C4-Alkylthiols with activity against Moraxella catarrhalis and Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.bmc.2011.09.030

Antimicrobial resistance represents a global threat to healthcare. The ability to adequately treat infectious diseases is increasingly under siege due to the emergence of drug-resistant microorganisms. New approaches to drug development are especially nee

Full text of DOI:10.1016/j.bmc.2011.09.030

Bioorganic & Medicinal Chemistry 19 (2011) 6842–6852
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
C4-Alkylthiols with activity against Moraxella catarrhalis
and Mycobacterium tuberculosis
Maya B. Kostova a, , Carey J. Myers a,à, Tim N. Beck a, Balbina J. Plotkin b, Jacalyn M. Green c,
Helena I.M. Boshoff d, Clifton E. Barry III d, Jeffrey R. Deschamps e, Monika I. Konaklieva a,
⇑
a Department of Chemistry, American University, Washington, DC 20016, USA
b Department of Microbiology and Immunology, Midwestern University, Downers Grove, IL 60515, USA
c Department of Biochemistry, Midwestern University, Downers Grove, IL 60515, USA
d Tuberculosis Research Section, LCID, NIAID, NIH, 33 North Drive, Bldg 33, Rm 2W20C, Bethesda, MD 20892, USA
e Naval Research Laboratory, Code 6930, 4555 Overlook Ave., 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:
Received 4 July 2011
Revised 11 September 2011
Accepted 19 September 2011
Available online 1 October 2011
Antimicrobial resistance represents a global threat to healthcare. The ability to adequately treat infec-
tious diseases is increasingly under siege due to the emergence of drug-resistant microorganisms. New
approaches to drug development are especially needed to target organisms that exhibit broad antibiotic
resistance due to expression of b-lactamases which is the most common mechanism by which bacteria
become resistant to b-lactam antibiotics. We designed and synthesized 20 novel monocyclic b-lactams
with alkyl- and aryl-thio moieties at C4, and subsequently tested these for antibacterial activity.
These compounds demonstrated intrinsic activity against serine b-lactamase producing Mycobacterium
tuberculosis wild type strain (Mtb) and multiple (n = 6) b-lactamase producing Moraxella catarrhalis
clinical isolates.
Keywords:
Mycobacterium tuberculosis
Moraxella catarrhalis
b-Lactams
Ó 2011 Elsevier Ltd. All rights reserved.
Antimicrobial resistance
1. Introduction
To address the growing need for more antimicrobials, new types
of b-lactam pharmacophores consisting of a series of C4-substi-
Discovered in 1928 by Fleming, penicillin was the first of the b-
lactam class of antibiotics which now includes members that are
natural, semi-synthetic and synthetic. Mechanistically, these com-
pounds act by inhibiting cell wall growth, thus cell division. This
occurs via inhibition of transpeptidases, otherwise referred to as
penicillin-binding proteins, which have been shown to catalyze
several reactions in the cross-linking of peptidoglycan strands.1 Be-
cause humans lack peptidoglycan, the selective toxicity of this
class of antibiotics is high and the compounds are generally safe
for humans. Unfortunately, bacteria now have several mechanisms
of resistance, including modification of penicillin-binding proteins
and expression of b-lactamases, which are enzymes that hydrolyt-
ically cleave the b-lactam ring thus inactivating the drug.1 Produc-
tion of b-lactamase is the most common mechanism by which
bacteria become resistant to b-lactam antibiotics. This is why the
development of new b-lactamase-resistant antimicrobials is
essential.
tuted monocyclic b-lactam compounds, were prepared in our lab-
oratories and tested against a range of bacteria including species
that elaborate b-lactamase. The design, synthesis and antibacterial
activities of these novel lactams are reported here.
2. Results and discussion
2.1. Design and synthesis
b-Lactams have long been known to be effective bactericidal
agents. In general, b-lactams are thought to exert their antibacte-
rial activity by specifically acylating enzymes containing serine
or cysteine as active-site nucleophiles. Until recently, it was gener-
ally accepted that for b-lactams to exert bactericidal activity they
must contain a scaffold which specifically has an ionizable group
at the lactam nitrogen within 3.6 Å of the b-lactam carbonyl car-
bon. However, there now appear to be exceptions to this scaffold
requirement since N-methylthiolated b-lactams possess inhibitory,
although not cidal, antimicrobial activity.2 In addition, it has been
demonstrated that essential bacterial serine or cysteine enzymes
can be inhibited by the positioning of leaving groups at C4, such
as those found in monocyclic b-lactams.3–6 Still others have shown
that hydrolysis of the b-lactam ring of the cephalosporins generate
⇑
Corresponding author. Tel.: +1 202 885 1777; fax: +1 202 885 1752.
E-mail address: mkonak@american.edu (M.I. Konaklieva).
Present address: Division of Chemical Therapeutics, The Sidney Kimmel
Comprehensive Cancer Center at Johns Hopkins, Baltimore, MD 21231, USA.
à
Present address: Jefferson College of Graduate Studies, Thomas Jefferson
University, Philadelphia, PA, USA.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.09.030

M. B. Kostova et al. / Bioorg. Med. Chem. 19 (2011) 6842–6852
6843
OAc
SAc
thiol intermediates that appear to specifically inhibit Bacillus cereus
SAc
H
metallo-b-lactamase.7 In addition, compounds carrying a free thiol
have been prepared as both serine and metallo-b-lactamase
inhibitors.8
HSAc
a
b
N
N
N
SO3H
O
O
O
H
2
1
3
The focus of this study was to design, synthesize and test novel
thio-b-lactams incorporating the knowledge gained by these previ-
ous studies. Towards this end, we prepared and determined the
antibacterial activity of monocyclic b-lactams having alkylthioe-
ther-, and arylthioether groups, thioacetate, and a variety of differ-
ent disulfide groups attached to the lactam’s C4.
Scheme 2. Synthesis of C4 thioacetyl b-lactams. Reagents and conditions: (a)
NaHCO3 in acetone/water, rt, 12 h, (2) 60%; (b) PySO3/Py, inert atmosphere, 80 °C,
2 h, (3) 26%.
OAc
H
SR
H
a
2.2. b-Lactams with alkyl sulfides/thiols at C4
N
N
HSR
O
O
The C3 unsubstituted b-lactams were prepared from commer-
cially available b-lactam 1 following the procedures of Clauss
et al.9 and Wasserman et al.,10 where the nucleophile (Nu) is a thiol
group (Scheme 1). b-Lactams unsubstituted at C3 were first syn-
thesized to test the hypothesis that a lack of substituent at C3
would generate a compound capable of inhibiting bacterial growth.
We then hypothesized that the thioacetate group could be
transformed into a free thiol, upon enzymatic hydrolysis by the
bacteria, thus, converting the pro-drug into an active antibacterial
compound. Thus the synthesis of b-lactam 3 (Scheme 2) was
performed.
The synthesis of S-acetyl b-lactam 2 has been previously de-
scribed by Clauss et al.9 using different multistep synthetic routes.
For our synthesis, commercially available 4-acetoxy-2-azetidinone
1 was used as the starting material to produce b-lactam 2 in a sin-
gle step. In the presence of thiolacetic acid the acetate group is re-
placed by a thioacetate to yield lactam 2 using the methods
described previously.10 b-Lactam 3 (Scheme 2) was obtained from
azetidinone 2 using a PySO3 complex in pyridine.
1
4
5
R = (CH2)2OH
R = (CH2)3OH
Scheme 3. Synthesis of unbranched alkylthio b-lactams. Reagents and conditions:
(a) NaHCO3 in acetone/water, rt, 12 h, (4, 5) 25%.
O
O
O
a
b
HO
S
S
8
HS
OH
6
7
SH
OAc
H
S
H
HO
N
N
c
O
O
9
1
Scheme 4. Synthesis of branched alkyl-thio b-lactams. Reagents and conditions: (a)
S2Cl2/CCl4, 55 °C, (7) 50%; (b) LAH, THF, (8) 70%; (c) NaHCO3 in acetone/water, rt,
12 h, (9) 60%.
Next unbranched C4 alkylthio-b-lactams were synthesized from
lactam 1 and commercially available ethyl and propyl alkylthiols
(Scheme 3).
To determine whether the branching of the sulfur substituent at
C4 had an effect on biological activity, we also synthesized
branched C4 alkylthio-b-lactams 9 and thiolalcohol 8 (Scheme 4).
The latter was prepared as described previously.14 Dialdehyde 7
was prepared from commercially available 2-ethylbutanal 6 fol-
lowing the procedure suggested by Roy et al.,15 where S2Cl2 was
added dropwise at 55 °C to a solution of the aldehyde 6 (Scheme 4).
Reduction of the latter with LiAlH4 in tetrahydrofuran (THF) led to
formation of thioalcohol 8. b-Lactam 9 (Scheme 4) was prepared
from 1, using the methods of Ghannoum et al.16
In addition to production of b-lactamases, many bacterial spe-
cies produce extracellular polysaccharide matrices (biofilm) which
protect the bacterial population from environmental assaults
including antibiotics.11 Compounds with bismuth-coordinated thi-
ols have been reported to possess potent antibacterial activity
against biofilm-producing bacteria.11–14 Synthesis of bismuth-
coordinated thiols is straightforward, requiring addition of bis-
muth nitrate to the thiol of interest in 1,2-propanediol.14,17 Based
on published studies of model compounds, the following struc-
tures are suggested for the investigated bismuth-coordinated C4
thio-b-lactams (Fig. 1).18,19
O
NH
O
S
O
Bi
S
O
S
S
Bi
O
NH
O
HN
O
HN
O
9
9
Figure 1. Suggested structures of bismuth-coordinated C4 thio-b-lactam 10.
attached at C4 through a one-carbon spacer were prepared. It has
been reported that a sulbactam analog possessing a –CH2CO2H
group at C3 exhibited a 10-fold improved inhibitory activity
against class C b-lactamases when compared to the parent com-
pound sulbactam.3 Following the synthetic procedure depicted be-
low, the synthesis of compounds 16, 17 and 18 was accomplished.
The overall design of the substituted b-lactams with sterically
hindered thiols is shown in Scheme 5. Aliphatic aldehyde 6 was re-
acted with sulfur monochloride to give disulfide-dialdehyde 7.15
For the synthesis of the imines 13 and 14, glycine ethyl and methyl
esters as hydrochloride salts were used. A procedure for prepara-
tion of the imines directly from the commercially available hydro-
chloride salts of the glycine esters was developed. This resulted in
successful synthesis of bis-imine 11 and monoimines 13 and 14
We next synthesized C3-substituted lactams. C3-Methoxy-
substituted b-lactams having branched alkylthio-substituents
OAc
Nu
base
Nu
N
N
N
acetone/ water
O
O
H
O
H
1
Scheme 1. Synthesis of C3 unsubstituted alkylthio-b-lactams.

6844
M. B. Kostova et al. / Bioorg. Med. Chem. 19 (2011) 6842–6852
O
O
O
N
N
11
O
O
O
N
O
O
O
O
d
a
b
NH2.HCl
RO
S
N
S
c
HS
S
S
O
12
6
7
O
OR
13 R = CH3CH2
14 R = CH3
S
S
O
O
MeO
O
S
S
OMe
N
N
O
O
N
N
15
O
O
e
S
S
MeO
SH
O
11
OEt
EtO
N
O
O
N
O
f
O
OR
S
S
OMe
O
e
18
N
S
S
O
O
16
R = CH3CH2
17 R = CH3
13 R = CH3CH2
14 R = CH3
O
RO
Scheme 5. Synthesis of b-lactams, where the sterically hindered thiol is attached through a carbon spacer to the b-lactam ring. Reagents and conditions: (a) S2Cl2/CCl4, 55 °C,
(7) 50%; (b) C6H6, Et3N, reflux, 2 days, (11) 89%; (c) CH2Cl2, Et3N, MgSO4, rt, 3 days, (13, 14) 85–90%; (d) MeOCH2COOH in C6H6, reflux, 12 h, (12) 27%; (e) MeOCH2COCl, Et3N, in
CH2Cl2, reflux, 12 h, (16, 17) 40–46%, (f) Zn/HCl in EtOH, reflux, inert atmosphere, (18) 70%.
(Scheme 5). Imines 13 and 14 were prepared in methylene chloride
at room temperature for 3 days. MgSO4 was used to absorb the
water released as a by-product of the reaction. Production of
bis-imine 11 was accomplished using benzene. In this case a
Dean–Stark apparatus was used to remove water formed during
the reaction. The bis-imine 11 was obtained after reflux when
maximum yield was obtained (2 days). Samples of the reflux were
taken periodically (every 4 h) and analyzed for product by NMR
over the course of 2 days. Stability of these imines was also moni-
tored by NMR. At RT, imines synthesized were stable for 48 h (i.e.,
no more than 10% was degraded). Thus, these compounds were
used within two days. Thioimine 12 was also prepared and charac-
terized (Scheme 5); this compound was stable and unreactive
toward [2+2] cycloaddition. We tested thioimine 12 to determine
whether building blocks of the final b-lactam structure would pos-
sess antibacterial activity. Monomeric b-lactams 16 and 17
(Scheme 5) were prepared using the corresponding monoimines
13 and 14. Using the same experimental conditions, we were
unable to synthesize b-lactam 15 from bis-imine 11. The configu-
ration of the main products 16, 17 and 18 from the [2+2] reaction
based on the coupling constant is cis, as expected for the Stauding-
er reaction taking place in a nonpolar solvent.20
18. Reduction of the disulfide bond of compound 16 was achieved
using Zn/HCl in ethanol under reflux and argon atmosphere. In all
cases the solvents used for the reduction were deoxygenated. All
attempts to neutralize the acid and bring the pH to 6 with sodium
bicarbonate, as described in the literature,26 led to opening of the
ring system. Therefore, the work-up procedure was simply done
by dilution with water and extraction with ethyl acetate. Flash
chromatography was used to purify the final product and afforded
a 70% yield of 18 as colorless oil.
A similar reaction scheme was used for the synthesis of b-lac-
tam 21 (Scheme 6). Commercially available aldehyde 19 was re-
acted with glycine ethyl ester–HCl in the presence of triethyl
amine at room temperature to give imine 20. Stirring the mixture
for 18 h resulted in a high yield (95%). The product was sufficiently
pure to be used for the preparation of lactam 21.
Syntheses of a variety of b-lactams with arylthio groups were
also accomplished. The choice of the arylthiol was based on the re-
ported monocyclic lactams as cysteine inhibitors.3 In order to
determine the optimal C4 substituent, lactams containing an
unsubstituted thiophenyl- (23, Scheme 7), thiophene- (25,
Scheme 7) group as well as those prepared by addition of 4-mer-
captophenol, benzene-1,4-dithiol and 4-aminobenzenethiol at C4
(Scheme 8), were also prepared. b-Lactams 22 and 24 (Scheme 7),
26–28 (Scheme 8) were synthesized from 4-acetoxy-2-azetidone 1,
using procedures described previously.10,27
Addition of the commercially available aromatic thiols to
b-lactam 1 led to the compounds of interest, that is, the arylthio-
b-lactams 26–28, 29–31, in two steps. Sulfur trioxide dimethyl-
formamide complex was successfully used for the sulfonation
reaction of the lactam nitrogen when aromatic thiols were present
Several procedures were explored in an attempt to produce b-
lactam 18. Successful synthesis of compound 18 required reduc-
tion of the disulfide group of 16 with Zn/HCl in an inert atmo-
sphere (Scheme 5). Conversion of disulfides to the corresponding
mercaptans was attempted with propanethiol, DTT, Ph3P–diox-
ane–water,21 polymer-bond Ph3P,22,23 Bu3P,24 ZnCl4/NaBH4,25 Zn/
glacial acidic acid,16 and Zn/HCl.26 Changes in the conditions and
the solvents of these reactions did not generate the desired product

M. B. Kostova et al. / Bioorg. Med. Chem. 19 (2011) 6842–6852
6845
MeO
S
O
S
O
S
H
NH2.HCl
EtO
S
N
N
S
b
a
O
S
O
O
21
19
20
O
O
Scheme 6. Synthesis of b-lactams containing alkyl disulfide at C4. Reagents and conditions: (a) CH2Cl2, Et3N, MgSO4, rt, overnight, (20) 95%; (b) MeOCH2COCl, Et3N, in CH2Cl2,
reflux, 2 h, (21) 58%.
OAc
S
H
S
SH
DMF.SO3/DMF
DMF.SO3/DMF
N
N
N
N
O
O
H
O
O
SO3H
1
1
23
22
S
S
S
OAc
H
S
H
S
HS
N
N
O
O
SO3H
25
24
Scheme 7. Synthesis of unsubstituted arylthio-b-lactams, overall yield 50%.
OAc
H
X
S
H
X
S
X
SH
DMF.SO3/DMF
N
N
N
O
X = OH, NH2, SH
O
SO3H
O
1
26 X = OH
27 X = NH2
28 X = SH
29 X = OH
30 X = NH2
31 X = SH
Scheme 8. Synthesis of substituted phenylthio b-lactams, overall yield 30–66%.
in the structure; for the aliphatic thiols, pyridine SOꢀ3 complex re-
sulted in better yields (50%), as compared to the DMFꢁSO3 complex
(30%). The alternative sulfonation of the functional groups of the
thiophenol at C4 as in compounds 29, 30 and 31 was not observed,
which was determined by NMR and X-ray crystallography.
While most synthetic products were sufficiently pure to be used
directly for the next step, some were first recrystallized. Purifica-
tion of lactam 28 using column chromatography led to formation
of a disulfide dimer. For a single -SH ‘monomer’ a molecular ion
peak [MꢀH]ꢀ with m/z 210 was present while a [MꢀH]ꢀ peak with
m/z 421 was detected for the dimer. The dimer/monomer ratio
after purification was observed to be 1:10.
Of the C4-thio-b-lactams compounds screened for activity com-
pounds 2, 3 and 29 demonstrated activity against at least five of
the six clinical b-lactam-producing M. catarrhalis isolates. In con-
trast, compounds 21–25 (Schemes 6 and 7) exhibited no inhibitory
activity. Since these compounds lack peripheral structural similar-
ities, we have no rationale for their lack of activity. The mechanism
of action of compounds 2, 3 and 29 may be different from the ‘clas-
sical’ b-lactams, since these compounds lack an ionizable group at
N1. Since glutathione could inactivate the thio group of the com-
pounds in vivo, we tested the C4-thio-b-lactams in the presence
of glutathione (1 mol equiv) which was added to each lactam-con-
taining disc just prior to testing. Overall, reduced glutathione did
not affect the biological activity of the C4-thio-b-lactams tested,
with the exception of compound 9, which was inactivated by the
presence of glutathione. This might be due to the formation of an
adduct of glutathione with lactam 9, whose 2-ethylbutanol group
could have been oxidized to 2-ethylbutanal in vitro. This adduct
might then be further modified to products, for example, S-
(hydroxymethyl)-glutathione, by an enzymatic reaction.30 In addi-
tion, the C4-thiol-b-lactams were tested in the presence of bismuth
nitrate, using an equimolar amount to that used in compound syn-
thesis. This was done because compounds with bismuth-coordi-
nated thiols have been reported to possess potent antibacterial
biofilm activity.11,14 The presence of bismuth had no effect on
the activity of any of the C4-thiol-b-lactams tested.
All of the compounds were prepared and subsequently tested
for antibacterial activity as racemates.
2.3. Antibacterial activity
Kirby-Bauer disc diffusion assays were initially used to screen
compounds for antibacterial activity.28 Clinical isolates of Moraxella
catarrhalis, (n = 5 or 6) a major cause of otitis media, sinusitis and
acute exacerbation of chronic obstructive pulmonary disease
(COPD), were screened. All M. catarrhalis strains produced b-lacta-
mase as documented by nitrocefin cleavage studies.29
Initially we screened precursors of the C4-thio-b-lactams
compounds for antibiotic activity (Table 1). Several of the C4-thio-
b-lactams compound precursors (7, 8, 12 and 16, Scheme 5) exhib-
ited activity against various M. catarrhalis clinical strains.
After initial screening of the C4-alkyl- and C4-aryl-thio b-lac-
tams using the Kirby Bauer disc diffusion assay (Tables 1 and 2),

6846
M. B. Kostova et al. / Bioorg. Med. Chem. 19 (2011) 6842–6852
Table 1
Table 3
Susceptibility of b-lactamase producing Moraxella catarrhalis clinical isolates to C4-
thio-b-lactam and precursers
Minimum bactericidal concentrations (lg/ml) of novel compounds against Moraxella
catarrhalis b-lactamase producing clinical isolates and Mycobacterium tuberculosis
(Mtb) H37Rv32,34
Compounds (10
l
g/disc)
Moraxella catarrhalis clinical isolates (n = 5) zone
of inhibitiona (mm)
Compounds
Moraxella catarrhalis
Mtb
1
2
3
4
5
1a
2
3
4
5
6
H37Rv
7
8
12
16
10
5
NE
20
10
3
2
NEb
NE
NE
NE
5
3
NE
NE
10
12
11
3
17
24
26
28
29
>500
>500
>500
250
>500
>500
<15.6
125
<15.6
500
<15.6
125
<15.6
500
<15.6
500
<15.6
500
<15.6
125
<15.6
>500
<15.6
125
200
100
>200
25
NE
>500
<15.6
<15.6
<15.6
<15.6
<15.6
>200
a
Kirby-Bauer disc diffusion assay. Zone size indicates area of bacterial growth
inhibition beyond the edge of the disc.
a
Clinical isolates of M. catarrhalis (n = 6).
b
NE = no effect.
that the –SO3 group at the lactam nitrogen is not necessary for
the biological activity of these lactams.
the minimum inhibitory concentration (MIC) and minimum bacte-
ricidal concentration (MBC) of the compounds was determined.
MIC and MBC levels for all compounds were congruent. Organisms
tested were the same as those used for the screening tests with the
addition of Mycobacterium tuberculosis (Mtb) which was tested as
previously described.29 The data in Table 3 indicates an improved
sensitivity of the microdilution method vs. the disc screening
method. Compounds were considered to have potential for thera-
Analysis of the effects various substituents on the aromatic ring
have on antimicrobial activity led to the conclusion that the hydro-
xyl group (26 and 29) is a better choice of substituent than the sulf-
33
hydryl and amino groups for bioactivity against M. catarrhalis.
With respect to Mtb, compound 28, with a p-dithiophenyl sub-
stituent (MBC of 25
Mtb than its phenol-containing counterpart 26 (MBC >200
l
g/mL), is significantly more active against
g/
l
peutic usage if their MBC was 6100 lg/mL. Compounds 4, 5, 18,
mL). This activity may be attributed to the highly lipophilic charac-
ter of the cell wall of Mtb or to a specific molecular target, interact-
ing with the thiol group of compounds 2 and 28.
and 22 were inactive against all organisms tested while com-
pounds 9, 16, 21, 23, 25, 27, 30, and 31 exhibited limited activity
(within 4-fold serial dilution of maximum concentration tested;
Both Kirby-Bauer disc diffusion assays and MIC/MBC were used
to screen compounds for antibacterial activity against non-b-lacta-
mase producing Escherichia coli, ATCC 25922, Pseudomonas aerugin-
osa, ATCC27853, Staphylococcus aureus, ATCC25923, Enterococcus
faecalis, ATCC29212 which are highly stable quality control strains
routinely used for antimicrobial testing. They also represent a
range of organisms, both Gram positive and Gram negative, that
cause clinically important infections. None of the synthesized com-
pounds affected the growth of E. coli, P. aeruginosa, S. aureus, or
E. faecalis.
500
pounds with the best activity against M. catarrhalis were 17 (four
of six strains had an MBC of <15.6 g/mL), and 26 and 29 (five of
six strains had an MBC of <15.6 g/mL). Some activity was also
measured for compounds 30 and 23 (125 g/mL; n = 4 and 6,
respectively). For Mtb, the compounds that showed the most po-
tent antimicrobial activity, were 2 (MBC = 25 g/mL) and 28
(MBC = 25 g/mL).
Compound 26, one of the most active b-lactams against
M. catarrhalis, possesses an aromatic ring with a hydroxyl group
at the p-position (see Scheme 8). The presence of a sulfonyl group
at the lactam nitrogen did not appear to improve the activity of the
compounds, for example, the activities of lactam 26 and lactam 29
are the same. Addition of the –SO3 group to lactam 2, leading to
lactam 3, which displayed activity in the agar diffusion assay, re-
duced biological activity by almost 50%. The fact that presence of
the –SO3 group in these b-lactams does not have any effect, or even
decreases b-lactam bioactivity in certain cases, clearly indicates
lg/mL for M. catarrhalis and 200 lg/mL for Mtb). The com-
l
l
l
l
l
3. Conclusion
We have designed, synthesized and tested a variety of novel
monocyclic b-lactams having alkyl- and aryl-thio-groups at C4.
Several compounds have shown specific antibacterial activity
against b-lactamase-producing Moraxella catarrhalis and Mycobac-
terium tuberculosis, while having no activity against other
Table 2
Effect of reduced glutathione on C4 thiol-b-lactam anti-Moraxella catarrhalis activity
Compounds (10
l
g/disc)
Moraxella catarrhalis clinical isolates (n = 6) zone of inhibition (mm)a
1b
2
3
4
5
6
2d
19
16
NEc
NE
NE
NE
NE
NE
NE
7
20
19
12
11
NE
NE
10
7
8
7
9
7
24
26
28
20
19
11
12
7d
NE
NE
NE
NE
NE
8
+GSHe
3
19
17
12
NE
NE
NE
NE
NE
NE
7
22
12
14
NE
NE
NE
NE
7
7
8
7
8
24
17
18
NE
NE
7
7
8
7
9
+GSH
9
+GSH
17
+GSH
28
+GSH
29
13
9
NE
NE
+GSH
30
+GSH
7
NE
NE
8
NE
NE
8
NE
NE
NE
NE
8
a
b
c
Kirby-Bauer disc diffusion assay. Zone size indicates area of bacterial growth inhibition beyond the edge of the disc.
1–6 = clinical isolates of Moraxella catarrhalis.
NE = no effect.
The data on the first line (numbers) is for the corresponding compound.
(GSH)—the corresponding compound in the presence of reduced glutathione.
d
e

M. B. Kostova et al. / Bioorg. Med. Chem. 19 (2011) 6842–6852
6847
representative non-b-lactamase-producing gram positive and
prepared. Stock solutions (100
lL) were further diluted to produce
gram negative bacteria. The bioactivity of our b-lactams appears
to be a function of the type of group, for example, aromatic at-
tached to the S-atom at C4, even though pronounced changes in
the lactam-C–S bond lengths and angles are not observed when
compared to similar compounds described in the literature. Inter-
estingly, the presence of a polar, charged group (–SO3), necessary
to secure binding in the enzyme active site in the ‘classical’ b-lac-
tams, is not required for the bioactivity of our compounds. In fact,
in certain cases the b-lactam thioethers unsubstituted at the lac-
tam nitrogen have higher bioactivity than those sulfonated at N1.
In addition, it is promising that these novel compounds have activ-
ity against b-lactamase producing bacteria. Current studies in our
laboratories are directed towards the synthesis of additional com-
pounds with increased activity against important human patho-
a total volume of 400 lL and a final concentration of 8 mM; a sam-
ple with the latter concentration was injected directly into the
instrument. The b-lactams and glutathione were dissolved in
methanol and in water, respectively. Methanol: water (60:40;
vol/vol) provided the optimal mobile phase for the study. Prior to
injection, a one to one molar ratio of the b-lactam and the glutathi-
one were mixed and incubated for varying periods of time (0 min,
15 min, 30 min, 1 h, 2 h, 4 h, and 24 h).
4.3. Synthesis
4.3.1. 4-Acetylthio-2-azetidinone (2)
4-Acetoxy-2-azetidinone (0.5 g, 4.0 mmol) was dissolved in
25 mL acetone/water (3:2). Thioacetic acid (0.3 mL, 4 mmol) and
sodium bicarbonate (1.3 g, 15 mmol) were added, and the resulting
solution was allowed to stir overnight. Sodium chloride was then
added until two layers formed. The solution was filtered and the
layers separated. The aqueous layer was extracted with AcOEt
(3 ꢂ 50 mL), the combined organic layers were dried with MgSO4,
filtered and evaporated under pressure. The residue was purified
on silica gel (CH2Cl2/hexanes, 9:1; v/v) to afford the desired prod-
uct as colorless oil (60%). The structure of the compound was spec-
troscopically characterized as described by Ernest et al.12 and
Clauss et al.8 1H NMR (400 MHz, CDCl3): d 6.37 (1H, br s), 5.24
(1H, dd, 1J = 5.2, 2J = 2.4 Hz), 3.46 (1H, ddd, 1J = 15.4, 2J = 5.3,
3J = 2.0 Hz), 2.97 (1H, ddd, 1J = 15.3, 2J = 2.3, 3J = 1.3 Hz), 2.39 (3H,
s). 13C NMR (125 MHz, CDCl3): d 196.20, 165.10, 49.90, 44.20,
30.95.
gens and
a better understanding of the structure–activity
relationships of these promising new antibacterial b-lactam
compounds.
4. Experimental
4.1. Equipment and materials
All air- or moisture-sensitive reactions were performed under
argon or nitrogen atmosphere using glassware that was pre-dried
in an oven at 120 °C overnight. All reactions were monitored by
thin-layer chromatography (TLC) using EM Reagent plates with
fluorescence indicator (SiO2-60, F254) and layer thickness:
250 m; purchased from EMD Chemicals, Inc. (Gibbstown, NJ). Un-
l
less otherwise noted, the compounds were detected under UV light
and iodine vapors.
4.3.2. 2-Acetylsulfanyl-4-oxo-azetidine-1-sulfonic acid tetra-n-
butylammonium salt (3)
Flash chromatography was performed by gradient elution from
silica gel columns (60 Å, particle size 40–75
ogies, Inc., Atlanta, GA).
l
m, Sorbent Technol-
4-Acetylthio-2-azetidinone (2) (1 g, 6.9 mmol) was dissolved in
15 mL of anhydrous pyridine and heated to 85 °C under argon. Two
equivalents of pyridine-sulfur trioxide (2.2 g, 14 mmol) were
added and the solution was stirred for 1.5 h to give a homogeneous
mixture that was then poured into 150 mL of 0.5 N KH2PO4. The
resulting solution was extracted with CH2Cl2 (1 ꢂ 100 mL) and
the same organic layer was back-washed with another 50 mL of
phosphate solution. The combined aqueous layers were treated
with 1 equiv (1.67 g) of TBAHS and extracted with AcOEt
(3 ꢂ 60 mL). The organic extracts were dried over MgSO4 and con-
centrated under low pressure. Flash chromatography (CH2Cl2/
AcOEt, 3:2 and CH2Cl2/AcOEt, 1:1; v/v) resulted in colorless oil
(26%). All attempts to crystallize the product were unsuccessful.
1H NMR (400 MHz, CDCl3): d 5.50 (1H, dd, 1J = 5.0, 2J = 2.3 Hz),
3.54 (1H, dd, 1J = 15.5, 2J = 5.2 Hz), 3.26 (8H, m), 2.29 (1H, dd,
1J = 15.4, 2J = 2.3 Hz), 2.33 (3H, s), 1.64 (8H, pentet, J = 7.6 Hz),
1.44 (8H, sextet, J = 7.3 Hz), 1.00 (12H, t, J = 7.3 Hz). 13C NMR
(125 MHz, CDCl3): d 195.36, 162.44, 58.67, 54.46, 46.36, 30.96,
Melt-Temp II melting point apparatus was used to determine
the melting points of the synthesized compounds. NMR spectra
(25 °C) were obtained at 400 MHz for 1H NMR and 125 MHz for
13C NMR with a Bruker 400 spectrometer (Billerica, MA) in CDCl3
or acetone-d6. In most cases, signals due to exchangeable protons
have been omitted. IR spectra were obtained as a thin film on NaCl
plates or in solid form (KBr standard) on a Shimadzu FT-IR-8300
(Columbia, MD). Elemental analyses were performed by Atlantic
Microlab, Inc., Norcross, GA. High Resolution Mass Spectrometry
(HRMS) was carried out by University of Iowa, (Iowa City, IA).
All other solvents, chemicals and enzymes were purchased from
Sigma-Aldrich (St. Louis, MO), Fisher Scientific (Pittsburgh, PA) and
Acros Organics (Geel, Belgium). Unless stated otherwise, solutions
in organic solvents were dried with anhydrous magnesium sulfate,
and concentrated under vacuum conditions using rotatory
evaporation.
23.94, 19.70, 13.72. IR (film) m
max: 1770, 1691 cmꢀ1. Anal. Calcd
4.2. LC–MS
for C21H42N2O5S2: C, 54.04; H, 9.07; N, 6.00. Exact mass Calcd for
C5H6NO5S2ꢀ: 223.9687 and C16H36N+: 242.2848; found by HRMS:
m/z (Mꢀ) 223.9679.
Mass spectra were measured on LC/MS 2010 Shimadzu mass
spectrometer (Columbia, MD), equipped with electrospray ioniza-
tion source. ESI/MS spectra were recorded mostly in the nega-
tive-ion setting. LC/MS grade methanol was purchased from
Fisher and used as received. Electrospray mass spectrometric anal-
ysis was performed using high purity nitrogen as the nebulizing
gas at a pressure of 80/80 kgf/cm2, with a gas-flow rate of 4.5 L/
min and the temperature of the probe at 250 °C. LC/MS analysis
was carried out without any backflow or drying gas, with a scan
speed of 2000 amu/s in the range of m/z 20–800. Each sample
4.3.3. 4-(3-Mercapto-propoxy)-azetidin-2-one (4)
4-Acetoxy-2-azetidinone (2 g, 15.49 mmol) was dissolved in
100 mL acetone/water (3:2). 2-Mercaptoethanol (1.18 mL,
17.04 mmol) and sodium bicarbonate (5.20 g, 49.6 mmol) were
added, and the resulting solution was allowed to stir overnight. So-
dium chloride was then added until two layers formed. The solu-
tion was filtered and the layers separated. The aqueous layer was
extracted with AcOEt (3 ꢂ 50 mL), and the combined organic layers
were dried with MgSO4, filtered and evaporated under pressure to
afford the desired product as colorless oil (48%). 1H NMR (400 MHz,
CDCl3): d 7.61 (1H, br s), 4.90–4.92 (1H, dd, 1J = 3.7, 2J = 2.4 Hz),
was introduced into the system at 3
l
L/injection.
test, stock
lL) of each b-lactam and glutathione were
For
the
b-lactam-glutathione
solutions
(6.5 ꢂ 10ꢀ6 mol in 400

6848
M. B. Kostova et al. / Bioorg. Med. Chem. 19 (2011) 6842–6852
4.06–4.08 (2H, t, J = 5.5 Hz), 3.7 (1H, m), 3.3 (1H, m), 2.96 (1H, s),
2.77–2.78 (2H, t, J = 3.1 Hz). 13C NMR (125 MHz, CDCl3) d: 167.65,
33.82, 61.98, 52.75, 45.17.
1H NMR (400 MHz, CDCl3): (9-desired thiol-conjugate) d 6.75
(1H, br s), 4.89 (1H, dd, 1J = 5.2, 2J = 2.6 Hz), 3.63 (2H, m), 3.41
(1H, ddd, 1J = 15.2, 2J = 5.2, 3J = 1.6 Hz), 2.86 (1H, ddd, 1J = 15.4,
2J = 2.3, 3J = 1.5 Hz), 2.45 (1H, br s), 1.66 (2H, q, J = 7.5 Hz), 1.64–
1.44 (2H, m), 3.36 (1H, ddd, 1J = 15.3, 2J = 5.1, 3J = 1.6 Hz), 0.95
(6H, dt, 1J = 7.4, 2J = 3.0 Hz); (9⁄) d 6.44 (1H, br s), 4,77 (1H, dd,
1J = 5.1, 2J = 2.5 Hz), 2.88 (1H, dt, 1J = 15.3, 2J = 2.0 Hz), 2.72 (2H, q,
J = 13.1 Hz), 1.74 (1H, br s), 1.66–1.52 (4H, m), 0.92 (6H, td,
1J = 18.0, 2J = 7.5 Hz). 13C NMR (125 MHz, CDCl3): (9) d 166.97,
67.64, 56.62, 48.32, 45.50, 26.40, 25.11, 7.83, 7.72; (9⁄) 165.93,
74.55, 53.75, 45.36, 40.96, 31.01, 30.04, 8.04, 7.86. IR (9) (film)
4.3.4. 4-(3-Hydroxy-propylsulfanyl)-azetidin-2-one (5)
4-Acetoxy-2-azetidinone (2 g, 15.49 mmol) was dissolved in
100 mL acetone/water (3:2). 3-Mercapto-1-propanol (1.47 mL,
17.04 mmol) and sodium bicarbonate (5.20 g, 49.6 mmol) were
added, and the resulting solution was stirred overnight. Sodium
chloride was then added until two layers formed. The solution
was filtered and the layers separated. The aqueous layer was ex-
tracted with AcOEt (3 ꢂ 50 mL), and the combined organic layers
were dried with MgSO4, filtered and evaporated under pressure
to afford the title compound as colorless oil (70%). 1H NMR
(400 MHz, CDCl3): d 7.69 (1H, br s), 4.81–4.83 (1H, dd, 1J = 2.5,
2J = 2.2 Hz), 3.67–3.73 (2H, t, J = 5.2 Hz), 3.35–3.41 (1H, m), 2.88–
2.92 (1H, m), 2.69–2.80 (2H, m), 1.80–1.91 (2H, m). 13C NMR
(125 MHz, CDCl3) d: 167.63, 60.49, 52.35, 45.26, 32.49, 27.04.
m
max: 3393, 1751, 1257 cmꢀ1; IR (9⁄) (film)
mmax: 1736, 1699,
1078 cmꢀ1. Anal. Calcd for C9H17NO2S: C, 53.17; H, 8.43; N, 6.89.
Found: C, 53.03; H, 8.47; N, 6.82.
4.3.8. {2-[1-(Ethoxycarbonylmethylimino-methyl)-1-ethyl-
propyldisulfanyl]-2-ethyl-butylideneamino}-acetic acid ethyl
ester (11)
Dialdehyde 7 (5 g, 19 mmol) and glycine ethyl ester hydro-
chloride (5.9 g, 42 mmol) were stirred in 80 mL of benzene. Tri-
ethylamine (5.8 mL, 42 mmol) was added and reaction was set
up with Dean–Stark apparatus and left to reflux for 2 days. The
progress of the reaction was monitored by NMR, by taking small
aliquots, evaporating the solvent and re-dissolving in CDCl3. The
mixture was filtered and the resulting solution was evaporated
under low pressure to result in (89%) amber viscous oil that
was used within 48 h without further purification. 1H NMR
(400 MHz, CDCl3): d 7.44 (2H, s), 4.24 (4H, s), 4.20 (4H, q,
J = 7.1 Hz), 1.79 and 1.69 (8H, m), 1.28 (6H, t, J = 7.2 Hz), 0.90
(12H, t, J = 7.4 Hz). 13C NMR (125 MHz, CDCl3): d 171.24, 169.83,
4.3.5. Ethyl-2-(1-ethyl-1-formyl-propyldisulfanyl)-
butyraldehyde (7)
2-Ethylbutanal (1.0 mol, 100 g) was dissolved in 70 mL of CCl4.
The solution was kept at 50–55 °C when fresh S2Cl2 (70 g, 0.5 mol)
was added dropwise. The reaction mixture was heated to 65 °C for
2 h and stirred overnight at 50 °C. The released HCl gas was dis-
placed by continuous air flow into the flask during the overnight
period. Crystallization from absolute ethanol resulted in white
crystals (50%, lit.19 70%). The product was further recrystallized
from acetone/ethanol, 1:1, v/v. The determined melting point of
71–72 °C was in agreement with previously published data (lit.20
mp: 71–72 °C). 1H NMR (400 MHz, CDCl3): d 9.03 (2H, s), 1.76
and 1.68 (8H, m), 0.89 (12H, t, J = 7.4 Hz). 13C NMR (125 MHz,
CDCl3): d 194.94, 65.63, 21.80, 8.15.
61.30, 60.98, 60.45, 25.16, 14.15, 8.26. IR (film)
mmax: 1736,
1653, 1186 cmꢀ1
.
4.3.9. (2-Ethyl-2-mercapto-butylideneamino)-acetic acid ethyl
ester (12)
4.3.6. 2-Ethyl-2-mercapto-butan-1-ol (8)
The compound was synthesized using protocol reported by
Bandarage et al.16 LiAlH4 (1 M, 38.2 mL) in THF (freshly distilled
over sodium) was added dropwise to a stirred solution of dialde-
hyde 7 (10 g, 38.17 mmol) in THF (70 mL). Additional portions of
LiAlH4 (3 ꢂ 100 mg) were added and the reaction mixture was stir-
red at room temperature under argon for 2 h. The mixture was
poured onto ice, treated with 3 M HCl (100 mL), and extracted with
AcOEt (3 ꢂ 100 mL). The combined organic layers were then dried
over MgSO4, filtered, and evaporated to yield colorless oil (70%),
which was used without further purification. 1H NMR (400 MHz,
CDCl3): d 3.50 (2H, s), 2.22 (1H, br s), 1.63 (4H, m), 1.32 (1H, s),
0.95 (6H, t, J = 7.4 Hz). 13C NMR (125 MHz, CDCl3): d 69.06, 55.03,
29.51, 8.42.
Methoxyacetic acid (2 equiv) was added to the freshly prepared
and filtered solution of diimine 11. The mixture was refluxed over-
night and evaporated under reduced pressure. The resulting brown
viscous oil was purified by flash chromatography using hexanes/
CH2Cl2 (1:1) to yield the desired product as yellow oil (1.8 g,
27%). 1H (400 MHz, CDCl3): d 7.72 (1H, br s), 4.44 (2H, d,
J = 4.5 Hz), 4.28 (2H, q, J = 7.1 Hz), 2.31 (1H, m, J = 4.7 Hz), 1.77
(2H, m), 1.62 (2H, m), 1.32 (3H, t, J = 7.2 Hz), 0.87 (6H, t,
J = 7.3 Hz). 13C NMR (125 MHz, CDCl3): d 210.54, 169.10, 61.97,
60.13, 46.94, 28.78, 14.13, 11.93. IR (film) m .
max: 1736, 1204 cmꢀ1
Anal. Calcd for C10H19NO2S: C, 55.26; H, 8.81; N, 6.44. Found: C,
55.41; H, 8.90, N, 6.43.
4.3.10. [2-Ethyl-2-(1-ethyl-1-formyl-propyldisulfanyl)-
4.3.7. 4-(2-Ethyl-2-mercapto-butoxy)-azetidin-2-one (9)
butylideneamino]-acetic acid ethyl ester (13)
4-Acetoxy-2-azetidinone (1 g, 8 mmol) was dissolved in 50 mL
acetone/water (3:2). Thioalcohol 8 (1.05 g, 8 mmol) and sodium
bicarbonate (4 equiv) were added, and the resulting solution was
allowed to stir overnight. Sodium chloride was then added until
two layers formed. The solution was filtered and the layers sepa-
rated. The aqueous layer was extracted with AcOEt (3 ꢂ 50 mL),
and the combined organic layers were dried with MgSO4, filtered
and evaporated under pressure. The residue was purified on silica
gel (CH2Cl2/AcOEt, 3:2; v/v) to yield the title compound as colorless
oil or white crystalline mass (60%) with mp 52–53 °C. Two differ-
ent products were isolated and identified by NMR and GC/MS.
The thiol and the alcohol were not products of the same reaction
mixture; they were isolated from different batches made under
the same reaction conditions. The reaction was repeated multiple
times with the intent to identify the reason for the formation of
two products. However, we were unable to explain this phenomenon.
Dialdehyde 7 (5 g, 19 mmol) and glycine ethyl ester hydro-
chloride (6.5 g, 47 mmol) were suspended in 50 mL of CH2Cl2. Tri-
ethylamine (6 mL, 43 mmol) and MgSO4 (10 g) were added and
the mixture was stirred at room temperature for 3 days. The sus-
pension was filtered and the solvent was evaporated to dryness.
The obtained product was a mixture of white crystalline mass
(unreacted amine and unwanted salts) and imine oil. A small por-
tion of acetone was used to dissolve the oil and separate it from
the crystals. Solvent evaporation resulted in yellow viscous oil
(90%), which was used without purification. 1H NMR (400 MHz,
CDCl3): d 8.93 (1H, s), 7.44 (1H, s), 4.22 (2H, s), 4.20 (2H, q,
J = 7.1 Hz), 1.79 and 1.68 (8H, m), 1.28 (3H, t, J = 7.1 Hz), 0.90
(6H, t, J = 7.4 Hz), 0.89 (6H, t, J = 7.4 Hz). 13C NMR (125 MHz,
CDCl3): d 195.57, 171.27, 169.87, 65.16, 61.22, 60.92, 60.48,
25.06, 21.56, 14.18, 8.30, 8.13. IR (film): mmax 1745, 1716,
1660 cmꢀ1
.

M. B. Kostova et al. / Bioorg. Med. Chem. 19 (2011) 6842–6852
6849
4.3.11. [2-Ethyl-2-(1-ethyl-1-formyl-propyldisulfanyl)-
butylideneamino]-acetic acid methyl ester (14)
118.06, 78.30, 61.55, 56.47, 40.88, 28.95, 24.21, 14.16, 13.39,
12.31. IR (film)
max: 1736, 1703, 1205 cmꢀ1. Anal. Calcd for
13H23NO4S: C, 53.95; H, 8.01; N, 4.84. Found: C, 53.90; H, 8.02;
N, 4.75.
m
Imine 14 was synthesized using glycine methyl ester hydro-
chloride following the procedure described for imine 13. The reac-
tion mixture was stirred for 3 days at room temperature resulting
in light yellow viscous oil (85%). 1H NMR (400 MHz, CDCl3): d 8.93
(1H, s), 7.44 (1H, s), 4.24 (2H, s), 3.74 (3H, s), 1.79 and 1.68 (8H, m),
1.28 (3H, t, J = 7.1 Hz), 0.90 (6H, t, J = 7.4 Hz), 0.89 (6H, t, J = 7.4 Hz).
13C NMR (125 MHz, CDCl3): d 195.56, 171.33, 170.49, 65.15, 61.12,
60.48, 51.98, 25.15, 21.54, 8.28, 8.12. IR (film): mmax 1751, 1713,
C
4.3.15. (2-Methyl-2-methyldisulfanyl-propylideneamino)-acetic
acid ethyl ester (20)
2-Methyldithiol-isobutyraldehyde 19 (0.5 g, 3 mmol) and gly-
cine ethyl ester hydrochloride (0.7 g, 5 mmol) were dissolved in
10 mL of CH2Cl2. Triethylamine (0.7 mL, 5 mmol) and 1.0 g of
MgSO4 were added and the reaction mixture was stirred overnight
at room temperature. The resulting solution was evaporated under
low pressure. Acetone (2 mL) was used to dissolve the oil and sep-
arate it from the crystals. Solvent evaporation resulted in viscous
oil (95%), which was used without further purification. 1H
1659 cmꢀ1
.
4.3.12. {2-[1-Ethyl-1-(1-ethyl-1-formyl-propyldisulfanyl)-
propyl]-3-methoxy-4-oxo-azetidin-1-yl}-acetic acid ethyl ester
(16)
Glycine mono-imine 13 (6 g, 17 mmol) and triethylamine
(4.8 mL, 35 mmol) were stirred into 100 mL of CH2Cl2. A second
solution, of methoxy-acetyl chloride (2.2 mL, 24 mmol) in 10 mL
of CH2Cl2, was added dropwise to the mixture. The reaction mix-
ture was refluxed overnight and then washed with a 5% aqueous
solution of NH4Cl (3 ꢂ 50 mL). The organic layer was dried with
MgSO4 and concentrated in vacuo. The [2+2] reaction yielded
5.7 g of crude mono-b-lactam. Flash chromatography of the prod-
uct (2:3 hexanes/CH2Cl2; 3:7 hexanes/CH2Cl2; v/v) afforded the
product as clear yellow oil (46%). 1H (400 MHz, CDCl3): d 9.23
(1H, s), 4.64 (1H, d, J = 5.3 Hz), 4.42 (1H, d, J = 18.1 Hz), 4.2 (2H,
q, J = 7.1 Hz), 3.93 (2H, d, J = 17.9 Hz), 3.57 (3H, s), 1.76 and 1.61
(8H, m), 1.3 (3H, t, J = 7.1 Hz), 0.97 (6H, t, J = 7.6 Hz), 0.91 (6H, t,
J = 7.6 Hz). 13C NMR (125 MHz, CDCl3): d 196.36, 169.21, 168.11,
85.42, 64.99, 64.13, 61.61, 59.87, 42.97, 27.26, 26.57, 22.23,
(400 MHz, CDCl3): d 7.55 (1H, s), 4.21 (4H, overlapping q
(J = 3.7 Hz) and s), 2.38 (3H, s), 1.50 (6H, s), 1.29 (3H, t,
J = 7.2 Hz). 13C NMR (125 MHz, CDCl3): d 170.78, 169.81, 61.33,
61.03, 53.17, 25.22, 24.06, 14.19. IR (film)
1186 cmꢀ1
mmax: 1744, 1663,
.
4.3.16. [3-Methoxy-2-(1-methyl-1-methyldisulfanyl-ethyl)-4-
oxoazetidin-1-yl]-acetic acid ethyl ester (21)
Imine 20 (0.75 g, 3 mmol) and triethylamine (0.8 mL, 5 mmol)
were stirred in 20 mL of CH2Cl2. Methoxy-acetyl chloride (0.4 mL,
4 mmol) was added dropwise and the reaction mixture was re-
fluxed for 2 h. The mixture was washed with 5% aqueous NH4Cl
(3 ꢂ 20 mL). The organic layer was dried with MgSO4 and concen-
trated to dryness. The [2+2] reaction yielded 0.78 g of crude mono-
b-lactam. Flash chromatography of the product (hexanes/
CH2Cl2,1:1 and 2:3; v/v) afforded the product as colorless oil
(58%). 1H (400 MHz, CDCl3): d 4.62 (1H, d, J = 5.3 Hz), 4.33 (1H, d,
J = 5.3 Hz), 4.19 (2H, dq, 1J = 7.1, 2J = 1.0 Hz), 4.40–4.05 (2H, dd,
1J = 37.4, 2J = 17.9 Hz), 3.58 (3H, s), 2.42 (3H, s), 1.45 (3H, s), 1.4
(3H, s), 1.28 (3H, t, J = 7.1 Hz). 13C NMR (125 MHz, CDCl3): d
167.45, 166.36, 83.28, 61.48, 59.96, 58.10, 50.92, 41.00, 23.71,
21.33, 14.19, 9.05, 8.92, 8.36, 7.96. IR (film)
mmax: 1767, 1736,
1713, 1204 cmꢀ1. Anal. Calcd for C19H33NO5S2: C, 54.39; H, 7.93;
N, 3.34. Found: C, 54.32; H, 7.93; N, 3.52.
4.3.13. {2-[1-Ethyl-1-(1-ethyl-1-formyl-propyldisulfanyl)-
propyl]-3-methoxy-4-oxo-azetidin-1-yl}-acetic acid methyl
ester (17)
23.06, 20.11, 12.59. IR (film) m
max: 1759, 1738, 1207 cmꢀ1. Anal.
Mono-b-lactam 17 was synthesized using the procedure de-
scribed for 16 using the corresponding mono-imine. The product
was purified on silica gel (CH2Cl2/AcOEt, 9:1; v/v) to afford the title
compound as clear yellow oil (40%). 1H (400 MHz, CDCl3): d 9.22
(1H, s), 4.64 (1H, d, J = 5.3 Hz), 4.50 (1H, d, J = 17.9 Hz), 4.17(1H,
d, J = 5.3 Hz), 3.96 (1H, d, J = 17.9 Hz), 3.76 (3H, s), 3.57 (3H, s),
1.76 and 1.61 (8H, m), 0.97 (6H, t, J = 7.6 Hz), 0.91 (6H, t,
J = 7.6 Hz). 13C NMR (125 MHz, CDCl3): d 196.37, 169.18, 168.56,
85.43, 65.00, 64.19, 59.89, 52.46, 42.82, 27.24, 26.57, 22.21,
Calcd for C12H21NO4S2: C, 46.88; H, 6.89; N, 4.56. Found: C,
46.73; H, 6.84; N, 4.62.
4.3.17. General procedure for synthesis of b-lactams containing
aromatic thiols, phenol and thiophene
Lactams 22 and 24, as well as 26–28 were prepared in a manner
similar to the synthesis of lactam 9. 4-Acetoxy-2-azetidone (1 g,
8 mmol) was stirred in 50 mL acetone/water (3:2) and 1.05 equiv
of the corresponding substituents (4-mercaptophenol, 4-amino-
thiophenol, 1,4-benzenedithiol, thiophenol, or 2-thiophene thiol,
respectively) was added. Sodium bicarbonate (4 equiv) was added
to the mixture, which was then stirred vigorously for 12 h in a
closed round bottom flask. Sodium chloride was subsequently
added to the solution and after the formation of two layers, the
mixture was filtered out and extracted with AcOEt (3 ꢂ 50 mL).
The combined organic layers were dried over MgSO4 and concen-
trated under vacuum. The crude material was purified by flash
chromatography or crystallized to give white crystals for all five
products in good yield (30–66%).
21.35, 9.05, 8.93, 8.36, 7.98. IR (film)
mmax: 1773, 1745, 1716,
1207 cmꢀ1. Anal. Calcd for C18H31NO5S2: C, 53.31; H, 7.70; N,
3.45. Found: C, 53.39; H, 7.73; N, 3.57.
4.3.14. [2-(1-Ethyl-1-mercapto-propyl)-3-methoxy-4-oxo-
azetidin-1-yl]-acetic acid ethyl ester (18)
b-Lactam (2.5 g) 16 were dissolved in EtOH/6 M HCl (120 mL,
5:1; v/v) in a three neck round bottom flask. Zn dust (10 g) was
added to the solution and the reaction mixture was refluxed over-
night under argon. The reaction mixture was left to cool to room
temperature and the solids were filtered out. The filtrate was con-
centrated under reduced pressure and diluted with water. The
acidic solution was extracted with AcOEt (3 ꢂ 60 mL). The com-
bined organic layers were dried with MgSO4, filtered, and evapo-
rated. Flash chromatography (hexanes/CH2Cl2 3:7; v/v) resulted
in colorless oil (70%). 1H (400 MHz, CDCl3): d 4.2 (2H, dq, 1J = 7.1,
2J = 2.7 Hz), 3.95 (2H, d, J = 10.2 Hz), 3.61 (3H, s), 3.56 (2H, d,
J = 4.5 Hz), 3.25 (1H, d, J = 10.2 Hz), 1.93 (1H, m), 1.8 (4H, m),
1.29 (3H, t, J = 7.2 Hz), 1.07 (3H, t, J = 7.3 Hz), 1.01 (3H, t,
J = 7.4 Hz). 13C NMR (125 MHz, CDCl3): d 172.08, 169.79, 152.00,
4.3.18. 4-Phenylsulfanyl-azetidin-2-one (22)
The crude product was crystallized from CH2Cl2 and hexanes to
afford white crystals (62%) with mp. 66–68 °C. 1H NMR (400 MHz,
CDCl3): d 7.46 and 7.37 (4H, m), 6.1 (1H, br s), 5.02 (1H, dd, 1J = 4.9,
2J = 2.3 Hz), 3.38 (1H, ddd, 1J = 15.2, 2J = 4.9, 3J = 1.9 Hz), 2.9 (1H,
ddd, 1J = 15.2, 2J = 2.3, 3J = 1.5 Hz). 13C NMR (125 MHz, CDCl3): d
165.56, 133.60, 131.21, 129.43, 128.76, 54.20, 45.40. IR (KBr) mmax
:
1766.7, 742.5, 690.5 cmꢀ1. Anal. Calcd for C9H9NOS: C, 60.31; H,
5.06; N, 7.81. Found: C, 60.26; H, 5.04; N, 7.81.

6850
M. B. Kostova et al. / Bioorg. Med. Chem. 19 (2011) 6842–6852
4.3.19. 4-(Thiophen-2-ylsulfanyl)-azetidin-2-one (24)
1H NMR (400 MHz, CDCl3): d 7.71 (2H, m), 7.31 (2H, m), 5.22
(1H, dd, 1J = 5.4, 2J = 2.6 Hz), 3.27 (8H, m), 3.15 (1H, dd, 1J = 15.2,
2J = 5.4 Hz), 2.62 (1H, dd, 1J = 15.2, 2J = 2.6 Hz), 1.64 (8H, m), 1.43
(8H, s, J = 7.3 Hz), 0.99 (12H, t, J = 7.3 Hz). 13C NMR (125 MHz,
CDCl3): d 162.80, 134.96, 130.63, 128.88, 128.28, 58.68, 58.41,
The crude product was crystallized from CH2Cl2 and hexanes to
afford white crystals (90%) with mp 57–58 °C. 1H NMR (400 MHz,
CDCl3): d 7.48 (1H, dd, 1J = 5.4, 2J = 1.2 Hz), 7.23 (1H, dd, 1J = 3.6,
2J = 1.2 Hz), 7.07 (1H, dd, 1J = 5.4, 2J = 3.6 Hz), 6.19 (1H, br s), 4.86
(1H, dd, 1J = 4.9, 2J = 2.3 Hz), 3.31 (1H, ddd, 1J = 15.3, 2J = 4.9,
3J = 1.9 Hz), 2.91 (1H, ddd, 1J = 15.2, 2J = 2.0, 3J = 1.6 Hz). 13C NMR
43.12, 23.97, 19.70, 13.71. IR (film) mmax
: 1766.7, 1247.8,
1049.2 cmꢀ1. Anal. Calcd for C25H44N2O4S2: C, 59.96; H, 8.86; N,
5.59. Exact mass Calcd for C9H8NO4S2ꢀ: 257.9895 and C16H36N+:
242.2848; found by HRMS: m/z (Mꢀ) 257.9903.
(125 MHz, CDCl3):
55.38, 44.72. IR (KBr)
d
165.52, 136.75, 131.82, 128.12, 127.49,
m
max: 1739.7, 968.2, 700.1 cmꢀ1. Anal. Calcd
for C7H7NOS2: C, 45.38; H, 3.81; N, 7.56. Found: C, 45.50; H,
3.76; N, 7.43.
4.3.25. 2-Oxo-4-(thiophen-2-ylsulfanyl)-azetidine-1-sulfonic
acid tetra-n-butylammonium salt (25)
4.3.20. 4-(4-Mercapto-phenoxy)-azetidin-2-one (26)
The product was crystallized from AcOEt/hexane to afford the
title compound as white crystals (55%) with mp 99–102 °C. 1H
NMR (400 MHz, CDCl3): d 7.53 (1H, dd, 1J = 7.5, 2J = 3.6 Hz), 7.40
(1H, dd, 1J = 5.3, 2J = 1.2 Hz), 7.02 (1H, dd, 1J = 5.3, 2J = 3.6 Hz),
5.06 (1H, dd, 1J = 5.3, 2J = 2.5 Hz), 3.27 (8H, m), 3.08 (1H, dd,
1J = 15.2, 2J = 5.3 Hz), 2.7 (1H, dd, 1J = 15.2, 2J = 2.7 Hz), 1.64 (8H,
m), 1.43 (8H, s, J = 7.4 Hz), 0.99 (12H, t, J = 7.3 Hz). 13C NMR
The crude product was crystallized from AcOEt/hexanes to af-
ford white crystals (66%) with mp 143–144 °C. 1H NMR
(400 MHz, acetone-d6): d 8.72 (1H, s), 7.66 (1H, br s), 7.39 (2H, d,
J = 8.6 Hz), 6.86 (2H, d, J = 8.6 Hz), 4.94 (1H, dd, 1J = 4.9,
2J = 2.3 Hz), 3.25 (1H, ddd, 1J = 14.9, 2J = 4.9, 3J = 1.9 Hz), 2.7 (1H,
ddd, 1J = 14.9, 2J = 2.2, 3J = 1.2 Hz). 13C NMR (125 MHz, acetone-
d6): d 165.68, 139.27, 137.59, 120.95, 117.04, 55.05, 45.63. IR
(125 MHz, CDCl3):
d 162.56, 137.97, 130.84, 128.06, 126.71,
(KBr)
m
max: 1732, 1695, 1580 cmꢀ1. Anal. Calcd for C9H9NO5S: C,
58.96, 58.62, 42.16, 23.96, 19.69, 13.72. IR (KBr) mmax: 1766.7,
55.37; H, 4.65; N, 7.17. Found: C, 55.35; H, 4.57; N, 7.20.
1051.1 cmꢀ1. Anal. Calcd for C23H42N2O4S3: C, 54.51; H, 8.35; N,
5.53. Found: C, 54.76; H, 8.44; N, 5.50.
4.3.21. 4-(4-Mercapto-phenylamino)-azetidin-2-one (27)
The crude product was purified on a silica gel column (CH2Cl2/
AcOEt, 1:1; v/v). The resulting yellow solid (51%) was a mixture of
two forms. The two forms were observed by NMR, where a single
compound as well as a formation of hydrogen bond between the
two structures was observed. The data reported here corresponds
to the single compound, mp 106–108 °C. 1H NMR (400 MHz, ace-
tone-d6): d 7.6 (1H, br s), 7.24 (2H, d, J = 8.6 Hz), 6.66 (2H, d,
J = 8.6 Hz), 4.98 (1H, br s), 4.85 (1H, dd, 1J = 4.9, 2J = 2.3 Hz), 3.22
(1H, ddd, 1J = 14.9, 2J = 4.9, 3J = 1.9 Hz), 2.67 (1H, ddd, 1J = 14.9,
2J = 2.2, 3J = 1.4 Hz). 13C NMR (125 MHz, acetone-d6): d 165.76,
4.3.26. 2-(4-Mercapto-phenoxy)-4-oxo-azetidine-1-sulfonic
acid tetra-n-butylammonium salt (29)
The product was purified on silica gel (AcOEt/MeOH, 9:1; v/v) to
afford the compound as white crystals (50%) with mp 148–150 °C.
1H NMR (400 MHz, acetone-d6): d 8.72 (1H, s), 7.64 (2H, d,
J = 8.6 Hz), 6.83 (2H d, J = 8.6 Hz), 4.93 (1H, dd, 1J = 5.4,
2J = 2.5 Hz), 3.45 (8H, m), 3.25 (1H, dd, 1J = 14.8, 2J = 5.5 Hz), 2.4
(1H, dd, 1J = 14.7, 2J = 2.5 Hz), 1.82 (8H, m), 1.43 (8H, s,
J = 7.4 Hz), 0.98 (12H, t, J = 7.4 Hz). 13C NMR (125 MHz, acetone-
d6): d 162.83, 159.42, 138.99, 119.40, 116.72, 59.29, 58.98, 42.39,
150.57, 135.68, 116.36, 115.57, 55.22, 45.41. IR (film)
m
max: 3462,
24.41, 20.34, 13.86. IR (KBr) mmax: 3205.5, 1776.3, 1043.4,
1747.4, 1627.8, 1595 cmꢀ1. Anal. Calcd for C9H10N2OS: C, 55.65;
H, 5.19; N, 14.42. Found: C, 55.43; H, 5.15; N, 14.28.
842.8 cmꢀ1. Anal. Calcd for C25H44N2O5S2: C, 58.11; H, 8.58; N,
5.42. Found: C, 57.84; H, 8.56; N, 5.39.
4.3.22. 4-(4-Mercapto-phenylsulfanyl)-azetidin-2-one (28)
The crude product was sufficiently pure to be used for the next
step of the synthesis. When purification was required column
chromatography (AcOEt/MeOH, 4:1; v/v) was performed to afford
the desired product as white amorphous solid (30%) with a mp
124–125 °C. 1H NMR (400 MHz, acetone-d6): d 7.92 (1H, br s),
7.51 (4H, m), 5.19 (1H, dd, 1J = 5.0, 2J = 2.3 Hz), 3.43 (1H, ddd,
1J = 15.1, 2J = 5.1, 3J = 2.2 Hz), 2.8 (1H, ddd, 1J = 15.1, 2J = 2.3,
3J = 1.2 Hz). 13C NMR (125 MHz, acetone-d6): d 165.75, 136.88,
4.3.27. 2-(4-Mercapto-phenylamino)-4-oxo-azetidine-1-
sulfonic acid tetra-n-butylammonium salt (30)
The product was purified on silica gel column (CH2Cl2/AcOEt,
1:9; v/v) to afford the title compound as white crystals (47%) with
mp 111–113 °C. This b-lactam was observed in two different forms
in the NMR: free compound and dimer structure formed as a result
of hydrogen bonding. The two forms were interchangeable when
the product was left in solvent for a few days. The data reported here
is for the dimer, as this was the prevalent form. 1H NMR (400 MHz,
acetone-d6): d 7.71 (2H, d, J = 8.5 Hz), 7.48 (2H, d, J = 8.6 Hz), 6.64
(4H, dd, 1J = 5.9, 2J = 8.5 Hz), 4.99 (1H, dd, 1J = 2.6, 2J = 5.4 Hz), 4.86
(1H, dd, 1J = 2.6, 2J = 5.4 Hz), 3.44 (16H, m), 3.08 (1H, dd, 1J = 14.8,
2J = 5.5 Hz), 2.95 (1H, dd, 1J = 14.7, 2J = 5.5 Hz), 2.86 (2H, br s), 2.43
(1H, dd, 1J = 14.6, 2J = 2.6 Hz), 2.41 (1H, dd, 1J = 14.6, 2J = 2.6 Hz),
133.61, 129.07, 129.16, 54.06, 46.24. IR (film) mmax
: 1745.4,
1712.7, 1571.9 cmꢀ1. Anal. Calcd for C9H9NOS2: C, 51.16; H, 4.29;
N, 6.63. Found: C, 51.37; H, 3.08; N, 6.54.
4.3.23. General procedure for preparation of mercapto-phenyl-
azetidine sulfonates
1.81 (16H, m), 1.43 (16H, s, J = 7.5 Hz), 0.98 (24H, t, J = 7.4 Hz). 13
C
b-Lactam (1.0 equiv) 22, 24 or 26–28 and sulfur trioxide–DMF
(5 equiv) were dissolved in 12 mL of anhydrous DMF. The mixture
was stirred for 2 h and poured into 0.5 N KH2PO4 (80 mL), followed
by addition of 1 equiv of TBAHS. The aqueous layer was extracted
with AcOEt (3 ꢂ 20 mL) and dried over MgSO4. The solvent was
evaporated under low pressure to afford solid or viscous oil that
was further purified.
NMR (125 MHz, acetone-d6): d 162.35, 162.14, 153.05, 150.21,
138.83, 137.36, 124.91, 120.69, 115.69, 115.28, 59.33, 59.28,
58.91, 58.83, 42.97, 42.19, 24.46, 20.37, 13.93. IR (KBr) mmax
:
3440.8, 1776.7, 1049.2 cmꢀ1. Anal. Calcd for C25H45N3O4S2: C,
58.22; H, 8.79; N, 8.15. Found: C, 58.01; H, 8.72; N, 8.06.
4.3.28. 2-(4-Mercapto-phenylsulfanyl)-4-oxo-azetidine-1-
sulfonic acid tetra-n-butylammonium salt (31)
4.3.24. 2-Oxo-4-phenylsulfanyl-azetidine-1-sulfonic acid tetra-
n-butylammonium salt (23)
The product was purified using column chromatography
(AcOEt/MeOH, 9:1; v/v) to afford the title compound as colorless
oil (46%). All attempts to crystallize this product were unsuccessful.
The product was purified using column chromatography
(AcOEt/MeOH, 8:2 and AcOEt/MeOH 7:3; v/v) to afford the title
compound as thick colorless oil (28%). The purification step was
challenging due to polymerization of the product on the silica gel
column and as a result the final yield was lower then expected.

M. B. Kostova et al. / Bioorg. Med. Chem. 19 (2011) 6842–6852
6851
1H NMR (400 MHz, acetone-d6): d 7.67 (4H, m), 5.14 (1H, m), 3.42
4.4.3. Crystallographic details for compound 29
(16H, m), 3.27 (1H, m), 2.54 (1H, m), 1.78 (16H, m), 1.43 (16H, m),
0.97 (24H, t, J = 7.4 Hz). 13C NMR (125 MHz, acetone-d6): d 162.29,
134.62, 132.80, 132.47, 59.10, 58.49, 44.02, 24.59, 20.47, 15.14. IR
The crystal of 2-(4-hydroxyphenylthio)-4-oxoazetidine-1-sulfo-
nate, 29, was triclinic in space group P1 with unit cell dimen-
ꢀ
sions a = 9.8191(13) Å, b = 10.5484(14) Å, c = 13.6158(18) Å,
a =
(film) mmax
:
1774.4, 1245.9, 1049.2 cmꢀ1
.
Anal. Calcd for
75.954(2)°, b = 89.389(2)°, and = 81.793(3)°. Data were 98.4%
c
C
25H44N2O4S3: C, 56.35; H, 8.32; N, 5.26. Exact mass Calcd for
complete to 25.79° h (approximately 0.82 Å) with an average
redundancy of 2.19.
ꢀ
C
25H44N2O4S3
:
289.9615 and C16H36N+: 242.2848; found by
HRMS: m/z (Mꢀ) 289.9630.
4.4. X-ray crystal structure determination
Single-crystal X-ray diffraction data on compounds 7, 22, 24, 26,
29, and 30 were collected at 113 K using Mo K
a radiation and a
Bruker APEX 2 CCD area detector. Crystals were prepared for data
collection by coating with high viscosity microscope oil (Paratone-
N, Hampton Research). The oil-coated crystal was mounted on a
Micro Mesh mount (MiteGen Inc.) and transferred immediately
to the cold stream (ꢀ160 °C) on the diffractometer. Corrections
were applied for Lorentz, polarization, and absorption effects. All
structures were solved by direct methods and refined by full-ma-
trix least squares on F2 values using the programs found in the
SHELXTL suite (Bruker, SHELXTL v6.10, 2000, Bruker AXS Inc., Madison,
WI). Parameters refined included atomic coordinates and aniso-
tropic thermal parameters for all non-hydrogen atoms. Hydrogen
atoms on carbons were included using a riding model [coordinate
shifts of C applied to H atoms] with C–H distance set at 0.96 Å.
Atomic coordinates for compounds 7, 22, 24, 26, 29, and 30 have
been deposited with the Cambridge Crystallographic Data Centre
(deposition numbers from 803445 to 803450). Copies of the data
can be obtained, free of charge, on application to CCDC, 12 Union
Road, Cambridge, CB2 1EZ, UK [fax: +44(0) 1223 336033 or e-mail:
deposit@ccdc.cam.ac.uk].
4.4.1. Crystallographic details for compound 15
4.4.4. Crystallographic details for compound 30
The crystal of the dialdehyde (2,20-disulfanediylbis(2-ethylbut-
anal)), 7, was monoclinic in space group P21/c with unit cell dimen-
sions a = 10.221(4) Å, b = 10.239(4) Å, c = 13.839(5) Å, and b =
97.906(6)°. Data were 97.8% complete to 29.18°h (approximately
0.73 Å) with an average redundancy of 2.8.
The crystal of 2-(4-aminophenylthio)-4-oxoazetidine-1-
ꢀ
sulfonate, 30, was triclinic in space group P1 with unit cell
dimensions a = 9.8030(4) Å, b = 10.5558(4) Å, c = 13.7240(6) Å,
a
= 76.2140(10)°, b = 89.6720(10)°, and c = 83.076(2)°. Data were
97.7% complete to 29.19° h (approximately 0.73 Å) with an average
redundancy of 2.07.
4.4.5. Crystallographic details for compound 22
The crystal of 4-(phenylthio)azetidin-2-one, 22, was monoclinic
in space group P21/c with unit cell dimensions a = 12.3179(4) Å,
b = 7.8615(3) Å, c = 9.1767(3) Å, and b = 101.7610(10)°. Data were
98.0% complete to 29.61° h (approximately 0.73 Å) with an average
redundancy of 3.9.
4.4.2. Crystallographic details for compound 27
The crystal of 4-(4-aminophenylthio)azetidin-2-one, 27, was
monoclinic in space group P21/c with unit cell dimensions
a = 5.7085(12) Å, b = 8.0637(17) Å, c = 20.094(4) Å, and b =
95.954(6)°. Data were 98.2% complete to 25.73° h (approximately
0.82 Å) with an average redundancy of 4.09.
4.4.6. Crystallographic details for compound 25
The crystal of 4-(thiophen-2-ylthio)azetidin-2-one, 25, was
monoclinic in space group P21/c with unit cell dimensions
a = 15.1939(9) Å,
b = 5.4702(3) Å,
c = 29.9729(17) Å,
and

6852
M. B. Kostova et al. / Bioorg. Med. Chem. 19 (2011) 6842–6852
b = 97.2650(10)°. Data were 99.4% complete to 28.17° h (approxi-
sample (10 lL) was taken and applied to a MH plate, to test
mately 0.75 Å) with an average redundancy of 3.87.
whether cells were dead (MBC) or simple not growing (MIC). The
MBC was defined as the lowest concentration of drug in which
no growth occured. All tests were performed in triplicate.
The antimycobacterial effects of the compounds against M.
tuberculosis were tested by the Alamar blue assay as detailed in
Kim et al.31
Acknowledgements
We express our sincere thanks and appreciation to the donors
of The Research Corporation Cottrell, for the financial support of
this research project. This work was funded in part, by the Ameri-
can University Research Grant and the Department of Chemistry at
American University, Washington, DC and by Midwestern Univer-
sity. We are grateful to Joyce Tjhio of Loyola University Stritch
School of Medicine for generously providing us with the Moraxella
catarrhalis clinical isolates. This research was supported in part by
the Intramural Research Program of the NIH, NIAID.
4.5. Biological assays
Moraxella catarrhalis clinical isolates (n = 6) used were b-lacta-
mase (nitrocefin-cleavage assay positive) producing.29 The clinical
isolates used were kindly provided by J. Tjhio (Loyola University
Stritch School of Medicine, Maywood, IL). Moraxella catarrhalis
antimicrobial activity was evaluated by B. J. Plotkin and J. M. Green
(Midwestern University, Downers Grove, IL). Mycobacterium tuber-
culosis (Mtb) H37Rv was used for all Mtb susceptibility determina-
tions. These determinations were done by Helena Boshoff and
Clifton Barry, III (Tuberculosis Research Section, NIAID, NIH Bethes-
da, MD). In addition, non-b-lactamase producing quality control
strains of Escherichia coli, ATCC 25922; Pseudomonas aeruginosa,
ATCC 27853; Staphylococcus aureus, ATCC 25923; Enterococcus fae-
calis, ATCC 29212 were tested for anti-microbial susceptibility as
described below. Inability to cleave nitrocefin was confirmed for
all ATCC quality control strains. All isolates were maintained at
ꢀ80 °C until use.
References and notes
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taining compound (10 lg compound tested/disc) were placed on
plates inoculated with bacteria. Discs were prepared by dissolving
compounds in methanol, applying the solution to the disc then
drying discs at room temperature. Each agar plate with discs was
incubated at 37 °C overnight and the zone of inhibition measured
(mm of no growth from edge of disc). Controls consisted of discs
with diluent (methanol) alone and penicillin (positive control).
MIC/MBC assays were performed using the microdilution broth
method in accordance with the guidelines of the Clinical and Lab-
oratory Standards Institute.28 Compounds were prepared in 95%
ethanol then diluted at least 10-fold into Mueller-Hinton (MH)
broth. Each diluted compound solution (100
serially diluted in MH (100 L/well; series of two-fold dilutions,
between 500 g/mL and 15.6 g/mL). Controls consisted of ethanol
lL/well) was then
l
l
l
without compound and penicillin similarly diluted. To each well
100
l
L/well, 5 ꢂ 105 cells/mL of an actively growing culture MH
broth was added. Plates were incubated at 37 °C for 48 h, and
scored for growth at 24 and 48 h for visible growth. The MIC was
defined as the lowest concentration of drug at which no growth
was observed. Minimal bactericidal growth was also measured in
these experiments; in wells where no growth was observed, a