Evaluation of N-phenyl-2-aminothiazoles for treatment of multi-drug resistant and intracellular Staphylococcus aureus infections

Article abstract of DOI:10.1016/j.ejmech.2020.112497

The increasing emergence of antibiotic-resistant bacterial pathogens calls for additional urgency in the development of new antibacterial candidates. N-Phenyl-2-aminothiazoles are promising candidates that possess potent anti-MRSA activity and could potentially replenish the MRSA antibiotic pipeline. The initial screen of a series of compounds in this novel class against several bacterial strains revealed that the aminoguanidine analogues possessed promising activities and superior safety profiles. The determined MICs of these compounds were comparable to, if not better than, those of the control drugs (linezolid and vancomycin). Remarkably, compounds 3a, 3b, and 3e possessed potent activities against multidrug resistant staphylococcal isolates and several clinically important pathogens, such as vancomycin-resistant enterococci (VRE) and Streptococcus pneumoniae. In addition, the compounds were superior to vancomycin in the rapid killing of MRSA and the longer post-antibiotic effects. Furthermore, low concentrations of compounds 3a, 3b, and 3e reduced the intracellular burden of MRSA by greater than 90%. Initial in vitro PK/toxicity assessments revealed that compound 3e was highly tolerable and possessed a low metabolic clearance rate and a highly acceptable half-life.

Full text of DOI:10.1016/j.ejmech.2020.112497

Journal Pre-proof
Evaluation of N-Phenyl-2-aminothiazoles for Treatment of Multi-Drug Resistant and
Intracellular Staphylococcus aureus Infections
Inas G. Shahin, Nader S. Abutaleb, Marwa Alhashimi, Asmaa E. Kassab, Khaled O.
Mohamed, Azza T. Taher, Mohamed N. Seleem, Abdelrahman S. Mayhoub
PII:
S0223-5234(20)30469-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112497
EJMECH 112497
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 December 2019
Revised Date: 16 February 2020
Accepted Date: 19 May 2020
Please cite this article as: I.G. Shahin, N.S. Abutaleb, M. Alhashimi, A.E. Kassab, K.O. Mohamed, A.T.
Taher, M.N. Seleem, A.S. Mayhoub, Evaluation of N-Phenyl-2-aminothiazoles for Treatment of Multi-
Drug Resistant and Intracellular Staphylococcus aureus Infections, European Journal of Medicinal
Chemistry, https://doi.org/10.1016/j.ejmech.2020.112497.
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Evaluation of N-Phenyl-2-aminothiazoles for Treatment of Multi-Drug
Resistant and Intracellular Staphylococcus aureus Infections
Inas G. Shahin‖a, Nader S. Abutaleb‖b, Marwa Alhashimib, Asmaa E. Kassabc, Khaled O.
Mohamedc, Azza T. Taherc,d, Mohamed N. Seleem b,e,* Abdelrahman S. Mayhoubf,g*
a
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, October
University for Modern Sciences and Arts, Giza 11787, Egypt.
b
Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue
University, West Lafayette, IN 47907, USA.
c
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy,
Cairo University, Cairo 11562, Egypt.
d
Department of Pharmaceutical Organic Chemistry, College of Pharmacy, October 6
University, 6-October, Giza, Egypt.
e
Purdue Institute of Inflammation, Immunology, and Infectious Disease, West Lafayette,
IN 47907, USA.
f
Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-
Azhar University, Cairo 11884, Egypt.
g
University of Science and Technology, Nanoscience Program, Zewail City of
Science and Technology, October Gardens, 6th of October, Giza 12578, Egypt.
Corresponding Authors
* MNS; email, mseleem@purdue.edu
* ASM; email, amayhoub@azhar.edu.eg
‖These two authors contributed equally

Abstract. The increasing emergence of antibiotic-resistant bacterial pathogens calls for
additional urgency in the development of new antibacterial candidates. N-Phenyl-2-
aminothiazoles are promising candidates that possess potent anti-MRSA activity and could
potentially replenish the MRSA antibiotic pipeline. The initial screen of a series of compounds in
this novel class against several bacterial strains revealed that the aminoguanidine analogues
possessed promising activities and superior safety profiles. The determined MICs of these
compounds were comparable to, if not better than, those of the control drugs (linezolid and
vancomycin). Remarkably, compounds 3a, 3b, and 3e possessed potent activities against
multidrug resistant staphylococcal isolates and several clinically important pathogens, such as
vancomycin-resistant enterococci (VRE) and Streptococcus pneumoniae. In addition, the
compounds were superior to vancomycin in the rapid killing of MRSA and the longer post-
antibiotic effects. Furthermore, low concentrations of compounds 3a, 3b, and 3e reduced the
intracellular burden of MRSA by greater than 90%. Initial in vitro PK/toxicity assessments
revealed that compound 3e was highly tolerable and possessed a low metabolic clearance rate
and a highly acceptable half-life.
Key words: Intracellular bacteria, post-antibiotic effect, methicillin-resistant Staphylococcus
aureus, bacterial resistance, vancomycin-resistant Enterococci.

1. Introduction. Based on the rapid emergence of global antimicrobial resistance (AMR), the
decreasing efficacy of current antibiotics, and their extensive effects on public health and the
global economy, immediate action is urgently needed to identify and develop new therapeutic
agents that will refresh the declining antibiotic pipeline. Over $20 billion are estimated to be
spent annually in the United States healthcare system to treat antibiotic-resistant infections.
Additionally, drug-resistant bacteria are infecting more than 7,500 people daily and cause more
than 35,000 deaths each year in the United States.[1] Most worrisome, resistance has been
reported to every known class of antibiotics.[2]
Methicillin-resistant Staphylococcus aureus (MRSA) poses a growing threat human health as
a global nosocomial endemic due to the development of concomitant resistance to the most
commonly used antibiotics, such as penicillins, aminoglycosides, macrolides, tetracycline,
chloramphenicol, and lincosamides.[3] Moreover, due to the emerging resistance to both
vancomycin and linezolid, the drugs of last resort for the treatment of staphylococcal infections,
the morbidity and mortality of MRSA have significantly increased, resulting in a substantial
economic burden and severe clinical consequences [4].
The World Health Organization (WHO) has declared both methicillin-resistant and
vancomycin-intermediate or -resistant Staphylococcus aureus (MRSA, VISA and VRSA,
respectively) as high priorities in the priority pathogen list of antibiotic-resistant bacteria [5].
According to the Centers for Disease Control and Prevention (CDC), MRSA is categorized as a
serious threat pathogen. Approximately 323,700 patients with MRSA infections were
hospitalized in 2017 in the United States, and these infections were associated with
approximately 10,600 deaths. [1] Moreover, up to 53 million people globally may be potential
carriers of MRSA, posing a high risk of infection to themselves and others. [6] Overall, S. aureus

is deemed the main culprit behind healthcare-associated infections (HAIs) in Europe and the
United States, most of which are caused by MRSA.[7-10] Thus, this highly evolving superbug
requires swift action to combat its infections.
In 2014, our group launched a project aiming to discover and develop new compounds with
antibacterial activity against multidrug-resistant bacteria. A highly active novel phenylthiazole
scaffold I (Figure 1) was discovered.[11] The main features that actively play a role in the
antibacterial activity were postulated to include a thiazole nucleus flanked by two main structural
components: a lipophilic moiety at C2 and a guanidine group at C5. [11] Despite its anti-MRSA
activity, the scaffold suffered from extensive hepatic metabolism. A pyrimidine junction was
inserted to conjoin the aminoguanidine tail with the thiazole core II in an attempt to further
improve the pharmacokinetic (PK) performance of the lead compound I (Figure 1).[12] This
pyrimidine ring enhanced the PK properties but conversely led to a decrease in its aqueous
solubility, therefore limiting its oral bioavailability and excluding it from the drug-like
category.[12] Accordingly, the next study aimed at increasing the topological polar surface area
(tPSA)/lipophilicity ratio by substituting the pyrimidine ring with the more hydrophilic
oxadiazole ring III.[13] The oxadiazole ring fulfilled the required physicochemical target, but
proved to be less active against MRSA than its pyrimidine analogue. Based on this information,
a new strategy was devised to increase the antibacterial activity without compromising the
augmented oral bioavailability. The hetero ring junction, a key element required to balance the
metabolic stability and the physicochemical properties, was further modulated into a thiazole
ring IV (Figure 1).[14] This modification yielded compounds with potent anti-MRSA activity
and hepatic metabolic stability, and most importantly, they eradicated intracellular MRSA.

NH
HN NHR
NH
NH
H2N
HN
N
N
NH
NH2
NHR
N
S
O
N
NH
HN
N
S
N
S
N
N
S
N
N
S
Ar
I
II
III
IV
Figure 1. The transitional stages in optimizing the ‘drug-likeness’ of the phenyl thiazole anti-
MRSA nucleus. Lipophilic component colored blue and cationic part colored red for the sake of
clarity.
Figure 2. Progress in development of novel antimicrobials and the aim of the present work.
In addition to the phenylthiazole nucleus, screening of an in-house library of
aminoguanidines connected with diverse aromatic structures provided a limited number of novel
structures (Figure 2).[15] Among the newly obtained leads, the N-phenyl-2-aminothiazole 3a
was one of the most potent compounds of those tested against MRSA USA300 (MIC = 2
µg/mL). Other compounds showed modest activities (MICs ranged from 8 to 16 µg/mL) (Figure
2).[15] Therefore, in the present study, the SAR of this novel lead compound 3a was studied by
focusing on the two sides of the structure. First, the aminoguanidine moiety was replaced with

the highly structurally related semicarbazide and its thio analogues. Second, the substitution of
the terminal phenyl with electronically different groups was achieved. The new compounds were
initially screened against MRSA and E. coli strains, and the metabolic stability of the most
promising analogue was investigated. Additionally, selected potent derivatives were further
subjected to bacteriological profiling.
2. Results and Discussion
2.1. Chemistry. The acetyl phenyl thiourea 1 was obtained by reacting 4-amino acetophenone
with potassium thiocyanate in presence of water that had been acidified with hydrochloric acid.
The base-promoted cyclisation of compound 1 with phenacyl bromides produced the thiazole
derivatives 2a-e, which are the main intermediates, from which the final products were afforded.
As shown in Scheme 1, the acetyl moiety in compounds 2a-e was replaced with various cationic
tails, such as aminoguanidine, semicarbazide, and thiosemicarbazide, and furnished the desired
final compounds 3a-e, 4a-e, and 5a-e, respectively.

The structures of compounds 2a-e, 3a-e, 4a-e, and 5a-e were supported by the results of
1
elemental analyses and spectroscopic data: IR, H NMR and 13C NMR. The structures of
compounds 2a-e were confirmed by the disappearance of the exchangeable signal of the NH2
protons at
δ
3.40 ppm and the appearance of the singlet signal corresponding to the CH of
7.35-8.13 ppm, unlike its precursor, compound 1.
thiazolyl moiety in the range between
δ
The IR spectra of target compounds 3a-e were characterized by the disappearance of the
1
C=O band, while the H NMR spectra showed the appearance of new exchangeable singlet
signals of NH at
δ 7.34-11.38 ppm and NH2 at δ 7.78-11.49 ppm. The IR spectra of compounds
4a-e showed new C=O bands at 1654-1693 cm-1, which were also confirmed by the presence of
1
signals in the 13C NMR spectra at
δ
158.1-162.8 ppm. The H NMR spectra revealed new

exchangeable NH singlet signals at
δ 8.86-10.66 ppm and NH2 at δ 4.29-6.55 ppm. Finally, the
structures of compounds 5a-e were confirmed by the appearance of new C=S bands in IR spectra
1
at 1083-1296 cm-1 and by 13C NMR peaks at
δ
178.8 -197.9 ppm. The H NMR spectra also
supported the attachment of the thiosemicarbazide moiety because a new exchangeable NH2
signal appeared at 4.50-8.15 ppm.
δ
2.2. Biological results and discussion
2.2.1. Antimicrobial activity
2.2.1.1. Initial screening against methicillin-resistant S. aureus, and E. coli strains. The initial
screening results revealed that the most active derivatives belonged to the aminoguanidine series
3a-e. Compounds 3a, 3b, and 3e exhibited the most potent activity against MRSA USA300, with
MIC values ranging from 2 to 4 µg/mL. Additionally, they exhibited one-fold higher MIC values
against tolC-mutant E. coli (deficient in tolC, an essential component of the AcrAB-TolC efflux
pump). They also exhibited a lower antibacterial activity against the wild-type E. coli (MICs = 8-
16 µg/mL). Thus, the lower activity of the compounds against E. coli may be attributed to their
efflux out of the bacterial cell. These 3 compounds were selected for further investigations.
Table 1. Initial screening of all the synthesized compounds against methicillin-resistant Staphylococcus
aureus and Escherichia coli isolates.
MICs (µg/mL)
Compounds/
E. coli
BW25113
(wild-type strain)
Control
antibiotics
E. coli JW55031
MRSA NRS384
(USA300)
(tolC-mutant)
>64
>64
>64
>64
>64
2
4
32
16
>64
>64
>64
>64
64
4
8
2
2
>64
>64
>64
>64
>64
8
8
4
4
2a
2b
2c
2d
2e
3a
3b
3c
3d

MICs (µg/mL)
Compounds/
Control
antibiotics
E. coli
BW25113
(wild-type strain)
E. coli JW55031
MRSA NRS384
(USA300)
(
tolC-mutant)
2
4
16
3e
>64
>64
>64
>64
32
>64
32
>64
>64
>64
1
>64
>64
>64
>64
32
>64
>64
>64
>64
>64
>64
>64
>64
>64
>64
NT
4a
4b
4c
4d
4e
5a
5b
5c
5d
>64
64
>64
>64
>64
NT1
0.25
5e
Vancomycin
Gentamicin
NT
0.25
1NT: not tested
2.2.1.2. Antibacterial activity of compounds 3a, 3b and 3e against a wide panel of clinical
staphylococcal isolates. After the initial screen, we investigated the activity of compounds 3a, 3b
and 3e against clinical isolates of methicillin-sensitive, methicillin-resistant and vancomycin-
resistant Staphylococcus aureus (MSSA, MRSA, and VRSA). As presented in Table 2, the
compounds maintained their potent activity against the tested MSSA, MRSA, and VRSA strains,
inhibiting their growth at concentrations ranging from 1 to 2 µg/mL. These compounds were
equipotent to vancomycin (MICs ranged from 1 to 2ꢀμg/mL against MSSA and MRSA) and
linezolid (MICs = 1-2ꢀμg/mL against MSSA and MRSA, except for MRSA NRS107).
Interestingly, they maintained their potency against linezolid-resistant and vancomycin-resistant
staphylococcal strains. Furthermore, compounds 3a, 3b, and 3e displayed bactericidal activity
against the tested strains, as their MBC values were equal to or one- to three-fold higher than
their corresponding MICs. Thus, these compounds were superior to linezolid, a frontline drug
used to treat staphylococcal infections that exhibits a bacteriostatic activity. As previously

established, chlorophenylthiazoles exert a profound effect on the antibacterial activity.[16]
Analogously, compound 3b emerged as the most potent analogue against the tested
staphylococcal strains.
Table 2. MICs and MBCs in µg/mL of compounds 3a, 3b and 3e against Staphylococcus aureus
clinical isolates.
Compounds/control antibiotics
Bacterial strains
Linezolid
Vancomycin
3a
3b
3e
MIC
MBC
MIC
MBC
MIC
MBC
MIC MBC MIC MBC
2
2
1
1
2
4
1
1
16
64
1
2
2
2
MSSA ATCC 6538
MSSA NRS 107
MRSA NRS119
1
2
2
2
2
2
2
4
4
2
2
8
2
4
1
1
1
1
1
1
1
1
1
1
2
4
1
1
2
2
2
2
2
2
2
2
4
2
2
2
2
2
64
1
> 64
32
1
2
MRSA NRS123
(USA400)
MRSA NRS 385
(USA500)
MRSA NRS 386
(USA700)
1
1
1
32
1
1
2
64
1
1
1
> 64
64
64
64
> 64
64
VRSA 10
VRSA 12
1
2.2.1.3. Spectrum of antibacterial activity. Furthermore, the spectra of antibacterial activity of
the three most potent derivatives was examined against a panel of clinically relevant Gram-
positive bacterial pathogens. Consistent with the results presented in Table 2, the compounds
maintained their notable antibacterial activity against different known Gram-positive pathogens
by inhibiting their growth at concentrations of 1 to 2 µg/mL (Table 3). Interestingly, they
exhibited potent activity against S. epidermidis, a common cause of skin infections, and
implanted medical device infections. Due to formation of strongly attached biofilms, S.
epidermidis infections are difficult to treat.[17, 18] Furthermore, the compounds retained their
superiority to vancomycin when screened against vancomycin-resistant enterococci (VRE), a
leading cause of nosocomial infections that causes 20-30% of VRE infections in the USA.[19] In

addition, the potent activities of compounds 3a, 3b and 3e were further extended to include
Listeria monocytogenes and Streptococcus pneumoniae. The MBCs of the tested compounds
were equal to or one- to three-fold higher than their corresponding MICs, indicating that the
compounds exhibited bactericidal activity against the tested strains.
Table 3. MICs and MBCs (µg/mL) of compounds 3a, 3b and 3e against clinically important
Gram-positive bacterial pathogens
Compounds/control antibiotics
Linezolid
Vancomycin
Bacterial Strains
3a
3b
3e
MIC MBC MIC MBC MIC MBC MIC MBC MIC
MBC
Methicillin-resistant
Staphylococcus epidermidis
NRS101
1
2
1
4
1
1
1
4
1
2
1
4
1
1
16
16
1
2
Vancomycin-resistant
Enterococcus faecalis
ATCC 51299
32
64
Vancomycin-resistant
Enterococcus faecium
ATCC 700221
Listeria monocytogenes
ATCC 19111
Cephalosporin-resistant
Streptococcus pneumoniae
ATCC 51916
2
1
2
4
4
4
1
1
2
2
2
4
2
1
2
4
2
4
1
≤0.5
1
16
16
32
>64
1
>64
1
1
2
Methicillin-resistant
Streptococcus pneumoniae
ATCC 700677
2
8
2
4
2
8
1
16
2
2
2.2.1.4 Time to kill assay. One of the most fundamental parameters to consider when designing a
therapeutic is the killing kinetics of the drug. Fast-acting drugs hold a significant advantage over
the slow-acting drugs because they are more efficacious in eradicating the infection. Although
vancomycin is the drug of choice for treating MRSA infections, its clinical efficiency is affected
by its slow bactericidal mode of action, which leads to difficulty in eradicating MRSA. A time to
kill assay was conducted with 5 × MIC against MRSA USA 400 to confirm the bactericidal
properties of the three most promising compounds (Figure 3). As mentioned above, vancomycin
exhibited a slow bactericidal activity. Vancomycin reduced the bacterial count by 3-log10 after 12
hours and completely eradicated it after 24 hours. Remarkably, the tested compounds exhibited

rapid bactericidal activity and required only two hours to reduce the bacterial count by 3-log10.
Compound 3b exhibited the most efficient bactericidal activity by completely eradicating the
high MRSA count within two hours. However, compounds 3a and 3e completely eliminated the
high inoculum after eight hours.
Figure 3. Killing kinetics of 3a, 3b and 3e (tested in triplicates at 5 × MIC) against methicillin-
resistant Staphylococcus aureus NRSA 123 (MRSA USA400) over a 24-h incubation period at
37°C. DMSO (solvent for the compounds) served as a negative control and vancomycin served
as a control drug. The error bars represent standard deviation values obtained from triplicate
samples used for each compound/antibiotic studied.
2.2.2. Post-antibiotic effect. The rapid bactericidal activity of N-phenyl-2-aminothiazoles
encouraged us to investigate the post-antibiotic effect (PAE) of these compounds. Drugs
exhibiting a long PAE are considered advantageous, as these agents may require fewer doses in
the clinic.[20] The PAEs of compounds 3b and 3e were tested against MRSA USA400. The time
required for bacteria exposed to each test agent to increase by 1-log10 relative to the untreated
control was determined. As shown in Table 4, vancomycin possessed a shorter PAE of 2 hours,

consistent with previous reports.[21, 22] and 3a showed the same PAE as vancomycin.
Conversely, compound 3e exhibited a long PAE of 6 hours, while compound 3b exhibited a
shorter PAE of 4 hours, exceeding the PAE of vancomycin.
Table 4. Post-antibiotic effect (PAE) of 3b, 3e and vancomycin (tested at 5 × MIC) against
methicillin-resistant Staphylococcus aureus NRS 123 (USA400).
PAE (hours)
Vancomycin
2
3a
3b
3e
2
4
6
2.2.3. Toxicity evaluation. The development of a safe antimicrobial agent is a top priority in any
research endeavor, where minimizing toxicity to host tissues must be carefully assessed.
Consequently, the three most auspicious compounds (3a, 3b, and 3e) were thoroughly examined
for cytotoxicity toward human colorectal adenocarcinoma (Caco-2) cells and human keratinocyte
(HaCaT) cells (Figures 4 & 5).
2.2.3.1 In vitro cytotoxicity assay using Caco-2 cells. The compounds displayed an acceptable
tolerability to Caco-2 cells at high concentrations. Compounds 3a and 3b were nontoxic to Caco-
2 cells at a concentration of 32 µg/mL, with approximately 100% cell viability recorded.
Nevertheless, compound 3e out-performed all the other compounds, as approximately 100% of
the cells were still viable after treatment with a concentration as high as 64 µg/mL (Figure 4).
These concentrations represent 16 to 64 times their MIC values against MRSA strains, providing
them a broad safety window.

Figure 4. Analyzing the toxicity of compounds 3a, 3b and 3e (tested in triplicates at 32, 64 and
128 µg/mL) against human colorectal cells (Caco-2) using the MTS assay. Results are presented
as percent viable cells relative to DMSO (negative control to determine a baseline measure for
the cytotoxic impact of each compound). The absorbance values represent an average of three
samples analyzed for each compound. Error bars represent standard deviation values. Data were
analyzed via a two-way ANOVA with post hoc Dunnett’s test for multiple comparisons. *
denotes a statistical difference (P<0.05) between values obtained for the compounds and the
DMSO.
2.2.3.2. In vitro cytotoxicity assay using HaCaT cells. Similar to the results obtained with Caco-2
cells, the tested compounds exhibited a good toxicity profile at high concentrations when
evaluated against HaCaT cells. All three compounds were nontoxic to HaCaT cells at a
concentration of 32 µg/mL, as approximately 100% of the cells were viable (Figure 5).

Figure 5. Analyzing the toxicity of compounds 3a, 3b and 3e (tested in triplicates at 32, 64 and
128 µg/mL) against human keratinocyte cells (HaCaT) using the MTS assay. Results are
presented as percent viable cells relative to DMSO (negative control to determine a baseline
measure for the cytotoxic impact of each compound). The absorbance values represent an
average of three samples analyzed for each compound. Error bars represent standard deviation
values. Data were analyzed via a two-way ANOVA with post hoc Dunnett’s test for multiple
comparisons. * denotes a statistical difference (P<0.05) between values obtained for the
compounds and the DMSO.
2.2.4. Intracellular infection of J774 cells with MRSA and treatment with compounds 3a, 3b and
3e. The rapid and potent bactericidal activity of N-phenyl-2-aminothiazoles, as well as the longer
post-antibiotic effect prompted us to investigate whether these compounds penetrate
macrophages infected with MRSA and kill the bacteria intracellularly. MRSA is capable of
multiplying within the phagolysosome after being taken up by macrophages, which allows the
bacteria to survive and reinfect the host.[23] Most antibiotics are inactive against intracellular
MRSA, including the frontline drugs vancomycin and linezolid.[24, 25] The progenitor
phenylthiazoles exhibited significant intracellular clearance activity.[26] This finding prompted
us to investigate the intracellular clearance activity of the newly synthesized compounds. First,
the compounds were tested for their cytotoxicity toward murine macrophages (J774).

Compounds 3b and 3e were nontoxic to J774 cells at concentrations up to 32 µg/mL, whereas
compound 3a was nontoxic at the concentration of 16 µg/mL, with 100% cell viability observed
(Figure 6A). Based on the results of the cytotoxicity assay, the compounds were tested for their
intracellular clearance activity at their nontoxic concentrations. As depicted in Figure 6B,
vancomycin was incapable of reducing the intracellular MRSA burden, consistent with previous
reports. Remarkably, compound 3e exhibited the highest intracellular clearance activity,
reducing the intracellular MRSA burden by approximately 2.49-log10, which is equivalent to a
99% reduction at 4× MIC (8 µg/mL). Even at a lower concentration (2× MIC), compound 3e still
managed to reduce the intracellular infection by 1.19-log10, which is equivalent to 93%
reduction. Compound 3a generated a 1.19-log10 reduction (93% reduction in CFU) at 4× MIC (8
µg/mL). This value was reduced to an approximately 1.07-log10 reduction (88% reduction) when
the concentration was decreased by one-fold (2× MIC). Compound 3b generated a 1.56-log10
reduction (an approximately 97% reduction) and 0.51-log10 reduction (an approximately 69%
reduction) at 4× and 2× MIC, respectively. Consequently, these three analogues not only
exhibited a more rapid bactericidal activity against MRSA than vancomycin but also surpassed
vancomycin in the ability to reduce the intracellular MRSA burden inside infected macrophages.
Figure 6A. In vitro analysis of the cytotoxicity of compounds 3a, 3b and 3e toward murine
macrophage (J774) cells using the MTS assay. Results are presented as average percentages of
viable mammalian cells relative to DMSO. Data were analyzed using a two-way ANOVA with

Dunnett’s post hoc test for multiple comparisons. * denotes a statistically significant difference
(P<0.05) between values obtained for the compounds and DMSO.
Figure 6B. Examination of the activity of compounds 3a, 3b and 3e toward the clearance of
intracellular MRSA present inside murine macrophage (J774) cells. Data are presented as log10
CFU/mL of intracellular MRSA USA400 inside infected murine macrophages after treatment
with 2 × MIC and 4 × MIC of compound 3a, 3b, 3e or vancomycin (tested in quadruplicate) for
24 h. Data were analyzed using a two-way ANOVA, followed by Dunnett’s post hoc test for
multiple comparisons (P < 0.05) utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla,
CA). Asterisks (*) denote statistically significant differences between treatment with compound
3a, 3b or 3e and vancomycin.
Multi-step resistance study against MRSA. Having demonstrated that N-phenyl-2-aminothiazoles
compounds could reduce the burden of MRSA inside macrophages, we proceeded to evaluate the ability
of MRSA to develop resistance against these compounds. A multi-step resistance selection test was
conducted against MRSA USA400. Interestingly, the MIC of 3e changed by 1-fold only after the
thirteenth passage without additional increase thereafter (Figure 7). The MIC values of 3b increased by
one-fold after the seventh passage and by three folds after the fourteenth passage. Moreover, the MIC
values of 3a increased one-fold after the fifth passage and another 3-fold-increase was observed after the
eleventh passage. These results indicate that MRSA is unlikely to form rapid resistance to N-Phenyl-2-
aminothiazoles, even after exposure to them for 14 passages. In contrast, MICs of ciprofloxacin, one of
the antibiotics used in the treatment of MRSA infections, rapidly increased. MIC of ciprofloxacin
increased by one-fold after the second passage and continued to increase up to a 127-fold increase by
passage 14.

150
100
50
3a
3b
3e
Ciprofloxacin
0
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
Passage Number
Figure 7. Multi-step resistance selection of N-Phenyl-2-aminothiazoles 3a, 3b and 3e and ciprofloxacin
against methicillin-resistant S. aureus USA400 (NRS123). Bacteria were serially passaged over a 14-day
period and the broth microdilution assay was used to determine the MIC of each test agent after each
successive passage. A four-fold shift in MIC would be indicative of bacterial resistance to the test agent.
2.2.5 Initial assessment of the physicochemical properties and pharmacokinetic parameters. One
of the most critical characters of candidate drugs that must be evaluated early in the drug
development process is the physicochemical properties. The physicochemical properties of the
most potent analogue, 3e, were explored. First, the aqueous solubility was analyzed using a
turbidometric solubility assay in phosphate-buffered saline. Solubility directly affects the
pharmacokinetic profiles of compounds, routes of administration and the formulation of
compounds into suitable dosage forms.[27] Compound 3e showed moderate aqueous solubility
compared with two FDA-approved drugs, rifampicin and tamoxifen (Table 5).
Table 5. Evaluation of solubility in phosphate-buffered saline (PBS)
Compound/Drug
Solubility limit
M)
cLogP
4.12
Note
(μ
26.7
183.3
2.1
3e
Reference with good solubility
Reference with limited solubility
Rifampicin
Tamoxifen
The solubility limit corresponds to the highest concentration of test compound at which no precipitate was detected (OD540
)

The antibacterial activity of the synthesized compounds is not the sole factor for them to be
considered successful candidates. The compounds must possess a set of drug-like properties. For
instance, compounds should possess an acceptable metabolic clearance rate to guarantee the
survival of the candidate compounds through phase 1 clinical trials in humans. The metabolic
stability and clearance rate of the most potent derivative, 3e, were evaluated through an
incubation with human liver microsomes to address these needs (Table 6). The intrinsic half-life
(t1/2) of compound 3e in the presence of NADPH was approximately 2 hours, which is
comparable to propranolol and higher than imipramine, drugs with a low clearance rate. Despite
the increase in its half-life in the absence of NADPH suggesting that the compound is mainly
metabolized by the co-factor CYP-450, compound 3e exhibited pronounced stability to hepatic
metabolism, with a clearance rate of less than 60 μL/min-mg. This result endows the new series
of N-phenyl-2-aminothiazoles an advantage over the old generation of phenylthiazoles that were
rapidly metabolized with a t1/2 of less than half an hour.[11]
Table 6. Analysis of the metabolic stability of compound 3e in human liver microsomes
Test
1
Test
Article
Mean CLint
Mean t1/2
(minutes)
Concentration
M)
Notes
(μ
L/min-mg)
578.3
211.1
48.2
(μ
High clearance
control
High clearance
control
Low clearance
control
Low clearance
control
Terfenadine
0.1
0.1
0.1
0.1
0.1
0.1
8.95
24.20
Verapamil
143.80
87.20
Propranolol
79.5
Imipramine
3e (with
Stable to
metabolism
Stable to
58.9
117.60
253.85
NADPH
3e (without
NADPH
1CLint, intrinsic clearance rate; t1/2, half-life
)
27.3
metabolism
)

3. Conclusions. The rapid spread of antimicrobial resistance has wreaked havoc in the
community, particularly with the associated lack of new antibiotics being introduced to the
market. MRSA represents one of the challenges in the healthcare system due to its increased
resistance to a wide range of antibiotics. Faced with this challenge, the mission of medicinal
chemists is to rejuvenate the current antibiotic armamentarium with new members that are less
susceptible to the ever-growing bacterial resistance. Therefore, the SAR of a series of N-phenyl-
2-aminothiazole derivatives as novel antibiotics was studied from both sides of the molecule: the
lipophilic motif and the nitrogenous head. Interestingly, the aminoguanidine series displayed the
best efficacy against MRSA. The three most active aminoguanidine derivatives, 3a, 3b and 3e,
displayed excellent activity against a larger panel of Gram-positive bacteria, including MSSA,
MRSA, VRSA, S. epidermidis, VRE, S. pneumoniae and L. monocytogenes. Moreover, the
compounds exhibited a rapid bactericidal activity and a longer PAE than vancomycin against
MRSA. These compounds also displayed an excellent tolerability in Caco-2 and HaCaT cells.
Furthermore, these compounds surpassed vancomycin in killing MRSA intracellularly.
Collectively, the N-phenyl-2-aminothiazole derivatives are promising anti-MRSA candidates.
Further investigations are warranted to clearly elucidate their antibacterial mechanisms of action
and to evaluate their activities in a suitable animal model.
4. Experimental Procedures
4.1. Chemistry
4.1.1. General. Melting points were obtained using a Stuart melting point apparatus and were
uncorrected. Microanalyses for C, H and N were performed at the Regional Center for Mycology
and Biotechnology, Al-Azhar University. IR spectra were recorded on Shimadzu IR 435
spectrophotometer (Shimadzu Corp., Kyoto, Japan) at the Faculty of Pharmacy, Cairo

University, Cairo, Egypt and values are presented in cm-1. 1H NMR spectra were recorded using
a Bruker 400 MHz (Bruker Corp., Billerica, MA, USA) spectrophotometer at the Faculty of
Pharmacy, Cairo University, Cairo, Egypt, and Varian Mercury-300BB 300 MHz (Varian Corp.,
Palo Alto, CA, USA) spectrophotometer at the Cairo University and Faculty of Science, Cairo
University, Cairo, Egypt. Tetramethylsilane (TMS) was used as an internal-standard, and
chemical shifts were recorded in ppm on the
δ scale and coupling constants (J) were reported in
13
Hz. C NMR spectra were recorded using a Bruker 100 MHz spectrophotometer at the Faculty
of Pharmacy, Cairo University, Cairo, Egypt and Varian Mercury-300BB 75 MHz at the Faculty
of Science, Cairo University, Cairo, Egypt. The progress of the reactions was monitored with
TLC using precoated aluminum sheet silica gel MERCK 60F 254. The spots were visualized
using a UV lamp. The solvent system used for this assay was ethyl acetate: hexane [7:3].
Compounds 1 and 2a were prepared as described in the literature.[28-30]
4.1.2. Synthesis of 1-(4-((4-phenylthiazol-2-yl)amino)phenyl)ethanone derivatives (2b-e).
Anhydrous sodium acetate (0.33 g, 4 mmol) was added to a heated solution of compound 1 (0.39
g, 2 mmol) in THF (25 mL), followed by the addition of a solution of the appropriate phenacyl
bromides (4 mmol) in THF. This mixture was heated under reflux for 5-6 h and the progress of
the reaction was monitored using TLC. Then, the reaction mixture was poured on 50 mL of ice
water, and the formed precipitate was filtered and crystallized from ethanol.
4.1.2.1. 1-(4-((4-(4-Chlorophenyl)thiazol-2-yl)amino)phenyl)ethanone (2b). Yellowish brown
1
crystals (0.54 g, 64.3%), m.p.198-200 °C; IR (KBr,
NMR (DMSO-d6, 400 MHz)
8.0 Hz, ArH), 7.83 (d, 2H, J = 8.0 Hz, ArH), 7.96-7.99 (dd, 4H, J = 3.2 Hz, 8.0 Hz, ArH), 10.76
ν
cm-1): 3275 (NH) and 1677 (C=O); H
δ
: 2.50 (s, 3H, CH3C=O), 7.50 (s, 1H, thiazolyl), 7.52 (d, 2H, J =

(s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6,100 MHz)
δ: 26.7 (CH3C=O), 105.5,
116.4, 127.8, 129.1, 130.2, 130.4, 132.6, 133.6, 145.5, 149.4 162.8(ArCs), 196.4 (C=O).
4.1.2.2. 1-(4-((4-(4-Methoxyphenyl)thiazol-2-yl)amino)phenyl)ethanone (2c). Yellow crystals
1
(0.8 g, 95.8 %), m.p. 212-214 °C; IR (KBr,
(DMSO-d6, 400 MHz)
ν
cm-1): 3271 (NH) and 1654 (C=O); H NMR
δ
: 2.50 (s, 3H, CH3C=O), 3.94 (s, 3H, OCH3), 7.19 (d, 1H, J = 8.6 Hz,
ArH), 7.64 (d, 2H, J = 8.6 Hz, ArH), 7.89 (d, 2H, J = 8.6 Hz, ArH), 7.95-7.98 (dd, 1H, J = 2.12,
8.6 Hz, ArH), 8.07 (d, 1H, J = 2.12 Hz, ArH), 8.10 (s, 1H, thiazolyl), 8.17 (d, 1H, J = 8.6 Hz,
ArH), 10.02 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6,100 MHz)
δ: 26.7
(CH3C=O), 56.8 (CH3O), 104.0, 114.5, 126.9, 129.0, 129.5, 130.4, 132.4, 144.3, 150.6, 159.4,
162.5 (ArCs), 196.5 (C=O).
4.1.4.3. 1-(4-((4-(4-Bromophenyl)thiazol-2-yl)amino)phenyl)ethanone (2d). Yellow solid (1.6 g,
1
83.2 %), m.p. 224-226 °C; IR (KBr,
300 MHz)
ν
cm-1): 3417 (NH) and 1678 (C=O); H NMR (DMSO-d6,
δ
: 2.50 (s, 3H, CH3C=O), 7.67 (d, 2H, J = 8.0 Hz, ArH), 7.74 (d, 2H, J = 8.0 Hz,
ArH), 7.86 (d, 2H, J = 8.0 Hz, ArH), 7.91 (d, 2H, J = 8.0 Hz, ArH), 8.13 (s, 1H, thiazolyl), 9.97
(s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6,75 MHz)
δ
: 26.3 (CH3C=O), 120.9, 127.1,
127.2, 128.9, 130.0, 131.6, 131.9, 132.0, 133.1, 143.8, 181.1 (ArCs), 196.4 (C=O).
4.1.2.4. 1-(4-((4-(p-Tolyl)thiazol-2-yl)amino)phenyl)ethanone (2e). Yellow solid (1 g, 63.3 %),
m.p. 170-172 °C; IR (KBr,
ν
cm-1): 3271 (NH) and 1654 (C=O); 1H NMR (DMSO-d6, 300 MHz)
δ
: 2.33 (s, 3H, CH3), 2.50 (s, 3H, CH3C=O), 7.24 (d, 2H, J = 8.0 Hz, ArH ), 7.35 (s, 1H,
thiazolyl), 7.81-7.85 (dd, 4H, J = 2.1Hz, 8.0 Hz, ArH), 7.97 (d, 2H, J = 8.0 Hz, ArH), 10.69 (s,
1H, NH, D2O exchangeable); 13C NMR (DMSO-d6,75 MHz)
δ
: 20.8 (CH3), 26.2 (CH3C=O),
103.3, 115.8, 125.6, 129.2, 129.7, 129.9, 131.6, 137.0, 145.2, 150.3, 162.1 (ArCs), 196.0 (C=O).

4.1.3. General procedure for synthesis of 2-(1-(4-((4-phenylthiazol-2-yl)amino)phenyl)
ethylidene)hydrazinecarboximidamide derivatives (3a-e) To a solution of the key intermediates
2a-e (1 mmol) in absolute ethanol (25 mL), 1 mL of concentrated HCl was added, followed by
dry aminoguanidine HCl (0.22g, 2 mmol). This mixture was heated under reflux for 20-24 h, the
progress of the reaction was monitored by TLC. The reaction mixture was rinsed with sodium
bicarbonate solution, the solid formed is filtered giving the pure compounds 3a-e.
4.1.3.1.
2-(1-(4-((4-Phenylthiazol-2-yl)amino)phenyl)ethylidene)hydrazinecarboximidamide
cm-1): 3545, 3410, 3244
(3a). Yellow solid (0.2 g, 60.8 %), m.p. 249-251 °C; IR (KBr,
ν
(NH/NH2), 1670, 1620, 1566 (C=N); 1H NMR (DMSO-d6, 300 MHz)
δ
: 2.41 (s, 3H, CH3), 7.87
(s, 1H, NH, D2O exchangeable), 7.88-7.92 (m, 5H, ArH), 8.15 (s, 1H, thiazolyl), 8.22 (d, 2H,
ArH), 8.26 (d, 2H, ArH), 11.38-11.49 (2s, 4H, NH, NH, NH2, D2O exchangeable); 13C NMR
(DMSO-d6,75 MHz) δ: 14.6 (CH3), 120.7, 121.9, 124.9, 127.2, 127.5, 137.7, 140.2, 144.1, 147.8,
150.1, 150.6, 156.0, 156.1 (ArCs + C=N); Anal. Calcd. for C18H18N6S (350.44): C, 61.69; H,
5.18; N, 23.98; found: C, 61.74; H, 5.20; N, 23.89.
4.1.3.2.
2-(1-(4-((4-(4-Chlorophenyl)thiazol-2-yl)amino)phenyl)ethylidene)hydrazine
carboximidamide (3b). Yellow solid (0.3 g, 67.7 %), m.p. 274-276 °C; IR (KBr,
3248, 3170 (NH/NH2), 1670, 1600, 1543 (C=N); 1H NMR (DMSO-d6, 300 MHz)
ν
cm-1): 3444,
δ
: 2.34 (s, 3H,
CH3), 7.43 (s, 1H, thiazolyl), 7.45 (d, 2H, J = 8.0 Hz, ArH), 7.78 (s, 1H, NH, D2O
exchangeable), 7.81 (d, 2H, J = 8.0 Hz, ArH), 7.92-7.99 (dd, 4H, J = 3.6 Hz, 8.0 Hz, ArH), 10.69
(s, 2H, NH2, D2O exchangeable), 11.14 (s, 1H, NH, D2O exchangeable), 11.15 (s, 1H, NH, D2O
13
exchangeable); C NMR (DMSO-d6,75 MHz)
δ
: 14.2 (CH3), 104.4, 116.2, 127.3, 127.7, 128.6,
129.4, 132.0, 133.3, 142.4, 148.8, 151.4, 155.9, 162.7 (ArCs+C=N); Anal. Calcd. for
C18H17ClN6S (384.89): C, 56.17; H, 4.45; N, 21.84; found: C, 56.20; H, 4.65; N, 21.66.

4.1.3.3.
2-(1-(4-((4-(4-Methoxyphenyl)thiazol-2-yl)amino)phenyl)ethylidene)hydrazine
carboximidamide (3c). Yellow solid (0.2 g, 63.15 %), m.p. 205-207 °C; IR (KBr,
3363, 3286 (NH/NH2), 1685, 1624, 1597 (C=N); 1H NMR (DMSO-d6, 300 MHz)
ν
cm-1): 3437,
δ
: 2.34 (s, 3H,
CH3), 3.94 (s, 3H, OCH3), 7.34 (s, 1H, NH, D2O exchangeable), 7.39 (d, 2H, J = 7.5 Hz, ArH),
7.44 (d, 2H, J = 7.5 Hz, ArH), 7.92 (d, 2H, J = 7.5 Hz, ArH), 7.95 (d, 2H, J = 7.5 Hz, ArH), 8.16
(s, 1H, thiazolyl), 10.67 (s, 2H, NH2, D2O exchangeable), 11.20 (s, 2H, 2NH, D2O
exchangeable); 13C NMR (DMSO-d6,75 MHz)
δ: 14.3 (CH3), 59.5 (OCH3), 103.6, 106.9, 116.1,
125.6, 127.7, 128.6, 129.3, 134.4, 142.4, 150.0, 151.3, 156.0, 162.5 (ArCs+ C=N); Anal. Calcd.
for C19H20N6OS (380.47): C, 59.98; H, 5.30; N, 22.09; found: C, 59.87; H, 5.23; N, 22.14.
4.1.3.4.
2-(1-(4-((4-(4-Bromophenyl)thiazol-2-yl)amino)phenyl)ethylidene)hydrazine
carboximidamide (3d). Yellow solid (0.3 g, 79.2 %), m.p. 172-174 °C; IR (KBr,
3228, 3155 (NH/NH2), 1685, 1627, 1585 (C=N); 1H NMR (DMSO-d6, 400 MHz)
ν
cm-1): 3375,
δ
: 2.43 (s, 3H,
CH3), 7.83 (d, 2H, J = 8.0 Hz, ArH), 7.92 (d, 2H, J = 8.0 Hz, ArH), 7.95 (s, 2H, NH2, D2O
exchangeable), 8.10 (s, 1H, thiazolyl), 8.13 (d, 2H, J = 8.0 Hz, ArH), 8.26 (d, 2H, J = 8.0 Hz,
ArH), 11.26 (s, 1H, NH, D2O exchangeable), 11.29 (s, 1H, NH, D2O exchangeable), 11.35 (s,
1H, NH, D2O exchangeable); 13C NMR (DMSO-d6,100 MHz)
δ: 14.8 (CH3), 122.5, 123.0,
125.5, 127.7, 128.1, 128.4, 131.6, 138.2, 140.8, 144.6, 148.3, 150.6, 156.5 (ArCs + C=N); Anal.
Calcd. for C18H17BrN6S (429.34): C, 50.36; H, 3.99; N, 19.57; found: C, 50.42; H, 3.73; N,
19.79.
4.1.3.5. 2-(1-(4-((4-(p-Tolyl)thiazol-2-yl)amino)phenyl)ethylidene)hydrazine carboximidamide
(3e). Yellow solid (0.3 g, 68.7 %), m.p. 205-207 °C; IR (KBr,
ν
cm-1): 3385, 3240, 3159
: 2.33 (s, 3H, CH3), 2.35
(NH/NH2), 1674, 1612, 1566 (C=N); 1H NMR (DMSO-d6, 300 MHz)
δ
(s, 3H, CH3), 7.23 (d, 2H, J = 8.0 Hz, ArH), 7.30 (s, 1H, thiazolyl), 7.78 (s, 2H, NH2, D2O

exchangeable), 7.80-7.82 (dd, 4H, J = 2.7 Hz, 8.0 Hz, ArH), 7.97 (d, 2H, J = 8.0 Hz, ArH), 10.59
(s, 1H, NH, D2O exchangeable), 11.13 (s, 2H, 2NH, D2O exchangeable); 13C NMR (DMSO-
d6,75 MHz) δ: 14.2 (CH3), 20.8 (CH3), 102.7, 116.1, 120.6, 125.5, 127.7, 129.2, 131.8, 136.9,
142.5, 150.1, 151.4, 155.9, 162.4 (ArCs+ C=N); Anal. Calcd. for C19H20N6S (364.47): C, 62.61;
H, 5.53; N, 23.06; found: C, 62.96; H, 5.30; N, 23.15.
4.1.4. General procedure for synthesis of 2-(1-(4-((4-phenylthiazol-2-yl)amino)phenyl)
ethylidene)hydrazinecarboxamide derivatives (4a-e). To a solution of 2a-e (1 mmol) in absolute
ethanol (25 mL), 1 mL of concentrated HCl was added, followed by dry semicarbazide HCl
(0.22g, 2 mmol). This mixture was heated under reflux for 20-24 h, the progress of the reaction
was monitored by TLC. The reaction mixture was rinsed with sodium bicarbonate solution, the
solid formed is filtered giving the pure compounds 4a-e.
4.1.4.1. 2-(1-(4-((4-Phenylthiazol-2-yl)amino)phenyl)ethylidene)hydrazinecarboxamide (4a).
Yellow solid (0.1 g, 37 %), m.p. 216-218 °C; IR (KBr,
ν
cm-1): 3464, 3390, 3275 (NH/NH2),
: 2.17 (s, 3H, CH3), 4.29 (s,
1
1678 (C=O), 1604, 1566 (C=N); H NMR (DMSO-d6, 400 MHz)
δ
2H, NH2, by D2O exchangeable), 7.31-7.34 (m, 1H, ArH), 7.38 (s, 1H, thiazolyl), 7.42-7.46 (m,
2H, ArH), 7.76 (d, 2H, J = 8.0 Hz, ArH), 7.86 (d, 2H, J = 8.0 Hz, ArH), 7.94 (d, 2H, J = 8.0 Hz,
ArH), 9.24 (s, 1H, NH, D2O exchangeable), 10.51 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO-d6,100 MHz) δ: 13.5 (CH3), 104.8, 116.3, 126.1, 127.3, 128.2, 129.1, 130.1, 134.7,
141.9, 145.7, 150.6, 158.2 (ArCs+ C=N), 162.6 (C=O); Anal. Calcd. for C18H17N5OS (351.43):
C, 61.52; H, 4.88; N, 19.93; found: C, 61.76; H, 4.62; N, 19.62.
4.1.4.2.
2-(1-(4-((4-(4-Chlorophenyl)thiazol-2-yl)amino)phenyl)ethylidene)hydrazine
cm-1): 3464, 3406,
3282 (NH/NH2), 1654 (C=O), 1593, 1535 (C=N); 1H NMR (DMSO-d6, 400 MHz)
carboxamide (4b). Yellow solid (0.1 g, 25.9 %), m.p. 140-142 °C; IR (KBr,
ν
δ
: 2.18 (s, 3H,

CH3), 6.30 (s, 2H, NH2, D2O exchangeable), 7.44 (s, 1H, thiazolyl), 7.48 (d, 1H, J = 8.0 Hz,
ArH), 7.51 (d, 1H, J = 8.0 Hz, ArH), 7.76 (d, 2H, J = 8.0 Hz, ArH), 7.85 (d, 1H, J = 8.0 Hz,
ArH), 7.88 (d, 1H, J = 8.0 Hz, ArH), 7.94-7.98 (m, 2H, ArH), 9.97 (s, 1H, NH, D2O
exchangeable), 10.66 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6,100 MHz)
δ: 13.7
(CH3), 116.8, 127.8, 127.8, 129.1, 130.1, 131.3, 133.6, 141.9, 145.7, 149.2, 158.0, 158.2 (ArCs+
C=N), 162.8 (C=O); Anal. Calcd. for C18H16ClN5OS (385.87): C, 56.03; H, 4.18; N, 18.15;
found: C, 56.25; H, 4.03; N, 18.34.
4.1.4.3.
2-(1-(4-((4-(4-Methoxyphenyl)thiazol-2-yl)amino)phenyl)ethylidene)hydrazine
cm-1): 3433, 3309,
3255 (NH/NH2), 1685 (C=O), 1585,1531(C=N); 1H NMR (DMSO-d6, 400 MHz)
carboxamide (4c). Yellow solid (0.2 g, 70.8 %), m.p. 225-227 °C; IR (KBr,
ν
δ: 2.14 (s, 3H,
CH3), 3.87 (s, 3H, OCH3), 6.47 (s, 2H, NH2, D2O exchangeable), 6.48-6.51 (m, 2H, ArH), 7.07
(d, 1H, J = 8.0 Hz, ArH), 7.49 (d, 1H, J = 8.0 Hz, ArH), 7.76-7.80 (m, 3H, ArH), 8.10 (d, 1H,
ArH), 9.27 (s, 1H, thiazolyl), 9.28 (s, 1H, NH, D2O exchangeable), 9.99 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6,100 MHz)
δ: 13.5 (CH3), 56.2 (CH3O), 106.5, 116.4, 116.7,
122.3, 126.8, 127.1, 125.9, 134.0, 135.5, 143.7, 146.2, 158.2 (ArCs+ C=N) , 163.4 (C=O). Anal.
Calcd. for C19H19N5O2S (381.45): C, 59.82; H, 5.02; N, 18.36; found: C, 59.63; H, 5.36; N,
18.59.
4.1.4.4.
2-(1-(4-((4-(4-Bromophenyl)thiazol-2-yl)amino)phenyl)ethylidene)hydrazine
cm-1): 3479, 3425,
3248 (NH/NH2), 1681 (C=O), 1581, 1523 (C=N); 1H NMR (DMSO-d6, 400 MHz)
carboxamide (4d). Yellow solid (0.2 g, 37.2 %), m.p. 157-159 °C; IR (KBr,
ν
δ
: 2.16 (s, 3H,
CH3), 6.55 (s, 2H, NH2, D2O exchangeable), 7.24-7.32(m, 1H, ArH), 7.38 (s, 1H, thiazolyl),
7.51-7.54 (m, 4H, ArH), 7.79 (d, 2H, J = 8.0 Hz, ArH), 7.90 (d, 1H, J = 8.0 Hz, ArH), 8.86 (s,
1H, NH, D2O exchangeable), 9.42 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6,100

MHz) δ: 13.6 (CH3), 113.9, 114.3, 117.0, 122.3, 127.7, 128.5, 130.6, 131.5, 135.1, 137.8, 143.7,
157.7 (ArCs+ C=N), 158.1 (C=O); Anal. Calcd. for C18H16BrN5OS (430.32): C, 50.24; H, 3.75;
N, 16.27; found: C, 50.52; H, 3.95; N, 16.38.
4.1.4.5.
2-(1-(4-((4-(p-Tolyl)thiazol-2-yl)amino)phenyl)ethylidene)hydrazinecarboxamide
(4e).Yellow solid (0.2 g, 43.8 %), m.p. 208-210 °C; IR (KBr,
ν
cm-1): 3464, 3421, 3232
: 2.18 (s, 3H,
1
(NH/NH2), 1693 (C=O), 1597, 1566 (C=N); H NMR (DMSO-d6, 400 MHz)
δ
CH3), 2.34 (s, 3H, CH3), 4.40 (s, 2H, NH2, D2O exchangeable), 7.24 (d, 2H, J = 8.0 Hz, ArH),
7.29 (s, 1H, thiazolyl), 7.75 (d, 2H, J = 8.0 Hz, ArH), 7.82 (d, 2H, J = 8.0 Hz, ArH), 7.86 (d, 2H,
J = 8.0 Hz, ArH), 9.25 (s, 1H, NH, D2O exchangeable), 10.53 (s, 1H, NH, D2O exchangeable);
13C NMR (DMSO-d6,100 MHz)
δ: 13.5 (CH3), 21.2 (CH3), 103.9, 116.3, 116.7, 126.0, 127.2,
129.7, 130.0, 131.3, 137.4, 145.0, 150.5, 158.1 (ArCs+ C=N) , 162.5 (C=O); Anal. Calcd. for
C19H19N5OS (365.45): C, 62.44; H, 5.24; N, 19.16; found: C, 62.77; H, 5.03; N, 19.39.
4.1.5. General procedure for synthesis of 2-(1-(4-((4-phenylthiazol-2-yl)amino)phenyl)
ethylidene)hydrazinecarbothioamide (5a-e). To a solution of 2a-e (1 mmol) dissolved in absolute
ethanol (25 mL), 1 mL of concentrated HCl was added, followed by dry thiosemicarbazide HCl
(0.25g, 2 mmol). This mixture was heated under reflux for 20-24 h, the progress of the reaction
was monitored by TLC. The reaction mixture was rinsed with sodium bicarbonate solution, the
solid formed is filtered giving the pure compounds 5a-e.
4.1.5.1. 2-(1-(4-((4-Phenylthiazol-2-yl)amino) phenyl) ethylidene) hydrazine carbothioamide
(5a). Yellow solid (0.3 g, 49.6 %), m.p. 229-231 °C; IR (KBr,
(NH/NH2), 1589, 1562 (C=N), 1249, 1087 (C=S); 1H NMR (DMSO-d6, 400 MHz)
ν
cm-1): 3417, 3317, 3228
δ
: 2.51 (s, 3H,
CH3), 4.72 (s, 2H, NH2, D2O exchangeable), 7.43 (s, 1H, thiazolyl), 7.77-7.90 (m, 5H, ArH),
7.94 (d, 2H, J = 8.0 Hz, ArH), 7.98 (d, 2H, J = 8.0 Hz, ArH), 10.96 (s, 1H, NH, D2O

exchangeable), 11.11 (s, 1H, NH, D2O exchangeable); 13C NMR (DMSO-d6,100 MHz)
δ:
14.1(CH3), 103.9, 116.6, 126.2, 128.0, 128.1, 129.1, 130.6, 134.9, 142.5, 148.1, 150.6, 163.0
(ArCs+ C=N), 179.0 (C=S); Anal. Calcd. for C18H17N5S2 (367.49): C, 58.83; H, 4.66; N, 19.06;
found: C, 58.65; H, 4.93; N, 19.46.
4.1.5.2.
2-(1-(4-((4-(4-Chlorophenyl)thiazol-2-yl)amino)phenyl)ethylidene)hydrazine
cm-1): 3348,
3240, 3159 (NH/NH2), 1592, 1535 (C=N), 1249, 1091 (C=S); 1H NMR (DMSO-d6, 400 MHz)
carbothioamide (5b). Yellow solid (0.2 g, 44.9 %), m.p. 221-223 °C; IR (KBr,
ν
δ
:
2.30 (s, 3H, CH3), 4.50 (s, 2H, NH2, D2O exchangeable), 7.49-8.29 (m, 9H, 8 ArH and 1H,
thiazolyl), 10.76 (s, 1H, NH, D2O exchangeable), 11.09 (s, 1H, NH, D2O exchangeable); 13C
NMR (DMSO-d6,100 MHz) δ: 17.0 (CH3), 116.9, 117.3, 127.8, 128.7, 129.1, 130.1, 131.0,
133.6, 144.8, 150.4, 151.4, 163.0 (ArCs+ C=N) , 196.5 (C=S); Anal. Calcd. for C18H16ClN5S2
(401.94): C, 53.79; H, 4.01; N, 17.42; found: C, 53.97; H, 4.21; N, 17.15.
4.1.5.3.
2-(1-(4-((4-(4-Methoxyphenyl)thiazol-2-yl)amino)phenyl)ethylidene)hydrazine
carbothioamide (5c). Yellow solid (0.1 g, 32.7 %), m.p. 202-204 °C; IR (KBr,
ν
cm-1): 3429,
1
3360, 3143 (NH/NH2), 1589, 1492 (C=N), 1269, 1095 (C=S),; H NMR (DMSO-d6, 400 MHz)
δ
: 2.26 (s, 3H, CH3), 3.88 (s, 3H, OCH3), 7.08 (d, 1H, J = 8.0 Hz, ArH), 7.52 (d, 1H, J = 8.0 Hz,
ArH), 7.85-7.90 (m, 4H, ArH), 8.06 (s, 2H, NH2, D2O exchangeable), 8.15-8.23 (m, 2H, ArH),
8.24 (s, 1H, thiazolyl), 10.13 (s, 1H, NH, D2O exchangeable), 10.17 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6,100 MHz)
δ: 14.3 (CH3), 55.6 (CH3O), 111.4, 112.3, 114.4,
121.9, 127.3, 128.0, 131.2, 131.9, 133.3, 147.0, 156.5, 179.1(ArCs+ C=N) , 181.4 (C=S); Anal.
Calcd. for C19H19N5OS2 (397.52): C, 57.41; H, 4.82; N, 17.62; found: C, 57.60; H, 4.55; N,
17.46.