A diaminocyclohexyl analog of SNS-032 with improved permeability and bioavailability properties

Article abstract of DOI:10.1016/j.bmcl.2008.09.073

The identification of a selective CDK2, 7, 9 inhibitor 4 with improved permeability is described. Compound 4 exhibits comparable CDK selectivity profile to SNS-032, but shows improved permeability and higher bioavailability in mice.

Full text of DOI:10.1016/j.bmcl.2008.09.073

Bioorganic & Medicinal Chemistry Letters 18 (2008) 5763–5765
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A diaminocyclohexyl analog of SNS-032 with improved permeability and
bioavailability properties
*
Ingrid C. Choong , Iana Serafimova, Junfa Fan, David Stockett, Erica Chan, Sravanthi Cheeti, Yafan Lu,
*
Bruce Fahr, Phuongly Pham, Michelle R. Arkin, Duncan H. Walker, Ute Hoch
Medicinal Chemistry, Sunesis Pharmaceuticals, 341 Oyster Point Boulevard, 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 4 June 2008
Revised 16 September 2008
Accepted 19 September 2008
Available online 24 September 2008
The identification of a selective CDK2, 7, 9 inhibitor 4 with improved permeability is described. Com-
pound 4 exhibits comparable CDK selectivity profile to SNS-032, but shows improved permeability and
higher bioavailability in mice.
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 and tran-
scriptional regulation.1–3 Cell-cycle regulatory CDKs include
cycD/CDK4 and cycD/CDK6, as well as cycE/CDK2, which sequen-
tially phosphorylate the retinoblastoma protein to facilitate
G1 ? S transition. CDK2 and CDK1 paired with cycA, and cycB/
CDK1 are required for orderly progression through S-phase and
the G2 ? M transition, respectively. Transcriptional CDKs include
cycH/CDK7 and cycT/CDK9. The CDK7 subunit of transcription fac-
tor II H (TFIIH) phosphorylates the carboxy-terminal domain (CTD)
of RNA polymerase II (pol II) on serine 5 at early stages of the tran-
scriptional cycle.4 CDK9, a member of the elongation factor P-TEFb,
phosphorylates serine 2 on the CTD to transition RNA pol II into
productive elongation.5 Inhibition of these kinases is predicted to
have the greatest effect on the expression of proteins with short
half-lives, many of which are encoded by antiapoptotic and growth
regulatory genes. Some of the CDKs are involved in both processes.
For example, CDK7 is a component of the CDK-activating kinase
(CAK). Full activation of CDKs 1, 2, 4, 5 and 6 requires phosphory-
lation by CAK; therefore, CDK7 serves as a ‘master regulator’ of the
cell cycle. Inappropriate activation of both cell-cycle and transcrip-
tional-regulatory CDKs can lead to unregulated proliferation,
avoidance of apoptosis, and the presence of genetic instability in
cancer cells. These attributes, which are among the hallmarks of
cancer, suggest that CDKs may be important targets for cancer
therapeutics. SNS-032 (Fig. 1, formerly BMS-387032) is such a
dual-acting CDK inhibitor, with potency and selectivity against
CDK2, 7 and 9 (Table 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. Bioavailability was limited by absorption rather
than extensive first-pass metabolism.6 Since SNS-032 is a substrate
of P-glycoprotein, 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 CDK2, 7, and 9 inhibitory activities, but with improved
permeability and lack of transporter-mediated efflux. We hypoth-
esized that improving the permeability of SNS-032 would possibly
provide an inhibitor with improved oral bioavailability. In order to
conserve the specificity and potency of SNS-032, we chose to main-
tain the general scaffold of SNS-032, but to replace the isonipecotic
amide fragment and explore N-alkyl instead of N-acyl moieties.
Inhibitors 2–10 (Tables 1 and 2) were synthesized as summa-
rized in Scheme 1. Intermediate 11 was prepared as reported pre-
NH
H
O
N
N
S
S
O
N
* Corresponding authors. Tel.: +1 650 619 6115.
E-mail addresses: ichoong2@gmail.com (I.C. Choong), uhoch@nektar.com (U.
1
Hoch).
Figure 1. SNS-032.
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.09.073

5764
I. C. Choong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5763–5765
viously.7 Treatment of 11 with NaNO2 in CH3CN provided 2-bro-
mothiazole 12 in 40–60% yield. Exposure of 12 to various primary
and secondary amines in dimethylacetamide at 110 °C afforded the
final products, 2–10.
Table 1
Inhibition of enzymatic CDK2 and cellular CDK9
O
R
S
N
S
N
All the compounds were screened in a CDK2/cycA8 biochemical
assay and a CDK9 high-content screen cellular assay9 and com-
pared against SNS-032 (Compound 1). Inhibition of CDK2/cycA
was readily achieved, consistent with CDK2/SNS-032 crystallogra-
phy which shows that the piperidinyl ring is directed into sol-
vent,10 and therefore replaceable. However, sub-micromolar
CDK9 cellular activity was only observed for the 1,4-diam-
inocyclohexyl substitution in 4 (Table 1).
Compound
R
IC50 (lM)
CDK2/cycAa
0.046
HCS CDK9b
0.458
NH
H
N
1 (SNS-032)
O
NH
H
N
Further exploration around 4 indicated a CDK9 preference for
trans-1,4-diaminocyclohexyl fragment over the cis-isomer 5, as
well as the 1,4-regioisomer vs the 1,3-isomer 7 (Table 1). Similar
SAR was not determined for SNS-032 amide series.
Compound 4 was further evaluated in in vitro studies, where it
was shown to have comparable activities against CDKs 2, 7, and 9
(Table 2). More significantly, 4 exhibited over 5-fold improved per-
meability in MDCK11 cells over 1 (Table 2). On the basis of the per-
meability data and acceptable liver microsomal stability,12 4 was
selected for pharmacokinetic studies.
Plasma concentration–time profiles after IV and PO administra-
tion of 5 and 10 mg/kg, respectively, of compounds 1 and 4 are
shown in Figure 2.13 Pharmacokinetic parameters are summarized
in Table 3. Pharmacokinetics after intravenous administration are
similar for 1 and 4. Compounds 1 and 4 show moderate to high
clearance, a large volume of distribution, and terminal half-lives
of 0.6 and 1.9 h, respectively. After oral administration of 10 mg/
kg, bioavailability of 1 was 14%, whereas 4 showed bioavailability
of 62%. Administration of 30 mg/kg compound 4, led to a more
than dose-linear increase in AUC (4.4-fold increase for a 3-fold in-
crease in dose), resulting in a bioavailability of 92%. The high bio-
availability was maintained at the next higher dose level of
45 mg/kg. Given the similar pharmacokinetics after intravenous
administration, the increased bioavailability of 4 compared to 1
may be the result of the improved permeability.
In summary, the incomplete bioavailability of compound 1
(SNS-032) in mice that resulted from poor absorption may be rem-
edied by improving the permeability of the compound. Through
replacing the N-isonipecotic fragment with the N-1,4-trans-diam-
inocyclohexyl fragment, we identified compound 4 which exhibits
comparable CDK selectivity profile to SNS-032, but shows im-
proved permeability and higher bioavailability in mice. Further
evaluation of compound 4 is required to determine the effects of
improved permeability on in vivo efficacy.
2
0.229
0.168
0.030
0.503
0.007
0.158
0.028
0.009
0.538
0.690
>10
>10
0.355
>10
3.5
H
N
3
H
N
4
NH2
NH2
H
N
5
H
N
6
OH
H
N
NH2
7
>10
>10
>10
>10
>10
H
N
OH
8
H
N
9
H
N
10
NH
NH2
11
a
IMAP enzymatic assay measuring inhibition of phosphorylation of fluorescein-
labeled substrate.
b
ArrayScan high-content cellular assay measuring phospho-ser2 of RNA pol II.
Table 2
In vitro profiles of 1 and 4
Compound
IC50
(l
M)a
MDCK
% Parent remaining after 30/60 min
CDK1/cycB
CDK2/cycA
CDK6/cycD
CDK7/cycH
CDK9/cycT
Papp (A À B)b
1
Efflux ratioc
HLM
MLM
1 (SNS-032)
4
0.480
0.763
0.038
0.020
ndd
>2
0.062
0.105
0.004
0.097
15
9.4
94/87
64/53
96/94
76/77
5.8
a
Except for CDK2/cycA, all enzymatic data obtained from Upstate.
Units = 10À6 cm/min.
b
c
Efflux ratio = Papp (B À A)/Papp (A À B).
d
No data.
NaNO2, CuBr2
O
O
O
NH2
CH3CN, 0 oC --> rt
40-60 %
Br
DMA, 110 oC
NR1R2
S
S
S
N
N
N
S
S
S
N
N
N
11
12
2-10
Scheme 1. Synthesis of N-alkyl analogs.

I. C. Choong et al. / Bioorg. Med. Chem. Lett. 18 (2008) 5763–5765
5765
Compound 1
Compound 4
a
b
10000
1000
100
10
10000
1000
100
10
1
1
0.1
0.1
0
1
2
3
4
5
6
7
8
0
1
2
3
4
5
6
7
8
Time (hr)
Time (hr)
Figure 2. Concentration vs time profiles of compounds 1 and 4 in mice after 5 mg/kg IV (closed circles) and 10 mg/kg PO (open circles). Concentrations are the averages of
three animals per timepoint.
Table 3
Pharmacokinetic parameters for 1 and 4
Compound
Dose (mg/kg)
Route
Cmax (ng/mL)
AUC (ng h/mL)
CL (mL/min/kg)
55
Vss (L/kg)
3
t1/2 (h)
%F
1 (SNS-032)
5
10
iv
po
2230
275
1510
434
0.6
0.5
14
4
5
10
30
45
iv
1660
411
2390
4600
1160
1430
6390
72
6
1.9
1.5
1.2
1.2
po
po
po
62
92
100
10,700
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.
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. Akoulitchev, S.; Makela, T. P.; Weinber, R. A.; Reinberg, D. Nature 1995, 377, 557.
5. Marshall, N. F.; Price, D. H. J. Biol. Chem. 1995, 270, 12335.
6. Kamath, A. V.; Chong, S. Cancer Chemother. Pharmacol. 2005, 55, 110.
7. 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 6,521,759, 2003.
8. Inhibition of CDK2-cyclinA was assayed with the IMAP (Molecular Devices)
florescence polarization assay and was used according to the manufacturer’s
12. MDCK II cells were cultured in DMEM supplemented with 10% FBS and 100 U
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
5 days. Prior to addition of the dosing solutions, MDCK monolayers were
washed with PBS. The bi-directional permeability studies were initiated by
adding an appropriate volume of DMEM media containing 2 lM compound to
guidelines. Briefly, 1 mM DTT and 100
IMAP tween reaction buffer. The final ATP concentration was 10
cyclinA was used at a final concentration of 2 nM (Cell Signaling, 7521) and the
substrate, FAM-H1-peptide, was used at final concentration of 100 lM
l
M sodium vanadate was added to the
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
l
M. CDK2/
a
(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.
9. 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.
10. Misra, R. N.; Xiao, H.; Kim, K. S.; Lu, S.; Han, W.; Barbosa, S. A.; Hunt, J. T.;
Rawlins, D. B.; Shan, W.; Ahmed, S. Z.; Qian, L.; Chen, B.; Zhao, R.; Bednarz, M.
S.; Kellar, K. A.; Mulheron, J. G.; Batorsky, R.; Roongta, U.; Kamath, A.; Marathe,
P.; Ranadive, S. A.; Sack, J. S.; Tokarski, J. S.; Pavletich, N. P.; Lee, F. Y. F.;
Webster, K. R.; Kimball, S. D. J. Med. Chem. 2004, 47, 1719.
*
*
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.
13. All animals used were handled according to Sunesis Pharmaceuticals, Inc.
institutional guidelines. Compounds 1 and 4 were dissolved in 2.1 mM tartaric
acid (pH 4.0) to obtain dosing solutions of 1, 3, or 4.5 mg/mL. Female, 6- to 8-
week-old CD-1 (3 mice/timepoint) received
a single IV dose of 5 mg/kg
compound 1 or 2 via the tail vein at 5 mL/kg or single po doses of 10, 30,
and 45 mg/kg via oral gavage at 10 mL/kg. After euthanasia, blood samples
were collected via cardiac puncture at 0.083 (iv), 0.25, 0.5, 1, 2, 3 (po), 4, 6, and
8 h postdose into a K2-EDTA tube and placed on ice until centrifugation to
harvest plasma. Plasma samples were stored frozen until analyzed by LC–MS/
MS. The lower limit of detection was 1 ng/mL. Back-calculated standards and
quality control samples (15, 125, and 500 ng/mL) did not deviate by more than
20% from their nominal concentration. Concentrations per timepoint were
averaged before estimating PK parameters.
11. 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