The Discovery of Raltegravir (MK-The Discovery of Raltegravir (MK-518), an Integrase Inhibitor for the 518), an Integrase Inhibitor for the

Treatment of HIV InfectionTreatment of HIV Infection

By Dr. Michael Rowley

IRBM, via Pontina Km 30,600, Pomezia, Rome 00040, Italy

Summarized by Qinglin Chefor Synta Chemistry Enhancement Program

Some Facts about HIV/AIDS

2.7 million in 20072.0 million in 2007Total in 2007 33 million

Schematic Illustration of HIV-1 Virion

Illustration of HIV-1 Virion

Lipid bilayerLipid bilayerViral envelopeViral envelope 72 spikes72 spikes

Structure of Hiv particle

Viral envelopeViral envelope

Image of HIV Infected T-Cells

Size of HIV Particle: ~100-150 nmSize of HIV Particle: ~100-150 nm

HIV-1 particles assembling at the surface of an HIV-1 particles assembling at the surface of an infected macrophageinfected macrophage

HIV Viral Life Cycle

CXCR4CXCR4CCR5CCR5

Intigrase inhibitor Intigrase inhibitor 0 until 20080 until 2008

Reverse Transcriiptase InhibitorReverse Transcriiptase Inhibitor11 (NRTI) + 4 (NtRTI)11 (NRTI) + 4 (NtRTI)

Protease inhibitor Protease inhibitor 99

The Chemistry of Integrase

HCV NS5B inhibitors

Finding the lead: From HCV NS5B to HIV Integrase inhibitors

HIV integrase & HCV NS5B

-share mechanistic similarities- Need two divalent metal ions for their phosphoryltransferase activities.

Compound 3: Dihydroxy pyrimidine acidLow permibility, Too polar

Compound 4: HCV NS5B inactiveHIV integrase IC50 85 nM

Finding the lead: From HCV NS5B to HIV Integrase inhibitors

HCV NS5B inhibitors

Discovery of L-870812 and L-870810Discovery of L-870812 and L-870810

Compound 5:Compound 5:•form form West PointWest Point•Improved PKImproved PK•Low potencyLow potency

Compound 4:Compound 4:•From From IRBMIRBM•high potencyhigh potency

From compound 4 & 5, after optimization, L-870810 was discovered at West Point

Con of (4): a. low activity in the cell-based assay > 5 M b. low bioavailability (~15%)

c. High plasma protein binding

Compound L-870810 (MK-624):Compound L-870810 (MK-624):•form form West PointWest Point•Discontinued after Phase II due to Discontinued after Phase II due to toxicity in dogs during long term toxicity in dogs during long term dosingdosing

Lead Optimization: Investigation of Benzyl AmideLead Optimization: Investigation of Benzyl Amide

1. NH is needed2. 1-2 methylene linker is required3. Heterocyle ok,

Potency correlated with Log P.1. Branching linker is no good.

1. NH is needed2. 1-2 methylene linker is required3. Heterocyle ok,

Potency correlated with Log P.1. Branching linker is no good.

Lead Optimization: Investigation on Benzyl SubstitutionLead Optimization: Investigation on Benzyl Substitution

Conclusion: 1. Para > meta > ortho2. 4-F > 4-Cl > 4-Br > 4-CF3 > others

Conclusion: 1. Para > meta > ortho2. 4-F > 4-Cl > 4-Br > 4-CF3 > others

After Extensive Investigation…..After Extensive Investigation…..

cell penetration, protein binding, PK and other parameterscell penetration, protein binding, PK and other parameters

N

N

OH

OH

O

HN

F

S

OH required

p-F optimal

requirednot vital

many substituentsallowed

can bemethylated

need O atomcan be carbonyl

Lead Optimization: 2-Substitution

Remining Issue:. Still very low activity in cell-based assay! Even in low serum level assay (10% FBS). Compound 35, Spread CIC95 >5μM

PhCH2

Compound 36: Adding a basic group confers cell activity

N

N

OH

OH

Ph

NMe2

HN

O

FN

N

OH

OH

PhHN

O

F

(35)

QI IC50 (nM) 200 Spread CIC95 10% FBS 0.31 Mspread CIC95 50% NHS >10

(36)

QI IC50 (nM) 50Spread CIC95 10% FBS > 5 spread CIC95 50% NHS >10

New Lead Compound 36:

-very good PK, high bioavailability: rat F 59%, dog F 93%-low clearance: rat 14 ml/min/kg, dog 0.5 ml/min/kg-protein binding (>99%)-Shift in a cell-based assay between low and high serumConditions is high!. activity in 50% NHS (> 10μM)

for CRAC chemists: THE IMPORTANCE OF PROTEIN BINDING

Lipophilic acid compound

Lipophilicity (logD) ↑

albumin binding ↑

Shift in cell-based assays and Protein Binding

Low protein binding low shift! But when <90%, shift < 4, good enough

Optimum protein binding range 80%~90%

Low protein binding High ClearanceNeed >80%

Direction 1 from compound 36: Simple Acyclic 2-Substituents

37: enzyme activity ok, But lower in FBS, due to low cell penetration

38: 1 methyl group FBS 10 times increase , and good NHS activity

39: 2 methyl groups, no chiral center, highly active both low and high serum

40, 41 ethyl or NH both detrimental

37: enzyme activity ok, But lower in FBS, due to low cell penetration

38: 1 methyl group FBS 10 times increase , and good NHS activity

39: 2 methyl groups, no chiral center, highly active both low and high serum

40, 41 ethyl or NH both detrimental

N

N

OH

OH

N

Ph

HN

O

F

QI IC50 (nM) 200 Spread CIC95 10% FBS 0.31 Mspread CIC90 50% NHS >10

(36)

N

N

OH

OH

R

HN

O

F

Compound 39, the First Candidate with development potential!

N

N

OH

OH

NHN

O

F

(39)

Not CYP450 inhibitor, not CYP3A4 inducer, no oxidative metabolismMajor Metabolite: 5-O-glucuronide

Direction 2 from compound 36: Simple Cyclic 2-Substituents

A basic nitrogen was tolerated!

2-NH > 3-NH > 4-NH

-NMe > -NH

A basic nitrogen was tolerated!

2-NH > 3-NH > 4-NH

-NMe > -NH

N

N

OH

OH

N

Ph

HN

O

F

QI IC50 (nM) 200 Spread CIC95 10% FBS 0.31 Mspread CIC90 50% NHS >10

(36)

N

N

OH

OHHN

O

F

N

R

An important discovery: N-methylpyrimidines

A side reaction?A side reaction?

An important discovery: N-methylpyrimidines

“it had two important improvements:The first was that the protein binding was considerably lower (a factor that we had struggled with in the dihydroxypyrimidine series) and,the second was that rat PK was improved, with better oral bioavailability and lower plasma clearance.

We reasoned that it would be easier to improve potency in a series with good protein binding and PK than the converse and turned our attention to the N-Me pyrimidinone series.”

“it had two important improvements:The first was that the protein binding was considerably lower (a factor that we had struggled with in the dihydroxypyrimidine series) and,the second was that rat PK was improved, with better oral bioavailability and lower plasma clearance.

We reasoned that it would be easier to improve potency in a series with good protein binding and PK than the converse and turned our attention to the N-Me pyrimidinone series.”

N-METHYLPYRIMIDINES WITH CYCLIC 2-SUBSTITUENTS

N-METHYLPYRIMIDINES WITH CYCLIC 2-SUBSTITUENTS

N-METHYLPYRIMIDINES WITH CYCLIC 2-SUBSTITUENTS

N

N

O

OH

N

HN

O

F

(50)

QI IC50 (nM) 20 Spread CIC95 10% FBS 0.04 Mspread CIC90 50% NHS 0.065

QI IC50 (nM) 440Spread CIC95 10% FBS 0.8 Mspread CIC90 50% NHS 1

N

N

O

OH

N

HN

O

F

(54)

O

Back to ACYCLIC BASIC SUBSTITUENTS on N-METHYLPYRIMIDINES

N

N

OH

OH

NHN

O

F

(39)

N-METHYLPYRIMIDINES WITH NON-BASIC POLOR SUBSTITUENTS

N-Me-PYRIMIDINES WITH NON-BASIC SUBSTITUENTS (cont.)

N

N

OH

OH

N

HN

O

F

N

N

OH

OHHN

O

S

N

N

OH

OHHN

O

S

(4)

F

QI I50 85 nM QI I50 10 nM

amide optim.

(24)

N

N

OH

OHHN

O

N

F

QI I50 200 nMSpead CIC95 0.31 M (10% FBS)

(36)Ph

2-subsitution

N

N

OH

OHHN

O

N

F

QI I50 50 nMSpead CIC95 0.06 M (10% FBS)Spead CIC95 0.078 M (50% NHS)

(39)(45)

QI I50 200 nMSpead CIC95 0.14 M (10% FBS)Spead CIC95 0.40 M (50% NHS)

N

N

O

OH

N

HN

O

F

(50)

QI I50 400 nMSpead CIC95 0.8 M (10% FBS)Spead CIC951 M (50% NHS)

methylation

cyclicacyclic

Protein Binding 95%rat PK F% 20Clearance 60

Protein Binding 55%rat PK F% 100Clearance 31

N

N

O

OH

N

HN

O

F

O(54)

QI I50 20 nMSpead CIC95 0.04 M (10% FBS)Spead CIC951 0.065 M (50% NHS)Protein Binding 55%rat PK F% 100Clearance 31

(+)

N

N

O

OHHN

O

F

N

QI I50 230 nMSpead CIC95 >1 M (10% FBS)Spead CIC95 >1 M (50% NHS)

N

N

O

OHHN

O

F

HN

QI I50 10 nMSpead CIC95 0.045 M (10% FBS)Spead CIC95 0.07 M (50% NHS)

O

O

N (66)

rat PK F% 28Clearance 16

rat PK F% 36Clearance 21

*

Evolution from Hit to Lead to preclinical candidate 66

RESISTANCE PROFILING

N

N

O

OHHN

O

F

(66)

HN

O

N

O

N

N

OH

N

HN

O

F

S O

O

L-870810

Fight is not over DISCOVERY OF RALTEGRAVIR

N

N

O

OHHN

O

F

HN

O

X

Y

N

N

O

OHHN

O

F

HN

O

O

N

(66)

DISCOVERY OF RALTEGRAVIR (cont.)

Low permeability

High Protein binding

RALTEGRAVIR , First-In-Class

N

N

O

OHHN

O

FHN

O

N

O

N

•QC IC50 10 nM•Spread CIC95 19 nM (10% FBS)•Spread CIC95 31 nM (50% NHS)•Protein Binding•PK F% 45% in rat, 69% in dog •Plasma clearance 1.8 rat, 11 dog•No CYP450 inhibitor•No CYP3A4 inducer•No UGT1A1 inhibitor•Resistance Profiling: excellent•Safety in human•Efficacy short term as monotherapy•long term in combination

The Synthesis of MK-518

HO CN H2N CNHN CNCbz H

NCbz

N

NH2

OH

CO2MeMeO2C

HN

Cbz

N

N

OH

CO2Me

OH

HN

Cbz

N

N

O

CO2Me

OHHN

Cbz

N

N

O

OHHN

O

F

H2,Pd/C

H2N

N

N

O

OHHN

O

FHN

N

N

O

OKHN

O

F

O

NN

O

(MK-518)

1). 2). KOH

MeI

WO 2006060712 (process for MK-518)

O

NN

OCl

StrekerAmine Synthesis

NH3

(97%)

CbzCl NH2OH

(86%) (88%)

(51.7%)(70%)(90%)

(96%) (91%)

S-1360 from GSKS-1360 from GSK-Licenced from Shionogi-Licenced from Shionogi-Diketoacid derivative-Diketoacid derivative-Low efficacy, extramely poor bioavailability-Low efficacy, extramely poor bioavailability-discontinued in Phase II-discontinued in Phase II

GS-9137 (elvitegravir) from GileadLicenced from Japan TobaccoMetabolised in part by Cyp3A4co-doing with ritonayir (a HIV protease inhibitor also inhibit Cyp3A4)

Phase III clinical trial Ongoing nowBMS compounds similar to RaltegravirBMS compounds similar to Raltegravir

N

N O

OH

NH

O

OHF

GSK-364735

Discontinued in Phase IIb due To hepatotoxicities

Other HIV Integrase Inhibitors

First-in-ClassFirst-in-ClassWhat do you think?What do you think?

First-in-ClassFirst-in-ClassWhat do you think?What do you think?

• Trial and error, learning curve, solid science,Trial and error, learning curve, solid science, be prepared of failure in the beginningbe prepared of failure in the beginning

• Competition. Not guaranteed winner among otherCompetition. Not guaranteed winner among other big pharmasbig pharmas

• Some luck is needed!Some luck is needed!

A Quote from Barbara Ehrenreich

“From the point of view of the pharmaceutical industry, the AIDS problem has already been solved. After all, we already have a drug which can be sold at the incredible price of $8, 000 an annual dose, and which has the added virtue of not diminishing the market by actually curing anyone.” Ehrenreich, Barbara

(from wikipedia)

In clinical trials patients taking Raltegravir achieved viral loads less than 50 copies per millitre sooner than those taking similarly potent Non-nucleoside Reverse Transcriptase Inhibitors or Protease Inhibitors. This statistically significant difference in viral load reduction has caused some HIV researchers to begin questioning long held paradigms about HIV viral dynamics and decay. Research into Raltegravir's ability to affect latent viral reservoirs and possibly aid in the eradication of HIV is currently ongoing.

Ongoing Clinical Trials: Depletion of Latent HIV in CD4 Cells

How difficult to get a first-in-class drug to the market from scratch

Raltegravir Potassium Salt: at a glance