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U NA-JliDs;J\I\I.HO

ADUL TS AND CHILDREN ESTIIVIATED TO SE LIVING

'\IVTTH HIVI'AIDS, E.ND 2003

iDe-.:o.ernber :20<:13

.2:'7

New HIV Infections in 2003: 5 millionDeaths due to HIV/AIDS in 2003: 3 millionPredicted HIV Infections in 2010: 100 million

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HIVTreatment

. HIV is a manageable but incurable disease

. Current arai therapies ali target 2 virai enzymes

. Reverse transciptasenucleoside (NRTI)

. nonnucleoside (NNRTI)

. Protease(PI)

. Current treatment guidelines recommend combination therapywith triple drug regimen for example:

. 2 NRTI's + 1 NNRTIor

2 NRTl's + 1 PI

Public.

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Public, dlsclosed

es in Treatment of HIV/AID_S

. Acute and Long Term Toxicities

. Non-Adherence

. Emergence of Resistant Viruses

. Resistance mitigates initial efficacy ANO durability ofantiretroviral therapy.. 780/0of treated patients resistant to one class. 50% of treated patients resistant to multiple classes. Increasing incidence of resistance in acute infection

<5% pre1999, 13%> in 1999, 20% in 2002

. Leads to therapy failure

=> Need for novel therapies

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Intearase Is Essential For Virai RecUcation

. HIV IntegraseIs one of three enzymes encoded by the viraigenome

. Like the other two, reverse transcriptase andprotease, HIV integrase is essential for viraireplication

Retroviruses lacking integrase or containing anon-functional integrase do not replicate

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The Chemistry of HIV Integrase

. oO.. Il..0.P

IO

O.

~ Mg2+-Ò)O \9""'-0.0"r'O. ~~ Mg2+ OO

OI

O"~"'O'

IO

°,

~~ O

dN ~ OH

I ~ O OHF o

O

--(0-O

-~-

IC50 = 0.01 uM

IO0..11..0-P

IO

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IO0..11..0.

PIO

Strand Transfer

°,

r\ ~ R

d"~/yy 'O

O '

I

~ ,0- - ", I - - '

F// ", : -M

g'2+

Mg2+

°,

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A mechanistically related enzY~~,,:~:J-ICVPolymerase

. An RNA dependent RNA Polymerase.00..110-

p'".°

t1A

0-

~o Mg2+"",,,0OH

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',,0 O'O .

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Oe

OHOH

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OHOH

() ~CI

ìC;ç~ O~OH~ o OHCN

CI

~ oCI

ìC;ç~ I ~ O

I ~ O I \~ O, 9----- \ 2+

CN '" Mg, ,CI Mg2+ICso= 0.04 uM

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HCV Poi

o OH

Jl ol .,OHPh'" ;r lf

O

ICso = 6 ~M

Biologically unstable- covalently binds to proteins

OH

Design.. N' ~

)l~Ph NO

O

EtO OH Chemically unstable-decarboxylates

OH

ICso = 30 ~MDoes not decarboxylateDoes not bind covalently to proteins

O O

ICso = 2 ~M

HCV Polymerase leadsfrom screening

PubUcl

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From Polvmerase to Inteara se

OH OHe

N~ ~ _O

Ph..JlN~ 0 e.

O

OHN'R

Bis anion at physiological pH- poor celi penetration

Lead for HIV-Integrase(inactive on HCVPolymerase)

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. Integrase Strand Transfer (QUICKIN ICso):Measures the ability of the test compound toinhibit the integration of preprocessed donorDNA fragment into target DNA.

. Virai Spread (CIC9s):A 3 day multiple-cycle infectivity assaymeasuring the inhibition of virai spread withH9/iiib virus in MT4 cells to >95% as detectedby p24 ELISA.

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. Complete inhibition of virai replication in manPlasma concentrations always > CIC95

Need potent compounds « 100 nM)

. Aiso need good Pharmacokinetics. High arai bioavailability (percentage of drug

absorbed when dosed orally)

Low clearance (rate at which drug is removedfrom the body)

This leads to long half life

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Plasma Pro te in Bindin

. Drugs bind to plasma protein. Protein binding reduces activity (Iess free drug)

. Measure activity in 100/0foetal bovine serum(minimum) and in 500/0 normal human serum(maximum)

10% FBS activity reflects 'celi penetration'

500/0NHS activity reflects protein binding

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The Lead Structure

OHI

Nr yOH ~,S~N~~~U O

QUICKIN ICso 80 nM

Rat Pharmacokinetics

Biovailability (F) Clearance Halt lite

15% 5 ml/min/kg 3h

.,-0H

N

~NH2\-1

S

[)-CNNH20H

MeOH

.OHI

N' ~ "OH

~N~O"\-~ O

RNH2

MeOH..

OH

N'

:çOH

~ I ~ H

ctN N'RS O

1) DMAD, CHCI3

2) Xylene, reflux

Library of 200 amides

Mechanism?

Public\

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C02Me

C02Me

N~cO'Me

~S~N~~ ClaisenV y .lfNH2

S ~ OH

~ I N'

III ..+C02Me

OH

N.0°H

/S~..Jl

V N C02Me

O

HN~O

,s~..A

V N C02Me

.....

C02Me

HN~cO'Me

lfNHO

=

~~ OMe

S N~O~~ CO,Me

I ~' O'

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Synthesis

NH20H...

MeOH

1) DMAD, CHCI3,

:çOH /~H

~~ p-Fluorobenzylamine H

~~N ~ O .. N ~Z--- N " MeOH Z--- N

O

541%

...

2) Xylene, reflux

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"OHN

'" JL NH27N~

IZ

473%

OH

OH ~F~~

(O

686%

XCN

NH3 (9)

XCNPhCH2OCOCI

XCN.. .-

OH MeOH NH2 Na2C03,H20 NHIZ

1 275% 385%

OH

OH OH

H2' Pd/C,MeOH

N/X; F HCHO,NaCNBH3I NX FH2NN/- "" I

. x: H I

MeOH, AcOH /N N NO O

7 100% 890%

Public.

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rrolidine s nthesis

HO,,,,,,,,~COOMe DAST, DC~ F~COOMe 1) NaOH 1N, MeOH..L~z -78Cto Lt. L~z 2) BOC20,NH4HC03

71%

F~CONH2

L~z

100%OH

TFAA, NEt3, DCM..

F~CN

L~z95%

1) NH20H HCI, Et3N,..

2) DMAD, CHCI3, reflux3) Xylenes, reflux4) Bz2O, Pyridine ~ N:IF N

NZ O

>25% (4 steps)

OMe

cisisomer

O

LiH, DMS

:]:OBZ 1) 4-Fluorobenzylamine O

.. M N ..dry diox:crne e

I

2) H2 Pd/C, MeOH, M~

F ~ 3) HCOH, NaCNBH4, Meo~N C02Me F ~

NZ NNMe

N-Me: OMe = 9:1

OH ~F~~

O

Public, dlsclosed

s nthesis

.O

d)ç~~FO70%

Chiral HPLC1) 4F-BnNH2, MeOH - product

2) TFA/DCM, 2h3) HCOH, NaCNBH3, AcONa 1h, MeOH

/OH

/°'ì[CO'MeOyN

NN INH2OH.HCI DMAD,CHCI3

ONH, CO,Meo-xylene,147°C. O NH2

..NBOC

TEA, EtOH NBOC reflux, 1.5 h NBOC

100% 78%

OH OH O

:):OH :):OBZ , ]c°BZN I Bz2O, Pyr, 3h N I LiH, DMS !°YN CO,Me °YN CO,Me

.. ." O N CO Medloxane, 58 C, 3h 2

NBOC NBOC NBOC

58% 62% 69% 4:1

ements

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Enzo SummaCristina GardeUiEster MuragliaPaola PacePhUJones

useppe CecereUweStephane SpieserOlaf KinzelMaria Rico FerreiraSteve HarperBarbara PaciniFederica OrvietoGiovanna PescatoreMarco FerraraMarco PomaMonica DonghiAlessia PetrocchiBenedetta CrescenziEmanuela NiziRita ScarpelliEmUia Di Francesco

Ralph lauferOdalys Gonzalez PazEdith MonteagudoAnna AlfieriFrancesca NaimoFabio BoneUiMaria VerdirameAlan BishopMarina TalianiMassimiliano Fonsi

Ivia Pescirina Grazianinalise Di Marco

Isabella MarcucciFabrizio FioreAlessandra De Pra

Daria HazudaBiUSchleif

StilmockJay GroblerMark Witmer

MillerPete FelockAbigail WolfeJoe VaccaJohnSteve Young

bertsonTerryNeviUeAnthony

ArmstrongDave