Phenylthiazole antibiotics: A metabolism-guided approach to overcome short duration of action

Article abstract of DOI:10.1016/j.ejmech.2016.11.042

Antibacterial resistance is a pressing global health challenge that necessitates the development of new therapeutic agents. Phenylthiazole antibacterial agents have been extensively studied, by our group, as a potential novel class of antibiotics to circumvent the scourge of antibacterial resistance. Previously, the phenylthiazole lead compound 1 was shown to possess potent activity against clinical isolates of methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA). The promising activity of this novel class of antibiotics is hampered by their short half-life due to rapid hepatic metabolism. In the present study, a metabolic methylene soft spot in the lead 1 was identified and replaced with an oxygen atom. The newly developed phenylthiazoles, with alkoxy side chains, demonstrate high metabolic stability (t_1/2> 4 h), while maintaining their potent anti-MRSA activity. Furthermore, compound 5p demonstrated a selective advantage over vancomycin with its ability to kill intracellular MRSA.

Full text of DOI:10.1016/j.ejmech.2016.11.042

Accepted Manuscript
Phenylthiazole antibiotics: A metabolism-guided approach to overcome short duration
of action
Eman Yahia, Haroon Mohammad, Tamer M. Abdelghany, Eman Fayed, Mohamed N.
Seleem, Abdelrahman S. Mayhoub
PII:
S0223-5234(16)30980-1
10.1016/j.ejmech.2016.11.042
EJMECH 9075
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 August 2016
Revised Date: 2 November 2016
Accepted Date: 20 November 2016
Please cite this article as: E. Yahia, H. Mohammad, T.M. Abdelghany, E. Fayed, M.N. Seleem, A.S.
Mayhoub, Phenylthiazole antibiotics: A metabolism-guided approach to overcome short duration of
action, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.042.
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ACCEPTED MANUSCRIPT
Graphical Abstract
HLM t1/2 = 28.8 min
HLM, CL = 80.3 mL/min-mg
> 4 h
< 9.6 mL/min-mg
int

ACCEPTED MANUSCRIPT
Phenylthiazole Antibiotics: A Metabolism-Guided Approach to
Overcome Short Duration of Action
1
,║
2,║
3
1
2,4
Eman Yahia, Haroon Mohammad, Tamer M. Abdelghany, Eman Fayed, Mohamed N. Seleem *
1
,5*
and Abdelrahman S. Mayhoub
1
Department of Pharmaceutical Organic Chemistry, College of Pharmacy, Al-Azhar University, Cairo
11884, Egypt
2
Department of Comparative Pathobiology, Purdue University, College of Veterinary Medicine, West
Lafayette, IN 47907, USA
3
Department of Pharmacology, College of Pharmacy, Al-Azhar University, Cairo 11884, Egypt
4
Purdue Institute for Inflammation, Immunology, and Infectious Diseases, West Lafayette, IN 479067,
USA
5
Biomedical Sciences, University of Science and Technology, Zewail City of Science and Technology,
Giza, Egypt
Corresponding Author.
*
For Abdelrahman S. Mayhoub: Phone 0020-100-771-5002; e-mail, amayhoub@azhar.edu.eg
For Mohamed N. Seleem: phone, 765-494-0763, email, mseleem@purdue.edu
*
Abbreviations: CFU, colony forming units; HaCaT, human keratinocytes.

ACCEPTED MANUSCRIPT
ABSTRACT. Antibacterial resistance is a pressing global health challenge that necessitates the
development of new therapeutic agents. Phenylthiazole antibacterial agents have been extensively
studied, by our group, as a potential novel class of antibiotics to circumvent the scourge of antibacterial
resistance. Previously, the phenylthiazole lead compound 1 was shown to possess potent activity against
clinical isolates of methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA).
The promising activity of this novel class of antibiotics is hampered by their short half-life due to rapid
hepatic metabolism. In the present study, a metabolic methylene soft spot in the lead 1 was identified
and replaced with an oxygen atom. The newly developed phenylthiazoles, with alkoxy side chains,
demonstrate high metabolic stability (t1/2 > 4h), while maintaining their potent anti-MRSA activity.
Furthermore, compound 5p demonstrated a selective advantage over vancomycin with its ability to kill
intracellular MRSA.
Keywords: MRSA, VRSA, Antibiotic-resistance

ACCEPTED MANUSCRIPT
1
. INTRODUCTION
Bacterial resistance to antibiotics is a significant challenge that claims the lives of more than 23,000
people each year in the United States alone.[1] A significant source of these fatalities can be attributed to
infections caused by a single pathogen, methicillin-resistant Staphylococcus aureus (MRSA). MRSA is
a leading source of skin, wound, and hospital-acquired infections. Treatment of these infections has
become more difficult with the emergence of clinical isolates of MRSA exhibiting resistance to first-line
antibiotics and agents of last resort, like vancomycin.[2] This highlights the urgent need to develop new
therapeutic agents to address the burden of MRSA infections.
Figure 1. Chemical structure of lead compound 1 and proposed metabolites.
In the last three years, extensive research from our group has focused on developing novel drug-
candidates for treatment of MRSA infections,[3-7] in addition to several other reports from other
groups.[8, 9] To date, potent phenylthiazole antibiotics we discovered were shown to possess
bactericidal activity against MRSA both in vitro and in vivo in a mouse model of MRSA skin
infection.[10] The topical in vivo study revealed that treatment with phenylthiazole 1 significantly
reduced bacterial load (2.36 log reduction in the wound) in infected mice, in a manner similar to the
1
0
conventional antibiotic mupirocin.[10] Though the initial series of phenylthiazole compounds possess
many desirable qualities as an antibacterial agent, one significant limitation is their stability to hepatic
metabolism, as discussed below.

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Preclinical drug metabolism studies play a key role in all modern drug discovery programs.
Characterization of the metabolic pathways of new chemical entities is an integral part of drug
discovery not only in the identification of initial lead compounds but also in the subsequent optimization
steps and drug-candidate selection.[11] From this perspective, and encouraged by the promising in vivo
result of our newly discovered phenylthiazoles,[10] the metabolites and pharmacokinetic profile of
compound 1 were determined (Figure 1) in order to move this project further for treatment of systemic
MRSA infections. Surprisingly, compound 1 was rapidly metabolized and cleared in human liver
microsomes (intrinsic clearance rate of 80.3 mL/min-mg) resulting in a very short half-life (<30 min)
(
Figure 1). The absence of the cofactor NADPH enhanced the half-life of compound 1 indicating this
compound most likely is subject to metabolism via the cytochrome P450 (CYP450) complex. In this
article, the metabolites of lead compound 1 were briefly studied using a rat model. Careful analysis of
the metabolites, in serum and urine, led to identification of a potential metabolic soft spot at a benzylic
methylene carbon. This article outlines our new metabolism-guided structural optimization of
phenylthiazole 1 with the objective of ameliorating the metabolic stability of this class of antibiotics.
2
. RESULTS AND DISCUSSION
.1. CHEMISTRY
2
Thioamide 2 was prepared by treatment of the corresponding commercially available p-
hydroxybenzamide with Lawesson’s reagent. The obtained yellow solid was then dissolved in absolute
ethanol and heated with a slight excess of α-chloroacetylacetone to yield phenylthiazole derivative 3
(Scheme 1). The latter compound was then allowed to react with different alkyl bromides to give
compounds 4a–p. Treatment of compounds 4a–p with aminoguanidine hydrochloride, in the presence of
a catalytical amount of lithium chloride, furnished the desired final products 5a–p (Scheme 1).

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1
2
3
2.2.
BIOLOGICAL RESULTS AND DISCUSSION
4
5
6
7
8
9
0
1
2
3
4
5
6
In order to rationally overcome the metabolic instability of lead compound 1, we attempted to initially
deduce how the phenylthiazoles bind to CYP450 in order to block the metabolic soft spots. However,
several crystal structures of CYP450 have revealed these enzymes possess extreme flexibility that
challenges attempts to apply computational tools without the support of relevant experimental data.[12]
Therefore, a rat was orally administered with lead compound 1, urine samples were collected over 24 h,
and all metabolites were analyzed using LC/MS/MS. In addition, a plasma sample was taken 6 h post-
dosing and fully analyzed using LC/MS/MS. The main detected metabolites, in both serum and urine,
relative to the unchanged drug, are presented in Figure 1. Mindful of those metabolites, compound 1
appears to undergo an extensive Phase I oxidation step, on one of its benzylic carbon atoms, followed
by glucuronation to yield metabolite M2. In order to capitalize on this finding, and in the absence of
detailed information on how compound 1 binds to CYP450, we proposed C1 of the lipophilic tail; i.e. n-
butyl group, as the metabolic soft spot (Figure 2). This hypothesis is bolstered by the fact that the active
site of CYP450 is surrounded by a highly hydrophobic seven phenylalanine cluster.[12]
1
1
1
1
1
1
1
1
7

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1
1
8
9
Figure 2. New design to overcome metabolic instability.
2
0
2
2
2
2
2
2
1
2
3
4
5
6
A rapidly metabolized benzylic carbon is a common problem in drug development that has been
previously encountered. Reported strategies to address this issue include adding two fluorine atoms, two
methyl groups or substitution with an oxygen atom.[13, 14] The latter option was selected in the present
study for two reasons: the accessibility of the synthetic protocol and the wide availability of commercial
alkyl halides that permitted a complete study of the structure-activity and structure-kinetic relationships
at this particular position (Figure 2).
2
2
7
8
Table 1. Antimicrobial activity (µg/mL) of tested phenylthiazoles against MRSA (2658 RCMB).
MIC ± SD
Entry
R
Compound
(µg/mL)
1
2
3
4
5
6
7
8
9
NA
ethyl
propyl
butyl
pentyl
hexyl
heptyl
octyl
nonyl
1
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
3.75 ± 0.0
> 30
> 30
15.63 ± 0.10
1.95 ± 0.10
0.35 ± 0.00
0.74 ± 0.00
15.63± 0.50
> 30
15.63± 0.50
7.81± 0.00
15.63± 0.10
7.81± 0.50
15.63± 0.50
> 30
1
1
1
1
1
1
1
1
1
0
1
2
3
4
5
6
7
8
isobutyl
isopentyl
4-methylpentyl
2-ethylbutyl
2-butenyl
methylcyclobutyl
cyclopentyl
cycloheptyl
NA
15.63± 0.10
0.98± 0.00
Vancomycin 1.95± 0.00

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2
9
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
At the outset of this study, the hypothesis of replacing the vulnerable methylene with an oxygen atom
was first tested with compound 5c that contains the same number of carbon atoms as the lead compound
1. Compound 5c showed a notable improvement in metabolic stability as expressed by its higher
microsomal half-life value (t1/2 > 4 h) and lower clearance rate as will be discussed below. However, its
anti-MRSA activity (MIC was 15.63 µg/mL) was diminished when compared to the lead compound 1
(MIC was 3.75 µg/mL) (Table 1). We next moved to comprehensively study the SARs at the lipophilic
tail in order to improve the antimicrobial potency of 5c. Therefore, a set of sixteen different alkyl
halides, including homologous, branched and cyclic derivatives, were tethered to the oxygen atom.
Table 1 presents the initial anti-MRSA screening results against MRSA 2658 RCMB. Generally
speaking, lower minimum inhibitory concentration (MIC) values were observed with side chains that
contained five to seven carbon units as seen with the pentyl, hexyl and heptyl derivatives 5d–5f. Briefly,
the pentyl derivative 5d has a MIC value similar to that of vancomycin and is one-fold better than the
lead compound 1 (MIC = 1.95 µg/mL, Table 1). By increasing the length of the side chain, the anti-
MRSA activity significantly improved. Both the hexyl 5e and heptyl 5f analogues were more potent
than vancomycin (Table 1). Interestingly, further increase in the methylene units of the alkyl chain
furnished less active compounds 5g and 5h with MIC values >15 µg/mL.
4
4
4
4
5
5
5
5
5
6
7
8
9
0
1
2
3
4
The second set of modifications included four branched derivatives 5i–5l. All branched derivatives were
less active than their corresponding normal analogues with the same number of carbon atoms; for
instance, the MIC value of the 4-methylpentyl derivative 5k was forty-five times more than that of n-
hexyl analogue 5e (Table 1). Lastly, the cyclic analogues 5n–5p were more active than the branched
ones; however, they were less active than their corresponding unbranched derivatives with the same
number of carbon atoms (Table 1). Of the cyclic analogues examined, the cycloheptyl derivative 5p was
found to possess the most potent anti-MRSA activity (MIC equal to 0.98 µg/mL).
In order to further examine the anti-MRSA activity of the new phenylthiazole series, the active
compounds were selected for further evaluation against an array of S. aureus isolates including

ACCEPTED MANUSCRIPT
5
5
5
6
methicillin-sensitive (NRS107), methicillin-resistant (MRSA), and vancomycin-resistant (VRSA)
Staphylococcus
aureus
(Table
2).
Table 2. The minimum inhibitory concentration (MIC in µg/mL) and the minimum bactericidal
concentration (MBC µg/mL) of tested phenylthiazoles screened against Staphylococcus aureus isolates.
S. aureus
NRS107
MRSA
NRS383
(USA200)
MRSA
NRS123
(USA400)
MRSA
NRS382
(USA100)
MRSA
NRS384
(USA300)
VRSA4
VRSA10
(
RN4220)
Compound
MI
C
-
-
4
2
4
16
32
16
16
-
32
8
4
MB
C
-
-
4
MI
C
32
64
1
0.5
1
4
8
8
4
32
16
2
2
2
MB
C
>64
>64
2
1
2
8
8
16
16
>64
>64
16
8
MI
C
-
-
4
MB
C
-
-
16
4
MI
C
32
-
2
1
MB
C
>128
-
2
1
2
8
16
8
2
32
128
4
4
16
<1
MI
C
-
-
4
MB
C
-
-
8
MI
C
-
-
4
2
4
16
16
16
16
-
32
8
4
MB
C
-
-
4
MI
C
-
-
4
MB
C
-
-
4
5
c
5
d
5
e
5f
2
8
2
4
2
4
2
4
2
4
2
4
2
4
5
5
g
h
8
2
16
32
16
16
-
32
8
8
16
16
16
16
-
32
8
4
16
>32
>32
32
-
>32
32
16
32
0.5
16
16
16
16
-
32
8
4
16
32
32
32
-
>32
8
16
16
1
16
32
32
16
-
32
8
4
16
16
16
16
-
32
8
4
16
32
32
16
-
32
8
4
5
i
8
8
2
32
16
2
2
2
<1
5
j
5
k
5
l
5
m
5
o
5
p
Linezolid
Vancomyci
n
2
1
32
1
32
0.5
2
0.5
2
0.5
2
>51
2
8
>
512
2
>51
2
16
>
512
0.5
Interestingly, some important changes in the SAR were noted including that potent anti-MRSA activity
was observed with longer side chains; i.e. compounds with six to eight carbon units (compounds 5e, 5f
and 5g) were the most active against isolates tested. Surprisingly, the pentyl derivative 5d, which was
active against the lab-reference strain (MRSA 2658 RCMB), was found to be inactive against the
MRSA USA200 clinical isolate. On the other hand, the octyl derivative 5g, which possessed negligible
activity against the MRSA 2658 RCMB reference strain, demonstrated potent anti-MRSA and anti-
VRSA activity with MIC values comparable to linezolid (Table 2). Furthermore, the alicyclic-
containing compounds 5p and 5o were among the most active compounds against the S. aureus clinical
isolates. Many of the clinical isolates selected for analysis exhibit resistance to more than one antibiotic
including mupirocin (NRS107), rifampicin (NRS107), erythromycin (USA300), tetracycline (USA300),
gentamicin (USA200), ciprofloxacin (USA100 and USA200), clindamycin (USA100 and USA200),
linezolid (NRS119), and vancomycin (VRSA4 and VRSA10). Given the phenylthiazole compounds

ACCEPTED MANUSCRIPT
retained their potent antibacterial activity against these different drug-resistant clinical isolates, this
indicates cross-resistance between our compounds and the antibiotics noted above is unlikely.
After confirming, the MIC values for the active compounds, we next moved to examine if the
compounds were bacteriostatic or bactericidal. To deduce this, the minimum bactericidal concentration
(MBC) was determined for the active compounds. As noted in Table 2, compounds 5e, 5f, and 5g
appear to be bactericidal against MRSA given the MBC values match or are one-fold higher than the
MIC values against the clinical isolates tested. The alicyclic-containing compounds 5p and 5o also
appear to be bactericidal against MRSA, though in a concentration-dependent manner. Against strains
USA200, USA300 (only 5p), and USA400, the MBC values are more than three-fold higher than the
MIC values; against the remaining strains examined, the MBC and MIC values match or are one-fold
higher.
Time-kill assay against MRSA. In order to confirm the bactericidal activity of the new analogues,
compounds 5e, 5f, 5g, and 5p (at 4 × MIC) were further examined against MRSA USA400 using a
standard time-kill assay. As presented in Figure 3, compound 5e produces a gradual reduction in MRSA
CFU, eventually generating a 3-log10 reduction after 24 h (confirming the compound is bactericidal).
Compounds 5f and 5g drastically reduce MRSA CFU within two hours (producing a near 4-log10
reduction, indicating bactericidal activity). However, 5f does not produce any additional reduction in
MRSA CFU even up to 48 h. 5g completely eliminates MRSA growth after 48 h. Compound 5p
gradually reduces MRSA CFU, requiring 48 h to produce a 3-log10 reduction (but is not capable of
completely reducing the burden of MRSA CFU to zero). Vancomycin requires 24 h to completely
eliminate MRSA. Given the behavior of these compounds differs from the first-generation
phenylthiazole compounds, the time-kill assay was repeated twice (once against MRSA USA400 and
once against MRSA USA300) to confirm the result (data not shown).

ACCEPTED MANUSCRIPT
Figure 3. Time-kill analysis of thiazole compounds 5e, 5f, 5g, 5p, and vancomycin against methicillin-resistant
Staphylococcus aureus (MRSA USA400) over a 24 h incubation period at 37 °C. DMSO served as a negative
control. The error bars represent standard deviation values obtained from triplicate samples used for each
compound/antibiotic studied.
Figure 4. Toxicity analysis of newly developed phenylthiazole compounds against human keratinocytes
(
HaCaT). Percent viable mammalian cells (measured as average absorbance ratio (test agent relative to DMSO))
for cytotoxicity analysis of tested compounds 5e, 5f, 5g, 5p, and 5o (tested in triplicate) at 8, 16, and 32 µg/mL
against HaCaT cells using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2
H-tetrazolium) assay. Dimethyl sulfoxide (DMSO) was used as a negative control to determine a baseline
measurement for the cytotoxic impact of each compound. The absorbance values represent an average of a
minimum of three samples analyzed for each compound. Error bars represent standard deviation values for the
absorbance values. A two-way ANOVA, with post hoc Dunnet’s multiple comparisons test, determined statistical
difference between the values obtained for each compound and DMSO (denoted by the asterisk) (P < 0.05).
Vancomycin has very low potential for keratinocyte toxicity.[15]
Cytotoxicity analysis of compounds against human keratinocytes (HaCaT). The most promising
compounds were next selected for preliminary cytotoxicity profiling. Hence, compounds 5e, 5f, 5g, 5p,
and 5o were assayed against a human keratinocyte (HaCaT) cell line to determine the potential toxic
effect to mammalian cells in vitro. As depicted in Figure 4, the most potent antibacterial compounds, 5e
and 5p, are not toxic to mammalian cells at concentrations up to 32 µg/mL (both are toxic at 64 µg/mL).

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This corresponds to a nearly 30-fold difference between the MIC values obtained against MRSA. When
comparing these values with the earlier generation of phenylthiazoles, a significant improvement in the
mammalian toxicity-to-MRSA MIC ratio, by a factor of 1.5, is observed.[4] Compounds 5g and 5o were
slightly more toxic and were tolerable up to 16 µg/mL. On the other hand, the high toxicity of the n-
heptyl derivative 5f (compound was nontoxic only up to 8 µg/mL), the most promising derivative in
terms of MIC values, may hinder its potential systemic application (as the dose of this agent would need
to be carefully monitored to minimize toxicity to host tissues).
Figure 5. Toxicity analysis and examination of clearance of intracellular MRSA present in murine
macrophage (J774) cells. Panel A) Percent viable mammalian cells (measured as average absorbance
ratio (test agent relative to DMSO)) for cytotoxicity analysis of thiazole compounds 5f and 5p (tested in
triplicate) at 8, 16, 32, and 64 µg/mL against J774 cells using the MTS 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Dimethyl sulfoxide (DMSO) was
used as a negative control to determine a baseline measurement for the cytotoxic impact of each
compound. The absorbance values represent an average of a minimum of three samples analyzed for
each compound. Error bars represent standard deviation values for the absorbance values. A one-way
ANOVA, with post hoc Dunnet’s multiple comparisons test, determined statistical difference between
the values obtained for each compound and DMSO (denoted by the asterisk) (P < 0.05). Panel B)
Percent reduction of MRSA USA400 colony forming units inside infected murine macrophage cells
after treatment with 16 µg/mL of either compound 5p or vancomycin (tested in triplicate) for 4, 8, 12,
and 24 h. Data were analyzed via a Student’s t-test (P < 0.05). Asterisks (*) represent significant
difference between treatment of J774 cells with 5p in comparison to vancomycin.

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Treatment of intracellular MRSA. The potent antibacterial activity of the new phenylthiazole
analogues against extracellular MRSA led us to investigate whether these compounds would be capable
of eradicating intracellular MRSA. S. aureus is capable of surviving inside host leukocytes as one
mechanism of evading the innate immune system.[16] This can lead to persistent recurring infections
particularly skin infections and chronic osteomyelitis.[17] Many antibiotics including vancomycin, are
ineffective at clearing intracellular S. aureus due to multiple factors including inability to cross the
plasma membrane (to gain entry into infected host cells), localization of antibiotic in relation to bacteria
inside host cells, inactivation of the antibiotic by factors inside the host cell (such as pH), and the
metabolic state of intracellular bacteria (may not be actively dividing thus rendering certain antibiotics
ineffective).[17]
In order to examine the ability of our phenylthiazole analogues to clear intracellular MRSA, toxicity of
two of the most potent analogues, 5f and 5p, was initially examined against murine macrophage cells
(J774) (Figure 5, panel A). The cycloheptyl 5p was found to not be toxic up to 16 µg/mL; however, its
corresponding open chain 5f was toxic to murine macrophage cells even at a concentration as low as 8
µg/mL. Due to its superior toxicity profile, the cycloheptyl 5p was selected to examine its ability to
clear intracellular MRSA harboring inside macrophage cells. As depicted in Figure 5 (panel B), after
eight hours 5p is unable to reduce MRSA CFU/mL when compared to untreated samples (indicating it is
not gaining entry into macrophages at a concentration sufficient to inhibit MRSA growth). However,
after 12 h a 48% reduction of MRSA is observed in infected macrophages. This number continues to
rise to 70% reduction of MRSA CFU in infected macrophages after 24 h of treatment. Vancomycin, as
expected, was not able to reduce the presence of MRSA inside infected J774 cells, even after 24 h of
treatment. The results collectively indicate compound 5p has the ability to gain entry into infected
macrophage cells (after 12 h of treatment) at a concentration high enough to significantly reduce the
burden of MRSA.
Examining phenylthiazole compounds’ ability to re-sensitize vancomycin-resistant S. aureus (VRSA)
to the effect of vancomycin. Resistance to vancomycin in S. aureus isolates is an emerging problem and

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has been associated with increased thickness of the bacterial cell wall and acquisition of resistance
genes, including vanA.[18] Given vancomycin is viewed as a key therapeutic ally in the treatment of
MRSA infections, the emergence of VRSA strains is particularly concerning. Identifying antibacterial
compounds capable of re-sensitizing VRSA to the effect of vancomycin would provide one potential
solution to address this issue.
We moved to examine if VRSA isolates initially subjected to a subinhibitory concentration (½ × MIC)
of two of the most potent analogues for a short duration (30 min) would sensitize these isolates to the
effect of vancomycin. The cycloheptyl 5p proved to be superior to 5f at re-sensitizing VRSA to the
effect of vancomycin. 5p produces a 256-fold improvement in vancomycin’s MIC against VRSA10 and
a 16-fold improvement against VRSA4 (Table 3). Though 5f does generate a four-fold improvement in
vancomycin’s MIC against VRSA4, it is unable to re-sensitize VRSA10 to the effect of vancomycin (no
improvement
in
MIC
of
vancomycin
observed),
(Table
3).
Table 3. The minimum inhibitory concentration (MIC in µg/mL) of vancomycin in the absence and
presence of a subinhibitory concentration (½ × MIC) of compounds 5f or 5p against vancomycin-
resistant S. aureus (VRSA)
VRSA strain
MIC of
vancomycin
MIC of
vancomycin
+
Fold re-
sensitization of
VRSA to
vancomycin
16-fold
256-fold
MIC of
vancomycin
Fold re-
sensitization of
VRSA to
vancomycin
4-fold
+
5f
128
>128
5
p
VRSA4
VRSA10
512
512
32
2
None
Table 4. Evaluation of metabolic stability of tested compounds, verapamil, and warfarin, in human liver
microsomes
Tested
NADPH-dependent
CLint (µL/min-mg)
NADPH-
dependent
t1/2 (min)
28.8
> 240
> 240
> 240
15.3
> 240
NADPH-Free
CLint
(µL/min-mg)
0.5
< 9.6
< 9.6
< 9.6
< 9.6
< 9.6
NADPH-Free
T1/2 (min)
Note
compound
1
80.3
< 9.6
< 9.6
< 9.6
151
>60
High clearance
Low clearance
Low clearance
Low clearance
High clearance
Low clearance
5
5
5
c
e
p
>240
>240
>240
>240
>240
Verapamil
Warfarin
< 9.6
CLint = microsomal intrinsic clearance; t1/2 = half-life
Pharmacokinetic profiling. Thus far, the newly developed alkoxy derivatives displayed improved
antibacterial activity, broader spectrum activity that includes both Gram +ve and weak Gram –ve (data

ACCEPTED MANUSCRIPT
for Gram –ve is not presented in this article) and an enhanced safety profile in relation to the early
st
developed phenylthiazoles. The main drawbacks of the 1 developed generation were their high
microsomal clearance and their poor penetrability through biological barriers. These two factors
st
confined the 1 generation phenylthiazoles to topical applications.
The above mentioned treatment of intracellularly infected macrophages indicates that the alkoxy
st
derivatives, unlike the 1 generation of phenylthiazoles, have an acceptable ability to penetrate
biological membranes. Our final analysis centered around confirming that the incorporation of the
oxygen atom in the alkyl side chain did in fact enhance the stability of the analogues to hepatic
metabolism. To assess this, compounds 5c, 5e and 5p were incubated with HLM in the presence and
absence of the cofactor NADPH (Table 4). All tested compounds mimic the behavior of warfarin,
indicating the compounds are highly metabolically stable, as they were cleared very slowly by human
liver microsomes (CLint < 9.6 µL/min-mg) (Table 4). The compounds possess excellent half-lives of
over 4 hours (Table 4). It is unclear if the alkoxy-containing phenylthiazoles are still a substrate of CYP-
mediated metabolism (as with the previous generation of phenylthiazole containing the alkyl side chain)
given the clearance rate and half-life are the same in the presence and absence of the cofactor NADPH.
Future studies will focus on addressing this point further.
3
. Conclusion
rd
th
Though several 3 and 4 generation antibiotics have successfully reached the clinic in the last
decade, most of them have already fallen prey to bacterial resistance. This has put pressure on the
community of medicinal chemists to enrich the currently available arsenal of antibiotics with new
scaffolds that are not susceptible to traditional bacterial resistance mechanisms. The recently discovered
phenylthiazoles class of antibiotics exhibited promising antibacterial effect against several highly
resistant strains including MRSA and VRSA, but with limited systemic applicability due to their ultra-
short t1/2 and high microsomal clearance rate. Analysis of the in vivo metabolites of the lead compound
1
indicated the potential presence of a metabolic soft spot at the benzylic methylene carbon.
Replacement of this methylene unit with an oxygen atom afforded a new generation of

ACCEPTED MANUSCRIPT
alkoxyphenylthiazoles that exhibited enhanced stability to hepatic metabolism, in addition to highly
promising antimicrobial activity.
Taken collectively, the antimicrobial activity, cytotoxicity and PK profiling of the cycloheptyl
derivative 5p possess balanced properties (PD/PK/Tox.) that make it a good candidate for further
examination for treatment of systemic MRSA and VRSA infections. Briefly, 5p possessed a MIC value
against MRSA 2658 RCMB that was one-fold better than vancomycin. This compound maintained its
antimicrobial activity when tested against different drug-resistant S. aureus isolates, including VRSA.
Furthermore, 5p demonstrated one more advantage over vancomycin, and all other glycoprotein
antibiotics, with its ability to kill intracellular MRSA. The ability of 5p to re-sensitize VRSA to the
effect of vancomycin indicates this compound has potential to prolong the utility of vancomycin in the
clinic
4
4
4
. EXPERIMENTAL SECTION
.1. Chemistry.
1
13
.1.1.
General. H NMR spectra were run at 300 MHz and C spectra were determined at 100
MHz in deuterated chloroform (CDCl ), or dimethyl sulfoxide (DMSO-d ) on a Varian Mercury VX-
3
6
4
00 NMR 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). The infrared spectra were recorded in potassium bromide disks on pye Unicam SP
3
300 and Shimadzu FT IR 8101 PC infrared spectrophotometer. 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. HPLC analyses were performed on an Agilent binary HPLC system (Model 1260)
equipped with a multiple wavelength absorbance UV detector set for 254 nM, and using a 5 µM C-18
reversed-phase column and methanol:water (4:1) as a mobile phase. All yields reported refer to isolated
yields.

ACCEPTED MANUSCRIPT
4
.1.2.
4-Hydroxybenzothioamide (2). [19] 4-Hydroxybenzamide (3.6 g, 26.5 mmol) was
dissolved in dry THF (100 mL), and then Lawesson’s reagent (11.77 g, 29.1 mmol) was added. The
reaction mixture was stirred at room temperature for 6 h. The solvent was then evaporated under
reduced pressure and the residue was partitioned between aq. NaHCO (0.2 M, 50 mL) and ethyl acetate
3
(
50 mL × 2). The organic solvent was separated and dried over anhydrous MgSO . The crude product
4
was further purified by silica gel flash chromatography, using EtOAc: Petroleum ether (85:15) to yield
the corresponding thioamide as a yellow solid (3.5 g, 87%); m.p = 172-176 °C.
4
.1.3.
2-(4-Hydroxyphenyl)-4-methy-5-acetylthiazole (3). 4-Hydroxybenzothioamide 2,[19]
(1 g, 6.5 mmol) and α-chloroacetylacetone (1.1 mL, 1.22 g, 9.1 mmol) were added to absolute ethanol
(20 mL). The reaction mixture was heated at reflux for 5 h. After removal of solvent under reduced
pressure, the oil residue was purified by silica gel chromatography using EtOAc-hexane (3:7) to provide
1
a yellow solid (0.85 g, 85%). H NMR (CDCl ) δ: 7.83 (d, J = 9 Hz, 2 H), 6.88 (d, J = 8.9Hz, 2 H), 4.87
3
+
(
brs., 1 H), 2.67 (s, 3 H), 2.53 (s, 3 H); EIMS m/z (rel intensity) 233 (M ,100); Anal. Calcd for
C H NO S (233): C, 61.78; H, 4.75; N, 6.00; found: C, 61.66; H, 4.79; N, 6.03.
12
11
2
4
.1.4.
4-[2-(4-Substituted)oxyphenyl)-4-methy-5-acetylthiazole derivatives (4a-p). To a
solution of compound 3 (0.46 g, 2 mmol), in dry DMF (5 mL), and anhydrous potassium carbonate (0.5
g, 3.4 mmol) and, an appropriate bromoalkyl halide (2 equivalent) was added. The mixture was heated
for 12 h at 80 °C, and then the reaction mixture was poured over crushed ice, filtered and washed with
water to yield the desired product. The isolated crude materials were further purified by silica gel
chromatography, using EtOAc-hexane (3:7) to provide the desired products. The physical characteristics
and spectral data of separated products are listed below:
4
.1.4.1. 2-(4-Ethyloxyphenyl)-4-methy-5-acetylthiazole (4a). Yellow solid (0.41 g, 90%): mp =
°
-1 1
5
5 C; IR (KBr), 1647 cm ; H NMR (400 MHz, CDCl
Hz, 2 H), 4.07 (q, J = 6.8 Hz, 2 H), 2.76 (s, 3 H), 2.55 (s, 3 H), 1.44 (t, J = 6.6 Hz, 3 H); C NMR
CDCl ) δ 189.9, 170.9, 155.0, 148.5, 131.4, 129.6, 127.8, 126.6, 69.2, 22.3, 16.6, 10.1; EIMS m/z (rel
3
) δ: 7.91(d, J = 8.8 Hz, 2 H), 6.94 (d, J = 8.8
1
3
(
3

ACCEPTED MANUSCRIPT
+
2
intensity) 261 (M ,100); Anal. Calcd for C14H15NO S (261): C, 64.34; H, 5.79; N, 5.36; found: C,
6
4.39; H, 5.81; N, 5.33.
.1.4.2. 2-(4-Propoxyphenyl)-4-methy-5-acetylthiazole (4b). Yellow solid (0.45 g, 98%): mp =
4
°
-1 1
8
0 C; IR (KBr) 1650 cm ; H NMR (400 MHz, CDCl ) δ: 7.91 (d, J = 9.2 Hz, 2 H), 6.94 (d, J = 8.8
3
Hz, 2 H), 3.98 (t, J = 6.6 Hz, 2 H), 2.76 (s, 3 H), 2.56 (s, 3 H), 1.84 (p, J = 6.6 Hz, 2 H), 1.05 (t, J = 7.4
1
3
Hz, 3 H); C NMR (100 MHz, CDCl ) δ 189.8, 171.0, 155.5, 148.2, 131.5, 129.6, 127.7, 126.7, 68.0,
3
+
2
6
6.0, 22.1, 16.8, 10.7; EIMS m/z (rel intensity) 275 (M ,100); Anal. Calcd for C15
2
H17NO S (275): C,
5.43; H, 6.22; N, 5.09; found: C, 65.22; H, 6.26; N, 5.15.
4
.1.4.3. 2-(4-Butoxyphenyl)-4-methy-5-acetylthiazole (4c). Yellow solid (0.39 g, 85%): mp =
°
-1 1
7
7 C; IR (KBr), 1641 cm ; H NMR (400 MHz, CDCl ) δ: 7.91 (d, J = 8.9 Hz, 2 H), 6.94 (d, J = 8.9
3
Hz, 2 H), 4.02 (t, J = 6.5 Hz, 2 H), 2.76 (s, 3 H), 2.55 (s, 3 H), 1.79 (p, J = 6.4 Hz, 2H), 1.55-1.46 (m,
1
3
2
1
H), 0.98 (t, J = 7.4 Hz, 3 H); C NMR (100 MHz, CDCl ) δ 190.0, 169.5, 159.7, 146.2, 130.9, 130.9,
3
+
28.7, 126.7, 69.9, 33.3, 31.7, 26.0, 18.4, 12.0; EIMS m/z (rel intensity) 301 (M ,100); Anal. Calcd for
C H NO S (289): C, 66.41; H, 6.62; N, 4.84; found: C, 66.55; H, 6.60; N, 4.78.
16
19
2
4
.1.4.4. 2-(4-Pentyloxyphenyl)-4-methy-5-acetylthiazole (4d). Yellow solid (0.32 g, 70%): mp
°
-1 1
=
66 C; IR (KBr), 1646 cm ; H NMR (400 MHz, CDCl
3
) δ: 7.91 (d, J = 8.8 Hz, 2 H), 6.94 (d, J = 8.8
Hz, 2 H), 4.01 (t, J = 6.6 Hz, 2 H), 2.76 (s, 3 H), 2.54 (s, 3 H), 1.81 (p, J = 6.8 Hz, 2 H), 1.49-1.34 (m, 4
1
3
H), 0.94 (t, J = 7.1 Hz, 3 H); C NMR (100 MHz, CDCl ) δ 190.3, 169.6, 159.9, 146.6, 130.7, 130.2,
3
+
1
29.0, 126.7, 70.7, 33.3, 31.7, 30.6, 22.4, 18.4, 13.7; EIMS m/z (rel intensity) 303 (M ,100); Anal.
Calcd for C H NO S (303): C, 67.30; H, 6.98; N, 4.62; found: C, 67.40; H, 7.00; N, 4.56.
1
7
21
2
4
.1.4.5. 2-(4-Hexyloxyphenyl)-4-methy-5-acetylthiazole (4e). Yellow solid (0.32 g, 70%): mp
°
-1 1
=
62 C; IR (KBr), 1647 cm ; H NMR (400 MHz, CDCl
3
) δ: 7.89 (d, J = 8.9 Hz, 2 H), 6.93 (d, J = 8.9
Hz, 2 H), 3.99 (t, J = 6.6 Hz, 2 H), 2.76 (s, 3 H), 2.54 (s, 3 H), 1.79 (p, J = 6.8 Hz, 2 H), 1.59 (p, J = 7.2
13
Hz, 2 H), 1.36-1.31 (m, 4 H), 0.90 (t, J = 7 Hz, 3 H); C NMR (100 MHz, CDCl
3
) δ 190.1, 169.6,
1
59.1, 146.7, 131.3, 130.4, 130.1, 129.9, 126.9, 69.7, 31.7, 31.1, 30.3, 29.2, 22.5, 18.5, 13.8; EIMS m/z

ACCEPTED MANUSCRIPT
+
(
2
rel intensity) 317 (M ,100); Anal. Calcd for C18H23NO S (317): C, 68.11; H, 7.30; N, 4.41; found: C,
6
7.98; H, 7.35; N, 4.44.
.1.4.6. 2-(4-Heptyloxyphenyl)-4-methy-5-acetylthiazole (4f). Yellow solid (0.32 g, 70%): mp
4
°
-1 1
=
100 C; IR (KBr), 1671 cm ; H NMR (400 MHz, CDCl ) δ: 7.88(d, J = 8.8 Hz, 2 H), 6.92 (d, J = 8.8
3
Hz, 2 H), 3.98 (t, J = 6.6 Hz, 2 H), 2.74 (s, 3 H), 2.53 (s, 3 H), 1.79 (p, J = 6.4 Hz, 2 H), 1.48-1.27 (m, 8
1
3
H), 0.88 (t, J = 6.8 Hz, 3 H); C NMR (100 MHz, CDCl ) δ 190.2, 169.6, 159.2, 146.7, 131.3, 130.3,
3
1
3
30.2, 129.6, 126.9, 71.4, 31.7, 31.1, 30.7, 29.2, 29.21, 22.6, 18.3, 14.0; EIMS m/z (rel intensity)
+
31(M ,100); Anal. Calcd for C19
H25NO
2
S (331): C, 68.85; H, 7.60; N, 4.23; found: C, 68.97; H, 7.54;
N, 4.10.
.1.4.7. 2-(4-Octyloxyphenyl)-4-methy-5-acetylthiazole (4g). Yellow solid (0.32 g, 70%): mp =
4
0
-1 1
1
10 C; IR (KBr), 1676 cm ; H NMR (400 MHz, CDCl ) δ: 7.90 (d, J = 8.8 Hz, 2 H), 6.94 (d, J = 8.8
3
Hz, 2H), 4.08 (t, J = 6.6 Hz, 2 H), 2.75 (s, 3 H), 2.55 (s, 3H), 1.80 (p, J = 6.4 Hz, 2 H), 1.50-1.29 (m, 10
13
H), 0.88 (t, J = 6.8 Hz, 3 H); C NMR (100 Hz, CDCl ) δ 189.8, 169.4, 159.4, 146.7, 130.6, 130.3,
3
+
1
28.9, 126.7, 70.8, 33.8, 32.1, 30.7, 29.6, 29.2, 22.7, 18.9, 14.1; EIMS m/z (rel intensity) 345 (M ,100);
Anal. Calcd for C20
2
H27NO S (345): C, 69.53; H, 7.88; N, 4.05; found: C, 69.40; H, 7.95; N, 4.01.
4
.1.4.8. 2-(4-Nonyloxyphenyl)-4-methy-5-acetylthiazole (4h). Yellow solid (0.47 g, 95%): mp
°
-1 1
=
145 C; IR (KBr), 1670 cm ; H NMR (400 MHz, CDCl ) δ: 7.89 (d, J = 8.6 Hz, 2 H), 6.93 (d, J = 8.6
3
Hz, 2 H), 3.99 (t, J = 6.6 Hz, 2 H), 2.75 (s, 3 H), 2.54 (s, 3 H), 1.62 (p, J = 6.8 Hz, 2 H), 1.62 (p, J = 6.8
1
3
Hz, 2 H), 1.49-1.28 (m, 10 H), 0.88 (t, J = 6.8 Hz, 3 H); C NMR (100 MHz, CDCl ) δ 190.2, 169.6,
3
1
59.4, 146.6, 130.7, 130.3, 129.0, 126.8, 71.6, 33.8, 32.1, 30.7, 29.4, 29.3, 29.2, 22.6, 18.5, 14.0; EIMS
+
m/z (rel intensity) 359 (M ,100); Anal. Calcd for C H NO S (359): C, 70.16; H, 8.13; N, 3.90; found:
2
1
29
2
7
0.06; H, 8.15; N, 3.93.
.1.4.9. 2-(4-Isobutoxyphenyl)-4-methy-5-acetylthiazole (4i). Yellow solid (0.37 g, 80%): mp
4
°
-1 1
=
3
70 C; IR (KBr), 1635 cm ; H NMR (400 MHz, CDCl ) δ: 7.91 (d, J = 8.9 Hz, 2 H), 6.94 (d, J = 8.9
Hz, 2 H), 3.77 (d, J = 6.5 Hz, 2 H), 2.76 (s, 3 H), 2.56 (s, 3 H), 2.17-2.14 (m, 1 H), 1.04 (d, J = 6.7 Hz, 6

ACCEPTED MANUSCRIPT
+
H); EIMS m/z (rel intensity) 289 (M ,100); Anal. Calcd for C16
H19NO
2
S (289): C, 66.41; H, 6.62; N,
4
.84; found: C, 66.48; H, 6.65; N, 4.79.
.1.4.10. 2-(4-Isopentoxyphenyl)-4-methy-5-acetylthiazole (4j). Yellow solid (0.39 g, 85%): mp
4
°
-1 1
=
72 C; IR (KBr) 1639 cm ; H NMR (400 MHz, CDCl ) δ: 7.91 (d, J = 8.9 Hz, 2 H), 6.94 (d, J = 8.9
3
Hz, 2 H), 4.04 (t, J = 6.6 Hz, 2 H), 2.76 (s, 3 H), 2.55 (s, 3 H), 1.89-1.86 (m, 1 H), 1.70 (q, J = 6.7 Hz, 2
+
H), 0.96 (d, J = 6.6 Hz, 6 H); EIMS m/z (rel intensity) 303 (M ,100); Anal. Calcd for C H NO S
1
7
21
2
(303): C, 67.30; H, 6.98; N, 4.62; found: C, 67.42; H, 6.97; N, 4.57.
4
.1.4.11. 2-(4-Methylpentyloxyphenyl)-4-methy-5-acetylthiazole (4k). Yellow solid (0.64 g,
°
-1 1
9
3
8%): mp = 73 C; IR (KBr), 1644 cm ; H NMR (400 MHz, CDCl ) δ: 7.90 (d, J = 8.8 Hz, 2 H), 6.94
(d, J = 8.8 Hz, 2 H), 3.99 (t, J = 6.8 Hz, 2 H), 2.75 (s, 3 H), 2.55 (s, 3 H), 1.8 (p, J = 6.8 Hz, 2 H), 1.65-
+
1
.56 (m, 2 H), 1.36-1.33 (m,1 H), 0.92 (d, J = 6.8 Hz, 6 H); EIMS m/z (rel intensity) 317 (M ,100);
Anal. Calcd for C H NO S (317): C, 68.11; H, 7.30; N, 4.41; found: C, 68.09; H, 7.33; N, 4.39.
1
8
23
2
4
.1.4.12. 2-(4-(2-Ethylbutyloxyphenyl)-4-methy-5-acetylthiazole (4l). Yellow solid (0.39 g,
°
-1 1
8
5%): mp = 40 C; IR (KBr), 1662 cm ; H NMR (400 MHz, CDCl ) δ: 7.89 (d, J = 8.8 Hz, 2 H), 6.94
3
(d, J = 8.8 Hz, 2 H), 3.89 (d, J = 6.0 Hz, 2 H), 2.75 (s, 3 H), 2.54 (s, 3 H), 1.68 (p, J = 6 Hz, 1 H), 1.53-
+
1
.41 (m, 4 H), 0.93 (t, J =7.6 Hz, 6 H); EIMS m/z (rel intensity) 317 (M ,100); Anal. Calcd for
C H NO S (317): C, 68.11; H, 7.30; N, 4.41; found: C, 67.99; H, 7.29; N, 4.36.
18
23
2
4
.1.4.13. 2-(4-(3-Butenyloxy)phenyl)-4-methy-5-acetylthiazole (4m). Yellow solid (0.32 g,
°
-1 1
7
0%): mp = 65 C; IR (KBr), 1647 cm ; H NMR (400 MHz, CDCl ) δ: 7.91 (d, J = 9.2 Hz, 2 H), 6.92
3
(d, J = 9.2 Hz, 2 H), 5.90 (m, 1 H), 5.15 (dd, J = 1.6, 6.0 Hz, 2 H), 4.07 (t, J = 6.4 Hz, 2H), 2.76 (s, 3
+
H), 2.55 (s, 3 H); EIMS m/z (rel intensity) 287 (M ,100); Anal. Calcd for C H NO S (287): C, 66.87;
1
6
17
2
H, 5.96; N, 4.87; found: C, 67.07; H, 5.88; N, 4.60.
.1.4.14. 2-(4-Cyclobutylmethoxyphenyl)-4-methy-5-acetylthiazole (4n). Yellow solid (0.41 g,
4
°
-1 1
7
3
0%): mp = 72 C; IR (KBr), 1645 cm ; H NMR (400 MHz, CDCl ) δ: 7.89 (d, J = 8.8 Hz, 2 H), 6.93
1
3
(
d, J = 8.8 Hz, 2 H), 3.97 (t, J = 6.4 Hz, 2 H), 2.75 (s, 3 H), 2.54 (s, 3 H), 2.18-1.83 (m, 7 H); C NMR
(
100 MHz, CDCl ) δ 189.9, 171.0, 155.4, 148.1, 131.7, 129.9, 127.6, 126.7, 71.2, 34.0, 24.5, 22.1, 18.0;
3

ACCEPTED MANUSCRIPT
+
2
EIMS m/z (rel intensity) 301 (M ,100); Anal. Calcd for C17H19NO S (301): C, 67.75; H, 6.35; N, 4.65;
found: C, 67.91; H, 6.36; N, 4.54.
4
.1.4.15. 2-(4-Cyclopentyloxyphenyl)-4-methy-5-acetylthiazole (4o). Yellow solid (0.41 g,
°
-1 1
9
0%): mp = 65 C; IR (KBr), 1635 cm ; H NMR (400 MHz, CDCl ) δ: 7.89 (d, J = 8.8 Hz, 2 H), 6.94
3
(d, J = 8.8 Hz, 2 H), 4.55 (p, J = 2.4 Hz, 1 H), 2.76 (s, 3H), 2.55 (s, 3 H), 1.95-1.56 (m, 8 H); EIMS m/z
+
(
rel intensity) 301 (M ,100); Anal. Calcd for C H NO S (301): C, 67.75; H, 6.35; N, 4.65; found: C,
1
7
19
2
6
7.60; H, 6.39; N, 4.77.
.1.4.16. 2-(4-Cycloheptyloxyphenyl)-4-methy-5-acetylthiazole (4p). Yellow solid (0.41 g,
4
°
-1 1
9
3
0%): mp = 84 C; IR (KBr), 1658 cm ; H NMR (400 MHz, CDCl ) δ: 7.89 (d, J = 8.8 Hz, 2 H), 6.94
(d, J = 8.8 Hz, 2 H), 4.54 (p, J = 4 Hz, 1 H), 2.76 (s, 3 H), 2.55 (s, 3 H), 2.06-1.61 (m, 12 H); EIMS m/z
+
(
rel intensity) 329 (M ,100); Anal. Calcd for C H NO S (329): C, 69.27; H, 7.04; N, 4.25; found: C,
1
9
23
2
6
9.45; H, 7.00; N, 4.19.
.1.5. 4-[2-(4-Substituted)oxyphenyl)-5-aminoguanidine-4-mathylthiazole derivatives (5a–
4
p). To a solution of the appropriate derivative of compound 4 (2 mmol) in absolute ethanol (5 mL),
aminoguanidine HCl (0.22 g, 2 mmol), anhydrous potassium carbonate (0.5 g, 3.4 mmol), anhydrous
lithium chloride (0.02 g) were added. The reaction mixture was heated at reflux for 12 h, and then
poured over crushed ice, filtered and washed with water to yield the desired product as crude solids.
Recrystallization from EtOH (95%) afforded the final products. The physical characteristics and spectral
data of separated products are listed below:
4
.1.5.1. 2-(1-(2-(4-Ethoxyphenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
°
1
carboximidamide (5a). Yellowish brown solid (0.27 g, 90%): mp = 270 C. H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.81 (d, J = 8.4 Hz, 2 H), 6.97 (d, J = 8.4 Hz, 2 H), 4.03 (q, J = 6.8 Hz, 2 H),
6
,
3
2
+
2
.57 (s, 3 H), 2.32 (s, 3 H), 1.29 (t, J = 6.6 Hz, 3 H); EIMS m/z (rel intensity): 318 (M ,100); HRMS
+
(
EI), m/z 317.1291 M , calcd for C H N OS 317.1310; HPLC purity 95.06%.
1
5
19
5
4
.1.5.2. 2-(1-(4-Methyl-2-(4-propoxyphenyl)thiazol-5-yl)ethylidene)hydrazine-1-
°
-1 1
carboximidamide (5b). Yellowish brown solid (0.21 g, 70%): mp > 300 C; IR (KBr) 1635 cm ; H

ACCEPTED MANUSCRIPT
3 2
NMR (400 MHz, DMSO-d6, CF CO D) δ: 7.82 (d, J = 8.8 Hz, 2 H), 7.01 (d, J = 8.8 Hz, 2 H), 3.97 (t, J
=
6.4 Hz, 2 H), 2.57 (s, 3 H), 2.34 (s, 3 H), 1.77-1.59 (m, 2 H), 0.96 (t, J = 7.2 Hz, 3 H); EIMS m/z (rel
+
+
intensity) 331 (M ,100); HRMS (EI), m/z 331.1481 M , calcd for C H N OS 331.1467; HPLC purity
1
6
21
5
9
7.17%.
.1.5.3. 2-(1-(2-(4-Butoxyphenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
4
°
1
carboximidamide (5c). Yellowish brown solid (0.25 g, 85%): mp = 300 C; H NMR (400 MHz,
DMSO-d6, CF CO D) δ: 7.82 (d, J = 8.8 Hz, 2 H), 7.01 (d, J = 8.8 Hz, 2 H), 4.02 (t, J = 6.4 Hz, 2 H),
.57 (s, 3 H), 2.33 (s, 3 H), 1.68 (p, J = 6.4 Hz , 2 H), 1.46-1.36 (m, 2 H), 0.90 (t, J = 7.6 Hz, 3 H);
3
2
2
+
+
23 5
EIMS m/z (rel intensity) 345 (M ,100); HRMS (EI), m/z 345.1623 M , calcd for C17H N OS 345.1623;
HPLC purity 95.20%.
4
.1.5.4. 2-(1-(4-Methyl-2-(4-(pentyloxy)phenyl)thiazol-5-yl)ethylidene)hydrazine-1-
°
1
carboximidamide (5d). Yellowish brown solid (0.21 g, 70%): mp = 165 C; H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.81 (d, J = 8.8 Hz, 2 H), 6.99 (d, J = 8.8 Hz, 2 H), 3.99 (t, J = 6.4 Hz, 2 H),
6
,
3
2
2
.52 (s, 3 H), 2.34 (s, 3 H), 1.70 (p, J = 6.4 Hz, 2 H), 1.40-1.28 (m, 4 H), 0.94 (t, J =7.1 Hz, 3 H); EIMS
+
+
25 5
m/z (rel intensity) 359 (M ,100); HRMS (EI), m/z 359.1776 M , calcd for C18H N OS 359.1780; HPLC
purity 95.70%.
4
.1.5.5. 2-(1-(2-(4-(Hexyloxy)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
°
1
carboximidamide (5e). Yellowish brown solid (0.23 g, 78%): mp = 200 C; H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.82 (d, J = 8.9 Hz, 2 H), 7.01 (d, J = 8.9 Hz, 2 H), 3.00 (t, J = 6.6 Hz, 2 H),
6
,
3
2
2
.57 (s, 3 H), 2.35 (s, 3 H), 1.68 (p, J = 6.8 Hz, 2H), 1.41-1.27 (m, 6 H), 0.85 (t, J = 6.8 Hz, 3 H); EIMS
+
+
m/z (rel intensity) 373 (M ,100); HRMS (EI), m/z 373.1957 M , calcd for C H N OS 373.1936; HPLC
19
27
5
purity 96.01%.
.1.5.6. 2-(1-(2-(4-(Heptyloxy)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
4
°
1
carboximidamide (5f). Yellowish brown solid (0.21 g, 70%): mp = 230 C. H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.88 (d, J = 8.8 Hz, 2 H), 7.01 (d, J = 8.8 Hz, 2 H), 4.00 (t, J = 6.4 Hz, 2 H),
6
,
3
2
2
.57 (s, 3 H), 2.34 (s, 3 H), 1.72 (p, J = 6.4 Hz, 2 H), 1.40-1.20 (m, 8 H), 0.84 (t, J = 6.8 Hz, 3 H); EIMS

ACCEPTED MANUSCRIPT
+
+
m/z (rel intensity) 388 (M ,100); HRMS (EI), m/z 387.2111 M , calcd for C20
H
29
N
5
OS 387.2093;
HPLC purity 95.09%.
4
.1.5.7. 2-(1-(4-Methyl-2-(4-(octyloxy)phenyl)thiazol-5-yl)ethylidene)hydrazine-1-
°
1
carboximidamide (5g). Yellowish brown solid (0.28 g, 95%): mp = 200 C. H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.81 (d, J = 7.2 Hz, 2 H), 7.01 (d, J = 7.2 Hz, 2 H), 4.08 (t, J = 6.6 Hz, 2 H),
6
,
3
2
+
2
.56 (s, 3 H), 2.33 (s, 3H), 1.69-0.89 (m, 15 H); EIMS m/z (rel intensity) 402 (M ,100); HRMS (EI),
+
m/z 401.2236 M , calcd for C21
H
31
N
5
OS 401.2249; HPLC purity 95.45%.
4
.1.5.8. 2-(1-(4-Methyl-2-(4-(nonyloxy)phenyl)thiazol-5-yl)ethylidene)hydrazine-1-
°
1
carboximidamide (5h). Yellowish brown solid (0.28 g, 95%): mp = 208 C. H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.84 (m, 2 H), 7.01 (m, 2 H), 4.00 (t, J = 6.4 Hz, 2 H), 2.56 (s, 3 H), 2.41 (s, 3
6
,
3
2
+
H), 1.33-1.20 (m, 14 H), 0.88 (t, J = 6.4 Hz, 3 H); EIMS m/z (rel intensity) 415 (M ,100); HRMS (EI),
+
m/z 415.2389 M , calcd for C H N OS 415.2406; HPLC purity 95.42%.
2
2
33
5
4
.1.5.9. 2-(1-(2-(4-isobutoxyphenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
°
1
carboximidamide (5i). Yellowish brown solid (0.24 g, 80%): mp = 265 C.; H NMR (400 MHz,
DMSO-d , CF CO D) δ: 7.91 (d, J = 8.9 Hz, 2 H), 6.94 (d, J = 8.9 Hz, 2 H), 3.77 (d, J = 6.5 Hz, 2 H),
.57 (s, 3 H), 2.43 (s, 3 H), 2.17-2.14 (m, 1 H), 1.04 (d, J = 6.7 Hz, 6 H); EIMS m/z (rel intensity) 345
6
3
2
2
+
+
(
M ,100); HRMS (EI), m/z 345.1633 M , calcd for C H N OS 345.1623; HPLC purity 95.10%.
17 23 5
4
.1.5.10. 2-(1-(2-(4-(Isopentyloxy)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
°
1
carboximidamide (5j). Yellowish brown solid (0.25 g, 85%): mp = 275 C; H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.82 (d, J = 8.9 Hz, 2 H), 7.03 (d, J = 8.9 Hz, 2 H), 4.05 (t, J = 6.4 Hz, 2 H),
6
,
3
2
2
.57 (s, 3 H), 2.34 (s, 3 H), 1.80-1.72 (m, 1 H), 1.61 (q, J = 6.7 Hz, 2 H), 0.96 (d, J = 6.6 Hz, 6 H);
+
+
27 5
EIMS m/z (rel intensity) 373 (M ,100); HRMS (EI), m/z 373.1914 M , calcd for C19H N OS 373.1936;
HPLC purity 95.35%.
4
.1.5.11. 2-(1-(4-Methyl-2-(4-((4-methylpentyl)oxy)phenyl)thiazol-5-yl)ethylidene)hydrazine-
°
1
1
-carboximidamide (5k). Yellowish brown solid (0.64 g, 98%): mp = 228 C. H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.81 (d, J = 8.8 Hz, 2 H), 7.07 (d, J = 8.8 Hz, 2 H), 3.98 (t, J = 6.8 Hz, 2 H),
6
,
3
2

ACCEPTED MANUSCRIPT
2
.56 (s, 3 H), 2.34 (s, 3 H), 1.70 (m, 4 H), 1.55 (m, 1 H), 0.85 (d, J = 6.4 Hz, 6 H); EIMS m/z (rel
+
+
intensity) 373 (M ,100); HRMS (EI), m/z 373.1944 M , calcd for C H N OS 373.1936; HPLC purity
1
9
27
5
9
6.18%.
.1.5.12. 2-(1-(2-(4-(2-Ethylbutoxy)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
4
°
1
carboximidamide (5l). Yellowish brown solid (0.25 g, 85%): mp = 210 C. H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.82 (d, J = 8.4 Hz, 2 H), 7.01 (d, J = 8.4 Hz, 2 H), 3.89 (d, J = 5.6 Hz, 2 H),
6
,
3
2
2
.57 (s, 3 H), 2.33 (s, 3 H), 1.65-1.56 (m, 1 H), 1.39 (p, J = 7.2 Hz, 4 H), 0.86 (t, J =7.2 Hz, 6 H); EIMS
+
+
27 5
m/z (rel intensity) 374 (M ,100); HRMS (EI), m/z 373.1942 M , calcd for C19H N OS 373.1936; HPLC
purity 96.44%.
4
.1.5.13. 2-(1-(2-(4-(But-3-en-1-yloxy)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
1
carboximidamide (5m). Yellowish brown solid (0.19 g, 65%): mp = 250 °C. H NMR (400 MHz,
DMSO-d6, CF CO D) δ: 7.82 (d, J = 8.8 Hz, 2 H), 7.02 (d, J = 8.8 Hz, 2 H), 5.84 (m, 1 H), 5.10 (dd, J =
.6, 0.8 Hz, 2 H) , 4.01 (t, J = 6.4 Hz, 2H), 2.57 (s, 3 H), 2.34 (s, 3 H); EIMS m/z (rel intensity) 343
3
2
6
+
+
(
M ,100); HRMS (EI), m/z 343.1485 M , calcd for C H N OS 343.1467; HPLC purity 95.98%.
17 21 5
4
.1.5.14. 2-(1-(2-(4-(Cyclobutylmethoxy)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-
°
1
1
-carboximidamide (5n). Yellowish brown solid (0.41 g, 70%): mp = 262 C. H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.81 (d, J = 8.4 Hz, 2 H), 7.00 (d, J = 8.4 Hz, 2 H), 3.97 (t, J = 6.4 Hz, 2 H),
6
,
3
2
+
2
3
.56 (s, 3 H), 2.33 (s, 3 H), 2.05-1.77 (m, 7 H); EIMS m/z (rel intensity) 357 (M ,100); HRMS (EI), m/z
+
57.1638 M , calcd for C18
H
23
N
5
OS 357.1623; HPLC purity 95.10%.
4
.1.5.15. 2-(1-(2-(4-(Cyclopentyloxy)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
°
1
carboximidamide (5o). Yellowish brown solid (0.41 g, 90%): mp = 266 C. H NMR (400 MHz,
DMSO-d CF CO D) δ: 7.80 (d, J = 8.8 Hz, 2 H), 6.97 (d, J = 8.8 Hz, 2 H), 4.84 (p, J = 2.4 Hz, 1 H),
6
,
3
2
+
2
3
.57 (s, 3 H), 2.36 (s, 3H), 1.95-1.56 (m, 8 H); EIMS m/z (rel intensity) 357 (M ,100). HRMS (EI), m/z
+
57.1640 M , calcd for C H N OS 357.1623; HPLC purity 95.12%.
1
8
23
5
4
.1.5.16. 2-(1-(2-(4-(Cycloheptyloxy)phenyl)-4-methylthiazol-5-yl)ethylidene)hydrazine-1-
°
1
carboximidamide (5p). Yellowish brown solid (0.41 g, 90%): mp = 265 C. H NMR (400 MHz,

ACCEPTED MANUSCRIPT
DMSO-d6, CF
3
CO
2
D) δ: 7.80 (d, J = 8.8 Hz, 2 H), 6.97 (d, J = 8.8 Hz, 2 H), 4.57 (m, 1 H), 2.57 (s, 3 H),
+
2
.34 (s, 3 H), 1.98-1.42 (m, 12 H); EIMS m/z (rel intensity) 385 (M ,100); HRMS (EI), m/z 385.1920
+
M , calcd for C H N OS 385.1936; HPLC purity 97.0%.
2
0
27
5
4
4
.2. Antimicrobial Testing
.2.1. Determination of Minimum Inhibitory Concentration (MIC) and Minimum
Bactericidal Concentration (MBC). Clinical isolates of MRSA and vancomycin-resistant
Staphylococcus aureus (VRSA) were obtained through the Network of Antimicrobial Resistance in
Staphylococcus aureus (NARSA) program.
The MIC of the newly synthesized compounds, linezolid (Selleck Chemicals, Houston, TX, USA),
and vancomycin (Gold Biotechnology, St. Louis, MO, USA) was determined using the broth
microdilution method in accordance with recommendations contained in the Clinical and Laboratory
Standards Institute guidelines.[20] Bacteria were cultured in Mueller-Hinton broth (Hardy Diagnostics,
Santa Maria, CA, USA) in a 96-well plate. Compounds, using triplicate samples, were added to the plate
and serially diluted. Plates were incubated at 37 °C for 18-20 h prior to determining the MIC. Plates
were visually inspected and the MIC was categorized as the concentration where no visible growth of
bacteria was observed. The minimum bactericidal concentration (MBC) of the active compounds was
tested by plating 5 µL from wells with no growth onto Tryptic soy agar plates. Plates were incubated at
3
7 ºC for 24 h before recording the MBC.
4
.2.2.
Time-kill assay. MRSA USA400 cells, in logarithmic growth phase (OD600 = 1.050),
6
were diluted to 3.76 × 10 colony-forming units (CFU/mL) and exposed to concentrations equivalent to
4
.0 × MIC (in triplicate) of tested compounds or vancomycin in Tryptic soy broth (TSB) (Becton,
Dickinson and Company, Sparks, MD, USA). Aliquots (100 µL) were taken from each sample after 0,
, 4, 6, 8, 10, 12, and 24 h, serially diluted in phosphate-buffered saline, and transferred to Trypticase
soy agar (TSA) (Becton, Dickinson and Company, Sparks, MD, USA) plates. Plates were incubated at
7 °C for at least 16 h before counting viable CFU/mL to determine the time required to reduce the
bacterial cell count by 3-log .
2
3
1
0

ACCEPTED MANUSCRIPT
4
.2.3.
In vitro cytotoxicity analysis. Human keratinocytes (HaCaT) and murine macrophage
(J774.A1) cells were cultured in Dulbeco’s modified Eagle’s medium (Sigma-Aldrich, St. Louis, MO,
USA) with 10% fetal bovine serum (USA Scientific, Inc., Ocala, FL, USA) at 37 °C with 5% CO2.
Controls received DMSO alone at a concentration equal to that in drug-treated cell samples. The cells
were incubated with compounds in a 96-well tissue-culture treated plate at 37 ºC and 5.0% CO
h prior to addition of the assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-
-(4-sulfophenyl)-2H-tetrazolium) (Promega, Madison, WI, USA). Absorbance readings were taken
2
for two
2
using the SpectraMax i3 Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA). The
quantity of viable cells after treatment with each compound was expressed as a percentage of the
viability of DMSO-treated control cells (average of triplicate wells ± standard deviation). The toxicity
data was analyzed via a one-way ANOVA, with post hoc Dunnet’s multiple comparisons test (P <
0
.05), utilizing GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).
.2.4. Intracellular infection of J774 cells with MRSA and treatment with phenylthiazole
4
compound 5p. Eradication of intracellular MRSA: Compounds 5f and 5p were initially assayed (at
concentrations of 8, 16, 32, and 64 µg/mL) against a murine macrophage (J774) cell line to determine
the potential toxic effect in vitro, as explained above. Compound 5p was selected for further analysis
given its superior toxicity profile. Murine macrophage cells (J774) were cultured in Dulbecco’s
Modified Eagle’s Medium (DMEM) supplemented with 10% FBS at 37 °C with CO (5%). J774 cells
2
were exposed to MRSA USA400 cells at a multiplicity of infection of approximately 100:1. One h post-
infection, J774 cells were washed with gentamicin (50 µg/mL) to kill extracellular MRSA. Compound
5
p and vancomycin, at a concentration equal to 16 µg/mL, were added. At specified time points (4, 8,
1
2, and 24 h), the test agents were removed; J774 cells were washed with gentamicin (50 µg/mL) and
subsequently lysed using 0.1% Triton-X 100. The solution was serially diluted in phosphate-buffered
saline and transferred to Tryptic soy agar plates in order to enumerate the viable number of MRSA
colony-forming units (CFU) present inside the J774 cells. Plates were incubated at 37 ºC for 18-22 h
before counting viable CFU/mL. Data are presented as percent reduction of MRSA CFU/mL in infected

ACCEPTED MANUSCRIPT
J774 cells in relation to the untreated control. The data was analyzed via a t-test (P < 0.05), utilizing
GraphPad Prism 6.0 (GraphPad Software, La Jolla, CA).
4
.2.5.
Examining phenylthiazole compounds’ ability to re-sensitize vancomycin-resistant
S. aureus (VRSA) to the effect of vancomycin. Mueller-Hinton broth was inoculated with either
5
VRSA4, or VRSA10 (5×10 CFU/mL), as described elsewhere in a previous study.[12] Aliquots (5 mL)
of the bacterial suspension were divided into microcentrifuge tubes and compounds 5f or 5p (at ½ ×
MIC) was introduced into each tube. After sitting at room temperature for 30 min, samples (1 mL) from
each tube were transferred to a new centrifuge tube prior to addition of vancomycin (at a concentration
equivalent to 128 µg/mL). Using a 96-well microtiter plate, rows 2-12 were filled with the remaining 4
mL bacterial suspension (containing the compound). 200-µL aliquots from tubes containing both the
compound and vancomycin were transferred to row 1 of the 96-well plate. After aspirating contents in
the first row 4-6 times, 100 µL was transferred from wells in row 1 to row 2. This process was repeated
to dilute the remaining wells containing no antibiotic. Untreated bacteria served as a control. The plate
was incubated at 37ºC for 20-22 h before the MIC was recorded. The MIC was categorized as the
concentration at which no visible growth of bacteria was observed in a particular well. A fold reduction
was calculated by comparing the MIC of vancomycin alone compared to the MIC of the antibiotic given
in combination with either 5f or 5p.
4
4
.3. Pharmacokinetic assays
.3.1. Human Microsomal Stability Analysis. The tested compounds were incubated in
duplicate with human liver microsomes at 37 ºC. The reaction contained microsomal protein in 100 mM
potassium phosphate, 2 mM NADPH, 3 mM MgCl , pH 7.4. A control was run for each test agent
2
omitting NADPH to detect NADPH-free degradation. At 0, 10, 20, 40, and 60 min, an aliquot was
removed from each experimental and control reaction and mixed with an equal volume of ice-cold Stop
Solution (methanol containing haloperidol, diclofenac, or other internal standard). Stopped reactions
were incubated at least ten minutes at -20 ºC, and an additional volume of water was added. The
samples were centrifuged to remove precipitated protein, and the supernatants were analyzed by

ACCEPTED MANUSCRIPT
LC/MS/MS to quantitate the remaining parent. Data were converted to % remaining by dividing by the
time zero concentration value. Data were then fit to a first-order decay model in order to determine the
half-life. Intrinsic clearance values were calculated from the half-life and the protein concentrations
using the following equation: CL = ln(2) /(T [microsomal protein]).
int
1/2
4
.3.2.
In Vivo Metabolic Study. This assay has been conducted at GRC bioequivalence center
(http://www.grc-me.com/pk_pd.html). Metabolites were studied using a male naïve Sprague−Dawley
(SD) rat, following Institutional Animal Care and Use Committee guidelines. Oral dosing of 50 mg/Kg
was administered by oral gavage in a vehicle containing 25% DMDO, 25% PEG 400, and 50% water.
The animal was kept in a cage supported with a urine collector and the urine was collected over 24 h. A
blood sample was taken after 6 h post dose into Vacutainer tubes containing EDTA-K2. Plasma was
isolated, and the concentration of metabolites relative to compound 1, was determined with LC/MS/MS
after extraction with ethyl acetate.
ASSOCIATED CONTENT
Supporting Information.
1
13
Copies of H NMR and C NMR are available free of charge via the Internet.
Author Contributions
∥
These two authors contributed equally to this work
Funding Sources.
This work was supported by US-Egypt Joint Grant, Grant ID# USC17:241 from both National Academy
of
Sciences
(NAS),
Washington,
USA,
and
the
Science
&
Technology Development Funds (STDF), Egypt.
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