The document describes the synthesis and biological evaluation of novel indolyl-isoquinolinyl/quinolinyl-pyrrole-2,5-diones and corresponding carbazole analogs as potential kinase inhibitors. The maleimide analogs showed potent and selective inhibition of PKC isoforms but weaker activity against D1-cdk4. In contrast, the carbazole analogs were potent and selective D1-cdk4 inhibitors with anticancer activity in human tumor cell lines. However, the maleimides displayed poor pharmacokinetic properties that may have limited their in vivo activity. Structure-activity relationships revealed the importance of nitrogen position in the isoquinoline ring for PKC selectivity and potency

1. SYNTHESIS OF NOVEL INDOLYL-
ISOQUINOLINYL/QUINOLINYL-
PYRROLE-2, 5-DIONES AND
CORRESPONDING CARBAZOLE
ANALOGS PROVIDE POTENT AND
SELECTIVE KINASE INHIBITORS
Scott E. Conner, Guoxin Zhu, Xun Zhou, Chuan Shih, Timothy
Burkholder, Harold B. Brooks, Charles D. Spencer, Scott A.
Watkins, Eileen Considine, Jack A. Dempsey, Cathy Ogg, Bharvin
Patel, Richard M. Schultz, Karen L. Huss, Ron Kaplan, Shehnaz
Khan, Bryan D. Anderson, and Robert M. Campbell.
Discovery Chemistry Research and Technologies, Lilly Research
Laboratories, A Division of Eli Lilly & Company
Lilly Corporate Center, Indianapolis, Indiana 46285, USA
Conner_Scott_E@lilly.com

2. OVERVIEW
• Find selective and potent kinase inhibitors
• Structure based design from known,
naturally occurring inhibitors like
staurosporine and Cdk2 complex, along
with conservation of ATP binding site
• Structurally novel kinase inhibitors
discovered by replacing one indole group
with heteroaryl group, while maintaining
the maleimide moiety for key protein
interactions

3. N
N
N
H
O
NH2
H
Lys33
O
O
HN
Leu83
O
O
Asp86
O
H
O
HN
H
STAUROSPORINE-Cdk2 COMPLEX
Glu81
Homma, Teruki, et al., J. Med. Chem. 2001, 44, 4615-4627.

4. RETROSYNTHETIC ANALYSIS OF
TARGETS
N
HN
ON
H
O
N
H
N
ON
H
O N
H
O
NH2
N
O
O
O
N
H
O
O
O
N
O
NH2
+
+
CASE 1
CASE 2
OR

5. INTERMEDIATE SYNTHESIS: INDOLES
N
H
N
H
N
N
H
N
N
H
O
NH2
N
H N
H
O
O
O
Indole 3-acetamides
Indole 3-glyoxalates
(i) (ii) (iii)
(iv)
(v) and (vi)
REAGENTS AND CONDITIONS
(i) N,N-dimethyleneammonium chloride, DCM; (ii) NaCN, DMF/H2O, reflux; (iii) K2CO3, H2O2, DMSO;
(iv) (a) (COCl)2, DCM, 0oC (b) NaOMe, -78oC - 25oC; (v) NH4OH, THF, 0oC-25oC;
(vi) 10% Pd(C), NaH2PO2*XH2O, dioxane/H2O, 100oC
70%95% 80%
90%
45%

6. INTERMEDIATE SYNTHESIS:
MONO-BROMOISOQUINOLINE
N
Br
N
NH2
N N
Br
Br
N
Br
N
Br
N N
Br
N
Br
N
N
Br2, AlBr3
Reflux
+ +
major product
Ag2SO4, H2SO4,
Br2
(1)
(2) +
(a) NaNO2, HBr
00C
(b) CuBr, HBr
750C
(3)
50%
+
major product
+

7. INTERMEDIATE SYNTHESIS:MONO-
BROMOISOQUINOLINE
•The swamping catalyst effect, halogenation of
isoquinoline reaction (1) produced mainly starting
material with the isolation of the three component
mixture shown. This reaction procedure was
modified from the literature, and it was unusually
complicated. Gordon, M., et al. J. Org. Chem., 1964, 29(2),
329-333.
•Attempts at using the acidic conditions in
reaction (2) also produced mainly starting
material, but some desired product was isolated.
Dreiding, A. S. , et al. Helv. Chem. Acta, 1985, 68, 1828-1834.
•The Sandmeyer reaction (3) was successful with
50% yield without distillation. Osborn et al., J. Chem
Soc., 1956, 4191-4204.

8. INTERMEDIATE SYNTHESIS:
QUINOLINE/ISOQUINOLINE
N
NH2
N
Br
N
O
O
O
N
H O
N
N
N
O
NH2
(i) (ii)
(iii)
(iv) (v)
(vi) and (vii)
REAGENTS AND CONDITIONS
(i) (a) NaNO2, HBr, 0oC (b) CuBr, HBr, 75oC; (ii) (a) nBuLi, THF, -78oC, (b) dimethyl oxalate;
(iii) (a) t-BuLi, -78oC, THF, (b) DMF; (iv) (a) LiCN, diethylcyanophosphonate, THF, 0oC (b) t-BuOH, SmI2, THF
(v) KOH, t-BuOH, reflux; (vi) 10% Pd(C), dioxane/H2O, NaH2PO2*XH2O, reflux; (vii) Me3Al, NH4Cl, toluene, 50oC
35%
40%
38%
50%

9. N
H
O
NH2
O
O
O
N
H
N
H
OO
N
H
N
H
OO
N
H
OO
N
H
1
3
4
2
10
11
12
13
56
N
H
OO
N
H
1
3
4
2
10
11
12
13
56
N
N
N
N
N
+
20 - 94%
KO-tBu, THF, 0oC-50oC
DDQ, p-TsOH, benzene, reflux
or
I2, hv, dioxane, reflux
68 - 88%
NITROGEN OUTSIDE NITROGEN INSIDE
General Reaction for Maleimide Synthesis
General Reaction for Maleimide to Carbazole Conversion
Faul, M., et al., Tet Lett., 1999, 40(6), 1109-1112.

10. Average IC50(uM) values, IC50 of
HCT-116 and NCI-H460 >6 uM
MALEIMIDES: EFFECT OF NITROGEN
POSITION AND KINASE SELECTIVITY
STRUCTURE
D1-Cdk4 ING >2 0.86 1.39 4.30
PKC-beta 12.93 2.32 0.04 0.74
PKC-alpha 15.00 2.32 0.12 1.60
PKC-epsilon >20 16.62 1.92 12.58
PKC-gamma >20 2.79 1.89 15.84
hGSK3-beta 9.32 1.61 0.13 2.49
STRUCTURE
D1-Cdk4 ING >2 >2 1.68
PKC-beta 0.84 1.12 0.04
PKC-alpha 1.56 4.00 0.30
PKC-epsilon 13.54 13.75 2.30
PKC-gamma >20 15.74 0.84
hGSK3-beta 1.19 5.70 1.10
N
H
O
N
H
N
O
N
H
O
N
H
O
N
N
H
O O
N
H N
N
H
O O
N
H
N
N
H
O O
N
H
N
C l H
N
N
H
O O
N
H
N
H
O O
N
H
N

11. Average IC50(uM) values
CARBAZOLES: ANTIPROLIFERATIVE
ACTIVITY
STRUCTURE
D1-Cdk4 ING >2 >2 0.68 >2
HCT 116 5.51 1.95 0.66 NT
NCI H460 4.04 0.93 0.31 NT
STRUCTURE
D1-Cdk4 ING >2 >2 0.07
HCT 116 0.43 3.59 0.13
NCI H460 1.91 >5 0.07
N
H
N
H
O
N
O
N
H
N
H
O O
N
N
H
N
H
O
N
O
N
H
N
H
O O
N
N
H
N
H
O O
N
N
H
N
N
H
O O
N
H
N
H
O O
N

12. Average IC50(uM) values
EFFECT OF INDOLE SUBSTITUTION
ON KINASE ACTIVITY
STRUCTURE
PKC-BETA 0.27 0.05 0.11 1.88
PKC-ALPHA 5.09 0.24 0.48 14.52
PKC-EPSILON 7.91 2.50 3.63 >2O
PKC-GAMMA 7.99 1.00 2.01 >20
hGSK3-BETA 3.06 0.99 4.63 0.09
STRUCTURE
PKC-BETA 0.03 0.04 0.07 0.08
PKC-ALPHA 0.42 0.41 0.38 0.63
PKC-EPSILON 1.39 1.76 3.04 6.73
PKC-GAMMA 2.32 1.00 1.51 2.07
hGSK3-BETA 0.48 0.54 3.77 1.39
N
H
O O
N
H
C H 3
N
C l H
N
H
O
O
N
CH3
N
N
H
O O
N
H
NO
CH3
N
H
O
N
H
CH3
OO
CH3
N
N
H
O O
N
H
F
N
ClH
N
H
O O
N
H
F N
N
N
H
O O
N
H
F
N
H
O O
N
HF
N

13. FUNCTIONAL DATA: CARBAZOLE
N
H
N
H
O O
N
G1(%) S(%) G2/M(%)
46.46 41.34 12.20
62.09 28.37 9.53
64.31 26.05 9.64
NCI H460
IC50 = 0.07 M
DOSE(M)
CTL
0.21
0.07
46.46
62.09
64.31
41.34
28.37
26.05
12.20
9.53
9.64
0% 20% 40% 60% 80% 100%
CTL
0.07
0.21
G1(%)
S(%)
G2/M(%)
0.07 0.21CTL
Channels
0 40 80 120 160 200
Number
0100200300400500600
Channels
0 40 80 120 160 200
Number
0200400600800
Channels
0 40 80 120 160 200
Number
0200400600800
ING Rb21 PKA FPr-5T MCF7 MB468 HCT-116 NCI- H460 Flow
0.074 0.02 0.359 0.150 0.600 <0.078 0.126 0.074 G1
All values are uM

14. MALEIMIDE: ANTIPROLIFERATIVE
ACTIVITY
NN
H
N
H
O O
D1-cdk4 ING HCT-116 NCI-H460
4.3 7.4 9.26
The other maleimides listed in this poster were either not tested due to low potency
in enzymatic assays or >5 uM in these antiproliferative assays.
All values are uM

15. MALEIMIDE: PK PROFILE
SINGLE DOSE STUDY
•rat female 30 mg/kg
AUC(0-24h) = 11.4 µM.hr
Cmax = 6.2 µM
•rat female 30 mg/kg
AUC(0-24h) = 0.12 µM.hr
Cmax = 0.01 µM
N
H
O O
N
H
N
N
H
O O
N
H
N
0
1
2
3
4
5
6
7
0 4 8 12 16 20 24
Time postdose (hr)
Plasmaconc.(µM)

16. RAT CORNEAL MICROPOCKET ASSAY:
VEGF-INDUCED ANGIOGENESIS & RESPONSE
VASCULAR AREA, pixels
0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000
TREATMENTGROUP
0
1
2
3
4
5
6
7
8
SURGICAL
CONTROL
VEGF
3 mg/kg
10 mg/kg
30 mg/kg
TREATMENT REGIMEN:
2x per day on days 1-10;
data collected on day 14.
N
H
O O
N
H
N
In Vitro
•Extent of Metabolism by
microsomes in rat 72%, humans 25%
•Aromatic Oxidation
•2D6 Activity is 19% inhibition at 10 uM
•3A4 Invovlement is 26% inhibition by
Ketoconazole at 0.5 uM
ANTIANGIOGENESIS
PROPERTIES
Teicher, B., et al., Investigational New Drugs, 2002, 20, 241-251.

17. SUMMARY
•The synthetic pathway and subsequent biological activity of a series of
indolyl-isoquinolinyl/quinolinyl-pyrrole-2, 5-diones and the corresponding
carbazole analogs are described
•The maleimide analogs display potent in vitro activity with good selectivity
among PKC isoforms and less potent activity against D1-cdk4
•They are particularly less active against D1-cdk4 in vivo in human tumor cell lines HCT-116
and NCI-H460
•Lack of good in vivo activity may be related to the PK properties of this class compound
•PK properties and PKC-beta activity are dramatically effected by the position of the
nitrogen in the isoquinoline analogs
•The carbazole analogs described are potent and selective D1-cdk4 inhibitors
with cellular activity by inhibiting cell growth in the human tumor cell lines
HCT-116 and NCI-H460
•They display specific G1 cell cycle arrest and selective inhibition of
phosphorylation of serine 780 on pRb, supporting the in vitro inhibitory action
of D1-cdk4
•The carbazole analogs described are less potent PKC isoform inhibitors
MALEIMIDES
CARBAZOLES

18. ACKNOWLEDGEMENTS
Discovery Chemistry LOB Cancer ADME
Guoxin Zhu Bob Campbell Beverly Teicher Pierre Gilbert
Scott Conner Karen Huss Enrique Alvarez Robert Cavalier
Xun Zhou Ron Kaplan Eileen Considine
Tim Burkholder B. Dirk Anderson Val Phares Biopharmaceutics
Michael Paal Shehnaz Khan Krishna Menon Beatrice Renson
Chuan Shih Qi Liu
Tom Engler Custom Synthesis Harold Brooks Legal
Jim Copp Charles Spencer Paul Darkes
Process Chemistry Bob Murff Scott Watkins Kirby Lee
Margaret Faul Pallab Goshal Eilen Considine
Kevin Sullivan Jack Dempsey
Cathy Ogg
Bharvin Patel
Richard Schultz
Robert Campbell