Design and synthesis of novel antimicrobials with activity against Gram-positive bacteria and mycobacterial species, including M. tuberculosis

Article abstract of DOI:10.1016/j.bmc.2013.10.011

The alarming increase in bacterial resistance over the last decade along with a dramatic decrease in new treatments for infections has led to problems in the healthcare industry. Tuberculosis (TB) is caused mainly by Mycobacterium tuberculosis which is responsible for 1.4 million deaths per year. A world-wide threat with HIV co-infected with multi and extensively drug-resistant strains of TB has emerged. In this regard, herein, novel acrylic acid ethyl ester derivatives were synthesized in simple, efficient routes and evaluated as potential agents against several Mycobacterium species. These were synthesized via a stereospecific process for structure activity relationship (SAR) studies. Minimum inhibitory concentration (MIC) assays indicated that esters 12, 13, and 20 exhibited greater in vitro activity against Mycobacterium smegmatis than rifampin, one of the current, first-line anti-mycobacterial chemotherapeutic agents. Based on these studies the acrylic ester 20 has been developed as a potential lead compound which was found to have an MIC value of 0.4 μg/mL against Mycobacterium tuberculosis. The SAR and biological activity of this series is presented; a Michael-acceptor mechanism appears to be important for potent activity of this series of analogs.

Full text of DOI:10.1016/j.bmc.2013.10.011

Bioorganic & Medicinal Chemistry 21 (2013) 7830–7840
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of novel antimicrobials with activity against
Gram-positive bacteria and mycobacterial species, including
M. tuberculosis
V. V. N. Phani Babu Tiruveedhula a, Christopher M. Witzigmann a, Ranjit Verma a, M. Shahjahan Kabir a,
Marc Rott c, William R. Schwan c, Sara Medina-Bielski c, Michelle Lane c, William Close c,
Rebecca L. Polanowski c, David Sherman d, Aaron Monte b, Jeffrey R. Deschamps e, James M. Cook a,
⇑
a Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, USA
b Department of Chemistry and Biochemistry, University of Wisconsin-La Crosse, La Crosse, WI 54601, USA
c Department of Microbiology, University of Wisconsin-La Crosse, La Crosse, WI 54601, USA
d Seattle Biomedical Research Institute and Department of Global Health, University of Washington, Seattle, WA 98109, USA
e Center for Bimolecular Science and Engineering, Naval Research Laboratory, Code 6930, Washington, DC 20375, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The alarming increase in bacterial resistance over the last decade along with a dramatic decrease in new
treatments for infections has led to problems in the healthcare industry. Tuberculosis (TB) is caused
mainly by Mycobacterium tuberculosis which is responsible for 1.4 million deaths per year. A world-wide
threat with HIV co-infected with multi and extensively drug-resistant strains of TB has emerged. In this
regard, herein, novel acrylic acid ethyl ester derivatives were synthesized in simple, efficient routes and
evaluated as potential agents against several Mycobacterium species. These were synthesized via a stereo-
specific process for structure activity relationship (SAR) studies. Minimum inhibitory concentration (MIC)
assays indicated that esters 12, 13, and 20 exhibited greater in vitro activity against Mycobacterium
smegmatis than rifampin, one of the current, first-line anti-mycobacterial chemotherapeutic agents.
Based on these studies the acrylic ester 20 has been developed as a potential lead compound which
Received 10 August 2013
Revised 3 October 2013
Accepted 10 October 2013
Available online 21 October 2013
Keywords:
Mycobacterium tuberculosis
Mycobacterium smegmatis
Anti-tuberculosis
Acrylic esters
was found to have an MIC value of 0.4 lg/mL against Mycobacterium tuberculosis. The SAR and biological
Michael-acceptor
activity of this series is presented; a Michael-acceptor mechanism appears to be important for potent
activity of this series of analogs.
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
their symptoms decrease long before the infection has been erad-
icated, allowing the bacteria to develop drug resistance, potentially
Surprisingly, tuberculosis (TB) is the second leading lethal infec-
tious disease in the world, following human immunodeficiency
virus (HIV). According to a recent global tuberculosis report by
the World Health Organization (WHO), TB caused 1.4 million
deaths in 2011 and 9 million newly infected cases are reported
each year.1 TB is a bacterial infection caused by the acid-fast bacil-
lus Mycobacterium tuberculosis. TB mainly infects the lungs (pul-
monary TB), although it can affect most organs in the body (extra
pulmonary TB) including the liver, brain and kidney.2 The tradi-
tional current first-line treatment of drug-sensitive TB infections
consists of a four-drug regimen that includes rifampin, isoniazid,
pyrazinamide, and ethambutol.3,4 This treatment requires a mini-
mum of 6 months to be effective.5 Due to the extended time course
of treatment many patients stop taking the medication as soon as
leading to multidrug-resistant (MDR) and extensively drug-
resistant (XDR) forms of TB. Treatment of these infections may
extend to 18–20 months.2 The ability to treat TB is further
confounded by co-infection with HIV leading to treatment failures
as well as a rise in transmission rates and mortality due to TB.
Without improvements one billion people will be newly infected,
there will be around 125 million people get sick, and 14 million
will die in the next 10 years.6–11 Consequently, the development
of new chemotherapeutic combinations for TB that eradicate the
disease quicker as well as are less complex, cheaper and have
fewer side effects are essential for the future.
In our continued efforts to develop new anti-mycobacterial
agents, a novel class of acrylic esters was synthesized.12–14 In early
efforts to increase the molecular diversity in this series of antimi-
crobial agents, certain acrylic acid ethyl esters such as 1 were syn-
thesized.15a This initial lead compound exhibited a promising MIC
⇑
Corresponding author. Tel.: +1 (414) 229 5856; fax: +1 (414) 229 5530.
E-mail address: capncook@uwm.edu (J.M. Cook).
of 16 lg/mL against Mycobacterium smegmatis, a safer surrogate of
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.10.011

V. V. N. Phani Babu Tiruveedhula et al. / Bioorg. Med. Chem. 21 (2013) 7830–7840
7831
Figure 1. Lead compounds.
the clinically significant TB causing mycobacteria. Consequently, 1
was assayed against the more virulent strain, M. tuberculosis,
to increase stability and to evaluate steric and electronic effects on
the bioavailability and potency (Scheme 1) of 1.
resulting in an MIC value of 25
l
g/mL.12
Further SAR studies on these compounds were carried out with
ligands which contained similar functionality. Hence, the sulfur
atom in 1 was replaced with the keto group at position B to furnish
ketones 12 and 13 (Scheme 2).17 This altered the electronic
character of the double bond of analog 1. These 4-oxo substituted
acrylic esters exhibited increased activity against Mycobacterium
smegmatis and M. tuberculosis (see Tables 1 and 2).
Presumably, the trans ester 13 is more stable in vivo than the cis
ester 12. Accordingly, a series of analogs were prepared to study
the importance of the double bond in regard to the increased po-
tency of 13. To evaluate the importance of the electronic character
of the double bond in keto ester 13, the saturated compounds 14
and 15 (Scheme 3) were synthesized as well as 19, 28, and 29, with
a benzene, cyclopropyl and epoxide ring in place of the double
bond, as illustrated in Scheme 4. To increase the hydrophobic
character of the molecule 13, a prenyl group was substituted for
the ethyl function (see Ref. 31 for a precedent) to provide alkyl es-
ter 17 (Scheme 3). The hydrogen bond acceptor properties of the
2. Results
The structure–activity relationship (SAR) study of lead com-
pound 1 provided valuable information regarding the basic struc-
tural requirements for anti-mycobacterial activity. In addition,
manipulations of the basic unit led to increased potency and stabil-
ity.15b In order to evaluate the effect of structural changes on anti-
mycobacterial activity, the esters of 1 at positions A, B, C, and D
were altered. First, in order to increase the hydrophobic interac-
tions of ester 1 with bacteria the ethyl ester was replaced with a
methyl cyclopropyl ester to give 4 (Scheme 1) at position D in 1.
To increase the stability as well as the water solubility of the ester
1, the acids 2 and 3 (Scheme 1) were prepared. This increased the
hydrophilic character of the molecule and the CLogP value went
from 5.7 to 4.7 in agreement with Lipinski’s rules and the classic
QSAR studies of Hansch.16 Various amides (5–11) were synthesized
O
O
O
SOCl2, 40 oC
20% KOH, rt
R
S
O
R
S
R
O
S
OH
6 - 8 h, 90 - 92%
, 4 h, 94%
OH
4
R = p-t-butyl benzene,
2
R = p-t-butyl benzene,
R = benzothiazole, 3
carbonyldiimidazole (CDI), amines,
toluene, 60 oC, 3-6 h, 88-94%
O
NR1R2
R
S
5-11
1
2
5
R = benzothiazole; R = R = isopropyl:
R = p-t-butyl
1
2
6
R = H; R = phenyl:
benzene;
R = p-t-butyl benzene; R1 = methyl; R2 = phenyl:
7
1
2
8
benzene; R = H; R = methyl:
butyl
R = p-t-
R = p-t-
R1 = R2 = methyl:
9
butyl benzene;
R = p-t-butyl benzene; R1 = R2 = isopropyl: 10
R = p-t-butyl benzene; R1 = H; R2 = cyclopropyl: 11
Scheme 1. Synthesis of cis acrylic acids, amides and ester.

7832
V. V. N. Phani Babu Tiruveedhula et al. / Bioorg. Med. Chem. 21 (2013) 7830–7840
OH
O O
O
O
NaHCO3, 18 h, rt
n-BuLi, RCHO,1 h
R
O
R
HC C
21
-78 ºC - rt, THF
DMSO/H2O (8:1)
60 - 70%
O
90% Z, 10% E
58 - 60%
O
rt H
26, R = p-t-butyl-benzene
27, R = benzo[b]thiophene
O
O
R
100% E
O
O
O O
O
O
O O
O
O
S
12
13
20
Scheme 2. Synthesis of 4-oxo substituted acrylic acid ethyl estersa. aZ and E isomers were separated by flash chromatography on silica gel.
Table 1
hydrogens appeared with a value of the coupling constant of
Minimum inhibitory concentrations (MIC) of acrylic acid ethyl ester analogs against
common bacterial species ( g/mL)
10.1 Hz and were readily correlated with the cis isomer with the
help of the available literature on acrylic esters and also confirmed
by X-ray crystallographic analysis (Fig. 2).15a,22,23 In the case of acid
3, the starting moiety in area A had been altered from t-butyl ben-
zene to benzothiazole. Due to the presence of the keto group adja-
cent to the double bond in keto ester 13, a similar hydrolysis
reaction was attempted with 20% KOH but resulted in the disap-
pearance of the alkene protons. The hydrolysis conditions were
modified and 16 was prepared from keto ester 13 in 90% yield
using K2CO3 in refluxing aqueous methanol, as illustrated in
Scheme 3. The cyclopropyl methyl ester 4 was prepared from the
acid using thionyl chloride and then addition of cyclopropylmethyl
alcohol in excellent yield 93% (Scheme 1).
Additional alteration of the ester moiety in 1 (area D) was
accomplished using carbonyldiimidazole (CDI) and the corre-
sponding amines in toluene at 60 °C giving amides 5–11 (Scheme 1)
in good to excellent yields 88–93%. The SAR of area B of 1 was
explored by introduction of the keto group in place of the sulphur
atom in 1 to furnish ketones 12 and 13 (individually). In order to do
this the 4-hydroxy-2-alkynoates 26 and 27 were prepared first by
the addition of n-butyllithium to propynoic acid ethyl ester at low
temperature À78 °C and the alkynic anion, which resulted, rapidly
added to the corresponding aldehydes (Scheme 2) in 58–60%
yield.24 Treatment with sodium bicarbonate as a catalyst for the re-
quired isomerization gave a mixture of cis and trans isomers 12 and
13 (9:1) which were readily separated by flash column chromatog-
raphy (overall yield 70%). Treatment of the mixture of 12 and 13
with anhydrous HCl(g) in ether gave complete conversion of cis
12 into trans 13 in excellent yield. In the presence of the benzothio-
phene heterocyclic ring in 20, the yield decreased to 60%
(Scheme 2).25
l
Compound
M. smegmatis
S. aureus ATCC
29213
B. cereus
E. coli ATCC
29522
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
24
25
28
29
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
4
>128
128
>128
128
>128
>128
>128
128
16
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
>128
128
>128
>128
16
>128
>128
32
>128
64
64
>128
8
8
>128
64
>128
>128
>128
>128
4
>128
>128
16
>128
>128
8
2
4
>128
>128
32
0.5
16
>128
1
>128
>128
64
NDb
NDb
NDb
NDb
NDb
NDb
NDb
>128
>128
NDb
NDb
NDb
NDb
>128
>128
>128
>128
1
128
NDb
64
32
Tetracyclinea
Rifampina
0.25
NDb
NDb
a
Positive control.
ND = not determined.
b
olefin in 13 were decreased via synthesis of an
a,b-unsaturated es-
Alteration of area C of 13 from alkene to alkane was slightly
more challenging. The classic route using Pd/C in EtOH with hydro-
gen gas (pressure at 20 psi) furnished saturated analog 15 instead
of the desired ketone 14 because the ketone was both benzylic and
allylic. However, the reduction procedure of trans ester 13 with
ter 18 (Scheme 4). To alter both the geometry of the molecule and
the Michael acceptor properties, the alkyne 25 was synthesized
(Scheme 4). It is well-known that acetylenic ketones do not under-
go Michael additions as rapidly as olefinic ketones or esters.18–21
26
TiCl3 gave ketone 14 in good yield 85% (Scheme 3). In the case
2.1. Chemistry
of thioalkyl 24, the standard Pd/C (H2) reduction was readily
executed (Scheme 4). Prenyl ester 17 was prepared by alkylation
of acid 16 with prenyl bromide 22 with cesium carbonate as the
base in DMF in 85% yield (Scheme 3). The synthesis of 25 was
accomplished using the Dess–Martin reagent on propargylic alco-
To study the SAR and establish the pharmacophoric unit of 1, as
mentioned earlier, the molecule was divided into four areas A, B, C,
and D (Fig. 1). To alter area D, the two esters represented by struc-
ture 1 were saponified to provide the corresponding carboxylic
acids 2 and 3 in excellent yields (91% and 92%), respectively, using
an aqueous solution of 20% KOH (Scheme 1). The stereochemistry
of acid 2 was assigned by 1H NMR. The characteristic olefinic
hol 26 in good yield (85%, Scheme 4). The
18 was synthesized by Suzuki palladium catalyzed cross
coupling reaction (Scheme 4) with the allylic bromide 23 and the
a,b-unsaturated analog
a

V. V. N. Phani Babu Tiruveedhula et al. / Bioorg. Med. Chem. 21 (2013) 7830–7840
7833
Table 2
Minimum inhibitory concentrations (MIC) of select compounds against additional mycobacterial species (
l
g/mL)
Compound
M. tuberculosis
M. fortuitum
M. kansasii
M. chelonae
M. avium
M. intracellulare
12
0.8
8
64
16
32
8
13
14
15
16
17
18
19
20
0.8
4
>128
32
>125
64
>128
>128
16
32
8
16
4
NDb
NDb
NDb
NDb
NDb
NDb
0.4
>128
128
NDb
>128
>128
>128
8
>128
32
>128
>128
NDb
>128
>128
>128
16
>128
>128
NDb
>128
>128
>128
4
NDb
32
>128
>128
8
24
25
28
29
NDb
NDb
NDb
NDb
1.2
>128
>128
64
>128
NDb
NDb
32
>128
>128
NDb
NDb
NDb
NDb
0.5
>128
>128
NDb
NDb
NDb
NDb
32
>128
>128
NDb
NDb
NDb
NDb
2
>128
>128
NDb
NDb
NDb
NDb
1
Ethambutola
Isoniazida
Rifampina
0.25
<0.03
a
Positive controls.
ND = not determined.
b
O
O
O
O
O
Pd/C, H2
rt,12 h, EtOH
80%
TiCl3, 10 min
rt,THF - H2O
85%
O
O
O
15
14
13
-H O(1:1),
5 h, 60 °C, 90%
K2CO3 MeOH
2
O
O
OH
O
Br
, KI
22
O
16
O
Cs2CO3, DMF, rt
2 h, 85%
17
Scheme 3. Synthesis of acrylic acid ester derivatives.
appropriate phenyl boronic acid.27 The benzene substituted com-
pound 19 was synthesized using a Friedel–Crafts acylation reaction
between phthalic anhydride and p-t-butyl benzene in the presence
of a Lewis acid (AlCl3).28 The subsequent acid was converted into
the ethyl ester with EtOH in the presence of a catalytic amount
of H2SO4 at 70 °C; activated molecular sieves (MS 4 Å) were used
for removal of water. Cyclopropanation of 13 to 28 was achieved
by the use of dimethylsulfoxonium methylide and trans epoxide
29 was prepared by the epoxidation of the trans olefin 13 with
alkaline hydrogen peroxide.29,30
include the Gram-positive species tested. However, replacement of
the ethyl ester (13) with a carboxylic acid (16) in position D again
destroyed anti-mycobacterial activity although some anti-
staphylococcal activity was retained. In the cis keto compound
12, replacement of the p-t-butyl phenyl group (12) with
a
benzo[b]thiophene moiety (20) in position A increased antibacte-
rial potency for all bacteria tested except Mycobacterium fortuitum
(Table 2). Saturation of the alkene bond in position C of the keto es-
ters (12 and 13) abolished all antibacterial activity (see 14 and 24).
Reduction of the C1 keto function (15) partially restored anti-
mycobacterial activity. However, anti-mycobacterial activity was
again abolished in the unsaturated alkene with the C1 keto group
fully reduced (18). When the alkene was replaced by either ben-
zene (19) or an alkyne (25) activity was abolished. Whereas, the
2.2. Biology
Structure–activity relationship (SAR) studies based on antimi-
crobial activity in a standard minimum inhibitory concentration
(MIC) assay, indicated 42% (10 of 24) of analogs tested showed equal
(3 of 24) or greater (7 of 24) potency than the positive control rifam-
cyclopropyl analog 28 showed decreased potency (MIC = 16 lg/
mL, Table 1) on M. smegmatis and epoxide ester 29 showed
moderate anti-staphylococcal activity but no activity against
mycobacterial species.
pin against M. smegmatis (MIC 6 64 lg/mL, Table 1). Anti-mycobac-
terial activity of 1 was abolished by alteration of position D to a
carboxylic acid (see MIC values for 2 and 3). Anti-mycobacterial
activity of 1 was retained when position D was altered to a cyclo-
propane (4), however, anti-mycobacterial activity was abolished
by the larger prenyl group (17). However, 17 showed potent
Of the compounds active against M. smegmatis, the three most
potent (12, 13, and 20) were also active against both the Gram-
positive bacteria tested (Table 1) as well as the other mycobacterial
species that were tested (Table 2), including M. tuberculosis. In fact,
the in vitro sub-lg/mL anti-mycobacterial activity of 12, 13, and 20
anti-staphylococcal activity (MIC = 0.5
l
g/mL) which is exciting
against M. tuberculosis indicated an increase in potency over the
lead compound 1 of 32- and 64-fold, respectively. Furthermore,
both 12 and 13 were 1.5-fold more active than ethambutol against
M. tuberculosis, whereas 20 was threefold more potent than this
current first line anti-tuberculosis drug.
via another study. An amide in position D either abolished (5, 6, 8,
and 11) or decreased (7, 9, and 10) anti-mycobacterial activity.
At position B, replacement of the sulfur atom with a keto group
(12 and 13) doubled the potency of 1 and extended the activity to

7834
V. V. N. Phani Babu Tiruveedhula et al. / Bioorg. Med. Chem. 21 (2013) 7830–7840
OH
B
O
OH
O
Pd(PPh3)4, Na2CO3
dioxane, 18 h, 105 °C
70%
Br
O
23
O
18
O
O
O
1. AlCl3, 10 °C, CH2Cl2, 30 min
2. conc. H2SO4 (cat), MS 4 Å
O
O
EtOH, 6 h, 70 °C
77%
O
19
S
O
O
Pd/C, H2, rt
O
S
O
EtOH, 12 h
24
1
70%
O
OH
26
Dess-Martin
O
O
CH2Cl2, rt, 2 h
85%
25
O
O
O
O
O
O
trimethyl sulfoxonium iodide
O
O
O
NaH, DMSO, 14 h, rt
60%
13
28
O
O
O
O
NaOH, H2O2
0 °C, 2 h
52%
O
29
O
13
Scheme 4. Synthesis of acrylic acid ethyl ester derivatives.
ester was transformed into the prenyl ester to give the lipophilic
3. Discussion
17, activity against M. smegmatis was completely eliminated,
although 17 was nearly as potent (MIC = 0.5
against S. aureus ATCC29213 as compared to the standard tetracy-
cline (MIC = 0.25 g/mL, Table 1) which is of interest in other stud-
lg/mL, Table 1)
The SAR studies clearly show that the structure of the most
potent compounds, 12, 13, and 20 contain an aromatic ring in area
A with a Michael acceptor scaffold in areas B, C, and D. In order to
study this effect, the saturated analogs (14, 15, 18), prenyl ester 17,
benzene compound 19, keto analog 20, alkyne 25, cyclopropyl
ester 28 and epoxide ester 29 were prepared. Alkyne 25 failed to
behave as a Michael acceptor because of the sp character in area
C as compared to the sp2 character in olefin 13. It is also possible
that the geometry of the molecule plays some role in activity from
sp2 hybridization to sp hybridization. To mimic the double bond
nature of the active compound 13, but limit the Michael acceptor
properties, the olefin in 13 was replaced by the benzene ring in
analog 19, cyclopropyl ring in analog 28 and epoxide ring in analog
29. The benzene analog 19 was inactive, presumably because the
Michael acceptor properties were decreased because of resonance
stabilization. However, the cyclopropyl ethyl ester 28 demon-
strated weak activity similar to the thio ester 1 on M. smegmatis,
whereas epoxide ester 29 showed moderate activity on Staphylo-
l
ies.12 It is not clear why the prenyl ester is not active since it has
been previously demonstrated that hydrophobicity was important
for very potent activity.31 Since mycolic acid surrounds the
mycobacterial cell, it is possible that the prenyl group of olefin
17 adheres to the mycolic acid bilayer and does not penetrate
the cell. Further work to explore this result is required. The methyl
cyclopropyl ester
4 was still active against M. smegmatis
(MIC = 16 g/mL, Table 1), but not active on other strains.
l
It is clear the Michael acceptor property of the active keto
targets 12, 13, and 20 is very important. In support of this, the ac-
rylic ester amides 5–11 were not active, presumably, because the
Michael acceptor properties of the olefin (area B) were decreased.
In modern medicine, many Michael acceptors are employed in the
clinic including several corticosteroids, antibiotics, antiviral and
anticancer drugs.32–39 Some Michael acceptor scaffolds have been
developed by accident to impart structural rigidity, for example,
corticosteroids, while others require Michael acceptors due to the
desired mechanism of action for some chronic diseases, such as
those used in antiviral and anticancer therapies. In drug discovery,
Michael acceptors are used to trap an active intermediate in the
biological cycle. One important component of such an intermediate
coccus aureus ATCC29213 (MIC = 32 lg/mL, Table 1) but no activity
against mycobacterial species. Saturated analogs 14, 15, and 18,
devoid of a keto function, were also prepared to examine the
importance of the Michael acceptor scaffold (area B–C) for activity.
The saturated analogs 14 and 15 were not active and loss of the
ketone in olefin 18 completely eliminated activity. When the ethyl

V. V. N. Phani Babu Tiruveedhula et al. / Bioorg. Med. Chem. 21 (2013) 7830–7840
7835
Figure 2. ORTEP view of the crystal structure of acrylic acid 2.
can be a free thiol. An example of this can be found in cysteine
protease inhibitors, which can be employed to help treat and pre-
vent many diseases including emphysema, stroke, viral infections,
cancer, Alzheimer’s disease, inflammation and arthritis.40–42 Aci-
fran,43 affinin,44 amcinonide,45 betamethasone,46 dexametha-
sone,47 are a few examples of Michael acceptor drugs used
clinically. Rifampin is also a Michael acceptor and, as mentioned,
is one of the current first line drugs in the tuberculosis treatment
regimen. These results suggest that Michael acceptor acrylates
12, 13, and 20 could potentially be developed into viable anti-
mycobacterial agents providing alternatives to current front line
therapies used in the treatment of TB, MRSA, and other less com-
mon infections caused by other Mycobacterium species. In unpub-
lished work, Schwan and co-workers have given mice a 300 mg/
kg dose of 20 and saw no overt toxic effects, showing that such
compounds may not be overtly cytotoxic in vivo. Much work must
be done to follow up these results and gain a clear understanding
of the mechanism of action for these extremely active compounds.
It is important to note that 12, 13, and 20 were not active toward
Escherichia coli or Pseudomonas aeruginosa indicating their mode of
action is not an indiscriminate interaction with bacteria.
compound 1 and threefold increase in potency over ethambutol.
Although the mechanism of action of the acrylic ethyl esters 12,
13, and 20 is not known at this time, experiments are underway
to see which biochemical pathway (if any) in the biogenesis of
TB is disrupted. These simple scaffolds warrant further study to
treat drug resistant antimicrobial strains including those related
to M. tuberculosis.52
These small molecules are easily and inexpensively synthesized,
even in multi-gram quantities, in comparison to other front-line
treatments. Further SAR studies to obtain greater potency, in addi-
tion to elucidation of the mode of action of the active compounds
are ongoing in our laboratories.
5. Experimental
5.1. Chemistry
All reactions were performed in oven-dried round-bottom
flasks under an argon atmosphere unless the reaction conditions
were supposed to contain water. Stainless steel syringes were used
to transfer air-sensitive liquids. Organic solvents were purified
when necessary by standard methods53 or purchased from
Sigma–Aldrich.™ All chemicals purchased from Sigma–Aldrich™
were employed as is, unless stated otherwise in regard to purifica-
tion. Silica gel (Dynamic Adsorbents, 230–400 mesh) for flash chro-
matography was utilized to purify the analogues. The 1H and 13C
NMR data were obtained on Bruker Spectrospin 300 MHz and GE
500 MHz instruments with chemical shifts in d (ppm) reported rel-
ative to TMS. The HRMS and GC/MS spectral data were determined
by the laboratory for mass spectrometry, University of Kansas,
Lawrence, KS 66045-7582, USA. Melting points were taken on a
Stuart melting point apparatus SMP3 manufactured by Barloworld
Scientific US Ltd. X-ray crystallographic studies were performed at
the Naval Research Laboratory, Code 6930, Washington, D. C.
20375, USA.
4. Conclusion
A new series of acrylic acids, including various amides, prenyl
and ethyl esters were synthesized by simple, cheap and efficient
synthetic routes as compared to those agents employed in first-line
therapies for TB, including rifampin.48–50 Due to their simple, un-
ique, and novel scaffold, these analogs have been evaluated and
demonstrated antimicrobial activity against a range of Gram-
positive bacteria including M. smegmatis and the pathogenic M.
tuberculosis. Keto analogs 12, 13, and 20 exhibited the most potent
antimicrobial activity; keto olefins 12 and 13 demonstrated an
eightfold greater activity against M. smegmatis than rifampin, one
of the primary anti-mycobacterial agents currently used to treat
TB (Table 1). Accordingly, 12 and 13 were assayed against other,
more virulent mycobacteria species, including M. tuberculosis. Both
5.2. General method for the synthesis of acids 2 and 3
analogs exhibited an MIC value of 0.8
(Table 2), indicating less potency than isoniazid (MIC = 0.25
Table 2) or rifampin (MIC = <0.03 g/mL, Table 2) but greater
potency than ethambutol (MIC = 1.2 g/mL, Table 2), all three of
which are part of the current first-line drug regimen for TB. Agents
l
g/mL against M. tuberculosis
l
g/mL,
To the ester 1 (0.1 mmol) was added 20% aq KOH (20 mL) and
the mixture was stirred at rt. The reaction progress was monitored
by TLC on a silica gel plate (10% EtOAc in hexane). After 6–8 h the
starting ester had disappeared on TLC and the mixture was cooled
to 0 °C. The acid was precipitated from the solution by addition of
cold aq 5% hydrochloric acid until the pH of the solution reached
1.5–2. The slurry which resulted was allowed to stir for 30 min
and the acid was filtered off under vacuum. The acid was dissolved
in a saturated aq solution of Na2CO3 (10 mL) and the aq layer was
extracted twice with DCM (15 mL) to remove impurities. The aq
layer was cooled to 0 °C while adjusting the pH to 1.5–2. The pure
acid precipitated and was filtered and dried in the air with yields
ranging from 90% to 92%.
l
l
which exhibit MIC values of less than 10 lg/mL are generally
considered clinically significant for further study.51 This activity
may signify a new mechanism of action for these readily available
small molecules. Analog 20 is unique in that it exhibits an MIC va-
lue of 4
than rifampin against this strain and at the same time is very
potent (1 g/mL) against S. aureus ATCC2913. The benzothiophene
analog 20 exhibited excellent activity against M. tuberculosis with
an MIC value of 0.4 g/mL, a 64-fold increase in activity over lead
lg/mL against M. smegmatis with 16-fold greater potency
l
l

7836
V. V. N. Phani Babu Tiruveedhula et al. / Bioorg. Med. Chem. 21 (2013) 7830–7840
5.2.1. (Z)-3-(4-(tert-Butyl)phenyl)thio)acrylic acid (2)
organic layer was dried (Na2SO4) and concentrated under
reduced pressure. Further purification was carried out by flash
column chromatography (silica gel) to yield a pure amide. The
yield was typically 88–94% depending on the amine.
The general method above was followed using ester 1 (215 mg,
0.1 mmol) which yielded 171 mg (92%) of acid 2 as a white
powder. 1H NMR (300 MHz, CDCl3): d 7.47–7.40 (m, 5H), 5.96–
5.93 (d, J = 10.1 Hz, 1H), 1.33 (s, 9H); 13C NMR (75 MHz, CDCl3): d
170.5, 149.1, 146.4, 132.1, 129.7, 125.4, 112.3, 40.7, 31.1. HRMS
(ESI) (M+H)+ calcd for C13H17O2S 237.0949; found 237.0942.
5.4.1. (Z)-3-(Benzo[d]thiazol-2-ylthio)-N,N-
diisopropylacrylamide (5)
The general method above was followed using acid 3 (237 mg,
0.1 mmol) and diisopropylamine (111 mg, 0.11 mmol) yielding
285 mg (89%) of amide 5. 1H NMR (300 MHz, CDCl3): d 8.51 (d,
J = 9.82 Hz, 1H), 8.12 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 7.8 Hz, 2H),
6.37 (d, J = 9.82 Hz, 1H), 3.93 (m, 2H), 1.27 (s, 12H); 13C NMR
(75 MHz, CDCl3): d 161.7, 156.8, 153.0, 145.3, 136.2, 125.3, 124.9,
121.6, 121.3, 116.8, 47.1, 22.3. HRMS (ESI) (M+H)+ calcd for
5.2.2. (Z)-3-(Benzo[d]thiazol-2-ylthio)acrylic acid (3)
The general method above was followed using benzothiazole
acrylic acid ethyl ester (266 mg, 0.1 mmol) which yielded 215 mg
(91%) of acid 3 as an off white powder. 1H NMR (300 MHz, CDCl3):
d 8.27–8.23 (d, J = 9.75 Hz, 1H), 8.12–8.09 (d, J = 7.5 Hz, 1H), 7.99–
7.96 (d, J = 7.95 Hz, 1H), 7.56 (t, J = 6.12 Hz, 1H), 7.47 (t, J = 7.5 Hz,
1H), 6.30–6.27 (d, J = 9.75 Hz, 1H); 13C NMR (75 MHz, CDCl3): d
167.8, 167.6, 152.4, 140.3, 135.4, 127.3, 125.8, 122.7, 121.1,
117.6. HRMS (ESI) (M+H)+ calcd for C10H8NO2S2 237.9996; found
237.9989.
C16H21N2OS2 321.1095; found 321.1088.
5.4.2. (Z)-3-((4-(tert-Butyl)phenyl)thio)-N-phenylacrylamide (6)
The general method above was followed using acid 2 (236 mg,
0.1 mmol) and aniline (102 mg, 0.11 mmol) yielding 289.5 mg
(93%) of amide 6. 1H NMR (300 MHz, CDCl3): d 7.69–7.03 (m,
9H), 7.26 (d, J = 10.17 Hz, 1H), 5.88 (d, J = 10.17 Hz, 1H), 1.29 (s,
9H); 13C NMR (75 MHz, CDCl3): d 169.7, 146.8, 145.1, 134.4,
132.0, 129.6, 129.3, 129.1, 125.6, 125.2, 121.7, 121.1, 112.5, 39.9,
31.4. HRMS (ESI) (M+H)+ calcd for C19H22NOS 312.1422; found
312.1411.
5.3. (Z)-Cyclopropylmethyl 3-((4-(tert-
butyl)phenyl)thio)acrylate (4)
To a stirred suspension of the acid 1 (236 mg, 0.1 mmol) in
DCM (10 mL) was added thionyl chloride (0.1 mL, 0.15 mmol).
The reaction mixture was allowed to heat to reflux for 2 h. The
reaction mixture was cooled to rt and the appropriate alcohol
(0.2 mmol) was added with stirring. The reaction was again
heated at reflux for 2 h. The reaction progress was monitored
by TLC (silica gel). After complete conversion of the starting acid
into the ester, the reaction solution was cooled to 0 °C and water
(5 mL) was added slowly. The reaction mixture was stirred fur-
ther for 15 min and the layers separated. The aq layer was ex-
tracted again with DCM (3 Â 10 mL). The combined organic
extracts were washed with brine (2 Â 15 mL) and this was
followed by cold water (10 mL). The organic layer was dried
(Na2SO4) and concentrated under vacuum to yield the crude es-
ter 4. This material was further purified by flash column chro-
5.4.3. (Z)-3-((4-(tert-Butyl)phenyl)thio)-N-methyl-N-
phenylacrylamide (7)
The general method above was followed using acid 2 (236 mg,
0.1 mmol) and N-methylaniline (118 mg, 0.11 mmol) yielding
296 mg (91%) of amide 7. 1H NMR (300 MHz, CDCl3):
d
7.61-–7.00 (m, 9H), 7.28 (d, J = 10.05 Hz, 1H), 5.90 (d,
J = 10.05 Hz, 1H), 2.77 (s, 3H), 1.29 (s, 9H); 13C NMR (75 MHz,
CDCl3): d 161.7, 148.1, 145.7, 134.2, 132.3, 129.9, 129.4, 129.1,
125.4, 125.1, 121.7, 121.3, 112.6, 39.2, 31.7, 30.2. HRMS (ESI)
(M+H)+ calcd for C20H24NOS 326.1579; found 326.1586.
matography (silica gel). The pure ester
4
(270 mg) was
5.4.4. (Z)-3-((4-(tert-Butyl)phenyl)thio)-N-methylacrylamide (8)
The general method above was followed using acid 2 (236 mg,
0.1 mmol) and N-methylamine (34 mg, 0.11 mmol) yielding
220 mg (88%) of amide 8. 1H NMR (300 MHz, CDCl3): d 7.51–7.35
(m, 4H), 7.28 (d, J = 10 Hz, 1H), 5.90 (d, J = 10 Hz, 1H), 2.84 (s,
3H), 1.29 (s, 9H); 13C NMR (75 MHz, CDCl3): d 163.9, 151.5,
150.4, 133.7, 131.2, 125.1, 114.5, 60.2, 34.5, 27.0. HRMS (ESI)
(M+H)+ calcd for C14H20NOS 250.1266; found 250.1259.
obtained in 93% yield as an oil. 1H NMR (300 MHz, CDCl3): d
7.64–7.61 (m, 4H), 7.58 (d, J = 10.38 Hz, 1H), 6.11 (d,
J = 10.38 Hz, 1H), 4.12 (d, 2H), 1.37 (s, 9H), 0.91–0.89 (m, 1H),
0.38–0.34 (m, 2H), 0.12–0.07 (m, 2H); 13C NMR (75 MHz, CDCl3):
d 168.1, 157.7, 141.4, 132.2, 126.4, 117.9, 114.9, 54.2, 34.2, 28.3,
11.3, 2.7. HRMS (ESI) (M+H)+ calcd for C17H23O2S 291.1419;
found 291.1428.
5.4. General method for the synthesis of amides 5–11
5.4.5. (Z)-3-((4-(tert-Butyl)phenyl)thio)-N,N-
dimethylacrylamide (9)
Acid (2 or 3, 0.1 mmol) and toluene (10 mL) were suspended
in a clean dry flask. The suspension was allowed to stir and
warmed to 60 °C under an inert atmosphere. The CDI (0.178 g,
0.11 mmol) was then added and the mixture allowed to stir
for 15 min which yielded a clear solution. The heating was dis-
continued and the reaction solution was allowed to cool to rt
under an inert atmosphere. The appropriate amine (0.11 mmol)
was dissolved in dry toluene (5 mL) and transferred to the reac-
tion flask. After completion of the addition, the reaction mixture
was stirred for 15 min at rt. The reaction mixture was then
heated to 45–60 °C and this temperature was maintained for
3–6 h. The progress of the reaction was followed using TLC (sil-
ica gel). On completion by analysis of the mixture by TLC, the
reaction mixture was cooled to rt and water (5 mL) was added
slowly. The reaction solution was allowed to stir for 10 min
and then diluted with EtOAc (10 mL). The layers were separated
and the aq layer was extracted with EtOAc (2 Â 5 mL). The com-
bined organic layers were washed with brine (2 Â 15 mL). The
The general method above was followed using acid 2 (236 mg,
0.1 mmol) and dimethylamine (50 mg, 0.11 mmol) which yielded
243.5 mg (92.5%) of amide 9. 1H NMR (300 MHz, CDCl3): d 7.67–
7.28 (m, 4H), 7.28 (d, J = 10.1 Hz, 1H), 5.90 (d, J = 10.1 Hz, 1H),
2.77 (s, 6H), 1.32 (s, 9H); 13C NMR (75 MHz, CDCl3): d 162.6,
151.2, 150.9, 133.3, 131.7, 125.0, 114.6, 60.7, 34.1, 27.6. HRMS
(ESI) (M+H)+ calcd for C15H22NOS 264.1422; found 264.1429.
5.4.6. (Z)-3-((4-(tert-Butyl)phenyl)thio)-N,N-
diisopropylacrylamide (10)
The general method above was followed using acid 2 (236 mg,
0.1 mmol) and diisopropylamine (111 mg, 0.11 mmol) which
yielded 300 mg (94%) of amide 10. 1H NMR (300 MHz, CDCl3): d
7.46–7.39 (m, 4H), d 7.28 (d, J = 10.1 Hz, 1H), 5.90 (d, J = 10.1 Hz,
1H), 3.93 (m, 2H), 1.36 (s, 9H), 1.12 (s, 12H); 13C NMR (75 MHz,
CDCl3): d 161.2, 151.4, 150.9, 132.1, 131.8, 125.3, 114.4, 45.6,
40.7, 34.5, 31.1, 21.4. HRMS (ESI) (M+H)+ calcd for C19H30NOS
320.2048; found 320.2040.

V. V. N. Phani Babu Tiruveedhula et al. / Bioorg. Med. Chem. 21 (2013) 7830–7840
7837
5.4.7. (Z)-3-((4-(tert-Butyl)phenyl)thio)-N-
cyclopropylacrylamide (11)
and trans ester 13 (6.5 mg, 7%). cis Ester 12 1H NMR (300 MHz,
CDCl3): d 7.89 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 6.89 (d,
J = 12.3 Hz, 1H), 6.27 (d, J = 12 Hz, 1H), 4.06 (q, J = 7.2 Hz, 2H),
1.35 (s, 9H), 1.08 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3): d
189.1, 165.7, 157.8, 136.6, 134.1, 132.2, 128.9, 125.9, 61.3, 35.3,
31.3, 14.2. HRMS (ESI) (M+Na)+, calcd for C16H20O3Na 283.1310;
found 283.1334. trans Ester 13 1H NMR (300 MHz, CDCl3): d 7.96
(d, J = 8.4 Hz, 2H), 7.93 (d, J = 15.6 Hz, 1H), 7.55 (d, J = 8.4 Hz, 2H),
6.90 (d, J = 15.6 Hz, 1H), 4.32 (q, J = 7.2 Hz, 2H), 1.38 (m, 12H);
13C NMR (75 MHz, CDCl3): d 189.0, 165.6, 157.8, 136.6, 134.1,
132.2, 128.9, 125.8, 61.3, 35.2, 31.0, 14.2. HRMS (ESI) (M+Na)+,
The general method above was followed using acid 2 (236 mg,
0.1 mmol) and cyclopropylamine (63 mg, 0.11 mmol) which
yielded 248 mg (90%) of amide 11. 1H NMR (300 MHz, CDCl3): d
8.07 (s, 1H), 7.46–7.39 (m, 4H), 7.28 (d, J = 10 Hz, 1H), 5.90 (d,
J = 10 Hz, 1H), 2.32 (m, 1H), 1.56 (s, 9H), 0.38–0.34 (m, 2H), 0.12–
0.07 (m, 2H); 13C NMR (75 MHz, CDCl3): d 165.4, 146.8, 145.1,
132.0, 129.5, 129.1, 116.7, 40.6, 31.4, 24.2, 7.4. HRMS (ESI)
(M+H)+ calcd for C16H22NOS 276.1422; found 276.1424.
5.5. General method for the preparation of propargylic alcohols
26 and 27
calcd for C16H20O3Na 283.1310; found 283.1348. When the
mixture of 12 and 13 was stirred with anhydrous HCl(g) in ether
it was completely converted into trans 13 with no formation of
the corresponding acid 16.
A round bottom flask was charged with anhydrous THF (5 mL)
and propynoic acid ethyl ester 21 (100 mg, 1.02 mmol) after which
it was cooled to À78 °C. Then n-BuLi (1.6 M of 0.8 mL, 1.22 mmol)
was added dropwise. After the addition the mixture which resulted
was stirred for 5 min and the appropriate aldehyde (1.02 mmol)
was added slowly. The solution which resulted was stirred for
1 h at À78 °C and then allowed to warm to rt. The reaction mixture
was then quenched with a saturated aq solution of NH4Cl, ex-
tracted with EtOAc (2 Â 10 mL) and then washed with brine. The
combined organic extracts were dried (Na2SO4) and concentrated
under reduced pressure. The crude oil was purified by silica gel
flash column chromatography (10–20% EtOAc in hexanes) to yield
pure alcohols.
5.6.1. (Z)-Ethyl 4-(benzo[b]thiophen-2-yl)-4-oxobut-2-enoate
(20)
The procedure (Section 5.6) was followed. The mixture of
4-benzo[b]thiophen-2-yl-4-hydroxy-but-2-ynoic acid ethyl ester
27 (157 mg, 0.6031 mmol), a 0.01 M solution of hydroquinone in
DMSO (0.6 mL, 0.0060 mmol) and NaHCO3 (10 mg, 0.1206 mmol)
in DMSO: H2O (8:1; 2 mL) was allowed to stir. Flash column chro-
matography on silica gel (2% EtOAc in hexane) provided enoate 20
(94 mg, 60% yield). 1H NMR (300 MHz, CDCl3): d 7.89 (m, 3H), 7.47
(m, 2H), 6.95 (d, J = 12.3 Hz, 1H), 6.36 (d, J = 12 Hz, 1H), 4.12 (q,
J = 6.9 Hz, 2H), 1.14 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d 187.2, 164.8, 143.0, 142.8, 138.9, 138.3, 131.0, 127.8, 127.6,
126.2, 125.3, 123.1, 61.3, 13.8. HRMS (ESI) (M+Na)+, calcd for C14-
H12O3SNa 283.0405; found 283.0432.
5.5.1. Ethyl 4-(4-(tert-butyl)phenyl)-4-hydroxybut-2-ynoate
(26)
The general method above was followed using t-butyl
benzaldehyde (0.17 mL, 1.02 mmol) which yielded 169 mg (60%)
of alcohol 26. 1H NMR (300 MHz, CDCl3): d 7.44 (m, 4H), 5.57 (s,
1H), 4.24 (q, J = 6.9 Hz, 2H), 1.33 (m, 12H); 13C NMR (75 MHz,
CDCl3): d 153.3, 152.2, 135.6, 126.5, 125.9, 86.04, 77.2, 64.2, 62.3,
34.7, 31.3, 14.0. HRMS (ESI) (M+Na)+, calcd for C16H20O3Na
283.1310; found 283.1309.
5.7. Ethyl 4-(4-(tert-butyl)phenyl)-4-oxobut-2-ynoate (25)
To alcohol 26 (0.5 g, 1.8 mmol) in dry CH2Cl2 (10 mL) was added
the Dess–Martin periodinane reagent (0.77 g, 1.8 mmol) at rt and
the reaction mixture was allowed to stir for 2 h. The volume of
the reaction mixture was increased by the addition of CH2Cl2
(10 mL). An aq solution (20 mL) containing sodium thiosulfate
(100 g/L) and sodium bicarbonate (100 g/L) was added and the
mixture which resulted was allowed to stir for 10 min The organic
phase was separated and washed with H2O (30 mL) and dried
(Na2SO4). The solvent was removed in vacuo. The residue was puri-
fied by flash column chromatography on silica gel (10% ethyl ace-
tate in hexane) to afford ketone 25 (0.39 g, 85%). 1H NMR
(300 MHz, CDCl3): d 8.07 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.4 Hz,
2H), 4.37 (q, J = 7.2 Hz, 2H), 1.39 (m, 12H); 13C NMR (75 MHz,
CDCl3): d 175.8, 159.4, 152.4, 133.2, 129.8, 125.9, 80.1, 80.0, 62.9,
35.4, 31.0, 13.9. HRMS (EI) (M)+, calcd for C16H18O3 258.1256;
found 258.1243.
5.5.2. Ethyl 4-(4-(benzo[b]thiophen-2-yl)-4-hydroxybut-2-
ynoate (27)
The general method above was followed using benzothiophene-
2-carboxaldehyde (165 mg, 1.02 mmol) which yielded 154 mg
(58%) of alcohol 27. 1H NMR (300 MHz, CDCl3): d 7.77 (m, 2H),
7.40 (s, 1H), 7.36 (m, 2H), 5.85 (d, J = 5.7 Hz, 1H), 4.28 (q,
J = 7.2 Hz, 2H), 1.33 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3):
d 155.8, 142.4, 140.1, 139.0, 125.1, 124.7, 124.4, 124.1, 122.8,
84.2, 77.9, 62.5, 60.9, 14.0. HRMS (ESI) (M+ H)+, calcd for
C14H13O3S 283.0585; found 283.0572.
5.6. The method for the preparation of enones 12 and 13
5.8. Ethyl 4-(4-(tert-butyl)phenyl)-4-oxobutanoate (14)
A round bottom flask was charged with propargylic alcohol 26
(100 mg, 0.3618 mmol), DMSO: H2O (8:1; 1.25 mL) and then
a solution of 0.01 M of hydroquinone in DMSO (0.36 mL,
0.0036 mmol) was added at 23 °C. Subsequently, solid NaHCO3
(6 mg, 0.0723 mmol) was added in one portion. After the addition
the solution which resulted was stirred for 18 h at 23 °C. The reac-
tion mixture was then diluted with H2O and brought to pH 3 [to
obtain pH 3 the phosphate buffer which was employed was pH
7.2 phosphate buffer and an aq solution of HCl (the solution of
1 N HCl was used to reduce the pH 7.2 to pH 3)]. The solution
which resulted was extracted with diethyl ether (2 Â 10 mL) and
the ether layer washed with brine. The combined organic extracts
were dried (Na2SO4) and concentrated under reduced pressure. The
crude oil was purified by silica gel flash column chromatography
(5% EtOAc in hexanes) to afford pure cis ester 12 (59 mg, 63%)
The trans ester 13 (40 mg, 0.153 mmol) was dissolved in
acetone (5 mL) and cold 20% TiCl3 solution (0.15 ml, 0.306 mmol)
was added dropwise with a syringe and the mixture was allowed
to stir for 10 min at rt. The solution was then poured into brine
(20 mL) and extracted with diethyl ether (2 Â 10 mL). The
combined extracts were dried (Na2SO4) and the solvent removed
under reduced pressure. The crude oil was purified by silica gel
flash column chromatography (10% EtOAc in hexanes) to afford
saturated analog 14 (34 mg, 85%). 1H NMR (300 MHz, CDCl3): d
7.95 (d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 4.18 (q, J = 7.2 Hz,
2H), 3.31 (t, J = 6.9 Hz, 2H), 2.77 (t, J = 6.6 Hz, 2H), 1.36 (s, 9H),
1.28 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3): d 197.8, 173.0,
156.9, 134.0, 128.0, 125.5, 60.6, 35.1, 33.2, 31.1, 28.3, 14.2. HRMS
(ESI) (M+H)+, calcd for C16H23O3 263.1647; found 263.1631.

7838
V. V. N. Phani Babu Tiruveedhula et al. / Bioorg. Med. Chem. 21 (2013) 7830–7840
5.9. Ethyl 4-(4-(tert-butyl)phenyl)butanoate (15)
tetrakis (300 mg, 0.26 mmol) was added to the round bottom flask.
The flask was then evacuated three times at rt and backfilled with
argon. The reaction mixture was allowed to stir for 15 min at rt
then phenyl boronic acid (1.84 g, 10.36 mmol) and Na2CO3
(2.75 g, 25.9 mmol) were added under a positive pressure of argon.
The reaction mixture which resulted was heated at reflux for 18 h
and then cooled to rt. It was then passed through a short bed of cel-
ite. The celite bed was washed with EtOAc (50 mL) and the com-
bined organic layers were washed with water (50 mL) and brine
(30 mL). The organic layer was dried (Na2SO4) and concentrated
under reduced pressure. The crude oil was purified by flash column
chromatography (5% EtOAc in hexanes) on silica gel to afford ester
18 (893 mg, 70%). 1H NMR (300 MHz, CDCl3): d 7.37 (d, J = 8.1 Hz,
2H), 7.14 (d, J = 7.8 Hz, 2H), 7.12 (d, J = 15.6 Hz, 1H), 5.85 (d,
J = 15.3 Hz, 1H), 4.21 (q, J = 7.2 Hz, 2H), 3.52 (d, J = 6.9 Hz, 2H),
1.35 (S, 9H), 1.31 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3): d
166.5, 149.6, 147.5, 134.7, 128.5, 125.6, 122.2, 60.2, 38.0, 34.4,
31.4, 14.3. HRMS (ESI) (M+Na)+, calcd for C16H22O2Na 269.1518;
found 269.1512.
A Parr hydrogenation bottle (50 mL) was charged with dry Pd/C
(10% by wt, 400 mg, 0.38 mmol) and the trans ester 13 (100 mg,
0.38 mmol) in ethanol (3 mL) was added. The mixture was
degassed under reduced pressure at rt and back filled with H2 (3
times) and then flushed with H2 and pressurized to the desired
pressure (20 psi) and stirred with H2 overnight at rt. The catalyst
was removed by filtration (celite) and the solid which remained
was washed with ethanol (3 Â 10 mL). The combined organic
layers were concentrated under reduced pressure to give an oil.
This oil was purified by silica gel flash column chromatography
(2% EtOAc in hexanes) to afford ester 15 (76 mg, 80%). 1H NMR
(300 MHz, CDCl3): d 7.33 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 8.1 Hz,
2H), 4.14 (q, J = 7.2 Hz, 2H), 2.64 (t, J = 7.5 Hz, 2H), 2.35 (t,
J = 7.5 Hz, 2H), 1.97 (m, 2H), 1.33 (s, 9H), 1.27 (t, J = 7.2 Hz, 3H);
13C NMR (75 MHz, CDCl3): d 173.6, 148.7, 138.3, 128.1, 125.3,
60.2, 34.6, 34.4, 33.8, 31.4, 26.5, 14.2. HRMS (ESI) (M+Na)+, calcd
for C16H24O2Na 271.1674; found 271.1657.
5.10. (E)-4-(4-(tert-Butyl)phenyl)-4-oxobut-2-enoic acid (16)
5.13. Ethyl 2-(4-(tert-butyl)benzoyl)benzoate (19)
To the trans ester 13 (105 mg, 0.4 mmol) in CH3OH (5 mL) was
added K2CO3 (0.279 mg, 2 mmol) in H2O (5 mL). The reaction
mixture was allowed to reflux for 5 h and then the CH3OH was
removed under reduced pressure. The residue was then cooled to
0 °C and brought to pH 2 with a solution of cold aq HCl (1 M).
The mixture, which resulted, was extracted with diethyl ether
(2 Â 15 mL). The combined extracts were washed with brine
(20 mL), dried (Na2SO4) and concentrated under reduced pressure
to furnish a pale green solid acid 16 (84.3 mg, 90%). Mp 118–
121 °C. 1H NMR (300 MHz, CDCl3): d 8.03 (d, J = 15.6 Hz, 1H),
7.98 (d, J = 7.97 Hz, 2H), 7.56 (d, J = 7.97 Hz, 2H), 6.91 (d,
J = 15.3 Hz, 2H), 1.38 (S, 9H); 13C NMR (75 MHz, CDCl3): d 188.8,
170.5, 158.2, 138.6, 133.8, 131.1, 128.9, 125.9, 35.3, 31.0. HRMS
(ESI) (M+H)+, calcd for C14H17O3 233.1178; found 233.1155. This
material was employed directly in the next experiment.
A round bottom flask was charged with tert-butyl-benzene
(1.04 mL, 6.7 mmol), phthalic anhydride (1 g, 6.7 mmol), anhy-
drous CH2Cl2 (10 mL) and cooled to 10 °C. Then AlCl3 (1.8 g,
13.4 mmol) was added portionwise under a positive pressure of
argon. The reaction mixture which resulted was then stirred for
15 min at 10 °C. The mixture was poured into an excess of ice-
water (100 mL) and the aq phase was extracted with CH2Cl2
(2 Â 30 mL). It was then washed with brine (50 mL). The organic
layer was dried (Na2SO4) and concentrated under reduced pressure
to furnish an acid (1.62 g, 85%) intermediate. The acid was then
dissolved in EtOH (10 mL) and a catalytic amount of H2SO4 as well
as activated MS (4 Å) were added. The mixture was allowed to stir
for 6 h. The EtOH was removed under reduced pressure and the
residue dissolved in EtOAc (30 mL). This organic solution was
washed with H2O (50 mL), brine (50 mL), dried (Na2SO4) and
concentrated under reduced pressure. The crude oil was purified
by silica gel flash column chromatography (10% EtOAc in hexanes)
to afford the benzoate 19 (1.9 g, 90%). 1H NMR (300 MHz, CDCl3): d
8.06 (m, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.58 (m, 2H), 7.45 (d,
J = 8.4 Hz, 2H), 7.38 (m, 1H), 4.10 (q, J = 6.9 Hz, 2H), 1.33 (S, 9H),
1.06 (t, J = 7.2 Hz, 3H); 13C NMR (75 MHz, CDCl3): d 196.7, 166.0,
156.9, 142.0, 134.6, 132.2, 130.1, 129.4, 127.7, 125.4, 61.4, 35.1,
31.1, 13.6. HRMS (ESI) (M+H)+, calcd for C20H23O3 311.1647; found
311.1649.
5.11. (E)-3-Methylbut-2-en-1-yl 4-(4-(tert-butyl)phenyl)-4-
oxobut-2-enoate (17)
The acid 16 (70 mg, 0.3 mmol) was dissolved in anhydrous DMF
(1 mL) and cesium carbonate (200 mg, 0.6 mmol) and KI (50 mg,
0.3 mmol) were added. The mixture which resulted was stirred
for 5 to 10 min at rt and then a solution of 1-bromo-3-methyl-
but-2-ene (22) in DMF (0.5 mL) was added with a syringe under
a positive pressure of argon. After the addition the mixture, which
resulted, was stirred for 2 h at rt. The reaction mixture was then
quenched with H2O and extracted with diethyl ether (2 Â 10 mL)
as well as washed with brine (2 Â 30 mL). The combined organic
extracts were dried (Na2SO4) and concentrated under reduced
pressure. The crude oil was purified by flash column chromatogra-
phy (5% EtOAc in hexanes) on silica gel to afford prenyl ester 17
(76.5 mg, 85%). 1H NMR (300 MHz, CDCl3): d 7.96 (d, J = 7.5 Hz,
2H), 7.93 (d, J = 15.3 Hz, 1H), 7.54 (d, J = 8.7 Hz, 2H), 6.90 (d,
J = 15.6 Hz, 1H), 5.43 (t, J = 7.5 Hz, 1H), 4.76 (d, J = 7.5 Hz, 2H),
1.79 (d, J = 9.9 Hz, 6H), 1.37 (S, 9H); 13C NMR (75 MHz, CDCl3): d
189.1, 165.7, 157.8, 139.9, 136.6, 134.1, 132.2, 128.9, 125.9,
118.1, 62.2, 35.3, 31.0, 25.8, 18.1. HRMS (ESI) (M+Na)+, calcd for
C19H24O3Na 323.1623; found 323.1627.
5.14. 3-(4-tert-Butyl-phenylsulfanyl)-propionic acid ethyl ester
(24)
The Parr hydrogenation bottle (500 mL) was charged with dry
Pd/C (10% by wt, 400 mg, 0.38 mmol), and thio ester 1 (100 mg,
0.38 mmol) in ethanol (3 mL).The mixture which resulted was
degassed under reduced pressure at rt and back filled with H2 (3
times) and then flushed with H2. It was pressurized to the desired
pressure (20 psi) with H2 and stirred overnight at rt. The catalyst
was removed by filtration (celite) and washed with ethanol
(3 Â 10 mL). The solvent was removed under reduced pressure.
The crude compound was purified by flash column chromatogra-
phy (2% EtOAc in hexanes) on silica gel to afford ester 24 (70 mg,
70%). 1H NMR (300 MHz, CDCl3): d 7.34 (s, 4H), 4.15 (q, J = 7.2 Hz,
2H), 3.15 (t, J = 7.5 Hz, 2H), 2.63 (t, J = 7.5 Hz, 2H), 1.33 (s, 9H),
1.27 (t, J = 6.9 Hz, 3H); 13C NMR (75 MHz, CDCl3): d 171.9, 150.0,
131.5, 130.5, 126.1, 60.7, 34.6, 34.5, 31.3, 29.5, 14.2. HRMS (ESI)
(M+Na)+, calcd for C15H22O2SNa 289.1238; found 289.1227.
5.12. (E)-Ethyl 4-(4-(tert-butyl)phenyl)but-2-enoate (18)
To a solution of ethyl 4-bromocrotonate 23 (0.9 mL, 5.18 mmol)
in anhydrous dioxane (10 mL), palladium triphenyl phosphine

V. V. N. Phani Babu Tiruveedhula et al. / Bioorg. Med. Chem. 21 (2013) 7830–7840
7839
5.15. (1R,2R)-Ethyl 2-(4-(tert-
butyl)benzoyl)cyclopropanecarboxylate (28)
0.1% casitone, 5.6
and 4 g/mL catalase) broth were used. The compounds were
diluted in assay media to 2Â the final test concentration and
25 L of these diluted compounds were transferred to 384-well
lg/mL palmitate, 0.5% bovine serum albumin
l
Trimethylsulfoxonium iodide (40 mg, 0.18 mmol) was added
portionwise to a slurry of sodium hydride (5 mg, 0.2 mmol) in
DMSO (1 mL). The mixture was stirred at rt until a completely clear
solution was obtained. The ester 13 was added dropwise and the
reaction mixture was then stirred for 14 h at rt. After completion,
the reaction mixture was poured on crushed ice (50 g) and the oily
product extracted with diethyl ether (2 Â 10 mL). The combined
organic extracts were washed with brine (2 Â 15 mL). The organic
layer was dried over MgSO4 and concentrated under vacuum to
yield the crude cyclopropyl ester. The crude compound was puri-
fied by flash column chromatography (5% EtOAc in hexanes) on sil-
ica gel to afford trans cyclopropyl ester 28 (25.2 mg, 60%). 1H NMR
(300 MHz, CDCl3): d 7.99 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H),
4.20 (q, J = 7.2 Hz, 2H), 3.20 (ddd, J = 5.7, 5.7, 9.3 Hz, 1H), 2.37 (ddd,
J = 5.7, 5.7, 9.6 Hz, 1H), 1.57 (m, 2H), 1.37 (s, 9H), 1.31 (t, J = 7.5 Hz,
3H); 13C NMR (75 MHz, CDCl3): d 196.6, 172.5, 157.2, 134.5, 128.3,
125.6, 61.1, 35.2, 31.1, 25.9, 24.5, 17.7, 14.2. HRMS (ESI) (M+H)+,
calcd for C17H23O3 275.1647; found 275.1639.
l
plates. Amikacin was included in the positive control wells in every
assay plate. Plates containing test compounds and positive control
compounds were transferred into the BSL3 facility for bacteria
addition and incubation. The bacterial stock was diluted to 1-
2 Â 105 CFU/mL in the assay medium, Middlebrook 7H12 broth
and 25 lL was plated over the compounds using a Thermo Scien-
tific Matrix WellMate, inside a Class 2A Biological Safety Cabinet.
Positive and negative control wells were included in each plate.
Plates were placed in stacks of two and incubated for 7 days at
37 °C with approximately 95% humidity. After 7 days of incubation,
autofluorescence of any test compounds was determined by
pre-reading the high dose plate by a bottom read for fluorescence
using a Perkin Elmer Envision plate reader at 535 nm excitation
and 590 nm emission. The assay plates were removed from the
incubator and allowed to equilibrate to room temperature.
Twenty-five microliters of Promega BacTiter-Glo™ Microbial Cell
Viability (BTG) reagent, one third of the final volume of the well,
was added using a WellMate. The plates were incubated for
20 min at room temperature, sealed with a Perkin Elmer clear Top-
Seal A and read from the top using luminescence on a Perkin Elmer
Envision. Rifampin, ethambutol, amikacin, isoniazid, and pyri-
methamine were used as positive controls.
5.16. (2R,3R)-Ethyl 3-(4-(tert-butyl)benzoyl)oxirane-2-
carboxylate (29)
A 6 N NaOH (0.1 mL) solution was added dropwise into a solu-
tion of 13 (100 mg, 0.38 mmol) and 30% H2O2 (0.05 mL) in EtOH
(5 mL) at 0 °C. The reaction mixture which resulted, was stirred
for 2 h at the same temperature after which water was added to
the reaction mixture and it was extracted with CH2Cl2
(2 Â 10 mL). The combined organic extracts were washed with
brine (2 Â 15 mL). The organic layer was dried (MgSO4) and con-
centrated under vacuum to yield the crude epoxide ethyl ester.
The crude compound was purified by flash column chromatogra-
phy (10% EtOAc in hexanes) on silica gel to afford epoxide ethyl es-
ter 29 (55 mg, 52%). 1H NMR (300 MHz, CDCl3): d 7.99 (d, J = 8.4 Hz,
2H), 7.55 (d, J = 8.4 Hz, 2H), 4.46 (d, J = 1.8 Hz, 1H), 4.32 (m, 2H),
3.70 (d, J = 1.8 Hz, 1H), 1.38 (m, 12H); 13C NMR (75 MHz, CDCl3):
d 191.3, 167.31, 158.5, 132.5, 128.6, 126.0, 62.3, 55.2, 53.0, 35.3,
31.0, 14.1. HRMS (ESI) (M+Na)+, calcd for C16H20O4Na 299.1259;
found 299.1248.
Acknowledgments
We wish to acknowledge financial assistance from the Research
Growth Initiative of the University of Wisconsin-Milwaukee
(J.M.C.). This work was supported in part by the National Institutes
of Health and the National Institute of Allergy and Infectious
Diseases, Contract No. HHSN272201100012I. X-ray crystallo-
graphic studies were carried out at the Naval Research Laboratory
and supported by NIDA-NRL Interagency Agreement Number Y1-
DA1101.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.10.011.
5.17. Microbiology
References and notes
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