Synthesis and structure activity relationship studies of novel Staphylococcus aureus Sortase A inhibitors

Article abstract of DOI:10.1016/j.ejmech.2010.05.024

Synthetic methods have been developed for lead Sortase A inhibitors identified from previous studies. Several derivatives of the lead inhibitor were synthesized to derive preliminary structure activity relationships (SAR). Different regions of the lead in

Full text of DOI:10.1016/j.ejmech.2010.05.024

European Journal of Medicinal Chemistry 45 (2010) 3752e3761
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and structure activity relationship studies of novel Staphylococcus
aureus Sortase A inhibitors
Bala Chandra Chenna, Jason R. King 1, Bidhan A. Shinkre 2, Amanda L. Glover 3,
*
Aaron L. Lucius, Sadanandan E. Velu
Department of Chemistry, University of Alabama at Birmingham, 901, 14th Street South, Birmingham, AL 35294-1240, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Synthetic methods have been developed for lead Sortase A inhibitors identified from previous studies.
Several derivatives of the lead inhibitor were synthesized to derive preliminary structure activity rela-
tionships (SAR). Different regions of the lead inhibitor that are critical for the enzyme activity have been
determined by systematic SAR studies. The E stereochemistry of the lead compound was found to be
critical for its activity. Replacement of the E double bond with Z double bond or a rigid triple bond
reduced the enzyme inhibitory activity in most cases. Reduction of the double bond to a CeC single bond
resulted in complete loss of activity. Amide carbonyl and NH groups were also found to be crucial for the
activity of this class of inhibitors, as well. The morpholine ring oxygen atom was also found to be an
important factor for the activity of the lead inhibitor. Preliminary SAR studies led to the identification of
compounds with improved enzyme inhibition. The most active compound was found to have an IC50
Received 24 November 2008
Received in revised form
10 May 2010
Accepted 11 May 2010
Available online 20 May 2010
Keywords:
Staphylococcus aureus
Sortase
Inhibitor
value of 58 mM against the enzyme.
Antibacterial
Structure activity relationship
Synthesis
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
strains make treatment options more difficult. MRSA strains were
isolated from 2% of staphylococcal infections in 1974 and from 63%
of staphylococcal infections in 2004. Many of the nosocomial MRSA
strains are multi-drug resistant, and even methicillin-sensitive
strains can be deadly. A recent report using population-based,
active case finding revealed that 94,360 invasive MRSA infections
occurred in the U.S. in 2005, and that the majority of these (58%)
occurred outside of the hospital [1].
Staphylococcus aureus causes a variety of human infections,
ranging from superficial abscesses to life threatening bacteremias.
Staphylococcal infections within the hospital and in the community
are increasing, and an ever-growing number of antibiotic-resistant
Notorious as a major source of nosocomial infections, S. aureus
has recently taken on a new role in causing an escalating number of
community-acquired infections in non-hospitalized persons
without predisposing risk factors. A single S. aureus clone, desig-
nated as USA300, causes the majority of community-acquired-
MRSA infections in the U.S., and its dissemination has been
observed globally [2e6]. Vancomycin is most commonly used for
treatment of systemic infections caused by MRSA. However, S.
aureus isolates with reduced susceptibility to vancomycin have
been reported since 1997 [7]. These isolates are also methicillin
resistant [8e10]. Because S. aureus cannot always be controlled by
antibiotics and MRSA isolates are becoming increasingly prevalent
in the community, additional control strategies and novel thera-
peutic approaches are sorely needed.
Abbreviations: Dabsyl, 4-(4-Dimethylaminophenylazo)-benzenesulfonyl; DIBAL,
Diisobutylaluminum hydride; DMAP, N,N-dimethylaminopyridine; DMF, N,N-
Dimethylformamide; EDAC, Ethyl (N, N-dimethylaminopropyl) carbodiimide;
EDANS, 5-[(2-aminoethyl)amino]naphthalene-1-sulfonyl; FRET, Fluorescence
Resonance Energy Transfer; IC50, Half maximal inhibitory concentration of inhib-
itor; MRSA, Methicillin resistant Staphylococcus aureus; MS, Mass spectrometry;
PCC, Pyridinium chlorochromate; Pd black, Palladium black; Pd/C, Palladium on
activated carbon; ppm, Parts per million; SAR, Structure activity relationship;
S. aureus, Staphylococcus aureus; SrtA, Sortase A; THF, Tetrahydrofuran; Thr, Thre-
onine; TLC, Thin layer chromatography; TMS, Tetramethylsilane.
* Corresponding author. Tel.: þ1 205 975 2478; fax: þ1 205 934 2543.
E-mail addresses: bala@uab.edu (B.C. Chenna), Jason.king@duke.edu (J.R. King),
bidhanshinkre@yahoo.com (B.A. Shinkre), alglover@crimson.ua.edu (A.L. Glover),
allucius@uab.edu (A.L. Lucius), svelu@uab.edu (S.E. Velu).
1
Current address: Department of Chemistry, Duke University, Box 90354,
Durham, NC 27708-0354, USA.
2
Current address: Syngene International, Bangalore, Karnataka 560099, India.
Current address: Department of Chemistry, The University of Alabama, Box
3
New approaches for the prevention and treatment of bacterial
infections require greater understanding of the molecular structure
870336, Tuscaloosa, AL 35487-0336, USA.
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.024

B.C. Chenna et al. / European Journal of Medicinal Chemistry 45 (2010) 3752e3761
3753
and mechanisms of the chosen intervention targets and of the
COOCH3
COOCH3
pathogenic role played by the target in the infection process.
Bacterial infections are complex and involve the action of a large,
sophisticated arsenal of virulence factors, many of which are
surface-bound or secreted. Gram-positive bacteria such as S. aureus
are endowed with a multitude of cell wall anchored proteins that
serve as an interface between the microbe and its host. Bacterial
sortases are cysteine transpeptidases that participate in secretion
and anchoring of many cell wall proteins by a mechanism
conserved in almost the entire class of Gram-positive bacteria.
Surface proteins can be attached to the bacterial surface in several
fashions [11,12]. Proteins that are covalently attached to the cell
wall share conserved regions known as the “sorting signal” or cell
wall anchors [13,14]. The sorting signal includes a conserved amino
acid motif, usually LPXTG. Precursor proteins are directed into
a secretory pathway by their N-terminal signal peptides. They are
translocated across the membrane and the signal peptide is cleaved
[14,15]. Then, the C-terminal sorting signal retains the protein in
the secretory pathway. The enzyme sortase acts at this point to
cleave the protein between the threonine (T) and the glycine (G) of
the LPXTG motif [11,16]. The carboxyl group of the Thr is then
amide-linked to the amino group of a “cross-bridge” peptide in the
lipid II precursor for cell wall synthesis [11,17]. Sortase-defective
strains of various pathogens were shown to be faulty in the display
of surface proteins and are less virulent [18,19]. In a number of
studies, individual sortase genes have been deleted and the loss-of-
sortase function has resulted in less virulence in several animal
models of the disease [18,20e24]. Hence, sortases are attractive
pharmacotherapeutic targets [12].
Currently, there have only been a few reports of specific sortase
inhibitors [25e28]. Recently, Oh et al. [29] identified a small-
molecule reversible inhibitor of SrtA with a low micromolar IC50
value by structurally modifying a lead compound identified by
random screening of a group of small-molecules. We have recently
identified an inhibitor (1) of S. aureus SrtA aided by in-silico virtual
screening (Fig. 1) [30]. We have conducted preliminary structure
activity relationship (SAR) studies with the goal of improving the
activity of inhibitor 1. This manuscript describes the synthesis of
inhibitor 1 and several of its derivatives and the results of their in
vitro enzymatic evaluation against SrtAD59, a fully active variant of
SrtA with 59 N-terminal residues removed.
H2,
O2N
N
O
H2N
N
O
Pd/C
EtOAc
3
2
O
O
N
X
X
OH
4a, X = S
4b, X = O
COOCH3
EDAC, DMAP
ClCH2CH2Cl
N
H
5a, X = S
5b, X = O
O
O
N
X
COOH
NaOH
CH3OH
THF
N
H
1, X = S
6, X = O
O
Scheme 1. Synthesis of inhibitor 1 and its furan analogue 6.
catalyst in anhydrous ethyl acetate to afford the corresponding
amino compound 3. Compound 3 was coupled with commercially
available trans-3-(thiophene-2-yl)acrylic acid (4a) in the presence
of ethyl(N,N-dimethylaminopropyl) carbodiimide (EDAC) and N,N-
dimethylaminopyridine (DMAP) in 1,2-dichloroethane to form the
amide compound 5a [31]. Basic hydrolysis of the ester methyl
group present in compound 5a afforded the inhibitor 1 as a white
crystalline solid.
We have synthesized several derivatives of inhibitor 1 in order
to derive preliminary structure activity relationship data. Their
activities against SrtAD were determined using a fluorescence
59
resonance energy transfer (FRET) assay which is a modification of
a previously reported procedure. In this assay, the IC50 values were
determined by monitoring the effect of the compounds on the
steady state cleavage of a model substrate peptide, Dabcyl-QAL-
PETGEE-EDANS [29,32,33]. Structures and IC50 values of the newly
synthesized derivatives of inhibitor 1 are given in Table 1.
Compound 6 is a furan analogue of inhibitor 1. Compound 6 was
synthesized by a similar procedure as described for the synthesis of
inhibitor 1 (Scheme 1). We have used trans-3-(furan-2-yl)acrylic
acid (4b) instead of trans-3-(thiophene-2-yl)acrylic acid (4a) in this
procedure. Compound 3 was coupled with commercially available
4b in the presence of EDAC and DMAP in 1,2-dichloroethane to
form the amide compound 5b. Basic hydrolysis of the ester present
in compound 5b afforded compound 6. Compound 6 along with the
synthetic intermediate methyl esters 5a and 5b were evaluated for
their enzyme inhibitory activity. The results of the enzyme assays
for these and all other compounds described in this report are
summarized in Table 1. Compound 6 did not show an enhancement
of activity. Instead, it was found to be less active compared to
2. Results and discussion
Inhibitor 1 was initially obtained from a commercial source in
milligram quantities. For SAR studies we needed this compound in
larger quantities. So, a general method for the synthesis of inhibitor
1 was developed in our laboratory. The synthetic procedures for all
analogues were modelled around this synthesis. Chemistry
employed for the synthesis of inhibitor 1 is outlined in Scheme 1.
Commercially available methyl 2-(N-morpholino)-5-nitro-
benzoate (2) was reduced using hydrogen in the presence of Pd/C
inhibitor 1 in the enzymatic assay (IC50 ¼ 181
mM). But, the methyl
ester intermediates (5a and 5b) showed improved inhibition as
compared to the corresponding acid derivatives 1 and 6. The
methyl ester derivative of the thiophene compound (5a) showed an
O
N
S
IC50 value of 71
compound (5b) showed a much improved inhibition at an IC50
value of 58 M. Although this increase in activity of methyl ester
mM and methyl ester derivative of the furan
O
m
OH
N
analogues is counter intuitive of the FlexX model which showed
a possible salt bridge interaction between the carboxylic acid and
Arg197 side chain, the increased activity may be explained using
the FlexX model of the methyl ester in the active site. This model
shows a similar fit of the molecule in the active site with the ester
O
H
Inhibitor 1, IC50 = 75 4.1 µM
Fig. 1. Structure and IC50 value of inhibitor 1.

3754
B.C. Chenna et al. / European Journal of Medicinal Chemistry 45 (2010) 3752e3761
Table 1
O
O
S. aureus SrtAD inhibition of synthesized derivatives of inhibitor 1.
59
H2 or
HCOONH4
N
N
X
O
X
O
Catalyst/
O Y Solvent
O Y
N
H
N
H
O
O
Catalyst
/Solvent
Yield
(%)
Reactant
X
Y
Product
S
S
O
O
H
CH3
H
CH3
Pd black/MeOH
Pd black/MeOH
Pd/C/EtOAc
69
82
88
1
5a
6
7
8
9
No
X
AeB
Stereochemistry
C
D
Y
R
IC50(m
M)a
Pd/C/EtOAc
5b
10
77
5a
5b
6
7
8
9
10
12
13
14
15
16a
16b
17a
17b
19
S
CH]CH
CH]CH
CH]CH
E
E
E
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NMe
NMe
NMe
NMe
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
HH
HH
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
COOCH3 71 ꢀ 2.5
COOCH3 58 ꢀ 4.9
O
O
S
Scheme 2. Synthesis of compounds 7e10.
COOH
COOH
181 ꢀ 14
CH2eCH2 eb
CH2eCH2 eb
CH2eCH2 eb
CH2eCH2 eb
>600
COOCH3 >600
COOH >600
COOCH3 >600
COOCH3 165 ꢀ 9
S
activity of the inhibitor 1. Our next goal was to examine the effect of
stereochemistry of the double bond on the activity. With this goal,
we prepared the Z isomer 15. In order to examine the influence of
a carbonecarbon triple bond on the activity, we prepared the
compound 13. Synthesis of compounds 13 and 15 is outlined in
Scheme 3.
O
O
S
S
S
S
S
O
S
O
S
S
S
O
S
O
S
O
S
O
S
C^C
C^C
eb
eb
Z
Z
E
E
E
E
E
E
E
E
E
E
E
E
E
COOH
183 ꢀ 12
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
CH]CH
COOCH3 61 ꢀ 4
COOH
154 ꢀ 5
COOCH3 571 ꢀ 43
COOCH3 514 ꢀ 39
The synthesis started from the known 3-(thiophen-2-yl)pro-
piolic acid 11, which is prepared according to the literature proce-
dure [34]. The acid 11 was coupled with the amino compound 3 in
the presence of EDAC and DMAP in 1,2-dichloroethane afforded the
alkyne ester 12. The ester group present in compound 12 was
hydrolyzed using NaOH in MeOH to afford the acetylenic acid 13.
The partial hydrogenation of the acetylenic ester 12 using Lindlar
Pd catalyst did not work as expected possibly due to the catalytic
poisoning effect of the thiophene ring already present in the
molecule. However, reduction worked smoothly to a cis double
bonded compound 14 when it was carried out with H2 in the
presence of Pd/C in ethyl acetate. The ester group present in
compound 14 was hydrolyzed using NaOH in MeOH to afford the cis
derivative 15.
COOH
COOH
>600
>600
COOCH3 249 ꢀ 9
20
COOH >600
24a
24b
25a
25b
26a
26b
27a
27b
28
CH2 COOCH3 92 ꢀ 5
CH2 COOCH3 131 ꢀ 14
CH2 COOH
CH2 COOH
181 ꢀ 12
463 ꢀ 19
73 ꢀ 2
O
O
O
O
O
CH2OH
CH2OH
CHO
CHO
CONH2
111 ꢀ 5.7
77 ꢀ 6.5
107 ꢀ 8.5
105 ꢀ 3.7
E
E
a
. IC50 values were determined by a Fluorescent Resonance Energy Transfer
(FRET) assay. Reported values are an average of 3e5 measurements.
b
. Not applicable.
Compounds 13, 15 and the intermediate esters, 12 and 14 were
all evaluated for their enzymatic activity (Table 1). The interme-
diate acetylenic compound 12 (165
mM) and the corresponding acid
13 (183 M) showed decreased inhibition of the enzyme compared
to the corresponding E alkene derivatives. The Z alkene ester
m
methyl group resting in a small hydrophobic pocket alongside the
central phenyl ring without completely destroying the electrostatic
interaction between the Arg197 side chain and ester group O atoms
of the inhibitor. The increased activity could be a result of these
additional hydrophobic interactions generated by the ester methyl
group with hydrophobic residues in the active site.
Compounds 7e10 were synthesized to evaluate the importance
of the double bond present in compounds 1 and 6 for their activity.
Compounds 7 and 9 are the saturated analogues of the carboxylic
acid derivatives 1 and 6. Compounds 8 and 10 are the saturated
analogues of the methyl ester derivatives 5a and 5b. Our assay
showed that all four saturated compounds 7e10 were inactive up to
3
S
COOH
EDAC, DMAP
ClCH2CH2Cl
11
O
O
S
S
N
N
NaOH
CH3OH
THF
COOCH3
COOH
a concentration of 600 mM showing that the double bond is crucial
13
12
N
H
N
H
for the activity of these inhibitors (Table 1). Synthesis of compounds
7e10 is outlined in Scheme 2. Hydrogenation of the thiophene
derivatives 1 and 5a using Pd/C as catalyst did not work, possibly due
tothe catalyst poisoning effect of the thiophene ring present in these
molecules. Use of a stronger catalyst like Pd black in the presence of
ammonium formate resulted in the hydrogenation of 1 and 5a to
corresponding hydrogenated compounds 7 and 8. Hydrogenation of
6 and 5b were carried out using H2 in the presence of Pd/C catalyst to
afford compounds 9 and 10 in good yields.
O
O
O
O
Pd/C
EtOAc
H2,
N
N
NaOH
CH3OH
THF
COOCH3
COOH
N
H
N
H
14
15
O
O
S
S
Our lead inhibitor 1 has a double bond with E stereochemistry.
We have already shown that the double bond is critical for the
Scheme 3. Synthesis of acetylenic acid, 13 and the cis derivative, 15.

B.C. Chenna et al. / European Journal of Medicinal Chemistry 45 (2010) 3752e3761
3755
O
COOCH3
COOCH3
N
O
Piperidine
THF
N
N
O2N
F
O2N
X
X
X
21
22
COOCH3
COOCH3
MeI
NaH
THF
O
N
16a, X = S
16b, X = O
CH3
N
H
5a, X = S
5b, X = O
COOCH3
O
O
OH
4a, X = S
4b, X = O
H2,
O
H2N
N
Pd/C
EDAC, DMAP
ClCH2CH2Cl
EtOAc
N
23
X
COOH
NaOH
N
CH3OH
N
N
17a, X = S
17b, X = O
CH3
X
X
THF
O
COOH
COOCH3
NaOH
CH3OH
THF
Scheme 4. Synthesis of N-methyl compounds 17a and 17b.
N
H
25a, X = S
25b, X = O
N
H
24a, X = S
24b, X = O
O
O
derivative 14 showed more or less similar inhibition (61
compared to the E derivative, 5a. However, the Z alkene acid 15
mM) as
Scheme 6. Synthesis of compounds 25a,b.
showed a decreased inhibition (154
mM) of the enzyme as
compared to E alkene acid, 1.
20 was inactive up to a concentration of 600
mediate ester 19 had a reduced activity at 249
m
m
M while the inter-
M.
We have synthesized compounds 17a and 17b in order to
examine the effect of interruption of possible H-bonding of the NH
group present in the inhibitors 1 and 6. Target compounds in this
case were compounds 17a and 17b where the amide N was meth-
ylated. Synthesis of the target compounds is outlined in Scheme 4.
Compound 5a and 5b were methylated using methyl iodide in
the presence of NaH in THF to afford the N-methylated esters 16a
and 16b. Basic hydrolysis of 16a and 16b using 1 N NaOH in MeOH
afforded the final acid products 17a and 17b, respectively.
The compounds 17a,b and intermediates 16a,b were evaluated
for their enzymatic activity in our assay (Table 1). As expected, all
four N-methyl derivatives showed decrease in inhibition as
compared to corresponding NH analogues. Compounds 16a and
We wanted to inspect the importance of the oxygen atom in the
morpholine ring of the inhibitors 1 and 6. With this objective, we
prepared the compounds 25a and 25b where the oxygen atom in
the morpholine ring is replaced with a carbon atom. Synthesis of
the compounds 25a and 25b is outlined in Scheme 6.
Commercially available methyl 2-fluoro-5-nitrobenzoate (21)
was treated with piperidine in THF to afford compound 22, which
upon hydrogenation using H2 in the presence of Pd/C gave the
amine 23. Compound 23 was coupled with trans-3-(thiophene-2-
yl)acrylic acid (4a) or trans-3-(furan-2-yl)acrylic acid (4b) in the
presence of EDAC and DMAP in 1,2-dichloroethane to form the
amides 24a or 24b. Basic hydrolysis of the ester methyl group
present in compounds 24a or 24b afforded the acids 25a or 25b.
Compounds 25a,b along with the intermediate esters, 24a-
b were all evaluated for their enzymatic activity (Table 1).
16b showed IC50 values 571
final acids 17a,b were inactive up to a concentration of 600
m
M and 514
m
M respectively and the
mM. This
demonstrates that the NH group of inhibitors 1 and 6 is very
important for their activity.
In order to examine the effect of amide carbonyl oxygen on the
activity of the lead compound, we have prepared compound 20 in
which the carbonyl group is replaced with a CH2 group. Synthesis of
compound 20 is outlined in Scheme 5.
Reductive amination of the known aldehyde 18 with the amine
3 in the presence of NaCNBH3 and ZnCl2 in MeOH afforded the ester
compound 19. Hydrolysis of the ester group present in compound
19 using NaOH in MeOH afforded the final acid product 20.
The compound 20 and intermediate 19 were evaluated for their
enzymatic activity (Table 1). Both compounds showed decreased
inhibition compared to corresponding amide analogues showing
that the amide carbonyl group is important for the activity. The acid
Carboxylic acid derivatives 25a (181
mM) and 25b (463 mM)
exhibited a decreased inhibition compared to corresponding mor-
pholine analogues. The ester intermediates 24a (92
(131
mM) and 24b
mM) also showed a similar decrease in activity compared to
corresponding morpholine analogues.
O
N
O
N
X
X
O
DIBAL
THF
OH
OMe
CH2Cl2
N
H
N
H
O
3
CHO
O
S
NaCNBH3, ZnCl2
5a, X = S
26a, X = S
26b, X = O
18
MeOH
5b, X = O
O
O
O
N
N
N
X
O
H
S
S
PCC
THF
COOH
COOCH3
NaOH
CH3OH
THF
N
H
N
H
N
H
27a, X = S
19
20
O
27b, X = O
Scheme 5. Synthesis of compound 20.
Scheme 7. Synthesis of compounds 26a,b and 27a,b.

3756
B.C. Chenna et al. / European Journal of Medicinal Chemistry 45 (2010) 3752e3761
¼ 490 nm. The fluorescence emission spectra for EDANS overlaps
O
d
O
very well with the absorption spectra of Dabcyl, where Dabcyl has
N
N
an absorption maximum, lmax,a ¼ 472 nm. As such, when the two
molecules are spatially close, as they are in the intact peptide, the
efficiency of transfer from EDANS to Dabcyl is high. Upon excitation
of EDANS little emission from the donor is observed because most
of the energy from fluorescence is transferred to the dabcyl
acceptor and lost through non radiative decay pathways. In
contrast, upon cleavage the newly formed fragments will diffuse
apart and the two peptide fragments will become spatially sepa-
rated and the FRET efficiency will diminish. Thus, the emission from
the EDANS fluorophore will precipitously increase. Because of these
properties, this FRET pair has been successfully employed to
monitor SrtA catalyzed peptide cleavage.
Typically, the EDANS fluorophore is excited at 350 nm and
fluorescence from EDANS is observed at 495 nm over a period of
time. However, to reduce the correction for the inner filter effect
we have chosen to monitor emission at 590 nm, where the
absorbance for either chromophore is insignificant. At the begin-
ning of the observation, fluorescence is low due to a high FRET
efficiency, but, as the peptide is cleaved the fluorescence increases
linearly. The slope of this linear increase is taken to be the initial
velocity, V0. Because of the large inner filter correction at high
concentrations of substrate, we have chosen to monitor the effect
of inhibitors on V0 at a select enzyme and substrate concentration,
where the correction is negligible. These experiments are per-
S
O
S
O
1. SOCl2
2. NH3
NH2
OH
N
H
N
H
28
O
O
1
Scheme 8. Synthesis of compound 28.
We have also made a few derivatives of inhibitor 1 by incor-
porating different substituents such as a eCH2OH (26a), eCHO
(27a) or eCONH2 (28) in the place of the carboxylic acid group of
inhibitors 1. Substitution with eCH2OH and eCHO groups did not
result in a major change in the activity (26a, IC50 ¼ 73
IC50 ¼ 77 M), while substitution with eCONH2 group resulted in
M)
mM and 27a,
m
a decrease in activity as compared to inhibitor 1 (28, IC50 ¼ 105
m
(Table 1). Derivatives of furan compound 6 incorporating substit-
uents such as a eCH2OH (26b) and eCHO (27b) in the place of the
carboxylic acid group were also made. These compounds showed
improved inhibition as compared to the parent furan compound, 6.
Compound 26b showed an IC50 value of 111
showed an IC50 value of 107 M. Synthesis of compounds 26a,b and
27a,b is outlined in Scheme 7.
mM, and compound 27b
m
Compounds 5a,b were reduced using DIBAL in a mixture of
anhydrous CH2Cl2 and THF to afford the alcohol derivatives 26a,b.
Oxidation of alcohols 26a,b using PCC in anhydrous THF afforded
the aldehydes, 27a,b. Synthesis of amide 28 is outlined in Scheme 8.
Compound 28 was prepared from inhibitor 1 by treatment with
SOCl2, followed by the treatment of the acid chloride produced with
ammonia.
Strikingly, the IC50 values determined for all of the active
compounds are well below the previously measured Km value of
5.5 mM for SrtA binding to the LPXTG peptide [32]. This suggests
that these inhibitors binde1e2 orders of magnitude tighter than the
LPXTG peptide and thus should be effective at blocking the
enzymes activity in vivo.
formed by mixing 5
mM SrtA with 20 mM Dabcyl-EDANS labelled
peptide in 0.5 cm cuvette. This mixture is excited at
a
lex ¼ 350 nm and fluorescence is observed at lem ¼ 590 nm. From
this experiment, a time course of fluorescence increase as a func-
tion of time is acquired. The slope of this line is considered the
maximum velocity in the absence of inhibitor. The identical
experiment is then performed in the presence of increasing
concentrations of inhibitor and the slopes are compared until
a 50% decrease in the slope is observed. The concentration of
inhibitor where 50% of this maximum velocity is observed is taken
to be the IC50. Each experiment was repeated at least three times
to ensure the reproducibility and calculate standard deviation of
the reported IC50 values.
3. Conclusions
4.2. General methods for synthesis
In conclusion, we have discovered a novel class of small-mole-
cule inhibitors of S. aureus Sortase A. Inhibitors were screened for
their activity against the enzyme using a FRET assay. Micromolar
inhibitors of the enzyme are identified. We have carried out
preliminary structure activity relationship studies that have resul-
ted in the identification of inhibitors with improved activity.
Regions of the molecular structure of the lead inhibitor that are
critical for its activity have been determined by systematic SAR
studies. Further SAR studies to refine the activity of the lead using
the information gained from these studies as well as attempts to
obtain high resolution inhibitor/Sortase A complex co-crystal
structures are currently in progress.
Solvent evaporations were carried out in vaccuo with a rotary
evaporator. Analytical samples were prepared by drying the
samples in vaccuo (0.2 mm Hg) in an Abderhalden drying appa-
ratus over P2O5 and ethyl acetate at reflux temperature. Thin layer
chromatography (TLC) was performed on silica gel plates with
fluorescent indicator (Whatmann, silica gel, UV254, 25 mm plates).
Spots were visualized by UV light (254 and 365 nm). All analytical
samples were single spots on TLC in at least two different solvent
systems. Purification by column and flash chromatography was
carried out using ‘BAKER’ silica gel (40 mm) in the solvent systems
indicated. The amount (weight) of silica gel for column chroma-
tography was in the range of 50e100 times the amount (weight)
of the crude compounds being separated. Melting points were
determined on a Mel-Temp II melting point apparatus and are
uncorrected. Proton nuclear magnetic resonance (1H NMR) and
carbon nuclear magnetic resonance (13C NMR) spectra were
recorded on a Brucker DPX 300 spectrometer using TMS as
4. Experimental section
4.1. Fluorescence resonance energy transfer (FRET) assay
To determine IC50 values for each potential inhibitor, we have
implemented the previously published fluorescence resonance
energy transfer (FRET) assay [32,33]. This FRET assay employs the
use of the donor and quencher pair, EDANS and Dabcyl, respec-
tively. We have purchased the peptide Dabcyl-QALPETGEE-EDANS
from a commercial source. The EDANS fluorophore has an excita-
tion wavelength, lex,d ¼ 336 nm and an emission maximum, lem,
internal standard. The values of chemical shifts (d) are given in
ppm and coupling constants (J) in Hz. Mass spectra were recorded
on a MicroMass Platform LCC instrument. Elemental analyses were
performed by Atlantic Microlab, Norcross, Georgia and the results
indicated by symbols for the elements were within ꢀ0.4% of
theoretical values. Anhydrous solvents used for reactions were

B.C. Chenna et al. / European Journal of Medicinal Chemistry 45 (2010) 3752e3761
3757
purchased in Sure-SealÔ bottles from Aldrich Chemical Company.
Other reagents were purchased from Aldrich or Fisher chemical
companies and used as received.
carefully adding 3 N HCl (e2.5 mL). The resulting white solid was
filtered and dried under vacuum to furnish pure compound 1.
(0.283 g, 98%); Mp 278e279 ꢁC; 1H NMR (DMSO-d6)
d 3.00e3.09
(m, 4H), 3.74e3.85 (m, 4H), 6.55 (d, 1H, J ¼ 15.3 Hz), 7.14 (dd, 1H,
J1 ¼ 3.6 Hz, J2 ¼ 5.1 Hz), 7.47 (d, 1H, J ¼ 3.3 Hz), 7.67 (s, 1H), 7.68 (d,
1H, J ¼ 3.0 Hz), 7.77 (d, 1H, J ¼ 15.3 Hz), 7.99 (dd, 1H, J1 ¼ 2.7 Hz,
J2 ¼ 8.7 Hz), 8.29 (d, 1H, J ¼ 2.7 Hz) and 10.4 (bs, 1H); 13C NMR
4.2.1. Methyl 5-amino-2-morpholinobenzoate (3)
To a solution of Methyl 5-nitro-2-morpholinobenzoate, 2 (1.0 g,
3.76mmol)inEtOAc(35mL)10%Pd/C (100mg)wasaddedandstirred
under an atmosphere of H2 from a balloon (e1 atm) for 12 h. TLC
analysis (1:1 EtOAc/CHCl3) revealed that the reaction is complete. The
catalyst was removed by filtration through a celite bed and the filtrate
was concentrated in vacuum to furnish 3 as a solid. (0.88 g, 99%); Mp
(DMSO-d6) d 52.7, 66.3, 120.3, 120.9, 123.8, 124.0, 125.4, 128.5, 128.7,
131.6, 133.7, 137.8, 139.6, 145.2, 163.4 and 166.4; MS (ES) m/z 357
(M ꢃ H); Anal Calcd for C18H18N2O4S: C, 60.32; H, 5.06; N, 7.82.
Found: C, 60.37; H, 5.14; N, 7.59.
121e122 ꢁC; 1H NMR (CDCl3)
d 2.91e2.97 (m, 4H), 3.60 (bs, 2H),
3.79e3.85 (m, 4H), 3.87 (s, 3H), 6.78 (dd, 1H, J1 ¼ 2.7 Hz, J2 ¼ 8.7 Hz),
4.2.5. 5-((E)-3-(Furan-2-yl)acrylamido)-2-morpholinobenzoic acid
(6)
Compound 6 was synthesized following a similar procedure as the
one used for preparation of compound 1 starting from 5b (0.200 g,
0.57 mmol) and 3 N. NaOH solution to afford the pure product as
a white solid. (0.192 g, 99%); Mp 281e282 ꢁC; 1H NMR (DMSO-d6)
6.95 (d, 1H, J ¼ 8.7 Hz) and 7.05 (d, 1H, J ¼ 2.7 Hz); 13C NMR (CDCl3)
d
51.7, 53.3, 67.1, 116.7, 118.8, 120.8, 127.0, 141.9, 143.7 and 168.0; MS
(ES) m/z 237 (M þ H); Anal Calcd for C12H16N2O3: C, 61.00; H, 6.83; N,
11.86. Found: C, 60.87; H, 6.81; N, 11.75.
4.2.2. Methyl 5-((E)-3-(thiophen-2-yl)acrylamido)-2-
morpholinobenzoate (5a)
d
3.01e3.11 (m, 4H), 3.75e3.86 (m, 4H), 6.59 (d, 1H, J ¼ 15.3 Hz),
6.60e6.69 (m,1H), 6.87(d,1H, J ¼ 3.6 Hz), 7.41 (d,1H, J ¼ 15.6 Hz), 7.68
(d, 1H, J ¼ 9.0 Hz), 7.84 (d, 1H, J ¼ 1.5 Hz), 8.00 (dd, 1H, J1 ¼ 2.7 Hz,
J2 ¼ 8.7 Hz), 8.29 (d,1H, J ¼ 2.7 Hz) and 10.4 (bs,1H); 13C NMR (DMSO-
To a solution of 3 (0.430 g, 1.82 mmol) in 1,2-dichloroethane
(20 mL) 3-(2-thienyl)acrylic acid 4a (0.337 g, 2.18 mmol), EDAC
(1.12 g, 5.82 mmol), and DMAP (0.022 g, 0.18 mmol) were added
and stirred for 16 h at room temperature. TLC analysis (1:1 EtOAc/
CHCl3) revealed that the reaction is complete. The reaction mixture
was diluted with CH2Cl2 (50 mL) and the organic layer was washed
with saturated NaHCO3 solution (2 ꢂ 25 mL), water (1 ꢂ 25 mL),
brine (1 ꢂ 25 mL) and dried over anhydrous Na2SO4. The drying
agent was filtered off and the solvent was concentrated in vaccuo to
afford the crude product which was crystallized form a mixture of
CHCl3 and hexanes to furnish 5a as a yellow solid. (0.62 g, 91%); Mp
d6)
d 52.8, 66.4, 112.5, 115.0, 118.9, 120.9, 123.8, 124.0, 125.4, 127.8,
137.8, 145.2, 145.4, 150.8, 163.6 and 166.4; MS (ES) m/z 341 (M ꢃ H);
Anal. (C18H18N2O5) C, H, N. Anal Calcd for C18H18N2O5: C, 63.15; H,
5.30; N, 8.18. Found: C, 63.06; H, 5.53; N, 7.89.
4.2.6. 5-(3-(Thiophen-2-yl)propanamido)-2-morpholinobenzoic
acid (7)
To a solution of compound 1 (0.071 g, 0.20 mmol) in a mixture of
MeOH (4 mL) and EtOAc (3 mL), HCOONH4 (0.188 g, 2.97 mmol) and
Pd black (40 mg) were added and refluxed for 6 h. TLC analysis (1:1
EtOAc/CHCl3) and mass spectrum revealed that the reaction was
complete. The catalyst was removed by filtration through a celite
bed, washed with a mixture of MeOH and CHCl3, and the filtrate
was concentrated in vaccuo to afford the crude product. The crude
product was purified by washing thoroughly with water to remove
the inorganic salts, filtered and dried under vacuum to furnish pure
product 7. (0.049 g, 69%); Mp 226e227 ꢁC; 1H NMR (DMSO-d6)
196e197 ꢁC; 1H NMR (CDCl3)
d 3.00e3.05 (m, 4H), 3.83e3.88 (m,
4H), 3.89 (s, 3H), 6.31 (d, 1H, J ¼ 15.3 Hz), 7.03e7.08 (m, 2H),
7.24e7.28 (m, 2H), 7.33e7.38 (m, 1H), 7.80 (d, 1H, J ¼ 8.7 Hz),
7.83e7.90 (m, 1H) and 7.92 (d, 1H, J ¼ 2.3 Hz); 13C NMR (CDCl3)
d
52.2, 53.1, 67.2, 119.3, 119.8, 123.2, 124.6, 124.9, 127.9, 128.2, 130.9,
132.5, 135.1, 139.8, 149.0, 163.7 and 167.5; MS (ES) m/z 373 (M þ H);
Anal Calcd for C19H20N2O4S: C, 61.27; H, 5.41; N, 7.52. Found: C,
60.98; H, 5.39; N, 7.45.
d
2.68 (t, 2H, J ¼ 7.5 Hz), 2.99e3.08 (m, 4H), 3.13 (t, 2H, J ¼ 7.5 Hz),
4.2.3. Methyl 5-((E)-3-(furan-2-yl)acrylamido)-2-
morpholinobenzoate (5b)
3.74e3.86 (m, 4H), 6.86e6.97 (m, 2H), 7.30 (dd, 1H, J1 ¼ 1.0 Hz,
J2 ¼ 4.9 Hz), 7.65 (d, 1H, J ¼ 8.4 Hz), 7.90 (dd, 1H, J1 ¼ 2.7 Hz,
J2 ¼ 8.7 Hz), 8.21 (d, 1H, J ¼ 2.4 Hz) and 10.2 (bs, 1H); 13C NMR
Compound 5b was synthesized, following a similar procedure as
the one used for preparation of 5a starting from compound 3
(0.426 g, 1.8 mmol), 3-(2-furyl)acrylic acid 4b (0.298 g, 2.16 mmol),
EDAC (1.10 g, 5.76 mmol), and DMAP (0.022 g, 0.182 mmol) to
obtain the pure product. (0.639 g, 99%); Mp 172e173 ꢁC; 1H NMR
(DMSO-d6)
d 24.9, 38.0, 52.7, 66.3, 120.8, 123.7, 123.8, 123.9, 124.7,
125.2, 126.9, 137.7, 143.4, 145.0, 166.3 and 170.1; MS (ES) m/z 361
(M þ H); Anal Calcd for C18H20N2O4S: C, 59.98; H, 5.59; N, 7.77.
Found: C, 60.15; H, 5.75; N, 7.64.
(CDCl3)
d 2.98e3.07 (m, 4H), 3.82e3.88 (m, 4H), 3.89 (s, 3H), 6.42
(d, 1H, J ¼ 15.0 Hz), 6.48 (dd, 1H, J1 ¼1.8 Hz, J2 ¼ 3.3 Hz), 6.60 (d, 1H,
J ¼ 3.3 Hz), 7.04 (d, 1H, J ¼ 9.0 Hz), 7.40 (bs, 1H), 7.46 (d, 1H,
J ¼ 1.5 Hz), 7.52 (d, 1H, J ¼ 15.0 Hz), 7.82 (d, 1H, J ¼ 7.2 Hz) and 7.91
4.2.7. Methyl 5-(3-(thiophen-2-yl)propanamido)-2-
morpholinobenzoate (8)
To a solution of compound 5a (0.100 g, 0.27 mmol) in MeOH
(5 mL), HCOONH4 (0.255 g, 4.05 mmol) and Pd black (0.05 g) were
added and refluxed for 6 h. TLC analysis (1:1 EtOAc/CHCl3) and
mass spectrum revealed that the reaction is complete. The catalyst
was removed by filtration through a celite bed, washed with
CHCl3, and the filtrate was concentrated in vaccuo. The residue
obtained was dissolved in CH2Cl2 (20 mL) and water (15 mL) was
added. The organic layer was separated and washed with satu-
rated NaHCO3 solution (2 ꢂ 15 mL), water (2 ꢂ 15 mL), brine
(1 ꢂ 15 mL) and dried (Na2SO4). The drying agent was filtered off
and the solvent was removed in vaccuo to obtain the crude
product. This crude product was purified by column chromatog-
raphy over Si gel (4 ꢂ 2 cm) using 1:5 EtOAc/CHCl3 as eluent to
afford the pure compound 8. (0.082 g, 82%); Mp 115e116 ꢁC; 1H
(s, 1H); 13C NMR (CDCl3)
d 52.1, 53.0, 67.1, 112.3, 114.4, 118.3, 119.6,
123.2, 124.6, 128.8, 132.7, 144.2, 148.8, 151.1 (2C), 164.1 and 167.6;
MS (ES) m/z 357 (M þ H); Anal Calcd for C19H20N2O5: C, 64.04; H,
5.66; N, 7.86. Found: C, 64.33; H, 5.63; N, 7.64.
4.2.4. 5-((E)-3-(Thiophen-2-yl)acrylamido)-2-morpholinobenzoic
acid (1)
To a solution of compound 5a (0.3 g, 0.81 mmol) in a mixture of
THF (1.5 mL) and MeOH (1.5 mL), 3 N NaOH solution (2.5 mL) was
added at room temperature and the resulting mixture was refluxed
gently for 45 min. TLC analysis (1:1 EtOAc/CHCl3) revealed that the
reaction is complete. The solvent was completely removed in vac-
cuo and the residue obtained was taken up in water (3 mL). The
resulting mixture was cooled to 0 ꢁC and acidified to pH e2 by
NMR (CDCl3)
d
2.70 (t, 2H, J ¼ 7.5 Hz), 2.97e3.05 (m, 4H), 3.27 (t,

3758
B.C. Chenna et al. / European Journal of Medicinal Chemistry 45 (2010) 3752e3761
2H, J ¼ 7.2 Hz), 3.81e3.87 (m, 4H), 3.88 (s, 3H), 6.86 (dd, 1H,
J1 ¼ 0.9 Hz, J2 ¼ 3.3 Hz), 6.90e6.96 (m, 1H), 7.01 (d, 1H, J ¼ 9.0 Hz),
7.11 (bs, 1H), 7.14 (dd, 1H, J1 ¼ 1.2 Hz, J2 ¼ 5.1 Hz), 7.64 (dd, 1H,
J1 ¼ 2.7 Hz, J2 ¼ 8.7 Hz) and 7.76 (d, 1H, J ¼ 2.7 Hz); 13C NMR
(4 mL, 1:1) medium to afford (0.03 g; 64%) as a white solid. Mp.
232 ꢁC (decomp.); 1H NMR (DMSO-d6)
d
3.04 (t, 4H, J ¼ 4.5 Hz), 3.79
(t, 4H, J ¼ 4.5 Hz), 7.21 (dd,1H, J1 ¼3.6 Hz, J2 ¼ 5.1 Hz), 7.64e7.67 (m,
2H), 7.85e7.89 (m, 2H), 8.28 (d, 1H, J ¼ 2.7 Hz) and 11.08 (s, 1H); 13C
(CDCl3)
d
25.5, 38.9, 52.0, 52.9, 67.0, 119.5, 123.2, 123.4, 124.6, 124.7
NMR (DMSO-d6) d 52.6, 66.4, 78.9, 87.8, 118.7, 121.4, 123.7, 124.5,
(2C), 126.8, 132.3, 143.0, 148.6, 167.4 and 170.1. MS (ES) m/z 375
(M þ H). Anal Calcd for C19H22N2O4S. 0.25H2O: C, 60.26; H, 5.99;
N, 7.40. Found: C, 60.27; H, 6.13; N, 7.22.
125.5, 128.3, 132.2, 136.3, 136.7, 146.0, 150.3, 166.4; MS (ESþ) m/z
357 (M þ H); Anal Calcd for C18H16N2O4S.0.25H2O: C, 59.90; H, 4.61;
N, 7.76. Found: C, 59.89; H, 4.56; N, 7.62.
4.2.8. 5-(3-(Furan-2-yl)propanamido)-2-morpholinobenzoic acid
(9)
4.2.12. (Z)-Methyl 5-(-3-(thiophen-2-yl)acrylamido)-2-
morpholinobenzoate (14)
To a solution of compound 6 (0.075 g, 0.22 mmol) in EtOAc
(15 mL) 10% Pd/C (0.02 g) was added and stirred under a hydrogen
atmosphere from balloon (e1 atm) for 25 min. TLC analysis (1:1
EtOAc: CHCl3) and 1H NMR of aliquot revealed that the reaction is
complete. (Note: Prolonged reaction times lead to hydrogenation of
furan ring). The catalyst was removed by filtration through a celite
bed and the filtrate was concentrated in vaccuo to afford the crude
product which was purified by column chromatography over Si gel
(4 ꢂ 2 cm) using EtOAc as eluent to afford pure compound 9 as
a white solid. (0.066 g, 88%); Mp 230e231 ꢁC; 1H NMR (DMSO-d6)
To a solution of 12 (0.042 g, 0.11 mmol) in EtOAc (6 mL) 10% Pd/C
(0.02 g) was added and stirred under H2 atmosphere for 30 min.
TLC examination (1:9 EtOAc/CHCl3) showed that the reaction is
complete. Catalyst was filtered off through a bed of celite. The
filtrate was concentrated in vaccuo to obtain the crude product
which was purified by flash chromatography over Si gel using 5%
EtOAc in CHCl3 to yield compound 14 as a yellow gum. (0.035 g,
82%); 1H NMR (CDCl3)
(d, 1H, J ¼ 12.6 Hz), 6.99e7.06 (m, 3H), 7.38e7.39 (m, 1H), 7.46e7.48
(m, 2H), 7.83 (m, 2H). 13C NMR (CDCl3)
52.2, 53.0, 67.1, 117.1, 199.7,
d 2.99e3.02 (m, 4H), 3.83e3.88 (m, 7H), 5.78
d
d
2.65 (t, 2H, J ¼ 7.5 Hz), 2.93 (t, 2H, J ¼ 7.3 Hz), 3.00e3.07 (m, 4H),
123.3, 124.8, 124.9, 126.6, 131.7, 132.4, 133.9, 134.8, 137.6, 148.9,
164.4, 167.6. MS (ESþ) m/z 373 (M þ H). Anal Calcd for C19H20N2O4S:
C, 61.27; H, 5.41; N, 7.52. Found: C, 61.28; H, 5.50; N, 7.50.
3.73e3.83 (m, 4H), 6.11 (dd,1H, J1 ¼1.2 Hz, J2 ¼ 2.1 Hz), 6.34 (dd,1H,
J1 ¼ 1.8 Hz, J2 ¼ 3.0 Hz), 7.51e7.52 (m, 1H), 7.65 (d, 1H, J ¼ 8.7 Hz),
7.89 (dd, 1H, J1 ¼ 2.7 Hz, J2 ¼ 8.7 Hz), 8.21 (d, 1H, J ¼ 2.7 Hz) and 10.2
(bs, 1H); 13C NMR (DMSO-d6)
d
23.2, 34.5, 52.8, 66.4, 105.2, 110.4,
4.2.13. (Z)-5-(3-(thiophen-2-yl)acrylamido)-2-morpholinobenzoic
acid (15)
120.8, 123.8, 123.9, 125.3, 137.8, 141.5, 145.0, 154.4, 166.4 and 170.2;
MS (ES) m/z 343 (M ꢃ H); Anal Calcd for C18H20N2O5: C, 62.78; H,
5.85; N, 8.13. Found: C, 62.50; H, 5.89; N, 8.01.
Compound 15 was prepared similar to compound 1 by hydrolysis
of compound 14 (0.029 g, 0.08 mmol) using 1 N. NaOH in a MeOH/
THF (2 mL, 1:1) to afford as a white solid. (0.018 g, 65% yield); Mp
4.2.9. Methyl 5-(3-(furan-2-yl)propanamido)-2-
190 ꢁC (decomp.); 1H NMR (DMSO-d6)
d 3.06 (bs, 4H), 3.80 (bs, 4H),
morpholinobenzoate (10)
6.02 (d, 1H, J ¼ 12.3 Hz), 7.08e7.11 (m, 1H), 7.19 (d, 1H, J ¼ 12.6 Hz),
7.52 (d, 1H, J ¼ 3.0 Hz), 7.68e7.71 (m, 2H), 7.98 (dd, 1H, J1 ¼ 2.1 Hz,
J2 ¼ 8.7 Hz), 8.38 (d,1H, J ¼ 2.1 Hz),10.59 (s,1H); 13C NMR (DMSO-d6)
Compound 10 was synthesized, following procedure used for the
preparation of compound 9 starting from compound 5b (0.136 g,
0.38 mmol) in EtOAc (15 mL) and 10% Pd/C (0.02 g) under a hydrogen
d
53.6, 67.2, 117.9, 121.7, 124.6, 124.9, 126.1, 127.5, 133.5, 134.7, 136.6,
atmosphere. (0.106 g, 77%); Mp 119e120 ꢁC; 1H NMR (CDCl3)
d
2.69
138.5, 138.7, 145.9, 165.2, 167.3; MS (ESþ) m/z 359 (M þ H); HRMS
(t, 2H, J ¼ 7.5 Hz), 2.97e3.03 (m, 4H), 3.07 (t, 2H, J ¼ 7.3 Hz),
3.81e3.87 (m, 4H), 3.88 (s, 3H), 6.07 (d,1H, J ¼ 3.3 Hz), 6.27e6.32 (m,
1H), 7.01 (d, 1H, J ¼ 9.0 Hz), 7.16 (bs, 1H), 7.32 (d, 1H, J ¼ 1.2 Hz), 7.65
(dd, 1H, J1 ¼ 2.7 Hz, J2 ¼ 8.7 Hz) and 7.78 (d, 1H, J ¼ 2.4 Hz); 13C NMR
Calcd for C18H18N2O4S: 358.0987. Found: 358.0980.
4.2.14. (E)-Methyl 5-(N-methyl-3-(thiophen-2-yl)acrylamido)-2-
morpholinobenzoate (16a)
(CDCl3)
d
23.7, 35.4, 52.0, 52.9, 67.1, 105.5, 110.2, 119.5, 123.2, 124.6,
To a solution of compound 5a (0.04 g, 0.11 mmol) in anhydrous
THF (10 mL), NaH (0.013 g, 60%; 0.33 mmol) was added and stirred
at 0 ꢁC under a N2 atm for 30 min. Iodomethane (0.03 mL,
0.4 mmol) was added to the reaction mixture and the solution was
stirred for 20 min at rt. MS analysis confirmed product formation
(m/z ¼ 387). The reaction was quenched with aqueous NaHCO3
(1 M, 20 mL). It was extracted with EtOAc (3 ꢂ 25 mL), washed with
water (2 ꢂ 25 mL) and brine (25 mL), and dried over Na2SO4. The
drying agent was filtered off and the filtrate was concentrated in
vaccuo to afford the crude product which was purified by flash
chromatography over Si gel (10 ꢂ 2 cm) using EtOAc/hexanes (1:1)
as eluent to afford compound 16a as a colorless oil. (0.043 g, 100%);
124.7, 132.3, 141.1, 148.7, 154.0, 167.5 and 170.3; MS (ES) m/z 357
(M ꢃ H). Anal Calcd for C19H22N2O5.0.25H2O: C, 62.92; H, 6.25; N,
7.72. Found: C, 62.60; H, 6.13; N, 7.59.
4.2.10. Methyl 5-(3-(thiophen-2-yl)propiolamido)-2-
morpholinobenzoate (12)
Compound 12 was prepared similar to compound 5a by the
treatment of methyl 4-amino-2-(4-morpholino)-benzoate
(0.542 g, 2.3 mmol) and 3-(thiophen-2-yl)propiolic acid, 11
(0.428 g, 2.8 mmol) in the presence of EDAC (1.42 g, 7.4 mmol) and
a DMAP (0.028 g, 0.23 mmol). The crude product was purified by
flash chromatography over Si gel using 10% EtOAc in CHCl3 as
eluent to afford compound 12 as oil. (0.407 g, 37%); 1H NMR (CDCl3)
3
1H NMR (CDCl3)
d 3.09e3.12 (m, 4H), 3.36 (s, 3H), 3.87e3.91 (m,
7H), 6.16 (d, 1H, J ¼ 15.3 Hz), 6.98 (dd, 1H, J1 ¼ 3.6 Hz, J2 ¼ 5.1 Hz),
7.07 (d, 1H, J ¼ 8.4 Hz), 7.15 (d, 1H, J ¼ 3.6 Hz), 7.25e7.29 (m, 2H),
7.66 (d,1H, J ¼ 2.7 Hz), 7.76e7.81 (d,1H, J ¼ 15 Hz); 13C NMR (CDCl3)
d
3.03 (t, 4H, J ¼ 4.5 Hz), 3.84e3.89 (m, 7H), 7.03e7.07 (m, 2H),
7.44e7.46 (m, 2H), 7.65 (bs, 1H), 7.77 (dd, 1H, J1 ¼ 2.7 Hz, J2 ¼ 9 Hz),
7.86 (d, 1H, J ¼ 2.7 Hz); 13C NMR (CDCl3)
d
52.3, 52.9, 67.1, 79.9, 87.4,
d 37.6, 52.3, 52.7, 66.9, 117.2, 119.7, 124.4, 127.4, 127.9, 130.1, 130.5,
119.7, 123.3, 124.6, 124.8, 127.5 (2C), 130.5, 131.9, 135.7, 149.2, 151.1,
167.5; MS (ESþ) m/z 371 (M þ H); Anal Calcd for C19H18N2O4S: C,
61.61; H, 4.90; N, 7.56. Found: C, 61.47; H, 4.88; N, 7.30.
131.8, 134.7, 136.9, 140.3, 151.4, 165.9, 167.1; MS (ESþ) m/z 387
(M þ H); Anal Calcd for C20H22N2O4S.0.5C6H14: C, 64.31; H, 6.80; N,
6.52 Found: C, 64.19; H, 6.56; N, 6.53.
4.2.11. 5-(3-(Thiophen-2-yl)propiolamido)-2-morpholinobenzoic
acid (13)
4.2.15. (E)-Methyl 5-(3-(furan-2-yl)-N-methylacrylamido)-2-
morpholinobenzoate (16b)
Compound 13 was prepared similar to compound 1 by hydro-
lysis of 12 (0.049 g, 0.13 mmol) using 1 N. NaOH in a MeOH/THF
Compound 16b was prepared in a similar fashion to compound
16a using compound 5b (0.048 g, 0.13 mmol), NaH (0.016 g, 60%;

B.C. Chenna et al. / European Journal of Medicinal Chemistry 45 (2010) 3752e3761
3759
0.4 mmol) and iodomethane (0.03 mL, 0.48 mmol) to afford
(0.044 g, 88% yield) as a colorless oil; 1H NMR (CDCl3)
3.10e3.12
J2 ¼ 3.0 Hz), 6.92e6.96 (m, 2H), 7.15 (d, 1H, J ¼ 4.8 Hz), 7.23 (d, 1H,
d
J ¼ 8.7 Hz), 7.52 (d,1H, J ¼ 3.0 Hz); 13C NMR (CDCl3)
d 45.6, 53.7, 67.0,
(m, 4H), 3.36 (s, 3H), 3.88e3.91 (m, 7H), 6.23 (d, 1H, J ¼ 15.1 Hz),
6.39e6.41 (m, 1H), 6.51 (d, 1H, J ¼ 3.4 Hz), 7.07 (d, 1H, 8.7 Hz), 7.28
(dd, 1H, J1 ¼ 2.7 Hz, J2 ¼ 8.7 Hz), 7.35 (d, 1H, J ¼ 1 Hz), 7.45 (d, 1H,
115.0,117.5, 123.3, 124.3,125.1, 125.6, 125.75,125.8, 127.4, 139.5, 141.7,
147.1, 167.5; MS (ES) m/z 345 (M þ H); Anal. Calc for C18H20N2O3S: C,
62.77; H, 5.85; N, 8.13. Found: C, 62.31; H, 5.83; N, 8.02.
J ¼ 15.2 Hz), 7.66 (d, 1H, J ¼ 2.7 Hz); 13C NMR (CDCl3)
d 38.0, 52.7,
53.1, 67.4, 112.5, 114.4, 116.3, 120.1, 124.8, 129.2, 130.9, 132.3, 137.4,
144.4, 151.8, 151.9, 166.6, 167.5; MS (ESþ) m/z 371 (M þ H); Anal
Calcd for C20H22N2O5: C, 64.85; H, 5.99; N, 7.56. Found: C, 64.27; H,
6.04; N, 7.39.
4.2.20. Methyl 5-nitro-2-(piperidin-1-yl)benzoate (22)
To a solution of Methyl 2-fluoro-5-nitrobenzoate, 21 (0.082 g,
0.41 mmol) and triethylamine (0.11 mL, 0.82 mmol) in THF, piper-
idine (0.054 g, 0.64 mmol) was added and the reaction mixture was
stirred at rt for 30 min. TLC (25% EtOAc in CHCl3) examination
revealed the completion of the reaction. The solvent was
completely removed and the residue was dissolved in CHCl3
(20 mL), washed with water (2 ꢂ 15 mL) and brine (15 mL), and
dried over anhydrous Na2SO4. Drying agent was filtered off and the
filtrate was concentrated to afford clean compound 22 (0.115 g,
4.2.16. (E)-5-(N-methyl-3-(thiophen-2-yl)acrylamido)-2-
morpholinobenzoic acid (17a)
Compound 17a was prepared similar to compound 1 by hydrolysis
of 16a (0.035 g, 0.09 mmol) using 1 N. NaOH in a MeOH/THF (2 mL,
1:1) to yield (0.026 g, 78%) as a white solid; Mp 220 ꢁC (decomp.); 1H
NMR (CDCl3)
d
3.14 (bs, 4H), 3.42 (s, 3H), 3.99 (bs, 4H), 6.14 (d, 1H,
100%) as a yellow gum. 1H NMR (CDCl3)
d 1.69e1.72 (m, 6H),
J ¼ 15.3 Hz), 6.99 (dd,1H, J1 ¼3.6 Hz, J2 ¼ 5 Hz), 7.18 (d,1H, J ¼ 3.1 Hz),
3.23e3.26 (m, 4H), 3.93 (s, 3H), 6.95 (d, 1H, J ¼ 9.3 Hz), 8.15 (dd, 1H,
7.26e7.29 (m,1H), 7.48e7.54 (m, 2H), 7.82 (d,1H, J ¼ 15.1 Hz), 8.20 (d,
J1 ¼ 2.7 Hz, J2 ¼ 9.3 Hz), 8.55 (d, 1H, J ¼ 2.7 Hz); 13C NMR (CDCl3)
1H, J ¼ 2.6 Hz); 13C NMR (CDCl3)
d
37.9, 54.0, 67.2, 117.2, 124.2, 127.1,
d 23.8, 25.6, 52.4, 52.6, 117.2, 119.5, 127.7, 128.8, 138.2, 156.3, 167.0;
128.1, 128.5, 130.8, 130.9, 133.3, 135.9, 140.4, 143.5, 149.1, 166.1, 166.2;
MS (ESþ) m/z 373 (M þ H); Anal Calcd for C19H20N2O4S: C, 61.27; H,
5.41; N, 7.52. Found: C, 61.34; H, 5.54; N, 7.34.
MS (ESþ) m/z 265 (M þ H); Anal. Calcd. for C13H16N2O4: C, 59.08; H,
6.10; N, 10.60. Found: C, 58.90; H, 6.29; N, 10.25.
4.2.21. Methyl 5-amino-2-(piperidin-1-yl)benzoate (23)
4.2.17. (E)-5-(3-(furan-2-yl)-N-methylacrylamido)-2-
morpholinobenzoic acid (17b)
Compound 17b was prepared similar to compound 1 by
hydrolysis of 16b (0.035 g, 0.095 mmol) using 1 N. NaOH in
a MeOH/THF (2 mL, 1:1) to afford (0.033 g; 97%) as oil; 1H NMR
To a solution of compound 22 (0.102 g, 0.38 mmol) in EtOAc
(5 mL) was added 10% Pd/C (37 mg) and stirred under a hydrogen
atmosphere (e1 atm) for 2 h. TLC analysis (25% EtOAc/CHCl3)
revealed that the reaction is complete. The catalyst was filtered off
on a celite bed and the filtrate was concentrated on vacuum to
furnish compound 23 (0.083 g, 93%) as a yellow oil. 1H NMR (CDCl3)
(CDCl3) d 3.05e3.15 (m, 4H), 3.42 (s, 3H), 3.90e4.02 (m, 4H), 6.24 (d,
1H, J ¼ 14.7 Hz), 6.42 (dd, 1H, J1 ¼ 1.8 Hz, J2 ¼ 3.4 Hz), 6.55 (d, 1H,
d 1.51e1.53 (m, 2H), 1.64e1.69 (m, 4H), 2.84e2.87 (m, 4H), 3.58 (bs,
J ¼ 3.4 Hz), 7.35 (s, 1H), 7.47e7.54 (m, 3H), 8.20 (d, 1H, J ¼ 2.9 Hz);
2H), 3.87 (s, 3H), 6.74 (dd, 1H, J1 ¼ 3 Hz, J2 ¼ 8.7 Hz), 6.92 (d, 1H,
13C NMR (CDCl3)
d
38.0, 54.0, 67.2, 112.7, 115.1, 115.7, 124.3, 127.1,
J ¼ 8.4 Hz), 7.02 (d, 1H, J ¼ 3 Hz); 13C NMR (CDCl3)
d 24.2, 26.5, 51.9,
130.1, 130.9, 133.4, 143.5, 144.6, 149.1, 151.7, 166.2, 166.4; MS (ESþ)
m/z 357 (M þ H); Anal Calcd for C19H20N2O5: C, 64.04; H, 5.66; N,
7.86. Found: C, 63.84; H, 6.06; N, 7.56.
54.7, 117.3, 119.2, 120.9, 126.7, 141.0, 145.7, 168.8; MS (ESþ) m/z 235
(M þ H); Anal. Calcd. for C13H18N2O2: C, 66.64; H, 7.74; N, 11.96;
Found C, 67.01; H, 7.59; N, 11.13.
4.2.18. (E)-Methyl 5-(3-(thiophen-2-yl)allylamino)-2-
morpholinobenzoate (19)
4.2.22. (E)-Methyl 5-(3-(thiophen-2-yl)acrylamido)-2-(piperidin-
1-yl)benzoate (24a)
To a solution of (E)-3-(thiophen-2-yl)acrylaldehyde, 18 (0.74 g,
5.32 mmol) in MeOH (30 mL). Methyl 5-amino-2-(4-morpholino)-
benzoate 3 (1.25 g, 5.32 mmol) was added and the mixture stirred at
rt for 30 min. A solution of ZnCl2 (0.362 g, 2.66 mmol) and NaCNBH3
(0.367 g, 5.85 mmol) in MeOH (20 mL) was added to the reaction
mixture and stirred at rt for 24 h. The reaction was then quenched
with water (20 mL) and solvents were completely evaporated in
vaccuo. The crude residue obtained was dissolved in CH2Cl2 (60 mL),
washed with water (3 ꢂ50 mL) and brine (1 ꢂ50 mL), and dried over
anhydrous Na2SO4. Drying agent was filtered off and the filtrate was
concentrated to afford the clean product 19 (1.68 g, 88% yield); Mp
Compound 24a was prepared similar to compound 5a by the
treatment of 23 (0.066 g, 0.28 mmol) and 3-(thiophen-2-yl)acrylic
acid, 4a (0.052 g, 0.34 mmol) in the presence of EDAC (0.162 g,
0.84 mmol) and a DMAP (0.005 g, 0.041 mmol) to yield (0.064 g,
62%) as a yellow gum. 1H NMR (CDCl3)
d 1.49e1.55 (m, 2H),
1.64e1.71 (m, 4H), 2.91 (t, 4H, J ¼ 5.1 Hz), 3.85 (s, 3H), 6.43 (d, 1H,
J ¼ 15.3 Hz), 6.93e7.01 (m, 2H), 7.15 (d, 1H, J ¼ 3.3 Hz), 7.28 (d, 1H,
J ¼ 6 Hz), 7.79e7.87 (m, 3H), 8.28 (bs, 1H); 13C NMR (CDCl3)
d 24.2,
26.2, 52.1, 54.1, 119.6, 119.9, 123.2, 124.2, 124.7, 127.6, 128.0, 130.5,
131.7, 134.5, 139.9, 150.1, 164.0, 168.4; MS (ESþ) m/z 371 (M þ H);
HRMS Calcd for C20H22N2O3S: 370.1351. Found: 370.1341.
131 ꢁC; 1H NMR (CDCl3)
d
2.94 (t, 4H, J ¼ 4.5 Hz), 3.82 (t, 4H,
J ¼ 4.5 Hz), 3.87e3.89 (m, 5H), 6.06e6.19 (m,1H), 6.70e6.76 (m, 2H),
4.2.23. (E)-Methyl 5-(3-(furan-2-yl)acrylamido)-2-(piperidin-1-yl)
benzoate (24b)
6.93e7.01 (m, 4H), 7.14 (d, 1H, J ¼ 5.1 Hz); 13C NMR (CDCl3)
d 46.2,
52.0, 53.7, 67.5, 114.9, 116.8, 121.3, 124.2, 124.8, 125.6, 126.4, 127.4,
127.6,141.9,143.3,143.7,168.5; MS (ESþ)m/z 359(M þ H);AnalCalcd
for C19H22N2O3S.0.25H2O: C, 62.87; H, 6.25; N, 7.72. Found: C, 62.74;
H, 6.13; N, 7.52.
Compound 24b was prepared similar to compound 5a by the
treatment of 23 (0.068 g, 0.29 mmol) and 3-(2-furyl)acrylic acid, 4b
(0.047 g, 0.34 mmol) in the presence of EDAC (0.14 g, 0.73 mmol)
and a DMAP (0.005 g, 0.041 mmol) to yield (0.081 g, 78% yield) as
a yellow gum. 1H NMR (CDCl3)
d 1.54e1.58 (m, 2H), 1.66e1.73 (m,
4.2.19. (E)-5-(3-(thiophen-2-yl)allylamino)-2-morpholinobenzoic
acid (20)
Compound 20 was prepared similar to compound 1 by hydrolysis
of19 (1.0g, 2.79mmol)using1 N.NaOHina MeOH/THF (14 mL,1:1)to
yield (0.745 g, 78% yield) as a white solid; Mp 208 ꢁC; 1H NMR (CDCl3)
4H), 2.92e2.96 (m, 4H), 3.88 (s, 3H), 6.44e6.49 (m, 2H), 6.56 (d, 1H,
J ¼ 3.3 Hz), 6.98 (d, 1H, J ¼ 9.3 Hz), 7.43 (d, 1H, J ¼ 1.8 Hz), 7.50 (d,
1H, J ¼ 15.3 Hz), 7.76e7.8 (m, 3H); 13C NMR (CDCl3)
d 24.6, 26.7,
52.7, 54.5, 112.6, 114.6, 119.2, 119.9, 123.6, 124.5, 125.1, 128.9, 132.2,
144.6, 150.4, 151.7, 164.6, 168.9; MS (ESþ) m/z 355 (M þ H); Anal
Calcd for C20H22N2O4: C, 67.78; H, 6.26; N, 7.90. Found: C, 67.90; H,
6.50; N, 7.70.
d
2.92e3.17 (m, 4H), 3.84e4.0 (m, 6H), 6.12 (dt, 1H, J1 ¼ 15.9 Hz,
J2 ¼ 5.7 Hz), 6.73 (d, 1H, J ¼ 16.5 Hz), 6.82 (dd, 1H, J1 ¼ 8.7 Hz,

3760
B.C. Chenna et al. / European Journal of Medicinal Chemistry 45 (2010) 3752e3761
4.2.24. (E)-5-(3-(thiophen-2-yl)acrylamido)-2-(piperidin-1-yl)
benzoic acid (25a)
4.2.28. (E)-N-(3-formyl-4-morpholinophenyl)-3-(thiophen-2-yl)
acrylamide (27a)
Compound 25a was prepared similar to compound 1 by
hydrolysis of 24a (0.056 g, 0.15 mmol) using 1 N. NaOH in a MeOH/
THF (2 mL, 1:1) to afford (0.048 g; 89%) as a white solid. Mp 239 ꢁC
To a solution of compound 26a (0.500 g, 0.44 mmol) in anhydrous
THF at 0 ꢁC, pyridinium chlorochromate (0.188 g, 0.87 mmol) was
addedandstirredfor30minandthenatroomtemperaturefor1h. TLC
analysis (1:1 EtOAc/CHCl3) revealed that the reaction was complete.
The solvent was removed under vacuum and the crude product
obtained was purified by column chromatography over Si gel using
EtOAc/CHCl3 (1:4) as the eluent to afford pure 27a. (0.107 g, 72%); Mp
(decomp.); 1H NMR (DMSO-d6)
d 1.61e1.63 (m, 2H), 1.75 (bs, 4H),
3.02e3.04 (m, 4H), 6.56 (d, 1H, J ¼ 15.6 Hz), 7.15 (t, 1H, J ¼ 3.6 Hz),
7.48 (d, 1H, J ¼ 3.3 Hz), 7.67e7.79 (m, 3H), 8.01 (dd, 1H, J1 ¼ 2.4 Hz,
J2 ¼ 8.7 Hz), 8.29 (d, 1H, J ¼ 2.4 Hz), 10.44 (s, 1H); 13C NMR (DMSO-
d6)
d
22.6, 26.0, 54.2, 120.8, 121.1, 124.1, 124.2, 125.9, 128.9, 129.2,
218e219 ꢁC; 1H NMR (DMSO)
d 2.94e3.03 (m, 4H), 3.72e3.82 (m, 4H),
132.0, 134.1, 138.8, 140.0, 145.5, 163.9, 167.0; MS (ESþ) m/z 357
(M þ H); Anal Calcd for C19H20N2O3S: C, 64.02; H, 5.66; N, 7.86.
Found: C, 63.69; H, 5.67; N, 7.62.
6.53 (d,1H, J ¼ 15.3 Hz), 7.11e7.17 (m,1H), 7.25 (d,1H, J ¼ 8.7 Hz), 7.45
(d,1H, J ¼ 3.3 Hz), 7.66 (d,1H, J ¼ 5.1 Hz), 7.74 (d,1H, J ¼ 15.6 Hz), 7.88
(dd,1H, J1 ¼2.4 Hz, J2 ¼ 8.7 Hz), 8.06 (d,1H, J ¼ 2.7 Hz),10.2 (s,1H) and
10.3 (bs, 1H); 13C NMR (CD3COCD3)
d 55.3, 67.4, 120.1, 121.0, 121.4,
4.2.25. (E)-5-(3-(furan-2-yl)acrylamido)-2-(piperidin-1-yl)benzoic
acid (25b)
Compound 25b was prepared similar to compound 1 by
hydrolysis of 24b (0.08 g, 0.23 mmol) using 1 N. NaOH in MeOH/
THF (2 mL, 1:1) to afford (0.078 g; 100%) as oil; 1H NMR (CD3OD)
126.8, 128.7, 129.1, 130.1, 131.7, 134.6, 136.1, 141.0, 152.5, 164.2 and
191.0; MS (ES) m/z 341 (M ꢃ H); Anal Calcd for C18H18N2O3S: C, 63.14;
H, 5.30; N, 8.18. Found: C, 63.39; H, 5.24; N, 8.00.
4.2.29. (E)-N-(3-formyl-4-morpholinophenyl)-3-(furan-2-yl)
acrylamide (27b)
d
1.82e2.07 (m, 6H), 3.66e3.73 (m, 4H), 6.55e6.75 (m, 3H), 7.47 (d,
1H, J ¼ 14.4 Hz), 7.63 (s, 1H), 7.89e8.16 (m, 2H), 8.59 (bs, 1H); 13C
27b was synthesized, following the procedure used for prepa-
ration of 27a, by the reaction of 26b (0.188 g, 0.57 mmol) and
pyridinium chlorochromate (0.247 g, 1.15 mmol) in anhydrous THF
(10 mL) to furnish the pure product. (0.115 g, 61%); Mp 231e232 ꢁC;
NMR (CD3OD)
d 22.9, 26.8, 58.6, 114.0, 116.7, 119.4, 124.2, 124.5,
126.9, 130.9, 140.6, 142.4, 146.9 (2C), 152.9, 167.3 (2C); MS (ESþ) m/z
341 (M þ H); HRMS Calcd for C19H20N2O4: 340.1423. Found:
340.1413.
1H NMR (DMSO-d6)
d 2.94e3.03 (m, 4H), 3.73e3.82 (m, 4H),
6.53e6.65 (m, 2H), 6.86 (d, 1H, J ¼ 3.6 Hz), 7.24 (d, 1H, J ¼ 9.0 Hz),
7.38 (d, 1H, J ¼ 15.6 Hz), 7.82 (d, 1H, J ¼ 1.5 Hz), 7.89 (dd, 1H,
J1 ¼ 2.5 Hz, J2 ¼ 8.8 Hz), 8.06 (d, 1H, J ¼ 2.7 Hz), 10.2 (s, 1H) and 10.3
4.2.26. (E)-N-(3-(Hydroxymethyl)-4-morpholinophenyl)-3-
(thiophen-2-yl)acrylamide (26a)
To a solution of compound 5a (0.500 g, 1.34 mmol) in a mixture
of anhydrous CH2Cl2 (35 mL) and THF (5 mL), DIBAL-H (6.7 mL, 1 M
in CH2Cl2, excess) was added and stirred at 0 ꢁC for 1 h. Then it was
allowed to attain room temperature at which it was stirred for an
additional 2 h. TLC analysis (1:1 EtOAc/CHCl3) revealed that the
reduction is complete. The reaction mixture was cooled to 0 ꢁC and
was quenched by careful addition of MeOH (3 mL) and 1 N. HCl
(0.5 mL). The solvent was removed under reduced pressure and the
residue obtained was dissolved in CHCl3 (300 mL) and washed with
water (2 ꢂ 75 mL), brine (1 ꢂ 150 mL) and dried (Na2SO4). The
drying agent was filtered off and the solvent was removed in vaccuo
to afford the crude product which was purified by flash column
chromatography on silica gel (20 ꢂ 3 cm) using EtOAc/hexanes
(3:1) as eluent to furnish compound 26a as a yellow solid. (0.354 g,
(bs, 1H); 13C NMR (DMSO-d6)
d 54.1, 66.2, 112.6, 114.8, 119.0, 119.1,
120.2, 126.0, 127.4, 128.2, 134.6, 145.3, 150.9, 151.2, 163.4 and 190.7;
MS (ES) m/z 325 (M ꢃ H); Anal Calcd for C18H18N2O4. 0.3H2O: C,
65.20; H, 5.65; N, 8.45. Found: C, 64.90; H, 5.35; N, 8.33.
4.2.30. 5-((E)-3-(Thiophen-2-yl)acrylamido)-2-
morpholinobenzamide (28)
To a solution of compound 1 (0.050 g, 0.14 mmol) in anhydrous
CH2Cl2 (4 mL) at room temperature a mixture of thionyl chloride
(0.2 mL) and anhydrous DMF (0.2 mL) was added and the resulting
mixture was stirred for 4 h at room temperature. The solvent was
removed under reduced pressure. The resulting gum was dissolved
in anhydrous CH2Cl2 (3 mL) and excess 50% aqueous ammonium
hydroxide (3 mL) was added to achieve basic pH (e12) and stirred at
room temperature for 2 h. The solvent was removed under reduced
pressure and the crude compound was purified by flash column
chromatography (20 ꢂ 1 cm) on silica gel using EtOAc/CHCl3 (1:1)
as eluent to furnish the pure amide 28. (0.013 g, 26%); 1H NMR
77%); Mp 184e185 ꢁC; 1H NMR (DMSO-d6)
d 2.79e2.83 (m, 4H),
3.65e3.75 (m, 4H), 4.54 (d, 2H, J ¼ 5.1 Hz), 5.12 (t, 1H, J ¼ 5.4 Hz),
6.58 (d, 1H, J ¼ 15.0 Hz), 7.02 (d, 1H, J ¼ 8.7 Hz), 7.13 (dd, 1H,
J1 ¼ 3.6 Hz, J2 ¼ 5.1 Hz), 7.42 (d, 1H, J ¼ 3.3 Hz), 7.60e7.74 (m, 4H)
and 10.1 (bs, 1H); 13C NMR (DMSO-d6)
d
52.7, 58.5, 66.6, 118.1, 119.0,
(DMSO-d6) d 2.86e2.94 (m, 4H), 3.71e3.78 (m, 4H), 6.55 (d, 1H,
119.1, 121.1, 128.3, 128.4, 131.1, 132.7, 135.0, 137.1, 139.8, 145.6 and
162.9; MS (ES) m/z 343 (M ꢃ H); Anal Calcd for C18H20N2O3S: C,
62.77; H, 5.85; N, 8.13. Found: C, 62.78; H, 5.95; N, 8.08.
J ¼ 15.3 Hz), 7.14 (dd, 1H, J1 ¼ 3.6 Hz, J2 ¼ 5.1 Hz), 7.20 (d, 1H,
J ¼ 8.7 Hz), 7.45 (d, 1H, J ¼ 3.0 Hz), 7.57 (bs, 1H), 7.66 (d, 1H,
J ¼ 5.1 Hz), 7.73 (d, 1H, J ¼ 15.6 Hz), 7.84 (dd, 1H, J1 ¼ 2.7 Hz,
J2 ¼ 8.7 Hz), 7.99 (d, 1H, J ¼ 2.7 Hz), 8.64 (bs, 1H) and 10.2 (bs, 1H);
4.2.27. (E)-3-(Furan-2-yl)-N-(3-(hydroxymethyl)-4-
13C NMR (DMSO-d6)
d 52.9, 66.4, 120.4, 120.8, 121.1, 122.0, 128.5
morpholinophenyl)acrylamide (26b)
(2C), 129.3, 131.3, 133.1, 135.1, 139.7, 146.2, 163.1 and 167.7; MS (ES)
Compound 26b was synthesized, following the procedure used
for preparation of 26a, by the reaction of 5b (0.500 g,1.40 mmol) and
DIBAL-H (7.02 mL, 1 M in CH2Cl2) in CH2Cl2 (35 mL) and THF (5 mL)
for 2 h at room temperature to furnish pure product. (0.372 g, 81%);
m/z 356 (M ꢃ H).
Acknowledgements
Mp 182e183 ꢁC; 1H NMR (DMSO e d6)
d
2.74e2.83 (m, 4H),
Authors wish to acknowledge the generous financial support
from the Department of Chemistry, University of Alabama at Bir-
mingham (UAB). Beginning Grant-in-Aid (AHA0865323E) and
Grant-in-Aid (AHA0855076E) from American Heart Association
Greater Southeast Affiliate are acknowledged. Breast Spore Pilot
grant from the UAB Comprehensive Cancer Center and a trans-
lational research grant from UAB Center for Clinical and Trans-
lational Science are also acknowledged.
3.65e3.74 (m, 4H), 4.54 (d, 2H, J ¼ 5.1 Hz), 5.12 (t, 1H, J ¼ 5.4 Hz),
6.58e6.67 (m, 2H), 6.82 (d,1H, J ¼ 3.3 Hz), 7.02 (d,1H, J ¼ 8.7 Hz), 7.35
(d,1H, J ¼ 15.3 Hz), 7.61e7.71 (m, 2H), 7.81 (d,1H, J ¼ 1.5 Hz) and 10.1
(bs, 1H); 13C NMR (DMSO e d6)
d 52.6, 58.5, 66.6, 112.5, 114.3, 118.2,
119.0, 119.1, 119.6, 126.8, 134.9, 137.0, 145.0, 145.5, 150.9 and 163.0;
MS (ES) m/z 327 (M ꢃ H); Anal Calcd for C18H20N2O4: C, 65.84; H,
6.14; N, 8.53. Found: C, 65.55; H, 6.27; N, 8.37.

B.C. Chenna et al. / European Journal of Medicinal Chemistry 45 (2010) 3752e3761
3761
peptidoglycan substrate for sortase-catalyzed surface protein anchoring. J.
Biol. Chem. 277 (2002) 16241e16248.
References
[18] I.M. Jonsson, S.K. Mazmanian, O. Schneewind, T. Bremell, A. Tarkowski, The
role of Staphylococcus aureus sortase A and sortase B in murine arthritis.
Microb. Infect. 5 (2003) 775e780.
[19] S.K. Mazmanian, G. Liu, E.R. Jensen, E. Lenoy, O. Schneewind, Staphylococcus
aureus sortase mutants defective in the display of surface proteins and in the
pathogenesis of animal infections. Proc. Natl. Acad. Sci. U S A. 97 (2000)
5510e5515.
[20] T.C. Barnett, J.R. Scott, Differential recognition of surface proteins in Strepto-
coccus pyogenes by two sortase gene homologs. J. Bacteriol. 184 (2002)
2181e2191.
[21] H. Bierne, S.K. Mazmanian, M. Trost, M.G. Pucciarelli, G. Liu, P. Dehoux,
L. Jansch, F. Garcia-del Portillo, O. Schneewind, P. Cossart, Inactivation of the
srtA gene in Listeria monocytogenes inhibits anchoring of surface proteins and
affects virulence. Mol. Microbiol. 43 (2002) 869e881.
[22] T.C. Bolken, C.A. Franke, K.F. Jones, G.O. Zeller, C.H. Jones, E.K. Dutton, D.
E. Hruby, Inactivation of the srtA gene in Streptococcus gordonii inhibits cell
wall anchoring of surface proteins and decreases in vitro and in vivo adhesion.
Infect. Immun. 69 (2001) 75e80.
[23] C. Garandeau, H. Reglier-Poupet, I. Dubail, J.L. Beretti, P. Berche, A. Charbit, The
sortase SrtA of Listeria monocytogenes is involved in processing of internalin
and in virulence. Infect. Immun. 70 (2002) 1382e1390.
[24] I.M. Jonsson, S.K. Mazmanian, O. Schneewind, M. Verdrengh, T. Bremell,
A. Tarkowski, On the role of Staphylococcus aureus sortase and sortase-cata-
lyzed surface protein anchoring in murine septic arthritis. J. Infect. Dis. 185
(2002) 1417e1424.
[25] S.S. Kang, J.G. Kim, T.H. Lee, K.B. Oh, Flavonols inhibit sortases and sortase-
mediated Staphylococcus aureus clumping to fibrinogen. Biol. Pharm. Bull. 29
(2006) 1751e1755.
[26] S.H. Kim, D.S. Shin, M.N. Oh, S.C. Chung, J.S. Lee, I.M. Chang, K.B. Oh, Inhibition
of sortase, a bacterial surface protein anchoring transpeptidase, by beta-
sitosterol-3-O-glucopyranoside from Fritillaria verticillata. Biosci. Biotechnol.
Biochem. 67 (2003) 2477e2479.
[27] S.W. Kim, I.M. Chang, K.B. Oh, Inhibition of the bacterial surface protein
anchoring transpeptidase sortase by medicinal plants. Biosci. Biotechnol.
Biochem. 66 (2002) 2751e2754.
[28] C.J. Scott, A. McDowell, S.L. Martin, J.F. Lynas, K. Vandenbroeck, B. Walker,
Irreversible inhibition of the bacterial cysteine protease-transpeptidase sor-
tase (SrtA) by substrate-derived affinity labels. Biochem. J. 366 (2002)
953e958.
[29] K.B. Oh, S.H. Kim, J. Lee, W.J. Cho, T. Lee, S. Kim, Discovery of diary-
lacrylonitriles as a novel series of small molecule sortase A inhibitors. J. Med.
Chem. 47 (2004) 2418e2421.
[30] B.C. Chenna, B.A. Shinkre, J.R. King, A.L. Lucius, S.V. Narayana, S.E. Velu,
Identification of novel inhibitors of bacterial surface enzyme Staphylococcus
aureus Sortase A. Bioorg. Med. Chem. Lett. 18 (2008) 380e385.
[31] J.S. Skotnicki, R.M. Kearney, A.L. Smith, Synthesis of secorapamycin esters and
amides. Tetrahedron Lett. 35 (1994) 197e200.
[32] R.G. Kruger, P. Dostal, D.G. McCafferty, Development of a high-performance
liquid chromatography assay and revision of kinetic parameters for the
Staphylococcus aureus sortase transpeptidase SrtA. Anal. Biochem. 326 (2004)
42e48.
[33] H. Ton-That, S.K. Mazmanian, K.F. Faull, O. Schneewind, Anchoring of surface
proteins to the cell wall of Staphylococcus aureus. Sortase catalyzed in vitro
transpeptidation reaction using LPXTG peptide and NH(2)-Gly(3) substrates. J.
Biol. Chem. 275 (2000) 9876e9881.
[34] D.H. Wadsworth, S.M. Geer, M.R. Detty, Preparation of arylpropiolate esters
from trichlorocyclopropenium cation and elaboration of the esters to
unsymmetrical 1,4-pentadiyn-3-ones and unsymmetrical tellurapyranones. J.
Org. Chem. 52 (1987) 3662e3668.
[1] R.M. Klevens, M.A. Morrison, J. Nadle, S. Petit, K. Gershman, S. Ray, L.
H. Harrison, R. Lynfield, G. Dumyati, J.M. Townes, A.S. Craig, E.R. Zell, G.
E. Fosheim, L.K. McDougal, R.B. Carey, S.K. Fridkin, Invasive methicillin-resis-
tant Staphylococcus aureus infections in the United States. J. Am. Med. Assoc.
298 (2007) 1763e1771.
[2] S.V. Kazakova, J.C. Hageman, M. Matava, A. Srinivasan, L. Phelan, B. Garfinkel,
T. Boo, S. McAllister, J. Anderson, B. Jensen, D. Dodson, D. Lonsway, L.
K. McDougal, M. Arduino, V.J. Fraser, G. Killgore, F.C. Tenover, S. Cody, D.
B. Jernigan, A clone of methicillin-resistant Staphylococcus aureus among
professional football players. N. Engl. J. Med. 352 (2005) 468e475.
[3] I.B. Gosbell, T. Barbagiannakos, S.A. Neville, J.L. Mercer, A.M. Vickery, F.
G. O’Brien, G.W. Coombs, M.J. Malkowski, J.C. Pearson, Non-multiresistant
methicillin-resistant Staphylococcus aureus bacteraemia in Sydney, Australia:
emergence of EMRSA-15, Oceania, Queensland and Western Australian MRSA
strains. Pathology 38 (2006) 239e244.
[4] M. Gilbert, J. MacDonald, D. Gregson, J. Siushansian, K. Zhang, S. Elsayed,
K. Laupland, T. Louie, K. Hope, M. Mulvey, J. Gillespie, D. Nielsen, V. Wheeler,
M. Louie, A. Honish, G. Keays, J. Conly, Outbreak in Alberta of community-
acquired (USA300) methicillin-resistant Staphylococcus aureus in people with
a history of drug use, homelessness or incarceration. Can. Med. Assoc. J. 175
(2006) 149e154.
[5] M.D. David, A.M. Kearns, S. Gossain, M. Ganner, A. Holmes, Community-
associated meticillin-resistant Staphylococcus aureus: nosocomial trans-
mission in a neonatal unit. J. Hosp. Infect. 64 (2006) 244e250.
[6] H.C. Baggett, T.W. Hennessy, K. Rudolph, D. Bruden, A. Reasonover, A. Parkinson,
R. Sparks, R.M. Donlan, P. Martinez, K. Mongkolrattanothai, J.C. Butler,
Community-onset methicillin-resistant Staphylococcus aureus associated with
antibiotic use and the cytotoxin Panton-Valentine leukocidin during a furun-
culosis outbreak in rural Alaska. J. Infect. Dis. 189 (2004) 1565e1573.
[7] W. Zhu, N.C. Clark, L.K. McDougal, J. Hageman, L.C. McDonald, J.B. Patel,
Vancomycin-resistant Staphylococcus aureus isolates associated with Inc18-
like vanA plasmids in Michigan. Antimicrob. Agents Chemother. 52 (2008)
452e457.
[8] S. Chang, D.M. Sievert, J.C. Hageman, M.L. Boulton, F.C. Tenover, F.P. Downes,
S. Shah, J.T. Rudrik, G.R. Pupp, W.J. Brown, D. Cardo, S.K. Fridkin, Infection with
vancomycin-resistant Staphylococcus aureus containing the vanA resistance
gene. N. Engl. J. Med. 348 (2003) 1342e1347.
[9] Anonymous, Staphylococcus aureus resistant to vancomycin-United States.
MMWR 51 (2002) 565e566.
[10] Anonymous, Vancomycin-resistant Staphylococcus aureuseNew York. MMWR
53 (2004) 322.
[11] W.W. Navarre, O. Schneewind, Surface proteins of gram-positive bacteria and
mechanisms of their targeting to the cell wall envelope. Microbiol. Mol. Biol.
Rev. 63 (1999) 174e229.
[12] P. Cossart, R. Jonquieres, Sortase, a universal target for therapeutic agents
against gram-positive bacteria? Proc. Natl. Acad. Sci.
U S A. 97 (2000)
5013e5015.
[13] V.A. Fischetti, V. Pancholi, O. Schneewind, Conservation of a hexapeptide
sequence in the anchor region of surface proteins from gram-positive cocci.
Mol. Microbiol. 4 (1990) 1603e1605.
[14] O. Schneewind, P. Model, V. Fischetti, Sorting of protein A to the staphylo-
coccal cell wall. Cell 70 (1992) 267e281.
[15] O. Schneewind, D. Mihaylova-Petkov, P. Model, Cell wall sorting signals in
surface proteins of gram-positive bacteria. EMBO J. 12 (1993) 4803e4811.
[16] W.W. Navarre, O. Schneewind, Proteolytic cleavage and cell wall anchoring at
the LPXTG motif of surface proteins in gram-positive bacteria. Mol. Microbiol.
14 (1994) 115e121.
[17] A.M. Perry, H. Ton-That, S.K. Mazmanian, O. Schneewind, Anchoring of surface
proteins to the cell wall of Staphylococcus aureus. III. Lipid II is an in vivo