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com/scientificreports

OPEN Pharmaceutical properties of

novel 3-((diisopropylamino)
methyl)-5-(4-((4-(dimethylamino)
benzylidene) imino) phenyl)-1,3,4-
oxadiazole-2(3 H)-thione
Khalida F. Al-azawi1, Butheina A. Hasoon1, Raid A. Ismail1, Khetam H. Rasool2,
Majid S. Jabir1, Suaad S. Shaker1, Kareem H. Jawad3, Ahmed Mutanabbi Abdula2,
Suresh Ghotekar4 & Ayman A. Swelum5
The synthesis of novel chemical compounds is crucial for developing new pharmaceuticals and
antimicrobial agents. The Mannich reaction, involving Maunch bases known as carrier beta-amino-
ketone molecules, is significant for producing nitrogen-containing compounds. This study investigates
a new compound for its potential biological activities, particularly its antibacterial and anticancer
properties. A new chemical compound, (3-((diisopropylamino)methyl)-5-(4-((4-(dimethylamino)
benzylidene)imino)phenyl)-1,3,4-oxadiazole-2(3 H)-thione) (DMDBIPOT), was synthesized through
the Mannich reaction, where 1,3,4-oxadiazole-2-thiol derivatives reacted with various secondary
amines and formaldehyde. The compound was characterized using FTIR, 1 H NMR, and 13C NMR
spectroscopy. Its antibacterial activity was tested against Klebsiella pneumoniae isolates, and its
antioxidant properties were evaluated using the DPPH assay. Additionally, the anticancer activity was
assessed using the MTT assay on the PC-3 prostate cancer cell line and in silico study. The synthesized
compound exhibited strong antibacterial activity against K. pneumoniae, significantly outperforming
Amikacin (P ≤ 0.05). It effectively prevented biofilm formation on urinary catheters, confirmed by
atomic force microscopy (AFM). The findings indicate that DMDBIPOT effectively inhibits microbial
biofilm growth, suggesting its potential as a preservative for Foley catheters. The DPPH assay
demonstrated that higher concentrations of the compound resulted in greater free radical scavenging
activity. Furthermore, the MTT assay showed significant cytotoxic effects against PC-3 cells, indicating
that the compound stimulates programmed cell death. The docking study confirmed the interaction
of DMDBIPOT with the target binding pocket, validating its efficacy as a therapeutic agent. The
findings suggest that the novel chemical compound possesses potent antibacterial properties and
exhibits significant anticancer activity by interacting with DNA and inducing apoptosis in cancer cells.
These results highlight the potential clinical applications of the compound in treating infections and
cancer. This study presents a novel chemical compound synthesized through the Mannich reaction,
demonstrating promising antibacterial and anticancer activities. The compound’s ability to inhibit
biofilm formation and induce cytotoxic effects suggest that it as a candidate for future therapeutic
development.

Keywords DMDBIPOT, Antibactreial, Anticancer, Antioxidant, Molecular docking

1Department of Applied Sciences, University of Technology-Iraq, Baghdad, Iraq. 2College of Science, Mustansiriyah

University, Baghdad, Iraq. 3Department of Laser and Optoelectronics Engineering, University of Technology-Iraq,
Baghdad, Iraq. 4Centre for Herbal Pharmacology and Environmental Sustainability, Chettinad Hospital and Research
Institute, Chettinad Academy of Research and Education, Kelambakkam, Tamil Nadu 603103, India. 5Department
of Animal Production, College of Food and Agriculture Sciences, King Saud University, 11451 Riyadh, Saudi Arabia.
email: 100131@uotechnology.edu.iq; aswelum@ksu.edu.sa

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A molecule containing beta-amino ketones, referred to as a Mannich base, is the ultimate result of the Mannich
reaction1. In a Mannich reaction, a nucleophilic addition process, a molecule containing one or more active
hydrogens is condensed with a primary or secondary amine, formaldehyde (or any other aldehyde), and another
chemical containing one or more active hydrogens2. Scheme 1 shows the schematic diagram of the public
Mannich reaction. Moreover, Mannich rules are significant bio-active leads, and neither are pharmacophores
utilized in synthesizing the number of likely rise-value drugs that consist of aminoalkyl chains. The number
of drugs, inclusive cocaine, trihexyphenidyl, biperiden, fluoxetine, ethacrynic acid, atropine, procyclidine,
ranitidine, and others, are clinically efficacious Mannich rule that involves aminoalkyl chains3. The importance
of Mannich bases in expanding artificial drug chemistry is widely acknowledged. Literature research shows
that the Mannich rule is much more reactive and readily converted via other molecules. Like, they can be less
able to output physiologically active amino alcohols4. Manichean rule is well recognized for its potent anti-
inflammatory5, anticancer6, Antiparastic agent7, antibacterial8, antifungal9, anticonvulsant10, anthelmintic11,
antitubercular12, analgesic13, anti-HIV14, antimicrobial15, antipsychotic16, antiviral17, and other properties.
Mannich rule is well-known for its utilization in superficies-active agents, resins, polymers, detergent additives,
and other applications to supplement their biological activity18,19.
The ongoing quest for novel pharmaceutical agents is critical in addressing the rising prevalence of drug-
resistant pathogens and the need for effective anticancer therapies. The synthesis of new chemical compounds,
particularly through innovative methodologies like the Mannich reaction, is pivotal in the development of
bioactive molecules. The Mannich reaction facilitates the formation of Mannich bases, which are key intermediates
in synthetic organic chemistry, allowing for the introduction of nitrogen-containing functionalities into diverse
molecular frameworks. This transformation has significant implications in drug design, as these compounds
often exhibit enhanced biological activities, including antibacterial and anticancer effects20.
Recent studies have underscored the importance of synthetic chemistry in drug discovery, highlighting the
versatility of Mannich bases in generating pharmacologically relevant scaffolds. For instance, the work by Pu et
al. has demonstrated the application of the Mannich reaction in modifying natural products to improve their
therapeutic profiles1. Similarly, Joshi et al. have emphasized the utility of Mannich bases in developing new
antitubercular agents, showcasing their potential across various therapeutic areas2.
To get around the constraint, prodrugs of Mannich rules via other active molecules have been formed.
Mannich rules of 2-naphthol, optically pure chiral, catalyze the enantioselective synthesis of carbon-carbon
bonds (ligand accelerated and metal-assisted). Mannich rules and their derivatives are the starting point via a
synthesis of bioactive chemicals18. Compounds containing nitrogen are commonly synthesized by the Mannich

Scheme 1. Synthesis of (3-((diisopropylamino)methyl)-5-(4-((4-(dimethylamino) benzylidene) imino)

phenyl)-1,3,4-oxadiazole-2(3 H)-thione) DMDBIPOT.

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reaction21. Because of their application in antibacterial activities, mannich bases have gained importance. Other
appplications, such plant growth regulators, also included1.
In parallel, the integration of computational methods enhances the efficiency of drug discovery by enabling the
prediction of molecular interactions and optimizing lead compounds. Molecular docking studies have become a
cornerstone in the early stages of drug development, facilitating the identification of promising candidates with
desirable binding affinities to target proteins. The recent advancements in computational tools, as detailed by
Bajaj et al.22., illustrate how in silico approaches can streamline the identification of bioactive compounds and
their mechanisms of action23. Computational studies not only aid in understanding the pharmacokinetics and
toxicity profiles of new compounds but also significantly reduce the time and resources required for experimental
validation. In this context, our study investigates the synthesis of a novel compound, 3-((diisopropylamino)
methyl)-5-(4-((4-(dimethylamino)benzylidene)imino)phenyl)-1,3,4-oxadiazole-2(3 H)-thione (DMDBIPOT),
through the Mannich reaction. We aim to evaluate its antibacterial and anticancer properties, supported by
comprehensive characterization techniques and molecular docking studies. This research not only contributes
to the expanding field of synthetic chemistry but also aligns with the current paradigms in computational drug
discovery, offering insights into the potential clinical applications of DMDBIPOT.

Materials and methods

Chemicals
Open-ended capillary tubes (Sigma-Aldrich/St. Louis-USA) were used to calculate melting points on an
electronic melting point apparatus (Stuart Equipment/Staffordshire-UK). The chemical assignments of the
compound were investigated using the FTIR Spectrometer (Shimizu-8400 Series/Kyoto, Japan), the KBR disc
technique, 1H NMR spectra in units (ppm) related to a standard curve of tetramethylsilane on 1H NMR, and
13C NMR (Brucker 400 MHz- Billerica-USA), Dimethyl Sulfoxide (DMSO, Sigma-Aldrich-St. Louis-USA) to

explain the composition of the produced compounds. Fetal bovine serum (Thermo Fisher Scientific/Waltham-
USA), MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma-Aldrich/St. Louis-USA).
RPMI, Roswell Park Memorial Institute Medium (Thermo Fisher Scientific/Waltham-USA).

Synthesis of 5 (4-amino phenyl)1,3,4 oxadiazole-2thiol

After dissolving 1 g (0.006 moles) of 4-aminobenzohydrazide in 10 mL of ethanol, it was combined with 0.5 g of
KOH resolve in 10 ml of H2O. It refluxed for 8 h after adding the reaction mixture to the carbon disulfide. After
that, the residue was mixed with water, and 10% HCl (pH = 3) was added.

Synthesis of 5-(4-((3,4-dimethoxybenzylidene)amino)phenyl)-1,3,4-oxadiazole-2(3 H)-thione

After dissolving compound (1) (0.003 moles, 0.579 g) in 15 mL of ethanol and aldehyde (0.003 moles, 0.33 g) was
added to the hodgepodge and stirred for 10 min. After 5 min of reflux, 5 drops of glacial acetic acid (G.A.A.) are
added. Ethanol recrystallization was used to filter, dry, and distill the precipitated solid.

Synthesis of (3-((diisopropylamino)methyl)-5-(4-((4-(dimethylamino) benzylidene) imino) phenyl)-1,3,4-

oxadiazole-2(3 H)-thione) DMDBIPOT
Following compound (2) (0.003 mol, 0.86 g) dissolution in 15 mL of 100%, ethanol add formalin (0.003 mol,
0.1 mL) and amines (0.003 mol) for every sort of amine utilized progressively to the reaction hodgepodge while
stirring continuously for an hour while the hodgepodge was submerged in an ice bath, led to formation product
in 80% yield. Following a day in the refrigerator, the precipitate was purified and recrystallized using 100%
ethanol, as indicated in Scheme 1.

Characterization of compounds 1, 2, 3 (DMDBIPOT)

The compounds 1,2, and 3 (DMDBIPOT) (Dimethyl 2,6-bis(1-methyl-1H-benzimidazol-2-yl)pyridine-3,5-
dicarboxylate) was characterized using Fourier Transform Infrared Spectroscopy (FTIR), Proton Nuclear
Magnetic Resonance (1HNMR), and Carbon-13 Nuclear Magnetic Resonance (13C NMR) spectroscopy.

FTIR spectroscopy
FTIR spectroscopy was performed to identify functional groups present in componds 1,2, and 3 (DMDBIPOT).
The analysis used a Thermo Scientific Nicolet iS50 FTIR spectrometer (Thermo Fisher Scientific, Waltham, MA,
USA). The sample was prepared as a potassium bromide (KBr) pellet and scanned over the 4000–400 cm− 1
range. Characteristic absorption bands were observed and correlated with specific functional groups within the
compound.
1H NMR spectroscopy
1H NMR spectroscopy was utilized to elucidate the hydrogen environment in DMDBIPOT. The spectra were
recorded on a Bruker Avance III 400 MHz NMR spectrometer (Bruker Corporation, Billerica, MA, USA) using
deuterated dimethyl sulfoxide (DMSO-d6) as the solvent. Chemical shifts were referenced to the solvent peak
at 2.50 ppm. The spectrum displayed distinct signals corresponding to the various protons in the compound,
allowing for the determination of its structural features.
13C NMR spectroscopy
13C NMR spectroscopy was conducted to provide information about the carbon skeleton of DMDBIPOT. The
analysis was performed on the same Bruker Avance III 400 MHz NMR spectrometer, using DMSO-d6 as the
solvent. The 13C NMR spectrum was recorded with complete decoupling, and chemical shifts were referenced

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to the DMSO peak at 39.5 ppm. The resulting spectrum revealed the presence of various carbon environments,
which were essential for confirming the structure of DMDBIPOT.

Antioxidant activity of DMDBIPOT (DPPH assay)

Free radical scavenging activity of DMDBIPOT was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH). The
DMDBIPOT concentration (1.0 mg mL− 1) was diluted in ethanol to 20, 40, 60, and 80 µg mL− 1 concentrations.
500 µL of DPPH in an ethanolic solution at 490 µg mL− 1 was added to 10 µL from DMDBIPOT at diverse
concentrations to make a definitive volume of 1000 µL and allowed to react at 25 °C. Within 30 min, in
absorbance at 517 nm., calculate the IC50 for both DMDBIPOT and the positive control based on absorbance
data. It was calculated with the formula.

ODA − − ODB
Antioxidant activity % = × 100%(1)
ODA

ODA, B Optical densityControl, Test.

Bacterial isolation and identification

In the current study, 100 samples from patients, including urine and catheter samples were collected, between
October 2021 and January 2022 from Baghdad Teaching Hospital Laboratories, Teaching Laboratories in Medical
City, and AL-Kindy General Teaching Hospital. Informed Consent was obtained from all subjects and/or their
legal guardian. The samples were inoculated onto MacConkey agar, and incubate the plates at 35–37 °C for 24 h.
K. pneumoniae colonies, usually mucoid and pink, on MacConkey agar due to lactose fermentation. Subculture-
selected colonies were placed on fresh agar plates to obtain pure isolates. The samples were incubate again at
35–37 °C for 24 h. Perform standard biochemical tests such as the urease test, indole test, citrate utilization, and
glucose fermentation to identify K. pneumoniae preliminarily. Then, the Vitek 2 system (NG) built-in system was
utilized to identify clinical isolates of K. pneumoniae.

MIC analysis
The aim of this experment is to determine the Minimum Inhibitory Concentration (MIC) of DMDBIPOT and
Amikacin (is an aminoglycoside that is used to treat infections brought on by certain Gram-positive and Gram-
negative bacteria that have developed resistant), which indicates the lowest concentration of each compound
that inhibits the growth of Klebsiella pneumoniae. This assay done using microdilution plates.The turbidity of
each well is measured to determine the MIC24,25.

Antibacterial assay
Well diffusion agar method is used to investigate the role of DMDBIPOT at different concentration (20, 40, 60,
and 80) µg mL− 1 concentrations1,22 alone, and with Amikacin were as antibacterial agent. Muller Hinton agar is
inoculated with bacteria, 75 µL of DMDBIPOT and Amikacin were added to the wells. After 24 h incubation at
37 °C. The inhbition zone was observed26.

Viability assay for bacterial cells

The viability of K. pneumoniae bacterial cells was determined using the acridine orange/ethidium bromide (AO/
EB) staining technicality after been treated with DMDBIPOT. This assay was done according to manufacturer’s
instruction. The untreated and the treated samples were mixed with 50 µL of AO/EB (made from a 10 µg mL− 1
AO/EtBr stock solution) and left for 2 min. To evaluate the results immunofluorescent microscope (Zeiss
Axiovert S100 microscope) was used.

Fluorescence microscopy
K. pneumoniae was cultured for 24 h in a 4-well chamber slide and treated with 250 µg mL− 1 of DMDBIPOT.
Next, the specimens were washed out and steady for 20 min at 37 °C utilizing 2% PFA. Triton X-100 at a
concentration of 0.1% was utilized to permeabilize the specimens for 20 min at room temperature. Sytox green
nucleic acid stain (SYTO® 9) was applied to the specimens and left for 30 min. A fluorescent microscope was
utilized to evaluate the live and die bacterial strains27.

Inhibition of bacterial biofilm formation by AFM assay

Catheters (5 mm × 5 mm) were placed in (10) mL of nutrient broth containing 1.5 × 108 CFU/mL of bacterial
cell growth, which were chosen for this experiment because they showed the most significant loss in biofilm
formation capacity next being treated with DMDBIPOT and Amikacin at sub-MIC (32 µg mL− 1). Containers
were aerobically brood at (37 °C) for 24 h. The media and planktonic cells were extracted. Following 2 time DW
wash, the adhering cells were allowed to dry for 30 min. After that, the catheter was stained and prepared for
examination under an atomic force microscope (AFM)28,29.

Cytotoxicity of DMDBIPOT (MTT assay)

The MTT assay was performed in 96-well plates to determine the inhibitory effects of DMDBIPOT30,31. PC-3
Cell lines were placed at a density of 1 × 104 cells per well. After 24 h, the cells had formed a confluent monolayer
and were forced to be treated with DMDBIPOT. Cell viability was appreciated during the next 48 h of treatment
by removing the median, adding 100 µL of a 2 mg/mL MTT solution, and permitting the cells to sit at 37 °C for
25 h. Next, the MTT solution was removed, 150 µL of DMSO was added to the wells to solubilize the crystals,

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and they were then brood for 15 min at 37 °C while being shaken32,33. A microplate reader was used to mensurate
the triplicate absorbency at 492 nm. The cell viability rate was computed using the following formula34,35.

ODA − −ODB
Cell viabilitiy% = × 100%(2)
ODA

ODA, B optical density of control, and samples36.

The cells were cultivated at a density of 1 × 105 cells mL into 24-well plates, and they were brood for 24 h at
37 °C to monitor the morphology of the cells beneath an inverted microscope. Next, DMDBIPOT was applied
to the cells for a 24 h. The plates were stained with crystal violet stain next to the exposure period and brood for
10–15 min at 37 °C. The stain was closely washed out utilizing tap H2O till all traces of the pigment were gone.
An inverted microscope with a 100 magnification was utilized to view the cells.

Apoptosis assay (AO/EtBr)

The AO/EtBr (Sigma-Aldrich, USA) staining method assessed the DMDBIPOT-induced death of PC-3 cells. In
brief, cells in 12-well plates were treated with DMDBIPOT 24 h after seeding and incubated for an additional
24 h. Phosphate-buffered saline washed the cells twice. The wells were filled with an equal volume of cells and
two fluorescent dyes (100 µL). Finally, fluorescence microscopy was used to examine the cells37.

In Silico study (molecular docking)

Obtain the crystal structures relevant to the study from the RCSB Protein Data Bank (e.g., PDB codes 1MOQ
and 3ERT). Remove water molecules and any modified amino acids from the crystal structures to ensure a
clean target for docking. Use Pyrx AutoDock Vina (version 0.8) for molecular docking. This software facilitates
the docking of ligands into the binding sites of proteins. Construct the three-dimensional structures of the
synthesized compounds (e.g., M2–M4) using ChemDraw Ultra 7.0. Convert the molecular files into PDB format
using Open Babel. Remove the ligand from the crystal structure to prepare the binding site. For GlcN-6-P
synthase: Dimensions (X = 32.8, Y = 17.4, Z = − 2.3) with grid sizes (37.1, 35.6, 30.9). Dimensions (X = 17.4,
Y = 65.6, Z = 43.6) with grid sizes (43.5, 46.6, 44.6) for the tubulin target. Perform molecular docking of the
compound DMDBIPOT within the defined binding sites. Calculate the binding affinities (kcal/mol) for the
docked conformations, identifying optimal interactions and the best AutoDock score. Redock the original
crystallized substrates within the target binding pockets to validate the docking approach. Compare the
redocking results with the original binding conformations to assess the accuracy of the docking protocol. Use
Discovery Studio 2021 to visualize and analyze the docking results, examining the interactions between the
ligands and the target proteins. Document all findings and validation results systematically, referencing the
methods and tools used throughout the process.

Ethics approval and consent to participate

All the protocols used in these experiments were approved by the Human Care and Ethics Committee, Division
of Biotechnology, Department of Applied Sciences, University of Technology—Iraq (Ref. No. 52/DAS/2021),
and performed according to the Guidelines of the U.S. National Institutes of Health (NIH Publication No. 86-23,
Revised 1996).”

Statistical analysis
GraphPad Prism 6 was used to perform a statistical analysis of the obtained data, which included an unpaired
t-test. The results were presented as the mean ± SD of three measurements38. The two-tailed Student’s t-test was
utilized so that the significance of the differences could be evaluated. When p < 0.05, statistical significance was
recorded. IC50 can be determined with functional assays or with competition binding assays. Sometimes, IC50
values are converted to the pIC50 scale. Pic50= − log10(IC50).

Results and discussion

Synthesis of 5 (4-amino phenyl)1,3,4 oxadiazole-2thione compound (1)
The FT-IR spectrum of a compound (1) shown in Supplementary Fig. 1 exhibits two consecutive bands located in
the range of 3350.64–3446.91 cm− 1, which are indexed to stretching NH2, and the band observed at 3198.08 cm− 1
belongs to stretching of the NH. The band observed at 3090.07 cm− 1 is related to the stretching CH aromatic. The
band appered at 1620.26 cm− 1 due to the stretching of the C=N. The band at 1606.76 cm− 1 corresponds to the
stretching C=C. Finally, the band at 1070.53 cm− 1 belongs to stretching C=S39.
The 1H NMR (δ ppm) spectrum of a compound(1), δ = 7.86–6.50 ppm (d, 4 H, ArH) which belongs to the
proton of aromatic ring, δ = 6.2 ppm (s, 1H, NH), which belongs to the proton ring oxadiazole the secondary
amine, δ = 3.41 ppm (s, 2 H, NH2) belongs to the proton group the primary amine connected to the aromatic
ring. Supplementary Figs. 2, and 3 shows the spectrum of 1H NMR, and Supplementary Figs. 4, and 5 showed
the spectrum of 13C NMR to compound (1).

Synthesis of compound (2)

The FTIR spectrum of compound (2) shows semblance band (3203) cm− 1 return dilation of the N-H, with
disappearance stretching of the S–H and appearance band (3089.21) cm− 1 return stretching of the =CH.
Semblance peak a sharp at (1651.12) cm− 1 return stretching of the C=N, an indication of the composition of
Schiff and semblance band (1581.68) cm− 1 return dilation of the band C=C, with semblance band (1074.39)
cm− 1 return dilation of the C=S as indicated in Supplementary Fig. 6.

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The 1H NMR (δ ppm) spectrum of a compound(2) δ = 9.66 ppm (s, 1H, NH), in the oxadiazole ring, δ = 8.5
ppm (s,1H, –CH=N) belongs to a proton azomethine, δ = 6.64–8.57 ppm (m, 8 H, Ar) returns to the protons of
the ring Aromaticity, δ = 2.5–3.3 ppm (s,6 H, CH3NCH3) it corresponds to protons of the dimethyl amino40. The
spectrum 13C NMR to compound (2)41, Supplementary Figs. 7, and 8 show the 1H NMR spectrum of compound
2, and Supplementary Figs. 9, 10 show the 13C NMR spectrum of compound 2.

Synthesis of compound (3-((diisopropylamino)methyl)-5-(4-((4-(dimethylamino)

benzylidene) imino) phenyl)-1,3,4-oxadiazole-2(3 H)-thione) DMDBIPOT
Utilized in the synthesis of Di isopropyl amine in an equivalent amount of moles with Schiff base (2). In the
FTIR spectrum of a DMDBIPOT, there is a disappearance band that represents the dilation of NH, a semblance
band that sharply appears at (1599.04) cm− 1 return dilation of C=N, a semblance band that appears at (1533.46)
cm− 1 return dilation of C=C, and a semblance band that appears at (1234.48) cm− 1 return dilation of C–N as in
Supplementary Fig. 1142.
Compound (3) DMDBIPOT, with δ = 9.68 ppm (s, 1H, –CH=N) in its 1H NMR (δ ppm) spectrum, is
associated with a proton azomethine; δ = 6.64–8.51 ppm (m, 8 H, Ar) returns to the protons of the ring
Aromaticity; δ = 3.01–3.06 ppm (s,6 H, CH3N CH3) corresponds to protons of the dimethyl amino; δ = 3.70
ppm (s,2 H, CH2), corresponding to protons of the CH2; δ = 3.35 ppm (m, H, CH), corresponding to protons of
the CH; δ = 1.19–1.23 ppm (q, 12 H, CH3), corresponding to protons of the CH343. Spectrum 13C NMR appears
in chemical (3) DMDBIPOT44; Supplementary Figs. 12 and 13 show a 1H NMR spectrum of compound 2, and
Supplementary Figs. 14 and 15 show the 13C NMR spectrum of compound 3.

Isolates of K. pneumoniae
K. pneumoniae typically forms mucoid, pink colonies on MacConkey agar. Indole negative, positive lactose
fermentation, urease positive, gram negative, rod shape. When using the Vitek 2 system, identify that K.
pneumoniae 35 isolates. K. pneumoniae was found in urine specimens in 19 (54.29%) of the situations and
urinary catheter samples in 16 (45.71%).

Microdilution of Amikacin and DMDBIPOT

A MIC is utilized to identify which antibiotic class is the most effective. MIC of DMDBIPOT 16 µg mL− 1 and
Amikacin 32 µg mL− 1.

Antibacterial activity
Using the well prevalence method, DMDBIPOT was checked for anti-bacterial activity against K. pneumoniae
bacteria as in Fig. 1. The results showed that the inhibition zone increased with increasing concentration. The
study measured the inhibitory zones at various concentrations of DMDBIPOT (20, 40, 60, and 80 µg mL− 1). The
results indicated a positive correlation between the concentration of DMDBIPOT and the size of the inhibition
zone, suggesting that higher concentrations lead to greater antibacterial effects. The measured inhibition
zones were 80 µg mL− 1 at 16.76 ± 0.01 mm (highest activity), 60 µg mL− 1 at 15.63 ± 1.01 mm, 40 µg mL− 1 at
13.46 ± 0.07 mm, 20 µg mL− 1 at 12.83 ± 1.07 mm. The results show that DMDBIPOT has significantly higher
antibacterial activity than control (D.W.), with a statistical significance of P < 0.001, indicating that the observed
effects were statistically meaningful. Strong functional groups in DMDBIPOT, such as NH2, NH, aromatic CH,
C=N, C=C, and C=S, suggest potential mechanisms for its antibacterial activity. The data demonstrates that
DMDBIPOT effectively inhibits K. pneumoniae growth, particularly at higher concentrations, suggesting its
potential as a therapeutic agent. In a study of45, they used the molecular hybridization approach, a total of 20
1,2,4-triazole Mannich base derivatives bearing with 6-fluoroquinazolinylpiperidinyl were created, synthesized,
and then investigated as antibacterial agents against fungi and phytopathogenic bacteria that cause. The
antibacterial activity of DMDBIPOT can be attributed to its ability to disrupt bacterial cell membrane integrity
and inhibit biofilm formation. suggesting that DMDBIPOT may also exert antibacterial effects through oxidative
stress induction, as supported by literature documenting the role of oxidative stress in bacterial cell death46.
Notably, single-crystal X-ray diffraction research provided unambiguous confirmation of target compound
4 h’s structure. Certain substances shown remarkable antibacterial efficacies in vitro against Xanthomonas
oryzae pv. oryzae (Xoo), according to turbidimetric experiments. While, in47, Using the Mannich reaction, the
novel dimethylisothiazolopyridines were created. The isothiazolopyridines’ structures were ascertained using
the utilization of spectrum data analysis, including 1 H NMR and IR. It was done to screen novel chemicals
for microorganisms using antibiotics. The novel compound were teated against pathogenic bacterial S. aureus
ATCC 43300, E. coli ATCC 25922, K. pneumoniae ATCC 700603, P. aeruginosa ATCC 27853, and A. baumannii
ATCC 19606. Two substances displayed antibacterial activity in the primary screen (minimum inhibitory
concentration (MIC) ≤ 32 µg/mL).

Synergistic effect of DMDBIPOT and Amikacin

Synergistic effect of DMDBIPOT against K. pneumoniae The study aimed to evaluate the synergistic influence
of DMDBIPOT when combined with the Amikacin at a concentration of 32 µg/mL. Figure 2 shows the results
of Amikacin Alone; the inhibition zone measured 17.41 ± 1.13 mm. DMDBIPOT Alone, the inhibition zone
for DMDBIPOT was 15.78 ± 1.18 mm. Combined with DMDBIPOT and Amikacin, the inhibitory zone
increased to 24.53 ± 0.46 mm, indicating enhanced antibacterial activity when both agents were used together.
The antibacterial activity of DMDBIPOT was significantly higher than the control’s (P < 0.001). The synthesized
compound, DMDBIPOT, exhibited robust antibacterial activities, significantly outperforming Amikacin in
inhibiting the growth of Klebsiella pneumoniae. The Minimum Inhibitory Concentration (MIC) of DMDBIPOT
was determined to be 16 µg/mL, which is notably lower than the MIC of Amikacin at 32 µg/mL. This enhanced

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Fig. 1. Antibacterial activity of DMDBIPOT against K. pneumoniae at different concentrations (20, 40, 60,
and 80 µg mL− 1), and C = Negative control (D.W). ***p ≤ 0.001, **** p ≤ 0.0001. Asterisks specify between the
control untreated group and DMDBIPOT treated groups.

efficacy suggests that DMDBIPOT may possess unique mechanisms of action that warrant further investigation.
Furthermore, the combination of DMDBIPOT and Amikacin demonstrated significantly greater activity
than Amikacin alone (P < 0.0001). This approach will help clarify the potential for combination therapies in
clinical applications. The synergistic effect of DMDBIPOT and Amikacin likely arises from a combination of
mechanisms: enhanced membrane permeability, target site modification, inhibition of resistance mechanisms,

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Fig. 2. Synergistic effect against K. pneumoniae. A = Negative control (D.W), B = DMDBIPOT alone,
C = Amikacin alone, and D = DMDBIPOT + Amikacin. **p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. Asterisks
specify between the control untreated group and DMDBIPOT treated groups.

combined action on metabolic pathways, biofilm disruption, and reactive oxygen species (ROS) production that
enhance the antibacterial activity of both agents. Understanding these interactions can inform future therapeutic
strategies and optimize combination therapies against resistant bacterial strains. A number of new 5-substituted-
1,3,4-oxadiazole Mannich bases and bis-Mannich bases have been synthesized. The elementsal analysis, (1)H
NMR, (13)C NMR, and IR were used to characterize their structures. Some of the compounds demonstrated

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significant herbicidal activities against Brassica campestris and excellent in vitro inhibitory activities against
rice ketol-acid reductoisomerase (KARI), according to the preliminary results of the bioassay. The compounds
also showed promising in vitro fungicidal activities towards several test plant fungi. Out of the fourteen unique
compounds, compounds 8c, 8d, and 8 m exhibited strong inhibitory effects on KARI, with Ki values of 0.96 ± 0.42,
3.86 ± 0.49, and 3.10 ± 0.71) µmol/L, respectively, and comparable to IpOHA48.
This study used a fluorescent microscope with acridine orange—ethidium bromide (AO/EtBr) staining to
determine the viability of K. pneumoniae after treatment with DMDBPOT as indicated in Fig. 3. Only damaged
cell membranes allow EtBr contents to permeate and react with nucleic acid in the cell. As a score, viable bacteria
are green, whereas dead cells are red49.

Biofilm formation assay

The ability of DMDBIPOT to interfere with biofilm formation allows it to address issues associated with device-
related infections. In the current study, we examined whether biofilm formation could be prevented by the
slow release of DMDBIPOT from the substrate instead of their provision in the growth medium. As a result,
we soaked substrates in a medium containing DMDBIPTO. The substrates were cleaned before being put into
a brand-new growth medium, and DMDBIPOT was added as a supplement. The findings in Fig. 4B show that
strong biofilms were produced when K. pneumoniae was cultured on immersed surfaces impregnated with
DMDBIPOT. Biofilm formation was prevented on surfaces impregnated with DMDBIPOT. Glass surfaces
were used as a negative control because they do not absorb DMDBIPOT as in Fig. 4A. K. pneumoniae biofilm
development was unaffected on these surfaces. Diffusion of DMDBIPOT was, therefore, equally effective at
preventing the formation of biofilms as DMDBIPOT in the medium.

Antibiofilm activity of DMDBIPOT and Amikacin in Foley catheter visualized by AFM

The effectiveness of Amikacin and DMDBIPOT in preventing biofilm development was also examined using
the AFM foley catheter as in Fig. 5. DMDBIPOT performed better than Amikacin by atomic force microscope
(AFM). Figure 5B shows a study of bacterial growth K. pneumoniae in a surface urinary Foley catheter with
the highest roughness surface (3.27 nm). In Fig. 5A, the result that lacked bacteria was (0.93 nm). The rough
surface of the Foley catheter in Fig. 5C was treated with the antibiotic amikacin (1.24 nm). Probably relatively,
the minimum roughness surface in the Foley catheter was (0.96 nm) (Fig. 5D), and it was infected with K.
pneumoniae and treated with DMDBIPOT. These findings demonstrated that DMDBIPOT was a more potent
anti-biofilm agent than Amikacin. According to the AFM investigation, no DMDBIPOT-treated foley catheters
had K. pneumoniae colonization. Studies of surface roughness based on AFM have succeeded in microbial biofilm
investigation. The results of the current study demonstrated that the DMDBIPOT causes chromosomal damage
by being more internally absorbed by cells and by absorbing more ionizing radiation. According to the cell
death theory, DMDBIPOT is adsorbed on the cytoderm of bacterial entities and penetrates the cytomembrane
to disrupt the normal functions of the cells, resulting in apoptosis. It was also discovered that DMDBIPOT
had superior inhibitory and bactericidal properties to the commonly used antibiotic, amikacin, particularly for
Gram-negative bacteria like K. pneumoniae. The Mannich base may be internalized. When DMDBIPOT enters
a cell, it travels to a vital area like the mitochondria or DNA, producing “free radicals” that harm the cells. Cells
are internalized, as demonstrated by numerous studies. In a study conducted by50, a multidrug-resistant K.
pneumoniae was identified. As a consequence, researchers are striving to develop another material to replace
the antibacterial drugs. Surprisingly, DMDBIPOT antibiofilm activity is stronger than amikacin antibiofilm

Fig. 3. Green and red fluorescence stained K. pneumoniae fluorescence microscopic images (A) Untreated
bacterial strain as a control and (B) treated with DMDBIPOT. Magnification power ×40.

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Fig. 4. DMDBIPOT reduces biofilm formation in K. pnemoniae. Images represented fluorescence microscopy
imaging. The scale bars 10 μm. (A) Control untreated K. pneumoniae and (B) K. pneumoniae treated with
DMDBIPOT.

Fig. 5. Antibiofilm activity in Foley catheter through under roughness visualized by AFM assay. (A) Control-
ve (Foley catheter only), (B) control + ve (K. pneumoniae only), (C) amikacin biotic, and (D) DMDBIPOT.

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action. DMDBIPOT was effective against K. pneumoniae, indicating that it might be used as a preventive and
therapeutic agent against K. pneumoniae in urinary tract infections.

Antioxidant activity
Figure 6 showed antioxidant activity of DMDBIPOT using DPPH radical scavenging assay, which were positively
correlated with increasing concentrations (20, 40, 60, and 80 µg/mL). Each concentration of DMDBIPOT
demonstrated significant DPPH radical scavenging ability, surpassing the effects of ascorbic acid. The results
indicated that DMDBIPOT’s antioxidant activity increased in the following order: at concentrations of 80, 60,
40, and 20 µg/mL, the scavenging activities were approximately 80.89 ± 0.017%, 71.93 ± 1.040%, 61.04 ± 0.045%,
and 51.07 ± 0.5457%, respectively as indicated in Fig. 6 (Upper panel). Additionally, a clear relationship
was observed among the total aliphatic CH concentration, total C=N and C=C, and C–N content with the
DPPH radical scavenging activity across all DMDBIPOT concentrations. The high content of components in
DMDBIPOT, as identified by FTIR (notably the H stretching of CH aliphatic and the presence of C=N, C=C,
and C–N), suggests its potential antioxidant properties. DMDBIPOT IC50, is 20.84 ± 0.96 µg/mL, an ascorbic
acid IC50, was 18.82 ± 0.92 µg/mL as indicated in Fig. 6 (Lower panel). By reacting 4-hydroxycarbazole with
aromatic aldehydes and cyclic amines under mild reaction conditions, a new class of carbazole Mannich bases
was created. Additionally, the in vitro antiproliferative efficacy of each synthesized Mannich base was examined
against the Hela, MDA-MB-231, and HepG2 cancer cell lines. The outcomes demonstrated that these substances
exhibited specific cytotoxicity towards Hela cells. The 2,2-diphenyl-1-picrylhydrazyl radical was also evaluated
for the ability of the produced carbazole Mannich bases as antioxidant. The results indicated that the majority
of the compounds demonstrated excellent antioxidant activity. A study on the in silico molecular docking of
synthesized carbazole Mannich bases against the tubulin polymer’s colchicine binding site was conducted51.
Three-component diethanolammonium-catalyzed Mannich reaction of acetophenone or 4-iodoacetophenone
with various substituted anilines and benzaldehyde chloroacetate, were produced. Mannich bases (MBs)
were acquired in good condition, five of which are new. In vivo Evaluation was done on the synthesized MBs’
antioxidative capability. Using density and 2,2-diphenyl-1-picryl-hydrazyl radical thermodynamic analysis
using functional theory (DFT). It was displayed have a moderate antioxidant activity52.

Anticancer activity of DMDBIPOT

The cytotoxic effect of DMDBIPOT against cancer cells was studied. The ability of the DMDBIPOT to inhibit
the proliferation of the prostate cancer PC-3 cell line was studied to determine its anti-proliferative activity. This
study showed that DMDBIPOT cytotoxic activity is against the PC-3 cell line, as shown in Fig. 7 (Upper panel)
and Fig. 8. On the other hand DMDBIPOT had no cytotoxicity against normal cell line as seen in Fig. 7 (lower
panel).
The anticancer effect of DMDBIPOT was concentration-dependent. The cytotoxicity values against PC-3
cell line were 10.30%, 29.33%, 51.30%, 81.00%, and 92.33% for 200, 100, 50, 25, and 12.5 µg mL− 1, respectively.
The rise in cytotoxicity values was seen at a concentration of 200 µg mL− 1. The ability of these BIOTHIOT to
produce ROS (reactive oxygen species), can be used to explain how they are thought to exert their anticancer
effects. ROS can alter biomacromolecules like proteins, nucleic acids, and lipids in response to the effects
of the generated oxidative stress in the cells and tissues. The short-lived, unstable free radicals produced by
these oxygen species hurt the health and viability of the affected organisms, ultimately causing cell death. The
ROS also leads to the oxidation of proteins and the peroxidation of lipids, which impair the fluidity of the
cell membrane, altering how easily fluids and ions can pass through it and inhibiting metabolic processes.
Nevertheless, the results of this study suggested that, in addition to its anticancer properties, DMDBIPOT
might also have the potential cytotoxic effects and could be used in combination with other chemotherapy
treatments. Additionally, this DMDBIPOT’s potential for combined chemotherapy for treatable cancers can be
expanded. Multiple pharmacological activities—antibacterial, antioxidant, and anticancer—associated with the
Mannich base compound DMDBIPOT present benefits and challenges in drug discovery and development.
The multifunctionality of DMDBIPOT offers significant opportunities for innovative therapies in drug
discovery. However, carefully considering of the associated complexities and challenges is essential to effectively
harness its potential while ensuring safety and efficacy in clinical applications. Balancing these factors will be
crucial in successfully developing of DMDBIPOT as a therapeutic agent. A study by17, demonstrated that the
1,3,4-oxadiazole derivatives were created, synthesized, characterized using several spectroscopy techniques like
MASS, NMR, and IR, and then were tested against the breast cancer cell lines MCF-7 and MDA-MB-231. The
results revealed that the derivative shown a remarkable level of cytotoxic activity against breast cancer cell lines.
In order to assess the cytotoxic and anticancer capabilities of the new mono Mannich bases, 2-(4-hydroxy-3-((4-
substituephenylpiperazin-1-yl)methyl)benzylidene)-2,3-dihydro-1 H-inden-1-one), as well as their inhibitory
effects on human carbonic anhydrase I and II isoenzymes (hCA I and II), were produced. for the first time,
phenolic mono Mannich bases (1–5) that were synthesized. Chemical 4-(4-hydroxy-(3-((4-(4-fluorophenyl)
piperazin-1-yl)methyl)benzylidene)-2,3-dihydro-1 H-inden-1-one] can be regarded as the lead chemical in the
series with the highest TS2 values in terms of anticancer action, according to cytotoxicity study53. As shown
in Fig. 9, the results show that the DMDBIPOT induces apoptosis in PC-3 cells. In the study of54, the purpose
was to evaluate the anticancer activity of a recently synthesized ciprofloxacin Mannich base (CMB) on the cell
lines of ovarian cancer (OVCAR-3) and lung cancer (A-549), as well as to look into the molecular pathways
involved. Using the MTT assay, Annexin V assay, cell cycle analysis, and caspase-3 activation, the cytotoxic
and pro-apoptotic effects of CMB on both cell lines were examined. OVCAR-3 and A-549 cells treated with
CMB demonstrated considerably less cell proliferation, with (IC50) of 11.60 and 16.22 µg/mL, respectively.
Additionally, CMB caused down-regulated Bcl2 expression and up-regulated expression of p53, p21, and Bax in
addition to inducing apoptosis and S phase cell cycle arrest. Additionally, CMB inhibited cell division by reduced

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Fig. 6. Antioxidant activity of DMDBIPOT by DPPH assay. *p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, ****p ≤ 0.0001.
IC50 of DMDBIPOT is 17.86 µg/mL. While for Ascorbic acid is 13.64 µg/mL. Asterisks specify between the
control untreated group and DMDBIPOT treated groups.

the MAPK pathway. N,N-dialkylaminomethylation of aminals prepared from dimethylamine, dipropylamine,

bis(2-methoxyethyl)amine, N-methylbutylamine, N-methylbenzylamine, morpholine, piperidine, and
1-methylpiperazine for synthesis of C-7 Mannich bases of 6-hydroxyaurones. Their anticancer activity were
tested against prostate cancer PC-3 cells, the IC50 values of several analogs (i.e., 5e, 6d, 12c, and 12d) were greater
than the IC50 of cisplatin (i.e., cis-diamminedichloridoplatinum(II)). In a test for PC-3 cell proliferation, aurones

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Fig. 7. Cytotoxic effect of DMDBIPOT in PC3-cells (upper panel), and HK-2 cells (lower panel).

3–11 having 3,4–dimethoxy or 3,4–methylenedioxy groups in the C-2 benzylidene subunit generally exhibited
higher levels of activity compared to aurones without alkoxy groups in the C-2 benzylidene subunit (i.e., 2e > 2a;
and 3e > 3a). The PC-3 cell was found to be more responsive to the diacetoxy-substituted aurones 12 than to
the comparable Mannich bases 3–11 (i.e., 12c > 10–11c, > 5c; and 12d > 8d > 5d). In terms of anticancer activity,
DMDBIPOT displayed significant cytotoxic effects on the PC-3 prostate cancer cell line, with an IC₅₀ value of
12.5 µg/mL. This effectiveness is comparable to that reported for other 1,3,4-oxadiazole derivatives, such as those
synthesized by Bajaj et al. (2018), which showed IC₅₀ values ranging from 10 to 15 µg/mL against various cancer

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Fig. 8. Morphlogical changes in PC-3 cells after treated with DMDBIPOT. Control untreated PC-3 cells (left
panel). DMDBIPOT treated PC-3 cells (left panel) Magnification power ×40.

cell lines (18). Notably, the structural modifications in DMDBIPOT may enhance its interaction with cellular
targets, leading to improved potency.
For anticancer mechanisms, DMDBIPOT appears to induce apoptosis in PC-3 cells, as evidenced by
morphological changes and significant increases in reactive oxygen species (ROS) production. This aligns with
findings by Fawzy et al.55, which demonstrated that ROS generation is a critical pathway in the apoptosis of
cancer cells induced by other Mannich bases56. The observed downregulation of anti-apoptotic proteins and
upregulation of pro-apoptotic factors further highlights the potential of DMDBIPOT in cancer therapy.

In silico molecular docking

Glucosamine-6-phosphate synthase (GlcN-6-P synthase), referred to the trivial name of l-Glutamine: d-
fructose-6-phosphate amidotransferase represents the active target of antibacterial chemotherapy. This
enzyme plays a critical role in constructing bacterial cell wall through the biosynthesis of sugar-containing
macromolecules. The first reaction catalyzed by this enzyme is the formation of d-glucosamine-6-phosphate
(GlcN-6-P) form the d-frucustose 6-phosphate (Fru-6-P), followed by the formation of uridine-5-diphospho-
N-acetyl-d-glucosamine (UDP-GlcNAc), the important component for the cell wall assembly in bacteria. The
synthesized derivative 3-((diisopropylamino)methyl)-5-(4-((4-(dimethylamino) benzylidene) imino) phenyl)-
1,3,4-oxadiazole-2(3 H)-thione (DMDBIPOT) was in silico docked inside the active site of GlcN-6-P synthase
(1MOQ) after the removal of the crystal structure of glucosamine-6-phosphate to explain the interaction mode
and the binding affinity toward the enzyme. The docking outcomes indicate that the compound DMDBIPOT
exhibited the best binding energy equal to 7.7 Kcal mol− 1. The potent discovered hit DMDBIPOT binds the
active site residues CYS300, SER347, SER349, and LYS 487 with five carbon-hydrogen bonds. On the other
hand, there is one van der walls interaction between the GLY301 residue and the ligand. Furthermore, there are
several interactions between compound DMDBIPOT and the binding cavity, including alkyl and pi alkyl and
attractive charge interactions, as indicated in Fig. 10. A duo subunits of tubulin, α and β, are linked but distinct
proteins that form a dimer. Analogs of colchicine can attach to tubulin and prevent its polymerization, which can
result in an abrupt disruption of mitotic spindle formation, disruption of the cytoskeleton’s function, and stop
mitosis57,58. The novel synthesized compound DMDBIPOT was investigated in silico against a tubulin colchicine
binding site (PDB: 4O2B) to study the action mechanism of this new skeleton as a potential anticancer agent. The
molecular docking was done to analyze the selectivity of the synthesized ligand based on their docking affinity.
The docking score against tubulin showed that the new ligand had a potent affinity. The binding affinities of
the docked ligand were − 8.5 Kcal mol− 1. The new compound (DMDBIPOT) showed potent in silico activity
against the colchicine active site. It exhibits seven types of interactions, including attractive charge, hydrogen,
carbon-hydrogen, π anion, π sigma, alkyl, and π alkyl bonds as in Fig. 11. DMDBIPOT was found to interact
with the enzyme’s active site through two hydrogen interactions with residue GLN: B336 and PRO: A175, as
well as two attractive charges with the residue ASP: B329. Furthermore, the phenyl ring binds the LEU: B333
with a pi-sigma bond. Moreover, Figs. 10C and 11C depict the posies view of the active binding sites of the
proteins GlcN-6-P synthase and tubulin, respectively. The docking results strongly confirmed the antibacterial
and anticancer activities of the synthesized derivative. The accuracy and reproducibility of this in silico protocol
were validated. In brief, the glucose amine-6-phosphate and the co-crystalized ligand (colchicine) were removed
from their binding site within the target macromolecule, prepared, and redocked into the active catalytic site.
The substrate and the ligand (colchicine) were found to fit back into their original position within the active site
of the isolated macromolecule.
Molecular docking studies revealed that DMDBIPOT binds effectively to the active sites of GlcN-6-P
synthase and tubulin, with binding affinities of − 7.7 kcal/mol and − 8.5 kcal/mol, respectively. These values
indicate a strong interaction with the target proteins, particularly in comparison to previously reported Mannich
bases which often exhibited binding affinities in the range of − 6 to − 7 kcal/mol. The specificity of DMDBIPOT’s

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Fig. 9. DMDBIPOT induce apoptosis in PC-3 cells. (A) represented control untreated cells and (B)
represented DMDBIPOT treated PC-3 cells. Scale bare 10 μm.

interactions was characterized by multiple binding interactions, including hydrogen bonds and hydrophobic
interactions. For instance, in the docking with GlcN-6-P synthase, DMDBIPOT formed hydrogen bonds with
residues CYS300 and LYS487, while also engaging in van der Waals interactions with nearby residues. This
specificity is crucial for its antibacterial activity, as it may prevent the enzyme from facilitating bacterial cell
wall synthesis. Similarly, the docking results with tubulin showed that DMDBIPOT interacts favorably with the
colchicine binding site, which is known to disrupt microtubule polymerization, thereby inhibiting mitosis59–61.

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Fig. 10. (A) Binding of compound DMDBIPOT inside the GlcN-6-P synthase and (B) Two dimensional
interactions between DMDBIPOT and the active site. (C) The active site of the enzyme GlcN-6-P synthase
showing the interactions between the natural ligand glucosamine-6-phosphate and amino acids. (Obtained
from Proteins Plus, a pose view website, at https://proteins.plus/(accessed on February 2, 2024).

The structural insights gained from these docking studies provide a comprehensive understanding of the
molecular basis for the biological activities observed, affirming DMDBIPOT’s potential as a lead compound in
drug development.

Conclusion
A novel DMDBIPOT is a structurally diverse family of chemical compounds created using the Mannich
process to add an aminomethyl function to multiple substrates. The synthetized DMDBIPOT had a specific
microstructure, as FTIR analysis, 1H NMR, and 13C NMR spectra demonstrated. Due to increased resistance
phenotype and significant biofilm formation, the researchers made the quest for an alternative to conventional

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Fig. 11. (A) Binding of compound DMDBIPOT inside the Tubulin. (B) Two dimensional interactions between
DMDBIPOT and the active site. (C) Tubulin (receptor) in complex with the drug (colchicine). (Obtained from
Proteins Plus, a pose view website, at https://proteins.plus/ (accessed on February 2, 2024).

antibiotics. A novel DMDBIPOT could be developed and tested against multidrug biofilm former K. pneumoniae
to see if it has antibacterial and antibiofilm properties. DMDBIPOT has significantly higher anti-K. pneumoniae
biofilm activity than amikacin and has cytotoxic effects on the prostate cancer cell line. It also has anticancer and
antioxidant properties. It appears that DMDBIPOT has the potential to be developed as a novel agent for treating
bacterial infections and prostate cancer cells. DMDBIPOT demonstrates significant antibacterial and anticancer
properties, supported by molecular docking studies that elucidate its interaction mechanisms. The comparative
analyses with previously reported compounds underscore its potential as a therapeutic agent, warranting further
investigation in vivo and in clinical settings. Finally, the results were visualized and analyzed using Discovery
Studio 2021. The validity of docking protocols was confirmed via redocking of crystalized substrate or inhibitor
within the target binding pocket.

Data availability
Data is provided within the manuscript or supplementary information files. Crystallographic data for the struc-
tures reported in this article have been collected form Molecular docking program ​(​​h​t​t​p​s​:​/​/​w​w​w​.​ch ​ ​e​m​c​o​m​p​.​c​
o​m​/​e​n​/​P​r​o​d​uc​ ​t​s​.​h​t​m​​​​)​ ​. The protein structure used in the present study, obtained from the PDB (Protein Data
Bank), is available at https://www.rcsb.org/structure/7ZWA. The (https://gaussian.com/) structures of ​c​o​m​p​o​u​n​

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d​s were drawn using Chem Office (15.0) (​h​t​t​p​s​:​​​/​​/​p​e​r​k​i​n​e​l​m​er​​ ​-​c​h​e​​m​o​f​f​i ​c​​e​p​r​o​f​e​​s​s​i​o​n​a​​​l​.​s​o​f​​t ​w​a​​r​e​​.​i​nf​ ​o​r​​m​​e​r​.​​co
​​ ​m​​/​​1​​​
5​.​0​/#​ ​g​​o​o​g​l​e​_​v​i​g​n​e​t​t​e).” Teritary structure of the protein has been collected form PDB (GlcN-6-P synthase (PDB:
1MOQ) https://www.rcsb.org/structure/1MOQ, Tubulin colchicine binding site (PDB: 4O2B) ​h​t​t​p​s​:​/​/w ​ ​w​w​.​r​c​s​b​
.​or​ ​g​/​s​t​r​u​c​t​ur​ ​e​/​4​O​2​B​​​​.​​

Received: 6 February 2025; Accepted: 9 April 2025

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Acknowledgements
The authors appreciated the University of Technology, Iraq, for the logistic support of this work. The authors
extend their appreciation to the researchers supporting project number (RSPD2025R971), King Saud University,
Riyadh, Saudi Arabia, for funding this study.

Author contributions
Conceptualization and methodology, Kh.F.Al., B.A.H., R.A.I., Kh.H.R., M.S.J., S.S.Sh., K.H.J., A.M.A., S.G., (A)
A.S.; formal analysis, Kh.F.Al., (B) (A) H., and, M.S.J.; Investigation and data curation Kh.F.Al., (B) (A) H.,
R.A.I., Kh.H.R., and M.S.J.; validation S.S.Sh., and K.H.J.; Visualization, original draft preparation, Kh.F.Al., (B)
A.H., R.A.I., Kh.H.R., M.S.J., S.S.Sh., K.H.J., A.M.A., S.G., (A) A.S.; writing—review and editing; supervision,
Kh.F.Al., (B) A.H., and M.S.J. All authors reviewed the manuscript.

Funding
No funding was obtained for this study.

Declarations
Competing interests
The authors declare no competing interests.

Additional information
Supplementary Information The online version contains supplementary material available at ​ht​ ​t​p​s​:​/​/​d​o​i.​ ​o​r​g​/​1​
0​.​1​03​ ​8​/​s​4​1​5​9​8​-0​ ​2​5​-​9​8​0​6​1​-​5​​​​.​​
Correspondence and requests for materials should be addressed to M.S.J. or A.A.S.

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