Preparation of novel aza-1,7-annulated indoles and their conversion to potent indolocarbazole kinase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2004.05.088

The synthesis of novel aza-1,7-annulated indoles was achieved and these were converted to indolocarbazoles that proved to be potent kinase inhibitors. These compounds were also evaluated in a human colon carcinoma cell line and proved to be good antiproliferative agents.

Full text of DOI:10.1016/j.bmcl.2004.05.088

Bioorganic & Medicinal Chemistry Letters 14 (2004) 3925–3928
Preparation of novel aza-1,7-annulated indoles and their
conversion to potent indolocarbazole kinase inhibitors
Rima S. Al-awar,a,* James E. Ray,a Kyle A. Hecker,a Sajan Joseph,a Jianping Huang,a
Chuan Shih,a Harold B. Brooks,b Charles D. Spencer,b Scott A. Watkins,b
Richard M. Schultz,b Eileen L. Considine,b Margaret M. Faul,c Kevin A. Sullivan,c
Stanley P. Kolis,c Michael A. Carrc and Faming Zhangd
aDiscovery Chemistry Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
bCancer Research Division, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA
cChemical Process Research and Development, Eli Lilly and Company, Indianapolis, IN 46285, USA
dScience and Technology, Eli Lilly and Company, Indianapolis, IN 46285, USA
Received 4 April 2004; revised 24 May 2004; accepted 24 May 2004
Available online
Abstract—The synthesis of novel aza-1,7-annulated indoles was achieved and these were converted to indolocarbazoles that proved
to be potent kinase inhibitors. These compounds were also evaluated in a human colon carcinoma cell line and proved to be good
antiproliferative agents.
Ó 2004 Elsevier Ltd. All rights reserved.
The complex machinery of the cell cycle is controlled by a
plethora of kinases that in turn regulate tumor sup-
pressor genes and transcription factors. The cyclins and
cyclin-dependent kinases, in particular, cyclinD1/cdk4
and cyclinE or A/cdk2 play an essential role in the
transition of cells from G1 to S phase. Several tumors,
such as colon, rectum, lung, and breast have been found
to have major alterations in this pathway.1 Several
structurally diverse molecular classes have been designed
and discovered as ATP competitive inhibitors of the
cyclin-dependent kinases.2 The 1,7-annulated indolo-
carbazole series of analogues (Fig. 1) derived from the
natural product Staurosporine was found to be effective
at inhibiting cyclinD1/cdk4 and arresting cells in the G1
phase of the cell cycle. These derivatives possessed potent
antiproliferative activity in the HCT-116 colon carci-
noma cell line, commensurate with their in vitro enzyme
inhibition activity.3
H
N
O
O
R
N
N
H
n
Figure 1.
was hoped that the aza-annulated carbazoles would
provide additional binding interactions within the ATP
active site of the enzyme and also allow for an additional
handle to possibly improve the solubility of the series. The
preparation of the desired glyoxylate esters and acetam-
ide indoles was accomplished in an efficient manner in 5–8
steps from a common starting material.4 Treatment of 7-
formylindole 1 with ethanolamine under reductive ami-
nation conditions followed by protection of the resulting
secondary amine with Boc anhydride gave intermediate 3.
Mesylation of the primary alcohol produced a good
leaving group that readily underwent displacement by the
indole nitrogen when subjected to NaH in DMF at 0 °C,
thus producing the key tetrahydrodiazepinoindole 5.
Follow-up efforts on this series led to the preparation of
the 1,7-aza-annnulated carbazoles described herein. It
Keywords: cdk4; Indolocarbazoles.
* Corresponding author. Tel.: +1-919-314-4205; fax: +1-919-314-4348;
e-mail: al-awar_rima_s@lilly.com
Preparation of the indole-3-glyoxylate ester 6 and its
condensation with indole-3-acetamide was accomplished
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.088

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R. S. Al-awar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3925–3928
using previously described methods to provide the de-
sired bis-indolylmaleimide 11a.5 Exploring the SAR
around the indole ring was a little more challenging in
that it was necessary to prepare acetamide 9 since the
intermediates 10b–f were only available as the glyoxylate
esters (Scheme 1).6
integrity of the carbamate.7 Condensation with the
variously substituted glyoxylate esters 10b–f provided
bis-indolylmaleimides 11b–f.
With the desired bis-indolylmaleimides 11a–f in hand it
was necessary to oxidize these to the indolocarbazoles
12a–f, which was accomplished in good yield with
Pd(OAc)2 (Scheme 2).
Although efficient conditions to prepare the acetamide
indoles of the all carbon annulated series had been re-
ported,3a it was still to be confirmed that the reaction
conditions would be compatible with the t-butoxycar-
bamate moiety of 8. Treatment of 6 with ammonium
hydroxide gave the corresponding amide in quantitative
yield. Surprisingly, ketoamide 8 readily underwent
reduction to 9 under the same previously reported
reaction conditions of 10% Pd/C in the presence of so-
dium hypophosphite but instead of refluxing conditions
the temperature was reduced to 65 °C to maintain the
Indolocarbazoles 12a–e were very potent inhibitors of
cyclinD1/cdk4 with activity ranging between 0.027 and
0.11 lM (Table 1). Although 12b and 12d showed some
selectivity against cyclinE/cdk2, only 12d and 12e were
18 and 29-fold selective for cdk4 over PKA. Neverthe-
less, they were all equally potent in the HCT-116 cell line
with activities ranging between 0.17 and 0.4 lM. Al-
though 12f was a very potent inhibitor of cdk4 it was not
as active in the carcinoma cell line showing only 3.5 lM
activity that might be due to reduced cell penetration.
b
a
It was hoped that substitution of the nitrogen of 12a could
potentially lead to more selective analogues and hence
13a–d were prepared to explore this hypothesis. Although
13a and 13b were more selective for cyclinD1/cdk4, only
13a had any cell activity despite the fact that 13b was a
potent inhibitor of cdk4. Amide 13c was also plagued
with the same issue of lacking cellular activity while
having enzyme inhibitory activity. The sterically hindered
tertiary amine 13d was, on the other hand, 10 and 25-fold
more selective for cdk4 over cdk2 and PKA, respectively,
while maintaining potent activity in the cell based assay.
N
H
NH
N
H
N
H
NBoc
CHO
3
OH
2
1
OH
d
e
c
N
H
N
N
NBoc
4
Boc
5
OMs
O
O
O
O
H2N
f,g
+
With these promising biological results, attention was
focused on exploring the effect of ring size on the activity
and selectivity of the aza series. The 8-membered aza-
annulated indole 19 was therefore prepared again from
N
N
H
7
N
6
H
N
Boc
O
O
h
R1
R2
H
N
O
O
O
O
N
N
H
NH2
R3
R1
R2
N
Boc
11a (R1=H, R2=H, 72%)
11b (R1=F, R2=H, 46%)
11c (R1=H, R2=F, 54%)
11d (R1=F, R2=F, 44%)
a,b
N
11a-f
N
N
H
R3
N
8
HCl.HN
Boc
11e (R1=H, R2=CF3, 91%)
11f (R1=H, R2=CF3,
12a (R1=H, R2=H, 34%)
12b (R1=F, R2=H, 31%)
12c (R1=H, R2=F, 62%)
12d (R1=F, R2=F, 30%)
i
R3=CH2OTIPS, 58%)
O
O
O
NH2
+
12e (R1=H, R2=CF3, 32%)
O
R1
f,g
12f (R1=H, R2=CF3
,
c
R3=CH2OH, 32%)
N
N
12a
R2
H
13a (R 4=SO2Me)
R3
9
H
N
d,e
N
13b (R4=SO2
)
N
10b (R1=F, R2=H)
10c (R1=H, R2=F)
10d (R1=F, R2=F)
10e (R1=H, R2=CF3)
10f (R1=H, R2=CF3
R3=CH2OH)
O
O
N
Boc
13c (R 4=COCH2NH2.HCl)
13d (R 4=Isopropyl)
f
,
N
N
H
N
R4
Scheme 1. Reagents and conditions: (a) ethanolamine, NaBH(OAc)3,
AcOH, DCE, 95%; (b) Boc2O, DAMP, THF, 100%; (c) MsCl, TEA,
CH2Cl2, 0 °C, 100%; (d) NaH, DMF, 0 °C, 97%; (e) oxalyl chloride,
NaOMe, CH2Cl2, 91%; (f) KOt-Bu, THF; (g) concd HCl, 44–91%; (h)
NH4OH, THF, 0 °C to rt, 95%; (i) 10% Pd/C, NaH2PO2, dioxane,
H2O, 65 °C, 93%.
Scheme 2. Reagents and conditions: (a) Pd(OAc)2, AcOH, 65 °C; (b)
4 M HCl, dioxane, CH2Cl2, 30–62%; (c) sulfonyl chloride, TEA,
CH2Cl2, 50–75%; (d) EDCI, HOBT, DMF, Boc-glycine, 35%; (e) 4 M
HCl, dioxane, CH2Cl2; (f) acetone, NaBH(OAc)3, AcOH, DCE, 90%.

R. S. Al-awar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3925–3928
3927
Table 1. Enzyme activity (cyclinD1/cdk4 and cyclinE/cdk2) and cell
based inhibition in HCT116 (colon) cell line for some prepared indo-
locarbazoles
dole-6-carboxylic acid tert-butyl ester, which was easily
converted to glyoxylate 19. Preparation of the final in-
dolocarbazole 21 was accomplished under similar con-
ditions used in the synthesis of 12a–e (Scheme 3).
Compds CyclinD1/
cdk4
CyclinE/
cdk2
IC50 lMa
PKA
IC50 lMa
Cytotoxicity
HCT-116
IC50 lM
IC50 lM
Evaluation of 12a and 21 revealed that a 3–6-fold
improvement in solubility was achieved with these aza-
1,7-annulated analogues when compared to the simple
all carbon parent compound (Fig. 1).
12a
12b
12c
12d
12e
12f
13a
13b
13c
13d
21
0.027
0.034
0.031
0.11
0.038
>0.2
0.016
>0.2
0.21
Nd
0.16
0.2
0.31
0.26
0.2
0.43
>2.0
>2.0
Nd
0.4
0.069
0.044
0.053
0.12
0.17
3.5
With the successful preparation of the 8-membered aza-
annulated indolocarbazole 21, attention was focused on
the synthesis of the 9-membered one. It was envisioned
that ring closing metathesis10 of the appropriate alk-
enes would yield the desired 9-membered aza annu-
lated indole. Indeed, alkene 24 underwent ring closing
metathesis when subjected to the Grubbs catalyst (ben-
zylidene-bis-(tricyclohexyl-phosphine)dichlororuthenium)
to give 25 in 61% yield. Alkene 25 was subsequently
reduced over hydrogen and platinum and converted to
the glyoxylate ester using oxalyl chloride followed by
NaOMe to provide the saturated 9-membered ring in-
dole 26 (Scheme 4). The precursor to the ring closing
reaction was obtained in three steps starting with a
reductive amination between 7-formylindole 1 and allyl-
amine to give 22. Protection of the amine and alkylation
of the indole nitrogen with allylbromide provided the
key intermediate 24 with the terminal alkenes set for
ring closing metathesis (Fig. 2).
>0.2
Nd
>2.0
>2.0
>20
>2.0
2.7
1.6
>10
>10
0.64
0.039
0.097
0.10
Nd
0.078
0.16
0.084
0.78
Nd
28
0.2
0.04
a Nd ¼ not determined.
7-formylindole in eight total steps starting with a Wittig
olefination,8 followed by borane assisted homologation
and oxidation to give alcohol 15.9 Mesylation of the
primary alcohol and its subsequent displacement with
ethanolamine provided the aminoalcohol 17. All that
remained to generate the desired ring was the protection
of the amine as a t-butoxycarbamate and the mesyla-
tion of the primary alcohol setting the stage for the
cyclization using previously described conditions thus
providing 4,5,7,8-tetrahydro-[1,4]diazocino-[7,8,1-hi]in-
c
a
b
1
a
c
b
1
N
H
N
H
N
H
N
H
NH
NBoc
14
15
OH
22
23
e,f
d
g,h
N
N
H
N
H
d
H
e,f
N
N
OMs
16
17
18
BocN
OMs
HN
NBoc
N
OH
Boc
H
N
H
N
O
O
24
25
O
O
O
O
O
O
O
O
i,j
7
+
N
g,h
N
N
H
N
N
N
7
+
H
N
Boc
N
N
19
20
N
Boc
Boc
Boc
26
27
H
H
N
N
O
O
O
O
k
l
20
i,j
N
N
27
H
N
N
H
21
N
28
HCl.HN
H.ClH
Scheme 3. Reagents and conditions: (a) MePh3PBr, KOt-Bu, THF,
93%; (b) BH3ÆTHF, H2O2, NaOH, 56%; (c) MsCl, TEA, CH2Cl2, 98%;
(d) ethanolamine, EtOH, D, 85%; (e) Boc2O, DAMP, THF, 85%; (f)
MsCl, TEA, CH2Cl2, 100%; (g) NaH, DMF, 0 °C, 79%; (h) oxalyl
chloride, NaOMe, CH2Cl2, 83%; (i) KOt-Bu, THF; (j) concd HCl; (h)
NH4OH, THF, 0 °C to rt, 95%; (k) Pd(OAc)2, AcOH, 65 °C; (l) 4 M
HCl, dioxane, CH2Cl2.
Scheme 4. Reagents and conditions: (a) allylamine, NaBH(OAc)3,
AcOH, DCE, 82%; (b) Boc2O, DAMP, THF, 100%; (c) NaH, allyl-
bromide, DMF, 0 °C, 91%; (d) Grubbs catalyst (benzylidene-bis-(tri-
cyclohexylphosphine)dichlororuthenium), D, CH2Cl2, 61%; (e) H2,
PtO2, EtOH, 95%; (f) oxalyl chloride, NaOMe, CH2Cl2, 91%; (i) KOt-
Bu, THF; (j) concd HCl, 43%; (k) Pd(OAc)2, AcOH, 60 °C, 50%; (l)
4 M HCl, dioxane, CH2Cl2, 100%.

3928
R. S. Al-awar et al. / Bioorg. Med. Chem. Lett. 14 (2004) 3925–3928
ꢀ
bond with Asp86 (2.7 A), an amino acid that is highly
conserved in many kinases including PKA, which shed
further light on the reduced selectivity of 28.
In conclusion, novel azaannulated indolocarbazoles of
varying ring sizes were prepared from 7-formylindole
and were readily converted to indolocarbazoles that are
potent inhibitors of cyclinD1/cdk4.
References and notes
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156.
3. (a) Al-awar, R. S.; Ray, J. E.; Hecker, K. A.; Huang, J.;
Waid, P. P.; Shih, C.; Brooks, H. B.; Spencer, C. D.;
Watkins, S. A.; Patel, B. R.; Stamm, N. B.; Ogg, C. A.;
Schultz, R. M.; Considine, E. L.; Faul, M. M.; Sullivan,
K. A.; Kolis, S. P.; John L. Grutsch, J. L.; Joseph S.
Bioorg. Med. Chem. Lett., 2004, 14, 3217–3220; (b) Long,
B. H.; Rose, W. C.; Vyas, D. M.; Matson, J. A.; Forenza,
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6. Glyoxylate ester 10f was prepared in three steps from
methyl indole-6-carboxylate. Reduction of the ester with
LiAlH4, protection of the resulting alcohol as silyl ether
with TIPSOTf and treatment with oxalyl chloride followed
by NaOMe.
7. Demopoulos, V. J. Synth. Commun. 1989, 19, 2585–2594.
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Figure 2. Key interactions of carbazole 28 bound to cdk2.
Condensation of 26 with acetamide indole 7 gave bis-in-
dolylmaleimide 27, which when oxidized over Pd(OAc)2
and deprotected using hydrogen chloride treatment yiel-
ded indolocarbazole 28 as the hydrochloride salt.
Indolocarbazoles 12a, 21, and 28 were found to all be
equally potent against cyclinD1/cdk4 and therefore the
ring size did not appear to affect the activity (Table 1).
Although 21 was 17-fold more selective for cdk4 versus
PKA, carbazole 28 was a 2-fold more potent inhibitor of
PKA. Nevertheless, both compounds were equally
cytotoxic when evaluated in the HCT-116 antiprolifer-
ation assay.
The indolocarbazole 28 was co-crystallized with cdk2
and was found not surprisingly to occupy the ATP
binding pocket. Several key hydrogen bond interactions
were identified whereby the N–H of the maleimide
moiety was hydrogen bonding to the carbonyl of Glu81
ꢀ
(2.8 A) and the carbonyl of the maleimide hydro-
gen bonded to the backbone amide group of Leu83
ꢀ
(2.5 A). In addition to these expected interactions, the
amine of the 9-membered ring also formed a hydrogen