Azetidinone-isothiazolidinones: Stereoselective synthesis and antibacterial evaluation of new monocyclic beta-lactams

Article abstract of DOI:10.1016/j.bmc.2010.03.045

A simple and efficient procedure for the stereoselective synthesis of new azetidinone-isothiazolidinones has been developed. New compounds were tested in vitro on a panel of Gram-positive and Gram-negative bacterial pathogens, some of them showing weak antibacterial activity.

Full text of DOI:10.1016/j.bmc.2010.03.045

Bioorganic & Medicinal Chemistry 18 (2010) 3053–3058
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Azetidinone-isothiazolidinones: Stereoselective synthesis and antibacterial
evaluation of new monocyclic beta-lactams
a
b,
a,
c
d
*
ˇ
´
´
ˇ
´
´
´
Helena Ceric , Marija Šindler-Kulyk , Mice Kovacevic , Mihaela Peric , Andreja Zivkovic
a
´
PLIVA Croatia Ltd, TAPI R&D, Chemical R&D, Prilaz baruna Filipovica 25, 10000 Zagreb, Croatia
b Department of Organic Chemistry, University of Zagreb, Maruli´cev trg 19, 10000 Zagreb, Croatia
c Center for Translational Medicine, School of Medicine, University of Zagreb, Šalata 3, 10000 Zagreb, Croatia
d PLIVA Croatia Ltd, Generics R&D, Analytics R&D, Prilaz baruna Filipovi´ca 25, 10000 Zagreb, Croatia
a r t i c l e i n f o
a b s t r a c t
Article history:
A simple and efficient procedure for the stereoselective synthesis of new azetidinone-isothiazolidinones
has been developed. New compounds were tested in vitro on a panel of Gram-positive and Gram-nega-
tive bacterial pathogens, some of them showing weak antibacterial activity.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 26 October 2009
Revised 12 March 2010
Accepted 20 March 2010
Available online 27 March 2010
Keywords:
Beta-lactam
Antibacterial activity
Azetidinone-isothiazolidinone
1. Introduction
trans-chloro-azetidinone derivatives 2 was formed.15 The ring
opening of sulfoxide derivatives 3 and rearrangement to isothiazo-
Beta-lactam antibiotics are still the main drugs to treat infec-
tions caused by bacteria. These molecules disturb the final step
of bacterial cell wall biosynthesis by inhibiting penicillin binding
proteins (PBPs) involved in the crosslinking of peptidoglycan
strands.1
lidinone-azetidinones 4 by thionyl chloride was mentioned in the
patent application.20
Cl
Pht
Pht
S
The first classes of monocyclic beta-lactam antibacterial agents
were isolated from natural sources in the late 1970s and early
1980s. The discovery of nocardicines and monobactams (Fig. 1)
demonstrated for the first time that a conformationally constrained
bicyclic structure was not necessary for antibacterial activity.2–5 Re-
search efforts in this class led to preparation of semi-synthetic and
synthetic monobactam analogues like Aztreonam and Carumonam
(Fig. 1), active mostly against Gram-negative bacteria. Work in this
area is still very active and new compounds are synthesized and re-
N
N
O
O
CO2R
CO2R
2
1
O
S
H/Br
N
H/Br
Cl
O
Br
Br
N
S
N
R
R
O
O
N
ported as inhibitors of D,D
-transpeptidase,6 beta-lactamase,7 mam-
O
H
malian elastase,8,9 fatty acid amide hydrolase10,11 and cholesterol
uptake.12 Some of the compounds out of this class have also shown
antifungal13 and antitubercular activity.14
4
3
One of commonly used methods for the preparation of monocy-
clic beta-lactams from natural penicillins is an electrophilic ring
opening of thiazolidine moiety by chlorinating agents.15–19 In the
Azetidinone-isothiazolidinones,
a new class of monocyclic
beta-lactams, represent very interesting structures with two
uncondensed heterocyclic rings that could both act as a potential
pharmacophore. As a part of our work on the transformation of
natural penicillins into new monocyclic beta-lactam deriva-
tives21,22 with potential biological activity, the synthesis of new
azetidinone-isothiazolidinones was investigated. In this paper we
report a simple and efficient procedure for the preparation of
case of phthalimido-penam esters 1,
a mixture of cis- and
* Corresponding author. Tel.: +385 1 4597242; fax: +385 1 4597250.
E-mail address: marija.sindler@fkit.hr (M. Šindler-Kulyk).
Currently retired.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.045

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H. Ceric et al. / Bioorg. Med. Chem. 18 (2010) 3053–3058
3054
LC/MS. 6-Phthalimido-penam amides 5–9 were prepared in good
R1
O
yields (76–94%) by amidation of previously synthesized23 6-phtha-
limido-penicillanic acid (6-PhtPA). Amidation was carried out by a
method commonly applied in organic synthesis and in beta-lactam
chemistry.24 The reaction of corresponding phthalimido-penam
amides 5–9 with sulfuryl chloride gave azetidinone-isothiazolidi-
nones 10–14 as a mixture of cis- and trans-diastereomers. The ob-
tained diastereomeric pairs 10–14 were separated by column
chromatography. The use of thionyl chloride for the ring opening
of phthalimido-penam amides, as mentioned in the literature,20
was not successful. The reaction did not proceed at room temper-
ature, while under heating a multicomponent complex mixture of
undesired products was obtained.
R1
O
HN
OH
R2
HN
N
O
N
SO3H
COOH
O
monobactams
nocardicines
NH2
S
NH2
S
N
N
N
O
N
O
O
O
O
Thiazolidine ring opening of penam amides 5–9 is regiospecific
and highly stereoselective (Scheme 2). Monitoring the reaction
course by LC/MS the formation of two intermediates was observed,
presumably cis- and trans-A, which undergo intramolecular cycli-
zation to cis- and trans-azetidinone-isothiazolidinones 10–14,
respectively. The ratio of the diastereoisomers, determined by LC/
MS, clearly shows the predominance of the trans-isomers which
could be explained by steric hindrance of the bulky phthalimide
(Pht) substituent on beta-lactam ring.
Benzyl esters of compounds trans-10–12 were transformed to
carboxylic acid derivatives trans-15–17 (Scheme 3) by treatment
with aluminium chloride in nitromethane.
All new compounds 5–17 were fully characterized by spectro-
scopic and analytical data. Amide derivatives 5–9 in 1H NMR spec-
tra show characteristic NH signals in the range of 8.1–9.2 ppm with
corresponding splitting pattern, which disappear after a D2O shake.
The structure of compounds 10–14 was evident from C2–H chem-
ical shifts to lower field, due to introduction of chlorine at that
NH2
HN
HN
CH3
O
HOOC
HOOC
N
N
SO3H
SO3H
O
O
Aztreonam
Carumonam
Figure 1. Natural and synthetic monocyclic beta-lactams.
new phthalimido-azetidinone-thiazolidinones 10–17, starting
from new penam amides, and evaluation of their antibacterial
activity.
2. Results and discussion
2.1. Chemistry
Preparation of new compounds 5–14 is depicted on Scheme 1.
Reaction course and formation of products was monitored by
O
Compound R1
R2
O
------------------------------------
O
N
O
Cl
1) Cl-COOEt
5, 10
6, 11
7, 12
8, 13
9, 14
Ph COOBn
Me COOBn
2)
N
O
H2N
R2
O
SO2Cl2
DCM
S
N
O
N
S
R1
O
H
H
COOBn
Bn
O
N
N
S
DCM, 0 °C
O
N
Me
Me
NH
OH
O
O
R2
R2
R1
R1
6-PhtPA
10-14
5-9
Scheme 1. Synthesis of new compounds 5–14.
Pht
Cl
Pht
Pht
Cl
S
Cl
S
Cl
S
N
N
O
N
SO2Cl2
O
O
NH
NH
NH
O
O
O
R2
R1
trans-A
cis-A
5-9
Compound R1
R2
cis:trans [%]
Pht
Cl
Pht
Cl
-------------------------------------------------------
10
11
12
13
14
Ph COOBn 0:100
N
N
Me COOBn
8:92
4:96
O
O
H
COOBn
S
S
N
O
N
O
H
Me
Bn
Me
6:94
14:86
R2
R2
R1
R1
cis-10-14
trans-10-14
Scheme 2.

´
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H. Ceric et al. / Bioorg. Med. Chem. 18 (2010) 3053–3058
and R2 on isothiazolidinone nitrogen favour antibacterial activity.
Compounds 14–17 that have no aromatic substituents on isot-
hiazolidinone nitrogen were generally inactive against all tested
strains, while some modest activity could be observed with com-
pounds 10–13, irrespective of their diastereomeric form. In line
with that, the removal of aromatic benzyl ester group in com-
pounds trans-10–12 into carboxylic acids trans-15–17 resulted in
loss of antibacterial activity. Compound 10, with two aromatic R1
and R2 substituents, showed the best inhibitory activity against se-
lected Gram-positive strains with the best growth inhibition of S.
Pht
Cl
Pht
Cl
Compound R1
-----------------------
AlCl3
N
N
10, 15
11, 16
12, 17
Ph
Me
H
O
O
MeNO2
DCM
S
S
O
N
O
N
COOH
COOBn
R1
R1
trans-15-17
trans-10-12
Scheme 3. Synthesis of new azetidinone-isothiazolidinones trans-15–17.
pneumoniae (MIC 8 lg/mL). Similar SAR was observed with some
previously described 4-amino-isothiazolidinone derivatives.25
These compounds were found to be chemically instable, that could
potentially contribute to the poor antibacterial activity.
position and thiazolidine ring opening. Isothiazolidinone ring clo-
sure by S–N bond formation was evident from the absence of NH
signals, present in amides 5–9. The configuration of compounds
trans-10–14 and trans-15–17 is based on the beta-lactam coupling
constants of 1.3–1.8 Hz, characteristic for trans-configuration. In
compounds 5–9 and cis-11–14 the coupling constants are
3.9–4.3 Hz, in accordance with the cis-configuration of the beta-
lactam ring.
3. Experimental
3.1. General procedures
All melting points were determined on Büchi B-540 apparatus
in capillary tubes and are uncorrected. IR spectra were recorded
on KBr discs on a Perkin–Elmer SpectrumGX FT-IR spectrophotom-
2.2. In vitro screen
eter with a resolution of 4 cmÀ1 1H and 13C NMR spectra were
.
New compounds 5–17 were tested in vitro on a panel of se-
lected Gram-positive ( Staphylococcus aureus, Enterococcus faecalis
and Streptococcus pneumoniae) and Gram-negative (Escherichia coli,
Haemophilus influenzae and Moraxella catarrhalis) bacterial strains
(Table 1). The selected set of strains represents well described ref-
erence strains obtained from ATCC bioresource center.
The activity of penicillin amides 5–9 against selected set of bac-
terial strains could not be precisely determined since the MICs
were generally higher than the standard cut-off value (128 lg/
recorded on Bruker Avance 300 and Bruker Avance 600 spectrom-
eters with TMS as an internal standard. Column chromatography
was carried out on silica gel Merck Kieselgel 60, 0.04–0.063 mm.
HPLC was performed on Agilent 1100 instrument with DAD detec-
tor and ESI LC/MSD trap on column Phenomenex Gemini C18 with
gradient elution (acetonitrile/ammonium acetate buffer, pH
7.4 = 20:80 to 90:10). High Resolution Mass Spectra (HRMS) were
recorded on Micromass Q-Tof micro™ instrument equipped with
the lock-spray (Waters Corporation, Manchester, UK). Elemental
analysis was carried out on Perkin–Elmer 2400 Series II CHNS/O
analyzer. Water content was determined by coulometric Karl
Fischer titration on Metrohm 831 KF Coulometer. Rotations were
recorded on polAAr31 polarimeter, at 20 °C in chloroform (com-
pounds 5–14) or methanol (compounds 15–17). Concentrations
are given in percentage (g/100 mL).
mL) used in our in vitro screen. These compounds could be consid-
ered as antibacterially inactive.
Several compounds from the azetidinone-isothiazolidinone
class showed some improved antibacterial activity compared to
penicillin amides. The activity of this class of compounds against
Gram-positive strains was somewhat superior when compared to
Gram-negative strains. It is evident that aromatic substituents R1
Table 1
Antibacterial activity of new compounds 5–17 in comparison to amoxycillin
Core structure
Compounda
R1
R2
MIC (lg/mL)
Bacterial strainsb
4
1
2
3
5
6
5
6
Ph
Me
COOBn
COOBn
>128
>128
Pht
S
7
8
9
H
H
Me
COOBn
Bn
Me
64–128
64–128
>128
N
O
NH
O
R2
R1
trans-10
trans-15
trans-11
cis-11
trans-16
trans-12
cis-12
trans-17
trans-13
cis-13
COOBn
COOH
COOBn
16
64
8
64
>128
32–64
32
>128
>128
>128
>128
Pht
Cl
Ph
Me
64
64–128
N
O
COOH
COOBn
>128
32–64
S
H
64
32
64
>128
>128
>128
>128
O
N
R2
COOH
Bn
>128
64
R1
H
64–128
16
128
trans-14
cis-14
Amoxycillin
Me
Me
64–128
0.5
4
4
1
0.5
0.015
a
Stability at testing conditions (pH 7.4; T = 35 °C) of all compounds was confirmed by LC/MS (Supplementary data).
E. faecalis ATCC 29212 (1); S. pneumoniae ATCC 6305 (2); M. catarrhalis ATCC 23246 (3); S. aureus ATCC 29213 (4); E. coli ATCC 25922 (5); H. influenzae ATCC 49247 (6).
b

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H. Ceric et al. / Bioorg. Med. Chem. 18 (2010) 3053–3058
3056
3.2. Chemistry
amide (9). Yield 85%; mp: 85–90 °C; [a]D = +330.7 (c 0.1, chloro-
form); IR/cmÀ1 (KBr): 3367, 2972, 1800, 1780, 1727, 1654, 1526,
1387, 719; 1H NMR/ppm (300 MHz, DMSO-d6): 1.09 (d, Me,
J = 6.2 Hz), 1.11 (d, Me, J = 6.4 Hz), 1.42 (s, Me), 1.67 (s, Me), 3.88
(m, 1H), 4.40 (s, C2–H), 5.56 (d, C5–H, J = 3.9 Hz), 5.74 (d, C6–H,
J = 3.9 Hz), 7.88–7.95 (m, 4H-Pht), 8.12 (d, NH, J = 7.7 Hz); 13C
NMR/ppm (150 MHz, DMSO-d6): 22.6, 22.7, 27.5, 31.8, 41.0, 59.2,
66.0, 67.3, 71.0, 124.0, 131.4, 135.5, 166.2, 166.7, 168.4; HRMS
calcd for C19H21N3O4S [M+Na]+: 410.1150. Found: 410.1151.
3.2.1. General procedure for the preparation of compounds 5–9
6-PhtPA (10.0 g, 28.9 mmol) was suspended in dichlorometh-
ane (DCM; 300 mL) and dissolved by the addition of triethylamine
(TEA; 4.0 mL, 28.9 mmol) with stirring at room temperature. The
solution was cooled to 0 °C and ethyl chloroformate (2.62 mL,
32.4 mmol) in DCM (150 mL) was added. The reaction mixture
was stirred for 1/2 h at 0 °C. Corresponding amine (29.9 mmol)
was dissolved in DCM (40 mL) (with the addition of eqTEA, if nec-
essary) and added drop wise to the reaction mixture during 1/2 h
at 0 °C. After 2 h of stirring at 0 °C, water (500 mL) was added to
the reaction mixture and the layers were separated. Organic layer
was washed with water (500 mL), dried over sodium sulfate and
solvent was evaporated under reduced pressure.
3.2.2. General procedure for the preparation of compounds
10–14
Corresponding compound 5–9 (1.0 mmol) was dissolved in
DCM (20 mL) and sulfuryl chloride (1.0 mmol, 82.9 lL) was added.
The reaction mixture was stirred for 1 h at room temperature and
water was added (20 mL). The layers were separated and organic
layer was washed with water (20 mL), dried over sodium sulfate
and solvent was evaporated under reduced pressure. Diastereo-
meric mixtures 10–14 were separated by column chromatography
(toluene/EtOA c 3:1) of the residue after evaporation to obtain cis-
and trans-diastereomers.
3.2.1.1. (S)-Benzyl 2-((2S,5R,6R)-6-(1,3-dioxoisoindolin-2-yl)-
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-heptane-2-car-
boxamido)-2-phenylacetate (5). Yield 76%; mp: 122–125 °C;
[a]
D = +360.1 (c 0.1, chloroform); IR/cmÀ1 (KBr): 3362, 1798,
1778, 1729, 1681, 1504, 1385, 1202, 1172, 720, 698; 1H NMR/
ppm (600 MHz, DMSO-d6): 1.42 (s, Me), 1.67 (s, Me), 4.70 (s, C2–
H), 5.14 and 5.17 (2d, CH2, J = 12.2 Hz), 5.53 (d, CH, J = 6.6 Hz),
5.56 (d, C5–H, J = 4.1 Hz), 5.75 (d, C6–H, J = 4.0 Hz), 7.22–7.33 (m,
5H-Ph), 7.39–7.43 (m, 5H-Ph), 7.88–7.93 (m, 4H-Pht), 9.19 (d,
NH, J = 6.4 Hz); 13C NMR/ppm (150 MHz, DMSO-d6): 27.2, 31.4,
56.3, 58.9, 65.8, 66.4, 67.1, 69.7, 123.6, 127.7, 127.8, 127.9, 128.3,
128.5, 128.8, 130.9, 135.0, 135.5, 166.2, 166.7, 167.7, 170.0; HRMS
calcd for C31H27N3O6S [M+Na]+: 592.1518. Found: 592.1546.
3.2.2.1. (S )-Benzyl 2-((S)-4-((2S,3R)-2-chloro-3-(1,3-dioxoisoin-
dolin-2-yl)-4-oxoazetidin-1-yl)-5,5-dimethyl-3-oxoisothiazoli-
din-2-yl)-2-phenylacetate (trans-10). Yield94%;mp147–150 °C;
[
a
]
D = À51.9 (c 0.1, chloroform); IR/cmÀ1 (KBr): 3375, 2921, 1797,
1720, 1668, 1497, 1394, 1169, 715, 698; 1H NMR/ppm (300 MHz,
CDCl3): 1.38 (s, Me), 1.67 (s, Me), 4.43 (s, 1H), 5.32 (s, CH2), 5.57 (d,
C3–H, J = 1.8 Hz), 6.19 (d, C2–H, J = 1.8 Hz), 6.22 (s, CH), 7.25–7.44
(m, 10H, Ph x 2), 7.75–7.94 (m, 4H-Pht); 13C NMR/ppm (75 MHz,
CDCl3): 22.1, 25.4, 54.6, 59.6, 64.3, 65.3, 67.4, 70.7, 123.9, 128.3,
128.4, 128.4, 128.5, 128.6, 128.8, 129.1, 129.2, 131.3, 131.4, 132.8,
134.7, 161.0, 166.2, 166.4, 168.8; HRMS calcd for C31H26N3O6SCl
[M+Na]+: 626.1129. Found: 626.1130.
3.2.1.2. (S)-Benzyl 2-((2S,5R,6R)-6-(1,3-dioxoisoindolin-2-yl)-
3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-car-
boxamido)propanoate (6). Yield 94%; mp: 65–70 °C;
[a] =
D
+290.9 (c 0.1, chloroform); IR/cmÀ1 (KBr): 3390, 1800, 1780,
1727, 1674, 1519, 1386, 1201, 720; 1H NMR/ppm (600 MHz,
DMSO-d6): 1.37 (d, Me, J = 7.3 Hz), 1.42 (s, Me), 1.68 (s, Me), 4.43
(pent, CH, J = 7.2 Hz), 4.53 (s, C2–H), 5.16 and 5.19 (2d, CH2,
J = 12.5 Hz), 5.57 (d, C5–H, J = 4.0 Hz), 5.76 (d, C6–H, J = 4.0 Hz),
7.36–7.40 (m, 5H-Ph), 7.90–7.95 (m, 4H-Pht), 8.78 (d, NH,
J = 7.1 Hz); 13C NMR/ppm (150 MHz, DMSO-d6): 16.5, 26.7, 30.6,
47.2, 58.2, 65.1, 65.6, 66.2, 70.0, 123.1, 127.3, 127.6, 128.0, 130.4,
134.5, 135.4, 165.7, 166.3, 167.6, 171.5; HRMScalcd for C26H25N3O6S
[M+Na]+: 530.1362. Found: 530.1352.
3.2.2.2. (S)-Benzyl 2-((S)-4-((2R,3R)-2-chloro-3-(1,3-dioxoisoin-
dolin-2-yl)-4-oxoazetidin-1-yl)-5,5-dimethyl-3-oxoisothiazoli-
din-2-yl)propanoate (cis-11). Yield 7%; mp 81–84 °C;
[a] =
D
+38.1 (c 0.1, chloroform); IR/cmÀ1 (KBr): 3488, 2930, 1794, 1775,
1728, 1686, 1456, 1391, 1310, 1202, 927, 722; 1H NMR/ppm
(600 MHz, DMSO-d6): 1.38 (d, Me, J = 7.1 Hz), 1.55 (s, Me), 1.64
(s, Me), 4.77 (s, 1H), 5.05 (q, CH, J = 7.1 Hz), 5.15 and 5.18 (2d,
CH2, J = 12.4 Hz), 5.89 (d, C3–H, J = 4.2 Hz), 6.73 (d, C2–H,
J = 4.2 Hz), 7.35–7.40 (m, 5H-Ph), 7.93–8.00 (m, 4H-Pht); 13C
NMR/ppm (150 MHz, DMSO-d6): 15.2, 23.9, 25.0, 51.9, 55.7, 59.9,
64.4, 67.2, 72.2, 124.4, 128.4, 128.7, 129.0, 131.3, 135.8, 136.0,
163.6, 167.4, 170.2; HRMS calcd for C26H24N3O6SCl [M+Na]+:
564.0972. Found: 564.0972.
3.2.1.3. Benzyl 2-((2S,5R,6R)-6-(1,3-dioxoisoindolin-2-yl)-3,3-
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxa-
mido)acetate (7). Yield 91%; mp: 93–97 °C; [a]D = +247.6 (c 0.1,
chloroform); IR/cmÀ1 (KBr): 3553, 1790, 1768, 1727, 1675, 1540,
1384, 1313, 1201, 719; 1H NMR/ppm (600 MHz, DMSO-d6): 1.43
(s, Me), 1.66 (s, Me), 4.00 (d, CH2, J = 5.8 Hz), 4.48 (s, C2–H), 5.15
(s, CH2), 5.55 (d, C5–H, J = 4.0 Hz), 5.74 (d, C6–H, J = 4.1 Hz), 7.34–
7.39 (m, 5H-Ph), 7.89–7.94 (m, 4H-Pht), 8.79 (t, NH, J = 5.8 Hz);
13C NMR/ppm (150 MHz, DMSO-d6): 27.2, 30.4, 40.6, 58.2, 65.3,
66.3, 66.3, 70.7, 123.6, 128.1, 128.1, 128.4, 130.8, 135.0, 135.7,
166.2, 167.4, 168.4, 169.3; HRMS calcd for C25H23N3O6S [M+Na]+:
516.1205. Found: 516.1207; Anal. Calcd for C25H23N3O6S Â
1.5H2O: C, 57.68; H, 5.03; N, 8.07; O, 23.05; S, 6.16. Found: C,
57.95; H, 5.21; N, 7.95; S, 6.22; KFq (water): 5.25% (calcd: 5.19%).
3.2.2.3. (S )-Benzyl 2-((S)-4-((2S,3R)-2-chloro-3-(1,3-dioxoisoin-
dolin-2-yl)-4-oxoazetidin-1-yl)-5,5-dimethyl-3-oxoisothiazoli-
din-2-yl)propanoate (trans-11). Yield 63%; mp 68–70 °C;
[
a
]
D = À45.0 (c 0.1, chloroform); IR/cmÀ1 (KBr): 3375, 2925, 1796,
1775, 1726, 1695, 1456, 1395, 1310, 1103, 962, 723; 1H NMR/
ppm (600 MHz, DMSO-d6): 1.37 (d, Me, J = 7.1 Hz), 1.59 (s, Me),
1.59 (s, Me), 4.51 (s, 1H), 5.05 (q, CH, J = 7.2 Hz), 5.16 and 5.18
(2d, CH2, J = 12.5 Hz), 5.58 (d, C3–H, J = 1.6 Hz), 6.43 (d, C2–H,
J = 1.6 Hz), 7.38–7.40 (m, 5H-Ph), 7.90–7.96 (m, 4H-Pht); 13C
NMR/ppm (150 MHz, DMSO-d6): 14.7, 21.8, 25.8, 50.9, 53.9, 63.2,
64.5, 66.6, 71.7, 123.6, 128.1, 128.2, 128.4, 131.2, 135.0, 135.4,
161.2, 166.1, 166.2, 169.7; HRMS calcd for C26H24N3O6SCl
[M+Na]+: 564.0972. Found: 564.0983.
3.2.1.4. (2S,5R,6R)-N -Benzyl-6-(1,3-dioxoisoindolin-2-yl)-3,3-
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carbox-
amide (8). Yield 91%; mp: 75–79 °C;
[a]D = +265.5 (c 0.1,
chloroform).22
3.2.2.4. Benzyl 2-((S)-4-((2R,3R)-2-chloro-3-(1,3-dioxoisoindo-
3.2.1.5. (2S,5R,6R)-6-(1,3-Dioxoisoindolin-2-yl)-N-isopropyl-3,3-
lin-2-yl)-4-oxoazetidin-1-yl)-5,5-dimethyl-3-oxoisothiazolidin-
dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-heptane-2-carbox-
2-yl)acetate (cis-12). Yield 3%; mp: 136–139 °C; [a]D = +26.7 (c

´
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H. Ceric et al. / Bioorg. Med. Chem. 18 (2010) 3053–3058
0.1, chloroform); IR/cmÀ1 (KBr): 3420, 2971, 1794, 1750, 1726,
1700, 1457, 1395, 1193, 1104, 963, 725; 1H NMR/ppm (600 MHz,
DMSO-d6): 1.63 (s, Me), 1.66 (s, Me), 4.29 and 4.34 (2d, CH2,
J = 17.9 Hz), 4.82 (s, 1H), 5.16 and 5.19 (2d, CH2, J = 12.4 Hz), 5.90
(d, C3–H, J = 4.1 Hz), 6.78 (d, C2–H, J = 4.3 Hz), 7.34–7.39 (m, 5H-
Ph), 7.93–8.00 (m, 4H-Pht); 13C NMR/ppm (150 MHz, DMSO-d6):
23.9, 24.6, 45.4, 55.4, 59.4, 63.2, 66.5, 71.4, 123.8, 128.1, 128.2,
128.4, 130.8, 135.3, 135.4, 163.1, 166.2, 167.1, 167.4; HRMS calcd
for C25H22N3O6SCl [M+Na]+: 550.0816. Found: 550.0823.
C2–H, J = 1.4 Hz), 7.90–7.95 (m, 4H-Pht); 13C NMR/ppm
(150 MHz, DMSO-d6): 19.7, 19.9, 21.7, 26.1, 44.8, 53.2, 64.1, 64.5,
71.7, 123.6, 131.2, 135.0, 161.2, 165.1, 166.2; HRMS calcd for
C19H20N3O6SCl [M+Na]+: 444.0761. Found: 444.0756.
3.2.3. General procedure for the preparation of compounds
15–17
Corresponding compound trans-10–12 (0.19 mmol) was dis-
solved in DCM (10 mL) and cooled to 0 °C. Aluminium chloride
(150 mg; 1.12 mmol) was dissolved in nitromethane (5 mL) and
added drop wise to the DCM solution. The reaction mixture was
stirred for 1/2 h at 0 °C. EtOAc (10 mL) was added and the solution
acidified with HCl, 0.1 M (15 mL). The layers were separated and
organic layer was washed with water (20 mL), dried over sodium
sulfate and solvent evaporated under reduced pressure. Com-
pounds trans-15–17 were purified by acid–base extractions in
water/DCM system by alkalizing with NaHCO3, satd to pH 8 and
acidifying with HCl, 1.2 M to pH 2.
3.2.2.5. Benzyl 2-((S)-4-((2S,3R)-2-chloro-3-(1,3-dioxoisoindo-
lin-2-yl)-4-oxoazetidin-1-yl)-5,5-dimethyl -3-oxoisothiazolidin-
2-yl)acetate (trans-12). Yield 60%; mp: 88–90 °C; [
a
]
D = À69.8 (c
0.1, chloroform); IR/cmÀ1 (KBr): 2968, 1795, 1750, 1726, 1700,
1456, 1396, 1192, 1104, 963, 725; 1H NMR/ppm (600 MHz,
DMSO-d6): 1.64 (s, Me), 1.67 (s, Me), 4.22 and 4.36 (d, CH2, J =
17.7 Hz), 4.58 (s, 1H), 5.18 (s, CH2), 5.57 (d, C3–H, J = 1.6 Hz), 6.43
(d, C2–H, J = 1.6 Hz), 7.37–7.39 (m, 5H-Ph), 7.89–7.96 (m, 4H-
Pht); 13C NMR/ppm (150 MHz, DMSO-d6): 22.4, 25.6, 45.4, 54.2,
63.0, 64.3, 66.4, 71.6, 123.6, 128.1, 128.2, 128.4, 131.2, 135.0,
135.5, 161.3, 166.2, 166.4, 167.5; HRMS calcd for C25H22N3O6SCl
[M+Na]+: 550.0816. Found: 550.0814.
3.2.3.1. (S)-2-((S)-4-((2S ,3R)-2-Chloro-3-(1,3-dioxoisoindolin-2-
yl)-4-oxoazetidin-1-yl)-5,5-dimethyl-3-oxoisothiazolidin-2-yl)-
2-phenylacetic acid (trans-15). Yield 65%; mp 111–114 °C;
[
a
]
D = À57.8 (c 0.1, methanol); IR/cmÀ1 (KBr): 3444, 2927, 1778,
3.2.2.6. 2-((2R,3R)-1-((S )-2-Benzyl-5,5-dimethyl-3-oxoisothiaz-
olidin-4-yl)-2-chloro-4-oxoazetidin-3-yl)isoindoline-1,3-dione
(cis-13). Yield 1%; mp 203–206 °C; [a]D = +17.3 (c 0.1, chloro-
1731, 1668, 1628, 1615, 1361, 1296, 703; 1H NMR/ppm (300
MHz, CDCl3): 1.39 (s, Me), 1.76 (s, Me), 4.35 (s, 1H), 5.61 (d, C3–H,
J = 1.6 Hz), 6.24 (s, 1H, CH(Ph)), 6.30 (d, C2–H, J = 1.6 Hz), 7.40 (s,
5H-Ph), 7.77–7.91 (m, 4H-Pht); 13C NMR/ppm (75 MHz, CDCl3):
22.3, 25.2, 54.5, 59.7, 64.6, 65.1, 70.8, 123.6, 128.5, 128.6, 128.7,
129.0, 129.2, 131.3, 131.5, 133.0, 161.1, 166.1, 166.4, 170.7; HRMS
calcd for C24H20N3O6SCl [M+Na]+: 536.0659. Found: 536.0655.
form); IR/cmÀ1 (KBr): 3309, 2972, 1789, 1772, 1724, 1686, 1456,
1389, 1202, 723; 1H NMR/ppm (600 MHz, DMSO-d6): 1.50 (s,
Me), 1.62 (s, Me), 4.56 and 4.60 (2d, CH2, J = 15.0 Hz), 4.88 (s,
1H), 5.92 (d, C3–H, J = 4.3 Hz), 6.85 (d, C2–H, J = 4.2 Hz), 7.29–7.38
(m, 5H-Ph), 7.93–8.00 (m, 4H-Pht); 13C NMR/ppm (150 MHz,
DMSO-d6): 24.0, 24.7, 47.7, 54.6, 59.3, 63.6, 71.6, 123.8, 127.9,
128.1, 128.5, 130.8, 135.3, 135.7, 163.0, 166.6; HRMS calcd for
C23H20N3O6SCl [M+Na]+: 492.0761. Found: 492.0762.
3.2.3.2. (S)-2-((S)-4-((2S ,3R)-2-Chloro-3-(1,3-dioxoisoindolin-2-
yl)-4-oxoazetidin-1-yl)-5,5-dimethyl-3-oxoisothiazolidin-2-yl)-
propanoic acid (trans-16). Yield 55%; mp 83–86 °C; [
a
]
D = À43.4
(c 0.1, methanol); IR/cmÀ1 (KBr): 3381, 2927, 1797, 1728, 1694,
1456, 1395, 1103, 722; 1H NMR/ppm (600 MHz, DMSO-d6): 1.32
(d, Me, J = 7.2 Hz), 1.59 (s, Me), 1.67 (s, Me), 4.53 (s, 1H), 4.88 (q,
CH, J = 7.2 Hz), 5.57 (d, C3–H, J = 1.4 Hz), 6.44 (d, C2–H, J = 1.3 Hz),
7.90–7.96 (m, 4H-Pht); 13C NMR/ppm (150 MHz, DMSO-d6): 14.7,
22.0, 25.5, 50.8, 53.9, 63.6, 64.4, 71.7, 123.6, 131.2, 135.0, 161.2,
166.0, 166.2, 171.4; HRMS calcd for C19H18N3O6SCl [M+Na]+:
474.0503. Found: 474.0506.
3.2.2.7. 2-((2S,3R)-1-((S )-2-Benzyl-5,5-dimethyl-3-oxoisothiaz-
olidin-4-yl)-2-chloro-4-oxoazetidin-3-yl)isoindoline-1,3-dione
(trans-13). Yield 54%; mp 70–72 °C; [
a
]
D = À43.3 (c 0.1, chloro-
form); IR/cmÀ1 (KBr): 2971, 1793, 1770, 1727, 1689, 1456, 1395,
1104, 722; 1H NMR/ppm (600 MHz, DMSO-d6): 1.55 (s, Me), 1.56
(s, Me), 4.45 and 4.67 (2d, CH2, J = 14.9 Hz), 4.62 (s, 1H), 5.59 (d,
C3–H, J = 1.6 Hz), 6.42 (d, C2–H, J = 1.6 Hz), 7.31–7.37 (m, 5H-Ph),
7.90–7.96 (m, 4H-Pht); 13C NMR/ppm (150 MHz, DMSO-d6): 22.5,
25.8, 47.8, 53.3, 63.7, 64.2, 71.6, 123.6, 127.8, 128.3, 128.5, 131.2,
135.0, 135.7, 161.3, 165.9, 166.2; HRMS calcd for C23H20N3O6SCl
[M+Na]+: 492.0761. Found: 492.0770.
3.2.3.3. 2-((S)-4-((2S,3R )-2-Chloro-3-(1,3-dioxoisoindolin-2-yl)-
4-oxoazetidin-1-yl)-5,5-dimethyl-3-oxoisothiazolidin-2-yl)ace-
tic acid (trans-17). Yield 29%; mp 84–88 °C; [
a
]
D = À56.9 (c 0.1,
methanol); IR/cmÀ1 (KBr): 3375, 2929, 1795, 1727, 1692, 1396,
1105, 723; 1H NMR/ppm (600 MHz, DMSO-d6): 1.65 (s, Me), 1.69
(s, Me), 3.98 and 4.21 (d, CH2, J = 17.7 Hz), 4.56 (s, 1H), 5.57 (d,
C3–H, J = 1.5 Hz), 6.43 (d, C2–H, J = 1.4 Hz), 7.90–7.97 (m, 4H-Pht);
13C NMR/ppm (150 MHz, DMSO-d6): 22.4, 25.5, 45.4, 54.1, 63.1,
64.2, 71.6, 123.6, 131.2, 135.0, 161.3, 166.2, 166.4, 168.9; HRMS
calcd for C18H16N3O6SCl [M+Na]+: 460.0346. Found: 460.0354.
3.2.2.8. 2-((2R,3R)-2-Chloro-1-((S)-2-isopropyl-5,5-dimethyl-3-
oxoisothiazolidin-4-yl)-4-oxoazetidin-3-yl)isoindoline-1,3-
dione (cis-14). Yield 8%; mp 102–105 °C; [a]D = +31.2 (c 0.1, chlo-
roform); IR/cmÀ1 (KBr): 3490, 2972, 1795, 1782, 1726, 1682, 1467,
1395, 1103, 720; 1H NMR/ppm (600 MHz, DMSO-d6): 1.09 (d, Me,
J = 6.7 Hz), 1.13 (d, Me, J = 6.5 Hz), 1.54 (s, Me), 1.65 (s, Me), 4.43
(sept, CH, J = 6.6 Hz), 4.76 (s, 1H), 5.89 (d, C3–H, J = 4.1 Hz), 6.78
(d, C2–H, J = 4.2 Hz), 7.92–7.98 (m. 4H-Pht); 13C NMR/ppm
(150 MHz, DMSO-d6): 19.3, 20.5, 23.7, 24.2, 45.0, 54.2, 59.3, 64.3,
71.7, 123.8, 130.8, 135.8, 163.1, 165.7, 166.4; HRMS calcd for
C19H20N3O6SCl [M+Na]+: 444.0761. Found: 444.0740.
3.3. In vitro antibacterial screen
The antibacterial testing was performed by determining the
MICs using the broth microdilution method as recommended by
the CLSI (formerly the National Committee for Clinical Laboratory
Standards)26 with minor modifications. Cation-adjusted Mueller–
Hinton broth (Difco Laboratories) was used and was supplemented
with 5% horse serum for S. pneumoniae while HTM (Haemophilus
Test Medium) was used for H. influenzae. Suspensions with a tur-
bidity equivalent to that of a 0.5 McFarland standard were pre-
pared by suspending the colonies from overnight agar cultures in
2 mL of sterile saline. The suspensions were further diluted in
3.2.2.9. 2-((2S,3R)-2-Chloro-1-((S)-2-isopropyl-5,5-dimethyl-3-
oxoisothiazolidin-4-yl)-4-oxoazetidin-3-yl)isoindoline-1,3-
dione (trans-14). Yield 42%; mp 116–119 °C; [
a
]D = À81.4 (c 0.1,
chloroform); IR/cmÀ1 (KBr): 3447, 2972, 1794, 1780, 1726, 1683,
1467, 1396, 1104, 721; 1H NMR/ppm (600 MHz, DMSO-d6): 1.11
(d, Me, J = 6.5 Hz), 1.13 (d, Me, J = 6.6 Hz), 1.60 (s, Me), 1.60 (s,
Me), 4.43 (m, CH), 4.45 (s, CH), 5.56 (d, C3–H, J = 1.5 Hz), 6.44 (d,

´
H. Ceric et al. / Bioorg. Med. Chem. 18 (2010) 3053–3058
3058
appropriate media to obtain a final inoculum of 1 Â 104 CFU/well.
The trays were incubated overnight in ambient air at 35 °C.
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Supplementary data
Supplementary data (copies of 1H and 13C NMR spectra of all the
compounds, 2D NMR spectra (COSY, HMQC, HMBC) of compound
cis-13 and LC/MS chromatograms of all the compounds) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmc.2010.03.045.
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