Cephalosporin Chemical Reactivity and It
Cephalosporin Chemical Reactivity and It
Cephalosporin Chemical Reactivity and It
implications
Ezequiel Perez-Inestrosaa, Rafael Suaua, Maria Isabel Montañeza,
Rebeca Rodriguezb, Cristobalina Mayorgab, Maria J. Torresb and
Miguel Blancab
Figure 1. Penicillin and cephalosporin chemical structures Figure 2. The penicillin conjugation to carrier proteins
Dihydrothiazine HH H
Thiazolidine R
ring N H H HS
HH H ring R1 HH H S R N
R N
N S O
S N N CO2 Na
O O O H
N N R2 O HN
O CO2 Na O Penicilloyl
O
CO2 Na CO2 Na
NH2
-lactam Penicillins Cephalosporins Carrier Carrier
ring protein protein
Immunochemistry: uncertainty about the Figure 3. The cephalosporins conjugation to carrier proteins
antigenic determinant
The immunological behavior of b-lactam antibiotics is
HH H
determined by their intrinsic chemical reactivity, which is R1
N S R1
H H HS
N
R2
Degradation
related to the ability of the carbonyl group to act as an O
N R2 O HN products
O CO2 Na
alkylating agent with the amino groups of proteins. CO2 Na
HN O
NH2 Carrier
Carrier protein
In penicillins, due to the chemical structure resulting protein
Figure 4. Chemical structures of cephalosporins with R2 side chains that cannot work as leaving groups
Figure 5. The two proposed mechanisms for the cephalosporins conjugation thorough the b-lactam ring opening
R1 HH H -R2 H H HS
N S R1 N
Degradation
O Concerted process O N products
N R2
O HN O CO2 Na
CO2 Na
NH2 Carrier
Carrier protein
protein
H H HS R2
R1 N
Not concerted -R2
O HN
process
HN O CO2 Na
Carrier
CPO
protein
Evidence for the R2 departure with the can apply in a double action way [24–28]. When the R2 is
opening of the b-lactam ring conformed as the inactive form of the drug, the action of
Several studies deal with the elimination of R2 by the b-lactam in the cephalosporin implies the release of
hydrolysis, aminolysis and hydrazinolysis of the cephalo- the drug in situ (Fig. 6).
sporins [17,18]. Theoretical and experimental evidence
suggests that the opening is a concerted process with the Beta-lactamases are used as biological markers for the
subsequent expulsion of the R2 [19–23]. No evidence has identification of pathogenic bacteria resistant to b-lactam
been shown for cephalosporoyl formation when R2 acts as antibiotics. Based upon the ability of the R2 to act as a
a good leaving group, like acetoxy or pyridinium. In other leaving group [29], cephalosporins can also be used as
cases the expulsion of the R2 group is not a concerted sensors to monitor processes or biological interactions
process with the opening of the b-lactam ring, although (Fig. 7).
this process may also occur in the presence of certain
enzymes. Consequently, the initial product of aminolysis of cephalo-
sporins (cephalosporoyl) is unstable, probably being
Kinetic studies combined with absorption and nuclear degraded with the rupture of the dihydrothiazine group
magnetic resonance spectroscopy have shown the struc- [30,31]. Apart from evidence of the R2 side chain extru-
ture of the opening of the b-lactam ring: cephalosporoyl sion, when this may act as a leaving group, no evidence is
[17,18]. Either in a concerted fashion or in stages, the yet available for the resulting chemical structure and
opening of the b-lactam ring leads to elimination of the it has not been possible to isolate and characterize
R2 when this is configured as a leaving group. The process the aminolysis products resulting from the scission of
is well documented chemically and this property has the dihydrothiazine moiety of cephalosporins (Fig. 8)
been used as a strategy to obtain cephalosporins that [32].
326 Drug allergy
R1 HH H H H HS
N R1 N
S + Active drug
O Inactive O N
N CO2 Na
drug HN O
O
CO2 Na -lactamase
-lactamase
Cephalosporins exert their biological activity by covalent binding to bacterial enzymes, opening of the b-lactam ring is accompanied by liberation of the
30 -substituent if that substituent can function as a leaving group.
H H H H HS
N HH H N N
N S O O O
O O O N
O -lactamase -
O O CO2 Na
N
O
CO2 Na +
Not fluorescent
-
O O O
Umbelliferone
(blue fluorescencel)
Cleavage triggers spontaneous elimination of any leaving group previously attached to the 30 -position.
The addition of water to the exocyclic double bond of be observed. This derivative can undergo opening of the
structure 1 (Fig. 8) to generate structure 2 (Fig. 8) was b-lactam ring by reaction with nucleophiles [31], facili-
recently described and this precursor is responsible for tating its conjugation to several carriers and the confor-
the degradation products of the dihydrothiazine ring [33]. mation of a new epitope. The isomerization of the double
bond to the 2,3 position results in equilibration of the
Several studies have described a high number of degra- reactivity of the two electrophilic centers of the molecule,
dation products of the different cephalosporins, depend- with a reduction in the reactivity of the carbonyl group of
ing on the chemical structure of the particular the b-lactam ring and, consequently, the possible com-
cephalosporin and the reaction conditions, mainly the petence of the 30 position enabling the formation of
pH [34,35]. The ease with which the R2 acetoxy group conjugates with a form such as structure 5 (Fig. 8) [37].
can be replaced by different nucleophiles and its reac-
tivity with nitrogen nucleophiles has been described In cephalosporins with nucleophilic groups at R1, such
[36]. Cephalosporins with some R2 side chains can thus as cephaloglycin, cefaclor, cephalexin, cefadroxil and
undergo reactions with the amino groups of the carrier cephradine, autoaminolytic reactions may occur to yield
proteins, not only via the carbonyl of the b-lactam ring, but the compound shown by structure 6 (Fig. 8), in which the
also via R2 substitution, giving forms such as structure 3 intramolecular opening of the b-lactam ring is followed
(Fig. 8). This mechanism of action enables the cephalo- by R2 exclusion, when this side chain can act as a good
sporins to bind to a carrier protein conforming a hapten- leaving group, for example in cephaloglycin. For cefaclor,
carrier conjugate in which the b-lactam structure is intact six different fluorescent products have been identified
and its capacity to be attached by a new nucleophile is with a form related to structure 7 (Fig. 8) [34]. When
decreased. These types of structures produce a new cefaclor reacts with nitrogen nucleophiles (Fig. 9) the
epitope in which the R2 side chain is not present. intramolecular aminolysis competes with the inter-
molecular process and the intermediate cephalosporoyl
Despite the facility for substitution of the R2 side chain structure 9 (Fig. 9) can react intramolecularly to yield
by sulphur and nitrogen nucleophiles, oxygen nucleo- a compound like that shown by structure 10 (Fig. 9)
philes do not react and no evidence exists for the direct [32], and the hypothesis of the adehyde of structure 11
formation of a lactone like structure 4 (Fig. 8). However, (Fig. 9) as a key intermediate in the formation of a
in water, a significant hydrolysis of the acetic ester to the fluorescent pyrazinone like structure 10 seems the most
corresponding alcohol, that lactonizes to structure 4, can plausible.
Cephalosporin chemical reactivity Perez-Inestrosa et al. 327
Figure 8. Possible pathways for the cephalosporin chemical reactivity with nucleophiles
CPO, cephalosporoyl.
Figure 9. Aminolysis of aminocephalosporins such as cefaclor giving a degradation product with a pyrazine nucleus
NH2 NH2
H H
N S NH2 N S
Cl N
O O
N HN H
O Cl O N
NH CO2 Na O N
CO2 Na (9) H
Cefaclor O
(10)
NH2
H N
N H
CHO N
O HO N
O O
NH
(11)
328 Drug allergy
Approaches to the chemical identification Figure 10. Proposed skeleton that remains linked to the carrier
of the epitope protein after chemical degradation in cephalosporin conju-
gation to carrier proteins
Very little information exists about allergenicity of
cephalosporins, especially regarding the structural differ-
ences between these compounds. Few studies have been R1 HH H
N H H HS R2
S R1 N
undertaken to identify which part of the molecule is O CPO
N R2 HN
recognized by specific IgE antibodies. In fact, much of O
O
CO2 Na
HN O
the data concerning the chemical nature of the allergenic CO2 Na
determinant have been produced with IgG or other NH2 Carrier chemical structure
Carrier protein
isotype human polyclonal antibodies [38]. In contrast protein
that...
Figure 11. Synthesized chemicals comprising the haptenic structure that are recognized by ceftriaxone and cefuroxime, respectively
N OCH3 N OCH3
H2 N N HH H H2 N N HH HH
N S N N
S CH3 S CH3 S CH3
O O O
N S N
O N O NH O NH ·
(12) (13)
CO2 Na N C4 H9 C4 H9
Ceftriaxone OH
O
NH2 NH2
HH HH
N N
HO CH3
O CH3
O
O NH
(14) (15) O NH
C4 H9 C4 H9
N OCH3 N OCH3 OH
HH H HH HH
N S N N
O O CH3 CH3
O O O
N O NH2
O O NH
(16) (17) O NH
Cefuroxime CO2 Na O C4 H9 C4 H9
Cephalosporin chemical reactivity Perez-Inestrosa et al. 329
The effect of the R2 side chain on the conjugation of 20 Waller RE. A method for determining free azide ions by automatic analysis
in the presence of a covalent cephalosporin azide. Analyst 1973; 98:535–
the carrier protein can be interpreted only from a 541.
kinetic perspective, such that an increase in the capacity 21 Bundgaard H. Chemical studies related to cephalosporin allergy. I. Kinetics of
of the R2 as a leaving group results in increased reactivity aminolysis of cephalosporins and effect of C-3 substituents on b-lactam
reactivity. Arch Pharm Chemi, Sci Ed 1975; 3:94–123.
for the attack of nucleophiles to the b-lactam ring, 22 Boyd DB, Hermann RB, Presti DE, et al. Electronic structures of cephalo-
increasing the facility and kinetics of the conjugation sporins and penicillins. 4. Modeling acylation by the beta-lactam ring. J Med
process. Chem 1975; 18:408–417.
23 Boyd DB, Lunn WHW. Electronic structures of cephalosporins and penicil-
lins. 9. Departure of a leaving group in cephalosporins. J Med Chem 1979;
Acknowledgement 22:778–784.
We thank Ian Johnston for the English version of the manuscript. 24 Mobashery S, Johnston M. Inactivation of alanine racemase by b-chloro
L-alanine released enzymatically from amino acid and peptide C10-esters of
deacetylcephalothin. Biochemistry 1987; 26:5878–5884.
25 Albrecht HA, Beskid G, Georgopapadakou NH, et al. Dual-action cephalo-
References sporins: cephalosporin 30 -quinolone carbamates. J Med Chem 1991; 34:
2857–2864.
1 Walsh C, editor. Antibiotics: actions, origins and resistance. Washington: 26 Grant JW, Smyth TP. Toward the development of a cephalosporin-based
ASM Press; 2003. dual-release prodrug for use in ADEPT. J Org Chem 2004; 69:7965–7970.
2 O’Callaghan CH. Description and classification of the newer cephalospo- 27 Veinberg G, Shestakova I, Vorona M, et al. Doxorubicin prodrug on the basis
rins and their relationship with the established compounds. J Antimicrob of tert-butyl cephalosporanate sulfones. Bioorg Med Chem Lett 2004;
Chemother 1979; 5:635–671. 14:1007–1010.
3 Bryskier A, Procyk T, Labro MT. Cefodizime, a new 2-aminothiazolyl cephalo- 28 Vrudhula VM, Kerr DE, Siemers NO, et al. Cephalosporin prodrugs of
sporin: physicochemical properties, toxicology and structureactivity relation- paclitaxel for immunologically specific activation by L-49-sFv-b-lactamase
ships. J Antimicrob Chemother 1990; 26 (Suppl C):1–8. fusion protein. Bioorg Med Chem Lett 2003; 13:539–542.
330 Drug allergy
29 Gao W, Xing B, Tsien RY, et al. Novel fluorogenic substrates for imaging 37 Holden KG. Cephalosporins. In: Katritzky AR, Rees CR, editors. Compre-
b-lactamase gene expression. J Am Chem Soc 2003; 125:11146–11147. hensive heterocyclic chemistry. Vol 7. Oxford: Pergamon Press; 1984. pp.
285–298.
30 Hamilton-Miller JMT, Richards E, Abraham EP. Changes in proton-magnetic-
resonance spectra during aminolysis and enzymic hydrolysis of cephalo- 38 Ahlstedt S, Kristofferson A. Immune mechanisms of induction of penicillin
sporins. Biochem J 1970; 116:385–395. allergy. In: Kallos P, editor. Recent trends in allergen and complement
31 Hamilton-Miller JMT, Newton GGF, Abraham EP. Products of aminolysis and research: progress in allergy. Basel: Karger Publishers; 1992. pp. 67–134.
enzymic hydrolysis of the cephalosporins. Biochem J 1970; 116: 371–384. 39 Harle DG, Baldo BA. Drags as allergens: an immunoassay for detecting IgE
32 Venemalm L. Pyrazinone conjugates as potencial cephalosporin allergens. antibodies to cephallosporins. Int Arch Allergy Appl immunol 1990; 92:439–
Bioorg Med Chem Lett 2001; 11:1869–1870. 444.
33 Baker K, Bleczinski C, Lin H, et al. Chemical complementation: a reaction- 40 Pham NH, Baldo BA. b-Lactam drug allergens: fine structural recognition
independent genetic assay for enzyme catalysis. Proc Natl Acad Sci U S A patterns of cephalosporin-reactive IgE antibodies. J Mol Recognit 1996;
2002; 99:16537–16542. 9:287–296.
34 Baertschi SW, Dorman DE, Occolowitz JL, et al. Isolation and structure 41 Baldo BA. Penicillins and cephalosporins as allergens-structural aspects
elucidation of the major degradation products of cefaclor formed under of recognition and cross-reaction. Clin Exp Allergy 1999; 29:744–
aqueous acidic conditions. J Pharm Sci 1997; 86:526–539. 749.
35 Indelicato JM, Norvilas TT, Pfeiffer RR, et al. Substituents effects upon the 42 Romano A, Mayorga C, Torres MJ, et al. Immediate allergic reactions to
base hydrolysis of penicillins and cephalosporins: competitive intramolecular cephalosporins: cross-reactivity and selective response. J Allegy Clin Immu-
nucleophilic amino attack in cephalosporins. J Med Chem 1974; 17:523– nol 2000; 106:1177–1183.
527.
43 Sanchez-Sancho F, Perez-Inestrosa E, Suau R, et al. Synthesis, characteriza-
36 Georg GI, editor. The organic chemistry of b-lactams. New York: VCH tion and immunochemical evaluation of cephalosporin antigenic determinants.
Publishers Inc; 1993. J Mol Reocgnit 2003; 16:148–156.