1,7-Annulated indolocarbazoles as cyclin-dependent kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.03.105

The synthesis and kinase inhibitory activity of a series of novel 1,7-annulated indolocarbazoles 6 and 16 is described. These compounds exhibited potent inhibitory activity against cyclin-dependent kinase 4 and good antiproliferative activity in a human colon carcinoma cell line.

Full text of DOI:10.1016/j.bmcl.2004.03.105

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3217–3220
1
,7-Annulated indolocarbazoles as cyclin-dependent
kinase inhibitors
Rima S. Al-awar,a,* James E. Ray, Kyle A. Hecker, Jianping Huang, Philip P. Waid,
a
a
a
a
a
b
b
Chuan Shih, Harold B. Brooks, Charles D. Spencer, Scott A. Watkins,
b
b
Bharvin R. Patel, Nancy B. Stamm, Catherine A. Ogg, Richard M. Schultz,
b
b
b
c
b
c
c
Eileen L. Considine, Margaret M. Faul, Kevin A. Sullivan, Stanley P. Kolis,
a
c
John L. Grutsch and Sajan Joseph
a
Discovery Chemistry Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
b
Cancer Research Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
c
Chemical Process Research and Development, Eli Lilly and Company, Indianapolis, IN 46285, USA
Received 10 January 2004; accepted 31 March 2004
Abstract—The synthesis and kinase inhibitory activity of a series of novel 1,7-annulated indolocarbazoles 6 and 16 is described.
These compounds exhibited potent inhibitory activity against cyclin-dependent kinase 4 and good antiproliferative activity in a
human colon carcinoma cell line.
Ó 2004 Elsevier Ltd. All rights reserved.
The cyclin-dependent kinases (cdks) are important reg-
ulators of the cell cycle. The serine/threonine specific
kinases, cdk2 and cdk4, play an essential role in the
transition from the G1 to S phase. The enzyme activity
of the cyclin-dependent kinases is controlled by their
partners the cyclins, cdk4 has cyclin D1 as a partner
whereas cdk2 is associated with both cyclin E and A in
mid and late G1, respectively. Stimulation of cells by
mitogens leads to induction of cyclin D1, which com-
plexes with cdk4 and phosphorylates and hence inacti-
vates the tumor suppressor gene pRb. The
retinoblastoma protein (Rb) thus releases the E2F
transcription factor, to which it normally binds, leading
to cell cycle progression. This pathway is altered in
many primary tumors such as: colon, rectum, lung,
breast, and prostate. Therefore, a cyclin D1/cdk4
selective, flavopiridol is undergoing clinical evaluation
3
for the treatment of several tumors. During our
screening to identify novel inhibitors of cdk4, we dis-
covered that indolocarbazole 1 (Fig. 2) was a potent
inhibitor of both cdk2 and 4 (IC50 ¼ 0.54 and 0.68 lM,
6
respectively). Therefore, we investigated this class of
molecules, that have long been identified for their bio-
logical activity, as potential inhibitors of the cyclin-
7
dependent kinases. Our initial efforts were focused on
the 1,7-annulated bisindolyl maleimides and their cor-
responding indolocarbazoles that are represented by
analogues 6a–d (Scheme 1).
Synthesis of the precursor bisindolyl maleimides 5, was
8
accomplished using previously described procedures
þ
inhibitor will modulate the growth of Rb tumors and
1
serve as an effective chemotherapeutic agent.
OH
O
O
O
NH
N
Cl
NH
Many structural classes have been found to inhibit
2
N
N
N
3
cyclin-dependent kinases, including the flavones, the
4
HO
HO
HO
5
N
H
paullones, and several purine based molecules, such as
olomoucine and roscovotine (Fig. 1). Although not very
N
H
N
Flavopiridol
Paullones
Olomoucine
*
0
Corresponding author. E-mail: rima@lilly.com
Figure 1.
960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.03.105

3218
R. S. Al-awar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3217–3220
H
N
Preparation of the indole-3-glyoxylate esters of 2a–d and
their condensation with the corresponding indole-3-
acetamides, provided the desired bis-indolylmaleimides
O
O
8
5
a–d. Oxidation of 5a–d to indolocarbazoles 6a–d was
performed in 50–70% yield using Pd(OAc) . Other
conditions previously reported for this transformation
ht with or without I or Pd/C, DDQ with or without
CCF , and CuCl ) were less
2
N
N
O
(
2
1
p-TsOH, PdCl
2
, Pd(O
2
3
)
2
2
effective.
Figure 2.
Indolocarbazoles 6a–d were equally potent against
cyclin D1/cdk4 and therefore the ring size did not
appear to affect the activity (Table 1). All compounds
were more selective for cdk4 versus cdk2 and equally
potent in the HCT-116 cell line with the exception of 6c,
which was 3–5-fold less active. Evaluation of substitu-
tion on the nonannulated indole ring indicated that the
O
O
O
O
R
NH
2
a,b
c,d
N
+
N
H
N
n
n
4
- and 5-positions did not tolerate any substitution
including a small fluoro group as demonstrated by
analogues 6e–i. With the exception of a methyl ester
group, substitution at position 6, contrary to positions 4
and 5, was well tolerated. The fluoro (6j), chloro (6k),
bromo (6l), and trifluoro methyl (6l) groups resulted in
very potent inhibitors of cyclin D1/cdk4 with selectivity
toward cyclin E/cdk2. In addition, they were equally
effective at inducing apoptosis in the HCT-116 colon
carcinoma cell line with activity ranging between 0.8 and
2.4 lM. The 3-pyridyl analogue 6o (IC50 ¼ 0.16 lM) was
about 3-fold more potent than the 4-pyridyl (6p)
2
a-d (n=1-4)
3a-d (n=1-4)
4
H
H
N
O
N
O
O
O
4
R
R 5
e
6
N
n
N
H
N
n
N
H
5a-d (n=1-4)
6a-d (n=1-4)
e-p (n=2)
6
Scheme 1. Reagents and conditions: (a) (COCl) , ether, )78 °C;
t
b) NaOMe, MeOH, 85–90%; (c) KO Bu, THF; (d) concd HCl, 60–
2
(IC50 ¼ 0.59 lM) in both the enzyme and cell based
(
assays.
9
0%; (e) Pd(OAc) , AcOH, D, 50–70%.
2
9
a
9b
Table 1. Enzyme activity (cyclin D1/cdk4 and cyclin E/cdk2) and cell
based inhibition in HCT116 (colon) cell line for some prepared indo-
locarbazoles
from the corresponding annulated indoles (2a, 2b,
2
9c
c, and 2d). The eight-membered annulated indole 2d
was prepared from 7-bromo indole via Stille coupling
with tributylvinyltin togive 8 followed by alkylation of
the indole nitrogen with 5-bromopentene to generate
di-alkene 9. Terminal alkenes 9 underwent ring closing
metathesis when subjected tothe Grubbs catalyst (benz-
ylidene-bis-(tricyclohexylphosphine)dichlororuthenium)
togive intermediate 10. Reduction of the alkene over
hydrogen and platinum provided the saturated eight-
membered ring indole 2d (Scheme 2).
Compds
R
Cyclin
D1/cdk4
Cyclin E/ Cytotoxicity
HCT-116
cdk2 IC50
,
IC50, lM lM
IC50, lM
6a
H
0.24
0.11
0.094
0.12
>2
>1
1.02
1.6
6
6
6
6
6
6
6
6
b
c
d
e
f
H
H
1.0
>2
na
5.16
1.25
na
H
4-F
5,6-Di F
5-F
5-Br
>2
>2
>2
>2
>2
na
na
na
na
g
h
i
na
na
5-CO
6-F
2
Me
na
na
a
b
6j
0.42
>0.2
>0.2
1.0
>2
na
2.37
0.78
2.3
6
6
6
6
6
6
1
k
6-Cl
6-Br
0.11
0.09
0.29
N
H
N
H
N
l
Br
m
n
o
p
4
6-CF
6-CO
3
1.48
na
7
8
9
2
Me
>2
6-(3-Pyridyl)
6-(4-Pyridyl)
na
0.16
0.59
na
na
1.13
3.29
0.34
2.26
0.23
0.17
0.32
0.37
0.6
c
d
0.052
0.17
na
na
N
N
16a
NH ÆHCl
Azetidine
2
1
1
1
1
1
1
1
6b
6c
6d
6e
6f
0.075
0.05
na
na
Pyrrolidine
Piperidine
Morpholine
0.087
0.075
na
na
1
0
2d
Thiomorpholine 0.09
0.065
0.079
na
na
Scheme 2. Reagents and conditions: (a) tributylvinyltin, PdPh
LiCl, PPh
, DMF, 90 °C, 80%; (b) NaH, 5-bromopentene, DMF, 0 °C,
5%; (c) Grubbs catalyst (benzylidene-bis-(tricyclohexylphosphine)-
dichlororuthenium), rt, CH Cl , 80%; (d) H , PtO , EtOH, 96%.
2 2
Cl ,
6g
6h
Piperazine
Diazepine
>10
7.6
3
na
9
2
2
2
2
na ¼ not applicable.

R. S. Al-awar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3217–3220
3219
With the accessibility of position 6 well established, we
tives 16b–h were potent inhibitors of cdk4 with IC50
s
ranging between 0.05 and 0.09 lM. This activity trans-
lated very well intothe cyt toxo icity assay with the
exceptions of 16g and 16h. Although they inhibit the
enzyme at 65 and 79 nM their activity in the HCT116
cell line was >10 and 7.6 lM, respectively. We specu-
lated the diamine salts might have influenced the cell
penetration abilities of these analogues and hence
influenced their antiproliferative activities.
turned our attention to utilizing this pocket to improve
the biopharmaceutical properties of the six-membered
indolocarbazole series. The synthesis of compounds 16
10
began with the condensation of glyoxylate ester 11 and
acetamide 12b togive the bis-ind ol ylmaleimide 13.
11
Indolocarbazole 14 was obtained under previously de-
scribed oxidation conditions and was converted to
benzyl bromide 15 using bromine and triphenyl phos-
phite. Subjecting bromide 15 toa variety of amines re-
sulted in analogues 16a–h with improved aqueous
solubility (Scheme 3).
In conclusion, we have demonstrated that 1,7-annulated
indoles are versatile building blocks for the preparation
of indolocarbazoles that are potent inhibitors of cyclin
D1/cdk4.
Acetamide 12b was prepared from glyoxylate ester 3b
via ketoamide 12a. Treatment of glyoxylate ester 3b
with ammonium hydroxide gave amide 12a in 85% yield,
which was subsequently reduced to 12b over 10% Pd/C
in the presence of sodium hypophosphite under reflux-
References and notes
11
ing conditions (Scheme 4).
1
. (a) Rao, N. R. Curr. Opin. Oncol. 1996, 8, 516; (b) Lingfei,
K.; Pingzhang, Y.; Zhengguo, L.; Jianhua, G.; Yaowu, Z.
Cancer Lett. 1998, 130, 93.
2. (a) Toogood, P. L. Curr. Opin. Chem. Biol. 2002, 6, 472;
(b) Fischer, L.; Endicott, J.; Meijer, L. Progr. Cell Cycle
Res. 2003, 5, 235; (c) Monaco, E. A., 3rd; Vallano, M. L.
Curr. Med. Chem. 2003, 10, 367; (d) Hardcastle, I. R.;
Golding, B. T.; Griffin, R. J. Annu. Rev. Pharmacol.
Toxicol. 2002, 42, 325; (e) Knockaert, M.; Greengard, P.;
Meijer, L. Trends Pharmacol. Sci. 2002, 23, 417.
The hydroxymethyl analogue 14 was about 3-fold
(
IC50 ¼ 0.052 lM) more potent than the amino methyl
derivative 16a (IC50 ¼ 0.17 lM). We found that deriva-
O
O
O
O
NH2
3. (a) Tan, A. R.; Swain, S. M. Seminars Oncol. 2002, 29, 77;
a,b
+
(
b) Filgueira de Azevedo, W., Jr.; Canduri, F.; Freitas da
Silveira, N. J. Biochem. Biophys. Res. Commun. 2002, 293,
66.
N
N
H
OTIPS
5
1
O
1
H
N
12b
H
N
4. (a) Gussio, R.; Zaharevitz, D. W.; McGrath, C. F.;
Pattabiraman, N.; Kellogg, G. E.; Schultz, C.; Link, A.;
Kunick, C.; Leost, M.; Meijer, L.; Sausville, E. A. Anti-
Cancer Drug Des. 2000, 15, 53; (b) Zaharevitz, D. W.;
Gussio, R.; Leost, M.; Senderowicz, A. M.; Lahusen, T.;
Kunick, C.; Meijer, L.; Sausville, E. A. Cancer Res. 1999,
9, 2566.
. (a) Haesslein, J.-L.; Jullian, N. Curr. Top. Med. Chem.
002, 2, 1037; (b) Legraverend, M.; Ludwig, O.; Leclerc,
O
O
O
c
d
N
N
H
N
N
H
OH
OTIPS
5
5
6
13
14
2
H
N
H
N
S.; Meijer, L. J. Heterocycl. Chem. 2001, 38, 299.
. (a) Jackson, J. R.; Gilmartin, A.; Imburgia, C.; Winkler,
J. D.; Marshall, L. A.; Roshak, A. Cancer Res. 2000, 60,
O
O
O
O
e
5
66; (b) Gilmartin, A. G.; Ho, M. L.; Imburgia, C. S.;
Roshak, A. K.; Lago, M. A. WO 0016781 A1 20000330,
2000; (c) Kleinschroth, J.; Schaechtele, C.; Hartenstein, J.;
Rudolph, C. Eur. Patent 410389 A1 19910130, 1991.
. (a) Sampath, D.; Shi, Z.; Plunkett, W. Mol. Pharmacol.
2002, 62, 680; (b) Johnson, L. N.; De Moliner, E.; Brown,
N. R.; Song, H.; Barford, D.; Endicott, J. A.; Noble, M.
E. M. Pharmacol. Therap. 2002, 93, 113; (c) Merchant, J.;
Tutsch, K.; Dresen, A.; Arzoomanian, R.; Alberti, D.;
Feierabend, C.; Binger, K.; Marnoccha, R.; Thomas, J.;
Cleary, J.; Wilding, G. Clin. Cancer Res. 2002, 8, 2193; (d)
Long, B. H.; Rose, W. C.; Vyas, D. M.; Matson, J. A.;
Forenza, S. Curr. Med. Chem.: Anti-Cancer Agent 2002, 2,
N
N
N
N
H
H
Br
R
1
5
16
7
t
Scheme 3. Reagents and conditions: (a) KO Bu, THF; (b) concd HCl,
7
3%; (c) Pd(OAc)
2
, AcOH, D, 81%; (d) (PhO)
3
P, Br
2
, pyridine, DMF,
)
15 °C tort; (e) amine (R), NMP, rt, 55–95%.
O
O
O
O
O
O
2
55; (e) Carrasco, C.; Vezin, H.; Wilson, W. D.; Ren, J.;
NH2
NH2
Chaires, J. B.; Bailly, C. Anti-Cancer Drug Des. 2001, 16,
99; (f) Tolcher, A. W.; Eckhardt, S. G.; Kuhn, J.;
Hammond, L.; Weiss, G.; Rizzo, J.; Aylesworth, C.;
Hidalgo, M.; Patnaik, A.; Schwartz, G.; Felton, S.;
Campbell, E.; Rowinsky, E. K. J. Clin. Oncol. 2001, 19,
a
b
N
N
N
3
b
12a
12b
2
937; (g) Senderowicz, A. M. Oncogene 2000, 19, 6600; (h)
Scheme 4. Reagents and conditions: (a) NH
4
OH, THF, 0 °C tort,
Zhu, G.; Conner, S. E.; Zhou, X.; Shih, C.; Li, T.;
Anderson, B. D.; Brooks, H. B.; Campbell, R. M.;
8
6%; (b) 10% Pd/C, NaH PO , dioxane, H O, D, 95%.
2
2
2

3220
R. S. Al-awar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3217–3220
Considine, E.; Dempsey, J. A.; Faul, M. M.; Ogg, C.;
9. (a) Paudler, W. W.; Shin, H. G. J. Heterocycl. Chem. 1969,
6, 415; (b) van Wijngaarden, I.; Hamminga, D.; van Hes, R.;
Standaar, P. J.; Tipker, J.; Tulp, M. T. M.; Mol, F.; Olivier,
B.; de Jonge, A. J. Med. Chem. 1993, 36, 3693; (c) Neiduzak,
T. R.; Boyer, F. E. Synth. Commun. 1996, 26, 3443.
Patel, B.; Schultz, R. M.; Spencer, C. D.; Teicher, B.;
Watkins, S. A. J. Med. Chem. 2003, 46, 2027; (i) Zhu, G.;
Conner, S.; Zhou, X.; Shih, C.; Brooks, H. B.; Considine,
E.; Dempsey, J. A.; Ogg, C.; Patel, B.; Schultz, R. M.;
Spencer, C. D.; Teicher, B.; Watkins, S. A. Bioorg. Med.
Chem. Lett. 2003, 13, 1231.
10. Glyoxylate ester 11 was prepared in three steps from
methyl indole-6-carboxylate. Reduction of the ester with
8
. (a) Faul, M. M.; Winneroski, L. L.; Krumrich, C. A.
Tetrahedron Lett. 1999, 40, 1109; (b) Faul, M. M.;
Winneroski, L. L.; Krumrich, C. A. J. Org. Chem. 1998,
4
LiAlH , protection of the resulting alcohol as a silyl ether
with TIPSOTf and treatment with oxalyl chloride followed
by NaOMe.
11. Demopoulos, V. J. Synth. Commun. 1989, 19, 2585.
63, 6053.