Targeting microbial resistance: Synthesis, antibacterial evaluation, DNA binding and modeling study of new chalcone-based dithiocarbamate derivatives

Article abstract of DOI:10.1016/j.bioorg.2019.01.001

New dithiocarbamate chalcone-based derivatives were synthesized, their structures were elucidated using different spectroscopic techniques. They were subjected to antimicrobial screening against selected Gram negative bacteria focusing on microbial resistance. Bacterial resistance was targeted via phosphoethanolamine transferase enzyme. Most of the synthesized compounds showed equal or higher activity to colistin standard. Compound 24 proved to be the most active candidate with MIC of 8 μg/ml against both Ps12 and K4 and MBC of 32 μg/ml against Ps12 and 16 μg/ml against K4 Molecular docking study showed that 20, 22, 24 and 25 had good binding affinity with active site residues via Thr280. DNA macromolecule was further targeted. Compounds 28 and 34 were recorded to have better DNA binding than doxurubucin with IC₅₀ of 27.48 and 30.97 μg/ml respectively, suggesting that it could have a role in their higher antibacterial effect. Their docking into DNA has shown a clear intercalation matching with antibacterial data. Pharmacokinetics parameters of active compounds showed that they have better absorption through GIT.

Full text of DOI:10.1016/j.bioorg.2019.01.001

Accepted Manuscript
Targeting Microbial Resistance: Synthesis, Antibacterial Evaluation, DNA
Binding and Modeling Study of New Chalcone-Based Dithiocarbamate Deriv-
atives
Marwa Ayman, Shahenda M. El-Messery, Elsayed E. Habib, Sara T. Al-
Rashood, Abdulrahman A. Almehizia, Hamad M. Alkahtani, Ghada S. Hassan
PII:
S0045-2068(18)30751-X
https://doi.org/10.1016/j.bioorg.2019.01.001
YBIOO 2705
DOI:
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
21 July 2018
29 December 2018
1 January 2019
Please cite this article as: M. Ayman, S.M. El-Messery, E.E. Habib, S.T. Al-Rashood, A.A. Almehizia, H.M.
Alkahtani, G.S. Hassan, Targeting Microbial Resistance: Synthesis, Antibacterial Evaluation, DNA Binding and
Modeling Study of New Chalcone-Based Dithiocarbamate Derivatives, Bioorganic Chemistry (2019), doi: https://
doi.org/10.1016/j.bioorg.2019.01.001
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Targeting Microbial Resistance: Synthesis, Antibacterial Evaluation, DNA
Binding and Modeling Study of New Chalcone-Based Dithiocarbamate
Derivatives
Marwa Ayman1, Shahenda M. El-Messery2#, Elsayed E. Habib3,4, Sara T. Al-Rashood5,
Abdulrahman A. Almehizia5, Hamad M. Alkahtani5, Ghada S. Hassan6*
1Faculty of Pharmacy, Mansoura University, P.O.Box 35516, Mansoura, Egypt
2Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura University, P.O.Box 35516, Mansoura, Egypt
3Department of Pharmaceutics and Pharmaceutical Technology, College of Pharmacy, Taibah University, Almadinah Almunawwarah,
344, Saudi Arabia.
4Department of Microbiology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
5Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, 11451, Saudi Arabia.
6Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, P.O.Box 35516, Mansoura, Egypt
* Corresponding author. Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University,P.O.Box
35516, Mansoura, Egypt. E-mail: ghadak25@yahoo.com. Tel, Fax: +20-50-2200242
# Corresponding author. Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Mansoura
University, P.O.Box 35516, Mansoura, Egypt. E-mail: selmessery@gmail.com Tel, Fax: +20-50-2200242
Abstract
New dithiocarbamate chalcone-based derivatives were synthesized, their structures were elucidated
using different spectroscopic techniques. They were subjected to antimicrobial screening against
selected Gram negative bacteria focusing on microbial resistance. Bacterial resistance was targeted via
phosphoethanolamine transferase enzyme. Most of the synthesized compounds showed equal or higher
activity to colistin standard. Compound 24 proved to be the most active candidate with MIC of 8 µg/ml against
both Ps12 and K4 and MBC of 32 µg/ml against Ps12 and 16 µg/ml against K4 Molecular docking study
showed that 20, 22,24 and 25 had good binding affinity with active site residues via Thr280. DNA
macromolecule was further targeted. Compounds 28 and 34 were recorded to have better DNA binding
than doxurubucin with IC50 of 27.48 and 30.97 µg/ml respectively, suggesting that it could have a role
in their higher antibacterial effect. Their docking into DNA has shown a clear intercalation matching
with antibacterial data. Pharmacokinetics parameters of active compounds showed that they have
better absorption through GIT.
Keywords: Synthesis; dithiocarbamate chalcones; Bacterial resistance; Molecular docking studies.
1

1. Introduction
Resistance of pathogenic bacteria towards already existing antibiotics has appeared extensively more
and more during the last decade. [1] Trials have been applied to face such resistance, either through
the use of antibiotic combinations, or through discovering new antibiotics that help repelling the
infectious diseases or through the use of natural products to decrease the effects of such drugs on
human bodies.[2, 3] The discovery of new lines of treatment may involve the use of new agents having
new modes of action, or combination of conventional antibiotics and suitable agents to treat the
infections produced by these pathogens. [3] The polymyxin colistin is a key antibiotic that acts as last
resort for infections treatment caused by multidrug resistant pathogens [4]. It acts through binding of
its positively-charged cyclic peptide to the negatively charged Lipid A headgroup. The colistin key
resistance mechanism occurs through the production of MCR-1 which is a plasmid-encoded
phosphoethanolamine transferase (PEA transferase) and it was isolated from many pathogens such as
Escherichia coli and Klebsiella pneumoniae. MCR-1 catalyses the transfer of positively charged PEA
transferase onto lipid A, which is incorporated to the outer membrane, and consequently decreases the
outer membrane negative charge and finally prevent the action of colistin producing pathogen
resistance to the latter.[4] So, to decrease the pathogen resistance to colistin, blocking of MCR-1 is
essential. Chalcones are compounds with simple structure that are formed through Claiesen-Schmidt
reaction between aldehydes and active methylene group in ketone under basic conditions. It is
considered to be important part of many biologically active agents. Chalcones and their derivatives
exert many activities such as antihypertensive, antitumor, anti-inflammatory and antioxidant
effects.[5] Naturally-occuring chalcones usually contain a variety of substituents such as hydroxyl,
methoxy, or phenyl groups, on the other hand, synthetic derivatives contain other substitution such as
halogen, nitro, and the benzene ring can be substituted by other aryl or heteroaryl rings. [6] Different
dithiocarbamate (DTC) ligands either connected to Zn (II) and Ni (II) complexes [7] or to different
amines [8] have showed significant antimicrobial activity[9]. Moreover dithiocarbamate moiety was
also included in carbapenem antibiotic chart 1 (A) and also in different metallic amtimicrobial
compounds as in chart 1 (B).[10, 11]
Moreover, it was concluded that the molecular hybridization technique of DTC with another
pharmacophore may be a successful method to discover a new promising antimicrobial candidate of
different physicochemical properties. Accordingly, they can be considered as lead to be used as
antimicrobial agents. [12]
2

OH
H
S
O
V
NH2
S
S
S
S
H
S
N
N
N
N
N
O
N
O
O
Cl
B
O
A
Chart 1: Structures of antimicrobial dithiocarbamates
In the present work, and in a continuation of our previous efforts to design and synthesize small
molecules of potential antimicrobial effect [13-16], a series of chalcone-based dithiocarbamates
derivatives has been designed using molecular hybridization technique gathering chalcones,
ditiocarbamate and amine moieties (chart 2) . They have been synthesized then characterized by
spectral data including IR, 1HNMR, 13CNMR, and mass spectrometry. The microbial resistance was
targeted through antibacterial evaluation of highly resistant gram-negative bacteria such as
Pseudomonas aeruginosa and Klebsiella pneumonia. The minimum inhibitory concentrations (MIC)
and minimum bactericidal concentrations (MBC) of the synthesized compounds were determined. To
study the mechanism of the antimicrobial activity obtained, two molecular targets were considered
PEA transferase inhibition as a first target focusing on bacterial resistance. A second goal targeting
DNA binding affinity relying on chalcone fragment of our compounds was attained. Docking studies
have been applied to understand the obtained results. ADMET study for the most active compounds
was also determined .
.
3

R1
O
N
S
Chalcone
X
S
X= N, O
Amines
thiocarbamate
R2
Potent antibacterial fragments
Interaction with DNA
Hybridization
X
Amine Drivatives
N
Different pattern
of substitution
Thiocarbamate Link
O
S
S
R2
Different pattern
of substitution
R1
Target Compounds
Chart 2: Representation of different active part of newly synthesized compounds
4

2. Results and Discussion
2.1. Chemistry
The synthetic procedures of the target compounds were illustrated in Scheme 1. Starting with
condensation reaction between acetophenone derivatives (1,2) and substituted aldehyde (3-7) to yield
the α,β-unsaturated compounds (8-17). [17a,b] The final compounds were obtained through two step
reaction, firstly, the reaction of compounds 8-17 with carbon disulphide in ethanol [17c], followed by
reaction of the resultant products –used without purification- with secondary amine either morpholine
or 1-benzyl piperazine producing the final compounds in good yields. (Scheme 1, Table 1). The
structures of the synthesized compounds have been confirmed through spectral data including IR,
13
1HNMR, CNMR and mass spectrometry. All synthesized compounds have shown a characteristic
C=S band in 950-1050 cm-1 range proving the inclusion of DCM group in all compounds accompanied
1
by characteristic N-CS band around 1530 cm-1. HNMR spectral data for compounds 20-29 showed
the appearance of morpholine protons either in the form of multiplets ranging from 2.34 – 3.50 or in
the form of two triplets ranging from 3.07 – 3.43 and 3.59 – 3.73. Similarly, for compounds 30-39, the
appearance of two benzylic protons in addition to the piperazine protons confirmed the structures of
the target compounds. The benzylic protons appeared in range of 3.43 – 4.22, and the piperazine
protons appeared either as multiplet peaks at range of 3.10 – 3.48, or as two triplets appearing at 3.10
– 3.11 and 3.52 - 3.58. The mass spectral data are consistent with the synthesized products through the
appearance of M+ for each compound under investigation.
O
H
O
CH3
O
10% alc. NaOH
R2
R2
+
R1
R1
: R1 = CH3
: R1 = OCH3
: R2 = 4-CH3
3
8-17
R
R
R
R
2 = 4-Cl
4:
5:
6:
7:
1
2 = 4-OCH3
2 = 3,4 di(OCH3)2
2 = 3,4,5 tri(OCH3)3
2
CS2
Ethanol
X
N
S
O
S
H
Ethanol
O
S
S
X
NH
+
R2
18: X = O
19: X = N-CH2-C6H5
R2
R1
R1
20-39
Scheme 1: Synthesis of the target compounds 20-39
5

Table 1: The physiochemical properties of the synthesized compounds 20-39
X
N
O
S
S
R2
R1
20-39
R1
CH3
R2
CH3
Cl
X
O
O
O
O
O
O
O
O
O
O
% Yield
75
Mp. oC
172-175
152-155
186-189
142-146
200-203
179-182
169-171
194-167
160-163
198-201
133-136
176-179
159-161
155-158
166-169
138-141
149-152
211-215
191-193
182-184
Molecular Formulaa
C22H25NO2S2 (399.57)
C21H22ClNO2S2 (419.98)
C22H25NO3S2 (415.57)
C23H27NO4S2 (445.59)
C24H29NO5S2 (475.62)
C22H25NO3S2 (415.57)
C21H22ClNO3S2 (435.98)
C22H25NO4S2 (431.57)
C23H27NO5S2 (461.59)
C24H29NO6S2 (491.62)
C29H32N2OS2 (488.71)
C28H29ClN2OS2 (509.12)
C29H32N2O2S2 (504.71)
C30H34N2O3S2 (534.73)
C31H36N2O4S2 (564.76)
C29H32N2O2S2 (504.71)
C28H29ClN2O2S2 (525.12)
C29H32N2O3S2 (520.71)
C30H34N2O4S2 (550.73)
C31H36N2O5S2 (580.76)
Comp. no.
20
CH3
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
55
62
88
49
90
57
76
69
80
84
93
61
74
59
66
83
CH3
OCH3
CH3
di- OCH3
tri- OCH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
CH3
Cl
OCH3
di- OCH3
tri- OCH3
CH3
N-CH2-C6H5
N-CH2-C6H5
N-CH2-C6H5
N-CH2-C6H5
N-CH2-C6H5
N-CH2-C6H5
N-CH2-C6H5
N-CH2-C6H5
N-CH2-C6H5
N-CH2-C6H5
CH3
Cl
CH3
OCH3
CH3
di- OCH3
tri- OCH3
CH3
CH3
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
OCH3
37
38
39
54
89
79
di- OCH3
tri- OCH3
aAnalysed for C,H,N; results were within ± 0.4 % of the theoretical values for the formulae given
2.2.
In vitro antibacterial activity
The synthesized compounds were tested for their in vitro antibacterial activity against selected
multidrug-resistant Gram negative clinical isolate, Pseudomonas aeruginosa (Ps12) and Klebsiella
pneumoniae (K4). The primary screen was carried out using the agar disc-diffusion method using
Müller-Hinton agar medium. The results of the preliminary antimicrobial testing of the synthesized
compounds and the antibacterial activity of the selected compound Colistin are shown in Table 2. The
results revealed that the majority of the synthesized compounds showed varying degrees of inhibition
against the tested microorganisms. The Bacterial isolates showed a moderate resistant to colistin which
considered to be more effective against the multidrug-resistant Gram negative bacteria. Compounds
6

20, 22, 25- 28, 33 - 37 showed good activity against the bacterial isolate. The most active compound
24 showed a very strong activity against both multidrug-resistant Gram negative clinical isolate,
Pseudomonas aeruginosa (Ps12) and Klebsiella pneumoniae (K4). The MIC and MBC for the most
active compounds against the same microorganism used in the primary screening was carried out using
the micro dilution susceptibility method in
Müller-Hinton Broth as shown in Table 3. Compound 24 showed very good MIC and MBC against
tested organisms. This results may explain that the multidrug-resistant Gram negative isolate produce
phosphoethanolamine transferase enzyme that is responsible for the resistant of these bacteria to the
most potent antibacterial agent, colistin and the tested compounds may be inhibited this enzyme so the
bacteria become highly sensitive to the tested compounds.
Table 2: Antibacterial activity of tested compounds (200 µg/8 mm disc) against selected multidrug-resistant
Gram negative clinical isolate, Pseudomonas aeruginosa (Ps12) and Klebsiella pneumoniae (K4).
Inhibition zone diameter (mm)
Comp. No.
Pseudomonas aeruginosa (Ps12)
Klebsiella pneumoniae (K4)
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
28
10
18
15
30
24
25
23
22
15
12
10
10
18
23
18
24
18
10
17
18
26
15
22
20
32
25
24
22
21
20
12
11
10
24
20
20
25
22
11
22
14
36
37
38
39
Colistin
Not active (8 mm), Weak activity (8-12 mm), Moderate activity (12-18 mm), Strong activity (> 18 mm). Solvent: DEMSO (8 mm).
7

Table 3: The Minimal Inhibitory Concentrations (MIC, µg/ml) and Minimal Bactericidal Concentrations
(MBC µg/ml) of compounds 20, 22, 24, 25. 26, 27, 28 33, 34 35, 36 and 37 in comparison with the broad
spectrum antibacterial drug, Colistin against selected multidrug-resistant Gram negative clinical isolate,
Pseudomonas aeruginosa (Ps12) and Klebsiella pneumoniae (K4).
Comp.
No.
Pseudomonas
aeruginosa (Ps12) pneumoniae (K4)
Klebsiella
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
16
32
> 64
ND
8
32
32
ND
32
64
32
> 64
64
ND
8
16
16
64
32
> 64
64
ND
32
ND
32
ND
64
ND
> 64
ND
16
64
32
20
22
24
25
26
32
ND
32
27
28
> 64
> 64
ND
32
> 64
> 64
ND
32
> 64
> 64
ND
> 64
ND
33
34
35
36
37
> 64
> 64
ND
Colistin
ND: Not Determined.
8

2.3. DNA Binding Assay
DNA is the pharmacologic target of many antimicrobial drugs currently in clinical use or in advanced
clinical trials. It was proved that small molecules can act as potent antimicrobials via DNA
intercalation. A colourimetric methyl green DNA displacement assay method was used to reversibly
bind to DNA, and the colored complex is stable at neutral pH, whereas free methyl green fades at this
pH value. [18] Incubation for 24 h, in the buffer was used for displacement reactions in this study,
results in virtually a complete loss of methyl green absorbance. In this study, compounds 20, 28, 34
,showed the highest affinity to DNA, compounds 20 and 23 with even equipotent activity to
doxorubusin while compounds 21 and 31 are the least DNA binding compounds other compounds
does not show DNA binding affinity although some of them have potent antibacterial activity which
could be attributed to another mechanism. (Table 4) Some of These results are in accordance with the
antimicrobial screening data and explain them,suggesting that binding with DNA may contribute the
activity of these compounds against bacterial infections.
Table 4: DNA/methyl green colourimetric assay of the DNA-binding compounds
DNA-active compound
DNA/methyl green (IC50 µg/ml)
31.27±1.8
39.70±2.1
81.12±3.5
44.81±2.3
36.14±1.9
27.48±1.5
63.24±2.7
78.03±3.2
83.26±3.9
30.97±1.7
48.20±2.4
67.35±3.1
55.79±2.6
DOX
20
21
22
23
28
29
30
31
34
35
38
39
9

2.3 Molecular Docking Study
Molecular docking and virtual screening have become an integral part of many modern structure-based
drug discovery efforts. The binding of small molecule ligands to large protein targets is central to
numerous biological processes. The accurate prediction of the binding modes between the ligand and
protein (the docking problem) is of fundamental importance in modern structure-based drug design.
[19, 20] MOE as a flexible docking program facilitates the prediction of favorable protein–ligand
complex structures with reasonable accuracy and speed. Phosphoethanolamine (PEA) trasferase and
DNA were targeted for docking to suggest the mechanism of antibacterial action
2.3.1 Phosphoethanolamine transferase enzyme:
Binding affinities and orientation of compounds at the calculated active site of the PEA trasferase
enzyme were determined. The protein–ligand complex was constructed based on the X-ray structure
PEA transferases belong to the alkaline phosphatase super-family. Many multidrug resistance (MDR)
gram-negative bacteria possess multiple members of this family of enzymes that are engaged in the
decoration of lipid A or the conserved inner core of the lipopolysaccharide [21,22]. To investigate
possible binding mode interactions, molecular docking studies were performed by domly docking
representative active antimicrobial compounds of our study into PEA transferase enzyme. The
recently-solved X-ray crystal structure of the PDB ID: 5FGN [23] was used as the starting model.
Structure of a lipid A PEA transferase suggests how conformational changes govern substrate binding
. The corresponding cocrystallized ligand show a network of hydrogen bonding with with Thr 280 Gln
91, Ala281, Asn, 106 and His 465 amino acid residues .(Fig. 1) It was reported that Thr.280 is a
considered a key aminoacid Especially concering binding. [23] All the compounds were found to
exhibit binding poses similar to those of cocrystallized ligands occupying the binding cavity and, to
varying extents, the gorge connecting it . Compound 20 was recorded to occur in active site
(characterized by our modeling experiment) binding to Thr 280 and His 465 by hydrogen bonding
between carbonyl oxygen and Met 103 and aromatic cationic bonding to Val 107 (Fig 2). Compound
22 showed Hydrogen bonding via thiocarbonyl sulphur to Thr 110 and another one through sulphur
atom to Asn 106 .Additional hydrogen bond is detected via morpholine ring oxygen to His 465 (Fig
3) while compound 24 has produced A net work of hydrogen bonding between carbon and oxygen
and both Asn 108 and Met 103 occupying all active site .(Fig. 4, 5) In addition to the sulphur atom
contribution by both hydrogen bonding tp Gln 91 and a arene-hydrogen tp Gln 91 too compound, 25
were connected to the enzyme via Thr 280, His 453, Ala 281, Ser 279 and His 465 through hydrogen
bonding (Fig. 6). The obtained results may suggest that the antibacterial activity of compounds 20, 22,
24 and 25 may occur via PEA transferase inhibition.
10

Figure 1: 2D binding mode and residues involved in the recognition of reference cocrystalized
ligand in phosphoethanolamine (PEA) transferase active site
Figure 2: 2D binding mode and residues involved in the recognition of 20 in PEA transferase
active site .
11

Figure 3: 2D binding mode and residues involved in the recognition of 22 in PEA transferase
active site .
Figure 4: 2D binding mode and residues involved in the recognition of 24 in PEA trasferase
active site
12

Figure 5: The aligned conformations of the most active compound 24 (IC50 0.004 M); occupying
phosphoethanolamine transferase active site
Figure 6: 2D binding mode and residues involved in the recognition of 25 in PEA transferase
active site.
2.3.2 Molecular Modeling Study & DNA Binding :
Molecular docking technique has been used as a tool to understand the practical results obtained from
DNA binding assay method to present a mechanistic study. They were docked into the binding site
region of the DNA mainly in a noncovalent fashion [13]. In the present study X-ray crystallographic
structure of the DNA dodecamerd (CGCAAATTTGCG) with a bifurcated hydrogen-bonded
conformation of the AT base pairs and its complex with distamycin A from the Protein Data Bank
(PDB code: 2DND) for the docking study [15]. DNA-binding affinity, as well as the preferred
orientation of the docking pose can be obtained from docking results. Doxorubicin formed a network
of hydrogen bonds (Fig. 7a) and it occupied all the pocket intercalating between the two DNA strands
(Fig. 7b). The lowest energy conformers of selected active and inactive DNA binders have been
obtained using conformational search (Fig 8) to interpretate the results we got, the proposed binding
13

mode of the represented DNA active binders 28 IC50 27.48±1.5 µg/ml and 34 IC50 30.97±1.7 µg/ml
showed that the aromatic planer moiety intercalates toward the minor groove between the double
strands of DNA (Fig 9 a,b). Hence the antibacterial activity of these compounds could be attributed
to DNA binding ability. On the other hand, the inactive candidates 21 and 31 were projecting out of
the double strands interpretating their DNA lower binding affinity (Fig 9 c,d).
.
(a)
(b)
Figure 7 : DNA minor groove binding of (a) Distamycin (yellow) with DNA, DNA is shown in ribbon
form. Distamycin, is shown in ball and cylinder form. Hydrogen-bonding (HB) interactions
of with the DNA bases (dashed lines). Numbers indicate the percent of HB
(a)
(c)
(b)
(d)
Figure 8: Lowest energy conformers of active compounds (a) 28 , (b) 34, and the least active
compounds (c) 21 and (d ) 31 with balls and cylinders.
14

(b)
(a)
(c)
(d)
Figure 9: Close-up view of active candidates 28 (a), 34 (b) and inactive candidates 21 (c), 31
(d) binding in the DNA minor groove, highlighting the “isohelical” conformation of
DNA active binders 28 and 34 to fit into minor groove
15

2.4 ADMET Analysis
The pharmacokinetic properties of most active antibacterial candidates of our study were calculated
theoretically by the use of online application PreADME https://preadmet.bmdrc.kr. The results
obtained revealed that all the tested compounds exerted excellent absorption through the intestine with
HIA values exceeding 97.5 %, in contrast for the standard compound that has no absorption through
the GIT because of its high polarity. In addition, they showed good binding with plasma protein that
can give indication about the diffusion and availability of the tested compounds. Compound 24 exerted
the best BBB penetration among the tested compounds; it has 10 times more penetration than the
standard compound that can give privilege to be used for brain infection. The cell permeability of the
compound 25 is higher than any other derivative. All the compounds proved to be non-carcinogen.
(Table 5).
Table 5: ADMET profile for compounds 24 – 28 and 34,36 and the control drug Colistin
Comp. HIA PPB BBB MDCK Carcinogenecity
24
25
26
27
28
34
36
98.99 91.33 0.31
97.91 95.20 0.03
97.71 96.09 0.11
98.55 95.84 0.22
99.19 93.30 0.37
97.90 87.88 0.15
98.16 90.18 0.07
2.070
55.42
34.59
16.97
1.370
0.060
5.29
Non-carcinogen
Non-carcinogen
Non-carcinogen
Non-carcinogen
Non-carcinogen
Non-carcinogen
Non-carcinogen
Non-carcinogen
Colistin 0.00 43.97 0.03
0.04
HIA : Human Intestinal Absorption, PPB: Plasma Protein Binding, BBB: Blood Brain Barrier, MDCK: Maden Darby Canine Kidney
2.5 Structure-Activity Correlation
In general, the synthesized compounds proved activity against the gram-negative microorganisms used
in the study. Most of them proved to be active than the standard drug used (Colistin). According to
the obtained antibacterial activity, most of the examined compounds revealed high degrees of
inhibition toward the tested organisms. By taking a closer look, it was found that the morpholine
analogs are the most potent series among the synthesized compounds; they exhibited potent activity
higher than the standard drug Colistin with inhibition zone diameter against both Pseudomonas
aeruginosa and Klebsiela pneumoniae ranging from 18 mm till reaching the maximum at 32 mm with
the most potent derivative 24. The latter compound is the most potent among all compounds with
inhibition zone diameter of 30 and 32 mm against the mentioned organisms respectively. The presence
of chloride atom decreased the activity of the analogs as in compounds 21 and 31 in both series, while
the other substitutents such as methyl and methoxy are only effective in the N-benzyl piperazine series
as in compounds 20 and 22. Furthermore, it was observed that most of the active compounds were
effective against Klebsiela pneumonia more than Pseudomonas aeruginosa with increased inhibition
16

zone diameters. In addition to compounds 25-28. On the other hand, replacement of morpholine with
benzyl piperazine retains the activity but with decrease in the potency as in compounds 33-37 and 39.
Furthermore, it was found that the presence of the chloride atom with mono-methoxy group is better
for activity as in case of compounds 26 and 36, while the presence of 2 methoxy groups leads to slight
decrease of potency as in case of compounds 27 and 37. It was also noticed that the introduction of
more than methoxy group retains the activity towards Klebsiella pneumonia while it abolishes the
activity towards Pseudomonas aeruginosa strains as in case of compounds 29, 38 and 39. Moreover,
the presence of methyl group either with the morpholine or the benzyl piperazine series completely
abolishes the activity as in case of compounds 21, 30 and 31.
3
Conclusion
A new series of chalcone-based dithiocarbamate derivatives was synthesized and evaluated for their
antibacterial activity against resistant gram-negative bacteria named Pseudomonas aeruginosa (Ps12)
and Klebsiella pneumoniae (K4). The results revealed the good activity of most of the synthesized
compounds in addition to the presence of potent derivatives that have activity higher than the standard
drug used in the antibacterial screening, Colistin. MIC and MBC for the active compounds were
determined and they are consistent with the results obtained from the preliminary screening. Molecular
DNA binding and PEA transferase enzyme were selected as targets for mechanism of action based on
structure based fragments from one side and the resistance issue from another side respectively.
Modeling studies revealed that compounds such as 20, 22, 24, and 25 have a good binding to PEA
trasferase which may contribute to higher activity against the selected resistant strains via Thr 280 .
Compounds 28 and 34 may excert their action via DNA intercalation as they have higher DNA binding
affinity than doxorubicin itself. Data obtained was supported by antimicrobial screening and DNA
binding assay in addition to docking study. Structure activity relationship has been performed.
Pharmacokinitic properties gives a good optimizing data regarding the better bioavailabilty characters
of synthesized compounds than colistin.
4
Experimental Section
The synthesis of the designed compounds was performed in Faculty of Pharmacy, Mansoura
University, Mansoura, Egypt. The antimicrobial screening was conducted in the department of
Micobiology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt. It was performed in
comparison with the broad spectrum antibacterial drug, Colistin against selected multidrug-resistant
Gram negative clinical isolate, Pseudomonas aeruginosa (Ps12) and Klebsiella pneumoniae (K4).
Molecular docking experiments were performed using ‘Molecular Operating Environment’ software
17

on Core i7 workstation. Melting points (°C) were determined on Mettler FP80 melting point
apparatus and are uncorrected. Microanalyses were performed on a Perkin-Elmer 240 elemental
analyzer. All of the new compounds were analyzed for C, H and N and agreed with the proposed
structures within ± 0.4% of the theoretical values.IR spectra were recorded in central lab of Fac. Of
Pharmacy, Mansoura Uniersity. 1H, 13C-NMR were recorded on a Joel 500 MHz FT spectrometer;
chemical shifts are expressed in  ppm with reference to TMS. Mass spectral (MS) data were
obtained on a Perkin Elmer, Clarus 600 GC/MS and Joel JMS-AX 500 mass spectrometers. Thin
layer chromatography was performed on precoated (0.25 mm) silica gel GF254 plates (E. Merck,
Germany), compounds were detected with 254 nm UV lamp. Silica gel (60230 mesh) was
employed for routine column chromatography separations. All the fine chemicals and reagents used
were purchased from Aldrich Chemicals Co, USA.
4.1 Chemistry
4.1.1 1-(Substituted phenyl)-3-oxo-3-(4-substituted phenyl)propyl morpholine-4-carbodithioate
(20-29).
A mixture of carbondisulfide (0.15 ml, 2.5 mM) and chalcone 8-17 (2.5 mM) was dissolved in
dichloromethane (10 ml) and the solution was cooled to 0 oC in an ice bath. Morpholine (0.19 g, 2.25
mM) was slowly added and the reaction mixture was stirred at 0 oC for 30 min. Then, the solution was
warmed to room temperature and stirred for another 24 h, and the reaction was monitored by TLC.
After the end of the reaction, solvents were removed under vaccum and the residue was purified by
column chromatography on silica gel (ethyl acetate–petroleum ether) affording compound
dithiocarbamates (Table 1).
3-Oxo-1,3-di-p-tolylpropyl morpholine-4-carbodithioate (20) :
1H-NMR (DMSO-d6); δ 1.04 (s, 3H, CH3), 2.40 (s, 3H, CH3), 3.38 (d, 2H, CH2- C=O), 3.44 – 3.48
(m, 8H, morpholine-H), 4.29 (t, 1H, J = 9.5, benzylic-H), 7.00 (d, 2H, J = 8.5, Ar-H), 7.36 (d, 2H, J =
8.0, Ar-H), 7.84 (d, 2H, J = 8.5, Ar-H), 8.05 (d, 2H, J = 8.0, Ar-H). 13C-NMR δ 15.2, 20.4, 35.2, 49.1,
53.0, 54.8, 62.1, 63.2, 119.2, 120.4, 121.1, 126.0, 128.5, 130.7, 135.9, 137.4, 139.6, 140.7, 144.0,
146.2, 185.9, 194.6. IR (cm -1): 1537 (N-CS), 1012 (C-S), MS m/z (%): 399.6 (15.2, M+).
1-(4-Chlorophenyl)-3-oxo-3-(p-tolyl)propyl morpholine-4-carbodithioate (21):
1H-NMR (DMSO-d6); δ 1.03 (s, 3H, CH3), 3.16 (d, 2H, CH2- C=O), 3.44-3.50 (m, 8H, morpholine-
H), 4.32 (t, 1H, J = 9.5, benzylic-H), 7.37 (d, 2H, J = 7.5, Ar-H), 7.52 (d, 2H, J = 7.5, Ar-H), 7.94 (d,
2H, J = 8.5, Ar-H), 8.07 (d, 2H, J = 8.0, Ar-H). 13C-NMR δ 21.0, 35.9, 50.2, 53.1, 53.9, 60.9, 64.1,
18

120.7, 122.9, 124.1, 126.0, 129.3, 130.7, 132.1, 133.8, 135.0, 141.2, 144.6, 145.2, 179.2, 185.6. MS
m/z (%): 419.9 (19.3, M+).
1-(4-Methoxyphenyl)-3-oxo-3-(p-tolyl)propyl morpholine-4-carbodithioate (22)
1H-NMR (DMSO-d6); δ 1.04 (s, 3H, CH3), 2.34 – 2.43 (m, 8H, morpholine-H), 2.48 (d, 2H, J = 3.0,
CH2- C=O), 3.41 (s, 3H, OCH3), 4.29 (t, 1H, J = 9.5, benzylic-H), 7.26 (d, 2H, J = 7.5, Ar-H), 7.36 (d,
2H, J = 8.0, Ar-H), 7.77 (d, 2H, J = 8.0, Ar-H), 8.05 (d, 2H, J = 8.5, Ar-H). 13C-NMR δ 23.5, 34.7,
49.8, 53.3, 54.6, 55.6, 63.1, 65.2, 119.2, 120.7, 121.6, 123.8, 126.9, 129.1, 130.6, 133.9, 136.0, 139.4,
142.1, 146.3, 169.7, 189.4. MS m/z (%): 415.6 (11.8, M+).
1-(3,4-Dimethoxyphenyl)-3-oxo-3-(p-tolyl)propyl morpholine-4-carbodithioate (23):
1H-NMR (DMSO-d6); δ 1.03 (s, 3H, CH3), 2.39 (d, 2H, J = 3.0, CH2- C=O), 3.45 – 3.48 (m, 8H,
morpholine-H), 3.84 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 4.28 (t, 1H, J = 9.5, benzylic-H), 7.02 (d, 2H,
J = 8.0, Ar-H), 7.37 (d, 2H, J = 8.5, Ar-H), 7.52 (s, 1H, Ar-H), 8.05 (d, 2H, J = 8.5, Ar-H). 13C-NMR
δ 22.7, 38.4, 50.9, 52.7, 53.6, 55.9, 56.3, 63.9, 65.7, 117.6, 119.4, 120.4, 124.0, 127.3, 128.4, 129.1,
133.4, 134.8, 136.7, 139.6, 145.1, 174.8, 183.4. IR (cm -1): 1530 (N-CS), 1005 (C-S), MS m/z (%):
445.6 (35.1, M+).
3-Oxo-3-(p-tolyl)-1-(3,4,5-trimethoxyphenyl)propyl morpholine-4-carbodithioate (24):
1H-NMR (DMSO-d6); 1.05 (s, 3H, CH3), 2.40 (d, 2H, J = 3.0, CH2- C=O), 3.07 (t, 4H, J = 9.5,
morpholine-H), 3.49 (s, 3H, OCH3), 3.70 (s, 3H, OCH3), 3.73 (t, 4H, J = 9.5, morpholine-H), 3.85 (s,
3H, OCH3), 4.28 (t, 1H, J = 9.0, benzylic-H), 7.22 (s, 1H, Ar-H), 7.37 (d, 2H, J = 8.0, Ar-H), 7.66 (s,
1H, Ar-H), 8.07 (d, 2H, J = 8.0, Ar-H). 13C-NMR δ 22.1, 38.7, 49.9, 50.4, 52.3, 55.6, 56.7, 59.1, 62.1,
64.9, 118.1, 120.4, 122.1, 126.7, 129.4, 132.0, 133.8, 135.1, 136.2, 137.5, 140.1, 145.3, 168.2, 179.4.
MS m/z (%): 475.6 (10.7, M+).
3-(4-Methoxyphenyl)-3-oxo-1-(p-tolyl)propyl morpholine-4-carbodithioate (25):
1H-NMR (DMSO-d6); δ 2.23 (s, 3H, CH3), 2.34 (d, 2H, J = 1.5, CH2- C=O), 3.34-3.48 (m, 8H,
morpholine-H), 3.86 (s, 3H, OCH3), 4.29 (t, 1H, J = 9.5, benzylic-H), 7.07 (d, 2H, J = 6.5, Ar-H), 7.27
(d, 2H, J = 8.0, Ar-H), 7.77 (d, 2H, J = 8.0, Ar-H), 8.15 (d, 2H, J = 9.5, Ar-H). 13C-NMR δ 20.6, 38.9,
50.1, 51.3, 53.7, 55.7, 63.0, 64.7, 120.4, 122.1, 123.9, 126.8, 129.8, 132.5, 136.2, 144.2, 146.1, 150.8,
152.6, 157.1, 169.5, 189.0. MS m/z (%): 415.6 (24.8, M+).
1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-3-oxopropyl morpholine-4-carbodithioate (26):
1H-NMR (DMSO-d6); δ 2.77 (d, 2H, J = 1.5, CH2- C=O), 3.46 - 3.55 (m, 8H, morpholine-H), 3.85 (s,
3H, OCH3), 4.28 (t, 1H, J = 9.5, benzylic-H), 7.08 (d, 2H, J = 8.5, Ar-H), 7.51 (d, 2H, J = 9.0, Ar-H),
7.91 (d, 2H, J = 8.0, Ar-H), 8.16 (d, 2H, J = 8.5, Ar-H). 13C-NMR δ 37.1, 49.3, 51.8, 54.9, 55.6, 62.4,
19

66.9, 119.0, 126.4, 128.3, 129.0, 131.2, 133.7, 135.8, 137.0, 138.6, 139.2, 143.2, 148.1, 159.5, 173.0.
MS m/z (%): 435.9 (17.4, M+).
1,3-Bis(4-methoxyphenyl)-3-oxopropyl morpholine-4-carbodithioate (27):
1H-NMR (DMSO-d6); δ 2.87 (d, 2H, J = 1.5, CH2- C=O), 3.48 (t, 4H, J = 9.5, morpholine-H), 3.61
(t, 4H, J = 9.5, morpholine-H), 3.81 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.29 (t, 1H, J = 10.0, benzylic-
H), 6.99 (d, 2H, J = 7.0, Ar-H), 7.08 (d, 2H, J = 9.0, Ar-H), 7.84 (d, 2H, J = 8.0, Ar-H), 8.15 (d, 2H, J
13
= 8.5, Ar-H). C-NMR δ 38.2, 47.5, 50.4, 53.7, 55.5, 57.6, 63.9, 66.0, 122.0, 124.7, 126.3, 129.4,
133.2, 134.1, 136.8, 137.6, 139.6, 144.3, 148.5, 155.4, 176.3, 190.6. MS m/z (%): 431.6 (13.3, M+).
1-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-3-oxopropyl morpholine-4-carbodithioate (28):
1H-NMR (DMSO-d6); δ 3.22 (d, 2H, CH2- C=O), 3.42 – 3.48 (m, 8H, morpholine-H), 3.81 (s, 3H,
OCH3), 3.86 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 4.28 (t, 1H, J = 10.0, benzylic-H), 7.01 (d, 2H, J =
7.5, Ar-H), 7.08 (d, 2H, J = 7.5, Ar-H), 7.67 (s, 1H, Ar-H), 8.17 (d, 2H, J = 10.5, Ar-H). 13C-NMR δ
36.2, 45.1, 49.7, 52.2, 55.5, 57.3, 59.8, 63.0, 66.8, 124.6, 125.4, 128.1, 130.8, 134.9, 135.0, 137.2,
139.0, 144.4, 145.6, 148.8, 150.7, 1168.4, 179.2. IR (cm -1): 1520 (N-CS), 1010 (C-S), MS m/z (%):
461.6 (9.8, M+).
3-(4-Methoxyphenyl)-3-oxo-1-(3,4,5-trimethoxyphenyl)propylmorpholine-4-carbodithioate(29):
1H-NMR (DMSO-d6); δ 2.85 (d, 2H, J = 7.5, CH2- C=O), 3.43 (t, 4H, J = 9.0, morpholine-H), 3.59
(t, 4H, J = 8.5, morpholine-H), 3.69 (s, 3H, OCH3), 3.86 (s, 9H, OCH3), 4.28 (t, 1H, J = 9.5, benzylic-
H), 7.10 (d, 2H, J = 9.0, Ar-H), 7.24 (s, 1H, Ar-H), 7.66 (s, 1H, Ar-H), 8.16 (d, 2H, J = 8.5, Ar-H).
13C-NMR δ 30.4, 45.4, 50.8, 52.0, 55.6, 57.8, 60.2, 62.7, 65.1, 69.4, 114.2, 117.3, 119.0, 123.6, 127.2,
128.5, 133.4, 137.0, 144.2, 147.1, 149.2, 153.6, 158.2, 168.4. MS m/z (%): 491.6 (17.2, M+).
4.1.2 1-(Substituted
phenyl)-3-oxo-3-(4-substituted
phenyl)propyl
4-benzylpiperazine-1-
carbodithioate (30-39).
A mixture of carbondisulfide (0.15 ml, 2.5 mM) and chalcone 8-17 (2.5 mM) was dissolved in
dichloromethane (10 ml) and the solution was cooled to 0 oC in an ice bath. 1-Benzyl piperazine (0.39
o
g, 2.25 mM) was slowly added and the reaction mixture was stirred at 0 C for 30 min. the reaction
procedures were continued as under 20-29 (Table 1).
3-Oxo-1,3-di-p-tolylpropyl 4-benzylpiperazine-1-carbodithioate (30):
1H-NMR (DMSO-d6); δ 2.34 (s, 3H, CH3), 2.39 (s, 3H, CH3), 2.54 (d, 2H, J = 7.5, CH2- C=O), 3.10
(t, 4H, J = 4.5, piperazine-H), 3.52 (t, 4H, J = 6.5, piperazine-H), 3.56 (s, 2H, benzylic-H), 4.36 (t, 1H,
20

J = 7.5, benzylic-H), 7.24 (d, 2H, J = 9.0, Ar-H), 7.28-7.33 (m, 5H, Ar-H), 7.40 (d, 2H, J = 8.5, Ar-
H), 7.77 (d, 2H, J = 7.5, Ar-H), 8.06 (d, 2H, J = 8.5, Ar-H). 13C-NMR δ 21.3, 21.8, 37.0, 49.1, 52.7,
52.9, 53.4, 53.8, 66.2, 124.8, 125.3, 126.2, 128.9, 132.7, 135.0, 138.6, 141.6, 144.0, 144.2, 145.3,
146.2, 147.9, 148.6, 149.5, 151.0, 155.3, 158.7, 169.0, 187.4.
1-(4-Methoxyphenyl)-3-oxo-3-(p-tolyl)propyl 4-benzylpiperazine-1-carbodithioate (31):
1H-NMR (DMSO-d6); δ 2.29 (s, 3H, CH3), 2.56 (d, 2H, CH2- C=O), 3.14 – 3.37 (m, 8H, piperazine-
H), 3.72 (s, 2H, benzylic-H), 4.26 (t, 1H, J = 7.0, benzylic-H), 7.21 (d, 2H, J = 8.5, Ar-H), 7.29-7.35
(m, 5H, Ar-H), 7.42 (d, 2H, J = 8.5, Ar-H), 7.60 (d, 2H, J = 8.0, Ar-H), 8.10 (d, 2H, J = 8.5, Ar-H).
IR (cm -1): 1525 (N-CSS), 1015 (C-S), MS m/z (%): 508.8 (23.8, M+).
1-(4-Chlorophenyl)-3-oxo-3-(p-tolyl)propyl 4-benzylpiperazine-1-carbodithioate (32):
1H-NMR (DMSO-d6); δ 2.21 (s, 3H, CH3), 2.54 (d, 2H, CH2- C=O), 3.11 – 3.33 (m, 8H, piperazine-
H), 3.43 (s, 2H, benzylic-H), 3.80 (s, 3H, OCH3), 4.25 (t, 1H, J = 7.5, benzylic-H), 7.07 (d, 2H, J =
8.0, Ar-H), 7.25 - 7.31 (m, 5H, Ar-H), 7.50 (d, 2H, J = 8.5, Ar-H), 7.66 (d, 2H, J = 8.0, Ar-H), 8.08
(d, 2H, J = 8.0, Ar-H). 13C-NMR δ 20.4, 37.6, 42.1, 53.0, 53.4, 53.7, 53.9, 55.6, 62.3, 123.4, 124.6,
126.1, 127.3, 130.0, 133.8, 134.2, 136.2, 139.9, 140.2, 141.8, 142.9, 144.8, 146.0, 148.7, 149.2, 153.7,
155.3, 167.9, 187.3. MS m/z (%): 504.7 (6.7, M+).
1-(3,4-Dimethoxyphenyl)-3-oxo-3-(p-tolyl)propyl 4-benzylpiperazine-1-carbodithioate (33):
1H-NMR (DMSO-d6); δ 2.25 (s, 3H, CH3), 2.49 (d, 2H, J = 5.5, CH2- C=O), 3.41-3.47 (m, 8H,
piperazine-H), 3.49 (s, 2H, benzylic-H), 3.80 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 4.29 (t, 1H, J = 6.5,
benzylic-H), 7.21 (d, 2H, J = 8.0, Ar-H), 7.30-7.33 (m, 5H, Ar-H), 7.52 (d, 2H, J = 8.5, Ar-H), 7.78
-1
(s, 1H, Ar-H), 8.05 (d, 2H, J = 7.5, Ar-H). IR (cm ): 1517 (N-CS), 1020 (C-S)MS m/z (%): 534.7
(19.3, M+).
3-Oxo-3-(p-tolyl)-1-(3,4,5-trimethoxyphenyl)propyl 4-benzylpiperazine-1-carbodithioate(34):
1H-NMR (DMSO-d6); δ 2.20 (s, 3H, CH3), 2.45 (d, 2H, J = 5.5, CH2- C=O), 3.22 - 3.36 (m, 8H,
piperazine-H), 3.80 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.96 (s, 2H, benzylic-H),
4.25 (t, 1H, J = 6.5, benzylic-H), 7.05 (d, 2H, J = 8.0, Ar-H), 7.22-7.37 (m, 5H, Ar-H), 7.58 (s, 1H,
Ar-H), 7.73 (s, 1H, Ar-H), 8.08 (d, 2H, J = 7.5, Ar-H). 13C-NMR δ 23.1, 36.8, 49.2, 52.7, 53.0, 53.6,
54.0, 55.5, 56.7, 60.1, 63.8, 123.7, 125.7, 127.4, 130.4, 133.6, 137.2, 139.0, 140.0, 141.5, 143.5, 142.8,
144.7, 146.3, 147.2, 149.1, 152.2, 155.6, 157.3, 164.2, 184.0. MS m/z (%): 564.8 (12.4, M+).
3-(4-Methoxyphenyl)-3-oxo-1-(p-tolyl)propyl 4-benzylpiperazine-1-carbodithioate (35):
21

1H-NMR (DMSO-d6); δ 2.25 (s, 3H, CH3), 2.43 (d, 2H, J = 6.0, CH2- C=O), 3.39 -3.48 (m, 8H,
piperazine-H), 3.86 (s, 3H, OCH3), 4.00 (s, 2H, benzylic-H), 4.30 (t, 1H, J = 6.5, benzylic-H), 7.07 (d,
2H, J = 7.5, Ar-H), 7.25-7.32 (m, 5H, Ar-H), 7.76 (d, 2H, J = 8.5, Ar-H), 7.68 (d, 2H, J = 8.0, Ar-H),
13
8.15 (d, 2H, J = 7.5, Ar-H). C-NMR δ 22.4, 37.2, 50.1, 52.6, 52.9, 53.7, 54.0, 55.6, 62.9, 119.2,
122.8, 124.5, 125.9, 126.8, 128.0, 133.4, 135.0, 137.5, 138.1, 139.9, 140.0, 142.1, 144.2, 145.2, 146.3,
148.1, 150.9, 157.6, 175.8. IR (cm -1): 1535 (N-CS), 1018 (C-S) MS m/z (%): 504.7 (31.7, M+).
1,3-bis(4-Methoxyphenyl)-3-oxopropyl 4-benzylpiperazine-1-carbodithioate (36):
1H-NMR (DMSO-d6); δ 2.49 (d, 2H, CH2- C=O), 3.34 - 3.44 (m, 8H, piperazine-H), 3.86 (s, 3H,
OCH3), 4.02 (s, 2H, benzylic-H), 4.29 (t, 1H, benzylic-H), 7.06 (d, 2H, J = 7.5, Ar-H), 7.23-7.31 (m,
5H, Ar-H), 7.52 (d, 2H, J = 8.0, Ar-H), 7.91 (d, 2H, J = 8.0, Ar-H), 8.17 (d, 2H, J = 7.0, Ar-H). 13C-
NMR δ 36.2, 49.3, 53.1, 53.3, 54.3, 54.8, 55.6, 63.0, 120.3, 120.9, 122.4, 123.6, 125.2, 127.8, 129.2,
133.1, 135.6, 137.5, 138.5, 140.2, 141.0, 144.9, 145.7, 146.6, 148.7, 149.2, 151.1, 168.9. MS m/z (%):
524.1 (9.2, M+).
1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-3-oxopropyl4-benzylpiperazine-1-carbodithioate(37):
1H-NMR (DMSO-d6); δ 2.48 (d, 2H, CH2- C=O), 3.11 (t, 4H, piperazine-H), 3.52 (t, 4H, piperazine-
H), 3.58 (s, 2H, benzylic-H), 3.80 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 4.33 (t, 1H, benzylic-H), 7.06
(d, 2H, J = 7.5, Ar-H), 7.25-7.34 (m, 5H, Ar-H), 7.68 (d, 2H, J = 8.5, Ar-H), 7.85 (d, 2H, J = 8.0, Ar-
H), 8.16 (d, 2H, J = 7.5, Ar-H). 13C-NMR δ 37.2, 48.9, 53.7, 53.8, 54.2, 54.6, 55.6, 56.1, 64.2, 120.6,
122.5, 125.7, 126.2, 129.4, 130.0, 131.2, 133.8, 138.7, 140.0, 141.0, 141.8, 143.2, 144.7, 145.6, 147.3,
149.8, 150.6, 157.4, 169.2. IR (cm -1): 1535 (N-CS), 1025 (C-S), MS m/z (%): 520.71 (15.2, M+).
1-(3,4-Dimethoxyphenyl)-3-(4-methoxyphenyl)-3-oxopropyl4-benzylpiperazine-1carbodithioate
(38):
1H-NMR (DMSO-d6); δ 2.49 (d, 2H, CH2- C=O), 3.22-3.41 (m, 8H, piperazine-H), 3.80 (s, 3H,
OCH3), 3.86 (s, 6H, OCH3), 4.22 (s, 2H, benzylic-H), 4.41 (t, 1H, benzylic-H), 7.01 (d, 2H, J = 8, Ar-
H), 7.23-7.36 (m, 5H, Ar-H), 7.38 (d, 2H, J = 8.5, Ar-H), 7.52 (s, 1H, Ar-H), 8.17 (d, 2H, J = 7.5, Ar-
H). MS m/z (%): 550.7 (20.8, M+).
3-(4-Methoxyphenyl)-3-oxo-1-(3,4,5-trimethoxyphenyl)propyl-4-benzylpiperazine-1-
carbodithioate (39):
1H-NMR (DMSO-d6); δ 2.54 (d, 2H, CH2- C=O), 3.10 (t, 4H, piperazine-H), 3.52 (t, 4H, piperazine-
H), 3.53 (s, 2H, benzylic-H), 3.69 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.86 (s, 6H, OCH3), 4.21 (t, 1H,
22

benzylic-H), 7.06 (d, 2H, J = 7.0, Ar-H), 7.21 (s, 1H, Ar-H), 7.24-7.34 (m, 5H, Ar-H), 7.87 (s, 1H, Ar-
H), 8.16 (d, 2H, J = 8.5, Ar-H). 13C-NMR δ 36.5, 49.8, 53.1, 53.5, 53.7, 54.0, 55.6, 56.4, 57.3, 60.2,
62.3, 120.3, 122.4, 124.1, 126.8, 127.8, 130.1, 131.4, 133.0, 136.7, 141.7, 143.7, 144.2, 146.7, 148.9,
150.1, 155.2, 168.9, 172.0. IR (cm -1): 1515 (N-CS), 1009 (C-S) MS m/z (%): 580.7 (13.2, M+).
4.2 Determination of in vitro antimicrobial activity
The primary screen was carried out using the agar disc-diffusion method1 using Müller-Hinton agar
medium. Sterile filter paper discs (8 mm diameter) were moistened with the compound solution in
dimethylsulphoxide of specific concentration 200 µg/disc, the antibacterial antibiotic Colistin (200
µg/disc) was carefully placed on the agar cultures plates that had been previously inoculated separately
with the microorganisms. The plates were incubated at 37oC, and the diameter of the growth inhibition
zones were measured after 24 hours including disc diameter. The Minimal inhibitory concentrations
(MIC) and the minimal bactericidal concentrations (MBC) for the compounds 22, 24, 25. 26, 27, 33,
35, 36 and 37 in comparison with the broad spectrum antibacterial drug, Colistin against selected
multidrug-resistant Gram negative clinical isolate, Pseudomonas aeruginosa (Ps12) and Klebsiella
pneumoniae (K4) were carried out using the microdilution susceptibility method in Müller-Hinton
Broth. The antibacterial drug, colistin, was dissolved in dimethylsulphoxide at concentration of 64
µg/ml. The twofold dilutions of the solution were prepared (64, 32, …, 0.5 µg/ml). The microorganism
suspensions at 106 CFU/ml (colony forming unit/ml) concentrations were inoculated to the
corresponding wells. The plates were incubated at 37 oC for 24 hours. The MIC values were determined
as the lowest concentration that inhibited the growth of the microorganism as detected by unaided eye
and the MBC values were determined by the lowest concentration that killed of the microorganism by
re-cultured on solid medium to verify the absence of growth.[24-25]
4.3 Colourimetric assay for DNA binding affinity
DNA methyl green (20 mg) was suspended in 100 ml of 0.05 M Tris-HCl buffer (pH 7.5) containing
7.5 mM MgSO4; the mixture was stirred at 37 °C with a magnetic stirrer for 24 h. Test samples 10,100,
1000 mg) were dissolved in ethanol in Ependoff tubes, solvent was removed under vacuum, and 200
µl of the DNA/methyl green solution were added to each tube. Samples were incubated in the dark at
ambient temperature. After 24 h, the final absorbance of the samples was determined at 642.5-645 nm.
Readings were corrected for initial absorbance and normalized as the percentage of the untreated
standard using Ethidium bromide as positive control. [18, 26].
4.4 Molecular modelling study
23

Initial
structures
for
the
active
molecule
were
constructed
Mechanics (MM) calculations in vacuo, bond dipole option for electrostatics, Polake–Ribiere
algorithm, and Root Mean Square Deviation (RMSD) gradient of 0.01 kcal/mol conformational
searching in torsional space was performed using the multiconformer method. Energy minima for the
above compounds were determined by a semi-empirical method AM1 (as implemented in MOE,
2009.10). The three-dimensional structures of the substituted dithiocarbamate chalcone derivatives,
which presented best and worst biological profiles, in their neutral forms were constructed using the
MOE of Chemical Computing Group Inc software. Lowest energy conformer of each new analogue
‘global-minima’ was docked into PEA transeferase enzyme-binding domain. Enzyme structure
starting coordinate code ID 5FGN was obtained from the Protein Data Bank of Brookhaven National
Laboratory [23]. All the hydrogens were added and enzyme structure was subjected to a refinement
protocol in which the constraints on the enzyme were gradually removed and minimized until the Root
Mean Square Deviation (RMSD) gradient was 0.01 kcal/mol A. The energy minimization was carried
out using the molecular mechanics force field ‘AMBER.’ The energy-minimized structure was used
for molecular modeling studies keeping all the heavy atoms fixed until a RMSD gradient of 0.05 kcal/
mol/ A˚ was reached. Ligand structures were built with MOE and minimized using the Molecular
Mechanics Force Field (MMFF94x) forcefield until a RMSD gradient of 0.05 kcal/ mol/A˚ was
reached. For each of chalcone analogue, energy minimizations were performed using 1000 steps of
steepest descent, followed by conjugate gradient minimization to a RMSD energy gradient of 0.01
Kcal/mol/ Å. The active site of the enzyme was defined using a radius of 10.0 Å around selected active
site for PEA transferase enzyme. Energy of binding was calculated as the difference between the
energy of the complex and individual energies of the enzyme and ligand . The docking was performed
using the alpha triangle placement method and the London dG scoring method. 300 results for each
ligand were generated, discarding the results with a RMSD value>3 A˚.19 the best scored result of the
remaining conformations for each ligand was further analyzed. The protein/ligand complexes were
minimized using the MMFF94x force field, until a RMSD gradient of 0.1 kcal mol/A˚ was reached
[27-32].
4.5 ADMET analysis
The pharmacokinetic properties of the most active compounds were calculated theoretically by the aid
of online PreADMET database. The most important parameters calculated are, HIA: Human Intestinal
Absorption, PPB: plama protein binding, BBB: permeability to the blood brain barrier, MDCK: Maden
Darby Canine Kidney. When the cell permeability is < 4 nm/s indicates low permeability, when it is
4-70 indicates medium permeability, if > 70 nm/s indicates high permeability. PPB indicates the action,
24

activity and efficacy of the tested compounds. The drug should not bind strongly to the PP to promote
good diffusion and transport to the agent through the target to exert its action when the value is > 90
% indicates the strong binding with the plasma protein.
5 Acknowledgements
The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at
King Saud University for its funding of this research through the Research Group Project no. RG-
1439-010
6 Declarations
The authors report no declarations of interest.
T. El Malah, H.F. Nour, A. A. Nayl, R. A. Elkhashab, F.M.E. Abdel-Megeid, M.M. Ali, Anticancer
evaluation of tris(triazolyl)triazine derivatives generated via click chemistry. Austr. J. Chem. 69(8)
(2016) 905-910
7
References
1- K.S Kaye, J.J Engemann, H.S Fraimow, E. Abrutyn, Pathogens resistant to antimicrobial
agents: epidemiology, molecular mechanisms, and clinical management. Infect.Dis,Clin.
NorthAm, 18 (2004) 467.
2- M. Stavri, L. J Piddock, S. Gibbons,
Novel inhibitory activity of the Staphylococcus
aureus NorA efflux pump by a kaempferol rhamnoside isolated from Persea lingue Nees
J. Antimicrob. Chemother. 59 (2007) 1247.
3- H. A Junio, A. A Sy-Cordero, K. A Ettefagh, J. T. Burns, K. T Micko, T. N. Graf, J. Richter,
R. E. Cannon, N. H. Oberlies, N. B. J Cech,. Isolation of berberine tannate using a
chromatography activity-guided fractionation from root bark of Berberis hispanica Boiss. &
Reut. Nat. Prod. 74, 2011; 1621.
4- S. Biswas, J.M Brunel, J. C Dubus, M. Reynaud-Gaubert, J.M. Rolain,, Colistin: an update on
the antibiotic of the 21st century Expert Rev. Anti. Infect. Ther. 2012;10:917-934.
5- D.K Mahapatra, S.K. Bharti, V. Asati. Chalcone scaffolds as antiinfective agents; Structural
and Molecular target perspectives. Eur. J. Med. Chem. 11 (2015) 496-524.
6- D.L Batovska, I.T. Todorova, Trends in utilization of the pharmacological potential of
chalcones. Curr. Clin.Pharmacolo., 5 (2010) 1-29.
7- A. H. Qasemi, J. Mansouri, Valizadeh, Antimicrobial activity of dithiocarbamate sodium salts
and their Zn (II) and Ni (II) complexes Research in Pharmaceutical Sciences, 7(5) (2012 )
25

8- N. A. J. Ibrahim, B. N. Ibrahim. Synthesis, structural and antibacterial studies of new
dithiocarbamate complexes of Sb(III) and Bi(III) The Malaysian Journal of Analytical
Sciences, 18 (2) (2014) 251 – 259.
9- C. L. Donnici, L. J. Nogueira, Araujo M. H., S. R. Oliveira, T. F. F. Magalhães, M.T.P Lopes,
A. C. Araújo e Silva, A. M. da Costa, F. Cleide, V. B. Martins, M. A. Stoianoff. In Vitro
Studies of the Activity of Dithiocarbamate Organoruthenium Complexes against Clinically
Relevant Fungal Pathogens Molecules 19 (2014) 5402-5420; doi:10.3390/molecules19045402
10- K. S. Kim, D. H. Oh, H. M. Choi, J.S. Bang, C.J. Ryu, J.H.Kim, M.C. Yoo, H.I. Yang.
Pyrrolidine dithiocarbamate, a NF-kappaB inhibitor, upregulates MMP-1 and MMP-13 in IL-
1beta-stimulated rheumatoid arthritis fibroblast-like synoviocytes. Eur. J. Pharmacol., 613
(2009) 167.
11- H. Imamura, N. Ohtake, H. Jona, A. Shimizu, M. Moriya, H. Sato, Y. Sugimoto, C. Ikeura,
H. Kiyonaga, M. Nakano, R. Nagano, S. Abe, K. Yamada, T. Hashizume, H. Morishima,
Dicationic dithiocarbamate carbapenems with anti-MRSA activity. Bioorg. Med. Chem., 9
(2001) 1571–1578
12- N. Lal, Dithiocarbamates: A Versatile Class of Compounds in Medicinal Chemistry Chemistry
& Biology Interface, 4 (2014) 321-340
13- S. M. El-Messery, E. E. Habib, S.T.A. Al-Rashood, G. S. Hassan, Synthesis, Antimicrobial ,
Antibiofilm Evaluation and Molecular Modeling Study of New Chalcone Linked Amines
Derivatives Journal of Enzyme Inhibition and Medicinal chemistry 33(1) (2018) 818–832.
14- R. Al-Harbi, M. Shaaban, R. Al-wegaisia, F. Moharram, O. Abd El-Rahmane, S. M. El-
Messery. Antimicrobial Activity and Molecular Docking of Tannins from Pimenta dioica
Letters in Drug Design & Discovery 14 (2017).
15- G. S. Hassan, S. M. El-Messery, A. Abbas. Synthesis and anticancer activity of new
thiazolo[3,2-a]pyrimidines: DNA binding and molecular modeling study, Bioorganic
Chemistry.74 (2017) 41-52.
16- S. A. El-Mowafy, K. H. Abd El Galil, S. M. El-Messery, and M. I. Shaaban. Aspirin an
Efficient Inhibitor of Quorum Sensing, Virulence and Toxins of Pseudomonas aeruginosa,
Microbial pathogenesis 74 (2014) 25-32.
17- (a) B.S. Holla, B.S. Rao, B.K. Sarojini, P.M. Akberali , One pot synthesis of thiazolodihydro-
pyrimidinones and evaluation of their anticancer activity, Eur. J. Med. Chem. 39 (2004) 777–
783. (b) I. Karaman, H. Gezegen, M.B. Guerdere, A. Dingil, M. Ceylan, Screening of
biological activities of a series of chalcone derivatives against human pathogenic
microorganisms, Chem. Biodivers. 7 (2010) 400–408. (c) A. Z. Halimehjani, K. Marjan and
A. Ashouri Synthesis of dithiocarbamate by Markovnikov addition reaction in aqueous
medium . Green Chem., 12 (2010) 1306–1310.
26

18- I. H. Eissa, A. M. El-Naggar, M. A. El-Hashash Design, synthesis, molecular modeling and
biological evaluation of novel 1H-pyrazolo[3,4-b]pyridine derivatives as potential anticancer
agents Bioorganic Chemistry 67 (2016) 43–56.
19- M. Kontoyianni , .L.M. McClellan, G.S. Sokol. Evaluation of docking performance:
comparative data on docking algorithms. J. Med. Chem. 47 (2004) 558–565.
20- R. Wang, Y. Lu, S. Wang Comparative evaluation of 11 scoring functions for molecular
docking. J Med Chem 46 (2003) 2287–2303.
21- B.D.Needham, M.S. Trent Fortifying the barrier: The impact of lipid A remodelling
on bacterial pathogenesis. Nat Rev Microbiol 11(7) (2013) 467–481.
22- S.N. Bartley, C.M. Kahler The glycome of Neisseria spp.: How does this relate to
pathogenesis? Pathogenic Neisseria: Genomics, molecular biology, and disease intervention,
eds Kahler CM, Davies JK 2014; (Caister Academic Press, Poole, UK), pp 114–145.
23- A Anandan, G L Evans, K. Condic-Jurkic, M. L. O’Mara, M. J. Constance , N. J. Phillips
A .Gary, S . Jarvis Siobhan. A. Wills Keith, Isabel Moraes, C. M. Stubbs Kahler and A.
Vrielink www.pnas.org/cgi/doi/10.1073/pnas.1612927114.
24- Clinical and Laboratory Standards Institute (CLSI). Performance standards for antimicrobial
susceptibility testing, CLSI approved standards M100-S15., Wayne, PA 2008.
25- P. R Murray, E. J Baron, M. A Pfaller, F. C. Tenover, R. H. Yolken, In Manual of Clinical
Microbiology; Wood, G. L.; Washington, J. A., Eds. Am. Soc. Microbiol.: Washington D.C.
1995
26- N.S. Burres , A. Frigo, R. R. Rasmussen, J.B. McAlpine.A colorimetric microassay for the
detection of agents that interact with DNA. J Nat Prod. 55 (1992) 1582-7.
27- N. L. Allinger, Conformational analysis. 130. MM2. A hydrocarbon force field utilizing V1
and V2 torsional terms. J. Am. Chem.Soc., 99 (1977) 8127-8134.
28- S. Profeta, & N. L. Allinger, Molecular mechanics calculations on aliphatic amines. J. Am.
Chem.Soc., 107 (1985) 1907-1918.
29- S. Kearsley & G. M. Smith, An alternative method for the alignment of molecular structures:
maximizing electrostatic and steric overlap. Tetrahedron Computer Methodology, 3 (1990)
615-633.
30- P. Labute, C. Williams, M. Feher, E. Sourial, & J. M. Schmidt, Flexible alignment of small
molecules. J. Med. Chem., 2001;44: 1483-1490.
31- B. J. Stockman, N. R. Nirmala, G. Wanger, T. J. Delcamp, M. T. DeYarman, & J. H.
Freisheim, Methotrexate Binds in a Non-Productive Orientation to Human Dihydrofolate
Reductase in Solution Based on NMR Spectroscopy. Federation of European Biochemical
Societies Letters, 283 (1991) 267-269.
32- V. Cody & C. H. Schwalbe, Structural characteristics of antifolate dihydrofolate reductase
enzyme interactions. Crystallography Reviews, 12 (2006) 301-333.
27

Captions:
Chart 1: Structures of antimicrobial dithiocarbamates
Chart 2: Representation of different active part of newly synthesized compounds against bacteria
Design of active
Scheme 1: Synthesis of the target compounds 20-39
Table 1: The physiochemical properties of the synthesized compounds 20-39
Table 2: Antibacterial activity of tested compounds (200 µg/8 mm disc) against selected multidrug-resistant
Gram negative clinical isolate, Pseudomonas aeruginosa (Ps12) and Klebsiella pneumoniae (K4).
Table 3: The Minimal Inhibitory Concentrations (MIC, µg/ml) and Minimal Bactericidal Concentrations
(MBC µg/ml) of compounds 20, 22, 24, 25. 26, 27, 28 33, 34 35, 36 and 37 in comparison with the
broad spectrum antibacterial drug, Colistin against selected multidrug-resistant Gram negative
clinical isolate, Pseudomonas aeruginosa (Ps12) and Klebsiella pneumoniae (K4).
Table 4: DNA/methyl green colourimetric assay of the DNA-binding compounds
Figure 1 2D binding mode and residues involved in the recognition of reference cocrystalized
ligand in phosphoethanolamine transferase enzyme active site
Figure 2: 2D binding mode and residues involved in the recognition of 20 in phosphoethanolamine
transferase enzyme active site.
Figure 3: 2D binding mode and residues involved in the recognition of 22 in phosphoethanolamine
transferase enzyme active site .
Figure 4: 2D binding mode and residues involved in the recognition of 24 in phosphoethanolamine
transferase enzyme active site
Figure 5: The aligned conformations of the most active compound 24 (IC50 0.004 M); occupying
phosphoethanolamine transferase enzyme active site
Figure 6: 2D binding mode and residues involved in the recognition of 25 in phosphoethanolamine
transferase enzyme active site.
Figure 7 : DNA minor groove binding of (a) Distamycin (yellow) with DNA, DNA is shown in
ribbon form. Distamycin, is shown in ball and cylinder form. Hydrogen-bonding (HB)
interactions of with the DNA bases (dashed lines). Numbers indicate the percent of HB
Figure 8: Lowest energy conformers of active compounds (a) 28 , (b) 34, and the least active
compounds (c) 21 and (d ) 31 with balls and cylinders
Figure 9: Close-up view of active candidates 28 (a), 34 (b) and inactive candidates 21 (c), 31 (d)
binding in the DNA minor groove, highlighting the “isohelical” conformation of DNA
active binders 28 and 34 to fit into minor groove
28

Research Highlights
 Synthesis of Dithiocarbamate chalcone-based derivatives scaffold.
 Antibacterial screening against selected Gram negative bacteria focusing on microbial resistance
were performed where most of the compounds showed equal or higher activity than colistin
standard.
 Compound 24 proved to be the most active candidate with MIC of 8 µg/ml against both Ps12 and
K4 and MBC of 32 µg/ml against Ps12 and 16 µg/ml against K4 respectively.
 .Compounds 28 and 34 were recorded to have DNA binding better than doxurubucin standard
with IC50 of 27.48 and 30.97 µg/ml respectively
 Compounds 20, 22,24 and 25 had good binding affinity to PEA transferase via Thr280, where 28
and 34 showed a clear intercalation between DNA double strands.
Graphical abstract
29