N/C-4 substituted azetidin-2-ones: Synthesis and preliminary evaluation as new class of antimicrobial agents

Article abstract of DOI:10.1016/j.bmcl.2006.10.064

A series of 3-chloro-4-(3-methoxy-4-acetyloxyphenyl)-1-[3-oxo-3-(phenylamino)propanamido] azetidin-2-ones 3a-g and 3-chloro-4-[2-hydroxy-5-(nitro substituted phenylazo)phenyl]-1-phenylazetidin-2-ones 6a-h were synthesized using appropriate synthetic route

Full text of DOI:10.1016/j.bmcl.2006.10.064

Bioorganic & Medicinal Chemistry Letters 17 (2007) 341–345
N/C-4 substituted azetidin-2-ones: Synthesis and preliminary
evaluation as new class of antimicrobial agents
Anand K. Halve,* Deepti Bhadauria and Rakesh dubey
School of Studies in Chemistry, Jiwaji University, Gwalior 474011, India
Received 22 June 2006; revised 28 September 2006; accepted 21 October 2006
Available online 26 October 2006
Abstract—A series of 3-chloro-4-(3-methoxy-4-acetyloxyphenyl)-1-[3-oxo-3-(phenylamino)propanamido] azetidin-2-ones 3a–g and
3-chloro-4-[2-hydroxy-5-(nitro substituted phenylazo)phenyl]-1-phenylazetidin-2-ones 6a–h were synthesized using appropriate syn-
thetic route. Structures of all the synthesized compounds were established on the basis of elemental analysis and spectroscopic data.
The antimicrobial activity of the synthesized compounds was screened against several microbes. Several of these molecules showed
potent antimicrobial activity against Bacillus anthracis, Staphyococcus aureus and Candida albicans and significant structure–activity
relationship (SAR) trends.
Ó 2006 Elsevier Ltd. All rights reserved.
Nitrogen heterocycles are the basis of many essential
pharmaceuticals and of many physiologically active nat-
ural products.1 The 2-azetidinone (b-lactam) ring system
is the common structural feature of a number of broad
spectrum b-lactam antibiotics, including penicillins,
cephalosporins, carbapenems, nocardicins and mono-
bactams, which have been widely used as chemothera-
peutic agents to treat bacterial infections and
microbial diseases.2,3 These molecules operate by form-
ing a covalent adduct with membrane-bound bacterial
transpeptidases, which are also known as penicillin
binding proteins (PBPs), involved in the biosynthesis
of cell walls.4,5 These mechanism-based inhibitors pre-
vent the construction of cell wall and eventually lead
to cell lysis and death. Moreover, due to their b-lacta-
mase inhibitory action, 2-azetidinone-based heterocycles
represent an attractive target of contemporary organic
synthesis.6 However, the efficacy of b-lactam antibiotics
has been overshadowed in the last 20 years by the emer-
gence of drug-resistant bacterial strains resulting from
evolutionary responses to the widespread overuse and
abuse of antibiotics in clinical traits.7 Consequently,
strategies to address this challenge lead to the design
of improved versions of b-lactams with novel modes of
action. It has been reported that introduction of differ-
ent substituents to four-membered b-lactam nucleus
tends to exert profound influence in conferring promis-
ing biological activities in these molecules.8–10
The recent discoveries of some 1,3,4-trisubstituted-b-
lactams as new potent cholesterol absorption inhibi-
tors,11 human cytomegalovirus protease inhibitors12
and thrombin inhibitors13 justify a renewed interest
in these compounds.14,15 Furthermore, interest in the
chemistry, synthesis and biology of the 2-azetidinone
pharmacophore continues to be fuelled by their
wide range of biological properties such as anti-
bacterial,16,17
antihyperglycemic,18
anti-tumour,19
anti-HIV,20 anti-inflammatory and analgesic activi-
ties.21 In addition, 2-azetidinones also display a broad
range of enzyme-inhibitory activities.22–26
Looking to the promising antimicrobial activity of some
2-azetidinone27,28 and 1,3-diketoamino29 analogues syn-
thesized earlier by us it was thought of interest to com-
bine all the above-mentioned biolabile heterocyclic rings
together in a molecular framework of hydrazones and
imines in order to enhance the additive effect toward
the biological activity. Keeping this in mind and in con-
tinuation of our earlier studies, we designed and synthe-
sized
3-chloro-4-(3-methoxy-4-acetyloxyphenyl)-1-[3-
oxo-3-(phenyl amino)propanamido] azetidin-2-ones
3a–g and 3-chloro-4-[2-hydroxy-5-(nitro substituted
phenylazo)phenyl]-1-phenylazetidin-2-ones 6a–h with
fascinating structural features. Moreover, in order to
assess the antimicrobial potentiality of 2-azetidinone
Keywords: Azetidin-2-ones; SAR; Pharmacophore; B. anthracis;
C. albicans.
*
Corresponding
drhalve_chemjiwaji@rediffmail.com
author.
Tel.:
+91
751
4016769; e-mail:
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.10.064

342
A. K. Halve et al. / Bioorg. Med. Chem. Lett. 17 (2007) 341–345
nucleus and to investigate the structure-activity-relation-
ship, the constructed molecules were screened for their
antibacterial and antifungal activities.
substituted malonanilic acid hydrazides were synthe-
sized by using reported methodology. The reactive imine
derivatives 5a–h were accessible via the reaction of an
equimolar quantity of 2-hydroxy-5-(nitro substituted
phenylazo) benzaldehyde 4a and b and appropriate aro-
matic amines in ethanol at room temperature, which
resulted in the formation of 2-hydroxy-5-(nitro substi-
tuted phenylazo) benzylidene anilines 5a–h in excellent
yield.31 2-Hydroxy-5-(nitrosubstituted phenylazo) ben-
zaldehydes 4 were prepared by diazotization of 3-nitro
aniline and 4-nitro aniline in acidic medium at 0–5 °C
and coupling with salicylaldehyde in basic medium at
0–5 °C, respectively.
The synthetic pathway includes an electrocyclisation
reaction of 1,3-diketoamino compounds 2a–g and imine
derivatives 5a–h to give direct access to the desired
3-chloro-4-(3-methoxy-4-acetyloxyphenyl)-1-[3-oxo-3-
(phenylamino)propanamido] azetidin-2-ones 3a–g and
3-chloro-4-[2-hydroxy-5-(nitro substituted phenylazo)-
phenyl]-1-phenylazetidin-2-ones
6a–h,
respectively
(Scheme 1). The approach involves the initial prepara-
tion of precursors, viz.; 1-(3000-methoxy-4000-acetyloxy-
benzalhydrazino)-3-(phenylamino)propan-1,3-diones
2a–g and substituted 2-hydroxy-5-(nitro substituted
phenylazo) benzylidene anilines 5a–h. Condensation of
3-methoxy-4-acetyloxy benzaldehyde 1 with substituted
malonanilic acid hydrazides in ethanol yielded the 1,3-
diketoamino analogs 2a–g.30 3-methoxy-4-acetyloxy
benzaldehyde 1 was prepared by the usual acetylation
of 3-methoxy-4-hydroxy benzaldehyde at 0–5 °C. The
Further, the initially formed 1,3-diketoamino mole-
cules 2a–g and reactive imines 5a–h underwent an
electrocyclisation reaction of chloroacetyl chloride
and triethyl amine in 1,4-dioxane afforded the target
compounds 3-chloro-4-(3-methoxy-4-acetyloxyphenyl)-
1-[3-oxo-3-(phenylamino)propanamido] azetidin-2-ones
3a–g and 3-chloro-4-[2-hydroxy-5-(nitro substituted
CHO
N
N
CHO
OH
O
H C
C
O
3
NO
OCH
2
3
4a,b
1
O
O
H2N
(i)
(i)
NH
HN
H N
2
2
R
(ii)
1
(ii)
R
2
R
O
O
NH
N
HN
N
CH
1
N
N
CH
R
O
H C
C
O
3
OCH
3
OH
NO
2
5a-h
2a-g
O
O
(iii)
Cl
(iv)
(iii)
Cl
(iv)
Cl
Cl
2
R
O
O
O
NH
HN
O
N
N
1
R
Cl
N
N
Cl
H
H
H
H
O
H C
C
3
OH
NO2
OCH
3
O
6a-h
Compound
3a-g
m
p-NO
-NO
4a
4b
2
2
Scheme 1. Reagents and conditions: (ii) EtOH, reflux, 2 h; (iv) Et3N, 1,4-dioxane, stirred 1 h, reflux, 8 h.

A. K. Halve et al. / Bioorg. Med. Chem. Lett. 17 (2007) 341–345
343
phenylazo)phenyl]-1-phenylazetidin-2-ones 6a–h in
good yield.32,33 Typically, ketenes are generated ther-
mally from acid chlorides in presence of triethyl
amine, which on subsequent reaction with reactive
imines yield the desired cis-azetidin-2-ones.34 Struc-
tures of newly obtained compounds have been ascer-
tained on the basis of their consistent IR, 1H NMR
and mass spectral assignments.35,36
anthracis. However, the growth inhibitory activities of
compounds 3d, 3f and 6c showed 2000 l g/ml MIC value
against A. fumigatus, 3c, 3a, 6g showed 2000 lg/ml MIC
value against A. niger and 6g compound showed 250 lg/
ml MIC value against C. albicans which is equal to stan-
dard drug fluconazole. On the contrary compound 3a,
3b, 3g, 6d, 6e, 6f and 6h were found to be inactive
against all the screened bacterial and fungal pathogens.
The m-nitro substituted compounds 6a–d showed better
activity than compounds 6e–h with nitro substituent
present on para position.
All the newly obtained azetidin-2-ones 3a–g and 6a–h
were assayed in vitro for their growth inhibitory activity
against pathogenic micro-organisms.The antibacterial
activity was tested by the disc-diffusion method37 using
selected Gram positive and Gram negative strains of
Staphyococcus aureus, Bacillus anthracis and Salmonella
typhi and compared to reference drug—chlorampheni-
col. The experimental results of antibacterial activity
indicated a variable degree of efficacy of the compounds
against different strains of bacteria (Table 1). We next
turned to examine the antifungal potentialities of azeti-
din-2-ones against pathogenic fungi viz.; Aspergillus
fumigatus, Aspergillus niger and Candida albicans by
broth-dilution assay.38 The minimum inhibitory concen-
tration (MIC) values were determined by comparison
with fluconazole as a reference standard and presented
in Table 1.
The results of antimicrobial screening clearly indicate
that the nature of substituents and their position on 2-
azetidinone nucleus affected the in vitro activity signifi-
cantly. All the three F, Cl and Br are classical bioisoster-
es so obey steric and electronic definition of classical
bioisosteres. Chloro substituent frequently appears in
many drugs and it follows the trend here also as chloro
substituted compounds seem to be more potent than flu-
oro- and bromo-containing compounds hence the inhib-
itory activities of p-fluoro and p-chloro substituents are
better than that of p-bromo substituent, suggesting that
some steric hindrance might be present around the para
position. Thus, p-chloro phenyl moiety and p-chloro
phenyl 1,3-diketo aminopropane functionality on nitro-
gen of 2-azetidinone nucleus is an essential part of the
pharmacophore.
The biological assay data have shown that screened
azetidin-2-ones 3a–g and 6a–h exhibited moderate to po-
tent activity against the tested microbes.
Further, changing the position of nitro group from clas-
sical meta to ortho or para position of phenyl ring re-
sults in complete reduction of activity suggesting that
the charge density is found to be greater at o, p than that
of meta position. Therefore, m-nitrophenylazo moiety at
C-4 of 2-azetidinone nucleus is also an essential part of
the pharmacophore. Additionally, the azetidine-2-one
derivatives (3c, 3d and 3f) substituted with the phenylc-
arbamoyl-acetylamino residue showed a more potent
In Table 1, the most potent compounds are 3c, 3d, 3f, 6b
and 6c. Among the series of compounds the best growth
inhibitory activity is of 3d compound against C. albi-
cans. Also 3c, 3f, 6b and 6c compounds have shown sub-
stantial activity against C. albicans. Furthermore
compounds 3c, 3d, 3f, 6a, 6b and 6c have moderate
inhibitory activity against S. aureus, S. typhi and B.
Table 1. The in vitro antimicrobial activity of azetidin-2-ones 3a–g and 6a–h
Compound
R1
R2
Inhibition zone diametersa
MIC (lg/ml)b
S. aureus
S. typhi
B. anthracis
A. fumigatus
A. niger
C. albicans
3a
3b
H
o-Cl
—
—
10
12
26
30
11
27
—
24
29
30
11
08
—
17
—
32
—
—
—
17
29
—
31
—
18
21
27
08
—
—
10
—
36
—
—
21
22
34
17
33
12
31
28
32
17
10
12
21
08
34
—
4000
2000
2000
1000
4000
1000
4000
2000
1000
2000
4000
—
4000
1000
2000
1000
4000
2000
4000
4000
500
1000
500
3c
3d
m-Cl
p-Cl
—
—
125
31.25
2000
62.5
1000
1000
62.5
125
3e
o-NO2
m-NO2
p-NO2
m-NO2
m-NO2
m-NO2
m-NO2
p-NO2
p-NO2
p-NO2
p-NO2
—
—
—
3f
3g
—
H
6a
6b
p-F
p-Cl
p-Br
H
6c
6d
1000
4000
4000
4000
2000
4000
—
—
6e
2000
2000
250
6f
6g
p-F
p-Cl
p-Br
—
4000
—
6h
Chloromycetin
Fluconazole
—
—
4000
—
—
—
2000
2000
250
Antibacterial susceptibility of compounds was measured in terms of zone of growth inhibitions.
(ꢀ) means no activity.
a Inhibition zone diameters in millimeters at 400 lg/ml concentration of compounds.
b The MIC value was defined as the lowest concentration of each chemical compound in the tubes (i.e., no turbidity) of inoculated fungi.

344
A. K. Halve et al. / Bioorg. Med. Chem. Lett. 17 (2007) 341–345
22. Ruhela, D.; Chatterjee, P.; Vishwakarma, R. A. Org.
Biomol. Chem. 2005, 3, 1043.
23. Buynak, J. D. Curr. Med. Chem. 2004, 11, 1951.
24. Silvaggi, N. R.; Kaur, K.; Adediran, S. A.; Pratt, R. F.;
Kelly, J. A. Biochemistry 2004, 43, 7046.
25. Moreira, R.; Santana, A. B.; Iley, J.; Joa, N.; Douglas, K.
T.; Horton, P. N.; Hursthouse, M. B. J. Med. Chem. 2005,
48, 4861.
26. Clader, J. W. Curr. Top. Med. Chem. 2005, 5, 243.
27. Halve, A. K.; Dubey, R.; Bhadauria, D.; Bhaskar, B.;
Gour, P. J. Indian Chem. Soc. 2006, 83, 386.
antimicrobial activity than the other derivatives 6a–h.
The chloro moiety of compound 3d improved their anti-
microbial activity significantly. In addition, we were able
to identify some interesting structure–activity relation-
ships around the phenyl moiety of phenylcarbamoyl-
acetylamino residue that of phenyl azophenyl residue
and N-phenyl residue on azetidinone ring. Our results
should encourage the synthesis of N/C-4 substituted
azetidine-2-one analogs to give new class of antimicrobi-
al agents.
28. Halve, A. K.; Gour, P.; Dubey, R.; Bhadauria, D.;
Bhaskar, B. J. Indian Chem. Soc. 2005, 82, 942.
29. Halve, A. K.; Gour, P.; Dubey, R.; Bhadauria, D.;
Bhaskar, B. Indian J. Chem. 2005, 44B, 2163.
Acknowledgments
30. Experimental: All melting points were measured in open
capillary and are uncorrected.The chemicals and reagents
used were of AR grade. The product mixtures were
analyzed by thin-layer chromatography (TLC) on silica
gel sheets. IR spectra (in cmꢀ1) were recorded on Perkin
Elmer 337 spectrometer. 1H NMR was recorded on a
Varian EM-390 MHz NMR spectrometer in DMSO-d6,
chemical shifts are given in d ppm using TMS as an
internal standard. Mass spectral analysis was performed
on Jeol SX-102 spectrometer using FAB technique.
General procedure for the synthesis of 1-(3000-methoxy-
4000-acetyloxy benzalhydrazino)-3-(phenylamino)propan-
1,3-diones (2a–g): In a 250 mL round-bottomed flask, an
equimolar solution (0.005 M) of compound 1 and substi-
tuted malonanilic acid hydrazides in EtOH was refluxed
for 2 h at room temperature and left aside in an ice bath.
The resulting precipitate was filtered, washed and recrys-
tallised from petroleum ether (60–80°C).
We gratefully acknowledge financial support from Uni-
versity Grants Commission, New Delhi, and are thank-
ful to the Dean, Birla Institute of Medical Research and
College of Life Sciences, India, for providing facilities of
biological screening.
References and notes
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substituted phenylazo) benzylidene anilines (5a–h): An
equimolar mixture (0.005 M) of compound 4a,b and
appropriate anilines in ethanol was refluxed for 2 h. The
reaction mixture was cooled in an ice bath and a drop of
sulfuric acid was added to it. The product obtained was
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32. General procedure for the synthesis of 3-chloro-4-(3-
methoxy-4-acetyloxyphenyl)-1-[3-oxo-3-(phenylamino)pro-
panamido] azetidin-2-ones 3a–g: In a closed vessel con-
taining compound 2a–g (0.001 M) in 20 mL of 1,4-dioxan,
0.095 mL of chloroacetyl chloride and 0.16 mL of trieth-
ylamine were added and the reaction mixture was stirred
at 50 °C for 1 h. The reaction mixture was then kept at
room temperature for 30 min and further refluxed for 8 h.
The filtrate was concentrated under reduced pressure and
poured into ice-cold water. The product 3a–g so obtained
was recrystallised from methanol as light brown crystals.
33. General procedure for the synthesis of and 3-chloro-4-[2-
hydroxy-5-(nitro substituted phenylazo)phenyl]-1-phenyl-
azetidin-2-ones 6a–h: In a closed vessel containing com-
pound 4 (0.001 M) in 20 mL 1,4-dioxan, 0.095 mL of
chloroacetyl chloride and 0.16 mL of triethylamine were
added and the reaction mixture was stirred at 50 °C for
1 h. The reaction mixture was then kept at room temper-
ature for 30 min and further refluxed for 8 h. The filtrate
was left for three days at room temperature and treated
with ice-cold water. The coloured solid obtained was
filtered, washed and recrystallised from 1:1 metha-
nol + chloroform mixture.
10. Slusarchyk, W. A.; Balton, S. A.; Huang, M. H. J. Med.
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35. Analytical data of compound 3a: mp (°C) 162–164, yield
68%: IR KBr (m cmꢀ1
OCOCH3), 1762 (C@O, four membered lactam), 1634,
) 3370 (N–H), 1770 (C@O,

A. K. Halve et al. / Bioorg. Med. Chem. Lett. 17 (2007) 341–345
345
2780 (C@O, 1,3-diketo), 1280, 1030 (C–O, OCH3), 1H
NMR (d) ppm 2.1 (s, 3H, OCOCH3), 3.8 (s, 3H, OCH3),
5.3 (d, 1H, J3,4 = 4.4 Hz, C4-H), 5.9 (d, 1H, J3,4 = 4.4 Hz,
C3-H), 6.9 (s, 1H, NH), 7.2 (s, 1H, NH),7.4–7.7 (m, 8H,
ArH). MS: Elemental analysis, found (calcd) (%) C, 55.04
(56.56): H, 4.19 (4.48); N, 9.03 (9.42).
The disc-bearing plates were incubated at 37 °C and
examined at 48 h for zone of inhibition, if any, around the
disc. Chloromycetin was used in assay as a standard
control drug. An additional negative control disc without
any sample but impregnated with equivalent amount of
solvent (DMF) was also used in the assay. The diameter of
inhibition zone is directly proportional to the degree of
sensitivity of bacterial strain and the concentration of
compound under test.
36. Analytical data of compound 6a: mp (°C) 188–190, yield
72%: IR KBr (m cmꢀ1) 3512 (O–H), 1760 (C@O, four-
membered lactam), 1578 (N@N), 1498 (N@O, asym), 1349
1
(N@O, sym), 1602, 1599, 1464 (C–C, ring str), H NMR
38. In broth dilution assay, different concentrations of com-
pound in the range from 15.62 to 8000 lg/mL in DMF
were prepared using Sabouraud’s Dextrose media in sterile
test tubes and suspension of fungal cultures was inoculat-
ed in them. These tube dilutions were incubated at 25 °C
for 72 h along with control. Fluconazole was also screened
under similar conditions as reference drug. The dilution
of a compound showing no visible growth of fungi was
taken as minimum inhibitory concentration (MIC) of
compound.
(d) ppm 4.2 (s, 1H, OH), 5.1 (d, 1H, J3,4 = 4.4 Hz, C4–H),
5.8 (d, 1H, J3,4 = 4.4 Hz, C3–H), 7.1–7.4 (m, 12H, ArH),
MS: Elemental analysis, found (calcd) (%) C, 61.23
(61.99): H, 3.18 (3.69); N, 13.41 (13.77).
37. In disc-diffusion assay, few colonies of organisms were
inoculated in 2–5 mL nutrient broth and grown for 2.5 h.
The agar plates were dried and inoculated by spreading
the bacterial suspension evenly over it. The sterile paper
discs (6 mm) impregnated with fixed dose viz., 400 lg/mL
of compound were placed on the preinoculated surface.