Synthesis and evaluation of 5-substituted 9-hydroxypyrrolo[3,4-c]carbazole- 1,3(2H,6H)-diones as check point 1 kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.09.042

The structure-activity relationship (SAR) of 5-substituted pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione derivatives 5 was investigated for their potential as Chk1 inhibitors for possible chemo- and radio-potentiators in anticancer chemotherapies. In silico vi

Full text of DOI:10.1016/j.bmc.2010.09.042

Bioorganic & Medicinal Chemistry 18 (2010) 7878–7889
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of 5-substituted 9-hydroxypyrrolo[3,4-c]carbazole-1,
3(2H,6H)-diones as check point 1 kinase inhibitors
Yuki Sako a, Satoshi Ichikawa a, , Akiko Osada b, Akira Matsuda a,
⇑
⇑
a Faculty of Pharmaceutical Sciences, Hokkaido University, Kita-12, Nishi-6, Kita-ku, Sapporo 060-0812, Japan
b Discovery & Development Laboratory I, Hanno Research Center, Taiho Pharmaceutical Co., Ltd, 1–27 Misugidai, Hanno, Saitama 357, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The structure–activity relationship (SAR) of 5-substituted pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione
derivatives 5 was investigated for their potential as Chk1 inhibitors for possible chemo- and radio-poten-
tiators in anticancer chemotherapies. In silico virtual screening helped to optimize the substituent on the
phenyl ring, and led to identification of the m-carbamoyl group among the 117 analogues tested. Further
optimization studies focusing on the docking model of 15 in the active site of Chk1 revealed that 32b
(IC50 = 2.8 nM) was a more potent inhibitor than UNC-01.
Received 7 September 2010
Revised 15 September 2010
Accepted 16 September 2010
Available online 22 September 2010
Keywords:
Check point
Ó 2010 Elsevier Ltd. All rights reserved.
Antitumor
Protein kinase
Structure-based drug design
1. Introduction
Several Chk1 inhibitors have entered Phase clinical trials.12–14
A
pyrrolo[3,4-c]carbazole motif is one of the representative scaffolds
for Chk1 inhibitors. UCN-01 (Fig. 1, 1), which is an indolocarbazole
natural product and currently in Phase II clinical trials15 in the US,
has been shown to enhance the therapeutic activity of DNA-dam-
aging agents in animal models.16 UCN-01, however, exhibited an
extremely long half-life that resulted from avid binding to a human
Many well-established cancer chemotherapies and radiothera-
pies exert their therapeutic effect through damage to DNA.
However, the activation of cell cycle checkpoints provides an
opportunity for cancer cells to repair DNA damage by reducing
the antitumor effect of DNA-damaging treatments.1 The G1 check-
point mediated by p53 is a major barrier to genomic instability, but
over half of all cancer cells are functionally defective for the p53
pathway.2 These tumor cells are more reliant on the S and G2/M
checkpoints to repair the damages. Premature mitotic entry of cells
with unrepaired DNA leads to mitotic catastrophe and/or apopto-
sis. Therefore drugs that abrogate the DNA damage-induced S
and G2/M checkpoints should selectively sensitize p53 deficient
cancer cells to anticancer therapies.1,3–5 The G2/M checkpoint is
initiated by two sensor kinases, ataxia telangiectasia (ATM) and
ATM- and Rad3-related (ATR), and two effector kinases, check
point kinase 1 (Chk1) and check point kinase 2 (Chk2). Among
them, Chk1 plays a crucial role in the G2/M checkpoint. Phosphor-
ylation of Cdc25A phosphatase by Chk1 inactivates Cdc25A, which
is responsible for activating the Cdc2 to regulate the cell-cycle
upon removal of the inhibitory phosphate at Tyr15. Consequently,
Chk1 inhibitors have been targeted as possible chemo- and radio-
potentiators.6–11
plasma protein,
a
1-acid glycoprotein.17 Although much effort has
been expanded to discover chemotypes different from the indoloc-
arbazole, most of these published Chk1 inhibitors originate from
hits discovered by high throughput screening (HTS) of large con-
ventional compound libraries,18–20 except for a few examples.21
The natural product staurosporine (2) did not show severe protein
binding in spite of its close similarity in chemical structure to UCN-
01, and isogranulatimide (3) is also known as a Chk1 inhibitor.
Therefore, the pyrrolo[3,4-c]carbazole motif is still a good scaffold
to develop novel Chk1 inhibitors.22 It has been reported that the
⇑
Corresponding authors. Tel.: +81 11 706 3763; fax: +81 11 706 4980 (S.I.); tel.:
+81 11 706 3228; fax: +81 11 706 4980 (A.M.).
E-mail addresses: ichikawa@pharm.hokudai.ac.jp (S. Ichikawa), matuda@
pharm.hokudai.ac.jp (A. Matsuda).
Figure 1. Structures of Chk1 inhibitors.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.042

Y. Sako et al. / Bioorg. Med. Chem. 18 (2010) 7878–7889
7879
of 11 with benzylamine using EDCI and DMAP in DMF gave 12 in
82% yield.
The Chk1 inhibitory activity of 10–12 was then evaluated with a
drug concentration of 10
carboxylic acid 11, and the benzyl amide 12 showed 1%, 46%, and
71% inhibition at 10 M drug concentrations, respectively, and
lM (Table 1). The t-butyl ester 10, the
l
the IC50 value of 12 was 9000 nM. Although the activity was very
weak, it was found that the N-benzylcarbamoyl group could be
introduced at the 5-position. The catalytic activity of most of the
protein kinases is regulated by the dynamics of the DFG-loop, the
conformation of which is called ‘DFG-in’ for the active and
‘DFG-out’ for the inactive form. This aspect consequently makes
it difficult to design an inhibitor to interact with this region.25
However, the catalytic activity of Chk1 was regulated by the
C-terminal domain, and the dynamics of the DFG-loop is quite
limited among the DFG-in vs. DFG-out.26 The properties of Chk1
allowed us to apply an in silico docking study in order to optimize
the substituent on the phenyl group. A total 117 analogues with
ortho-, para-, or meta-substituted N-phenyl-, -benzyl-, or -pheneth-
ylcarbamoyl groups (Fig. 3) were docked to the active site of the
public domain crystal structure published for Chk1 (PDB accession
code 1nvq)26 using the Glide program,27–29 and the docked struc-
tures were ranked by GlideScore. The m-carbamoylbenzyl deriva-
tive 15 (Scheme 1) showed the best score (GlideScore = ꢀ11.34).
The docking structure indicated additional interactions of the
carbamoyl moiety with Asp148 within the DFG-loop as shown in
Figure 4a. In order to confirm the prediction, 15 was synthesized
as shown in Scheme 1. As control compounds, the m-cyano deriv-
ative 13 and the p-carbamoyl derivative 16 were also prepared.
These were predicted to show low affinities to Chk1 (Glide-
Score = ꢀ5.90 for 13, ꢀ10.36 for 16, respectively). Condensation
of 11 with the corresponding amines gave the amides (80% for
13, 69% for 14). Hydration of the nitrile group of 13 and 14 pro-
vided the carbamoyl derivatives 15 and 16, respectively. The
Chk1 inhibitory activity of 15 was improved to 83% inhibition at
Figure 2. Structures of pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione derivatives.
4-phenyl substituted pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione
derivative 4 (Fig. 2) exhibited potent Chk1 and Wee1 inhibitory
activity.23 X-ray crystal structure analysis of complex 4 and the
kinase domain of Chk1 revealed that the phenyl group at the
4-position fit inside the hydrophobic pocket.23a This structure also
indicated that the environment around the 5-position was close to
a DFG (aspartic acid-phenylalanine-glycine) loop and a charged re-
gion, which binds to the phosphate group of ATP and catalyzes
transfer of the phosphate to the substrate serine residue. It has
been suggested that a relatively large substituent might be intro-
duced, which could interact with amino acid residues around these
regions. This observation prompted us to investigate the structure–
activity relationship (SAR) of 5-substituted pyrrolo[3,4-c]carba-
zole-1,3(2H,6H)-dione derivatives 5. Here we report the synthesis
and SAR of 5.
2. Results and discussion
2.1. First generation inhibitors
We began our investigation by introducing an N-benzylcarba-
moyl group as a substituent at the 5-position because of ease of
modification as described later. First, compound 12 was synthe-
sized to examine whether its introduction would still allow Chk1
inhibitory activity (Scheme 1). Bromoindolylmaleimide 8, which
was obtained from dibromomaleimide (6) and the magnesium salt
of indole (7),24 was cross-coupled with t-butyl acrylate by the Heck
10 lM concentration and its IC50 value was 448 nM, which was a
20-fold increase in the activity compared to that of 12. On the
other hand, the m-cyano derivative 13 and the p-carbamoyl deriv-
ative 16 reduced the potency (48% for 13 and 27% for 16 at 10 lM
drug concentrations). A further docking study using the global en-
ergy-minimized complex structure of 15 and the active site of
Chk1 (PDB accession code 1nvq) indicated additional interactions
reaction to give 9 in 88% yield. Photochemical 6p-electron cycliza-
tion followed by oxidative aromatization gave the desired pyrrol-
o[3,4-c]carbazole-1,3(2H,6H)-dione 10 in 42% yield. Deprotection
of the t-Bu group afforded the carboxylic acid 11. Condensation
Scheme 1.

7880
Y. Sako et al. / Bioorg. Med. Chem. 18 (2010) 7878–7889
Table 1
in order to increase the inhibitory activity of 15. This design simul-
taneously allowed us to improve the solubility of 15. At the time
our program began, it was reported that introducing a hydroxyl
group at the 9-position increased the binding affinity for Chk1.23
Therefore, the corresponding 9-hydroxy derivatives were also
synthesized.
Inhibitory activity of 5-substituted pyrrolo[3,4-c]carba-
zole-1,3-(2H,6H)-diones
a
Compound
Inhibitory activity
at 10 lM (%)
IC50 (nM)
10
11
12
13
l4
1
46
71
48
46
83
27
ND
ND
The synthesis of a series of analogues is shown in Scheme 2.
Bromination of 3-methylbenzoic acid 17 by NBS and (BzO)2 gave
the bromide 18, which was substituted by NaN3 to afford the azide
19. After the carboxylate group of 19 was protected with a t-Bu
group, the azide group was reduced by catalytic hydrogenation,
and the liberated amine was acylated with acryloyl chloride to give
21, a precursor to the substituent introduced at the 5 position of
the analogues. Heck reaction of 8 or 2230 with 21 cleanly provided
23a or 23b in 82% and 83% yield, respectively. Photocyclization
followed by aromatization gave the pyrrolo[3,4-c]carbazole-
1,3(2H,6H)-dione 24a in 73% and 24b in 73% yield, respectively.
Deprotection of the t-Bu group of 24 was effected by aqueous
TFA (quant. for 25a, 90% for 25b). The O-methyl group at the
9-position of 25b was removed at this stage by heating with pyrid-
inium hydrochloride at 200 °C to give 25c. The carboxylic acids 25a
and 25c were then coupled with either mono-Boc protected
ethylenediamine or propylenediamine to give 26a–27b, respec-
tively (45% for 26a, 84% for 26b, 57% for 27a, 89% for 27b). The
Boc protecting groups were removed by 4 N HCl to afford the
N-aminoalkyl analogues 28a–29b (94% for 28a, 86% for 28b, 88%
for 29a, 75% for 29b) as HCl salts. Compounds 28a–29b were trea-
ted with N,N0-Boc2-thiourea to give 30a–31b, the Boc deprotection
of which provided the N-guanidinoalkyl analogues 32a–33b (75%
for 32a, 70% for 32b, 70% for 33a, 88% for 33b) as HCl salts.
The Chk1 inhibitory activity was then evaluated, and the results
are summarized in Table 2. Overall, introducing positively charged
substituents at the m-carbamoyl group on the phenyl ring im-
proved the Chk1 inhibitory activity up to 3.6-fold (15 vs 32a).
The impact of the hydroxyl group at the 9-position was significant
and all the 9-hydroxy analogues exhibited superior activity to the
corresponding 9-unsubstituted analogues by a factor of 5–44.
Compound 32b was the most potent inhibitor with an IC50 value
of 2.8 nM, which was twice as strong as that of UCN-01. Introduc-
tion of two substituents, namely the 9-hydroxyl and the
N-guanidinoethyl groups increased the inhibitory activity by a
magnitude of 160 compared to that of 15. The length of the alkyl
group linking the amino or guanidine group to the m-carbamoyl
moiety did not significantly influence the inhibitory activity except
in the case of 32b. To rationalize the observed SAR from a 3D struc-
tural perspective, 32b was docked in the active site of Chk1 in a
manner similar to that of 15. This model suggested that a very
similar binding mode to that reported for 4 with Wee 1 (Fig. 6a,
9000
ND
ND
448
ND
15
16
Values are an average of three separate determina-
tions; variation was generally 15%.
a
IC50: concentration of drug (nM) to inhibit the
phosphorylation of a Cdc25 substrate peptide by Chk1.
Figure 3. Chemical structures used for virtual screening by GlideÒ
.
of the carbamoyl moiety with Lys132 in addition to that with
Asp148 as shown in Figure 4b. These results clearly demonstrate
the contribution of the m-carbamoyl group to the Chk1 binding.
Compound 15 was further screened against a panel of 30 kinases
at 0.1 lM concentration as shown in Figure 5. Compound 15 was
relatively selective for the Calmodulin kinase (CaMK) family and
was suggested to be a potential scaffold to design an inhibitor of
the CaMK family including Chk1.
2.2. Second generation inhibitors
The docking model of 15 with Chk1 indicated that there are
negatively charged residues, which are associated with binding
to the phosphate group of ATP, around the introduced benzyl
group of 15. This model suggested that only one of the hydrogen
atoms of the m-carbamoyl group makes a contact with Asp148
while the other hydrogen atom is free from interaction with
Chk1. Considering these observations, we introduced a positively
charged substituent, which can interact with the negatively
charged residues, on the nitrogen atom of the m-carbamoyl group
Figure 4. Docking model of 15 and Chk1. (a) Docking pose obtained by a virtual screening with Glide. (b) Proposed model for the global energy-minimum conformation of the
complex 15 and Chk1 by conformation search using Macromodel.

Y. Sako et al. / Bioorg. Med. Chem. 18 (2010) 7878–7889
7881
Figure 5. Kinase panel assay. Inhibitory activity of 15 at 0.1 lM was measured.
PDB code: 3biz), with matching occupancy of the binding pocket
and key hydrogen bonds to the hinge domain (Glu85 and the
hydrogen atom at the 2-position, and Cys87 and the carbonyl oxy-
gen at the 3-position and the 9-hydroxyl group) as shown in Figure
6b. The substituent introduced at the 5-position made a contact
with Asp148 in the DFG-loop; however, the mode of interaction
was different from suggested for 15 (Fig. 4b). The orientation of
the phenyl ring was distorted, and the m-carbamoyl group at the
phenyl ring interacted only with Asp148. However, the guanidine
group interacted with the two oxygen atoms of Asp148 through
a hydrogen bond network.
pling constant). Assignment was based on 1H–1H COSY, HMBC and
HMQC NMR spectra. FABMS was obtained on a JEOL JMS-HX101 or
JEOL JMS-700TZ. Analytical thin layer chromatography (TLC) was
performed on Merck silica gel 60F254 plates. Normal-phase col-
umn chromatography was performed on Merck silica gel 5715 or
Kanto Chemical silica gel 60 N (neutral). Flash column chromatog-
raphy was performed on Merck silica gel 60.
4.2. 4-[(2-tert-Butoxycarbonyl)vinyl]-3,4-dihydro-3-(1H-indol-
3-yl)-1H-pyrrole-2,5-dione (9)
An ideal Chk1 inhibitor would show no cytotoxicity or off-target
effects in use as a single agent. None of the compounds listed in
Table 2 showed any cytotoxicity against human acute lymphoma
A mixture of 8 (1.9 g, 6.4 mmol), t-butyl acrylate (1.9 mL,
12.7 mmol), Pd(OAc)2 (143 mg, 0.6 mmol) and Bu3N (1.7 mL,
7.1 mmol) in DMF (65 mL) was stirred at 60 °C for 2 h. The mixture
was partitioned between AcOEt (150 mL) and H2O (100 mL), and
the organic layer was washed with brine (200 mL ꢁ 2), dried over
Na2SO4, filtered and concentrated. The residue was purified by col-
umn chromatography (SiO2, 5 ꢁ 15 cm, hexane/AcOEt = 3:1) to give
9 (1.9 g, 88%) as a red solid. 1H NMR (500 MHz, DMSO-d6) d 12.20 (br
s, 1H, indole-NH), 11.14 (br s, 1H, imide-NH), 7.94 (s, 1H, H-20), 7.56
(d, 1H, H-40, J = 8.1 Hz), 7.52 (d, 1H, CH@CHCO2But, J = 16.1 Hz), 7.50
(d, 1H, H-70, J = 8.1 Hz), 7.21 (t, 1H, H-50, J = 6.9 Hz), 7.14 (t, 1H, H-60,
J = 6.9 Hz), 6.78 (d, 1H, CH@CHCO2But, J = 16.1 Hz), 1.39 (s, 9H, t-bu-
tyl); 13C NMR (125 MHz, DMSO-d6) d 171.8, 171.1, 165.2, 138.0,
136.9, 132.5, 132.2, 125.1, 122.9, 122.6, 121.1, 120.6, 112.7, 104.9,
80.3, 27.7; ESIMS-LR m/z 338 [M+H]+; ESIMS-HR calcd for
CCR-CEM and ML-1a cells up to 10 lM drug concentrations. Preli-
minary results to evaluate the potential of 28b as a chemo-poten-
tiator revealed that 28b showed a very weak combination activity
with the DNA-damaging agent, SN-38, at a drug concentration of
5 lM, resulting in up to fourfold sensitization of the IC50 value
when 28b was co-administered with SN-38 to HeLa cells.
3. Conclusion
Here we investigated the structure–activity relationship (SAR) of
5-substituted pyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione derivatives
5. In silico virtual screening helped optimize the substituent on the
phenyl ring, identifying the m-carbamoyl group among 117 ana-
logues. Further optimization studies focusing on the docking model
of 15to theactivesiteofChk1werepursuedanditwasfoundthatthe
inhibitor 32b (IC50 = 2.8 nM) was more potent than UNC-01.
C19H18N2O4 338.3572, found 338.1249.
4.3. 5-tert-Butoxycarbonyl-1,3-dihydropyrrolo[3,4-c]carbazole-
1,3(2H,6H)-dione (10)
A solution of 9 (1.5 g, 4.3 mmol) in THF (170 mL) was irradiated
with a medium-pressure mercury lamp (400 W) under oxygen
atmosphere for 16 h. The solvent was removed, and the residue
was triturated from hexane/AcOEt to give 10 (630 mg, 42%) as a
yellow solid. 1H NMR (500 MHz, DMSO-d6) d 12.13 (br s, 1H, in-
dole-NH), 11.41 (br s, 1H, imide-NH), 8.82 (d, 1H, H-10,
J = 6.9 Hz), 8.17 (s, 1H, H-4), 7.82 (d, 1H, H-7, J = 8.1 Hz), 7.57 (t,
1H, H-9, J = 6.9 Hz), 7.33 (t, 1H, H-8, J = 6.9 Hz), 1.65 (s, 9H, t-butyl);
13C NMR (125 MHz, DMSO-d6) d 169.7, 169.6, 164.5, 143.4, 142.3,
129.6, 128.8, 124.7, 122.7, 121.0, 120.7, 120.4, 119.4, 116.1,
113.0, 82.2, 27.9; ESIMS-LR m/z 336 [M+H]+; ESIMS-HR calcd for
4. Experimental
4.1. General experimental methods
NMR spectra were obtained on a JEOL EX270, JEOL GX270, JEOL
AL400 or JEOL ECA500, and were reported in parts per million (d)
relative to tetramethylsilane (0.00 ppm) as internal standard
otherwise noted. Coupling constant (J) was reported in hertz
(Hz). Abbreviations of multiplicity were as follows; s: singlet, d:
doublet, t: triplet, q: quartet, m: multiplet, br: broad. Data were
presented as follows; chemical shift (multiplicity, integration, cou-
C19H16N2O4 336.3413, found 336.1101.

7882
Y. Sako et al. / Bioorg. Med. Chem. 18 (2010) 7878–7889
Scheme 2.
4.4. 5-Carboxy-1,3-dihydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-
dione (11)
1H, indole-NH), 11.39 (s, 1H, imide-NH), 8.83 (d, 1H, H-10,
J = 7.9 Hz), 8.21 (s, 1H, H-4), 7.82 (d, 1H, H-7, J = 8.0 Hz), 7.57 (t,
1H, H-9, J = 6.9 Hz), 7.31 (t, 1H, H-8, J = 6.9 Hz); 13C NMR
(125 MHz, DMSO-d6) d 169.7, 169.6, 166.8, 143.3, 142.2, 129.7,
128.7, 124.7, 122.9, 121.0, 120.8, 120.6, 119.5, 116.1, 113.0;
ESIMS-LR m/z 280 [M+H]+; ESIMS-HR calcd for C15H8N2O4
280.2350, found 280.0476.
Compound 10 (322 mg, 0.91 mmol) was treated with TFA
(25 mL) at room temperature for 3 h. The precipitate was filtered
and washed with H2O to give 11 (218 mg, 80%) as a yellow solid.
1H NMR (500 MHz, DMSO-d6) d 13.85 (br s, 1H, CO2H), 12.01 (s,

Y. Sako et al. / Bioorg. Med. Chem. 18 (2010) 7878–7889
7883
Table 2
(2 mL) and EtOH (1 mL) was stirred at room temperature for
22 h. The mixture was partitioned between AcOEt (30 mL) and
H2O (20 mL ꢁ 2). The organic layer was washed with brine
(30 mL), dried over Na2SO4, filtered and concentrated. The residue
was triturated from hexane/AcOEt to give 6 (8 mg, 0.020 mmol,
53%) as a yellow solid. 1H NMR (270 MHz, DMSO-d6) d 12.10 (br
s, 1H, indole-NH), 11.35 (br s, 1H, imide-NH), 9.66 (br s, 1H,
amide-NH), 8.80 (d, 1H, H-10, J = 8.0 Hz), 8.42 (s, 1H, H-4), 7.89
(s, 1H, H-20), 7.80 (d, 1H, H-7, J = 8.0 Hz), 7.73 (t, 1H, H-40,
J = 7.4 Hz), 7.56–7.53 (m, 3H, H-50, H-60, and NH2), 7.41 (t, 1H, H-
9, J = 7.6 Hz), 7.32 (t, 1H, H-8, J = 7.4 Hz), 4.61 (d, 2H, CH2,
J = 8.0 Hz); 13C NMR (125 MHz, DMSO-d6) d 170.6, 166.5, 141.8,
132.9, 131.5, 131.3, 130.2, 128.8, 125.0, 121.0, 120.8, 119.4,
119.4, 118.6, 114.1, 113.7, 111.8, 42.7; FABMS-LR m/z 435
[M+Na]+; FABMS-HR calcd for C23H16N4O4Na 435.1091, found
435.1112.
Inhibitory activity of 5-substituted pyrrolo[3,4-c]carbazole-1,3-(2H,6H)-diones
R1
R2
IC50 (nM)
a
Compound
15
H
H
OH
H
OH
H
OH
H
OH
H
448
336
31
251
46
123
2.8
235
22
28a
28b
29a
29b
32a
32b
33a
33b
UCN-01
CH2CH2NH3Cl
CH2CH2NH3Cl
CH2CH2CH2NH3Cl
CH2CH2CH2NH3Cl
CH2CH2NHC(NH)NH3Cl
CH2CH2NHC(NH)NH3Cl
CH2CH2CH2NHC(NH)NH3Cl
CH2CH2CH2NHC(NH)NH3Cl
5.6
4.7. 5-(4-Cyanobenzylaminocarbonyl)-1,3-dihydropyrrolo[3,4-
c]carbazole-1,3(2H,6H)-dione (14)
Values are an average of three separate determinations; variation was generally 15%.
a
IC50: concentration of drug (nM) to inhibit the phosphorylation of a Cdc25
substrate peptide by Chk1.
According to the procedure for the preparation of 13, 14 (13 mg,
0.037 mmol, 69%) was obtained from 11 (15 mg, 0.054 mmol) and
4.5. 5-(3-Cyanobenzylaminocarbonyl)-1,3-dihydropyrrolo[3,4-
c]carbazole-1,3(2H,6H)-dione (13)
p-cyanobenzylamine hydrochloride (13 mg, 0.081 mmol) as
a
yellow solid. 1H NMR (270 MHz, DMSO-d6) d 12.16 (s, 1H, indole-
NH), 11.37 (s, 1H, imide-NH), 9.70 (t, 1H, amide-NH, J = 5.7 Hz),
8.84 (d, 1H, H-10, J = 8.0 Hz), 8.42 (s, 1H, H-4), 7.90–7.30 (m, 7H,
H-7, H-8, H-9, H-20, H-30, H-50, and H-60), 4.63 (d, 2H, CH2,
J = 5.7 Hz); 13C NMR (125 MHz, DMSO-d6) d 170.9, 170.8, 166.6,
146.1, 132.9, 129.0, 128.7, 128.5, 125.0, 121.2, 120.4, 120.3,
119.5, 119.4, 118.5, 114.0, 113.9, 110.1, 43.1; ESIMS-LR m/z 417
[M+Na]+; ESIMS-HR calcd for C23H14N4O3Na 417.0944, found
417.0971.
A
mixture of 11 (15 mg, 0.054 mmol), EDCI (15 mg,
0.081 mmol), DMAP (9.8 mg, 0.081 mmol) and m-cyanobenzyl-
amine hydrochloride (13 mL, 0.081 mmol) in DMF (500 L) was
l
stirred at room temperature for 5 days. The mixture was parti-
tioned between AcOEt (50 mL) and H2O (20 mL ꢁ 2), and the or-
ganic layer was washed with 1 N aqueous HCl (50 mL), saturated
aqueous NaHCO3 (50 mL) and brine (50 mL), dried over Na2SO4, fil-
tered and concentrated. The residue was triturated from hexane/
AcOEt to give 13 (17 mg, 0.43 mmol, 80%) as a yellow solid. 1H
NMR (270 MHz, DMSO-d6) d 12.16 (s, 1H, indole-NH), 11.37 (s,
1H, imide-NH), 9.70 (t, 1H, amide-NH, J = 5.7 Hz), 8.84 (d, 1H, H-
10, J = 8.0 Hz), 8.42 (s, 1H, H-4), 7.90–7.30 (m, 7H, H-7, H-8, H-9,
H-20, H-40, H-50, and H-60), 4.63 (d, 2H, CH2, J = 5.7 Hz); 13C NMR
(125 MHz, DMSO-d6) d 170.6, 166.5, 141.8, 132.9, 131.5, 131.3,
130.2, 129.0, 128.8, 125.0, 121.0, 120.8, 119.4, 119.4, 118.6,
113.7, 111.8, 42.7; ESIMS-LR m/z 417 [M+Na]+; ESIMS-HR calcd
for C23H14N4O3Na 417.0946, found 417.0976.
4.8. 5-(4-Carbamoylbenzylaminocarbonyl)-1,3-dihydropyrrolo-
[3,4-c]carbazole-1,3(2H,6H)-dione (16)
According to the procedure for the preparation of 15, 16 (7 mg,
0.018 mmol, 48%) was obtained from 14 (15 mg, 0.038 mmol) as a
yellow solid. 1H NMR (270 MHz, DMSO-d6) d 12.16 (br s, 1H,
indole-NH), 11.35 (br s, 1H, imide-NH), 9.69 (br s, 1H, amide-
NH), 8.84 (d, 1H, H-10, J = 7.9 Hz), 8.45 (s, 1H, H-4), 7.92–7.77
(m, 4H, aromatic), 7.63–7.32 (m, 5H, aromatic, NH2), 4.63 (d, 2H,
CH2, J = 5.6 Hz); 13C NMR (125 MHz, DMSO-d6) d 170.8, 170.8,
166.6, 142.1, 132.9, 128.7, 128.5, 125.0, 121.2, 120.4, 120.3,
119.5, 119.4, 118.5, 112.4, 114.0, 113.9, 110.1, 43.1; ESIMS-LR m/
z 435 [M+Na]+; ESIMS-HR calcd for C23H16N4O4 435.1076, found
435.10697.
4.6. 5-(3-Carbamoylbenzylaminocarbonyl)-1,3-dihydropyrrolo-
[3,4-c]carbazole-1,3(2H,6H)-dione (15)
A mixture of 13 (15 mg, 0.038 mmol), 35% aqueous H2O2 (17
0.18 mmol) and 6 N aqueous NaOH (2.2 L, 0.013 mmol) in THF
lL,
l
Figure 6. Structural comparison of 4 and 32b. (a) X-ray crystal structure of the 4 and Wee1 active site (PDB code: 3biz). (b) Docking model of 32b and Chk1 active site.
Proposed model for the global energy-minimum conformation of the complex was calculated by conformation search using Macromodel.

7884
Y. Sako et al. / Bioorg. Med. Chem. 18 (2010) 7878–7889
4.9. 3-(Bromomethyl)benzoic acid (18)
1H, H-5, J = 7.8 Hz), 6.27 (dd, 1H, CH2@CH, J = 9.7, 17.1 Hz), 6.12
(dd, 1H, CH2@CH, J = 2.3, 17.2 Hz), 5.62 (dd, 1H, CH2@CH, J = 2.3,
9.8 Hz), 4.77 (s, 2H, CH2), 1.52 (s, 9H, t-butyl); 13C NMR
(125 MHz, DMSO-d6) d 164.9, 164.7, 139.9, 132.0, 131.5, 131.4,
128.7, 127.8, 127.6, 125.6, 80.8, 41.9, 27.8; ESIMS-LR m/z 261
[M+H]+; ESIMS-HR calcd for C15H19NO3 261.1364, found 261.1333.
A mixture of m-toluic acid (10.1 g, 73.5 mmol), N-bromosuccini-
mide (14.6 g, 80.8 mmol) and benzoyl peroxide (356 mg,
1.47 mmol) in MeCN (800 mL) was gently heated under reflux for
4 h. After concentrated, the residue was dissolved in Et2O (300 mL)
and cooled to ꢀ78 °C, and the precipitate was filtered off. The filtrate
was concentrated in vacuo. Repeating this procedure twice gave 18
(12.2 g, 79%) as a white solid. 1H NMR (500 MHz, DMSO-d6) d 8.01 (s,
1H, H-2), 7.86 (d, 1H, H-6, J = 8.1 Hz), 7.67 (d, 1H, H-4, J = 7.5 Hz),
7.49 (t, 1H, H-5, J = 7.8 Hz), 4.77 (s, 2H, CH2); 13C NMR (125 MHz,
DMSO-d6) d 166.9, 138.7, 133.7, 131.3, 130.1, 129.2, 129.1, 33.7;
ESIMS-LR m/z 214, 216 [M+H]+; ESIMS-HR calcd for C8H7BrO2
213.9629, found 213.9617.
4.13. tert-Butyl 3-{2E-3-[2,5-dihydro-4-(1H-indol-3-yl)-2,5-
dioxo-1H-pyrrol-3-yl]acrylamidomethyl} benzoate (23a)
A mixture of 8 (389 mg, 1.3 mmol), 21 (701 mg, 2.7 mmol),
Pd(OAc)2 (31 mg, 0.13 mmol) and Bu3N (479 lL, 2.0 mmol) in
DMF (15 mL) was stirred at 60 °C for 5 h. The mixture was parti-
tioned between AcOEt (80 mL) and H2O (50 mL ꢁ 2), and the or-
ganic layer was washed with brine (100 mL), dried over Na2SO4,
filtered and concentrated. The residue was purified by column
chromatography (SiO2, 3 ꢁ 12 cm, hexane/AcOEt = 3:1) to give
23a (523 mg, 82%) as a red solid. 1H NMR (500 MHz, DMSO-d6) d
12.11 (s, 1H, indole-NH), 11.08 (br s, 1H, imide-NH), 8.95 (br s,
1H, amide-NH), 7.87 (s, 1H, H-2), 7.77 (s, 1H, H-l), 7.55 (d, 1H, H-
40, J = 8.0 Hz), 7.50–7.39 (m, 4H, aromatic), 7.47 (d, 1H,
CH@CHCONH, J = 16.4 Hz), 7.20 (d, 1H, CH@CHCONH, J = 16.3 Hz),
7.18 (t, 1H, H-50, J = 7.5 Hz), 7.10 (t, 1H, H-60, J = 7.5 Hz), 4.38 (d,
2H, CH2, J = 5.8 Hz), 1.53 (s, 9H, t-butyl); 13C NMR (125 MHz,
DMSO-d6) d 172.5, 171.9, 165.4, 165.3, 140.4, 137.3, 132.4, 131.9,
131.9, 129.2, 129.0, 128.4, 128.4, 128.1, 125.8, 124.8, 123.2,
121.4, 121.1, 113.0, 105.2, 81.3, 42.7, 28.3; ESIMS-LR m/z 494
[M+Na]+; ESIMS-HR calcd for C27H25N3O5Na 494.1682, found
494.1662.
4.10. 3-(Azidomethyl)benzoic acid (19)
A
mixture of 18 (12.0 g, 55.7 mmol) and NaN3 (5.4 g,
83.7 mmol) in DMF (600 mL) was stirred at room temperature
for 2 h. The mixture was partitioned between AcOEt (500 mL)
and H2O (300 mL), and the organic layer was washed with brine
(300 mL ꢁ 2), dried over Na2SO4, filtered and concentrated. The
residue was purified by column chromatography (SiO2, 3 ꢁ
12 cm, hexane/AcOEt = 3:1) to give 19 (9.8 g, quant.) as a white
solid. 1H NMR (500 MHz, DMSO-d6) d 7.94 (s, 1H, H-2), 7.91 (d,
1H, H-6, J = 8.1 Hz), 7.60 (d, 1H, H-4, J = 7.6 Hz), 7.51 (t, 1H, H-5,
J = 7.9 Hz), 4.54 (s, 2H, CH2); 13C NMR (125 MHz, DMSO-d6) d
167.1, 136.3, 132.8, 131.3, 129.2, 129.1, 129.1, 53.1; ESIMS-LR
m/z 177 [M+]; ESIMS-HR calcd for C8H7N3O2 177.0536, found
177.0336.
4.14. tert-Butyl 3-{2E-3-[2,5-dihydro-4-(5-methoxy-1H-indol-3-
yl)-2,5-dioxo-1H-pyrrol-3-yl]acrylamidomethyl} benzoate (23b)
4.11. tert-Butyl 3-(Azidomethyl)benzoate (20)
A mixture of 19 (9.8 g, 55.3 mmol), EDCI (15.9 mg, 82.9 mmol)
and DMAP (10.1 g, 82.9 mmol) in t-BuOH (300 mL) and DMF
(200 mL) was stirred at room temperature for 4 h. The mixture
was concentrated in vacuo to remove t-BuOH. The mixture was
partitioned between AcOEt (400 mL) and H2O (200 mL), and the or-
ganic layer was washed with 1 N aqueous HCl (300 mL), saturated
aqueous NaHCO3 (300 mL) and brine (300 mL), dried over Na2SO4,
filtered and concentrated. The residue was purified by column
chromatography (SiO2, 3 ꢁ 10 cm, hexane/AcOEt = 6:1) to give 20
(13.6 g, 94%) as a brown oil. 1H NMR (500 MHz, DMSO-d6) d 7.89
(s, 1H, H-2), 7.86 (d, 1H, H-6, J = 8.1 Hz), 7.60 (d, 1H, H-4,
J = 7.6 Hz), 7.52 (t, 1H, H-5, J = 7.8 Hz), 4.54 (s, 2H, CH2), 1.52 (s,
9H, t-butyl); 13C NMR (125 MHz, DMSO-d6) d 164.6, 136.3, 132.8,
131.8, 129.0, 128.8, 128.7, 80.9, 53.1, 27.7; ESIMS-LR m/z 233
[M+H]+; ESIMS-HR calcd for C12H15N3O2 233.1158, found
233.0558.
According to the procedure for the preparation of 23a, 23b
(128 mg, 83% as a red solid) was obtained from 2230 (100 mg,
0.31 mmol), 21 (123 mg, 0.47 mmol), Pd(OAc)2 (7 mg, 0.31 mmol),
Bu3N (112 l
L, 0.47 mmol). 1H NMR (500 MHz, DMSO-d6) d 12.03 (s,
1H, indole-NH), 11.08 (br s, 1H, imide-NH), 8.98 (br s, 1H,
amide-NH), 7.88–776 (m, 3H, aromatic), 7.58 (d, 1H, CH@CHCONH,
J = 15.4 Hz), 7.50–7.39 (m, 4H, aromatic), 7.15 (d, 1H,
CH@CHCONH, J = 15.5 Hz), 6.83 (dd, 1H, benzyl-5, J = 7.8 Hz), 4.39
(d, 2H, CH2, J = 6.3 Hz), 3.76 (s, 3H, methyl), 1.50 (s, 9H, t-butyl);
13C NMR (125 MHz, DMSO-d6) d 172.0, 171.5, 164.9, 164.9, 154.6,
139.9, 136.7, 131.9, 131.6, 131.4, 128.9, 128.6, 127.8, 127.6,
127.2, 125.8, 122.9, 113.3, 113.1, 104.9, 102.1, 80.8, 54.8, 42.2,
27.8; FABMS-LR m/z 524 [M+Na]+; FABMS-HR calcd for
C28H27N3O6Na 524.1809, found 524.1809.
4.15. 5-[(3-tert-Butoxycarbonyl)benzylaminocarboyl]-1,3-
dihydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione (24a)
4.12. tert-Butyl 3-Acrylamidomethylbenzoate (21)
A solution of 23a (554 mg, 1.1 mmol) in THF (170 mL) was irradi-
atedfor1 daywithamedium-pressuremercurylamp(400 W)under
oxygen atmosphere. The solvent was removed, and the residue was
triturated from hexane/AcOEt to give 14 (403 mg, 0.8 mmol, 73%) as
a yellow solid. 1H NMR (500 MHz, DMSO-d6) d 12.17 (s, 1H, indole-
NH), 11.36 (s, 1H, imide-NH), 9.72 (t, 1H, amide-NH, J = 5.2 Hz),
8.84 (d, 1H, H-10, J = 8.0 Hz), 8.44 (s, 1H, H-4), 7.94 (s, 1H, H-60),
7.82 (d, 1H, H-7, J = 8.0 Hz), 7.80 (d, 1H, H-40, J = 8.0 Hz), 7.65 (d,
1H, H-20, J = 8.0 Hz), 7.56 (t, 1H, H-9, J = 7.2 Hz), 7.48 (t, 1H, H-8,
J = 7.4 Hz), 7.32 (t, 1H, H-30, J = 8.0 Hz), 4.64 (d, 2H, CH2, J = 5.7 Hz),
1.50 (s, 9H, t-butyl); 13C NMR (125 MHz, DMSO-d6) d 169.9, 169.9,
165.7, 164.9, 142.8, 142.2, 139.9, 132.0, 131.4, 128.7, 128.5, 128.0,
127.6, 124.6, 122.9, 120.7, 120.3, 119.4, 119.0, 117.9, 113.0, 80.8,
42.5, 27.8; ESIMS-LR m/z 469 [M+H]+; ESIMS-HR calcd for
A mixture of 20 (1.06 g, 4.55 mmol) and Pd(OH)2 (130 mg) in
THF (400 mL) was vigorously stirred under H2 atmosphere for
1 h. The insoluble was filtered off through a Celite pad, and the fil-
trate was concentrated in vacuo. The residue in CH2Cl2 (50 mL) was
treated with acryloyl chloride (481 lL, 5.92 mmol), Et3N (824 lL,
5.92 mmol) at room temperature for 2 h. The mixture was parti-
tioned between AcOEt (200 mL) and H2O (200 mL). The organic
layer was washed with 1 N aqueous HCl (200 mL), saturated aque-
ous NaHCO3 (200 mL) and brine (200 mL), dried over Na2SO4, fil-
tered and concentrated. The residue was purified by column
chromatography (SiO2, 1.5 ꢁ 18 cm, hexane/AcOEt = 6:1) to give
21 (745 mg, 68% over
2 steps) as a
white solid. 1H NMR
(500 MHz, DMSO-d6) d 8.95 (t, 1H, amide-NH, J = 6.3 Hz), 7.79 (s,
1H, H-2), 7.77 (s, 1H, H-6), 7.50 (d, 1H, H-4, J = 8.0 Hz), 7.44 (t,
C27H23N3O5 469.1635, found 469.1633.

Y. Sako et al. / Bioorg. Med. Chem. 18 (2010) 7878–7889
7885
4.16. 5-[(3-tert-Butoxycarbonyl)benzylaminocarboyl]-9-meth-
oxy-1,3-dihydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione (24b)
7.21 (dd, 1H, H-8, J = 2.2, 8.0 Hz), 4.64 (d, 2H, CH2, J = 5.7 Hz); 13C
NMR (125 MHz, DMSO-d6) d 170.0, 167.4, 165.4, 149.9, 143.2,
140.0, 137.0, 132.1, 130.9, 128.7, 128.6, 128.3, 127.9, 122.9, 120.3,
119. 8, 118.8, 118.1, 117.7, 113.8, 106.5, 42.6; FABMS-LR m/z 452
[M+Na]+; FABMS-HR calcd for C23H15N3O6Na 452.1243, found
452.1169.
According to the procedure for the preparation of 24a, 24b
(73 mg, 73% as a yellow solid) was obtained from 23b (100 mg,
0.31 mmol). 1H NMR (500 MHz, DMSO-d6) d 12.03 (s, 1H, indole-
NH), 11.32 (s, 1H, imide-NH), 9.71(t, 1H, amide-NH, J = 5.7 Hz),
8.42 (s, 1H, H-4), 8.40 (d, 1H, H-9, J = 2.0 Hz), 7.99 (s, 1H, H-60),
7.83 (d, 1H, H-7, J = 7.7 Hz), 7.73 (d, 1H, H-40, J = 7.8 Hz), 7.66 (d,
1H, H-20, J = 7.8 Hz), 7.48 (t, 1H, H-30, J = 7.8 Hz), 7.21 (dd, 1H, H-8,
J = 2.2, 7.7 Hz), 4.64 (d, 2H, CH2, J = 5.7 Hz), 3.76 (s, 3H, methyl),
1.51 (s, 9H, t-butyl); 13C NMR (125 MHz, DMSO-d6) d 167.0, 165.7,
164.9, 154.0, 143.1, 140.0, 137.0, 132.0, 131.4, 128.7, 128.6, 128.0,
127.6, 128.1, 122.4, 120.3, 119.8, 119.0, 118.0, 117.7, 113.7, 106.5,
80.8, 55.5, 42.5, 27.8; EIMS-LR m/z 499 [M+]; EIMS-HR calcd for
4.20. 5-{3-[(2-tert-Butoxycarbonylamino)ethylcarbamoyl]-
benzylaminocarboyl}-1,3-dihydropyrrolo[3,4-c]carbazole-
1,3(2H,6H)-dione (26a)
A mixture of 25a (40 mg, 0.096 mmol), EDCI (28 mg, 0.15 mmol),
DMAP (18 mg, 0.15 mmol) and N-Boc-ethylenediamine hydrochlo-
ride (23 mg, 0.15 mmol) in DMF (1.5 mL) was stirred at room tem-
perature for 2 days. The mixture was partitioned between AcOEt
(40 mL) and H2O (20 mL ꢁ 2). The organic layer was washed with
1 N aqueous HCl (40 mL), saturated aqueous NaHCO3 (40 mL) and
brine (40 mL), dried over Na2SO4, filtered and concentrated. The res-
idue was triturated from hexane/AcOEt to give 26a (23 mg, 45%) as a
yellow solid. 1H NMR (500 MHz, DMSO-d6) d 12.16 (s, 1H, indole-
NH), 11.35 (br s, 1H, imide-NH), 9.72 (br s, 1H, 5-CONH), 8.84 (d,
1H, H-10, J = 8.0 Hz), 8.46 (s, 1H, H-4), 8.46 (t, 1H, 50-CONH,
J = 5.7 Hz), 7.88 (s, 1H, H-60), 7.82 (d, 1H, H-7, J = 8.0 Hz), 7.72 (d,
1H, H-40, J = 8.1 Hz), 7.57 (d, 1H, H-20, J = 8.1 Hz), 7.57 (t, 1H, H-9,
J = 7.2 Hz), 7.43 (t, 1H, H-8, J = 7.4 Hz), 7.32 (t, 1H, H-30, J = 8.1 Hz),
6.89 (t, 1H, NHBoc, J = 5.7 Hz), 4.64 (d, 2H, CH2, J = 5.7 Hz), 3.27
(dd, 2H, NHCH2CH2NHBoc, J = 5.7, 6.3 Hz), 3.08 (dd, 2H,
NHCH2CH2NHBoc, J = 5.7, 6.3 Hz), 1.34 (s, 9H, t-butyl); 13C NMR
(125 MHz, DMSO-d6) d 170.0, 169.9, 167.0, 166.4, 165.7, 155.8,
142.9, 142.2, 139.6, 134.6, 130.2, 128.5, 128.3, 126.7, 125.6, 124.6,
123.0, 120.7, 120.4, 119.4, 119.1, 118.0, 112.9, 77.7, 42.8, 28.2; FAB-
MS-LR m/z 578 [M+Na]+; FABMS-HR calcd for C30H29N5O6Na
578.2045, found 578.2026.
C28H25N3O6 499.1753, found 499.1771.
4.17. 5-[(3-Carboxy)benzylaminocarboyl]-1,3-dihydropyrrolo-
[3,4-c]carbazole-1,3(2H,6H)-dione (25a)
Compound 24a (322 mg, 0.89 mmol) was treated with TFA
(25 mL) at room temperature for 3 h. The precipitate was filtered
and washed with H2O to give 25a (218 mg, quant.) as a yellow solid.
1H NMR (500 MHz, DMSO-d6) d 13.56 (br s, 1H, CO2H), 12.17 (s, 1H,
indole-NH), 11.36 (s, 1H, imide-NH), 9.73 (t, 1H, amide-NH,
J = 5.2 Hz), 8.84 (d, 1H, H-10, J = 8.0 Hz), 8.45 (s, 1H, H-4), 7.99 (s,
1H, H-60), 7.84 (d, 1H, H-7, J = 8.0 Hz), 7.82 (d, 1H, H-40, J = 7.9 Hz),
7.65 (d, 1H, H-20, J = 7.9 Hz), 7.56 (t, 1H, H-9, J = 7.2 Hz), 7.49 (t, 1H,
H-8, J = 7.4 Hz), 7.32 (t, 1H, H-30, J = 7.9 Hz), 4.64 (d, 2H, CH2,
J = 5.7 Hz); 13C NMR (125 MHz, DMSO-d6) d 170.4, 170.4, 167.8,
166.2, 143.4, 142.7, 140.5, 132.6, 131.4, 129.2, 129.0, 128.8, 128.5,
123.5, 121.2, 120.9, 119.9, 119.5, 118.4, 113.4; FABMS-LR m/z 412
[M+H]+; FABMS-HR calcd for C23H15N3O5 413.1012, found 412.0867.
4.18. 5-[(3-Carboxy)benzylaminocarboyl]-9-methoxy-1,3-di-
hydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione (25b)
4.21. 5-{3-[(2-tert-Butoxycarbonylamino)ethylcarbamoyl]-
benzylaminocarboyl}-9-hydroxy-1,3-dihydropyrrolo[3,4-c]-
carbazole-1,3(2H,6H)-dione (26b)
According to the procedure for the preparation of 25a, 25b
(40 mg, 90%) was obtained from 24b (50 mg, 0.1 mmol) as a yellow
solid. 1H NMR (500 MHz, DMSO-d6) d 12.98 (br s, 1H, CO2H), 12.04
(s, 1H, indole-NH), 11.33 (s, 1H, imide-NH), 9.70 (t, 1H, amide-NH,
J = 5.7 Hz), 8.42 (s, 1H, H-4), 8.40 (d, 1H, H-10, J = 2.2 Hz), 7.98 (s,
1H, H-60), 7.83 (d, 1H, H-7, J = 7.8 Hz), 7.73 (d, 1H, H-40, J = 8.0 Hz),
7.66 (d, 1H, H-20, J = 8.0 Hz), 7.48 (t, 1H, H-30, J = 8.0 Hz), 7.21 (dd,
1H, H-8, J = 2.2, 7.8 Hz), 4.64 (d, 2H, CH2, J = 5.7 Hz), 3.75 (s, 3H,
methyl); 13C NMR (125 MHz, DMSO-d6) d 167.0, 167.3, 165.7,
154.1, 143.2, 140.0, 137.0, 132.1, 130.9, 128.7, 128.6, 128.3, 127.9,
122.4, 120.3, 119.8, 118.9, 118.0, 117.7, 113.8, 106.5, 55.5, 42.6; FAB-
MS-LR m/z 443 [M]+; FABMS-HR calcd for C24H17N3O6 443.1143,
found 443.1169.
According to the procedure for the preparation of 26a, 26b
(67 mg, 84% as an orange solid) was obtained from 25c (60 mg,
0.15 mmol) and N-Boc-ethylenediamine hydrochloride (34 mg,
0.22 mmol). 1H NMR (500 MHz, DMSO-d6) d 11.96 (s, 1H, indole-
NH), 11.29 (br s, 1H, imide-NH), 9.67 (br s, 1H, 5-CONH), 9.30 (s,
1H, OH), 8.89 (t, 1H, 50-CONH, J = 5.3 Hz), 8.46 (s, 1H, H-4), 8.24 (d,
1H, H-10, J = 2.0 Hz), 7.88 (s, 1H, H-60), 7.78 (d, 1H, H-7, J = 8.0 Hz),
7.62 (d, 1H, H-40, J = 8.0 Hz), 7.57 (d, 1H, H-20, J = 8.0 Hz), 7.45 (dd,
1H, H-8, J = 2.0, 8.0 Hz), 7.03 (t, 1H, H-30, J = 8.0 Hz), 6.89 (t, 1H,
NHBoc, J = 5.7 Hz), 4.64 (d, 2H, CH2, J = 5.7 Hz), 3.49 (dd, 2H,
NHCH2CH2NHBoc, J = 5.7, 6.3 Hz), 2.98 (dd, 2H, NHCH2CH2NHBoc,
J = 5.3, 6.3 Hz), 1.34 (s, 9H, t-butyl); 13C NMR (125 MHz, DMSO-d6)
d 170.0, 167.9, 166.0, 165.8, 165.5, 155.8, 147.8, 145.3, 142.9,
142.2, 139.6, 134.6, 130.2, 128.5, 128.3, 126.7, 125.6, 124.6, 123.0,
120.7, 120.4, 119.4, 119.1, 118.0, 112.9, 77.70, 42.8, 28.2; FABMS-
LR m/z 571 [M+H]+; FABMS-HR calcd for C30H29N5O7 571.2124,
found 571.2169.
4.19. 5-[(3-Carboxy)benzylaminocarboyl]-9-hydroxy-1,3-di-
hydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione (25c)
A mixture of 25b (100 mg, 0.22 mmol) and pyridinium hydro-
chloride (1.0 g) was heated at 200 °C under a CaCl2 drying tube with
stirring for 30 min. The mixture was partitioned between AcOEt
(80 mL) and H2O (60 mL ꢁ 2). The organic layer was washed with
1 N aqueous HCl (60 mL) and brine (40 mL), dried over Na2SO4, fil-
tered and concentrated. The residue was triturated from hexane/
AcOEt to give 32 (78 mg, 80%) as an orange solid. 1H NMR
(500 MHz, DMSO-d6) d 13.59 (br s, 1H, CO2H), 11.89 (s, 1H, indole-
NH), 11.30 (s, 1H, imide-NH), 9.69 (t, 1H, amide-NH, J = 5.7 Hz),
9.30 (s, 1H, OH), 8.42 (s, 1H, H-4), 8.27 (d, 1H, H-10, J = 2.3 Hz),
7.91 (s, 1H, H-60), 7.81 (d, 1H, H-7, J = 8.0 Hz), 7.73 (d, 1H, H-40,
J = 7.9 Hz), 7.66 (d, 1H, H-20, J = 7.9 Hz), 7.48 (t, 1H, H-30, J = 7.9 Hz),
4.22. 5-{3-[(3-tert-Butoxycarbonylamino)propylcarbamoyl]-
benzylaminocarboyl}-1,3-dihydropyrrolo[3,4-c]carbazole-
1,3(2H,6H)-dione (27a)
According to the procedure for the preparation of 26a, 27a
(31 mg, 57% as a yellow solid) was obtained from 25a (40 mg,
0.096 mmol) and N-Boc-propylenediamine hydrochloride (25 mg,
0.15 mmol). 1H NMR (500 MHz, DMSO-d6) d 12.16 (s, 1H, indole-
NH), 11.36 (s, 1H, imide-NH), 9.70 (t, 1H, 5-CONH, J = 5.7 Hz), 8.84
(d, 1H, H-10, J = 8.0 Hz), 8.46 (s, 1H, H-4), 8.44 (t, 1H, 50-CONH,

7886
Y. Sako et al. / Bioorg. Med. Chem. 18 (2010) 7878–7889
J = 5.7 Hz), 7.88 (s, 1H, H-60), 7.83 (d, 1H, H-7, J = 8.0 Hz), 7.72 (d, 1H,
H-40, J = 8.0 Hz), 7.57 (d, 1H, H-20, J = 7.9 Hz), 7.56 (t, 1H, H-9,
J = 7.2 Hz), 7.43 (t, 1H, H-8, J = 7.4 Hz), 7.33 (t, 1H, H-30, J = 8.0 Hz),
6.80 (t, 1H, NHBoc, J = 5.8 Hz), 4.64 (d, 2H, CH2, J = 5.7 Hz), 3.23 (dd,
2H, NHCH2CH2CH2NHBoc, J = 5.8, 6.8 Hz), 2.94 (dd, 2H,
NHCH2CH2CH2NHBoc, J = 5.7, 6.8 Hz), 1.59 (m, 2H, NHCH2CH2CH2-
NHBoc), 1.34 (s, 9H, t-butyl); 13C NMR (125 MHz, DMSO-d6) d
169.8, 169.8, 166.1, 165.6, 155.5, 142.1, 139.6, 134.6, 130.1, 128.4,
128.2, 126.6, 125.4, 124.5, 122.9, 120.3, 119.3, 119.0, 118.5, 117.9,
112.8, 77.4, 42.7, 37.7, 36.6, 30.1, 28.2; FABMS-LR m/z 592
[M+Na]+; FABMS-HR calcd for C31H31N5O6Na 592.2201, found
592.2229.
(d, 1H, H-10, J = 2.2 Hz), 7.92 (s, 1H, H-60), 7.90 (br s, 3H, NH3),
7.79 (d, 1H, H-7, J = 8.0 Hz), 7.62 (d, 1H, H-40, J = 8.0 Hz), 7.58 (d,
1H, H-20, J = 8.0 Hz), 7.45 (t, 1H, H-8, J = 8.0 Hz), 7.03 (t, 1H, H-30,
J = 8.0 Hz), 4.64 (d, 2H, CH2, J = 5.3 Hz), 3.49 (m, 2H,
NHCH2CH2NH3), 2.98 (dd, 2H, NHCH2CH2NH3, J = 5.3, 6.3 Hz); 13C
NMR (125 MHz, DMSO-d6) d 170.0, 169.9, 166.7, 165.8, 151.8,
143.2, 139.7, 136.1, 134.1, 130.4, 128.5, 128.3, 126.7, 125.8,
122.1, 120.2, 118.7, 118.4, 117.6, 113.3, 108.9, 42.7, 38.6, 37.1,
34.1; ESIMS-LR m/z 472 [M+H]+; ESIMS-HR calcd for C25H22N5O5
472.1621, found 472.1612.
4.26. 5-{3-[3-(Amino)propylcarbamoyl]benzylaminocarboyl}-
1,3-dihydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione hydro-
chloride (29a)
4.23. 5-{3-[(3-tert-Butoxycarbonylamino)propylcarbamoyl]-
benzylaminocarboyl}-9-hydroxy-1,3-dihydropyrrolo[3,4-c]-
carbazole-1,3(2H,6H)-dione (27b)
According to the procedure for the preparation of 28a, 29a
(3.9 mg, 88% as a yellow solid) was obtained from 27a (5.1 mg,
According to the procedure for the preparation of 26a, 27b
(73 mg, 89% as an orange solid) was obtained from 25c (60 mg,
0.15 mmol) and N-Boc-propylenediamine hydrochloride (36 mg,
0.21 mmol). 1H NMR (500 MHz, DMSO-d6) d 11.98 (s, 1H, indole-
NH), 11.29 (br s, 1H, imide-NH), 9.70 (br s, 1H, 5-CONH), 9.29 (s,
1H, OH), 8.90 (t, 1H, 50-CONH, J = 5.3 Hz), 8.46 (s, 1H, H-4), 8.24 (d,
1H, H-10, J = 2.2 Hz), 7.87 (s, 1H, H-60), 7.78 (d, 1H, H-7, J = 8.0 Hz),
7.62 (d, 1H, H-40, J = 8.0 Hz), 7.57 (d, 1H, H-20, J = 8.0 Hz), 7.45 (t,
1H, H-8, J = 8.0 Hz), 7.03 (t, 1H, H-30, J = 8.0 Hz), 6.89 (t, 1H, NHBoc,
J = 5.7 Hz), 4.64 (d, 2H, CH2, J = 5.3 Hz), 3.39 (dd, 2H, NHCH2-
CH2CH2NHBoc, J = 5.7, 6.3 Hz), 3.03 (dd, 2H, NHCH2CH2CH2NHBoc,
J = 5.3, 6.3 Hz), 1.78 (m, 2H, NHCH2CH2CH2NHBoc), 1.34 (s, 9H, t-bu-
tyl); 13C NMR (125 MHz, DMSO-d6) d 169.8, 167.8, 166.1, 165.6,
155.5, 147.1, 143.1, 142.1, 139.6, 134.6, 130.1, 128.4, 128.2, 126.6,
125.4, 124.5, 122.9, 120.3, 119.3, 119.0, 118.5, 117.9, 112.8, 77.4,
42.7, 37.7, 36.6, 30.1, 28.2; FABMS-LR m/z = 585 [M+H]+; FABMS-
HR calcd for C31H31N5O7 585.6123, found 585.6169.
7.1 l
mol). 1H NMR (500 MHz, DMSO-d6) d 12.16 (s, 1H, indole-
NH), 11.37 (s, 1H, imide-NH), 9.72 (t, 1H, 5-CONH, J = 5.7 Hz), 8.85
(d, 1H, H-10, J = 8.0 Hz), 8.70 (br s, 1H, 50-CONH), 8.46 (s, 1H, H-4),
7.90 (s, 1H, H-60), 7.90 (br s, 3H, NH3), 7.83 (d, 1H, H-7, J = 7.9 Hz),
7.76 (d, 1H, H-40, J = 8.0 Hz), 7.56 (d, 1H, H-20, J = 8.0 Hz), 7.56 (t,
1H, H-9, J = 7.4 Hz), 7.45 (t, 1H, H-8, J = 7.5 Hz), 7.33 (d, 1H, H-30,
J = 8.0 Hz), 4.64 (d, 2H, CH2, J = 5.7 Hz), 3.32 (dt, 2H, NHCH2-
CH2CH2NH3, J = 6.3, 7.4 Hz), 2.80 (dd, 2H, NHCH2CH2CH2NH3,
J = 5.7, 7.4 Hz), 1.79 (m, 2H, NHCH2CH2CH2NH3); 13C NMR
(125 MHz, DMSO-d6) d 170.0, 169.9, 166.5, 165.8, 165.7, 142.9,
142.2, 134.3, 130.4, 128.5, 128.4, 126.6, 124.6, 123.0, 120.8, 120.7,
120.4, 119.4, 119.1, 118.0, 112.9, 42.7, 36.8, 36.3, 27.9; FABMS-LR
m/z 470 [M+H]+; FABMS-HR calcd for C26H24N5O4 470.1834, found
470.1840.
4.27. 5-[3-(3-Aminopropylcarbamoyl)benzylaminocarboyl]-9-
hydroxy-1,3-dihydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione
hydrochloride (29b)
4.24. 5-{3-[(2-Amino)ethylcarbamoyl]benzylaminocarboyl}-
1,3-dihydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione hydro-
chloride (28a)
According to the procedure for the preparation of 28a, 29b
(48 mg, 75% as an orange solid) was obtained from 25c (72 mg,
0.12 mmol). 1H NMR (500 MHz, DMSO-d6) d 11.88 (s, 1H, indole-
NH), 11.30 (br s, 1H, imide-NH), 9.67 (br s, 1H, 5-CONH), 9.29 (s,
1H, OH), 8.70 (t, 1H, 50-CONH, J = 5.3 Hz), 8.46 (s, 1H, H-4), 8.26 (d,
1H, H-10, J = 2.0 Hz), 7.92 (s, 1H, H-60), 7.90 (br s, 3H, NH3), 7.76 (d,
1H, H-7, J = 8.0 Hz), 7.63 (d, 1H, H-40, J = 8.0 Hz), 7.56 (d, 1H, H-20,
J = 8.0 Hz), 7.44 (t, 1H, H-8, J = 8.1 Hz), 7.03 (t, 1H, H-30, J = 8.1 Hz),
4.62 (d, 2H, CH2, J = 5.3 Hz), 3.30 (dd, 2H, NHCH2CH2CH2NH3,
J = 5.3, 6.3 Hz), 2.80 (dd, 2H, NHCH2CH2CH2NH3, J = 5.3, 6.3 Hz),
1.79 (m, 2H, NHCH2CH2CH2NH3); 13C NMR (125 MHz, DMSO-d6) d
170.0, 169.9, 166.5, 165.8, 151.8, 143.2, 139.7, 136.1, 134.4, 130.3,
128.5, 128.3, 126.6, 125.6, 122.1, 120.3, 120.1, 118.7, 118.4, 117.6,
113.4, 108.9, 42.7, 37.7, 36.2, 27.3; EIMS-LR m/z 486 [M+H]+; FAB-
MS-HR calcd for C26H24N5O5 486.1768, found 486.1777.
A solution of 26a (10 mg, 0.017 mmol) in THF (1 mL) was trea-
ted with 4 N HCl in AcOEt (1 mL) at room temperature for 4.5 h.
The mixture was concentrated in vacuo, and the residue was tritu-
rated from hexane/AcOEt to give 28a (8.1 mg, 0.016 mmol, 94%) as
a yellow solid. 1H NMR (500 MHz, DMSO-d6) d 12.16 (s, 1H, indole-
NH), 11.37 (s, 1H, imide-NH), 9.74 (t, 1H, 5-CONH, J = 5.9 Hz), 8.84
(d, 1H, H-10, J = 8.0 Hz), 8.70 (br s, 1H, 50-CONH), 8.46 (s, 1H, H-4),
7.91 (s, 1H, H-60), 7.90 (br s, 3H, NH3), 7.83 (d, 1H, H-7, J = 8.0 Hz),
7.78 (d, 1H, H-40, J = 8.0 Hz), 7.56 (d, 1H, H-20, J = 7.9 Hz), 7.56 (t,
1H, H-9, J = 7.2 Hz), 7.45 (t, 1H, H-8, J = 7.7 Hz), 7.33 (d, 1H,
H-30, J = 7.4 Hz), 4.65 (d, 2H, CH2, J = 5.9 Hz), 3.48 (m, 2H,
NHCH2CH2NH3), 2.95 (m, 2H, NHCH2CH2NH3); 13C NMR
(125 MHz, DMSO-d6) d 170.0, 169.9, 166.8, 165.7, 142.9, 142.2,
139.7, 134.1, 130.5, 128.5, 128.5, 128.3, 126.8, 125.8, 124.6,
123.0, 120.7, 120.3, 119.4, 119.1, 118.0, 112.9, 42.7, 38.6, 37.1;
FABMS-LR m/z 456 [M+H]+; FABMS-HR calcd for C25H22N5O4
456.1659, found 456.1646.
4.28. 5-{3-[2-(Di-tert-butoxycarbonylguanidino)ethylcarba-
moyl]benzylaminocarboyl}-1,3-dihydropyrrolo[3,4-c]car-
bazole-1,3(2H,6H)-dione (30a)
A mixture of 28a (10 mg, 0.018 mmol), di-Boc-thiourea (6.7 mg,
4.25. 5-[3-(2-Aminoethylcarbamoyl)benzylaminocarboyl]-9-
hydroxy-1,3-dihydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione
hydrochloride (28b)
0.023 mmol), HgBr2 (8.6 mg, 0.023 mmol) and Et3N (14 lL,
0.092 mmol) in DMF (1 mL) was stirred at room temperature for
2 h. Insoluble was filtered off through a Celite pad, and filtrate was
concentrated. The residue was partitioned between AcOEt (30 mL)
and H2O (20 mL ꢁ 2). The organic layer was washed with brine
(40 mL), dried over Na2SO4, filtered and concentrated. The residue
was triturated from hexane/AcOEt to give 30a (12 mg, 84%) as an or-
ange solid. 1H NMR (500 MHz, DMSO-d6) d 12.16 (s, 1H, indole-NH),
11.47 (s, 1H, NHBoc), 11.36 (s, 1H, imide-NH), 9.70 (t, 1H, 5-CONH,
According to the procedure for the preparation of 28a, 28b
(50 mg, 86% as an orange solid) was obtained from 25c (66 mg,
0.11 mmol). 1H NMR (500 MHz, DMSO-d6) d 11.89 (s, 1H, indole-
NH), 11.30 (br s, 1H, imide-NH), 9.67 (br s, 1H, 5-CONH), 9.30 (s,
1H, OH), 8.69 (t, 1H, 50-CONH, J = 5.3 Hz), 8.40 (s, 1H, H-4), 8.26

Y. Sako et al. / Bioorg. Med. Chem. 18 (2010) 7878–7889
7887
J = 6.0 Hz), 8.84 (d, 1H, H-10, J = 8.1 Hz), 8.56 (t, 1H, 50-CONH,
J = 6.0 Hz), 8.45 (s, 1H, H-4), 8.44 (t, 1H, guanidine-NH, J = 6.2 Hz),
7.87 (s, 1H, H-60), 7.82 (d, 1H, H-7, J = 8.1 Hz), 7.70 (d, 1H, H-40,
J = 8.0 Hz), 7.56 (d, 1H, H-20, J = 8.0 Hz), 7.55 (t, 1H, H-9, J = 8.1 Hz),
7.42 (t, 1H, H-8, J = 8.1 Hz), 7.32 (t, 1H, H-30, J = 8.0 Hz), 4.63 (d, 2H,
CH2, J = 6.0 Hz), 3.47 (dt, 2H, CONHCH2CH2NH, J = 6.2, 6.8 Hz), 3.39
(dt, 2H, CONHCH2CH2NH, J = 6.1, 6.7 Hz), 1.41 (s, 9H, t-butyl), 1.39
(s, 9H, t-butyl); 13C NMR (125 MHz, DMSO-d6) d 170.0, 169.9,
166.6, 165.7, 155.8, 151.9, 142.9, 142.2, 139.6, 134.6, 130.3, 128.5,
128.3, 126.7, 125.5, 124.6, 122.9, 120.7, 120.4, 119.4, 119.1, 118.0,
112.9, 82.8, 79.2, 78.21 42.8, 38.8, 28.0, 27.6; ESIMS-LR m/z 698
[M+H]+; ESIMS-HR calcd for C36H40N7O8Na 698.2938, found
698.2929.
NH), 11.27 (s, 1H, H-r), 11.35 (s, 1H, imide-NH), 9.69 (t, 1H, 5-CONH,
J = 5.5 Hz), 9.30 (s, 1H, OH), 8.54 (t, 1H, 50-CONH, J = 5.3 Hz), 8.46 (s,
1H, H-4), 8.44 (t, 1H, OH, J = 5.0 Hz), 8.24 (d, 1H, H-10, J = 2.1 Hz),
7.88 (s, 1H, H-60), 7.75 (d, 1H, H-7, J = 8.1 Hz), 7.63 (d, 1H, H-40,
J = 8.0 Hz), 7.59 (d, 1H, H-20, J = 8.0 Hz), 7.45 (t, 1H, H-8, J = 8.1 Hz),
7.03 (t, 1H, H-30, J = 8.0 Hz), 4.63 (d, 2H, CH2, J = 5.5 Hz), 3.39 (m,
2H, CONHCH2CH2CH2NH), 3.39 (m, 2H, CONHCH2CH2CH2NH), 1.69
(m, 2H, CONHCH2CH2CH2NH), 1.42 (s, 9H, t-butyl), 1.33 (s, 9H, t-bu-
tyl); 13C NMR (125 MHz, DMSO-d6) d 170.0, 170.0, 166.4, 166.3,
165.8, 163.2, 162.4, 155.4, 152.0, 151.8, 143.2, 139.7, 136.1, 130.2,
128.5, 128.4, 126.7, 125.5, 122.1, 120.3, 120.1, 118.7, 118.4, 117.6,
113.4, 108.9, 82.9, 78.2, 42.7, 38.0, 36.6, 30.1, 28.2, 27.7; FABMS-LR
m/z 728 [M+H]+; FABMS-HR calcd for C37H42N7O9 728.3063, found
728.3081.
4.29. 5-{3-[2-(Di-tert-butoxycarbonylguanidino)ethylcarba-
moyl]benzylaminocarboyl}-9-hydroxy-1,3-dihydropyrrolo-
[3,4-c]carbazole-1,3(2H,6H)-dione (30b)
4.32. 5-{3-[2-(Guanidino)ethylcarbamoyl]benzylaminocar-
boyl}-1,3-dihydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione
hydrochloride (32a)
According to the procedure for the preparation of 30a, 30b
(19 mg, 54% as an orange solid) was obtained from 28b (25 mg,
0.051 mmol). 1H NMR (500 MHz, DMSO-d6) d 11.88 (s, 1H, in-
dole-NH), 11.29 (s, 1H, H-r), 11.31 (s, 1H, imide-NH), 9.70 (t, 1H,
5-CONH, J = 5.3 Hz), 9.29 (s, 1H, OH), 8.52 (t, 1H, 50-CONH,
J = 5.3 Hz), 8.48 (s, 1H, H-4), 8.44 (t, 1H, guanidine-NH,
J = 5.0 Hz), 8.24 (d, 1H, H-10, J = 2.0 Hz), 7.88 (s, 1H, H-60), 7.75
(d, 1H, H-7, J = 8.1 Hz), 7.61 (d, 1H, H-40, J = 8.0 Hz), 7.60 (d, 1H,
H-20, J = 8.0 Hz), 7.45 (t, 1H, H-8, J = 8.1 Hz), 7.03 (t, 1H, H-30,
J = 8.0 Hz), 4.63 (d, 2H, CH2, J = 5.3 Hz), 3.43 (m, 2H,
CHNHCH2CH2NH), 3.39 (m, 2H, CONHCH2CH2NH), 1.45 (s, 9H,
t-butyl), 1.31 (s, 9H, t-butyl); 13C NMR (125 MHz, DMSO-d6) d
170.0, 169.9, 166.6, 165.8, 163.1, 155.7, 151.8, 143.2, 139.6,
136.2, 134.6, 139.2, 128.5, 128.6, 126.7, 125.5, 122.1, 120.3, 118.7
118.3, 117.6, 113.4, 108.9, 82.8, 78.2, 42.7, 38.8, 28.0, 27.6; FAB-
MS-LR m/z 714 [M+H]+; FABMS-HR calcd for C36H40N7O9
714.2892, found 714.2897.
According to the procedure for the preparation of 28a, 32a
(2.4 mg, 75% as an orange solid) was obtained from 30a (4.5 mg,
6.5 l
mol). 1H NMR (500 MHz, DMSO-d6) d 12.16 (s, 1H, indole-
NH), 11.37 (s, 1H, imide-NH), 9.71 (t, 1H, 5-CONH, J = 6.0 Hz),
8.84 (d, 1H, H-10, J = 8.1 Hz), 8.62 (t, 1H, 50-CONH, J = 6.0 Hz),
8.46 (s, 1H, H-4), 7.90 (s, 1H, H-60), 7.82 (d, 1H, H-7, J = 8.1 Hz),
7.74 (d, 1H, H-40, J = 8.0 Hz), 7.58 (d, 1H, H-20, J = 8.0 Hz), 7.55 (t,
1H, H-9, J = 8.1 Hz), 7.46 (t, 1H, H-8, J = 8.1 Hz), 7.33 (t, 1H, H-30,
J = 8.0 Hz), 6.81 (br s, 3H, NH3), 4.64 (d, 2H, CH2, J = 6.0 Hz), 3.36
(m, 2H, CONHCH2CH2NH), 3.28 (m, 2H, CONHCH2CH2NH); 13C
NMR (125 MHz, DMSO-d6) d 169.7, 168.9, 167.4, 165.5, 142.7,
141.1, 139.6, 134.6, 130.2, 128.5, 128.3 126.6, 125.6, 124.6, 122.7,
120.6, 120.3, 119.4, 119.1, 118.0, 112.9, 79.2, 42.7, 38.5, 36.6;
ESIMS-LR m/z 498 [M+H]+; ESIMS-HR calcd for C26H24N7O4
498.1889, found 498.1887.
4.33. 5-{3-[2-(Guanidino)ethylcarbamoyl]benzylaminocar-
boyl}-9-hydroxy-1,3-dihydropyrrolo[3,4-c]carbazole-1,3-
(2H,6H)-dione hydrochloride (32b)
4.30. 5-{3-[3-(Di-tert-butoxycarbonylguanidino)propylcarba-
moyl]benzylaminocarboyl}-1,3-dihydropyrrolo[3,4-c]carba-
zole-1,3(2H,6H)-dione (31a)
According to the procedure for the preparation of 28a, 32b
(8.1 mg, 70% as an orange solid) was obtained from 30b (15 mg,
0.021 mmol). 1H NMR (500 MHz, DMSO-d6) d 11.90 (s, 1H, in-
dole-NH), 11.30 (br s, 1H, imide-NH), 9.67 (br s, 1H, 5-CONH),
9.30 (s, 1H, OH), 8.64 (t, 1H, 50-CONH, J = 5.3 Hz), 8.40 (s, 1H,
H-4), 8.26 (d, 1H, H-10, J = 2.0 Hz), 7.90 (s, 1H, H-60), 7.75 (d, 1H,
H-7, J = 8.0 Hz), 7.63 (d, 1H, H-40, J = 8.0 Hz), 7.58 (d, 1H, H-20,
J = 8.0 Hz), 7.45 (t, 1H, H-8, J = 8.0 Hz), 7.41–6.77 (br s, 4H, guani-
dine), 4.62 (d, 2H, CH2, J = 5.7 Hz), 3.39 (dd, 2H, CONHCH2CH2NH,
J = 5.7, 6.3 Hz), 2.28 (dd, 2H, CONHCH2CH2NH, J = 5.7, 6.3 Hz); 13C
NMR (125 MHz, DMSO-d6) d 170.0, 169.9, 166.8, 165.8, 157.0,
151.8, 143.2, 139.8, 136.1, 134.2, 130.5, 128.5, 128.4, 126.6,
125.6, 122.1, 120.3, 118.7, 118.4, 117.6, 113.4, 108.9, 42.7, 40.4,
38.6, 30.7; FABMS-LR m/z 514 [M+H]+; FABMS-HR calcd for
According to the procedure for the preparation of 30a, 31a
(17 mg, 80% as an orange solid) was obtained from 29a (15 mg,
0.029 mmol). 1H NMR (500 MHz, DMSO-d6) d 12.16 (s, 1H, in-
dole-NH), 11.47 (s, 1H, H-r), 11.35 (s, 1H, imide-NH), 9.69 (t, 1H,
5-CONH, J = 6.0 Hz), 8.84 (d, 1H, H-10, J = 8.1 Hz), 8.54 (t, 1H,
50-CONH, J = 6.0 Hz), 8.46 (s, 1H, H-4), 8.44 (t, 1H, guanidine-NH,
J = 8.0 Hz), 7.88 (s, 1H, H-60), 7.82 (d, 1H, H-7, J = 8.1 Hz), 7.72 (d,
1H, H-40, J = 8.0 Hz), 7.56 (d, 1H, H-20, J = 8.0 Hz), 7.55 (t, 1H, H-9,
J = 8.1 Hz), 7.44 (t, 1H, H-8, J = 8.1 Hz), 7.32 (t, 1H, H-30,
J = 8.0 Hz), 4.64 (d, 2H, CH2, J = 6.0 Hz), 3.34 (m, 2H, CON-
HCH2CH2CH2NH), 3.25 (m, 2H, CONHCH2CH2CH2NH), 1.69 (m,
2H, CONHCH2CH2CH2NH), 1.42 (s, 9H, t-butyl), 1.33 (s, 9H, t-butyl);
13C NMR (125 MHz, DMSO-d6) d 169.9, 169.9, 166.3, 165.7, 163.1,
155.4, 152.0, 142.9, 142.2, 139.6, 134.7, 130.2, 128.5, 128.3,
126.6, 125.5, 124.6, 122.9, 120.7, 120.4, 119.4, 119.1, 117.9,
112.9, 82.8, 79.2, 78.2, 42.7, 37.9, 36.5, 31.3, 29.0, 28.3, 28.0,
27.6; ESIMS-LR m/z 712 [M+H]+; ESIMS-HR calcd for C37H42N7O8
712.3073, found 712.3095.
C26H24N7O5 514.1821, found 514.1989.
4.34. 5-{3-[3-(Guanidino)propylcarbamoyl]benzylaminocar-
boyl}-1,3-dihydropyrrolo[3,4-c]carbazole-1,3(2H,6H)-dione
hydrochloride (33a)
4.31. 5-{3-[3-(Di-tert-butoxycarbonylguanidino)propylcarba-
moyl]benzylaminocarboyl}-9-hydroxy-1,3-dihydropyrrolo-
[3,4-c]carbazole-1,3(2H,6H)-dione (31b)
According to the procedure for the preparation of 28a, 33a
(3.2 mg, 70% as an orange solid) was obtained from 31a (7.1 mg,
9.8 l
mol). 1H NMR (500 MHz, DMSO-d6) d 12.16 (s, 1H, indole-
NH), 11.37 (s, 1H, imide-NH), 9.72 (t, 1H, 5-CONH, J = 6.0 Hz),
8.84 (d, 1H, H-10, J = 8.1 Hz), 8.55 (t, 1H, 50-CONH, J = 6.0 Hz),
8.46 (s, 1H, H-4), 7.89 (s, 1H, H-60), 7.82 (d, 1H, H-7, J = 8.1 Hz),
7.74 (d, 1H, H-40, J = 8.0 Hz), 7.58 (d, 1H, H-20, J = 8.0 Hz), 7.54 (t,
According to the procedure for the preparation of 30a, 31b
(19 mg, 59% as an orange solid) was obtained from 28b (25 mg,
0.049 mmol). 1H NMR (500 MHz, DMSO-d6) d 11.92 (s, 1H, indole-

7888
Y. Sako et al. / Bioorg. Med. Chem. 18 (2010) 7878–7889
1H, OH, J = 8.1 Hz), 7.44 (t, 1H, H-8, J = 8.1 Hz), 7.33 (t, 1H, H-30,
J = 8.0 Hz), 6.88 (br s, 3H, NH3), 4.64 (d, 2H, CH2, J = 6.0 Hz), 3.28
(dt, 2H, COHCH2CH2CH2NH, J = 6.3, 6.8 Hz), 3.12 (s, 1H, guani-
dine-NH), 3.11 (dt, 2H, CONHCH2CH2CH2NH, J = 6.0, 6.8 Hz), 1.69
(m, 2H, CONHCH2CH2CH2NH); 13C NMR (125 MHz, DMSO-d6) d
169.9, 169.9, 166.4, 165.7, 142.9, 142.1, 139.6, 134.6, 130.2,
128.5, 128.3 126.6, 125.6, 124.6, 122.9, 120.7, 120.3, 119.4, 119.1,
118.0, 112.9, 79.2, 42.7, 38.5, 36.6, 28.7; ESIMS-LR m/z 512
[M+H]+; ESIMS-HR calcd for C27H26N7O4 512.2079, found
512.2095.
7. Mobility sift assay for kinase panel assay
To test the enzyme selectivity of compounds, a kinase panel as-
say was carried out using ProfilerPro Kits (Caliper Life Sciences,
Inc., Hopkinton, MA). The procedure was according to the instruc-
tion manual of ProfilerPro kit 1 and 2. In 384-well plates, 16 lL of
1.625ꢁ concentration compounds was first added to the enzyme
plates, followed by a 15-min preincubation at room temperature.
10
l
L of a 2.6ꢁ mixture of peptide substrate and ATP in 2.6ꢁ ki-
nase buffer was added to initiate the reaction. Each assay was
run at the apparent Km (Km,app) concentration of ATP and at a fixed
low concentration (1.5
stopped by the addition of Termination buffer. For percent inhibi-
tion screening, compounds were tested in duplicate at both 0.1
and 10 M. After the reactions were stopped, the plates were read
lM) of peptide for 90 min. Reactions were
4.35. 5-{3-[3-(Guanidino)propylcarbamoyl]benzylaminocar-
boyl}-9-hydroxy-1,3-dihydropyrrolo[3,4-c]carbazole-1,3-
(2H,6H)-dione hydrochloride (33b)
lM
l
on a Caliper EZReder using a four-sipper chip and separation con-
ditions that were optimized for each kinase. Product-to-sum ratios,
indicative of percent conversion from substrate to product, were
taken as the assay signal.
According to the procedure for the preparation of 28a, 33b
(10.1 mg, 88% as an orange solid) was obtained from 31b (15 mg,
0.020 mmol). 1H NMR (500 MHz, DMSO-d6) d 11.88 (s, 1H, indole-
NH), 11.30 (br s, 1H, imide-NH), 9.67 (br s, 1H, 5-CONH), 9.29 (s,
1H, OH), 8.55 (t, 1H, 50-CONH, J = 5.3 Hz), 8.40 (s, 1H, H-4), 8.26 (d,
1H, H-10, J = 2.0 Hz), 7.88 (s, 1H, H-60), 7.76 (d, 1H, H-7, J = 8.0 Hz),
7.63 (d, 1H, H-40, J = 8.0 Hz), 7.54 (d, 1H, H-20, J = 8.0 Hz), 7.44 (t,
1H, H-8, J = 8.0 Hz), 7.05 (t, 1H, H-30, J = 8.0 Hz), 7.41–6.77 (br s,
4H, guanidine-NH), 4.62 (d, 2H, CH2, J = 5.7 Hz), 3.34 (dd, 2H,
COHCH2CH2CH2NH, J = 5.7, 6.3 Hz), 3.14 (dd, 2H, COHCH2CH2-
CH2NH, J = 5.3, 6.3 Hz), 1.70 (m, 2H, COHCH2CH2CH2NH); 13C NMR
(125 MHz, DMSO-d6) d 170.0, 169.9, 166.4, 165.8, 156.9, 151.8,
143.2, 139.7, 136.1, 134.6, 130.2, 128.5, 128.3, 126.6, 125.6, 122.1,
120.2, 120.0, 118.7, 118.3, 117.6, 113.3, 108.9, 42.7, 38.4, 36.5,
28.7; ESIMS-LR m/z 528 [M+H]+; ESIMS-HR calcd for C27H26N7O5
528.1983, found 528.2183.
8. Cell culture
CCRF-CEM and ML-1a cells were grown in RPMI 1640 supple-
mented with 10% fetal bovine serum (FBS) and HeLa cells were
grown in MEM supplemented with 10% FBS, 1% nonessential amino
acids at 37 °C in a 5% CO2 incubator.
9. Cell cytotoxicity assay
SN-38 was purchased from LKT Laboratories. All the data gener-
ated were the result of three separate experiments performed in
triplicate. Exponential growing cells were seeded in 96-well plates.
After 24 h, the cells were treated with SN-38 with or without the
various doses of the test compounds for 48 h. After the treatment,
Cell counting Kit-8 reagents (Dojindo Laboratories) that measured
the amount of live cell were added to the incubated cells and al-
lowed to develop for 2 h and the absorbance at 450 nm was mea-
sured using a microplate reader from BIO-RAD. The control value
corresponding to untreated cells was taken as 100% and the viabil-
ity of treated samples was expressed as a percentage of the control.
IC50 values were determined as concentrations that reduced cell
viability by 50%.
5. Molecular docking
UCN-01/the active site of Chk1 complex structure (PDB code:
1nvq) was used for the docking. The docking calculations were per-
formed using the Schrödinger software suite with default settings
if not indicated otherwise. For protein preparation, all the crystal-
lographic water molecules were removed, hydrogens were added,
and bond orders were assigned using Maestro’s Protein Prepara-
tion Wizard (Maestro ver. 8.6). The added hydrogen atoms were
minimized with all heavy atoms fixed using the OPLS2001 force
field. For each of the three structures, energy grids were built using
the default value of protein atom scaling. This box was centered
around the centroid of the UCN-01. Docking was performed using
Schrödinger Glide.26–28 Calculation was conducted using the stan-
dard-precision (SP) mode with default settings. As for global
energy-minimized conformation search, key binding site side resi-
dues were refined in the presence of inhibitor by a 100,000-step
MCMM conformational search in MacroModel 9 (Schrodinger,
LLC, New York, NY).
Acknowledgment
We thank Ms. S. Oka (Center for Instrumental Analysis, Hokka-
ido University) for measurement of mass spectra.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.09.042.
6. Chk1 kinase assay
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