selektivní kinázové inhibitory na bázi pyrazolo[1,5-a]pyrimidinu dr. kamil paruch...
Post on 27-Dec-2015
220 Views
Preview:
TRANSCRIPT
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%
Bioorg. Med. Chem. Lett. 2007, 17, 6216.9
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
Bioorg. Med. Chem. Lett. 2007, 17, 6216.10
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
Bioorg. Med. Chem. Lett. 2007, 17, 6216.11
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.
Bioorg. Med. Chem. Lett. 2007,17, 6220.19
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.
Bioorg. Med. Chem. Lett. 2007,17, 6220.20
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
Rat PK (IV/PO, 2/10 mpk)
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
Rat PK (IV/PO, 5/10 mpk)
PO AUC = 34.5 uM.hr
IV t½ = 5.9 hrs
Monkey PK (IV/PO, 1/5 mpk)
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
Rat PK (IV/PO, 5/10 mpk)
AUC = 28 uM.hr
IV t½ = 2.1 hr
Monkey PK (IV/PO, 2/10 mpk)
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
Bioorg. Med. Chem. Lett. 2011, 21, 471.32
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
top related