Discovery and characterization of potent thiazoles versus methicillin- and vancomycin-resistant Staphylococcus aureus

Article abstract of DOI:10.1021/jm401905m

Methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) infections are growing global health concerns. Structure-activity relationships of phenylthiazoles as a new antimicrobial class have been addressed. We present 10 thiazole derivatives that exhibit strong activity against 18 clinical strains of MRSA and VRSA with acceptable PK profile. Three derivatives revealed an advantage over vancomycin by rapidly eliminating MRSA growth within 6 h, and no derivatives are toxic to HeLa cells at 11 μg/mL.

Full text of DOI:10.1021/jm401905m

Brief Article
pubs.acs.org/jmc
Discovery and Characterization of Potent Thiazoles versus
Methicillin- and Vancomycin-Resistant Staphylococcus aureus
Haroon Mohammad,† Abdelrahman S. Mayhoub,‡,∥ Adil Ghafoor,† Muhammad Soofi,†
Ruba A. Alajlouni,† Mark Cushman,‡ and Mohamed N. Seleem*,†
†Department of Comparative Pathobiology, Purdue University College of Veterinary Medicine, West Lafayette, Indiana 47906,
United States
‡Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University College of Pharmacy and the Purdue Center
for Cancer Research, West Lafayette, Indiana 47906, United States
S
* Supporting Information
ABSTRACT: Methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA) infections are growing global
health concerns. Structure−activity relationships of phenylthiazoles as a new antimicrobial class have been addressed. We present
10 thiazole derivatives that exhibit strong activity against 18 clinical strains of MRSA and VRSA with acceptable PK profile. Three
derivatives revealed an advantage over vancomycin by rapidly eliminating MRSA growth within 6 h, and no derivatives are toxic
to HeLa cells at 11 μg/mL.
INTRODUCTION
■
Methicillin-resistant Staphylococcus aureus (MRSA) is a rapidly
expanding global health concern. It is currently the most
common pathogen linked to patients with skin and soft-tissue
infections.1Apart from the high mortality and rapid trans-
mission rates, MRSA infections result in an estimated $3 billion
to $4 billion of additional health care costs per year.2 Resistance
has also emerged to therapeutic agents once deemed to be the
drugs of choice in treating MRSA infections, such as
vancomycin3 and linezolid.4 Researchers and clinical-care
providers are thus facing an increasingly difficult challenge
trying to construct novel antimicrobials and new therapeutic
options to treat MRSA-related infections.
The thiazole ring is a key structural component for a wide
Figure 1. Chemical structures of lead 1a and 4b (removal of the
cationic moiety) and 1b (removal of the lipophilic alkane side chain).
spectrum of therapeutic agents including anticonvulsants,5
anticancer,6,7 antiviral,8 and antibacterial agents.9,10 In this
study, whole-cell screening assays of libraries of substituted
thiazoles and thiadiazoles identified a novel lead compound that
displayed notable antibacterial activity against MRSA. The lead
1a (Figure 1) consists of a thiazole central ring connected to
optimizations were focused on the lipophilic side chain at
thiazole-C2 of the lead compound in an attempt to enhance the
antimicrobial activity of the lead compound against MRSA and
VRSA. Chemical modifications reported here involved building
two unique structural features: a cationic element at the C5
position and a lipophilic moiety at the C2 position. These two
structural components have been hypothesized to contribute to
Received: August 7, 2013
the antibacterial activity of the lead compound. Structural
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
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a
a focused library of phenylthiazoles with different lipophilic
moieties at the phenyl para position to define the structure−
activity relationships (SARs) at the thiazole-C2 position in a
rigorous way. Our objectives were to investigate the antimicrobial
activities of the thiazole derivatives against MRSA and VRSA,
ascertain the killing kinetics of MRSA in vitro by the lead
compound and two derivatives, determine the cytotoxic impact of
the derivatives on mammalian cells in vitro, and investigate the
pharmacokinetics (namely, solubility, permeability, and metabolic
stability) of the thiazole compounds.
Scheme 2. Preparation of 1h
CHEMISTRY
■
Thiazole ethyl ketone derivatives 4a−g were prepared in
moderate yields by heating thioamides 3a−g, obtained by
treatment of the corresponding amides with Lawesson’s reagent
in dry THF, with 3-chloropentane-2,4-dione in absolute
ethanol (Scheme 1). The methyl ketones 4a−g were gently
a
Reagents and conditions: (a) absolute ethanol, 3-chloropentane-2,4-
dione, heat to reflux, 12 h, 67%; (b) aminoguanidine hydrochloride,
absolute ethanol, heat to reflux, 24 h, 40%.
a
Scheme 1. Preparation of 1a−g
a
Scheme 3. Preparation of 7 and 8
a
Reagents and conditions: (a) (i) SOCl2, heat to reflux, 2 h, (ii)
NH4OH, 0−23 °C, 2−5 h, (iii) Lawesson’s reagent, dry THF, 50−60 °C,
5−24 h; (b) absolute ethanol, 3-chloropentane-2,4-dione, heat to
reflux, 12 h, 63%; (c) aminoguanidine hydrochloride, absolute EtOH,
heat to reflux, 24 h.
a
Reagents and conditions: (a) absolute ethanol, 3-chloropentane-2,4-
dione, heat to reflux, 12 h, 58%; (b) cyclohexene, PdAcO2, Et3N,
DMF, 80 °C, 5 h, 39%; (c) aminoguanidine hydrochloride, absolute
ethanol, heat to reflux, 24 h; (d) H2, Pd/C, methanol, 23 °C, 24 h,
38−42%.
heated with aminoguanidine hydrochloride in the presence of
lithium chloride as a catalyst to afford hydrazinecarboximid-
amide derivatives 1a−g (Scheme 1). Similarly, the final
products 1h, 7, 8, and 12 were obtained using a similar
synthetic protocol (Schemes 2−4).
two (1c) to three (1a) to four (1d) methylene units, there was
a consistent improvement in the MIC values observed against
all MRSA strains tested. However, additional lengthening of the
alkyl side chain appeared to nullify the improvement observed
in the antimicrobial activity, as the MIC for 1e (containing six
methylene units) nearly matched or exceeded the values
obtained for 1d. This result held true, as an increase to eight
methylene units (1f) resulted in an MIC that nearly matched or
exceeded the MIC attained for 1a. Altogether this indicates that
an alkyl side chain with four methylene units exhibits the
BIOLOGICAL RESULTS AND DISCUSSION
■
The 10 substituted thiazole compounds we synthesized
inhibited growth of 18 different strains of MRSA and VRSA
at 0.4−5.5 μg/mL (Table 1). The lead 1a inhibited the growth
of MRSA strains at 1.4−5.5 μg/mL. Subsequently synthesized
derivatives demonstrated a 2- to 5-fold improvement in the
MIC values. Initially, the effect of increasing the length of the
alkyl side chain, through insertion of methylene units, was
explored. As the length of the alkyl side chain increased from
B
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Journal of Medicinal Chemistry
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a
in inhibiting growth of VISA and VRSA strains. Compounds 7, 8,
and 12 also proved to be more effective at eliminating growth of
MRSA NRS119, a strain resistant to linezolid (a drug of last resort
in the treatment of MRSA infections), and several strains resistant
to multiple antibiotic classes including lincosamides, aminoglyco-
sides, fluoroquinolones, and macrolides (USA100, USA200, and
USA500). In addition to this, all 10 compounds exhibited excellent
activity against MRSA USA300, a strain responsible for most cases
of community-acquired MRSA (CA-MRSA) and MRSA skin and
soft tissue infections (SSTIs) in the U.S.11,12
Scheme 4. Preparation of 12
A drawback of several commercial antimicrobials used to
treat MRSA infections, including vancomycin and linezolid, is
that they are only capable of inhibiting bacterial growth (but do
not kill the bacteria) or that they exhibit a very slow bactericidal
effect resulting in difficulty in clearing the infection.13,14 Thus, a
compound that demonstrates the ability to rapidly kill MRSA is
highly desirable, since it limits the possibility of developing
bacterial resistance/tolerance. We studied the rate at which the
compounds were able to eliminate MRSA (USA300) in vitro
in a time-kill assay. The results (Figure S1) indicate that at
3.0 × MIC, 1a, 1d (derivative that contains one more
methylene unit in the alkyl side chain), and 8 (derivative that
replaces the alkyl side chain with a cyclohexane ring) are
bactericidal. However, the rate of clearance of MRSA
(USA300) varies among the three compounds. Compound
1d mimics the action of 1a, rapidly eliminating MRSA
completely within 2 h. This would appear logical, as 1a and
1d are similar in structure, the major difference resulting from
the number of methylene units contained in the alkyl side
chain. Compound 8 requires more than double the time (6 h)
to logarithmically reduce MRSA colony forming units (CFUs)
to zero. Though 8 appears to be more potent compared to 1a
and 1d (when comparing MIC values), the last two appear
capable of clearing MRSA colonies (albeit at a higher
concentration) more rapidly. Vancomycin was not able to
reduce the number of CFU by 3 log10 within a 12 h window.
Collectively, this indicates that the thiazole compounds possess
a selective advantage over vancomycin in terms of rate of
elimination of MRSA cells. This information is clinically
a
Reagents and conditions: (a) (i) H2NOH HCl, DMSO, 100 °C,
20 min; (ii) NaOH, H2O2, 12 h; (iii) Lawesson’s reagent, THF, 23 °C,
12 h; (b) absolute ethanol, 3-chloropentane-2,4-dione, heat to reflux,
12 h, 49%; (c) aminoguanidine hydrochloride, absolute ethanol, heat
to reflux, 24 h, 45%.
optimum potency against MRSA and addition of methylene
units to the alkyl side beyond four units will not significantly
enhance the antimicrobial activity of the lead compound.
Replacement of the linear alkyl side chain with a branched
alkane (1g) produced mixed results. There was a modest
improvement in the MIC values against six MRSA strains
(1.0 μg/mL for 1g compared to 1.4 μg/mL for 1a) and a nearly
2- to 5-fold enhancement in the MIC for five additional strains.
Substitution of the alkyl side chain with a more bulky fused ring
system (1h) did not significantly enhance the activity of the
derivative against the MRSA strains tested. However, replacement
of the alkyl side chain with conformationally restricted analogues
(7, 8, and 12) demonstrated the most consistent, significant
improvement in the MIC obtained relative to the lead 1a (2- to
4-fold improvement against 16 MRSA strains tested).
The MIC values obtained for 7, 8, and 12 on multiple
occasions matched or were lower than the values of antibiotic
vancomycin against the MRSA strains tested. Furthermore, all
10 thiazole compounds proved to be more potent than vancomycin
Table 1. Antimicrobial Activities (μg/mL) of Modified Thiazole Compounds Screened against Staphylococcus aureus
MIC SD (μg/mL) of thiazole compounds and vancomycin against S. aureus
strain
1a
1c
1d
1e
1f
1g
1h
7
8
12
VAN
MRSA ATCC 43300 2.8
VISA ATCC 700699 1.4
0
0
0
0
0
0
0
0
0
0
0
0
0
1.2
0
0.8
0
3.0
0
0
0
0
2.2
1.1
0
0
1.9
1.6 0.6 3.2 1.1 0.5 0.2 0.7
2.6 1.1 0.8 0.3 0.7
0
3.8
0
1.2
0
1.5
0
0
0
0.6
0.6
0
0
0.7
2.9
0
0
0
0
0
0
1.9 0.7 0.5 0.2 0.7
1.9 0.7 0.7 0.3 1.5
VISA HIP07256
VISA LIM 3
1.4
1.4
1.4
1.4
1.4
1.4
1.4
5.5
2.8
1.4
1.4
2.9 1.3 1.9
0
0.5 0.2 2.9
1.6 0.7 0.5
0
0
0.7
1.1
0
0
0
0
0
0
0
0
0
1.6 0.6 1.9
0
0
0
0
0
0
0
0.8 0.3 0.5 0.2 0.6
0.6 0.6 0.2 1.2
1.7 1.0 1.5
0
0
2.9
0.7
MRSA NRS107
MRSA NRS108
MRSA NRS119
MRSA USA400
MRSA NRS194
MRSA USA100
MRSA USA200
MRSA USA300
MRSA USA500
MRSA USA700
MRSA USA800
MRSA USA1000
MRSA USA1100
VRSA
1.2
1.2
0
0
0.5
1.0 0.4 1.1
1.0
1.9
1.9
1.9
0
0
0
0
1.9
1.9
3.8
1.9
0
0.9 0.3 3.0
0
0
0
0
4.4
2.2
4.4
4.4
0
0
0
0
0
2.0 0.7 0.7
0.6
1.9 0.7 0.9 0.3 1.5
1.2 0.9 0.3 1.5
0.6
0.6
0
0
0.7
0.7
0
1.2 0.4
0
0.8 0.3 0.7
1.4 0.9 0.7
0
0
1.9 0.7 1.1
1.6 0.7 1.1
1.9 0.7 1.1
0
0
0
0
0
3.0
2.6 1.1 1.9
1.3 0.6 1.9
0.8 0.3 0.7
1.0 0.3 0.7
3.0 2.6 2.2
1.2 0.4 2.2
2.5 0.9 2.2
2.0 0.7 1.2 0.4
0.4
1.0 0.3 0.6 0.2
2.6 1.1 2.6 1.1 1.0 0.3 1.2 0.4 0.6
1.3 0.6 1.9 1.2 0.7
0
0
1.2
0
1.1
0
0
0
1.9 0.7 1.1
3.0
0
3.7 1.3 1.3 0.6 2.6 1.1 1.0 0.3 0.9 0.6 1.6 0.7 1.0 0.4
1.8 0.8 1.2
0
0.9 0.3 4.0 1.7 1.8 0.6 1.0
0
2.6 1.1 1.2
0
1.5
0
0
0.8 0.3 1.0 0.4
1.2 0.7
2.3 0.8 1.9 0.7 0.7 0.3 1.5
0
1.8 0.6 1.3 0.6 1.9
0
0
0
0
0.8 0.3 0.7
0
0
1.4
2.8
1.4
0
0
0
1.6 0.7 1.1
1.6 0.7 1.1
0
0
0
2.5 0.9 2.2
0
0
0
1.0
1.9
0
0
1.9
1.9
0.8 0.3 0.9 0.6 0.8 0.3 0.6 0.2
1.5
1.5
0
0
2.2
2.2
0.6
1.2
0
0
0.7
1.5
0
0
0.6
0.6
0
0
0.7
0
1.2
0
1.1
1.6 0.6 1.9
185.5
0
C
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Journal of Medicinal Chemistry
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significant, as it would impact the size and timing of the dose
given to patients with an infection caused by MRSA.
reserpine (31.3 μM) and nearly 4 times the concentration of
the cancer drug tamoxifen (15.6 μM) as presented in Table S3.
Taking into consideration that both drugs are administered
orally, the solubility properties of phenylthiazoles are highly
promising.
In addition to this, combination therapy using multiple
antibiotics to treat MRSA infections is commonly used in
clinical practice. Antibiotics that are bacteriostatic or exhibit a
slow bactericidal effect (such as vancomycin)13 are often paired
with antibiotics exhibiting a rapid bactericidal effect (such as
rifampin) to limit the emergence of bacterial strains with reduced
susceptibility to vancomycin.13 As the thiazole compounds
presented here exhibit a rapid bactericidal effect against MRSA,
analysis of synergy between the thiazole compounds and
commercial antimicrobials (such as vancomycin and linezolid)
for potential use in combination therapy would be an interesting
avenue to further explore.
The cytotoxicity assay (Figure S2) confirmed that all of the
compounds are selective for bacterial cell inhibition over
mammalian cells. All compounds tested were not toxic to the
HeLa human tumor cell line up to 11 μg/mL; this
concentration is more than 20-fold higher than the MIC for
the most potent thiazole derivatives (8 and 12). Irrespective of
the modification made to the alkane side chain of the lead
compound (addition of methylene units or substitution with a
cyclic moiety), the subsequent derivatives maintained a good
toxicity profile when tested against HeLa cells.
Physicochemical properties, including solubility and perme-
ability, of potential therapeutic agents are critical factors that
need to be explored early in drug development. Though a
compound proves potent against a target organism during in
vitro studies and exhibits limited toxicity to cultured
mammalian cells, the drug candidate can fail in animal and
human studies if the drug is poorly soluble in aqueous solutions
or is incapable of passing through cellular barriers. Analysis of
the hydrogen bonding potential and lipophilicity of a
compound can lend valuable insight into potential solubility
and permeability issues. After documentation of the strong
antimicrobial activity of the thiazole compounds and
determination of their limited toxicity against mammalian
cells, it was critical to establish whether the compounds possess
potential solubility and permeability issues. By use of Lipinski’s
rule of 5 and topological polar surface area (TPSA) as guidelines,
the results in Table S2 demonstrate that all 10 thiazole
compounds possess clogP and TPSA values that are associated
with good solubility and permeability qualities. Two derivatives
(1e and 1f) violate one parameter of the rule of 5 with each
derivative possessing a clogP above 5. These derivatives contain
the longest linear alkyl chain (six and eight methylene units for
1e and 1f, respectively) connected to the phenylthiazole nucleus.
This result supports the notion that an ideal thiazole side chain
should have four methylene units, as compounds possessing an
alkyl side chain with more than four methylene units exhibit a
decrease in the antimicrobial activity against MRSA and pose
potential solubility issues.
The Caco-2 permeability assay revealed the lead 1a
surprisingly exhibited very limited permeability across the
membrane from the apical (A) to basolateral (B) direction as
demonstrated in Table S4, maybe because of the slight acidic
experimental conditions. Compound 1a exhibits a higher
apparent permeability coefficient (Papp) in the basolateral to apical
direction (Papp = 2.2 × 10−6 cm/s) which mimics the result
obtained with the control drug ranitidine (Papp = 1.2 × 10−6 cm/s
in the B−A direction compared to 0.2 × 10−6 cm/s in the A−B
direction). Transporters in the membranes can enhance or reduce
the permeability of a compound. The presence of efflux
transporters on the apical surface of epithelial cells in the
intestine may play a role in preventing the absorption of
the thiazole compounds and passage through Caco-2 cells. The
efflux ratio of >2 for the lead thiazole compound supports the
notion that the compound may be a substrate for an efflux
transporter (possibly P-glycoprotein which is a potential reason
for the higher rate of transfer of compound from the B to A
direction). To confirm if this was the case, 1a was analyzed
using Madin−Darby canine kidney (MDCK) cells transfected
with a gene overexpressing multidrug resistance protein 1 (MDR1),
also referred to as P-glycoprotein 1 (Pgp). As presented in
Table S5, a higher rate of transfer of 1a is observed in the B to A
direction (Papp = 3.6 × 10−6 cm/s) compared to the A to B
direction (Papp = 0.7 × 10−6 cm/s), consistent with what is
observed with the Caco-2 permeability assay. The efflux ratio
determined from the MDCK-MDR1 permeability assay for 1a is
5.0, indicating that the compound may be subject to the effect of
Pgp. An increasing number of hydrogen bond acceptors (oxygen
and nitrogen atoms) has been shown to increase the likelihood of
Pgp efflux of drugs.15 Thus, constructing derivatives of the lead
thiazole compound focusing on modifications to the cationic
head (where the hydrogen bond acceptor groups are present) is
one mechanism to enhance permeability. A delicate balance
between addition or substitution of functional groups would
need to be achieved to ensure that permeability is enhanced
without reducing the solubility profile of the thiazole compounds.
Another method to enhance passage of the thiazole compounds
across the intestinal membrane is to use a higher concentration
of the lead compound, especially if the compound is a substrate
for efflux transporters. The Caco-2 assay utilized a low
concentration (10 μM) of the lead compound. The concen-
tration of a drug in the gastrointestinal lumen after an oral dose is
typically 50−100 μM.15 Thus, testing the lead compound at a
higher concentration is necessary to confirm if the poor
permeability observed in Caco-2 assay is potentially due to the
low concentration of compound used in the assay. A higher
concentration may help the thiazole compound to effectively
cross the intestinal barrier, as efflux transporters will eventually
become saturated in a concentration-dependent manner
permitting compound that has passively diffused across the
membrane to reach the portal circulation. Taken collectively, the
permeability profile of the thiazole compounds is initially
promising and can be potentially improved by modifying the
structure of the lead compound or increasing the concentration
of the compound used.
To confirm if the thiazole compounds possess good
physicochemical properties as predicted, lead 1a was analyzed
using the Caco-2 permeability assay, MDCK-MDR1 perme-
ability assay, and a solubility screen utilizing phosphate buffered
saline (PBS). The solubility screen was used to determine the
highest concentration of lead 1a in comparison with three
control drugs that was able to dissolve in PBS before precipitate
formed. The PBS solubility screen indicates that 1a possesses
acceptable solubility compared to the reference tested drugs.
When compared to drugs with poor aqueous solubility, 1a was
soluble at twice the concentration of the antihypertensive drug
In addition to testing the solubility and permeability
characteristics of the lead 1a, the metabolic stability of 1a
D
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Journal of Medicinal Chemistry
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solvent was evaporated under reduced pressure, and the residues were
partitioned between aqueous NaHCO3 (2 M, 25−50 mL) and ethyl
acetate (25−75 mL). The organic solvent was separated and dried
over anhydrous MgSO4. After solvent evaporation, the crude products
were further purified by silica gel flash chromatography, using hexane−
ethyl acetate (4:1), to yield the corresponding thioamides as yellow
solids (55−57%) in the desired purity degree. Characterizations of
3a−g are listed in Supporting Information.
Preparation of Methyl Ketones 4a−i. General Procedure.
Thiobenzamides 3a−i (2−10 mmol) and 3-chloropentane-2,4-dione
(1.4 equiv) were added to absolute ethanol (10−30 mL). The
mixtures were heated at reflux for 12−24 h. After evaporation of
solvent under reduced pressure, the brown residues were collected and
purified by silica gel flash chromatography, using hexane−ethyl acetate
(9:1), to yield 4a−i in the desired purity. Compounds 4a16 and 4b17
are previously reported. Characterizations of 4c−i are listed in
Supporting Information.
Preparation of Hydrazinecarboximidamides 1a−h, 7, 8, and
12. General Procedure. The ketone derivative 4a−h, 5, 6, or 11
(1−10 mmol) was dissolved in absolute ethanol (10−50 mL).
Aminoguanidine hydrochloride (1 equiv) and a catalytic amount of
LiCl (5−20 mg) were added. The mixtures were 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 absolute methanol to afford the desired compounds
as solids. Compound 1a16 is previously reported. Physical properties
and spectroscopic data of hydrazinecarboximidamides 1a−h, 7, 8, and
12 are listed below.
was investigated using human liver microsomes. As Table S6
demonstrates, 1a is subject to metabolism by the liver with a
microsomal intrinsic clearance of 80.3 μL min−1 mg−1 and a
half-life of 28.8 min. These values align with the values obtained
with verapamil (the metabolized control drug) rather than for
warfarin (nonmetabolized control drug). Removing the cofactor
NADPH significantly reduced the metabolism of 1a, indicating
that these metabolic processes are NADPH-dependent.
In conclusion, the identification of novel antimicrobial agents
to treat an array of infections caused by methicillin-resistant and
vancomycin-resistant S. aureus requires a multifold approach
from whole-cell screening of chemical libraries to rational drug
design. We present the exciting discovery of a lead
antimicrobial compound, identified from whole-cell screening
of a library of thiazole and thiadiazole compounds, that is
capable of inhibiting growth of 18 strains of MRSA and VRSA.
The lead compound consists of a thiazole central ring
connected to two structural elements critical for activity,
namely, a cationic element at the C5 position and a lipophilic
moiety at the C2 position. A focused library of derivatives
containing modifications to the lipophilic moiety was
constructed to enhance the antimicrobial activity against
MRSA and VRSA. The lead compound and nine derivatives
are capable of inhibiting growth of 18 different clinical isolates
of MRSA and VRSA at 0.5−3.0 μg/mL. The antibacterial
spectrum of some analogues surpassed that of several known
antibiotics in eliminating the growth of highly resistant strains
such as MRSA NRS119, a strain resistant to linezolid (a drug of
last resort in treatment of MRSA infections), and several other
strains resistant to multiple antibiotic classes including
vancomycin, lincosamides, aminoglycosides, fluoroquinolones,
and macrolides (VRSA, USA100, USA200, and USA500).
Furthermore, the lead compound and two derivatives exhibit a
rapid bactericidal effect, eliminating MRSA growth in vitro
within 6 h. In addition to this, six derivatives, including the
three most potent compounds against MRSA, are not toxic.
The 10 thiazole compounds were predicted to have good
solubility and permeability characteristics based on the criteria set
forth by Lipinski’s rule of 5. However, analysis of permeability of
the lead 1a via the Caco-2 and MDCK-MDR1 assays indicated
that the compound had poor permeability from the apical to
basolateral surface of the membrane (possibly due to the effect of
the Pgp efflux transporter). We confirmed that the lead
compound does not target the integrity of the bacterial cell
wall or cytoplasmic membrane (data not published); further
explanation of the molecular target of the thiazole compounds
will be presented in a future study. The characterization of the
novel thiazole compounds presents an intriguing step in the
development of a novel class of therapeutic agents effective for
treating MRSA and VRSA infections.
2-[1-(4-Methyl-2-phenylthiazol-5-yl)ethylidene]hydrazine-
carboximidamide (1b). Yellowish white solid (124 mg, 61%): mp
1
195−196 °C. H NMR (DMSO-d6) δ 11.65 (brs, 1 H), 8.88 (brs,
1 H), 7.91−7.88 (m, 4 H), 7.48 (m, 3 H) 2.60 (s, 3 H), 2.43 (s, 3 H);
13C NMR (DMSO-d6) δ 165.79, 160.11, 156.96, 153.33, 147.96,
133.51, 131.53, 130.24, 126.90, 19.27, 19.03; ESIMS m/z (rel intensity)
274 (MH+, 100); HRESIMS, m/z 274.1128 MH+, calcd for C13H16N5S
274.1126; HPLC purity (methanol/water, 1:1), 95.44%.
2-{1-[4-Methyl-2-(4-propylphenyl)thiazol-5-yl]ethylidene}-
hydrazinecarboximidamide (1c). Yellowish white solid (100 mg,
55%): mp 256−257 °C. 1H NMR (DMSO-d6) δ 11.47 (brs, 1 H), 7.80
(d, J = 8.1 Hz, 2 H), 7.76 (brs, 3 H), 7.29 (d, J = 8.1 Hz, 2 H), 2.58
(s, 3 H), 2.55 (t, J = 7.8 Hz, 2 H), 2.41 (s, 3 H), 1.60 (m, 2 H), 0.88 (t,
J = 7.5 Hz, 3 H); 13C NMR (DMSO-d6) δ 165.01, 156.80, 153.28,
148.14, 146.06, 131.21, 130.14, 126.95, 126.88, 37.94, 24.71, 19.14,
19.05, 14.54; ESIMS m/z (rel intensity) 316 (MH+, 100); HRESIMS,
m/z 316.1590 MH+, calcd for C16H22N5S 316.1596; HPLC purity
(methanol/water, 1:1), 97.09%.
2-{1-[4-Methyl-2-(4-pentylphenyl)thiazol-5-yl]ethylidene-
hydrazinecarboximidamide (1d). Yellow solid (54 mg, 50%): mp
210 °C. 1H NMR (DMSO-d6) δ 11.41 (brs, 1 H), 7.81 (d, J = 7.8 Hz,
2 H), 7.78 (brs, 3 H), 7.31 (d, J = 7.8 Hz, 2 H), 2.62 (m, 5 H), 2.41
(s, 3 H), 1.57 (m, 2 H), 1.27 (m, 4 H), 0.84 (t, J = 7.5 Hz, 3 H); 13C
NMR (DMSO-d6) δ 165.43, 156.16, 152.71, 147.59, 145.73, 130.57,
130.47, 129.51, 126.49, 35.25, 31.21, 30.66, 22.26, 18.52, 18.45, 14.24;
ESIMS m/z (rel intensity) 344 (MH+, 100); HRESIMS, m/z 344.1913
MH+, calcd for C18H26N5S 244.1909; HPLC purity (methanol/water,
1:1), 95.12%.
2-{1-[4-Methyl-2-(4-heptylphenyl)thiazol-5-yl]ethylidene-
hydrazinecarboximidamide (1e). Yellow solid (151 mg, 53%): mp
233−235 °C. 1H NMR (DMSO-d6) δ 11.43 (brs, 1 H), 7.80 (m, 5 H),
7.30 (d, J = 8.1 Hz, 2 H), 2.61 (m, 5 H), 2.42 (s, 3 H), 1.55 (t, J = 6.6
Hz, 2 H), 1.24 (m, 8 H), 0.82 (t, J = 6.6 Hz, 3 H); 13C NMR (DMSO-
d6) δ 165.98, 156.86, 153.25, 148.09, 146.28, 131.17, 131.06, 130.07,
126.89, 35.88, 32.15, 31.57, 29.54, 29.43, 23.00, 19.19, 19.05, 14.86;
ESIMS m/z (rel intensity) 372 (MH+, 100); HRESIMS, m/z 372.2228
MH+, calcd for C20H30N5S 372.2222; HPLC purity (methanol/water, 1:1),
99.31%.
EXPERIMENTAL SECTION
■
Chemistry. Detailed procedures are in Supporting Information.
The purities of tested compound are ≥95% (HPLC). HPLC analyses
were performed on a Waters binary HPLC system (model 1525, 20 μL
injection loop) equipped with a Waters dual wavelength absorbance
UV detector (model 2487) set for 254 nm and using a 5 μm C-18
reverse phase column.
Preparation of Thioamides 3a−g. General Procedure.
Thioamides 3a−g (1−5 mmol), which were obtained by treatment
of their corresponding carboxylic acids 2a−g with thionyl chloride
followed by gradual addition to ammonia solution, and Lawesson’s
reagent (1.2 equiv) were added to dry THF (15−40 mL). The
reaction mixtures were stirred at room temperature for 5−12 h. The
2-{1-[4-Methyl-2-(4-nonylphenyl)thiazol-5-yl]ethylidene-
hydrazinecarboximidamide (1f). Yellow solid (133 mg, 55%): mp
203−206 °C. 1H NMR (DMSO-d6) δ 11.32 (brs, 1 H), 7.79 (d, J = 8.1
Hz, 2 H), 7.60 (brs, 3 H), 7.28 (d, J = 8.1 Hz, 2 H), 2.58 (m, 5 H),
E
dx.doi.org/10.1021/jm401905m | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Brief Article
2.41 (s, 3 H), 1.55 (m, 2 H), 1.22 (m, 12 H), 0.82 (t, J = 6.9 Hz, 3 H);
13C NMR (DMSO-d6) δ 165.80, 156.87, 153.45, 147.99, 146.28,
131.60, 131.19, 129.44, 126.25, 36.34, 35.29, 31.60, 30.98, 29.28,
29.19, 28.99, 22.42, 18.46, 18.39, 14.27; ESIMS m/z (rel intensity) 400
(MH+, 100); HRESIMS, m/z 400.2540 MH+, calcd for C22H34N5S
400.2535; HPLC purity (methanol/water, 1:1), 95.40%.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors 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. This work was supported by
the Showalter Research Trust (Grant 205994) and the Purdue
Research Foundation (2012−2013 PRF).
2-{1-[2-(4-(tert-Butyl)phenyl)-4-methylthiazol-5-yl]-
ethylidene}hydrazinecarboximidamide (1g). Off-white solid
(155 mg, 67%): mp 252−253 °C. 1H NMR (DMSO-d6) δ 11.41
(brs, 1 H), 7.83 (d, J = 8.4 Hz, 2 H), 7.65 (brs, 3 H), 7.51 (d, J =
8.4 Hz, 2 H), 2.59 (s, 3 H), 2.41 (s, 3 H), 1.29 (s, 9 H); 13C NMR
(DMSO-d6) δ 165.90, 156.75, 154.40, 153.34, 148.22, 131.09, 130.94,
127.02, 126.76, 35.60, 31.81, 19.13, 19.05; ESIMS m/z (rel intensity)
330 (MH+, 100); HRESIMS, m/z 330.1755 MH+, calcd for C17H24N5S
330.1752; HPLC purity (methanol/water, 1:1), 96.86%.
2-{1-[4-Methyl-2-(naphthalen-2-yl)thiazol-5-yl]ethylidene}-
hydrazinecarboximidamide (1h). Yellow solid (80 mg, 40%): mp
288−290 °C. 1H NMR (DMSO-d6) δ 11.39 (brs, 1 H), 8.50 (s, 1 H),
8.08−7.94 (m, 4 H), 7.73 (brs, 3 H), 7.58 (m, 2 H), 2.64 (s, 3 H), 2.44
(s, 3 H); 13C NMR (DMSO-d6) δ 165.86, 156.73, 153.56, 148.19,
134.70, 133.75, 131.77, 130.94, 129.91, 129.59, 128.71, 128.41, 128.07,
126.44, 124.20, 19.06, 17.18; ESIMS m/z (rel intensity) 324 (MH+,
100); HRESIMS, m/z 324.1179 MH+, calcd for C17H18N5S 324.1283;
HPLC purity (methanol/water, 1:1), 95.99%.
2-{1-[2-(4-(1-Cyclohexenyl)phenyl)-4-methylthiazol-5-yl]-
ethylidene}hydrazinecarboximidamide (7). Yellow solid (58 mg,
42%): mp 213−215 °C. 1H NMR (DMSO-d6) δ 11.29 (brs, 1 H), 7.82
(d, J = 8.1 Hz, 2 H), 7.37 (d, J = 8.1 Hz, 2 H), 7.05 (brs, 3 H), 5.74
(m, 2 H), 2.80 (m, 1 H), 2.58 (s, 3 H), 2.37 (s, 3 H), 2.20−2.06 (m,
3 H), 1.82−1.71 (m, 3 H); 13C NMR (DMSO-d6) δ 164.46, 157.32,
151.22, 149.83, 146.15, 132.04, 131.07, 128.02, 127.11, 126.79, 126.31,
42.01, 32.85, 29.43, 25.59, 18.49, 17.77; ESIMS m/z (rel intensity) 354
(MH+, 100); HRESIMS, m/z 354.1759 MH+, calcd for C19H24N5S
354.1752; HPLC purity (methanol/water, 1:1), 97.50%.
2-{1-[2-(4-Cyclohexylphenyl)-4-methylthiazol-5-yl]-
ethylidene}hydrazinecarboximidamide (8). Yellow solid (42 mg,
38%): mp 273−276 °C. 1H NMR (DMSO-d6) δ 11.27 (brs, 1 H), 7.80
(d, J = 8.4 Hz, 2 H), 7.50 (brs, 3 H), 7.33 (d, J = 8.4 Hz, 2 H), 2.58
(s, 3 H), 2.57 (m, 1 H), 2.40 (s, 3 H), 1.76 (m, 5 H), 1.37 (m, 4 H);
13C NMR (DMSO-d6) δ 166.88, 156.35, 152.19, 150.97, 147.98,
ABBREVIATIONS USED
■
MIC, minimum inhibitory concentration; CFU, colony forming
unit; PK, pharmacokinetics
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130.33, 130.20, 127.13, 126.04, 44.34, 33.86, 26.35, 25.62, 16.70,
16.20; ESIMS m/z (rel intensity) 356 (MH+, 100); HRESIMS, m/z
356.1912 MH+, calcd for C19H26N5S 356.1909; HPLC purity
(methanol/water, 1:1), 98.09%.
2-{1-[2-([1,1′-Biphenyl]-4-yl)-4-methylthiazol-5-yl]-
ethylidene}hydrazinecarboximidamide (12). Yellow solid (104 mg,
1
45%): mp 278−280 °C. H NMR (CD3OD) δ 8.00 (d, J = 9.0 Hz,
2 H), 7.73 (d, J = 9.0 Hz, 2 H), 7.66 (d, J = 9.0 Hz, 2 H), 7.45 (t, J =
9.0 Hz, 2 H), 7.36 (t, J = 9.0 Hz, 1 H), 2.66 (s, 3 H), 2.42 (s, 3 H); 13C
NMR (CD3OD) δ 166.40, 155.92, 152.80, 148.24, 143.22, 139.60, 131.49,
130.29, 128.59, 127.16, 127.04, 126.47, 126.34, 16.74, 16.26; ESIMS m/z
(rel intensity) 350 (MH+, 100); HRESIMS, m/z 350.1435 MH+, calcd for
C19H20N5S 350.1439; HPLC purity (methanol/water, 1:1), 95.96%.
ASSOCIATED CONTENT
* Supporting Information
■
S
Synthetic procedures, characterization, yields, and physical and
spectral data of 3f, 4c−i, 5−8, 11, and 12 and biological tests.
This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Phone: 765-494-076. Fax: 765-496-2627. E-mail: mseleem@
purdue.edu.
■
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Roghmann, M.-C. Skin and soft tissue infections caused by methicillin-
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2007, 13, 1195−1200.
Present Address
∥A.S.M.: Department of Organic Chemistry, College of
Pharmacy, Al-Azhar University, Cairo, Egypt.
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