Indenopyrazoles as novel cyclin dependent kinase (CDK) inhibitors [1]

Full text of DOI:10.1021/jm0100032

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1334
J . Med. Chem. 2001, 44, 1334-1336
Letters
In d en op yr a zoles a s Novel Cyclin
Dep en d en t Kin a se (CDK) In h ibitor s
David A. Nugiel,* Ana-Marie Etzkorn,
Anup Vidwans, Pamela A. Benfield,
Michael Boisclair, Catherine R. Burton, Sarah Cox,
Phillip M. Czerniak, Deborah Doleniak, and
Steven P. Seitz
F igu r e 1. High-throughput lead A3915.
The DuPont Pharmaceuticals Company, Box 80336,
Wilmington, Delaware 19880-0336
analogue generation. The original procedure for prepar-
ing tricarbonyl compounds such as 2 used sodium
methoxide in refluxing toluene.7 We found that NaH in
DMF at 90 °C gave much better results for this
transformation. The tricarbonyl intermediate 2 was
then treated with hydrazine to give the indenopyrazole
3 as a single regioisomer. The acetamide group provides
an additional benefit during the treatment with hydra-
zine to form the pyrazole. An intramolecular hydrogen
bond between the acetamide NH and the adjacent
carbonyl affects this group’s reactivity, and subsequent
attack by hydrazine occurs at the other carbonyl group,
leading to the one isomeric indenopyrazole shown in
Scheme 1. This initial assignment was by 2D NMR
experiments and subsequently confirmed by X-ray
crystallography.8 Removal of the acetamide group was
accomplished with concentrated HCl in refluxing metha-
nol.
Received J anuary 4, 2001
In tr od u ction . A fundamental process in biology is
the division of cells mediated by the cell cycle. This
process ensures the controlled production of subsequent
generations of cells with defined biological function.
Overexpression of the tumor promoting components or
the loss of tumor suppressing products can lead to
unregulated cellular proliferation and the generation of
tumors.1
Cyclin dependent kinases (cdks) play a key role in
regulating the cell cycle machinery. Each kinase associ-
ates with a specific regulatory cyclin and together make
up the active catalytic moiety. The coordinated activity
of these kinases guides cells through the replication
process and ensures the vitality of each subsequent
generation.2
An increasing body of evidence has shown a link
between tumor development and cdk-related malfunc-
tions.3 This evidence has led to an intense search for
small molecule inhibitors of the cdk family as an
approach to cancer chemotherapy.4 Several disclosures
have discussed small molecules with cdk inhibitory
activity.5 To date, only two compounds (flavopiridol and
UCN-01) have entered into clinical trials as cancer
therapeutics based on this cdk inhibition mechanism.6
We disclose here a new structural class of potent and
selective cdk inhibitors, which are active against trans-
formed cell lines and show in vivo activity in a human
xenograft mouse model.
Resu lts a n d Discu ssion . The aniline intermediate
4 was useful in preparing additional analogues in this
series,9 and the SAR of selected analogues is presented
in Table 1.10 There is a good correlation between the
amide substituent’s size and its activity against both
Sch em e 1a
High-throughput screening (HTS) of our compound
collection against cdk4/D1 gave several interesting
compounds including A3915 (Figure 1). Subsequent
screening of this hit against other cdks as well as other
relevant kinases showed the compound to be selective
for the cdk family. We began a medicinal chemistry
effort around A3915 in an attempt to improve its
potency against the cdk family while maintaining
selectivity against our panel of kinases.
Ch em istr y. The synthesis of these compounds is
shown in Scheme 1. The starting point for the synthesis
was dimethyl 3-nitrophthalate readily converted to the
acetamide 1 in two high-yielding steps. We chose the
acetamide as an aniline protecting group due to its
ability to withstand the harsh basic conditions of the
triketone formation reaction. It could then be removed
under acidic conditions to provide aniline 4 for further
a
Reagents and conditions: (a) 1. H2, Pd/C, 2. Ac2O, pyridine,
25 °C, 79%; (b) 2 equiv of NaH, DMF, 90 °C, 30%; (c) hydrazine,
cat. p-TsOH, EtOH, reflux, 50%; (d) conc. HCl, MeOH, reflux, 96%.
10.1021/jm0100032 CCC: $20.00 © 2001 American Chemical Society
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Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9 1335
Ta ble 1. Enzymatic and Cellular Activity for Selected
Compounds
family and are active in cell culture against a trans-
formed colon cell line (HCT116). In addition, these
compounds demonstrate in vivo activity by reducing
tumor growth in a human xenograft mouse model in a
dose-dependent manner. Additional studies are under-
way to improve the potency of these compounds and to
optimize the compound’s physical properties to facilitate
additional in vivo experiments.
k4/D1
IC50
k2/E
IC50
HCT 116 AG1523
IC50
IC50
Su p p or tin g In for m a tion Ava ila ble: Complete experi-
mental details for 1, 2, 3, and 4. This material is available
free of charge via the Internet at http://pubs.acs.org.
cmpd
R
(µM)a
(µM)a
(µM)b
(µM)b
5
3
6
7
8
9
H
0.2
0.45
35
45
0.48
>250
0.08
0.27
1.4
2.1
0.038
41
0.69
2.7
>63
Me
i-Pr
t-Bu
Refer en ces
2.9
7.5
>28
(1) Pardee, A. B. G1 Events and Regulation of Cell Proliferation.
Science 1989, 246, 603-8.
4-NH2C6H4CH2
Ph
0.4
>24
N/Mc
N/Mc
(2) (a) Meijer, L.; Guidet, S.; Philippe, M. Progress in Cell Cycle
Research. Plenum: New York, 1997; Vol. 3. (b) Sherr, C. J .
Mammalian G1 Cyclins. Cell 1993, 73 (6), 1059-65. (c) Draetta,
G. Cell Cycle Control in Eukaryotes: Molecular Mechanisms of
cdc2 Activation. Trends Biochem. Sci. 1990, 15 (10), 378-83.
(3) (a) Draetta, G.; Pagano, M. Cell Cycle Control and Cancer. Annu.
Rep. Med. Chem. 1996, 31, 241-248. (b) Hartwell, L. H.; Kastan,
M. B. Cell Cycle Control and Cancer. Science 1994, 266, 1821-
28. (c) Hunter, T.; Pines, J . Cyclins and Cancer. II: Cyclin D
and CDK Inhibitors Come of Age. Cell 1994, 79, 573-582. (d)
Sielecki, T. M.; Boylan, J . F.; Benfield, P. A.; Trainor, G. L.
Cyclin-Dependent Kinase Inhibitors: Useful Targets in Cell
Cycle Regulation J . Med. Chem. 2000, 43 (1), 1.
a
Values correspond to n ) 2. For assay conditions, see ref 13.
Values correspond to n ) 2. For assay conditions, see ref 14. c N/
b
M ) not measured.
Ta ble 2. In Vivo Antitumor Activity for Compound 5
tumor
growth
dose ip
tumor wt inhibition
no.
cmpd
control
(-flavopiridol
mitomycin C
5
5
5
(mg/kg/day)a (mg ( SE)
(%)b
survivedc
1069 ( 107
(4) (a) Meijer, L.; Kim, S.-H. Chemical Inhibitors of Cyclin-Depend-
ent Kinases. Methods Enzymol. 1997, 283, 113-128. (b) Cole-
man, K. G.; Lyssikatos, J . P.; Ynag, B. V. Chemical Inhibitors
of Cyclin-Dependent Kinases. Annu. Rep. Med. Chem. 1997, 32,
171-179.
7.5
1
30
10
3
538 ( 76
455 ( 40
608 ( 54
871 ( 52
1171 ( 40
50
58
43
19
0
5/8
8/8
8/8
8/8
8/8
(5) (a) Vesely, J .; Havlicek, L.; Strnad, M.; Blow, J . J .; Donella-
Deana, A.; Pinna, L.; Letham, D. S.; Kato, J .-Y.; Detivaud, L.;
Leclerc, S.; Meijer, L. Inhibition of Cyclin-Dependent Kinases
by Purine Analogues. Eur. J . Biochem. 1994, 224, 771-786. (b)
Gray, N. S.; Wodicka, L.; Thunnissen, A.-M.; Norman, T. C.;
Kwon, S.; Espinoza, F. H.; Morgan, D. O.; Barnes, G.; Leclerc,
S.; Meijer, L.; Kim, S.-H.; Lockhart, D. J .; Schultz, P. G.
Exploiting Chemical Libraries, Structure, and Genomics in the
Search of Kinase Inhibitors. Science 1998, 281, 533-538. (c)
Schultz, C.; Link, A.; Leost, M.; Zaharevitz, D. W.; Gussio, R.;
Sausville, E. A.; Meijer, L.; Kunick, C. Paullones, a Series of
Cyclin-Dependent Kinase Inhibitors: Synthesis, Evaluation of
CDK1/Cyclin B Inhibition, and in Vitro Antitumor Activity. J .
Med. Chem. 1999, 42, 2909-2919. (d) Barvian, M.; Boschelli,
D. H.; Cossrow, J .; Dobrusin, E.; Fattaey, A.; Fritsch, A.; Fry,
D.; Harvey, P.; Keller, P.; Garrett, M.; La, F.; Leopold, W.;
McNamara, D.; Quin, M.; Trumpp-Kallmeyer, S.; Toogood, P.;
Wu, Z.; Zhang, E. Pyrido[2,3-d]pyrimidin-7-one Inhibitors of
Cyclin-Dependent Kinases. J . Med. Chem. 2000, 43 (24), 4606-
4616.
(6) (a) Sedlacek, H. H.; Czech, J .; Naik, R.; Kaur, G.; Worland, P.;
Losiewicz, M.; Parker, B.; Carlson, B.; Smith, A.; Senderowicz,
A.; Sausville, E. Flavopiridol (L86-8275, NSC-649890), a New
Kinase Inhibitor for Tumor Therapy. Int. J . Oncol. 1996, 9,
1143-1168. (b) Senderowicz, A. M.; Headlee, D.; Stinson, S. F.;
Lush, R. M.; Kalil, N.; Villalba, L.; Hill, K.; Steinberg, S. M.;
Figg, W. D.; Tompkins, A.; Arbuck, S. G.; Sausville, E. A. Phase
I Trial of Continuous Infusion Flavopiridol, a Novel Cyclin-
Dependent Kinase Inhibitor, in Patients with Refractory Neo-
plasms. J . Clin. Oncol. 1998, 16, 2986-2999. (c) Akinaga, S.;
Sugiyama, K.; Akiyama, T. UCN-01 (7-hydroxystaurosporine)
and other indolocarbazole compounds: a new generation of anti-
cancer agents for the new century? Anti-Cancer Drug Des. 2000,
15 (1), 43-52.
(7) (a) Kilgore, L. B.; Ford, J . H.; Wolfe, W. C. Insecticidal Properties
of 1,3-Indandiones Ind. Eng. Chem. 1942, 34 (4), 494-7. (b)
Antitumor Formulations Containing Indeno[1,2-]pyrazoles. Mor-
ishita Pharmaceutical Co.; J P 60130521, 850712. Application:
J P-83-240686.
(8) Chang, C.-H. Unpublished results of an indenopyrazole analogue
bound to the ATP-binding pocket of cdk2.
(9) Selective acylation of the aniline amine in the presence of the
free pyrazole NH was accomplished using an excess of the
desired acid chloride in dioxane with aqueous saturated NaHCO3
as the base at 45 °C for 1 h. Standard workup and recrystalli-
zation from ethanol gave the desired products in good yield. The
formamide group of compound 5 was introduced by heating
aniline 4 in neat formic acid at 100 °C for 1 h. After the solvent
was evaporated and the residue was recrystallized from ethanol,
compound 5 was obtained in 80% yield.
a
b
Dosing schedule: Q1DX14. For assay conditions, see ref 15.
c Refers to acute drug toxicity not long-term survival.
cdk4 and cdk2. Small aliphatic groups provide com-
pounds with good cdk inhibitory activity (3, 5). Branch-
ing at the R-carbon (6, 7) as well as a phenyl substituent
(9) reduced activity. By adding a methylene bridge we
were able to expand the size and character of the amide
substituents while maintaining good activity (8). In
general, the compounds are more active against cdk2
compared to cdk4. One example (8) is 10-fold selective,
favoring cdk2. All analogues maintain good selectivity
against other kinases from our selectivity panel.11 The
cellular activity of these compounds was also routinely
good against a transformed colon cell line (HCT116).
Here too the cellular activity parallels the cdk inhibitory
activity. We also tested the compounds for their cyto-
toxic effects in a normal fibroblast cell line (AG1523).
The compounds have minimal influence on the mortality
of this cell line, indicating a promising therapeutic
window for this series against transformed cell lines.
We chose compound 5 for further in vivo evaluation
and dosed this compound in a human xenograft mouse
model. The compound was given ip once a day for 14
days, and the tumor size was evaluated at the end of
the experiment based on weight. As shown in Table 2,
there is a dose dependent response to the tumor growth
inhibition. At 30 mg/kg, compound 5 had an effect on
tumor growth approaching that of flavopiridol.12 In
addition, all eight animals survived the dosing regimen
and appeared healthy and normal at the end of the
experiment.
Con clu sion . We disclosed a new structural class of
cyclin dependent kinase inhibitors. These compounds
are selective for the cdk related serine/threonine kinase

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1336 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 9
Letters
(10) Spectroscopic data for selected compounds: 5: mp 280 °C; 1H
NMR (300 MHz, DMSO) 13.6 (s, 1 H), 10.35 (s, 1 H), 8.5 (s, 1
H), 8.2 (d, 8 Hz, 1 H), 8.1 (d, 8 Hz, 2 H), 7.5 (t, 8 Hz, 1 H), 7.2
(d, 8 Hz, 1 H), 7.05 (d, 8 Hz, 2 H), 3.8 (s, 3 H). CIMS m/e Calcd
for C18H14N3O3: 320.1035. Found: 320.1040. Anal. Calcd for
C18H13N3O3: C, 67.71; H, 4.10; N, 13.16. Found: C, 67.55; H,
4.22; N, 13.00. 6: mp 288 °C; 1H NMR (300 MHz, DMSO) 13.6
(s, 1 H), 10.2 (s, 1 H), 8.2 (d, 8 Hz, 1 H), 8.1 (d, 8 Hz, 2 H), 7.5
(t, 8 Hz, 1 H), 7.2 (d, 8 HZ, 1 H), 7.05 (d, 8 Hz, 2 H), 3.8 (s, 3 H),
and unlabeled ATP. The GST-Rb labeled protein is sequestered
on a GSH-Sepharose bead suspension, washed, and resuspended
in scintillant, and the 32P activity was detected in a scintillation
counter. The compound concentration which inhibits 50% of the
kinase activity was calculated for each compound.
(14) We examined the effect of these compounds on cultured HCT116
cells and determined their effect on cell-cycle progression by the
colorimetric cytotoxcity test using sulforhodamine B (Skehan et
al. J . Natl. Cancer Inst. 1990, 82, 1107-12). HCT116 cells are
cultured in the presence of test compounds at increasing
concentrations. At selected time points, groups of cells are fixed
with trichloroacetic acid and stained with sulforhodamine B
(SRB). Unbound dye was removed by washing, and protein-
bound dye was extracted for determination of optical density.
(15) HCT116 human colon carcinoma cells were injected subcutane-
ously at a concentration of 1 × 107 cells/animal into the inguinal
region of female nude mice. The test drugs were administered
intraperitoneally once a day, every day, for 14 days beginning
seven days following cell injection. On day 15 postdrug admin-
istration, tumors were removed and weighed and the tumor
growth inhibition index (%) was determined according to the
following formula: tumor growth inhibition index (%) ) (1- mean
net tumor weight of experimental group/mean net tumor weight
of control group) × 100.
2.6 (m,
C
1 H), 1.5 (d, 15 Hz, 6 H). CIMS m/e Calcd for
21H20N3O3: 362.1505. Found: 362.1535. 8: mp 283 °C; 1H NMR
(300 MHz, DMSO) 13.6 (s, 1 H), 10.2 (s, 1 H), 8.2 (d, 8 Hz, 1 H),
8.1 (d, 8 Hz, 2 H), 7.5 (t, 8 Hz, 1 H), 7.2 (m, 3 H), 7.05 (d, 8 Hz,
2 H), 6.5 (d, 9 Hz, 2 H), 5.0 (s, 2 H), 3.8 (s, 3 H), 3.2 (s, 2 H).
CIMS m/e Calcd for C25H21N4O3: 425.1614. Found: 425.1643.
(11) Values were measured for these compounds against PKA, PKC,
and c-Abl. All had IC50 values greater than 250 µM.
(12) The solubility of compound 5 limited us from dosing at concen-
trations higher than those presented in Table 2.
(13) The in vitro assays employ cell lysates from insect cells express-
ing either of the kinases and subsequently their corresponding
regulatory units. The cdk/cyclin lysate is combined in a microti-
tre-type plate along with a kinase compatible buffer, 32P-labeled
ATP at a concentration of 50 mM, a GST-Rb fusion protein, and
the test compound at varying concentrations. The kinase reac-
tion is allowed to proceeded with the radiolabled ATP, and then
it is effectively stopped by the addition of a large excess of EDTA
J M0100032