiff’s bases and their deri-
vatives are recognized due to their biological activities,
mainly as antimicrobial agents [12–15], antivirus agents
[16], antioxidants [17], radical inhibitors [18], antitumor
2
3
Department of Chemistry, Abdul Wali Khan University Mardan,
Mardan 23200, Pakistan
H. E. J. Research Institute of Chemistry, International Center for
Chemical and Biological Sciences, University of Karachi,
Karachi 75270, Pakistan
4
5
Department of Chemistry, Bacha Khan University Charsadda,
Charsadda 24420 Khyber Pakhtunkhwa, Pakistan
Department of Organic Chemistry, Martin-Luther University
Halle-Wittenberg, Kurt-Mothes-Str. 2, D-06120 Halle (Saale),
Germany

Medicinal Chemistry Research (2021) 30:712–728
713
Scheme 1 Reagents and conditions: a SOCl , MeOH, 0 °C, overnight (2a); b NaCO , H O, Boc, EtOAc (3a); c NH NH .H O, MeOH, rt, 24 h
2
3
2
2
2
2
(4a); and d aldehydes, EtOH, rt, 24 h (5a–5l)
agents [19, 20], carbonic anhydrase (CA) inhibitors [21],
xanthine oxidase inhibitors [22], antibacterial [21, 23, 24],
plant-growth regulators [25], free radical scavengers
such as peptic ulcers, gastric lesion, and intestinal cancer
[50, 51].
α-Glucosidase enzyme catalyzes the hydrolysis of α-
glucosidal bond of complex carbohydrates and releases the
monosaccharide (α-D-glucose), which is absorbable in
small intestine [52, 53]. Hyperactivity of α-glucosidase
enzyme causes elevation in blood glucose in diabetic
patients known as hyperglycemia [17]. Inhibition of α-
glucosidase enzyme by Schiff bases is one way to treat type
II diabetes mellitus by suspending the absorption of glucose
in intestine [54]. Acarbose, miglitol, and voglibose are the
drugs which are being clinically used for the treatment of
type II diabetes mellitus.
[
26], trypsin inhibitors [27], inhibitors of cartilage matrix
degeneration [28], 5-HT6 antagonists [29], and anti-
inflammatory agents [29, 30].
CAs (EC 4.2.1.1) are zinc enzymes strongly involved in
regulating cell homeostasis, intracellular pH, fluid secretion,
ion transport, and biosynthetic reactions by catalyzing the
reversible hydration of carbon dioxide (CO ) to bicarbonate
2
3
−
+
ion (HCO ) and proton (H ) [31–37]. These enzymes are
common in almost all organisms, from simple to complex
[
38, 39]. Notably, in tumors, the extracellular pH is more
acidic than intracellular pH [40]. In order to maintain the
pH, tumor cells generate ion transportations via CA
enzymes and ionic channels in between extra and intracel-
lular compartments [40–42]. In brain malignant, tumor cells
express CA II [43–45], similar to that of renal cancer cell
line and pancreatic cell carcinomas [43, 45]. With that, CA
II has also been involved in glaucoma, epilepsy, leukemia,
and cystic fibrosis [46, 47]. Due to the significant role of
CAs in life threating diseases, it is important to inhibit the
activity of this enzyme to obtain a therapy.
The current study aims to explore CA II, α-glucosidase,
and urease the inhibition for this purpose, compounds (5a–
5l and 10a–10k) were synthesized from two amino acids
(L-β-phenylalanine and L-leucine). The newly synthesized
heteroaromatic Schiff bases were evaluated against CA II,
α-glucosidase, and urease inhibition to check their poten-
cies. In addition, the in vitro enzyme inhibitory results were
further confirmed with the aid of molecular docking studies.
On the other hand, Schiff bases, an important class of
compounds, having azomethine (C=N) functional group are
known for urease inhibition and are extensively used as
mixture with crop fertilizers to inhibit the urea decom-
position by soil bacteria [48, 49]. The therapeutic demand
of urease inhibition is very important as it has a potential
role in curing diseases such as peptic ulcer, urolithiasis,
pyelonephritis, and hepatic coma caused by ureolytic bac-
teria e.g., Helicobacter pylori, Yersinia enterocolitica, and
Proteus mirabilis. Urease facilitates the colonial growth of
H. pylori by elevating the stomach local pH thereby
allowing the bacteria H. pylori to cause gastric infection
Results and discussion
Chemistry
Different amino acid Schiff bases (5a–5l and 10a–10k)
were synthesized through esterification of L-
β-phenylalanine (1a) and L-leucine (6a) in methanol
(MeOH) by adding thionyl chloride, followed by protection
of the amino group as tert-butyloxycarbonyl (Boc). The
resulting hydrazides (4a and 9a) were then reacted with
different benzaldehyde derivatives to furnish different
L-α-phenylalanine hydrazones and L-leucine hydrazones

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Medicinal Chemistry Research (2021) 30:712–728
Scheme 2 Reagents and conditions: a SOCl , MeOH, 0 °C, overnight (7a); b NaCO , H O, Boc, EtOAc (8a); c NH NH .H O, MeOH, rt, 24 h
2
3
2
2
2
2
(9a); and d aldehydes, EtOH, rt, 24 h (10a–10k)
derivatives (Schemes 1 and 2; Table 1). The structures of
the resulting compounds were confirmed using different
spectroscopic techniques.
observed. The higher inhibition of 5d can be accounted for
the arrangements of functional groups attached to phenyl
ring. The higher activity of 5d than 5i (IC = 37.2 µM) and
5
0
5
e (IC = 41.0 µM) is likely due to the replacement of
50
Enzyme inhibition
hydroxyl group with methoxy group at meta and para
positions of the phenyl ring. Similarly, a higher inhibition of
compound 5a (IC = 30.1 µM) is seen when compared
with compounds 5j‒5l; a fact that can be attributed to the
replacement of pyridine ring with phenyl ring.
The newly synthesized amino acid assembled hybrid
structures (5a–5l and 10a–10k) were evaluated against CA
II, α-glucosidase, and urease inhibitory activity. The in vitro
studies results are summarized in Table 1. The results
demonstrate a clear variation in the inhibitory activities
ranging from 11.8 to 122.2 µM, 83.3 to 367.8 µM, and 88.2
to 427.3 µM against CA II, α-glucosidase, and urease
enzymes, respectively. The structural–activity relationship
of the compounds was investigated on the basis of attach-
ment of the functional (electron withdrawing and donating)
groups at different positions of phenyl ring of R group.
5
0
Similarly, among the leucine hydrazones, compound 10a
has displayed an excellent inhibitory activity having an IC50
value of 12.5 µM), followed by 10b (IC = 20.5 µM) and
5
0
10g (IC = 49.8 µM), while 10h (IC = 82.1 µM) and 10d
5
0
50
(IC = 106.5 µM) exhibited weak activities when com-
5
0
pared with standard acetazolamide (IC = 18.2 µM) (Table
5
0
1). Compound 10a (IC = 12.5 µM) exerted a stronger
5
0
inhibition than other compounds in the series, which is
possibly due to the presence of pyridine ring. When com-
paring compounds 10b and 10g, the higher activity of 10b
is likely due to the presence of hydroxyl group at ortho
position of the phenyl ring. A comparison between 10d with
10g revealed a decrease in inhibition of 10d, which is
possibly by the replacement of hydroxyl group with meth-
oxy group attached at the para position of the phenyl ring.
Finally, compound 10h possesses better inhibition when
compared with 10d due to the presence of methoxy group at
the meta position of the phenyl ring.
Structure–activity relationship of CA II enzyme
In case of phenylalanine hydrazine derivatives, eight com-
pounds were found active against CA II. Among the active
compounds, 5b was the most potent with IC50 value of
1
1.8 µM, followed by 5d (IC = 13.4 µM) and 5c (IC =
50 50
1
5.8 µM) even superior than standard acetazolamide
(
IC = 18.2 µM), whilst compounds 5a (IC = 30.1 µM),
50 50
5
e (IC = 41.0 µM), and 5i (IC = 37.2 µM) showed pro-
50 50
mising inhibition comparable to standard acetazolamide.
Compounds 5g and 5h showed weak activities, while rest of
compounds found to be inactive (Table 1). The higher
activity of compound 5b could be attributed to the presence
of hydroxyl group at ortho position of phenyl ring. When
comparing 5b with 5c, the decrease in inhibition is due to
the position of methoxy group at para position of the
phenyl ring. A variation in activities between 5c and 5d is
Structure–activity relationship of α-glucosidase
enzyme
In the preliminary screening of phenylalanine hydrazones
against α-glucosidase, six compounds were found to be more
active than standard acarbose. The compound 5k has
demonstrated the highest activity with an IC50 value of

Medicinal Chemistry Research (2021) 30:712–728
715
Table 1 Enzyme inhibitory activities of compounds 5a–5l and 10a–10k
Compounds
R
IC50 ± S.E.M (µM)
Carbonic anhydrase II
α-Glucosidase
Urease
5
5
5
5
5
a
b
c
30.1 ± 0.18
11.8 ± 1.33
15.8 ± 0.61
13.4 ± 0.37
41.0 ± 2.34
NA
304.5 ± 2.17
173.7 ± 3.2
NA
N
269.8 ± 3.40
166.7 ± 2.20
NA
HO
d
e
NA
270.7 ± 2.60
NA
OCH3
OCH3
5
5
f
NA
NA
NA
H CO
3
g
84.5 ± 2.19
176.7 ± 1.90
128.6 ± 0.16
HO
OH
5
5
5
h
i
122.2 ± 3.16
37.2 ± 1.28
NA
NA
427.3 ± 4.11
OH
317.4 ± 2.60
NA
OCH3
OCH3
j
NA
NA
Cl

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Medicinal Chemistry Research (2021) 30:712–728
Table 1 (continued)
Compounds
R
IC50 ± S.E.M (µM)
Carbonic anhydrase II
α-Glucosidase
Urease
NA
5
k
NA
87.1 ± 0.51
O N
2
5
l
NA
167.8 ± 2.53
NA
NO2
1
0a
0b
12.5 ± 0.42
20.5 ± 0.62
NA
320.6 ± 1.40
NA
N
1
354.4 ± 2.41
HO
1
1
0c
NA
NA
NA
NA
0d
106.5 ± 2.58
367.8 ± 1.10
OCH3
OCH3
1
1
0e
0f
NA
NA
367.7 ± 2.74
351 ± 3.61
NA
H CO
3
88.2 ± 2.27
HO
OH
1
1
0g
0h
49.8 ± 1.68
82.1 ± 2.48
NA
NA
NA
OH
271.7 ± 1.43
OCH3
OCH3

Medicinal Chemistry Research (2021) 30:712–728
Table 1 (continued)
717
Compounds
R
IC50 ± S.E.M (µM)
Carbonic anhydrase II
α-Glucosidase
Urease
NA
1
0i
NA
83.3 ± 1.13
Cl
1
1
0j
NA
NA
NA
NA
308.3 ± 2.68
O N
2
0k
NA
NO2
Standard
Standard
Standard
Acetazolamide
Acarbose
18.2 ± 1.23
–
–
–
–
942 ± 0.74
–
Thiourea
–
20.8 ± 0.75
R aldehydes, NA not active
8
7.1 µM, followed by 5c, 5l, and 5g with IC values of 166.7,
10i is possibly due to the presence of chloro group as an
electron-withdrawing group at para position of the phenyl
group. When 10f is compared with 10e, the higher inhibi-
tion of 10f is due to the replacement of dimethoxy with
dihydroxy groups at the ortho and para positions of the
phenyl group. Likewise, when 10f is compared with 10b, a
slight difference was observed indicating that the presence
of –OH group at ortho position is crucial in the series.
Similarly, when compound 10d is compared with 10e, a
decrease in inhibition of 10e (IC = 397.7 µM) is observed
50
167.8 and 176.7 µM, respectively (Table 1). However, com-
pounds 5e (IC = 270.7 µM) and 5i (IC = 317.4 µM)
exhibited weak inhibition when compared with standard
acarbose (IC = 942 µM). Compound 5k was found to be the
most active compound of the series, about 11 times better than
the standard. This enhanced activity is likely due to the pre-
sence of nitro group as electron-withdrawing substituent at
ortho position of the phenyl ring. Compound 5l having nitro
group at para position has resulted in a significant reduction in
α-glucosidase inhibition (IC = 167.8 µM). This suggests that
50
50
50
5
0
probably due to the presence of an extra –OCH group at
50
3
the inhibitory activity of α-glucosidase enzyme could be
increased with an electron-withdrawing substituent at ortho
position. A slight change in activity was observed between
compounds 5c and 5g. On the other hand, replacing nitro
group at para position of Schiff base moiety (compound 5l)
with –OMe group (compound 5e) (IC = 270.7 µM), has
meta position of the phenyl ring.
Structure–activity relationship of urease enzyme
The in vitro testing against urease suggested that compound 5g
(in case of phenylalanine hydrazine derivatives) was found the
most active having IC50 value of 128.6 µM, followed by 5b
(IC = 173.7 µM), while compounds 5a (IC = 304.5 µM)
50
significantly lowered the α-glucosidase inhibitory activity.
Likewise, when comparing 5e with 5i, a higher inhibition of
50
50
5e is likely due to the presence of two methoxyl groups at
and 5h (IC = 427.3 µM) exhibited weak inhibition when
50
meta and para positions of phenyl ring. In general, the
electron-withdrawing groups at ortho position of phenyl group
are playing a crucial role in the α-glucosidase inhibition.
Whereas, among the leucine hydrazone derivatives, 10i
exerted higher inhibitory activity with IC50 value of
compared with the standard thiourea (IC = 20.8 µM) (Table
50
1). The higher activity of compound 5g is due to the presence
of two hydroxyl groups at ortho and para positions of the
phenyl ring. The lower inhibition of 5a is possibly due to
the replacement of substituted phenyl with pyridine ring. When
8
3.3 µM, then 10f, 10b, 10d, and 10e with IC50 values of
comparing 5g with 5h (IC = 427.3 µM), the decrease in
50
3
51.0, 354.4, 367.8, 397.7 µM, respectively (Table 1).
inhibition is due to the absence of –OH group at ortho position
which further confirm that –OH group substituted at ortho
position of the phenyl ring is playing crucial role against urease
Compound 10i has displayed the most potent activity
against α-glucosidase enzyme when compared with other
derivatives of the series (10a–10k). The higher inhibition of
enzyme in the series. Compounds having –OMe, –NO , and
2

718
Medicinal Chemistry Research (2021) 30:712–728
Fig. 1 The 3D structure of α-glucosidase is shown in complex with the
docked conformation of the most active compound (10i). The
protein–ligand interactions are highlighted in the box. The ligand (10i)
is shown in green stick model, active site amino acids are depicted in
cyan stick model. Hydrogen bonds are shown in green lines
halo substituted phenyls did not show urease inhibition, which
indicates the importance of the hydroxyl group in this series.
In case of leucine hydrazones, compound 10f (IC =
specific targets including CA II, α-glucosidase, and urease
enzymes. Compound 10i was found to be potent against
α-glucosidase with IC = 83.3 µM. The docked view of 10i
50
50
8
8.2 µM) was found to be the most active, whilst compounds
showed that the amino group and the carbonyl oxygen of the
compound mediate strong hydrogen bonds with the side
chains of Glu276 and His348, respectively. Moreover, the
compound has also displayed highest docking score
(−11.41). The binding interactions of 10i are displayed in
Fig. 1. Compound 5K also demonstrated biological activities
in range of IC = 87.1 µM. The docked view of 5K showed
10h (IC = 271.7 µM), 10j (IC = 308.3 µM), and 10a
5
0
50
(IC = 320.6 µM) exerted weak inhibition when compared to
50
the standard (Table 1). The higher activity of 10f is due to the
presence of two hydroxyl groups at ortho and para positions
of the phenyl group, which further justify that the –OH group
substituted at ortho and para positions as stated above.
5
0
that the carbonyl oxygen and the nitro group of the com-
pound formed hydrogen bonds with a surrounded water
molecule and the side chain of His348. Therefore, we can
predict that His348 plays important role in the binding and
stabilization of active inhibitors in the active site of α-glu-
cosidase [55, 56]. While compounds 5c, 5l, and 5g exhibited
moderate inhibitory activities in range of IC =
166–176 µM against α-glucosidase. The amino group of 5c
mediated bidentate interactions with the side chain of
Glu276. Compound 5l exhibited hydrogen bonding with the
side chain of Arg312 and a water molecule, while the
hydroxyl group of compound 5g formed hydrogen bond with
the side chain of Glu304, and the amino group of compound
interacted with a water molecule. In addition, 5g also
mediated hydrophobic interaction with the phenyl ring of
Phe157. Compounds 5b, 5e, 5i, 10f, 10b, 10d, and 10e were
among the least active hits. The docked view of these
compounds showed that the amino group of 5b and 5e
formed hydrogen bond with the side chain of Glu276, while
5i, 10f, 10b, 10d, and 10e interacted with only water
molecules. The binding interactions and docking scores of
the compounds are tabulated in Table 2. The 2D interactions
of the active hits are shown in Fig. S73.
Redocking results
Redocking experiment was performed to evaluate the per-
formance of docking program. The substrate molecule of
α-glucosidase was taken from model, while cocrystalized
inhibitors of urease and CA II were extracted from their
PDB files and redocked in the active site of their respective
protein files. After docking, root mean square deviation
50
(
RMSD) was automatically calculated by software between
the docked and undocked conformations of compounds.
The RMSD for of α-glucosidase substrate was 0.49 Å, while
RMSD for CA II and urease inhibitors were 0.65 and
0
.224 Å, respectively. The redocking results assured that the
docking program can produce reliable results. The redocked
conformations of compounds are presented in Fig. S1
(
Supporting Information).
Molecular docking and interaction analysis of active
hits in α-glucosidase, urease, and CA II
Molecular docking of active inhibitors was carried out to
predict their mode of binding and interactions with their

Medicinal Chemistry Research (2021) 30:712–728
719
Table 2 Molecular docking
Compounds
Docking score
Binding interactions
results of α-glucosidase
inhibitors
Ligand
Receptor
Bonds
Distance (Å)
1
5
5
5
5
0i
k
c
−11.41
−11.20
−9.76
−9.54
−9.36
N19
O24
O38
O54
N17
N17
O21
O54
O53
N39
C10
N17
N17
N17
N39
N19
N22
O21
O24
C45
O24
N19
N22
OE2-GLU276
NE2-HIS348
O-HOH1174
NE2-HIS348
OE1-GLU276
OE2-GLU276
O-HOH1102
N-ARG312
HBD
HBA
HBA
HBA
HBD
HBD
HBA
HBA
HBD
HBA
H-pi
2.57
2.59
2.80
2.88
2.38
2.57
2.64
3.13
3.24
2.91
3.52
3.19
3.03
2.99
2.80
2.94
2.70
2.55
2.99
3.57
3.01
2.85
2.80
l
g
OE2-GLU304
O-HOH1174
6-ring-PHE157
OE2-GLU276
OE1-GLU276
OE2-GLU276
O-HOH1174
O-HOH1228
O-HOH1174
O-HOH1174
O-HOH1102
5-ring-HIS245
O-HOH1102
O-HOH1228
O-HOH1174
5
5
b
e
−8.21
−8.06
HBD
HBD
HBD
HBA
HBD
HBA
HBA
HBA
H–π
5
1
i
−7.63
−7.44
0f
1
0b
−7.21
1
1
0d
0e
−6.58
−6.56
HBA
HBD
HBA
HBA and HBD: hydrogen bond donor
Fig. 2 The 3D structure of urease is presented in complex with the
docked conformation of the most active compound (10f). The
protein–ligand interactions are highlighted in the box. 10f is shown in
olive green stick model, active site amino acids are depicted in
magenta stick model. Hydrogen bonds are shown in green lines.
Metal–ligand interactions are shown in black dotted lines
When tested in urease inhibitory assay, compound 10f
exhibited highest inhibitory potential with IC = 88.2 ±
groups of the compound interacted with the Ni atoms pre-
sent in the active site. While amino groups of the compound
formed hydrogen bonding with the main chain carbonyl
oxygen of Ala636 and a water molecule [56]. The hydroxyl
group of 5b donated a hydrogen bond to the side chains of
50
2
.27 µM, while compounds 5b, 10h, 5a, 10j, 10a, and 5h
depicted moderate-to-least biological activities. The docked
view of 10f (Fig. 2) demonstrated that both the carbonyl

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Medicinal Chemistry Research (2021) 30:712–728
Table 3 Molecular docking
results of urease inhibitors
Compounds
Docking score
Binding interactions
Ligand
Receptor
Bonds
Distance (Å)
1
0f
−18.21
O21
O24
N17
N19
N17
O38
O38
O52
O52
NI842
Metal
Metal
HBD
HBD
HBD
HBA
HBA
HBD
HBD
π–π
1.89
2.06
2.38
3.26
2.97
2.54
1.90
3.39
3.39
2.78
2.59
2.87
3.48
3.18
3.2
NI841
O-HOH884
O-ALA636
O-MET637
NE2-HIS407
NE2-HIS409
OD1-ASP633
OD2-ASP633
5-ring-HIS519
NE2-HIS492
O-HOH1178
NH2-ARG609
NE2-HIS593
5-ring-HIS409
5-ring-HIS407
5
5
1
g
−15.55
−15.24
−15.12
b
6
-ring
0h
N22
HBA
HBA
HBA
HBA
π–π
O24
5
1
1
5
a
−15.08
−12.28
−9.06
−8.27
N39
0j
0a
h
O21
6-ring
6-ring
π–π
2.31
HBA and HBD: hydrogen bond donor
Asp633, while His519 provide π–π interaction to the
compound. Similarly, the side chain of His492 and a water
molecule interacted with the nitrogen and carbonyl oxygen
of 10h. Moreover, Arg609 and His593 provide hydrogen
bond to the nitrogen and carbonyl oxygen of 5a and 10j,
respectively. While the side chain of His409 and His407
provide π–π interaction to the ring of 10a and 5h, respec-
tively. The docking results of active inhibitors are tabulated
in Table 3. The 2D interactions of the active hits at the
active site of urease are presented in Fig. S74.
hydrogen bond with the Phe94. The binding interactions and
docking results of CA II inhibitors are tabulated in Table 4.
The binding mode of the most active hit (5b) is presented in
Fig. 3. The 2D interactions of the active hits within the active
site of CA II are demonstrated in Fig. S75.
Conclusion
In summary, a series of new amino acid hybrid Schiff bases
(5a–5l and 10a–10k) were synthesized and their CA II,
α-glucosidase, and urease inhibitor activities were evaluated
in vitro. Among the phenylalanine hydrazine derivatives,
compounds 5b (11.8 µM) proved to be the most potent against
CA II enzyme, while 10i (83.3 µM) and 10f (88.2 µM) were
found to be the most active scaffolds against α-glucosidase and
urease, respectively. The structure–activity relationship was
discussed. Some compounds proved to be more potent than
the control groups. Furthermore, the molecular docking studies
showed that all the active compounds accommodate well in
the active site of these enzymes by interacting with key amino
acids. These results should be evaluated in vivo to further
assess their potential as enzymes inhibitory drugs.
Among the tested compounds, 5b, 10a, 5d, and 5c
exhibited highest inhibitory potential on CA II inhibitory
assay. The binding modes of those inhibitors in the active site
of CA II showed that the carbonyl moieties of 5b, 10a, and
5
d mediated hydrogen bonding with a water molecule, while
amino nitrogen of 5b, 5d, and 5c interacted with the side
chain of Thr198, His93, and Gln91, respectively, [57].
However, the ring nitrogen of 10a and 5a mediated hydrogen
boding with the side chain of His95. Similarly, the amino and
carbonyl groups of 10b interacted with the side chain of
Thr198 and a water molecule. Furthermore, compounds 5i
and 5e only formed water mediated bridging within the
active site, however, the amino and –OH group of 10g
interacted with a water molecule and Phe94, respectively.
The docked view of 10h depicted that the carbonyl group of
compound formed multiple hydrogen bonds with the Thr197
and Thr198. Similarly, the carbonyl group of 10g interacted
with Thr197 and a water molecule, while His93 stabilized
the compounds 10g, 10d, and 5h through hydrophobic
interaction. In addition, 10d and 5h also interacted with water
molecule in the vicinity. Moreover, 5h also formed a
Experimental
General
All reagents were obtained from Sigma-Aldrich chemical
company, Germany. Solvents used for chemical reactions

Medicinal Chemistry Research (2021) 30:712–728
721
Table 4 Molecular docking
Compounds
Docking score
Binding interactions
results of carbonic anhydrase II
inhibitors
Ligand
Receptor
Bonds
Distance (Å)
5
5
a
−5.15
−4.54
N24
N12
O11
O11
N10
O11
N12
O11
NE-HIS95
OG1-THR198
HOH462
HBA
HBD
HBD
HBA
HBD
HBA
HBD
HBA
π–π
2.75
2.96
2.96
2.79
3.07
2.92
2.71
2.83
2.86
2.56
2.60
2.82
3.95
3.12
3.87
3.15
2.84
3.08
2.66
3.22
4.42
3.01
2.85
2.87
3.33
3.21
3.25
2.79
b
HOH462
5
5
5
c
d
e
−3.92
−4.24
−3.15
OE1-GLN91
N-THR197
HOH279
HOH466
6
-ring
6-ring-PHE129
OG1-THR197
HOH462
5
5
5
1
1
1
1
1
g
−3.60
−3.02
−3.54
−6.28
−4.96
−4.80
−4.55
−2.66
−5.07
O11
O11
O15
HBA
HBA
HBA
π–H
h
HOH466
6
-ring
NE2-HIS95
NE2-HIS95
OG1-THR198
HOH466
i
O29
HBA
π–H
6
-ring
0a
0b
0d
0g
0h
O12
N21
N8
HBA
HBA
HBD
HBA
HBA
H–π
NE2-HIS95
OG1-THR198
HOH466
O9
O9
HOH279
N7
5-ring-HIS93
HOH279
N7
HBD
HBD
HBA
HBA
HBD
HBA
Metal
O26
O9
O-PHE94
N-THR197
N-THR198
OGSER64
HOH340
O9
Standard
N9
O11
O11
ZN260
HBA and HBD: hydrogen bond donor
Fig. 3 The 3D structure of CA II is presented in complex with the
docked conformation of the most active compound (5b). The
protein–ligand interactions are highlighted in the box. The inhibitor is
shown in olive green stick model, active site amino acids are depicted
in coral stick model. Hydrogen bonds are shown in black lines

722
Medicinal Chemistry Research (2021) 30:712–728
were purified and dried by standard procedures. Melting
point was determined using Stuart SMP10 digital melting
point apparatus). The masses of the compounds were con-
firmed from high-resolution electrospray ionization mass
spectrometry (Agilent 6530 LC Q-TOF, country of origin
anhydrous MgSO and evaporated under reduced pressure
4
to obtain Boc-phenylalanine methyl ester (91%). Same
procedure was used for the synthesis of Boc-leucine methyl
ester (yield, 92%) using anhydrous Na CO (1.2 g,
2
3
12 mmol), water (30 mL), and Boc (2.1 g, 10 mmol).
Third step involved the formation of Boc-phenylalanine
hydrazide. Hydrazine monohydrate (2 mL, 32 mmol) was
added to a solution of Boc-phenylalanine methyl ester
(2.5 g, 9.1 mmol) and methanol (20 mL) and stirred under
room temperature for 24 h. The reaction mixture was
thoroughly monitored by TLC. After reaction completion,
the solvent was evaporated under reduced pressure and
the product was washed with methanol (89%). Similarly,
the same procedure was used for the synthesis of Boc-
leucine hydrazide using hydrazine monohydrate (2 mL,
32 mmol) and methanol (25 mL).
1
USA/EU, made in Singapore). The NMR spectra ( H-NMR
1
3
(
600 MHz) and C-NMR (150 MHz)) were recorded on
nuclear magnetic resonance (BRUKER, Zürich, Switzer-
land) spectrometer using the solvent peaks as internal
reference (CDCl , δ : 7.26; δ : 77.2–76.8; DMSO-d , δ :
3
H
C
6
H
2
.49; δ : 40.0–39.1). The following abbreviations: s: sing-
C
let, d: doublet, dd: doublet of doublet, t: triplet, m: multiplet,
J: coupling constant were used to explain NMR signals.
Chemical shifts were expressed in parts per million (δ)
values and coupling constants (J) are given in Hertz (Hz).
ATR-Tensor 37 spectrophotometer (Bruker, Ettlingen,
Baden-Württemberg, Germany) was used the determination
of infrared (IR) spectra. All reactions were monitored by
thin-layer chromatography (TLC). TLC plates were visua-
lized under the UV light at 254 and 366 nm. Solvents for
chromatography were of commercial grade and distilled
prior to use.
Tert-butyl(S, E)-(3-oxo-1-phenyl-3-(2-(pyridine-2-
ylmethylene)hydrazineyl)propyl)carbamate (5a)
2-Pyridinecarboxaldehyde (32.1 mg, 0.3 mmol) was added to a
stirred solution of Boc-phenylalanine hydrazide (55.4 mg,
0.2 mmol) in EtOH (5 mL) at room temperature for 24 h. The
General procedure for the synthesis of amino acids
Schiff bases derivatives
progress of the reaction was monitored with TLC. After
completion reaction, the product (5a) was filtered, and washed
with n-hexane for removing excess amount of aldehydes (Yield
88%). The same procedure was used for the synthesis of other
compounds (5b–5l) using different types of benzaldehydes
moities (0.3 mmol). Similarly, same benzaldehydes were used
for the synthesis of compounds (10a–10k) using Boc-leucine
hydrazide (50 mg, 0.2 mmol) in 5 mL EtOH at room tem-
perature for 24 h. All compounds were obtained in good yields
ranging from 78 to 94%. The structures of all compounds were
Amino acid Schiff bases were synthesized in four steps
following published protocol [58]. L-β-phenylalanine
(
1.65 g, 10 mmol)) and L-leucine (1.3 g, 10 mmol) were
used as a starting materials for the synthesis of amino acid
Schiff bases (5a–5l and 10a–10k). Esterification of car-
boxylic group was done by the addition of thionyl
chloride (2.1 mL, 15 mmol) slowly to a solution of L-
β-phenylalanine (1.65 g, 10 mmol) and methanol (20 mL)
at 0 °C. The reaction solution was stirred (at room tem-
perature) for overnight. Reaction was thoroughly
observed by TLC for assuring that all amount of amino
acid changed to ester or not. After reaction completion,
the organic solvent was evaporated under vacuum and the
resultant product was filtered. Same procedure was used
for the esterification of L-leucine (1.3 g, 10 mmol) using
1
13
established by HRMS, IR, and H- and C-NMR.
Light yellow powder; Yield: 88%; m.p. 130–135 °C; FT-
−
1
IR (solid, cm ): 3321, 3250, 1666, 1514, 1461, 1377,
1
1335, 1290, 1233, 1160; H-NMR (600 MHz, DMSO-d ): δ
6
8.19 (1H, s, NH), 8.59 (1H, CH=N), 7.99, 8.01 (2H, m),
7.99, 7.87 (2H, m), 7.58–7.13 (5H, m), 5.09 (1H, br.s, NH-
Boc), 4.20 (1H, br.s, CH–NH), 2.77, 2.97 (2H, m), 1.30
1
3
(9H, s); C-NMR (150 MHz, DMSO-d ): δ 28.1, 36.6,
6
SOCl (2.1 mL, 15 mmol) and methanol (15 mL).
52.7, 78.2, 119.5, 124.2, 126.3, 128.1, 129.1, 136.8, 137.7,
143.8, 147.2, 149.5, 153.0, 155.4, 168.7, 173.4; HRMS
(ESI ) m/z: 369.1846 [M+H] .
2
Second step was to protect the amino group through the
reaction of tert-Boc reagent. In this step, amino acid methyl
ester (1.75 g, 9.8 mmol) was added to a mixture of anhy-
drous sodium carbonate (1.2 g, 12 mmol) and water (40 mL)
in the round bottom flask (100 mL). Then, tert-Boc (2.1 g,
+
+
Tert-butyl(S,E)-(3-(2-(2-hydroxybenzylidene)hydrazineyl)-3-
oxo-1-phenylpropyl)carbamate (5b)
1
0 mmol) was dissolved in ethyl acetate (20 mL) and then
added to that solution and the reaction was allowed to stir
for 24 h at room temperature. The progress of the reaction
was checked by TLC. When the reaction was completed,
adjusts PH at 5.5 with oxalic acid and then extracted the
desire product through EtOAc. The solvent was dried over
White powder; Yield: 86%; m.p. 115–120 °C; FT-IR (solid,
−
1
cm ): 3333, 2975, 1663, 1614, 1520, 1367, 1264, 1162,
1
1092; H-NMR (CDCl , 600 MHz): δ 10.99 (1H, s, NH),
3
8.03 (1H, CH=N), 7.29‒7.16 (5H, m), 7.05 (2H, d, J =
7.2 Hz), 9.68 (2H, d = 7.8 Hz), 6.80 (1H, t, J = 7.2 Hz),

Medicinal Chemistry Research (2021) 30:712–728
723
5
2
2
1
1
.68 (1H, br.s, NH-Boc), 4.51 (1H, br.s, CH–NH), 3.22‒
Tert-butyl(S,E)-(3-(2-(2,5-dimethoxybenzylidene)
hydrazineyl)-3-oxo-1-phenylpropyl)carbamate (5f)
.98 (2H, m), 1.37 (9H, s); 13C-NMR (150 MHz, CDCl ): δ
3
8.3, 38.5, 55.2, 80.7, 117.0, 117.6, 119.7, 127.0, 128.5,
29.3, 131.3, 132.1, 136.3, 149.4, 151.2, 156.2, 157.8,
Light yellow powder; Yield: 89%; m.p. 160–165 °C; FT-IR
+
+
−1
58.5, 167.9, 172.9; HRMS (ESI ) m/z: 384.1908 [M+H] .
(solid, cm ): 3342, 3248, 1659, 1512, 1453, 1415, 1368,
1
1
309, 1257, 1219, 1164, 1090; H-NMR (600 MHz,
Tert-butyl(S,E)-(3-(2-(2-hydroxy-4-methoxybenzylidene)
DMSO-d ): δ 8.50 (1H, s, NH), 8.30 (1H, CH=N), 7.35
6
hydrazineyl)-3-oxo-1-phenylpropyl)carbamate (5c)
(1H, d, J = 2.4 Hz), 7.31‒7.23 (5H, m), 7.10 (1H, d, J =
8
.4 Hz), 6.99 (1H, dd, J = 8.4, 2.4 Hz), 5.03 (1, d, J =
Yellow amorphous powder; Yield: 80%; m.p. 166–170 °C;
6.0 Hz, NH-Boc), 4.16 (1H, br.s, CH–NH), 3.79 (3H, s,
−
1
FT-IR (solid, cm ): 3341, 3246, 1659, 1607, 1572, 1513,
OCH ), 3.73 (3H, s, OCH ), 3.00–2.992 (1H, m), 2.82–2.73
3
3
1
13
1
6
7
6
447, 1373, 1343, 1306, 1277, 1163; H-NMR (CDCl ,
(1H, m), 1.32 (9H, s); C-NMR (150 MHz, DMSO-d ): δ
3
6
00 MHz): δ 10.39 (1H, s, NH), 7.94 (1H, CH=N),
.23–7.15 (5H, m), 6.92 (1H, d, J = 9.0 Hz), 6.40 (1H, s),
.33 (1H, dd = 8.4, 1.6 Hz), 5.85 (1H, br.s, NH-Boc), 4.53
28.2, 36.2, 56.3, 55.5, 55.4, 78.1, 108.6, 113.6, 117.7,
126.3, 128.1, 129.0, 129.3, 138.9, 142.2, 152.2, 152.3,
+
+
153.3, 168.3, 173.1; HRMS (ESI ) m/z: 428.2195 [M+H] .
(
2
1H, d, J = 6.6 Hz, CH–NH), 3.74 (3H, s, OCH ), 3.13‒
3
.98 (2H, m), 1.37 (9H, s); 13C-NMR (150 MHz, CDCl3):
δ 28.3, 38.6, 55.3, 52.3, 80.6, 101.3, 106.9, 126.9, 127.1,
Tert-butyl(S,E)-(3-(2-(2,4-dihydroxybenzylidene)
hydrazineyl)-3-oxo-1-phenylpropyl)carbamate (5g)
1
1
28.5, 129.5, 132.1, 149.4, 151.1, 156.2, 159.8, 160.5,
+
62.8, 163.0, 167.8, 172.6; HRMS (ESI ) m/z: 414.2033
Dark brown solid powder; Yield: 84%; m.p. 110–120 °C;
+
−1
[
M+H] .
FT-IR (solid, cm ): 3214, 2976, 1666, 1621, 1505, 1450,
1
1
366, 1316, 1230, 1159; H-NMR (600 MHz, DMSO-d ): δ
6
Tert-butyl(S,E)-(3-(2-(4-hydroxy-3-methoxybenzylidene)
hydrazineyl)-3-oxo-1-phenylpropyl)carbamate (5d)
8.27 (1H, s, NH), 9.95 (1H, CH=N), 7.27‒7.26 (5H, br.s),
7.18 (1H, s), 6.33 (2H, d, J = 9.0 Hz), 4.19 (1H, dd, J = 9.0,
4
.8 Hz, CH–NH), 2.96–2.72 (2H, m), 1.30 (9H, s); 13C-
Colorless solid; Yield: 82%; m.p. 118–123 °C; FT-IR
NMR (150 MHz, DMSO-d ): δ 28.2, 37.4, 39.9, 40.0, 52.7,
6
−
1
(
solid, cm ): 3339, 2972, 1663, 1595, 1515, 1456, 1376,
55.0, 78.2, 102.5, 107.7, 110.4, 126.3, 128.1, 131.1, 137.9,
148.4, 155.3, 158.1, 159.4, 160.6, 167.7, 172.4; HRMS
1
1
9
7
274, 1164, 1124, 1030; H-NMR (CDCl , 600 MHz): δ
3
+
+
.53 (1H, s, NH), 7.94 (1H, CH=N), 7.23‒7.15 (5H, m),
.02 (1H, d = 1.6 Hz), 6.90 (1H, dd, J = 7.8, 1.6 Hz), 6.84
(ESI ) m/z: 400.1862 [M+H] .
(
1H, d, J = 7.8 Hz), 5.43 (1H, br.s, NH-Boc), 4.44 (1H, t,
Tert-butyl(S, E)-(3-(2-(4-hydroxybenzylidene)hydrazineyl)-3-
J = 7.8 Hz, CH–NH), 3.94 (3H, s, OCH ), 3.25‒2.96 (2H,
oxo-1-phenylpropyl)carbamate (5h)
3
m), 1.38 (9H, s); 13C-NMR (150 MHz, CDCl ): δ 28.3,
3
3
1
1
8.5, 52.5, 56.1, 76.7, 79.6, 107.6, 114.0, 123.0, 125.8,
26.7, 128.2, 129.3, 145.1, 147.0, 149.2, 155.2, 167.8,
73.3; HRMS (ESI ) m/z: 414.2021 [M+H] .
Colorless solid; Yield: 85%; m.p. 190–200 °C; FT-IR
−
1
(solid, cm ): 3324, 1689, 1649, 1594, 1504, 1432, 1369,
+
+
1
1332, 1258, 1206, 1163; H-NMR (600 MHz, DMSO-d ): δ
6
8.07 (1H, s, NH), 7.87 (1H, CH=N), 7.50 (2H, dd, J = 7.8,
Tert-butyl (S,E)-(3-(2-(4-methoxybenzylidene)hydrazineyl)-
-oxo-1-phenylpropyl)carbamate (5e)
2.4 Hz), 7.29‒7.22 (5H, br.s), 6.82 (2H, dd, J = 8.4,
3
2.4 Hz), 5.04 (1H, br.s, NH-Boc), 4.19 (1H, dd, J = 9.0,
1
3
4
.8 Hz, CH–NH), 2.96‒2.73 (2H, m), 1.30 (9H, s); C-
Yellow amorphous powder; Yield: 83%; m.p. 173–178 °C;
NMR (150 MHz, DMSO-d ): δ 28.2, 36.5, 37.4, 39.8, 52.9,
6
−
1
FT-IR (solid, cm ): 3330, 3250, 1659, 1601, 1516, 1452,
55.1, 78.1, 115.8, 124.9, 126.3, 128.1, 128.8, 129.1, 129.3,
138.0, 143.8, 147.2, 155.4, 159.7, 168.0, 172.8; HRMS
1
1
374, 1309, 1252, 1164, 1024; H-NMR (600 MHz,
+
+
CDCl ): δ 9.08 (1H, s, NH), 7.62 (1H, CH=N), 7.62‒7.56
(ESI ) m/z: 384.1894 [M+H] .
3
(
2H, dd, J = 8.4 Hz), 7.58‒7.13 (5H, m), 6.91–6.85 (2H,
dd, J = 8.4 Hz), 5.29 (1H, br.s, NH-Boc), 4.40 (1H, dd, J =
.8, 7.2 Hz, CH–NH), 3.84 (3H, s, OCH ), 5.29 (1H, br.s,
Tert-butyl(S,E)-(3-(2-(3,4-dimethoxybenzylidene)
hydrazineyl)-3-oxo-1-phenylpropyl)carbamate (5i)
7
3
13
CH–NH), 3.21‒3.01 (2H, m), 1.39 (9H, s); C-NMR
150 MHz, CDCl ): δ 28.3, 38.6, 52.3, 55.3, 79.6, 114.0,
(
Colorless solid; Yield: 91%; m.p. 168–173 °C; FT-IR
3
−
1
1
1
3
25.9, 126.0, 126.7, 128.7, 129.4, 129.5, 136.5, 144.4,
(solid, cm ): 3324, 1690, 1649, 1595, 1511, 1433, 1368,
+
1
48.5, 155.1, 161.4, 167.5, 173.1; HRMS (ESI ) (m/z):
1331, 1257, 1204, 1163; H-NMR (600 MHz, CDCl3):
+
98.2077 [M+H] .
δ 9.14 (1H, s, NH), 7.61 (1H, CH=N), 7.40 (1H, s),

724
Medicinal Chemistry Research (2021) 30:712–728
7
(
.28‒7.21 (5H, br.s), 7.07 (1H, dd, J = 8.4, 2.4 Hz), 6.84
1H, d, J = 8.4 Hz), 5.28 (1H, br.s, NH-Boc), 4.41 (1H, dd,
J = 7.8, 7.2 Hz, CH–NH), 3.95 (6H, s, 2 × OCH ),
Tert-butyl (S,E)-(4-methyl-1-oxo-1-(2-(pyridine-2-
ylmethylene)hydrazineyl)pentan-2-yl)carbamate (10a)
3
13
3
.25–2.95 (2H, m), 1.39 (9H, s); C-NMR (150 MHz,
White powder; Yield: 82%; m.p. 67–72 °C; FT-IR (solid,
−
1
DMSO): δ 28.3, 38.5, 52.5, 55.9, 56.1, 78.1, 108.2, 110.4,
1
1
cm ): 2962, 1671, 1509, 1463, 1366, 1241, 1162, 1043;
1
22.3, 126.2, 126.7, 128.7, 129.3, 136.6, 144.7, 149.4,
H-NMR (CDCl , 600 MHz): δ 10.12 (1H, s, NH), 8.56
3
51.3, 155.2, 167.5, 173.2; ESI-HRMS (m/z): 428.2181
(1H, dd, J = 10.8, 4.2 Hz), 7.99 (1H, s, CH=N), 7.96 (1H,
d, J = 7.8 Hz), 7.68 (1H, t, J = 7.2 Hz), 7.25 (1H, br.s),
+
[
M+H] .
5
.39 (1H, br.s, NH-Boc), 4.29 (1H, br.s, CH–NH), 1.80
Tert-butyl(S,E)-(3-(2-(4-chlorobenzylidene)hydrazineyl)-3-
oxo-1-phenylpropyl)carbamate (5j)
(1H, br.s), 1.53 (1H, br.s), 1.65 (1H, m), 1.40 (9H, s), 1.04
(6H, d, J = 6.6 Hz); C-NMR (150 MHz, CDCl ): δ 21.6,
3
1
3
2
3.3, 28.3, 42.1, 49.7, 80.0, 120.0, 124.2, 136.5, 145.2,
+
Colorless solid; Yield: 89%; m.p. 186–192 °C; FT-IR
149.1, 152.8, 155.7, 175.7; HRMS (ESI ) m/z: 334.20
[M+H] .
−
1
+
(
1
solid, cm ): 3338, 1669, 1596, 1527, 1432, 1369, 1314,
1
264, 1169, 1054, 1018; H-NMR (DMSO-d , 600 MHz):
6
δ 8.13 (1H, s, NH), 7.87 (1H, CH=N), 7.61 (2H, dd, J =
.0, 2.4 Hz), 7.43 (2H, dd, J = 9.0, 2.4 Hz), 7.20–7.13
5H, m), 4.98 (1H, br.s, NH-Boc), 4.13 (1H, br.s,
Tert-butyl(S,E)-(1-(2-(2-hydroxybenzylidene)hydrazineyl)-4-
methyl-1-oxopentan-2-yl)carbamate (10b)
9
(
1
3
CH–NH), 2.85‒2.66 (2H, m), 1.20 (9H, s); C-NMR
150 MHz, DMSO): δ 28.2, 36.6, 52.7, 78.1, 126.3, 128.1,
Light Yellow solid powder; Yield: 86%; m.p. 80–90 °C;
FT-IR (solid, cm− ): 2962, 1666, 1615, 1525, 1489,
1
(
1
1
1
28.7, 129.1, 130.8, 132.9, 133.2, 134.3, 137.9, 142.1,
1362, 1270, 1239, 1204, 1160; H-NMR (CDCl3,
+
45.6 155.4, 168.6, 173.3; HRMS (ESI ) m/z: 424.1403
600 MHz): 10.77 (1H, s, NH), 8.15 (1H, s, CH=N), 7.20
(1H, t, J = 7.2 Hz), 7.05 (1H, dd, J = 8.4, 4.2 Hz), 6.85
3
5
37
+
(
Cl ), 426.1408 (Cl ) [M+Na] .
(
1H, d, J = 7.8 Hz), 6.75 (1H, t, J = 8.4, 6.0 Hz), 5.73
Tert-butyl(S,E)-(3-(2-(2-nitrobenzylidene)hydrazineyl)-3-
(1H, br.s, NH-Boc), 4.30 (1H, br.s, CH–NH), 1.74 (1H,
oxo-1-phenylpropyl)carbamate (5k)
m), 1.72 (1H, br.s), 1.65 (1H, m), 1.41 (9H, s), 0.95 (6H,
1
3
d, J = 6.6 Hz); C-NMR (150 MHz, CDCl ): δ 22.8,
3
Dark yellow amorphous powder; Yield: 87%; m.p.
23.3, 28.3, 40.9, 52.2, 80.7, 117.0, 119.2, 130.9, 131.7,
−
1
+
1
1
95–200 °C; FT-IR (solid, cm ): 2973, 1668, 1521, 1339,
149.4, 151.1, 156.6, 158.5, 169.2; HRMS (ESI ) m/z:
1
+
260, 1220, 1169, 1056; H-NMR (600 MHz, DMSO-d ):
350.1920 [M+H] .
6
δ 8.60 (1H, s, NH), 7.37 (1H, CH=N), 8.07 (1H, t, J = 9.0,
8
7
5
.4 Hz), 8.20 (1H, d, J = 7.8 Hz), 7.82 (1H, dd, J = 8.4,
.8 Hz), 7.67 (1H, t, J = 7.8, 7.2 Hz), 7.30–7.17 (5H, m),
.06 (1H, br.s, NH-Boc), 4.23 (1H, br.s, CH–NH), 2.98
Tert-buty(S,E)-(1-(2-(4-hydroxy-3-methoxybenzylidene)
hydrazineyl)-4-methyl-1-oxopentan-2-yl)carbamate (10c)
1
3
(
1H, m), 2.82 (1H, m), 1.30 (9H, s); C-NMR (150 MHz,
DMSO): δ 28.2, 36.5, 52.7, 78.1, 124.7, 126.3, 127.8,
Colorless solid; Yield: 84%; m.p. 120–125 °C; FT-IR
−
1
(solid, cm ): 2960, 1663, 1594, 1511, 1461, 1428, 1370,
1
1
1
28.5, 129.2, 130.5, 133.7, 138.2, 142.3, 148.1, 155.4,
1276, 1162, 1119, 1035; H-NMR (600 MHz, CDCl ): δ
3
+
+
68.9, 173.5; HRMS (ESI ) m/z: 435.1363 [M+Na] .
9.24 (1H, s, NH), 7.67 (1H, s), 7.34 (1H, s, CH=N), 6.97
(
1H, d, J = 7.8 Hz), 6.89 (1H, d, J = 7.8 Hz), 6.85 (1H, d,
Tert-butyl(S,E)-(4-methyl-1-(2-(2-nitrobenzylidene)
J = 7.8 Hz), 5.91 (1H, br.s, NH-Boc), 5.22 (1H, br.s,
hydrazineyl)-1-oxopentan-2-yl)carbamate (5l)
CH–NH), 3.88 (3H, s, OCH ), 1.85 (1H, m), 1.80 (1H, m),
3
1
3
1
.68 (1H, m), 1.42 (9H, s), 0.94 (6H, d, J = 6.6 Hz); C-
Yellow solid powder; Yield: 94%; m.p. 201–208 °C; FT-IR
NMR (150 MHz, CDCl ): δ 22.7, 24.9, 28.3, 42.4, 49.8,
3
−
1
(
1
7
solid, cm ): 2973, 1668, 1521, 1339, 1260, 1220, 1169,
55.9, 56.1, 79.5, 107.2, 114.0, 123.2, 126.0, 144.7, 149.1,
1
+
+
056; H-NMR (600 MHz, DMSO-d ): δ 8.37 (1H, s, NH),
155.8, 175.0; HRMS (ESI ) m/z: 380.2064 [M+H] .
6
.37 (1H, CH=N), 8.07 (1H, t, J = 9.0, 8.4 Hz), 8.31 (2H, t,
J = 9.0 Hz), 7.96 (2H, dd, J = 8.4 Hz), 7.32‒7.26 (4H, m),
Tert-butyl(S,E)-(1-(2-(4-methoxybenzylidene)hydrazineyl)-4-
methyl-1-oxopentan-2-yl)carbamate (10d)
7
.21 (1H, m), 5.13 (1H, br.s, NH-Boc), 4.26 (1H, br.s,
1
3
CH–NH), 2.99 (1H, m), 2.87 (1H, m), 1.32 (9H, s); C-
NMR (150 MHz, DMSO): δ 28.2, 36.7, 37.2, 39.3, 40.0,
Light brown amorphous powder; Yield: 88%; m.p.
−
1
5
1
2.7, 55.4, 78.1, 124.0, 126.3, 127.7, 128.1, 129.2, 137.9,
40.6, 141.0, 144.5, 147.7, 155.4, 169.2, 173.7; HRMS
92–100 °C; FT-IR (solid, cm ): 2961, 1665, 1604, 1562,
1
1508, 1460, 1365, 1304, 1246, 1163, 1028; H-NMR
+
+
(
ESI ) m/z: 435.1363 [M+Na] .
(CDCl , 600 MHz): δ 9.44 (1H, s, NH), 7.82 (1H, s,
3

Medicinal Chemistry Research (2021) 30:712–728
725
CH=N), 7.58 (2H, d, J = 8.4 Hz), 6.88 (2H, d, J = 8.4 Hz),
Tert-butyl(S,E)-(1-(2-(3,4-dimethoxybenzylidene)
5
.22 (1H, br.s, NH-Boc), 4.22 (1H, br.s, CH–NH), 3.82
hydrazineyl)-4-methyl-1-oxopentan-2-yl)carbamate (10h)
(
1
3H, s, OCH ), 1.82 (1H, m), 1.70 (1H, m), 1.62 (1H, m),
.42 (9H, s), 0.94 (6H, d, J = 6.6 Hz); C-NMR (150 MHz,
3
13
Colorless solid; Yield: 79%; m.p. 99–105 °C; FT-IR (solid,
−
1
CDCl ): δ 21.7, 22.7, 28.3, 42.2, 49.2, 55.3, 79.2, 114.1,
cm ): 2961, 1667, 1602, 1572, 1510, 1460, 1418, 1389,
3
1
1
4.2, 114.3, 126.2, 128.8, 129.4, 144.4, 161.4, 175.2;
1364, 1265, 1237, 1162, 1052; H-NMR (DMSO-d6,
600 MHz): δ 11.12 (1H, s, NH), 7.88 (1H, s, CH=N), 7.28
+
+
HRMS (ESI ) m/z: 364.2227 [M+H] .
(1H, s), 7.00 (1H, d, J = 8.4 Hz), 6.87 (1H, d, J = 8.4 Hz),
Tert-butyl(S,E)-(1-(2-(2,5-dimethoxybenzylidene)
hydrazineyl)-4-methyl-1-oxopentan-2-yl)carbamate (10e)
4.92 (1H, br.s, NH-Boc), 4.01 (1H, dd, J = 8.4, 6.0 Hz,
CH–NH), 4.01 (3H, s, OCH ), 3.78 (3H, s, OCH ), 1.72
3
3
(
1H, m), 1.63 (1H, m), 1.52 (1H, m), 1.36 (9H, s), 0.99 (3H,
1
3
Colorless solid; Yield: 84%; m.p. 88–95 °C; FT-IR (solid,
d, J = 6.6 Hz), 0.88 (3H, d, J = 6.6 Hz);
C-NMR
−
1
cm ): 2959, 1669, 1495, 1462, 1425, 1362, 1253, 1218,
(150 MHz, DMSO): δ 21.7, 22.9, 28.3, 39.4, 40.1, 49.4,
1
1
164, 1111, 1042; H-NMR (DMSO-d , 600 MHz): δ 11.23
52.0, 55.2, 78.0, 107.6, 111.5, 121.5, 127.0, 143.3, 147.0,
6
+
+
(
7
1H, s, NH), 8.28 (1H, s, CH=N), 7.30 (1H, d, J = 1.8 Hz),
.06 (2H, d, J = 8.4 Hz), 4.92 (1H, br.s, NH-Boc), 3.99
1H, br.s, CH–NH), 3.99 (3H, s, OCH ), 3.79 (3H, s,
149.0, 155.4, 174.1; HRMS (ESI ) m/z: 394.1983 [M+H] .
(
Tert-butyl(S,E)-(1-(2-(4-chlorobenzylidene)hydrazineyl)-4-
3
OCH ), 1.71 (1H, m), 1.61 (1H, m), 1.50 (1H, m), 1.44 (9H,
s), 0.96 (6H, d, J = 6.6 Hz); C-NMR (150 MHz, DMSO):
δ 21.2, 22.9, 28.2, 39.5, 40.1, 49.4, 52.0, 55.3, 78.0, 108.5,
methyl-1-oxopentan-2-yl)carbamate (10i)
3
13
Colorless solid; Yield: 85%; m.p. 151–155 °C; FT-IR (solid,
−1
1
13.4, 117.5, 122.8, 138.7, 142.1, 152.1, 155.6, 174.3;
cm ): 2958, 1667, 1523, 1489, 1458, 1392, 1366, 1319,
+
+
1
HRMS (ESI ) m/z: 394.2340 [M+H] .
1279, 1236, 1091, 1054, 1012; H-NMR (600 MHz, DMSO-
d6): δ 11.30 (1H, s, NH), 7.96 (1H, s, CH=N), 7.70 (2H, dd,
J = 8.4 Hz), 7.50 (2H, dd, J = 8.4 Hz), 6.87 (1H, d, J =
8.4 Hz), 4.93 (1H, br.s, NH-Boc), 4.02 (1H, dd, J = 8.4,
Tert-butyl(S,E)-(1-(2-(2,4-dihydroxybenzylidene)
hydrazineyl)-4-methyl-1-oxopentan-2-yl)carbamate (10f)
5.4 Hz, CH–NH), 1.70 (1H, m), 1.61 (1H, m), 1.50 (1H, m),
Dark brown solid powder; Yield: 82%; m.p. 101–110 °C;
1.36 (9H, s), 0.94 (3H, d, J = 6.6 Hz), 0.88 (3H, d, J =
−
1
13
FT-IR (solid, cm ): 2966, 1667, 1622, 1511, 1457, 1366,
6.6 Hz); C-NMR (150 MHz, DMSO-d ): δ 21.3, 22.9, 28.3,
6
1
1
8
321, 1235, 1162; 1H-NMR (DMSO-d , 600 MHz): δ
1.22 (1H, s, NH), 8.28 (1H, s, CH=N), 7.24 (1H, d, J =
.4 Hz), 7.05 (1H, d, J = 8.4 Hz), 6.30 (1H, s), 4.83 (1H,
39.5, 40.1, 49.3, 52.0, 79.2, 128.2, 128.9, 133.2, 134.3, 142.0,
6
+
35
145.5, 155.5, 174.4; HRMS (ESI ) m/z: 390.1563 (Cl ),
392.1540 (Cl ) [M+Na] .
3
7
+
br.s, NH-Boc), 4.00 (1H, br.s, CH–NH), 1.69 (1H, m),
1
6
3
1
3
.61 (1H, m), 1.52 (1H, m), 1.36 (9H, s), 0.91 (6H, d, J =
.6 Hz); C-NMR (150 MHz, DMSO): δ 21.2, 24.3, 28.2,
Tert-butyl(S, E)-(4-methyl-1-(2-(2-nitrobenzylidene)
hydrazineyl)-1-oxopentan-2-yl)carbamate (10j)
1
3
9.5, 51.8, 78.0, 102.5, 107.7, 110.3, 127.9, 131.2, 142.2,
+
48.4, 155.4, 158.2, 160.6, 168.6; HRMS (ESI ) m/z:
Light yellow powder; Yield: 80%; m.p. 164–168 °C; FT-IR
+
−1
66.1768 [M+H] .
(solid, cm ): 2973, 1668, 1521, 1339, 1260, 1220, 1169,
1
1
056; H-NMR (600 MHz, DMSO-d ): δ 8.38 (1H, s, NH),
6
Tert-butyl(S,E)-(1-(2-(4-hydroxybenzylidene)hydrazineyl)-4-
methyl-1-oxopentan-2-yl)carbamate (10g)
8.67 (1H, s, CH=N), 8.06 (2H, t, J = 8.4 Hz), 7.80 (1H, t,
J = 7.8, 7.2 Hz), 7.66 (1H, dd, J = 7.2 Hz), 4.91 (1H, br.s,
NH-Boc), 4.03 (1H, dd, J = 8.4, 6.0 Hz, CH–NH), 1.71 (1H,
m), 1.63 (1H, m), 1.54 (1H, m), 1.37 (9H, s), 0.92 (3H, d,
Light brown amorphous powder; Yield: 89%; m.p.
−
1
13
1
1
6
05–112 °C; FT-IR (solid, cm ): 2965, 1661, 1604, 1511,
J = 6.6 Hz), 0.87 (3H, d, J = 6.6 Hz); C-NMR (150 MHz,
1
444, 1366, 1239, 1161, 1055, 1022; H-NMR (DMSO-d ,
00 MHz): δ 11.26 (1H, s, NH), 7.85 (1H, s, CH=N), 7.49
DMSO-d ): δ 21.2, 24.3, 28.2, 39.7, 49.4, 52.1, 78.1, 124.7,
6
6
127.4, 128.6, 130.5, 133.6, 138.6, 142.1, 148.2, 155.5,
+
+
(
2H, d, J = 8.4 Hz), 6.84 (2H, d, J = 8.4 Hz), 4.92 (1H, br.s,
174.7; HRMS (ESI ) m/z: 401.1812 [M+Na] .
NH-Boc), 4.00 (1H, t, J = 8.4, 5.4 Hz, CH–NH), 1.71 (1H,
m), 1.61 (1H, m), 1.51 (1H, m), 1.44 (9H, s), 0.95 ((3H, d,
J = 6.6 Hz), 0.88 (3H, d, J = 6.6 Hz); C-NMR (150 MHz,
DMSO): δ 21.7, 22.9, 28.3, 39.4, 40.1, 49.3, 51.9, 78.0,
Tert-butyl(S,E)-(4-methyl-1-(2-(4-nitrobenzylidene)
hydrazineyl)-1-oxopentan-2-yl)carbamate (10k)
13
1
1
15.7, 125.1, 128.3, 143.5, 147.2, 155.4, 159.5, 159.5,
74.0; HRMS (ESI ) m/z: 350.2070 [M+H] .
Light yellow powder; Yield: 82%; m.p. 153–158 °C; FT-IR
(solid, cm ): 2973, 1668, 1521, 1339, 1260, 1220, 1169,
+
+
−1

726
Medicinal Chemistry Research (2021) 30:712–728
1
1
8
056; H-NMR (DMSO-d , 600 MHz): δ 8.10 (1H, s, NH),
test compounds (0.5 mmol), and 55 substrate urea
(100 mmol) at 30 °C for 15 min. To each vial, phenolic
reagent (A) 45 μL and 70 μL of alkali reagent (B) were
added [56]. Phenolic reagent (A) was a combination of 1%
w/v phenol and 0.005% w/v sodium nitroprusside. While
Alkali reagent (B) was of composition (0.5% w/v NaOH
and 0.1% w/v NaOCl). To observe urease inhibition of new
derivatives, method published by Weatherburn was used
considering release of ammonia upon hydrolysis. Absor-
bance values were taken after 50 min using microplate
reader (xMark™ Microplate Spectrophotometer, BIO-
RAD). After 50 min, the increasing absorbance at 630 nm
was measured in a microplate reader (xMark™ Microplate
Spectrophotometer, BIO-RAD). Each reaction was per-
formed in triplicate using a volume of 200 μL at end. As a
standard urease inhibitor, we have used thiourea.
6
.36 (1H, s, CH=N), 8.32 (2H, d, J = 9.0 Hz), 7.96 (1H, d,
J = 8.4 Hz), 7.92 (1H, d, J = 8.4 Hz), 4.97 (1H, br.s, NH-Boc),
.07 (1H, dd, J = 8.4, 6.0 Hz, CH–NH), 1.72 (1H, m), 1.63
1H, m), 1.52 (1H, m), 1.38 (9H, s), 0.97 (3H, d, J = 6.6 Hz),
.90 (3H, d, J = 6.6 Hz); C-NMR (150 MHz, DMSO): δ
1.4, 22.9, 28.2, 39.7, 40.0, 49.3, 52.1, 78.1, 79.2, 124.0,
27.5, 140.8, 144.4, 147.7, 155.5, 174.8; HRMS (ESI ) m/z:
01.1478 [M+Na] .
4
(
0
2
1
4
13
+
+
CA II inhibition assay
The total reaction mixture comprised of 20 µL (0.5 mmol/
well) of test compounds (10% DMSO in total), then 140 µL
of HEPES–Tris buffer (20 mmol, pH = 7.4), 20 µL of pur-
ified bovine erythrocyte CA II (1 mg/mL, 0.1 units/well)
prepared in buffer, and finally 20 µL substrate 4-nitrophenyl
acetate (4-NPA, 0.7 mmol) were added to attain final
volume of 200 µL/well [59]. Enzyme (EC 4.2.1.1, Sigma-
Aldrich, St. Louis, MO, USA) along with tested compounds
were incubated for 15 min in a 96-well plate. Then, the
reaction was started by adding 20 µL of substrate (4-nitro-
phenyl acetate) and continuously monitoring the rate
The percent inhibition was calculated from the formula
given below
%
Inhibition ¼ 100 À ðOD test=OD controlÞ Â 100:
Molecular docking
(
velocities) of product formation for 30 min with the inter-
Molecular docking of active inhibitors was performed on
their respective targets by Molecular Operating Environment
software (MOE, 2014.09). The X-ray crystallographic
structures of CA II (PDB ID: 1BN1) and urease (PDB ID:
4H9M) were retrieved from RCSB Protein Data Bank. The
homology model α-glucosidase was developed by using the
primary sequence of yeast (S. cerevisiae) from UniProtKB
vals of 1 min, at 25 °C by using a microplate reader (Bio-
Rad, Molecular Devices, CA, USA).
The percent inhibition was calculated from the formula
given below
%
Inhibition ¼ 100 À ðOD test=OD controlÞ Â 100:
(http://www.uniprot.org/) with accession ID = P53341.
α-Glucosidase inhibition assay
Homology search was built by SwissModel server using the
crystallographic structure of S. cerevisiae isomaltase (3AJ7)
as a template, which depicted 72.4% sequence similarity
with our query. Later, model was refined by energy mini-
mization (up to 0.1 RMS gradients). The refined structure
was scrutinized by Ramachandaran plot which showed that
86.7, 12.3, 0.6, and 0.4% residues are present in most
favored, allowed, generously allowed, and generously dis-
allowed regions (Ala278 and Thr566, which are not a part of
active site), respectively. Thus, the model is good to be used
in docking studies. The 3D structures of protein targets were
prepared for molecular docking by 3D protonate command
of MOE, which add hydrogens and atomic charges on pro-
teins using MMFF94x force field (RMSD gradient =
This study was carried out by following the published
methodology [55, 60] with slight modifications. In this
assay, test compounds (20 µL/well) of different concentra-
tions, α-glucosidase enzyme solution (20 µL/well) from
Saccharomyces cerevisiae, and phosphate-buffered saline
buffer (pH 6.8) (135 µL/well), were employed to a 96-well
plate followed by incubation at 37 °C for 15 min and blank
absorbance without the substrate was recorded. Changes in
absorbance were recorded for 30 min at 400 nm after
addition of substrate (25 µL).
The percent inhibition was calculated from the formula
given below
.1 kcal × mol− Å ). For docking, active site was defined
1
−1
0
%
Inhibition ¼ 100 À ðOD test=OD controlÞ Â 100:
around the 3 Å vicinity of the cocrystalized ligands of CA II
and urease and the docked substrate of α-glucosidase model.
The molecular docking of newly synthesized amino acid
clubbed Schiff bases was carried out by applying the triangle
matcher algorithm and London dG scoring function (MOE,
2014.09). The docking protocol was tested by redocking
experiments for each target prior to the docking of newly
Urease inhibition protocol
The in vitro assay of urease inhibition was performed in
total 200 µL reaction by incubating 25 µL urease enzyme (1
unit/well) from Canavalia ensiformis (Jack bean), 5 µL of

Medicinal Chemistry Research (2021) 30:712–728
727
synthesized inhibitors. The best docked solution of each
compound against its specific target was selected on the
basis of docking score and binding interactions [61].
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Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
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