Phenylthiazoles with nitrogenous side chain: An approach to overcome molecular obesity

Article abstract of DOI:10.1016/j.ejmech.2019.111593

A novel series of phenylthiazoles bearing cyclic amines at the phenyl-4 position was prepared with the objective of decreasing lipophilicity and improving the overall physicochemical properties and pharmacokinetic profile of the compounds. Briefly, the pi

Full text of DOI:10.1016/j.ejmech.2019.111593

Journal Pre-proof
Phenylthiazoles with nitrogenous side chain: An approach to overcome molecular
obesity
Mohamed M. Elsebaei, Nader S. Abutaleb, Abdulrahman A. Mahgoub, Daoyi Li,
Mohamed Hagras, Haroon Mohammad, Mohamed N. Seleem, Abdelrahman S.
Mayhoub
PII:
S0223-5234(19)30727-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.111593
EJMECH 111593
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 May 2019
Revised Date: 11 July 2019
Accepted Date: 5 August 2019
Please cite this article as: M.M. Elsebaei, N.S. Abutaleb, A.A. Mahgoub, D. Li, M. Hagras, H.
Mohammad, M.N. Seleem, A.S. Mayhoub, Phenylthiazoles with nitrogenous side chain: An approach
to overcome molecular obesity, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.111593.
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2019 Published by Elsevier Masson SAS.

One extra Nitrogen for a molecule,
one giant leap for the properties

Phenylthiazoles with Nitrogenous Side Chain: An Approach to Overcome Molecular
Obesity
a
b‖
a
b
Mohamed M. Elsebaei , Nader S. Abutaleb , Abdulrahman A. Mahgoub , Daoyi Li , Mohamed
a
b
b,c**
a,d*
Hagras , Haroon Mohammad , Mohamed N. Seleem
and Abdelrahman S. Mayhoub
a.
Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar
University, Cairo 11884, Egypt.
b.
Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue
University, West Lafayette, IN 47907, USA.
c.
Purdue Institute of Inflammation, Immunology, and Infectious Disease, West
Lafayette, IN 47907, USA.
d.
University of Science and Technology, Nanoscience Program, Zewail City of
th
Science and Technology, October Gardens, 6 of October, Giza 12578, Egypt.
Corresponding Authors
*
* MNS; email, mseleem@purdue.edu
ASM; e-mail, amayhoub@azhar.edu.eg
*
ǁ
These two authors contributed equally

Abbreviations. CFU, colony forming units; CL, clearance rate; Cmax, maximum plasma
concentration; DME, dimethoxyethane; MIC, minimum inhibitory concentration; MBC,
minimum bactericidal concentration; MRSA, methicillin-resistant Staphylococcus aureus; PK,
pharmacokinetics; t , half-life; V , volume of distribution; Tmax, time required to reach the
1
/2
d
maximum plasma concentration.

Abstract. A novel series of phenylthiazoles bearing cyclic amines at the phenyl-4 position was
prepared with the objective of decreasing lipophilicity and improving the overall
physicochemical properties and pharmacokinetic profile of the compounds. Briefly, the
piperidine ring (compounds 10 and 12) provided the best ring size in terms of antibacterial
activity when tested against 16 multidrug-resistant clinical isolates. Both compounds were
superior to vancomycin in the ability to eliminate methicillin-resistant Staphylococcus aureus
(MRSA), residing within infected macrophages and to disrupt mature MRSA biofilm.
Additionally, compounds 10 and 12 exhibited a fast-bactericidal mode of action in vitro.
Furthermore, the new derivatives were 160-times more soluble in water than the previous lead
compound 1b. Consequently, compound 10 was orally bioavailable with a highly-acceptable
pharmacokinetic profile in vivo that exhibited a half-life of 4 hours and achieved a maximum
plasma concentration that exceeded the minimum inhibitory concentration (MIC) values against
all tested bacterial isolates.

1
. Introduction
Our group launched a project in 2014 that aimed to discover and develop a new chemical
scaffold to develop a novel series of compounds with antibacterial activity against multidrug-
resistant bacteria. In the past five years, more than 500 arylthiazole derivatives have been
synthesized and their antibacterial profiles were thoroughly investigated.[1-14] The compounds
exhibited potent antibacterial activity against pertinent Gram-positive bacterial pathogens of
clinical interest including methicillin-resistant S. aureus, drug-resistant Streptococcus
pneumoniae, and vancomycin-resistant enterococci (VRE) but were inactive against Gram-
negative bacteria. MRSA, S. pneumoniae, and VRE are responsible for an array of non-invasive
and systemic bacterial infections that are often resistant to treatment by more than one antibiotic.
Altogether, these three bacterial pathogens have been linked to more than one million antibiotic-
resistant infections and nearly 20,000 deaths annually in the United States of America alone[15].
Using transposon mutagenesis study followed by biochemical assays, it was proved that
undecaprenyl diphosphate phosphatase (UppP) is the main bacterial target for our phenylthiazole
antibacterial agents.[16]
Initial inspection of the structure-activity-relationships (SAR) of the arylthiazole
compounds led us to cyclize the terminal n-butyl moiety in order to improve the lead
compound’s antibacterial potency [9]. However, as the antibacterial activity of the compound’s
improved, the modifications incorporated resulted in deterioration of the physicochemical
properties and pharmacokinetic profile of the new analogs (Figure 1). In brief, cyclization of the
n-butyl side chain of the initial lead compound 1a lowered the MIC value against several MDR-
staphylococcal strains by a factor of 5 [9]. Simultaneously, the tremendous increase in
lipophilicity, as indicated by the clogP value, lowered the aqueous solubility limit from 65

µg/mL, for 1a, to around 3 µg/mL, for 1b. A direct consequence of this decrease in solubility
was difficulty in developing a suitable formulation for 1b to administer either orally or
parenterally.
Conformationally-
restricted side
chain
Conformationally-
restricted MORE
polar side chain
P r e v i o u s W o r k
Figure 1. Overview of the aim of the present work.
GeneralDesignofThisWork
Advances in formulation technology (such as hot melt extrusion [17, 18] and spray-
drying [19]) can aid in solubilizing obese molecules to permit their absorption into systemic
circulation. However, these advanced methods hinder excretion of obese molecules from the
human body as metabolic enzymes must work aggressively to increase the polarity of these
obese xenobiotics, in order to excrete them [20]. In addition to reduced aqueous solubility,
compounds exhibiting a logP value above 3.5 are usually associated with increased off-target
side effects [21]. Therefore, we hypothesized that decreasing the lipophilic capacity of the
phenylthiazole compounds would reduce off-target effects and eventually improve the
compounds’ aqueous solubility and overall pharmacokinetic profile. To address this point, the
cyclohexyl group in the lead compound 1b was replaced with a cyclic amine, as depicted in
Figure 1, and the analogs synthesized were evaluated for their antibacterial activity,
improvement in aqueous solubility, and in vivo pharmacokinetic profile.

Scheme 1
NH2
NH
O
S
HN
N
O
S
S
NH2
N
N
S
N
a
b
c
Cl
2
N
N
Cl
3
4a-p
5-20
N
N
N
N
N
N
N
4a, 5
4b, 6
4c, 7
4d, 8
4e, 9
4f, 10
4g, 11
4h, 12
4i, 13
4j, 14
4k, 15
Br
N
N
4l, 16
O
HO
N
N
N
N
4m, 17
4n, 18
4o, 19
S
N
N
N
S
N
N
N
N
N
4p, 20
Reagents and conditions: (a) Absolute EtOH, 3-chloro-2,4-pentandion, heat to
reflux, 6 h, (b) Pd(OAc) (5% mol), X-phos (10% mol), KOtBu (2.5 equiv.), appropriate
2
sec-amine, DME, heat at 200°C for 3 h; (c) aminoguanidine HCl, EtOH, conc. HCl,
heat to reflux, 3 h.
2
. Results and Discussion
2
.1. Chemistry. Treatment of para-chlorobenzothioamide (2) with α-chloroacetylacetone
yielded the key starting compound 3, as reported previously.[22] The corresponding sec-amine
derivatives 4a-p were obtained using the Buchwald synthetic protocol (scheme 1). In the first
thrust, we used Cu-catalyzed C-N cross coupling protocol; i.e., copper(I) iodide and ʟ-proline,

but the isolated yield was terribly low (< 5%). So that, we switched to the Pd-catalyzed protocol,
in which Pd(II) and X-Phos ligand in a polar aprotic solvent provided the best yields of
compounds 4a-p (Scheme 1). Condensation of 4a-p with aminoguanidine hydrochloride yielded
the final products 5-20 (Scheme 1).
2
2
.2. Biological Results and Discussion.
.2.1. Antibacterial activity of new analogs against MRSA. The newly synthesized analogs
were initially screened against the highly-pathogenic strain, MRSA USA300 [23-25]. The 6-
membered piperidine side chain (compound 10) represented the best ring size as it afforded the
most potent analog from this series (MIC value of 4 µg/mL) (Table 1), which means that the
target protein cannot accommodate, at this particular position, for more than 6-membered ring.
The SAR appears straightforward as it pertains to the ring size. Briefly, shrinking the nitrogenous
side chain gradually impaired the compounds’ anti-MRSA activity as the MIC value increased to
1
6 µg/mL 4-times for the pyrrolidine-containing derivative 7 and was completely nullified for
the azetidine-containing analog 5. Similarly, expanding the nitrogenous ring size provided the
hexamethyleneimine- and heptamethyleneimine-containing derivatives 18 and 19 with MIC
values ≥ 128 µg/mL (Table 1). This observation confirms our previous hypothesis that the active
site of the targeted receptor cannot accommodate rings larger than 6-membered ones.
Further attempts to increase the polarity of the side chain by using the more polar
nitrogenous rings morpholine and piperazine resulted in less active derivatives (compounds 14
and 16). Substitution with methyl groups yielded the methylpiperidines 11-13 with high
variability in MIC values in which the 2-methyl and 4-methylpiperidine-containing derivatives
1
1 and 13 were 2 to 4-times less active than the unsubstituted piperidine 10 (Table 1). On the
other hand, the 3-methyl substitution (compound 12) maintained the same anti-MRSA inhibitory

potency as compound 10. The MIC for the control antibiotics linezolid and vancomycin against
MRSA USA300 was 1 µg/mL.
Table 1. Initial screening of the newly synthesized arylthiazole analogs against methicillin-
resistant Staphylococcus aureus NRS384 (MRSA USA300).
Compounds/
Control
Antibiotics
Compounds/
Control
Antibiotics
MIC
µg/mL)
MIC
(µg/mL)
Side chain
Side chain
(
5
6
7
8
9
>128
64
14
15
16
17
18
64
8
16
>128
32
>128
16
>128
1
1
0
1
4
8
19
20
128
>128
1
1
2
3
4
Linezolid
_
_
1
1
16
Vancomycin
2
.2.2. Preliminary toxicological and pharmacokinetic evaluation. The most promising
compounds, 10 and 12, were tested to evaluate our hypothesis that the substitution of the
cyclohexyl moiety in 1b with cyclic amines would positively impact the physicochemical
properties and PK behavior of the compounds. Before advancing to an in vivo PK study,
compounds 10 and 12 were assessed for toxicity to mammalian cells, and both compounds
exhibited a good safety profile against human colorectal cells
(Caco-2) and human keratinocytes
(HaCaT) (Figure 2, Panel A). Compound 12 was non-toxic to Caco-2 cells up to 32 µg/mL. This
concentration is 8-times higher than the compound’s corresponding MIC value against MRSA
USA300. Compound 10 was non-toxic to Caco-2 cells up to 64 µg/mL, which is 16-times higher

than the compound’s MIC against MRSA USA300. Similar results were observed when the
compounds were tested against human keratinocytes (HaCaT) (Figure 2, Panel B).
Figure 2. Toxicity analysis of compounds 10 and 12 (tested in triplicate at 32, 64 and 128
µg/mL) against A) human colorectal (Caco-2, panel A) and B) human keratinocytes
(HaCaT,
panel
B)
using
the
MTS
3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) 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 sample standard deviation values. The
data were analyzed via two-way ANOVA with post-hoc Dunnett’s test for multiple comparisons
(P<0.05).
Physicochemical properties of candidate antibacterial agents are critical to evaluate early
in drug development [26, 27]. Promising compounds with potent in vitro activity against a target
pathogen may fail in clinical trials due to poor aqueous solubility and permeability. Thus, after
confirming compounds 10 and 12 possessed good antibacterial activity and to safety to
mammalian cells, we investigated the compounds’ aqueous solubility profile. Both compounds
exhibited a more than 160-fold increase in aqueous solubility relative to the first-generation lead
compound 1b (Table 2). The notable improvement observed supports the first part of our
hypothesis that the new chemical modification, presented here, on the phenylthiazole scaffold
would positively impact the compounds’ physicochemical properties. Next, we tested the second
part of our hypothesis, investigating the impact of incorporating the piperidine ring as part of the
lipophilic side chain, on the compound’s PK profile. In this vein, a PK study in rats was

performed to assess the distribution of compound 10 in the bloodstream after oral administration.
Compound 10 was selected for in vivo PK evaluation due to its superior aqueous solubility and
safety profile. The PK curve (Figure 3) of compound 10 demonstrated it reached a maximum
plasma concentration (Cmax) of 10 µg/mL (2.5 × MIC vs. MRSA USA300) ~90 minutes after
administration with a biological half-life of 4 hours. Moreover, the plasma concentration of
compound 10 remained above the MIC value for MRSA USA300 for nearly 7 hours. This
represents a marked improvement relative to the first-generation phenylthiazoles which were
metabolically unstable resulting in short half-lives and rapid hepatic clearance.[9] The PK study
suggests that the piperidine-containing derivative 10 is suitable for oral administration.
Table 2. Evaluation of aqueous solubility limit of compounds 1a, 1b, 10 and 12 in phosphate-
buffered saline (PBS).
1
Compounds Solubility limit (µM)
1
a
65
2.7
480
435
1b
1
0
2
1
1
Solubility limit corresponds to the highest concentration of test compound where no precipitate
was detected (OD540).
1
1
2
0
8
6
4
2
0
T1/2 (h)
Vd L
4.00
4.42
0.76
9.70
1.30
CL L/hr
Cmax ug/mL
Tmax h
0
2
4
6
8
10
12
14
TIME (H)
Figure 3. The pharmacokinetic profile of compound 10 after oral administration of a single
dose of 50 mg/Kg in rat.

2
.2.3. Comprehensive antibacterial profiling of compounds 10 and 12. After confirming our
hypothesis that piperidine-containing phenylthiazoles (represented by compounds 10 and 12)
exhibit a markedly improved physicochemical profile compared to previously-synthesized
analogs, we assessed the antibacterial profile of compounds 10 and 12 against a panel of
multidrug-resistant staphylococcal isolates. Both compounds inhibited growth of the tested
strains at concentrations ranging from 4 to 8 µg/mL and exhibited bactericidal activity (as the
MBC values were equal to or one-fold higher than the MIC values) (Table 1S). Furthermore, the
two compounds maintained the same potent bactericidal activity against other clinically-relevant
Gram-positive bacterial species including vancomycin-resistant enterococci (VRE), multidrug-
resistant Streptococcus pneumoniae and Listeria monocytogenes (Table 2S).
In order to confirm this bactericidal mode of action against MRSA, a time-kill assay was
utilized. The new series of phenylthiazoles was superior to vancomycin in terms of the time
required to exert bactericidal activity in vitro. Vancomycin reduced the burden of MRSA by 3-
log within 12 hours and required 24 hours to reduce the burden of MRSA below the limit of
1
0
detection (Figure 4). In contrast, the piperidine-containing phenylthiazoles 10 and 12 required
only 4 hours to reduce the burden of MRSA below the limit of detection (Figure 4). It has been
postulated that the slow bactericidal effect of antibiotics such as vancomycin can result in
difficulty in clearing a bacterial infection [28, 29]. Thus, agents such as compounds 10 and 12
that can rapidly kill MRSA are highly desirable

Figure 4. Killing kinetics of compounds 10 and 12 and vancomycin (tested in triplicate at 5
MIC) against methicillin-resistant Staphylococcus aureus (MRSA USA400) over a 24-
×
o
hour incubation period at 37 C. DMSO (solvent for the compounds) served as a negative
control. The error bars represent standard deviation values obtained from triplicate samples used
for each compound/antibiotic studied.
The rapid, potent bactericidal activity of compounds 10 and 12 propelled us to investigate
if these compounds could gain entry inside macrophages infected with S. aureus to reduce the
burden of bacteria. The success of S. aureus as a highly-pathogenic microorganism is not only
attributed to extensive release of different virulence factors but to its ability to evade host innate
immune responses as well. Virulence factors released by S. aureus are capable of interfering with
leukocyte recruitment and inhibiting production of complement factors and antimicrobial
peptides [30-32]. Furthermore, S. aureus produces toxins, like leukocidin A/B, that are able to
specifically target and kill phagocytes [33, 34]. Additionally, it has been reported that
intracellular MRSA can replicate within the phagolysosome after phagocytosis by macrophages
thus permitting the organism to survive and re-infect the host [35]. Most antibiotics are unable to
target intracellular bacteria due to: 1) low levels of intracellular accumulation as in the case of
linezolid, β-lactams, and gentamicin; 2) inactivation/loss of activity due to the acidic pH within
macrophages, as with aminoglycosides; and 3) binding to lysosomal contents, as in the case of
oritavancin [36, 37]. Like most antibiotics, vancomycin, the drug of last resort for treatment of

MRSA infections fails to gain entry inside infected macrophages and must be used at high
concentrations to achieve significant activity against intracellular S. aureus [38, 39]. The
previously reported intracellular clearance activity of some compounds in our series of
phenylthiazole antibiotics[1, 3] encouraged us to investigate the ability of the newly-synthesized
compounds to reduce the burden of intracellular MRSA present within macrophages. Compound
1
0 was selected for this experiment based on its superior safety profile to the murine
macrophages used for the intracellular infection experiment (Figure 5A). As depicted in Figure
, after 24 hours incubation, phenylthiazole 10 (at 2 × MIC) generated a 1.18-log10 reduction
equivalent to 93.2% reduction) of intracellular MRSA. This was superior to vancomycin which
5
(
was unable to reduce the burden of intracellular MRSA. This result indicates that in addition to
compound 10 exhibiting more rapid bactericidal activity against MRSA in vitro compared to
vancomycin, the compound possesses an additional advantage over vancomycin in the ability to
reduce the burden of intracellular MRSA within infected macrophages.
Figure 5. Effect of compound 10 and vancomycin to reduce intracellular MRSA present
inside murine macrophages (J774). Data are presented as log10 colony forming units of MRSA
USA400 per mL inside infected murine macrophages after treatment with 2×MIC of either
compound 10 or vancomycin (tested in quadruplicates) for 24-hours. Data were analyzed via
one-way ANOVA, with post hoc Dunnet’s test for multiple comparisons (P < 0.05), utilizing
GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA). The asterisk (*) represents a significant
difference between the treatment of J774 cells with compound 10 in comparison to vancomycin.

2
.2.4. Evaluation of the anti-biofilm activity of compounds 10 and 12. Targeting factors that
contribute to bacterial virulence and colonization of host tissues represents an alternative
approach currently being pursued to counter the emergence of bacterial resistance to antibiotics
[40]. One example of this is the identification and development of molecules capable of
inhibiting or eradicating bacterial biofilms [41]. Biofilms can form on the surfaces of indwelling
catheters and implanted medical devices [42, 43]. These biofilms can lead to fatal bloodstream
infections which are associated with substantial treatment costs [44]. Many invasive infections
caused by S. aureus develop from bacterial biofilms formed on the surface of implanted medical
devices [42, 45]. This problem could be further exacerbated as bacteria embedded within
biofilms exhibit increased resistance to antibiotics [3, 46]. Surgical intervention through physical
replacement of the infected medical implant is the primary option currently available to
cliniciansbut is assoictaed with multiple risks and complications [47]. Identifying molecules
capable of disrupting adherent biofilm from these devices would provide a suitable alternative to
surgical intervention. Previous reports have demosntrated that phenylthiazoles are able to disrupt
pre-formed bacterial biofilms [3, 5, 8, 48]. Thus compounds 10 and 12 were examined for their
ability to disrupt pre-formed, well-established staphylococcal biofilm using the microtiter plate
biofilm formation assay[8]. The piperidine-containing derivatives 10 and 12 were both superior
to vancomycin in their ability to disrupt the highly-resistant MRSA USA300 biofilm (Figure 6).
Compound 10 exhibited the highest biofilm eradication activity as it eradicated about 72% of
MRSA300 biofilm mass at 4 × MIC (16 µg/mL), while its 3-methyl analog 12 disrupted about
5
0% of MRSA USA300 biofilm mass, at the same concentration. On the other hand, vancomycin

at same concentration was unable to effectively penetrate and disrupt the mature biofilm because
of its large molecular structure and polar nature, in agreement with previous reports [8, 49].
Figure 6. Disruption of mature MRSA biofilm by compounds 10, 12 and vancomycin at 4 ×
MIC. The data are presented as percent disruption of MRSA USA300 mature biofilm in relation
to DMSO (the solvent for the compounds that served as a negative control). Vancomycin served
as the control antibiotic. The values represent an average of four samples analyzed for each
compound/antibiotic. Error bars represent standard deviation values. An asterisk (*) denotes
statistical significance (P < 0.05) between results for test compounds relative to vancomycin
analyzed via an unpaired Student’s t-test.
3
. Conclusion. Previously we have reported that cyclization of the n-butyl side chain of lead
compound 1a provided the more potent derivative 1b. Though potency improved, compound 1b
was highly lipophilic and could not be tested in an in vivo model. The present study focused on
increasing the drugability of the phenylthiazoles by controlling their physicochemical properties
so that they can be formulated and tested in an animal model. This aim was achieved by using
cyclic amines as the terminal side chain. Out of all tested derivatives, the piperidine and its 3-
methyl analog (compounds 10 and 12) provided the most promising candidates when tested
against sixteen MDR clinical isolates. The new piperazine-containing phenylthiazoles 10 and 12
outperformed vancomycin in different aspects: being effective in disrupting MRSA biofilm
mass, displaying efficient intracellular clearance activity against MRSA residing inside infected

macrophages, and exhibiting a fast-bactericidal activity against MRSA in vitro. More
importantly, this series of antibacterial agents, represented by compound 10, showed a
significantly improved pharmacokinetic profile relative to the first-generation compounds. In this
regard, compound 10 was orally absorbed and achieved a maximum plasma concentration more
than two times higher than the compound’s MIC values against MRSA. Additionally, unlike the
metabolically unstable first-generation phenylthiazoles, compound 10 exhibited a high degree of
metabolic stability with a half-life of 4 hours. The promising results obtained for compound 10
warrant further investigation in suitable animal models of MRSA infection, as part of a future
study.
4
4
4
. Experimental
.1. Chemistry
1
13
.1.1. General. H NMR spectra were run at 400 MHz and C spectra were determined at 100
MHz in deuterated dimethyl sulfoxide (DMSO-d ) on a Varian Mercury VX-400 NMR
6
spectrometer. Chemical shifts are given in parts per million (ppm) on the delta (δ) scale.
Chemical shifts were calibrated relative to those of the solvents. Flash chromatography was
performed on 230-400 mesh silica. The progress of reactions was monitored with Merck silica
gel IB2-F plates (0.25 mm thickness). Mass spectra were recorded at 70 eV. High resolution
mass spectra for all ionization techniques were obtained from a FinniganMAT XL95. Melting
points were determined using capillary tubes with a Stuart SMP30 apparatus and are uncorrected.
All yields reported refer to isolated yields. Compound (3) was prepared as reported.[22]
4
.1.2. 1-(2-(4-(Sec-amine derivatives-1-yl)phenyl-4-methylthiazol-5-yl)ethan-1-one (4a-p).
General procedure: to dry DME (15 mL) and a few drops of distilled water in a 75-mL sealed
tube compound 3 (300 mg, 1 mmol), palladium acetate (13 mg, 5 mol%), 2-

dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl (X-phos) (56 mg, 10 mol%) and potassium
tert-butoxide (336 mg, 2.5 equiv.). After the reaction mixture was purged with dry nitrogen gas
o
for 15 min at 100 C, appropriate Sec. amines (3 equiv.) were added. The sealed tube was then
o
heated and stirred at 200 C for 3 h and monitored by thin-layer chromatography (TLC). After
completion of the reaction, the reaction mixture was poured on water then extracted with DCM
(
3 × 15 mL) then dried over MgSO , the organic materials were then concentrated under reduced
4
pressure. The crude materials were purified via silica gel flash column chromatography using
hexane-ethyl acetate (8:2) as yellowish viscous oil.
4
.1.2.1. 1-(2-(4-(Azetidin-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one (4a). Orange oil (110
1
mg, 33.8%); H NMR (DMSO-d )
δ
: 7.98 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4 Hz, 2H), 2.98 (t, J
6
1
3
=
8 Hz, 4H), 2.69 (s, 3H), 2.43 (s, 3H), 1.98-1.97 (m, 2H); C NMR (DMSO-d ) δ: 191.05,
6
1
2
4
67.38, 161.69, 148.35, 143.23, 133.04, 132.98, 121.93, 52.53, 24.61, 18.08, 16.88; MS (m/z);
+
72 (M , 78.46%).
.1.2.2. 1-(2-(4-(3-Hydroxyazitidin-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one (4b).
1
Yellowish oil (95 mg, 27.6%); H NMR (DMSO-d
6
)
δ: 7.89 (d, J = 8.4 Hz, 2H), 7.68 (d, J = 8.4
Hz, 2H), 4.56 (brs, 1H), 4.27-4.24 (m, 1H), 4.15 (dd, J = 12.1, 3.4, Hz, 2H), 3.62 (d, J = 12.2, 6.2
1
3
Hz, 2H), 2.59 (s, 3H), 2.47 (s, 3H); C NMR (DMSO-d )
δ
: 192.95, 168.24, 163.66, 155.53,
6
+
1
49.84, 132.27, 127.19, 119.51, 64.44, 59.23, 31.36, 18.54; MS (m/z); 288 (M , 44.61%).
4
.1.2.3. 1-(4-Methyl-2-(4-pyrrolidin-1-yl)phenyl)thiazol-5-yl)ethan-1-one (4c). Dark brown
1
oil (120 mg, 35%); H NMR (DMSO-d )
δ
: 7.88 (d, J = 8.8 Hz, 2H), 7.49 (d, J = 8.8 Hz, 2H),
6
1
3
3
.52 (t, J = 6.8 Hz, 4H), 2.59 (s, 3H), 2.33 (s, 3H), 1.7 (t, J = 6.8 Hz, 4H); C NMR (DMSO-d )
6
δ
: 191.45, 165.24, 160.14, 148.33, 143.93,133.14, 132.92, 121.91, 53.55, 29.47, 18.55, 16.61;
+
MS (m/z); 286 (M , 54.20%).

4
.1.2.4. 1-(4-Methyl-2-(4-(thiazolidin-3-yl)phenyl)thiazol-5-yl)ethan-1-one (4d). Yellow oil
1
(
(
(
96 mg, 26.4%); H NMR (DMSO-d )
δ
: 7.97 (d, J = 8.1 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H), 4.51
6
1
3
s, 2H), 3.74 (t, J = 6.4 Hz, 2H), 3.19 (t, J = 6.4 Hz, 2H), 2.62 (s, 3H), 2.48 (s, 3H); C NMR
DMSO-d6)
δ
: 191.66, 163.46, 158.83, 136.63, 132.44, 131.97, 126.14, 119.31, 60.26, 52.44,
+
2
9.86, 18.55, 16.76; MS (m/z); 304 (M , 45.03%).
4
.1.2.5. 1-(2-(4-(1
H-Imidazol-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one (4e). Brown oil
1
(
89 mg, 26.3%); H NMR (DMSO-d
6
)
δ: 7.91 (d, J = 8.4 Hz, 2H), 7.68 (s, 1H), 7.57 (d, J = 8.4
1
3
Hz, 2H), 7.33 (d, J = 6.8 Hz, 1H), 6.98 (d, J = 6.8 Hz, 1H), 2.59 (s, 3H), 2.32 (s, 3H); C NMR
(
DMSO-d
6
)
δ: 192.15, 164.34, 162.73, 136.93, 133.07, 132.94, 132.12, 131.91, 128.13, 124.63,
+
1
22.33, 29.86, 17.65; MS (m/z); 283 (M , 100%).
4
.1.2.6. 1-(4-Methyl-2-(4-(piperidin-1-yl)phenyl)thiazol-5-yl)ethan-1-one (4f). Yellow oil
1
(
120 mg, 33.5%); H NMR (DMSO-d )
δ
: 8.02 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H),
6
1
3
2
.71 (s, 3H), 2.57 (s, 3H), 2.52 (t, J = 6.4 Hz, 4H), 1.57-1.51 (m, 4H), 1.39-1.36 (m, 2H); C
NMR (DMSO-d6)
δ
: 191.35, 167.92, 158.56, 133.83, 132.54, 132.26, 127.25, 125.61, 53.10,
+
3
0.64, 25.94, 24.04, 18.45; MS (m/z); 300 (M , 40.55%).
4
.1.2.7. 1-(4-Methyl-2-(4-(2-methylpiperidin-1-yl)phenyl)thiazol-5-yl)ethan-1-one (4g).
1
Brown oil (95 mg, 25.3%); H NMR (DMSO-d )
δ
: 7.98 (d, J = 8 Hz, 2H), 7.52 (d, J = 7.2 Hz,
6
2
H), 3.83-3.81 (m, 1H), 3.16-2.86 (m, 2H), 2.54 (s, 3H), 2.38 (s, 3H), 1.60-1.22 (m, 6H), 1.05 (d,
1
3
J = 6.8 Hz, 3H); C NMR (DMSO-d6)
δ
: 191.15, 168.12, 163.17, 158.83, 132.39, 132.54,
+
1
1
4
27.34, 126.08, 54.66, 53.39, 34.62, 30.69, 26.34, 24.76, 20.41, 18.31; MS (m/z); 314 (M ,
5.74%).
.1.2.8. 1-(4-Methyl-2-(4-(3-methylpiperidin-1-yl)phenyl)thiazol-5-yl)ethan-1-one (4h).
1
Yellow oil (120 mg, 32%); H NMR (DMSO-d
6
)
δ
: 8.02 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4 Hz,

2
H), 2.80-2.61 (m, 2H), 2.71 (s, 3H), 2.58 (s, 3H), 2.14-1.46 (m, 7H), 0.87 (d, J = 6.4 Hz, 3H);
1
3
C NMR (DMSO-d6)
δ: 191.34, 168.02, 158.89, 133.53, 133.22, 132.24, 127.11, 125.92, 60.35,
+
5
2.40, 48.01, 32.62, 30.99, 25.64, 18.94, 16.77; MS (m/z); 314 (M , 23.14%).
4
.1.2.9. 1-(4-Methyl-2-(4-(4-methylpiperidin-1-yl)phenyl)thiazol-5-yl) ethan-1-one (4i).
1
Light-yellow oil (150 mg, 40%); H NMR (DMSO-d )
δ: 7.88 (d, J = 8.4 Hz, 2H), 7.51 (d, J =
6
8
.4 Hz, 2H), 2.84 (m, 4H), 2.59 (s, 3H), 2.31 (s, 3H), 2.20-2.12 (m, 4H), 1.61-1.11 (m, 1H), 0.90
1
3
(
d, J = 6.4 Hz, 3H); C NMR (DMSO-d
6
)
δ
: 191.33, 168.84, 158.13, 147.33, 143.30, 132.44,
+
1
32.28, 122.31, 52.75, 34.71, 30.29, 26.24, 24.31, 16.84; MS (m/z); 314 (M , 21.14%).
4
.1.2.10. 1-(4-Methyl-2-(4-(4-methylpiperazin-1-yl)phenyl)thiazol-5-yl)ethan-1-one (4j).
1
Orange oil (160 mg, 42.5%); H NMR (DMSO-d )
δ: 8.01 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.4
6
1
3
Hz, 2H), 3.62-3.54 (m, 4H), 2.72 (s, 3H), 2.59 (s, 3H), 2.52-2.49 (m, 4H), 2.16 (s, 3H); C NMR
(DMSO-d6)
δ
: 191.18, 167.73, 158.63, 147.67, 143.62, 133.14, 132.43, 127.41, 55.15, 52.15,
+
4
6.01, 31.04, 18.74; MS (m/z); 315 (M , 24.14%).
4
.1.2.11. 1-(2-(4-(4-Bromopiperidin-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one (4k).
1
Yellow oil (200 mg, 44%); H NMR (DMSO-d
6
)
δ
: 7.49 (d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.4 Hz,
2
H), 4.43-4.12 (m, 1H), 3.62 (t, J = 6.4 Hz, 4H), 2.49 (s, 3H), 2.27 (s, 3H), 2.25-1.89 (m, 4H);
1
3
C NMR (DMSO-d6) δ: 191.41, 161.54, 147.41, 144.43, 136.11, 132.04, 126.56, 124.12, 53.35,
+
+2
3
4.41, 29.34, 18.34, 16.31; MS (m/z); 378 (M , 18.69%), 380 (M , 19.36%).
4
.1.2.12. 1-(4-Methyl-2-(4-morpholinophenyl)thiazol-5-yl)ethan-1-one (4l). Brown oil (150
1
mg, 41.6%); H NMR (DMSO-d )
δ
: 7.79 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H), 3.62 (t, J
6
13
=
6.4 Hz, 4H), 3.28 (t, J = 6.4 Hz, 4H), 2.64 (s, 3H), 2.36 (s, 3H); C NMR (DMSO-d6) δ:
1
91.53, 162.34, 154.91, 147.43, 132.88, 132.14, 126.16, 124.72, 64.15, 52.41, 28.15, 16.78; MS
+
(
m/z); 302 (M , 40.69%).

4
.1.2.13. 1-(4-Methyl-2-(4-thiomorpholinophenyl)thiazol-5-yl)ethan-1-one (4m). Yellow oil
1
(
110 mg, 28.9%); H NMR (DMSO-d )
δ
: 8.22 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 3.92
6
1
3
(
t, J = 6.4 Hz, 4H), 3.75 (t, J = 6.4 Hz, 4H), 2.69 (s, 3H), 2.36 (s, 3H); C NMR (DMSO-d ) δ:
6
1
91.73, 166.34, 160.91, 157.73, 143.88, 133.14, 132.96, 122.62, 52.85, 28.85, 26.64, 16.78; MS
+
(
m/z); 318 (M , 42.69%).
4
.1.2.14. 1-(2-(4-(Azepan-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one (4n). Dark-brown oil
1
(
100 mg, 26.6%); H NMR (DMSO-d
6
)
δ
: 8.26 (d, J = 8.4 Hz, 2H), 7.46 (d, J = 8.4 Hz, 2H),
1
3
4
1
1
4
6
.43-4.38 (m, 4H), 2.52 (s, 3H), 2.46 (s, 3H), 1.55-0.96 (m, 8H); C NMR (DMSO-d ) δ:
90.95, 166.62, 160.86, 147.83, 143.74, 132.86, 132.25, 122.61, 53.19, 28.36, 26.64, 24.04,
+
8.35; MS (m/z); 314 (M , 27.60%).
.1.2.15. 1-(2-(4-(Azocan-1-yl)phenyl)-4-methylthiazol-5-yl)ethan-1-one (4o). Brown oil (110
1
mg, 28%); H NMR (DMSO-d )
δ: 7.98 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.4 Hz, 2H), 4.45 (t, J =
6
1
3
5
1
1
4
.4 Hz, 4H), 2.62 (s, 3H), 2.46 (s, 3H), 1.89-0.86 (m, 10H); C NMR (DMSO-d ) δ: 191.35,
6
68.62, 163.86, 142.83, 133.74, 133.06, 126.65, 121.04, 53.26, 30.64, 30.02, 26.16, 24.84,
+
8.35; MS (m/z); 328 (M , 4.60%).
.1.2.16. 1-(4-Methyl-2-(4-(octahydroisoquinolin-2(1
H
)-yl)phenyl)thiazol-5-yl)ethan-1-one
1
(
4p). Dark-brown oil (133 mg, 31.4%); H NMR (DMSO-d )
δ
: 7.88 (d, J = 8.4 Hz, 2H), 7.51 (d,
6
J = 8.4 Hz, 2H), 2.57-2.51 (m, 2H), 2.49-2.42 (m, 2H), 2.57 (s, 3H), 2.31 (s, 3H), 2.20-0.89 (m,
1
3
1
5
4
2H); C NMR (DMSO-d )
δ
: 191.15, 160.42, 147.46, 133.33, 132.84, 128.76, 126.72, 124.62,
6
+
9.44, 53.58, 43.01, 42.68, 33.22, 30.34, 26.64, 18.41, 17.65; MS (m/z); 354 (M , 18.20%).
.1.3.
2-(1-(2-(4-(Substituted
sec-amine-1-yl)phenyl)-4-methylthiazol-5-
yl)ethylidene)hydrazine-1-carboximidamide 5-20. General procedure: Acetylphenylthiazole
derivatives 4a-p (0.31 mmol) were dissolved in absolute ethanol (15 mL), concentrated

hydrochloric acid (1 mL), aminoguanidine hydrochloride (175 mg, 1.5 mmol, 5 equiv.), were
added. The reaction mixture was heated at reflux for 2 h. The solvent was concentrated under
reduced pressure, then poured in crushed ice and neutralized with sodium carbonate to pH 7-8,
and the formed precipitated was collected by filtration, washed with copious amount of water.
Crystallization from absolute ethanol afforded the desired products as solids.
4
.1.3.1.
2-(1-(2-(4-(Azetidin-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
1
carbo- ximidamide (5). Yellow solid (90 mg, 75%); mp = 170-171 °C. H NMR (DMSO -d
6
)
δ
:
1
1.49 (brs, 1H), 7.77 (d, J = 8.4 Hz, 2H), 7.21 (d, J = 8.4 Hz, 2H), 4.11 (brs, 3H), 3.52 (t, J = 5.2
1
3
Hz, 4H), 2.62 (s, 3H), 2.49 (s, 3H), 2.13-2.02 (m, 2H); C NMR (DMSO-d
6
) δ: 169.08, 165.42,
1
58.83, 147.51, 133.06, 132.61, 132.18, 126.94, 125.97, 49.76, 31.11, 18.44, 16.37; HRMS (EI)
+
+
m/z 328.1478 M , calc. for C H N S 328.1470 M ; Anal. Calc. for: C H N S (328): C, 58.51;
16
20
6
16 20
6
H, 6.14; N, 25.59%; Found: C, 58.53; H, 6.09; N, 25.60%.
4
.1.3.2.
2-(1-(2-(4-(3-Hydroxyazetidin-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)
1
hydrazine-1-carboximidamide (6). Orange solid (105 mg, 88%); mp = 191-192 °C. H NMR
(
DMSO -d
6
)
δ
: 7.65 (d, J = 8.4 Hz, 2H), 7.05 (brs, 1H), 6.68 (d, J = 8.4 Hz, 2H), 6.21 (brs, 3H),
5
2
1
.48 (brs, 1H), 3.81-3.78 (m, 1H), 3.75 (dd, J = 11.3, 3.4 Hz, 2H), 3.70 (dd, J = 11.4, 6.2 Hz,
1
3
H), 2.51 (s, 3H), 2.36 (s, 3H); C NMR (DMSO-d )
δ: 168.77, 162.42, 158.83, 155.42, 148.89,
6
+
33.17, 132.78, 127.13, 126.77, 63.35, 58.43, 31.11, 18.49; HRMS (EI) m/z 344.1399 M , calc.
+
for C H N OS 344.1419 M ; Anal. Calc. for: C H N OS (344): C, 55.79; H, 5.85; N,
1
6
20
6
16 20
6
2
4.40%; Found: C, 55.81; H, 5.91; N, 24.48%.
4
.1.3.3.
2-(1-(4-Methyl-2-(4-(pyrrolidin-1-yl)phenyl)thiazol-5-yl)ethylidene)hydrazine-1-
1
carboximidamide (7). Brown solid (174 mg, 74%); mp = 166-168 °C. H NMR (DMSO -d
6
)
δ
:
7
.77 (d, J = 7.2 Hz, 2H), 7.50 (d, J = 7.2 Hz, 2H), 5.52 (brs, 2H), 5.48 (brs, 2H), 3.64-3.57 (m,

1
3
4
1
H), 2.57 (s, 3H), 2.31 (s, 3H), 1.71-1.62 (m, 4H); C NMR (DMSO-d
6
) δ: 162.57, 161.14,
48.74, 143.19, 136.08, 133.16, 132.67, 126.22, 124.18, 52.49, 23.67, 18.73, 16.55; HRMS (EI)
+
+
m/z 342.1641 M , calc. for C H N S 342.1627M ; Anal. Calc. for: C H N S (342): C, 59.62;
1
7
22
6
17 22
6
H, 6.48; N, 24.54%; Found: C, 59.66; H, 6.52; N, 24.61%.
4
.1.3.4.
2-(1-(4-Methyl-2-(4-(thiazolidin-3-yl)phenyl)thiazol-5-yl)ethylidene)hydrazine-1-
1
carboximidamide (8). Yellow solid (67 mg, 65.7%); mp = 177-179 °C. H NMR (DMSO -d )
δ
:
6
8
.05 (d, J = 8.4 Hz, 2H), 7.81 (brs, 4H), 7.72 (d, J = 8.4 Hz, 2H), 4.65 (s, 2H), 3.23 (t, J = 6.4
1
3
Hz, 2H), 2.81 (t, J = 6.4 Hz, 2H), 2.62 (s, 3H), 2.45 (s, 3H); C NMR (DMSO-d
6
)
δ
: 168.54,
1
1
64.94, 158.74, 147.79, 137.18, 132.16, 131.87, 127.12, 125.64, 62.33, 55.45, 31.27, 22.43,
+
+
8.65; HRMS (EI) m/z 360.1185 M , calc. for C H N S 360.1191 M ; Anal. Calc. for:
1
6
20
6 2
C H N S (360): C, 53.31; H, 5.59; N, 23.31%; Found: C, 53.35; H, 5.63; N, 23.36%.
16
20
6 2
4
.1.3.5. 2-(1-(2-(4-(1
H-Imidazol-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
1
carboximidamide (9). Brown solid (71 mg, 59%); mp = 183-185 °C. H NMR (DMSO -d )
δ
:
6
7
.89 (d, J = 8.4 Hz, 2H), 7.68 (s, 1H), 7.54 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 7.2 Hz, 1H), 6.93 (d,
1
3
J = 7.2 Hz, 1H), 5.66 (brs, 2H), 5.54 (brs, 2H), 5.22 (s, 2H), 2.58 (s, 3H), 2.32 (s, 3H); C NMR
DMSO-d : 165.25, 158.63, 154.63, 134.93, 134.74, 133.52, 132.91, 128.53, 127.43, 126.85,
22.46, 124.33, 30.50, 18.55; HRMS (EI) m/z 339.1250 M , calc. for C H N S 339.1266 M ;
(
6
) δ
+
+
1
1
6
17
7
Anal. Calc. for: C H N S (339): C, 56.62; H, 5.05; N, 28.89%; Found: C, 56.70; H, 5.09; N,
1
6
17
7
2
8.94%.
4
.1.3.6.
2-(1-(4-Methyl-2-(4-(piperidin-1-yl)phenyl)thiazol-5-yl)ethylidene)hydrazine-1-
1
carboximidamide (10). Yellow solid (86 mg, 72.8%); mp = 192-194 °C. H NMR (DMSO -d )
6
δ
: 11.51 (brs, 1H), 8.01 (d, J = 8.4 Hz, 2H), 7.82 (brs, 3H), 7.64 (d, J = 8.4 Hz, 2H), 3.55-3.49
1
3
(
6
m, 4H), 3.02-3.95 (m, 4H), 2.62 (s, 3H), 2.42 (s, 3H), 1.84-1.76 (m, 2H); C NMR (DMSO-d )

δ
: 167.84, 160.23, 156.42, 147.39, 133.10, 132.27, 127.53, 125.94, 119.87, 53.47, 31.91, 26.23,
+
+
2
4.91, 18.75; HRMS (EI) m/z 356.1795 M , calc. for C H N S 356.1783 M ; Anal. Calc. for:
18 24 6
C H N S (356): C, 60.65; H, 6.79; N, 23.57%; Found: C, 60.69; H, 6.85; N, 23.62%.
18
24
6
4
.1.3.7.
2-(1-(4-Methyl-2-(4-(2-methylpiperidin-1-yl)phenyl)thiazol-5-yl)ethylidene)
1
hydrazine-1-carboximidamide (11). Yellow solid (65 mg, 55.5%); mp = 189-191 °C. H NMR
(DMSO -d6)
δ: 7.89 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 5.78 (brs, 4H), 3.74-3.58 (m,
2
H), 3.52-3.49 (m, 1H), 2.59 (s, 3H), 2.33 (s, 3H), 1.61-1.19 (m, 6H), 1.06 (d, J = 4.8 Hz, 3H);
1
3
C NMR (DMSO-d
6
)
δ
: 162.59, 161.15, 148.75, 143.44, 137.43, 133.19, 132.78, 126.33,
+
1
24.91, 54.82, 53.26, 34.63, 26.22, 24.96, 20.13, 18.79, 16.69; HRMS (EI) m/z 370.1944 M ,
+
calc. for C H N S 370.1940 M ; Anal. Calc. for: C H N S (370): C, 61.59; H, 7.07; N,
1
9
26
6
19 26
6
2
2.68%; Found: C, 61.62; H, 7.11; N, 22.74%.
4
.1.3.8.
2-(1-(4-Methyl-2-(4-(3-(methylpiperidin-1-yl)phenyl)thiazol-5-yl)ethylidene)
1
hydrazine-1-carboximidamide (12). Yellow-white solid (80 mg, 68%); mp = 195-196 °C. H
NMR (DMSO -d6)
δ
: 7.79 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 5.77 (brs, 2H), 5.47 (brs,
2
1
1
H), 3.27 (d, J = 9.6 Hz, 2H), 3.19-3.09 (m, 2H), 2.61 (s, 3H), 2.46 (s, 3H), 2.12-1.36 (m, 5H),
1
3
.56 (d, J = 6.4 Hz, 3H); C NMR (DMSO-d
6
) δ: 168.51, 163.25, 158.75, 147.33, 132.78,
32.11, 127.53, 125.87, 122.61, 54.72, 53.26, 34.73, 31.22, 25.71, 24.56, 18.53, 16.89; HRMS
+
+
(
EI) m/z 370.1930 M , calc. for C H N S 370.1940 M ; Anal. Calc. for: C H N S (370): C,
19 26 6 19 26 6
6
4
1.59; H, 7.07; N, 22.68%; Found: C, 61.62; H, 7.11; N, 22.74%.
.1.3.9.
2-(1-(4-Methyl-2-(4-(4-(methylpiperidin-1-yl)phenyl)thiazol-5-yl)ethylidene)
1
hydrazine-1-carboximidamide (13). Yellow solid (90 mg, 76.9%); mp = 201-202 °C. H NMR
6
(DMSO -d ) δ: 11.41 (brs, 1H), 7.78 (d, J = 8 Hz, 2H), 7.51 (brs, 3H), 7.27 (d, J = 8 Hz, 2H),
2
.61 (s, 3H), 2.31 (s, 3H), 2.18 (t, J = 12 Hz, 4H), 1.62 (t, J = 10.8 Hz, 4H), 1.32-1.27 ( m, 1H),

1
3
0
1
3
7
4
.89 (d, J = 6.4 Hz, 3H); C NMR (DMSO-d
6
) δ: 161.61, 160.19, 148.72, 143.41, 136.18,
32.90, 132.02, 126.10, 124.47, 52.52, 34.65, 30.16, 22.34, 18.75, 16.39; HRMS (EI) m/z
+
+
70.1940 M , calc. for C H N S 370.1933 M ; Anal. Calc. for: C H N S (370): C, 61.59; H,
1
9
26
6
19 26
6
.07; N, 22.68%; Found: C, 61.62; H, 7.11; N, 22.74%.
.1.3.10.
2-(1-(4-Methyl-2-(4-(4-(4-methylpiperazin-1-yl)phenyl)thiazol-5-yl)ethylidene)
1
hydrazine-1-carboximidamide (14). Orange solid (77 mg, 65%); mp = 175-176 °C. H NMR
6
(DMSO -d ) δ: 7.99 (d, J = 8.8 Hz, 2H), 7.57 (d, J = 8.8 Hz, 2H), 5.75 (brs, 4H), 4.75-4.69 (m,
1
3
4
1
1
H), 2.52 (s, 3H), 2.45 (s, 3H), 2.31-2.25 (m, 4H), 2.21 ( s, 3H); C NMR (DMSO-d
6
) δ: 168.28,
65.09, 158.65, 146.11, 143.58, 133.45, 132.88, 127.21, 125.93, 56.01, 46.03, 45.13, 30.28,
+
+
8.55; HRMS (EI) m/z 371.1901 M , calc. for C H N S 371.1892 M ; Anal. Calc. for:
1
8
25
7
C H N S (371): C, 58.19; H, 6.78; N, 26.39%; Found: C, 58.25; H, 6.84; N, 26.45%.
18
25
7
4
.1.3.11.
2-(1-(2-(4-(4-Bromopiperidin-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)
1
hydrazine-1-carboximidamide (15). Yellow solid ( 96 mg, 84%); H NMR (DMSO-d )
δ
: 7.49
6
(d, J = 8.4 Hz, 2H), 7.02 (d, J = 8.4 Hz, 2H), 5.21 (brs, 2H), 5.07 (brs, 2H), 4.62-4.54 (m, 1H),
1
3
3
1
2
4
4
4
.42 (t, J = 6.4 Hz, 4H), 2.49 (s, 3H), 2.27 (s, 3H), 2.25-1.89 (m, 4H); C NMR (DMSO-d
6
) δ:
62.54, 160.96, 148.41, 144.53, 136.21, 132.04, 131.83, 126.56, 124.12, 53.55, 48.11, 34.51,
+
+
9.44, 18.34, 16.21; HRMS (EI) m/z 434.0890 M , calc. for C H BrN S 434.0888 M ,
1
8
23
6
+
36.0868 M Anal. Calc. for: C H BrN S (435): C, 49.66; H, 5.32; N, 19.30%; Found: C,
1
8
23
7
9.70; H, 5.35; N, 19.37%.
.1.3.12.
2-(1-(4-Methyl-2-(4-morpholinophenyl)thiazol-5-yl)ethylidene)hydrazine-1-
1
carboximid-amide (16). Brown solid (87 mg, 73%); mp = 186-187 °C. H NMR (DMSO -d )
δ
:
6
8
4
.01 (d, J = 8.4 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 5.65 (brs, 2H), 5.48 (brs, 2H), 3.87-3.81 (m,
1
3
6
H), 3.65-3.60 (m, 4H), 2.71 (s, 3H), 2.57 (s, 3H); C NMR (DMSO-d ) δ: 167.28, 162.49,

1
54.75, 147.37, 142.18, 133.98, 132.45, 126.21, 124.95, 64.11, 52.23, 27.18, 16.55; HRMS (EI)
+
+
m/z 358.1595 M , calc. for C H N OS 358.1576 M ; Anal. Calc. for: C H N OS (358): C,
1
7
22
6
17 22
6
5
6.96; H, 6.19; N, 23.45%; Found: C, 57.01; H, 6.25; N, 23.55%.
4
.1.3.13.
2-(1-(4-Methyl-2-(4-thiomorpholinophenyl)thiazol-5-yl)ethylidene)hydrazine-1-
1
carboximid-amide (17). Yellow solid (68 mg, 58%); mp = 194-196 °C. H NMR (DMSO -d )
δ
:
6
8
.25 (d, J = 8.4 Hz, 2H), 7.75 (d, J = 8.4 Hz, 2H), 5.60 (brs, 4H), 3.98-3.91 (m, 4H), 3.81-3.77
1
3
(
m, 4H), 2.69 (s, 3H), 2.41 (s, 3H); C NMR (DMSO-d
6
) δ: 162.11, 160.09, 147.75, 143.37,
1
36.78, 132.45, 132.05, 126.41, 124.85, 56.23, 30.23, 18.38, 16.65; HRMS (EI) m/z 374.1349
+
+
M , calc. for C17
H
22
N
6
S
2
374.1347 M ; Anal. Calc. for: C17
22 6 2
H N S (374): C, 54.52; H, 5.92; N,
2
2.44%; Found: C, 54.61; H, 5.99; N, 22.51%.
4
.1.3.14.
2-(1-(2-(4-(Azepan-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
1
carboximid-amide (18). Brown solid (65 mg, 55.5%); mp = 166-168 °C. H NMR (DMSO -d )
6
δ
6
1
1
: 7.98 (d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 5.53 (brs, 2H), 5.45 (brs, 2H), 4.35 (t, J =
1
3
.8 Hz, 4H), 2.71 (s, 3H), 2.44 (s, 3H), 1.95-1.05 (m, 8H); C NMR (DMSO-d )
δ
: 167.49,
6
64.45, 158.36, 149.75, 136.93, 132.79, 132.18, 127.23, 125.11, 53.26, 31.71, 26.33, 24.70,
+
+
26 6
8.69; HRMS (EI) m/z 370.1923 M , calc. for C19H N S 370.1940 M ; Anal. Calc. for:
C H N S (370): C, 61.59; H, 7.07; N, 22.68%; Found: C, 61.67; H, 7.11; N, 22.74%.
19
26
6
4
.1.3.15.
2-(1-(2-(4-(Azocan-1-yl)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
1
carboximid-amide (19). Brown solid (60 mg, 51%); mp = 167-169 °C. H NMR (DMSO -d )
δ
:
6
7
.78 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 5.76 (brs, 2H), 5.56 (brs, 2H), 4.44 (t, J = 6.8
1
3
Hz, 4H), 2.75 (s, 3H), 2.44 (s, 3H), 1.95-1.09 (m, 8H), 0.88 (d, J = 6.4 Hz, 2H); C NMR
(
DMSO-d
6
)
δ
: 163.49, 161.75, 148.86, 143.45, 136.43, 133.29, 129.48, 126.23, 124.41, 47.11,
+
3
4.21, 30.13, 26.10, 25.76, 18.49, 16.66; HRMS (EI) m/z 384.2094 M , calc. for C20
H
28
N
6
S

+
3
84.2096 M ; Anal. Calc. for: C20
H, 7.38; N, 21.93%.
.1.3.16.
28 6
H N S (384): C, 62.47; H, 7.34; N, 21.85%; Found: C, 62.55;
4
2-(1-(4-Methyl-2-(4-(octahydroisoquinolin-2(1H)-yl)phenyl)thiazol-5-
yl)ethylidene) hydrazine-1-carboximidamide (20). Yellow solid (69 mg, 59%); mp = 177-179
1
°
C. H NMR (DMSO -d )
δ
: 7.85 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 5.76 (brs, 2H),
6
5
2
1
.68 (brs, 2H), 2.72 (t, J = 10.8 Hz, 2H), 2.68 (d, J = 8.8 Hz, 2H), 2.59 (s, 3H), 2.31 (s, 3H),
1
3
.22-0.92 (m, 12H); C NMR (DMSO-d
6
) δ: 162.11, 160.06, 148.96, 143.77, 136.48, 132.53,
29.39, 126.49, 124.17, 59.32, 56.89, 47.69, 43.95, 34.25, 30.60, 26.60, 26.17, 18.65, 16.57;
+
+
HRMS (EI) m/z 410.2265 M , calc. for C22
30 6 30 6
H N S 410.2253 M ; Anal. Calc. for: C22H N S
(410): C, 64.36; H, 7.37; N, 20.47%; Found: C, 64.40; H, 7.41; N, 20.52%.
4
4
.2. Microbiological assays
.2.1. Bacterial strains, mammalian cell lines and antibiotics. Bacterial strains used in this
study were obtained from Biodefense and Emerging Infections Research Resources Repository
BEI Resources) and the American Type Culture Collection (ATCC). Human colorectal
(
adenocarcinoma (Caco-2) cell line, human keratinocyte cell line (HaCaT) and murine
macrophage (J774) cells were purchased from American Type Culture Collection (ATCC).
Linezolid (Chem-impex International, Wood Dale, IL, USA) and vancomycin hydrochloride
(
Gold Biotechnology, St. Louis, MO, USA), were purchased from commercial vendors.
Phenylthiazole compounds were prepared in a stock concentration of 10 mg/mL in DMSO.
.2.2. Determination of MICs and MBCs of the new phenylthiazole compounds against
4
Staphylococcus aureus and other multidrug-resistant Gram-positive bacterial species. The
broth microdilution method was utilized to test the antibacterial activity of the new
phenylthiazole compounds against a panel of clinically-important S. aureus strains and Gram-

positive bacteria. The minimum inhibitory concentration (MIC) of teste compounds and control
antibiotics (linezolid, and vancomycin) was determined using the broth microdilution method
against methicillin-sensitive Staphylococcus aureus (ATCC 6538 and NRS107), methicillin-
resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus
(VRSA) clinical isolates according to the guidelines outlined by the Clinical and Laboratory
Standards Institute (CLSI) [50]. Bacterial strains were grown aerobically overnight on tryptone
soy agar plates at 37° C. Afterwards, a bacterial solution equivalent to 0.5 McFarland standard
was prepared and diluted in cation-adjusted Mueller-Hinton broth (CAMHB) to achieve a
5
bacterial concentration of about 5 × 10 CFU/mL and seeded in 96-well plates. Compounds and
control drugs were added in the first row of the 96-well plates and serially diluted along the
plates. Plates were then, incubated aerobically at 37° C for 18-20 hours. MICs reported in here
are the minimum concentration of the compounds and control drugs that could completely inhibit
the visual growth of bacteria. The minimum bactericidal concentration (MBC) of these
compounds was tested by plating 4 µL from wells with no growth onto Tryptic soy agar plates.
Plates were incubated at 37 ºC for 18-20 hours before recording the MBC. The MBC was
categorized as the lowest concentration that reduced bacterial growth by 99.9%.
4
.2.3. In vitro cytotoxicity analysis of compounds 10 and 12 against human colorectal,
human keratinocyte and murine macrophage cells. Phenylthiazoles 10 and 12 were assayed
for potential cytotoxicity against a human colorectal adenocarcinoma (Caco-2) cell line, human
keratinocyte cell line (HaCaT) and murine macrophage (J774) cells, as described previously [5].
Briefly, tested compounds were incubated with caco-2 and HaCaT cells for 2 hours, and with
J774 cells for 24 hours. Then, cells were incubated with MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-

carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) reagent for 4 hours before measuring
absorbance values (OD ).
4
90
4
.2.4. MRSA biofilm eradication assessment. Phenylthiazole compounds 10 and 12 were
examined for their ability to eradicate pre-formed, well established mature staphylococcal
biofilm using the microtiter plate biofilm formation assay as described in previous reports [8,
5
1]. An overnight culture of MRSA USA300 (NRS384) was diluted 1:100 in culture medium
(Tryptic soy broth + 1% glucose). For detection of biofilm eradication, bacterial suspension was
added, and plates were incubated at 37 °C for 24 hours in order to form a strong adherent
biofilm. The bacterial suspension (planktonic cells) was removed and compounds were added at
concentrations ranging from 256 to 2 µg/mL in Tryptic soy broth. Plates were incubated at 37 °C
for 24 hours. In order to quantify biofilm mass, the bacterial suspension was removed, and wells
were washed with phosphate-buffered saline to remove planktonic bacteria. An aliquot of 0.1%
crystal violet was added to each well to stain the attached biofilm mass. After 30 minutes, wells
were washed with sterile water and dried. Wells were de-stained using 100% ethanol prior to
quantifying biofilm mass using a spectrophotometer (OD595). Data are presented as percent
eradication of MRSA USA300 biofilm for each test agent relative to the negative (DMSO)
control wells. Data were analyzed using unpaired t test (P < 0.05).
4
.2.5. Intracellular infection of J774 cells with MRSA and treatment with compound 10.
The ability of compound 10 and vancomycin (at 2 × MIC) to reduce the burden of intracellular
MRSA USA400 inside murine macrophage (J774) cells was evaluated. In brief, murine
macrophage cells (J774) were cultured in Dulbecco’s Modified Eagle Medium (DMEM)
supplemented with 10% FBS at 37°C with CO
2
(5%). J774 cells were exposed to MRSA
USA400 cells at a multiplicity of infection of approximately 10:1. After 1 hour of infection, J774