Synthesis and antibacterial evaluation of a novel series of synthetic phenylthiazole compounds against methicillin-resistant Staphylococcus aureus (MRSA)

Article abstract of DOI:10.1016/j.ejmech.2015.03.015

Methicillin-resistant Staphylococcus aureus infections are a significant global health challenge in part due to the emergence of strains exhibiting resistance to nearly all classes of antibiotics. This underscores the urgent need for the rapid development of novel antimicrobials to circumvent this burgeoning problem. Previously, whole-cell screening of a library of 2,5-disubstituted thiazole compounds revealed a lead compound exhibiting potent antimicrobial activity against MRSA. The present study, conducting a more rigorous analysis of the structure-activity relationship of this compound, reveals a nonpolar, hydrophobic functional group is favored at thiazole-C2 and an ethylidenehydrazine-1-carboximidamide moiety is necessary at C5 for the compound to possess activity against MRSA. Furthermore, the MTS assay confirmed analogs 5, 22d, and 25 exhibited an improved toxicity profile (not toxic up to 40 μg/mL to mammalian cells) over the lead 1. Analysis with human liver microsomes revealed compound 5 was more metabolically stable compared to the lead compound (greater than eight-fold improvement in the half-life in human liver microsomes). Collectively the results presented demonstrate the novel thiazole derivatives synthesized warrant further exploration for potential use as future antimicrobial agents for the treatment of multidrug-resistant S. aureus infections.

Full text of DOI:10.1016/j.ejmech.2015.03.015

European Journal of Medicinal Chemistry 94 (2015) 306e316
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and antibacterial evaluation of a novel series of synthetic
phenylthiazole compounds against methicillin-resistant
Staphylococcus aureus (MRSA)
Haroon Mohammad a, P.V. Narasimha Reddy b, Dennis Monteleone b,
,
*
Abdelrahman S. Mayhoub c, Mark Cushman b, Mohamed N. Seleem a
a Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, IN 47907, USA
b Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University College of Pharmacy, and the Purdue Center for Cancer Research,
West Lafayette, IN 47907, USA
c Department of Organic Chemistry, Al Azhar University, Cairo 11884, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Methicillin-resistant Staphylococcus aureus infections are a significant global health challenge in part due
to the emergence of strains exhibiting resistance to nearly all classes of antibiotics. This underscores the
urgent need for the rapid development of novel antimicrobials to circumvent this burgeoning problem.
Previously, whole-cell screening of a library of 2,5-disubstituted thiazole compounds revealed a lead
compound exhibiting potent antimicrobial activity against MRSA. The present study, conducting a more
rigorous analysis of the structureeactivity relationship of this compound, reveals a nonpolar, hydro-
phobic functional group is favored at thiazole-C2 and an ethylidenehydrazine-1-carboximidamide
moiety is necessary at C5 for the compound to possess activity against MRSA. Furthermore, the MTS
Received 20 January 2015
Received in revised form
4 March 2015
Accepted 6 March 2015
Available online 7 March 2015
Keywords:
Antimicrobials
Drug-resistance
assay confirmed analogs 5, 22d, and 25 exhibited an improved toxicity profile (not toxic up to 40 mg/mL
to mammalian cells) over the lead 1. Analysis with human liver microsomes revealed compound 5 was
more metabolically stable compared to the lead compound (greater than eight-fold improvement in the
half-life in human liver microsomes). Collectively the results presented demonstrate the novel thiazole
derivatives synthesized warrant further exploration for potential use as future antimicrobial agents for
the treatment of multidrug-resistant S. aureus infections.
Methicillin-resistant Staphylococcus aureus
(MRSA)
Thiazole compounds
HEK293 toxicity
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
children [13,14]. Moreover, CA-MRSA infections are typically asso-
ciated with more severe morbidity and mortality than their HA-
Methicillin-resistant Staphylococcus aureus (MRSA) infections
remain a significant public health challenge globally. Though re-
ports have indicated the incidence of healthcare-associated MRSA
(HA-MRSA) infections have diminished [1,2], transmission of
community-associated MRSA (CA-MRSA) infections, primarily
strains USA300 and USA400 [3], has continued to present major
problems amongst a diverse population including healthcare
workers [4], prison inmates [5,6], military service personnel [6],
contact sport athletes [7,8], homeless individuals [9], intravenous
drug users [9,10], tattoo recipients [11], neonates [12], and young
MRSA counterparts [15]. While CA-MRSA is a leading cause of
skin and soft-tissue infections [16,17], MRSA has also been associ-
ated with more complicated medical diseases including necrotizing
pneumonia [18], osteomyelitis [19], and sepsis [20], leading to over
11,000 deaths annually [21].
A recent study has estimated the total annual burden upon so-
ciety for treatment of CA-MRSA infections alone may exceed US$13
billion [22]. Part of the associated cost is due to failure of current
antimicrobials to treat certain clinical isolates of MRSA that have
developed resistance to these therapeutic agents. Indeed, clinical
isolates of both CA-MRSA and HA-MRSA have been documented
that exhibit resistance to an array of different antibiotic classes
* Corresponding author. Department of Comparative Pathobiology, Purdue Uni-
versity College of Veterinary Medicine, 625 Harrison St., West Lafayette, IN 47907,
USA.
including the b-lactams [23], macrolides [24], quinolones [25,26],
tetracyclines [27], and lincosamides [27]. Further exacerbating the
problem, strains have emerged which exhibit resistance to first-line
E-mail address: mseleem@purdue.edu (M.N. Seleem).
http://dx.doi.org/10.1016/j.ejmech.2015.03.015
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

H. Mohammad et al. / European Journal of Medicinal Chemistry 94 (2015) 306e316
307
antibiotics (such as mupirocin [27,28] for the treatment of MRSA
skin infections) and drugs deemed agents of last resort (such as
linezolid [29,30] and vancomycin [31]). Prudent use and develop-
ment of effective antimicrobials is a critical step to alleviate com-
plications and costs associated with MRSA infections. Therefore
there is an urgent need for the development of novel therapeutic
agents and treatment strategies to circumvent this significant
global health issue.
Utilizing whole-cell screening of a library of substituted thia-
zoles, our research group identified a novel lead thiazole compound
that possesses potent antimicrobial activity against clinically rele-
vant isolates of MRSA, vancomycin-intermediate S. aureus (VISA),
and vancomycin-resistant S. aureus (VRSA) [32]. The basic structure
of the lead 1 consists of a central thiazole ring connected to two
distinct moieties e a lipophilic side chain at C2 and a cationic amino
group at C5. The objectives of the present study were to construct a
series of analogs to the lead 1 (Table 1) with modifications to the
functional groups at both the thiazole-C2 and C5 positions to more
rigorously ascertain the structureeactivity relationships of these
compounds against a diverse array of HA-MRSA and CA-MRSA
isolates, identify new derivatives exhibiting an improved toxicity
profile against mammalian cells, and to enhance the metabolic
stability profile of the lead 1.
Fig. 1. Chemical structure of the lead compound 1.
and 6, respectively, with aminoguanidine hydrochloride in the
presence of a catalytic amount of lithium chloride in absolute
ethanol (Scheme 1).
The amide derivatives 10e13 were prepared in quantitative
yields by reacting the 4-butylphenylthiazole acid chloride inter-
mediate 8 [34] with the appropriate amines in THF, as illustrated in
Scheme 2. Compound 16 was synthesized in three steps, starting
with the formation of the amide derivative 9 by way of reacting the
acid chloride intermediate 8 with ammonium hydroxide in THF at
room temperature. The amide intermediate 9 was then heated in
thionyl chloride to give the nitrile intermediate 14, which upon
subsequent treatment with NaN3 in the presence of iodine gave the
tetrazole-containing thiazole derivative 16 as shown in Scheme 2.
The nitrile intermediate 14 was also treated with hydroxylamine
hydrochloride in absolute ethanol with a catalytic amount of po-
tassium carbonate to afford the thiazole derivative 15.
2. Chemistry
The detailed synthetic protocols and spectral data of the lead 1
(Fig. 1) in addition to all intermediates have been reported else-
where [32,33]. All thiazole compounds were dissolved in dimethyl
sulfoxide (DMSO) (SigmaeAldrich, St. Louis, MO, USA) to achieve a
stock 10 mM solution.
The phenylthiazolyl methyl ketone derivative 18 was prepared
by treatment of the commercially available 4-aminothiobenzamide
17 with 3-chloro-2,4-pentanedione in absolute ethanol. Synthesis
of the hydrazinecarboximidamide derivative 19 was achieved by
treatment of the phenylthiazolyl methyl ketone derivative 18 with
aminoguanidine hydrochloride in the presence of a catalytic
amount of lithium chloride (Scheme 3).
The (4-iodophenyl)thiazole derivative 3 was prepared by heat-
ing a mixture of the commercially available 4-iodothiobenzamide 2
and 3-chloro-2,4-pentanedione in absolute ethanol, as illustrated
in Scheme 1. The phenylthiazolyl methyl ketone derivatives 4 and 6
were prepared via the Sonogashira cross coupling of the (4-
iodophenyl)thiazole derivative 3 with commercially available 1-
hexyne and 1-nonyne, respectively, in DMF using
a bis(-
Phenylthiazole methylketone derivatives 21aed and 24 were
prepared via the Suzuki-Miyaura cross coupling of the (4-
iodophenyl)thiazole derivative 3 with the commercially available
phenylboronic acid derivatives 20aed and 23, respectively, in the
presence of a catalytic quantity of palladium(II) acetate and (2-
biphenyl)dicyclohexylphosphine ligand, as shown in Scheme 4.
Synthesis of the hydrazinecarboximidamide derivatives 22aed and
25 was achieved by treatment of phenylthiazole methylketone
derivatives 21aed and 24, respectively, with aminoguanidine hy-
drochloride in the presence of lithium chloride as a catalyst
(Scheme 4).
triphenylphosphine)palladium(II) dichloride catalyst, copper(I) io-
dide co-catalyst, and caesium carbonate base (Scheme 1). The
hydrazinecarboximidamide derivatives 5 and 7 were synthesized
by treatment of the phenylthiazolyl methyl ketone derivatives 4
Table 1
Minimum inhibitory concentration (MIC) of thiazole
compounds against methicillin-resistant Staphylo-
coccus aureus (MRSA) ATCC 43300.
Analog
MIC (mg/mL)
1 (lead)
5
7
1.3
1.4
12.6
3. Biological results and discussion
9
10
11
12
>35.1
>45.8
>42.4
44.5
3.1. Antibacterial activity of thiazole compounds and vancomycin
against MRSA, VISA, and VRSA
13
15
16
19
21b
21c
22a
22b
22c
22d
24
44.2
To ascertain the structureeactivity relationships of the lead
thiazole compound more thoroughly, derivatives were initially
constructed with modifications to the thiazole-C5 cationic moiety
(keeping the lipophilic alkane side chain at thiazole-C2 intact).
Substitution of the ethylidenehydrazine-1-carboximidamide of the
lead 1 with moieties such as a tetrazole (16), an amide derivative
(9e13), or a hydroxamidine (15) results in complete abolishment of
antimicrobial activity against MRSA (minimum inhibitory concen-
>37.0
>38.3
>36.9
>39.8
>46.2
>46.7
5.9
3.3
6.3
>43.9
1.6
tration (MIC) > 35.1
mg/mL (Table 1). This trend continues when the
25
amino moiety is replaced with a ketone in derivatives 21bec and

308
H. Mohammad et al. / European Journal of Medicinal Chemistry 94 (2015) 306e316
Scheme 1. Reagents and conditions: (a) 3-chloro-2,4-pentanedione, EtOH, reflux, 24 h; (b) 1-hexyne, PdCl2(PPh3)2, CuI, Cs2CO3, DMF, sealed tube, 65 ꢀC, 15 h; (c) 1-nonyne,
PdCl2(PPh3)2, CuI, Cs2CO3, DMF, sealed tube, 70 ꢀC, 15 h; (d) aminoguanidine HCl, LiCl, EtOH, reflux, 24 h.
Scheme 2. Reagents and conditions: (a) 30% aq NH4OH, THF, rt, 24 h; (b) biguanidine hydrochloride, Et3N, THF, 24 h; (c) aminoguanidine hydrochloride, Et3N, THF, 24 h; (d) (R)-
(ꢁ)-3-amino-1,2-propanediol, THF, rt, 24 h; (e) N,N-dimethylethylenediamine, THF, rt, 48 h; (f) thionyl chloride, reflux, 7 h; (g) NH2OH HCl, K2CO3, EtOH, 78 ꢀC, 24 h; (h) NaN3, I2,
DMF, 120 ꢀC, 15 h.

H. Mohammad et al. / European Journal of Medicinal Chemistry 94 (2015) 306e316
309
Scheme 3. Reagents and conditions: (a) 3-chloro-2,4-pentanedione, EtOH, reflux, 20 h; (b) aminogaunidine hydrochloride, LiCl, EtOH, reflux, 24 h.
Scheme 4. Reagents and conditions: a) Pd(OAc)2, (2-biphenyl)dicyclohexylphosphine, K3PO4, toluene, 90 ꢀC, 24 h; b) aminoguanidine hydrochloride, LiCl, EtOH, reflux, 24 h.
24. Interestingly, derivatives 22bec and 25 (consisting of the
same cationic head group as the lead 1 but with substitutions to
the linear alkane side chain at thiazole-C2 identical to those in
compounds 21bec and 24) retain antimicrobial activity; among
the groups studied thus far, the ethylidenehydrazine-1-
carboximidamide is the only one to retain potency at this posi-
tion of the structural series and we therefore retained it in all future
analogs.
antimicrobial activity, with both compounds possessing
MIC > 36.9 g/mL (Table 1). Replacement of the alkane side chain
with hydrophobic, polar substituents such as an acetyl group (22d,
g/mL), a fluoride (22b), or a trifluoromethyl group (22c)
results in the compounds possessing antimicrobial activity, but
with a MIC higher than the parent compound. On the other hand,
substitution of the alkane side chain with a nonpolar, hydrophobic
a
m
MIC ¼ 6.3
m
moiety, such as an alkyne (5 with MIC of 1.4
mg/mL) or naphthalene
Modifications made to the linear alkane side chain at thiazole-
C2 revealed hydrophobic, nonpolar moieties at this position are
preferred for the compound to retain potent antimicrobial activity.
The presence of a hydrophilic, polar group, such as an amine (19) or
alcohol (22a) at this position, results in complete loss of
(25 with MIC of 1.6 g/mL) functional group, results in derivatives
m
with potent antimicrobial activity (nearly identical MIC to the lead
1). Once again this confirms that a more nonpolar, hydrophobic
functional group is needed at the C5 position for the thiazole
compounds to possess potent antibacterial activity. This is in

310
H. Mohammad et al. / European Journal of Medicinal Chemistry 94 (2015) 306e316
agreement with previously reported findings where alkane,
cycloalkane, cycloalkene, and arene substitutions at thiazole-C2
resulted in compounds with stronger activity against MRSA [32].
Interestingly, extending the alkyne length from a hexyne (5) to a
nonyne (7) group results in diminished anti-MRSA activity with the
12
10
8
DMSO
1
6
MIC increasing nine-fold from 1.4
mg/mL to 12.6
mg/mL. This is
5
4
similar to what was previously found with lengthening of the
alkane side chain at thiazole-C2; increasing the alkane side chain
beyond four methylene units resulted in a drastic reduction in
antimicrobial activity of the compounds. Future studies examining
decreasing the alkyne side chain length at thiazole-C5 and its effect
on anti-MRSA activity warrant further exploration. Additionally,
repositioning the nonpolar moiety (at the ortho and meta positions
of the phenyl substituent connected to C2 on the thiazole ring)
would be of interest to assess if the para position plays a crucial role
in the antimicrobial activity of the compounds.
25
Vancomycin
2
0
0
2
4
6
8
10 12 14 16 18 20 22 24
Time (h)
Fig. 2. Time-kill analysis of thiazole compounds 1, 5, 25, and vancomycin against
methicillin-resistant Staphylococcus aureus (MRSA USA300) over a 24 h incubation
period at 37 ꢀC. DMSO served as a control. The error bars represent standard deviation
values obtained from triplicate samples used for each compound/antibiotic studied.
After confirming that five derivatives (5, 22bed and 25)
possessed strong antimicrobial activity against a single strain of
MRSA, we next assessed their activity against an array of clinically
relevant multidrug-resistant HA-MRSA and CA-MRSA strains as
well as vancomycin-intermediate (VISA) and vancomycin-resistant
(VRSA) S. aureus isolates. All five compounds maintained their ac-
tivity (with MICs identical or two-fold higher than those reported
against MRSA ATCC43300) against MRSA isolates exhibiting resis-
tance to mupirocin (NRS107), linezolid (NRS119), erythromycin
(USA300), tetracycline (USA300), ciprofloxacin (USA500), clinda-
mycin (USA500), and gentamicin (USA500) (Table 2); this indicates
cross-resistance between these antibiotics and the thiazole com-
pounds is unlikely to occur. The thiazole derivatives also exhibited
potent activity against strains of MRSA (USA300 and USA400)
responsible for the majority of MRSA-related skin and soft tissue
infections in North America [3,35]. Additionally, analogs 5 (MIC
we next turned our attention to assessing whether these com-
pounds were bacteriostatic or bactericidal. Antimicrobial agents
that are bactericidal, as opposed to their bacteriostatic counter-
parts, are thought to help patients recover more rapidly from in-
fections, resulting in a better clinical outcome [36]. To assess if the
thiazole compounds were bacteriostatic or bactericidal, the mini-
mum bactericidal concentration (MBC) was determined. As Table 2
presents, all six thiazole compounds tested exhibited MBC values
that were identical to or two-fold higher than their MIC values. The
results mimic those of vancomycin, a known bactericidal antibiotic,
indicating the thiazole compounds are bactericidal.
3.2. Time-kill analysis of most potent thiazole analogs against
MRSA USA300
between 1.3 and 2.6
and 25 (MIC of 1.6
than vancomycin (MIC of 3.0
tested. Furthermore, while all three VRSA strains exhibited resis-
tance to vancomycin (MIC > 190.2 g/mL), the lead thiazole (1) and
the five most potent derivatives retained their antimicrobial ac-
tivity with MIC values ranging from 0.7 g/mL (for 1) to 6.7 g/mL
(for 22c). Finding alternative therapeutic options (such as these
thiazole compounds) to vancomycin and linezolid, agents of last
resort for treatment of severe MRSA infections, is critical to address
the burden of these challenging infections.
m
g/mL), 22b (MIC between 2.9 and 5.9
g/mL) proved to be similar in activity or better
g/mL) against two VISA isolates
mg/mL),
To confirm the thiazole compounds are in fact bactericidal
agents against MRSA, a time-kill analysis was performed. MRSA
USA300 cells in late logarithmic growth were treated with 3 ꢂ MIC
of the lead thiazole (1), the two most potent derivatives (5 and 25),
or vancomycin. Interestingly, a simple substitution at thiazole-C2
from an alkane/alkyne (1/5) to the more conformationally-
restricted naphthalene analog (25), results in a dramatic shift in
the rate of bacterial killing by the thiazole compounds. As Fig. 2
demonstrates, compounds 1 and 5 completely eradicate MRSA
growth within 4 h while compound 25 requires 10 h to achieve the
same effect. Though all three compounds possess nearly identical
MIC values, the structural modifications made at thiazole-C2
significantly affect the rate of bacterial killing observed for each
compound against MRSA.
m
m
m
m
m
Subsequent to establishing that the lead compound and the five
most active analogs exhibited potent antimicrobial activity against
a diverse spectrum of CA-MRSA, HA-MRSA, VISA, and VRSA isolates,
Table 2
Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of thiazole compounds 1, 5, 22bed, 25 and vancomycin against seven methicillin-
resistant (MRSA), three vancomycin-intermediate (VISA), and three vancomycin-resistant Staphylococcus aureus (VRSA) strains.
S. aureus strain
1
5
22b
MIC
22c
22d
MIC
25
Vancomycin
MIC
MIC
MBC
MIC
MBC
MBC
MIC
MBC
MBC
MIC
MBC
MBC
<1.5
<1.5
0.7
NRS107 (MRSA)
NRS119 (MRSA)
NRS194 (MRSA)
USA300 (MRSA)
USA 400 (MRSA)
USA500 (MRSA)
ATCC 43300 (MRSA)
NRS1 (VISA)
NRS19 (VISA)
NRS37 (VISA)
VRS1 (VRSA)
VRS4 (VRSA)
2.6
2.6
1.3
1.3
1.3
1.3
1.3
1.3
1.3
2.6
0.7
0.7
0.7
2.6
2.6
1.3
1.3
1.3
1.3
1.3
2.6
2.6
2.6
0.7
1.3
1.3
1.4
1.4
1.4
1.4
1.4
1.4
1.4
0.7
1.4
1.4
1.4
1.4
2.8
1.4
1.4
1.4
1.4
1.4
1.4
1.4
0.7
1.4
1.4
1.4
2.8
2.8
2.9
5.9
5.9
5.9
5.9
5.9
5.9
2.9
5.9
2.9
2.9
2.9
2.9
5.9
11.7
11.7
5.9
11.7
5.9
5.9
2.9
5.9
5.9
3.3
6.7
6.7
6.7
6.7
3.3
3.3
3.3
3.3
3.3
6.7
6.7
6.7
6.7
6.7
13.3
6.7
13.3
3.3
3.3
3.3
3.3
3.3
6.7
6.7
6.7
6.3
6.3
6.3
6.3
6.3
6.3
6.3
6.3
12.5
3.1
3.1
1.6
6.3
12.5
12.5
6.3
6.3
6.3
6.3
6.3
6.3
12.5
6.3
3.1
3.1
6.3
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6
1.6
3.2
3.2
3.2
1.6
1.6
1.6
1.6
1.6
3.2
1.6
1.6
1.6
1.6
3.2
3.2
3.2
<1.5
<1.5
0.7
0.7
0.7
0.7
0.7
3.0
<1.5
3.0
0.7
0.7
0.7
0.7
3.0
<1.5
3.0
>190.2
>190.2
>190.2
2.9
2.9
2.9
>190.2
>190.2
>190.2
VRS5 (VRSA)

H. Mohammad et al. / European Journal of Medicinal Chemistry 94 (2015) 306e316
311
Table 3
While all three thiazole compounds exhibit the ability to elim-
inate MRSA growth completely within 10 h, vancomycin requires
24 h to achieve the same result. This is similar to what has been
reported elsewhere regarding vancomycin's slow bactericidal ac-
tivity [37]. Rapid bactericidal activity is considered to be a critical
factor in slowing the emergence of bacterial resistance to an anti-
microbial agent and is important clinically in preventing an infec-
tion from spreading [36]. Additionally, bactericidal agents have
been shown both clinically and through in vivo studies to be su-
perior to bacteriostatic agents for the treatment of certain invasive
diseases such as endocarditis [38]. Thus these thiazole compounds
may have the potential to be utilized in a wide array of clinically
important MRSA diseases from skin and soft tissue infections to
systemic infections such as endocarditis.
Evaluation of metabolic stability of thiazole compound 5, verapamil, and warfarin in
human liver microsomes.
Compound/ NADPH-
Drug tested dependent
NADPH-
NADPH-free CLint NADPH-free
dependent
(
mL/min/mg)
T1/2 (min)
a
b
CLint
(m
L/min/mg) T1/2 (min)
5
3.7
213
0.0
>240
10.8
>240
0.0
0.0
0.0
>240
>240
>240
Verapamil
Warfarin
a
CLint ¼ microsomal intrinsic clearance.
T1/2 ¼ half-life.
b
there is a significant improvement in the toxicity profile of these
novel analogs when compared to the lead compound.
3.3. Toxicity analysis of potent thiazole derivatives against
mammalian cells
3.4. Metabolic stability analysis of compound 5
Previously, microsomal stability analysis of the lead 1 revealed
this compound was metabolized fairly rapidly (intrinsic clearance
Selective toxicity is an important property that both approved
antibiotics and novel antimicrobial compounds must possess. The
ability for antimicrobial agents to exhibit their activity on the target
microorganism while not causing harm to host (mammalian) tis-
sues is important to ascertain early in the drug discovery process.
Previously, the lead thiazole (1) was found to be nontoxic to human
rate of 80.3
mL/min/mg and half-life of 28.8 min) via a NADPH-
mediated process (such as via the cytochrome P450 system) [32].
As compound 5 demonstrated nearly identical antimicrobial ac-
tivity to the lead compound, we were curious to assess if the sub-
stitution of an alkane side chain with an alkyne at thiazole-C2
would enhance the metabolic stability of the compound, prevent-
ing its conversion to potentially inactive metabolites. Using pooled
human liver microsomes, 5, similar to the parent compound, was
found to be metabolized via a NADPH-mediated process (intrinsic
cervical (HeLa) cells at a concentration of 11 mg/mL [32]. A principal
objective of the present study was to develop new analogs of the
lead that exhibited an improved/more selective toxicity profile. To
assess this, the lead compound and five most potent derivatives
against MRSA (5, 22bed and 25) were screened against a human
embryonic kidney (HEK293) cell line using the MTS assay. Fig. 3
clearance rate of 3.7 mL/min/mg as compared to 0.0 mL/min/mg in
the absence of the cofactor, NADPH) (Table 3). Interestingly, the
slower clearance rate correlates with an improved half-life for
compound 5 (as compared to the lead compound) that exceeds 4 h.
This marked improvement in the metabolic stability of the thiazole
compound is important as it has the potential to positively impact
the pharmacokinetic profile of this compound, reduce the fre-
quency of doses needed to be administered for treatment (fewer
doses leads to improved patient compliance), while also ensuring
the active drug circulates within the patient's system to assist with
treating and clearing an infection. Additionally, compounds that are
metabolically stable are less susceptible to experiencing issues
pertaining to toxicity and drugedrug interactions caused by me-
tabolites [39]. The metabolic stability analysis combined with the
enhanced toxicity profile of compound 5 (as compared to the lead)
warrants further analysis of this compound as a potential novel
antibiotic for the treatment of MRSA infections.
presents the results garnered. At a concentration of 40 mg/mL, the
lead 1 and compounds 22b and 22c proved to be toxic to
mammalian cells. However, three of the novel analogs e com-
pounds 5 (alkynyl side chain), 22d (p-acetylbenzyl), and 25 [p-(1-
naphthyl)] e exhibit an improved toxicity profile compared to the
lead 1 at the tested concentration. This concentration (40 mg/mL)
represents a 25- (for 25) to 28-fold (for 5) difference between the
MIC values determined against MRSA for these compounds. Thus
140
120
100
80
60
4. Conclusion
40
We present herein a novel series of 2,5-disubstituted thiazole
compounds exhibiting potent activity against clinically relevant
isolates of MRSA, VISA, and VRSA. A rigorous analysis of the
structureeactivity relationships of these analogs reveals the eth-
ylidenehydrazine-1-carboximidamide head group (at thiazole-C5)
and a nonpolar, hydrophobic moiety (at thiazole-C2) are critical
for the thiazole compound's antibacterial action. Three derivatives
with substitutions at thiazole-C2 (an alkyne, p-acetylbenzene, and
p-naphthalene) demonstrate an improved toxicity profile against
mammalian cells compared to the lead compound. Furthermore,
the alkyne substitution results in a compound that is more meta-
bolically stable as assessed via human liver microsomes. Collec-
tively, the results present critical information necessary for further
analysis and development of these thiazole compounds as novel
antimicrobial agents for use in treatment of infections caused by
multidrug-resistant S. aureus.
20
0
1
5
25
22c
22b
22d
DMSO
Compound Number
Fig. 3. Percent viable mammalian cells (measured as average absorbance ratio (test
agent relative to DMSO)) for cytotoxicity analysis of thiazole compounds 1, 5, 22bed,
and 25 at 40 mg/mL against HEK293 cells using the MTS 3-(4,5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. 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. An unpaired t-test, P ꢃ 0.05, demonstrated statistical
difference between the values obtained for compounds 1, 22b and 22c relative to the
cells treated with DMSO.

312
H. Mohammad et al. / European Journal of Medicinal Chemistry 94 (2015) 306e316
5. Material and methods
5.1.4. 1-(4-Methyl-2-(4-(non-1-yn-1-yl)phenyl)thiazol-5-yl)ethan-
1-one (6)
The thiazole derivative 3 (750 mg, 2.19 mmol), 1-nonyne
1H NMR spectra were recorded in CDCl3 or DMSO-d6 using a
300 MHz spectrometer. Chemical shifts are reported in units of
ppm on the delta (d) scale and coupling constants (J) are reported in
(1.44 mL, 8.76 mmol), PdCl2(PPh3)2 (76.6 mg, 0.11 mmol), cop-
per(I) iodide (41.6 mg, 0.22 mmol) and Cs2CO3 (1.42 g, 4.38 mmol)
were added to a sealed tube under argon for 10 min, and then DMF
(7.5 mL) was added. The tube was once again evacuated and purged
with argon for 5 min and heated to 70 ꢀC for 15 h. The tube was
allowed to cool to room temperature, and the solids were removed
by filtration and the filter cake was extracted with additional CHCl3
(50 mL). The combined filtrate and extracts were concentrated
under vacuum and extracted with EtOAc (2 ꢂ 50 mL) and washed
with water (2 ꢂ 50 mL). After evaporation of the solvent under
reduced pressure, the residue was collected and purified by flash
chromatography (SiO2, hexanes-EtOAc, 8.8:1.2) to yield the desired
compound 6 as a dark green oil (772 mg, 100%); 1H NMR (300 MHz,
units of Hz. The following splitting abbreviations are used:
s ¼ singlet, d ¼ doublet, t ¼ triplet and m ¼ multiplet. All melting
points were recorded using capillary tubes on a Mel-Temp appa-
ratus and are not corrected. Mass spectral analyses were performed
at the Purdue University Campus-Wide Mass Spectrometry Center.
Reagents and solvents were purchased from commercial vendors
and were used as received without further purification, unless
otherwise stated.
5.1. Procedure for synthesis of thiazole compounds and
intermediates
CDCl3)
d
7.9 (t, J ¼ 6.8 Hz, 2H), 7.46 (d, J ¼ 8.4 Hz, 2H), 2.76 (s, 3H),
2.55 (s, 3H), 2.44 (t, J ¼ 7.1 Hz, 3H), 1.63 (t, J ¼ 7.7 Hz, 2H), 1.30 (m,
5.1.1. 1-(2-(4-Iodophenyl)-4-methylthiazol-5-yl)ethanone (3)
J ¼ 3.3 Hz, 9H), 0.9 (q, J ¼ 6.2 Hz, 4H).
4-Iodothiobenzamide (2, 3.80 mmol) and
a-chloropenta-
nedione (0.611 mg, 4.56 mmol) were added to absolute ethanol
(50 mL). The reaction mixture was heated at reflux for 24 h. After
removal of solvent under reduced pressure, the residue was pu-
rified by silica gel chromatography using hexaneseethyl ace-
tate (7:3) to provide the desired compound as light orange
solid (0.800 g, 62%): mp 105e106 ꢀC. 1H NMR (300 MHz, CDCl3)
5.1.5. (E)-2-(1-(4-Methyl-2-[4-(non-1-yn-1-yl)phenyl]thiazol-5-yl)
ethylidene)hydrazinecarboximidamide (7)
The thiazole derivative 6 (140 mg, 0.41 mmol), aminoguanidine
hydrochloride (90.85 mg, 0.83 mmol), and a catalytic amount of LiCl
(5 mg) were added to absolute ethanol (10 mL). The reaction
mixture was heated at reflux for 24 h. After evaporation of the
solvent under reduced pressure, the crude residue was extracted
with CHCl3/MeOH (90:10, 2 ꢂ 40 mL) and washed with water
(2 ꢂ 50 mL) and brine (40 mL). The extracts were pooled together,
dried over Na2SO4 and stripped of solvents under reduced pressure.
The residue was suspended in CHCl3/hexanes (50:50, 50 mL) and
filtered through Whatman filter paper to afford the desired product
(7) as a yellow-white solid (200 mg, 100%): mp 255e260 ꢀC dec, 1H
d
7.81 (d, J ¼ 6.6 Hz, 2H), 7.70 (d, J ¼ 6.6 Hz, 2H), 2.77 (s, 3H), 2.56
(s, 3H).
5.1.2. 1-(2-(4-(Hex-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)
ethanone (4)
1-(2-(4-Iodophenyl)-4-methylthiazol-5-yl)ethanone (3, 0.5 g,
1.45 mmol), 1-hexyne (0.373 g, 7.73 mmol), cesium carbonate
(0.947 g, 2.91 mmol), dichloro-bis(triphenylphosphine)palladiu-
m(II) (0.051 g, 0.072 mmol) and CuI (0.027 g, 0.145 mmol) were
dissolved in DMF (6 mL). The reaction mixture was purged with
argon for 20 min. The sealed tube was closed, placed in an oil bath
and stirred at 65 ꢀC for 15 h. The reaction mixture was filtered
through celite, and the celite was washed with chloroform (50 mL).
The organic phase was washed with 1% hydrochloric acid (30 mL),
water (3 ꢂ 40 mL) and brine (30 mL). The organic layer was dried
over Na2SO4, concentrated and purified by silica gel flash column
chromatography using hexaneseethyl acetate (8:2) to provide the
desired compound as yellow syrup (0.400 g, 92.5%): IR (film) 1945,
NMR (300 MHz, DMSO-d6)
d
7.88 (d, J ¼ 8.3 Hz, 3H), 7.49 (d,
J ¼ 8.4 Hz, 3H), 2.60 (s, 3H), 2.49 (q, J ¼ 1.7 Hz, 5H),1.26 (s,12H), 0.85
(s, 4H); ESIMS m/z (rel intensity) 395 (Mþ, 57).
5.1.6. 2-(4-Butylphenyl)-4-methylthiazole-5-carboxamide (9)
Acid chloride 8 [34] (0.200 g, 0.682 mmol) was dissolved in THF
(20 mL) and then 30% aq NH4OH (10 mL) was added. The reaction
mixture was stirred at room temperature for 24 h. The THF was
removed on a rotary evaporator and the crude product was
extracted with EtOAc (2 ꢂ 25 mL) and washed with water
(2 ꢂ 20 mL) and brine (20 mL). The combined organic layer was
dried over Na2SO4 and concentrated to afford the amide 9 (0.185 g)
in quantitative yield: mp 160e161 ꢀC. IR (KBr) 3245, 1691, 1611,
1675, 1111, 819, 666 cmꢁ1 1H NMR (300 MHz, CDCl3)
; d 7.90 (d,
J ¼ 8.5 Hz, 2H), 7.46 (d, J ¼ 8.3 Hz, 2H), 2.76 (s, 3H), 2.56 (s, 3H), 2.45
(t, J ¼ 6.9 Hz, 2H),1.59 (m, 4H), 0.97 (t, J ¼ 7.3 Hz, 3H); ESIMS m/z (rel
intensity) 298 (MHþ, 100).
1121, 846, 665 cmꢁ1; 1H NMR (300 MHz, CDCl3)
d
7.84 (d, J ¼ 8.2 Hz,
2H), 7.26 (d, J ¼ 8.2 Hz, 2H), 5.79 (br s, 2H), 2.74 (s, 3H), 2.66 (t,
J ¼ 7.5 Hz, 2H), 1.63 (m, 2H), 1.39 (m, 2H), 0.94 (t, J ¼ 7.3 Hz, 3H);
APCIMS m/z (rel intensity) 275 (MHþ, 100); HPLC purity 97.89% (1%
TFA in MeOH:H2O e 90:10).
5.1.3. (Z)-2-(1-(2-(4-(Hex-1-yn-1-yl)phenyl)-4-methylthiazol-5-yl)
ethylidene) hydrazinecarboximidamide (5)
The thiazole derivative 4 (200 mg, 0.673 mmol) was dissolved in
absolute ethanol (10 mL), and aminoguanidine hydrochloride
(0.088 mg, 0.808 mmol) and a catalytic amount of LiCl (5 mg) were
added. The reaction mixture was heated at reflux for 24 h. The
solvent was evaporated under reduced pressure. The crude product
was purified by crystallization from 70% methanol and then
recrystallized from methanol to afford the desired compound as a
yellow solid (80 mg, 46%): mp 253e254 ꢀC. IR (KBr) 3329, 2227,
5.1.7. 2-(4-Butylphenyl)-N-(N-carbamimidoylcarbamimidoyl)-4-
methylthiazole-5-carboxamide (10)
Acid chloride 8 (0.200 g, 0.682 mmol) was dissolved in THF
(20 mL) and then biguanidine hydrochloride (0.467 g, 3.41 mmol)
followed by triethylamine (0.344 g, 3.41 mmol) were added. The
reaction mixture was stirred at room temperature for 24 h. The THF
was removed on a rotary evaporator and the crude product was
extracted with EtOAc (2 ꢂ 30 mL) and washed with water
(2 ꢂ 20 mL) and brine (20 mL). The combined organic layer was
dried over Na2SO4, concentrated and purified by silica gel flash
column chromatography using chloroformemethanol (9.5:0.5) to
provide the desired compound as a yellow solid (0.070 g, 30%): mp
150e151 ꢀC. IR (KBr) 3312,1694,1655,1148, 823, 666 cmꢁ1; 1H NMR
1678, 1143, 836, 657 cmꢁ1 1H NMR (DMSO-d6)
; d 11.35 (br s, 1H),
7.88 (d, J ¼ 8.1 Hz, 2H), 7.69 (br s, 3H), 7. 49 (d, J ¼ 8.1 Hz, 2H), 2.60
(s, 3H), 2.44 (s, 3H), 2.41 (t, J ¼ 7.1 Hz, 2H), 1.48 (m, 4H), 0.93 (t,
J ¼ 7.1 Hz, 3H); ESIMS m/z (rel intensity) 354 (MHþ, 100); HRESIMS
calcd for C19H24N5S 354.1509 (MHþ), found 354.1514; HPLC purity
98.07% (1% TFA in MeOH:H2O e 85:15).

H. Mohammad et al. / European Journal of Medicinal Chemistry 94 (2015) 306e316
313
(300 MHz, CDCl3)
d
7.89 (br s, 2H), 7.25 (br s, 2H), 2.80 (s, 3H), 2.67
d
7.83 (d, J ¼ 8.1 Hz, 2H), 7.27 (d, J ¼ 8.1 Hz, 2H), 2.67 (s, 3H), 2.64 (m,
(t, J ¼ 7.2 Hz, 2H), 1.60 (m, 2H), 1.38 (m, 2H), 0.94 (t, J ¼ 7.1 Hz, 3H);
2H),1.63 (m, 2H),1.38 (m, 2H), 0.94 (t, J ¼ 7.3 Hz, 3H); ESIMS m/z (rel
ESIMS m/z (rel intensity) 359 (MHþ, 65), 341 (MHþ-NH3, 68);
intensity) 256 (Mþ, 33), 213 (MþꢁC3H7, 100).
HRESIMS calcd for
C
17H23N6OS m/z 359.1248 (MHþ), found
5.1.12. (Z)-2-(4-Butylphenyl)-N0-hydroxy-4-methylthiazole-5-
carboximidamide (15)
359.1251; HPLC purity 95.16% (1% TFA in MeOH:H2O e 90:10).
5.1.8. 2-(2-(4-Butylphenyl)-4-methylthiazole-5-carbonyl)
hydrazinecarboximidamide (11)
A mixture of the thiazole derivative 14 (0.19 g, 0.74 mmol), hy-
droxylamine hydrochloride (0.07 g, 1 mmol) and K2CO3 (0.102 g,
0.74 mmol) in absolute EtOH (15 mL) was stirred at room tem-
perature for 1 h and then heated at reflux overnight. The EtOH was
removed under reduced pressure and the resulting crude residue
was purified by flash column chromatography (SiO2, hexanes-
EtOAc, 9:1) to afford the product 15 as an off white to light yel-
low solid (35.5 mg, 17%): mp 135e137 ꢀC. 1H NMR (300 MHz, CDCl3)
Acid chloride 8 (0.200 g, 0.682 mmol) was dissolved in THF
(20 mL) and then aminoguanidine hydrochloride (0.377 g,
3.41 mmol) followed by triethylamine (0.344 g, 3.41 mmol) were
added. The reaction mixture was stirred at room temperature for
24 h. The THF was removed on a rotary evaporator and the crude
product was extracted with EtOAc (2 ꢂ 30 mL) and washed with
water (2 ꢂ 20 mL) and brine (20 mL). The combined organic layer
was dried over Na2SO4, concentrated and purified by silica gel flash
column chromatography using hexaneseethyl acetate (4:6) to
provide the desired compound as a yellow solid. (0.090 g, 40%): mp
d
7.81 (d, J ¼ 8.05 Hz, 2H), 7.25 (t, J ¼ 5.7 Hz, 2H), 2.63 (t, J ¼ 7.6 Hz,
5H), 1.59 (q, J ¼ 7.4 Hz, 2H), 1.34 (p, J ¼ 11.2 Hz, 2H), 0.92 (t,
J ¼ 7.28 Hz, 3H); ESIMS m/z (rel intensity) 290 (Mþ, 100).
174e175 ꢀC. IR (KBr) 3322, 1698, 1658, 1462, 1155, 856, 665 cmꢁ1
;
5.1.13. 2-(4-Butylphenyl)-4-methyl-5-(1H-tetrazol-5-yl)thiazole
(16)
1H NMR (300 MHz, CDCl3)
d
7.87 (d, J ¼ 8.0 Hz, 2H), 7.24 (d,
J ¼ 8.0 Hz, 2H), 2.78 (s, 3H), 2.67 (t, J ¼ 7.6 Hz, 2H), 1.63 (m, 2H), 1.39
(m, 2H), 0.94 (t, J ¼ 7.3 Hz, 3H); ESIMS m/z (rel intensity) 332 (MHþ,
100); HRESIMS calcd for C16H22N5OS m/z 332.1112 (MHþ), found
332.1210; HPLC purity 96.57% (1% TFA in MeOH:H2O e 90:10).
I2 (20 mg) was added to a mixture of nitrile (14, 0.2 g,
0.781 mmol) and NaN3 (0.076 g, 1.17 mmol) and the mixture was
stirred at 120 ꢀC for 15 h. After completion of the reaction, EtOAc
(15 mL) and 4 M HCl (10 mL) were added and the mixture was
stirred vigorously for 10 min. The organic layer was separated and
the aqueous layer was extracted with EtOAc (2 ꢂ 10 mL).
The combined organic layer was washed with brine (4 ꢂ 15 mL),
dried over Na2SO4, concentrated and purified by silica gel flash
column chromatography using hexane-ethyl acetate (5:5) to
provide the desired compound as a light yellow solid (0.085ꢁg1,
5.1.9. (R)-2-(4-Butylphenyl)-N-(2,3-dihydroxypropyl)-
4methylthiazole-5-carboxamide (12)
A mixture of the acid chloride derivative 8 (0.25 g, 0.9 mmol)
and (R)-(ꢁ)-3-amino-1,2-propanediol (0.154 g, 1.7 mmol) in THF
(15 mL) were stirred at room temperature for 48 h. The THF was
removed under reduced pressure and the resulting crude oil was
extracted with chloroform (2 ꢂ 50 mL) and the extract was washed
with water (2 ꢂ 50 mL) and brine (50 mL). The combined extracts
were dried over Na2SO4 (10 g), filtered and concentrated under
vacuum. The residue was purified by flash column chromatography
(SiO2, CHCl3eMeOH, 9.4:0.6) to afford the product 12 as dark red
crystals (0.17 g, 57%): mp 93e95 ꢀC. 1H NMR (300 MHz, CDCl3)
37%): mp 170e171 ꢀC. IR (KBr) 1825, 1415, 1098, 844, 664 cm
;
1H NMR (300 MHz, CDCl3)
d
7.84 (d, J ¼ 8.1 Hz, 2H), 7.22 (d,
J ¼ 8.1 Hz, 2H), 2.85 (s, 3H), 2.64 (t, J ¼ 7.5 Hz, 2H), 1.63 (m, 2H),
1.35 (m, 2H), 0.93 (t, J ¼ 7.3 Hz, 3H); ESIMS m/z (rel intensity) 300
(MHþ, 100); HRESIMS calcd for C15H18N5S m/z 300.1267 (MHþ),
found 300.1270; HPLC purity 98.25% (1% TFA in MeOH:H2O e
90:10).
d
7.82 (d, J ¼ 8.2 Hz, 2H), 7.24 (d, J ¼ 7.2 Hz, 4H), 6.32 (s, 1H), 3.89 (d,
J ¼ 5.0 Hz, 1H), 3.65 (m, J ¼ 5.2 Hz, 4H), 2.73 (s, 3H), 2.63 (t,
J ¼ 7.6 Hz, 2H), 1.61 (t, J ¼ 7.9 Hz, 2H), 1.35 (q, J ¼ 7.5 Hz, 2H), 0.92 (t,
J ¼ 6.1 Hz, 3H); ESIMS m/z (rel intensity) 349 (MHþ, 100).
5.1.14. 1-(4-Methyl-2-(4-aminophenyl)thiazol-5-yl)ethanone (18)
4-Aminothiobenzamide (0.6 g, 3.80 mmol) and
a-chlor-
opentanedione (0.611 mg, 4.56 mmol) were added to absolute
ethanol (50 mL). The reaction mixture was heated at reflux for 24 h.
After removal of solvent under reduced pressure, the residue was
purified by silica gel chromatography using hexaneseethyl acetate
(6:4) to provide the desired compound as light brown solid
(0.920 g, 97%): mp 204e205 ꢀC. IR (KBr) 3334, 1745, 1637, 1145, 865,
5.1.10. 2-(4-Butylphenyl)-N-[(dimethylamino)methyl]-4-
methylthiazole-5-carboxamide (13)
A mixture of acid chloride derivative 8 (0.4 g, 1.44 mmol) and
N,N-dimethylethylenediamine (0.63 mL, 5.7 mmol) in THF (15 mL)
were stirred at room temperature for 48 h. The THF was removed
under reduced pressure and the resulting residue was purified by
flash chromatography (SiO2, CHCl3eMeOH, 9.3:0.7) to afford the
product 13 as a pink solid (0.123 g, 24%): mp 79e81 ꢀC. 1H NMR
666 cmꢁ1; 1H NMR (300 MHz, CDCl3)
(d, J ¼ 8.5 Hz, 2H), 2.66 (s, 3H), 2.53 (s, 3H).
d
7.89 (d, J ¼ 8.5 Hz, 2H), 7.07
(300 MHz, CDCl3)
d
7.83 (d, J ¼ 8.2 Hz, 2H), 7.25 (d, J ¼ 9.7 Hz, 4H),
5.1.15. (E)-2-(1-(4-Methyl-2-(4-aminophenyl)thiazol-5-yl)
ethylidene) hydrazinecarboximidamide (19)
3.49 (t, J ¼ 5.8 Hz, 2H), 2.72 (s, 3H), 2.63 (t, J ¼ 7.6 Hz, 2H), 2.55 (t,
J ¼ 5.9 Hz, 2H), 2.3 (s, 6H), 1.61 (t, J ¼ 7.8, 2H), 1.36 (t, J ¼ 7.4, 2H),
0.92 (t, J ¼ 7.3 Hz, 3H); ESIMS m/z (rel intensity) 346 (MHþ, 100).
The thiazole derivative 18 (0.250 g, 0.954 mmol) was dissolved
in absolute ethanol (50 mL), and aminoguanidine hydrochloride
(0.125 g, 1.14 mmol) and a catalytic amount of LiCl (15 mg) were
added. The reaction mixture was heated at reflux for 24 h. The
solvent was evaporated under reduced pressure. The crude product
was purified by crystallization from 70% methanol and then
recrystallized from methanol to afford the desired compound as a
yellow solid (0.175 g, 58%): mp > 280 ꢀC. IR (KBr) 3402, 1705, 1665,
5.1.11. 2-(4-Butylphenyl)-4-methylthiazole-5-carbonitrile (14)
Amide 9 (0.400 g, 1.45 mmol) was dissolved in thionyl chloride
(20 mL) and the solution was heated to reflux for 7 h. Thionyl
chloride was removed under reduced pressure, EtOAc (30 mL) was
added and the mixture was washed with saturated aqueous
NaHCO3 (2 ꢂ 15 mL) and water (2 ꢂ 15 mL). The organic layer was
dried over Na2SO4, concentrated and purified by silica gel flash
column chromatography using hexaneseethyl acetate (9:1) to
provide the desired compound as yellow syrup (0.300 g, 81%): IR
1156, 826, 665 cmꢁ1 1H NMR (300 MHz, DMSO-d6)
; d 11.44 (br s,
1H), 7.81 (d, J ¼ 8.4 Hz, 2H), 7.75 (br s, 3H), 7.00 (d, J ¼ 8.4 Hz, 2H),
2.58 (s, 3H), 2.41 (s, 3H); ESIMS m/z (rel intensity) 289 (MHþ, 100);
HRESIMS calcd for C13H17N6S m/z 289.1123 (MHþ), found 289.1120;
HPLC purity 96.58% (1% TFA in MeOH:H2O e 90:10).
(KBr) 2246, 1456, 1122, 841, 665 cmꢁ1 1H NMR (300 MHz, CDCl3)
;

314
H. Mohammad et al. / European Journal of Medicinal Chemistry 94 (2015) 306e316
5.1.16. 1-(2-(40-Hydroxy-[1,10-biphenyl]-4-yl)-4-methylthiazol-5-
yl)ethanone (21a)
J ¼ 8.3 Hz, 2H), 7.74 (d, J ¼ 8.4 Hz, 2H), 7.59 (d, J ¼ 8.6 Hz, 2H), 6.88
(d, J ¼ 8.6 Hz, 2H), 2.70 (s, 3H), 2.56 (s, 3H); ESIMS m/z (rel intensity)
366 (MHþ, 100); HRESIMS calcd for C19H20N5OS 366.1045 (MHþ),
found 366.1048; HPLC purity 96.11% (1% TFA in MeOH:H2O e
90:10).
Iodide 3 (0.172 g, 0.5 mmol), the 4-hydroxyphenyl boronic acid
(20a, 0.205 g, 1.5 mmol), tripotassium monophosphate (0.424 g,
2 mmol) and (2-biphenyl)dicylohexylphosphine (18 mg) were
dissolved in dry toluene (15 mL) and the solution was purged with
argon for 20 min. Pd(OAc)2 (25 mg, 0.24 mmol) was added to the
reaction mixture and the reaction mixture was heated at 90 ꢀC
under argon for 24 h. Ethyl acetate (40 mL) was added to reaction
mixture, which was then washed with water (2 ꢂ 25 mL). The
combined organic layer was dried over Na2SO4, concentrated and
purified by flash column chromatography (EtOAc:hexanes 3:7 to
0.9:9.1) to afford the desired compound as yellow solid (0.150 g,
93%): mp 212e214 ꢀC. IR (KBr) 3356, 1745, 1123, 856, 665 cmꢁ1; 1H
5.1.20. (E)-2-(1-(2-(40-Fluoro-[1,10-biphenyl]-4-yl)-4-
methylthiazol-5-yl)ethylidene)hydrazinecarboximidamide (22b)
Compound 21b (0.1 g, 0.321 mmol) was dissolved in absolute
ethanol (50 mL) and aminoguanidine hydrochloride (0.064 g,
0.588 mmol) and a catalytic amount of LiCl (10 mg) were added.
The reaction mixture was heated at reflux for 24 h. The solvent was
evaporated under reduced pressure. The crude product was puri-
fied by crystallization from 70% methanol, and then recrystallized
from methanol to afford the desired compound as a light yellow
solid (0.077 g, 65%): mp 273e274 ꢀC. IR (KBr) 3308,1687,1645,1146,
NMR (300 MHz, CDCl3)
d
7.86 (d, J ¼ 8.3 Hz, 2H), 7.51 (d, J ¼ 8.3 Hz,
2H), 7.38 (d, J ¼ 8.5 Hz, 2H), 6.79 (d, J ¼ 8.6 Hz, 2H), 2.64 (s, 3H), 2.45
(s, 3H); ESIMS m/z (rel intensity) 309 (Mþ, 100).
823, 666 cmꢁ1; 1H NMR (300 MHz, DMSO-d6)
d 11.73 (br s, 1H), 8.00
(d, J ¼ 8.3 Hz, 2H) 7.81 (m, 8H), 7.32 (m, 2H), 2.62 (s, 3H), 2.44 (s,
5.1.17. 1-(2-(40-Fluoro-[1,10-biphenyl]-4-yl)-4-methylthiazol-5-yl)
ethanone (21b)
3H); ESIMS m/z (rel intensity) 368 (MHþ, 100); HRESIMS calcd for
C
19H19FN5S m/z 368.1245 (MHþ), found 368.1251; HPLC purity
Iodide 3 (0.172 g, 0.5 mmol), 4-fluorophenyl boronic acid (20b,
0.209 1.5 mmol), tripotassium monophosphate (0.424 g, 2 mmol)
and (2-biphenyl)dicylohexylphosphine (18 mg) were dissolved in
dry toluene (15 mL) and the solution was purged with argon for
20 min. Pd(OAc)2 (25 mg, 0.24 mmol) was added to the reaction
mixture and the reaction mixture was heated at 90 ꢀC under argon
for 24 h. Ethyl acetate (40 mL) was added to reaction mixture,
which was then washed with water (2 ꢂ 25 mL). The combined
organic layer was dried over Na2SO4, concentrated and purified by
flash column chromatography (EtOAc:hexanes 3:7 to 0.9:9.1) to
afford the desired compound as an off-white solid (0.140 g, 90%):
mp 127e128 ꢀC. IR (KBr) 2956, 1689, 1123, 819, 659 cmꢁ1; 1H NMR
95.78% (1% TFA in MeOH:H2O e 90:10).
5.1.21. (E)-2-(1-(4-Methyl-2-(40-(trifluoromethyl)-[1,10-biphenyl]-
4-yl)thiazol-5-yl)ethylidene)hydrazinecarboximidamide (22c)
Compound 21c (0.1 g, 0.277 mmol) was dissolved in absolute
ethanol (50 mL) and aminoguanidine hydrochloride (0.064 g,
0.588 mmol) and a catalytic amount of LiCl (10 mg) were added.
The reaction mixture was heated at reflux for 24 h. The solvent was
evaporated under reduced pressure. The crude product was puri-
fied by crystallization from 70% methanol, and then recrystallized
from methanol to afford the desired compounds as yellow solid
(0.088 g, 76%): mp 269e270 ꢀC. IR (KBr) 3583, 3307, 1678, 1145, 821,
(300 MHz, CDCl3)
d
8.05 (d, J ¼ 7.6 Hz, 2H), 7.64 (m, 4H), 7.23 (d,
665 cmꢁ1; 1H NMR (300 MHz, DMSO-d6)
d 11.78 (br s, 1H), 8.05 (d,
J ¼ 7.5 Hz, 2H), 2.79 (s, 3H), 2.58 (s, 3H); ESIMS m/z (rel intensity)
J ¼ 8.4 Hz, 2H), 7.98 (d, J ¼ 8.2 Hz, 2H), 7.90 (m, 8H), 2.62 (s, 3H),
2.45 (s, 3H); ESIMS m/z (rel intensity) 418 (MHþ, 100); HRESIMS
calcd for C20H19F3N5S m/z 418.1423 (MHþ), found 418.1420; HPLC
purity 96.10% (1% TFA in MeOH:H2O e 90:10).
312 (Mþ, 100); HPLC purity 98.50% (1% TFA in MeOH:H2O e 90:10).
5.1.18. 1-(4-Methyl-2-(40-(trifluoromethyl)-[1,10-biphenyl]-4-yl)
thiazol-5-yl)ethanone (21c)
Iodide 3 (0.172 g, 0.5 mmol), 4-trifluromethylphenyl boronic
acid (20c, 0.228 g, 1.5 mmol), tripotassium monophosphate
(0.424 g, 2 mmol) and (2-biphenyl)dicylohexylphosphine (18 mg)
were dissolved in dry toluene (15 mL) and the solution was purged
with argon for 20 min. Pd(OAc)2 (25 mg, 0.24 mmol) was added to
the reaction mixture and the reaction mixture was heated at 90 ꢀC
under argon for 24 h. Ethyl acetate (40 mL) was added to reaction
mixture, which was then washed with water (2 ꢂ 25 mL). The
combined organic layer was dried over Na2SO4, concentrated and
purified by flash column chromatography (EtOAc:hexanes 3:7 to
0.9:9.1) to afford the desired compound as an off-white solid
(0.150 g, 80%): mp 116e117 ꢀC. IR (KBr) 2959, 1938, 1650, 1111, 819,
5.1.22. (E)-2-(1-(2-(40-Acetyl-[1,10-biphenyl]-4-yl)-4-
methylthiazol-5-yl)ethylidene)hydrazinecarboximidamide (22d)
Compound 11d (0.150 g, 0.472 mmol) was dissolved in absolute
ethanol (50 mL) and aminoguanidine hydrochloride (0.064 g,
0.588 mmol) and a catalytic amount of LiCl (10 mg) were added.
The reaction mixture was heated at reflux for 24 h. The solvent was
evaporated under reduced pressure. The crude product was puri-
fied by crystallization from 70% methanol, and then recrystallized
from methanol to afford the desired compound as a light yellow
solid (0.062, 35%): mp > 280 ꢀC. IR (KBr) 3402, 1715, 1655, 1446,
1125, 856, 665 cmꢁ1 1H NMR (300 MHz, DMSO-d6)
; d 11.68 (br s,
1H), 8.10 (d, J ¼ 8.2 Hz, 4H), 7.91 (m, 6H), 7.80 (d, J ¼ 8.2 Hz, 2H),
2.72 (s, 3H), 2.58 (s, 3H), 2.40 (s, 3H); ESIMS m/z (rel intensity) 392
(MHþ, 100); HRESIMS calcd for C21H22N5OS m/z 392.1165 (MHþ),
found 392.1169.
659 cmꢁ1; 1H NMR (300 MHz, CDCl3)
d
8.09 (d, J ¼ 8.3 Hz, 2H), 7.72
(m, 6H), 2.80 (s, 3H), 2.58 (s, 3H); ESIMS m/z (rel intensity) 361 (Mþ,
100); HPLC purity 98.75% (1% TFA in MeOH:H2O e 90:10).
5.1.19. (E)-2-(1-(2-(40-Hydroxy-[1,10-biphenyl]-4-yl)-4-
5.1.23. 1-(4-Methyl-2-(4-(naphthalen-1-yl)phenyl)thiazol-5-yl)
ethanone (24)
methylthiazol-5-yl)ethylidene)hydrazinecarboximidamide (22a)
Compound 21a (0.150 g, 0.485 mmol) was dissolved in absolute
ethanol (50 mL) and aminoguanidine hydrochloride (0.064 g,
0.588 mmol) and a catalytic amount of LiCl (10 mg) were added.
The reaction mixture was heated at reflux for 24 h. The solvent was
evaporated under reduced pressure. The crude product was puri-
fied by crystallization from 70% methanol, and then recrystallized
from methanol to afford the desired compound as a light yellow
solid (0.115 g, 66%): mp 252e254 ꢀC. IR (KBr) 3398,1695,1655,1142,
Iodide 3 (0.172 g, 0.5 mmol), 1-naphalene boronic acid (23,
258 g, 1.5 mmol), tripotassium monophosphate (0.424 g, 2 mmol)
and (2-biphenyl)dicylohexylphosphine (18 mg) were dissolved in
dry toluene (15 mL) and the solution was purged with argon for
20 min. Pd(OAc)2 (25 mg, 0.24 mmol) was added to the reaction
mixture and the reaction mixture was heated at 90 ꢀC under argon
for 24 h. Ethyl acetate (40 mL) was added to reaction mixture,
which was then washed with water (2 ꢂ 25 mL). The combined
organic layer was dried over Na2SO4, concentrated and purified by
855, 665 cmꢁ1; 1H NMR (300 MHz, DMSO-d6)
d 9.73 (s, 1H), 8.02 (d,

H. Mohammad et al. / European Journal of Medicinal Chemistry 94 (2015) 306e316
315
flash column chromatography (EtOAc: hexane 3:7 to 0.9:9.1) to
afford the desired compound as a light brown solid (0.170 g, 99%):
mp 141e142 ꢀC. IR (KBr) 2287, 1670, 1006, 801, 663 cmꢁ1; 1H NMR
were collected after 0, 2, 4, 6, 8, 10, 12, and 24 h of incubation at
37 ꢀC and subsequently serially diluted in PBS. Bacteria were then
transferred to TSA plates and incubated at 37 ꢀC for 18e20 h before
viable CFU/mL was determined.
(300 MHz, CDCl3)
d
8.11 (d, J ¼ 6.5 Hz, 2H), 7.90 (m, 3H), 7.61 (m,
7H), 2.82 (s, 3H), 2.60 (s, 3H); ESIMS m/z (rel intensity) 344 (MHþ,
100); HRESIMS calcd for C22H18NOS m/z 344.1123 (MHþ), found
344.1125.
5.2.4. In vitro cytotoxicity analysis
Compounds 1, 5, 22bed and 25 were assayed at concentrations
of 5 mg/mL, 10 mg/mL, 20 mg/mL, and 40 mg/mL against a human
5.1.24. (E)-2-(1-(4-Methyl-2-(4-(naphthalen-1-yl)phenyl)thiazol-
5-yl)ethylidene)hydrazinecarboximidamide (25)
embryonic kidney (HEK293) cell line to determine the potential
toxic effect to mammalian cells in vitro. Cells were cultured in
Dulbeco's modified Eagle's medium (SigmaeAldrich, St. Louis, MO,
USA) with 10% fetal bovine serum (USA Scientific, Inc.) 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 the
compounds in a 96-well plate at 37 ꢀC and 5% CO2 for 2 h prior to
addition of the assay reagent MTS 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)
(Promega, Madison, WI, USA). Absorbance readings (at OD490) were
taken using a kinetic microplate reader (Molecular Devices, Sun-
nyvale, CA, USA). The quantity of viable cells after treatment with
each compound was expressed as a percentage of the viability of
DMSO-treated control cells.
Compound 24 (0.1 g, 0.291 mmol) was dissolved in absolute
ethanol (50 mL) and aminoguanidine hydrochloride (0.064 g,
0.588 mmol) and a catalytic amount of LiCl (10 mg) were added.
The reaction mixture was heated at reflux for 24 h. The solvent was
evaporated under reduced pressure. The crude product was puri-
fied by crystallization from 70% methanol, and then recrystallized
from methanol to afford the desired compound as a yellow solid
(0.057, 49%): mp 241e242 ꢀC. IR (KBr) 3299, 2300, 1675, 1618, 1142,
800, 664 cmꢁ1; 1H NMR (300 MHz, DMSO-d6)
d 11.63 (br s,1H), 8.08
(m, 5H), 7.65 (d, J ¼ 5.1 Hz, 1H); 7.61 (m, 9H), 2.64 (s, 3H), 2.44 (s,
3H); ESIMS m/z (rel intensity) 400 (MHþ, 100); HRESIMS calcd for
C
23H22N5S m/z 400.1323 (MHþ), found 400.1327; HPLC purity
95.55% (1% TFA in MeOH:H2O e 85:15).
5.2.5. Microsomal stability analysis
5.2. Biological characterization of thiazole compounds
The metabolic stability analysis of analog 5 was performed as
described previously [32]. Compound 5 was incubated in duplicate
with human liver microsomes at 37 ꢀC. The reaction contained
microsomal protein in 100 mM potassium phosphate, 2 mM
NADPH, 3 mM MgCl2, pH 7.4. A control was run for each test agent
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 ꢀC, and an additional volume of water was added.
The samples were centrifuged to remove precipitated protein, and
the supernatants were analyzed by LC/MS/MS to quantitate the
remaining parent. Data were converted to % remaining by dividing
by the time zero concentration value. Data were fit to a first-order
decay model to determine half-life. Intrinsic clearance was calcu-
lated from the half-life and the protein concentrations as follows:
5.2.1. Bacterial strains and reagents
Clinical isolates of MRSA, VISA, and VRSA were obtained through
the Network of Antimicrobial Resistance in S. aureus (NARSA)
program. In addition, MRSA ATCC 43300 was obtained from the
American Type Cultural Collection (Manassas, VA, USA). Vanco-
mycin hydrochloride powder was purchased commercially (Gold
Biotechnology Inc., St. Louis, MO, USA) and dissolved in DMSO to
prepare a 10 mM stock solution.
5.2.2. Determination of minimum inhibitory concentration (MIC)
and minimum bactericidal concentration (MBC) against MRSA,
VISA, and VRSA strains
The MICs of the thiazole compounds and vancomycin against
seven clinical isolates of MRSA, three clinical isolates of VISA, and
three clinical isolates of VRSA were determined using the broth
microdilution method in accordance with the recommendations
contained in the CLSI guidelines [40]. Bacteria were prepared in
phosphate-buffered saline (PBS) to achieve a McFarland standard of
0.5. The solution was subsequently diluted 1:300 in Mueller-Hinton
broth (MHB) to reach a starting inoculum of 1 ꢂ 105 colony-forming
units (CFU/mL). Bacteria were then transferred to a 96-well mi-
crotiter plate. Thiazole compounds and vancomycin were added (in
triplicate) to wells in the first row of the microtiter plate and then
serially diluted along the vertical axis. The plate was incubated at
37 ꢀC for 18e20 h before the MIC was determined as the lowest
concentration where visible growth of bacteria was not observed.
CLint ¼ ln(2)/(T1/2 [microsomal protein])
5.2.6. Statistical analysis
All statistical analysis was performed using the unpaired t-test
(P < 0.05) utilizing GraphPad Prism 6 software. Data for both the
time-kill assay and toxicity analysis of the tested compounds are
presented as mean standard deviation (as depicted by the error
bars).
The MBC was determined by plating 5 mL from wells on the 96-
well microtiter plate (where the MIC was determined), where no
growth was observed, onto Tryptic soy agar (TSA) plates. The TSA
plates were then incubated at 37 ꢀC for 18e20 h before the MBC
was determined. The MBC was classified as the concentration
where ꢄ99% reduction in bacterial cell count was observed.
Acknowledgments
The authors would like to thank the Network of Antimicrobial
Resistance in Staphylococcus aureus (NARSA) program supported
under NIAID/NIH Contract # HHSN272200700055C for providing
MRSA strains used in this study. ASM is supported by the Center of
Special Studies, Bibliotheka Alexandria, Egypt.
5.2.3. Time-kill analysis of thiazole compounds 1, 5, and 25 and
vancomycin against MRSA
MRSA USA300 cells in late logarithmic growth phase were
diluted to ~1 ꢂ 108 colony-forming units (CFU/mL) and exposed to
concentrations equivalent to 3 ꢂ MIC (in triplicate) of thiazole
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