The rational design, synthesis, and antimicrobial investigation of 2-Amino-4-Methylthiazole analogues inhibitors of GlcN-6-P synthase

Article abstract of DOI:10.1016/j.bioorg.2020.103781

A series of novel 2-Amino-4-Methylthiazole analogs were developed via three-step reaction encompassing hydrazine-1-carboximidamide motif to combat Gram-positive and Gram-negative bacterial and fungal infections. Noticeably, the thiazole-carboximidamide de

Full text of DOI:10.1016/j.bioorg.2020.103781

Journal Pre-proofs
The Rational Design, Synthesis, and Antimicrobial Investigation of 2-Ami-
no-4-Methylthiazole Analogues Inhibitors of GlcN-6-P Synthase
Abdelsattar M. Omar, Saleh Ihmaid, EL-Sayed E. Habib, Sultan S. Althagfan,
Sahar Ahmed, Hamada S Abulkhair, Hany E. A. Ahmed
PII:
S0045-2068(20)30409-0
DOI:
https://doi.org/10.1016/j.bioorg.2020.103781
YBIOO 103781
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Bioorganic Chemistry
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Please cite this article as: A.M. Omar, S. Ihmaid, E.E. Habib, S.S. Althagfan, S. Ahmed, H.S. Abulkhair, H. E. A.
Ahmed, The Rational Design, Synthesis, and Antimicrobial Investigation of 2-Amino-4-Methylthiazole
Analogues Inhibitors of GlcN-6-P Synthase, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.
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The Rational Design, Synthesis, and Antimicrobial Investigation of 2-Amino-4-Methylthiazole Analogues Inhibitors of
GlcN-6-P Synthase
Abdelsattar M. Omara,b*¥, Saleh Ihmaid , EL-Sayed E. Habib , , Sultan S. Althagfan , Sahar Ahmed , Hamada S Abulkhair , Hany E. A.
c
d,e
f
c,g
h
Ahmedc,h*¥
a
Pharmaceutical Chemistry Department, Faculty of Pharmacy, King Abdul-Aziz University, Jeddah 21589, Saudi Arabia
b
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Nasr City 11884, Cairo, Egypt
c
Pharmacognosy and Pharmaceutical Chemistry Department, Pharmacy College, Taibah University, Al-Madinah Al-Munawarah 41477, Saudi Arabia
d
Department of Pharmaceutics and Pharmaceutical Technology, College of Pharmacy, Taibah University, Al-Madinah Al-Munawarah 344, Saudi Arabia.
e
Department of Microbiology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
f
Clinical and Hospital Pharmacy Department, Taibah University, College of Pharmacy, Taibah University, Al-Madinah Al-Munawarah 41477, Saudi Arabia
g
Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
h
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Al-Azhar University, Nasr City 11884, Cairo, Egypt
*
Corresponding author.
E-mail address: asmansour@kau.edu.sa, jan_25_misr@yahoo.com
¥
These authors contributed equally to this work
Abstract
A series of novel 2-Amino-4-Methylthiazole analogs were developed via three-step reaction encompassing hydrazine-1-carboximidamide motif to
combat Gram-positive and Gram-negative bacterial and fungal infections. Noticeably, the thiazole-carboximidamide derivatives 4a-d displayed
excellent antimicrobial activity and the most efficacious analogue 4d with MIC/MBC values of 0.5 and 4 μg/mL, compared to reference drugs with very
low toxicity to mammalian cells, resulting in a prominent selectivity more than 100 folds. Microscopic investigation of 4d biphenyl analogue showed
cell wall lysis and promote rapid bactericidal activity though disrupting the bacterial membrane. In addition, an interesting in vitro investigation against
GlcN-6-P Synthase Inhibition was done which showed potency in the nanomolar range. Meanwhile, this is the first study deploying a biomimicking
strategy to design potent thiazole-carboximidamides that targeting GlcN-6-P Synthase as antimicrobial agents. Importantly, Molecular modeling
simulation was done for the most active 4d analogue to study the interaction of this analogue which showed good binding propensity to glucosamine
binding site which support the in vitro data.
Keywords: Thiazole; aminoguanidine; antimicrobial activity, GlcN-6-P Synthase Inhibition.
1
. Introduction
The need for novel antibacterial agents has intensified due to the continuing emergence of resistant bacteria limiting the utility of many drug therapies
[1-3]. At the last few years, FDA have approved only seven new chemical entities as systemic antibacterials and, of these, only two drugs ,linezolid and
daptomycin, work through novel modes-of-action compared to the previous antibiotics [4]. The marketplace includes many active antibacterial drugs
against resistant strains because of either the increase in potency or decrease susceptibility to the metabolic degradations [5-7]. However, the resistance
mechanisms are in increase and more expected in the future time. Nowadays, antibiotic-resistant bacteria accounts for 700,000 infections worldwide,
and are responsible for over 25,000 deaths per year in the European Union only, which World Health Organization (WHO) identified as a major public
health issue of the 21st century [8, 9]. Thiazole ring having a strong S-C-N fragment which is a member of the family of five-membered heterocyclic
compounds; has been the subject of many different types of research to date. Thiazole ring is found to be a common structural component in many
biological agents. On the other hand, natural compounds such as thiamine (known as vitamin B1), thiamine pyrophosphate (TPP), epothilone, bacitracin,
penicillin antibiotics and a wide range of synthetic drug groups contain thiazole moiety [7]. Previous research has shown that it is an undeniable fact
that thiazole based-compounds had effective different pharmacological importance [10-17]. Thiazole-based recent drugs have been extensively studied
for their antibacterial and antifungal activities [18-21]. Glucosamine-6-phosphate (GlcN-6-P) synthase has been proposed as a potential target for design
of antibacterial and antifungal agents. GlcN-6-P synthase catalyzes the first step in the biosynthesis of D-glucosamine-6-phosphate, which is a precursor
of a number of amino sugar containing macromolecules, including chitin and mannoproteins in fungi, peptidoglycan and lipopolysaccharides in bacteria
and glycoproteins in mammals [22, 23]. This enzyme was previously reported to be inhibited by a naturally occurring compound tetaine and peptide
1

A190095, and also by a number of chemically obtained compounds, especially electrophilic ones [24]. In fact, not all of these inhibitors are selective;
consequently, they have ability to react with other enzymes in the fungal cell. The most effective and selective compounds, which are the strong
3
inactivators of GlcN-6-P synthase, are the analogues of L-glutamine (the substrate of the enzyme) and one of these compounds is N -4-metoxyfumaroyl-
(S)-2,3-diaminopropanoic acid (FMDP) [23]. Despite the effectiveness of the enzyme inhibition, the FMDP inhibitor shows none or very poor
antimicrobial activity caused by the high polarity of the inhibitor molecule. Different trials were done for improving its antimicrobial and antifungal
activity by increasing of the lipophilicity character through synthesis of ester and amide derivatives [25-27]. It was reported that polyguanidines have
attracted considerable attention for their antibacterial and antifungal activities [28-30]. The supposed mechanism of action of guanidine compounds is
based on the strong electrostatic interaction between their positive charges and the electronegative envelope of bacteria. This immediate binding to the
components of the cytoplasmic membrane or the cell wall leads to the loss of bio-functions of phospholipids or to the disruption of the membrane and
eventually the cell death [31, 32]. In our research work here, we proposed molecular hybridization of an interesting thiazole moiety linked to hydrophilic
guanidine part and terminal lipophilic part mediated by imine hydrogen bonding linker, Figure 1. This molecular design is explained in the view of the
fragments to the glucosamine substrate of the GlcN-6-P synthase and the capability to bind similarly in the enzyme pocket. In Figure 1, the amidine
moiety looks like the phosphate group and thiazole to the tetrahydropyran that accommodation the same binding regions that might responsible to
inhibitory effect of the scaffold derivatives.
2

Target scaffold
Glucosamine-6-phosphate
Hydrophilic
O
O P O
O
HO
N
O
O
S
N
H N
N
3
Lipophilic
H
HO
OH
O
HO
H N
O
O P O
O
3
HO
OH
N
HN
N
NH
S
N
N
NH2
H
Compounds
Steric contributions
GlcN-6-P synthase
Hydrogen bond acceptor
Molecular surface of aligned substrate with
proposed inhibitor
Figure 1. Rational discussion of study including the mapping of target scaffold inhibitor to that with the enzyme substrate and the binding site.
2
2
. Results and discussions
.1. Chemistry
2
.1.1. Synthesis and Characterization
2
-Benzylidenehydrazine-1-carbothioamides (2a-e) were synthesized by condensation of substituted aromatic aldehyde (1) with thiosemicarbazide (2)
1
13
in ethanol (Scheme 1). The physical property, IR, H NMR, C NMR and MS in addition to the microanalysis (see Experimental section) are in support
1
13
with the structures of the new compounds (2a-e) were confirmed by the analysis of IR, H NMR and C NMR spectroscopy in addition to the
1
3
microanalysis (see Experimental section). C NMR spectrum showed a requisite signal at δ = 177.71 ppm assignable to (C=S) group in support of the
thione tautomeric form. The substituted 1-(2-(2-drevatives benzylidenehydrazineyl)-4-methylthiazol-5-yl)ethan-1-ones (3a-e) were prepared by heating
a mixture of the substituted 2-benzylidenehydrazine-1-carbothioamides (2a-e) and 3-chloro-2,4-pentanedione in absolute ethanol, as illustrated in
1
13
(
Scheme 1). The structures of the new products (3a-e) were confirmed by the analysis of their IR, H NMR and C NMR spectroscopy in addition to
the microanalysis (see Experimental section). The structures of the synthesized compounds (3a-e) were established on the basis of their analytical and
3

-1
spectral data. The infrared spectra of compounds (3a-e) displayed sharp absorption band at 1666-1695 cm which is a characteristic of the carbonyl
1
group (C=O), The representative H NMR spectrum of compounds (3a-e) (DMSO-d ) showed two singlet signals at  = 2.40 and 2.50 ppm, assigned
6
1
3
to the two CH protons. C NMR spectrum showed a requisite signal at δ = 189.34 ppm assignable to (C=O) group.
3
O
NH2
S
O
O
H
N
NH2
HN
N
S
HN
N
N
H N
2
O
H
S
Cl
Ethanol/Reflux
Ar
Ethanol/Acetic acid
Reflux
Ar
Ar
1
2a-e
3a-e
Compound
Ar
a
b
c
d
e
4-lodophenyl
4-Hydroxy, 3-Methoxyphenyl
3-Hydroxynaphthalene
Biphenyl-4-yl
3-Bromo-4,5-dimethoxy
Scheme 1. Synthesis of target compounds (2a-e) and (3a-e)
The hydrazinecarboximidamide derivatives (4a-e) (Scheme 2). In this procedure a suspension of compounds (3a-e) in ethanol and aminoguanidine
hydrochloride in the presence of a catalytic amount of lithium chloride which was then refluxed for 24 hours. At the completion of the reaction the
work-up gave compounds yeilds; 4a (85%), 4b (83%), 4c (77%), 4d (66%), and 4e (87%). The structure of the compoundsd (4a-e) were confirmed by
1
13
the analysis of their IR, H NMR, C NMR spectroscopy and LCMS in addition to microanalysis (see Experimental section).
NH2
HN
N
O
NH
S
S
HN
N
N
H
N
HN
N
N
NH2
H N
2
NH
Ethanol/LiCl
Ar
Ar
4
a-e
3a-e
Compound
Ar
4-lodophenyl
4-Hydroxy, 3-Methoxyphenyl
3-Hydroxynaphthalene
Biphenyl-4-yl
a
b
c
d
e
3-Bromo-4,5-dimethoxy
Scheme 2. Synthesis of hydrazinecarboximidamide derivatives (4a-e)
2
2
.2. Biological screening
.2.1. Antibacterial screening
All the newly synthesized compounds (3a-e) and (4a-e) and their precursors (2a-e) were screened for their antibacterial and antifungal activities via the
agar diffusion well method [33] by recording the inhibition zones, and for the most effective derivatives, the minimum inhibitory concentrations (MIC)
4

and the minimum bactericidal concentrations (MBC) were determined by the serial dilution method [34]. The antimicrobial evaluation was done for all
the three series of compounds varied from the benzylidenehydrazine-1-carbothioamide to the acetyl thiazole and passing to the final products thiazole-
hydrazine-1-carboximidamide for clarifying the role of each reaction step in the activity and further showing the role of thiazole with and without the
terminal carboximidamide. The activity of the synthesized compounds was tested against three Gram-positive bacteria (Staphylococcus aureus IFO
3
060, Bacillus subtilis IFO 3007 and Micrococcus luteus IFO 3232), two Gram-negative bacteria (Escherichia coli IFO 3301 and Pseudomonas
aeuroginosa IFO 3448), and three pathogenic fungi (Candida albicans IFO 0583, Aspergillus oryzae IFO 4177 and Aspergillus niger IFO 4414) with
commercially available antibiotics from 3 different classes were used as positive controls; ampicillin, ciprofloxacin, and fluconazole, with GlcN-6-P
synthase inhibitor FMDP1 for comparison [23]. The observed antimicrobial data of the target compounds and the reference drugs; the inhibition zone
(IZ) and the minimum inhibitory and bactericidal concentrations (MIC, MBC) are given in Tables 1 and 2. The observed antimicrobial data in vitro
indicated that a close to 90% of the tested compounds exhibited appreciable bacterial and fungal inhibition with inhibition zone range (10-36 mm) in
comparison to the reference drugs.
In general, the synthesized compounds were more active against Gram-positive microorganism compared to the Negative ones. As shown in Table1,
Compounds 2a-e and 3a-e showed moderate antibacterial activity against Gram-positive microorganism and weak activity against Gram-negative ones,
with almost no activity against most of the tested fungal strains. On the other hand, the hydrazinecarboximidamide derivatives (4a-e) showed moderate
to strong activity against Gram-positive tested microorganism with IZ from 12-36 mm. With respect to the activity against Gram negative microorganism
compounds 4a-e showed weak to moderated antimicrobial activity. Among this group Compound 4c and 4d were found to be the most active against
all tested strains, except P. aeruginosa. Compound 4d was the most active against Staphylococcus aureus with an inhibition zone (IZ = 34 mm) greater
than that of the standard drug ampicillin (IZ = 28 mm) and equal to that of ciprofloxacin (IZ = 34 mm), this compound also showed high activity against
Bacillus subtilis with an inhibition zone (IZ =36 mm) which is greater than the standard drug ampicillin (IZ = 30 mm) and almost equal to that of
ciprofloxacin (IZ =38 mm) at the same tested concentration. In the case of the antibacterial activity against the Gram-positive bacteria, most of the novel
final compounds were found to be comparable in activity to the reference drugs. Most of the hydrazinecarboximidamide derivatives described here
revealed activity that was found to have good MIC and MBC values (0.5–16 µg/mL), as shown in Table 2. The best MIC and MBC value observed for
4
d with same value as the reference drug ciprofloxacin and even better values compared to that of Ampicillin followed by compound 4c. A direct
comparison could be made between highly active derivatives 4a-e and less active ones 3a-e a good correlation between activity and structural features
could obtained. First and most profound effect was obtained after the addition of the hydrophilic group (guanidine) moiety significantly enhance the
activity 4a-e compared to 3a-e. However, the 4a-e derivatives showed difference antibacterial activity within the same group. The Lipophilicity is an
important physicochemical parameter in the development of an antibacterial agent, and the aromatic ring structures produce additional affinity through
hydrophobic interaction to the bacterial membrane and this could account for the high affinity for the gram positive compared to the gram-negative
ones. From the structural point of view, it seems that the presence of the biphenyl ring at imine side chain of the thiazole ring produce derivative 4d
with the most promising antibacterial activity (Scheme 2). Also it revealed that the presence of polar group on hydrophobic side chain decrease the
antibacterial activity as shown for 4c compared to the most active one 4d. Taken together, these data support the contention that both guanidine moiety
and the terminal aryl moiety are required to get the high observed antibacterial activity.
2
.2.2. Antifungal screening
Preliminary screening of the tested compound revealed good antifungal activity on the most with the final derivatives 4a-e, with moderate activity was
produce against Aspergillus niger and strong activity against both Candida albicans and Aspergillus oryzae. The best activity was observed for
Compounds 4d with IZ of 36 mm followed by 4c (IZ = 34 mm) then 4e (IZ = 26 mm), compared to the 21-mm IZ of the standard drug fluconazole
against Candida albicans, same trend of activity was also observed against Aspergillus oryzae (Table1). The MIC and MBC values support the high
5

potency of the hydrazinecarboximidamide derivatives (4a-e) against the tested fungal species, compounds 4a-d give better activity compared to the
reference drug fluconazole (Table 2).
Table 1. Antimicrobial activity of the synthetic compounds (Inhibition Zone, IZ, diameter (mm)) (200 g/8 mm disc).
ID
Diameter of Inhibition Zone (mm)
Gram negative
Gram positive
Fungi
S. aureus B. subtilis M. luteus E. coli
P. aeruginosa C. albicans A. oryzae A. niger
Benzylidenehydrazine-1-carbothioamides
2
a
10
24
16
18
14
11
22
18
20
15
-
12
12
14
10
-
12
-
14
-
-
10
-
12
-
13
25
-
-
-
-
-
-
-
-
-
-
-
-
-
2
b
2
c
2
d
2
e
(4-Methylthiazol-5-yl)ethan-1-ones
3
a
12
14
14
15
16
14
16
16
16
18
-
-
-
-
10
16
-
-
-
-
12
-
-
-
-
14
-
-
-
-
12
-
-
-
-
-
3
b
12
12
11
12
3
c
3
d
3
e
((4-Methylthiazol-5-yl)ethylidene)hydrazinecarboximidamides
4
a
18
20
25
34
20
20
24
34
36
22
16
12
20
18
15
14
16
17
18
16
12
-
14
10
10
18
14
34
36
26
18
10
26
25
22
15
-
16
18
18
4
b
4
c
4
d
4
e
Reference drugs
A
C
F
28
34
NT
30
38
NT
25
32
NT
24
38
NT
22
36
NT
NT
NT
21
NT
NT
22
NT
NT
24
-
-
-
(-) Not active (<8 mm), Weak activity (8-12 mm), Moderate activity (12-16 mm), Strong activity (> 18 mm).
Solvent: DMSO (8 mm). A; Ampicillin, C; Ciprofloxacin, F; Fluconazole.
NT: Not tested.
Table 2. The minimum inhibitory and bactericidal concentrations (MIC, MBC in µg/mL) of the compounds initially screened against S. aureus, B.
subtilis, C. albicans, and A. oryzae strains.
ID
MIC/MBC Gram positive bacteria
Fungi
S. aureus B. subtilis C. albicans A. oryzae
MIC
MBC
2± 1.12
4± 0.55
2± 0.14
4± 0.11
8± 1.20
16± 1.41
NT
NT
2
b
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
8± 0.11
8± 0.10
4± 1.13
8± 0.14
2± 0.01
4± 1.11
1± 0.15
2± 0.13
4± 0.10
8± 0.52
2± 0.11
4± 0.04
1± 0.07
2± 0.12
NT
4± 0.11
8± 0.14
2± 0.10
4± 0.04
1± 0.09
2± 0.10
0.5± 0.15
1± 0.11
2± 0.10
4± 0.26
1± 0.31
2± 0.20
0.5± 0.11
1± 0.14
NT
4± 2.10
8± 1.10
8± 0.23
8± 0.54
4± 1.05
8± 2.25
4± 0.33
8± 0.51
8± 1.10
16± 0.27
NT
NT
NT
NT
8± 0.32
16± 0.34
64± 0.23
8± 0.47
16± 1.05
8± 1.32
8± 2.11
4± 0.35
8± 1.60
4± 0.33
8± 0.44
8± 1.48
16± 0.81
NT
NT
NT
NT
4± 0.22
8± 0.36
64± 0.25
4
a
4
b
4
c
4
d
4
e
A
C
F
MBC
MIC
NT
NT
64± 0.51
6
4± 0.13
FMDP1
MBC
64± 0.10
64± 0.26
64± 0.41
64± 0.33
-
-
Results are mean values from at least three experiments; (NT) Not Tested (not significant IZ).
1
FMDP “N3-(4-methoxyfumaroyl)-
L
-2,3-diaminopropanoic acid” Data taken from reference [23]
6

-
A; Ampicillin, C; Ciprofloxacin, F; Fluconazole.
2
.2.2. Antibacterial Activity of selected compounds 4a-d against a panel of highly resistant strains
To further probe the activity of antimicrobial diamines 4a-d as representative examples, these compounds were evaluated for the ability to kill human
pathogenic strains; two Gram-positive bacteria including; methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile (C.D), two Gram-
negative bacteria including; two Escherichia coli isolates (E.C TM, E.C WT) in vitro. The data obtained are compared to control drugs (gentamicin,
linezolid, vancomycin, and Metronidazole). The observed minimum inhibitory concentration (MIC) antimicrobial data of the compounds and the
reference drugs are given in Tables 3. The results of the in vitro antimicrobial screening revealed that, the series of 4a-d exhibited promising Gram-
positive antibacterial and low Gram-negative inhibitory effects. Table 2 showed that the compounds 4d has best activity towards Gram-positive bacteria
(E.C TM) with MIC 32 μg/mL. Whereas, the other derivatives 4a-c showed weak inhibitory activity against both of bacteria, MIC 140 μg/mL. This
performance is consistent with the results of antimicrobial assay on standard strains. SAR analysis proved that derivative with biphenyl lipophilic tail could inhibit
the more resistant MRSA type.
Table 3. The minimum inhibitory concentration (MIC in µg/mL) of the compounds initially screened against methicillin-resistant Staphylococcus
aureus, Escherichia coli and Clostridium difficile isolates.
Comp. ID
S. aureus
NRS 384 (MRSA USA 300)
E. coli
JW55031
E. coli BW525113
(wild type strain)
C. difficile
(ATCCBAA1870)
(TolC mutant strain)
4
a
128
128
128
32
1
1
130
130
130
128
8
NT
1
NT
145
140
130
130
65
NT
1
NT
150
150
150
150
NT
1
4
b
4
c
4
d
Linezolid
Vancomycin
Gentamicin
Metronidazole
NT
NT
NT
0.25
1NT: Not tested
2
.2.2. Cytotoxicity assay
The cytotoxicity of compound 4d was also monitored in two mammalian cell lines, the HEK293T human kidney embryonic cell line and the A549
human alveolar basal epithelial adenocarcinoma cell line. The results of these studies are shown in Table 4. The selectivity index (SI) in each cell line
was the ratio of the IC value of cytotoxicity against a given human cell line divided by the MIC values for given bacterial strains. Compound 4d
5
0
possessed a favorable SI between 0.5 and 100 against S. aureus, B. subtilis, C. albicans, and MRSA isolates. The results overall indicated that compound
is selectively kill bacterial cells other than the human cells in most of cases. The SI against MRSA was lower (either 0.55 or 1.56), suggesting that
additional structure optimization is required to identify a compound that kills MRSA strain with an acceptable SI.
Table 4. Cytotoxicity of Compound 4d in immortalized human keratinocytes and normal human diploid fibroblasts
Organism type
MBC/MIC,
Selectivity indexa
μg/mL
HaCaT
WI38
17.70
35.4
Staphylococcus aureus IFO 3060
Bacillus subtilis IFO 3007
Candida albicans IFO 0583
MRSA USA 300
1
0.5
4
49.97
99.94
12.49
1.56
4.42
32
0.55
7

a
Selectivity index = IC /MIC. The IC value of compound 4d against normal HaCaT and WI38 cells were 49.97 and 17.70 μg/mL respectively. The IC value =
5
0
50
50
the concentration that reduced HaCaT or WI38 cell viability by 50%.
2
.2.3. Mechanism of Action of target compounds
The overall theory analysis of mechanism of action of these analogues are owing to their structure which bears variable lipophilic parts and terminal
hydrophilic one. The lipophilic systems were in favor of assembly on the membrane due to the lipophilicity aiding in the embedding of the compound
inside the cell. This embedding made this compound as a false substrate for the target enzyme and use it instead of glucosamine leading to inhibition of
cell wall. We hypothesized that the thicker, peptidoglycan-rich Gram-positive cell wall was prevented more than Gram-negative one and this was clear
from the observed antimicrobial data. In addition, the mechanism of action of compound 4d was investigated and fast killing activity was an indicative
of the membrane-targeting activities, which could be mediated by an electrostatic interaction between the positively charged central nitrogens in 4d and
other analogues, and the negatively charged bacterial membrane. This hypothesis is consistent with the fact that positively charged antibacterial peptides
can disrupt bacterial membrane integrity and depolarize bacterial membranes [32]. To test this hypothesis, Nikon Eclipse Ci, Japan microscopy was
subsequently used to visualize bacterial cell morphology at high resolution and provide direct evidence of the membrane effects caused by 4d. Untreated
S. aureus (Figure 2A) and C. albicans (Figure 2C) possessed an intact cell envelope with both a well-defined peptidoglycan cell wall and intact
membrane. Following treatment with 4d at 2× MIC for 30–60 min, S. aureus formed numerous spherical, double layered mesosome-like structures
(Figure 2A). Cellular debris of completely lysed cells was also observed after the treatment with 3 (Figure 2B). Similarly, C. albicans treated with 4d
at 2× MIC (15 min) exhibited extensive membrane damage, as illustrated by the appearance of lysed cells and the release of cell contents (Figure 2B
and 2D).
Figure 2. Confocal microscopy experiments performed on A) untreated S. aureus and B) Treated S. aureus ,C) Untreated C. albicans D) treated C.
albicans, with 2× its’ MIC 4d analog and stained with Gram-stain using Nikon Eclipse Ci, Japan microscope. All images have been captured using a
×
100 oil immersion objective. The areas of membrane damage and cell content release are marked with black arrows and clear from morphological
analyses.
8

2
.3. In vitro GlcN-6-P Synthase activity analysis
Glucosamine 6-P Synthase is considered an essential enzyme involved in the peptidoglycan N-acetyl-glucosamine (NAG) precursor. In this work, we
introduced novel compounds bearing polar amidine motif similar to glucosamine and assumed to accommodate similar interactions as the substrate does
with the enzyme. Hence, in vitro inhibitory analysis of the novel compounds was done, and the results are shown in Table 5. As shown in Table 5, the
inhibitory activity of all synthesized compounds was evaluated against the GlcN-6-P Synthase, displaying IC50 in the micromolar range with high
comparative results as that obtained for gentamicin. Highest inhibitory activity was obtained by 3e which produce 50% inhibition of the enzyme at 1.25
µM for compound 3e. Compounds 4a-e consider the best activity when compared to both gentamicin as well as FMDP ‘’the selective in activator of
GlcN-6-P Synthase’’. This improvement in the inhibitory activity of the hydrazinecarboximidamide derivatives (4a-e) could be account due to the
impact of the guanidine moiety which would interact with the enzyme on the same manner as the phosphate group do. Out of these final compounds,
compound 4c and 4d give the best inhibitory activity which in turn very supportive to their antibacterial activity observed before as seen in Table 1,
and 2 which also supported by their docking interaction Figure 3.
Table 5. In vitro inhibitory GlcN-6-P Synthase activity data for new compounds.
Compounds IC (µM)
5
0
2
a
8.63±0.21
77.14±1.93
15.26±0.38
2
b
2
c
2
2
d
e
8.20±0.10
7.18±0.10
3
a
5.81±0.14
4.66±0.12
17.19±0.43
3.72±0.093
1.25±0.031
3
b
3
c
3
d
3
e
4
a
4.06±0.10
5.15±0.12
3.87±0.097
3.47±0.087
4.93±0.12
4
b
4
c
4
d
4
e
Gentamicin 3.41±0.08
1
FMDP
4.03 ± 0.26
-
-
Data are presented as average IC ± SD (µM) values for at least three experiments.
FMDP data was reported [23].
5
0
1
2
.3. Molecular properties and pharmacokinetic profiling
PK properties such as absorption, distribution, metabolism, excretion and toxicity (ADMET) profiling of compounds were determined using the pkCSM
ADMET descriptors algorithm protocol [35-37]. Two important chemical descriptors correlate well with PK properties, the 2D polar surface area
(PSA_2D, a primary determinant of fractional absorption) and the lipophilicity levels in the form of atom-based LogP. The absorption of drugs depends
on factors including membrane permeability [indicated by colon cancer cell line (Caco-2)], intestinal absorption, skin permeability levels, P-glycoprotein
substrate or inhibitor. The distribution of drugs depends on factors that include the blood–brain barrier (logBB), CNS permeability, and the volume of
distribution (VDss). Metabolism is predicted based on the CYP models for substrate or inhibition (CYP2D6, CYP3A4, CYP1A2, CYP2C19, CYP2C9,
CYP2D6, and CYP3A4). Excretion is predicted based on the total clearance model and renal OCT2 substrate. The toxicity of drugs is predicted based
9

on AMES toxicity, hERG inhibition, hepatotoxicity, and skin sensitization. These parameters were calculated for potent 4c, 4d, and 2b and inactive 2e
analogs compared to marketed drugs ampicillin and fluconazole and checked for compliance with their standard ranges. After calculating the ADMET
properties (Table 6), we can verify that the ligands 2b, 4c, 4d and the reference ampicillin have a low percentage of human intestinal absorption
(69.96%, 71.15%, 79.77%, 45.09% respectively), as they have very hydrophilic groups in their chemical structures, which would make it difficult to
pass through the biological membranes [37]. Conversely, 2e and fluconazole have human intestinal absorption values above 90%, demonstrating that
these compounds may have good oral bioavailability when tested experimentally [37]. Analyzing the central nervous system (CNS) permeability, 2e,
4
c and 4d have good penetration in the CNS (Log PS> -2.0), while 2b, ampicillin and fluconazole are unable to penetrate the CNS (Log PS <-3.0) [37].
These data may help that 2b, ampicillin and fluconazole present lower side effects in the (CNS) compare 2e, 4c and 4d, contributing to a better tolerability
among patients. It was also possible to observe that only 4d is able to inhibit the cytochrome P450, CYP3A4 isoform, the main enzyme responsible for
drug metabolism. This possibly occurs because these ligands exhibit high lipophilicity and molecular size, thus favoring the inhibition of this enzyme
and may lead to interactions with other drugs metabolized by this CYP and may cause serious adverse effects [37]. The excretion was evaluated as Total
Clearance. This parameter is related to bioavailability and is important in determining dosage rates to achieve steady state concentrations. Our data
demonstrated that 2e, 4c, ampicillin and fluconazole exhibit the higher Total Clearance values when compared to other ligands. Thus, these compounds,
due to their high hydrophilicity, could be excreted rapidly by the kidneys, requiring shorter administration intervals to maintain desired therapeutic
concentrations. The last parameter analyzed in our studies was hepatotoxicity. In Table 6, we can verify that no ligand showed hepatotoxic effects
except 4c. Therefore, these ligands of better interaction with TcTR may not have a physiological or pathological event that could be associated with
hepatic injury, suggesting that these compounds may be well tolerated by the liver when they are tested in a biological environment especially the 4d
analogue.
Table 6. Predicted ADMET properties of selective target compounds.
Parameter
2e
Molecular properties
318.2 225.3 381.5 391.5 349.4
2b
4c
4d
Ampicillin Fluconazole
Molecular Weight
306.3
0.74
5
7
1
LogP
1.6
4
0.6
3
2.9
5
3.8
6
0.31
4
Rotatable Bonds
Acceptors
Donors
4
4
7
6
5
2
3
5
4
3
Surface Area
113
93
160
167
143
123
Absorption
Water solubility
Caco2 permeability
-3.196 -2.197 -3.036 -3.06
-2.43
-2.788
1.213
95.275
-2.756
No
0.696 0.665 -0.724 -0.531 0.398
91.068 69.968 71.157 79.771 45.097
-3.133 -3.407 -2.735 -2.735 -2.735
Intestinal absorption (human)
Skin Permeability
P-glycoprotein substrate
P-glycoprotein I inhibitor
P-glycoprotein II inhibitor
No
No
No
No
No
No
Yes
No
Yes
Yes
Yes
Yes
No
No
No
No
No
Distribution
VDss (human)
-0.276 -0.245 1.07
0.416 0.519 0.009
-0.591 -0.631 -1.532 -1.337 -0.861
-2.973 -3.004 -2.596 -2.383 -3.145
Metabolism
0.961 -1.114
0 0.759
-0.112
0.269
-1.088
-3.235
Fraction unbound (human)
BBB permeability
CNS permeability
CYP2D6 substrate
CYP3A4 substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
No
No
Yes
No
No
No
No
No
No
No
No
No
Yes
Yes
Yes
No
No
Yes
No
No
No
Yes
No
No
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
Yes
No
No
No
No
No
No
Excretion
Total Clearance
Renal OCT2 substrate
0.059
No
-0.03 0.138 -0.056 0.327
0.37
No
No
Toxicity
No
No
Yes
No
AMES toxicity
Max. tolerated dose (human)
hERG I inhibitor
No
0.86
No
No
Yes
No
No
0.316
No
0.747 0.451 0.459 1.222
No No Yes No
1
0

hERG II inhibitor
No
No
Yes
Yes
No
Yes
Oral Rat Acute Toxicity (LD )
Oral Rat Chronic Toxicity (LOAEL) 1.971
Hepatotoxicity
Skin Sensitization
T. Pyriformis toxicity
Minnow toxicity
3.25
2.636 2.423 2.195 1.826
2.227
1.079
Yes
No
0.298
3.547
5
0
1.6
No
Yes
0.2
1.779 1.725 1.594
No
Yes
1.251
2.07
No
No
Yes
No
Yes
No
0.289 0.293 0.285
2.318 5.586 5.383 3.411
-
http://biosig.unimelb.edu.au/pkcsm/prediction
2
.4. Molecular docking studies
For the docking calculations, only the most promising active analogue 4d and pdb structure 2VF5 of bacterial Glucosamine-6-phosphate synthase in
complex with glucosamine-6- phosphate were utilized by the Autodock 4.2.3 package [38] as a receptor model for interaction analysis. Ligand was
built by means of MOE2012 package [39], the geometry was optimized with the CHARMm forcefield and then prepared for docking calculations with
the python scripts available in the Autodock package. For the compound 4d, 50 runs were performed, and the resulting poses were clustered with 1.8 Å
tolerance. Lamarckian GA was used for the conformational space search with initial population set to 150, and fitness function evaluations set to
2
5000000. The most abundant low energy clusters were selected for analysis. As shown in Figure 3, the docking simulations suggested that the
compound 4d interact with the active site in a fashion similar to the glucosamine bound substrate observed in the reported crystal structure of the target
enzyme, Figure 3. Firstly, the placement of substrate in the complex, Figure 3A, consists of a long, narrow tunnel, leading to a cavity that contains the
phosphate terminal polar part surrounded by set of hydrophilic amino acid residues showing group of hydrogen bonding interactions.
The group of thiazole-based analogues as GlcN-6-P synthase inhibitors mimic the transition state of the reaction taking place in the enzyme’s C-terminal
sugar isomerizing domain which consider to be transition state inhibitor which is known to be more effective type of inhibitors. The interaction map of
target compound 4d exhibited pivotal hydrogen bonding of amidine motif to the corresponding amino acid residues; Ser347 and Gln348 with consistent
distance 3.01 Å compared to glucosamine-6-phosphate in the polar site, Figure 3B. In addition, 2-amino thiazole fragment as antimicrobial scaffold
shared the interaction with its chelation part through Sulphur and amino atoms with Glu488 that stabilize the compound within the binding pocket.
Moreover, terminal lipophilic part of biphenyl accommodates the hydrophobic pocket formed of Leu601, Val605, and Val399 that correctly oriented
within the substrate sugar, Figure 3C. Overall, the docking simulation of potent analog 4d could explain the in vitro activity of the thiazole design
involving two essential part; lipophilic aromatic and terminal polar amidine parts which contributes strongly in the substrate pocket.
A
B
1
1

C
Figure 3. Docking modes of active compounds in the binding pocket of GlcN-6-P synthase. Interactions between the protein (PDB ID: 2VF5), the
interactions of compound 4d and the target substrate are shown as dotted lines; the residues as colored codes as presented in the software; and the ligands as
black line models. (A) 2D Predicted binding mode of substrate with GlcN-6-P synthase. (B) 2D Predicted binding mode of 4d inhibitor with GlcN-6-P synthase.
(C) 3D aligned map of 4d (blue sticks) and glucosamine-6-phosphate (red sticks) inside the pocket.
3
. Conclusions
In this study, we designed and synthesized a series of thiazole derivatives as potential GlcN-6-P synthase inhibitors. Most of the derivatives including
acetylthiazole 3a-e and thiazole-carboximidamide 4a-e target the enzyme and inhibit it through competing with the glucosamine substrate and the IC values
5
0
were determined to be in the micromolar range. One of these compounds 4d exhibit significant antimicrobial activity with MIC/MBC 1-8 µg/mL with
prominent pharmacokinetic profile compared to reference marketed drugs. Interestingly, the mechanism of such derivatives on cell wall was imaged
and linked to the disruption of peptidoglycan formation. Computational docking studies were done for binding interaction profile for the potent 4d compound
as a representative example of this class and confirming the role of each fragment in target activity. Moreover, the cytotoxicity profiling reported that none of
the compounds have normotoxicity activity against human normal cells. Molecular docking. Finally, the compounds identified here can be considered as initial
leads for the development of improved antibacterial agents.
4
4
. General Chemistry
.1. Chemistry
The starting reagents and solvents were purchased from the Aldrich chemical company and were used as received. The melting points were determined
1
13
1
with a Mel-Temp apparatus and are uncorrected. All the new compounds were characterized by the H-NMR and C-NMR, using a H-NMR (400
1
3
MHz) and C-NMR (100 MHz) spectra, and were measured in DMSO at room temperature. The chemical shifts (δ) were reported in ppm to a scale
calibrated for tetramethylsilane (TMS), which is used as an internal standard. The IR spectra were recorded on a KBr disc on a Schimadzu 8201 PC,
−
1
FTIR spectrophotometer ( max in cm ). Melting points (Mp) were determined in open capillary tubes using Electrothermal apparatus (Stuart, UK)
and are uncorrected. HPLC-Mass Spectrometry were performed on Agilent 1100 / ZQ MSD including C18 column and diod-array UV detector. The
mobile phase (containing 0.01 M ammonium acetate) was gradient starting from 20% acetonitrile/80% water to 80% acetonitrile/20% water. Purities
are reported according to percentage of Peak Areas at wavelength 254 nm. The follow up of the reactions and the check of the purity of the compounds
were made by the TLC on silica gel-protected aluminum sheets (Type 60 GF254, Merck), and the spots were detected by exposure to a UV-lamp at λ
2
54 nm for a few seconds.
4
.1.1. General procedure for the synthesis of substituted 2-benzylidenehydrazine-1-carbothioamides (2a-e)
1
2

The appropriate aldehyde (1.1 mmol) and thiosemicarbazide (1.0 mmol) was dissolved in ethanol (10 ml) with addition of glacial acetic acid (0.2
mmol). The reaction mixture was refluxed for 5-6 hours and then cooled to room temperature. The resulting precipitate was filtered, washed by ether
and recrystalized from ethanol to obtain the corresponding thio-semicarbazones, (2a-e). All the compounds were characterized by spectral techniques
and recrystallized from absolute ethanol to give the pure products (2a-e).
2
-(4-Iodobenzylidene)hydrazinecarbothioamide (2a)
-1
1
Yellow crystals from EtOH (83%), m.p. 145-146 °C; IR (KBr) cm :  3401, 3180 (N-H, NH ), 1602 (C=N); H-NMR (DMSO-d 400 MHz) δ (ppm):
max
2
6
1
3
7
.59-8.23 (4H, m, Ar-H + 2H, NH +1H, C-H=), 11.48 (1H, s, NH, D O exch.); C-NMR (DMSO-d 100 MHz) δ (ppm): 96.86, 129.53, 129.50, 134.10,
2 2 6
1
37.92, 137.95, 141.71, 178.26 (C=S) ppm; HRMS (m/z) 306.0556 [M+H]+, calcd 305.1386; Anal. Calcd for C H IN S: C, 31.49; H, 2.64; N, 13.77.
8
8
3
Found: C, 31.48; H, 2.64; N, 13.76%.
2
-(4-Hydroxy-3-methoxybenzylidene)hydrazinecarbothioamide (2b)
-1
1
Red crystals from EtOH (78%), m.p. 150-152 °C; IR (KBr) cm :  3480 (OH), 3250, 3180 (N-H, NH ), 1605 (C=N); H-NMR (DMSO-d 400 MHz)
max
2
6
1
3
δ (ppm): 3.82 (3H, s, OCH ), 7.59-8.23 (3H, m, Ar-H + 2H, NH , 1H, C-H=), 9.56 (1H, bs, OH), 11.26 (1H, s, NH, D O exch.); C-NMR (DMSO-d
6
3
2
2
1
00 MHz) δ (ppm): 56.17, 109.62, 115.63, 122.85, 126.01, 143.46, 148.51, 149.22. 177.71 (C=S) ppm; HRMS (m/z) 225.0566 [M+H]+, calcd 225.0566;
Anal. Calcd for C H N O S: C, 47.99; H, 4.92; N, 18.65. Found: C, 47.98; H, 4.95; N, 18.65%.
9
11
3
2
2
-[(2-Hydroxynaphthalen-1-yl)methylidene]hydrazinecarbothioamide (2c)
-1
1
Orange crystals from EtOH (92%), m.p. 136-137 °C; IR (KBr) cm :  3375 (OH), 3250, 3154 (N-H, NH ), 1602 (C=N); H-NMR (DMSO-d 400
max
2
6
1
3
MHz) δ (ppm): 7.19-9.03 (6H, m, Ar-H + 2H, NH , 1H, C-H=), 10.56 (bs, 1H, OH, D O exch.), 11.41 (1H, s, NH, D O exch.); C-NMR (DMSO-d
6
2
2
2
1
00 MHz) δ (ppm): 110.25, 118.76, 123.46, 123.98, 128.44, 128.53, 129.18, 131.93, 133.06, 143.52, 157.12, 177.63 (C=S) ppm; HRMS (m/z) 246.0695
+
[
M+H] , calcd 245.0617; Anal. Calcd for C H N OS: C, 58.76; H, 4.52, N, 17.13. Found: C, 58.75; H, 4.55; N, 17.15 %.
1
2
11
3
2
-(Biphenyl-4-ylmethylidene)hydrazinecarbothioamide (2d)
-1
1
Orange crystals from EtOH (75%), m.p. 133-134 °C; IR (KBr) cm :  3320, 3154 (N-H, NH ), 1610 (C=N); H-NMR (DMSO-d 400 MHz) δ (ppm):
max
2
6
1
3
7
1
.44-8.28 (9H, m, Ar-H + 2H, NH , 1H, C-H=), 11.53 (1H, s, NH, D O exch.); C-NMR (DMSO-d 100 MHz) δ (ppm): 126.00, 126.10, 126.21, 127.20,
2 2 6
27.26, 127.29, 128.38, 128.53, 132.60, 132.62, 138.67, 140.65, 141.27, 177.18 (C=S) ppm; HRMS (m/z) 256.0913 [M+H]+, calcd 255.0824; Anal.
Calcd for C H N S: C, 65.85, H, 5.13, N, 16.46. Found: C, 65.82; H, 5.15; N, 16.65%.
1
4
13
3
2
-(3-Bromo-4,5-dimethoxybenzylidene)hydrazinecarbothioamide (2e)
-1
1
Orange crystals from EtOH (92%), m.p. 136-137 °C; IR (KBr) cm :  3150, 3105 (N-H, NH ), 1607 (C=N); H-NMR (DMSO-d 400 MHz) δ (ppm):
max
2
6
1
3
3
.80 (3H, s, OCH ), 3.90 (3H, s, OCH ), 7.15-8.36 (2H, m, Ar-H + 2H, NH , 1H, C-H=), 11.52 (1H, s, NH, D O exch.); C-NMR (DMSO-d 100 MHz)
3 3 2 2 6
δ (ppm): 56.47, 58.21, 109.60, 115.65, 125.38, 141.77, 149.06, 151.51, 178.14 (C=S) ppm; HRMS (m/z) 318.1979 [M+H]+, calcd 318.1902; Anal.
Calcd for C H BrN O S: C, 37.75, H, 3.80, N. 13.21. Found: C, 37.51; H, 3.30; N, 13.18%.
1
0
12
3
2
4
.1.2. General procedure for the synthesis of substituted 1-(2-(2-drevatives benzylidenehydrazineyl)-4-methylthiazol-5-yl)ethan-1-ones (3a-e)
The thiosemicarbazone derivatives (2a-e) (1.0 mmol) and α-chloropentanedione (1.1 mmol) were dissolved in ethanol (10 ml). The reaction mixture
was refluxed for 1-3 hours and then was cooled to room temperature. The resulting precipitate was filtered, washed with cold ethanol and recrystallized
from ethanol to obtain the corresponding 1-(2-(2-drevatives benzylidenehydrazineyl)-4-methylthiazol-5-yl)ethan-1-ones (3a-e) in analytically pure
form. All the compounds were characterized by spectral techniques and the data confirm the proposed structures.
a
1
-(2-(2-(4-Iodobenzylidene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (3a)
1
3

-1
1
Antique white solid from EtOH (85%). m.p. 189-191°C; IR (KBr) cm :  3401, 3180 (N-H), 1665 (C=O), 1602 (C=N); H-NMR (DMSO-d 400
max
6
MHz) δ (ppm): 2.41 (s, 3H, CH ), 2.51 (s, 3H, C=O-CH ), 7.48 (d, J- 8.08 Hz, 2H), 7.82 (d, J- 8.08 Hz, 2H), 8.05 (s, 1H, CH=), 12.63 (bs, 1H, N-H,
3
3
1
3
D O exch.); C NMR (DMSO-d 100 MHz) δ (ppm): 17.54, 29.98, 97.04, 105.12, 128.99, 129.06, 133.97, 138.18, 138.20, 143.32, 156.74, 170.65,
2
6
1
1
1
89.60; HRMS (m/z) 386.9704 [M+H]+, calcd 385.2233; Anal. Calcd for C H IN OS: C, 40.53, H, 3.14, N, 10.91. Found: C, 40.51; H, 3.20; N,
13 12 3
0.90%.
-(2-(2-(4-Hydroxy-3-methoxybenzylidene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (3b)
-1
1
Light yellow from EtOH (88% yield). m.p. 226-229 °C; IR (KBr) cm :  3480 (OH), 3250, 3180 (NH), 1666 (C=O), 1612 (C=N); H-NMR (DMSO-
max
d 400 MHz) δ (ppm): 1.96 (s, 3H, CH ), 2.40 (s, 3H, C=O-CH ), 3.82 (s, 3H), 8.10 (s, 1H, CH=), 7.97 (s, 1H), 7.47 (d, J-8.0, 1H), 6.85 (d, J-8.0, 1H),
6
3
3
1
3
1
1
5
1
0.87 (s, 1H, OH, D O exch.), 11.88 (s, 1H, NH, D O exch.); C NMR (DMSO-d 100 MHz) δ (ppm): 17.24, 29.63, 66.80, 109.97, 118.83, 122.91,
2 2 6
24.05, 128.31, 131.70, 133.18, 147.71, 158.10, 167.50, 189.20; HRMS (m/z) 306.0868 [M+H]+, calcd 305.3522; Anal. Calcd for C H N O S: C,
1
4
15
3
3
5.07, H, 4.95, N, 13.76. Found: C, 55.10; H, 4.91; N, 13.75%.
-(2-(2-((2-Hydroxynaphthalen-1-yl)methylene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (3c)
-1
1
Light yellow from EtOH (78% yield). m.p. 226-229 °C; IR (KBr) cm :  3380 (OH), 3250, 3180 (NH), 1695 (C=O), 1607 (C=N); H-NMR (DMSO-
max
d 400 MHz) δ (ppm): 2.47 (s, 3H, CH ), 2.38 (s, 3H, C=O-CH ), 8.10 (s, 1H, CH=), 7.24-8.63 (m, 6H, Ar), 10.87 (bs, 1H, OH, D O exch), 11.70 (s,
6
3
3
2
1
3
1
H, NH, D O exch.); C NMR (DMSO-d 100 MHz) δ (ppm): 17.30, 28.97, 110.04, 113.54, 116.92, 123.91, 125.05, 128.31, 130.70, 132.88, 140.22,
2 6
1
45.71, 156.51, 159.24, 168.50, 189.34; HRMS (m/z) 326.0919 [M+H]+, calcd 325.3849; Anal. Calcd for C H N O S: C, 62.75, H, 4.65, N, 12.91.
17 15 3 2
Found: C, 62.71; H, 4.63; N, 12.95%.
1
-(2-(2-(Biphenyl-4-ylmethylene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (3d)
-1
1
Light green solid from EtOH (80% yield). m.p. 242-245 °C; IR (KBr) cm :  3320, 3154 (NH), 1695 (C=O), 1607 (C=N); H-NMR (DMSO-d 400
max
6
1
3
MHz) δ (ppm): 2.51 (s, 3H,), 2.46 (s, 3H, C=O-CH ), 8.15 (s, 1H, CH=), 7.78-7.39 (m, 9H, Ar), 12.58 (s, 1H, NH D O exch.); C NMR (DMSO-d
6
3
2
1
1
1
1
00 MHz) δ (ppm): 16.93, 27.80, 125.98, 126.82, 127.31, 128.54, 129.22, 129.51, 130.70, 131.05, 132.6, 140.45, 144.12, 145.71, 156.51, 159.24,
68.50, 189.34; HRMS (m/z) 335.1092 [M+H]+, calcd 335.4228; Calcd for C H N OS: C, 68.03, H, 5.11, N, 12.53. Found: C, 68..03; H, 5.09; N,
1
9
17
3
2.49%.
-(2-(2-(3-Bromo-4,5-dimethoxybenzylidene)hydrazinyl)-4-methylthiazol-5-yl)ethanone (3e)
-1
1
Light yellow solid from EtOH (88% yield). m.p. 177-180 °C; ; IR (KBr) cm :  3150, 3105 (NH); H-NMR (DMSO-d 400 MHz) δ (ppm): 2.40 (s,
max
6
1
3
3
H), 2.50 (s, 3H, CH ), 3.80 (3H, s, OCH ), 3.83 (3H, s, OCH ), 8.34 (s, 1H,C-H=), 7.39-7.24 (m, 2H, Ar), 11.52 (1H, s, NH D O exch.); C NMR
3 3 3 2
(
DMSO-d 100 MHz) δ (ppm): 17.20, 28.98, 62.58, 110.25, 115.83, 122.91, 124.05, 143.3, 145.31, 153.51, 156.10, 168.34, 189.68; HRMS (m/z)
6
3
98..9939 [M+H]+, calcd 398.2748; Anal. Calcd for C H BrN O S: C, 45.24, H, 4.05, N, 10.55. Found: C, 45.52; H, 3.99; N, 10.63%.
15 16 3 3
4
.1.3.
General
procedure
for
the
synthesis
of
substituted
2-(1-(2-(-2-(4-benzylidene)hydrazinyl)-4-methylthiazol-5-
yl)ethylidene)hydrazinecarboximidamides (4a-e)
The thiazole derivatives (3a-e) (0.954 mmol) was dissolved in absolute ethanol (50 mL), and aminoguanidine hydrochloride (1.14 mmol) and a catalytic
amount of LiCl (15 mg) were added. The reaction mixture was heated at reflux for 24 hours. The solvent was evaporated under reduced pressure. The
crude product was purified by crystallization from 70% methanol and then recrystallized from methanol to afford the desired compound.
2
-(1-(2-((E)-2-(4-Iodobenzylidene)hydrazinyl)-4-methylthiazol-5 yl)ethylidene)hydrazinecarboximidamide (4a)
-1
1
Light yellow solid (65% yield): mp 273-274 °C (MeOH). IR (KBr) cm :  3401, 3180 (NH, NH ), 1602 (C=N); H-NMR (DMSO-d 400 MHz) δ
max
2
6
(
ppm): 2.38 (s, 3H, CH ), 2.48 (s, 3H, CH ), 7.65-8.25 (m, 4H, Ar, 2H, NH , 1H, NH), 8.84 (s, 1H, CH=), 11.36 (bs, 1H, NH, D O exch.), 12.20 (bs,
3 3 2 2
1
3
1
H, NH, D O exch.); C NMR (DMSO-d 100 MHz) δ (ppm): 17.20, 19.53, 97.31, 130.65, 130.87, 132.43, 132.64, 137.74, 137.75, 143.53, 155.63,
2 6
1
4

1
56.54, 158.51, 170.58; HRMS (m/z) 442.0266 [M+H]+, calcd 441.2932; Anal. Calcd. For C H IN S: C, 38.10; H, 3.65; N, 22.22; Found: C, 38.14;
14 16 7
H, 3.62; N, 22.25.
2
-(1-(2-((E)-2-(4-Hydroxy-3-methoxybenzylidene)hydrazinyl)-4-methylthiazol-5-yl)ethylidene)hydrazinecarboximidamide (4b)
-1
1
Light yellow solid (66% yield): mp 252-254 °C (MeOH). IR (KBr) cm :  3451 (OH), 3401, 3180 (NH, NH ), 1608 (C=N); H-NMR (DMSO-d
max
2
6
4
1
1
00 MHz) δ (ppm): 1.96 (s, 3H, CH ), 2.49 (s, 3H, CH ), 3.81 (s, 3H, OCH ), 6.84-7.59 (m, 3H, Ar, 2H, NH , 1H, NH), 8.10 (s, 1H, CH=), 9.55 (bs,
3 3 3 2
1
3
H, OH, D O exch.), 10.87 (bs, 1H, NH, D O exch.), 11.88 (bs, 1H, NH, D O exch.); C NMR (DMSO-d 100 MHz) δ (ppm): 17.22, 19.43, 56.10,
2
2
2
6
12.15, 117.21, 123.21, 131.10, 132.45, 143.41, 149.35, 151.10, 155.66, 156.51, 158.52, 170.45; HRMS (m/z) 362.1354 [M+H]+, calcd 361.4221;
Anal. Calcd. For C H N O S: C, 49.85; H, 5.30; N, 27.13; Found: C, 49.81; H, 5.25; N, 27.15.
1
5
19
7
2
2
-(1-(2-((E)-2-((2-Hydroxynaphthalen-1-yl)methylene)hydrazinyl)-4-methylthiazol-5-yl)ethylidene)hydrazinecarboximidamide (4c)
-1
1
Light yellow solid (35% yield): m.p. 262-263 °C (MeOH). IR (KBr) cm :  3351 (OH), 3201, 3180 (NH, NH ), 1605 (C=N); H-NMR (DMSO-d
max
2
6
4
1
1
00 MHz) δ (ppm): 2.34 (s, 3H, CH ), 2.49 (s, 3H, CH ), 7.35-8.21 (m, 6H, Ar, 2H, NH , 1H, NH), 8.31 (s, 1H, CH=), 10.25 (bs, 1H, OH, D O exch.),
3 3 2 2
1
3
0.93 (bs, 1H, NH, D O exch.), 11.93 (bs, 1H, NH, D O exch.); C NMR (DMSO-d 100 MHz) δ (ppm): 17.12, 19.52, 106.94, 107.26, 111.61, 115.82,
2
2
6
23.51, 125.40, 126.756.10, 112.15, 117.21, 123.21, 131.10, 132.45, 143.41, 149.35, 151.10, 155.66, 156.51, 158.52, 170.45; HRMS (m/z) 382.1405
[
M+H]+, calcd 381.1372; Anal. Calcd. For C H N O S: C, 49.85; H, 5.30; N, 27.13; Found: C, 49.81; H, 5.25; N, 27.15.
15 19 7 2
2
-(Biphenyl-4-ylmethylene)hydrazinyl)-4-methylthiazol-5-yl)ethylidene)hydrazinecarboximidamide (4d)
-1
1
Light yellow solid (66% yield): mp 262-264 °C (MeOH). IR (KBr) cm :  3401, 3180 (NH, NH ), 1604 (C=N); H-NMR (DMSO-d 400 MHz) δ
max
2
6
(
ppm): 1.96 (s, 3H, CH ), 2.49 (s, 3H, CH ), 6.84-7.59 (m, 9H, Ar, 2H, NH , 1H, NH), 8.10 (s, 1H, CH=), 10.87 (bs, 1H, NH, D O exch.), 11.88 (bs,
3 3 2 2
1
3
1
1
5
2
H, NH, D O exch.); C NMR (DMSO-d 100 MHz) δ (ppm): 17.22, 19.43, 112.15, 117.21, 123.21, 127.26, 127.62, 128.93, 129.43, 131.10, 132.45,
2 6
43.41, 149.35, 151.10, 155.66, 156.51, 158.52, 170.45; HRMS (m/z) 392.1613 [M+H]+, calcd 391.4926; Anal. Calcd. For C H N S: C, 61.36; H,
2
0
21
7
.41; N, 25.04; Found: C, 61.33; H, 5.39; N, 25.01.
-(3-bromo-4,5-dimethoxybenzylidene)hydrazinyl)-4-methylthiazol-5-yl)ethylidene)hydrazinecarboximidamide (4e)
-1
1
Orange solid (58% yield): mp 242-244 °C (MeOH). IR (KBr) cm :  3401, 3180 (NH, NH ), 1602 (C=N); H-NMR (DMSO-d 400 MHz) δ (ppm):
max
2
6
1
.96 (s, 3H, CH ), 2.49 (s, 3H, CH ), 3.85 (s, 3H, OCH ), 3.81 (s, 3H, OCH ), 6.84-7.59 (m, 2H, Ar, 2H, NH , 1H, NH), 8.10 (s, 1H, CH=), 10.87 (bs,
3 3 3 3 2
1
3
1
H, NH, D O exch.), 11.88 (bs, 1H, NH, D O exch.); C NMR (DMSO-d 100 MHz) δ (ppm): 17.22, 19.43, 56.10, 112.15, 117.21, 123.21, 131.10,
2 2 6
1
32.45, 143.41, 149.35, 151.10, 155.66, 156.51, 158.52, 170.45; HRMS (m/z) 454.5421 [M+H]+, calcd 454.3447; Anal. Calcd. For C H BrN O S:
16 20 7 2
C, 42.30; H, 4.44; N, 21.58; Found: C, 42.35; H, 4.39; N, 21.61.
4
4
.2. Biological assays procedures
.2.1. Antimicrobial screening
The disc diffusion method was utilized for antimicrobial evaluation of the tested compounds. The microorganism inoculums were uniformly spread,
using sterile cotton swabs on a sterile Petri dish with malt extract agar (for fungi) and nutrient agar (for bacteria). One hundred cubic millimeters of
each sample was added to each well (10-mm-diameter holes were cut in the agar gel, 20 mm apart from one another). The systems were incubated for
o
o
2
4-48 hr. at 37 C (for bacteria) and at 28 C (for fungi) recommended by Clinical and Laboratory Standards Institute (CLSI) [40] with the positive
control of clinically antimicrobial drugs ampicillin, ciprofloxacin, fluconazole, and GlcN-6-P synthase inhibitor FMDP1 [23]. After incubation, the
microorganism’s growth was observed. The inhibition zones of the bacterial and fungal growth were measured in millimeters. The tests were performed
in triplicate experiments [35,36]. Minimal inhibitory concentration (MIC, µg/mL) referred to the lowest concentration of new compounds that required
to completely arrest the growth of microbes. Minimal bactericidal concentration (MBC, µg/mL) referred to the lowest concentration of new compounds
that required to completely kill microbes [34]. All results are mean values from at least three experiments.
1
5

4
.2.1.1. Antibacterial assays
The prepared compounds were evaluated for their in vitro antibacterial activities against three Gram--positive bacteria (Staphylococcus aureus IFO
3
060, Bacillus subtilis IFO 3007 and Micrococcus luteus IFO 3232), two Gram--negative bacteria (Escherichia coli IFO 3301 and Pseudomonas
5
aeuroginosa IFO 3448). The bacterial suspension was adjusted with sterile saline to a concentration of 1 × 10 CFU (Colony Forming Unit). Initially
the compounds were dissolved in DMSO to prepare the stock solutions, then the tested compounds and reference drugs were prepared in Mueller-Hinton
broth [41] to obtain the required (two-fold serial dilutions) eight wanted concentrations 0.5 to 64 μg/mL. These dilutions were inoculated and incubated
at 37 ºC for 24 h.
4
.2.1.2 Antifungal assays
The newly synthesized compounds were evaluated for their in vitro antifungal activities against three pathogenic fungi (Candida albicans IFO 0583,
Aspergillus oryzae IFO 4177 and Aspergillus niger IFO 4414). A spore suspension in sterile distilled water was prepared from one day old culture of
-1
the fungi of the fungi growing on Sabouraud agar (SA) media. The final spore concentration was 1-5 × 1033 spore mL . From the stock solutions of
the tested compounds and reference antifungal drug Fluconazole, dilutions in sterile RPMI 1640 medium were made resulting in eight wanted
concentrations (0.5 to 64 μg/mL) of each tested compound. These dilutions were inoculated and incubated at 28 ºC for 48 h.
4
.2.1.3. Antibacterial assay against resistant strains
The minimum inhibitory concentrations (MICs) of the tested compounds and control drugs; linezolid, vancomycin, gentamicin (antibiotics) and
fluconazole and 5-fluorocytosine (5-FC) (antifungal drugs) were determined using the broth microdilution method, according to guidelines outlined by
the Clinical and Laboratory Standards Institute [42] against clinically-relevant bacterial (methicillin-resistant Staphylococcus aureus (MRSA),
Escherichia coli, Clostridium difficile and Neisseria gonorrhea strains) and fungal (Candida albicans) strain. S. aureus and E.coli were grown
aerobically overnight on tryptone soy agar plates at 37° C. difficile was grown anaerobically on brain heart infusion supplemented agar at 37° C for 48
hours. N. gonorrhea was grown on Brucella broth supplemented with yeast extract, neopeptone, hematin, pyridoxal and NAD at 37° C for 24 hours in
presence of 5% CO . C. albicans was grown aerobically overnight on yeast peptone dextrose (YPD) agar plate at 35° C. Afterwards, a bacterial solution
2
equivalent to 0.5 McFarland standard was prepared and diluted in cation-adjusted Mueller-Hinton broth (CAMHB) (for S. aureus and E. coli) to achieve
5
a bacterial concentration of about 5 × 10 CFU/mL. C. difficile was diluted in brain heart infusion supplemented broth, supplemented with yeast extract,
5
hemin and vitamin K to achieve a bacterial concentration of about 5 × 10 CFU/mL. N. gonorrhea was diluted in Brucella broth supplemented with
6
yeast extract, neopeptone, hematin, pyridoxal and NAD to achieve a bacterial concentration of about 1 × 10 CFU/mL. C. albicans was diluted in
Roswell Park Memorial Institute (RPMI 1640) medium with glutamine and without bicarbonate (GIBCO by Life Technologies, Green Island, NY,
USA) which was buffered to pH 7.0 with 0.165 M of [3-(N-morpholino) propanesulfonic acid] (MOPS) (dot scientific inc., Burton, MI, USa) to achieve
3
a fungal concentration of about 1.5 × 10 CFU/mL. Compounds and control drugs were added in the first row of the 96-well plates and serially diluted
with the corresponding media containing bacteria/fungi. Plates were then, incubated aerobically at 37° C for 18-20 hours (for S. aureus and E. coli).
C.albicans was incubated aerobically at 37° C for 24 hours. MICs defined as the minimum concentration of the compounds and control drugs that could
completely inhibit the visual growth of bacteria/fungi [43].
4
.2.1.4. GlcN-6-P synthase activity determination
A standard incubation mixture consisted of 10ꢀmM Fru-6-P, 10ꢀmMꢀL-Gln, 1ꢀmM EDTA, 1ꢀmM DTT and appropriately diluted enzyme preparation in
buffer D (final protein concentration 0.5–1.0ꢀμg/mL). The reaction started by adding the enzyme, the mixture was incubated at 37ꢀ°C for 30ꢀmin, and
the reaction was terminated by boiling for 1ꢀmin. The concentration of GlcN-6-P formed was determined using a modified Elson-Morgan procedure
[44]. One unit of the specific activity (U) was defined as an amount of the enzyme that catalyzed formation of 1ꢀμmol GlcN-6-Pꢀmin−1ꢀmgꢀprotein−1.
1
6

For the determination of kinetic constants, the enzyme activity was determined at variable (0–10ꢀmM) initial concentrations of appropriate substrates.
Data were plotted as Lineweaver-Burk plots. For the studies on Inhibition of GlcN-6-P synthase and its muteins activity by UDP-GlcNAc, the incubation
mixtures contained constant amounts of substrates (10ꢀmM Fru-6-P, 10ꢀmM L-Gln), variable concentrations of UDP-GlcNAc (0–5ꢀmM) and Glc-6-P,
1
0ꢀmM, when indicated [45].
4
.3. Molecular modeling
The newly synthesized compounds were docked into the active site of bacterial glucosamine-6-phosphate synthase (PDB ID: 2VF5) that used as receptor
model. The AutoDock 3.0 [38] and the MOE software [39] were used for all the docking calculations. The AutoDock Tools package was employed to
generate the docking input files and to analyze the docking results. A grid box size of 90×90×90 points with a spacing of 0.375 Å between the grid
points was generated that covered almost the entire protein surface. Ligands were built by means of the MOE builder interface, their geometries were
optimized with the CHARMm forcefield[46] and then prepared for docking calculations with the python scripts available in the Autodock package. For
each ligand 50 runs were performed, and the resulting poses were clustered with 1.8 Å tolerance. Lamarckian GA was used for the conformational space
search with initial population set to 150, and fitness function evaluations set to 25000000. The most abundant low energy clusters were selected for
analysis. The protein-ligand interaction plots were generated, using MOE 2012.10 [39]. The quantum mechanical calculations and the surface molecular
orbitals were generated by the simulation module in the MOE software.
Acknowledgment
This project was funded by the Deanship of Scientific Research (DSR), King Abdul-Aziz University, Jeddah, under grant No. (DF-655-166-1441).
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2
0

Graphical abstract
The Rational Design, Synthesis, and Antimicrobial Investigation of 2-Amino-4-Methylthiazole Analogues Inhibitors of
GlcN-6-P Synthase
A series of novel 2-Amino-4-Methylthiazole analogs were developed via three-step reaction encompassing hydrazine-1-carboximidamide motif to
combat Gram-positive and Gram-negative bacterial and fungal infections. Noticeably, the thiazole-carboximidamide derivatives 4a-d displayed
excellent antimicrobial activity and the most efficacious analogue 4d with MIC/MBC values of 0.5 and 4 μg/mL, compared to reference drugs with
very low toxicity to mammalian cells, resulting in a prominent selectivity more than 100 folds. Microscopic investigation of 4d biphenyl analogue
showed cell wall lysis and promote rapid bactericidal activity though disrupting the bacterial membrane.
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. A series of novel thiazole-based derivatives were synthesized and evaluated for their antimicrobial inhibiting activity.
. Antimicrobial activity, GlcN-6-P Synthase inhibitory activity were tested
. Molecular docking analyses was done for SAR interpretation.
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All authors have participated in (a) conception and design, or analysis and interpretation of the data; (b) drafting the article or revising it critically for
important intellectual content; and (c) approval of the final version.
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This manuscript has not been submitted to, nor is under review at, another journal or other publishing venue.
The following authors have affiliations with organizations with direct financial interest in the subject matter discussed in the manuscript:
Author’s name
Affiliation
Hany Emary Ali Ahmed
Taibah University
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