Structure Activity Relationship

Antibacterial Agents Structure Activity Relationships André Bryskier MD

1

Lille - December 7 th 2004

Natural compounds

Antibacterial Agents

Synthetic compounds

– – – – – – – –

Beta-lactams Aminoglycosides Macrolides Streptogramin Lincosamines Peptides Mupirocin Ansamycins

– – – – – – – – – –

Benzyl pyrimidines Sulphonamides Sulfones Furans 4-quinolones Oxazolidinones Nitroxoline Penem Fosfomycin Anti-TB

Antibiotic resistance

1941

2000

3

Antibiotic era

Pre antibiotic era

Research in anti-infectives

Research up mid - 80s

•Enlarge the antibacterial spectrum • Enhance the antibacterial activity (e.g. cefotaxime) • Improve the pharmacokinetics (e.g. roxithromycin , clarithromycin, azithromycin) 4

Research after mid - 80s

•Overcome bacterial resistance

Purification

e.g Penicillin G Erythromycin A Kanamycin

Semi synthetic ß-lactams Macrolides Aminoglycosides

5

Structure-Activity-Relationships WHY

Antibacterial activity in vitro and in vivo (?) Bacterial resistance Bactericidal activity Toxicity-tolerance Optimisation of chemical strcutures Improvement of physicochemical properties Pharmacodynamics Pharmacokinetics. 6

Antibacterial agents

Future

Existing compounds

New molecule entities

Alterations chemical structures

7

Screening " Chemical " Natural products

New targets

Optimization of existing non-antibacterials

Improvement of physicochemical properties

Exemple : water- solubility

N

(1)

N N

N H

H3C

Norfloxacin

8

Pefloxacin (IV formulation)

Improvement of physicochemical properties Amino acid

Trovafloxacin

(2)

Alatrovafloxacin (IV formulation) Amino acid

Ceftizoxime

(3)

Prodrug

N

H2 N

N

S

R

N H

Amino acid 9

S

ASN-924

Fluoroquinolones

Fluoroquinolones History of quinolones Pipemidic acid 7

N

7-piperazinyl moiety

N

(partial crosscross-resistance With oxolonic acid) acid )

F

Nalidixic acid (1962)

Oxolinic acid (quinoline)

Piromedic acid

Fluoroquinolones

6

Flumequine

7

N

Pirrolidinyl moiety (+ ve activity) 11

Fluoroquinolones Definition

O

COOH

Synthetic antibacterial agents Pharmacophore : pyrridone-ß-carboxylic acid

R N

Auxopharmacophore : fused aromatic ring appended substituents

R1

12

Fluoroquinolones

O COOH

(CH3)2N N

Ro 13-5478

Monocyclic derivative

13

Fluoroquinolones Structure - activity relationships

Classification Microbiology Pharmacokinetics Adverse events

14

Fluoroquinolones Classifications

Chemical classification Biological classification

15

Fluoroquinolones Chemical classification Group I

Group II

Monocyclic derivatives

Bicyclic derivatives

Group III

Group IV

Tricyclic derivatives

Tetracyclic derivatives

Ro-145478 Group II B

Pentacyclic ring

Ro-149578 KB-5246 Group II A

Hexacyclic ring

Group IIC

III A

Heptacyclic ring

Non-fluorinated

III B

Fluorinated III B1

T - 14097

FD 501 FD 103 Tioxic Oxolinic acid Miloxacin EN 272 DJ 6783 Droxacin

16

Ofloxacin Levofloxacin Flumequine Abufloxacin Rufloxacin S-25932 QA-241 DN 9494 A-62824 MF 961 Marbofloxacin Verbafloxacin Pazufloxacin CP 91121 WQ 0835 KRQ 10099

III B2

Prulifloxacin

Fluoroquinolones Chemical classification (bicyclic derivatives ) Group II Group IIB

Group IIA -hexacyclic ring

IIA-1 1.8 naphthyridine

No fluor Nalidixic acid

17

IIA-2 Pyrido [2,3-b] pyrimidine

6-Fluor Pipemidic acid Piromedic acid 7-piperazinyl

7-pyrrolidinyl

Other

Enoxacin A-57132

AT 3295 AT 3765 Tosufloxacin A-65485 BMY 43738 BMY 41802 U-91939E PD 131112 (Cl 990) Gemifloxacin DC-756

BMY 40062 E-3499 Trovafloxacin Ecenofloxacin CP 99433

IIA-3 Quinoline

Fluoroquinolones Chemical classification - Bicyclic derivatives Group II Group II B

Group IIA -six membered-ring

IIA-1

IIA-3

IIA-2

6-Fluor

18

7-piperazinyl

7-pyrrolidinyl

7-pyrrylyl

7-azetidinyl

Bicyclic

Other

Norfloxacin Pefloxacin Amifloxacin Difloxacin A – 56620 Fleroxacin Sparfloxacin Lomefloxacin Temafloxacin CS 940 Grepafloxacin Gatifloxacin DW-116 NSFQ-104 NSFQ-105 AMQ-4

PD 117596 PD 117558 PD 124816 A 57132 OPC 17080 Merafloxacin Clinafloxacin WQ 2128 A-80556 WQ 1197 SYN 987 SYN 1193 SYN 1253 S-32730 Sitafloxacin Alumafloxacin Y-688 DC-756

Pirfloxacin (Irloxacin) E 3624 E 3485 . . .

Esteves series WQ 2724 WQ 2743 WQ 2756 WQ 2765

Moxifloxacin Bay y-3118 KRQ 10196 KRQ 10099

Y-25024 Binfloxacin Y-26611 Balofloxacin BAY y 3118 SYN 987 S-31076 KRQ 10196 Y-34867

des (6) fluorinated

6-NH2

Acroxacin WIN 35439 Piroxacin Garenoxacin DX-619

MF 5137

Fluoroquinolones Tricyclic derivatives

O F

R2

COOH

R1

R2

Flumequine

CH3

H

Methyl flumequine

CH3

CH3

N

N

Abufloxacin

CH3

Verbufloxacin

CH3

N

R1

Benzoquinazoline derivatives

19

N H3CN

Fluoroquinolones Tricyclic derivatives

O

R1

F

COOH

Ofloxacin

4’-methyl piperazinyl

Levofloxacin

4’-methyl piperazinyl

Neuquinoron

H2N

CP 92121

Pyridine

N

R2 O

R1

20

R2

Fluoroquinolones O F

R7

COOH

X8

N1 R

7-position

21

Bicyclic

Piperazinyl

Pyrrolidinyl

Trovafloxacin Moxifloxacin Danafloxacin Garenoxacin

Ciprofloxacin Clinafloxacin Lomefloxacin Nadifloxacin Norfloxacin Sitafloxacin Fleroxacin Ofloxacin Sparfloxacin Grepafloxacin Gatafloxacin Levofloxacin

Azetidinyl

Pyrryl

Piperidinyl

Pyridinyl

Morpholine

E 4695 E 4767 E 4633

Irloxacin

Balofloxacin

WIN 52773

Y-26611 Y-25024

Fluoroquinolones R5

O

R6

R7

COOH

X8

N R1

Substituents at position 8 C-F

CH3

C-Cl

C-Br

Sparfloxacin Alumofloxacin Clinafloxacin WQ 2743 Lomefloxacin Sitafloxacin Fleroxacin WQ 2724 KRQ 10196 WQ 3034

22

N

COCHF 2

Enoxacin CS-940 Tosufloxacin CI 990 Gemifloxacin Trovafloxacin Ecenofloxacin

C-OCH3

CH2

Gatifloxacin Pazufloxacin Y-688 S-32730 Balofloxacin Moxifloxacin Garenoxacin DC-456 Y-34867

Ciprofloxacin Temafloxacin Pefloxacin Norfloxacin Grepafloxacin

Fluoroquinolones O R6

R7

COOH

X

N R1

Substituents at NN -1

23

Ethyl (C2 H5 )

Fluoro ethyl (C2 H4F)

Cyclopropyl (c-C3 H5 )

Pefloxacin Norfloxacin Enoxacin Lomefloxacin

Fleroxacin

Ciprofloxacin Grepafloxacin Y-688 S-32730 Alumofloxacin Gemifloxacin Moxifloxacin FD 501 FD 103 Ecenofloxacin CI 990 Balofloxacin CS 940 T-3811 KRQ 10196

Fluorocyclopropyl

4'-F-pyridyl

Sitafloxacin DX-619

DW 116

Fluorophenyl (4'F -C3H 5)

Difluorophenyl (2',4'-F-C3H 5)

Methyl amino (NH-CH3)

t-butyl

Difloxacin

Temafloxacin E-4868 Trovafloxacin

Amifloxacin

t-C4 H6 40062

5'-amino 2',4'F pyridinium WQ 3034 WQ 2724 WQ 2743

Oxetane

WQ 175 WQ 1197 WQ 1101

Fluoroquinolones

R5

O

COOH

F

R7

X8

N

R1

24

Substituents at C-5 NH2

CH3

OCH3

Sparfloxacin WQ 0175 PD 124816 SYN 987 FD 501 FD 103 KRQ 10196

Grepafloxacin BMY 43748

SYN 1193 SYN 1253

Fluoroquinolones Prodrugs O F

R7

COOH

N

N R1

R1 A 70826

R7

R’-aminoacid

2’4’ difluorophenyl

L-norval-norval

N

HN

R’ N

PD 131628

Cyclopropyl

L-alanyl HN

R’

H N

Alatrovafloxacin

2’4’ difluorophenyl

L-ala-L-ala HN

R H

25

Fluoroquinolones Trovafloxacin enantiomers

N

N

R

In vitro activity In vivo activity Pharmacokinetics (animal) +++

+++

+++

+++

+

+

H2N

H2N

26

Fluoroquinolones Enantiomers O F

COOH

N N

O H

H3C

27

In vitro activity

Pharmacokinetics

CH3

Ofloxacin

Less active

No change

CH3

Levofloxacin

2-4 x

No change

CH3

d-ofloxacin

Inactive (MIC > 128 mg/l)

No change

Fluoroquinolones Co drugs COOH O NH2

O F

COO

N

O

N

C S N

N

N H

S C

Ro 23-9484

N N

F

OCH3

H3C

OH NH N S

O N

HOOC

28

O

O

O

FCE 26600

Antibacterial activity

Fluoroquinolones Antibacterial activity Antibacterial activity R5

Outer membrane penetration DNA gyrase mutagenesis

F

Antibacterial spectrum

R7

O

COOH

X8

N

R1

Anaerobes 30

Fixation sites

Global antibacterial activity

Fluoroquinolones Antibacterial activity

R5

O

C-6 fluorine (5)

F

COOH

(6)

7-substituent N-1 substituent

(7)

R7

X8

N

(1)

(8)

C-8 substituent R1

31

C-5 substituent

Fluoroquinolones Antibacterial activity

O

COOH

4 3

2

N1

R1

32

Minimal requirement . double bond in 2-3 must be reduced . free ketone in position 4 . free carboxylic group in position 3 . N-1 has to be substituted

Fluoroquinolones Antibacterial activity

R5

F

" gyrase inhibition " cell penetration.

C6 R7

33

C-6 fluorine enhances

Fluoroquinolones Antibacterial activity

R5

N

F

N

(piperazinyl)

R

Best moiety against Gram-negative bacteria

C7 R7

X8

H2N N

(pyrrolidinyl)

R

Best moiety against Gram-positive cocci

34

Fluoroquinolones Antibacterial activity O

COOH

N1

N1

R1

Fluorocyclopropyl Cyclopropyl > 2’4’ difluorophenyl > t-butyl > oxetane > butyl > ethyl F

C2H 5

F O F

35

Sitafloxacin DX-619

Ciprofloxacin

Temafloxacin

BMY 10062

WQ 1107

Norfloxacin

Fluoroquinolones Antibacterial activity

R5

Additive activity against Gram-positive cocci

F

NH2 > OH > H e.g. : NH2 ... sparfloxacin CH3 ... grepafloxacin

C5 R7

36

X8

Fluoroquinolones Antibacterial activity

Control anaerobe activity

F

X8 R7

37

X8

C-Cl = C-F = CO-CH3 > CH > N e.g. : C-Cl ... clinafloxacin C-F ... sparfloxacin

Fluoroquinolones Extend the antibacterial activity

Gram-positive bacteria

S. pneumoniae MRSA

38

Anaerobes

Targeted bacteria

M. tuberculosis H. pylori

Fluoroquinolones Targeted indications mycobacteria Mycobacteria Structure - activity relationships have been extensively study Some fluoroquinolones are active in vitro and in clinical trials against M. tuberculosis M. leprae

1,8 naphthyridone are inactive. 39

Fluoroquinolones Targeted indications O

Mycobacteria

O

F COOH

R7

X

N R1

MIC (mg/l)

40

R1

X

R7

M. fortuitum

M. tuberculosis

PD 163753

Cyclopropyl

C-Br

3'-methyl piperazinyl

≤ 0.03

0.76

PD 161144

Cyclopropyl

C-OCH3

4'-ethyl

≤ 0.03

0.39

PD 163048

tert –butyl

N

3'-methyl piperazinyl

0.03

0.78

PD 163049

tert –butyl

N

3', 5' dimethyl piperazinyl

0.03

0.78

PD 161148 Ciprofloxacin

Cyclopropyl Cyclopropyl

C-OCH3 CH2

3'-ethyl piperazinyl piperazinyl

0.03 0.06

0.10 0.25

Sparfloxacin

Cyclopropyl

C-F

3', 5' dimethyl piperazinyl

0.06

0.06

Fluoroquinolones Targeted indications H. pylori

Two compounds Natural compounds Y-34967

41

Fluoroquinolones Targeted indications H. pylori In vitro activity - H. pylori MIC50 (mg/l) O F

COOH

N

N

O

OCH3

N

Y-34867 Levofloxacin Sparfloxacin Amoxicillin Clarithromycin

In vivo (murine infection - H. pylori 1907)

CH3

MIC (mg/l)

CH3

Y-34867

42

0.025 0.39 0.20 0.012 0.025

Control Y-34867

0.025

Amoxicillin Clarithromycin

0.39 0.05

Dose (mg/kg bid day 7) 3 10 30 30 100

Clearance (%) 0 100 100 100 0 80 From Sukurai et al, 1998

Piperazinyl derivatives

Pyrrolidinyl derivatives

Bicyclic derivatives

7

X3

N

N

R1

7

N

N X1

R2 X2 Temafloxacin Sparfloxacin Levofloxacin Gatifloxacin Grepafloxacin

Tosufloxacin Clinafloxacin

S. pneumoniae 43

Trovafloxacin Moxifloxacin

Respiratory Quinolones

S. pneumoniae

Ciprofloxacin Norfloxacin Lomefloxacin Pefloxacin Ofloxacin Enoxacin Fleroxacin 44

+ Levofloxacin Moxifloxacin Gatifloxacin Sitafloxacin Gemifloxacin Garenofloxacin

Pharmacokinetics

Fluoroquinolones Pharmacokinetics

R5

O

C-8 substituent COOH

F

R7

X8

N

R1

46

C-7 substituent

Fluoroquinolones Pharmacokinetics R5

O

COOH

F

R7

X8

N

R1

C-8 substituent :oral absorption C-7 substituent :metabolism and oral absorption C-3 substituent :iron chelation 47

Fluoroquinolones Pharmacokinetics

O

O O OH

C3 N R1

48

C3

Reduce oral absorption (interactions with antiacids, milk...) divalent cations : Ca++, Fe++, Zn++

Metal cations, antacids, anti-ulcers ?

Al2+, Mg2+, Ca2+, Fe2+ and other cations formchelate complexes with fluoroquinolones . Cation R5

O

O

R6 3 4

R7

OH

N R1

? 49

Reduce bioavailability of fluoroquinolones .

Fluoroquinolones Pharmacokinetics

C7

Improve oral absorption and water solubility eg. norfloxacin versus pefloxacin Site of metabolism for C-7 piperazinyl derivative.

50

Fluoroquinolones Pharmacokinetics

R5 F

C8 R7

X8

Substituent at C-8 may improved oral absorption C-F, C-Cl > C-OCH3, > CH

51

Fluoroquinolones Pharmacokinetics N

N

or HN

N H3C

Metabolism : 15-90% (nine metabolites)

C7 metabolism Metabolism : < 5% except grepafloxacin H3C N

N

or HN

HN

CH3

52

CH3

Adverse events

Fluoroquinolones Adverse events

Cutaneous rash Minor Gastric pain events Diarrhea

54

Specific

Adverse events

Phototoxicity CNS QTc prolongation Tendinopathies Hypoglycemia Hepatic injuries Urticaria

Fluoroquinolones Adverse events

R5

O

C-7 substituent COOH

F

C-8 substituent N-1 substituent

R7

X8

N

R1

55

Fluoroquinolones Adverse events

R5

GABA binding (CNS-tolerability)

F

piperazine > pyrrole

R7

Theophylline interaction pyrrole > piperazine R7

X8

Genetic toxicity pyrrole > piperazine

Solubility

56

Fluoroquinolones Adverse events

R5

Phototoxicity

F

C-F > C-Cl > N > CH > C-OCH3, C-CF

X8

Genetic toxicity C-F > C-Cl > C-OCH3 > N > CH R7

57

X8

Water solubility

Fluoroquinolones Adverse events

O

COOH

Control theophylline cyclopropyl > ethyl > 2’,4’-difluorophenyl > C2H4F

N1

Genetic toxicity N1

R1

58

cyclopropyl = t-butyl > 2.4’- difluorophenyl > ethyl

Fluoroquinolones Adverse events

59

Phototoxicity

Solubility

Genetic toxicity

Theophylline

CNS

N-1

-

-

+

+

-

C-5

+

-

+

-

-

C-6

-

-

-

-

-

C-7

-

++

++

+

++

X-8

++

++

++

-

-

Fluoroquinolones Clastogenicity

Concentration (mg/l) causing 50% cytotoxicity

60

Ofloxacin

= 500

Norfloxacin

= 500

Temafloxacin

= 500

Fleroxacin

= 500

Ciprofloxacin

330

Sparfloxacin

370

Tosufloxacin

120

Merafloxacin

190

Fluoroquinolones Topoisomerase II activity

ID50 (mg/l) DNA gyrase from E. coli KL-16

topoisomerase II from thymus

Ofloxacin

0.76

1870

Ciprofloxacin

0.13

155

Levofloxacin

0.78

280

Enoxacin

1.72

93

Merafloxacin

3.55

64

Nalidixic acid

23.00

325

Compound

61

Fluoroquinolones Affinity for GABA receptors

O F

COOH

R

IC50 (M)

H

> 10-3

HN

N

1.8 x 10-5

H3C

N

N

1.0 x 10-3

H3C

N

N

> 10-3

N

N

> 10-3

N

> 10-3

N

R O

CH2 H5C2

62

CO

GABA receptors IC50 (M) Norfloxacin Enoxacin Ofloxacin Ciprofloxacin Tosufloxacin Fleroxacin Sparfloxacin Levofloxacin Sitafloxacin BAY y 3118 63

Without NSAID

4-biphenylacetate

1.4 x 10-5 1.4 x 10-4 1.0 x 10-3 7.6 x 10-5 5.7 x 10-4 7.6 x 10-4 9.1 x 10-4 > 10-3 1.0 x 10-3 > 10-3

< 10-8 1.1 x 10-7 8.3 x 10-7 3.0 x 10-8 1.2 x 10-4 1.0 x 10-4 5.2 x 10-5 3.5 x 10-4 3.6 x 10-4 2.2 x 10-4

Affinity for the GABA receptor of fluoroquinolones

Adapted from HORI

Fluoroquinolones Phototoxicity

Substituents C-F

Phototoxicity +

C-Cl 8 R7

X

N

N C-H

Chemical structure

R1

C-F C-OCH3

64

-

Adapted from Domagala

Fluoroquinolones Photosensibility

Highest no effect phototoxic dosage (mg/kg)

65

Ciprofloxacin

> 300

Cl 990

> 300

Ofloxacin

> 300

Fleroxacin

> 300

Norfloxacin

> 300

Clinafloxacin

> 300

Tosufloxacin

> 100

Sparfloxacin

> 100

Neuroquino??

> 300

Lomefloxacin

> 300

Temafloxacin

300

Cl 938

300

Fleroxacin

172

Bay y 3118

172

Enrofloxacin

100

Murafloxacin

100

Phototoxicity ?

Method : ear swelling of mice after UV-A irradiation and quinolone administration X

O F

COOH

8-F

8

N

X

O-CH3

N

-H 8 CH3

66

Dose (mg/kg) N 200 800 3.1 12.5 50 50 200 800

6 6 6 6 6 6 6 6

Inflammation 0/6 0/6 0/6 3/6 5/6 0/6 6/6 6/6

From Maratuni et al, 1993

Fluoroquinolones Photocarcino-genotoxicity Mice SKH-1 (hairless) - 1.5 hours / day of 25 J/cm2 of UV-A for 78 weeks T50 % (weeks) Carcinoma Tumors 8-methoxy psoralen

67

4

++

Lomefloxacin

16

+++

Fleroxacin

28

(+)

Ofloxacin

> 50

-

Ciprofloxacin

> 50

-

Nalidixic acid

> 50

-

Fluoroquinolones Mutagenicity O F

H3C

N

COOH

N

N O

R1

ID50 (mg/l)

R2

Compound

R1

R2

MIC (µg/ml)

DNA gyrase

topoisomerase II

Levofloxacin

CH3

H

0.025

0.38

1380

DR-3354

H

CH3

1.56

4.70

2550

Ofloxacin

CH3

0.05

0.75

1870

DN-9494

=CH2

0.05

0.70

64

0.10

3.10

178

DL-8165 68

H

H

Fluoroquinolones Toxicity-tolerance : cardiotoxicity

R5

O

R6

R8

COOH

X8

N R1

Bulky substituents at C - 5seem to be responsible for cardiotoxicity (e.g : sparfloxacin : C5 = NH2) 69

Fluoroquinolones and QTc effect in humans

Agent Sparfloxacin Grepafloxacin Gatifloxacin Gemifloxacin Levofloxacin

70

Route of administration PO PO PO IV PO / IV PO PO

QTc Prolongation (mean ± sd - msec) 10.3 ± 27.6 8 6 ± 26 12.1 2.9 ± 16.5 5 ± 25.6 4.6 ± 2.3

Owens, RC Jr. Pharmacotherapy. 2001 ; 21 : 301 - 319.

Theophylline (rat) R7

% of inhibition of 1,3 DMU N

HN

N

47 <1

O N

2

HN

N H3C

71

CO N

6

Conclusion

Fluoroquinolones Difficult to predict Increased difficulties in synthesis Tolerance

Medical need Overcome ciprofloxacin resistance (S. aureus, P. aeruginosa....)

New concept Targeted clinical indication : e.g .... (Helicobacter pylori, mycobacteria) 73

Fluoroquinolones Expand the clinical indications

Intra abdominal infections Lower respiratory tract intections Upper respiratory tract infections

74

Fluoroquinolones Future - New avenues Development of new chemical structures Improvement in vitro activity correlated with clinical outcome Increased problems in the field of side effects.

New classifications of quinolones Extend the antibacterial activity Expand the clinical indications Overcome ciprofloxacin - resistance. 75

Fluoroquinolones Garenoxacin O

O OH

N HN OCHF 2 H3C

No 6-fluorine 7-dihydro iso indanyl Less activity on cartilage than other fluoroquinolones 76

GAR

TRO

CIP

S. pneumoniae

0.03

0.12

1.0

E. coli B. fragilis

0.03 0.12

0.03 0.12

0.01 2.0

ß-lactams

77

ß-lactam Classification OH

X R1 N

Penems

O COOH

R1

H N

R

H N

N O

R1

H N

N

N O

R2

X

O COOH

monocyclic ß-lactams

Penams R1

H N

X

N O

Cephems 78

R2 COOH

ß-lactam Penams

Group I

Group II

Group III

Penicillin G

Penicillin M

Group IV

6-α-OH-penicillin α carboxy penicillins IIA

Amidino penicillin

Group VII Oxi imino penicillin Oxi imino penicillin

Temocillin

IIB Ampicillin Amoxicillin N-acyl penicillins

Azlocillin Mezlocillin Piperacillin Apalcillin 79

Group V Group VI

Carbenicillin Ticarcillin Sulbenicillin

Mecillinam BRL 44154

BRL 44154

Cephems

Cephalosporin C

Discovered in 1953 (Newton & Abraham) Isolated from Cephalosporium acremonium (Brotzu, 1945) Chemical structure was elucidated in 1959 1969 : -7 amino cephalosporinic acid (7-ACA)

81

Cephems Wave of parenteral cephems

7

ACA (1960)

Cephalothin Cephaloridine (1964)

82

Cephems Classification Chemical classification (1) - Modification of the ring

R2

H N

X1

X1

N O

R

Cephalosporin

S

Oxacephems

O

Carbacephems

CH3

COOH R2

H N

X1

X1 N O

R COOH

83

-iso-2 cephalosporin

S

-iso-3 oxacephems

O CH3

Cephems Discovery of cephalosporin

Mould from C. acremonium (1945)

Cephalosporin C (1953) Hydrolysis

- amino cephalosporinic acid (7 7 -

84

ACA) (1960)

Cephems Classification Chemical classification (2) - Modification substituents R

OCH3

H N

S

R1

NHCHO

H N

S

N

N R2

O

O

R2

COOH

COOH

Cephamycins

R1

H N

Cephabicins

S

R1 N O

R COOH

85

Cephalosporins

Aryl — CH2 — Aryl — CH — ¦ X Aryl — CH ¦ X—R

Cephems Microbiological classification

Cephems could be divided according to their antibacterial spectrum in three groups limited spectrum : I and II Broad spectrum : III, IV and V Narrow spectrum : VI and VII

86

Group I : Cephalothin, cephaloridine

Active against penicillinase - producing S. aureus Other cephems from group I show marginal antibacterial activities Group I cephems show moderate anti Gram - negative activities.

87

Group I : Cephems

Designed to overcome S. aureus resistant in penicillin G First cephalosporins to bear at C-3 an heterocycle moiety Cephaloridine

N+

R

N

N

N

Cefazolin

COOH S

S

CH3

Activity against Gram-negative bacilli (Enterobacteriaceae) and stability to ß-lactamase hydrolysis - equivalent to that of ampicillin.

88

Cephems Group II

Designed to increase the antibacterial activity against Gram - negative bacilli (enterobacteriaceae). Increase stability to ß - lactamase hydrolysis.

89

Cephems - Group II

R

H N

H

Cephalosporins

N O

R

H N

Cephamycins

N O

90

CH3

Group II-Cephems Limited spectrum cephems, stable to broad spectrum ß-lactamases Less active against S. aureus (penicillinase-producing strains) than group I compounds More active against Enterobacteriaceae than group I cephems. Cephem Cefuroxime was the first derivative with an oxime side-chain. C

CO

N H

S

N O

OCH3

N O

R COOH

91

Cephems - Group II In vitro activity

MIC (mg/l) Cefuroxime

92

Cefamandole Cefoxitin

E. coli

1.0

1.0

4.0 (TEM-1)

K. pneumoniae

2.0

0.5

4.0 (CEZ-R)

S. marcescens

64.0

> 64.0

16.0

H. influenzae

0.5

1.0

2.0

S. aureus peni-R

1.0

1.0

1.0

S. pneumoniae

0.12

0.25

2.0

Group III - cephems Cephems which belong to group III have two or more of the following characteristics . 2-amino-5-thiazolyl ring N

H2N

S

. broad antibacterial spectrum . MIC50 values = 1.0 mg/l for H. influenzae, Neisseria spp, S. pneumoniae, S. pyogenes, Enterobacteriaceae (non producing class I ß-lactamases or ESBL) . good stability to hydrolysis by plasmid mediated broad spectrum ß-lactamases . good antipseudomonal activity.

93

Group III - cephems Group III is the most important group All the molecules are chemically related to cefotaxime All have a 2-amino-5-thiazolyl ring.

Alkoxy amino side chain at C-7

N

H2N

94

C

CO

N S

O

R

Group III - cephems

R

S

N R COO- Na-

R

Ceftizoxime

-H

Cefotaxime

-CH3OCOCH3 OH

Ceftriaxone

N

Cefodizime O

C3 Side - chain

N

N

Cefmenoxime N CH3

95

N

Cefuzonam N

S

CH2COOH

S

S

N S

Cephems Group III - Five subgroups A to E

Group III

96

IIIA

IIIB

IIIC

IIID

IIIE

Cefotiam Cefoperazone Cefpimizole Cefpiramide

Cefotaxime Ceftizoxime Cefodizime Ceftriaxone Ceftazidime Cefmenoxime Cefuzonam

Moxalactam Flomoxef

KT 3767 KT 3919

RU 45978 RU 46069 Ceftioxide CM 40874

Cephems - Group III Chemical modifications C-7 oxime side-chain O N OR

— CH3 Standard

97

Other

OH Better anti Gram-positive activity

Cephems - Group III Chemical modifications C-7 : methoxyimino side-chain (cefuroxime) C

CO

H N

N N

OCH3

O

O

C-3 : N-methyl tetrazol thio moiety (cefamandole) Br

H N

S

N O

CH2

S

N

N N

COOH N

98

CH3

Cephems - Group III Chemical innovation - 7moiety C

2-amino 5-thiazolyl ring

5-amino 2-thiadiazolyl ring

N

N N

H2N

H2N

S

• • 99

S

Improve antipneumococcal activity Decrease anti Enterobacteriaceae activity

Cephems - Group III Chemical structure Innovation 2-amino 5-thiazolyl ring N

+ H2N

Oxime side-chain

S

O N O

Discovery

100

N+

R

R

Antipseudomonas activity

Group III - cephems Evolution

Strep 1 : to improve the antibacterial activity extend the antibacterial spectrum " Cefotaxime Strep 2 : to improve the pharmacokinetic profile " Ceftriaxone Strep 3 : acquisition of new properties : immunorestoration " Cefodizime

101

Group III - cephems Improve the antibacterial activity MIC (mg/l) Cefamandole S. pneumoniae

0.25

0.12

E. coli Ampi-S

1.00

0.03

E. coli Ampi-R K. pneumoniae cefazolin-S

16.00 0.50

0.12 0.12

K. pneumoniae cefazolin-R

> 128.00

0.12

32.00

0.03

C. freundii

8.00

0.12

Indd + Proteus

8.00

0.12

S. marcescens

> 64.00

0.12

H. influenzae ß-

1.00

0.03

H. influenzae ß+

8.00

0.06

Enterobacter spp

102

Cefotaxime

Group III - cephems Long-acting cephem : ceftriaxone Elimination halfhalf - life (min) (rats) H3C

N

OH

N

35 O

H3C

N

O

N

OCH3

N

12 O

N

H3C

O

N N

10 O

H3C

N

O

N

O

N

O

N

10 O

103

Cefodizime Structure-activity-relationships (pharmacokinetics) MIC (mg/l) T ½ (h) N

AUC (mg.h/l)

CH3 COOH

0.27

8.5

0.31

1.8

0.52

15.2

1.28

40.7

0.30

21.4

S N

CH2

CH3 COOH

S CH3 COOH N

CH3

S CH3 N

S

CH3 COOH

Cefodizime

Cefotaxime

104

Group III - cephems Cephalosporin BRM : cefodizime

N

C

CO

H N

CH3

S N

N S

OCH3

N

S

O

S COONa +

105

CH3COONa +

Cephems Group III - oxa-1 -cephem

R1

R1

OCH3 CO

N H

Latamoxef

O N N

CH

O

N

Flomoxef

F2

CH

S

CH2

N COOH

-CH3

COOH

N

S

O

-CH2-CH2OH

NH2 OC

R2

2355-S

S F

106

R2

CH2

-CH2-CH2OH

Cephems Group III - oxa-1 -cephem Flomoxef is more active than latamoxef against Gram-positive cocci MIC50 (mg/l)

S. aureus S. epidernidis S. pneumoniae

Latamoxef

Flomoxef

Cefotaxime

6.25

0.39

1.56

25.00

1.56

1.56

1.56

0.10

= 0.025

Latamoxef and flomoxef share tho same in vitro activity against Gram-negative bacilli Latamoxef is responsible of disuliram-like syndrome and hyprothrombinemia (N-methyl substituent) and bleeding (α carboxylic group at C-7). 107

Cephems - Group IV

These compounds have been designed overcome class I producing strains within Enterobacteriaceae.

108

Cephems Group IV - Definition

Group III definition - 3’quaternary ammonium moiety C Activity against Enterobacteriaceae producing class -1 ß - lactamase.

109

Cephems Group IV - Antibacterial activity

Enhance activity against Enterobacteriaceae producing class I ß - lactamase (Amp C) Mechanism of action Some compound retain good anti Gram - positive activity (cefpirome, cefozopran) Hydrolysis by ESBL.

110

Cephems Group IV - Classification C-3' quaternary ammonium cephems C-7

C-3

2-Amino-5-thiazolyl

5-Amino-2-thiadiazolyl

Ceftazidime Cefpirome Cefepime Cefquinone Cefoselis DQ-2556 CS-461 ME-1228 MT-520 MT-382 ME-1221 ME-1220

Cefclidin Cefozopran Cefluprenam FK 518 YM 40220

DN 9550 ICI 194008 ICI 193428 TOC-39 TOC-50 CP 6679 111

L-640876 L-642946 L-652813

Cephems - Group VI - 3quaternary ammonium cephem C are zwitterionic compounds N

H2N

C

CO

H N

O

N S

R

N+

N O COO-

Negative charge (SO- or COO -)

Dianionic cephems Ceftazidime Cefsulodin

112

Negative charge (OCH3)

Zwitterionic cephems Cefpirome Cefepime Cefoselis Cefclidin Cefluprenam

Cephems - Group IV Mechanism of action

Velocity through outer membrane

113

+

Poor affinity to ß-lactamase

+

Strong affinity to PBPS

Cephems Group IV - Weaknesses

Hydrolysis by ESBL Variable activity against P. aeruginosa Short elimination half- life (≈ 2 hours).

114

Cephems Group V

Designed to overcome resistance due to ESBL, producing strains Increase in vitro activity against P. aeruginosa

115

Cephems - Group V

Chemical modification

HO

OH N

OH R CH3

O

OH

Catechol moiety fixes on the oxime chain

116

Pyrridone fixes on the oxime chain

Cephems Group V - Classification Cephems

Catecholes Group V-1

Group V-1A

Group V-1B

Group V-2A

Group V-2B

C-7

C-3

C-7

Ro 091428 Gr 69153 M-14659 E-0702 BRL 41897A RU 59863 LK 10517 Ro 44-3949 LB 10522

CP 6162

SPD 391 SPD 411 KP 736 MT 0703S

C-3 Bo 1236 Bo 1341 Merck derivatives

117

Hydroxypyridones Group V-2

RU 59863 N

O

H2N

H N

S

S

N

N+

N O

O

O

O

HO OH

O-

Staphylococcus spp

ß-lactamases F

Pharmacokinetics

118

OH

P. aeruginosa

Cephems - Group V

Antibacterial activity . overcome ESBL . original additional mechanism of action : Fe2+ chelation.

Wearknesses . metabolism . tolerance (?) . cost of production.

119

Cephems - Group VI

Investigations . . . .

120

In vitro .... MIC Bactericidal activity Affinity for PBP2a In vivo

Cephems - Group VI

121

VA

VB

Anti pseudomonas

Anti MRSA

Cefsulodin

RWJ RWJ BAL 9141 LY CP TAK-599

Cephems - Group VI

Cefsulodin . Dianionic compound : derived from sulbenicillin

Anti pseudomonal activity

CH

CO

N H

S

SO3N+

N O COO-

122

CO

NH2

Cephems group VI Cephems Group VI VIA

VIB

(antipseudomonal)

(anti-MRSA)

Cefsulodin

123

RWJ 54428 (MC 021079) TAK-599 (T -91825) Cefprozan derivatives S-3578 BMS-247243 Benzotiophene comp LY-206 763 BAL-9141 CP-0467 SM-197436 OPC 20011 TOC-39 RWG 333 441 CP 5068 ME 1209 (CP 6679) PGE 673410 PGE 9739390 CB 181963 (BC 1175) RWJ 442831 LB 11058

Figure 57

Cephems - Group VI Cephems designed for an anti MRSA activity OH N+H4

N H N

S O

N

O

NH2

N O

NH2

S

MC 02331

S

H2N

COOH O

F

NH2

O N H N

X

S

LY 274858 N

O

N O

H2N

X

S

O N

O 124

O

O-Na+

CH3

Cephems - Group VI Antibacterial activity

MIC (mg/l) MRSA heterogenous LY 274858 Methicillin Nafcillin

125

IC50 (mg/l)

MRSA homogenous

PBP 2’

1.0

1.9

3.5

256.0

> 52

456.0

32.0

128

200.0

Cephems Pharmacokinetics classification (1)

Apparent elimination half- life (three groups) Subdivision : elimination route

126

Cephems Pharmacokinetics classification Cephems T 1/2 (h)

Group I <1h

Group II 1h-3h

Urinary elimination Cephaloridine Cefacetrile Cephalothin Cephradine

127

Bile (> 20 %)

Cefotaxime Cefoperazone Ceftizoxime Cefmenoxime Ceftazidime Cefuzonam Cefpirome Cefepime Cefclidin Cefozopran Flomoxef Latamoxef Cefluprenam

Group III 3h-8h

Urinary elimination

Bile (> 20 %)

Cefodizime

Cefotetan Ceftriaxone Cefpiramide

Cephems - Group II Improvement of antibacterial activity Fixed on numerous cephems within group II and III Side effect doe to the methyl group : disulfiram-like and hypothrombinemia removal of CH3 group

N

N

N R

128

N

N

CH3?????

CH3?????

??????????

??????????

Cephems - Group VI Conclusion

Powerfull successful research to meet medical needs Ü S. aureus peni-R Ü Gram-negative bacilli Ü MRSA

129

Penems

Penem

OH

Synthetic compounds

S

H3 C

H N

- lactams. ß

O COOH

131

Antibacterial agents

Penem Classification X Group I

Group II

Group III

Penem

Carbapenem

Oxapenem

OH

A Sch 29482 Sch 34343 X

H3C

B HR 664 R N O COOH

C Ritipenem FCE 25199 FCE 21420 CGP 31608 CGP 39866 A D Faropenem TMA 3176 FCE 24362

132

Group IIA

Group II B

Group II C

Group II D

CH2

1-ß-methyl

Polycyclic

Others

Imipenem L 695256 BMY 25174 Panipenem SYN-513

Meropenem Biapenem Lenapenem S 4661 CL 190294 CL 188624 CL 191121 B 2502 A DX-8739 Ertapenem

GV 118819 S 903012

BMS 181139 Ro 403485

Penem Classification OH

X

H3C

R N O COOH

R

Group IA

Group IB

Group IC

Group ID

Group IE

Thiopenem

Oxypenem

Alkylpenem

Arylpenem

Aminopenem

Ritipenem FCE 25190 FCE 21420 CGP 31608 CGP 39068 A

Furopenem FCE 24362 TMA 3176

SCH 29182 SCH 34143 Sulopenem

133

HR 654

Penem

Synthetic compounds

New compounds

134

Penem R SCH 29482

-S-C2H5

SCH 34343

-S-C2H4OCONH2 S

Sulopenem

S+ O

OH S

Zeneca derivatives NH

S

H3C

HR 664

CONH 2

O

R N O COOH

CGP 31608

-CH2-NH2

Ritipenem

-CH2OCONH2

FCE 21420

-CH2OCOCH3

FCE 24964

-CH2OCH3

Faropenem H

O N

TMA 3176 FCE 24362 135

CH

N+

Penem

Ester (R 2)

Parent compound

OH CH3

FCE 25199 S

O

R1

SUN A 0026

N O COO-R2

136

FCE 24964

O

O

Fropenem

CP 65207

-CO-C(CH3)3

Sulopenem

Ritipenem-acoxil

-CH2OCOCH3

Ritipenem

TMA-230

-CH2OCOCH3

TMA-3176

Penem Structure-activity

OH

Antibacterial activity S

H3C

H N O

ß-lactamase stability

137

COOH

Penem Structure-activity

Penem nucleus displays an antibacterial activity equal to that of ampicillin against ampi-S strains The stereochemistry of the 6-side-chain is compulsary C-2 side-chain retains the antibacterial activity.

138

Penem Hydrolysis by DHP-1 OH H

H

OH

H3C

S

N

HOOC

OH

COOH S H3C

M1 (SUN 9609)

DHP-1 O

N O

H

Lung, kidney (dog, human) OH

COOH H H3C

OH S

Hydrolytic site HOOC

N

COOH

M2 (SUN 9608) 139

Penem Dioxolenone ester : metabolism

N

COO

CH3-CO-CO-CH3 O

O

O

diacetyl

Gastro intestinal tract/liver CH3COH-CO-CH3 acetoine

Liver CH3COH-COH-CH3 2, 3 butenadiol 140

Penem André, je suis vraiment désolé....... mais je ne vois rien du tout pour la figure page « Fro-penem 3 »

141

Macrolides

Macrolides Three waves of macrolides

1st Erythromycin A Oleandomycin Spiramycin

2d Roxithromycin Clarithromycin Azithromycin Dirithromycin

3d Ketolides 143

Objectives • S. aureus peni-R

• Atypical microorganisms • Improvement of pharmacokinetics

• Overcome resistance to erythromycin A • Enhance activity against Gram-positive bacteria

Macrolides Second wave of molecules

Target

Increase absorption Good stability in acid conditions No enhancement of in vitro activity against common pathogens Increase in vitro activity against atypical pathogens. 144

Macrolide

145

Erythromycin A

Saccaropolyspora erythrea

1952

Oleandomycin

Streptomyces antibioticus

1954

Spiramycin(s)

Streptomyces ambofaciens

1954

Josamycin

Streptomyces narbonensis var. josamyceticus vova sp

1957

Midecamycin

Streptomyces mycarofaciens

1971

Tylosin

Streptomyces fradiae

1961

Macrolide Erythromycin A O H3C

CH3

9 8 11

HO

OH OH

12

6

CH3

H3C H3C

O

O

CH3

CH3 O

O

N CH3

CH3 OH

O CH3

O CH3

CH3 146

O CH3

OH

Macrolide

O

OH

H H3C

9

[H+]

8

CH3

7 6

HO

Erythromycin A

147

H

H3C

CH3

9

H3C

CH3 8

O

O

CH3

7 6

Hemicetal

CH3

[H+]

CH3

H2O

Didehydro-erythromycin A

Macrolide

Concentrations (plasma) 2'-ester erythromycin A

148

100 mg/L

5 mg/L

2'-propionyl

69

247

2'-ethylsuccinyl

39

60

2'-acethyl

39

170

2'-butyryl 2'-valeryl

188 492

353 478

Macrolide

Erythromycin A Cmax (mg/L) Tmax (h) T ½ (h) AUC0-8 (mg.h/L)

149

0.9 ± 0.2 2.6 ± 0.4 1.5 ± 0.1 4.5 ± 1

Anhydroerythromycin A 0.3 ± 0.1 2±0 3.3 ± 2.4 4.1 ± 2.4

Macrolide Weak and link points of erythronolide A

Weak point

CH3

Linking point 1 O

8

OH

9

H3C 6

Linking point 2

HO

12

H3C

150

OH

H3C

CH3 O

R

Macrolide O F

H3C

8

CH3

HO HO

OH 6

H3C

CH3

Flurithromycin

O

O

H3C

H3C

H3C

X 12

O

X = O : Davercin X = N : Carbamate R other 151

CH3

8

HO H3C

CH3

8

HO

11

O

O

12

OCH3

OH

11 6

6

Erythromycin A

Clarithromycin

CH3

Macrolide Semisynthetic derivatives of 9 -erythromycin A OR H3C

N N

9a 9

alkylation azalide O

Beckman rearrangement

R

roxithromycin

N

9

reduction

NH2

9

intermediate synthesis derivative (9-oxime) O

erythromycylamine

weak point 9

erythromycin A 152

dirithromycin

Macrolide O

O

CH3 O

N CH3 H3C

O(CH2 )2

O

CH2

H3C

N 9

O

CH3

8

HO

10

OH OH

11

H3C

CH3 H3C

Dirithromycin

O

O CH3

CH3 O CH3 H2N

H3C

O CH3 N CH3

OH 9

HO

O

O

CH3 O CH3

10 11

Erythromycylamin

CH3

Roxithromycin

153

O

Macrolide

CH3

H3C

H3C

N CH3

CH3 R

R

CH3 8a

N

9a

8a-azalide

HO CH3 O

OCH3

CH3 CH3

CH3

O O

Azithromycin

154

CH3

HO

H3C

9a-azalide

O O

O

Erythromycin A

HO

CH3

N

O 9

H3C HO

CH3

N

OH CH3

Macrolide

CH3 X

N-10-methyl azalide

R

X=H R = CH3 N Lactam X=O R=H

14-membered ring azalides

155

Macrolide

CH3

CH3 O H3C

OCH3

HO

CH3

OCH3

H3C

[H+ ] (translactonisation)

CH3 O

HO H3C

H3C

Clarithromycin

156

Pseudoclarithromycin

Macrolide

H3C

H3C N

N O-Desosamine

O-Desosamine

HO

HO

O

CH3

O

[H+ ] OH L-cladinose cleavage

OH O CH3

Azithromycin

157

CP 66458

Macrolide 2'-esters erythromycin A

OCH3 CH3

5

O CH3 O CH3 N

CH3

D-desosamine

158

R

Sale

Acistrate

COCH3

CH3(CH2)16COOH -

Ethylsuccinate Estolate

OCCH2CH3

C12H25OSO 3H

Propionate

OCCH2CH3

-

RV-11

OCCH2CH3

HS—CH—COOH

¦

2'

O

2'-ester

R

H2C—COOH

Ketolides

Ketolides Definition

Semisynthetic derivatives of erythromycin A Lack of α - L - cladinose Highly stable in acidic environment Overcome erythromycin A resistance (MLSB inducible, efflux) Unable to induce MLSB resistance.

160

ketolides

Third wave of molecules

Target

Same pharmacokinetic profile Activity against erythromycin A resistant isolates.

161

ketolides

Target

Same pharmacokinetic profile of macrolides Activity against erythromycin A resistant isolates 162

Bacterial resistance to antibacterials

β-lactams : penicillin G, amoxicillin, cephems

Other antibacterials

Co-trimoxazole Rifampicin Tetracycline Chloramphenicol 163

S. pneumoniae

Macrolides Lincosamides Streptogramins

Fluoroquinolones

Spread of bacterial resistance

Gram -positive S. pneumoniae • S. pyogenes • Enterococcus spp • S. aureus • Coagulase negative staphylococci •

164

Other •

M. tuberculosis

•H. pylori

Gram -negative Enterobacteriaceae • P. aeruginosa •

Ketolides In vitro activity of narbomycin

165

Ketolides Natural ketolides O

O CH3

C H3

HO

CH3

CH 3

6

6

H 3C

H3C O

O 3

O

3

O O

O CH3

H 3C

O

O

O CH 3

N

OH

Picromycin

166

O NZ

OH

Narbomycin

Ketolides

Natural derivatives Picromycin Narbomycin

Semi synthetic derivatives

15-membered

GW 581506X

167

14-membered Telithromycin HMR 3004 Cethromycin HMR 3562 HMR 3787 HMR 3832 TE-802 TE-810 CP 654743

Ketolides Telithromycin

3

O-cladinose

HMR 3004 HMR 3787 HMR 3832 HMR 3562 Cethromycin

Ketolide

3

•O

168

Aventis Pharma

Abbott

TE 802, TE 810 Taisho RWJ 415 663 RWJ 415 667

RW Johnson and pharmaceutical

CP 654 743

Pfizer

Ketolides They are composed of three specific chemical structures

3-keto function (lack of L-cladinose) Side-chain C11-C12 Side chain C6

169

3

170

KETO FUNCTION

Ketolides

New medicinal chemical entities Semisynthetic compounds obtained from erythromycin A Removal of the L - cladinose (neutral sugar), and oxidation of the 3 - hydroxyl (OH) yields to a -3 keto group (ketolide). 3

3

O

cladinose removal

171

3

OH

Oxidation

O (= ketolide) - keto 3

Ketolides 3-keto function 3-keto function imparts the following biological properties 3

O

Antibacterial activities against erm-containing-Gram-positive cocci Absence of ability to induce MLSB resistance High stability in acidic environment.

172

Stability in acidic environment

pH 1.0 at 37°C

Telithromycin

100

% of activity

80 60 40

Azithromycin

20

Clarithromycin 0 0

173

1

2

3

4

5

6 Time (h)

Interaction with human motilin receptor

IC50 (µM) (inhibition of motilin binding ) Telithromycin

> 100µM (23 % inhibition at 100 µM)

Clarithromycin

≥ 100 µM (54 % inhibition at 100 µM)

Erythromycin A < 3 µM (60 % inhibition at 3 µM)

174

No inducer of MLSB resistance 1.2

1.2

1.0

1.0

0.8

0.8

0.6

0.6

0.4

0.4

Ery/Ery

0.2

Ery/Ery

0.2

RU 69874/Ery

TEL/Ery 0

C/Ery 0

0

C/Ery

0.91 1.50 1.91 2.19 2.49 2.82 3.32 3.82 4.67 5.57 6.32

0 0.91 1.50 1.91 2.19 2.49 2.82 3.32 3.82 4.67 5.57 6.32

N

N N

O H3C

N

CH3

H3C

C

O

O

N

Telithromycin

CH 3

CH3

H3C O

C

O

O CH3

N

CH3

H3C O OH

O

O

H3C

O O

O CH3

O CH3

CH3 H3C

N CH3

RU 69874 OH

CH3

O O

N

CH3

O

O H3C

CH3

175

N

O

CH3 HO O OCH3

H3C

N CH3

C11 -

176

C12 carbamate residue

Bacterial resistance to antibacterials

β-lactams : penicillin G, amoxicillin, cephems

Other antibacterials

Co-trimoxazole Rifampicin Tetracycline Chloramphenicol 177

S. pneumoniae

Macrolides Lincosamides Streptogramins

Fluoroquinolones

Clarithromycin... ...carbamates analogues R A-61795

H

A-62514

N H

O

A-66173 N

N H

R N

A-66005 O N

A-64239

A-66321 178

CH2

Spread of bacterial resistance

Gram -positive S. pneumoniae • S. pyogenes • Enterococcus spp • S. aureus • Coagulase negative staphylococci •

179

Other •

M. tuberculosis

•H. pylori

Gram -negative Enterobacteriaceae • P. aeruginosa •

Ketolides C11-C12 carbamate substituted side chain Carbamate residue In vitro activity Pharmacokinetics O

Pharmacodynamics

R

Intracellular kinetics

N 11

O

Efflux Mechanism of action Tolerance- toxicity.

180

12

O

Research in anti-infectives

Ketolides

New chemical entities

3

O

N

C11 – C12 carbamate ketolide

O

11 12

Optimisation

O

R N

Substituted carbamate ketolide 181

11

O

12

O

Innovation

pyridyll imidazolyl N N

butyl N

R

C

N

N

11

11

O 12

O

Carbamate ketolide 182

O

12

O

telithromycin

Ketolides C11-

C12 carbamate substituted side chain Imidazolyl ring

Quinoline ring

N O

N

O N HC

CH HN O N

CH3

C

O

OCH3

N

CH3

3

3

O

CH

3

N

Pyridyl ring

CH

HC

3

3

O

O

HMR 3004

183

Telithromycin

Mode of action Significant of different mode of action A-2058

5S rRNA

A-2058

V 5S rRNA

ERY

TEL

cladinose

A-752

O

II

No link with domain V

Resistance to erythromycin A, azithromycin, roxithromycin, clarithromycin, 184

V

A-752

II

Link with domain II

Telithromycin retains activity against erythromycin A-resistant organisms

Ketolides C11-

C12 carbamate substituted side chain (1) Pharmacokinetics in mice Concentrations (mg/l) at Cmax Tmax (mg/l) (h)

0.25* 0.50

Telithromycin

4.67

0.50

HMR 3004

1.70

0.25

* sampling time (hours)

185

1.0

2.0

3.0

4.0

3.97

4.67 3.52

1.60

1.70

0.90

1.70

0.90 1.00

0.20

0.10

0.06 Craig, 1996

Ketolides C11-

C12 carbamate substituted side chain (2) Pharmacokinetics in mice

Cmax (mg/l)

Tmax (h)

AUC0-24h (mg.h/l)

Telithromycin

4.67

0.50

17.92

HMR 3004

1.67

0.25

2.47 Craig, 1996

186

Ketolides R N

C11 -C12 carbamate residue

11

O

12

O

Comparative human pharmacokinetics after a single oral dose 600 mg N

N N N

HMR 3004

HMR 3647 N

Cmax (mg/l) 0.16 ± 0.30 Tmax (h) 1.75 AUC 0-8 (mg.h/l) 0.59 ± 0.11 C24h (mg/l) ND t1/2 (h) 2.25 ± 0.16 187

Cmax (mg/l) Tmax (h) AUC 0-8 (mg.h/l) C24h (mg/l) t1/2 (h)

0.90 ± 0.13 1.5 4.09 ± 0.55 0.01 ± 0.01 11.0 ± 1.90

Telithromycin

Metabolites (1)

CH2 OH N N

O CH3 C

O

C N

N OCH3

N H3C

C

CH3 O

RU 78849 (N-propyl carboxylic)

COOH

N

N

N

O

N

O-

OH O H3C

RU 76584 (N-pyridine oxide)

C N

CH3

N O

H3C

Telithromycin

O

H3C

N

CH3

H

RU 72365 (N-monodemethyl desosamine) 188

Telithromycin

Disposition

Systemic circulation (blood stream) Intestinal secretion

Bile

Kidney

• 11.8 % telithromycin • 5.6 % metabolites

Metabolism to a : Liver

Absorption Gastrointestinal tract

Urine (17.4 %)

• • • • •

RU 76363 12.6 % RU 72365 2.97 % RU 78849 2.22 % RU 76584 2.03 % Others

(Via bile)

Formation enzyme non CYP CYP3A CYP3A CYP3A/1A

Feces (75.6 %) • 20.2 % telithromycin • 55.4 % metabolites

Metabolites expressed as relative radioactivity in circulation (metabolite AUC / telithromycin AUC)

189

Structure activity relationships

N N

O HC

3

9

C

O

• Mode of action • Reduced efflux

11

O

CH

3

N 12

H3C

• • • •

C H3 O

O H3C

CH3

3

O

O

O OH

CH3

• High stability in acidic pH • Overcoming MLSB • Non inducer of MLSB resistance 190

N

CH

3

O CH

3

H3C

N CH3

Enhanced in vitro activity Intracellular accumulation Pharmacokinetics Tolerance

Macrolide resistance

14-membered ring macrolide

O

Phosphorylation

HO

P O

OH

E. coli Nocardia spp

CH2 OH

Glycosylation HO

O 1'''

CH3

HO

O

OH 6

191

O CH3 O

2'

N(CH 3)2

O

CH3

HO

Streptomyces antibioticus Streptomyces Vendargensis Nocardia spp

Macrolide resistance

Ring hydrolysis by esterases

HO 11

H3C

12 13

HO

O

1

H3C

Hydrolysis esterases

O

CH3

Erythromycin A

Described in S. aureus - E. coli OH

OH CH3

H3C O

192

Macrolide resistance Erythromycin A binding site

O CH2

O

O P

O

Ribosome methylase Erne OH

Adenine 2058 in 23 S rRNA

CH3

N

N

N

HO

193

N

N

N

O

H3C

S-adenosyl-L-methionine

NH2

P

Transfert of methyl group in adenosyl methionine

N O

Erythromycin

N

No binding

CH H2 O

HO

O

erm gene

N

OH

Dimethylated A-2058 (A ⇒ G)

Adapted from K. O'hara, 2000

Macrolides Mechanism of resistance CH

3

O

R2

R1

O

O

7

5

3

HO

CH3

8

OH H3C HO

CH3 6

9

O

5

H3C

4

CH 3

3

O O

R1

2 1

H3C

OH

O

OH

15 14

HO

CH3

O

R1 : α-L cladinose R2 : D-desosamine 194

OH

R2

Esterase

13

H3C

12

11

10

9

OH CH3

CH3

CH3

2

CH3

C 1

O