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Therapeutic Potentials of β‐Lactam: A Scaffold for New Drug Development

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DOI: 10.1002/9781119708841.ch4

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 59

Therapeutic Potentials of β-Lactam

A Scaffold for New Drug Development
Biswa Mohan Sahoo*1 and Bimal K. Banik2
1
Roland Institute of Pharmaceutical Sciences, Berhampur, Odisha, India
2
Department of Mathematics and Natural Sciences, College of Sciences and Human Studies, Prince Mohammad Bin Fahd University,
Al Khobar, Kingdom of Saudi Arabia

4.1 ­Introduction

Antibiotics are medications used for the treatment and prevention of bacterial infections. They
may be either bacteriostatic or bactericidal, depending on whether they inhibit the growth of
­bacteria or kill them. They are generally produced by one microorganism and kill or inhibit the
growth of other microorganisms. The term “antibiotic” was first introduced by Selman Waksman
in 1942. On the basis of the spectrum of antimicrobial activity, antibiotics may be of two types:
narrow-spectrum or broad-spectrum. Based on their chemical structures, they are categorized into
β-lactam antibiotics (e.g. benzyl penicillin, ampicillin, amoxicillin) and non-β-lactam antibiotics
(e.g. tetracycline, streptomycin) [1, 2].
β-Lactam antibiotics are considered broad-spectrum and contain a β-lactam ring in their molec-
ular structure. For example, 2-azetidinone (Figure 4.1) is the simplest β-lactam, and is present in
the structures of penicillin (penams), cephalosporins (cephems), monobactams, and carbapen-
ems, among others (Figure 4.2) [1, 3].
The absorption bands due to vibrational stretching found in the infrared (IR) spectra of these
substances (in the region of 1870–1640 cm−1) confirm the presence of a C═O group in the β-lactam
ring. The unstrained β-lactams have an IR spectrum absorption at 1600 cm−1 only, whereas the
strained β-lactams have an IR absorption at 1735–1765 cm−1. In proton nuclear magnetic reso-
nance (1H-NMR) spectra, protons present on C-3 and C-4 appear in different regions. Substituents
with electronegative properties (e.g. halogen) decrease the electron density around the protons,
which leads to deshielding of the protons [4].

4.2 ­History of Development

β-Lactam antibiotics are frequently used as chemotherapeutic agents due to their broad spec-
trum of in vitro and in vivo activities with wide therapeutic index [4, 5]. The first β-lactam anti-
biotic was synthesized by Staudinger in 1907, but their use began with the development of
penicillin by the Scottish scientist Alexander Fleming in 1928; Fleming discovered a mold

Synthetic Approaches to Nonaromatic Nitrogen Heterocycles, First Edition.

Edited by Ana Maria M. M. Faisca Phillips.
© 2021 John Wiley & Sons Ltd. Published 2021 by John Wiley & Sons Ltd.
60 4 Therapeutic Potentials of β-Lactam

HN C

Figure 4.1 Structure of the 2-azetidinone ring.

R HN
H H R2 Figure 4.2 Structure of β-lactam
S CH3 R1
antibiotics.
O N R3
CH3 N
O O
OH COOH
O
Penicillins Carbapenems

O R2
S O O OH H
N S HN S
O O O
H2 N N N N
H R1
N O
O
OH O OH

O
Monobactam Cephalosporins

OH Figure 4.3 Structures of 6-APA and Ezetimibe.

OH
H
H2 N S CH3

N CH3 N
O F O
COOH
6-APA Ezetimibe
F

(Penicillium notatum) that inhibits the growth of Staphylococcus bacteria [6]. In 1940, penicillin
was isolated and tested on mice by a research group at Oxford University. It was mass produced
by a fermentation process and was used by US soldiers in 1941, during the Second World War.
Dorothy Crowfoot-Hodgkin confirmed its structure by X-ray crystallography in 1945. By 1950,
the first total synthesis of ­penicillin and some analogs had been achieved by the chemist John
Sheehan at MIT.
In 1948, Cephalosporin-C was isolated from the fungus Cephalosporium acremonium, found in
seawater by Giuseppe Brotzu. In 1950, 6-aminopenicillanic acid (6-APA) was discovered and semi-
synthetic penicillins were developed (Figure 4.3). Since 1960, various novel β-lactam antibiotics
produced by microorganisms have been discovered [7]. Ampicillin and carbenicillin were first
used clinically in the 1960s. Broad-spectrum antibiotics, such as cephalosporins, monobactams
and carbapenems, were developed in the 1980s [8].
A research group at the Schering-Plough Corporation identified a novel compound,
Ezetimibe, capable of lowering the level of total cholesterol and low-density lipoprotein
(LDL; bad cholesterol) in the blood. Ezetimibe is helpful for patients who cannot reduce their
cholesterol levels by diet and exercise. It was approved in the USA by the Food and
Drug Administration (FDA) and also in Japan and marketed as Zetia worldwide in 2002
(Figure 4.3) [9].
 4.3 ­Classification of β-Lactams 61

4.3 ­Classification of β-Lactams

A) β-Lactams are classified as follows, based on their core ring structures [10]:
1) β-Lactams fused to saturated five-membered rings
a) β-Lactams containing thiazolidine rings, e.g. penams.
b) β-Lactams containing pyrrolidine rings, e.g. carbapenams.
c) β-Lactams fused to oxazolidine rings, e.g. oxapenams or clavams.
2) β-Lactams fused to unsaturated five-membered rings
a) β-Lactams containing 2,3-dihydrothiazole rings, e.g. penems.
b) β-Lactams containing 2,3-dihydro-1H-pyrrole rings, e.g. carbapenems.
3) β-Lactams fused to unsaturated six-membered rings
a) β-Lactams containing 3,6-dihydro-2H-1,3-thiazine rings, e.g. cephems.
b) β-Lactams containing 1,2,3,4-tetrahydropyridine rings, e.g. carbacephems.
c) β-Lactams containing 3,6-dihydro-2H-1,3-oxazine rings are named oxacephems.
4) β-Lactams not fused to any other ring, e.g. monobactams.
B) β-Lactams are also classified based on their spectrum of activity, as shown in Figure 4.4.

4.3.1 Penicillins
Penicillins act as bactericidal β-lactam antibiotics when administered at therapeutic doses. Many
are useful in treating a range of infections caused by certain susceptible bacteria, such as strepto-
cocci, staphylococci, Clostridium, Neisseria, and Listeria. Penicillins are classified as narrow-spec-
trum or broad-spectrum (Figure 4.5). Narrow-spectrum penicillins are further classified into two
types: penicillinase-susceptible (e.g. penicillin-G and penicillin-V) and penicillinase-resistant (e.g.
methicillin, oxacillin, cloxacillin, dicloxacillin, and floxacillin). Penicillinase-resistant penicillins
are not inactivated by the enzyme penicillinase; they are thus used to treat resistant strains of
staphylococci. Aminopenicillins, like ampicillin and amoxicillin, belong to the group of penicilli-
nase-sensitive antibiotics [10].

Beta-Lactam Antibiotics

Penicillins Cephalosporins
Miscellaneous

Narrow Broad Narrow Broad Carbapenems Monobactams

Spectrum Spectrum Spectrum Spectrum

1st Generation 2nd, 3rd, 4th & 5th

Generation
Penicillinase Penicillinase
Susceptible Resistance

Figure 4.4 General classifications of β-lactam antibiotics.

62 4 Therapeutic Potentials of β-Lactam

Penicillins

Source Route of Spectrum of Resistance to Resistance to

Administration Activity Enzymes Acids

Natural Oral Narrow Spectrum Resistance to Acid Stable

Penicillin-G Penicillin-V Methicillin Beta-Lactamase Penicillin-V
Penicillin-V Amoxicillin Oxacillin Methicillin Amoxicillin
Semisynthetic Ampicillin Nafcillin Nafcillin Ampicillin
Cloxacillin Oxacillin Dicloxacillin Oxacillin Oxacillin
Dicloxacillin Parenteral Broad Spectrum Dicloxacillin Dicloxacillin
Methicillin Penicillin-G Amoxicillin Cloxacillin Cloxacillin
Nafcillin Methicillin Ampicillin Non-Resistance to Acid Unstable
Oxacillin Nafcillin Intermediate Spectrum Beta-Lactamase Penicillin-G
Carbencillin Penicillin-V Penicillin-G Methicillin
Piperacillin Penicillin-G Penicillin-V Nafcillin
Extended Spectrum Amoxicillin Carbencillin
Carbencillin Ampicillin Piperacillin
Piperacillin Carbencillin Ticarcillin

Figure 4.5 Therapeutic classification of penicillins.

Advantages of penicillins as antibiotics

●● Bactericidal against sensitive strains.

●● Excellent tissue penetration.

●● Nontoxic.

●● Effective in the treatment of bacterial infections.

●● Inexpensive compared to other antibiotics.

●● Newer penicillins are resistant to the acidic pH of the stomach, e.g. penicillin V and broader-

spectrum penicillins like ampicillin and amoxicillin.

Disadvantages of penicillins as antibiotics
●● Most are destroyed by gastric acid.
●● Lack of activity against Gram-negative bacteria.

●● Intramuscular administration of drugs is painful.

●● Short duration of action.

●● Many patients experience gastrointestinal (GI) tract upsets.

Clinical uses
●● Urinary tract infections (UTIs).

●● Respiratory tract infections (RTIs).

●● Meningitis.

●● Gonorrhea and syphilis.

●● Typhoid and bacillary dysentery.

●● Bone and joint infections.

 4.3 ­Classification of β-Lactams 63

●● Bronchitis and pneumonia.

●● Skin and soft-tissue infections.

4.3.2 Cephalosporins
Cephalosporins are bactericidal β-lactam antibiotics derived from the fungus C. acremonium
[11]. They produce their action by disrupting peptidoglycan formation in the bacterial cell wall.
They are classified as narrow-spectrum or broad-spectrum. Narrow-spectrum cephalosporins
are further categorized as first-generation (e.g. cefadroxil, cephalexin, cephradine, cefazedone,
and cefazoline) and broad-spectrum cephalosporins as second- (e.g. cefaclor, cefuroxime, cefo-
tetan, and cefprozil), third- (e.g. cefixime, cefotaxime, ceftazidime, and ceftriaxone), fourth-
(e.g. cefepime and cefquinome), or fifth-generation (e.g. ceftobiprole, ceftaroline, and
ceftolozane). Based on the route of administration, cephalosporins are classified as oral cepha-
losporins (cephalexin, cephradine, cefadroxil, cefaclor, cefixime) or parenteral cephalosporins.
Cefuroxime, cefoxitin, and cefamandole are parenterally administered cephalosporins that are
resistant to β-lactamase, whereas cephalothin, cephapirin, and cefazedone are parenterally
administered but are sensitive to it [8]. The therapeutic profile of cephalosporins is shown in
Table 4.1 [8].
Advantages of cephalosporins as antibiotics
●● Relatively stable to acidic conditions.
●● Relatively stable to β-lactamase activity.

●● Improved pharmacokinetic properties, i.e. oral absorption.

●● Broad spectrum of activity.

●● Nontoxic.

●● Increased activity against resistant strains.

●● Relatively low risk of allergic reactions.

●● Increased tolerance after parenteral administration.

Table 4.1 List of cephalosporins and their therapeutic profile [8].

Resistant Resistant toward

Name of Spectrum of toward β-lactamase Route of Plasma protein
cephalosporins activity acids activity administration binding (%)

Cephalexin Broad Yes Poor Oral 5–15

Cephradine Broad Yes Poor Oral and 8–17
Parenteral
Cephadroxil Broad Yes Poor Oral 20
Cefaclor Broad Yes Poor Oral 22–25
Cefotetan Extended No Good Parenteral 78–91
Cefuroxime Extended No Good Oral and 33–50
Parenteral
Cefixime Extended Yes Good Oral 65
Cefotaxime Extended No Good Parenteral 30–51
Cefepime Extended No Good Parenteral 16–19
64 4 Therapeutic Potentials of β-Lactam

Disadvantages of cephalosporins as antibiotics

●● Difficult to absorb orally.

●● Low potency for the treatment of UTIs.

●● Difficult to isolate and purify.

Clinical uses
●● Allergic reactions.
●● UTIs.

●● RTIs.

●● Soft-tissue infections.

●● Staphylococcal infections.

●● Septicemia and surgical prophylaxis.

●● Meningitis.

●● Gonorrhea and typhoid.

4.3.3 Carbapenems
Carbapenems have a broad spectrum of antibacterial activity against various microorganisms, includ-
ing Gram-positive and Gram-negative, aerobic, and anaerobic bacteria. Like other β-lactam antibiotics,
the carbapenems bind to penicillin-binding proteins and disrupt the structural ­integrity and growth of
the bacterial cell wall. Imipenem, developed by Merck, was the first to be used in the clinic. Other exam-
ples of carbapenems include biapenem, doripenem, meropenem, and ertapenem [12–14].

4.3.4 Monobactams
Monobactams are effective only against aerobic Gram-negative bacteria such as Neisseria and
Pseudomonas. Examples include aztreonam, tigemonam, nocardicin-A, and tabtoxin [15].

4.4 ­Structure and Nomenclature

β-Lactams contain as their basic structural component a cyclic amide, from which their name
arises. The β-lactam ring has four atoms (Figure 4.6). It is named as such because the nitrogen
atom is attached to the β-carbon atom relative to the carbonyl group in the four-membered
ring [16].
Penams contain a β-lactam ring fused with a five-membered saturated ring, in which one of the
atoms is sulfur, whereas cephems contain a β-lactam ring fused with a six-membered ring, in
which one of the atoms is again sulfur (Figure 4.7) [16]. Chemically, penam is 4-thia-1-azabicy-
clo[3.2.0]heptanes-7-one, penem is 4-thia-1-azabicyclo[3.2.0]hept-2ene-7-one, and cephem is
5-thia-1-azabicyclo[4.2.0]oct-2-ene-8-one.
The monobactams represent a subgroup of β-lactam antibiotics in
α β which the β-lactam ring is not fused to any other ring (Figure 4.8). They
are relatively effective against aerobic Gram-negative bacteria such as
N Neisseria and Pseudomonas. Chemically, a monobactam is 1-azacyclobu-
O H tan-4-one. The structure of a carbapenem is similar to that of a penam,
β−Lactam ring
but the sulfur atom is replaced by a carbon atom at position 1 of the
Figure 4.6 Structure ­five-membered unsaturated ring (Figure 4.8). Chemically, a carbapenem
of the β-lactam ring. is 1-azabicyclo[3.2.0]hept-2-ene-7-one [15, 17].
 4.6 ­Synthesi 65

Figure 4.7 Structures of penam, penem, and cephem. 4 4

5
S
6 5 S 6 5 S 7 6
4
3 3
N N 8
N 3
7 1 7 1 1
2 2 O
O O 2
Penam Penem Cefem

4
There are two different systems followed in numbering these 3 2 6 5

structures. According to the United States Pharmacopeia (USP), NH N

3

4 7
the position of the nitrogen atom is 1 and that of the sulfur atom is
1 1 2
O O
4, whereas the Chemical Abstracts system assigns the sulfur atom Monobactam Carbapenem
as 1 and the nitrogen atom as 4, as presented in Figure 4.9.
Figure 4.8 Structures of
carbapenam and
monobactam.
4.5 ­Stereochemistry
H 1
HN 6 5 R
Penicillins contain three chiral carbon atoms, at positions C-3, C-5, S
CH3
and C-6. The β-lactam hydrogens at C-5 and C-6 have a cis relation-
2
O 7
N 3 CH3
4
ship, and the acylamino side chain at C-6 is trans to the carboxylic O
OH
acid group at C-3. All synthetic and semisynthetic ­penicillins have Penicillins O
the same absolute configuration (3S : 5R : 6R), like the natural peni-
cillins. All naturally occurring penicillins are d
­ extrorotatory [18]. Figure 4.9 Numbering of a
penicillin structure with
Cephalosporins possess two chiral centers at positions C-6 and C-7 stereocenters.
with a cis-configuration, and between C-3 and C-4 there is a double
bond conjugated to the β-lactam nitrogen (Figure 4.10) [18, 19].
The penicillin antibiotics available for clinical treatment today are listed in Table 4.2 [10], and
the cephalosporin antibiotics are listed in Table 4.3 [10].

4.6 ­Synthesis

The first synthetic β-lactam was prepared by Hermann Staudinger. The method involved a non-
photochemical cycloaddition reaction between an imine and a ketene (Figure 4.11) [20].
In 2014, Doyle and coworkers reported a one-pot, multicomponent Staudinger synthesis of
β-lactams from azides and two diazo compounds (Figure 4.12). The reaction proceeds via a

Figure 4.10 Numbering of a

cephalosporin. R1
Acylamino Dihydrothiazine
H H
substituents HN S Acetyloxy group
1
O 7
6 2

8 5 3
N 4 O CH3
O
β-Lactam ring O
OH
O

7-Amino cephalosporanic acid

66 4 Therapeutic Potentials of β-Lactam

Table 4.2 Clinically available penicillin antibiotics [10].

R HN
S
CH3
O N CH3
O
COOH

Generic name Chemical name R

Penicillin-G Benzyl penicillin CH2

Phenoxymethyl-
Penicillin-V OCH2
penicillin

Penicillin-X p-Hydroxybenzyl penicillin HO CH2

H2 H
C C
Penicillin-F 2-Pentyl penicillin H3 C C CH2
H

Penicillin-K n-Heptyl penicillin CH3(CH2)7

H3CO OCH3
Methicillin 2,6-Dimethoxyphenyl penicillin

O CH3
Nafacillin 2-Ethoxy-1-naphthyl penicillin

CH3
Oxacillin 5-Methyl-3-phenyl-4-isoxazolyl penicillin
O
N

Cl
CH3
Cloxacillin 5-Methyl-3-(2-chlorophenyl)-4-isoxazolyl penicillin
O
N

Cl
CH3
5-Methyl-3-(2,6-dichlorophenyl)-4-isoxazolyl
Dicloxacillin
penicillin N
O

Cl

d-α-Amino-benzyl CH
Ampicillin
penicillin NH2

HO CH
Amoxicillin d-α-Amino-p-hydroxy benzyl penicillin
NH2

CH
Carbenicillin α-Carboxybenzyl penicillin
COOH

Ticarcillin α-Carboxy-3-thienyl penicillin CH

COOH
 4.6 ­Synthesi 67

Table 4.3 Clinically available cephalosporin antibiotics [10].

R1
H
NH S
O
N
O R2

O OR3

Name R1 R2 R3 Generation

H
C
Cephalexin CH3 H First
NH2

H
C
Cefradine CH3 H First
NH2

H
HO C
Cefadroxil CH3 H First
NH2

H
C
Cefaclor Cl H Second
NH2

OCOCH3
O C H
Cefuroxime O C
CH2OCNH2 Second
NOCH3 CH3

C CH2
Fixime H 2N N C H H Third
NOCH2COOH

S
O
Cefotaxime H 2N N C
CH2OCCH3 H Third
NOCH2COOH

N
Cefepime H2 N N C
H3 C CH2
H Fourth
NOCH3

S
H2
N C
Cefpirome H2 N N C H Fourth
NOCH3

N O
H2 N N N NH
Ceftobiprole H Fifth
S NH O

N O CH3
HO H N
N N
HO P
Ceftaroline CH3 N H Fifth
O S N
S S
68 4 Therapeutic Potentials of β-Lactam

r­ hodium acetate-catalyzed reaction between the aryldiazoacetate and the organic azide to form an
imine. Finally, Wolff rearrangement of the diazoacetoacetate enone forms a stable ketene, which
reacts with the imine to produce the β-lactam [21].
The mechanism of β-lactam synthesis has two steps (Figure 4.13). The first involves nucleophilic
attack by the imine nitrogen on the carbonyl carbon, generating a zwitterion as intermediate. Electron-
donating groups on the imine facilitate this step, while electron-withdrawing groups hinder the attack.
The second step involves intramolecular nucleophilic ring closure [22]. Although the Staudinger reac-
tion remains one of the most popular classical methods, many different procedures have been described
for the construction of the four-membered β-lactam ring [23, 24]. Recent developments are discussed in
other chapters of this book; for example, catalytic [2 + 2] cycloaddition reactions are reviewed in
Chapter 3, and the Kinugasa reaction (the cycloaddition of terminal alkynes with nitrones) in Chapter 17.
6-Amino penicillanic acid (6-APA) is the core part of a penicillin. It is produced from the fermen-
tation brew of the Penicillium mold [25] and is used as the precursor for the preparation of various
semisynthetic penicillins, such as penicillin-G and penicillin-V (Figure 4.14) [26].

4.6.1 Production of Penicillins

The industrial production of penicillin is mostly classified into two processes, namely the upstream
process and the downstream process [27]. An upstream process utilizes any technology that leads
to the synthesis of a product. It includes exploration, development, and production. The extraction
and purification of a biotechnological product from a fermentation is called downstream process-
ing (Figure 4.15).
The strains of Penicillium are grown in a nutrient medium containing a sugar, such as glucose,
sucrose, lactose, maltose, or starch. The fungus can be cultured in one of three ways: by the surface

Ph O
Ph Ph [2+2] Cycloaddition Ph
N + C C O
N
Ph Ph Ph Ph
Imine Ketene Beta-Lactam

Figure 4.11 Staudinger synthesis of a β-lactam.

N3
O O O PMP O
Rh2(OAc)4 N
Ph
Ph OMe + OMe + Ph COOMe
N2 N2 MeOOC
OMe Ph

Figure 4.12 One-pot multicomponent Staudinger synthesis of β-lactams.

O
O Ph O Ph
Ph Ph
C N N N
+
Ph Ph
Ph Ph Ph Ph Ph
Ph
Ketene Imine Intermediate Beta-Lactam

Figure 4.13 Reaction mechanism of β-lactam synthesis.

 4.6 ­Synthesi 69

Figure 4.14 Preparation of semisynthetic H

H 2N
penicillins. S
CH3
N CH3
O
COOH
6-APA

CH2COCl Acylation OCH2COOH

–HCl –H2O
2-phenyl-acetylchloride 2-phenoxyacetic acid

H H H H
N N
S O S
CH3 CH3
O N O N
CH3 CH3
O O
COOH COOH
penicillin-G penicillin-V

Culture Medium

Fermentation

Centrifugation

Filtration

Solvent Extraction Precipitation Crystallization

Figure 4.15 Production processes for penicillins.

culture method, by the submerged culture method, or in bran cultures. Surface culture is an old
method in which the fungus is grown on the surface of a liquid medium without agitation. After
an appropriate incubation period, penicillin is extracted from the medium. Nowadays, penicillin is
produced by the submerged method, in which the fungus is allowed to grow in a liquid medium
that is vigorously aerated and agitated [28]. After an appropriate incubation period, the penicillin
is separated from the medium. In the bran method, moist bran is used as substrate and the result-
ant penicillin is extracted from it.

4.6.1.1 Biosynthesis of Penicillin-G

The mechanism of the formation of the β-lactam ring of penicillins and cephalosporins is not
clearly known. Information about the biosynthesis of benzylpenicillin from Penicillium chrys-
ogenum was obtained by Morecombe and Young in 1975, when they showed that cysteine
70 4 Therapeutic Potentials of β-Lactam

HOOC CH CH2 CH2 CH2 COOH is incorporated into this antibiotic with retention of its
NH2
3-pro-R-hydrogen and loss of its 3-pro-S‑hydrogen [29].
L-α-aminoadipic acid (α−AAA)
Biosynthesis of penicillin-G by P. chrysogenum proceeds
H
H 2N C CH2 SH
in the following way. The β-lactam and thiazolidine ring
COOH of penicillin are formed by the condensation of l-cysteine
(L-Cysteine)
and l-valine (Figure 4.16). Biosynthesis occurs in a nonri-
L-α-aminoadipyl cysteine (α-AAA-Cys)
bosomal process by means of a dipeptide composed of (α-
CH3
H2 N
H
C C CH3
AAA) and α-cysteine. Subsequently, l-valine is connected
H
COOH (L-Valine) via an epimerization reaction, which results in the forma-
L-α-aminoadipylcysteinylvaline tion of a tripeptide. The first product resulting from the
cyclization of the tripeptide that can be isolated is isopen-
Cyclase Cyclization in two steps
icillin N. Benzyl penicillin is produced in an exchange of
CH3
AAA HN HC
S α-AAA with activated phenylacetic acid.
CH3
N
COOH
O
4.6.1.2 Biosynthesis of Cephalosporins
Isopenicillin-N
Biosynthesis of cephalosporins proceeds from α-(α-
CH2COCoA aminoadipyl)–l-cysteinyl-d-valine to isopenicillin-N, as in
Acyltransferase
α−AAA + CoA
the case of benzyl penicillin [30, 31] (Figure 4.17). In the
next stage, penicillin N is produced by transformation of
H S
CH3
the l-α-AAA side chain into the d-form via the action of a
H2COC N HC
N
CH3
very labile racemase. After ring expansion to deacetoxy
O COOH cephalosporin by an expandase reaction, hydroxylation via
Penicillin-G a dioxygenase to deacetyl cephalosporin C occurs.
Figure 4.16 Biosynthesis of Acetylation of cephalosporin-C by an acetyl coenzyme A
penicillin-G. (acetyl-CoA)-dependent transferase is the end point of the
biosynthetic pathway in fungi.

4.7 ­Mode of Action of β-Lactam Antibiotics

Antibiotics containing a β-lactam ring produce their drug action by forming a covalent adduct with
membrane-bound bacterial peptidoglycan transpeptidases, also known as penicillin-binding pro-
teins (PBPs) [32–34]. Peptidoglycan is a carbohydrate composed of two units: NAMA (N-acetyl
muramic acid) and NAGA (N-acetyl glucosamine). Generally, the NAMA units contain a peptide
side chain, which is cross-linked from the l-Lys residue to the terminal d-Ala-d-Ala link on a
neighboring NAMA unit (Figure 4.18).
Transpeptidase enzymes are responsible for cross-linking the NAG and NAM moieties on the
peptidoglycan layer surrounding the periplasmic space and membrane of bacteria. The transpepti-
dase enzymes are acylated by β-lactams, which prevents bacterial cell-wall synthesis. This results
in a weakened cell wall, which leads to osmotic lysis of the bacterial cell. Therefore, these antibiot-
ics act as bactericidal agents.
The amide part of the β-lactam ring is reactive mainly due to the presence of ring strain and a
conformational arrangement that does not allow the lone pair of the nitrogen to interact with the
double bond of the carbonyl group. β-Lactams acylate the hydroxyl group on the serine residue of
the PBP active site in an irreversible manner (Figure 4.19). This reaction is accelerated by the for-
mation of an oxyanion that stabilizes the tetrahedral intermediate and thereby reduces the transi-
tion-state energy [34].
 4.7 ­Mode of Action of β-Lactam Antibiotics 71

HOOC CH CH2 CH2 CH2 COOH

NH2
L-α-aminoadipic acid (α–AAA)
H
H 2N C CH2 SH
COOH
(L-Cysteine)

L-α-aminoadipyl cysteine (α-AAA-Cys)

CH3
H
H2 N C C CH3
H
COOH (L-Valine)
L-α-aminoadipylcysteinylvaline

Cyclase Cyclization in two steps

CH3
S
AAA HN HC CH3
N
O COOH

Isopenicillin-N
Racemase

Penicillin-N
Ring formation

S
α AAA HN
N
O CH3
COOH
Deacetoxycephalosporin-C

O
S H3C C CoA S
α AAA HN α AAA HN
N N
O CH2OH O CH2OCOCH3
COOH COOH
Deacetylcephalosporin-C Cephalosporin-C

Figure 4.17 Biosynthesis of cephalosporins.

4.7.1 Mechanisms of Resistance

Resistance is the ability of a microorganism to overcome the effects of an antimicrobial agent.
Resistance may be either intrinsic or acquired. Intrinsic resistance is present before exposure to
antimicrobial therapy, whereas acquired resistance is developed due to exposure to antibiotics.
Generally, antibiotic resistance is accelerated by the misuse or overuse of antibiotics. Care should
be taken at each level of the health care system to reduce the impact and limit the spread of resist-
ance. To prevent and control the spread of antibiotic resistance, individuals should use antibiotics
only when prescribed by certified health care professionals.
There are three main mechanisms adopted by bacteria against β-lactam compounds: (i) produc-
tion of β-lactamase enzymes; (ii) alteration in drug binding to PBP; and (iii) alteration in cell
­permeability [34, 35].
Microorganisms develop resistance to β-lactam antibiotics by synthesizing a β-lactamase
enzyme, which attacks the β-lactam ring to produce inactive metabolites (Figure 4.20) [33–36]. To
72 4 Therapeutic Potentials of β-Lactam

H
R N

O –
O NH COO

Acyl D-Ala-D-Ala
(Cell wall precursor)

Glycan N-acetyl muramic acid N-acetyl glyucosamine N-acetyl muramic acid N-acetyl glyucosamine
Part (NAMA) (NAGA) (NAMA) (NAGA)

L-alanine L-alanine

Peptide D-glutamine D-glutamine Peptide

Chain Chain
Peptide interbridge
L-lysine L-lysine
Gly
Gly
Gly
D-alanine Gly Gly D-alanine

Figure 4.18 Structure of the bacterial peptidoglycan.

R R
S S CH3
CH3
O N
OH N CH3 CH3
O SER O
SER COOH COOH
H

R
S CH3
O
SER HN CH3
O
COOH

Figure 4.19 Mechanism of action of β-lactam drugs.

H H
(a) R HN S
R HN S CH3
CH3
Beta-Lactamase
O N O HN
CH3 CH3
O
CO2
OH OH
O O
Penicillins Inactive metabolites

(b) R2 R2
H H
HN HN S
S
O O
Beta-Lactamase
N N
R1 R1
O
CO2
O OH O OH

Cephalosporins Inactive metabolites

Figure 4.20 Mechanisms of resistance toward β-lactam antibiotics [21].

 4.7 ­Mode of Action of β-Lactam Antibiotics 73

Figure 4.21 Structure of β-lactamase inhibitors.

H HO O
O OH S CH3

N N CH3
O O
OH OH
O O

Clavulanic acid Sulbactam

overcome this resistance, β-lactam antibiotics are currently administered with β-lactamase inhibi-
tors, such as clavulanic acid, tazobactam, or sulbactam (Figure 4.21).

4.7.2 Metabolism and Degradation

Penicillins are metabolized in humans primarily to penicilloic acids (Figure 4.22) [37]. Liver is their
main site of inactivation. Penicilloic acids are produced by hydrolysis of the β-lactam ring. This reac-
tion occurs slowly in aqueous solution but rapidly in the presence of β-lactamase, an enzyme pro-
duced by some bacteria. But the extent of metabolism depends on the structure of the penicillin. In
the case of the phenoxypenicillin series, phenoxymethylpenicillin (penicillin-V) is unstable, whereas
the higher-homolog penicillins are more stable. Similarly, in the case of the isoxazolyl series, oxacil-
lin is unstable, but the presence of halogen in the phenyl ring increases the stability. After intramus-
cular administration, carbenicillin becomes stable in the body, ampicillin is fairly stable, and benzyl
penicillin is unstable. Therefore, it is essential to determine the content of penicilloic acid in the

Figure 4.22 Chemical degradation of penicillin [37]. HOOC S H 2N

H
HC CH3 S CH3
N N CH3 N
C CH3
O
R OH OH
O O
Penillic acids 6-APA

Acidic pH Amidase
(HCl)
H
R HN S CH3

O N
CH3
O
OH
O
Penicillins

Alkaline pH (NaOH ) Beta-Lactamase or Penicillinase

Hydrolysis
H
R HN S CH3

O C HN
O CH3
OH
OH
O
Penicilloic acids

–CO2 Decarboxylation

H
R HN S CH3

O HN
CH3

OH
O
Penilloic acids
74 4 Therapeutic Potentials of β-Lactam

R R
H H H H
HN S HN S
O β -Lactamase O
N O CH3 O HN O CH3
O OH
O O
O OH O OH
Cephalosporins Cephalosporic acid
H
R
H H
H2O Acylase HN S
O
N OH
H H O
H2 N S
O OH
N O CH3 Desacetylcephalosporins
O
O
O OH Acid Lactonization
7-Amino cephalosporanic acid (7-ACA)

H H2 O R
HN S
H2 N S O
N
N O
O O
O O
O Inactive lactone
Desacetyl-7-ACA-lactone (Inactive)

Figure 4.23 Chemical degradation of cephalosporins [38].

urine during estimation of the total absorption of penicillin. The clearance of penicilloic acids from
the body takes place more slowly when compared to that of penicillins. Thus, the increase in stabil-
ity of penicillins – which have a slower rate of renal clearance – inside the body leads to high con-
centrations of the drug in the blood stream. The reactive nature of the β-lactam ring system in
penicillins and other antibiotics makes it susceptible to various degradative processes.
Cephalosporins undergo different hydrolytic degradation reactions, which depend on the
nature of the individual structures (Figure 4.23) [38]. Among 7-acylaminocephalosporanic acid
derivatives, the 3-acetoxylmethyl group is the most reactive site. In addition to its reactivity to
nucleophilic displacement reactions, the acetyloxy group readily undergoes solvolysis in
strongly acidic solutions to form desacetyl-cephalosporin derivatives. Then it undergoes lac-
tonization to produce desacetyl-cephalosporin lactones, which are inactive. The 7-acylamino
group of cephalosporins can be hydrolyzed under enzymatic conditions (i.e. by acylases).
Following hydrolysis or solvolysis of the 3-acetoxymethyl group, 7-ACA also lactonizes under
acidic conditions.

4.8 ­Structure–Activity Relationship (SAR) Studies

A structure–activity relationship (SAR) is the relationship between the chemical structure of a

drug molecule and its pharmacological activity. The study of SARs facilitates the determination
of the chemical group responsible for inducing a target biological effect in an organism. Such
studies help modify the effect or the potency of a drug via changes to its chemical
structure [39].
 4.8 ­Structure–Activity Relationship (SAR) Studie 75

4.8.1 SAR of Penicillins cha

in
side Cis-stereocentre
ino
In penicillins, the fusion of a β-lactam ring with a thiazo- Ac
yla
m
H H
* 1
lidine ring is essential for antibacterial activity R HN S
6 5 CH3
(Figure 4.24). Binding of the β-lactam ring with penicilli- O
7
N
4 2

CH3
nase transpeptidase is the main factor responsible for its
3
O
β-Lactam ring
ability to cross the bacterial cell wall. The increase in strain O
OH
of the β-lactam ring leads to an increase in antibacterial oiety
id m
c ac
oxyli
activity. In the case of the thiazolidine ring, the oxidation Carb

of sulfur to sulfone or sulfoxide causes loss of activity.

Figure 4.24 Structure of a penicillin
●● The presence of a free carboxylic acid group at the C-3 molecule.
position is essential. Esterification of the carboxylic acid
group produces prodrugs that increase lipophilicity and acid stability. These prodrugs undergo
cleavage to generate active penicillin in vivo. Examples include pivampicillin and becampicillin.
●● The introduction of arylalkyl or aryloxyalkyl in the side chain of penicillin increases acid stabil-
ity and improves oral absorption.
●● The replacement of the acyl side chain with a hydroxymethyl group increases antibacterial activ-
ity against Gram-negative bacteria.
●● The presence of a bulky group on the acyl side chain at C-6 minimizes the degradation of the
drug by β-lactamases.
●● Introduction of a C-6 α-methoxy group increases the stability against β-lactamases without loss
of potency.
●● Substituents at the chiral centers at positions C-3 and C-6 are trans to each other, whereas sub-
stituents at C-5 and C-6 are cis to each other. Modification of these configurations results in loss
of activity.
●● The d-isomer is two to eight times more active than the l-isomer. Ampicillin is an example [39].

4.8.2 SAR of Cephalosporins

●● The cis-configuration at positions C-6 and C-7 is essential for antibacterial activity (Figure 4.25).
●● Fusion of the β-lactam ring with the dihydrothiazine ring results in a bicyclic system, which
increases ring strain and is necessary for antibacterial activity.
●● The 7-amino cephalosporanic acid (7-ACA) acts as a precursor for the production of semisyn-
thetic cephalosporins.
●● Modifications at position C-3 influence the pharmacokinetic properties.
●● Replacement of the acetoxy group at position C-3 with CH3 and Cl results in orally active
compounds.

Figure 4.25 Structure of a cephalosporin.

Acylamino
R1 Dihydrothiazine
H H
substituents HN S Acetyloxy group
O 6 1
* 7
8 5
2

3
N 4 O CH3
O
β -Lactam ring OH O
O

7-Amino cephalosporanic acid

76 4 Therapeutic Potentials of β-Lactam

●● The acetyloxy group at position C-3 is essential. It acts as a leaving group when the molecule
binds to transpetidase.
●● The allylic acetyloxy group at position C-3 is not necessary for activity.
●● Replacement of S with O increases the acylating power and thereby the activity (e.g.
oxacepam).
●● Replacement of sulfur with a methylene group causes an increase in chemical stability, resulting
in a long half-life (e.g. loracarbef).
●● The olefinic linkage at positions C-3 and C-4 is essential for antibacterial activity. Upon satura-
tion of the double bond, activity is lost.
●● The presence of a carboxylic acid (─COOH) group at position C-4 is essential for binding. Its
esterification produces ester prodrugs that increase the oral bioavailability, lipophilicity, and acid
stability of the cephalosporin. Examples include cefuroxime axetil and cefodoxime proxetil.
●● Introduction of a methoxy group at position C-7 increases the resistance toward hydrolysis by
β-lactamases.
●● Oxidation of sulfur to sulfoxide (S═O) or sulfone (O═S═O) destroys the antibacterial activity.
●● Acylation of the amino group at position C-7 increases the potency of the drug against Gram-
positive bacteria but decreases that against Gram-negative bacteria.
●● The presence of ─NH in the acylamino side chain increases the acid stability of the β-lactam ring.
●● Substitutions on the aromatic ring (phenyl) increase lipophilicity and provide high Gram-
positive activity [40, 41].

4.9 ­Toxicity

There are several β-lactam antibiotics that cause different adverse side effects. The use of β-lactam
antibiotics may cause allergic reactions like urticaria, bronchoconstriction, and other conditions
such as immune-mediated haemolytic anemia and intravascular hemolysis. Some β-lactam antibi-
otics are neurotoxic, nephrotoxic, or genotoxic, and some are toxic to the urogenital system. The
toxicity of β-lactam antibiotics depends mainly on the route of administration and the dose.
β-Lactam antibiotics can trigger epilepsy or seizures, due to the presence of the β-lactam ring,
which binds to the gamma-aminobutyric acid (GABA) receptors in the brain. Known adverse side
effects of β-lactam antibiotics are listed in Table 4.4 [42].

Table 4.4 Adverse side effects of β-lactam antibiotics [42].

Name Effect Type of toxicity

Penicillin Seizures Moderate

Piperacillin Nephrotoxicity Moderate
Amoxicillin Genotoxicity Severe
Cefazolin Nephrotoxicity Mild
Cefuroxime Retinal toxicity Mild
Ceftriaxone Nephrotoxicity Mild
Cefotiam Seizures Mild
 4.10 ­Therapeutic Potential 77

4.10 ­Therapeutic Potentials

The β-lactam nucleus is the core structural feature of various classes of therapeutic agents with a
broad spectrum of activity and low toxicity. The development of numerous synthetic and semisyn-
thetic β-lactam antibiotics by the pharmaceutical industry is due to the increase in the resistance
of bacteria toward β-lactams and the need for novel drugs with more specific antibacterial activi-
ties. β-Lactam antibiotics are commonly used for the treatment of various infectious diseases.
These antibiotics are effective against Gram-positive and Gram-negative bacteria. The combina-
tion of β-lactam antibiotics with β-lactamase inhibitors is preferable, because this increases their
effectiveness against the more resistant bacteria. The higher the version of the antibiotic, the
greater its sensitivity against organisms (i.e. the greater the susceptibility of the microorganisms to
it). For example, among the different generations of cephalosporins, the order of sensitivity is
fourth > third > second > first generation. β-Lactam antibiotics exhibit diverse biological activities,
acting as antibacterial, antifungal, antitubercular, antiviral, anticancer, anti-inflammatory,
enzyme-inhibitory, anticonvulsant, and antidiabetic drugs, among other effects.
It is essential to make clinicians aware that the proper use of antibiotic therapy will vary according
to the infection stage. It has been proposed by the Directorate General Health Services of the
Government of India that the sale of antibiotics over the counter (OTC) without prescription should
be prohibited. The proper implementation of antibiotic policies should be followed. The World
Health Organization (WHO) has offered the free WHONET as a means of solving this issue [43].

4.10.1 β-Lactams as Antibacterial Drugs

Variya and colleagues performed the synthesis of 1,3,4-oxadiazoles 1 bearing a 2-azetidinone ­moiety
and investigated their potential as antibacterial agents [44]. The synthesis of (5-(pyridine-4-yl)-1,3,4-
oxadiazol-2-yl)2-((3-chloro-2-oxo-4-phenylazetidin-1-yl)amino)-ethan-thioate derivatives was car-
ried out by subjecting 1,3,4-oxadiazoles in sequence to reaction with chloroacetyl chloride, hydrazine
hydrate (NH2-NH2), and different benzaldehydes (Figure 4.26). The compounds obtained in this
manner were screened for antibacterial activity against several microorganisms, including
Staphylococcus aureus (Gram-positive), Bacillus subtilis (Gram-positive), Pseudomonas aeruginosa
(Gram-negative), and Escherichia coli (Gram-negative). Some (1b, 1c, 1f, 1j) exhibited significant
antibacterial activity in comparison with the standard drug, ampicillin (Table 4.5) [44].
Farooqui and Shafakat reported the synthesis of 3-(acetoxymethyl-7-(2-(7-methyl-2-p-
tolylimidazo[1,2-a]pyridine-3yl) acetamide-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carbaylic
acid derivatives (Figure 4.27) [45]. The compounds obtained (2a–f) were screened for their anti-
bacterial activity against three microorganisms – E. coli (Gram-negative), Salmonella typhi (Gram-
negative), and S. aureus (Gram-positive) – by the agar dilution method, using streptomycin as
standard and dimethyl sulfoxide as solvent. The activity of the compounds was tested against dif-
ferent bacteria and compared with the standard drug, streptomycin, at a concentration of 100 μg/
ml. The results are presented in Table 4.6 in terms of the size of the zone of inhibition (mm) [45].

4.10.2 β-Lactams as Antifungal Agents

Gupta and Halve carried out the synthesis and antifungal screening of novel azetidin-2-ones
(β-lactam) 3 (Figure 4.28) [46]. The compounds synthesized exhibited potent antifungal activity
against Aspergillus niger and Aspergillus flavus (Table 4.7).
78 4 Therapeutic Potentials of β-Lactam

O
O Cl
CS2, KOH O Cl S O
N N SH N Cl
NHNH2 C2H5OH Et3N, DMF
N N N N O
–HCl
Isonicotinohydrazide 5-(Pyridin-4-yl)-1,3,4-oxadiazole-2-thiol S-5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-yl 2-chloroethanethioate

–HCl NH2NH2, C2H5OH

O S
N NHNH2
N N O
S-5-(Pyridin-4-yl)-1,3,4-oxadiazol-2-yl
2-hydrazinylethanethioate

O
C2H5OH, AcOH
R = p-Cl, p-F, m-OCH3, m-NO2, H
o-OH, p-OH, p-CH3, 2,4-(Cl)2 R
R

O S N
N N
H
N N O

O Et3N
Cl R
Cl
Cl

O S N
N N O
H
N N O
1a-j

Figure 4.26 Synthesis of target compounds 1a–j.

Table 4.5 Antibacterial activity of target compounds 1a–j [44].

Gram-positive bacteria Gram-negative bacteria

Comp. Substituent (R) S. aureus B. subtilis P. aeruginosa E. coli

1a H + ++ + −
1b p-Cl +++ − +++ +++
1c 2,4-(Cl)2 +++ − − +++
1d o-OH − ++ + −
1e p-OH ++ + + +
1f p-CH3 + + ++ ++
1f m-NO2 +++ + +++ +++
1h o-OCH3 + ++ + +
1i p-OCH3 + ++ ++ ++
1j p-F +++ + ++ +++
Ampicillin − +++ ++ ++ +++

−, inactive (zone of inhibition <6 mm); +, slightly active (ZOI 6–9 mm); ++, moderately active (ZOI 9–12 mm);
+++, highly active (ZOI >12 mm).
 4.10 ­Therapeutic Potential 79

H3 C N H3 C N
Ethylchloroformate
CH3 CH3
N N
DCM, Et3N

O O
O
OH O
H2 N S OEt

N R1
O DCM, Et3N

O R

H3 C N
CH3
N

HN S

N R1
O

O R
2a-e

Figure 4.27 Synthesis of target compounds 2a–e.

Table 4.6 Antibacterial activity of target compounds 2a–e [45].

Zone of inhibition (mm)

Entry R R1 E. coli S. typhi B. megaterium

2a H CH2OCOCH3 20 18 15
2b H CH2═CH2 17 15 13
2c H CH3 14 13 10
2d CH2═CH2 ─CH─(C6H5)2 13 13 14
2e H CH2─CH═CH2 15 14 15
Standard Streptomycin 30 25 24

4.10.3 β-Lactams as Antiviral Agents

The human cytomegalovirus (HCMV) is a ubiquitous member of the herpes virus family. Most
infections are asymptomatic, but when they occur in individuals whose immune system has been
weakened by a disease, such as late-stage cancers and AIDS, or by immunosuppressive therapy
following organ transplantation, HCMV symptoms can be severe.
In 1990, Skiles and McNeil synthesized a novel spiro-β-lactam 4, making a successful entry into the
class of antiviral β-lactams (Figure 4.29) [47]. The precursor was obtained as a mixture of diastereoiso-
meric β-lactams, which could be separated by column chromatography. The (Z)-N-aryl-2-azetidinone
thus obtained was subsequently converted in two steps into spiro indolinone 4, which was found to be
a good inhibitor of both the poliovirus and human rhinovirus 3C-proteinases (IC50 = 20 μg/ml). All
picornaviruses studied produce a 3C-proteinase, which is required for virus maturation.
80 4 Therapeutic Potentials of β-Lactam

O
CHO N S NHR1
O
EtOH O
R + H2N S NHR1

OCH3 O R
OCH3

(C2H5)3N ClCH2COCl

O
O
Cl N S NHR1
O

R
OCH3

3a-d

Figure 4.28 Synthesis of target compounds with antifungal activity 3a–d.

Table 4.7 In vitro antifungal activity of target compounds 3a–d [46].

Zone of inhibition (mm)

Comp. R R1 A. niger A. flavus

3a p-OH H 10 11
S
3b p-OH 10 14
N

3c p-OH H3 C O
N 11 14

N
N CH3
3d p-OH 10 13
H3C

Std. Terbiforce 14 48

4.10.4 β-Lactams as Anticancer Agents

Banik and colleagues explored the anticancer activity of monocyclic β-lactams such as compound 5
(Figure 4.30) against various cancer cell lines, including leukemia and colon carcinoma cell lines [48, 49].
The effect of the peri hydrogen present in 5 was found to be significant in the control of the stereo-
chemistry of the resulting β-lactam. The presence of an acetoxy group in the β-lactam ring potenti-
ated the anticancer activity.
Azetidin-2-ones 6, substituted at positions 1, 3, and 4 on the azetidinone ring, were obtained by
O’Boyle and coworkers via a Staüdinger reaction [50]. The structure of these substances was based
on that of the natural product combretastatin A-4, a tubulin-binding diaryl stilbene known to have
potent anticancer activity against several cell lines, with the four-membered ring replacing a dou-
ble bond. The compounds were evaluated for antiproliferative, cytotoxic, and tubulin-binding
 4.10 ­Therapeutic Potential 81

O O N OCH3
Na / C2H5OH NH2C6H4OCH3
C6H5Cl, toluene toluene
N O N O N O
H

CH3OCH2COCl
Et3N, CH2ClCH2Cl

H3CO O H3CO O H3CO O

O i) (NH4)2Ce(NO3)6
N N N
S O CH3CN / H2O +
N O ii) p-CH3PhSO2Cl N O N O
KOH / Bu4NBr / THF OCH3 OCH3

4 (Z) (E)
CH3

Figure 4.29 Synthesis of antiviral spiroindolinone-β-lactams.

activity. The thienyl derivatives 6a and 6b displayed the highest potency

in human MCF-7 breast cancer cells, with IC50 values of 7 and 10 nM,
respectively – similar to that of combretastatin A-4 (Figure 4.31). It was
O
also shown that the target of these substances is indeed tubulin, with N

an IC50 value of 1.37 μM being obtained in binding studies. 5

Ph OAc

Figure 4.30 A β-lactam

4.10.5 β-Lactams as Antitubercular Drugs with anticancer activity.
β-Lactam derivatives of the antibiotic isoniazid (i.e. isonicotinylhy-
drazide, a first-line antituberculosis drug) were synthesized by Hussain and colleagues, with the
aim of developing novel drugs [51]. These new substances, N-[3-chloro-4-(aryl)-2-oxoazetidin-1-
yl]-pyridine-4-carboxamides, were screened for antimycobacterial and anticonvulsant activity.
Moderate to significant anticonvulsant activity was observed. Compounds 7 showed significant
antimycobacterial activity against the standard strain H37Rv and two other clinical strains isolated
from patients suffering from ­pulmonary tuberculosis (Figure 4.32). Isoniazid was used for
comparison.
Nikalje and colleagues reported a microwave-assisted synthesis to obtain N-(3-chloro-2-oxo-4-
substituted-azetidin-1-yl)isonicotinamide derivatives (7 or 8), which were evaluated as antimyco-
bacterial agents against Mycobacterium tuberculosis H37Rv; they also reported a quantitative
structure–activity relationship (QSAR) study (Figure 4.33) [52]. Significant activity was found in
some cases. A comparative study on the effect of the substitution pattern of the aryl and heteroaryl
substituents at C-4 toward the antimycobacterial activity showed that compounds bearing elec-
tron-donating groups were more active and that the presence of electron-withdrawing groups low-
ered the activity. The most active compound was 7c, which displayed an activity (minimum
inhibitory concentration [MIC] = 50 μg/ml) comparable to that of the standard drug, rifampicin.
The QSAR model indicated that the thermodynamic descriptors (molar refractivity), the electronic
descriptors (dipole moment), the principal moment inertia, and so on play important roles in the
antimycobacterial activities of these compounds.
A series of related analogs, 3-chloro-1-{[2-(6-nitro-1H-indazol-1-yl)ethyl]amino}-4-(substituted-
phenyl)-2-azetidinones 9, were described by Samadhiya and colleagues (Figure 4.34) [53]. They
82 4 Therapeutic Potentials of β-Lactam

OH OH Figure 4.31 β-lactams with anticancer activity.

OMe OMe

S S

N N
O OMe O OMe

6a 6b
OMe OMe
MeO MeO

N Figure 4.32 β-Lactams with antitubercular properties.

H
N Ar
C 7a: Ar = 4-HOPh
N
7b: Ar = 4-MePh
O 7c: Ar = 4-MeOPh
O Cl 7d: Ar = 4-NMe2Ph

N Figure 4.33 β-Lactams with antitubercular activity.

H
N Ar 8a: Ar = 4-Cl-Ph
C N 8b: Ar = 4-NO2Ph
O 8c: Ar = 2-HOPh
7c: Ar = 4-MeOPh
O Cl

N Figure 4.34 Azetidinones with antitubercular

N activity.
O 2N N 9a: Ar = Ph 9f: Ar = 3-NO2Ph
CH2 9b: Ar = 4-Cl-Ph 9g: Ar = 2-Br-Ph
9c: Ar = 3-Cl-Ph 9h: Ar = 3-Br-Ph
NH
9d: Ar = 2-Cl-Ph 9i: Ar = 4-Br-Ph
N 9e: Ar = 4-NO2Ph
O Ar

Cl

were prepared in four steps from 6-nitro-1H-indazole. Their antitubercular activity against M. tuber-
culosis (H37Rv strain) was studied and compared with that of isoniazid and of rifampicin. MICs in
the range 1.25–2.50 mg/ml were obtained. The results revealed that the nitro group-containing
­compounds were more active than those bearing chlorine or bromine substituents. The activity as a
function of the substituent decreased in the order NO2 > Cl > Br > H.

4.10.6 β-Lactams as Hypoglycemic Agents

Diabetes mellitus is a metabolic disorder resulting from a defect in insulin secretion, insulin action,
or both. Insulin deficiency in turn leads to chronic hyperglycemia with disturbances of carbohy-
drate, fat, and protein metabolism.
Reddy and colleagues described the synthesis of a series of N1-benzothiazolyl-3-chloro-1,5,6-
triazaspiro[3.4]oct-6-en-2-ones, prepared via the stepwise condensation of 3-methyl-5-phenyl-
1-pyrazolone with a 2-aminobenzothiazole, cyclization of the resulting Schiff base with ClCOCH2Cl
to form the β-lactam, and final condensation with several primary and secondary amines (Figure 4.35)
[54]. The biological activity of the products, including their antidiabetic activity, was evaluated.
Inhibition of α-amylase activity, measured by a colorimetric assay, in which acarbose was used as the
control for comparison, was significant in some cases. Compounds 10a and 10b showed activity
 4.10 ­Therapeutic Potential 83

comparable to that of the standard. For 10a, IC50 = 120 μg/ml; N O

for 10b, IC50 = 130 μg/ml; for acarbose, IC50 = 63 μg/ml. F S N Cl

Goel and colleagues studied the effect of monocyclic NH
Ar N Ph
β-lactams on hypoglycemic activity. The compounds tested N
(11) were synthesized via [2 + 2] cycloaddition (Staudinger) H 3C

reactions of imines with ketenes (Figure 4.36) [55]. The anti- 10a: Ar = p-Toluidine
10b: Ar = β-Phenylethylamine
hyperglycemic effect of the compounds was evaluated in
alloxan-induced diabetes in rats, by monitoring blood glucose Figure 4.35 Antidiabetic azetidinones.
and liver glycogen contents.

4.10.7 β-Lactams as Anticonvulsant Agents S

Convulsion is manifested by abnormal, involuntary contrac- N

O CH2CONH(CH2)5CH2 Cl
tion of the muscles. It is observed in seizure disorders. It occurs
11
mainly as a result of abnormal electrical activity in the brain.
There are several different types of generalized seizures: Figure 4.36 β-lactam with
absence seizures (petit mal), tonic–clonic or convulsive sei- potential for the treatment of
hypoglycemia.
zures (grand mal), atonic seizures (drop attacks), and tonic,
clonic, and myoclonic seizures [56]. Srivastava and colleagues
reported the ­synthesis of new carbazolyl-thiadiazol-2-oxo-­
azetidines 12 and evaluated them for anticonvulsant activity N N
O
N
(Figure 4.37) [57]. S N

12 Cl
Ar

4.10.8 β-Lactams as Hypocholesterolemic Agents

Figure 4.37 β-lactam with
Elevated lipid levels are one of the major risk factors of ath- anticonvulsant properties.
erosclerosis and related cardiovascular diseases, as well as
stroke, which is directly connected to mortality and morbidity. Lowering lipid levels is thus one
of the key approaches used to prevent coronary heart diseases and stroke. Therefore, there is a
need to design, synthesize, and evaluate new drug molecules for antihyperlipidemic activity.
Jain and colleagues synthesized a series of novel 2-azetidinones 13, analogs of ezetimibe, a selec-
tive cholesterol absorption inhibitor, used in therapy to reduce blood LDL-C levels (Figure 4.38)
[58]. The new thienopyrimidine derivatives of β-lactam were designed to combine the structural
features of the azetidin-2-one moiety with potentially antihyperlipidemic 2-substituted thieno[2,3-
d]-pyrimidin-4-ones. Their lipid-lowering activity was evaluated on Wistar albino rats. Some
showed significant effects comparable to the standard drug gemfibrozil, when administered at the
same dose; 13a, R1/R2 = ─(CH2)4─, R3 = 2-CH3 was the most
O
active, causing significant lipid-lowering effects including R3
R1
reducing serum levels of cholesterol and triglycerides in test NH

animals. R2 S N N

13 O

4.10.9 β-Lactams as Analgesic and Anti-Inflammatory R1, R2 = aryl, cycloalkyl, H

R3 = alkyl, halogen
Agents
Figure 4.38 Thieno-pyrimidine
A series of 1-(2-(1H-benzimidazol-2-yl) phenyl)–3–chloro–4- β-lactam derivatives with
(un/substituted-phenyl)azetidin-2-ones 14 were synthesized lipid-lowering activity.
84 4 Therapeutic Potentials of β-Lactam

by Chhajed and Upasani (Figure 4.39) [59]. They were screened for analgesic and anti-inflamma-
tory activities on acetic acid-induced writhing in mice and carrageenan-induced paw edema in
rats. One of the compounds, 14, R = 2-NO2, exhibited better analgesic and anti-inflammatory
properties than the standard drug nimesulide: analgesic activity = 46% at 20 mg/kg bw; anti-
inflammatory activity = 66.5% at 20 mg/kg bw.

4.10.10 β-Lactams as Anti-Parkinsonian Agents

Parkinson’s disease is one of the most common neurological and degenerative disorders. It affects
mostly the motor system, causing resting tremor, slowness of movements (bradykinesia), difficulty
in initiating movements (akinesia), rigidity, gait disturbance, and postural instability, as well as
nonmotor dysfunctions. In Parkinson’s disease there is a progressive loss of neurons within the
substantia nigra and other brain structures that produce dopamine, and the appearance of intracy-
toplasmic inclusions composed of aggregates of the protein α-synuclein, known as Lewy bodies.
Parkinsonian symptoms may also occur in the cerebellum.
S. Kumar and colleagues reported the synthesis of quinazolinone derivatives 15, 3-amantadinyl-
2-[(4-(substituted-phenyl)-3-chloro-2-oxoazetidin-1-yl)methylamino]quinazolin-4(3H)-ones,
which were screened for anti-parkinsonian activity (Figure 4.40) [60]. These compounds exhibited
almost equipotent antitremor activity as l-dopa, the amino acid l-3,4-dihydroxyphenylalanine,
used in the symptomatic treatment of Parkinson’s disease, usually combined with other drugs. The
best results in the azetidinone series were obtained with the compound bearing a 3,4-dimethoxy-
phenyl substituent (X = Br). Compound 15 was also tested for an approximate LD50, which was
found to be greater than 1000 mg/kg.

4.10.11 β-Lactams as Antimalarial Agents

β-Lactams and some derivatives were synthesized by reacting different imines with a special ketene
derived from nendo-5-norbornene-2,3-dicarboxyloylglycine via a [2 + 2] ketene imine cycloaddi-
tion (Figure 4.41). The β-lactams were then treated with 1-azido-4-nitrobenzene to afford β-lactam-
triazole hybrids. The compounds thus synthesized were tested against the chloroquine-resistant
Plasmodium falciparum K14 strain. Compounds 16a and 16b showed IC50s 20 μM, while 16c, 16d,
17a, and 17b exhibited IC50s 30 μM [61].

R
N N
R = H, 2-Cl, 4-Cl, 4-F,
NH
O Cl 2-OH, 4-OH, 2-NO2, 4-OMe

14

Figure 4.39 β-lactam derivatives with analgesic and anti-inflammatory properties.

N
X H R R = H, 2-Cl, 2-Br, 2-OMe, 3,4-OMe,
N 4-OH/3-OMe
N N X = H, Br
15
O Cl

Figure 4.40 β-lactam derivatives with anti-parkinsonian activity.

  ­Reference 85

H O O H O
H DMF O
+ NH2 H
HO
O N
OH
O O

Et3N, CH2Cl2 N R2
TsCl R1

N3

N H O H O
N
R1 O 2N R1
N H H H H
N toluene, reflux N
N N
O2N 16 R2 16 R2
O O
O O

17a: R1 = 4-Cl-C6H4, R2 = 4-MeO-C6H4 16a: R1 = 4-Cl-C6H4, R2 = 4-MeO-C6H4

17b: R1 = 4-Cl-C6H4, R2 = 3,4-dimethoxyphenethyl 16b: R1 = 2-Cl-C6H4, R2 = 4-(i-Pr)-C6H4
16c: R1 = 4-(NMe2)-C6H4, R2 = 4-(NMe2)-C6H4
16d: R1 = 4-Cl-C6H4, R2 = 3,4-diMeO-C6H4

Figure 4.41 β-Lactam derivatives with antimalarial activity.

4.11 ­Conclusion

β-Lactam antibiotics are used clinically for the treatment of various infectious diseases.
Understanding of the mechanism of action of these antibiotics and of the mechanisms of resist-
ance to them has increased dramatically in the past few years. Therefore, efforts are underway to
discover their structure–function relationships. Drug molecules are designed based on rational
drug design by considering both the structures of the targets and of the antibiotics (PBPs) and their
active sites. To reduce or overcome bacterial resistance toward β-lactam antibiotics, there is an
increasing demand for the development of combination therapies using β-lactam and β-lactamase
inhibitors.

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