anticancer agents farnesyl transferase inhibitors - sar (structure activity relationship)

ANTICANCER AGENTS

FARNESYL TRANSFERASE INHIBITORS

Patrick: An Introduction

to Medicinal Chemistry 5e Chapter 21

1

1. Ras Protein

Notes

•Signalling protein that is crucial to cell growth and division

•Abnormal form is present in 30% of cancers

•Prevalent in colonic and pancreatic cancers

•Abnormal Ras is coded by a mutated ras gene

•Small G-protein

•Ras binds GDP in resting state and GTP in active state

•Active Ras normally autocatalyses hydrolysis of GTP back to

GDP

•Abnormal Ras fails to hydrolyse GTP

•Abnormal Ras remains permanently active

•Three human Ras proteins (H-Ras, N-Ras and K-Ras) 2

2. Farnesyl transferase

Notes

•Zinc metalloproteinase

•Catalyses attachment of a farnesyl group to Ras

•Hydrophobic farnesyl group anchors Ras to the inner part of

the cell membrane

•Farnesylation is necessary for Ras to become activated during

signal transduction

•Inhibition of farnesyl transferase should inhibit this process

3

PPO

farnesyl diphosphate

Ras

HN

NH

HN

NH

O

O

O

OH

OHS

S

MetVal

Cys FTase Ras

HN

NH

HN

NH

O

O

O

OH

OS

S

Further

processing

Ras

HN

OMe

O

S

Methyl ester


Enzyme mechanism

4


Notes

•Farnesyl diphosphate (FPP) binds to the active site first

•FPP aids binding of Ras protein to the active site

•Magnesium and iron ions are present as cofactors

•Magnesium ion interacts with the pyrophosphate group

•Results in a better leaving group

•Iron ion interacts with the thiol group of cysteine

•Results in thiol acting as a better nucleophile

5

3. FT Substrates

C-a-a-X

Substrate

•C = cysteine

• a = valine, isoleucine or leucine

•X = methionine, glutamine or serine

Substrates share a terminal tetrapeptide moiety called the CaaX

peptide

6

4. FT Inhibitors

Aims

•Good inhibitory activity vs enzyme

•Ability to cross the cell membrane to reach the enzyme

•Metabolic stability

•Aqueous solubility

•Oral absorption

•Favourable pharmacokinetic properties

7

4. FT Inhibitors

Notes

•Inhibitors were developed to mimic the terminal tetrapeptide

moiety - CaaX peptide

•Tetrapeptides having Phe next to X act as inhibitors

•Serve as lead compounds

C-a-a-X

Substrate

C-a-Phe-X

Inhibitor

•C = cysteine

• a = valine, isoleucine or leucine

•X = methionine, glutamine or serine

8

5. Lead compound

H2NNH

HN

NH

OH

O

O

HS

SMe

O

O

Cys

Val

Phe

Met

Disadvantages •Terminal carboxylic acid likely to be ionised - bad for absorption

•Peptide bonds are susceptible to enzyme-catalysed hydrolysis

•Poor stability to digestive or metabolic enzymes (e.g. aminopeptidases)

9

Lead compound

H2NNH

HN

NH

OH

O

O

HS

SMe

O

O

Cys

Val

Phe

Met

6. Drug design

Notes

•Modifications carried out to remove peptide nature - peptidomimetics

•Ester masks polar carboxylic acid or carboxylate ion - acts as prodrug

•Methyleneamino link replaces N-terminal peptide bond

•Methyleneamino link introduces a resistance to aminopeptidases

•Peptide bond isostere introduced to mimic central peptide bond

•Isostere should be capable of mimicing any binding interactions

•Isostere should be stable to enzyme-catalysed hydrolysis

H2NNH

XY

NH

OR

O

O

HS

SMe

Peptidomimetic

Methylene-

amino link

H2NNH

XY

NH

OR

O

O

HS

SMe

Peptidomimetic

Ester

Methylene-

amino link

H2NNH

XY

NH

OR

O

O

HS

SMe

Peptidomimetic

Peptide bond

isostere

Ester

Methylene-

amino link

H2NNH

XY

NH

OR

O

O

HS

SMe

Peptidomimetic

10

7. Examples of FT Inhibitors

H2NNH

HS

NH

O

OR

O

SMe

R=H FTI 276

R=iPr FTI 277

Terminal

amino group

Thiol Aromatic

substituent

Notes

•Thiol group forms important interactions with the zinc ion cofactor

•Methyleneamino link is stable to aminopeptidases

•Aromatic substituent is important for inhibitory activity

•Aromatic ring acts as a peptide bond isostere

•Terminal amino group is ionised

•Terminal amino group forms an ionic bond to the phosphate group of FPP

•Terminal carboxylate group is important to binding

Stable

methylene-

amino link

H2NNH

HS

NH

O

OR

O

SMe

Peptide bond

isostere

Stable

methylene-

amino link

H2NNH

HS

NH

O

OR

O

SMe

11


H2NNH

ONH

OR

O

O

HS

SO2Me

Sulfone

Aromatic

substituent

Terminal

amino group

Thiol

R=H L739750

R=iPr L744832

Notes

•Thiol group forms important interactions with the zinc ion cofactor

•Methyleneamino link is stable to aminopeptidases

•Aromatic substituent is important for inhibitory activity

•Methyleneoxy group acts as the peptide bond isostere

•Terminal amino group is ionised

•Terminal amino group forms an ionic bond to the phosphate group of FPP

•Terminal carboxylate group is important to binding

•Sulfone increases activity over a methylthio group

Peptide bond

isostere

Stable

methylene-

amino link

H2NNH

ONH

OR

O

O

HS

SO2Me

12

AZD-3409

HN

NH

O

O

O

F

NH

S

O

N

SMe

Pyrrolidine

Aromatic

substituent


Notes

•Acts as a prodrug

•Thiol and carboxylic acid groups are both masked in the prodrug

•Lowers the toxicity risk of the thiol group

•Protects the thiol from possible metabolism

•Pyrrolidine ring introduces conformational rigidity

•Potent inhibitor (Ki < 1 nM)

•Also inhibits geranylgeranyltransferase which catalyses prenylation with

geranylgeranyl diphosphate

•Agents inhibiting both enzymes are potentially advantageous

Masking

group

Masking

group

AZD-3409

HN

NH

O

O

O

F

NH

S

O

N

SMe

Peptide bond

isostere

Masking

group

Masking

group

AZD-3409

HN

NH

O

O

O

F

NH

S

O

N

SMe

13

O

SN

NN

NH

O

NC

S

Structure I

IC50 1.4 nM


Notes

•Non-peptide inhibitor

•Imidazole ring acts as the zinc ligand

•Decreases the risk of toxicity due to a free thiol group

Imidazole ring

O

SN

NN

NH

O

NC

S

Structure I

IC50 1.4 nM

14

N

Br

N

Br

Cl

O

N NH2

O

Lonafarnib

IC50 1.9 nM


Notes


•Developed from lead compound discovered by screening compound libraries

•10,000 times more active than the lead compound

•No ligand for the zinc cofactor is present!

15



•Developed from lonafarnib by structure-

based drug design

•Imidazole ring introduced as zinc ligand

•Aromatic ring introduced as a steric

shield vs metabolism

N

N

N

Cl

O O

N

O

NN

Me

Sch 226374

IC50 0.36 nM

N

Br

N

Br

Cl

O

N NH2

O

Lonafarnib

IC50 1.9 nM

Steric

shield

N

N

N

Cl

O O

N

O

NN

Me

Sch 226374

IC50 0.36 nM

Imidazole

ring

Steric

shield

N

N

N

Cl

O O

N

O

NN

Me

Sch 226374

IC50 0.36 nM

16

•Strategy - variation of

substituents

•Activity increases with

addition of N-methyl

substituent

N

HN

Cl

N

O

Cl

II; IC50 35 nM

N

MeN

Cl

N

O

Cl

III; IC50 15 nM

8. Development of Tipifarnib

Imidazole

Quinolone

N

HN

Cl

N

O

I; IC50 180 nM

17

•Lead compound

•Identified from screening

compound libraries

•Imidazole ring present - zinc

ligand

•Both aromatic rings are

important to activity

•Strategy - variation of

substituents

•Activity increases with

introduction of a meta-chloro

substituent

N

HN

Cl

N

O

Cl

II; IC50 35 nM

N

MeN

Cl

N

O

Cl

III; IC50 15 nM

MeN

ClN

N

O

Cl

Me

IV; IC50 2.5 nM

•Strategy - variation of ring substitution

•Activity increases


Imidazole

Quinolone

N

HN

Cl

N

O

I; IC50 180 nM

18

N

MeN

Cl

N

O

Cl

III; IC50 15 nM

MeN

ClN

N

O

Cl

Me

IV; IC50 2.5 nM

MeN

Cl

ClN

N

Me

H2N

O

Tipifarnib; IC50 0.6 nM

•Extension strategy

•Extra functional group

•Extra binding interactions

•Activity increases


Imidazole

Quinolone

N

HN

Cl

N

O

I; IC50 180 nM

N

HN

Cl

N

O

Cl

II; IC50 35 nM

19

9. Other Factors

Notes

•FT-Inhibitors show potential as anticancer agents

•Anticancer activity may not necessarily be due solely to FT-inhibition

•FTIs inhibit farnesylation of H-Ras, N-Ras and K-Ras

•But N-Ras and K-Ras can by prenylated by GGTase

•GGTase provides alternative mechanism of attaching Ras to cell membranes

•FTIs still have anticancer activity in cells expressing excess K-Ras

•Inhibition of FT may affect other cellular processes other than Ras

20

1. Matrix metalloproteinases

Notes

•Zinc-dependent enzymes

•Play an important role in metastasis

•Destructive enzymes

•Play a role in the breakdown, normal turnover and remodelling

of the extracellular matrix

•Play a role in releasing VEGF from storage depots in

extracellular matrix

•Four types - collagenases, gelatinases, stromelysins and

membrane type

21

ANTICANCER AGENTS MATRIX METALLOPROTEINASE INHIBITORS


Reaction catalysed by collagenases

Notes

•Proteins are substrates for these enzymes

•Enzyme hydrolyses the peptide bond between glycine and isoleucine

N

HN

HN

O

O

Protein

Me

NH

O

Protein

H

N

HN

O Me

NH

O

Protein

H

H

OH

HN

O

Protein

22

OH H

Glu

O O O

Ala

Zn2+


Binding interactions

Notes

•Zinc ion interacts with the susceptible carbonyl oxygen

•Activates the peptide bond for hydrolysis

•NH of the susceptible peptide forms a hydrogen bond to alanine

•Bridging water molecule present between zinc cofactor and glutamate residue

•Bridging water acts as a nucleophile for hydrolysis

•Bridging water is activated by glutamate and zinc

P1'

P2'

N

HN

HN

O

O

Me

NH

O

H

23

2. Matrix metalloproteinase inhibiters

Notes

•Potential agents for inhibiting angiogenesis and metastasis

•First-generation inhibitors are based on the structure of the substrate

•Susceptible peptide bond is replaced with a stable transition-state isostere

•Hydrophobic substituents are present to fit the enzyme subsites

•Substituents mimicking P1’ or P2’ (right-hand side inhibitors) are better than

substituents mimicking P1 or P2 (left-hand-side inhibitors)

•Functional group(s) are included to form H-bonds with the enzyme backbone

•A group is included to form a strong interaction with the zinc ion cofactor

•Zinc ligands commonly used - thiol, carboxylate or hydroxamic acid

24


Notes

•Orally active synthetic compound

•Undergoing phase III clinical trials for breast and prostrate cancer

•Hydroxamic acid acts as the zinc ligand

•Hydroxymethylene group acts as the transition-state isostere

•Isostere replaces the NH of the previously susceptible peptide bond

•Hydroxyl group of the isostere is good for aqueous solubility

NH

HN

NH

CH3O

O

OH O

O

H

Marimastat

t-Butyl group

Hydroxamic acid

Transition-state

isostere

NH

HN

NH

CH3O

O

OH O

O

H

25


NH

HN

NH

CH3O

O

OH O

O

H

Marimastat

t-Butyl group

Hydroxamic acid

Transition-state

isostere

NH

HN

NH

CH3O

O

OH O

O

H

•t-Butyl group acts as a steric shield to protect N-terminal amide from

hydrolysis

•t-Butyl group desolvates peptide bonds

•No desolvation penalty for binding

Notes

26

NH

HN

NH

CH3O

O

OH O

O

H


Marimastat - binding interactions

Notes

•Three hydrophobic substituents fit enzyme sub pockets

•Substituents bind using van der Waals interactions

•Hydroxamic acid acts as a bidentate ligand for zinc

•Hydroxamic acid NH acts as a hydrogen bond donor

•The alcohol group is good for activity and solubility

•The alcohol group is directed away from the protein surface and forms a

hydrogen bond to water

S1'

S2'

S3'

Zn2+

HBD

P1’

P2’

P3’

27


BMS 275291

Notes

•Thiol group acts as a zinc ligand

•Substituents (P1-P3’) fit four enzyme sub pockets

•Right-hand half is identical to marimastat

NH

HN

NH

CH3HS

O

O

O

N

N

OO

P1

P1'

P2'

P3'Thiol

Hydantoin ring

28


Design aims for second-generation inhibitors

•Reduce or eliminate peptide character

•Increase selectivity

•Decrease side effects

•Increase aqueous solubility

•Increase resistance to peptidases in gastrointestinal tract

29


Second-generation inhibitors

Notes

•Hydroxamic acid acts as a zinc ligand

•Two substituents fit enzyme sub pockets

•Decreased peptide character

•Lack of selectivity

•Isopropyl group acts as a steric shield

•Thought to protect hydroxamic acid from metabolism

Steric shield

NNH

O

HO

SO

O

OMe

N

P1'

P2'

CGS 27023A

30



Notes on prinomastat

•Isopropyl substituent is incorporated into a ring - rigidification

•Less conformations are available

•Results in increased activity

•Pyridine ring allows extra binding interactions - extension strategy

Steric shield

Pyridine

Prinomastat

S

NNH

O

HO

SO

O

O

NP1'

NNH

O

HO

SO

O

OMe

N

P1'

P2'

CGS 27023A

Rigidification

Prinomastat

S

NNH

O

HO

SO

O

O

NP1'

31


Zinc

ligand

BAY 12 9655

O

O

SO

Cl

P1'


Notes

•The carboxylate group acts as the ligand for the zinc ion

•Entered clinical trials

32

anticancer agents farnesyl transferase inhibitors - sar (structure activity relationship)

Health & Medicine