Novel, potent and selective cyclin D1/CDK4 inhibitors: Indolo[6,7-a]pyrrolo[3,4-c]carbazoles

Article abstract of DOI:10.1016/S0960-894X(03)00461-X

The synthesis and CDK inhibitory properties of a series of indolo[6,7-a]pyrrolo[3,4-c]carbazoles is reported. In addition to their potent CDK activity, the compounds display antiproliferative activity against two human cancer cell lines. These inhibitors also effect strong G1 arrest in these cell lines and inhibit Rb phosphorylation at Ser780 consistent with inhibition of cyclin D1/CDK4.

Full text of DOI:10.1016/S0960-894X(03)00461-X

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2261–2267
Novel, Potent and Selective Cyclin D1/CDK4 Inhibitors:
Indolo[6,7-a]pyrrolo[3,4-c]carbazoles
Thomas A. Engler,a,* Kelly Furness,a Sushant Malhotra,a Concha Sanchez-Martinez,b
Chuan Shih,a Walter Xie,a Guoxin Zhu,a Xun Zhou,a Scott Conner,a Margaret M. Faul,c
Kevin A. Sullivan,c Stanley P. Kolis,c Harold B. Brooks,a Bharvin Patel,a
Richard M. Schultz,a Tammy B. DeHahn,a Kashif Kirmani,a Charles D. Spencer,a
Scott A. Watkins,a Eileen L. Considine,a Jack A. Dempsey,a Catherine A. Ogg,a
Nancy B. Stamm,a Bryan D. Anderson,a Robert M. Campbell,a Vasu Vasudevana
and Michelle L. Lytlea
aLilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
bLilly Spain S.A., Avda de la Industria 30, 28108 Alcobendas, Madrid, Spain
cChemical Process Research and Development, Eli Lilly and Company, Indianapolis, IN 46285, USA
Received 10 February 2003; revised 2 May 2003; accepted 5 May 2003
Abstract—The synthesis and CDK inhibitory properties of a series of indolo[6,7-a]pyrrolo[3,4-c]carbazoles is reported. In addition
to their potent CDK activity, the compounds display antiproliferative activity against two human cancer cell lines. These inhibitors
also effect strong G1 arrest in these cell lines and inhibit Rb phosphorylation at Ser780 consistent with inhibition of cyclin D1/
CDK4.
# 2003 Elsevier Science Ltd. All rights reserved.
Cell cycle progression leading to cell division and pro-
liferation is strictly controlled by the timed expression
of various cyclins which associate with specific cyclin-
dependent kinases (CDKs).1,2 For example, passage
through the G1 phase of the cell cycle is regulated by
cyclin D1 and CDK4/6. These kinases regulate the
release of the E2F family of transcription factors
through hyperphosphorylation of the retinoblastoma
tumor suppressor protein (Rb). Cyclin D1/CDK4/6 is in
turn controlled by the p16 family of tumor suppressor
genes. Aberrations of the Rb signalling pathway are
frequent in human cancers.3 Such aberrations include
deletion or mutation of the Rb gene, mutations in p16
or CDK4, or overexpression or upregulation of cyclin
D1 and/or CDK4. Another regulator of cyclin D1 is
b-catenin and elevated levels of this protein are also
associated with many cancers.4 For these reasons,
inhibitors of D1/CDK4,2 particularly selective inhib-
itors,5 have attracted much attention recently as poten-
tial therapeutic agents.
A variety of CDK inhibitors have been reported2,5,6
including the indolo[2,3-a]carbazoles 1a, 1b, 2 and rela-
ted structures (Fig. 1).7 We have been interested in
exploring novel analogues of these indolocarbazoles in
which one indole moiety is replaced by different aryl
groups.6 Herein we report the activity of indolo[6,7-
a]pyrrolo[3,4-c]carbazoles 3 as a new class of potent and
selective inhibitors of cyclin D1/CDK4.
Indolocarbazoles 3 were prepared as shown in Scheme 1.
Condensation of indole-7- or indole-3-acetamides (6
and 9) with indole-3- or 7-glyoxylates (7 and 8) accord-
ing to the procedure reported by Faul8 gave maleimides
10 that were cyclized photochemically9 in the presence
of DDQ or iodine to give 3. Indole-7-acetamides 6
were prepared from commercially available indole-7-
carboxaldehyde 4 via a modification10c of the Kurihara
*Corresponding author. Tel.: +1-317-433-2885; fax: +1-317-276-
1177; e-mail: engler_thomas@lilly.com
0960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00461-X

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T.A.Engler et al./ Bioorg.Med.Chem.Lett.13 (2003) 2261–2267
nitrile with Br(CH2)3OAc (NaH/DMF) followed by
hydrolysis with KOH/t-BuOH.
The kinase inhibitory activities of carbazoles 3 against
cyclin D1/CDK4 and cyclin E/CDK2 were evaluated in
enzymatic assays by measuring phosphorylation of
RbING and Rb21 according to standard protocols
(Table 1).13,14 Lower IC50’s were consistently observed
with the more physiologically relevant Rb21 substrate13
compared to RbING. Inhibition of PKA (histone) was
also measured to preliminarily assess selectivity. In
addition, effects on cell proliferation in vitro were
determined in two human carcinoma cell lines, HCT-
116 (colon) and NCI-460 (lung).15
The data in Table 1 reveals that the parent compound
3a is a potent CDK inhibitor and displays anti-
proliferative activity. Methyl or ethyl groups are toler-
ated at N-5 (3b, c), but some loss in cellular potency is
found with the latter; larger substituents at this position
are significantly less active (>1 mM CDK4 IC50, data
not shown). Fluorine substitution at C-9 and C-10 gives
compounds 3d and 3h with good enzymatic activity, but
lacking antiproliferative properties. Significant decrea-
ses in enzymatic potency are observed when other sub-
stituents were introduced at C-9/C-10 (3e–g, i–l,
respectively). The position tolerating the widest range of
substituents in terms of CDK inhibition is C-11 (3m–w),
and to a lesser extent C-12 (3z–3cc). However, a number
of these compounds do not exhibit activity in cells,
likely due to their poor aqueous solubility. Addition of
hydroxyalkyl groups to C-12 or N-13 (3bb–cc, 3kk–oo)
gives potent inhibitors and cellular activity is retained
provided that hydrogen is present at C-1. Kinetic ana-
lysis of a number of the inhibitors, 3v, 3bb, 3ll revealed
them to be reversible ATP-competitive inhibitors (data
to be published elsewhere).
Figure 1. Chemical structures for (+)-Staurosporine (1a), UCN-01
(1b), arcyriaflavin A (2) and indolo[6,7-a]pyrrolo[3,4-c]carbazoles (3).
procedure,10 and indole-7-glyoxylates 8 were produced
from 7-bromoindoles 5a–e. Bromoindole 5a was com-
mercially available and 5b–e were produced from the
corresponding 2-bromoanilines by the method of Suga-
sawa.11 Indole-3-glyoxylates 7a were prepared from the
corresponding indoles by reaction with oxalyl chloride
followed by treatment with NaOMe; indoleglyoxylate
7b was prepared by alkylation of indole with
Br(CH2)3OTBDMS (NaH/DMF) followed by glyoxyl-
ation. The 7-(2-hydroxyethyl)indole precursor to 7c was
prepared via a Bartoli synthesis12 using the TBDMS-
ether of 2-nitrophenyl-ethanol. Indole-7-carbox-
aldehyde was converted into 7d by the sequence (i)
Wadworth–Emmons
reaction
with
(EtO)2-
Compounds bearing substituents at C-1 and C-2 have
also been examined (3dd–3jj). Good enzymatic and cel-
lular activity is found for those incorporating a sub-
stituent at C-1 (3dd–3jj). However, activity is
significantly lowered with C-1 and N-13 di-substitution
(3qq–3ss). In the latter, one of the two substituents is
likely forced out of plane due to steric interaction with
P(O)CH2CO2Et (KOtBu/THF), (ii) hydrogenation (Pd/
C, THF/MeOH), (iii) LAH reduction (THF) and (iv)
TBDMSCl, imidazole (THF) to give the TBDMS-ether
of 7-(3-hydroxypropyl)indole followed by glyoxylation
as described above. N-(3-hydroxypropyl)-indole-3-acet-
amide 9b was prepared by alkylation of indole-3-aceto-
.
Scheme 1. (a) NaH, DMF, (MeO)2SO2 or EtI; (b) (i) (EtO)2P(O)CN, [LiCN THF], THF; (ii) tBuOH; (iii) Sml2; (c) KOH, tBuOH; (d) tBuLi, THF,
ꢀ78 ꢁC, MeO2CCO2Me; (e) KOtBu, DMF or THF; (f) hn, DDQ or I2, dioxane or EtOAc.

T.A.Engler et al./ Bioorg.Med.Chem.Lett.13 (2003) 2261–2267
2263
the other making it difficult to fit into the narrow ATP
binding pocket of the enzyme.
Synthesis of the aminoalkyl-indolocarbazoles 11/12 is
shown in Scheme 2. Conversion of (hydroxyalkyl)
indolo-maleimides 15 into carbazoles 11/12 was effected
in one of three ways. (1) The hydroxyalkyl side chain in
15 was converted into a bromoalkyl group followed by
oxidative photocyclization to bromoalkylcarbazole 17.
Reaction of 17 with a variety of amines afforded 11/12.
(2) Alternatively, amine displacement on 16 to give
aminoalkylmaleimides 18 could be effected prior to the
photocyclization to 11/12. (3) Finally, oxidative photo-
cyclization of 15 produced hydroxyalkyl-carbazole 3
that could then be subjected to hydroxy to bromide
exchange followed by amine displacement to yield 11/
12. Tactically, the first route (15!16!17!11/12) was
favored due to the ease of handling 16/17 relative to
the other intermediates (higher solubility in organic
solvents and ease of purification). Similar transforma-
Although in most cases there appears to be a slight
selectivity for CDK4 versus CDK2, the carbazoles 3
inhibit both kinases potently. However, they exhibit
good selectivity with respect to PKA.
The aqueous solubility of indolocarbazoles 3 was poor;
for example, aqueous solubility of 3v at various pHs
was <1 mg/mL. Data in Table 1 established that large
substituents [e.g., (CH2)3OH] are well tolerated at N-13
and C-12. For further SAR, we chose to focus on
introducing groups to these two positions to improve
aqueous solubility of the platform. A summary of the
aminoalkyl-substituted indolocarbazoles studied is
shown in Figure 2.
Table 1. Kinase inhibitory activity (IC50, mM) against D1/CDK4, E/CDK2 and PKA, and antiproliferative activity (IC50, mM) of indolocarbazoles
1–3
Compd
R5
R13
X
Z
CDK4
(RbING
CDK4
(Rb21
CDK2
(RbING
)
PKA
HCT-116
H460
)
)
a
1a
2
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
3p
3q
3r
3s
3t
3u
3v
3w
3x
3y
0.059
0.140
0.036
0.047
0.059
0.071
0.171
0.155
>2
0.084
>2
>2
0.393
0.519
0.038
0.038
0.076
0.029
0.048
0.101
0.037
0.033
0.067
0.053
0.044
0.187
0.123
0.093
0.062
0.082
0.168
0.028
0.067
0.032
0.054
0.050
0.042
0.092
0.066
0.096
0.134
0.047
0.054
0.092
0.673
—
—
—
—
>2
>2
>2
>20
>20
>20
—
0.85
1.44
2.11
4.52
>2
4.71
>10
>10
>10
—
—
0.59
0.163
0.022
0.022
0.019
0.081
0.136
0.216
—
0.897
0.064
0.075
—
>2
2.86
>2
—
>0.2
—
H
CH3
CH2CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
9-F
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.43
1.17
>10
>10
3.52
>10
>10
>10
—
7.85
3.35
>10
>10
0.72
3.51
>10
9-Br
9-CH2OH
9-OCH3
10-F
—
>20
0.039
—
—
10-Br
—
—
4.06
10-CF3
10-CH3
10-OCH3
11-F
—
7.04
2.87
>10
>10
1.08
>10
>10
3.84
3.51
0.17
0.67
2.16
1.80
2.35
>10
6.52
17.1
1.00
1.13
3.02
1.98
3.49
1.90
1.13
0.58
1.43
8.78
0.75
1.36
1.20
4.07
1.62
4.70
3.85
>10
2.39
0.407
0.680
0.011
<0.013
0.009
0.011
0.018
0.022
0.017
0.009
0.036
0.006
0.010
0.378
0.025
—
0.040
0.075
0.040
0.002
0.016
0.018
—
0.006
0.007
0.025
0.012
0.042
0.090
0.024
0.012
0.045
—
1.07
>2
0.009
0.075
0.225
0.010
0.320
0.360
0.006
—
0.125
0.112
0.324
—
0.328
—
>20
>20
>20
>20
>20
>20
>20
0.949
2.35
11.4
>20
>20
11-Br
11-CF3
11-CN
11-CH3
11-CH2CH3
11-OH
11-OH
11-OCH3
11-OCH3
11-OCH2CH3
11-OBn
11-OBn
12-Br
3.41
7.49
0.23
1.08
1.92
1.09
3.20
CH3
H
CH3
CH3
H
—
3.29
—
>10
>10
13.8
2.71
—
2.35
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
CH3
CH2CH3
CH3
CH3
CH3
CH3
CH3
CH3
3z
3aa
3bb
3cc
3dd
3ee
3ff
3gg
3hh
3ii
3jj
3kk
3ll
3mm
3nn
3oo
3pp
3qq
3rr
3ss
12-OCH3
12-(CH2)2OH
12-(CH2)3OH
H
>2
>20
0.210
0.558
0.070
0.086
0.067
0.035
0.023
0.057
>2
>20
>20
>20
—
13.2
>20
>20
>20
>20
>2
1-CH3
1-F
1-OCH3
2-OCH3
1-CH3
1-CH3
1-CH3
H
H
H
H
H
1-CH3
1-CH3
1-CH3
1-F
2.26
2.90
0.69
0.71
1.02
1.30
2.42
1.07
1.13
1.77
1.32
0.50
H
H
H
11-F
11-OCH3
11-CF3
H
H
H
H
>2
(CH2)3OH
(CH2)3OH
(CH2)3OH
(CH2)2OH
(CH2)3OH
CH3
(CH2)3OH
(CH2)4OH
(CH2)3OH
0.008
0.067
0.400
0.101
0.113
0.267
—
H
H
5.31
11-OCH3
11-OCH3
H
1.62
1.04
3.90
—
11.7
—
>10
4.75
>10
2.48
H
H
H
>2
0.346
—
0.448
—
—
aNot tested.

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T.A.Engler et al./ Bioorg.Med.Chem.Lett.13 (2003) 2261–2267
tions employing 7-(o-hydroxyalkyl)-indolomaleimides
19 afforded the C-12 substituted carbazoles 13/14
(Scheme 3).
of 14m again indicated a reversible ATP-competitive
inhibitor (data to be published elsewhere). Compared to
the indolo[6,7-a]pyrrolo[3,4-c]carbazoles lacking ami-
noalkyl side chains, 11–14 were significantly more
selective with respect to E/CDK2, with many showing
>20–40-fold selectivity and some >40-fold (13a, 12d,
14l). Interestingly, the scaffolds bearing amine h usually
showed the greatest inhibition of CDK2. Regarding
inhibition of other kinases, 11–14 displayed typically
little activity for PKA. Similarly, most 11–12 exhibited
only modest inhibition of CaMKII. In contrast, the
C-12 substituted compounds 13 were more potent inhi-
bitors of CaMKII and the homologous analogues 14
were somewhat less active against this kinase. However,
considering the higher Km of ATP for CDK4 relative to
these other kinases (>10-fold),16 the intracellular selec-
tivity (mM ATP) is likely considerably higher than in
vitro IC50s suggest. Compound 14m was chosen for
further scrutiny against a diverse set of kinases. IC50s
against GSK-3b (>20 mM), MLK-7 (9.97 mM), TGF-b
RII (>20 mM), p38a-MAPK (20.2 mM) and AKT-1
(5.48 mM) indicated comparatively little activity. Addi-
tional profiling against a wider range of both serine/
threonine and tyrosine kinases (Table 3) showed little
inhibition at inhibitor concentrations of 500 nM except
for Blk and Lyn. For the latter, only modest inhibition
was observed at 100 nM 14m.
Indolocarbazoles 11–14 were all potent inhibitors of
D1/CDK4, with the C-12 substituted analogues
demonstrating the highest activity, as indicated most
strikingly by the Rb21 data (IC50sꢂ69 nM). Repre-
sentative data is presented in Table 2. Kinetic analysis
Most indolocarbazoles 3, and 11–14 were also anti-
proliferative agents (Tables 1 and 2).15 Cell cycle effects
in HCT-116 cells of a number of these compounds were
then examined by flow cytometry (Table 4),17 and
inhibition of Rb phosphorylation at the CDK4 specific
Ser780 residue18 was investigated by Western Blot ana-
lysis. These studies showed significant G1 cell cycle
arrest and strong inhibition of Rb phosphorylation,
consistent with inhibition of cyclin D1/CDK4.14c
Figure 2. Aminoalkyl-substituted indolo[6,7-a]pyrrolo[3,4-c]carbazoles.
Scheme 2. (a) PPh3, CBr4, DMF or CH2Cl2: THF; (b) hn, DDQ or I2, dioxane or EtOAc; (c) NHR2, DMF, 65 ꢁC.

T.A.Engler et al./ Bioorg.Med.Chem.Lett.13 (2003) 2261–2267
2265
A primary driver for the SAR summarized in Figure 2
was to improve the aqueous solubility of the indolo-
carbazole platform. Several of these aminoalky-sub-
stituted indolocarbazoles were converted in situ to their
methanesulfonate salts in D5W. Solubilities of >6 mg/
mL were attained and these solutions were stable for
more than one week at room temperature (Table 4).
This compares very favorably with solubilities found
with non-basic analogues (ꢂ1 mg/mL).
In summary, the discovery and SAR of a new series of
potent CDK inhibitors based on the indolo[6,7-a]pyr-
rolo[3,4-c]carbazole scaffold is described. Simple deriva-
tives are potent inhibitors of both cyclin D1/CDK4 and
cyclin E/CDK2, but generally inactive or only modestly
active against PKA. However, attachment of aminoalkyl
substituents significantly improves the CDK4 inhibitory
activity relative to CDK2 and dramatically increases
aqueous solubility. In vitro characterization reveals that
the antiproliferative activity exhibited by these agents is
consistent with inhibition of cyclin D1/CDK4.
Scheme 3. (a) PPh3, CBr4, DMF or CH2Cl2: THF; (b) hn, DDQ or I2,
dioxane or EtOAc; (c) NHR2, DMF, 65 ꢁC.
Table 2. Kinase inhibitory activity (IC50, mM) against D1-CDK4, E-CDK2, CaMKII and PKA and antiproliferative activity (IC50, mM) of
representative indolocarbazoles 11-14
Compd
n
NR2
CDK4
(RbING
CDK4
(Rb21
CDK2
(RbING
)
PKA
CaMKII
HCT-116
H460
)
)
11e
11f
11h
11m
2
2
2
2
e
f
h
0.084
0.135
0.035
0.063
0.023
0.033
0.021
0.023
0.942
0.928
0.089
0.902
>20
>20
3.74
18.0
6.12
>20
2.69
3.74
1.47
0.89
3.80
2.47
1.31
1.05
0.527
1.17
m
12b
12d
12g
12h
12j
3
3
3
3
3
3
b
d
g
h
j
0.077
0.048
0.095
0.072
0.069
0.190
0.013
0.004
0.014
0.028
0.021
0.047
1.00
2.10
1.23
0.432
0.716
1.26
3.38
3.05
3.13
11.60
0.732
>20
1.96
1.26
0.45
1.55
1.69
2.02
2.44
1.13
0.90
1.28
1.63
2.11
2.81
2.93
3.15
>20
>20
12k
k
>20
13a
13b
13c
13d
13e
13f
13h
13j
13k
13m
2
2
2
2
2
2
2
2
2
2
a
b
c
d
e
f
h
j
0.047
0.051
0.049
0.045
0.039
0.045
0.033
0.054
0.082
0.053
0.013
0.005
0.015
0.008
0.023
0.011
0.010
0.015
0.024
0.005
0.972
0.796
2.03
0.978
0.685
1.06
0.265
1.28
1.80
>20
5.89
12.9
0.081
0.025
0.301
0.046
0.090
0.062
0.095
0.177
0.403
0.049
>10
1.00
1.04
0.20
0.42
2.12
0.17
1.63
3.41
1.23
>10
1.35
1.11
0.14
0.21
2.47
0.33
1.91
3.41
2.82
2.99
4.58
9.14
6.12
>20
>20
9.81
k
m
0.450
14g
14h
14j
14l
14m
3
3
3
3
3
g
h
j
l
m
0.052
0.040
0.056
0.034
0.049
0.035
0.019
0.043
0.024
0.019
1.67
0.567
1.33
1.50
1.03
16.6
8.31
>20
14.1
2.93
0.382
0.266
0.516
0.174
0.216
1.38
0.77
2.82
0.95
1.05
1.48
1.10
1.68
0.37
1.67
Table 3. Kinase Profiling of 14m against 39 kinases; data is presented as activity (% control) in the presence of 100/500 nM of inhibitora,b
Kinase
% Control
Kinase
% Control
Kinase
% Control
c-Raf
112/107
95/87
103/90
100/98
107/109
92/90
91/84
86/70
99/88
104/65
94/84
ROCK-II
CK2
Lck
PRAK
Fyn
ZAP-70
CHK2
PRK2
PKCbII
PKCg
Blk
90/62
99/94
98/60
86/50
92/74
134/107
84/53
97/88
96/59
105/84
74/14
108/102
89/63
CDK5/p35
CK1
CSK
IKKb
Lyn
PKCy
Syk
p70S6K
CHK1
AMPK
JNK3
94/62
98/89
104/116
120/106
78/18
82/58
94/68
93/85
86/70
98/95
MEK1
MAPK2
MKK6
SAPK3
MSK1
MKK4
MKK7b
JNK1a1
PDK1
SGK
PKCa
MAPKAP-K2
105/97
100/91
83/42
100/84
94/104
CaMKIV
Cyclin E/CDK3
cSRC
Cyclin A/CDK2
a[ATP] at Km where known or 10 mM.
bMean of duplicate assays.

2266
T.A.Engler et al./ Bioorg.Med.Chem.Lett.13 (2003) 2261–2267
Table 4. G1 arrest and inhibition of Rb (Ser780) phosphorylation in HCT-116 cells, and aqueous solubility of selected indolocarbzoles 3, 11–14
Compd
G1 Arresta (fold increase over control)
% Inhibition of Rb Phosphorylationa (Ser780)
Solubilityb
(mg/mL)
@1ꢃIC50
@3ꢃIC50
@1ꢃIC50
@3ꢃIC50
c
3b
3t
3v
3bb
3ff
3gg
3ii
3oo
11h
11m
12b
12h
13b
13d.TFA
13e.TFA
13f
1.4
2.2
2.6
1.4
1.8
1.2
2.0
2.4
3.1
1.8
1.9
1.8
1.9
1.8
1.5
1.8
1.5
1.2
2.0
2.9
2.5d
—
63
37
90
60
56
71
83
66
71
67
58
57
52
68
65
69
57
69
78
45
97
95
97
92
77
86
95
95
92
95
67
78
55
69
73
78
88
80
87
83
—
—
—
—
—
—
—
—
6.0
7.8
—
8.6
—
—
—
8.5
8.7
—
—
8.9
—
2.7
2.1
2.2
2.2
—
—
2.0
2.5e
2.7
2.2
—
—
2.1
2.2
2.3
—
13j
13j.HCl
13m
14m
3.4
aAfter 24 h incubation.
bAs MsOH salts formed in situ in D5W after 1 week.
cNot reported.
dAt 2.4ꢃIC50
.
.
eAt 2.6ꢃIC50
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