Selektivní kinázové inhibitory na bázi pyrazolo [1,5-a]p yrimidinu

Selektivní kinázové inhibitory na bázi pyrazolo[1,5-a]pyrimidinu

Dr. Kamil Paruch

Schering-Plough Research Institute, Kenilworth, NJ, USA Ústav chemie, Masarykova univerzita, Brno

• CDK inhibitor SCH 727965 (dinaciclib)• CHK1 inhibitor SCH 900776

CDKs participate in cell cycle progression and cellular transcription

Sausville, EA. Trends Molec Med 8: S32, 2002Sausville, EA. Trends Molec Med 8: S32, 2002

1

Targeting the cell cycle and CDKs

• Inhibition of CDK activity is an attractive therapeutic concept

• Anticipated effects, based on mechanism, include:

Tumor cell-specific apoptosis Reversible inhibition of proliferation in normal cells

• Phenotypes confirmed by extensive experimental validation (siRNA, dominant negatives and reference inhibitors)

• Activity versus CDK2 and CDK1 shown to be essential for efficacy

2

Target candidate profile

1. Potent, selective inhibitor of CDK2 and CDK1

2. Broad spectrum of activity (in vitro and in vivo)

3. Differential effects on tumor versus normal cells

Apoptosis in tumors Transient arrest in normal tissues Non-genotoxic MOA

4. Acceptable therapeutic index

Superior to competition and standards of care

5. Suitable for iv formulation and infusion

6. Reversible and manageable side-effects

3

CDK2 vs. GSK3

Selectivity issue

4

Lead finding

•Early effort: synthesis based on molecular modeling: chemotypes capable of binding to the protein backbone (e.g. indazoles) - acceptable in vitro activity (< 50 nM), poor potency in the cell.

•Variety of compound libraries purchased to improve hit rate in kinase inhibitor programs.

•CDK2 actives found in Biofocus kinase-directed libraries.

N

N

N

ClCl

HN

N

Compound A

CDK2 IC50 = 0.50 uM

Pyrazolo[1,5-a]pyrimidines

NN

NF

HN

N

Compound B

CDK2 IC50 = 0.8 uM

Imidazo[1,2-a]pyrazines

5

Proposed binding modes

NN

H O

H

Leu 83

O

NN

NN

NH

Cl

CO2H

NHHO

NN

H O

H

Leu 83

O

N

N

NNH N

Cl

Cl

3

5

7

Compound A

CDK2 IC50 = 0.50 uM

Purvalanol B

CDK2 IC50 = 0.009 uM

• Initial SAR Plan: Increase potency by incorporating 3-substitution.

Crystal structure of A in CDK2

6

3-Substitution improves potency

Follow-up SAR plan: evaluate similar heterocyclic cores:

N

N

HN

Br

NN

N

HN

Br

N N

HN

Br

N

N

N

HN

N

ClCl

Compound A

CDK2 IC50 = 0.50 uM

Thym. IC50 = 21 uM

N

N

N

HN

Br

N

Cl Compound A1

CDK2 IC50 = 0.003 uM

Thym. IC50 = 0.48 uM

7

O O

OCH3

NH

N

NH2

AcOH

R

HN

N N

O

RDMAP

pyr.

POCl3

+

N

N N

Cl

R

NBS

CH3CN

N

N N

Cl

R BrN

NH2

DIPEAdioxane

N

N N

HN

R Br

N

IC50 = 0.011uMthym. IC50 = 0.39 uM

1 2 3 4

5

80%85%

95%

91%R = H: 6

Synthesis of pyrazolo[1,5-a]pyrimidines

Bioorg. Med. Chem. Lett. 2007, 17, 6216.8

Synthesis of pyrazolo[1,5-a]pyridines

7

N

PhSO

OOH2N

CH2Cl2 8

N+

Ph

H2N

OR-

CO2Et

K2CO3, DMF

air/O2

1.

2. H2SO4, heatN N

Ph

9

n-BuLi

ICH2CH2IN N

Ph

10I

N

Ph Ph

H

Pd(OAc)2

BINAP

Cs2CO3

PhCH3

N N

Ph

11

N

Ph Ph

NBS

CH3CNN N

Ph

12

N

Ph Ph

Br

NH2OH

NaOAc

N

CHO

ZnCl2then NaBH3CN

N N

HN

Br

N13

IC50 = 1.87 uMIC50 = 0.011uMthym. IC50 = 0.39 uM

pyrazolopyrimidine

39% 55%

76%

80%80%

65%


BrO

RN

N

CONH2

+CH3CN HN

N

N+

R

O CH3

Br-

N

NH

HNN

N

R

O

pyr.

POCl3

NN

N

R

Cl

NBS

CH3CNNN

N

R

Cl

BrN

NH2

DIPEA

dioxane

NN

N

R

HN

Br

N

R = H: 20IC50 = 0.44uMthym. IC50 = 2.0 uM

IC50 = 0.011uMthym. IC50 = 0.39 uM

pyrazolopyrimidine

14 15 16 17

1819

80% 77%

40%

96%

78%

Synthesis of imidazo[1,2-a]pyrazines


N

H2NNO2

Br 1. red.

2. Br CHO

K2CO3

N

N

Br

NH2

N

CHO

ZnCl2then NaBH3CN

N

N

HN

N

PhB(OH)2

Pd(PPh3)4

K3PO4

DME, H2O

N

N

NH2

21 22 23

AcCl

pyr.

N

N

NHAc

24

NBS

CH3CN

N

N

NHAc

25

Br

Br

HCl

EtOH

26

IC50 = 0.70 uMIC50 = 0.011uMthym. IC50 = 0.39 uM

pyrazolopyrimidine

57%

49%

91%

78%

89%

93%

Synthesis of imidazo[1,2-a]pyridines


CDK2 activity in various heterocyclic cores

N

N

HN

Br

NN

N

HN

Br

N N

HN

Br

< <

N

N N

HN

Br

<<

• focus on SAR of pyrazolo[1,5-a]pyrimidines

12

Validating the concept

Does the lead series give the desired activity in cell culture?

Can we derive a therapeutic index?

Can the desired activity be demonstrated in vivo?

13

Initial lead compound

N

N

N

HN

Br

F

N+

O–

35

7

In Vitro IC50’s (uM)

CDK2/E = 0.031

CDK2/A = 0.030

CDK1 = 0.045

CDK4 = 0.045

MAPK = 1.2

GSK3b = 0.15

3A4, 2D6, 2C9, 2C19 > 30 uM

In-Cell, thy uptake, A2780

IC50 (10% FBS) = 0.16 uM

IC90 (10% FBS) = 0.29 uM

hERGRb:17% @ 1.5 uM

Rb: 44% @ 5 uM

VC: IC50 = 3.2 uM

Mouse PK (IV/PO, 5 mpk)

AUC = 8.4 uM.hr

Cmax = 5.8 uM

IV t1/2 = 2.4 hrs

(NOEL)

Rat PK (IV/PO, 5/10 mpk)

PO AUC = 34.5 uM.hr

Cmax = 4.4 uM

IV t1/2 = 5.9 hrs

clearance = 18 mL/min/kg

(IV, PO NOEL)

• Efficacy: A2780 (ovarian): 96% I @ 40 mpk, qd; MiaPaCa (pancreatic): 77% I @ 40 mpk, qd.

• Increasing exposure with increasing dose; physical properties appear to limit Cmax.

• Poor solubility has precluded development of an IV formulation.

Monkey PK (IV/PO, 1/5 mpk)

PO AUC = 29.9 uM.hr

Cmax = 2.5 uM

IV t1/2 = 11.0 hrs

(IV: NOEL; PO: 2/3 diarrhea)

Compound 27

14

Compound 27 Continuous Exposure Assays

WS1 (Normal)A2780 (Tumor)

• Cell cultures show very distinct responses

In-cell activity/therapeutic index

Compound 27 Pulsed Exposure Assays

• Expose cells to vehicle, IC50, IC90, 2 x IC90 for 24 hrs• Wash out compound, follow recovery of culture• Support of efficacy (putative trough/exposures)

A2780 ApoptosisWS1 Cell cycle delay/arrest

15

Program progression

Is the current lead appropriate to progress for further evaluation?

What are the key issues?

What approach do we take to resolve them?

16

Improving the potency of the lead compound

3-Position:• Tight SAR.• Br, Cl, ethyl optimal.• Kinase selectivity domain.

7-Position: • Solvent exposed; wide variety of functionality tolerated.• Potency improvements with modulation of physical characteristics.• Substitution influences selectivity.

5-Position:• Variety of polar/nonpolar functionality tolerated.• Greatest potential for increases in potency.• High affinity for –OH substitution.• Substitution influences selectivity.

17

3-Substitution

# R CDK2 IC50 Thy. IC50 # R CDK2 IC50 Thy. IC50

8 Cl; o-F

0.021 uM 0.8 uM 17 CF3

0.71 ---

9 Br

0.011 0.48 18 OH ; 4-pyr. 2.7 ---

10 CN

; 4-pyr. 0.62 --- 19 OCH3

3.8 ---

11 CH3

0.072 1.8

20 OH

0.45 ---

12

0.008 0.76

21 N(CH3)2 34 ---

13

1.2 ---

22 NH2

13.4 ---

14

0.071 2.4

23 CN 0.049 3.5

15

0.37 --- 24 0.13 1.3

16

0.048 1.1 25

1.3 ---

N

N

N

HN

R

N

• Tight SAR; only small hydrophobic substituents tolerated.• 3-Ethyl similar to 3-Br.

Bioorg. Med. Chem. Lett. 2007,17, 6220.18

7-Substitution

# R= CDK2 Thy. RR AUC # R= CDK2 Thy. RR AUC

26 N+ O–

0.034 0.14 17.7

32

NNH

0.014 uM 0.052 uM 3.9 uM.hr

27 N

N

0.032 0.28 6.0 33 H

N

O

0.013 0.024 1.7

28 N

N

0.005 0.18 1.1

34 (o-F)

N NS

0.18 0.029 0.2

29

N

OH

0.016 0.1 0.4

35 (o-F)

N

S

0.19 0.069

30 N

OH

0.019 0.12 1.8 36

NH2S

OO

0.018 0.15 0.5

31

OEt

N+ O–

0.018 0.026 11.4

37 NH2

0.008 0.09 1.1

N

N

N

HN

Br

R

• Solvent exposed; wide variety of functionality tolerated.• Potency improvements with modulation of physical characteristics.


5-Substitution: aryl

# R= CDK2 IC50 Thy. IC50 # CDK2 IC50 Thy. IC50 RR AUC

48

0.011 uM 0.38 uM N-oxide 48A 0.034 uM 0.14 uM 17.7 uM.hr

49

F

0.011 0.48 N-oxide 7 0.031 0.21 15.0

50

Cl

0.003 0.5 N-oxide 50A 0.011 0.17 7.9

51

F

0.037 1.2 N-oxide 51A 0.09 0.30 11.4

52 NC

0.017 0.34 N-oxide 52A 0.047 0.15 tbd

53 S

0.013 0.75 N-oxide 53A 0.035 0.22 9.2

54 O

0.008 0.48 N-oxide 54A 0.016 0.16 12.0

55 N

S

0.032 1.6

R

N

N

N

HN

Br

N

• Variety of polar/nonpolar functionality tolerated.

• Greatest potential for increases in potency.


5-Substitution: alkyl

# R= CDK2 IC50 Thy. IC50 # CDK2 IC50 Thy. IC50 RR AUC

56

0.018 0.52 N-oxide 56A 0.12 uM 0.87 uM ---

57

0.017 0.95 N-oxide 57A 0.09 0.7 ---

58 OH

0.038 1.4

59

0.020 1.3

60

0.013 0.2 N-oxide 60A 0.015 0.12 5.8 uM.hr

61 HN

0.016 0.16 N-oxide 61A 0.025 3.1 0.0

62 O

0.021 0.38 N-oxide 62A 0.028 1.4

63 HN

0.008 0.045 N-oxide

R

N

N

N

HN

Br

N

21

Compounds with improved potency/PK

N

N

N

HN

Br

N+O–

O

Compound 31

CDK2/A IC50 = 0.018 uM

Thy. IC50 = 0.025 uM


AUC = 15 uM.hr

IV t½ = 3.6 hr

Monkey PK (IV/PO, 0.5/1 mpk)

AUC = 10 uM.hr

t½ = 12 hrs

N

N

N

HN

Br

N+

O–

F

Compound 27

CDK2/A IC50 = 0.030 uM

Thy. IC50 = 0.16 uM


PO AUC = 34.5 uM.hr

IV t½ = 5.9 hrs


PO AUC = 29.9 uM.hr

IV t1/2 = 11.0 hrs

N

N

N

HN

Br

N+O–

O

Compound 28

CDK2/A IC50 = 0.016 uM

Thy. IC50 = 0.16 uM


AUC = 28 uM.hr

IV t½ = 2.1 hr


AUC = 71 uM.hr

t½ = 12 hrs

22

5-Position: alcohol substitutionR

N

N

N

HN

Br

N

F

Compound 70

CDK2 IC50 = 0.011 uM

Thy. IC50 = 0.48 uM

Compound 71

CDK2 IC50 = 0.063 uM

Thy. IC50 = 2.5 uM

Compound 72

CDK2 IC50 = 0.027 uM

Thy. IC50 = 0.26 uM

Compound 73

CDK2 IC50 = 0.0004 uM

Thy. IC50 = 0.006 uM

N

OH

N NOH

HN

Compound 74

CDK2 IC50 = 0.063 uM

Thy. IC50 = 1.1 uM

Compound 75

CDK2 IC50 = 0.018 uM

Thy. IC50 = 0.85 uM

Compound 76

CDK2 IC50 = 0.006 uM

Thy. IC50 = 0.2 uM

Compound 77

CDK2 IC50 = 0.0003 uM

Thy. IC50 = 0.002 uM

HN

HN

OH

HN

OH

• Alcohol substitution improves potency and solubility, but increases rate of clearance and decreases exposure

23

• Series with distinct properties were identified:

Compound 7: average potency, good PK Compound 31: improved in-cell potency, excellent PK Compound 73A: high potency, rapid clearance

• How do they compare to known CDK inhibitors?

BMS-387032: average potency, rapid clearance Flavopiridol: average potency, rapid clearance

• How do we identify the optimal profile (potency, PK)?

What is the optimal profile for a CDK inhibitor?

N

N

N

N

HN

Br

OH

N+

O–

N

N

N

HN

Br

N+

O–

O

S

N

S

NO

NH

O

NH

Compound 31 Compound 73A BMS-387032

24

In vivo screening paradigm • Goal: identification of optimal profile

• Readout: efficacy below MTD in mouse

“Efficacy Arm”“Efficacy Arm”“Efficacy Arm”

“Safety Arm”“Safety Arm”“Safety Arm”

Test Compounds

Mouse mini-tox(ip, qdx7)

MTD determination

Efficacy(A2780 xenograft

ip, qdx7)

Establish effectivedose/plasma concentration

Single Dose Mouse PK(IP)

Evaluate Index

SuperiorCompounds

Rising Dose Rat/Dog (iv)Confirm index/multiples

Candidate

Rat mini-tox/PK(iv)

MTD determination

Evaluate exposuresand multiples

“Efficacy Arm”“Efficacy Arm”“Efficacy Arm”“Efficacy Arm”“Efficacy Arm”“Efficacy Arm”“Efficacy Arm”

“Safety Arm”“Safety Arm”“Safety Arm”“Safety Arm”“Safety Arm”“Safety Arm”“Safety Arm”

Test Compounds

Mouse mini-tox(ip, qdx7)

MTD determination

Efficacy(A2780 xenograft

ip, qdx7)

Establish effectivedose/plasma concentration

Single Dose Mouse PK(IP)

Evaluate Index

SuperiorCompounds

Rising Dose Rat/Dog (iv)Confirm index/multiples

Candidate

Rat mini-tox/PK(iv)

MTD determination

Evaluate exposuresand multiples

25

Structure Compound #

CDK2 (CDK1)

IC50 [uM]

In-Cell IC50 [uM]

mouse IP MTD [mpk]

MED* (%I)

TI**

AUC (uM.hr)

Cmax (uM)

N

N

N

HN

N+

O–

Br

O

31 0.018 (0.12)

0.025 5

<5 mpk (68%)

1-2

10 @ 5 mpk

9 @ 5 mpk

N

N

N

N

HN

N+

O–

Br

OH

73A 0.001

(0.008) 0.003 20

<4 mpk (73%)

~5

1.4@ 5 mpk

6 @ 5 mpk

NH

O

NH

S

N

S

N

O

BMS-387032 0.011

(0.068) 0.039 50

25 mpk (58%)

2-3

6 @ 20 mpk

8 @ 20 mpk

Initially evaluated compounds

*MED – Minimum Effective Dose (>50% inhibition in A2780 efficacy); **TI (therapeutic index) = MTD/MED; MTD: dose (ip, qdx7) giving 20% BW loss.

• Amino alcohol series (high potency, short t½) gives best therapeutic index.• High potency, short t½ contrary to conventional wisdom What is the rationale for the activity seen with compound 73A? 26

Structure Compound #

CDK2 (CDK1)

IC50 [uM]

In-Cell IC50 [uM]

mouse IP MTD [mpk]

MED* (%I)

TI

AUC (uM.hr)

Cmax (uM)

N

N

N

N

HN

N+

O–

Br

OH

73A 0.001

(0.008) 0.003 20

<4 mpk (73%)

~5

1.4 @ 5 mpk

6 @ 5 mpk

N

N

N

N

HN

N+

O–

OH

SCH 727965 0.001

(0.006) 0.004 60

3 mpk** (48%)

>10

1.4 @ 5 mpk

2 @ 5 mpk

Identification of SCH 727965

*MED – Minimum Effective Dose (>50% inhibition in A2780 efficacy)

N

N N

N

HNOH

NO

N

N N

HN

NO

HOvs.

ACS Med. Chem. Lett. 2010, 1, 204.• in vivo differentiation of compounds with nearly identical in vitro profile27

In Vitro

A/CDK2 IC50 = 0.001 uMB/CDK1 IC50 = 0.004 uMp35/CDK5 IC50 = 0.001 uM

D/CDK4 IC50 = 0.1 uMH/CDK7 IC50 = 0.07 uMGSK3b IC50 = 0.78 uMMAPK IC50 = 4.1 uM

SCH 727965

SCH 727965: in vitro properties

N

N

N

N

HNOH

N+

O–

In Cell

IC50 (A2780) = 0.004 uMMarker IC50 (phospho-Rb) = ~0.01 uM

2 hour apoptosis = 0.025 uM (BMS-387032 = 1 uM)

Cell Line Survey (106 cell lines, NCI-60 plus others)

Multiple assays (FACS, markers, viability, apoptosis)100% arrest; >85% apoptosis.

Predominant tumor cell response - apoptosisNormal cell response (WS-1) - cell cycle arrest, little apoptosis

UBI Kinase Profiler – No issuesPanLabs – No issuesGPCR – No issues

CYP – No issueshERG (VC) – 5% @ 1 uMInvest. CV – No issues up to ~0.37 uM

CDK2/cyclin A + SCH 727965Mol. Cancer Ther. 2010, 9, 2344.

28

In vivo efficacy: SCH 727965 is active < MTD A2780 (ovarian cancer) RPMI 8226 (multiple myeloma)

• same model used for preclinical studies of Velcade• complete cures in 20 mpk group (no regrowth of tumor) A549 (lung cancer)

• complete cures in 20 mpk group (no regrowth of tumor)• no effect on RBC, platelets• mild reversible myelosupression

29

Antimetabolites induce activation (and phosphorylation) of CHK1 kinase

CHK1 inhibition + HU (or gemcitabine or cytarabine) induce DNA damage and cell death

N

N N

N

HN

N

N N

R5

R7

R6

R3

CHK1 inhibitor?

OH

NOCHK1 IC50 : 25 000 nM

CDK2 IC50 = 1 nM

SCH 727965

CHK1 inhibitorstemplate-based approach

30

R5

O O

OCH3

NH

N

NH2

+

N

N N

R5

NHR7

R3

N

N N

R5

NHR7

R3

R5

O O

OCH3

NH

N

NH2

+

R6

CDK2 CHK1kinase kinase

R6

N

N N

OHO O

OEt

NH

N

NH2

+

N

N N

NHR7

R3

2 stepsR3

R3

O O

OEt

NH

N

NH2

+

R3

BocNN

N N

OH

R3BocN

O O

OEt

NH

N

NH2

+BocNN

N N

OH

BocN

R3 = H, alkyl, aryl

31

N

N N

OH

BocN N

N N

NHR7

BocN

R6

N

N N

NHR7

BocN

R6

R3

alkyl, (het)aryl

N

N N

R

BocN

R = OH, Cl, NH2

NBS

CH3CN

N

N N

R

BocNBr

N

N N

R

BocN

R = OH, NH2

t-BuNH2

Br2N

N N

R

BocN

R = OH, NH2

Br

N

N N

NH2

BocN

Br

5 steps N

N N

NH2

HN

NN

NN

CHK1 IC50 = 100 nMCDK2 IC50 = 22 000 nM

N

N N

NH2

HN

R6

R3


N

N N

N

HN

N

N N

NH2

HN

NN

NN

CHK1 IC50 = 100 nMCDK2 IC50 = 22 000 nM

OH

NOCHK1 IC50 : 25 000 nM

CDK2 IC50 = 1 nM

SCH 727965

CHK1 inhibitorstemplate-based approach

N

N N

NH2

HN

Br

NN

CHK1 IC50 < 10 nM

SCH 900776

SCH 900776 + HU (or Gem or Ara-C) induce DNA damage and cell death in vivo

Mol. Cancer Ther. 2011, doi:10.1158/1535-7163.MCT-10-0324. 33

CHK1 small molecule inhibitor + HU (or Gem or Ara-C) induce DNA damage and cell death

O N

F

FHO

HON

O

NH2

gemcitabine• inhibits RNR (as triphosphate)• gets incorporated into DNA• relatively toxic• limited stability

O N

F

FHO

ON

O

NH2

R

gem analogs (WO 2009/061781 A1)• gem-like phenotype in combination with CHK1 inhibitor• no DNA incorporation

current effort• compounds with targeted biological activity

synthetic lethal treatment

CHK1 + DNA polymerase- inhibition induce DNA damage and cell death

Taricani, L.; Shanahan, F.; Parry, D. Cell Cycle 2009, 8, 482.

34

The End

Selektivní kinázové inhibitory na bázi pyrazolo [1,5-a]p yrimidinu

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