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, 2002
Sausville, 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
Selectivity issue
CDK2 vs. GSK3b
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.
Pyrazolo[1,5-a]pyrimidines
N
Cl
Cl
N
F
N N
Compound A
CDK2 IC50 = 0.50 uM
HN
N
5
Imidazo[1,2-a]pyrazines
N
N
Compound B
CDK2 IC50 = 0.8 uM
HN
N
Proposed binding modes
Cl
HO
NH
N
N
N
N
H
N
Cl
Cl
5
N
CO2H
3
N
H
O
O
N
H
Leu 83
7
N
N
N
H
H
N
O
O
N
N
H
Leu 83
Crystal structure of A in CDK2
Purvalanol B
CDK2 IC50 = 0.009 uM
Compound A
CDK2 IC50 = 0.50 uM
• Initial SAR Plan: Increase potency by incorporating 3-substitution.
6
3-Substitution improves potency
Br
N
Cl
Cl
N
N N
HN
Compound A
CDK2 IC50 = 0.50 uM
Thym. IC50 = 21 uM
Cl
HN
N
N
Follow-up SAR plan: evaluate similar
heterocyclic cores:
Br
Br
Br
N
N N
HN
7
N N
N
N
HN
N
HN
N
Compound A1
CDK2 IC50 = 0.003 uM
Thym. IC50 = 0.48 uM
Synthesis of pyrazolo[1,5-a]pyrimidines
R
NH2
R
OCH3 +
O
N
H
O
N
AcOH
85%
R
DMAP
N N
N
N N
pyr.
O
Cl
POCl3
80%
3
2
1
H
N
4
NBS
CH3CN
NH2
R
95%
Br
N
N
N N
DIPEA
dioxane
91%
HN
R = H: 6
N
R
N
Br
N N
Cl
5
IC50 = 0.011uM
thym. IC50 = 0.39 uM
Bioorg. Med. Chem. Lett. 2007, 17, 6216.
8
Synthesis of pyrazolo[1,5-a]pyridines
1.
O
H2N O S
Ph
O
7
K2CO3, DMF
Ph
air/O2
Ph
N
H2N
CH2Cl2
CO2Et
N+
OR-
8
Ph
n-BuLi
N N
2. H2SO4, heat
9
39%
N N
ICH2CH2I
55%
I
Ph
HN
13
NH2OH
ZnCl2
NaOAc
Br
Ph
NBS
N N
N
80%
H
Cs2CO3
Ph
Ph
PhCH3
Ph
N N
CH3CN
N
80%
12
N
IC50 = 1.87 uM
9
N
then NaBH3CN
65%
BINAP
76%
Br
N N
Pd(OAc)2
Ph
N
CHO
10
Ph
Ph
11
pyrazolopyrimidine
IC50 = 0.011uM
thym. IC50 = 0.39 uM
Bioorg. Med. Chem. Lett. 2007, 17, 6216.
Synthesis of imidazo[1,2-a]pyrazines
N
O
R
Br
R
N
N
+
N
CONH2
15
14
HN
CH3CN
80%
O
R
N
H
Br-
N+
CH3
N
HN

77%
N
O
17
16
pyr.
POCl3
40%
NH2
Br
R
N
N
N
DIPEA
dioxane
N
78%
R = H: 20
IC50 = 0.44uM
thym. IC50 = 2.0 uM
10
NBS
R
N
N
N
HN
Br
R
N
N
Cl
19
CH3CN
96%
N
N
Cl
18
pyrazolopyrimidine
IC50 = 0.011uM
thym. IC50 = 0.39 uM
Bioorg. Med. Chem. Lett. 2007, 17, 6216.
Synthesis of imidazo[1,2-a]pyridines
Br
N
H2N
1. red.
2. Br
NO2
21
Br
N
CHO
K2CO3
57%
PhB(OH)2
N
NH2
22
Pd(PPh3)4
K3PO4
DME, H2O
91%
AcCl
N
N
NH2
N
N
pyr.
78%
NHAc
24
23
NBS
CH3CN
89%
Br
CHO
N
N
N
Br
HCl
N
HN
ZnCl2
26
N
IC50 = 0.70 uM
11
then NaBH3CN
49%
EtOH
93%
N
NHAc
25
pyrazolopyrimidine
IC50 = 0.011uM
thym. IC50 = 0.39 uM
Bioorg. Med. Chem. Lett. 2007, 17, 6216.
CDK2 activity in various heterocyclic cores
Br
N N
HN
Br
Br
N
<
<
N
HN
N
N
HN
N
<<
Br
N N
N
HN
• 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
Br
5
N
F
3
N N
7
HN
N+
O–
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
hERG
Rb:17% @ 1.5 uM
Rb: 44% @ 5 uM
VC: IC50 = 3.2 uM
Compound 27
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)
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)
• 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.
14
In-cell activity/therapeutic index
Compound 27 Continuous Exposure Assays
A2780 (Tumor)
WS1 (Normal)
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)
• Cell cultures show very distinct responses
15
A2780
WS1
Apoptosis
Cell cycle delay/arrest
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.
5-Position:
• Variety of polar/nonpolar functionality
tolerated.
• Greatest potential for increases in potency.
• High affinity for –OH substitution.
• Substitution influences selectivity.
7-Position:
• Solvent exposed; wide variety of functionality tolerated.
• Potency improvements with modulation of physical characteristics.
• Substitution influences selectivity.
17
3-Substitution
R
N
• Tight SAR; only small hydrophobic substituents tolerated.
• 3-Ethyl similar to 3-Br.
N N
HN
N
#
R
CDK2 IC50
Thy. IC50
#
8
Cl
0.021 uM
0.8 uM
17
0.011
0.48
18
0.62
---
19
0.072
1.8
20
12
0.008
0.76
21
13
1.2
---
22
14
0.071
2.4
23
15
0.37
---
16
0.048
1.1
; o-F
Br
9
10
CN
; 4-pyr.
R
CDK2 IC50
Thy. IC50
0.71
---
OH ; 4-pyr.
2.7
---
OCH3
3.8
---
0.45
---
34
---
13.4
---
0.049
3.5
24
0.13
1.3
25
1.3
---
CF3
OH
11
18
CH3
N(CH3)2
NH 2
CN
Bioorg. Med. Chem. Lett. 2007,17, 6220.
7-Substitution
Br
N
• Solvent exposed; wide variety of functionality tolerated.
• Potency improvements with modulation of physical characteristics.
N N
HN
#
26
R
R=
N+
CDK2
Thy.
RR AUC
#
0.034
0.14
17.7
32
R=
CDK2
Thy.
RR AUC
0.014 uM
0.052 uM
3.9 uM.hr
0.013
0.024
1.7
0.18
0.029
0.2
0.19
0.069
0.018
0.15
0.5
0.008
0.09
1.1
O–
NH
N
0.032
0.28
6.0
N
O
N
28
0.005
0.18
1.1
34 (o-F)
S
N
N N
OH
29
H
N
33
N
27
0.016
0.1
0.4
35 (o-F)
S
N
N
30
N
0.019
0.12
1.8
36
S
OH
NH2
O O
–
31
N+
O
0.018
OEt
19
0.026
11.4
37
NH2
Bioorg. Med. Chem. Lett. 2007,17, 6220.
Br
R
5-Substitution: aryl
N
N N
• Variety of polar/nonpolar functionality tolerated.
HN
• Greatest potential for increases in potency.
N
#
R=
48
CDK2 IC50
Thy. IC50
#
CDK2 IC50
Thy. IC50
RR AUC
0.011 uM
0.38 uM
N-oxide 
48A
0.034 uM
0.14 uM
17.7 uM.hr
0.011
0.48
N-oxide 
7
0.031
0.21
15.0
0.003
0.5
N-oxide 
50A
0.011
0.17
7.9
0.037
1.2
N-oxide 
51A
0.09
0.30
11.4
0.017
0.34
N-oxide 
52A
0.047
0.15
tbd
0.013
0.75
N-oxide 
53A
0.035
0.22
9.2
0.008
0.48
N-oxide 
54A
0.016
0.16
12.0
0.032
1.6
F
49
Cl
50
F
51
52
NC
53
54
S
O
N
55
20
S
Bioorg. Med. Chem. Lett. 2007,17, 6220.
5-Substitution: alkyl
Br
R
N
N N
HN
N
#
R=
CDK2 IC50
Thy. IC50
56
0.018
0.52
57
0.017
0.95
0.038
1.4
0.020
1.3
0.013
OH
58
59
60
61
62
63
21
HN
O
HN
#
CDK2 IC50
Thy. IC50
RR AUC
N-oxide 
56A
0.12 uM
0.87 uM
---
N-oxide 
57A
0.09
0.7
---
0.2
N-oxide 
60A
0.015
0.12
5.8 uM.hr
0.016
0.16
N-oxide 
61A
0.025
3.1
0.0
0.021
0.38
N-oxide 
62A
0.028
0.008
0.045
N-oxide 
1.4
Compounds with improved potency/PK
Br
Br
N
F
O
HN
HN
N+ –
O
N N
N N
N N
HN
22
N
Br
N
N+ –
O
N+ –
O
O
Compound 27
CDK2/A IC50 = 0.030 uM
Thy. IC50 = 0.16 uM
Compound 28
CDK2/A IC50 = 0.016 uM
Thy. IC50 = 0.16 uM
Compound 31
CDK2/A IC50 = 0.018 uM
Thy. IC50 = 0.025 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
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
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
Br
R
5-Position: alcohol substitution
N
N N
HN
N
OH
OH
N
N
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
H
N
H
N
H
N
H
N
OH
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
OH
Compound 77
CDK2 IC50 = 0.0003 uM
Thy. IC50 = 0.002 uM
• Alcohol substitution improves potency and solubility, but increases rate of clearance
and decreases exposure
23
What is the optimal profile for a CDK inhibitor?
• 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)?
Br
Br
N
N
N
N
N N
N N
HN
OH
O
S
HN
S
N
N
+
O–
+
N
O
N
H
O–
O
Compound 31
24
Compound 73A
BMS-387032
NH
In vivo screening paradigm
• Goal: identification of optimal profile
• Readout: efficacy below MTD in mouse
Test Compounds
Mouse mini-tox
(ip, qdx7)
MTD determination
Efficacy
(A2780 xenograft
ip, qdx7)
Establish effective
dose/plasma
concentration
“Efficacy Arm”
Single Dose Mouse PK
(IP)
Evaluate Index
MTD determination
Evaluate exposures
and multiples
Superior
Compounds
Rising Dose Rat/Dog (iv)
Confirm index/multiples
Candidate
25
Rat mini-tox/PK
(iv)
“Safety Arm”
Initially evaluated compounds
Structure
Compound #
CDK2
(CDK1)
IC50
[uM]
31
0.018
(0.12)
In-Cell IC50
[uM]
mouse
IP MTD
[mpk]
MED*
(%I)
TI**
AUC
(uM.hr)
Cmax
(uM)
10 @ 5
mpk
9@5
mpk
1.4@ 5
mpk
6@5
mpk
6 @ 20
mpk
8 @ 20
mpk
Br
N
N N
HN
N
0.025
5
<5 mpk
(68%)
 1-2
+
O
–
O
Br
N
N
N N
OH
73A
HN
0.001
(0.008)
0.003
20
<4 mpk
(73%)
 ~5
+
N
O–
N
O
S
O
S
N
N
H
BMS-387032
NH
0.011
(0.068)
0.039
50
25 mpk
(58%)
 2-3
*MED – Minimum Effective Dose (>50% inhibition in A2780 efficacy); **TI (therapeutic index) = MTD/MED; MTD: dose (ip, qdx7) giving 20% BW loss.
26
• 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?
Identification of SCH 727965
Structure
Compound #
CDK2
(CDK1)
IC50
[uM]
In-Cell IC50
[uM]
mouse
IP MTD
[mpk]
MED*
(%I)
 TI
AUC
(uM.hr)
Cmax
(uM)
1.4 @ 5
mpk
6@5
mpk
1.4 @ 5
mpk
2@5
mpk
Br
N
N
N N
OH
73A
HN
0.001
(0.008)
0.003
20
<4 mpk
(73%)
 ~5
N+
N
O–
N
N N
OH
SCH 727965
HN
0.001
(0.006)
0.004
60
3 mpk**
(48%)
 >10
N+
O–
*MED – Minimum Effective Dose (>50% inhibition in A2780 efficacy)
N
N
N
N N
OH
HN
vs.
N
27
N N
O
HO HN
N
• in vivo differentiation of compounds
with nearly identical in vitro profile
O
ACS Med. Chem. Lett. 2010, 1, 204.
SCH 727965: in vitro properties
In Vitro
N
N
N N
OH
HN
SCH 727965
N+
O–
A/CDK2 IC50 = 0.001 uM
B/CDK1 IC50 = 0.004 uM
p35/CDK5 IC50 = 0.001 uM
UBI Kinase Profiler – No issues
PanLabs – No issues
GPCR – No issues
D/CDK4 IC50 = 0.1 uM
H/CDK7 IC50 = 0.07 uM
GSK3b IC50 = 0.78 uM
MAPK IC50 = 4.1 uM
CYP – No issues
hERG (VC) – 5% @ 1 uM
Invest. CV – No issues up to ~0.37 uM
In Cell
IC50 (A2780) = 0.004 uM
Marker 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 - apoptosis
Normal cell response (WS-1) - cell cycle arrest, little apoptosis
CDK2/cyclin A + SCH 727965
28
Mol. Cancer Ther. 2010, 9, 2344.
In vivo efficacy: SCH 727965 is active < MTD
A2780 (ovarian cancer)
A549 (lung cancer)
RPMI 8226 (multiple myeloma)
• same model used for preclinical studies of Velcade
• complete cures in 20 mpk group (no regrowth of tumor)
• complete cures in 20 mpk group
(no regrowth of tumor)
• no effect on RBC, platelets
• mild reversible myelosupression
29
CHK1 inhibitors
template-based approach
N
N
R5
N N
OH
R3
N N
R6
HN
R7
SCH 727965
CHK1 IC50 : 25 000 nM
CDK2 IC50 = 1 nM
N
N
CHK1 inhibitor?
O
Antimetabolites induce activation (and phosphorylation) of CHK1 kinase
CHK1 inhibition + HU (or gemcitabine or cytarabine) induce DNA damage and cell death
30
NH2
R5
OCH3 +
O
N
H
O
O
N
H
O
O
+
N
H
O
OEt
O
+
N
H
O
R3
N
R3
N
2 steps
NHR7
OH
BocN
R3
N
N N
N
OH
R3 = H, alkyl, aryl
NH2
BocN
OEt
O
31
O
+
N
H
N
BocN
R3
N
N N
N N
N
CHK1
kinase
CDK2
kinase
N N
R6
NHR7
NH2
R3
BocN
N
R5
NH2
R3
OEt
N N
NHR7
NH2
OCH3 +
R3
N
N
R6
R5
R5
N
N N
OH
BocN
BocN
N
N N
BocN
N
N N
R6
N N
R6
NHR7
NHR7
OH
R3
N
alkyl, (het)aryl
BocN
N
BocN
NBS
N N
Br
N
N N
CH3CN
R
R
R = OH, Cl, NH2
BocN
N
BocN
Br2
N N
N
N N
Br
t-BuNH2
R
R
R = OH, NH2
R = OH, NH2
N
BocN
5 steps
N
Br
N N
NH2
32
Bioorg. Med. Chem. Lett. 2011, 21, 471.
HN
R3
N
R6
N N
NH2
HN
N
N
N N
N
N
NH2
CHK1 IC50 = 100 nM
CDK2 IC50 = 22 000 nM
CHK1 inhibitors
template-based approach
N
N
HN
N
OH
N
HN
N N
N N
N
HN
N
NH2
N
N
Br
N N
NH2
SCH 900776
SCH 727965
CHK1 IC50 : 25 000 nM
CDK2 IC50 = 1 nM
N
N
N
O
CHK1 IC50 = 100 nM
CDK2 IC50 = 22 000 nM
SCH 900776 + HU (or Gem or Ara-C) induce DNA damage
and cell death in vivo
33 Mol. Cancer Ther. 2011, doi:10.1158/1535-7163.MCT-10-0324.
CHK1 IC50 < 10 nM
CHK1 small molecule inhibitor + HU (or Gem or Ara-C) induce DNA damage and cell death
NH2
O
HO
N
F
HO
NH2
N
O
F
R
O
O
F
HO
gemcitabine
• inhibits RNR (as triphosphate)
• gets incorporated into DNA
• relatively toxic
• limited stability
N
N
O
F
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-a inhibition induce
DNA damage and cell death
Taricani, L.; Shanahan, F.; Parry, D. Cell Cycle 2009, 8, 482.
34
The End