Synthesis, structure-activity relationship, and biological studies of indolocarbazoles as potent cyclin D1-CDK4 inhibitors

Article abstract of DOI:10.1021/jm0256169

Novel substituted indolocarbazoles were synthesized, and their kinase inhibitory capability was evaluated in vitro. 6-Substituted indolocarbazoles 4 were found to be potent and selective D1/CDK4 inhibitors. 4d and 4h exhibited potent and ATP-competitive D

Full text of DOI:10.1021/jm0256169

J . Med. Chem. 2003, 46, 2027-2030
2027
Syn th esis, Str u ctu r e-Activity
Rela tion sh ip , a n d Biologica l Stu d ies of
In d oloca r ba zoles a s P oten t Cyclin
D1-CDK4 In h ibitor s
Guoxin Zhu,* Scott E. Conner, Xun Zhou,
Chuan Shih, Tiechao Li, Bryan D. Anderson,
Harold B. Brooks, Robert Morris Campbell,
Eileen Considine, J ack A. Dempsey,
F igu r e 1.
Margaret M. Faul, Cathy Ogg, Bharvin Patel,
Richard M. Schultz, Charles D. Spencer,
Beverly Teicher, and Scott A. Watkins
Sch em e 1. Synthesis of the Unsymmetrical
Indolocarbazoles
Lilly Research Laboratories,
A Division of Eli Lilly & Company, Lilly Corporate Center,
Indianapolis, Indiana 46285
Received November 21, 2002
Abstr a ct: Novel substituted indolocarbazoles were synthe-
sized, and their kinase inhibitory capability was evaluated in
vitro. 6-Substituted indolocarbazoles 4 were found to be potent
and selective D1/CDK4 inhibitors. 4d and 4h exhibited potent
and ATP-competitive D1/CDK4 activities with IC50 values of
76 and 42 nM, respectively. Both compounds had high
selectivity against the other kinases. These D1/CDK4 inhibi-
tors inhibited tumor cell growth, arrested tumor cells at the
G1 phase, and inhibited pRb phosphorylation.
a
(a) KOtBu, THF, room temperature; (b) DDQ, solvent, p-TsOH,
In tr od u ction . Cancer has been recognized as a
disease of uncontrolled cell proliferation. Consequently,
the genes that regulate cell proliferation have been the
targets of cancer chemotheraphy. The cell cycle is a
series of highly regulated processes that result in the
duplication of a cell.1 Much evidence suggests that cell
cycle progression is controlled by the sequential activa-
tion of a series of cyclin-dependent kinases (CDKs). This
family of serine/threonine protein kinases is composed
of a catalytic subunit (CDK) that is present throughout
the cell cycle and an activating subunit (cyclin) that is
present only at specific stages of the cell cycle.2 The
importance of CDKs in cell cycle regulation and the
frequent documented deregulation of CDKs and their
modulators in cancer have stimulated great research
efforts in this area.3 In particular, the D type cyclins,
D1, D2, and D3, associated with CDK 4 and 6 are
believed to play a critical role early in the G1 phase of
the cell cycle. These complexes phosphorylate the ret-
inoblastoma protein and inactivate its ability to act as
a transcriptional repressor in a complex with E2F.4a
Thus, inhibitors of these cyclin-dependent kinases that
stop uncontrolled tumor cell growth are expected to be
promising new therapeutic agents for the treatment of
cancer.
reflux, or I2, benzene, hν.
against CDK1 and/or CDK2.7-17 Owing to recent strong
evidence of the link between D1/CDK4 activity and
many types of tumors, there is significant interest in
searching for selective CDK4 inhibitors. However, a few
selective CDK4 inhibitors have recently been reported
in the literature.18-25
High-throughput screening of the Lilly compound
collection against the cyclin D1/CDK4 enzyme complex
yielded several interesting scaffolds, including SB-
210878 (Figure 1), with an IC50 of 0.54 µM in the D1/
CDK4 kinase assay. Structurally, SB-210878 belongs to
the indolocarbazole family, and subsequent testing of
other indolocarbazoles indicated that the naturally
occurring arcyriaflavin A (Figure 1) also showed moder-
ate inhibitory activity against D1/CDK4. Because of our
interest in obtaining selective and potent CDK4 inhibi-
tors, we began a medicinal chemistry approach around
this lead with the help of a molecular modeling study
using a CDK4 homology model. In this report, we
discuss the synthesis and structure-activity relation-
ships of unsymmetrical substituted indolocarbazoles as
D1/CDK4 inhibitors.
Ch em istr y. The general synthetic approach to the
unsymmetrically substituted indolocarbazole compounds
4 is outlined in Scheme 1. The key step in the synthesis
of these compounds involved the cyclization of bisin-
dolylmaleimide 3 to the corresponding indolocarbazole
4. This was accomplished by DDQ-mediated oxidative
cyclization in the presence of a catalytic amount of
p-toluenesulfonic acid using benzene, toluene, or diox-
ane as solvent.26 Alternatively, the cyclization of 3 could
be carried out under photochemical conditions using
During the past decade, several scaffolds were dis-
covered with inhibitory activity against CDK.4 Of these
small-molecule CDK inhibitors, three compounds (fla-
vopiridol,5 UCN-01,6a and roscovitine6b) have entered
into clinical development as cancer therapeutics. De-
spite the disclosure of many potent CDK inhibitors in
the literature, most of them are potent inhibitors
* To whom correspondence should be addressed. Phone: 317-433-
3697. Fax: 317-277-2035. E-mail: Zhu_Guoxin@lilly.com.
10.1021/jm0256169 CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/19/2003

2028 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Letters
Ta ble 1. Effect of Bromine Substitution on D1/CDK4 Activity29
Ta ble 2. Kinase Inhibitory Activity of 6-Substituted
and Kinase Selectivity
Indolocarbazoles
IC50 (µM)
D1-CDK4 E-CDK2 B-CDK1
CamKII
PKA
RB21 RB21 histone autocamtide histone
4a
4b
4c
4d
4e
H
0.16 0.53
1.1
0.3
19.4
2.1
>20.0
<0.125
0.058
<1.25
12.4
>2.0
IC50 (µM)
4-Br
5-Br
6-Br
7-Br
0.08
4.4
0.22
4.3
0.103
>20.0
>20.0
>20.0
compd
D1-CDK4 E-CDK2
CamKII
autocamtide histone
PKA
4
X
R1 R2
Y
RB21
RB21
0.076
16.2
0.52
19.4
11.1
4f
Br
H
H
H
H
H
H
H
H
Me
H
Me
H
H
H
H
H
H
H
H
H
H
0.074
0.103
0.042
0.072
0.226
1.135
0.134
0.63
nt
0.174
0.092
>2.0
0.914
0.082
>2.0
0.236
>2.0
>2.0
>2.0
>2.0
>2.0
>2.0
>2.0
>2.0
>2.0
4g
4h
4i
4j
4k
4l
F
F
0.141
0.144
0.278
>1.0a
>2.0a
2.287a
>2.0a
iodine as an oxidant in a benzene/methanol solvent
combination.27 3 was prepared in good yield via base-
promoted condensation of an appropriately substituted
indolyl 3-acetamide 1 and an indolyl 3-glyoxylate 2 in
the presence of KOtBu in THF based on Faul’s proce-
dure.28 In some cases, dehydration of the hydroximide
intermediates was slow under basic conditions, so
concentrated hydrochloric acid was added to accelerate
the dehydration of this intermediate. Both 1 and 2 are
readily available by a simple transformation from a
variety of commercially available indoles following
literature procedures.28
Cl
Me
CF3
OMe
Br
H
H
4m
OMe
a
RbING as substrate.
Ta ble 3. Cellular Activity for Substituted Indolocarbazoles
compd
4a
4b
4d
4f
4g 4h
4i
4j
4k
4l 4m
HCT-116
IC50 (µM)
NCI-H460
IC50 (µM)
0.85 0.26 2.23 2.36 3.53 0.76 2.47 4.54 1.65 1.79 2.64
0.59 0.71 2.76 1.23 2.60 0.53 1.62 4.22 1.35 1.18 1.78
Resu lts a n d Discu ssion . Selective In h ibition of
D1/CDK4 by In d oloca r ba zole An a logu es. The initial
medicinal chemistry effort was focused on evaluating
the effect of substitution at the different positions of the
indole in the indolocarbazole on the D1/CDK4 inhibitory
activity as well as selectivity against the other kinases.
The D1/CDK4 enzyme inhibitory activities of indolocar-
bazoles 4 was determined by measuring the phospho-
rylation of Rb protein.29 These compounds were also
tested in other kinase inhibitory assays by measuring
the phosphorylation of the corresponding substrates
(e.g., E-CDK2 using Rb21 as substrate, B-CDK1 and
PKA using histone as substrates, CamKII using auto-
camtide as substrate, and GSK3â using pCREB as
substrate). Staurosporine, the well-known kinase in-
hibitor, was used as a standard compound for the
assays. Table 1 summarizes the enzyme inhibitory
activity of indolocarbazoles substituted by bromine at
the different indole positions. 4c and 4e with bromine
at the 5- and 7-position, respectively, of one indole led
to poor inhibitory activity against D1/CDK4. While
bromine at the 4- and 6-position of the indole in the
indolocarbazole are well tolerated for D1/CDK4 inhibi-
tory activity, both 4b and 4d showed a 2-fold activity
increase against D1/CDK4 compared to parent indolo-
carbazole, arcyriaflavin A. Besides the potent activity
against D1/CDK4, compound 4b with a bromine at the
4-position of an indole was also very potent against
other kinases (e.g., IC50 of 220 nM for E-CDK2, 300 nM
for B-CDK1, 58 nM for CamKII, and 103 nM for PKA).
However, 4d with a bromine at the 6-position of an
indole was significantly more selective against other
kinases tested; 4d was 7-fold less active against cyclin
E/CDK2. Similar trends were also found when fluorine
or chlorine was used instead of bromine as the substi-
tuted group on one of the indoles. 4a -e were found to
be inactive against GSK3â at 20 µM. Thus, substitution
on the 5- or 7-positions of the indolocarbazole decreases
D1/CDK4 inhibitory activity and the indolocarbazole
analogues with a substitution at the 4- or 6-position
improve D1/CDK4 activity. However, 6-substituted in-
dolocarbazoles gave better kinase selectivity than the
corresponding 4-substituted analogues.
After finding out that the 6-bromo-substituted indolo-
carbazole 4d had the most potent D1/CDK4 enzyme
inhibitory activity with the desired selectivity profile
within the kinase panel tested, we conducted an SAR
study on the 6-position of the indole by introducing
different functional groups at this position. The enzy-
matic kinase activities of these 6-substituted indolocar-
bazoles are summarized in Table 2. As shown in Table
2, a variety of substituents at the 6-position of the indole
was well tolerated for maintaining D1/CDK4 activity;
all of them exhibited potent D1/CDK4 inhibitory activ-
ity. In general, these compounds were more potent
against D1/CDK4 compared to E/CDK2 and PKA.
However, the selectivity against CamKII was more
sensitive to the nature of the substituent group. Sub-
stituting the 6-position of one indole in the carbazoles
with fluorine (4g), methyl (4j), or methoxy (4l) led to
potent CamKII activity. By introducing a methyl at one
of the indole nitrogen R2 positions, we were able to
improve the potency of D1/CDK4 and selectivity against
CamKII (4g vs 4h in Table 2). A combination of 6-bromo
and 6′-methoxy substitution (4m ) was also evaluated.
Although D1/CDK4 activity was diminished, better
selectivity against other panel kinases was observed.
4f-m were found to be much less active against GSK3â
compared with their inhibitory activity against D1/
CDK4. They were found to be inactive against GSK3â
at 5 µM.
Kinetic studies showed that 4d ,g,i,l exhibited pure
competitive inhibition with respect to ATP; thus, certain
6-substituted indolocarbazoles were potent and selective
ATP competitive inhibitors of D1/CDK4.
In h ibition of Tu m or Cell P r olifer a tion a n d Ef-
fect on Cell Cycle a n d p R b P h osp h or yla t ion . In
addition to the kinase activity discussed above, the
synthesized indolocarbazoles were also studied for their

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2029
Ta ble 4. pRb Phosphorylation Inhibition by Indolocabazolesa
compd
4b
4f
4g
4h
4i
4k
4l
4m
HCT-116 IC50 (µM)
% pRb inhibition @ IC50
% pRb inhibition @2 IC50 56
0.26 2.36 3.53 0.76 2.47 1.65 1.79 2.64
33
86
85
66
89
82
86
72
92
82
97
66
82
85
93
a
Colon carcinoma cells (HCT-116) treated with compounds at
1× and 3× antiproliferation IC50 concentration for 24 h.
noma cells (HCT-116) cells were treated with com-
pounds at 1× and 3× antiproliferation IC50 concentra-
tions for 24 h, followed by the Western blot analysis.
As shown in Table 4, these compounds potently inhib-
ited phosphorylation of RbS780, demonstrating that they
inhibit cellular CDK4 activity. These specific observa-
tions of the G1 cell cycle arrest and inhibition of
phosphorylation of serine 780 on pRb indicate that these
indolocarbazoles are potent CDK4 inhibitors.
Finally, selected compounds were studied in the
human colon carcinoma (HCT-116) xenograft to evalu-
ate their antitumor effects. These compounds caused a
certain degree of tumor growth delay in this model (data
not included). These results indicated that these small
molecular D1/CDK4 inhibitors might have therapeutic
potential in cancer treatment.
In summary, we have disclosed 6-substituted indolo-
carbazoles representing a new class of D1/CDK4 inhibi-
tors. These compounds are selective and ATP competi-
tive D1/CDK4 inhibitors and are capable of inhibiting
cell growth in the human tumor cell lines HCT-116 and
NCI H460. In addition, the specific G1 cell cycle arrest
and selective inhibition of phosphorylation of serine 780
on pRb are consistent with the in vitro D1/CDK4
inhibitory activity.
F igu r e 2. Cell cycle analysis of the cell lines treated for 24 h
with D1/CDK4 inhibitors 4d and 4h : (red) G1; (white) S; (blue)
G2/M.
antiproliferative activity in a variety of tumor cell
lines.30 The properties of these indolocarbazole ana-
logues in in vitro cellular activities in human colon
carcinoma (HCT-116) and non-small-cell lung carcinoma
(NCI-460) are shown in Table 3. Indolocarbazole ana-
logues 4 inhibited cell growth in the HCT-116 and NCI-
H460 tumor cell lines with IC50 values from 0.26 to 4.54
µM.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails for the assays and the final compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
It is clear that CDK4 plays a critical role in the G1-S
transition of the cell cycle by phosphorylating Rb. It is
also known that the Ser-780 residue on Rb is specifically
phosphorylated by CDK4.31 Thus, inhibition of cellular
CDK4 activity will result in the inhibition of Rb phos-
phorylation on Ser-780 and cell cycle arrest in the G1
phase. To investigate whether these CDK4 inhibitors
will produce G1 cell cycle arrest and inhibition of Rb
phosphorylation, we evaluated the effects of these
compounds by cell cycle analysis and a phosphorylation
assay in tumor cells.
A cell cycle inhibition study was conducted by treating
HCT-116 and NCI-460 cells with different concentra-
tions of the CDK inhibitors for 24 h and then analyzing
them by flow cytometry. As exemplified in Figure 2,
treatment of the tumor cells with these indolocarbazole
D1/CDK4 inhibitors results in a significant accumula-
tion of cells in the G1 population and decreases the S
and G2/M populations in a dose-responsive manner. In
most cases, G1 arrest occurred in the same concentra-
tion range as that for the corresponding cell growth
inhibition (cellular inhibition IC50).
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