Modifications of the isonipecotic acid fragment of SNS-032: Analogs with improved permeability and lower efflux ratio

Article abstract of DOI:10.1016/j.bmcl.2008.09.099

Modifications of the isonipecotic acid fragment of SNS-032 results in analogs which are more permeable and lower effluxed than SNS-032. The enantiomerically pure synthesis and the in vivo profile of analog 20 is described.

Full text of DOI:10.1016/j.bmcl.2008.09.099

Bioorganic & Medicinal Chemistry Letters 18 (2008) 6236–6239
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Modifications of the isonipecotic acid fragment of SNS-032: Analogs
with improved permeability and lower efflux ratio
*
Junfa Fan , Bruce Fahr, David Stockett, Erica Chan, Sravanthi Cheeti, Iana Serafimova, Yafan Lu,
*
*
Phuongly Pham, Duncan H. Walker, Ute Hoch , Ingrid C. Choong
Sunesis Pharmaceuticals, 341 Oyster Point Blvd, South San Francisco, CA 94080, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 24 June 2008
Revised 26 September 2008
Accepted 29 September 2008
Available online 2 October 2008
Modifications of the isonipecotic acid fragment of SNS-032 results in analogs which are more permeable
and lower effluxed than SNS-032. The enantiomerically pure synthesis and the in vivo profile of analog 20
is described.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
CDK
Cyclin
SNS-032
Kinase
The CDKs (cyclin-dependent kinases) are a family of serine/
threonine protein kinases that, in conjunction with their cyclin
(cyc) partners, play key roles in cell-cycle progression (CDK2, 4, 6
and 7) and transcriptional regulation (CDK7 and 9).1–3 Inappropri-
ate activation of both cell-cycle and transcriptional-regulatory
CDKs can lead to unregulated proliferation, avoidance of apoptosis,
and the presence of genetic instability in cancer cells. These attri-
butes, which are among the hallmarks of cancer, suggest that CDKs
may be important targets for cancer therapeutics.4–6
tein, this efflux transporter may be responsible for limiting its
absorption. With an interest in a potential oral CDK program, we
sought to develop a backup inhibitor to SNS-032 with comparable
CDK 2, 7, and 9 activities, but with improved permeability and lack
of transporter-mediated efflux. Previous identification of 2 demon-
strated that improving permeability resulted in significant higher
bioavailabilities in mice.8 Described here is a concurrent effort to
identify a series which had both good permeability and lowered
efflux.
SNS-032 (1, formerly BMS-387032, Fig. 1) is a dual-acting CDK
inhibitor, with potency and selectivity against CDKs 2, 7 and 9 (Ta-
ble 3). SNS-032 is currently in a phase 1 clinical trial for multiple
myeloma and chronic lymphocytic leukemia as an intravenous
agent. Early reports showed that SNS-032 has oral bioavailability
of about 31% in rats. The incomplete oral bioavailability has been
shown to be the result of poor absorption rather than extensive
first-pass metabolism.7 Since SNS-032 is a substrate of P-glycopro-
In early efforts, we demonstrated that we could improve per-
meability and eliminate efflux by masking the isonipecotic second-
ary amine of 1 as a tertiary amine, sulfonamide or amide (data not
shown). However, these compounds proved to be very unstable to
liver microsomes, so we turned our attention to blocking potential
P-glycoprotein binding with the secondary amine function through
substitutions around the isonipecotic or nipecotic ring system.
Inhibitors 3–9 (Table 1) were synthesized as summarized in
Scheme 1. Intermediate 10 was prepared as previously reported.9
Under standard coupling conditions, treatment of 10 with different
acids provided resulting amides in good yields. For N-Boc-pro-
tected analogs, deprotection with 4.0 N HCl in dioxane at 50 °C
afforded the final products. The acids used above were commer-
cially available or readily prepared according to the literature.9,10
Specifically, 7–9 were synthesized through platinum reduction of
the corresponding nicotinic acids to provided the saturated nipe-
NH
H
H
O
N
O
N
N
N
S
S
S
S
O
NH2
N
N
1, SNS-032
2
Figure 1. SNS-032 and 2.
11
cotic acids as mixtures of cis-enantiomers.
All the compounds were evaluated for inhibition of
CDK2/cycA12 enzymatic activity, inhibition of CDK9 phosphoryla-
tion of ser2 on RNA pol II13 and permeability in MDCK cells.14
* Corresponding authors. Tel.: +1 650 619 6115 (I.C. Choong).
E-mail addresses: jfan@sunesis.com (J. Fan), uhoch@nektar.com (U. Hoch),
ichoong2@gmail.com (I.C. Choong).
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.099

J. Fan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6236–6239
6237
Table 1
In vitro profile of analogs 3–9
H
O
N
R
N
S
S
O
N
Compound
R
IC50
(lM)
MDCK
% Parent Remaining @ 30/60 min
CDK2/cycAa
0.046
HCS CDK9b
0.55
Papp (A–B)c
Efflux ratiod
15
HLM
MLM
1
3
4
5
1.0
94/87
96/94
NH
NH
NH
0.015
0.0030
0.017
1.5
1.3
4.5
3.2
37
13
16
NDe
NDe
NDe
24/1
0.68
0.81
60/41
33/7
NH
6
7
8
0.020
0.095
0.014
0.14
0.55
1.6
7.5
25
6.0
3.0
2.0
2.5
83/74
12/4
68/44
7/2
N
H
N
H
0.37
0.27
24
71/49
60/39
26/8
49/27
N
H
9
14
N
H
a
IMAP enzymatic assay measuring inhibition of phosphorylation of fluorescein-labeled substrate.
b
C
d
e
ArrayScan high-content cellular assay measuring phospho-ser2 of RNA Pol II.
Units = 10À6 cm/min.
Efflux ratio = Papp (B–A)/Papp (A–B).
No data.
Table 2
In vitro profile of four isomers of 9
H
O
N
R
N
S
S
O
N
Compound
R
IC50
(
l
M)
MDCK
% remaining @ 30/60 min
HLM MLM
CDK2/Aa
0.14
HCS CDK9b
Papp (A–B)c
Efflux ratiod
9
0.27
14
2.5
6.3
2.1
6.0
2.0
60/39
57/29
61/35
70/45
54/25
49/27
50/33
32/8
N
H
18
19
20
0.036
0.0050
0.020
0.20
3.2
10
26
4.8
15
N
H
0.77
0.12
4.1
N
H
35/13
5/0
N
H
21
N
H
a
IMAP enzymatic assay measuring inhibition of phosphorylation of fluorescein-labeled substrate.
ArrayScan high-content cellular assay measuring phospho-ser2 of RNA Pol II.
Units = 10À6 cm/min.
b
C
d
Efflux Ratio = Papp (B–A)/Papp (A–B).

6238
J. Fan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6236–6239
Table 3
In vitro profile of 20
Compound
IC50
(l
M)a
MDCK
% Parent remaining @ 30/60 min
CDK1/cycB
CDK2/cycA
CDK4/cycD
CDK7/H
CDK9/T
Papp (A-B)b
Efflux Ratioc
HLM
MLM
1
20
0.48
0.12
0.038
0.020
0.93
0.25
0.062
0.11
0.0040
0.0040
1.0
4.8
15
6.0
94/87
70/45
96/94
35/13
a
Except for CDK2/A, all enzymatic data obtained from Upstate.
Units = 10À6 cm/min.
Efflux ratio = Papp (B–A)/Papp (A–B).
B
c
the same route using (R)-3-bromo-2-methyl-propan-ol as the
starting material.
H
O
N
a or b
O
N
NH2
R
N
S
S
The in vitro profile of the four isomers of 9 is summarized in Ta-
ble 2. Isomers 19 and 20 retained both biochemical activity against
CDK2 and cellular activity in the HCS assay. More significantly, iso-
mer 19 showed good permeability and low efflux. On the basis of
the permeability data and acceptable liver microsomal stability
(Table 3),14,15 20 is a potential candidate for pharmacokinetic
studies.
S
S
N
O
N
10
3-9
Scheme 1. Synthesis of analogs. Reagents and conditions: (a) acid, DMAP, EDCI,
DCM, rt; (b) a, then 4.0 M HCl in dioxane, 50 °C, 2 h.
In summary, through modifications of the isonipecotic acid
fragment of SNS-032, we identified compound 20 which exhibits
comparable CDK selectivity profile to SNS-032, but shows im-
proved permeability and lower efflux. Pharmacokinetic studies will
determine bioavailability in mice.
Disappointingly, flanking substituents around the isonipecotic
amine did not improve permeability or decrease efflux (com-
pounds 3–5). However, since nipecotic amide 6 showed good per-
meability and only moderate efflux, we decided to explore
incorporating substituents on the nipecotic ring. Indeed, com-
pounds incorporating a methyl substituent at various positions
around the ring demonstrated improved permeability and lowered
efflux (7–9). Given that 9 demonstrated the highest stability in
mouse liver microsomes while maintaining CDK2/cycA and CDK9
activity, we synthesized the discrete enantiomers as well as the
two other isomers not represented in the enantiomeric mixture
of 9 to identify which isomer(s) had the most promising in vitro
profile.
Synthesis of the four single isomers of 9 is summarized in
Scheme 2. Commercially available (S)-3-bromo-2-methyl-propan-
ol was oxidized with Dess–Martin periodinane to give aldehyde
12, which was condensed with secondary amine under standard
reductive amination conditions to afford tertiary amine 13. Intra-
molecular alkylation of 13 with LDA at low temperature provided
diasteroisomers 14 and 15. Exchanging protecting groups followed
by hydrolysis provided N-Boc protected nipecotic acid intermedi-
ates 16 and 17. Standard amide coupling conditions provided dias-
teroisomers 18 and 19, which were easily separated by column
chromatography. Isomers 20 and 21 were synthesized following
References and notes
1. Sausville, E. A. Trends Mol. Med. 2002, 8, 32.
2. Shapiro, G. I. J. Clin. Oncol. 2006, 24, 1770.
3. Marshall, N. F. et al J. Biol. Chem. 1996, 271, 27176.
4. Review see Giannis, A. et al Angew. Chem. Int. Ed. 2003, 42, 2122.
5. Akoulitchev, S.; Makela, T. P.; Weinber, R. A.; Reinberg, D. Nature 1995, 377,
557.
6. Marshall, N. F.; Price, D. H. J. Biol. Chem. 1995, 270, 12335.
7. Kamath, A. V.; Chong, S. Cancer Chemother. Pharmacol. 2005, 55, 110.
8. Choong, I. C.; Serafimova, I.; Fan, J.; Stockett, D.; Chan, E.; Cheeti, S.; Lu, Y.; Fahr,
B.; Pham, P.; Arkin, M. R.; Walker, D. H.; Hoch, U. Bioorg. Med. Chem. Lett., 2008,
doi:10.1016/j.bmcl.2008.09.073.
9. Kim, K. S.; Kimball, D. S.; Cai, Z.; Rawlins, D. B.; Misra, R. N.; Poss, M. A.;
Webster, K. R.; Hunt, J. T.; Han, W. U.S. Patent 2003, 6(521), 759.
10. Klein, S. I.; Molino, B. F.; Czekaj, M.; Gardner, C. J.; Chu, V.; Brown, K.; Sabatino,
R. D.; Bostwick, J. S.; Kasiewski, C.; Bentley, R.; Windisch, V.; Perrone, M.;
Dunwiddie, C. T.; Leadley, R. J. J. Med. chem. 1998, 2492.
11. Zacharie, B.; Moreau, N.; Dockendorff, C. J. J. Org. Chem. 2001, 66, 5264.
12. Inhibition of CDK2-cyclinA was assayed with the IMAP (Molecular Devices)
fluorescence polarization assay and was used according to the
manufacturer’s guidelines. Briefly, 1 mM DTT and 100
lM sodium vanadate
was added to the IMAP tween reaction buffer. The final ATP concentration
Br
O
a
b
c
O
H
Br
Br
OCH3
OH
N
Bn
11
12
13
O
O
O
O
d,e,f
OCH3
OCH3
OH
OH
NH
+
+
N
N
N
N
Bn
Bn
Boc
16
Boc
14
15
17
g,h
H
H
O
N
O
NH
N
N
+
S
S
N
S
S
O
O
N
N
18
19
Scheme 2. Synthesis of single isomers of 9. Reagents and conditions: (a) Dess–Martin periodinane, dichloromethane, 95%; (b) BnNHCH2CH2CO2CH3, NaHB(OAc)3,
dichloroethane, HOAc, 75%; (c) LDA, ether, HMPA, À78 °C, 80%; (d) Pd-C, H2, 95%; (e) (Boc)2O, 1.0 M NaOH, dioxane, 97%; (f) 1.0 M LiOH, CH3OH/H2O, 90%; (g) DMAP, EDCI, 10,
dichloromethane, 87%; (h) 4.0 N HCl in dioxane, 50 °C, 2 h, 90%.

J. Fan et al. / Bioorg. Med. Chem. Lett. 18 (2008) 6236–6239
6239
was 10
Signaling, 7521) and the substrate, FAM-H1-peptide, was used at
concentration of 100 (Molecular Devices, R7439). Compounds were
serial diluted in an 11-point titration and IC50s, the concentration required
to inhibit enzyme activity by 50%, was determined for each compound
under the assay conditions described.
l
M. CDK2/cyclinA was used at a final concentration of 2 nM (Cell
either the apical (apical to basolateral transport) or basolateral (basolateral to
apical transport) side of the monolayer. Aliquots were taken from both the
apical and basolateral compartments after incubation for 3 h in a humidified
CO2 incubator at 37 °C. Samples were diluted with acetonitrile containing
internal standard (verapamil) and SNS-032 concentrations were analyzed
using a LC-MS/MS method. The permeability coefficient (Papp) was calculated
a
final
lM
*
*
13. HCT116 cell lines were obtained from ATCC. Array Scan: HCT116 cells were
treated for 16 h with serial dilutions of compound and fixed and permeabilized
with 100% MeOH. The cells were then stained with either anti-RNA polymerase
II serine2 (Abcam #ab5095) antibody in combination with AlexaFluor 488 anti-
rabbit IgG secondary antibody (Invitrogen #A11008). The cell nuclei were
stained using Hoechst 33342 (Invitrogen #3570). Fluorescence levels in the
cells were then analyzed by HCS using a Cellomics ArrayScan instrument.
14. MDCK II cells were cultured in DMEM supplemented with 10% FBS and 100
units antibiotic solution (Cellgro) per milliliter. Cells were seeded into wells of
12-transwell plates at a seeding density of 50,000 cells/well and cultured for
five days. Prior to addition of the dosing solutions, MDCK monolayers were
washed with PBS. The bi-directional permeability studies were initiated by
as follows: Papp = (1/A) C0 (dQ/dt), with A representing the surface are of the
membrane, C0 the initial concentration, and dQ/dt the drug flux.
15. Microsomal stability assays were performed in 100 mM sodium phosphate
buffer, pH 7.4, containing 3.3 mM MgCl2, 0.5 mg/mL liver microsomal
protein,
1 lM test article, and 1 mM NADPH. The reaction mixture was
allowed to equilibrate to 37 °C for 10 min before reactions were started by
addition of NADPH. Aliquots were removed immediately, 30 min, and
60 min after addition of NADPH. Reactions were stopped by addition of
acetonitrile containing internal standard (verapamil). Samples were placed
on ice until centrifugation (4100g, 10 min) to remove protein content and
analyzed by LC-MS/MS. Incubations with lidocaine and dextromethorphan
served as positive controls and indicated that reactions functioned
properly.
adding an appropriate volume of DMEM media containing 2 lM compound to