parp-1 inhibitors in oncology

G.Wells | 7 June 2011

PARP-1 Inhibitors in Oncology

The Discovery and Development of CEP-9722,an Orally Active Prodrug for the Treatment of Cancer


Rationale for PARP-1 Inhibitors in Oncology

• PARP comprises a family of at least 14 related enzymes some of which play a pivotal role in DNA repair.

• The primary member, Poly (ADP-ribose) polymerase 1 (PARP-1), is a nuclear enzyme that catalyzes the synthesis of poly-ADP ribose chains from NAD+ as part of the DNA repair process.

• Signals and recruits other proteins to repair damaged DNA and can be activated by single strand breaks in DNA.

• Inhibitors of PARP-1 have shown promise in oncology through potentiation of the anti-tumor activity of radiation or chemotherapeutic DNA damaging agents.


PARP-1 - Background

• Inverse correlation of PARP-1 activity with the degree of cell differentiation. Tumors have increased PARP activity as compared to the corresponding normal tissue.

• PARP inhibitors as chemopotentiators: Benzamides, Isoquinolones, Nicotinamide derivatives in vitro and in vivo.

• Potentiates the activity of TMZ, bleomycin, cisplatin, and radiation in human and murine tumor models


Role of PARP-1 in DNA Repair

N

C

N

C

A. de Murcia & M. de Murcia (1994) TIBS 19, 172

DNA Damage

ADP-ribose

PARP Activation

NAD+ ATP



N

C

N

C


DNA Damage

ADP-ribose

PARP Activation

NAD+ ATP

DNA damage repaired



N

C

N

C


DNA Damage

ADP-ribose

PARP Activation

NAD+ ATP

DNA damage repaired

Healthy cell



N

C

N

C


PARP Inhibitor

DNA damage persists

DNA Damage

ADP-ribose

PARP Activation

NAD+

ATP

Damage repaired,Healthy cell



N

C

N

C


PARP Inhibitor

DNA damage persists

DNA Damage

ADP-ribose

PARP Activation

NAD+

ATP

Damage repaired,Healthy cell

Apoptosis,Cell death


PARP activity, NAD+, and ATP levels are interdependent

Ha, H. C.; Neurobiology of Disease 7, 225–239 (2000)


Clinical Path Forward

• Glioblastoma w/TMZ - # 1 Choice

o Poor single agent response rate (~5%) allows for clear improvement using combination therapy with p.o. TMZ

o Reasonable size and duration of clinical trials with TMZ

o Unmet therapeutic need for GBM; other therapeutic indications for TMZ- sarcoma, melanoma, colon carcinoma and CNS lymphoma

o Substantial supportive pre-clinical data with TMZ

• Therapeutic Endpoints: Improved Response Rate and “Time to Event” outcome


N

NH2

O

Ribose

Ribose Adenine

PP

+

NAD+

Known PARP inhibitors mimic nicotinamide binding at the NAD+ site and are planar aromatic ring systems containing a bidentate H-bonding group

PARP-1 inhibitors based on NAD+ substrate

N OO

H

NH2

NH2

O

NH2

4-ANI3-AB

Ki = 4M Ki ~ 150nM


N

X

NO

H

H

R

O

NN O

H

R

N O

N

O

H

Me2N

Pfizer/Agouron - Indoles/Benzimidazoles

Iconix - PhenanthridinonesGuilford - Phthalazinones

Earlier Competitor PARP-1 Scaffolds


NH

CH3

O

NH2

NH2

O

PD128763

Ki = 70nM

3-AB

o inc. radiation sensitivity of Chinese hamster V79 cellso Radiat Res; 126(3), 367 (1991)

NH

N

NH2

O

OH

NH

NH

O

F

NH

NU-1085 AG14699

+ crystal structure+ modelling

Ki = 6nM Ki < 5nM

o potentiates TMZ and TP growth inhib. in human tumor cell lineso Clin Cancer Res; 6, 2860 (2000)

Proof of Concept

o Clinical trials candidate


NH2

O

NH

N

NH

ABT-888 (Veliparib)

o Potentiates TMZ, Cisplatin, radiation in syngeneic and xenograft tumor models o Clin Cancer Res; 13, 2728 (2007)

o Completing Phase 2 trials w/TMZ

o Irreversible inhibitoro Excellent Phase 2 resultso First PARP inhibitor in Phase 3 trialso Failed primary endpoint

NH2

O

I

O2N

BSI-201 (Iniparib)

(Sanofi-Aventis)

N

N

O

F

O

NHN

O

AZD-2281 (Olaparib)

o Cancelled Phase 3 trials for breast cancero Commencing Phase 3 for ovarian cancer

NN

NH

ONH2

HCl

MK-4827

o Phase 1 trial for various cancerso Well toleratedo Commencing Phase 2 for Mantle Cell Lymphoma

In the Clinic


Early CEP Library Screening Hit - Pyrrolocarbazole Imide

N

NO O

H

H12

34

5 7

A

B CD

E

CEP-3498 I C50= 35 nM

• Screening of Cephalon’s internal library identified a pyrrolocarbazole as a potent inhibitor of PARP-1.

• Improvements Needed: Potency Cellular Permeability/Activity Solubility


N OO

H

NH2

4-ANI

PARP-1 Modeling

Apply structure-based design to optimize potency and cell activity

Obtained 2PAX from PDB (Protein Data Bank) - Catalytic Fragment Of Poly(ADP-Ribose) Polymerase complexed with 4-amino-1,8-naphthalimide Chicken PARP (PARP-CF), 87% homologous with the human form


Ala898

Glu988

Gly863

Tyr907

3-position

Modeling of CEP-3499 with PARP-CF

NH

NH

O

CEP-3499

Wells, Bihovsky; BMCL, 16, 1151 (2006)


PARP Inhibitor Discovery Flow

In Vitro Cytotoxicity Assays (PARP inhibitors + Chemotherx.)

In Vivo Chemo-Potentiation Studies

GBMs /TMZ, HT-29/Irinotecan

Significant shift in tumor versus normal cell kill versus chemotherx. alone

In Vivo PAR Accumulation Assay

No enhanced humanmyelotoxicity in vitro

Biochemical efficacy in vivo

Cmpd Scale-Up

Significant potentiation of anti-tumor efficacy versus chemotherx. alone; acceptable systemic tolerability.

Go/No GoDecision

PK and Tolerability in Rodents

rh PARP Inhibition Assay

PC12 cells/H2O2 insultAssay for Inhibition of NAD+ Depletion

In vitro and in vivo evaluation on normal tissues; clinical chemistry and histopathology

IC50 < 50 nM

50% recovery @ < 1 uM>90% max. recovery

Criteria


General Route to Pyrrolocarbazoles

XO

R1

R2

NH

NH

OO

R1

R2

NH

NH

X

NH

OO

O

R

R R

NC CO2EtNH

O O

X

R2

R1

R

(X = NH, S, O)

(Key Intermediates)

> Heteroaryl analogs - Indole - Benzofuran - Benzothiophene

> Carbazole Imide analogs> Right-hand modifications

> Carbazole Lactam analogs

(X = NH, S, O)

+


PARP-1 Activity of Pyrrolocarbazole Lactam Isomers

CEP Structure PARP IC50 (nM)

Imide (CEP-3498)

35

7-oxo(CEP-3499)

90

5-oxo(CEP-3500)

~10, 000

NH

NH

O5

NH

NH

O 7

NH

NH

OO

• PARP activity resides in the 7-oxo isomer, the 5-oxo is inactive for PARP

• Imide CEP-3498 is 3-fold more potent than the 7-oxo lactam CEP-3499 (enhanced H-bonding?)


Pyrrolocarbazole Right-Hand Modifications

NH R1

R2

NH

O O

CEP R1, R2 PARP IC50 (nM)

3498 35

5558 ~10,000

2520 > 10,000

5848 ~ 10,000O

• The cyclopentyl ring is critical for potency


PARP-1 Inhibition of Pyrrolocarbazoles

NH R1

R2

NH

O O

CEP R1 R2 PARP IC50 (nM)

3498 -CH2CH2CH2- 35

1526 H H ~ 10, 000

5653 Methyl Methyl 700

5674 Methyl H 5000

5729 H Methyl ~2000

5706 Ethyl Propyl > 10,000

• Ring-opened analogs showed decreased potency

Tao, Wells; BMCL; 16, 938 (2006)


PARP-1 Inhibition of Benzofuran/Benzothiophene Analogs

X

NH

O O

CEP X PARP IC50 (nM)

3498 NH 35

6297 S > 10, 000

6373 O > 10, 000

• Indole N-H an essential binding function for potency• Corresponding N-Me also inactive


PARP-1 Inhibition of Truncated Analogs

CEP Structure PARP IC50 (nM)

6012 40

6011 2220

5775 750

NH

NH

O O

NH

O O

NH

O O

NH

• Des-Aryl CEP-6012 was equipotent with carbazole CEP-3498• SAR supports the model and the importance of the N-H interaction


Indole-Cyclopentyl Series

R IC50 (nM)

H 40

Br 5

CN 6

CH2NH2 89

N

N

OO

H

H

R

Tao, Wells; BMCL; 16, 938 (2006)

• Smaller MW - potentially improved physical properties• Single digit nM leads


SAR of Methoxy Analogs

NAD+ Rec. PC12 cells

R CEP IC50 (nM) Conc. For 50%

Rec. (uM)

H 3498 35 >30

2-OMe 8062 224 3.0

3-OMe 8091 32 1.0

4-OMe 8983 20 0.3

3,4-OMe 9712 21 <1.0

N

N

OO

H

H

R

12

34

• Data confirms model for 3- and 4-substituents for optimal activity

• CEP-8983 is a potent, high permeability compound (PAMPA = 7.3 x 10-6 cm/sec)


3-Aminoalkyl-1-Carba-Series

R CEP IC50 (nM) NAD+ rec. @1 M

CH2NH2 6800 22 73%

CH2NMe2 7264 20 69%

CH2NEt2 7273 17 97%

CH2NHEt 7271 32 102%

CH2NnPr2 7272 16 104%

CH2NnBu2 7317 28 50%

CH2NBn2 7318 293 40%

CH2NC4H8 7826 35 83%

CH2CH2NH2 7828 36 49%

N

N

OO

H

H

R

12

34

• Generally good solubility, potency and cell activity• CEP-6800-HCl demonstrated >10 mg/mL aq. solubility• CEP-6800 showed toxicity, low brain levels, poor PK


3-Alkoxy-1-Carba-Series

R CEP IC50 (nM) NAD+ rec. 1 M

OH 7958 27 58%

OAc 7957 33 59%

OMe 8091 32 41%

OCH2CH2OEt 8323 59 27%

OCH2CH2NEt2 8371 88 21%

OCH2CH2CH2NEt2 8349 100 25%

OCH2CH2NC4H8O 8969 22 17%

N

N

OO

H

H

R

12

34

• Evaluated amino and ether alkyl spaced groups

• Morpholino-CEP-8969 showed good enzyme activity and solubility, but low cellular activity


CEP R PARP

IC50 (nM) NAD+ Rec. in

PC12 cells (1 M)

H2O sol. (mg/mL)

9010

10

53%

>10

9013

30

52%

>10

9016

22

44%

---

9027

15

58%

>10

3-Carbamate and Amide Derivatives

/

O

N

N

O

O

N

HN

/

NN O

O

/OH

N

N

OO

H

H

R

NN

NN O

O

N

H

/

H

Wells, Bihovsky; BMCL, 16, 1151 (2006)

• Identified potent, cell permeable inhibitors with good water solubility• Low brain levels observed with carbamates


General Synthesis of Alkoxy Analogs

NH O

NH

OH

NH

NH

O O

NH

NH

OO

NH

(iPr)3SiO (iPr)3SiO(iPr)3SiO

R-OR-O

1) nBuLi, CO2

THF, -78oC

2) tBuLi

3)

Et2O

AcOH

RX, CsF

AcCN, 50 oC AcOH, r.t.

p-Chloranil

AcOH, 95 oC


Summary SAR

N

N

O

H

H

O

4-Alkoxy important - potency - cell activity

3-Substitution - potency - solubility

Substitution not tolerated

N-H essential(O or S inactive)

Cyclopentylrequired

7-oxo required


PARP-1 model for 1-Aza analog of CEP-8983

• Favorable H-bond postulated between 1-aza group and amide of Met890


Synthetic Approach to Aza-analog CEP-8315

N NH

O

N NH

OH

NH

O O

N NH

NH

OO

N NH

1) nBuLi, CO2

THF, -78oC

2) tBuLi

3)

Et2O

AcOH

p-Chloranil

AcOH, 95 oC

CEP-8315 (IC50= 3 nM)

• Further confirms model, binding pose• Most potent analog prepared


Proposed 1-Aza-4-Alkoxy Series

N

NO O

H

H

O

N N

NO O

H

H

N N

NO O

H

OR

H

CEP-8983

(IC50 = 20 nM)

CEP-8315

(IC50 = 3 nM)

• Good cellular activity • Better potency

• Best of both worlds?


1-Aza-4-Alkoxy Synthetic Challenges

N NH

N NH

Cl

N NH

OR

N NH

OH

1) MCPBA, DME

2) aq. K2CO3 (pH 9)

3) POCl3, reflux

4) aq.K2CO3 (pH 9)

(50%)

5% NaOH/ROH

160-180oC, 18-24h

(20-50%)

NaOHo Expen$iveo Limited supply

• Chloride displacement tricky, requires sealed (“bomb”) reactor

• Ethers thermally sensitive, give variable yields and purity profile


1-Aza-4-Alkoxy Series Synthetic Challenges

X N

R

HX N

R

H

R X % Yield

H C 70-80OMe C 60-70 H N 40-50OMe N 0-10____________________

1. n-BuLi/THF, then CO2

2. t-BuLi, then c-pentanone

3. HCl

• Series requires alternate approach


1-Aza-4-Alkoxy Series Synthetic Challenges

N NSO2Ph

OR

N NSO2Ph

I

OMe

N NSO2Ph

OMeO

OH

N NSO2Ph

OMeO

DN N

SO2Ph

OMeO

B(OCH3)2

N NSO2Ph

SnMe3

OMe

t-BuLi, THF, -78oC;

then c-pentanone

(0-10%)

t-BuLi, THF-78oC - 0oC; then D2O

(50-60%)

t-BuLi, THF-78oC - 0oC;then (MeO)3B

LDA, THF-78oC - 0oC;then Me3SnCl

LDA, THF, TMEDA, -20oC;then I2

(40-60%)


Diene Problem “Solved”

N N

OR

SO2Ph

IN N

H

ORSnBu3

N N

NO O

H

H

OR

(5-steps from 7-azaindole)

cat. PdCl2(PPh3)2

DMF, 90oC;then NaOH, EtOH

(~50%)(2 steps)

(~50%)

• Suitable method for small (mg-gm) quantities


SAR of 1-Aza analogs

CEP X R IC50 (nM) % NAD+ rec.

(1 M)

3498 CH H 35 46% (30 M)

8315 N H 3 50%

9222 N 3-Me 2 46%

9667 N 3-OMe 4 44%

9397 N 4-OMe 4 94%

N

NX

OO

H

H

R

1

2

34

• 1-Aza group confers order-of-magnitude greater potency• 3- and 4-substituents tolerated – opportunity for solubility, improved PK


SAR of 1-Aza-4-Subst’d- analogs

CEP R IC50 (nM) % NAD rec. 1

M

9397 OMe 4 94%

9890 OEt 5 62%

9955 OCH2CH2OMe 10 74%

9956 OCH2CH2OEt 11 63%

9891 Cl 9 87%

9371 NMe2 121 42%

N

NN

OO

H

H

R

1

2

34

• Non-basic 4-substituents well-tolerated

• Aza-series ultimately discontinued due to synthetic challenges, non-scalability, expense, and poor solubility


OH

NH

O

NH

Si

O

O

NH

Si

OH

O

NH

Si

NH

OMe

(i-Pr)3SiCl

ImidazoleDMF

then CO2

CH3I ,CsF

CH3CN

(95%)

n-BuLi, THF, -65oC t-BuLi, THF, -65oC

then

(92%)

(91%)

(92%)

HOAc, Et2O

Synthetic Process for Drug Candidate CEP-8983


NH

OMe

NH

NH

O

OOMeN

HOO

NH

NH

O

OOMe

HOAc

p-Chloranil

HOAc

CEP-8983

(80%) (94%)

o MW = 306o Poor solubility (<<1mg/mL)o Difficult purification (DMF/Al2O3)

Synthetic Process for CEP-8983 (cont.)


NH

NH

O

OOMe

N N

NH

NO

OOMe

NH N

CEP-8983 CEP-9722

CH2O, EtOH

(96%)

o MW = 306o Poor solubility (<<1mg/mL)o Difficult purification (DMF/Al2O3)

o MW = 418o Crystallized from THF/hexaneso Stable solido Aqueous soluble salts

Synthetic of Prodrug CEP-9722

• Other related prodrug analogs (amide, sulfonamide, carbamate, urea, N,O-aminal) were more or less stable and/or soluble


N

N

N

N

OO

H

R

R CEP Solubility (pH 4.2)

Ascorbic Acid (mg/mL)

T90 Stability (hrs)

OMe 9722 ~20 18

OCH2CH2OMe 16345 >40 >20

OCH2CH2OEt 16346 8 22

Solubility and Aqueous Stability of N-Methylpiperazinyl Aminal Prodrug Analogs

• CEP-16345 and CEP-9722 met solubility and stability criteria at a pH sufficient for I.V. delivery in the clinic


Stable, Soluble Mannich Base Gluconates

N

N

O

X

H

H

OO

N

N

N

N

O

X

H

OON N H

HO2C

OH

OH

OH

OH

OH

, CH2O, EtOH

+ 2

CEP-8983 (X = C)CEP-9397 (X = N)

CEP-9722 (X = C)CEP-10306 (X = N)

(then gluconic acid)

• Gluconic acid salt gave optimal aqueous solubility and stability

• Decomposes in-vivo to CEP-8983/9397, formaldehyde, and methylpiperazine


Single Crystal Structure of CEP-9722

• Confirms bonding of prodrug moiety at imide, not indole


N

N

OO

H

H

R4

hERG Structure-Activity Relationships: 4-Alkoxy SAR

• Diether substitution at the 4-position reduced hERG channel activity in patch clamp assay

R4 CEP PARP IC50

(nM)

hERG IC50 (M)

OMe 8983 20 2.1

OCH2CH2OMe 9274 11 82

OCH2CH2OEt 9430 10 22


Dose Escalation Study with CEP-9722/8983 in Rats

• i.v. administration of CEP-9722 (3, 10, 30 mg/kg dose equivalents) to rats showed dose related increases in plasma level exposure of CEP-8983


The Effects of CEP-8983 and CEP-9397 on Temozolomide Mediated Toxicity in U251MG Cells

0 50 100 150 200 250 3000

50

100

TMZ alone

TMZ+0.1 M CEP-8983TMZ+0.3 M CEP-8983TMZ+1.0 M CEP-8983TMZ+3.0 M CEP-8983TMZ +10 M CEP-8983

*

*

***

*******

**

****

**p0.01; ***p0.1- TMZ alone as compared to TMZ + 1.0 M CEP-8983;*p0.05; **p0.01; ***p0.001- TMZ alone as compared to TMZ + 3.0 MCEP-8983; *p0.05; **p0.01; ****p0.0001- TMZ alone as compared toTMZ + 10.0 M CEP-8983 by Mann Whitney Rank Sum test or t-test whereappropriate.

TMZ (M)

Rel

ativ

e C

ell

Nu

mb

er (

%)

0 50 100 150 200 250 3000

50

100

TMZ alone

TMZ+0.1 M CEP-9397TMZ+0.3 M CEP-9397TMZ+1.0 M CEP-9397TMZ+3.0 M CEP-9397

TMZ +10 M CEP-9397

***************

*************

**

**

*

**

*p0.05; **p0.01 - TMZ alone as compared to TMZ + 0.1 M CEP-9397; *p0.05; **p0.01***p0.001 - TMZ alone as compared to TMZ + 0.3 M CEP-9397; *p0.05; ***p0.001****p0.0001 - TMZ alone as compared to TMZ + 1.0 M CEP-9397; *p0.05; ***p0.001;****p0.0001- TMZ alone as compared to TMZ + 3.0 M CEP-9397; *p0.05; ***p0.001;****p0.0001- TMZ alone as compared to TMZ + 10.0 M CEP-9397 by Mann Whitney RankSum test or t-test where appropriate.

TMZ (M)

Rel

ativ

e C

ell

Nu

mb

er (

%)

• CEP-8983 and CEP-9397 potentiated the growth inhibitory effects of TMZ in U251MG cells


0 100 200 3000

50

100

TMZ alone


********

**

*

***

**

******

********* *

**

****

**

********

TMZ (M)

Rel

ativ

e C

ell

Nu

mb

er (

%)

0 100 200 3000

50

100

TMZ alone


**

**

***

****

**

*

**

***

***

**

**

***

******

********

*******

TMZ (M)

Rel

ativ

e C

ell

Nu

mb

er (

%)

*p≤0.5, ** p≤0.01-TMZ alone as compared to TMZ + 0.3 µM CEP-8983; * p≤0.5 ** p≤0.01, **** p≤0.0001- p≤0.01-TMZ alone as compared to TMZ + 1.0 µM CEP-8983; *** p≤0.001 **** p≤0.0001- p≤0.01-TMZ alone as compared to TMZ + 3.0 µM CEP-8983; **p≤0.01****p≤0.0001-TMZ alone as compared to TMZ + 10.0 µM CEP-8983 by Mann-Whitney Rank Sum Test or t-test where appropriate.

*p≤0.5, ** p≤0.01-TMZ alone as compared to TMZ + 0.1 µM CEP-9397 *p≤0.5, ** p≤0.01-TMZ alone as compared to TMZ + 0.3 µM CEP-9397; * p≤0.5 ** p≤0.01, *** p≤0.001- p≤0.01-TMZ alone as compared to TMZ + 1.0 µM CEP-9397; ** p≤0.01 *** p≤0.001 **** p≤0.0001- p≤0.01-TMZ alone as compared to TMZ + 3.0 µM CEP-9397; **p≤0.01, ***p≤0.001, ****p≤0.0001-TMZ alone as compared to TMZ + 10.0 µM CEP-9397 by Mann-Whitney Rank Sum Test or t-test where appropriate.

The Effects of CEP-8983 and CEP-9397 on Temozolomide Mediated Toxicity in NB1691 Cells

• CEP-8983 and CEP-9397 potentiated the growth inhibitory effects of TMZ in TMZ-resistant tumor cell lines


Phase 1 commenced June 2009

Open-label study to evaluate the safety, pharmacokinetics, and pharmacodynamics as single-agent oral therapy and as combination therapy with temozolomide in patients with advanced or metastatic solid tumors.

Expected completion May-June 2011

Phase 2 IND filed Jan 2011

Evaluate safety and tolerability of maximum tolerated dose (MTD) found in Phase 1, and investigate CEP-9722 oral efficacy as a single agent.

Additional combination studies with Gemcitabin/Cisplatin planned

Expected completion July 2013

CEP-9722 Advanced to Clinical Trials


Oncology

Candace Burns Jennifer Grobelny Kathryn Hunter Sonya Pritchard Hugh Zhao Susan Jones-Bolin Bruce Ruggeri

Acknowledgements

Chung Ho Park Dandu Reddy Sankar Chatterjee Ron Bihovsky Gregory Wells

Chemistry

Mary Birchler Laura Gwinn Jean Husten Bruce Jones

Biochemistry

Seetha Murthy Damaris Rolon-Steele Kelli Zeigler Lisa Aimone Mark Ator

Jim Diebold Ming Tao Derek Dunn Allison Zulli Bob Hudkins

Fox Chase Cancer Center

Andres Klein-Szanto


Extra slides


N

NN

NN

O

ONH2

NN

NHN

ONH2

NH

H2O

(-CO2)

TMZ MTIC

TMZ – Hydrolysis gives active form

Temozolomide is not directly active but undergoes rapid nonenzymatic conversion at physiologic pH to the reactive compound 5-(3-methyltriazen-1-yl)-imidazole-4-carboxamide (MTIC). The cytotoxicity of MTIC is thought to be primarily due to alkylation of DNA. Alkylation (methylation) occurs mainly at the O6 and N7 positions of guanine.


29th AnnualJ.P. Morgan Healthcare ConferenceJanuary 10-12, 2011

Cephalon Oncology Pipeline


Wang; Am J Cancer Res; 1(3):301-327 (2011)

parp-1 inhibitors in oncology

Business

inhibitors of parp

role of parp

oncology parp

ani parp

parp inhibitor dna damage

potent inhibitor of

parp imide cep

trials o