linezolid case study

Linezolid – a true small molecule antibiotic

Alexei Pushechnikov, Ph.D.

Disney group

January 13, 2009

Outline

• Introduction

• Synthetic Approaches

• Activity

• Mode of Action

• Conclusions

2

Introduction 3

Ford, C. W.; Zurenko, G. E.; Barbachyn, M. R. Cur. Drug Targets 2001, 1, 181-199Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008, 51, 1981-1990

NO

O

NHAc

XO

X = C : DuP 721X = S : DuP 105

NO

O

OH

SO

H2N O

NO

O

Cl

S(O)n

E.I. du Pont de Nemours & Co.Slee, A.M. et al 1987

DuPont’s SAR of the oxazolidinone pharmacophore:

importance of the N-aryl group

optimal activity of a C-5 acetamidomethyl group

C-5 (S)-configuration required for antibacterial activity

electron-withdrawing groups in the aryl para- position provided optimal activity

additional substitutions at the aryl ortho- position or C-4 of the oxazolidinone ring had a detrimental or indifferent effect on the antibacterial activity

NO

O

NHAc

O

Introduction 4

N NX(CH2)n

O

OR3

R4

R1

R2

HN R

O

Brickner, S.J.; Hutchinson, D.K.; Barbachyn, M.R.; Manninen, P.R.; Ulanowicz, D.A.; Garmon, S.A.; Grega, K.C.; Hendges, S.K.; Toops, D.S.; Ford, C.W.; Zurenko, G.E. J. Med. Chem. 1996, 39, 673

Barbachyn, M.R.; Brickner, S.J.; Hutchinson, D.K.

WO95/07271 The Upjohn Company (Filed on April 1994)

N

O

O

NHAc

N

E-3709

N

O

O

NHAc

U-97457

N

OHO

N

O

O

NHAc

N

F

N

N

O

O

NHAc

N

F

O

U-100592 U-100766

O

HO

Introduction 5

Barbachyn, M. R.; Ford, C. W. Angew. Chem. Int. Ed. 2003, 42, 2010-2023

Generalized testing scheme for oxazolidinones development

Analogues In Vitro testing - Intrinsic activity

- Activity against resistant strains- Spectrum of activitySAR

In Vivo testing - Acceptable in vivo activity

- Route of administration

Pharmacokinetics/Toxicology - Useful blood levels

- Frequency of dosing - Acceptable toxicity profile

Further Biological Characterization - Mechanism of action

- Pharmacodynamics - Tissue penetration

Clinical Trials Registration

Introduction 6

N

O

O

NHAc

N

F

O

Linezolid(PNU-100766)

Drug-like compounds (Lipinski’s rule)

* Not more than 5 hydrogen bond donors : 1

* Not more than 10 hydrogen bond acceptors : 6 (8)

* A molecular weight under 500 daltons : 337

* A partition coefficient logP less than 5 : 0.9

* Number of atoms from 20 to 70 : 44

* Number of rotatable bonds less than 10 : 5

* Polar surface area (PSA) less than 140 : 91

Computed Properties - Chem3D Properties Broker

Has been approved by the FDA in 2000 under the trade name Zyvox

Good solubility : 3.7 mg/mL in pH 7 phosphate buffer

The oral bioavailability : 100% (rapid and complete absorption)

The excretion : 20–30% of the dose found in the urine as the parent drug


Introduction 7

NO

O

NHAc

NO

O

NHAc

O O

PNU-82965 PNU-85055 PNU-85112

NO

O

NHAc

N

O

HO

NO

O

NHAc

O

PNU-86093H

N O

O

NHAc

N

E-3709

NN

F

O

CO2H

HN

Ciprofloxacin

N

F

O

CO2H

N

Win-57273

N O

O

NHAc

N

N

(F)H

H(F)R1

R2

R3

NO

O

NHAcPNU-97457

N

O

HO

NO

O

NHAc

N

F

O

Introduction 8

N

O

O

NHAc

N

F

N

Eperezolid(PNU-100592)

O

HO

N

O

O

NHAc

N

F

S

PNU-100480

N

O

O

NHAc

N

F

N

PNU-97665

NC

N

O

O

NHAc

F

PNU-97786

OHN

Pharmacia&Upjohn’s revised SAR :

N-aryl group required for activity

electron-donating nitrogen atom well tolerated and often improves safety profile

C-5 (S)-configuration necessary for antibacterial activity

C-5 acetamidomethyl group essential for good activity

fluorination of phenyl ring often improves antibacterial activity/efficacy

Synthetic approaches 9


F

F

NO2 HN O

DIPEAEtOAc, reflux

F

N

NO2

O

1. H2, Pd/C

2. CbzCl, NaHCO3 Acetone/water

F

N

NHCbz

O

1. n-BuLi, THF, -78°C

2. O

OO

H

F

N

N

O

OO

OH

1. MsCl, Et3N DCM

2. NaN3, DMF

F

N

N

O

OO

N3

1. H2, Pd/C

2. Ac2O Pyridine

Linezolid

(>99.8% ee)


1. n-BuLi, THF, -78°C

2. O

OO

HArHN O

O

RN

ArO

RO

LiO

HH

H O O

ArN

ORO OLi

HO

OONAr

O

H

O

O

RO

ONAr

O

H

OH

RO

O

+




F

F

NO2

HN

OF

N

NH2

O2. H2, Pd/C

CbzCl

K2CO3

F

N

NHCbz

O

F

N

N

O

O

O

HO

1. NsCl, Et3N

2. NH4OH, MeOH 45°C

Linezolid

1.

Cl OHHO H

1.

2. t-BuOK3. LDA

F

N

N

O

O

O

H2N

Ac2O

Process scale synthesis


OH

OHOH

OHOH

OH O

OO

O 1. ArNH2

2. CDI

O

OO

O

N

N

Ar

ArO

O

dil. HCl

O

O

OH

HO

N

NO

Ar

O

Ar

1. Pb(OAc)4/THF

2. NaBH4/MeOHO

HO

N

OAr

O

N3

N

OAr

1. MsCl, Et3N

2. NaN3/DMSOO

AcHN

N

OAr

SH

O

Lohray, B. B.; Baskaran, S.; Rao, B. S.; Reddy, B. Y.; Rao, I. N. Tetrahedron Lett. 1999, 40, 4855-4856

Activity 13In vitro activities of linezolid and vancomycin, Minimum Inhibitory Concentration (mg/L)

Staph

yloco

ccus

aur

eus

Staph

yloco

ccus

epi

derm

idis

Strept

ococ

cus p

neum

onia

e

Strept

ococ

cus p

yoge

nes

Enter

ococ

cus f

aeciu

m

E. faec

ium

- VR

Enter

ococ

cus f

aeca

lis

E. faec

alis

- VR

Haem

ophi

lus i

nflu

enza

e

Mor

axel

la ca

tarrh

alis

Bacte

roid

es fr

agilis

Pepto

strep

toco

ccus

spp.

Coryn

ebac

teriu

m sp

p.

Clostr

idiu

m sp

p.0

2

4

6

8

10

12

14

16

18

Linezolid

Vancomycin

MIC

90


Activity 14


0

5

10

15

20

25

30

35

40

45

50

>100

Linezolid

Vancomycin

ED

50

In vivo activities of linezolid and vancomycin, Effective Dose (mg/kg)

Linezolid – orally Vancomycin - subcutaneously

Activity 15Activities against Mycobacterium tuberculosis

In Vitro

Line

zolid

Epere

zolid

PNU-100

480

Isoni

azid

0

0.2

0.4

0.6

0.8

1

1.2

MIC

90

, mg

/L

Late Con-trols

Linezolid Eperezolid PNU-100480

Isoniazid0

1

2

3

4

5

6

7

8

9Spleen

Lung

Lo

g1

0 C

FU

/org

an

In Vivo

Treatment was started 1 day after the mice received 7x106 viable mycobacteria.

Cynamon,M.H.; Klemens,S.P.; Sharpe,C.A.; Chase,S. Antimicrob. Agents Chemother. 1999, 43, 1189-1191

Activity 16

• Linezolid lacked significant effects against most Gram-negative pathogens

including E. coli, Klebsiella pneumoniae, and Proteus penni

• Despite the observed in vitro activity, linezolid was ineffective against the

Moraxella catarrhalis and H. influenzae, even at concentrations above the MIC

• However, in the absence of cell membranes and cell walls the oxazolidinones

were very active in inhibiting E. coli protein synthesis

• Making the E. coli transmembrane pump nonfunctional made whole E. coli cells

sensitive to linezolid both in vitro and in vivo

• The lack of Gram-negative activity is the result of the presence of

transmembrane pumps which, along other molecules, pump oxazolidinones out

of the cell faster than they can accumulate

Activity 17Pharmacokinetic Profile

Oral bioavailability 100%

Food effectSlight decrease in rate but not extent of absorption

Volume of distribution 40-50L

Protein binding 0.31

Peak concentration 1-2h after oral dose

375 mg 12 mg/L

625 mg 18 mg/L

Elimination half-life

oral 5.5h

intravenous 4.5h

Excretion

renal 30%

liver 70%

Active metabolites 50%

Excreted unchanged in urine 30%

Activity 18

• Linezolid behaves as a cidal drug in vivo although it is clearly static for

staphylococci and enterococci in the test tube

• Linezolid is generally considered to be well tolerated in humans. Most common side

effects in the clinical trials were (percent incidence) :

– diarrhea (2.8-11%)

– nausea (3.4-9.6%)

– headache (0.5-11.3%)

• With longer term usage of linezolid (>2 weeks), there is an association of reversible

myelosuppression (anemia, thrombocytopenia, leukopenia, or pancytopenia)

• Linezolid is a weak, reversible, and nonselective inhibitor of Monoamine Oxidase. A

risk of serotonin toxicity is anticipated with linezolid. Avoidance of large quantities of

food with a high tyramine level (aged cheese, beer, or red wine) along with

administering linezolid is suggested

Lawrence, K.R.; Adra, M.; Gillman, P.K. Clin. Infect. Dis. 2006, 42, 1578-1583Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008, 51, 1981-1990

Mode of Action 19

Swaney,S.M.; Aoki,H.; Ganoza,M.C.; Shinabarger,D.L. Antimicrob. Agents Chemother. 1998, 42, 3251-3255Clemett,D.; Markham,A. Drugs 2000, 59, 815-827

Mode of Action 20

Lin,A.H.; Murray,R.W.; Vidmar,T.J.; Marotti,K.R. Antimicrob. Agents Chemother. 1997, 41, 2127-2131

[14C]Eperezolid binding to E. coli ribosomes.

(A) Total ribosomes; (B) 50S subunits; (C) 30S subunits.

▓, total binding; █, specific binding.

Mode of Action 21

Lin,A.H.; Murray,R.W.; Vidmar,T.J.; Marotti,K.R. Antimicrob. Agents Chemother. 1997, 41, 2127-2131

Binding to ribosomes.

Competition by various concentrations of unlabelled antibiotics.

(A) [14C]eperezolid binding; (B) [14C]chloramphenicol binding.

●, eperezolid; ■, linezolid; ▲, chloramphenicol; ▼, lincomycin.

Eperezolid and linezolid bind to the 50S ribosomal subunit with Kd ~20 M

Mode of Action 22

Lin,A.H.; Murray,R.W.; Vidmar,T.J.; Marotti,K.R. Antimicrob. Agents Chemother. 1997, 41, 2127-2131Thompson, J.; O’Connor, M.; Mills, J.A.; Dahlberg, A.E. J. Mol. Biol. 2002, 322, 273–279

• Oxazolidinones block translation initiation, binding to a site on the 50S subunit closely

related to the chloramphenicol and lincomycin binding site and near the interface with

the 30S subunit.

• The resulting distorted site may prevent the correct positioning of the 30S initiation

complex from forming the 70S initiation complex and hence inhibit translation initiation.

N

O

CH3

H3C

NH

CHHO

CH3

O

OH

CH

SCH3

OH

HO

Lincomycin

OH

O2N

HN

O

Cl

Cl

OH

Chloramphenicol

Mode of Action 23

Kloss,P.; Xiong,L.; Shinabarger,D.L.; Mankin, A.S. J. Mol. Biol. 1999, 294, 93-101Xiong,L.; Kloss,P.; Douthwaite, S.; Andersen, N.M.; Swaney,S.; Shinabarger,D.L.; Mankin, A.S. J. Bacteriol. 2000, 182, 5325-5331

• Location of linezolid resistance

mutations in E. coli 23S rRNA

(central loop of domain V and the

neighboring regions).

• E. coli linezolid resistance

mutations are shown by arrows

(thickness proportional to the level

of linezolid resistance for each

mutation).

• Marked positions of nucleotide

substitutions that confer linezolid

resistance in H. halobium (boxed)

and in S. aureus and E. faecalis

(circled).

Mode of Action 24

Leach, K.L., Swaney, S.M., Colca, J.R., McDonald, W.G., Blinn, J.R., Thomasco, L.M., Gadwood, R.C., Shinabarger, D., Xiong, L., Mankin, A.S. Mol. Cell 2007, 26, 393-402Wilson, D.N.; Nierhaus, K.H. Mol. Cell 2007, 26, 460-462

Orientation of Linezolid at the Peptidyltransferase Center of the Ribosome. (A) Model for the binding position of linezolid (Lnz, red) with respect to nucleotides (blue) at the

E. coli PTC. (B) Relative position of linezolid (red) compared to chloramphenicol (Cam, green). PTC

nucleotides are shown as green surface representation.

A B

Mode of Action 25

Linezolid (cyan) and CCA-Phe (gold) binding to H50S. Linezolid molecule occupied the A-site and CCA-Phe occupied the P-site (PDB code 3CPW)

Superposition of the structure of linezolid (cyan) with the structures of A-site (orange) and P-site (green) substrate analogues bound to H50S

Ippolito, J.A.; Kanyo, Z.F.; Wang, D.; Franceschi, F.J.; Moore, P.B.; Steitz, T.A.; Duffy, E.M. J. Med. Chem. 2008, 51, 3353-3356

Mode of Action 26

Wilson, D.N.; Schluenzen, F.; Harms, J.M.; Starosta, A.L.; Connell, S.R.; Fucini, P. Proc. Natl. Acad. Sci. USA 2008, 105, 4673-4678

The binding site of oxazolidinones.Linezolid bound to the Deinococcus radiodurans 50S ribosomal subunit.

Oxazolidinones induce an A/O state recognized by LepA. Relative position of linezolid (red), P-tRNA (cyan), A-tRNA (pale green), LepA (maroon density and ribbon) and A/L-tRNA (blue).

Mode of Action 27

Wilson, D.N.; Schluenzen, F.; Harms, J.M.; Starosta, A.L.; Connell, S.R.; Fucini, P. Proc. Natl. Acad. Sci. USA 2008, 105, 4673-4678

Events during normal translation (A–D), compared with the effect of the linezolid (red) during translation (E–H).

Conclusions 28

Linezolid has been approved in the U.S.

• for the treatment of nosocomial and communityacquired pneumonia

caused by S. aureus (methicillin-susceptible or MRSA) or S.

pneumoniae (penicillin-susceptible or multidrug-resistant strains) and

vancomycin-resistant E. faecium (including concurrent bacteremias)

• for use in children and newborns against Gram-positive infections

• for treatment of complicated skin and skin-structure infections

including those due to MRSA (including Gram-positive bacterial

diabetic foot infections (MRSA) without concomitant osteomyelitis)

• the only approved agent for treatment of hospital-acquired MDR

S.pneumoniae infections and is the first and only oral drug approved

for the treatment of VRE infections.

Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008, 51, 1981-1990

Conclusions 29

• Early 1993 - first synthesis

• April 1995 - entered phase I trials

• 1996 - initiated phase II studies

• January of 1998 - phase III trials began

• April 18, 2000 - approved by the FDA

• January of 2008 - has been used in an estimated 3 million

patients

Brickner, S.J.; Barbachyn, M.R.; Hutchinson, D.K.; Manninen, P.R. J. Med. Chem. 2008, 51, 1981-1990

linezolid case study

Documents

drug targets2001

activity16 linezolid

activity route

parent drug

antibacterial activityc

effective dose mgkg

optimal activity importance

synthetic approaches12lohray