The synthesis of phenylalanine-derived C5-substituted rhodanines and their activity against selected methicillin-resistant Staphylococcus aureus (MRSA) strains

Article abstract of DOI:10.1016/j.ejmech.2010.09.045

A series of rhodanine compounds containing various substituents at the N3- and C5-positions were synthesized and their in vitro activity against a panel of clinically relevant MRSA strains was determined. The anti-MRSA activity of compounds 21 (MIC = 3.9

Full text of DOI:10.1016/j.ejmech.2010.09.045

European Journal of Medicinal Chemistry 45 (2010) 5827e5832
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
The synthesis of phenylalanine-derived C5-substituted rhodanines and their
activity against selected methicillin-resistant Staphylococcus aureus (MRSA)
strains
Diane Hardej, Charles R. Ashby Jr., Nikhil S. Khadtare, Shridhar S. Kulkarni, Satyakam Singh,
*
Tanaji T. Talele
Department of Pharmaceutical Sciences, College of Pharmacy and Allied Health Professions, St. John’s University, 8000 Utopia Parkway, Jamaica, NY 11439, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of rhodanine compounds containing various substituents at the N3- and C5-positions were
Received 26 April 2010
Received in revised form
14 September 2010
Accepted 20 September 2010
Available online 25 September 2010
synthesized and their in vitro activity against a panel of clinically relevant MRSA strains was determined.
The anti-MRSA activity of compounds 21 (MIC ¼ 3.9
MBC ¼ 7.8
biotics penicillin G (MIC ¼ 31.25
vancomycin (MIC ¼ 0.97
m
g/mL, MBC ¼ 7.8
m
g/mL) and 22 (MIC ¼ 1.95
mg/mL,
m
g/mL) was significantly greater than that of the lead compounds, 1e3 and reference anti-
m
g/mL) and ciprofloxacin (MIC ¼ 7.8
m
g/mL) and comparable to that of
m
g/mL). Compounds 21 and 22 were found to be bactericidal at only 2e4-fold
higher than their MIC concentrations. In addition, their MIC values remained unchanged in the presence
or absence of 10% serum. Overall, the results suggest that compounds 21 and 22 may be of potential use
in the treatment of MRSA infections.
Keywords:
MRSA
Rhodanine
Serum
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
other genes, has allowed MRSA to become resistant to many different
classes of antibiotics [16,17]. In contrast, CA-MRSA strains harbor
Methicillin-resistant Staphylococcus aureus (MRSA) has become
endemic in most hospitals and health care facilities in Western
nations. In the United States, MRSA infections cause an estimated
19,000 deaths per annum [1]. The two major types, hospital (HA)-
and community-acquired (CA) MRSA, are significantly different in
their epidemiological and genetic characteristics [2e4]. HA-MRSA
occurs in patients with specific risk factors [5e7], whereas CA-MRSA
can occur in healthy individuals that do not have predisposing factors
[8e10]. Typically, HA-MRSA infections occur in the urine, lungs,
bloodstream and surgical sites whereas CA-MRSA primarily produces
skin and skin structure infections [4,11]. The HA-MRSA strains
contain the mobile class I, II and III staphylococcus chromosome
SCCmec IV [18], a smaller and more mobile SCCmec than classes IeIII
and this may contribute to its widespread dissemination [19,20]. The
CA-MRSA strains, like HA-MRSA, are broadly resistant to
and macrolide/azalide antimicrobials but responsive to certain non-
-lactam antibiotics [3,21]. However, resistance rates are increasing
b-lactam
b
and there are other limitations in the use of these drugs. Thus, given
the widespread dissemination and mortality caused by MRSA, the
synthesis and development of new drugs is imperative.
Rhodanine-based molecules have been shown to be small mole-
cule inhibitors of numerous targets such as hepatitis C virus (HCV)
NS3 protease[22], aldosereductase[23,24],
b-lactamase [25], UDP-N-
acetylmuramate/ -alanine ligase [26], penicillin binding protein
L
cassettes mec (SCCmec) and resistance to the
due to the encoding of the penicillin binding protein (PBP) 2a by the
mecA gene [12e14]. In contrast, the resistance to non- -lactams is
due to the presence of transposons and other genes. PBP2a has an
elevated rate constant for dissociation and decreased acylation rate
constant and these together significantly decrease the acylation of
b
-lactam antibiotics is
(PBP) [27], antidiabetic agents [28], cathepsin D [29] and histidine
decarboxylase [30]. In addition, we have reported that rhodanine
analogs are inhibitors of the HCV enzyme NS5B polymerase [31]. In
a search for new antibacterial compounds from our in-house rho-
danine library, we initially identified three rhodanine derivatives [1:
b
MIC ¼ 62.5
mg/mL; 2: MIC ¼ 250
mg/mL and 3: MIC ¼ 125
mg/mL]
PBP2a, producing
b-lactam resistance [15]. This, in combination with
exhibiting modest activity against selected MRSA strains (Chart 1).
Here, we report the subsequent synthesis, anti-MRSA activity and
SAR optimization focusing on N3- and C5-positions of the rhodanine
core of these lead compounds. A variety of strains of S. aureus were
used for this study and were obtained from the AmericanType Tissue
* Corresponding author. Tel.: þ1 718 990 5405; fax: þ1 718 990 1877.
E-mail address: talelet@stjohns.edu (T.T. Talele).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.09.045

5828
D. Hardej et al. / European Journal of Medicinal Chemistry 45 (2010) 5827e5832
Chart 1. Structures of the anti-MRSA rhodanine hits.
Collection (ATTC). Strains were selected to represent the scope of
Staphylococcus variants that might be present in a number of clinical,
community and quality control situations. ATCC strain 34404 is
commonly used as a quality control organism for susceptibility
testing. Strains 700698, 700787, and BAA-39, represent nosocomial
or hospital-acquired strains from distant global locations and body
sites (i.e., pneumonia patient from Japan, blood culture from New
York and nasal culture from Hungary, respectively). Strain BAA-1680
represents a community-acquired MRSA (CA-MRSA) strain from the
skin of a patient from Michigan, U.S.A.
Although the SAR of rhodanine derivatives has been studied
extensively, there is a paucity of information about the contribution
of the amino acid side chain at N3-position and uniquely
substituted arylidenes at C5-position to their antibacterial action
[25,27,32e34]. We hypothesized that the phenylalanine or other
hydrophobic amino acid substituents at N3-position would
increase the likelihood of penetration through bacterial cell wall
and therefore potentially improving their anti-MRSA activity.
Scheme 1. Synthesis of compounds 14–18 and 20–22.
lead compounds 1e3. In an effort to rapidly obtain SAR data, we
decided to purchase structural analogs of compounds 1e3.
Compounds 5e13 were purchased from SigmaeAldrich, whereas
compounds 14e18 and 20e22 were synthesized and tested for
their in vitro activity against a panel of MRSA strains together with
reference antibiotics ciprofloxacin, vancomycin and penicillin G.
Following the initial hits with rhodanines 1e3, commercially
available rhodanine analogs (compounds 5e13) were obtained for
exploration of the SAR around the N3- and C5-position of the rho-
danine scaffold, leading to the development of SAR data (Table 1)
2. Results and discussion
2.1. Chemistry
A group of rhodanine analogs (14e18 and 20e22) was synthe-
sized according to Scheme 1. Rhodanine-3-acetic acid (4) was
subjected to Knoevenagel condensation with varied aldehydes to
form the target compounds (14e18) as per the procedure reported
by us previously [31]. The phenylalanine-derived rhodanine inter-
mediate (19) needed for the preparation of target compounds
and identification of
12, MIC ¼ 15.6 g/mL) as described below. Compounds 5e8 con-
tained the -isopropylacetic acid substituent at N3-position of the
a relatively potent analog (compound
m
a
rhodanine ring, as well as a series of substituted C5-benzylidenes.
While 2-chlorobenzylidene (compound 5), 4-fluorobenzylidene
(compound 6) and naphthyledene-1-yl (compound 7) analogs each
20e22 was synthesized by first cyclizing the
L-phenylalanine with
CS2 and sodium -chloroacetate to form 19, using a previously
a
exhibiting MIC value of 62.5 mg/mL were as active as lead compound
reported procedure [35]. The resulting rhodanine intermediate 19
was subjected to Knoevenagel condensation with varied aldehydes
as mentioned above to form the target compounds 20e22. The
Knoevenagel condensation reaction with aromatic aldehydes
provided only the Z isomer, as determined by the chemical shift of
the methine proton ranging from 7.7 to 8.5 as a singlet (except
cinnamylidene analog 16 which showed a doublet) whereas the
methine proton of the Z isomer of cyclohexylmethylidene analog
(18) appeared at 6.9 instead of at the calculated chemical shift of 6.3
for the E isomer [28,36]. This downfield movement of methine
proton in (Z) C5-benzylidene and alkylmethylidene analogs was
reported to be due to the deshielding effect of the adjacent carbonyl
group [28,36]. The synthesized compounds 14e18 and 20e22,
along with commercially available rhodanine analogs 5e13, were
subjected to evaluation against various strains of MRSA (Table 1).
1, 2e4-fold improvement in inhibitory activity was observed in the
presence of the 2,4-dichlorobenzylidene substituent (compound 8,
MIC ¼ 15.6
mg/mL). Based on these findings, we obtained compounds
9e13 that had a 2,4-dichlorobenzylidene substituent at the C5-
position and various substituents at the N3-position of the rhodanine
ring. The bulky
a
-n-propylacetic acid (compound 9, MIC ¼ 15.6
-methylthioethylacetic acid (compound 10, MIC ¼ 31.25
-isobutylacetic acid (compound 11, MIC ¼ 15.6
g/mL) analogs
exhibited either comparable or a 2-fold improvement in MIC values.
In contrast, the less bulky -methylacetic acid analog (compound 13,
MIC ¼ 125 g/mL) exhibited a significant decrease in activity.
m
g/
mL),
mL),
a
a
mg/
mg/mL) and
a-benzylacetic acid (compound 12, MIC ¼ 15.6
m
a
m
Based on the SAR information gathered, we synthesized the
next series of rhodanines in the hopes of identifying analogs with
greater potency. We chose rhodanine-3-acetic acid 4 as the scaffold
for further SAR exploration, focusing on variations at the C5-benzyli-
dene substituent. The 4-methoxybenzylidene (compound 14,
2.2. Anti-MRSA activity
MIC ¼ 250
mL) and styryl (compound 16, MIC ¼ 250
a 4-fold decrease in activity versus the lead compound 1 clearly
m
g/mL), quinolinylidene-4-yl(compound15, MIC¼ 250
mg/
An in vitro evaluation of in-house rhodanine analogs [31] against
MRSA strains resulted in the identification of the moderately potent
m
g/mL) analogs exhibited

D. Hardej et al. / European Journal of Medicinal Chemistry 45 (2010) 5827e5832
5829
Table 1
Anti-MRSA activity of compounds 1e3, 5e18 and 20e22.
R
O
HOOC
N
Ar
S
S
Compda
R
Ar
MIC m
g/mLb
MRSA
ATCC
MRSA
MRSA
ATCC
MRSA
ATCC
MRSA CA
ATCC
ATCC
34404
700787
700698
BAA-39
BAA-1680
1
2
3
5
6
7
8
9
-H
-H
-H
-i-Pr
-i-Pr
-i-Pr
-i-Pr
-n-Pr
-methylthioethyl
-i-But
-benzyl
-Me
-H
-H
-H
-H
2-nitrophenyl
4-nitrophenyl
62.5
250
125
ND
62.5
250
125
62.5
250
125
62.5
250
125
62.5
250
125
ND
ND
62.5
15.6
15.6
31.25
15.6
15.6
ND
250
250
250
31.25
31.25
62.5
3.9
1.95
7.8
3,4-dichlorophenyl
2-chlorophenyl
4-fluorophenyl
naphthalene-1-yl
2,4-dichlorophenyl
2,4-dichlorophenyl
2,4-dichlorophenyl
2,4-dichlorophenyl
2,4-dichlorophenyl
2,4-dichlorophenyl
4-methoxyphenyl
quinolin-4-yl
styryl
3-phenoxypheny
cyclohexyl
2-nitrophenyl
3,4-dichlorophenyl
3-phenoxyphenyl
62.5
62.5
62.5
15.6
15.6
31.25
15.6
15.6
125
250
250
250
31.25
31.25
125
3.9
3.9
62.5
62.5
62.5
15.6
15.6
31.25
15.6
15.6
125
250
500
250
31.25
31.25
62.5
3.9
3.9
7.8
62.5
62.5
62.5
15.6
15.6
31.25
15.6
15.6
125
250
500
250
31.25
31.25
62.5
3.9
3.9
7.8
ND
62.5
15.6
15.6
31.25
15.6
15.6
10
11
12
13
14
15
16
17
18
20
21
22
Cipro
Vanco
Pen-G
ND
200
200
250
31.25
31.25
125
3.9
1.95
7.8
-H
-benzyl
-benzyl
-benzyl
62.5
1.95
31.25
ꢁ0.97
ꢁ0.97
ꢁ0.97
ꢁ0.97
31.25
15.6
31.25
62.5
a
Compounds 1e3 were synthesized previously [31], compounds 5e13 were acquired from SigmaeAldrich whereas compounds 14e22 were synthesized in this report, ND:
not determined.
b
Results of average values obtained from two independent experiments in duplicates; Cipro ¼ ciprofloxacin, Vanco ¼ vancomycin, Pen-G ¼ penicillin G.
demonstrating the important role of electron withdrawing groups on
C5-benzylidene moiety in influencing the anti-MRSA activity. The
replacement of the 2-nitrobenzylidene moiety in lead compound 1
analogs, we hypothesized that these compounds may have the
propensity to bind avidly to serum proteins and may influence their
anti-MRSA activity. The binding of the molecules to serum proteins
can significantly reduce their free concentrations in plasma,
thereby potentially decreasing their antimicrobial efficacy. The
common indirect approach to evaluate the propensity of a molecule
to bind serum proteins is the determination of its MIC in the
presence of serum. In order to evaluate our serum protein binding
hypothesis, we determined MIC values of the most potent
compounds, 8, 21 and 22 against all MRSA strains in the presence of
10% fetal bovine serum (FBS). The results indicated that the MIC
values for these compounds were identical in the presence and
absence of 10% FBS. This finding suggests that these compounds do
not significantly bind to serum proteins and this would increase the
amount of free drug and thus the anti-MRSA activity.
with 3-phenoxybenzylidene (compound 17, MIC ¼ 31.25
mg/mL) and
g/mL) moieties
with cyclohexylidene (compound 18, MIC ¼ 31.25
m
produced a 2-fold increase in activity. Subsequently, we used
a molecular hybrid approach, where we chose compound 12 carrying
a-benzyl group at N3 substituent, while varying the C5-position
substituents present in compounds 1, 3 and 17. The molecular hybrid
of compounds 12 and 1 resulted in a new analog (compound 20,
MIC ¼ 125
mg/mL) with 8-fold decreased activity compared to 12 and
only 2-fold less active compared to 1. However, when this approach
was utilized for compounds 12 and 3, it resulted in a new analog
(compound 21, MIC ¼ 3.9
mg/mL) with a 4- and 30-fold improvement
in activity compared to the respective parent compounds. Finally, the
applicationof themolecularhybridapproachtocompounds12 and17,
resulted in the most potent analog of the entire series (compound 22,
The MIC values obtained for ciprofloxacin, vancomycin and
penicillin G against the various MRSA strains in this study are
consistent with previous studies. For example, the MIC value for
MIC ¼ 3.9
mg/mL against MRSA ATCC 700787, MRSA ATCC 700698 and
MRSA ATCC BAA-39 and MIC ¼ 1.95
mg/mL against MRSA ATCC 34404,
vancomycin is typically 0.5e2
(MIC ¼ ꢀ0.97 g/mL). As expected, the MIC values for penicillin G
were relatively high (15.6e62.5 g/mL) and this is consistent with
the fact that MRSA strains are resistant to penicillin G. The MIC value
for ciprofloxacin (7.8 g/mL for all strains, except ATCC 700787,
which was 62.5 g/mL) is similar to that of earlier report [38].
mg/mL [21], similar to our results
MRSA CA ATCC BAA-1680).
m
Ligands binding to site II (also called the indole-benzodiazepine
site) on human serum albumin frequently contain aromatic
carboxylic acids with a negatively charged acidic group at one end
of the molecule located away from a hydrophobic center (e.g., non-
steroidal anti-inflammatory drugs [NSAIDs]) [37]. Since these
structural features (hydrophobic group at C5-position and the
acidic function at the N3-position) are present in our rhodanine
m
m
m
However, the in vitro susceptibility of MRSA strains is highly variable
and furthermore, resistance to the fluoroquinolone antibiotics is
increasing in CA- and HA-MRSA. Consequently, they are not

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D. Hardej et al. / European Journal of Medicinal Chemistry 45 (2010) 5827e5832
routinely recommended for use in the treatment of MRSA. The
activity of compounds 21 and 22 against the CA-MRSA strain used in
this study is important as it is a USA300 genotype. The USA300,
a highly virulent strain, is characterized by its widespread dissem-
ination and greater likelihood to produce necrotizing pneumonia,
invasive skin and skin structure infections and sepsis [2,39]. In
addition, compounds 21 and 22 were active against MRSA BAA-39,
a strain that has in vitro resistance to clindamycin, erythromycin,
gentamicin, tobramycin, imipenem, tetracycline, amoxicillin and
five different cephalosporins. This suggests that these compounds
maybe effective against MRSA strains that aremulti-drugresistance.
It has been reported that several arylalkylidene rhodanines interact
with PBPs [27]. In addition, all MRSA strains harbor the mec gene that
codes for the PBP2a protein [12,18]. Therefore, it is possible that
these inhibitors may be blocking functionality of PBP2a, although
this remains to be proven experimentally.
Hinton II cation-adjusted broth was purchased from Teknova
(Hollister, CA). Mueller Hinton agar was purchased from HiMedia
(India). N,N-dimethyl formamide (DMF) was purchased from USB
Corporation (Cleveland, OH), and fetal bovine serum from Atlanta
Biologicals (Atlanta, GA). Antibiotics were purchased as follows:
vancomycin hydrochloride and penicillin G (potassium salt) from
Calbiochem (LaJolla, CA) and ciprofloxacin from USP (Rockville,
MD). Plates used from microdilution techniques were purchased
from BectoneDickinson (FranklinLakes, NJ).
4.2. General procedure for the preparation of 5-benzylidene
rhodanine-3-acetic acid derivatives (14e18)
Rhodanine-3-acetic acid 4 (0.3 g, 1.57 mmol) was added to
a three neck reaction flask equipped with a reflux condenser con-
taining 6 mL anhydrous toluene, followed by the addition of
ammonium acetate (1.60e3.20 mmol) and 1.57 mmol of the cor-
responding aromatic aldehydes. The reaction mixture was refluxed
for 1e2 h under inert conditions and the reaction was monitored by
TLC. The reaction mixture was evaporated under vacuum, and the
residue was dissolved in water. The aqueous solution was then
acidified with concentrated HCl and the precipitated product was
extracted into ethyl acetate, dried over sodium sulfate and evapo-
rated under vacuum to obtain the purified target compounds 14e18
(compounds 20e22 were purified by column chromatography).
3. Conclusions
We report the synthesis and anti-MRSA activity of a series of
glycine and phenylalanine-derived rhodanine analogs. The SAR
study clearly suggests the presence of the hydrophobic side chains
of valine (compound 8), leucine (compound 11) and phenylalanine
(compounds 12, 21e22) at the N3-position and the electron defi-
cient benzylidene moiety at C5-position of the rhodanine scaffold
improves the activity of these compounds against MRSA. This could
be due to these substituents increasing their ability to penetrate the
bacterial cell wall. The antibacterial activity of compounds 21 and
22 against a panel of MRSA strains was significantly greater than
that of the reference antibiotics penicillin G and ciprofloxacin. Most
importantly, compound 22 exhibited only a 2e4-fold higher MIC
value than that of vancomycin. The anti-MRSA activity of the most
potent compounds 8, 21 and 22 remained unchanged in the
absence or presence of 10% FBS. Currently, the mechanism of action
of the compounds tested in this study is unknown. Compounds 21
and 22 did produce a bactericidal action on selected MRSA strains.
Typically, antibiotics can produce a bactericidal effect by inhibiting
cell wall synthesis or DNA synthesis. Thus, it is possible that the
bactericidal compounds in this study could produce their anti-
MRSA activity via these mechanisms, although this remains to be
proven. We conclude from this study that the phenylalanine-
derived compounds 21 and 22 are promising templates for the
development of new drugs to treat MRSA infections.
4.3. 2-(5-(4-Methoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)
acetic acid (14)
Yellow solid. Yield ¼ 0.39 g, (81%), mp 210e214 ꢃC (lit. mp
190e193 ꢃC) [40]; Rf ¼ 0.37 (DCM:MeOH 90:10), 1H NMR (DMSO-
d6)
d
3.84 (3H, s), 4.72 (2H, s), 7.13 (2H, d, J ¼ 4.4 Hz), 7.66 (2H, d,
J ¼ 5.2 Hz), 7.86 (1H, s, CH]), 13.43 (1H, s); 13C NMR (DMSO-d6)
d
45.5, 56.1, 115.7, 118.9, 125.8, 133.6, 134.6, 162.2, 166.9, 167.8, 193.7.
Anal. Calcd. for C13H11NO4S2: C, 50.47; H, 3.58; N, 4.53. Found: C,
50.61; H, 3.49; N, 4.44.
4.4. 2-(5-(4-Quinolinylidene)-4-oxo-2-thioxothiazolidin-3-yl)
acetic acid quinolin-4-yl (15)
Yellow solid. Yield ¼ 0.42 g, (84%), mp 97e102 ꢃC; Rf ¼ 0.35
(DCM:MeOH 90:10), 1H NMR (DMSO-d6)
d 4.44 (2H, s), 7.73 (1H, d,
J ¼ 4.64 Hz), 7.79 (1H, t, J ¼ 7.66 Hz), 7.95 (1H, t, J ¼ 7.64 Hz), 8.18
(1H, d, J ¼ 8.20 Hz), 8.29 (1H, d, J ¼ 8.12 Hz), 8.51 (1H, s, CH]), 9.10
4. Experimental
(1H, d, J ¼ 4.68 Hz), 13.34 (1H, s); 13C NMR (DMSO-d6)
d 47.8, 120.3,
124.4,125.6,127.1,128.3,130.7,138.7,148.5,150.9,166.3,167.8,174.4,
3
4.1. Chemistry-general
193.6. Anal. Calcd. for C14H11NO3S2. /4 H2O: C, 52.39; H, 3.37; N,
8.15. Found: C, 52.65; H, 3.57; N, 7.83.
Melting points (mp) were determined on a Thomas-Hoover
capillary melting point apparatus and are uncorrected. The reagents
for organic synthesis were purchased from Aldrich Chemical Co.
(Milwaukee, WI), TCI America (Portland, OR), Alfa Aesar (Ward Hill,
MA), and Acros Organics (Antwerp, Belgium) and were used as
received. All compounds were checked for homogeneity by TLC
using silica gel as a stationary phase. NMR spectra were recorded on
a Bruker 400 Avance DPX spectrometer (1H at 400 MHz and 13C at
100 MHz) outfitted with a z-axis gradient probe. The chemical shifts
4.5. 2-(5-(Cinnamylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic
acid styryl (16)
Yellow solid. Yield ¼ 0.37 g, (77%), mp 220e223 ꢃC (lit. mp
>217 ꢃC) [41]; Rf ¼ 0.34 (DCM:MeOH 94:6), 1H NMR (DMSO-d6)
4.45 (2H, s), 7.16 (1H, m), 7.45 (4H, m), 7.64 (1H, d), 7.74 (2H, d, CH]
and ArCH]), 13.44 (1H, s); 13C NMR (DMSO-d6)
d 45.4, 123.6, 124.0,
128.7, 129.4, 130.7, 134.9, 135.9, 146.5, 166.2, 167.8, 193.4. Anal.
Calcd. for C14H11NO3S2. 1/5H2O: C, 54.53; H, 3.70; N, 4.54. Found: C,
54.53; H, 3.34; N, 4.63.
for 1H and 13C are reported in parts per million (
d ppm) downfield
from tetramethylsilane (TMS) as an internal standard. The 1H NMR
data are reported as follows: chemical shift, multiplicity (s) singlet,
(d) doublet, (t) triplet, (m) multiplet. The C, H, and N analyses were
performed by Atlantic Microlabs, Inc., (Norcross, GA) and the
observed values were within ꢂ0.4% of calculated values.
4.6. 2-(5-(3-Phenoxybenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)
acetic acid (17)
All strains of MRSA (BAA-39, BAA-1680, 34404, 700698 and
700787) were purchased from ATCC (Manassas, VA). Mueller
Brown solid. Yield ¼ 0.45 g, (87%), mp 178e180 ꢃC [31]; Rf ¼ 0.62
(DCM:MeOH 80:20), 1H NMR (DMSO-d6)
d 4.73 (2H, s), 7.10 (2H, d,

D. Hardej et al. / European Journal of Medicinal Chemistry 45 (2010) 5827e5832
5831
J ¼ 8.52 Hz), 7.22 (2H, m), 7.28 (1H, s), 7.45 (3H, t, J ¼ 7.68 Hz), 7.58
(1H, t, J ¼ 7.98 Hz), 7.90 (1H, s, CH]), 13.46 (1H, s); 13C NMR
5.85 (1H, s), 7.08 (2H, d, J ¼ 7.84 Hz), 7.18 (8H, m), 7.37 (1H, d,
J ¼ 7.68 Hz), 7.44 (2H, t, J ¼ 7.2 Hz), 7.55 (1H, t, J ¼ 7.88 Hz), 7.79 (1H,
(DMSO-d6)
d
48.4,119.7,120.0, 120.9,124.2,124.6,125.6, 130.7,131.6,
s, CH]), 13.52 (1H, s); 13C NMR (DMSO-d6)
d 29.0, 33.1, 58.2, 119.3,
132.0, 135.4, 156.2, 158.0, 167.1, 167.3, 193.3. Anal. Calcd. for
C18H13NO4S2. 1/2H2O: C, 56.83; H, 3.71; N, 3.68. Found: C, 57.10; H,
3.75; N, 3.72.
120.0, 121.0, 121.6, 123.0, 124.2, 125.3, 126.8, 128.3, 129.0, 130.3,
131.3, 133.3, 134.5, 136.5, 140.8, 155.8, 157.6, 166.3, 168.7, 192.6. Anal.
Calcd. for C25H19NO4S2. 1/5C6H14: C, 65.78; H, 4.51; N, 2.93. Found:
C, 65.48; H, 4.89; N, 2.79.
4.7. 2-(5-(Cyclohexylidene)-4-oxo-2-thioxothiazolidin-3-yl)acetic
acid (18)
4.12. Test for antibacterial activity
Brown solid. Yield ¼ 0.39 g, (86%), mp 138e141 ꢃC (lit. mp
209e211 ꢃC) [36]; Rf ¼ 0.89 (DCM:MeOH 80:20),1H NMR (DMSO-d6)
All purchased and synthesized compounds were tested for their
in vitro antibacterial activity against all strains of MRSA by per-
forming a microdilution minimal inhibitory concentration (MIC)
technique. Initially, a 2 mg/mL concentration of a compound was
made by dissolving it in N,N-dimethyl formamide (DMF) and then
adding cation-adjusted Mueller Hinton broth. The quantity of DMF
added never exceeded 0.5% of the total media volume. The MIC
assays were carried out in 96 well sterile plates. Serial dilution of
d
1.28 (5H, m), 1.66 (6H, m), 4.66 (2H, s), 6.94 (1H, d, J ¼ 9.52, CH]),
13.40 (1H, s); 13C NMR (DMSO-d6)
d 25.1, 25.5, 30.8, 31.7, 41.5, 45.3,
72.8,124.3,144.6,165.5,167.8,194.2. Anal. Calcd. for C12H15NO3S2: C,
50.51; H, 5.30; N, 4.91. Found: C, 50.81; H, 5.44; N, 4.80.
4.8. Preparation of 2-(4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (19)
the compounds was made in Mueller Hinton broth (100
mL).
Appropriate cell concentrations of each strain of MRSA were made
in Mueller Hinton broth by adjusting the turbidity to a 0.5 McFar-
lane Standard and then adding 0.2 mL of the suspension to 40 mL of
fresh Mueller Hinton Broth. One hundred microliters of cell
suspension was added to each well. Control wells contained
Mueller Hinton broth and cell suspension but no compound. Each
compound was tested against all strains of MRSA minimally in
triplicate. The MIC of each compound was recorded as the well
showing no turbidity when compared to control wells.
In a round-bottomed flask equipped with a magnetic stirrer,
phenylalanine (5 g, 30.3 mmol) was dissolved with sodium
hydroxide (1.21 g, 30.3 mmol) in water (25 mL). Then, carbon
disulfide (2.3 g, 30.3 mmol) was added to the reaction mixture,
which was stirred vigorously overnight. An aqueous solution of
sodium chloroacetate (3.51 g, 30.3 mmol) was added and stirring
was continued at 23 ꢃC for 3 h. Then the reaction mixture was
acidified with dilute HCl until pH 1.0 and refluxed overnight. The
reaction mixture was neutralized with saturated NaHCO3 solution.
The resultant solution was acidified again with dilute HCl. The
cyclized product was extracted in ethyl acetate, dried over anhy-
drous sodium sulfate and evaporated under vacuum and the
residue was purified by column chromatography to afford a brown
liquid. Yield ¼ 2.55 g, (30%); Rf ¼ 0.7 (DCM:MeOH 95:05), 1H NMR
MBCs were established by extending the MIC procedure to the
evaluation of bactericidal activity. After 24 h, 5 mL was drawn from
the wells and spotted onto suitable agar plates. The plates were
incubated at 37 ꢃC overnight. The MBC read 18 h later was defined
as the lowest concentration of compound or standard reference
antibiotics that resulted in 0.1% survival in the subculture. All the
experiments were performed in triplicate.
(DMSO-d6)
d 3.41 (2H, m), 4.27 (2H, m), 5.65 (1H, s), 7.14 (1H, m),
7.17 (3H, t, J ¼ 6.82), 7.23 (1H, m), 13.37 (1H, s).
Acknowledgements
4.9. 2-(5-(2-Nitrobenzylidene)-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (20)
The financial support and resources provided by the College of
Pharmacy of St. John’sUniversity are gratefully acknowledged. We
thank Dr. Mark L. McLaughlin from the Department of Chemistry,
University of South Florida and the Department of Drug Discovery,
H. Lee Moffitt Cancer Center & Research Institute, Tampa, Florida for
his help in interpretation of the spectral data.
Yellow solid. Yield ¼ 0.087 g, (25%), mp 170e175 ꢃC; Rf ¼ 0.68
(DCM:MeOH 95:5), 1H NMR (DMSO-d6)
d 3.48 (2H, m), 5.87 (1H, s),
7.21 (5H, m), 7.69 (1H, d, J ¼ 7.52 Hz), 7.75 (1H, t, J ¼ 7.78 Hz), 7.87
(1H, t,J ¼ 7.44 Hz), 8.04 (1H, s, CH]), 8.22 (1H, d, J ¼ 8.08 Hz), 13.59
(1H, s); 13C NMR (DMSO-d6)
d 33.6, 58.9, 115.6, 117.9, 122.0, 125.6,
126.1, 127.2, 128.8, 129.0, 129.4, 130.0, 131.3, 132.1, 135.2, 148.4,
166.2, 169.1, 193.6. Anal. Calcd. for C19H14N2O5S2. 2 CH2Cl2: C, 43.17;
H, 3.10; N, 4.79. Found: C, 43.33; H, 3.09; N, 4.98.
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