Lead identification to generate isoquinolinedione inhibitors of insulin-like growth factor receptor (IGF-1R) for potential use in cancer treatment

Article abstract of DOI:10.1016/j.bmcl.2008.04.044

Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer therapy. Efforts in our "Hit to Lead" group have generated a novel series of submicromolar IGF-1R inhibitors based on a isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process. The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.

Full text of DOI:10.1016/j.bmcl.2008.04.044

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3641–3645
Lead identification to generate isoquinolinedione inhibitors
of insulin-like growth factor receptor (IGF-1R)
for potential use in cancer treatment
Scott C. Mayer,a,* Annette L. Banker,a Frank Boschelli,d Li Di,a Mark Johnson,b
Cynthia Hess Kenny,c Girija Krishnamurthy,c Kristina Kutterer,c Franklin Moy,b
Susan Petusky,a Malini Ravi,c Diane Tkach,d Hwei-Ru Tsouc and Weixin Xub
aWyeth Research, Chemical & Screening Sciences, 865 Ridge Road, Monmouth Junction, Princeton, NJ 08852, USA
bWyeth Research, Chemical & Screening Sciences, Cambridge, MA, USA
cWyeth Research, Chemical & Screening Sciences, Pearl River, NY, USA
dWyeth Research, Oncology, Pearl River, NY, USA
Received 7 March 2008; revised 18 April 2008; accepted 21 April 2008
Available online 25 April 2008
Abstract—Insulin-like growth factor receptor (IGF-1R) is a growth factor receptor tyrosine kinase that acts as a critical mediator of
cell proliferation and survival. This receptor is over-expressed or activated in tumor cells and is emerging as a novel target in cancer
therapy. Efforts in our ‘‘Hit to Lead’’ group have generated a novel series of submicromolar IGF-1R inhibitors based on a
isoquinolinedione template originating from a Lance enzyme HTS screen. Chemical triage and parallel synthesis incorporating
focused library arrays were instrumental in moving these investigations through the Wyeth exploratory medicinal chemistry process.
The strategies, synthesis, and SAR behind this interesting kinase scaffold will be described.
Ó 2008 Elsevier Ltd. All rights reserved.
Receptor tyrosine kinases are proven targets for ther-
apeutic intervention in human cancer.1 As a member
of this class of signaling molecules, the insulin-like
growth factor receptor tyrosine kinase (IGF-1R) is a
pivotal element of a signaling pathway involved in cell
growth, proliferation, and survival.2 It is highly re-
lated to the insulin receptor (IR) yet plays a different
role in organism development, being responsible for
normal growth and development as opposed to glu-
cose homeostasis.3 Several studies indicate that the
IGF-1 receptor is overexpressed in human cancers,
and plays a essential role in tumorigenesis.4 Several
experimental approaches to reducing IGF-1R levels
have been reported, and the results suggest that tumor
cell proliferation, survival and sensitivity to cytotoxic
agents are dependent on the presence of IGF-1R.
These effects are believed to result from downregulat-
ing PI3K and Ras pathways.5 Inhibitors of IGF-1R
kinase activity may therefore be suitable agents for
inhibition of human tumor growth.
Efforts from our ‘‘Hit to Lead’’ group were aimed at
developing selective small molecule inhibitors of IGF-
1R for the treatment of cancer. The most significant
challenge for the team would be obtaining selectivity
over IR due to homology of these two receptors (IGF-
1R has 84% sequence identity to IR in the kinase
domain).6
A high-throughput screen (HTS) using a Lance en-
zyme assay7 was performed generating 5359 confirmed
hits. Through chemical triage and follow-on assays,
full hit characterization was completed in a few
months generating a novel kinase template; the isoqu-
inolinedione series met all criteria as a viable lead and
provided an unique scaffold for SAR development
[compound 1 (Fig. 1)].8 Along with chemical triage
and data-mining, input from structural biology helped
to fully characterize this lead series. Fluorescence data
Keywords: IGFR inhibitor; Isoquinolinedione; ‘Hit to Lead’ investigations.
*
Corresponding author. Tel.: +1 732 274 4457; fax: +1 732 274
4505; e-mail: mayers@wyeth.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.04.044

3642
S. C. Mayer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3641–3645
orientations of this small molecule lead in the ATP-
H
N
binding pocket of the IGFR kinase domain. Fig. 3
displays the key interactions to the kinase determined
from the co-crystal structure: (a) Glu1080s backbone
carbonyl oxygen and 1’s core NH, (b) Met1082’s
backbone NH and 1’s core carbonyl oxygen and (c)
Met1082’s backbone carbonyl oxygen and 1’s anilino
NH.10
N
Br
O
NH
O
1
Figure 1. Representative compound from isoquinolinedione cluster.
Data-mining and clustering techniques using the Wyeth
compound database were crucial in determining key
functional areas of the molecule; replacement of the bro-
mine of 2 (analog of 1) with the 6-anilino linkage of 5
(Table 1) provided enhanced activity for IGF-1R versus
CDK4, the program from which our equity originated8
(i.e., 2: ꢀ3-fold selective for CDK4 and 5: ꢀ7-fold selec-
tive for IGF-1R; also 1: ꢀ10-fold selective for CDK4).
Using structure-based design techniques, the initial
libraries (analogs 3 and 4, and 6–30) established the
SAR around this anilino group with compounds 5, 8,
and 15 being the most potent initially in this series of
IGFR inhibitors. No clear electronic preference was
apparent and from molecular modeling analysis, it was
rationalized that potency was directly proportional to
anilino-NH binding to the protein backbone; the more
potent analogs provided optimum alignment of anili-
˚
no-NH with ASP1153 (i.e., ꢀ3.74 A distance for 5, 8,
Figure 2. Fluorescence binding experiment: Graph displays fluores-
cence intensity of 1 lM IGF-1R with compound 1. Compound 1 binds
to IGF-1R with a KD of 614 nM.
and 15).
From analogs 3 and 4 in Table 1, it was apparent that
a basic amine in the tailpiece region of the molecule
was crucial for inhibitory activity. Our final library
(analogs 31–41) was designed to further establish this
importance, highlighting that many different tertiary
amines were tolerated. Table 1 also summarizes selec-
tivity versus IR for selected analogs; however, to this
point, no apparent window of selectivity has been
achieved. Interestingly, even our most potent IGFR
analog (39) showed only a slight preference for IGFR
versus IR. With such small differences in the kinase
backbone between IGF-1R and IR [Thr1083 (IR
Ala) and Arg1084 (IR His)], we are in the process
of trying to better understand the minor selectivity
preference of 39 with the hopes of obtaining future
analogs with a more desirable selectivity window. In
addition, aqueous solubility (ꢀ1 mg/mL at pH 7.4
for 39) still needs to be addressed for this series to
meet our desired lead profile.
indicated 1 bound to IGF-1R in the submicromolar
range (Fig. 2, KD = 0.614 lM).9 In addition, co-crystal
and molecular modeling studies of 1 provided similar
Compounds were prepared by a five-step sequence out-
lined in Scheme 1.11 To generate the isoquinolinedione
core (42), diethyl carbonate was reacted with 4-bromo-
2-methyl-benzoic acid in the presence of LDA followed
by heating with urea in 1,2-dichlorobenzene at elevated
temperature. Further heating this intermediate with
triethyl orthoformate in DMF:Ac2O (4:1) provided com-
pound 43. Phenylamino-tails A or B (see Scheme 2) were
added to this intermediate followed by addition of the
appropriate substituted aniline to afford the final prod-
ucts (see Table 1).
Figure 3. Co-crystal structure of compound 1 in IGF-1R. The key
interactions are: (a) Glu1080’s O and 1’s NH, (b) Met1082’s NH and
1’s O, and (c) Met1082’s O and 1’s NH.
In summary, following a Lance enzyme HTS of the
corporate database, full hit characterization of the po-

S. C. Mayer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3641–3645
Table 1. IGFR inhibitory activity and selectivity data versus IR
3643
H
N
R1
R2
O
NH
O
R1
R2
IGFR IC50 (lM)
Sel. ratio (IR/IGFR)b
a
Compound
1
CH2-pyrrolidine
CH2-piperidine
H
Br
Br
2.42
2.89
0.57
ntc
2
3
m-Cl-aniline
m-MeO-aniline
Aniline
>10
>10
ntc
4
H
ntc
5
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-piperidine
CH2-NMe2
0.455
4.14
0.87
ntc
ntc
6
o-Me-aniline
m-Me–aniline
p-Me-aniline
o-OMe-aniline
m-OMe-aniline
p-OMe-aniline
o-CN-aniline
m-CN-aniline
p-CN-aniline
m-Ac-aniline
p-Ac-aniline
o-Cl-aniline
7
>10
8
0.498
0.610
1.08
1.14
ntc
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
ntc
0.971
4.20
1.13
ntc
ntc
ntc
1.33
ntc
0.438
5.89
1.66
0.72
ntc
ntc
m-Cl-aniline
p-Cl-aniline
m-F-aniline
2.39
ntc
>10
ntc
2.63
ntc
m-CF3-aniline
m-NH2-aniline
>10
>10
ntc
ntc
p-N(CH3)2-aniline
p-CONH2-aniline
p-CO2Et-aniline
3-OMe-5-CF3-aniline
5-NH-(1-quinoline)
5-NH-indole
>10
ntc
0.957
>10
>10
ntc
ntc
ntc
5.20
ntc
>10
ntc
3,4 ring -(CH2)3-aniline
3,4 ring -OCH2O-aniline
Br
7.93
0.894
7.17
ntc
ntc
0.58
0.75
0.83
1.0
0.62
0.48
0.69
0.87
1.6
0.85
0.64
CH2-piperidine
N-Me-piperazine
3,5-di-Me-piperazine
CH2–NMe2
3-furyl
Ph-SO2NH
0.715
0.829
0.629
0.920
1.39
3-furyl
Aniline
CH2–NMe2
CH2–NMe2
CH2–NMe2
m-MeO-aniline
p-MeO-aniline
m-Ac-aniline
0.566
0.887
0.319
0.540
1.16
N-Me-piperazine
3,5-Di-Me-piperazine
CH2–NEt2
m-Ac-aniline
m-Ac-aniline
p-Me-aniline
a Concentration inducing 50% inhibition of IGFR (N = 2–3).8
b Selectivity ratio (IC50 IR inhibition/IC50 IGFR inhibition).
c nt, not tested in IR inhibition assay.
tential equity generated a novel kinase template as an
IGFR inhibitor. Chemical triage and parallel synthesis
incorporating focused libraries helped to establish the
SAR around this isoquinolinedione core. The key ani-
lino functionality provided compounds (i.e., 5) with
submicromolar activity; however, future analogs
around compounds such as 39 will need to be designed
with the aim of hopefully improving its selectivity ver-
sus IR.
Acknowledgments
We are grateful to the Discovery Analytical Chemistry
department of Wyeth Research for elemental analyses,
1H NMR, and mass spectroscopy and Dr. John
A. Butera, Dr. John Ellingboe, Mr. Eric Gundersen,
Ms. Lori Miller, Ms. Joan Sabalski, and Mr. Gengcheng
Yang for scientific discussions.

3644
S. C. Mayer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3641–3645
4. (a) Chng, W. J.; Gualberto, A.; Fonseca, R. Leukemia
2006, 20(1), 174; (b) Stro¨mberg, T.; Ekman, S.; Girnita,
L.; Dimberg, L. Y.; Larsson, O.; Axelson, M.; Lennarts-
Br
O
Br
a, b
NH
CO2H
¨
son, J.; Hellman, U.; Carlson, K.; Osterborg, A.; Vander-
O
c
42
kerken, K.; Nilsson, K.; Jernberg-Wiklund, H. Blood
2006, 107(2), 669; (c) Breuhahn, K.; Longerich, T.;
Schirmacher, P. Oncogene 2006, 25(27), 3787; (d) Fang,
J.; Zhou, Q.; Shi, X.; Jiang, B. Carcinogenesis 2007, 28(3),
713.
H
N
R1
O
5. (a) Furstenberger, G.; Senn, H.-J. Lancet—Oncology
¨
R2
O
Br
O
d, e
2002, 3, 298; (b) Blum, G.; Gazit, A.; Levitzki, A. J.
Biol. Chem. 2003, 278, 40442; (c) Pollak, M. N.; Schern-
hammer, E. S.; Hankinson, S. E. Nat. Rev. Cancer 2004, 4,
505; (d) Garber, K. J. Nat. Cancer Inst. 2005, 97, 790; (e)
Yeh, A. H.; Bohula, E. A.; Macaulay, V. M. Oncogene
2006, 25(50), 6574; (f) Sachdev, D.; Hartell, J. S.; Lee, A.
V.; Zhang, X.; Yee, D. J. Biol. Chem. 2004, 279(6), 5017;
(g) Sachdev, D.; Li, S.-L.; Hartell, J. S.; Fujita-Yamagu-
chi, Y.; Miller, J. S.; Yee, D. Cancer Res. 2003, 63(3), 627;
(h) Ho¨pfner, M.; Sutter, A. P.; Huether, A.; Baradari, V.;
NH
NH
43
O
O
Scheme 1. Reagents and conditions: (a) i—LDA, diethyl carbonate,
THF; ii—H2O; iii—H+; (b) 1,2-dichlorobenzene, urea, 150 °C; (c)
triethylorthoformate, DMF:Ac2O(4:1), 125 °C; (d) phenylamino-tail A
or B (see Scheme 2), DMF, 110 °C; (e) substituted aniline, KOt-Bu,
NMP, [1,10-bis(diphenylphosphino)- ferrocene]dichloro-palladium (II)
complex with CH2Cl2 (1:1), IPrÆHClÆPd.
Scherubl, H. World J. Gastroenterol. 2006, 12(35), 5635;
¨
(i) Ho¨pfner, M.; Huether, A.; Sutter, A. P.; Baradari, V.;
Schuppan, D.; Scherubl, H. Biochem. Pharmacol. 2006,
71(10), 1435.
6. De Meyts, P.; Whittaker, J. Nat. Rev. Drug Discov. 2002,
1, 769.
¨
Phenylamino-tail A
R1
X
Cl
a, b
7. IGF-1R and IR kinase assays: The catalytic domains of
IGF-1R or IR were cloned described in Pautsch et al.12
into pAcG2T [BD Biosciences (San Jose, CA)]. GST-IGF-
1R and GST-IR fusion proteins were isolated by gluta-
thione bead capture, eluted, incubated in 20 mM Tris–
HCl, pH 7.5, 0.2 M NaCl, 0.01 M MgCl2 and 7.5 mM
ATP for 12 min. followed by thrombin cleavage, purifi-
cation on a glutathione column and MonoQ fractionation.
IGF-1R or IR catalytic domains phosphorylated on all
three tyrosines in the activation loop as determined by
mass spectrometry were then purified on a Superdex 200
column. This ‘‘tris’’ phosphorylated protein was used in
enzyme assays at 0.6 ng per reaction in 50 mM Hepes, pH
7.5, 0.01 M MgCl2with 150 lg/mL of bovine serum
albumin (Sigma #A-8918) and peptide at a final concen-
tration of 1 lg/mL (Biotin–NH2–TRDIYETDYYRK–
OH) for 90 min at room temperature. The final ATP
concentration was 100 lM.
NH2
NH2
NO2
Phenylamino-tail B
R1
F
c, b
NH2
NO2
Scheme 2. Reagents and conditions: (a) substituted amine XH, THF,
Et3N; (b) 10% Pd/C, H2, MeOH; (c) substituted amine R1H, DMF,
K2CO3.
8. Originally came from our in-house CDK4 program equity
–Tsou; H.-R.; Otteng, M.; Tran, T.; Floyd, M. B. Jr.;
Reich, M.; Birnberg, G.; Kutterer, K.; Ayral-Kaloustian,
S.; Ravi, M.; Nilakantan, R.; Grillo, M.; McGinnis, J. P.;
Rabindran, S. K. J. Med. Chem., in press.
9. Jamieson, E. R.; Jacobson, M. P.; Barnes, C. M.; Chow,
C. S.; Lippard, S. J. J. Biol. Chem. 1999, 274, 12346.
10. RCSB Protein Data Bank (PDB) deposition number
2ZM3.
References and notes
1. (a) Slamon, D. J.; Leyland-Jones, B.; Shak, S.; Fuchs,
H.; Paton, V.; Bajamonde, A.; Fleming, T.; Eiermann,
W.; Wolter, J.; Pegram, M.; Baselga, J.; Norton, L. N.
Engl. J. Med. 2001, 344(11), 783; (b) Romond, E. H.;
Perez, E. A.; Bryant, J.; Suman, V. J.; Geyer, C. E., Jr.;
Davidson, N. E.; Tan-Chiu, E.; Martino, S.; Paik, S.;
Kaufman, P. A.; Swain, S. M.; Pisansky, T. M.;
Fehrenbacher, L.; Kutteh, L. A.; Vogel, V. G.; Visscher,
D. W.; Yothers, G.; Jenkins, R. B.; Brown, A. M.;
Dakhil, S. R.; Mamounas, E. P.; Lingle, W. L.; Klein,
P. M.; Ingle, J. N.; Wolmark, N. N. Engl. J. Med. 2005,
353(16), 1673; (c) Johnston, J. B.; Navaratnam, S.; Pitz,
M. W.; Maniate, J. M.; Wiechec, E.; Baust, H.;
Gingerich, J.; Skliris, G. P.; Murphy, L. C.; Los, M.
Curr. Med. Chem. 2006, 13(29), 3483; (d) Haber, D.
A.; Bell, D. W.; Sordella, R.; Kwak, E. L.; Godin-
Heymann, N.; Sharma, S. V.; Lynch, T. J.; Settleman, J.
Cold Spring Harb. Symp. Quant. Biol. 2005, 70, 419.
2. Randhawa, R.; Cohen, P. Mol. Genet. Metab. 2005, 86(1–
2), 84.
11. Experimental: The synthesis of compound 39 described
here serves as a representative example of the synthetic
methodology used in this paper.
Step 1: 4-Bromo-2-carboxylmethyl-benzoic acid. To a
stirring solution of diisopropylamine (47.4 g, 465 mmol) in
dry THF at ꢁ78 °C was added dropwise n-butyl lithium
(37.3 g, 581 mmol). Mixture was stirred at ꢁ78 °C for
0.5 h and then allowed to warm to 25 °C for 5 min causing
a yellow suspension to form. Suspension was cooled to
ꢁ78 °C.
4-Bromo-2-methyl-benzoic
acid
(25.0 g,
116 mmol) and diethylcarbonate (10.5 g, 116 mmol) were
dissolved together in 100 ml of dry THF. This solution
was added dropwise to the reaction mixture over 30 min
causing a deep reddish-brown color. The resulting mixture
was stirred at ꢁ78 °C for 1 h and then allowed to warm to
room temperature causing a precipitate to form. Mixture
3. Blakesley, V. A.; Scrimgeour, A.; Esposito, D.; Le Roith,
D. Cytokine Growth Factor Rev. 1996, 7(2), 153.

S. C. Mayer et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3641–3645
3645
was stirred overnight at room temperature and then
cooled in an ice bath. 400 mL of water was slowly added
to the mixture keeping the internal temperature below
20 °C causing two layers to form. The layers were
separated. The organic layer was extracted with 150 ml
of H2O, and all aqueous layers were combined. Aqueous
layers were acidified with concd HCl causing an off-white
solid to form. This solid was filtered and washed with
200 ml of H2O to afford the desired product (18.6 g,
71.8 mmol, 62%); 1H NMR (DMSO-d6) d 3.51 (s, 2H),
7.32 (d, J = 2.0 Hz, 1H), 7.39 (dd, J = 1.7, 8.5 Hz, 1H),
7.51 (d, J = 8.4 Hz, 1H).
Step 2: 6-Bromo-4H-isoquinoline-1,3 dione (42). 4-Bro-
mo-2-carboxylmethyl-benzoic acid (5.00 g, 19.0 mmol)
and urea (2.45 g, 40.8 mmol) were suspended in 150 ml
of 1,2-dichlorobenzene. This mixture was heated to 150 °C
forming a homogeneous mixture. Temperature was main-
tained for 2 h during which time a yellow precipitant
formed. Mixture was cooled to room temperature and
filtered. Residue was washed with 100 ml of ethyl acetate,
100 ml of methanol, and 100 ml of water to afford the
product as a yellow solid (3.80 g, 15.8 mmol, 83%); 1H
NMR (DMSO-d6) d 4.01 (s, 2H), 7.65–7.69 (m, 2H), 7.89
(d, J = 8.7 Hz, 1H), 11.36 (s, 1H).
(990 mg, 5.18 mmol) were suspended in 20 ml of dimeth-
ylformamide. Mixture was heated at 110 °C for 2 h
forming a homogeneous mixture. Mixture was cooled to
room temperature and reduced on a rotovap to yield an
oil. The residue was diluted with H2O and stirred for
10 min. The resulting solid was filtered off and washed
with H2O and excess Et2O to provide the product as a
yellowish-brown solid (1.98 g, 86%); 1H NMR (DMSO-
d6) d 2.19 (s, 3H), 2.42–2.47 (m, 4H), 3.10–3.16 (m, 4H),
6.95 (d, J = 9.0 Hz, 2H), 7.33 (dd, J = 1.7, 8.5 Hz, 1H),
7.43 (d, J = 9.0 Hz, 2H), 7.86 (d, J = 8.5 Hz, 1H), 8.38
(d, J = 1.74 Hz, 1H), 8.84 (d, J = 12.9 Hz, 1H), 11.28 (s,
1H), 12.54 (d, J = 12.9 Hz, 1H); mass spectrum [(+)ESI],
m/z 441/443 (M+H)+.
Step 5: 4-{4-(4-methyl-piperazin-1-yl)-phenylamino]-meth-
ylene} -6-m-acetylphenylamino-4H-isoquinoline-1,3-dione
(39).
6-Bromo-4-{4-(4-methyl-piperazin-1-yl)-phenyl-
amino]methylene}-4H- isoquinoline-1,3-dione (300 mg,
0.680 mmol), 3-amino-acetophenone (184 mg, 1.36 mmol),
allylchloro{1,3-bis(2,6-di-propylphenyl)imidazol-2- yili-
dine]palladium(II) (IPrÆHClÆPd, 78 mg, 0.136 mmol), [1,10-
bis(diphenylphosphino)-ferrocene]dichloro-palladium (II)
complex with CH2Cl2 (1:1) (56 mg, 0.0680 mmol), and
potassium-t-butoxide (229 mg, 2.04 mmol) were all dis-
solved in 3.0 ml of N-Methyl-2-pyrolidinone. Mixture was
heated at 150 °C in a microwave for 15 min using normal
absorption and fixed hold on. The resulting solution was
purified by silica gel chromatography (eluent: 1–20%
MeOH:CHCl3) followed by preparatory plate chromatog-
raphy (eluent: 10% MeOH:CHCl3) to afford the product as
a brownish solid (29 mg, ꢀ10%), mp 268–270 °C; 1H NMR
(DMSO-d6) d 2.19 (s, 3H), 2.36–2.48 (m, 4H), 2.54 (s, 3H),
3.04–3.16 (m, 4H), 6.88–6.99 (m, 3H), 7.29 (d, J = 9.0 Hz,
2H), 7.39–7.48 (m, 2H), 7.49–7.54 (m, 2H), 7.73 (s, 1H), 7.85
(d, J = 8.7 Hz, 1H), 8.46 (d, J = 12.8 Hz, 1H), 8.83 (s, 1H),
10.89 (s, 1H), 12.28 (d, J = 12.9 Hz, 1H); mass spectrum
[(+)ESI], m/z 496 (M+H)+; Anal. Calcd for C29H29
N5O3Æ0.5CH2Cl2: C, 65.85; H, 5.62; N, 13.02 Found: C,
66.00; H, 4.99; N, 12.72.
Step 3: 6-Bromo-4-methoxymethylene-4H-isoquinoline-1,3-
dione (43). 6-Bromo-4H-isoquinoline-1,3-dione (120 mg,
0.500 mmol) and trimethyl orthoformate (106 mg,
1.00 mmol) were suspended in 1.25 ml of a 1:4 ratio mixture
of acetic anhydride and dimethylformamide. Mixture was
heated at 125 °C for 2 hours causing a yellow solid to form.
Mixture was cooled to room temperature and filtered.
Residue was washed with 20 ml of ethyl ether to afford the
1
product as a yellow solid (109 mg, 0.380 mmol, 77%); H
NMR (DMSO-d6) d 4.25 (s, 3H),7.60 (dd, J = 1.9, 8.4 Hz,
1H), 7.96 (d, J = 8.5 Hz, 1H), 8.07 (s, 1H), 8.36 (d,
J = 1.9 Hz, 1H), 11.38 (s, 1H); mass spectrum [(+)ESI], m/
z 282/284 (M+H)+.
Step 4: 6-Bromo-4-{4-(4-methyl-piperazin-1-yl)-phenyl-
amino]methylene}-4H- isoquinoline-1,3-dione. 6-Bromo-
4-methoxymethylene-4H-isoquinoline-1,3-dione (1.46 g,
5.18 mmol) and 4-(4-methyl-piperazin-1-yl)-phenylamine
12. Pautsch, A.; Zoephel, A.; Ahorn, H.; Spevak, W.; Hau-
ptmann, R.; Nar, H. Structure 2001, 9(10), 955.