Synthesis and structure-activity relationships of N-6 substituted analogues of 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones as inhibitors of Wee1 and Chk1 checkpoint kinases

Article abstract of DOI:10.1016/j.ejmech.2007.07.016

A series of N-6 substituted 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H)-diones were prepared from N-substituted (5-methoxyphenyl)ethenylindoles. The target compounds were tested for their ability to inhibit the G2/M cell cycle checkpoint kinases, Wee1 and Chk1. Analogues with neutral or cationic N-6 side chains were potent dual inhibitors. Acidic side chains provided potent (average IC₅₀ 0.057 μM) and selective (average ratio 223-fold) Wee1 inhibition. Co-crystal structures of inhibitors bound to Wee1 show that the pyrrolo[3,4-c]carbazole scaffold binds in the ATP-binding site, with N-6 substituents involved in H-bonding to conserved water molecules. HT-29 cells treated with doxorubicin and then target compounds demonstrate an active Cdc2/cyclin B complex, inhibition of the doxorubicin-induced phosphorylation of tyrosine 15 of Cdc2 and abrogation of the G2 checkpoint.

Full text of DOI:10.1016/j.ejmech.2007.07.016

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 43 (2008) 1276e1296
http://www.elsevier.com/locate/ejmech
Original article
Synthesis and structureeactivity relationships of N-6 substituted
analogues of 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H )-
diones as inhibitors of Wee1 and Chk1 checkpoint kinases
b
c
c
Jeff B. Smaill a, , Edward N. Baker , R. John Booth , Alexander J. Bridges ,
James M. Dickson b, Ellen M. Dobrusin c, Ivan Ivanovic b, Alan J. Kraker c, Ho H. Lee a,
Elizabeth A. Lunney d, Daniel F. Ortwine c, Brian D. Palmer a, John Quin III c,
Christopher J. Squire b, Andrew M. Thompson a, William A. Denny a
*
a Auckland Cancer Society Research Centre, School of Medical Sciences, The University of Auckland, Private Bag 92019, Auckland 1020, New Zealand
b Laboratory of Structural Biology, School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1020, New Zealand
c Pfizer Global Research and Development, Michigan Laboratories, 2800 Plymouth Road, Ann Arbor, MI 48105-1047, USA
d Pfizer Global Research and Development, La Jolla Laboratories, 10614 Science Center Drive, La Jolla, CA 92121, USA
Received 16 April 2007; received in revised form 12 July 2007; accepted 12 July 2007
Available online 6 August 2007
Abstract
A series of N-6 substituted 9-hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H )-diones were prepared from N-substituted (5-methoxy-
phenyl)ethenylindoles. The target compounds were tested for their ability to inhibit the G2/M cell cycle checkpoint kinases, Wee1 and
Chk1. Analogues with neutral or cationic N-6 side chains were potent dual inhibitors. Acidic side chains provided potent (average IC50
0.057 mM) and selective (average ratio 223-fold) Wee1 inhibition. Co-crystal structures of inhibitors bound to Wee1 show that the
pyrrolo[3,4-c]carbazole scaffold binds in the ATP-binding site, with N-6 substituents involved in H-bonding to conserved water molecules.
HT-29 cells treated with doxorubicin and then target compounds demonstrate an active Cdc2/cyclin B complex, inhibition of the doxorubicin-
induced phosphorylation of tyrosine 15 of Cdc2 and abrogation of the G2 checkpoint.
Ó 2007 Elsevier Masson SAS. All rights reserved.
Keywords: Phenylpyrrolocarbazole; Wee1; Chk1; G2/M checkpoint abrogation
1. Introduction
mediated phosphorylation [3e5]. Thus inhibition of Wee1
and/or Chk1 kinases can result in abrogation of the G2 check-
point induced by DNA damage, preferentially enhancing the
cytotoxic effects of DNA damaging agents within p53-
negative cells (which lack the ability to repair DNA damage
at a functional G1/S checkpoint).
While several small molecule inhibitors of Chk1 have been
reported, including the indolocarbazole staurosporine (1) [4],
UCN-01 (2) [4], Go6976 (3) [6] and isogranulatimide (4) [7],
the only substantive reports of Wee1 inhibitors concern 6-
phenylpyrido[2,3-d]pyrimidin-7(8H )-ones, e.g., PD 166285
(5) [2] and phenyl-substituted analogues [8]. These compounds
inhibit a significant number of kinases, most notably c-Src.
There is currently wide interest in the development of drugs
that can modulate the cell cycle checkpoints in eukaryotic
cells. A key control mechanism at the G2/M checkpoint is
inhibitory phosphorylation of Cdc2 Tyr15, mainly by the reg-
ulatory kinase Wee1 [1,2]. Removal of this inhibitory phos-
phate is mediated by the phosphatase Cdc25C, which itself
is inactivated (following DNA damage) by Chk1/Chk2
* Corresponding author. Tel.: þ64 9 3737 599 x 86626; fax: þ64 9 3737
502.
E-mail address: j.smaill@auckland.ac.nz (J.B. Smaill).
0223-5234/$ - see front matter Ó 2007 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2007.07.016

J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
1277
We recently reported that a series of 4-phenylpyrrolocarba-
zoles (e.g., 6) were potent inhibitors of Wee1 (IC50 97 nM)
and Chk1 (IC50 47 nM), without concomitant c-Src activity
[9]. Structureeactivity studies around this lead showed that
small lipophilic groups (e.g., Cl) at the 20-position of the pen-
dant phenyl ring enhanced selectivity for Wee1, by increasing
potency for Wee1 kinase (compare compounds 6 and 7 in
Table 1) and lowering activity against the Chk1 enzyme.
Larger lipophilic N-6 substituents were also found to improve
selectivity for Wee1, again by reducing Chk1 activity (com-
pare compounds 7, 10 and 11, Table 1). Since these substitu-
tions did not appreciably alter Wee1 activity, this improved
Wee1 selectivity was considered to be due to intolerance of
hydrophobic bulk at this site in the Chk1 enzyme rather than
favourable lipophilic interactions with Wee1. A general draw-
back of this series of Wee1 inhibitors was their poor aqueous
solubility, and the above result suggested the possibility of
utilising the N-6 site for the incorporation of hydrophilic,
solubilising functionality without loss of potency. In this
paper, we explore the influence of a variety of neutral, basic
and acidic solubilising groups at the N-6 position of the 9-
hydroxy-4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H )-diones
on solubility, selectivity of Wee1 inhibition and G2/M check-
point abrogation in vitro.
electrophiles, typically alkyl bromides, using sodium hydride
as the base, to give for example the N-substituted indoles
(71aed and 75aec) (Scheme 1). Deprotection of the tert-
butyldimethylsilyl-protected alcohols (71aed) or tetrahydro-
pyranyl-protected alcohols (75aec) was carried out at this
stage or after the DielseAlder and aromatisation steps that
form the 4-phenylpyrrolocarbazole ring system. To this end
acid-promoted deprotection of 71c and 75b,c gave 72 and
76b,c, respectively. DielseAlder reaction of the N-substituted
dienes (71a,b,d, 72, 75a, 76b,c) with maleimide was performed
in refluxing toluene, with a catalytic amount of tin (II) chloride,
to give the respective DielseAlder adducts as mixtures of dia-
stereomers. These were aromatised directly using either excess
activated MnO2 or 2e5 equivalents of DDQ to give the N-6
substituted 4-phenylpyrrolocarbazoles, which were further sub-
jected to acid-promoted deprotection where necessary to give
73aed and 77aec. Finally, 9-O-demethylation to give com-
pounds 12e17 of Table 1 was achieved using either pyridine hy-
drochloride at 200 ꢀC or 2e10 equivalents of BBr3 in CH2Cl2 at
room temperature.
Similarly, alkylation of the diene (70b) with acrylonitrile
and epibromohydrin gave nitrile 78 and epoxide 79, respec-
tively (Scheme 2). Treatment of the epoxide 79 with methyl-
amine and dimethylamine gave 80 and 81, respectively.
DielseAlder reaction of 78, 80 and 81 followed by aromatisa-
tion and 9-O-demethylation then gave compounds 22, 57 and
58 of Table 1.
H
N
H
N
H
N
O
O
R
O
O
We have previously reported [9] that late stage alkylation at
the N-6 position could be achieved selectively by protection of
the N-2 position with a 2,4-dimethoxybenzyl group, such as in
4-phenylpyrrolocarbazole 85 (Scheme 3). Potassium carbonate
mediated alkylation of 85 with allyl bromide gave 86, which
was dihydroxylated with osmium tetroxide in the presence
of N-methylmorpholine-N-oxide to give diol 87. Imide depro-
tection was achieved with trifluoroacetic acid and anisole at
90 ꢀC. Subsequent 9-O-demethylation with BBr3 gave com-
pound 26 of Table 1.
Compounds 27e53 of Table 1, bearing basic N-6 substitu-
ents tethered with an ethyl or propyl side chain, were synthes-
ised by amine displacement of mesylates 88aec and bromides
89aec, respectively, via both singleton and array synthesis
methods (Scheme 4). Mesylates 88aec were prepared by reac-
tion of the ethyl alcohols (77aec) with methanesulfonyl chlo-
ride, followed by 9-O-demethylation with BBr3. Interestingly,
application of the same procedure to the propyl alcohols
(73aec) gave predominantly the propyl bromides (89aec), re-
sulting from displacement of mesylate by bromide during the
BBr3 mediated demethylation. In instances where this dis-
placement was incomplete, an additional treatment with lith-
ium bromide in ethyl acetate at 50 ꢀC was performed on the
mixture to increase the yield of bromide.
N
N
N
N
Me
N
H
N
N
O
4
CN
MeO
NHMe
3
1: R = H; 2: R = OH
Cl
H
N
1
3
N
O
O
Cl
HN
O
N
N
O
9
HO
4
Me
N
5
6
H
6
NEt2
2. Chemistry
The 4-phenylpyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione ring
system was generally constructed as reported previously [9]
through DielseAlder reactions between maleimide and phenyl-
substituted dienes, followed by aromatisation. Introduction of
side chains at what is eventually the carbazole N-6 position
was best performed on the phenyl-substituted dienes (70aed)
[9,10] usually isolated as an E/Z mixture of alkene isomers, prior
to construction of the 4-phenylpyrrolocarbazole ring system.
This prevents an alkylation regioselectivity problem as the im-
ide N-2 position is the preferred site of alkylation of the intact
chromophore. Thus, 70aed [9] were reacted with various
Reaction of 89b with sodium cyanide in DMSO gave com-
pound 24 of Table 1 (Scheme 5), while reaction with sodium
methoxide in refluxing methanol gave the corresponding methyl
ether together with concomitant imide ring opening and subse-
quent anhydride formation, resulting in 90. Treatment of 90 with
ammonium formate at 140 ꢀC gave compound 25 of Table 1.

1278
J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
Table 1
Inhibitory activity of 4,6-disubstituted 9-hydroxypyrrolo[3,4-c]carbazole-1,3(2H,6H )-diones
H
N
O
O
3
Z
2
4
HO
5
6
N
R
No
R
Z
IC50 (mM)
Solb
Calc. pKac
Synd
Wee1a
Chk1a
Ratio
Neutral N-6 side chains
6e
H
H
0.097
0.011
0.028
0.14
0.047
0.48
7e
H
2-Cl
2,6-diCl
H
0.44
1.2
40
43
8e
9e
H
Me
0.056
0.22
0.40
3.9
593
10e
11e
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
Me
n-Bu
2-Cl
2-Cl
H
0.057
0.059
0.025
0.045
0.008
0.20
35
(CH2)2OH
(CH2)2OH
(CH2)2OH
(CH2)3OH
(CH2)3OH
(CH2)3OH
(CH2)3OH
(CH2)2CONH2
(CH2)2CONH2
(CH2)2CONH2
(CH2)2CN
(CH2)2COOMe
(CH2)3CN
(CH2)3OMe
CH2CH(OH)CH2OH
0.088
0.42
0.58
3.5
9.3
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
2-Cl
2,6-diCl
H
<3
<3
<3
<3
<3
<3
<3
73
0.30
19
59
0.60
0.059
0.17
0.41
2-Cl
2,6-diCl
2-OMe
H
0.009
0.007
0.030
0.021
0.006
0.33
0.018
0.035
0.054
0.36
1.7
9.0
1.1
2-Cl
2,6-diCl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
<3
<3
<3
0.015
0.030
0.033
0.027
0.023
0.20
0.20
13
6.7
7.0
6.7
3.3
0.23
0.18
<3
12
0.077
Basic N-6 side chains
(CH2)2NMe2
(CH2)2NMe2
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
H
0.20
0.054
0.11
0.85
0.28
0.77
4.3
0.27
1.1
18
9.4
9.4
9.4
7.4
7.4
7.4
7.1
7.1
7.1
10.6
10.6
10.6
9.6
9.6
9.6
7.7
7.7
7.7
7.2
7.2
7.2
7.9
7.9
7.9
4.9
4.9
4.9
8.9
S
2-Cl
2,6-diCl
H
0.096
0.17
0.14
8.5
7.4
14
A
A
S
(CH2)2NMe2
5.0
2.0
(CH2)2Nmorpholide
(CH2)2Nmorpholide
(CH2)2Nmorpholide
(CH2)2Nimidazolide
(CH2)2Nimidazolide
(CH2)2Nimidazolide
(CH2)3NHMe
2-Cl
2,6-diCl
H
0.064
0.11
0.23
12
39
1.1
2.9
28
<3
A
A
S
0.25
0.27
3.4
2-Cl
2,6-diCl
H
0.092
0.12
0.28
A
A
S
<3
0.002
0.012
0.005
0.021
0.029
0.096
0.38
1.1
0.01
0.17
0.05
0.06
0.29
0.69
1.3
(CH2)3NHMe
(CH2)3NHMe
2-Cl
2,6-diCl
H
0.069
0.11
0.36
5.2
7.3
A
A
S
(CH2)3NMe2
(CH2)3NMe2
(CH2)3NMe2
2-Cl
2,6-diCl
H
0.10
0.14
A
A
S
(CH2)3Nmorpholide
(CH2)3Nmorpholide
(CH2)3Nmorpholide
(CH2)3Nimidazolide
(CH2)3Nimidazolide
(CH2)3Nimidazolide
(CH2)3N(4-Mepiperazine)
(CH2)3N(4-Mepiperazine)
(CH2)3N(4-Mepiperazine)
(CH2)3NHPh
0.29
<3
<3
2-Cl
2,6-diCl
H
0.071
0.064
0.11
15
92
0.33
1.0
22
0.67
10
27
2.3
A
A
S
5.9
0.036
0.055
1.3
2-Cl
2,6-diCl
H
0.054
0.059
0.30
A
A
A
A
A
S
3.2
28
0.20
0.83
1.7
2-Cl
2,6-diCl
H
0.082
0.062
0.093
0.074
0.067
0.17
0.21
4.7
(CH2)3NHPh
(CH2)3NHPh
(CH2)2CONH(CH2)2NMe2
2-Cl
2,6-diCl
H
64
11
A
A
S
0.73
0.027
0.16
>60

J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
1279
Synd
Table 1 (continued)
No
R
Z
IC50 (mM)
Solb
Calc. pKac
Wee1a
Chk1a
0.053
0.75
Ratio
1.5
54
0.05
0.52
55
56
57
58
(CH2)2CONH(CH2)2NMe2
(CH2)2CONH(CH2)2NMe2
CH2CH(OH)CH2NHMe
CH2CH(OH)CH2NMe2
2-Cl
0.035
0.014
0.057
0.058
>60
>60
8.9
8.9
9.5
8.9
S
S
S
S
2,6-diCl
2-Cl
2-Cl
0.003
0.030
>60
Acids/acid isosteres/misc.
59
60
61
62
63
64
65
66
67
68
69
a
(CH2)2CO2H
(CH2)2CO2H
(CH2)2CO2H
H
0.023
0.009
0.39
0.41
4.4
18
>60
>60
33
4.6
4.6
4.6
4.6
5.0
5.3
S
S
S
S
S
S
S
S
S
S
S
2-Cl
2,6-diCl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
2-Cl
489
>26
209
1143
67
>10
(CH2)2CONHSO2Me
(CH2)2CONHSO2Ph
(CH2)2Ctetrazole
(CH2)2Striazole
(CH2)2SOtriazole
(CH2)2SO2triazole
(CH2)3CO2H
0.012
0.007
0.021
0.024
0.009
0.019
0.013
0.016
2.5
8.0
>60
47
1.4
>60
3
0.42
0.12
0.077
0.12
0.63
18
13
18
16
4.1
9.2
39
6.6
4.8
5.4
(CH2)3Ctetrazole
>60
IC50: concentration of drug (mM) to inhibit the phosphorylation of a model polyornithineetyrosine copolymer by Wee1 kinase and of a GSTeCdc25 substrate
by Chk1 kinase. For active compounds, values are an average of two or more separate determinations; variation was generally ꢁ30%.
b
High throughput aqueous solubility screen (mg/mL) where <3 ¼ poor solubility, 3e60 ¼ moderate solubility, >60 ¼ acceptable solubility.
c
Calculated using ACDLabs log P suite version 5.0 with the chromophore set as carbazole.
S, singleton synthesis; A, array synthesis.
Ref. [9].
d
e
MeO
MeO
MeO
Cl
Cl
i
ii
Z
N
H
N
N
for c
Z
OTBDMS
70
71
72
OH
a: Z=H
a: Z=H
iii,ii
b: Z=2-Cl
b: Z=2-Cl
iii
c: Z=2,6-diCl
c: Z=2,6-diCl
d: Z=2-Cl-6-OMe
for a,b,d
d: Z=2-Cl-6-OMe
H
H
N
N
O
O
O
vi
for a,b,c
O
iv
MeO
HO
MeO
Z
Z
N
N
N
Z
73
15: Z=H
OH
OH
OTHP
75
16: Z=2-Cl
17: Z=2,6-diCl
a: Z=H
b: Z=2-Cl
a: Z=H
iii,vii
for a
74 Z=2-Cl-6-OMe
18: Z=2-OMe
v
b: Z=2-Cl
c: Z=2,6-diCl
c: Z=2,6-diCl
d: Z=2-Cl-6-OMe
H
H
for b,c
vii
N
N
O
O
O
O
MeO
iii
iv
MeO
HO
N
Z
Z
N
N
Z
77
OH
OH
OH
76
a: Z=H
12: Z=H
b: Z=2-Cl
13: Z=2-Cl
b: Z=2-Cl
c: Z=2,6-diCl
14: Z=2,6-diCl
c: Z=2,6-diCl
Scheme 1. (i) NaH, DMF, Br(CH2)3OTBDMS, rt; (ii) 1e3 N HCl, MeOH/co-solvent, rt; (iii) (a) maleimide, cat. SnCl2, toluene, reflux; (b) MnO2, p-dioxane, reflux
or DDQ, toluene/p-dioxane, reflux; (iv) BBr3, CH2Cl2, 0 ꢀC to rt or pyridine$HCl, 200 ꢀC; (v) H2, Pd/C, MeOH/EtOAc, rt; (vi) NaH, DMF, Br(CH2)2OTHP, rt;
(vii) p-TsOH, MeOH, 50 ꢀC or 2 N HCl, MeOH/THF, rt.

1280
J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
MeO
H
H
N
iv
v
N
i
O
O
70b
of Scheme 1
O
O
N
R
MeO
HO
Cl
Cl
Cl
78: R=(CH2)2CN
N
R
N
R
79
: R=
O
22: R=(CH2)2CN
ii
82: R=(CH2)2CN
iii
80: R=CH2CH(OH)CH2NHMe
81: R=CH2CH(OH)CH2NMe2
57: R=CH2CH(OH)CH2NHMe
58: R=CH2CH(OH)CH2NMe2
83: R=CH2CH(OH)CH2NHMe
84: R=CH2CH(OH)CH2NMe2
Scheme 2. (i) DBU, CH3CN, acrylonitrile, rt (for 78); or NaH, DMF, epibromohydrin, rt (for 79); (ii) MeNH2, THF, 60 ꢀC; (iii) Me2NH, THF, 60 ꢀC; (iv) (a)
maleimide, cat. SnCl2, toluene, reflux; (b) MnO2, p-dioxane, reflux or DDQ, toluene/p-dioxane, reflux; (v) BBr3, CH2Cl2, 0 ꢀC to rt or pyridine$HCl, 200 ꢀC.
Two-step oxidation of alcohols 73aec with DesseMartin
periodinane and then sodium chlorite gave the carboxylic
acids (91aec) (Scheme 6). Conversion of these to the acid
chlorides with oxalyl chloride/DMF, followed by reaction
with ammonia or N,N-dimethylethylenediamine, gave the pri-
mary amides (92aec) and solubilised amides (93aec), respec-
tively. 9-O-Demethylation of the primary amides (92aec) was
best achieved with BBr3, giving compounds 19e21 of Table 1
(pyridine hydrochloride at 200 ꢀC caused significant amide
hydrolysis). Conversely, 9-O-demethylation of the solubilised
amides (93aec) was best achieved with pyridine hydrochlo-
ride at 200 ꢀC, giving compounds 54e56 of Table 1 (amide
hydrolysis appeared to be slower, while formation of the
HBr salt of starting material and subsequent insolubility was
problematic when BBr3 was used). 9-O-Demethylation of
91aec with either BBr3 or pyridine hydrochloride at 200 ꢀC
gave compounds 59e61 of Table 1. Standard esterification
of 60 in methanolic HCl gave compound 23 of Table 1. Alkyl-
ation of the diene 70b [9] with ethyl bromobutyrate, followed
by DielseAlder reaction with maleimide, aromatisation with
DDQ and ester saponification gave the acid 94. Subsequent
9-O-demethylation with BBr3 gave compound 68 of Table 1.
Reaction of the nitriles (22 and 24) with azidotrimethyl tin
in DMF/toluene at reflux gave tetrazoles 64 and 69 of Table
1, respectively. Mesylation of the alcohol 77b to give mesylate
97 and subsequent displacement with 1H-1,2,4-triazole-5-thiol
in p-dioxane at reflux gave the sulfur-linked triazole (98). 9-O-
Demethylation of this with BBr3 gave compound 65 of Table 1.
Oxidation of 65 with 35% hydrogen peroxide in THF contain-
ing glacial acetic acid at either room temperature or for a
prolonged period at 50 ꢀC gave compounds 66 and 67 of
Table 1, respectively.
3. Results and discussion
3.1. Isolated enzyme activity
Table 1 gives the structures and enzyme inhibitory activities
of 9-hydroxy-4-phenylpyrrolocarbazole analogues bearing
a wide range of N-6 substituents. The goal of this work was
to understand the influence of N-6 substitution on the potency
and selectivity of Wee1/Chk1 inhibition, while providing com-
pounds with sufficient solubility to be studied for their ability
1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide
hydro-
73a-c
77a-c
chloride (EDCI$HCl)-promoted coupling of methanesulfona-
mide and benzenesulfonamide with 91b in the presence of
3 equivalents of 4-dimethylaminopyridine, gave the acyl sul-
fonamides (95 and 96, respectively) (Scheme 7). BBr3 deme-
thylation of these gave compounds 62 and 63 of Table 1.
of Scheme 1
of Scheme 1
i, ii, iii
i, ii
H
H
N
N
O
O
O
O
MeO
N
OMe
H
N
HO
HO
O
O
Z
Z
N
N
O
HO
O
88
iii
89
OMs
Br
Cl
N
MeO
a: Z=H
a: Z=H
b: Z=2-Cl
c: Z=2,6-diCl
OH
OH
b: Z=2-Cl
c: Z=2,6-diCl
Cl
N
R
iv
85: R=H
86: R=CH2CH=CH2
87: R=CH2CH(OH)CH2OH
26
i
ii
compounds 27-53 of Table 1
Scheme 3. (i) K2CO3, DMF, allyl bromide, 90 ꢀC; (ii) OsO4, NMMO, acetone/
water, rt; (iii) (a) TFA, anisole, 90 ꢀC; (b) BBr3, CH2Cl2, 0 ꢀC to rt.
Scheme 4. (i) MsCl, Et3N, THF, 0 ꢀC; (ii) BBr3, CH2Cl2, 0 ꢀC to rt; (iii) LiBr,
EtOAc, 50 ꢀC; (iv) R1R2NH, DMA, rt to 80 ꢀC.

J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
1281
H
H
N
N
O
O
O
O
i
91b
89b
i
MeO
HO
of Scheme 6
of Scheme 4
Cl
NHSO2R
Cl
N
N
ii
22
of Scheme 2
95: R=CH3
96: R=Ph
CN
24
O
O
24
O
O
iii
of Scheme 5
iii
ii
HO
compound 25
of Table1
Cl
N
77b
compounds 62-64, 69 of Table 1
of Scheme 1
OMe
90
H
iv
O
N
O
O
Scheme 5. (i) NaCN, DMSO, rt; (ii) NaOMe, MeOH, reflux; (iii) NH4OAc,
140 ꢀC.
H
N
O
RO
v
MeO
Cl
N
N
to abrogate the G2/M checkpoint in vitro. Wee1 selective and
dual Wee1/Chk1 analogues were desired for comparative pur-
poses. We have previously reported [9] that substitution of the
4-phenyl ring with 20-chloro or 20,60-dichloro substituents in-
creases potency for Wee1, and selectivity over Chk1. While
a broad range of phenyl substituents was explored in the
N
Cl
OSO2Me
N
(O)nS
N
H
98: n=0, R=CH3
65: n=0, R=H
66: n=1, R=H
67: n=2, R=H
97
ii
vi
vii
73a-c of Scheme 1
H
N
Scheme 7. (i) CH3SO2NH2 (PhSO2NH2), DMAP, EDCI$HCl, DMF, rt; (ii)
BBr3, CH2Cl2, 0 ꢀC to rt; (iii) Me3SnN3, DMF/toluene, reflux; (iv) MsCl,
Et3N, THF, 0 ꢀC; (v) 1H-1,2,4-triazole-5-thiol, Et3N, p-dioxane, reflux; (vi)
35% H2O2, HOAc, THF, rt; (vii) 35% H2O2, HOAc, THF, rt to 50 ꢀC.
O
O
i
MeO
H
ii
N
O
Z
O
N
present study our findings were consistent with the previously
reported SAR for the 4-phenyl ring. Therefore only phenyl, 20-
chloro and 20,60-dichloro are reported herein, with the notable
exception of a single 20-methoxy analogue (18), which was
successfully co-crystallised with a construct of Wee1 provid-
ing valuable information about interactions between the kinase
ATP-binding site and the 20-methoxy and N-6-substituents (see
later).
As noted above, previous studies had suggested that larger
lipophilic N-6 substituents allowed retention of nanomolar
Wee1 activity, while also improving selectivity with respect
to Chk1 activity (compare compounds 7, 10 and 11). Com-
pounds 12e26 bearing neutral substituents of increased polar-
ity also showed retention of nanomolar Wee1 activity, but
these substituents were less effective at retaining high selectiv-
ity over Chk1 (compare 11 with 16, 24 and 25). A number of
analogues bearing alcohol (14, 16 and 17) or primary amide
(20) functionalities possessed very potent activity for inhibi-
tion of Wee1 (IC50 values of 6e9 nM), but these neutral polar
groups did not confer significant increases in aqueous
solubility.
92
MeO
a: Z=H
CONH2
b: Z=2-Cl
Z
N
c: Z=2, 6-diCl
91
iv
a: Z=H
COOH
b: Z=2-Cl
iv
c: Z=2, 6-diCl
iii
compounds
19-21, 54-56,
59-61, 68
H
N
O
O
iv
of Table 1
MeO
Z
N
O
iv
H
93
a: Z=H
NMe2
b: Z=2-Cl
N
H
c: Z=2, 6-diCl
N
O
O
MeO
v
70b
of Scheme 1
Cl
COOH
N
94
Compounds 27e58 explore a more extensive range of cat-
ionic N-6 side chains, again in combination with 20-chloro and
20,60-dichloro substitution of the 4-phenyl ring. Introduction of
a cationic N-6 side chain resulted in similar or modestly re-
duced (<2-fold) Wee1 potency, when compared to the corre-
sponding N-6 methyl substituted compound. The nature of
Scheme 6. (i) (a) DesseMartin periodinane, THF, rt; (b) NaClO2, 2-methyl-2-
butene, NaH2PO4, t-BuOH/THF/water, rt; (ii) (a) ClCOCOCl, DMF, THF, rt;
(b) NH3, THF, rt; (iii) (a) ClCOCOCl, DMF, THF, rt; (b) Me2N(CH2)2NH2,
THF, rt; (iv) pyridine$HCl, 200 ꢀC or BBr3, CH2Cl2, 0 ꢀC to rt; (v) (a)
Br(CH2)3COOEt, NaH, DMF, rt; (b) maleimide, cat. SnCl2, toluene, reflux;
(c) DDQ, p-dioxane, reflux; (d) 2 M KOH, MeOH, rt.

1282
J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
the pendant amine, in terms of steric bulk or pKa, had little in-
fluence. Conversely, increased inhibition of Chk1 was noted
with small, strongly basic amine substituents. Methylamine
pendant on a three carbon chain provides a particular potency
advantage (see compounds 36e38). It is interesting to note
that the known Chk1 inhibitory natural product staurosporine
(1) and its derivative UCN-01 (2) also possess a C-3 methyl-
amine motif (as part of a glycosyl moiety) in this relative
position. The reported crystal structure of UCN-01 bound in
the ATP-binding site of Chk1 [11] indicates that the glycosyl
moiety is positioned in the ribose-binding pocket, with the
protonated methylamine residue forming hydrogen bonds to
Glu91 and Glu134. It seems likely that a similar interaction
between the methylamine moieties of compounds 36e38
and the Chk1 protein are providing the potency advantage
observed for these compounds. Compounds 54e58 of Table 1
represent an attempt to combine the preferred alcohol and
amide functionalities discussed above with cationic side
chains capable of providing improved solubility. These com-
pounds were potent dual inhibitors of Wee1 and Chk1, with
the exception of 56, which was a selective Wee1 inhibitor
(54-fold). The aqueous solubility of amines 27e53 at pH 6.9
was generally poor, while the combination side chains 54e58
gave improved solubility. Most of the amines did show
improved solubility when measured at lower pH (data not
shown), with compound 55, for example, having a solubility
of 1.2 mg/mL at pH 5.4 (acetate buffer).
Compounds 59e64 and 67e69 of Table 1 contain acid and
acid isostere bearing side chains, with calculated pKa values
ranging from 4.6 to 6.6. Compounds 65 and 66 are intermediates
in the synthesis of compound 67 and were also included for ki-
nase screening. The C-2 carboxylic acids (59e61) explored the
effect of phenyl ring substitution in this series, while compounds
62e69 contained the 20-chlorophenyl substitution pattern that
provides the highest Wee1 potency. As a sub-class, the com-
pounds bearing an acidic side chain (59e64, 67e69) had the
highest potency against Wee1 (average IC50 0.057 mM) and
the highest Chk1/Wee1 ratios (average ratio 223-fold). This
finding is consistent with unfavourable electrostatic interactions
between the acidic side chains of the inhibitors and the carbox-
ylate side chains of Glu91 and Glu134 present in this region of
the Chk1 protein [11]. Several of these compounds also had
good solubilities.
3.2. Crystal structures of N-6 substituted inhibitors
bound to Wee1 kinase
Fig. 1. Crystal structures of compound 6 [12] (a), 18 (b) and 59 (c) bound in
the ATP-binding site of a construct of Wee1 kinase. Important hydrogen bonds
between the ligand, protein and ordered water molecules in the active site are
indicated with dotted orange lines (for interpretation of the references to col-
our in this figure legend, the reader is referred to the web version of this
article).
˚
We have previously reported the structure, at 1.8 A resolu-
tion, of an inhibitor bound co-crystal of 6 and the Wee1291e575
construct containing residues 291e575 of the human Wee1
enzyme [12]. The pyrrolocarbazole ring system binds in the
ATP-binding site of the kinase with the pendant 4-phenyl
ring twisted out of the plane of the chromophore (Fig. 1a).
Key hydrogen bond contacts are made between the 9-hydroxyl
group (donor) and Cys379(CO), the 1-carbonyl group (accep-
tor) and Cys379(NH), the 2-NH imide group (donor) and
Glu377(CO) and the 3-carbonyl group (acceptor) and
Asn376 side chain. Of particular relevance to this discussion
is that the carbazole 6-NH group donates an H-bond to the first
of an ordered lattice of three water molecules that link through
to Ser383. The 5-fold loss of potency observed following

J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
1283
simple alkyl substitution at this N-6 position (compare 7 with
10 and 11) is consistent with unfavourable disruption of this
lattice. We determined the co-crystal structures of both the
N-6 propyl alcohol 18 (Fig. 1b) and the N-6 propanoic acid
basic (compounds 31, 37, 55, 58) and acidic moieties (com-
pounds 62, 64, 68, 69). The compounds selected ranged
from 6-fold Chk1 selective to 209-fold Wee1 selective.
The compounds were evaluated in four in vitro assays
measuring target modulation and G2 checkpoint abrogation
in HT-29 cells (p53-negative) following sub-lethal DNA dam-
age by doxorubicin. The histone H1 kinase assay (HKA) has
been employed by Suganuma et al. to characterise the G2/M
checkpoint abrogation of peptide inhibitors of human Chk1
[3]. This assay indirectly measures the effect of test com-
pounds on the kinase activity of the Cdc2/cyclin B complex
for phosphorylation of histone H1, one of its physiological
substrates. Thus strong phosphorylation of histone H1 repre-
sents an active Cdc2/cyclin B complex, where Tyr15 on
Cdc2 is non-phosphorylated. Wee1 inhibition blocks the
phosphorylation of Tyr15 on Cdc2, directly resulting in an
active Cdc2/cyclin B complex, while Chk1 inhibition blocks
phosphorylation of the phosphatase Cdc25C, ensuring it
remains active to cleave the phosphate from Tyr15 of Cdc2,
also resulting in an active Cdc2/cyclin B complex. The IC50
gives the concentration of drug required to permit a 50% acti-
vation of the Cdc2/cyclin B complex in cells, compared to
controls (Table 2). This is reported as 50% inhibition of the
regulatory kinases of the Cdc2/cyclin B complex. The mitotic
index assay used a polyclonal antibody to quantify the
M-phase specific histological markers, which were used to
determine the fraction of cells (% of total) found in mitosis
(Table 2). Thirdly, Western blots were used to directly assess
the Tyr15 phosphorylation (PY15) on Cdc2 kinase (the natural
59 (Fig. 1c) with the Wee1291e575 construct, to 2.2 A and
˚
˚
1.9 A resolution, respectively. Both inhibitors form identical
H-bond contacts from the pyrrolocarbazole chromophore to
the protein as does 6. As predicted by modelling studies [9]
the 20-methoxy group on the 4-phenyl ring of 18 is directed to-
wards a lipophilic pocket bounded by Ile374, Lys328 and
Val313. In both structures, the presence of an N-6 substituent
has resulted in displacement of the first two ordered water
molecules seen in the structure of 6. Displacement of the sec-
ond ordered water by the terminal oxygen functionality in the
N-6 side chain places this oxygen atom (in both inhibitors) in
a position to form an H-bond contact with the remaining third
ordered water, which bridges to Ser383. This finding is consis-
tent with the most potent N-6 substituted Wee1 inhibitors of
Table 1 generally possessing an H-bond acceptor as the fourth
atom of the substituent chain (compare 10 with 16, 20, 22, 60,
63, 66).
3.3. Cell-based assays for target modulation
To study the influence of the N-6 substituents on cellular
activity, a subset of compounds from Table 1 were selected.
The 4-phenyl substituent was kept constant as 20-chloro (on
average the most potent for Wee1 inhibition). The N-6 substit-
uents included neutral hydrophilic (compounds 16, 20, 22),
Table 2
In vitro data for selected N-6-substituted 4-(2-chlorophenyl)-9-hydroxypyrrolo[3,4-c]carbazole-1,3(2H,6H )-diones
H
N
O
O
HO
Cl
N
R
No
R
IC50 (mM)
Mitotic indexd (%)
Wee1a
Chk1a
HKAb
PY15c
16
20
22
31
37
55
58
62
64
68
69
a
(CH2)3OH
(CH2)2CONH2
0.009
0.006
0.015
0.064
0.069
0.035
0.058
0.012
0.021
0.013
0.016
0.17
0.054
0.20
0.77
0.012
0.053
0.030
2.5
0.65
>3.4
>3.4
1.7
0.25e2.5
>10
62 at 1.25 mM
47 at 2.50 mM
53 at 2.50 mM
65 at 2.50 mM
59 at 1.25 mM
65 at 2.50 mM
81 at 2.50 mM
Not active
(CH2)2CN
(CH2)2Nmorpholide
(CH2)3NHMe
<0.25
0.25
0.71
1.3
<0.25
<0.25
0.25
(CH2)2CONH(CH2)2NMe2
CH2CH(OH)CH2NMe2
(CH2)2CONHSO2Me
(CH2)2Ctetrazole
(CH2)3COOH
>3.4
>3.4
>3.4
>3.4
>3.4
0.25
2.5
1.4
Not active
30 at 2.50 mM
Not active
0.12
0.63
>10
>10
(CH2)3Ctetrazole
As for Table 1.
Histone H1 kinase assay; IC50 is the concentration of drug (mM) to inhibit the phosphorylation of histone H1 by Cdc2/cyclin B in HT-29 cells.
IC50 is the approximate concentration of drug required to produce a 50% reduction in phosphorylation of tyrosine 15 on Cdc2 kinase in HT-29 cells (from
b
c
western blots).
d
Fraction of doxorubicin pre-treated HT-29 cells (% of total) found in mitosis after treatment with an abrogator at the stated concentration.

1284
J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
Wee1 substrate) using a phosphospecific antibody (the signal
was normalised to the total amount of Cdc2 detected). Here
the IC50 value gives the approximate concentration of drug re-
quired to produce a 50% reduction of this phosphorylation in
HT-29 cells, compared to controls (Table 2). Finally, the clo-
nogenic survival of HT-29 cells that were treated with selected
compounds of Table 1 (at a range of doses), with and without
doxorubicin, was also measured (Fig. 2).
Compounds with neutral N-6 side chains (16, 20, 22) had
poor HKA activity, with the exception of 16, which had an
IC50 of 0.65 mM. Compound 16 also gave a robust abrogation
of the G2 checkpoint at a concentration of 1.25 mM, as mea-
sured in the mitotic index assay, with 62% of doxorubicin-
damaged cells found in mitosis. This compound inhibited
Cdc2 Tyr15 phosphorylation with an IC50 between 0.25 and
2.5 mM and gave a 45% reduction in clonogenic survival se-
lectively in doxorubicin treated HT-29 cells at a non-toxic
dose of 1 mM (Fig. 2).
Compounds with basic N-6 side chains (31, 37, 55, 58)
showed improved HKA activity. This is likely to be due to im-
proved solubility and cellular uptake. All of these compounds
showed potent inhibition of Cdc2 Tyr15 phosphorylation with
approximate IC50s of 0.25 mM or less, and abrogation of the
G2 checkpoint in combination with doxorubicin. In the latter
assay, the dual Wee1/Chk1 inhibitor 37 was the most dose-po-
tent compound, giving 59% of cells in mitosis at 1.25 mM,
while 31, 55 and 58 gave 65, 65 and 81% of cells in mitosis,
respectively, at 2.5 mM (Table 2). Compounds 31 (12-fold
Wee1 selective) and 55 (dual Wee1/Chk1 inhibitor) were stud-
ied in the clonogenic survival assay. Both showed significant
cytotoxicity as single agents at the highest dose tested
(10 mM), while compound 31 appeared to have the better ther-
apeutic index, giving a 77% reduction in clonogenic survival
selectively in doxorubicin treated HT-29 cells at a non-toxic
dose of 2.5 mM (Fig. 2).
of Cdc2 Tyr15 with approximate IC50s of 0.25 mM and
2.5 mM, respectively, compounds 68 and 69 were inactive
in this assay. All the compounds were essentially inactive
in the mitotic index assay. Compounds 62, 63 and 69 were
also evaluated in the clonogenic survival assay; both 62
and 63 were inactive over all concentrations tested, while
69 was too toxic as a single agent to draw any conclusions
on G2 checkpoint abrogation (Fig. 2). The general lack of
activity for this anionic sub-class of compounds across
a range of cellular assays may be due to poor cellular perme-
ability or protein binding, typical of acid moieties.
3.4. Kinase selectivity
Selected compounds were screened against a panel of 28
kinases at a concentration of 10 mM [13], as shown in
Table 3. Compounds 62 and 69 bearing acidic N-6 side chains
were the most selective for Wee1. Compounds bearing
alcohol and amine N-6 side chains (7, 16, 17, 36, 37, 55)
were less selective, showing inhibition of a number of other
kinases. As a class, the N-6 substituted 9-hydroxypyr-
rolo[3,4-c]carbazole-1,3(2H,6H )-diones appear also to be
inhibitors of MAPK-activated protein kinase 1 (MAPKAP-
K1a), AMP-activated protein kinase (AMPK) and checkpoint
kinase 1 (Chk1) (as described above). A number of com-
pounds tested also showed activity against 3-phosphoinosi-
tide-dependent protein kinase 1 (PDK1), phosphorylase
kinase and lymphocyte kinase (Lck). Representative com-
pounds were also screened against c-Src, and as reported
previously [9] for compound 6, nearly all were inactive
(IC50s: 7, >50; 26, 2.5; 27, 31; 33, 27; 39, 5.6; 42, 46; 48,
17; 59, 13 mM). Introduction of side chains at the N-6 posi-
tion of the 4-phenylpyrrolocarbazole chromophore allows ac-
cess to the ribose-binding pocket within the ATP-binding
domain. It would appear that appending polar functionality
in this region from a relatively flexible chain might be
counter-productive in terms of kinase selectivity. This lack
of selectivity may in part be responsible for the single agent
Compounds with acid and acid isostere N-6 side chains
(62, 64, 68, 69) were uniformly inactive in the cell-based HKA
assay. While 62 and 64 showed inhibition of phosphorylation
120.0
100.0
80.0
60.0
40.0
20.0
0.0
without DOX with DOX
Compounds of Table 1 (µM)
Fig. 2. Soft agar clonogenic assay of HT-29 cells treated with compounds of Table 1, with and without doxorubicin.

J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
1285
Table 3
Kinase counter-screening of representative compounds
Kinasea
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28
7
49 54 84 52 69 53 95 13 93 58
6
72 58
0
0
26 19 44 35 29
12 37 36 38
8
4
4
2
101
8
11 33 38 17
28 38 79
22 21 14
2
1
4
16 13 36 83 50 49 18 72 NT 93 25 58 33 20
4
11 104 18
5
17 72 14 43 25 13
2
14
6
90 33 21 65 12 11 55 11 26 37 13
6
95
8
4
36
9
11 50 81 83 77 95
5
0
4
100
71
3
3
76 49
5
0
0
0
12
2
5
7
6
61 20
72 22
0
0
0
1
5
4
62
88
1
1
13 82 14 29 45
27 52 10 28 30
37 20 42 51 73 61 51 62
55 60 114 99 111 108 125 106
102
5
8
20
90 27 98
39 24 15 41 51 93 100
111 32
8
92 48 78 46
62 21 54 79 83 89 62 96 46 102 81 55 77 32 60 63 67 52 17 58 18
69 12 101 88 87 99 96 110 11 100 59 66 48 63 13 31
66 112 14
7
1
100 72 16 60 54 22 27
103 61 17 45 27 94 29
3
1
Kinase activity at 10 mM as a % of non-treated control, where red ¼ 20% or less; yellow ¼ 21e50%; and green ¼ 51% or greater (actual values in cell) (for in-
terpretation of the references to colour in this legend, the reader is referred to the web version of this article). Kinases tested [13]: (1) MKK1; (2) MAPK2/ERK2;
(3) JNK/SAPK1c; (4) SAPK2a/p38; (5) SAPK2b/p38b2; (6) SAPK3/p38g; (7) SAPK4/p38d; (8) MAPKAP-K1a; (9) MAPKAP-K2; (10) MSK1; (11) PRAK; (12)
PKA; (13) PKCa; (14) PDK1; (15) PKBa; (16) SGK; (17) S6K1; (18) GSK3b; (19) ROCK-II; (20) AMPK; (21) Chk1; (22) CK2; (23) PHOS. kinase; (24) Lck;
(25) CSK; (26) CDK2/cyclinA; (27) CK1; (28) DYRK1A.
cytotoxicity observed for some of these compounds in the
clonogenic survival assay described above.
5. Experimental protocols
Combustion analyses were performed by the Microchemi-
cal Laboratory, University of Otago, Dunedin, NZ. Analyses
indicated by the symbols of the elements or functions were
within ꢁ0.4% of the theoretical values. Melting points were
determined using an Electrothermal Model 9200 digital
melting point apparatus, and are as read. NMR spectra were
measured on a Bruker DRX-400 spectrometer or a Bruker
Avance-400 spectrometer, and are referenced to Me4Si. High
resolution mass spectra were recorded on a Varian VG-70SE
spectrometer at nominal 5000 resolution. Liquid chromatogra-
phy-mass spectrometry (LCMS) was performed on an Agilent
1100 LC system interfaced with an Agilent MSD mass detec-
tor. Mass detection was performed with an APCI source, using
simultaneous positive and negative ion acquisition. Compound
purities were determined by HPLC using simultaneous diode
array UV detection. Unless otherwise indicated, compounds
were purified by flash column chromatography on Silica gel
60 support (Scharlau, 230e400 mesh ASTM), using the indi-
cated eluents.
4. Conclusions
The influence of a range of neutral, basic and acidic solu-
bilising groups at the N-6 position of the 9-hydroxypyr-
rolo[3,4-c]carbazole-1,3(2H,6H )-dione class of Wee1/Chk1
kinase inhibitors has been investigated in combination with
previously described optimal substitutions in the 4-phenyl
ring [9]. As would be expected, the most basic or acidic side
chains provided the biggest improvements in aqueous solubil-
ity. Compounds bearing neutral (hydrophilic) or amine side
chains were dual inhibitors of Wee1 and Chk1, although this
could be influenced by appropriate substitution in the 4-phenyl
ring, with 20-chloro and 20,60-dichloro analogues, respectively,
being successively more Wee1 selective. Compounds with
acidic side chains were the most selective for Wee1 over
Chk1. Crystal structures of compounds 6, 18 and 59 bound
in the ATP-binding domain of Wee1 kinase have highlighted
an organized lattice of water molecules bridging from the car-
bazole 6-NH group of the unsubstituted inhibitor 6 through to
Ser383. Favourable displacement of (or H-bonding interaction
to) water molecules in this lattice by an N-6 side chain pro-
vides improved potency for inhibition of Wee1 kinase. Exam-
ples of both N-6 alcohol and N-6 amine bearing compounds
(but not the acidic compounds), at concentrations ranging
from 0.25 to 2.5 mM, resulted in an active Cdc2/cyclin B com-
plex in doxorubicin treated HT-29 cells, capable of phosphor-
ylating histone H1, abrogated the G2 checkpoint and inhibited
Cdc2 Tyr15 phosphorylation. They also produced a reduction
in clonogenic survival selectively in doxorubicin treated HT-29
cells. Counter-screening in a panel of 28 kinases indicated sig-
nificant off-target potency. Further studies to improve the aque-
ous solubility, cellular activity and kinase counter-screening
profile of this class of Wee1/Chk1 kinase inhibitors will be
reported in due course.
5.1. Procedures of Scheme 1
5.1.1. Procedure 1: sodium hydride mediated
indole alkylation
Sodium hydride (1.05 g of a 50% dispersion in mineral
oil, 0.022 mol) was added to a solution of 2-[(E,Z )-2-(2-
chlorophenyl)ethenyl]-5-methoxy-1H-indole (70b) [9] (4.13 g,
0.014 mol) in DMF (30 mL) and the solution was stirred at
room temperature for 5 min. (3-Bromopropoxy)-tert-butyldi-
methylsilane (4.04 g, 0.016 mol) was added and stirring was
continued for 2 h. The solution was diluted with water and ex-
tracted with EtOAc. The combined organic layers were
washed well with brine and the organic phase was dried
over anhydrous Na2SO4. The drying agent was removed by
filtration and the solution was concentrated to dryness, to

1286
J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
give 1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-2-[(E,Z )-2-
(2-chlorophenyl)ethenyl]-5-methoxy-1H-indole (71b) as an
oily solid (4.98 g), which was used without further
purification.
concentrated to dryness, adsorbed onto silica and chromato-
graphed. Elution with EtOAc/petroleum ether (1:1), followed
by EtOAc, gave 16 (0.90 g, 93%), which crystallised from
THF/petroleum ether as a yellow/orange powder; mp 291e
1
294 ꢀC. H NMR [(CD3)2SO] d 11.05 (br s, 1H), 9.36 (s,
5.1.2. Procedure 2: DielseAlder reaction
then aromatisation with MnO2
1H), 8.38 (d, J ¼ 2.5 Hz, 1H), 7.74 (s, 1H), 7.59e7.54 (m,
2H), 7.52e7.42 (m, 3H), 7.14 (dd, J ¼ 8.7, 2.5 Hz, 1H), 4.62
(m, 1H), 4.49 (t, J ¼ 6.8 Hz, 2H), 3.41 (m, 2H), 1.90 (m,
2H). Anal. C23H17ClN2O4$1/4H2O (C, H, N).
A mixture of 71b (4.98 g, 0.011 mol), maleimide (1.83 g,
0.019 mol) and powdered SnCl2 (40 mg, 0.21 mmol) in toluene
(80 mL) was heated at reflux for 16 h. The cooled solution was
added to aqueous NaHCO3 (100 mL) and extracted with EtOAc.
The crude DielseAlder adduct was then concentrated to dryness
and re-dissolved in p-dioxane (100 mL), before activated MnO2
(20 g, 0.23 mol) was added and the resulting suspension was
heated at reflux for 8 h. Filtration through Celite, concentration
under reduced pressure and chromatography on silica, eluting
with MeOH/EtOAc (1:9), gave 6-(3-{[tert-butyl(dimethyl)-
silyl]oxy}propyl)-4-(2-chlorophenyl)-9-methoxypyrrolo[3,4-
c]carbazole-1,3(2H,6H )-dione (4.87 g, 67% from 70b) as a
5.1.5. Procedure 5: DielseAlder reaction
then aromatisation with DDQ
Alkylation of 5-methoxy-2-[(E,Z )-2-phenylethenyl]-1H-in-
dole (70a) [10] (1.93 g, 7.74 mmol) with 2-(2-bromoethoxy)-
tetrahydro-2H-pyran, according to Procedure 1, gave crude
5-methoxy-2-[(E,Z )-2-phenylethenyl]-1-[2-(tetrahydro-2H-
pyran-2-yloxy)ethyl]-1H-indole (75a) which was reacted with
maleimide (0.79 g) following Procedure 2 to give a crude
DielseAlder adduct, that was used directly. To the crude
DielseAlder adduct, in toluene/p-dioxane (4:1, 250 mL) was
added DDQ (2.83 g, 12.5 mmol). The resulting solution was
heated at reflux for 3 h. The cooled solution was diluted
with aqueous NaHCO3 (500 mL) and extracted with EtOAc.
The combined organics were washed with aqueous NaHCO3
(several times) and then brine, before being dried over anhy-
drous Na2SO4 and evaporated to dryness to give a crude
mixture of 6-(2-hydroxyethyl)-9-methoxy-4-phenylpyrrolo[3,
4-c]carbazole-1,3(2H,6H )-dione (77a) (and partially THP pro-
tected material) that was used directly.
1
yellow solid; mp (Et2O) 199e201 ꢀC. H NMR [(CD3)2SO]
d 11.12 (br, 1H), 8.52 (d, J ¼ 2.6 Hz, 1H), 7.75 (s, 1H), 7.66
(d, J ¼ 8.9 Hz, 1H), 7.57 (dd, J ¼ 8.3, 2.2 Hz, 1H), 7.5e7.4
(m, 3H), 7.30 (dd, J ¼ 8.9, 2.6 Hz, 1H), 4.53 (t, J ¼ 6.4 Hz,
2H), 3.90 (s, 3H), 3.55 (t, J ¼ 5.8 Hz, 2H), 1.94 (m, 2H), 0.77
(s, 9H), ꢂ0.06 (s, 3H); which was used directly.
5.1.3. Procedure 3: TBDMS deprotection
5.1.3.1. 4-(2-Chlorophenyl)-6-(3-hydroxypropyl)-9-methoxy-
pyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (73b). Treatment
of the above TBDMS-protected alcohol (4.87 g, 8.85 mmol)
with 3 N HCl (50 mL) in 1:1 THF/MeOH (200 mL) at room
temperature for 2 h was followed by removal of most of the
solvents under reduced pressure. The residue was extracted
with EtOAc, washed well with water, and concentrated to
a volume of 60 mL. Petroleum ether was added to precipitate
the product, which was filtered off and triturated several times
with Et2O. The solid was crystallised from THF/petroleum
ether to give 73b (3.77 g, 88%) as a yellow powder; mp
5.1.6. Procedure 6: THP deprotection
5.1.6.1. 6-(2-Hydroxyethyl)-9-methoxy-4-phenylpyrrolo[3,4-c]-
carbazole-1,3(2H,6H )-dione (77a). The crude mixture of
77a obtained above was then dissolved in MeOH (100 mL)
and treated with p-toluenesulfonic acid monohydrate (30 mg,
0.16 mmol) warming at 50 ꢀC for 3 h. The resulting solution
was diluted with water and extracted with EtOAc, then the
product was adsorbed onto silica and chromatographed, elut-
ing with EtOAc/hexane (1:2 to 1:1), to give 77a as a yellow
powder (0.51 g, 17%); mp 262e264 ꢀC. 1H NMR
[(CD3)2SO] d 11.09 (br s, 1H), 8.56 (d, J ¼ 2.6 Hz, 1H),
7.83 (s, 1H), 7.66 (m, 3H), 7.47 (m, 3H), 7.28 (dd, J ¼ 9.0,
2.6 Hz, 1H), 4.86 (t, J ¼ 5.5 Hz, 1H), 4.55 (t, J ¼ 5.3 Hz,
2H), 3.90 (s, 3H), 3.78 (m, 2H). Anal. C23H18N2O4 (C, H, N).
1
228e230 ꢀC. H NMR [(CD3)2SO] d 11.12 (br s, 1H), 8.52
(d, J ¼ 2.5 Hz, 1H), 7.80 (s, 1H), 7.70 (d, J ¼ 8.9 Hz, 1H),
7.58 (dd, J ¼ 8.1, 2.2 Hz, 1H), 7.53e7.42 (m, 3H), 7.31 (dd,
J ¼ 8.9, 2.5 Hz, 1H), 4.63 (br s, 1H), 4.53 (t, J ¼ 6.9 Hz,
2H), 3.91 (s, 3H), 3.40 (m, 2H), 1.91 (m, 2H). Anal.
C24H19ClN2O4 (C, H, N).
5.1.4. Procedure 4: boron tribromide demethylation
5.1.7. Procedure 7: pyridine hydrochloride demethylation
5.1.4.1. 4-(2-Chlorophenyl)-9-hydroxy-6-(3-hydroxypropyl)-
pyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (16). Boron tri-
bromide (11.5 mL of a 1.0 M solution in CH2Cl2, 0.011 mol)
was added to a stirred solution of 73b (1.00 g, 2.30 mmol)
in dry CH2Cl2 (100 mL) under N2 at 0 ꢀC, and then the mix-
ture was stirred at room temperature for 3 h. Saturated aqueous
NaHCO3 solution was added and the solution was diluted with
water and extracted with EtOAc. The organic phase was dried
(Na2SO4), the drying agent was removed and the solution was
5.1.7.1. 9-Hydroxy-6-(2-hydroxyethyl)-4-phenylpyrrolo[3,4-c]-
carbazole-1,3(2H,6H)-dione (12). Compound 77a (135 mg,
0.35 mmol) was heated in molten pyridine hydrochloride
(15 g) at 200 ꢀC under N2. After 15 min, the reaction mixture
was cooled and diluted with water to precipitate the product,
which was collected by filtration, washed with water and
dried, then chromatographed on silica gel, eluting with
EtOAc/hexane, to give 12 (90 mg, 69%) as an orange powder;
mp 298e301 ꢀC. 1H NMR [(CD3)2SO] d 11.04 (br s, 1H), 9.31

J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
1287
(br s, 1H), 8.40 (d, J ¼ 2.5 Hz, 1H), 7.79 (s, 1H), 7.64 (m, 2H),
7.55 (d, J ¼ 8.8 Hz, 1H), 7.47 (m, 3H), 7.10 (dd, J ¼ 8.8,
2.5 Hz, 1H), 4.85 (t, J ¼ 5.3 Hz, 1H), 4.51 (t, J ¼ 5.4 Hz,
2H), 3.77 (m, 2H). Anal. C22H16N2O4$3/4H2O (C, H), N:
calcd, 7.26; found, 6.76.
4.54 (t, J ¼ 5.2 Hz, 2H), 3.91 (s, 3H), 3.77 (m, 2H). Anal.
C23H16Cl2N2O4 (C, H, N).
5.1.11. 4-(2,6-Dichlorophenyl)-9-hydroxy-6-(2-hydroxy-
ethyl)pyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (14)
Demethylation of 77c (0.10 g, 0.22 mmol) with BBr3, fol-
lowing Procedure 4, gave 14 (49 mg, 50%) as an orange/yellow
5.1.8. 4-(2-Chlorophenyl)-6-(2-hydroxyethyl)-9-methoxypy-
rrolo[3,4-c]carbazole-1,3(2H,6H )-dione (77b)
1
powder; mp 254e257 ꢀC. H NMR [(CD3)2SO] d 11.10 (br s,
Alkylation of 2-[(E,Z )-2-(2-chlorophenyl)ethenyl]-5-me-
thoxy-1H-indole (70b) [9] (1.92 g, 6.77 mmol) with sodium
hydride (0.39 g of 60% dispersion in mineral oil, 8.12 mmol)
and 2-(2-bromoethoxy)tetrahydro-2H-pyran (1.56 g, 7.44
mmol) following Procedure 1, gave crude 2-[(E,Z )-2-(2-
chlorophenyl)ethenyl]-5-methoxy-1-[2-(tetrahydro-2H-pyran-2-
yloxy)ethyl]-1H-indole (75b) which was dissolved in MeOH/
THF (1:1, 120 mL) and treated with 2 N HCl (15 mL), stirring
at room temperature for 3 h. The solution was then diluted with
saturated aqueous NaHCO3 and concentrated under reduced
pressure to precipitate crude 2-{2-[(E,Z )-2-(2-chlorophenyl)
ethenyl]-5-methoxy-1H-indol-1-yl}ethanol (76b) (1.90 g,
86%), which was collected by filtration and dried in vacuo.
This solid was reacted directly with maleimide and aromatised
with MnO2 for 18 h, following Procedure 2 described above, to
give 77b (72%) as a yellow powder; mp 255e257 ꢀC. 1H NMR
[(CD3)2SO] d 11.10 (br s, 1H), 8.52 (d, J ¼ 2.6 Hz, 1H), 7.80 (s,
1H), 7.71 (d, J ¼ 9.0 Hz, 1H), 7.59e7.56 (m, 1H), 7.52e7.43
(m, 4H), 7.29 (dd, J ¼ 9.0, 2.6 Hz, 1H), 4.84 (t, J ¼ 5.0 Hz,
1H), 4.53 (t, J ¼ 5.2 Hz, 2H), 3.90 (s, 3H), 3.77 (m, 2H). Anal.
C23H17ClN2O4 (C, H, N).
1H), 9.35 (s, 1H), 8.36 (d, J ¼ 2.4 Hz, 1H), 7.80 (s, 1H), 7.61
(m, 3H), 7.50 (dd, J ¼ 8.7, 7.3 Hz, 1H), 7.13 (dd, J ¼ 8.8,
2.4 Hz, 1H), 4.82 (t, J ¼ 5.5 Hz, 1H), 4.49 (t, J ¼ 5.2 Hz, 2H),
3.75 (m, 2H). Anal. C22H14Cl2N2O4$1/2H2O (C, H, N).
5.1.12. 6-(3-Hydroxypropyl)-9-methoxy-4-phenylpyrrolo-
[3,4-c]carbazole-1,3(2H,6H )-dione (73a)
Alkylation of 5-methoxy-2-[(E,Z )-2-phenylethenyl]-1H-in-
dole (70a) [10] (6.85 g, 27.5 mmol) with 3-bromopropoxy-
tert-butyldimethylsilane, following Procedure 1, gave crude
1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-5-methoxy-2-
[(E,Z )-2-phenylethenyl]-1H-indole (71a) which was reacted
successively with maleimide and DDQ following Procedures
2 and 5, respectively, to give crude 6-(3-{[tert-butyl(dimethyl)-
silyl]oxy}propyl)-9-methoxy-4-phenylpyrrolo[3,4-c]carbazole-
1,3(2H,6H )-dione. This product was dissolved in MeOH
(300 mL) containing 1 N HCl (50 mL), and the solution was
kept at room temperature for 3 h, then partitioned between water
and EtOAc. The organic layer was dried and evaporated, and the
residue was chromatographed on silica gel, eluting with CH2Cl2
to CH2Cl2/EtOAc (7:3), to give 73a (2.55 g, 23%); mp (Et2O)
241e243 ꢀC. 1H NMR [(CD3)2SO] d 11.10 (br s, 1H), 8.56 (d,
J ¼ 2.6 Hz, 1H), 7.82 (s, 1H), 7.67 (m, 3H), 7.47 (m, 3H),
7.30 (dd, J ¼ 9.0, 2.6 Hz, 1H), 4.66 (t, J ¼ 4.9 Hz, 1H), 4.55
(t, J ¼ 6.9 Hz, 2H), 3.90 (s, 3H), 3.39 (m, 2H), 1.93 (m, 2H).
Anal. C24H20N2O4 (C, H, N).
5.1.9. 4-(2-Chlorophenyl)-6-(2-hydroxyethyl)-
9-hydroxypyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (13)
Deprotection of 77b with BBr3, following Procedure 4, gave
13 (87%) as a yellow/orange powder; mp 265 ꢀC (dec). 1H NMR
[(CD3)2SO] d 11.04 (br s, 1H), 9.33 (br s, 1H), 8.38 (d,
J ¼ 2.4 Hz, 1H), 7.75 (s, 1H), 7.60e7.56 (m, 2H), 7.52e7.43
(m, 4H), 7.12 (dd, J ¼ 8.8, 2.4 Hz, 1H), 4.83 (t, J ¼ 5.5 Hz,
1H), 4.49 (t, J ¼ 5.2 Hz, 2H), 3.77 (dt, J ¼ 5.5, 5.2 Hz, 2H).
Anal. C22H15ClN2O4$1/2H2O (C, H, N).
5.1.13. 9-Hydroxy-6-(3-hydroxypropyl)-
4-phenylpyrrolo-[3,4-c]carbazole-1,3(2H,6H )-dione (15)
Demethylation of 73a (60 mg, 0.15 mmol) with BBr3 at
room temperature for 3 h, following Procedure 4, gave 15
(45 mg, 78%); mp 274e276 ꢀC. 1H NMR [(CD3)2SO]
d 11.05 (br s, 1H), 9.33 (s, 1H), 8.41 (d, J ¼ 2.4 Hz, 1H),
7.77 (s, 1H), 7.64 (m, 2H), 7.56 (d, J ¼ 8.8 Hz, 1H), 7.46
(m, 3H), 7.12 (dd, J ¼ 8.8, 2.4 Hz, 1H), 4.64 (t, J ¼ 4.9 Hz,
1H), 4.51 (t, J ¼ 6.9 Hz, 2H), 3.39 (m, 2H), 1.92 (m, 2H).
Anal. C23H18N2O4 (C, H, N).
5.1.10. 4-(2,6-Dichlorophenyl)-6-(2-hydroxyethyl)-9-metho-
xypyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (77c)
Alkylation of 2-[(E )-2-(2,6-dichlorophenyl)ethenyl]-5-
methoxy-1H-indole (70c) [9] (3.0 g, 10.6 mmol) with 2-(2-
bromoethoxy)tetrahydro-2H-pyran, according to Procedure 1,
gave crude 2-[(E )-2-(2,6-dichlorophenyl)ethenyl]-5-methoxy-
1-[2-(tetrahydro-2H-pyran-2-yloxy)ethyl]-1H-indole
(75c).
5.1.14. 4-(2,6-Dichlorophenyl)-6-(3-hydroxypropyl)-
Treatment of this with 2 N HCl in MeOH/THF, following Pro-
cedure 3, gave crude 2-{2-[(E )-2-(2,6-dichlorophenyl)
ethenyl]-5-methoxy-1H-indol-1-yl}ethanol (76c) which was
reacted directly with maleimide (1.26 g) according to Proce-
dure 2 and aromatised with DDQ following Procedure 5. Trit-
uration in MeOH then gave 77c as a yellow solid (2.14 g,
9-methoxypyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (73c)
Alkylation of 2-[(E )-2-(2,6-dichlorophenyl)ethenyl]-5-me-
thoxy-1H-indole (70c) [9] (3.0 g, 9.43 mmol) with 3-bromo-
propoxy-tert-butyldimethylsilane, according to Procedure 1,
gave crude 1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-2-[(E )-
2-(2,6-dichlorophenyl)ethenyl]-5-methoxy-1H-indole
(71c)
1
44%); mp 229e231 ꢀC. H NMR [(CD3)2SO] d 11.17 (br s,
which was dissolved in MeOH/CH2Cl2 (3:1, 300 mL) and
treated with 1 N HCl (60 mL). This solution was stirred at
room temperature for 2 h, then diluted with saturated
aqueous NaHCO3 and concentrated under reduced pressure
1H), 8.50 (d, J ¼ 2.6 Hz, 1H), 7.86 (s, 1H), 7.72 (d,
J ¼ 9.0 Hz, 1H), 7.62 (m, 2H), 7.51 (dd, J ¼ 8.9, 7.4 Hz,
1H), 7.31 (dd, J ¼ 9.0, 2.6 Hz, 1H), 4.83 (t, J ¼ 5.3 Hz, 1H),

1288
J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
to precipitate crude 3-{2-[(E )-2-(2,6-dichlorophenyl)ethenyl]-
5-methoxy-1H-indol-1-yl}-1-propanol (72), which was col-
lected by filtration and dried in vacuo. Crude 72 thus obtained
was reacted directly with maleimide (1.20 g) and then aroma-
tised with DDQ, following Procedures 2 and 5, respectively.
Chromatography on silica, eluting with MeOH/CH2Cl2
(2:98e5:95), gave 73c (2.4 g, 54%) as a yellow powder; mp
3H), 3.42 (m, 2H), 1.88 (m, 2H). Anal. C24H19ClN2O5 (C,
H, N).
5.1.18. 9-Hydroxy-6-(3-hydroxypropyl)-4-(2-methoxyphenyl)-
pyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (18)
A solution of 74 (0.20 g, 0.44 mmol), potassium acetate
(0.20 g) and 5% Pd/C in EtOAc/MeOH (50 mL, 1:1) was
hydrogenated at 60 psi for 7 h. The catalyst was filtered
off and the filtrate concentrated to dryness, partitioned be-
tween EtOAc and water, and evaporated. The residue was
chromatographed on silica, eluting with EtOAc/petroleum
ether (2:1), to give 18 (51%) as an orange powder (from
THF/petroleum ether); mp 296e300 ꢀC. 1H NMR
[(CD3)2SO] d 10.93 (br s, 1H), 9.31 (br s, 1H), 8.38 (d,
J ¼ 2.4 Hz, 1H), 7.69 (s, 1H), 7.55 (d, J ¼ 8.8 Hz, 1H),
7.42 (m, 1H), 7.35 (dd, J ¼ 7.5, 1.7 Hz, 1H), 7.13e7.08
(m, 2H), 7.05 (dd, J ¼ 7.2, 7.2 Hz, 1H), 4.62 (t,
J ¼ 4.8 Hz, 1H), 4.48 (t, J ¼ 6.8 Hz, 2H), 3.68 (s, 3H),
3.40 (m, 2H), 1.90 (m, 2H). Anal. C24H20N2O5 (C, H, N).
1
254e256 ꢀC. H NMR [(CD3)2SO] d 11.18 (br s, 1H), 8.51
(d, J ¼ 2.6 Hz, 1H), 7.86 (s, 1H), 7.72 (d, J ¼ 9.0 Hz, 1H),
7.63 (m, 2H), 7.51 (dd, J ¼ 8.8, 7.4 Hz, 1H), 7.34 (dd,
J ¼ 9.0, 2.6 Hz, 1H), 4.62 (t, J ¼ 5.0 Hz, 1H), 4.54 (t,
J ¼ 6.9 Hz, 2H), 3.91 (s, 3H), 3.42 (m, 2H), 1.90 (m, 2H).
FABMS found Mþ: 468.0630, 470.0628, 472.0626.
C24H18Cl2N2O4 requires 468.0644, 470.0614, 472.0585.
5.1.15. 4-(2,6-Dichlorophenyl)-9-hydroxy-6-(3-hydroxypro-
pyl)pyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (17)
Demethylation of 73c (0.36 g, 0.77 mmol) with BBr3, ac-
cording to Procedure 4, gave 17 (0.32 g, 92%) as an orange/
yellow powder; mp 215e218 ꢀC. 1H NMR [(CD3)2SO]
d 11.12 (br s, 1H), 9.39 (s, 1H), 8.37 (d, J ¼ 2.5 Hz, 1H),
7.80 (s, 1H), 7.61 (m, 3H), 7.51 (dd, J ¼ 8.7, 7.2 Hz, 1H),
7.16 (dd, J ¼ 8.8, 2.5 Hz, 1H), 4.61 (t, J ¼ 5.0 Hz, 1H), 4.49
(t, J ¼ 6.9 Hz, 2H), 3.42 (m, 2H), 1.88 (m, 2H). Anal.
C23H16Cl2N2O4 (C, H, N).
5.2. Procedures of Scheme 2
5.2.1.
1H-indol-1-yl}propanenitrile (78)
solution of 2-[(E )-2-(2-chlorophenyl)ethenyl]-5-me-
3-{2-[(E )-2-(2-Chlorophenyl)ethenyl]-5-methoxy-
A
5.1.16. 4-(2-Chloro-6-methoxyphenyl)-6-(3-hydroxypropyl)-
thoxy-1H-indole (70b) [9] (1.5 g, 5.25 mmol) in CH3CN
(30 mL) was treated with acrylonitrile (2.42 mL, 36.8 mmol)
and 1,8-diazabicyclo[5.4.0]undec-7-ene (20 drops). The re-
sulting solution was stirred at room temperature under nitro-
gen for 18 h, then diluted with water and extracted with
EtOAc. The organic layer was dried (Na2SO4), the drying
agent was removed and the solution was concentrated to dry-
ness. Chromatography on silica, eluting with EtOAc/hexane
(1:1), followed by trituration in MeOH gave 78 (1.36 g,
77%) as an off-white solid; mp 136e138 ꢀC. 1H NMR
[(CD3)2SO] d 8.05 (dd, J ¼ 7.9, 1.5 Hz, 1H), 7.50 (m, 4H),
7.41 (m, 1H), 7.33 (m, 1H), 7.04 (d, J ¼ 2.4 Hz, 1H), 6.90
(s, 1H), 6.81 (dd, J ¼ 9.0, 2.4 Hz, 1H), 4.68 (t, J ¼ 6.6 Hz,
2H), 3.77 (s, 3H), 2.93 (t, J ¼ 6.6 Hz, 2H). Anal.
C20H17ClN2O (C, H, N).
9-methoxypyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (73d)
Reaction
of
2-[(E,Z )-2-(2-chloro-6-methoxyphenyl)
ethenyl]-5-methoxy-1H-indole (70d) [9] with 3-bromopro-
poxy-tert-butyldimethylsilane following Procedure 1, gave
1-(3-{[tert-butyl(dimethyl)silyl]oxy}propyl)-2-[(E,Z )-2-(2-
chloro-6-methoxyphenyl)ethenyl]-5-methoxy-1H-indole (71d),
which was used without further purification. Reaction of 71d
with maleimide and then with DDQ, following Procedures 2
and 5, respectively, gave 6-(3-{[tert-butyl(dimethyl)silyl]
oxy}propyl)-4-(2-chloro-6-methoxyphenyl)-9-methoxypyr-
rolo[3,4-c]carbazole-1,3(2H,6H )-dione, which was reacted di-
rectly with 2 N HCl in THF/MeOH (4:1) according to
Procedure 3, to give 73d (76%) as an orange powder; mp
1
224e227 ꢀC. H NMR [(CD3)2SO] d 11.05 (br s, 1H), 8.51
(d, J ¼ 2.6 Hz, 1H), 7.75 (s, 1H), 7.69 (d, J ¼ 8.9 Hz, 1H),
7.44 (dd, J ¼ 8.2, 8.2 Hz, 1H), 7.31 (dd, J ¼ 8.9, 2.6 Hz,
1H), 7.18 (d, J ¼ 8.2 Hz, 1H), 7.13 (d, J ¼ 8.2 Hz, 1H), 4.63
(t, J ¼ 4.9 Hz, 1H), 4.51 (t, J ¼ 6.8 Hz, 2H), 3.91 (s, 3H),
3.68 (s, 3H), 3.41 (m, 2H), 1.89 (m, 2H). Anal. C25H22N2O5
(C, N), H: calcd, 5.15; found, 4.53.
5.2.2. 3-(4-(2-Chlorophenyl)-9-methoxy-1,3-dioxo-2,3-dihy-
dropyrrolo[3,4-c]carbazol-6(1H )-yl)propanenitrile (82)
Reaction of 78 (1.30 g, 3.86 mmol) with maleimide
(0.49 g), and aromatisation of the product with MnO2 follow-
ing Procedure 2, gave crude material that was chromato-
graphed on silica gel, eluting with EtOAc/hexane (1:1).
Trituration from MeOH gave 82 (1.33 g, 80%) as a yellow
5.1.17. 4-(2-Chloro-6-methoxyphenyl)-9-hydroxy-6-(3-hydro-
xypropyl)pyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (74)
1
Deprotection of 73d with BBr3 in CH2Cl2 for 90 min, fol-
lowing Procedure 4, gave 74 (64%) as an orange powder; mp
270e273 ꢀC. 1H NMR [(CD3)2SO] d 11.01 (br s, 1H), 9.35 (br
s, 1H), 8.37 (d, J ¼ 2.4 Hz, 1H), 7.70 (s, 1H), 7.58 (d,
J ¼ 8.7 Hz, 1H), 7.44 (dd, J ¼ 8.3, 8.3 Hz, 1H), 7.20e7.11
(m, 3H), 4.61 (m, 1H), 4.47 (t, J ¼ 6.9 Hz, 2H), 3.68 (s,
powder; mp 287e288 ꢀC. H NMR [(CD3)2SO] d 11.16 (br
s, 1H), 8.53 (d, J ¼ 2.6 Hz, 1H), 8.02 (s, 1H), 7.83 (d,
J ¼ 9.0 Hz, 1H), 7.59 (m, 1H), 7.49 (m, 3H), 7.33 (dd,
J ¼ 9.0, 2.6 Hz, 1H), 4.86 (t, J ¼ 6.7 Hz, 2H), 3.91 (s,
3H), 3.04 (t, J ¼ 6.7 Hz, 2H). Anal. C24H16ClN3O3$3/4H2O
(C, H, N).

J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
1289
5.2.3. 3-(4-(2-Chlorophenyl)-9-hydroxy-1,3-dioxo-2,3-dihy-
dropyrrolo[3,4-c]carbazol-6(1H )-yl)propanenitrile (22)
Demethylation of 82 (0.15 g, 0.35 mmol) with 1 N BBr3 in
CH2Cl2, according to Procedure 4, followed by trituration in
MeOH, gave 22 (0.13 g, 89%) as an orange/yellow powder;
from EtOAc/hexane, gave 58 (165 mg, 40%) as a yellow pow-
der; mp 225e228 ꢀC. 1H NMR [(CD3)2SO] d 11.04 (br s, 1H),
9.34 (br s, 1H), 8.38 (d, J ¼ 2.4 Hz, 1H), 7.75 (s, 1H), 7.57 (m,
2H), 7.47 (m, 3H), 7.13 (dd, J ¼ 8.8, 2.4 Hz, 1H), 4.91 (br s,
1H), 4.50 (m, 1H), 4.34 (m, 1H), 4.00 (m, 1H), 2.35 (m,
1H), 2.24 (m, 1H), 2.17 (s, 6H). Anal. C25H22ClN3O4$1/
2H2O (C, H, N).
1
mp 332e336 ꢀC. H NMR [(CD3)2SO] d 11.10 (br s, 1H),
9.42 (br s, 1H), 8.39 (d, J ¼ 2.4 Hz, 1H), 7.96 (s, 1H), 7.69
(d, J ¼ 8.8 Hz, 1H), 7.58 (m, 1H), 7.48 (m, 3H), 7.15
(dd, J ¼ 8.8, 2.4 Hz, 1H), 4.82 (t, J ¼ 6.7 Hz, 2H), 3.02 (t,
J ¼ 6.7 Hz, 2H). Anal. C23H14ClN3O3 (C, H, N).
5.3. Procedures of Scheme 3
5.3.1. 6-Allyl-4-(2-chlorophenyl)-2-(2,4-dimethoxybenzyl)-
9-methoxypyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (86)
To a solution of 4-(2-chlorophenyl)-2-(2,4-dimethoxy-
benzyl)-9-methoxypyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione
(85) [9] (150 mg, 0.28 mmol) in DMF (10 mL) under nitrogen
was added K2CO3 (0.39 g, 2.80 mmol) and allyl bromide
(72 mL, 0.84 mmol). The resulting suspension was warmed
to 90 ꢀC with stirring for 3 h, then diluted with water and ex-
tracted with EtOAc. The organic phase was dried (Na2SO4),
the drying agent was removed, and the solution was concen-
trated to dryness. Chromatography on silica, eluting with
EtOAc/hexane (1:2), followed by crystallisation from Et2O/
hexane, gave 86 (90 mg, 57%) as a yellow powder; mp
171e173 ꢀC. 1H NMR [(CD3)2SO] d 8.51 (d, J ¼ 2.6 Hz,
1H), 7.84 (s, 1H), 7.67 (d, J ¼ 8.9 Hz, 1H), 7.58 (m, 1H),
7.53e7.46 (m, 3H), 7.32 (dd, J ¼ 8.9, 2.6 Hz, 1H), 6.97 (d,
J ¼ 8.4 Hz, 1H), 6.57 (d, J ¼ 2.4 Hz, 1H), 6.45 (dd, J ¼ 8.4,
2.4 Hz, 1H), 5.99 (m, 1H), 5.18 (d, J ¼ 4.9 Hz, 2H), 5.14
(dd, J ¼ 10.3, 1.3 Hz, 1H), 4.99 (dd, J ¼ 17.2, 1.3 Hz, 1H),
4.69 (s, 2H), 3.90 (s, 3H), 3.80 (s, 3H), 3.72 (s, 3H). Anal.
C33H27ClN2O5 (C, H, N).
5.2.4. 4-(2-Chlorophenyl)-9-hydroxy-6-[2-hydroxy-3-
(methylamino)propyl]pyrrolo[3,4-c]carbazole-1,3(2H,6H )-
dione (57)
Alkylation of 2-[(E )-2-(2-chlorophenyl)ethenyl]-5-me-
thoxy-1H-indole (70b) [9] (0.25 g, 0.88 mmol) with epibro-
mohydrin, following Procedure 1, gave crude epoxide 79,
which was used directly. The epoxide 79 was dissolved in
THF (2 mL) and aqueous methylamine (0.5 mL, 40% solu-
tion) was added. The resulting solution was heated at 60 ꢀC
for 6 h, then diluted with water and extracted with EtOAc to
give crude 1-{2-[(E )-2-(2-chlorophenyl)ethenyl]-5-methoxy-
1H-indol-1-yl}-3-(methylamino)-2-propanol (80). This mate-
rial was triturated in Et2O/hexane and reacted directly with
maleimide (0.13 g, 1.5 mmol), following Procedure 2 (with
the addition of p-dioxane as a co-solvent). DDQ aromatisation
of the crude DielseAlder adduct, according to Procedure 5,
gave
4-(2-chlorophenyl)-6-[2-hydroxy-3-(methylamino)-
propyl]-9-methoxypyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione
(83), which was demethylated with pyridine hydrochloride,
according to Procedure 7. Chromatography on silica, eluting
with EtOAc/MeOH/Et3N (4:1:trace), and then crystallisation
from EtOAc/hexane gave 57 (61 mg, 15%) as a yellow pow-
der; mp 188e191 ꢀC. 1H NMR [(CD3)2SO] d 10.9 (br s,
1H), 9.36 (br s, 1H), 8.37 (d, J ¼ 2.4 Hz, 1H), 7.77 (s, 1H),
7.58 (m, 2H), 7.47 (m, 3H), 7.13 (dd, J ¼ 8.8, 2.4 Hz, 1H),
5.09 (br s, 1H), 4.50 (m, 1H), 4.34 (m, 1H), 3.97 (m, 1H),
2.77e2.63 (m, 2H), 2.28 (d, J ¼ 2.1 Hz, 3H). Anal.
C24H20ClN3O4$2/3H2O (C, H, N).
5.3.2. 4-(2-Chlorophenyl)-6-(2,3-dihydroxypropyl)-2-(2,4-
dimethoxybenzyl)-9-methoxypyrrolo[3,4-c]carbazole-
1,3(2H,6H )-dione (87)
To a solution of 86 (80 mg, 0.14 mmol) in acetone/water
(4:1, 20 mL) was added N-methylmorpholine N-oxide
(33 mg, 0.28 mmol) and osmium tetroxide (176 mL, 4% solu-
tion in water, w0.028 mmol). The reaction mixture was stirred
at room temperature for 18 h, then diluted with 1 N HCl and
extracted with EtOAc. The organic phase was dried, the drying
agent was removed, and the solution was concentrated to dry-
ness. Chromatography on silica, eluting with EtOAc/hexane
(2:1), followed by crystallisation from Et2O/hexane, gave 87
(80 mg, 94%) as a yellow powder; mp 151e156 ꢀC. 1H
NMR [(CD3)2SO] d 8.50 (d, J ¼ 2.6 Hz, 1H), 7.82 (s, 1H),
7.69 (br d, J ¼ 9.0 Hz, 1H), 7.58 (m, 1H), 7.52e7.46 (m,
3H), 7.30 (dd, J ¼ 9.0, 2.6 Hz, 1H), 6.95 (d, J ¼ 8.4 Hz, 1H),
6.57 (d, J ¼ 2.4 Hz, 1H), 6.44 (dd, J ¼ 8.4, 2.4 Hz, 1H), 5.02
(d, J ¼ 5.0 Hz, 1H), 4.87 (br s, 1H), 4.69 (s, 2H), 4.55 (m,
1H), 4.38 (m, 1H), 3.89 (m, 4H), 3.80 (s, 3H), 3.72 (s, 3H),
3.41 (m, 2H). Anal. C33H29ClN2O7$1/4H2O (C, H, N).
5.2.5. 4-(2-Chlorophenyl)-6-[3-(dimethylamino)-
2-hydro-xypropyl]-9-hydroxypyrrolo[3,4-c]carbazole-
1,3(2H,6H )-dione (58)
Epoxide 79 (0.88 mmol) was dissolved in THF (2 mL) and
aqueous dimethylamine (0.5 mL, 40% solution) was added.
The resulting solution was heated at 60 ꢀC for 6 h, then diluted
with water and extracted with EtOAc to give 1-{2-[(E )-2-(2-
chlorophenyl)ethenyl]-5-methoxy-1H-indol-1-yl}-3-(dimethy-
lamino)-2-propanol (81). This crude material was triturated in
Et2O/hexane and then reacted directly with maleimide (0.13 g,
1.5 mmol), following Procedure 2. DDQ aromatisation of the
crude DielseAlder adduct, according to Procedure 5, gave
4-(2-chlorophenyl)-6-[3-(dimethylamino)-2-hydroxypropyl]-
9-methoxypyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione
(84),
5.3.3. 4-(2-Chlorophenyl)-6-(2,3-dihydroxypropyl)-
which was demethylated with pyridine hydrochloride follow-
ing Procedure 7. Chromatography on silica, eluting with
EtOAc/MeOH/Et3N (4:1:trace), followed by crystallisation
9-hydroxypyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (26)
To a solution of 87 (75 mg, 0.13 mmol) in anisole (0.5 mL)
was added TFA (2.0 mL). The reaction vessel was sealed and

1290
J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
heated to 90 ꢀC in an oil bath for 18 h before the TFA was re-
moved under reduced pressure. The residue was diluted with
water and extracted with Et2O (three times). The combined or-
ganic extracts were washed thoroughly with 1 N KOH and
brine, then dried over anhydrous Na2SO4 and concentrated
in vacuo. The aqueous layer was acidified by the addition of
concentrated HCl, extracted with Et2O and worked up as
above. The combined crude material was triturated in Et2O/
hexane and then dissolved in CH2Cl2 (20 mL) and demethy-
lated with BBr3, according to Procedure 4. Chromatography
of the product on silica, eluting with EtOAc/hexane (1:1 to
1:0), gave 26 (31 mg, 57%) as an orange powder; mp 305e
309 ꢀC. 1H NMR [(CD3)2SO] d 11.04 (br s, 1H), 9.33 (s,
1H), 8.38 (d, J ¼ 2.4 Hz, 1H), 7.73 (s, 1H), 7.57 (m, 2H),
7.47 (m, 3H), 7.13 (dd, J ¼ 8.8, 2.4 Hz, 1H), 4.99 (d,
J ¼ 5.5 Hz, 1H), 4.83 (t, J ¼ 5.4 Hz, 1H), 4.50 (m, 1H), 4.32
(m, 1H), 3.87 (br s, 1H), 3.45e3.36 (m, 2H). Anal.
C23H17ClN2O5 (C, H, N).
Chromatography of the product on silica gel, eluting with
EtOAc/hexane (1:1 to 4:1), gave 88c (0.95 g, 83%); mp
1
(EtOAc/hexane) 255e260 ꢀC. H NMR [(CD3)2SO] d 11.16
(br s, 1H), 9.43 (br s, 1H), 8.38 (d, J ¼ 2.5 Hz, 1H), 7.91 (s,
1H), 7.63 (m, 3H), 7.51 (dd, J ¼ 8.7, 7.3 Hz, 1H), 7.16 (dd,
J ¼ 8.9, 2.5 Hz, 1H), 4.85 (t, J ¼ 4.9 Hz, 2H), 4.53 (t,
J ¼ 4.9 Hz, 2H), 2.88 (s, 3H). Anal. C23H16Cl2N2O6S (C, H, N).
5.4.1.4. 6-(3-Bromopropyl)-9-hydroxy-4-phenylpyrrolo[3,4-c]-
carbazole-1,3(2H,6H )-dione (89a). Alcohol 72a (0.50 g,
1.25 mmol) was treated with MsCl in THF/Et3N and the crude
product was demethylated with BBr3 (0 ꢀC for 18 h), follow-
ing Procedure 8. This product was dissolved in EtOAc
(100 mL) containing LiBr (1.0 g), and the solution was kept
at 50 ꢀC for 2 h, then worked up and chromatographed on sil-
ica gel, eluting with EtOAc/hexane (1:1 to 4:1), to give 89a
(0.54 g, 97%); mp (EtOAc/hexane) 280e282 ꢀC. 1H NMR
[(CD3)2SO] d 11.07 (s, 1H), 9.36 (s, 1H), 8.41 (d,
J ¼ 2.2 Hz, 1H), 7.83 (s, 1H), 7.66 (m, 2H), 7.58 (d,
J ¼ 8.8 Hz, 1H), 7.47 (m, 3H), 7.14 (dd, J ¼ 8.8, 2.2 Hz,
1H), 4.57 (t, J ¼ 6.9 Hz, 2H), 3.53 (t, J ¼ 6.5 Hz, 2H), 2.32
(m, 2H). Anal. C23H17BrN2O3 (C, H, N).
5.4. Procedures of Scheme 4
5.4.1. Procedure 8: preparation of C-2 mesylates/C-3
bromides for amine displacement
5.4.1.5. 6-(3-Bromopropyl)-4-(2-chlorophenyl)-9-hydroxypyr-
rolo[3,4-c]carbazole-1,3(2H,6H )-dione (89b). Reaction of al-
cohol 73b with MsCl, followed by demethylation with BBr3,
following Procedure 8, gave 89b (81%) as an orange powder;
5.4.1.1.
2-(9-Hydroxy-1,3-dioxo-4-phenyl-2,3-dihydropyr-
rolo[3,4-c]carbazol-6(1H )-yl)ethyl methanesulfonate (88a).
Alcohol 77a (0.21 g, 0.53 mmol) in dry THF (30 mL) and
Et3N (0.37 mL) under nitrogen was cooled to 0 ꢀC and treated
dropwise with MsCl (45 mL, 0.58 mmol). After 1 h, the reac-
tion was partitioned between saturated aqueous NaHCO3 and
EtOAc. The organic layer was evaporated and the crude prod-
uct was demethylated with BBr3, following Procedure 4.
Chromatography of the product on silica gel, eluting with
EtOAc/hexane (1:1 to 4:1), gave 88a (0.19 g, 80%); mp
1
mp 278 ꢀC (dec). H NMR [(CD3)2SO] d 11.08 (s, 1H), 9.39
(s, 1H), 8.39 (d, J ¼ 2.4 Hz, 1H), 7.81 (s, 1H), 7.60 (d,
J ¼ 8.7 Hz, 1H), 7.60e7.58 (m, 1H), 7.53e7.43 (m, 3H),
7.16 (dd, J ¼ 8.7, 2.4 Hz, 1H), 4.55 (t, J ¼ 6.9 Hz, 2H),
3.56e3.49 (m, 2H), 2.34e2.24 (m, 2H). Anal.
C23H16BrClN2O3 (C, H, N).
1
(EtOAc/hexane) 271e276 ꢀC. H NMR [(CD3)2SO] d 11.08
5.4.1.6. 6-(3-Bromopropyl)-4-(2,6-dichlorophenyl)-9-hydroxy-
(s, 1H), 9.37 (br s, 1H), 8.40 (d, J ¼ 2.4 Hz, 1H), 7.86 (s,
1H), 7.67 (m, 2H), 7.60 (d, J ¼ 8.8 Hz, 1H), 7.46 (m, 3H),
7.13 (dd, J ¼ 8.8, 2.4 Hz, 1H), 4.86 (t, J ¼ 4.8 Hz, 2H), 4.57
(t, J ¼ 4.8 Hz, 2H), 2.97 (s, 3H). FABMS found [M þ H]þ:
451.0958. C23H18N2O6S requires 451.0964.
pyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione
(89c). Alcohol
73c (0.77 g, 1.68 mmol) was mesylated and demethylated
with BBr3, according to Procedure 8. This product was dis-
solved in EtOAc (100 mL) containing LiBr (1.0 g), and the
solution was kept at 50 ꢀC for 2 h, then worked up. Chroma-
tography on silica gel, eluting with EtOAc/hexane (1:1 to
4:1), gave 89c (0.70 g, 80%); mp (EtOAc/hexane) 273e
5.4.1.2. 2-(4-(2-Chlorophenyl)-9-hydroxy-1,3-dioxo-2,3-dihy-
dropyrrolo[3,4-c]carbazol-6(1H )-yl)ethyl methanesulfonate
(88b). A solution of alcohol 77b (1.10 g, 2.61 mmol) was me-
sylated and demethylated with BBr3 (7 h) according to Proce-
dure 8, then chromatography of the product on silica gel,
eluting with EtOAc/hexane (1:1 to 3:1), gave 88b (1.05 g,
65%) as a yellow solid; mp (EtOAc/hexane) 266 ꢀC (dec).
1H NMR [(CD3)2SO] d 11.09 (br s, 1H), 9.40 (s, 1H), 8.39
(d, J ¼ 2.5 Hz, 1H), 7.84 (s, 1H), 7.62 (d, J ¼ 8.9 Hz, 1H),
7.58 (m, 1H), 7.48 (m, 3H), 7.14 (dd, J ¼ 8.9, 2.5 Hz, 1H),
4.85 (t, J ¼ 5.0 Hz, 2H), 4.54 (m, 2H), 2.93 (s, 3H).
1
276 ꢀC. H NMR [(CD3)2SO] d 11.15 (br s, 1H), 9.42 (s,
1H), 8.38 (d, J ¼ 2.4 Hz, 1H), 7.87 (s, 1H), 7.62 (m, 3H),
7.51 (dd, J ¼ 8.9, 7.6 Hz, 1H), 7.18 (dd, J ¼ 8.8, 2.4 Hz,
1H), 4.56 (t, J ¼ 6.9 Hz, 2H), 3.51 (t, J ¼ 6.7 Hz, 2H), 2.27
(m, 2H). Anal. C23H15BrCl2N2O3 (C, H, N).
5.4.2. Procedure 9: amine displacement of C-2 mesylates/
C-3 bromides
5.4.2.1. 6-[3-(Dimethylamino)propyl]-9-hydroxy-4-phenylpyr-
rolo[3,4-c]carbazole-1,3(2H,6H )-dione (39). A solution of
89a (0.12 g, 0.27 mmol) in dimethylacetamide (4 mL) was
treated with NHMe2 (25 mol equivalents, 0.85 mL of a 40%
aqueous solution) and the reaction was stirred in a sealed ves-
sel at 80 ꢀC for 18 h, then diluted with water. The resulting
5.4.1.3. 2-(4-(2,6-Dichlorophenyl)-9-hydroxy-1,3-dioxo-2,3-di-
hydropyrrolo[3,4-c]carbazol-6(1H )-yl)ethyl methanesulfonate
(88c). Alcohol 77c (1.0 g, 2.2 mmol) was mesylated and de-
methylated with BBr3 (30 h), according to Procedure 8.

J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
1291
solution was acidified with conc. HCl and the pH was then ad-
justed to ca. 9 with solid K2CO3. The resulting precipitate was
either collected by filtration and washed with water, or ex-
tracted with EtOAc, dried, and the organic layer evaporated.
Chromatography on silica gel, eluting with MeOH/CH2Cl2
(1:9 to 1:4), gave 39 (52 mg, 47%) as a yellow powder; mp
found [M þ H]þ: 437.1088, 435.1090. C24H20ClN2O4 requires
437.1082, 435.1112.
5.6. Procedures of Scheme 6
5.6.1. Procedure 10: DesseMartin/sodium chlorite oxidation
DesseMartin periodinane (1.91 g, 4.5 mmol) was added to
a stirred solution of 73a (1.20 g, 3.0 mmol) in THF (100 mL)
under nitrogen. After 1 h at room temperature, a solution of
saturated aqueous Na2S2O8 and saturated aqueous NaHCO3
(1:1, 100 mL) was added, and the mixture was stirred vigor-
ously for 15 min, then extracted with EtOAc. The organic
layer was dried (Na2SO4) and evaporated to dryness. The
crude aldehyde was dissolved in t-BuOH/THF (9:1, 230 mL)
and treated with 2-methyl-2-butene (12.0 mmol, 6.0 mL of
a 2 M solution in THF), followed by sodium chlorite
(1.09 g, 12.0 mmol) and NaH2PO4 (2.5 g, 18.0 mmol) in water
(100 mL) and t-BuOH (4 mL). The resulting solution was
stirred at room temperature for 18 h, then diluted with brine
and extracted with EtOAc. The organic layer was dried and
evaporated, and the residue was chromatographed on silica
gel, eluting with EtOAc/MeOH (1:0 to 9:1), to give 3-(9-me-
thoxy-1,3-dioxo-4-phenyl-2,3-dihydropyrrolo[3,4-c]carbazol-
6(1H )-yl)propanoic acid (91a) (0.86 g, 69%), which was used
directly; mp (EtOAc/hexane) 252e255 ꢀC. 1H NMR
[(CD3)2SO] d 12.2 (br s, 1H), 11.13 (br s, 1H), 8.55 (d,
J ¼ 2.6 Hz, 1H), 7.89 (s, 1H), 7.68 (m, 3H), 7.47 (m, 3H),
7.28 (dd, J ¼ 9.0, 2.6 Hz, 1H), 4.73 (t, J ¼ 6.6 Hz, 2H), 3.90
(s, 3H), 2.76 (t, J ¼ 6.6 Hz, 2H).
1
(EtOAc/hexane) 185e189 ꢀC. H NMR [(CD3)2SO] d 11.05
(s, 1H), 9.34 (s, 1H), 8.41 (d, J ¼ 2.4 Hz, 1H), 7.78 (s, 1H),
7.64 (m, 2H), 7.56 (d, J ¼ 8.9 Hz, 1H), 7.47 (m, 3H), 7.14
(dd, J ¼ 8.9, 2.4 Hz, 1H), 4.48 (t, J ¼ 6.6 Hz, 2H), 2.16 (t,
J ¼ 6.5 Hz, 2H), 2.08 (s, 6H), 1.90 (m, 2H). Anal.
C25H23N3O3$1/4H2O (C, H, N).
Compounds 27e38, 40e53 were similarly prepared
according to Procedure 9 (Supplementary material).
5.5. Procedures of Scheme 5
5.5.1. 4-(4-(2-Chlorophenyl)-9-hydroxy-1,3-dioxo-2,3-
dihydropyrrolo[3,4-c]carbazol-6(1H )-yl)butanenitrile (24)
To a solution of 89b (0.13 g, 0.27 mmol) in DMSO (2 mL)
was added a solution of NaCN (15 mg, 0.30 mmol) in DMSO
(2 mL) dropwise over 15 min. After 30 min the reaction was
diluted with water and given a standard EtOAc work-up. Chro-
matography on silica, eluting with EtOAc/hexane (1:4 to 1:1),
followed by crystallisation from EtOAc/hexane, gave 24
(68 mg, 59%) as an orange powder; mp 262e266 ꢀC. 1H
NMR [(CD3)2SO] d 11.08 (br s, 1H), 9.39 (s, 1H), 8.39 (d,
J ¼ 2.4 Hz, 1H), 7.83 (s, 1H), 7.59 (m, 2H), 7.49 (m, 3H),
7.15 (dd, J ¼ 8.8, 2.4 Hz, 1H), 4.50 (t, J ¼ 7.3 Hz, 2H), 2.57
(m, 2H), 2.07 (m, 2H). FABMS found [M þ H]þ: 430.0927,
432.0916. C24H16ClN3O3 requires 430.0958, 432.0929.
5.6.2. Procedure 11: primary carboxamide formation
5.5.2. 4-(2-Chlorophenyl)-9-hydroxy-6-(3-methoxypropyl)-
pyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (25)
5.6.2.1. 3-(9-Methoxy-1,3-dioxo-4-phenyl-2,3-dihydropyrrolo-
[3,4-c]carbazol-6(1H )-yl)propanamide (92a). To a solution
of 91a (100 mg, 0.24 mmol) in dry THF (20 mL) under nitrogen
was added one drop of DMF, followed by oxalyl chloride
(84 mL, 0.96 mmol) dropwise. The resulting solution was stirred
at room temperature for 2 h, then evaporated to dryness invacuo.
Dry benzene (20 mL) was added to the residue and the suspen-
sion was again evaporated to dryness in vacuo, then dissolved in
dry THF (20 mL) and flushed with nitrogen. To this solution was
added a saturated solution of ammonia gas in THF (20 mL). The
resulting solution was stirred at room temperature for 1 h, then
diluted with brine and extracted with EtOAc. The organic layer
was dried and evaporated, and the residue was chromatographed
on silica gel, eluting with CH2Cl2/MeOH (95:5), to give 92a
A solution of 89b (50 mg, 0.103 mmol) and sodium methox-
ide (35.2 mg, 0.65 mmol) in MeOH (0.5 mL) and p-dioxane
(8 mL) was refluxed for 3 h. The solution was acidified with
2 N HCl, then extracted with EtOAc. The organic layer was
dried (Na2SO4), the drying agent was removed and the solution
was concentrated to dryness and then chromatographed on
silica. Elution with EtOAc gave 4-(2-chlorophenyl)-9-hydroxy-
6-(3-methoxypropyl)-1H-furo[3,4-c]carbazole-1,3(6H )-dione
1
(90) as an orange powder which was used directly. H NMR
[(CD3)2SO] d 9.57 (br s, 1H), 8.24 (d, J ¼ 2.3 Hz, 1H), 8.00
(s, 1H), 7.66e7.61 (m, 2H), 7.58e7.48 (m, 3H), 7.22 (dd,
J ¼ 9.0, 2.3 Hz, 1H), 4.56 (t, J ¼ 6.7 Hz, 2H), 3.22 (t,
J ¼ 6.0 Hz, 2H), 3.13 (s, 3H), 2.02 (m, 2H).
1
(61 mg, 62%) as a yellow powder; mp 266e270 ꢀC. H NMR
Compound 90 was added to molten ammonium acetate
(10 g) at 140 ꢀC and the mixture was warmed at this temper-
ature for 3 h. Water was added and the resulting precipitate
was filtered off, adsorbed onto silica from a THF solution,
and chromatographed. Elution with EtOAc/petroleum ether
(1:1) gave 25 (32 mg, 71%) an orange powder; mp 260e
[(CD3)2SO] d 11.11 (br s, 1H), 8.55 (d, J ¼ 2.6 Hz, 1H), 7.87
(s, 1H), 7.68 (m, 3H), 7.48 (m, 3H), 7.33 (br s, 1H), 7.29 (dd,
J ¼ 9.0, 2.6 Hz, 1H), 6.85 (br s, 1H), 4.71 (t, J ¼ 6.5 Hz, 2H),
3.90 (s, 3H), 2.60 (t, J ¼ 6.5 Hz, 2H). Anal. C24H19N3O4$1/
2H2O (C, H, N).
1
262 ꢀC. H NMR [(CD3)2SO] d 11.07 (br s, 1H), 9.42 (br s,
5.6.3. 3-(9-Hydroxy-1,3-dioxo-4-phenyl-2,3-dihydropy-
rrolo[3,4-c]carbazol-6(1H )-yl)propanamide (19)
Reaction of 92a (56 mg, 0.14 mmol) with molten pyridine
hydrochloride, according to Procedure 7, followed by
1H), 8.39 (d, J ¼ 2.4 Hz, 1H), 7.69 (s, 1H), 7.60e7.44 (m,
5H), 7.15 (dd, J ¼ 8.8, 2.4 Hz, 1H), 4.49 (t, J ¼ 6.6 Hz, 2H),
3.22 (t, J ¼ 6.0 Hz, 2H), 3.13 (s, 3H), 1.99 (m, 2H). FABMS

1292
J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
chromatography on silica, eluting with EtOAc/hexane (4:1) to
EtOAc/MeOH (9:1), gave 19 (11 mg, 20%) as an orange pow-
der; mp 284e288 ꢀC. 1H NMR [(CD3)2SO] d 11.05 (br s, 1H),
9.33 (br s, 1H), 8.40 (d, J ¼ 2.5 Hz, 1H), 7.82 (s, 1H), 7.67 (m,
2H), 7.56 (d, J ¼ 8.8 Hz, 1H), 7.47 (m, 3H), 7.34 (br s, 1H),
7.12 (dd, J ¼ 8.8, 2.5 Hz, 1H), 6.86 (br s, 1H), 4.67 (t,
J ¼ 6.5 Hz, 2H), 2.58 (t, J ¼ 6.5 Hz, 2H). FABMS found
[M þ H]þ: 400.1307. C23H17N3O4 requires 400.1297.
diluted with water and extracted with EtOAc. Chromatography
of the product on silica, eluting with EtOAc/hexane (1:1), fol-
lowed by crystallisation from EtOAc/hexane, gave 23 (25 mg,
70%) as an orange powder; mp 218e220 ꢀC. 1H NMR
[(CD3)2SO] d 11.07 (br s, 1H), 9.39 (br s, 1H), 8.38 (d,
J ¼ 2.5 Hz, 1H), 7.79 (s, 1H), 7.57 (m, 2H), 7.51e7.44 (m,
3H), 7.14 (dd, J ¼ 8.8, 2.5 Hz, 1H), 4.71 (t, J ¼ 6.8 Hz, 2H),
3.48 (s, 3H), 2.83 (t, J ¼ 6.8 Hz, 2H). Anal.
C24H17ClN2O5$3/4H2O (C, H, N).
Compounds 20 and 21 were similarly prepared according to
Procedures 10, 11, and 4 (Supplementary material).
5.6.7.
4-(4-(2-Chlorophenyl)-9-methoxy-1,3-dioxo-2,3-
5.6.4. Procedure 12: solubilised carboxamide formation
dihydropyrrolo[3,4-c]carbazol-6(1H )-yl)butanoic acid (94)
Alkylation of 2-[(E,Z )-2-(2-chlorophenyl)ethenyl]-5-me-
thoxy-1H-indole (70b) [9] (0.40 g, 1.41 mmol) with NaH
(0.14 g of a 50% dispersion in mineral oil, 2.82 mmol) and
ethyl bromobutyrate in DMF (20 mL), according to Procedure
1, followed by reaction with maleimide and aromatisation with
DDQ, following Procedures 2 and 5, respectively, gave the
crude ester product. This crude product was dissolved in
MeOH (100 mL) and treated with 2 M KOH (2 mL) at room
temperature for 2 h. Dilution with water and acidification
with 1 N HCl gave a precipitate that was collected by filtra-
tion, dried and chromatographed on silica gel, eluting with
EtOAc. Trituration of the purified fractions with Et2O/hexane,
gave 94 as a yellow powder (0.10 g, 16%); mp 251e254 ꢀC.
1H NMR [(CD3)2SO] d 12.12 (br s, 1H), 11.12 (br s, 1H),
8.53 (d, J ¼ 2.6 Hz, 1H), 7.85 (s, 1H), 7.72 (d, J ¼ 9.0 Hz,
1H), 7.58 (m, 1H), 7.53e7.45 (m, 3H), 7.32 (dd, J ¼ 9.0,
2.6 Hz, 1H), 4.50 (t, J ¼ 7.3 Hz, 2H), 3.91 (s, 3H), 2.30 (m,
2H), 1.97 (m, 2H). FABMS found [M þ H]þ: 463.1025,
465.1034. C25H19ClN2O5 requires 463.1061, 465.1031.
5.6.4.1. N-[2-(Dimethylamino)ethyl]-3-(9-methoxy-1,3-dioxo-
4-phenyl-2,3-dihydropyrrolo[3,4-c]carbazol-6(1H )-yl)propa-
namide (93a). Reaction of 91a (100 mg, 0.24 mmol) with
oxalyl chloride, according to Procedure 11, gave the crude
acid chloride which was dissolved in dry THF (20 mL), flushed
with nitrogen, and treated with N,N-dimethylethylenediamine
(0.105 mL, 0.96 mmol) at room temperature for 2 h. The reac-
tion was diluted with water, basified with solid K2CO3, and
extracted with EtOAc. The organic layer was dried and evapo-
rated, and the residue was chromatographed on silica gel,
eluting with EtOAc/MeOH/Et3N (1:0:0 to 3:1:trace), to give
93a (85 mg, 73%) as a yellow powder; mp (EtOAc/hexane)
1
206e210 ꢀC. H NMR [(CD3)2SO] d 11.11 (br s, 1H), 8.54
(d, J ¼ 2.6 Hz, 1H), 7.81 (s, 1H), 7.73 (t, J ¼ 5.6 Hz, 1H),
7.67 (m, 3H), 7.48 (m, 3H), 7.29 (dd, J ¼ 8.9, 2.6 Hz, 1H),
4.73 (t, J ¼ 6.3 Hz, 2H), 3.89 (s, 3H), 2.93 (dd, J ¼ 6.8,
5.6 Hz, 2H), 2.59 (t, J ¼ 6.3 Hz, 2H), 1.90 (s, 6H), 1.84 (t,
J ¼ 6.8 Hz, 2H). Anal. C28H28N4O4$H2O (C, H, N).
5.6.5. N-[2-(Dimethylamino)ethyl]-3-(9-hydroxy-1,3-dioxo-
4-phenyl-2,3-dihydropyrrolo[3,4-c]carbazol-6(1H )-yl)-
propanamide (54)
5.6.8. 4-(4-(2-Chlorophenyl)-9-hydroxy-1,3-dioxo-2,3-
dihydropyrrolo[3,4-c]carbazol-6(1H )-yl)butanoic acid (68)
Demethylation of 94 (32 mg, 0.07 mmol) with 1 N BBr3 in
CH2Cl2, following Procedure 4, gave 68 (6 mg, 19%) as an or-
ange powder; mp 274e277 ꢀC. 1H NMR [(CD3)2SO] d 12.0 (v
br s, 1H), 11.06 (br s, 1H), 9.38 (br s, 1H), 8.38 (d, J ¼ 2.4 Hz,
1H), 7.80 (s, 1H), 7.60 (d, J ¼ 8.9 Hz, 1H), 7.58 (m, 1H),
7.52e7.43 (m, 3H), 7.14 (dd, J ¼ 8.9, 2.4 Hz, 1H), 4.45 (t,
J ¼ 7.5 Hz, 2H), 2.27 (m, 2H), 1.95 (m, 2H). FABMS found
[M þ H]þ: 449.0874, 451.0879. C24H17ClN2O5 requires
449.0904, 451.0875.
Compound 93a (70 mg, 0.14 mmol) was demethylated with
pyridine hydrochloride at 200 ꢀC for 10 min, following Proce-
dure 7. The reaction mixture was diluted with water, basified
with conc. ammonia and extracted with EtOAc. The organic
layer was dried and evaporated, and the residue was chromato-
graphed on silica gel, eluting with EtOAc/MeOH/Et3N (1:0:0
to 3:1:trace), to give 54 (35 mg, 53%); mp (EtOAc/hexane)
1
176e180 ꢀC. H NMR [(CD3)2SO] d 11.06 (br s, 1H), 9.35
(br s, 1H), 8.39 (d, J ¼ 2.5 Hz, 1H), 7.77 (s, 1H), 7.74 (t,
J ¼ 5.6 Hz, 1H), 7.66 (m, 2H), 7.53 (d, J ¼ 8.8 Hz, 1H), 7.48
(m, 3H), 7.12 (dd, J ¼ 8.8, 2.5 Hz, 1H), 4.69 (t, J ¼ 6.4 Hz,
2H), 2.94 (m, 2H), 2.58 (t, J ¼ 6.4 Hz, 2H), 1.91 (s, 6H),
1.87 (m, 2H). Anal. C27H26N4O4$3/4H2O (C, H, N).
Compounds 55, 56, and 59e61 were similarly prepared ac-
cording to Procedures 11, 12 and 7 (or 4) (Supplementary
material).
5.7. Procedures of Scheme 7
5.7.1. Procedure 13: acylsulfonamide formation
5.7.1.1. N-[3-(4-(2-Chlorophenyl)-9-methoxy-1,3-dioxo-2,3-
dihydropyrrolo[3,4-c]carbazol-6(1H )-yl)propanoyl]methane-
sulfonamide (95). To a stirred solution of 91b (0.20 g,
0.45 mmol), 4-dimethylaminopyridine (DMAP) (165 mg,
1.35 mmol) and methanesulfonamide (86 mg, 0.90 mmol) in
DMF (10 mL) under nitrogen was added 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (EDCI$HCl)
(259 mg, 1.35 mmol). The resulting mixture was stirred at
room temperature for 18 h and then diluted with water,
5.6.6. Methyl 3-(4-(2-chlorophenyl)-9-hydroxy-1,3-dioxo-2,3-
dihydropyrrolo[3,4-c]carbazol-6(1H )-yl)propanoate (23)
Gaseous HCl was bubbled through a stirred solution of 60
(35 mg, 0.08 mmol) in MeOH (10 mL) for 30 s. The resulting
solution was stirred for 30 min at room temperature, then

J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
1293
acidified by the addition of 1 N HCl, and extracted with
EtOAc. The organic phase was dried (Na2SO4), the drying
agent was removed, and the solution was concentrated to dry-
ness. Chromatography on silica, eluting with EtOAc/hexane
(4:1) to EtOAc/MeOH (1:0 to 9:1), gave 95 (151 mg, 64%)
to dryness. Crystallisation from EtOAc/hexane gave 97
1
(0.96 g, 74%) as a yellow solid; mp 254 ꢀC (dec). H NMR
[(CD3)2SO] d 11.15 (s, 1H), 8.53 (d, J ¼ 2.6 Hz, 1H), 7.90
(s, 1H), 7.74 (d, J ¼ 9.0 Hz, 1H), 7.58 (m, 1H), 7.48 (m,
3H), 7.33 (dd, J ¼ 9.0, 2.6 Hz, 1H), 4.89 (t, J ¼ 5.0 Hz, 2H),
4.56 (m, 2H), 3.91 (s, 3H), 2.94 (s, 3H). FABMS found
[M þ H]þ: 499.0694, 501.0696. C24H19ClN2O6S requires
499.0731, 501.0701.
1
as a yellow powder; mp 293e295 ꢀC. H NMR [(CD3)2SO]
d 11.76 (br s, 1H), 11.14 (br s, 1H), 8.52 (d, J ¼ 2.6 Hz,
1H), 7.87 (s, 1H), 7.73 (d, J ¼ 9.0 Hz, 1H), 7.58 (m, 1H),
7.53e7.46 (m, 3H), 7.32 (dd, J ¼ 9.0, 2.6 Hz, 1H), 4.74 (t,
J ¼ 6.9 Hz, 2H), 3.91 (s, 3H), 3.08 (s, 3H), 2.81 (t,
J ¼ 6.9 Hz, 2H). Anal. C25H20ClN3O6S (C, H, N).
5.7.5. 4-(2-Chlorophenyl)-9-methoxy-6-[2-(4H-1,2,4-triazol-
3-ylsulfanyl)ethyl]pyrrolo[3,4-c]carbazole-1,3(2H,6H )-
dione (98)
5.7.2. N-[3-(4-(2-Chlorophenyl)-9-hydroxy-1,3-dioxo-2,3-
dihydropyrrolo[3,4-c]carbazol-6(1H )-yl)-
propanoyl]methanesulfonamide (62)
A solution of 97 (0.25 g, 0.50 mmol), 1H-1,2,4-triazole-5-
thiol (76 mg, 0.75 mmol) and Et3N (2.0 mL) in p-dioxane
(50 mL) under nitrogen was heated at reflux for 48 h, then di-
luted with water and extracted with EtOAc. The organic phase
was dried, the drying agent was removed and the solution was
concentrated to dryness. Chromatography on silica, eluting
with EtOAc/hexane (1:1 to 1:0), followed by crystallisation
from EtOAc/hexane, gave 98 (0.24 g, 95%) as a yellow pow-
der; mp 211e213 ꢀC. 1H NMR [(CD3)2SO] d 14.09 (br s, 1H),
11.13 (br s, 1H), 8.52 (d, J ¼ 2.6 Hz, 1H), 8.43 (br s, 1H), 7.88
(s, 1H), 7.80 (d, J ¼ 9.0 Hz, 1H), 7.57 (m, 1H), 7.48 (m, 3H),
7.31 (dd, J ¼ 9.0, 2.6 Hz, 1H), 4.83 (t, J ¼ 7.4 Hz, 2H), 3.91 (s,
3H), 3.52 (t, J ¼ 7.4 Hz, 2H). Anal. C25H18ClN5O3S$1/4H2O
(C, H, N).
Reaction of 95 (130 mg, 0.25 mmol) with BBr3, according
to Procedure 4, followed by chromatography on silica, eluting
with MeOH/CH2Cl2 (5:95e1:9), gave 62 (83 mg, 66%) as an
orange powder; mp 290e296 ꢀC. 1H NMR [(CD3)2SO]
d 11.75 (br s, 1H), 11.08 (br s, 1H), 9.38 (s, 1H), 8.38 (d,
J ¼ 2.4 Hz, 1H), 7.80 (s, 1H), 7.59 (m, 2H), 7.53e7.46 (m,
3H), 7.14 (dd, J ¼ 8.9, 2.4 Hz, 1H), 4.69 (t, J ¼ 6.9 Hz,
2H), 3.04 (s, 3H), 2.76 (t, J ¼ 6.9 Hz, 2H). Anal.
C24H18ClN3O6S$3/4H2O (C, H, N).
Compound 63 was similarly prepared according to Proce-
dures 13 and 4 (Supplementary material).
5.7.3. Procedure 14: tetrazole formation
5.7.6. 4-(2-Chlorophenyl)-9-hydroxy-6-[2-(4H-1,2,4-triazol-
3-ylsulfanyl)ethyl]pyrrolo[3,4-c]carbazole-1,3(2H,6H )-
dione (65)
5.7.3.1. 4-(2-Chlorophenyl)-9-hydroxy-6-[2-(1H-tetrazol-5-yl)-
ethyl]pyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione
(64). To
Reaction of 98 (0.25 g, 0.50 mmol) with BBr3 in CH2Cl2,
following Procedure 4, and chromatography of the product
on silica gel, eluting with EtOAc/THF (1:0 to 9:1 to 1:1), fol-
lowed by trituration in MeOH, gave 65 (0.22 g, 90%) as an or-
a solution of 22 (0.68 g, 1.63 mmol) in toluene/DMF (5:1,
120 mL) was added azidotrimethyl tin (0.67 g, 3.26 mmol).
The resulting solution was heated at reflux for 24 h, then a fur-
ther portion of azidotrimethyl tin (0.34 g, 1.63 mmol) was
added. After a further 24 h at reflux, the reaction was diluted
with water and extracted with EtOAc. Chromatography on sil-
ica, eluting with EtOAc/hexane (3:1) to EtOAc/MeOH (9:1),
followed by crystallisation from EtOAc/hexane, gave 64
(0.54 g, 72%) as an orange powder; mp 230e234 ꢀC (dec).
1H NMR [(CD3)2SO] d 11.06 (br s, 1H), 9.37 (br s, 1H),
8.36 (d, J ¼ 2.4 Hz, 1H), 7.61 (s, 1H), 7.56 (m, 1H), 7.48
(m, 4H), 7.10 (dd, J ¼ 8.8, 2.4 Hz, 1H), 4.82 (t, J ¼ 6.9 Hz,
2H), 3.32 (obscured t, J ¼ 6.9 Hz, 2H). Anal.
C23H15ClN6O3$3½H2O (C, H, N).
1
ange powder; mp 314e319 ꢀC. H NMR [(CD3)2SO] d 14.10
(br s, 1H), 11.07 (br s, 1H), 9.39 (s, 1H), 8.42 (br s, 1H), 8.38
(d, J ¼ 2.3 Hz, 1H), 7.83 (s, 1H), 7.68 (d, J ¼ 8.8 Hz, 1H),
7.57 (m, 1H), 7.48 (m, 3H), 7.13 (dd, J ¼ 8.8, 2.3 Hz, 1H),
4.79 (t, J ¼ 7.4 Hz, 2H), 3.50 (t, J ¼ 7.4 Hz, 2H). Anal.
C24H16ClN5O3S$2/5MeOH (C, H, N).
5.7.7. 4-(2-Chlorophenyl)-9-hydroxy-6-[2-
(1H-1,2,4-triazol-5-ylsulfinyl)ethyl]pyrrolo[3,4-c]-
carbazole-1,3(2H,6H )-dione (66)
To a solution of 65 (70 mg, 0.14 mmol) in THF (30 mL)
and glacial HOAc (8 mL) was added 35% H2O2 (2 mL). The
resulting solution was stirred at room temperature for 10 h,
then poured onto solid NaHCO3, diluted with water, and ex-
tracted with EtOAc. The combined organic layers were dried
(Na2SO4), partially concentrated under reduced pressure,
then adsorbed directly onto silica and chromatographed, elut-
ing with MeOH/CH2Cl2 (5:95e1:9). Trituration in Et2O gave
5.7.4. 2-(4-(2-Chlorophenyl)-9-methoxy-1,3-dioxo-2,3-dihy-
dropyrrolo[3,4-c]carbazol-6(1H )-yl)ethyl-
methanesulfonate (97)
Alcohol 77b (1.10 g, 2.61 mmol) was dissolved in dry THF
(80 mL) under nitrogen. The resulting solution was cooled to
0 ꢀC and Et3N (2.0 mL) was added, followed by MsCl
(263 mL, 3.40 mmol) dropwise. After 30 min a further portion
of MsCl (50 mL) was added and then, after another 30 min, the
reaction was diluted with saturated aqueous NaHCO3 and ex-
tracted with EtOAc. The organic layer was dried (Na2SO4), the
drying agent was removed and the solution was concentrated
1
66 (29 mg, 41%) as an orange powder; mp 237e242 ꢀC. H
NMR [(CD3)2SO] d 14.7 (br s, 1H), 11.09 (br s, 1H), 9.41
(br s, 1H), 8.69 (br s, 1H), 8.38 (d, J ¼ 2.3 Hz, 1H), 7.79 (d,
J ¼ 9.6 Hz, 1H), 7.60e7.46 (m, 5H), 7.15 (dd, J ¼ 8.8,

1294
J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
2.3 Hz, 1H), 4.89 (m, 2H), 3.71 (m, 2H). Anal.
C24H16ClN5O4S$1/5Et2O (C, H, N).
plate was incubated at 37 ꢀC for 4 h. The plate was removed
from the incubator and spun in a Beckman GS-6R Centrifuge
for 10 min at 800 rpm and 4 ꢀC. The media was removed and
the plate surface dried by blotting. To each well 100 mL of
PBS was added. The plate was spun as above. The PBS was
removed from the plate and the plate surface was dried. To
each well 20 mL of lysis buffer (50 mM Hepes pH 7.5,
250 mM NaCl, 0.1% NP 40, 10 mM b-glycerophosphate,
1 mM NaF, 1 mM EDTA, 1 mM pefabloc, 1 mM DTT,
0.1 mM sodium orthovanadate, 10 mg/mL aprotinin, 20 mM
leupeptin) was then added, followed by medium-speed rocking
at 4 ꢀC for 45 min. After lysis, 30 mL of kinase assay buffer
(50 mM Hepes, 22 mM MgCl2, 1 mM DTT, 166.7 ng/mL his-
tone H1, 83 mM ATP, 0.033 mCi/mL [g-33P] ATP) was added.
The plate was incubated on a 32 ꢀC plate warmer for
25 min. The kinase reaction was stopped by adding 80 mL of
100 mM EDTA, pH 7.8, to each well. The lysate was har-
vested onto a pre-wetted Wallac P30 filtermat (Wallac
1450e523, glass fiber filter with negatively charged P30 active
groups, size 90 ꢃ 120 mm) using 75 mM H3PO4 for 10 s, fol-
lowed by a 10 s aspiration step. The filtermat was placed in
a 75 mM H3PO4 bath and shaken gently for 10 min at room
temperature, then placed within the fold of a single sheet of
paper towel, and subjected to microwaves on high power for
2e3 min, or until the filtermat was dry. The filtermat was
placed in a sample bag (Wallac 1450-432), 5 mL nonaqueous
scintillation fluid was added to the sample bag and the bag was
sealed. The samples were read in a Wallac 1450 MicroBeta
Liquid Scintillation Counter. The data shown in Table 1 dem-
onstrate the cellular effect of a Wee1 and/or Chk1 inhibitor on
a physiological substrate of Cdc2/cyclinB, a complex which
itself is a substrate of Wee1 kinase.
5.7.8. 4-(2-Chlorophenyl)-9-hydroxy-6-[2-(1H-1,2,4-tria-
zol-5-ylsulfonyl)ethyl]pyrrolo[3,4-c]carbazole-1,3(2H,6H )-
dione (67)
To a solution of 65 (60 mg, 0.12 mmol) in THF (30 mL)
and glacial HOAc (10 mL) was added 35% H2O2 (2 mL).
The resulting solution was stirred at room temperature for
30 h, then additional 35% H2O2 (2 mL) was added and the
temperature was increased to 50 ꢀC for 18 h. The reaction
was then poured onto solid NaHCO3, diluted with water, and
extracted with EtOAc. The combined organic layers were
dried, partially concentrated under reduced pressure, then ad-
sorbed directly onto silica and chromatographed, eluting with
EtOAc/hexane (1:1 to 1:0). Crystallisation from EtOAc/hex-
ane gave 67 (45 mg, 72%) as an orange powder; mp 301e
1
304 ꢀC. H NMR [(CD3)2SO] d 14.9 (br s, 1H), 11.07 (br s,
1H), 9.38 (br s, 1H), 8.60 (s, 1H), 8.34 (d, J ¼ 2.4 Hz, 1H),
7.71 (s, 1H), 7.56 (m, 2H), 7.48 (m, 3H), 7.12 (dd, J ¼ 8.8,
2.4 Hz, 1H), 4.89 (m, 2H), 4.03 (m, 2H). Anal.
C24H16ClN5O5S$1/4EtOAc (C, H, N).
5.7.9. 4-(2-Chlorophenyl)-9-hydroxy-6-[3-(1H-tetrazol-5-yl)-
propyl]pyrrolo[3,4-c]carbazole-1,3(2H,6H )-dione (69)
A solution of 24 (60 mg, 0.14 mmol) in toluene/DMF (5:1,
35 mL) was treated with azidotrimethyl tin (57 mg,
0.28 mmol) for 72 h, according to Procedure 14. Chromatogra-
phy on silica gel, eluting with EtOAc/hexane (3:1) to EtOAc/
MeOH (9:1), gave 69 (29 mg, 44%); mp (THF/hexane) 269e
1
272 ꢀC. H NMR [(CD3)2SO] d w16 (v br s, 1H), 11.07 (s,
1H), 9.38 (br s, 1H), 8.39 (d, J ¼ 2.5 Hz, 1H), 7.84 (s, 1H),
7.61 (d, J ¼ 8.9 Hz, 1H), 7.57 (m, 1H), 7.53e7.43 (m, 3H),
7.16 (dd, J ¼ 8.9, 2.5 Hz, 1H), 4.59 (t, J ¼ 7.1 Hz, 2H), 2.93
(m, 2H), 2.20 (m, 2H). Anal. C24H17ClN6O3$3/4H2O$1/
4THF (C, H, N).
5.10. Detection of MPM-2 in cultured HT-29
cells ꢁ potential checkpoint abrogators, doxorubicin,
and nocodazole
5.8. Determinations of enzyme inhibition
This assay uses polyclonal antibody to quantitate the M-
phase specific histological markers in the determination of
a mitotic index (fraction of cells found in mitosis). HT-29 cells
were grown in Dulbecco’s Modified Eagle Medium with high
glucose, supplemented with 1 mM sodium pyruvate, 2 mM
L-glutamine, 16 mM HEPES, 8 mM MOPS, and 10% fetal
bovine serum. They were incubated at 37 ꢀC in an atmosphere
of 5% CO2 and 100% relative humidity. Cells were seeded in
96-well tissue culture plates at 100 mL per well at a concentra-
tion of 4 ꢃ 104 per mL. The cells were allowed to attach and
begin growing for 24 h. To the test cells 100 mL of doxorubicin
(DOX) at 2 mM (final ¼ 1 mM) was added and the cells were
incubated for 1 h as above. Following the incubation, the
plates were washed twice with growth media. The media
was replaced with 100 mL fresh media and incubated for a fur-
ther 16 h. Serial 2-fold dilutions of potential abrogators were
added to the test wells. The rows that received nocodazole
(NOC) have 2 mL NOC added at 2.5 mg/mL. The plates were
then incubated for an additional 6 h. Following the incubation,
the plates were centrifuged for 5 min at 4 ꢀC and 200G.
Wee1 and Chk1 isolated enzyme inhibition assays were
performed as reported previously [9].
5.9. Cdc2 histone H1 kinase assay for G2 checkpoint
abrogation
This cellular assay is a measure of the effect of the test
compounds on the activity of the Cdc2/cyclin B complex on
one of its physiological substrates, histone H1. HT-29 cells,
2 ꢃ 104 per well (NUNCLONÔ cat no. 163320, 96-well
plate), were plated in 171 mL media [Dulbecco’s Modified Ea-
gle’s Medium 4500 mg/L Glucose (DME High Glucose), 1%
penicillin and streptomycin, 2% L-glutamate, 10% FBS]. The
plate was incubated at 37 ꢀC for 24 h. To each well 9 mL of
a 5 mM doxorubicin (DOX) solution was added (250 nM final
concentration) and incubated at 37 ꢀC for an additional 16 h.
Next, 20 mL of 500 ng/mL nocodazole was added per well, im-
mediately followed by addition of 5 mL test compound. The

J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
1295
To each well 100 mL of methanol/acetic acid in a ratio 3:1, was
added directly and left at room temperature for 15 min. The
media/fixative mixture was removed by suction and replaced
with ice cold ethanol/acetic acid in a 20:1 ratio. The plates
were stored at 4 ꢀC until stained. The fixed cells were stained
for MPM-2 using the Upstate Biotechnology MPM-2 rhoda-
mine detection kit as per the manufacturer’s instructions.
Briefly, the cells were washed with Dulbecco’s phosphate
buffered saline (DPBS) with calcium and magnesium, fol-
lowed by incubation with blocking buffer, consisting of 8%
bovine serum albumen (BSA) for a minimum of 1 h. The cells
were then washed with PBS one time and treated overnight at
4 ꢀC with primary antibody at 5 mg/mL (100 mL per well), di-
luted using DPBS with 1% BSA. The cells were then washed
twice for 15 min each with DPBS and then incubated with
a 1:1000 dilution of rhodamine conjugated goat antimouse
IgG in DPBS with 1% BSA for 1 h. The secondary antibody
was washed off in three 30 min washes with DPBS. The cells
were then counterstained using the Molecular Probes Myco-
flour kit to stain nuclei following the instructions for the kit.
The stained cells were viewed under fluorescence microscopy
using fluorescence filters suitable for detecting rhodamine and
DAPI stains. Images were captured using a Spot digital camera
and analyzed using ImageQuant to quantitate total nuclei and
to count MPM-2 positive cells.
calcium and magnesium and the rinse combined with the
scraped cells. The cells were centrifuged at 200G at 4 ꢀC
for 5 min. The supernatant was discarded and the pellets re-
suspended in 100 mL DPBS. The cell suspension was then
transferred to 1.5 mL eppendorf centrifuge tubes and centri-
fuged at 4 ꢀC for 4 min at 4000 rpm. After the supernatant
was removed, the pellet was frozen on dry ice and stored
at ꢂ80 ꢀC. The pellets were thawed on ice prior to lysis
with the lysis buffer, ELB (2.5 mM HEPES (7.5), 150 mM
NaCl, 25 mM NaF and 0.5% NP40 supplemented with 1 mM
AEBSF, 1 mM sodium orthovanadate, and 1 mM dithiothre-
tol) and complete protease inhibitor cocktail tablets (Roche
Biochemicals). The tablets were dissolved in 2 mL distilled
water and diluted 1:25 in the lysis buffer. The pellets were
suspended in 100 mL complete lysis buffer and incubated on
ice for 30 min. Following lysis, the suspension was centri-
fuged at 14,000 rpm for 15 min at 4 ꢀC. The supernatant liq-
uid was collected and the protein concentration determined
using the Pierce BCA protein assay kit as per the manufactur-
er’s instructions. The protein concentration was adjusted to
3 mg/mL with DPBS. The samples were then diluted 1:1
with Invitrogen 2 ꢃ Triseglycine sample buffer supplemented
with 50 mL/mL 2-mercaptoethanol, boiled for 3 min, and
stored frozen at ꢂ20 ꢀC. Thirty micrograms of protein per
lane were run on Novex pre-cast 12%, 1.5 mm, 10-well,
Triseglycine polyacrylimide gels using Novex running buffer
and Invitrogen (see Blue Plus 2) molecular weight standards.
The gels were run at 100 V for 30 min, then 125 V for 1.5 h.
The proteins were transferred to 0.45 mm pore nitrocellulose
membranes using Novex transfer buffer and the Novex X-
Cell II blot module. The nitrocellulose membranes were
blocked overnight at room temperature. The blocking buffer
was 5 mM Tris (8.0), 150 mM NaCl, 0.1% Tween 20,1 mM
NaF, 10 mM glycerolphosphate, 100 mM sodium orthovana-
date, and 3% bovine serum albumen. After blocking, the
gels were cut with a razor blade and treated with biotinylated
antiphosphotyrosine 15, diluted 1:5000 in blocking buffer.
The membranes were incubated for 2 h at room temperature
with constant rocking. The antibody solution was removed
and the membranes were washed three times for 20 min
each with TNT buffer. TNT buffer consisted of 50 mM Tris
(8.0), 150 mM NaCl, and 0.1% Tween 20. Secondary antibody
was then added in blocking buffer (Neutravidin HRP at
1:40,000). The blots remained in secondary antibody for 1 h
at room temperature, followed by three 20 min washes with
TNT buffer. Protein bands were detected using the Amersham
Pharmacia ECL detection kit and Kodak Bio Max film as per
the manufacturer’s instructions. The phosphotyrosine 15
membranes were stripped using the Chemicon International
Re-Blot kit as per the manufacturer’s instructions. The blots
were then washed twice with TNT buffer and once with block-
ing buffer for 20 min each. Anti Cdc2 (cdk1; Labvision Cor-
poration) were diluted 150 mL per 50 mL blocking buffer and
incubated with the blots for 2 h at room temperature, followed
by three 20 min washes in TNT buffer. The secondary anti-
body was Bio Rad goat antimouse HRP and was diluted
1:10,000 in blocking buffer before a 1 h incubation with the
5.11. Western blot determination of phosphotyrosine
15 on Cdc2
These western blot assays measure the phosphorylation
state of the physiological substrate of Wee1: tyrosine 15 on
Cdc2 kinase. This is accomplished by means of a phosphospe-
cific antibody whose signal is normalised by comparison with
the total amount of Cdc2 detected in the samples. HT-29 cells
were grown in Dulbecco’s Modified Eagle Medium with high
glucose, supplemented with 1 mM sodium pyruvate, 2 mM
L-glutamine, 16 mM HEPES, 8 mM MOPS, and 10% fetal
bovine serum. The cells were incubated at 37 ꢀC, in 5%
CO2, and 100% relative humidity. Cells were grown and
treated in 6-well tissue culture plates. Cells were seeded in
3 mL media at a concentration of 2 ꢃ 105 per mL. Once
seeded, the cells were allowed to attach 24 h. All treatments
were done in duplicate wells. The wells that were treated
with doxorubicin (DOX) were exposed to 1 mM DOX for
1 h. DOX was dissolved in sterile distilled water. After the
1 h incubation, the cells were washed twice with 2 mL media
and then incubated in 3 mL media for 16 h. After the 16 h in-
cubation, the cells were treated with various concentrations of
abrogator ꢁ nocodazole (NOC) at 50 ng/mL. Abrogators were
dissolved in dimethylsulfoxide (DMSO) at a concentration of
10 mM and diluted with growth medium before being added
to the cells and NOC was dissolved at 1 mg/mL in DMSO
and diluted with growth medium before administration to
the cells. The cells were incubated 6 h with the abrogator
and NOC. The duplicate wells were scraped, on ice, and com-
bined in a 15 mL centrifuge tube. The wells were rinsed with
Dulbecco’s phosphate buffered saline (DPBS) without

1296
J.B. Smaill et al. / European Journal of Medicinal Chemistry 43 (2008) 1276e1296
blots at room temperature. Three 20 min washes preceded
ECL detection.
The Wee1 complex structures were determined by molecular
replacement using the previously published structure as initial
model and refinement with Refmac [14]. Refinement pro-
ceeded with the replacement of molecule 6 by molecule 18
or 59 as appropriate. Tabulated details of the data collection,
structure solution and refinement can be found in Supplemen-
tary material. Compound 18 X-ray coordinates; PDB ID 2IO6.
Compound 59 X-ray coordinates; PDB ID 2IN6.
5.12. Clonogenic survival assay in HT-29
cells ꢁ doxorubicin
This cellular assay is a measure of the toxicity of the test
compounds in the absence and presence of DNA damage in-
duced by a conventional chemotherapeutic agent. HT-29 cells
were grown in Dulbecco’s Modified Eagle Medium with high
glucose, supplemented with 1 mM sodium pyruvate, 2 mM
L-glutamine, 16 mM HEPES, 8 mM MOPS, and 10% fetal
bovine serum. The cells were incubated at 37 ꢀC in an atmo-
sphere of 5% CO2 and 100% relative humidity. Two or three
T-75 tissue culture flasks were seeded at about 50% confluency
in 30 mL media and incubated for approximately 24 h. Doxo-
rubicin (DOX) (dissolved at 5 mM in distilled water (dH2O))
was added to the flasks to a final concentration of 1 mM or
500 nM. One flask received no DOX. The cells were allowed
to incubate with the DOX for 1 h. All flasks were then washed
twice with 20 mL media and allowed to incubate for a further
16 h in 30 mL media. The stock agar (3.2% Seaplaque GTG
Agarose (BioWhittaker Molecular Applications)) was sus-
pended in dH2O and autoclaved for 20 min. The agar was
melted prior to use in a microwave oven. The bottom agar
was a 1:4 dilution of the stock agar in media, with enough fetal
bovine serum added to bring the solution to 10%. One milliliter
was plated in each well of a 6-well tissue culture plate and the
plates were allowed to harden. The cloning agar (a 1:8 dilution
of stock agar in media with fetal bovine serum added to 10%)
was prepared and held at 40 ꢀC until used. The cells were tryp-
sinized using Trypsin/EDTA and their concentration adjusted
to 7.5 ꢃ 104 cells/mL with media. Into sterile 15 mL plastic
centrifuge tubes 100 mL of each cell suspension was placed.
Twenty-five microliters of each test compound was added to
appropriate tubes, followed by the addition of 5 mL of warm
cloning agar. The tubes were mixed well and 2 mL of the
agar/cell suspension was added to duplicate wells of the 6-
well plates that were coated with agar earlier. The plates
were swirled and placed in the refrigerator for 5 min. After
the plates returned to room temperature, they were incubated
for 10 to 14 days, until colonies were visible. The colonies
were stained with INT ( p-iodonitrotetrazolium violet) (dis-
solved in dH2O at 1 mg/mL and filter sterilized). To each
well 1 mL INT was added and the plates were incubated over-
night at 37 ꢀC, 5% CO2, and 100% relative humidity. The col-
onies were counted using a Hamamatsu video imaging system
and ImageQuant software.
Acknowledgements
We would like to thank ONYX Pharmaceuticals and Dr.
Peter Toogood for helpful discussions. We also thank the
Cancer Society Auckland and the Maurice Wilkins Centre
for Molecular Biodiscovery for continued support.
Appendix. Supplementary material
Additional experimental procedures, characterisations, and
combustion analytical data. Tabulated details of the data col-
lection, structure solution and refinement for compounds 18
and 59 bound to Wee1 kinase. This material is available online
at www.sciencedirect.com. Supplementary material associated
with this article can be found in the online version, at
doi:10.1016/j.ejmech.2007.07.016.
References
[1] J. Li, Y. Wang, Y. Sun, T.S. Lawrence, Radiat. Res. 157 (2002) 322e330.
[2] Y. Wang, J. Li, R.N. Booher, A. Kraker, T. Lawrence, W.R. Leopold,
Y. Sun, Cancer Res. 61 (2001) 8211e8217.
[3] M. Suganuma, T. Kawabe, H. Hori, T. Funabiki, T. Okamoto, Cancer
Res. 59 (1999) 5887e5891.
[4] A.N. Tse, G.K. Schwartz, Cancer Res. 64 (2004) 6635e6644.
[5] N.R. den Elzen, M.J. O’Connell, EMBO J. 23 (2004) 908e918.
[6] E.A. Kohn, C.J. Yoo, A. Eastman, Cancer Res. 63 (2003) 31e35.
[7] X. Jiang, B. Zhao, R. Britton, L.Y. Lim, D. Leong, J.S. Sanghera,
B.S. Zhou, E. Piers, R.J. Anderson, M. Roberge, Mol. Cancer. Ther. 3
(2004) 1221e1227.
[8] B.D. Palmer, J.B. Smaill, G.W. Rewcastle, E.M. Dobrusin, A. Kraker,
C.W. Moore, R.W. Steinkamp, W.A. Denny, Bioorg. Med. Chem. Lett.
15 (2005) 1931e1935.
[9] B.D. Palmer, A.M. Thompson, J.R. Booth, E.M. Dobrusin, A.J. Kraker,
H.H. Lee, E.A. Lunney, L.H. Mitchell, D.F. Ortwine, J.B. Smaill,
L.M. Swan, W.A. Denny, J. Med. Chem. 49 (2006) 4896e4911.
[10] J. Kleinscroth, J. Hartenstein, C. Schachtele, C. Rudolph, D.J. Dooley, G.
Weinheimer, U.S. Patent 4,912,107, 1990.
[11] B. Zhao, M.J. Bower, P.J. McDevitt, H. Zhao, S.T. Davis, K.O. Johanson,
S.M. Green, N.O. Concha, B.-B.S. Zhou, J. Biol. Chem. 277 (2002)
46609e46615.
[12] C. Squire, J.M. Dickson, I. Ivanovic, E.N. Baker, Structure 13 (2005)
541e550.
5.13. Crystallography
[13] S.P. Davies, H. Reddy, M. Caivano, P. Cohen, Biochem. J. 351 (2000)
95e105.
Wee1 protein was expressed, purified, complexed, crystal-
lised, and X-ray data collected as described previously [12].
[14] G.N. Murshudov, A.A. Vagin, E.J. Dodson, Acta Crystallogr. D 53
(1997) 240e255.