Design, synthesis, and biological activity of 5,10-dihydro-dibenzo[b,e][1, 4]diazepin-11-one-based potent and selective Chk-1 inhibitors

Article abstract of DOI:10.1021/jm070105d

A novel series of 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-ones have been synthesized as potent and selective checkpoint kinase 1 (Chk1) inhibitors via structure-based design. Aided by protein X-ray crystallography, medicinal chemistry efforts led to the

Full text of DOI:10.1021/jm070105d

4162
J. Med. Chem. 2007, 50, 4162-4176
Design, Synthesis, and Biological Activity of
5,10-Dihydro-dibenzo[b,e][1,4]diazepin-11-one-Based Potent and Selective Chk-1 Inhibitors
Le Wang,* Gerard M. Sullivan, Laura A. Hexamer, Lisa A. Hasvold, Reema Thalji,† Magdalena Przytulinska, Zhi-Fu Tao,
Gaoquan Li, Zehan Chen, Zhan Xiao, Wen-Zhen Gu, John Xue, Mai-Ha Bui, Philip Merta, Peter Kovar, Jennifer J. Bouska,
Haiying Zhang, Chang Park, Kent D. Stewart, Hing L. Sham, Thomas J. Sowin, Saul H. Rosenberg, and Nan-Horng Lin
R47B, Cancer Research, Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, Abbott Park, Illinois 60064
ReceiVed January 26, 2007
A novel series of 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-ones have been synthesized as potent and
selective checkpoint kinase 1 (Chk1) inhibitors via structure-based design. Aided by protein X-ray
crystallography, medicinal chemistry efforts led to the identification of compound 46d, with potent enzymatic
activity against Chk1 kinase. While maintaining a low cytotoxicity of its own, compound 46d exhibited a
strong ability to abrogate G2 arrest and increased the cytotoxicity of camptothecin by 19-fold against SW620
cells. Pharmacokinetic studies revealed that it had a moderate bioavailabilty of 20% in mice. Two important
binding interactions between compound 46b and Chk1 kinase, revealed by X-ray cocrystal structure, were
hydrogen bonds between the hinge region and the amide bond of the core structure and a hydrogen bond
between the methoxy group and Lys38 of the protein.
Introduction
(UCN-01), which is reported to be in phase II clinical trials.12
Synthetic Chk1 inhibitors with various core structures have also
been reported in literature and in patent applications. For
example, PF-00394691, bearing diazepinoindolone as the core,
showed potent enzymatic activity against Chk1, with a Ki of
0.34 nM.13 It also potentiated antitumor activity of irinotecan
in Colo205 mouse xenograft. CHIR-124 is a benzoimidazole-
quinolinone based Chk1 inhibitor with potent activity against
the Chk1 enzyme (IC50 ) 0.3 nM).14 A patent application filed
by Millennium disclosed pyrazole-based tricyclic Chk1 inhibi-
tors.15 A series of N-aryl-N′-pyrazinylurea-based Chk1 inhibitors
have been disclosed recently through patent applications and
publications.16
Human Chk1 is a nuclear protein of 476 amino acids. It
contains a highly conserved N-terminal kinase domain (residues
1-265), a flexible linker region, and a less-conserved C-terminal
region that may negatively regulate Chk1 kinase activity. The
crystal structures of the human Chk1 kinase domain and its
complexes with AMP-PNP, an ATP analogue, staurosporine,
UCN-01, and SB-218078 have been solved.17 It appears that
the active binding site of Chk1 can be roughly divided into four
regions. The highly conserved kinase hinge region consists of
Cys87 and Glu85. Tyr20, Lys38, Glu55, and Asp148 are the
major components of the polar region. There is also a ribose
pocket made of hydrophilic amino acids, such as Glu91, Glu134,
and Asn135. A solvent-exposed region is located only a few
amino acids away from the hinge region. Thus, a solid
foundation for structure-based inhibitor design has been laid
out.
Checkpoint kinase 1 (Chk1) is a serine/threonine kinase that
is closely involved in the regulation of the cell cycle.1 Upon
damage by either DNA damaging agents or ionic irradiation,
two major weapons for current cancer treatment, Chk1 is
activated via activation of the upstream ATM/ATRa pathway.2
Activation of Chk1 results in phosphorylation of Cdc25A at
Ser123 and several other serine residues and Cdc25C at Ser216.
The phosphorylation of Cdc25 thus inhibits dephosphorylation
and activation of cyclin-dependent kinases (CDKs) via the
ubiquitin-proteosome pathway, arresting DNA-damaged cells
at various checkpoints so that the cells’ repair mechanism can
be carried out to avoid mitotic catastrophe.3 While normal cells
can depend on G1 checkpoint arrest to repair their damaged
DNA,4 cancer cells with p53 deficiency can only use the S and
G2 checkpoints for DNA repair.5 It is rationalized that the
inhibition of Chk1 would abrogate the S/M or the G2/M
checkpoint so that cancer cells would enter mitosis/chromosome
condensation prematurely with damaged DNA, eventually
leading to cell death.6 Chk1 knock-down experiments, either
with Chk1 antisense or siRNA, revealed a significant enhance-
ment of cytotoxicity of chemotherapeutics against p53-deficient
cancer cell lines.7 Thus, Chk1 inhibitors may be useful in cancer
therapy as sensitizing agents.8
Several natural products are found to be potent Chk1
inhibitors, such as indolocarbazole analogues,9 isogranulatim-
ide,10 debromohymenialdisine,11 and 7-hydroxystaurosporine
* To whom correspondence should be addressed. Phone: (847) 935-
4952. Fax: (847) 935-5165. E-mail: le.wang@abbott.com.
8-Chloro-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one, com-
pound 1a (A-45444; Chart 1), was identified to be weakly active
against human Chk1 kinase during a high-throughput screen.
The co-X-ray structure of Chk1 kinase and compound 1a
revealed several important binding characteristics. The amide
bond of compound 1a formed two hydrogen bonds with the
carbonyl and NH of Cys87 in the hinge region. The chlorine
pointed to the solvent front and did not appear to have any
interaction with the binding pocket. There was also a large space
between the right-hand side phenyl ring with the carbonyl group
and the polar region in the binding site. We felt that compound
† Summer student from the Department of Chemistry at U.C., Berkeley.
Current address: GlaxoSmithKline Co. in Philadelphia.
a Abbreviations: AMP-PNP, adenyl-5′-yl imidodiphosphate; ATM, ataxia
telangiectasia mutated; ATR, ataxia telangiectasia and Rad3 related; CPT,
camptothecin; DCI, direct chemical ionization, dppf, 1,1′-bis(diphenylphos-
phino)ferrocene; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic
acid; FACS, fluorescence-activated cell sorting; HATU, O-(7-azabenzotrizol-
1-yl)-N,N,N′,N′ tetramethyluronium hexalfluorophosphate; HEPES, N-(2-
hydroxyethyl)piperazine-N′-2-ethanesulfonic acid; HTRF, homogeneous
time-resolved fluorescence; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-car-
boxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; RNase, ribonu-
clease; SEMCl, 2-(trimethylsilyl)ethoxymethyl chloride; siRNA, small
interfering ribonucleic acid; TMSCHN2, (trimethylsilyl)diazomethane.
10.1021/jm070105d CCC: $37.00 © 2007 American Chemical Society
Published on Web 07/21/2007

Design, Synthesis, and ActiVity of Chk-1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4163
Scheme 3a
Chart 1
Scheme 1a
a Reagents and conditions: (i) Bis(pinacolato)diboron, Pd2(dba)3, PCy3,
KOAc, dioxane, 85 °C; (ii) ArBr, Pd(PPh3)4, CsF, MeOH/DME (1:2).
19, synthesized in a similar manner to that of compound 16,
was coupled with various aryl bromides to give compounds 20-
24, as shown in Scheme 3.
The synthesis of compounds 31a-c, 31f,g, 33, and 35
proceeded as shown in Scheme 4. An Ullmann-type aryl
amination between nitroaniline 26 and methyl 4-chloro-2-
iodobenzoate in the presence of copper and K2CO3 resulted in
formation of diaryl amine 28 in good to excellent yields. The
nitro group of compound 28 were reduced by hydrogenation
using platinum on carbon to provide diamines 29, which were
cyclized under acidic conditions in refluxing toluene to form
compounds 30. During the cyclization, the benzyl ethers were
cleaved to give the corresponding phenols (30b and 30d).
Alternatively, diamines 29a and 29f,g were hydrolyzed and
cyclized in the presence of HATU to give compounds 30a and
30f,g, respectively. The Suzuki coupling of compounds 30 with
pinacol arylboronate 16 afforded compounds 31b,c and 31f,g.
Treatment of the Suzuki coupling product from 30a and 16 with
HCl in MeOH and THF resulted in compound 31a. The phenol
30d was allowed to react with MeI in acetone to give compound
32, which was coupled with 16 to afford compound 33. The
hydroxyl group of 30 was protected with SEMCl and then
reacted with 16 to give compound 34. Deprotection followed
by alkylation with 4-chloromethylpyridine yielded compound
35.
Scheme 5 details the formation of compounds 39 and 41.
The amino group para to the nitro group of 36 reacted with
compound 27 exclusively in the presence of K2CO3 and copper.
It seemed that the base, K2CO3, did not just function to
neutralize HI, since without it, a mixture was generated.
Reduction followed by acid-catalyzed cyclization resulted in
compound 37. Compound 38, obtained from the Suzuki reaction
of compounds 37 and 16, was allowed to react with 3-chloro-
propanesulfonyl chloride in pyridine to provide the correspond-
ing sulfonamide, which was cyclized upon treatment of sodium
ethoxide in ethanol to yield 39. Reaction of compound 37 with
4-chlorobutanoyl chloride in pyridine followed by treatment of
sodium ethoxide in ethanol resulted in compound 40. Compound
41 was prepared by the Suzuki coupling of 40 and 16.
a Reagents and conditions: (i) Cu, PhCl, reflux; (ii) MeOH/DME (1:2),
CsF, Pd(PPh3)4, ArB(OH)2, reflux.
Scheme 2a
a Reagents and conditions: (i) Bis(pinacolato)diboron, Pd2(dba)3, Cy-
Map,
KOAc,
dioxane,
85 °C; (ii) MeOH/DME (1:2), Pd(PPh3)4, 4b, CsF; (iii) Pd/C, H2, MeOH.
1a was a good starting point for our Chk1 program in that it
was structurally simple and had interesting binding character-
istics with the Chk1 enzyme. Furthermore, despite the fact that
5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one has been known
for a long time, it was never used as a temple for a kinase
inhibitor. In this paper, we wish to report the synthesis, SAR
study, and pharmacokinetic evaluation of a series of Chk1
inhibitors based on 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-
one. Through the X-ray cocrystal structures, the binding modes
of several potent Chk1 inhibitors will be discussed as well.
Chemistry
The synthesis of compounds 5-14 and 25 is illustrated in
Scheme 1. Thus, compounds 4a,b were obtained in modest
yields by heating compounds 2a or 2b with compound 3 in
chlorobenzene in the presence of copper.18 The Suzuki coupling
of 4a and 4b with various phenyl boronic acids yielded the
desired compounds 5-14 and 25.
Scheme 2 depicts the synthesis of compounds 17 and 18.
Compound 16, a key intermediate made from 15 using pal-
ladium-mediated borylation,19 was coupled with compound 4b
to give compound 17, which was then reduced by hydrogenation
to afford compound 18. Alternatively, the pinacol arylboronate
Compounds 46a-h were prepared as shown in Scheme 6.
Nitroanilines 42 were coupled with compound 27 using a
palladium-mediated diaryl amine formation protocol to give
compounds 43 in excellent yields. It appeared that the reaction
temperature was critical for a clean transformation in that the
chlorine became quite reactive at higher temperatures, leading

4164 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Wang et al.
Scheme 4a
a Reagents and conditions: (i) Cu, K2CO3, PhCl, reflux; (ii) Pt/C, H2, MeOH; (iii) (a) LiOH, THF, H2O; (b) H3O+; (c) HATU, Et3N, DMF, for 29a, 29f,
and 29g; (iv) p-TsOH‚H2O, PhCH3, Dean-Stark trap, for 29b, 29c, and 29d; (v) Pd(OAc)2, Cy-Map, MeOH/DME (1:2), CsF, 16; (vi) HCl, THF, MeOH;
(vii) K2CO3, acetone, MeI; (viii) SEMCl, i-Pr2NEt, CH2Cl2, MeCN; (ix) K2CO3, DMF, 4-chloromethylpyridine.
to the formation of several byproducts. Hydrogenation of 43
followed by acid-catalyzed cyclization in refluxing toluene led
to formation of compounds 44. Further modifications such as
Suzuki coupling and amide formation gave final compounds
46a-h.
Schemes 7 and 8 describe the synthesis of two close analogues
of 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one. N-Methyla-
tion of compound 28e gave compound 47. Preparation of
compound 49 was conducted in a similar manner to that of
compound 31c. Acid 50 was sequentially treated with SOCl2
and TMSCHN2 to form a diazo ketone, which underwent a
Wolff rearrangement in the presence of silver benzoate and
triethylamine in MeOH to give the homologized methyl ester
51. Displacement of the fluorine of 51 with methyl 4-chloro-
2-hydroxyl-benzoate led to the formation of biaryl ether 52.
Compound 54 was synthesized in a similar manner to that of
compound 45a.
demonstrated by compounds 6-14. Replacement of the methoxy
group with a slightly larger ethoxy group caused more than a
20-fold loss in activity, as shown by compounds 7 and 8,
suggesting that the space around the methoxy group was limited.
It was somewhat gratifying to see that the introduction of a
hydroxyl group next to the methoxy group gave rise to
compound 14 with a 6-fold increase in the enzymatic activity
compared to compound 7. Encouraged by this result, a number
of functional groups were investigated (compounds 17-24). It
appeared that electron-withdrawing groups were important for
the activity. Compound 17, bearing a strong electron-withdraw-
ing nitro group next to the methoxy, showed the most potent
activity against the Chk1 enzyme. Introduction of an amine
group rendered compound 18 completely inactive. Not surpris-
ingly, because position 9 of 1a had been shown to point away
from the polar region, compound 25, a regioisomer of 14, did
not exhibit any enzymatic activity.
Once we identified the best substituent, 3-methyl-4-nitrophe-
nyl, for the polar region of the active binding site of the Chk1
enzyme, we moved our attention to the other phenyl ring,
pointing to the solvent-exposed region according to the X-ray
cocrystal structure. The results are listed in Table 2. It was clear
that the space around R1 was rather limited because even a small
hydroxyl group caused a significant loss in enzymatic activity,
as shown by compounds 17 and 31a. Because the amide bond
Results and Discussion
Position 8 of the compound 1a was explored first because
the X-ray cocrystal structure with the active site of the Chk1
enzyme had shown a large space between this position and the
polar region of the active site. The results are summarized in
Table 1. It appeared that substitution at the meta-position of
the benzene ring was important for the enzymatic activity, as

Design, Synthesis, and ActiVity of Chk-1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4165
Scheme 7a
Scheme 5a
a Reagents and conditions: (i) MeI, NaH, DMF; (ii) (a) Pt/C, H2, MeOH;
(b) p-TsOH‚H2O, PhCH3, Dean-Stark trap; (iii) Pd(OAc)2, Cy-Map,
MeOH/DME (1:2), CsF, 16.
Scheme 8a
a Reagents and conditions: (i) (a) Cu, K2CO3, PhCl, reflux; (b) Pt/C,
H2, MeOH; (c) p-TsOH‚H2O, PhCH3, Dean-Stark trap; (ii) Pd(OAc)2, Cy-
Map, MeOH/DME (1:2), CsF, 16; (iii) 3-chloropropyl sulfonyl chloride,
pyridine; (iv) NaOEt, EtOH, reflux; (v) 4-chlorobutanoyl chloride, pyridine;
(vi) NaOEt, EtOH, rt, overnight.
Scheme 6a
a Reagents and conditions: (i) (a) SOCl2; (b) TMSCHN2; (c) silver
benzoate, Et3N, MeOH; (ii) K2CO3, DMA, methyl 4-chloro-2-hydroxyl-
benzoate, 80 °C; (iii) (a) Pt/C, H2, MeOH; (b) p-TsOH‚H2O, PhCH3, Dean-
Stark trap; (iv) Pd(OAc)2, Cy-Map, MeOH/DME (1:2), CsF, 16.
such as a methoxy, without a significant loss in activity, as
demonstrated by compounds 33. However, a larger group at
position 5 caused a 10-fold drop in activity as shown by
compounds 17 and 35. In contrast to the limited tolerability at
positions 2 and 5, a wide variety of functional groups were
accommodated at both position 3 and position 4. It seemed that
a five-membered ring lactam or sultam conferred better activity
relative to the amino group at position 2, as suggested by
compounds 38, 39, and 41. A slightly more potent compound
45a was obtained when position 3 was occupied by a methyl
acetate compared to compound 17. Change of the ester to amides
did not affect the enzymatic activity regardless of their sizes,
as suggested by compounds 46b and 46c. Compound 46e, a
homolog of 46b, exhibited slightly better potency. The center
NH functionality was very important for the activity. Even small
changes, such as replacement of the NH group with either a
NCH3 or an oxygen atom, resulted in a dramatic loss in activity.
This was likely caused by a change in the dihedral angle of the
two phenyl rings when X was either NCH3 or an oxygen atom.
The cellular activities of potent Chk1 inhibitors were evalu-
ated in two routine assays. An MTS assay was used to determine
the antiproliferative EC50 of compounds against HeLa cells both
alone and with doxorubicin (at 150 nM), a topoisomerase II
inhibitor known to induce G2/M checkpoint arrest. An ideal
Chk1 inhibitor should not have any toxicity by itself. However,
when it is utilized with DNA-damaging agents, such as
doxorubicin, it should show high antiproliferative activity. A
FACS analysis was also performed to monitor Chk1 specific
and nonspecific cellular activities of potent inhibitors. In this
assay, H1299 cells were treated with 0.5 to 10 µM of a Chk1
inhibitor in the presence or absence of 500 nM doxorubicin. A
a Reagents and conditions: (i) 27, Pd(OAc)2, dppf, Cs2CO3, PhCH3,
95 °C; (ii) (a) Pt/C, H2, MeOH; (b) PhCH3, p-TsOH‚H2O, Dean-Stark
trap; (iii) Pd(OAc)2, Cy-Map, MeOH/DME (1:2), CsF, 16; (iv) (a) LiOH,
MeOH/H2O; (b) HCl; (c) HATU, Et3N, DMF, amines.
of the core forms two hydrogen bonds with the hinge binding
site, the steric bulk of a substituent next to the NH of the amide
bond is detrimental to that interaction, resulting in a great loss
in activity. Position 5 was able to tolerate some small groups,

4166 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Table 1. Structure-Activity Relationship for the Polar Regiona
Wang et al.
a IC50 value was calculated from 11 concentrations. All the compounds were assayed once unless indicated by the number of the replicates shown in
parentheses. The assay’s reproducibility was within (50%.
specific Chk1 inhibitor was expected not to alter cell distribu-
tions at all doses as a single agent. In the presence of
doxorubicin, a Chk1 inhibitor was expected to abrogate G2/M
arrest induced by doxorubicin in a dose-dependent manner.
Therefore, cell population in G2/M phase would decrease after
the treatment of Chk-1 inhibitor, while the cell populations in
other phases, such as apotosis, G1, and S, would increase
substantially (see Figure 1 in the Supporting Information). The
results are detailed in Table 3. Although compound 41 showed
a reasonable combination activity in the MTS assay, its ability
to abrogate G2 arrest was quite weak, as suggested in the FACS
analysis. Compound 39 showed a potent combination activity.
However, it also had significant toxicity as a single agent.
Compound 45a exhibited an interesting property: potent single
agent, but weaker combination activity. Change from an N,N-
dimethyl amide to a 4-morpholinyl phenyl amide removed the
single agent activity, as shown by compounds 46b and 46c.
Installation of a gem-dimethyl functionality gave rise to
compound 46d with a significantly increased combination
activity while maintaining its low single agent activity. Com-
pound 46d also exhibited a better capability of abrogating G2
arrest relative to compound 46c as demonstrated in the FACS
analysis. Compound 46e showed the most potent abrogating
ability. However, closer examination revealed that it caused
significant alteration of cell distribution as a single agent just
above 10 µM. This made compound 46e much less desirable.
A homolog of compound 46c, compound 46f, did not show any
improvement. Compound 31f showed good cellular activities
both in the MTS assay and the FACS analysis. However,
replacement of the NH2 group with a morpholinyl group resulted
in compound 31g with significant non-Chk1 toxicity as sug-
gested by the FACS analysis. It is worthwhile to point out that
compound 46h, a regioisomer of compound 46d, had weaker
cellular activities even though their enzymatic activities were

Design, Synthesis, and ActiVity of Chk-1 Inhibitors
Table 2. Structure-Activity Relationship for the B-Ringa
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4167
a
IC50 value was calculated from eleven concentrations. All the compounds were assayed once unless indicated by the number of the replicates shown
in parentheses. The assay’s reproducibility was generally within ( 50%.
Several potent Chk1 inhibitors were further evaluated using
a potentiation MTS assay in which the antiproliferative EC50’s
of a DNA-damaging agent, CPT, were measured in the presence
of a fixed concentration of Chk1 inhibitor against SW620 cells.
The results are listed in Table 4. It was clear that compound
46d was the best for potentiating CPT compared to the others.
The EC50 of CPT against SW620 was around 381 nM. In the
presence of 3 µM and 10 µM of compound 46d, the EC50’s of
CPT were measured to be 42 nM and 20 nM, a 12- and 19-fold
increase, respectively. The potentiation of SW620 cells to CPT
by compound 46d was confirmed by a soft agar colony growth
assay that was believed to be the best in vitro cell-based system
for predicting in vivo activity. Compound 46d itself did not
show any inhibition of colony growth at concentrations up to
10 µM. In the presence of 10 µM of compound 46d in the soft
agar, the SW620 cells became much more sensitive to CPT.
Figure 1. X-ray cocrystal structures of 1a (red) and 46b (green) bound
The EC50 of CPT itself was measured to be 1.75 nM. In the
presence of 10 µM of compound 46d, the EC50 decreased to
0.224 nM, suggesting a 9-fold potentiation. Even at a lower
concentration of compound 46d (3 µM), a 6.5-fold potentiation
was also observed in this assay format (see Figure 3 in the
Supporting Information).
to Chk-1 active site.
comparable. When FACS analysis was run with camptothecin
(CPT) instead of doxorubicin, compound 46d also showed
potent ability to abrogate S phase arrest induced by CPT (see
Figure 2 in the Supporting Information).

4168 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Table 3. Cellular Activity of Potent Chk1 Inhibitorsa
Wang et al.
Figure 2. X-ray cocrystal structure around Chk-1 active site with the
surface of the protein and 46b.
Table 4. Potentiation Assay for Potent Chk1 Inhibitorsa
potentiation
potentiation
ratio (3 µM)
ratio (10 µM)
46d
46e
46f
46h
31d
12 (3)
11
6
11
7
19 (3)
9.7
6.8
13
8
a All the compounds were assayed once unless indicated by the number
of the replicates shown in parentheses.
Table 5. Selectivity Profiles of Compounds 46c and 46da
IC50 (µM)
46c
46d
serine/threonine
kinase panel
AMPK
>50
>10
Aur2
cTAK
Emk
Chk2
Akt1
CDC2
CK2
ERK2
Gsk3b
Ikkb
8.62
3.65
13.86
13.45
0.88
>50
Nd
5.0
Nd
Nd
>50
>10
>10
>50
>10
>10
>10
>10
>10
5.8
a Each EC50 value for MTS was performed with 16 concentrations, and
each assay point was determined in duplicate. Each EC50 value for
FACS analysis was performed with six concentrations, and each assay
point was determined once. All the compounds were assayed once unless
indicated by the number of the replicates shown in parentheses. Reproduc-
ibility for both MTS and FACS assays was generally within (50%. b Not
determined.
MAPKAPKA
PKA
PKCδ
PKCγ
Plk1
Rsk2
SGK
SRC
Nd
>10
>10
>10
>10
>10
>10
>10
>10
>50
>50
>50
Nd
Nd
>50
>50
tyrosine kinase
panel
The selectivity profiles of compounds 46c and 46d were
evaluated against a panel of Ser/Thr kinases in the radiometric
assay using 5 µM ATP. The results are summarized in Table 5.
Compound 46c was rather weak against the kinases screened,
except Akt1, which still showed a selectivity of more than 100-
fold. Compound 46d was essentially inactive against all the
kinases tested. This high degree of selectivity may be one of
the reasons that contributed to its superior potentiating capability
and low single agent toxicity.
The pharmacokinetic study of several potent Chk1 inhibitors
was conducted in mice. The results are listed in Table 6. When
dosed intraperitoneally, compound 46d exhibited a good drug
concentration in blood as suggested by an AUC of 14.9. It also
showed a reasonable absorption when dosed orally with a 20%
bioavailability. Compounds 46e and 46f did not have a good
AUC when dosed intraperitoneally. It is interesting to note that
cKit
CSF1R
Flt1
Flt3
Kdr
FGFR1
EGFR
PDGFR
31.8
Nd
>10
>10
>10
>10
>10
>10
>10
>10
>50
>50
>50
>50
Nd
Nd
a Both compounds were assayed once. The assay’s reproducibility was
within (50%.
poor oral absorption was observed when the 4-morpholinyl
phenyl amide moiety was moved from position 3 to position 4,
as shown by compounds 46h and 46d.
X-ray Crystallography. A number of potent Chk1 inhibitors
were soaked into the native Chk1 crystals and their complex
structures were determined by X-ray crystallography. Figure 1

Design, Synthesis, and ActiVity of Chk-1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4169
Table 6. Pharmacokinetic Properties of Selected Chk1 Inhibitorsa
IP
IV
PO
AUC
AUC
Cl
Vd
t1/2
AUC
Cmax
Tmax
F
No.
(µmol‚hr/L)
(µmol‚hr/L)
(L/hr/kg)
(L/kg)
(h)
(µmol‚hr/L)
(µM)
(h)
(%)
46d
46e
46f
31g
46h
14.9
3.9
2.8
18.8
7.5
3.4
1.45
3.40
1.6
2.2
0.39
2.0
20
Ntb
Ntb
Ntb
2.4
2.0
5.0
1.7
0.16
0.09
0.67
2
a At least two animals were used for each dosing group. The dose for the oral and IP groups were 10 mg/kg. The dose for the IV group was 3 mg/kg;
the parameters reported here were the averages of the animals. b Not tested.
shows the complex structures of Chk1 compound 1 and 46b.
Figure 2 shows compound 46b in the Chk1 active binding site,
with boundaries of the protein and compound 46b. As expected,
the amide bond of the 5,10-dihydro-dibenzo[b,e][1,4]diazepin-
11-one formed two hydrogen bonds, with the backbone protein
in the hinge region. Specifically, one hydrogen bond formed
between the NH of the amide bond and the carbonyl group of
Cys87 with a distance of 2.66 Å. The other was between the
carbonyl group of the amide and NH of Cys87 with a distance
of 2.54 Å. This hinge binding pattern was different from that
of N-aryl-N′-heterocycle urea-based Chk1 inhibitors. The car-
bonyl of the amide bond of the urea formed a hydrogen bond
with the NH of Cys87, while the NH of the amide bond formed
a hydrogen bond with the carbonyl of Glu85.20 The same hinge
binding properties were also observed in a series of 3-ethylidene-
1,3-dihydro-indol-2-one-based Chk1 inhibitors.21 The 3-methoxy
group played an important role in conferring the activity. The
Chk1 complexed with compound 46d was also solved. Both
compound 46b and compound 46d shared the same binding
characteristics except for the 4-morpholinyl phenyl amide
moiety. Its electron density could not be detected in the X-ray
cocrystal structure, suggesting that it did not have any solid
interaction with the protein and moved around in the solvent-
exposed region.
Conclusion
A novel series of 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-
one-based Chk1 inhibitors have been identified. Aided by X-ray
cocrystal structures, the medicinal chemistry efforts led to the
discovery of a number of potent and selective Chk1 inhibitors.
Specifically, compound 46d was shown to have the best cellular
profiles. It did not possess any single-agent toxicity, yet it
increased the activity of CPT by 19-fold in a potentiation MTS
assay against SW620 cells. Compound 46d exhibited high
selectivity against a number of Ser/Thr kinases. It also had
moderate oral absorption with a bioavialability of 20% in mice.
Several important binding interactions between compound 46b
and the Chk1 kinase were revealed by X-ray cocrystal structure,
which paved the way for further exploration.
+
oxygen of the methoxy hydrogen-bonded strongly with NH3
of Lys38 in the polar region with a distance of 3.08 Å. The
methyl of the methoxy was in the vicinity of Leu84 with a
distance of 3.99 Å, suggesting a perfect van der Waals contact.
This may explain why even a slightly larger ethoxy could not
be tolerated at this position. The role of the nitro group in the
binding pocket was not entirely clear. It may have a π-electron
stacking interaction with the phenyl ring of Tyr20 in the polar
region because the distance between one of the oxygen atoms
of the nitro group and the phenyl was around 3.6 Å. It may
also exert activity via its strong electron-withdrawing ability
because Val23 was directly over the 3-methoxy-4-nitrophenyl
moiety with a distance of about 3.95 Å.
The nonamide NH of 5,10-dihydro-dibenzo[b,e][1,4]diazepin-
11-one did not appear to have any direct interaction with the
protein. However, it was essential for the activity in that it
provided a perfect dihedral angle between the aryl rings. Because
this type of molecule is rather rigid, a small change to that
dihedral angle would cause a substantial alteration to the part
of the molecule that interacted with the polar region, leading to
a significant loss in activity, as demonstrated by compounds
49 and 54.
Experimental Section
All commercially available solvents and reagents were used
without further treatment as received unless otherwise noted. DMF
(N,N-dimethylformamide), DME (1,2-dimethoxyethane), methylene
chloride, toluene, methanol, and THF (tetrahydrofuran) were
commercial anhydrous solvents from Aldrich. FT-NMR spectra
were obtained on Bruker MHz 300 MHz (75 MHz for 13C) or 500
MHz (1250 MHz for 13C) spectrometers. Elemental analyses were
performed by Quantitative Technologies, Inc., Whitehouse, NJ. Thin
layer chromatography (TLC) was performed on Kiesegel 60 F254
plates (Merck) using reagent grade solvents. Flash chromatography
was performed on Merck silica gel 60 (230-400 mesh) using
reagent grade solvents. All reactions were performed under a
nitrogen atmosphere. Reverse-phase Prep high performance liquid
chromatography (HPLC) was performed by a Gilson HPLC
machine equipped with a C18-coated column (Phenomenex, Luna,
10 µ, 250 × 21.2 mm), eluting with gradient 0-70% acetonitrile
in 0.1% TFA water.
N,N-Dimethyl acetamide of compound 46b pointed to the
solvent-exposed region. It is worthwhile to point out that,
although there was a strong hydrogen bond observed between
the carbonyl of the acetamide and the hydroxyl group of Tyr86,
as the distance of 2.68 Å suggested, compound 46b was only
slightly more potent than compound 17. The X-ray structure of
General Procedure for Preparation of Compound 5-14 and
25. 3-Bromo-5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-one
(4b). A mixture of 4-bromo-2-chlorobenzoic acid (2.35 g, 10 mmol),
1,2-benzenediamine (1.08 g, 10 mmol), and copper (0.63 g, 10
mmol) in chlorobenzene (150 mL) was heated to reflux for 48 h

4170 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Wang et al.
and filtered. The filter cake was washed with ethyl acetate several
times. The combined filtrates were concentrated in vacuo, and the
residue was purified by flash column chromatography on silica gel
with eluting 4:1 hexanes/ethyl acetate to provide 1.20 g (22%) of
the title compound. MS (DCI) m/e 289 (M + H)+, 307 (M +
1.56 Hz, 1H), 7.27 (dd, J ) 8.27, 1.72 Hz, 1H), 6.90-7.02 (m,
4H). Anal. (C20H13N3O‚0.3MeOH) C, H, N.
3-(4-Cyano-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]diazepin-
11-one (10). Compound 10 was prepared in a similar manner to
the synthesis of compound 14 by substituting 4-cyanohenylboronic
acid for 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenol in 80% yield. MS (DCI) m/e 312 (M + H)+, 329 (M +
1
NH4)+; H NMR (500 MHz, DMSO-d6) δ 9.90 (s, 1H), 8.02 (s,
1H), 7.62 (d, J ) 8.4 Hz, 1H), 7.24 (d, J ) 1.8 Hz, 1H), 7.06 (dd,
J ) 8.4, 1.8 Hz, 1H), 6.92-6.95 (m, 4H).
1
NH4)+; H NMR (500 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.10 (s,
3-(4-Hydroxy-3-methoxyphenyl)-5,10-dihydro-11H-dibenzo-
[b,e][1,4]diazepin-11-one (14). A mixture of compound 4b (116
mg, 0.4 mmol), 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaboro-
lan-2-yl)phenol (150 mg, 0.6 mmol), Pd(PPh3)4 (23 mg, 0.02 mmol),
and CsF (121 mg, 0.8 mmol) in DME (20 mL) and methanol (10
mL) was heated to reflux for 16 h. After cooling, the reaction
mixture was partitioned between ethyl acetate and water. The
aqueous layer was extracted with additional ethyl acetate. The
combined organic layer was washed with brine, dried (MgSO4),
filtered, and concentrated in vacuo. The residue was purified by
flash column chromatography on silica gel eluting with 1:1 hexanes/
ethyl acetate to provide 0.108 g (81%) of the desired product. MS
(DCI) m/e 333 (M + H)+, 350 (M + NH4)+; 1H NMR (500 MHz,
DMSO-d6) δ 9.90 (s, 1H), 8.02 (s, 1H), 7.62 (d, J ) 8.4 Hz, 1H),
7.24 (d, J ) 1.8 Hz, 1H), 7.15-7.18 (m, 2H), 7.08 (dd, J ) 8.1,
1.5 Hz, 1H), 6.97-7.02 (m, 5H), 3.86 (s, 3H). Anal. (C20H16N2O3‚
0.4MeOH) C, H, N.
1H), 7.98 (d, J ) 8.42 Hz, 1H), 7.94 (s, 1H), 7.88 (d, J ) 7.8 Hz,
1H), 7.80 (d, J ) 8.11 Hz, 1H), 7.69-7.72 (m, 1H), 7.35 (d, J )
1.56 Hz, 1H), 7.27 (dd, J ) 8.27, 1.72 Hz, 1H), 6.90-7.02 (m,
4H). Anal. (C20H13N3O ‚0.3MeOH) C, H, N.
3-(2-Acetyl-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]diazepin-
11-one (11). Compound 11 was prepared in a similar manner to
the synthesis of compound 14 by substituting 2-acetylphenylboronic
acid for 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenol in 73% yield. MS (DCI) m/e 329 (M + H)+, 346 (M +
1
NH4)+; H NMR (500 MHz, DMSO-d6) δ 9.84 (s, 1H), 7.95 (s,
1H), 7.72 (d, J ) 8.11 Hz, 1H), 7.64-7.66 (m, 1H), 7.58-7.61
(m, 1H), 7.49-7.53 (m, 1H), 7.40 (d, J ) 6.55 Hz, 1H), 6.90-
7.00 (m, 5H), 6.81 (dd, J ) 8.11, 1.56 Hz, 1H), 2.25 (s, 3H). Anal.
(C21H16N2O2 ‚0.4MeOH) C, H, N.
3-(3-Acetyl-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]diazepin-
11-one (12). Compound 12 was prepared in a similar manner to
the synthesis of compound 14 by substituting 3-acetylphenylboronic
acid for 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenol in 78% yield. MS (DCI) m/e 329 (M + H)+, 346 (M +
3-Phenyl-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one (5).
Compound 5 was prepared in a similar manner to the synthesis of
compound 14 by substituting benzeneboronic acid for 2-methoxy-
4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol in 67% yield.
1
NH4)+; H NMR (500 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.19 (s,
1
MS (DCI) m/e 287 (M + H)+, 304 (M + NH4)+; H NMR (500
1H), 7.99-8.03 (m, 2H), 7.92 (d, J ) 8.59 Hz, 1H), 7.81 (d, J )
8.29 Hz, 1H), 7.67-7.68 (m, 1H), 7.39 (d, J ) 1.84 Hz, 1H), 7.27
(dd, J ) 8.13, 1.69 Hz, 1H), 6.90-7.04 (m, 4H), 2.66 (s, 3H).
Anal. (C21H16N2O2‚0.35MeOH) C, H, N.
MHz, DMSO-d6) δ 9.81 (s, 1H), 7.93 (s, 1H), 7.77 (d, J ) 8.11
Hz, 1H), 7.63 (d, J ) 7.18 Hz, 2H), 7.48-7.51 (m, 2H), 7.40-
7.43 (m, 1H), 7.30 (d, J ) 1.56 Hz, 1H), 7.19 (dd, J ) 8.11, 1.56,
1H), 6.89-7.02 (m, 4H). Anal. (C19H14N2O‚0.1MeOH) C, H, N.
3-(2-Methoxy-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]diazepin-
11-one (6). Compound 6 was prepared in a similar manner to the
synthesis of compound 14 by substituting 2-methoxy-
phenylboronic acid for 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenol in 90% yield. MS (DCI) m/e 317 (M +
H)+, 334 (M + NH4)+; 1H NMR (500 MHz, DMSO-d6) δ 9.78 (s,
1H), 7.85 (s, 1H), 7.70 (d, J ) 8.11 Hz, 1H), 7.36-7.39 (m, 1H),
7.28 (dd, J ) 7.49, 1.56 Hz, 1H), 7.12-7.13 (m, 2H), 6.88-7.05
(m, 6H), 3.78 (s, 3H). Anal. (C20H16N2O2) C, H, N.
3-(4-Acetyl-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]diazepin-
11-one (13). Compound 13 was prepared in a similar manner to
the synthesis of compound 14 by substituting 4-acetylphenylboronic
acid for 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenol in 61% yield. MS (DCI) m/e 329 (M + H)+, 346 (M +
NH4)+; 1H NMR (500 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.07 (d, J
) 8.42 Hz, 2H), 7.99 (s, 1H), 7.78-7.81 (m, 3H), 7.38 (d, J )
1.87 Hz, 1H), 7.26 (dd, J ) 8.27, 1.72 Hz, 1H), 6.90-7.03 (m,
4H), 2.62 (s, 3H). Anal. (C21H16N2O2‚0.3MeOH) C, H, N.
2-Bromo-5,10-dihydro-11H-dibenzo[b,e][1,4]diazepin-11-
one (4a). Compound 4a was prepared in a similar manner to the
synthesis of compound 4b by substituting 5-bromo-2-chloro-benzoic
acid for 4-bromo-2-chlorobenzoic acid in 22% yield. MS (DCI)
m/e 333 (M + H)+, 350 (M + NH4)+; 1H NMR (500 MHz, DMSO-
d6) δ 9.95 (s, 1H), 8.03 (s, 1H), 7.74 (d, J ) 2.37 Hz, 1H), 7.49
(dd, J ) 8.65, 2.54 Hz, 1H), 6.91-6.98 (m, 4H).
3-(3-Methoxy-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]diazepin-
11-one (7). Compound 7 was prepared in a similar manner to the
synthesis of compound 14 by substituting 3-methoxy-
phenylboronic acid for 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-
dioxaborolan-2-yl)phenol in 84% yield. MS (DCI) m/e 317 (M +
H)+, 334 (M + NH4)+; 1H NMR (500 MHz, DMSO-d6) δ 9.81 (s,
1H), 7.91 (s, 1H), 7.77 (d, J ) 8.11 Hz, 1H), 7.39-7.42 (m, 1H),
7.30 (d, J ) 1.56 Hz, 1H), 7.19-7.21 (m, 2H), 7.16 (d, J ) 2.18
Hz, 1H), 6.89-7.02 (m, 5H), 3.83 (s, 3H). Anal. (C20H16N2O2) C,
H, N.
2-(4-Hydroxy-3-methoxy-phenyl)-5,10-dihydro-dibenzo[b,e]-
[1,4]diazepin-11-one (25). Compound 25 was prepared in a similar
manner described for the synthesis of compound 14 by substituting
compound 4a for compound 4b in 73% yield. MS (DCI) m/e 333
3-(3-Ethoxy-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]diazepin-
11-one (8). Compound 8 was prepared in a similar manner to the
synthesis of compound 14 by substituting 3-ethoxyphenylboronic
acid for 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenol in 89% yield. MS (DCI) m/e 331 (M + H)+, 348 (M +
1
(M + H)+, 350 (M + NH4)+; H NMR (500 MHz, DMSO-d6) δ
9.86 (s, 1H), 9.00 (s, 1H), 7.88 (s, 1H), 7.87 (d, J ) 2.5 Hz, 1H),
7.61 (dd, J ) 8.42, 2.5 Hz, 1H), 7.10 (d, J ) 2.18 Hz, 1H), 7.05
(d, J ) 8.42 Hz, 1H), 6.89-7.02 (m, 5H), 6.83 (d, J ) 8.42 Hz,
1H), 3.84 (s, 3H). Anal. (C20H16N2O3) C, H, N.
1
NH4)+; H NMR (500 MHz, DMSO-d6) δ 9.81 (s, 1H), 7.89 (s,
2-(3-Methoxy-4-nitro-phenyl)-4,4,5,5-tetramethyl-[1,3,2]diox-
aborolane (16). A mixture of PCy3 (33.6 mg, 0.12 mmol) and Pd2-
(dba)3 (23 mg, 0.025 mmol) in dioxane (6.0 mL) purged with
nitrogen was stirred at room temperature under nitrogen for 30 min.
To this red solution, 4-chloro-2-methoxy-1-nitro-benzene (188 mg,
1.0 mmol), bis(pinacolato)diboron (279 mg, 1.1 mmol), and
potassium acetate (147 mg, 1.5 mmol) were added. The reaction
mixture was subjected to a vacuum/nitrogen cycle several times
and stirred at 85 °C for 20 h. After cooling, the reaction mixture
was partitioned between ethyl acetate and water. The aqueous layer
was extracted with additional ethyl acetate. The organic layers were
combined, dried (MgSO4), filtered, and concentrated in vacuo. The
1H), 7.76 (d, J ) 8.11 Hz, 1H), 7.37-7.40 (m, 1H), 7.31 (d, J )
1.56 Hz, 1H), 7.19-7.20 (m, 2H), 7.15 (d, J ) 2.18 Hz, 1H), 6.89-
7.02 (m, 5H), 4.01 (q, J ) 6.97 Hz, 2H), 1.36 (t, J ) 7.02 Hz,
3H). Anal. (C21H18N2O2) C, H, N.
3-(3-Cyano-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]diazepin-
11-one (9). Compound 9 was prepared in a similar manner to the
synthesis of compound 14 by substituting 3-cyanophenylboronic
acid for 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenol in 72% yield. MS (DCI) m/e 312 (M + H)+, 329 (M +
1
NH4)+; H NMR (500 MHz, DMSO-d6) δ 9.86 (s, 1H), 8.10 (s,
1H), 7.98 (d, J ) 8.42 Hz, 1H), 7.94 (s, 1H), 7.88 (d, J ) 7.8 Hz,
1H), 7.80 (d, J ) 8.11 Hz, 1H), 7.69-7.72 (m, 1H), 7.35 (d, J )

Design, Synthesis, and ActiVity of Chk-1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4171
residue was purified by flash chromatography on silica gel eluting
with 1:4 ethyl acetate/hexane to give 170 mg of the desired product
(61%). Alternatively, the residue was dissolved in 1 mL of CH2-
Cl2 and treated with 10 mL of hexanes. The suspension was stirred
overnight, and the solid was collected to give analytically pure
product. MS (DCI/NH3) m/e 409 (M + H)+; MS (DCI) m/e 279
2H), 7.34-7.36 (m, 2H), 7.28 (d, J ) 1.6 Hz, 1H), 7.27 (d, J )
1.6 Hz, 1H), 6.91-7.03 (m, 4H), 3.93 (s, 3H), 3.81 (s, 3H). Anal.
(C22H18N2O4) C, H, N.
2-Methoxy-4-(11-oxo-10,11-dihydro-5H-dibenzo[b,e][1,4]-
diazepin-3-yl)benzamide (24). Compound 24 was prepared in a
similar manner to the synthesis of compound 14 by using compound
19 and 4-chloro-2-methoxybenzamide in 49% yield. MS (DCI) m/e
360 (M + H)+, 377 (M + NH4)+; 1H NMR (500 MHz, DMSO-d6)
δ 9.89 (s, 1H), 8.00 (s, 1H), 7.91 (d, J ) 8.1 Hz, 1H), 7.79 (d, J
) 8.0 Hz, 1H), 7.70 (s, 1H), 7.60 (d, J ) 1.2 Hz, 1H), 7.36 (d, J
) 1.8 Hz, 1H), 7.33 (s, 1H), 6.89-7.04 (m, 4H), 4.00 (s, 3H).
Anal. (C21H17N3O3‚0.15CH2Cl2) C, H, N.
1
(M)+, 297 (M + NH4)+; H NMR (500 MHz, DMSO-d6) δ 7.84
(d, J ) 7.8 Hz, 1H), 7.46 (s, 1H), 7.38 (d, J ) 7.8 Hz, 1H), 3.95
(s, 3H), 1.33 (s, 12H).
3-(3-Methoxy-4-nitro-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]-
diazepin-11-one (17). Compound 17 was prepared in a similar
manner to the synthesis of compound 14 by substituting compound
16 with 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-
phenol in 76% yield. MS (DCI) m/e 362 (M + H)+, 379 (M +
NH4)+; 1H NMR (500 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.00-8.02
(m, 2H), 7.80 (d, J ) 8.0 Hz, 1H), 7.53 (d, J ) 1.8 Hz, 1H), 7.33-
7.36 (m, 2H), 7.30 (dd, J ) 8.1, 1.7 Hz, 1H), 6.90-7.03 (m, 4H),
4.03 (s, 3H). Anal. (C20H15N3O4‚0.4MeOH) C, H, N.
Representative Procedure for the Formation of Diaryl Amine
from Methyl 2-Chloro-4-iodobenzoate and Substituted 2-Ni-
troanilines (General Method A). Methyl 4-Chloro-2-(5-mor-
pholino-2-nitrophenylamino)benzoate (28g). A mixture of methyl-
2-chloro-4-iodobenzoate (0.81 g, 2.73 mmol), 5-morpholino-2-
nitroaniline (0.61 g, 2.73 mmol), K2CO3 (0.395 g, 2.86 mmol), and
copper (0.180 g, 2.86 mmol) in chlorobenzene (50 mL) was heated
to reflux for 48 h and filtered. The filter cake was washed with
ethyl acetate several times. The combined filtrates were concentrated
in vacuo, and the residue was purified by flash column chroma-
tography on silica gel eluting with 4:1 hexanes/ethyl acetate to
provide 0.80 g (75%) of the title compound. The yield for this
transformation varied from 40% to 80%, depending on the
3-(4-Amino-3-methoxy-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]-
diazepin-11-one (18). A mixture of compound 17 (50 mg) and
5% palladium on carbon (20 mg) in MeOH (15 mL) was equipped
with a balloon of hydrogen gas and stirred at room temperature.
After the reaction was over, the solution was filtered through a
pack of Celite. The solvent was removed and the residue was
purified by reverse-phase prep HPLC to give the desired product
as a TFA salt (27%). MS (DCI) m/e 332 (M + H)+, 349 (M +
1
substrates. MS (DCI) m/e 392 (M + H)+, 409 (M + NH4)+; H
1
NH4)+; H NMR (500 MHz, DMSO-d6) δ 9.78 (s, 1H), 7.92 (s,
NMR (500 MHz, DMSO-d6) δ 11.04 (s, 1H), 8.06 (d, J ) 9.51
Hz, 1H), 7.95 (d, J ) 8.59 Hz, 1H), 7.66 (d, J ) 2.15 Hz, 1H),
7.12 (dd, J ) 8.59, 2.15 Hz, 1H), 6.87 (d, J ) 2.45 Hz, 1H), 6.74
(dd, J ) 6.67, 2.61 Hz, 1H), 3.87 (s, 3H), 3.69-3.71 (m, 4H),
3.34-3.36 (m, 4H).
1H), 7.77 (d, J ) 8.29 Hz, 1H), 7.31 (s, 1H), 7.20-7.24 (m, 2H),
7.16 (d, J ) 1.84 Hz, 1H), 6.94-7.10 (m, 3H), 6.72 (d, J ) 8.29
Hz, 1H), 4.70 (s, 1H), 3.72 (s, 1H). Anal. (C20H17N3O2‚TFA) C,
H, N.
3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-5H-dibenzo-
[b,e][1,4]diazepin-11(10H)-one (19). Compound 19 was prepared
in a similar manner to the synthesis of compound 16 by substituting
4-chloro-2-methoxy-1-nitro-benzene with compound 4b in 92%
Representative Procedure for the Formation of 5H-Dibenzo-
[b,e][1,4]diazepin-11(10H)-one (General Method B). 3-Chloro-
7-morpholino-5H-dibenzo[b,e][1,4]diazepin-11(10H)-one (30f).
A mixture of compound 28g (0.60 g, 1.53 mmol) and platinum on
carbon (0.1 g) in MeOH (20 mL) was equipped with a balloon of
hydrogen gas and stirred at room temperature. After uptake of H2
was complete, the solution was filtered through a pad of Celite.
The filtrate was concentrated in vacuo, and the residue was treated
with LiOH‚H2O (0.32 g, 7.67 mmol), MeOH (10 mL), and H2O
(10 mL). The reaction mixture was heated under reflux for 6 h.
After cooling, the reaction mixture was neutralized to pH ) 4 with
10% HCl. The mixture was extracted with ethyl acetate several
times. The combined organic layers were washed with brine, dried
(MgSO4) and concentrated in vacuo. The residue was dissolved in
DMF (10 mL), and the solution was treated with HATU (0.70 g,
1.83 mmol) and Et3N (0.62 g, 6.12 mmol). The reaction mixture
was stirred for 1 h at room temperature. It was partitioned between
water and ethyl acetate. The aqueous layer was extracted with
additional ethyl acetate. The combined organic layers were washed
with brine, dried (MgSO4), and concentrated under reduced pressure.
The residue was triturated with 1:4 ethyl acetate/hexanes (10 mL)
overnight to give 0.302 g of the desired product (60%). The yield
for this transformation varied from 60% to 81%, depending on the
1
yield. MS (DCI) m/e 337 (M + H)+, 354 (M + NH4)+; H NMR
(500 MHz, DMSO-d6) δ 9.86 (s, 1H), 7.82 (s, 1H), 7.69 (d, J )
7.8 Hz, 1H), 7.42 (s, 1H), 7.16 (d, J ) 7.8 Hz, 1H), 7.01-7.03 (m,
1H), 6.88-6.97 (m, 3H), 1.30 (s, 12H).
2-Methoxy-4-(11-oxo-10,11-dihydro-5H-dibenzo[b,e][1,4]-
diazepin-3-yl)-benzonitrile (20). Compound 20 was prepared in
a similar manner to the synthesis of compound 14 by using
compound 19 and 4-chloro-2-methoxy-benzonitrile in 41% yield.
1
MS (DCI) m/e 342 (M + H)+, 359 (M + NH4)+; H NMR (500
MHz, DMSO-d6) δ 9.88 (s, 1H), 7.98 (s, 1H), 7.84 (d, J ) 7.8 Hz,
1H), 7.80 (d, J ) 8.1 Hz, 1H), 7.42 (s, 1H), 7.33-7.35 (m, 2H),
7.29 (dd, J ) 8.1, 1.6 Hz, 1H), 6.90-7.02 (m, 4H), 4.02 (s, 3H).
Anal. (C21H15N3O2‚0.46EtOAc) C, H, N.
3-(4-Chloro-3-methoxy-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]-
diazepin-11-one (21). Compound 21 was prepared in a similar
manner to the synthesis of compound 14 by using compound 19
and 1-chloro-4-iodo-2-methoxy-benzene in 74% yield. MS (DCI)
m/e 362 (M + H)+, 379 (M + NH4)+; 1H NMR (500 MHz, DMSO-
d6) δ 9.84 (s, 1H), 7.95 (s, 1H), 7.78 (d, J ) 8.0 Hz, 1H), 7.53 (d,
J ) 8.0 Hz, 1H), 7.34 (d, J ) 1.8 Hz, 1H), 7.31 (m, 1H), 7.20-
7.25 (m, 2H), 6.89-7.03 (m, 2H), 3.96 (s, 3H). Anal. (C20H15-
ClN2O2) C, H, N.
3-(4-Acetyl-3-methoxy-phenyl)-5,10-dihydro-dibenzo[b,e][1,4]-
diazepin-11-one (22). Compound 22 was prepared in a similar
manner to the synthesis of compound 14 by using compound 19
and 1-(4-chloro-2-methoxy-phenyl)-ethanone in 51% yield. MS
(ESI) m/e 359 (M + H)+; 1H NMR (300 MHz, DMSO-d6) δ 9.88
(s, 1H), 7.99 (s, 1H), 7.79 (d, J ) 8.14 Hz, 1H), 7.70 (d, J ) 8.14
Hz, 1H), 7.27-7.36 (m, 3H), 6.91-7.01 (m, 5H), 4.00 (s, 3H),
2.56 (s, 3H). Anal. (C22H18N2O3‚0.4MeOH) C, H, N.
1
substrates. MS (DCI) m/e 330 (M + H)+; H NMR (500 MHz,
DMSO-d6) δ 9.70 (s, 1H), 7.89 (s, 1H), 7.67 (d, J ) 8.42 Hz, 1H),
7.04 (d, J ) 1.87 Hz, 1H), 6.92 (dd, J ) 8.58, 2.03 Hz, 1H), 6.83
(d, J ) 8.42 Hz, 1H), 6.55-6.58 (m, 2H), 3.71-3.73 (m, 4H),
2.99-3.01 (m, 4H).
Representative Procedure for the Formation of 5H-Dibenzo-
[b,e][1,4]diazepin-11(10H)-one (General Method C). A mixture
of methyl 2-(2-amino-4-(benzyloxy)phenylamino)-4-chlorobenzoate
29b (0.72 g, 1.88 mmol), prepared from 28b by hydrogenation as
described in the synthesis of compound 30f, and TsOH‚H2O (0.72
g, 3.76 mmol) in toluene (100 mL) was equipped with a Dean-
Stark trap. The reaction mixture was heated under reflux for 2 days.
The solvent was removed under reduced pressure, and the residue
was dissolved in ethyl acetate. The organic layer was washed with
saturated NaHCO3 and brine, dried (MgSO4), filtered, and concen-
trated in vacuo. The residue was purified by column chromatog-
raphy on silica gel eluting with 1:1 hexanes/ethyl acetate to provide
Methyl 2-Methoxy-4-(11-oxo-10,11-dihydro-5H-dibenzo[b,e]-
[1,4]diazepin-3-yl)benzoate (23). Compound 23 was prepared in
a similar manner to the synthesis of compound 14 by using
compound 19 and methyl 4-chloro-2-methoxybenzoate in 52%
1
yield. MS (DCI) m/e 375 (M + H)+, 392 (M + NH4)+; H NMR
(500 MHz, DMSO-d6) δ 9.85 (s, 1H), 7.97 (s, 1H), 7.76-7.80 (m,

4172 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Wang et al.
0.32 g (65%) of the title compound along with corresponding benzyl
ether (35%). The yield for this transformation varied from 40% to
77%, depending on the substrates. MS (DCI) m/e 260 (M)+, 278
(M + NH4)+; 1H NMR (500 MHz, DMSO-d6) δ 9.79 (s, 1H), 9.15
(s, 1H), 7.56 (s, 1H), 7.71 (s, 1H), 7.65 (d, J ) 8.4 Hz, 1H), 7.03
(d, J ) 2.2 Hz, 1H), 6.87 (dd, J ) 8.4, 2.2 Hz, 1H), 6.77 (d, J )
8.7 Hz, 1H), 6.44 (d, J ) 2.5 Hz, 1H), 6.38 (dd, J ) 8.4, 2.5 Hz,
1H).
3-Chloro-6-methoxy-5H-dibenzo[b,e][1,4]diazepin-11(10H)-
one (32). A mixture of 30d (54 mg, 0.2 mmol), prepared using
general methods A and C from 26d, iodomethane (14 µL, 0.22
mmol), and K2CO3 (34.5 mg, 0.25 mmol) in acetone (4 mL) was
stirred overnight at room temperature, diluted with ethyl acetate,
washed with water and brine, dried (MgSO4), filtered, and
concentrated. The residue was purified by column chromatography
on silica gel to give 33 mg (58%) of compound 32. MS (ESI) m/e
1
275 (M + H)+; H NMR (300 MHz, DMSO-d6) δ 9.91 (s, 1H),
Representative Procedure for the Suzuki Coupling between
Aryl Chlorides and Pinacol Arylboronates (General Method D).
3-(3-Methoxy-4-nitrophenyl)-7-morpholino-5H-dibenzo[b,e][1,4]-
diazepin-11(10H)-one (31e). A mixture of compound 30f (50 mg,
0.15mmol), compound 16 (84 mg, 0.30 mmol), Pd(OAc)2 (5.6 mg,
0.025 mmol), Cy-Map (19.7 mg, 0.05 mmol), and CsF (68 mg,
0.45 mmol) in DME (4 mL) and MeOH (2 mL) was heated under
reflux for 12 h. After cooling, the reaction mixture was partitioned
between water and ethyl acetate. The aqueous layer was extracted
with additional ethyl acetate. The combined organic layers were
washed with brine, dried (MgSO4), filtered, and concentrated in
vacuo. The residue was purified by column chromatography on
silica gel eluting with 2:3 hexanes/ethyl acetate to provide 35 mg
(52%) of the title compound. The yield for this transformation
varied from 20% to 70%, depending on the substrates. MS (ESI)
7.66 (d, J ) 8.48 Hz, 1H), 7.34 (s, 1H), 7.24 (d, J ) 2.03 Hz, 1H),
6.87-6.96 (m, 2H), 6.74 (dd, J ) 8.14, 1.36 Hz, 1H), 6.61 (dd, J
) 8.14, 1.36 Hz, 1H), 3.83 (s, 3H).
6-Methoxy-3-(3-methoxy-4-nitrophenyl)-5H-dibenzo[b,e][1,4]-
diazepin-11(10H)-one (33). The title compound was prepared using
general method D from 32 in 50% yield. MS (ESI) m/e 392 (M +
H)+; 1H NMR (300 MHz, DMSO-d6) δ 9.92 (s, 1H), 8.01 (d, J )
8.48 Hz, 1H), 7.80 (d, J ) 8.48 Hz, 1H), 7.54 (d, J ) 1.70 Hz,
2H), 7.38 (dd, J ) 8.48, 1.70 Hz, 1H), 7.34 (dd, J ) 8.31, 1.86
Hz, 1H), 7.20 (s, 1H), 6.89 (t, J ) 8.14 Hz, 1H), 6.73-6.78 (m,
1H), 6.63 (dd, J ) 7.97, 1.19 Hz, 1H). Anal. (C21H17N3O5‚0.02CCl4)
C, H, N.
3-(3-Methoxy-4-nitrophenyl)-6-((2-(trimethylsilyl)ethoxy)-
methoxy)-5H-dibenzo[b,e][1,4]diazepin-11(10H)-one (34). A mix-
ture of 30d (1.3 g, 5.03 mmol), SEMCl (1.07 mL, 6.04 mmol) and
diisopropylethylamine (DIPEA; 1.3 mL, 7.55 mmol) in CH2Cl2 (10
mL) and CH3CN (20 mL) was stirred overnight at room temper-
ature. Additional SEMCl (360 µL) and DIPEA (450 µL) were added
and stirring was continued for about 8 h. The reaction mixture was
concentrated, diluted with ethyl acetate, washed with water and
brine, dried (MgSO4), filtered, concentrated, and purified by column
chromatography on silica gel using a mixture of hexanes and ethyl
acetate to give 623 mg (32%) of 3-chloro-6-((2-(trimethylsilyl)-
ethoxy)methoxy)-5H-dibenzo[b,e][1,4]diazepin-11(10H)-one. MS
(ESI) m/e 391 (M + H)+; 1H NMR (300 MHz, DMSO-d6) δ 9.95
(s, 1H), 7.67 (d, J ) 8.48 Hz, 1H), 7.38 (s, 1H), 7.26 (d, J ) 2.03
Hz, 1H), 6.95 (dd, J ) 8.48, 2.03 Hz, 1H), 6.83-6.88 (m, 2H),
6.65 (dd, J ) 7.12, 2.37 Hz, 1H), 5.29 (s, 2H), 3.69-3.75 (m,
2H), 0.84-0.90 (m, 2H), -0.07- -0.05 (m, 9H).
1
m/e 447 (M + H)+; H NMR (500 MHz, DMSO-d6) δ 9.70 (s,
1H), 8.01 (d, J ) 8.42 Hz, 1H), 7.86 (s, 1H), 7.79 (d, J ) 8.42 Hz,
1H), 7.53 (d, J ) 1.56 Hz, 1H), 7.33-7.36 (m, 2H), 7.30 (dd, J )
8.27, 1.72 Hz, 1H), 6.85 (d, J ) 8.73 Hz, 1H), 6.62 (d, J ) 2.49
Hz, 1H), 6.56 (dd, J ) 8.73, 2.81 Hz, 1H), 4.03 (s, 3H), 3.71-
3.73 (m, 4H), 3.00-3.02 (m, 4H). Anal. (C24H22N4O5‚0.45 TFA)
C, H, N.
9-Hydroxy-3-(3-methoxy-4-nitrophenyl)-5H-dibenzo[b,e][1,4]-
diazepin-11(10H)-one (31a). 3-(3-Methoxy-4-nitrophenyl)-9-
(methoxymethoxy)-5H-dibenzo[b,e][1,4]diazepin-11(10H)-one (15
mg), which was prepared from 26a using the general methods A,
C, and D, was dissolved in THF/MeOH 1:1 (0.5 mL), then 2 drops
of concentrated HCl was added and the reaction was heated at 70
°C for 30 min. The reaction was then cooled, concentrated, and
purified by reverse-phase prep HPLC to give the product (3 mg,
23%) as a white solid. MS (DCI/NH3) m/e 378 (M + H)+; 1H NMR
(400 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.36 (s, 1H), 8.01 (d, J )
8.6 Hz, 1H), 7.95 (s, 1H), 7.81 (d, J ) 8.3 Hz, 1H), 7.53 (s, 1H),
7.37 (s, 1H), 7.34 (d, J ) 8.6 Hz, 1H), 7.31 (d, J ) 8.3 Hz, 1H),
6.80 (dd, J ) 8.0, 8.0 Hz, 1H), 6.51 (d, J ) 8.0 Hz, 2H), 4.03 (s,
3H).
Compound 34 was prepared using the general method D from
3-chloro-6-((2-(trimethylsilyl)ethoxy)methoxy)-5H-dibenzo[b,e]-
[1,4]diazepin-11(10H)-one. MS (ESI) m/e 508 (M + H)+; 1H NMR
(300 MHz, DMSO-d6) δ 9.97 (s, 1H), 8.00 (d, J ) 8.48 Hz, 1H),
7.80 (d, J ) 8.14 Hz, 1H), 7.51-7.57 (m, 2H), 7.29-7.44 (m,
2H), 7.24 (s, 1H), 6.80-6.94 (m, 2H), 6.67 (dd, J ) 6.44, 3.05
Hz, 1H), 5.30 (s, 2H), 4.03 (s, 3H), 3.66-3.76 (m, 2H), 0.78-
0.86 (m, 2H), -0.17 (s, 9H).
8-Hydroxy-3-(3-methoxy-4-nitrophenyl)-5H-dibenzo[b,e][1,4]-
diazepin-11(10H)-one (31b). The title compound was prepared
using general methods A, C, and D from 26b. MS (DCI) m/e 378
3-(3-Methoxy-4-nitrophenyl)-6-(pyridin-4-ylmethoxy)-5H-
dibenzo[b,e][1,4]diazepin-11(10H)-one (35). A mixture of 34 (275
mg, 0.54 mmol) in THF (6 mL), MeOH (4 mL), and 5 drops of
concentrated HCl was stirred at room temperature overnight. The
solvents were removed under reduced pressure, and the residue was
purified with reverse-phase prep HPLC to give 65 mg of 6-hydroxy-
3-(3-methoxy-4-nitrophenyl)-5H-dibenzo[b,e][1,4]diazepin-11(10H)-
1
(M + H)+; H NMR (500 MHz, DMSO-d6) δ 9.80 (s, 1H), 9.12
(s, 1H), 8.00 (d, J ) 8.42 Hz, 1H), 7.78 (d, J ) 8.11 Hz, 1H), 7.66
(s, 1H), 7.52 (d, J ) 1.52 Hz, 1H), 7.32-7.35 (m, 2H), 7.26 (dd,
J ) 8.27, 1.72 Hz, 1H), 6.82 (d, J ) 8.73 Hz, 1H), 6.46 (d, J )
2.5 Hz, 1H), 6.38 (dd, J ) 8.58, 2.65 Hz, 1H), 4.03 (s, 3H). Anal.
(C20H15N3O5‚0.5CH3OH) C, H, N.
1
8-Methoxy-3-(3-methoxy-4-nitrophenyl)-5H-dibenzo[b,e][1,4]-
diazepin-11(10H)-one (31c). The title compound was prepared
using general methods A, C, and D from 26c. MS (DCI) m/e 392
one. MS (ESI) m/e 378 (M + H)+; H NMR (300 MHz, DMSO-
d6) δ 9.84 (s, 1H), 8.00 (d, J ) 8.48 Hz, 1H), 7.79 (d, J ) 8.14
Hz, 1H), 7.56 (d, J ) 1.70 Hz, 2H), 7.30-7.43 (m, 3H), 7.05 (s,
1H), 6.73 (t, J ) 7.97 Hz, 1H), 6.58 (dd, J ) 8.14, 1.36 Hz, 1H),
6.48 (dd, J ) 7.97, 1.19 Hz, 1H), 4.04 (s, 3H).
1
(M + H)+; H NMR (500 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.01
(d, J ) 8.29 Hz, 1H), 7.78-7.81 (m, 2H), 7.52 (d, J ) 1.53 Hz,
1H), 7.32-7.35 (m, 2H), 7.28 (dd, J ) 8.29, 1.53 Hz, 1H), 6.94
(d, J ) 8.29 Hz, 1H), 6.57-6.61 (m, 2H), 4.03 (s, 3H), 3.67 (s,
3H). Anal. (C21H17N3O5‚2H2O‚TFA) C, H, N.
A mixture of 6-hydroxy-3-(3-methoxy-4-nitrophenyl)-5H-dibenzo-
[b,e][1,4]diazepin-11(10H)-one (20 mg, 0.053 mmol), 4-(chlorom-
ethyl)pyridine hydrochloride (13 mg, 0.080 mmol) and K2CO3 (22
mg, 0.159 mmol) in DMF (1 mL) was heated at 50 °C overnight.
The reaction mixture was partitioned between ethyl acetate and
water. The organic layer was washed with brine, dried (MgSO4),
and concentrated in vacuo. The residue was triturated with MeOH.
The solid was collected by filtration, washed with MeOH, and
redissolved in a mixture of MeOH and 4 N HCl in dioxane. After
several minutes, a precipitate formed and was collected to give 14.5
7-Amino-3-(3-methoxy-4-nitrophenyl)-5H-dibenzo[b,e][1,4]-
diazepin-11(10H)-one (31d). The title compound was prepared
using general methods A, B, and D from 26f. MS (DCI) m/e 377
1
(M + H)+; H NMR (300 MHz, DMSO-d6) δ 9.51 (s, 1H), 8.01
(d, J ) 8.42 Hz, 1H), 7.77 (d, J ) 8.11 Hz, 1H), 7.69 (s, 1H), 7.51
(d, J ) 1.87 Hz, 1H), 7.36 (d, J ) 1.87 Hz, 1H), 7.33 (dd, J )
8.42, 1.87 Hz, 1H), 7.29 (dd, J ) 8.11, 1.87 Hz, 1H), 6.66 (d, J )
8.42 Hz, 1H), 6.25 (d, J ) 2.18 Hz, 1H), 6.16 (dd, J ) 8.42, 2.50
Hz, 1H), 4.90 (s, 2H), 4.03 (s, 3H). Anal. (C20H16N4O4‚0.6EtOAc)
C, H, N.
1
mg of 35 as an HCl salt (54%). MS (ESI) m/e 469 (M + H)+; H
NMR (300 MHz, DMSO-d6) δ 10.03 (s, 1H), 8.79 (d, J ) 6.44
Hz, 2H), 8.03 (d, J ) 8.48 Hz, 1H), 7.96 (d, J ) 5.76 Hz, 2H),

Design, Synthesis, and ActiVity of Chk-1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4173
7.82 (d, J ) 8.14 Hz, 1H), 7.54 (dd, J ) 5.09, 1.70 Hz, 2H), 7.35-
7.42 (m, 3H), 6.89 (t, J ) 8.14 Hz, 1H), 6.76-6.82 (m, 1H), 6.67-
6.71(m,1H),5.46(s,2H),4.03(s,3H).Anal.(C26H20N4O5‚HCl‚0.8CH2-
Cl2) C, H, N.
8-Amino-3-chloro-5H-dibenzo[b,e][1,4]diazepin-11(10H)-
one (37). The title compound was prepared using general methods
A and C from compound 36. MS (DCI) m/e 259 (M)+, 277 (M +
NH4)+; 1H NMR (500 MHz, DMSO-d6) δ 9.69 (s, 1H), 7.64 (d, J
) 8.4 Hz, 1H), 7.56 (s, 1H), 7.00 (d, J ) 1.9 Hz, 1H), 6.85 (dd, J
) 8.4, 1.9 Hz, 1H), 6.65 (d, J ) 8.1 Hz, 1H), 6.20-6.21 (m, 2H),
4.81 (s, 2H).
was collected by filtration to give 154 mg of compound 40 (94%).
1
MS (DCI) m/e 328 (M + H)+; H NMR (500 MHz, DMSO-d6) δ
9.93 (s, 1H), 8.06 (s, 1H), 7.72 (d, J ) 8.48 Hz, 1H), 7.37 (d, J )
2.50 Hz, 1H), 7.28 (dd, J ) 8.73, 2.50 Hz, 1H), 7.09 (d, J ) 1.87
Hz, 1H), 6.98 (d, J ) 8.73 Hz, 1H), 6.95 (dd, J ) 8.42, 1.87 Hz,
1H), 3.77 (t, J ) 7.02 Hz, 2H), 2.49 (t, J ) 7.95 Hz, 2 H), 2.04-
2.10 (m, 2H).
3-(3-Methoxy-4-nitrophenyl)-8-(2-oxopyrrolidin-1-yl)-5,10-di-
hydro-11H-dibenzo[b,e][1,4]diazepin-11-one (41). The title com-
pound was prepared using general method D from compound 40
in 28% yield. MS (DCI) m/e 445 (M + H)+; 1H NMR (500 MHz,
DMSO-d6) δ 9.90 (s, 1H), 8.00-8.02 (m, 2H), 7.81 (d, J ) 8.29
Hz, 1H), 7.63 (d, J ) 1.53 Hz, 1H), 7.32-7.35 (m, 3H), 7.28-
7.31 (m, 1H), 7.25 (dd, J ) 8.75, 2.30 Hz, 1H), 7.00 (d, J ) 8.90
Hz, 1H), 4.03 (s, 3H), 3.74 (t, J ) 6.90 Hz, 2H), 2.46 (t, J ) 8.13
Hz, 2 H), 2.01-2.08 (m, 2H). Anal. (C24H20N4O5‚0.62CH2Cl2) C,
H, N.
Representative Procedure for the Formation of Diaryl Amine
from Methyl 2-Chloro-4-iodobenzoate and Substituted 2-Ni-
troanilines Mediated by Palladium (General Method E). Methyl
4-Chloro-2-{[4-(2-methoxy-2-oxoethyl)-2-nitrophenyl]amino}-
benzoate (43a). A mixture of compound 42a (4.21 g, 20 mmol),
compound 27 (5.93 g, 20 mmol), Pd(OAc)2 (0.224 g, 1 mmol),
dppf (1.11 g, 2 mmol), and Cs2CO3 in toluene (80 mL) was
degassed via vacuum/nitrogen cycle several times. The reaction
mixture was heated at 95 °C for 16 h. After cooling to ∼80 °C,
the reaction mixture was filtered through a pack of silica gel and
washed with additional 3:7 ethyl acetate/hexanes until all the desired
product was washed off. The solvents were removed under reduced
pressure, and the residue was recrystallized in EtOH (100 mL) to
give 5.4 g of compound 43a. The mother liquid was concentrated
and purified by column chromatography on silica gel eluting with
85:15 hexanes/ethyl acetate to provide additional 1.2 g of 43a (total
yield: 87%). MS (DCI) m/e 379 (M + H)+, 396 (M + NH4)+; 1H
NMR (300 MHz, DMSO-d6) δ 10.84 (s, 1H), 8.10 (d, J ) 2.1 Hz,
1H), 7.96 (d, J ) 8.5, Hz, 1H), 7.67 (d, J ) 8.5 Hz, 1H), 7.60 (dd,
J ) 8.8, 2.0 Hz, 1H), 7.46 (d, J ) 2.1 Hz, 1H), 7.11 (dd, J ) 8.5,
2 Hz, 1H), 3.69 (s, 3H), 3.81 (s, 2H), 3.65 (s, 3H).
Methyl (3-Chloro-11-oxo-10,11-dihydro-5H-dibenzo[b,e][1,4]-
diazepin-8-yl)acetate (44a). The title compound was prepared using
general method C from compound 43a in 78% yield. MS (DCI)
m/e 317 (M + H)+, 334 (M + NH4)+; 1H NMR (500 MHz, DMSO-
d6) δ 9.88 (s, 1H), 8.03 (s, 1H), 7.68 (d, J ) 8.7 Hz, 1H), 7.06 (d,
J ) 2.2 Hz, 1H), 6.91-6.93 (m, 2H), 6.86-6.87 (m, 2H), 3.60 (s,
3H), 3.54 (s, 2H). Anal. (C23H19N3O6‚0.1CH2Cl2) C, H, N.
Methyl [3-(3-Methoxy-4-nitrophenyl)-11-oxo-10,11-dihydro-
5H-dibenzo[b,e][1,4]diazepin-8-yl]acetate (45a). The title com-
pound was prepared using general method D from compound 44a
in a 78% yield. MS (ESI) m/e 432 (M - H)-; 1H NMR (500 MHz,
DMSO-d6) δ 9.87 (s, 1H), 7.98-8.01 (m, 2H), 7.78 (d, J ) 8.4
Hz, 1H), 7.51 (d, J ) 1.6 Hz, 1H), 7.30-7.34 (m, 2H), 7.29 (dd,
J ) 8.3, 1.7 Hz, 1H), 6.96 (d, J ) 7.8 Hz, 1H), 6.85-6.87 (m,
2H), 4.02 (s, 3H), 3.59 (s, 3H), 3.53 (s, 2H).
8-Amino-3-(3-methoxy-4-nitrophenyl)-5H-dibenzo[b,e][1,4]-
diazepin-11(10H)-one (38). The title compound was prepared using
general method D from compound 37 in 60% yield. MS (DSI) m/e
1
377 (M + H)+; H NMR (500 MHz, DMSO-d6, TFA salt) δ 9.91
(s, 1H), 7.94 (d, J ) 8.3 Hz, 1H), 7.87 (s, 1H), 7.73 (d, J ) 8.0
Hz, 1H), 7.45 (s, 1H), 7.27-7.28 (m, 2H), 7.23 (d, J ) 8.0 Hz,
1H), 6.88 (d, J ) 8.0 Hz, 1H), 6.58-6.61 (m, 2H), 3.96 (s, 3H).
Anal. (C20H16N4O4‚0.5EtOAc) C, H, N.
8-(1,1-Dioxo-1l6-isothiazolidin-2-yl)-3-(3-methoxy-4-nitro-
phenyl)-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one (39). A
mixture of 38 (60 mg, 0.159 mmol) and 3-chloropropane-1-sulfonyl
chloride (34 mg, 0.191 mmol) in pyridine (2 mL) was stirred at
room temperature overnight. The reaction mixture was partitioned
between ethyl acetate and water. The organic layer was washed
with aqueous 4 N HCl and brine, dried (MgSO4), filtered, and
concentrated under reduced pressure. The residue was purified with
reverse-phase prep HPLC to give 65 mg of 3-chloro-N-(3-(3-
methoxy-4-nitrophenyl)-11-oxo-10,11-dihydro-5H-dibenzo[b,e][1,4]-
diazepin-8-yl)propane-1-sulfonamide (68%). MS (DCI) m/e 517 (M
1
+ H)+; H NMR (500 MHz, DMSO-d6) δ 9.98 (s, 1H), 9.70 (s,
1H), 8.01-8.02 (m, 2H), 7.80 (d, J ) 8.24 Hz, 1H), 7.52 (s, 1H),
7.30-7.35 (m, 3H), 6.98 (d, J ) 8.24 Hz, 1H), 6.94 (d, J ) 2.14
Hz, 1H), 6.83 (dd, J ) 8.54, 2.44 Hz, 1H), 4.03 (s, 3H), 3.71 (t, J
) 3.71 Hz, 2H), 3.13-3.15 (m, 2H), 2.06-2.13 (m, 2H).
Sodium ethoxide solution was prepared by adding sodium metal
(24 mg, 1.0 mmol) to EtOH (10 mL) at room temperature. To this
solution was added 3-chloro-N-(3-(3-methoxy-4-nitrophenyl)-11-
oxo-10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-8-yl)propane-1-
sulfonamide (51 mg, 0.1 mmol). The reaction mixture was heated
under reflux for 2 h. After cooling, the reaction mixture yielded a
yellow precipitate that was collected by filtration to give 34 mg of
compound 39 (72%). MS (DCI) m/e 481 (M + H)+; 1H NMR (500
MHz, DMSO-d6) δ 9.93 (s, 1H), 8.00-8.02 (m, 2H), 7.81 (d, J )
8.42 Hz, 1H), 7.53 (d, J ) 1.56 Hz, 1H), 7.34-7.35 (m, 2H), 7.30
(dd, J ) 8.26, 1.72 Hz, 1H), 7.02 (d, J ) 8.42, Hz, 1H), 6.92 (d,
J ) 2.18 Hz, 1H), 6.88 (dd, J ) 8.42, 2.50 Hz, 1H), 4.03 (s, 3H),
3.63 (t, J ) 6.39 Hz, 2H), 3.46 (t, J ) 7.49 Hz, 2H), 2.34-2.41
(m, 2H). Anal. (C23H20N4O6‚0.1CH2Cl2) C, H, N.
3-Chloro-8-(2-oxopyrrolidin-1-yl)-5,10-dihydro-11H-dibenzo-
[b,e][1,4]diazepin-11-one (40). Compound 37 (0.41 g, 1.58 mmol)
in pyridine (3 mL) was treated with 4-chlorobutanoyl chloride
(0.267 g, 1.90 mmol) in CH2Cl2 (3 mL) dropwise at room
temperature. The reaction was stirred overnight. It was partitioned
between ethyl acetate and water. The organic layer was washed
with aqueous 4 N HCl and brine, dried (MgSO4), filtered, and
concentrated in vacuo. The residue was purified by column
chromatography on silica gel eluting with 2:3 hexanes/ethyl acetate
to provide 0.345 g of 4-chloro-N-(3-chloro-11-oxo-10,11-dihydro-
5H-dibenzo[b,e][1,4]diazepin-8-yl)butanamide (60%). MS (DCI)
2-[3-(3-Methoxy-4-nitrophenyl)-11-oxo-10,11-dihydro-5H-
dibenzo[b,e][1,4]diazepin-8-yl]-N,N-dimethylacetamide (46b). A
mixture of 45a (0.864 g, 2 mmol) and LiOH‚H2O (0.335 g, 8 mmol)
in MeOH (20 mL) and water (20 mL) was heated under reflux
overnight. The reaction mixture was neutralized to pH ) 4 and
extracted with ethyl acetate twice. The combined organic layers
were washed with brine, dried (MgSO4), filtered, and concentrated
in vacuo to give 0.75 g of [3-(3-methoxy-4-nitrophenyl)-11-oxo-
10,11-dihydro-5H-dibenzo[b,e][1,4]diazepin-8-yl]acetic acid that
was used for the next reaction without further purification (89%).
A mixture of [3-(3-methoxy-4-nitrophenyl)-11-oxo-10,11-dihy-
dro-5H-dibenzo[b,e][1,4]diazepin-8-yl]acetic acid (83 mg, 0.2
mmol), HATU (92 mg, 0.24 mmol), dimethyl amine hydrochloride
(32 mg, 0.4 mmol), and Et3N (81 mg, 0.8 mmol) in DMF (3 mL)
was stirred overnight. The reaction mixture was partitioned between
ethyl acetate and water, and the aqueous layer was extracted with
additional ethyl acetate. The combined organic layers were washed
with brine, dried (MgSO4), filtered, and concentrated in vacuo. The
1
m/e 365 (M + H)+; H NMR (500 MHz, DMSO-d6) δ 9.95 (s,
1H), 9.88 (s, 1H), 7.95 (s, 1H), 7.67 (d, J ) 8.48 Hz, 1H), 7.27 (d,
J ) 2.03 Hz, 1H), 7.18 (dd, J ) 8.48, 2.37 Hz, 1H), 7.05 (d, J )
2.03 Hz, 1H), 6.87-6.93 (m, 2H), 3.68 (t, J ) 6.61 Hz, 2H), 2.43
(t, J ) 7.29 Hz, 2 H), 1.97-2.05 (m, 2H).
Sodium ethoxide solution was prepared by adding sodium metal
(138 mg, 0.6 mmol) to EtOH (15 mL) at room temperature. To
this solution was added 4-chloro-N-(3-chloro-11-oxo-10,11-dihydro-
5H-dibenzo[b,e][1,4]diazepin-8-yl)butanamide (182 mg, 0.5 mmol).
A precipitate started to form several minutes later. The reaction
mixture was stirred overnight at room temperature, and the solid

4174 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17
Wang et al.
residue was purified by reverse phase prep HPLC to give 62 mg
Methyl 4-Chloro-2-(methyl(2-nitrophenyl)amino)benzoate (47).
Compound 28e (0.26 g, 0.85 mmol) in DMF (8.0 mL) was treated
with 95% NaH (35 mg, 1.38 mmol), followed by iodomethane (0.25
g, 1.67 mmol). The reaction was stirred at room temperature
overnight. The reaction was diluted with water and extracted with
ethyl acetate. The organic layer was washed with brine and dried
over Na2SO4. The product (0.27 g, 100%) was recovered as a yellow
solid. MS (DCI/NH3) m/e 323, 321 (M + H)+; 1H NMR (400 MHz,
DMSO-d6) δ 7.78 (dd, J ) 7.78, 1.36 Hz, 1H), 7.63-7.69 (m,
1H), 7.45 (d, J ) 8.14 Hz, 1H), 7.35 (d, J ) 7.46 Hz, 1H), 7.29
(d, J ) 2.03 Hz, 1H), 7.22 (t, J ) 7.63 Hz, 1H), 7.15 (dd, J )
8.14, 2.03 Hz, 1H), 3.35 (s, 3H), 3.34 (s, 3H).
3-Chloro-5-methyl-5H-dibenzo[b,e][1,4]diazepin-11(10H)-
one (48). The title compound was prepared using general methods
C and D from compound 47. MS (DCI/NH3) m/e 259, 261 (M +
H)+; 1H NMR (300 MHz, DMSO-d6) δ 10.28 (s, 1H), 7.63 (d, J )
8.5 Hz, 1H), 7.23-7.04 (m, 6H), 3.28 (s, 3H).
3-(3-Methoxy-4-nitrophenyl)-5-methyl-5H-dibenzo[b,e][1,4]-
diazepin-11(10H)-one (49). The title compound was prepared using
general method D from compound 48 in 46% yield. MS (DCI/
NH3) m/e 376 (M + H)+; 1H NMR (300 MHz, DMSO-d6) δ 10.29
(s, 1H), 8.00 (d, J ) 8.2 Hz, 1H), 7.75 (d, J ) 7.9 Hz, 1H), 7.58
(s, 1H), 7.46 (m, 3H), 7.22 (d, J ) 7.9 Hz, 1H), 7.13 (m, 1H), 7.07
(m, 2H), 4.04 (s, 3H), 3.40 (s, 3H).
1
of the title compound (71%). MS (ESI) m/e 445 (M - H)-; H
NMR (300 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.01 (d, J ) 8.48 Hz,
1H), 7.97 (s, 1H), 7.79 (d, J ) 8.14 Hz, 1H), 7.52 (d, J ) 1.70 Hz,
1H), 7.32-7.36 (m, 2H), 7.28-7.32 (dd, J ) 8.14, 1.70 Hz, 1H)
6.95 (d, J ) 7.80 Hz, 1H), 6.80-6.85 (m, 2H), 4.03 (s, 3H), 3.55
(s, 2H), 2.97 (s, 3H), 2.81 (s, 3H). Anal. (C24H22N4O5‚0.3H2O) C,
H, N.
2-[3-(3-Methoxy-4-nitrophenyl)-11-oxo-10,11-dihydro-5H-
dibenzo[b,e][1,4]diazepin-8-yl]-N-[4-(4-morpholinyl)phenyl]ac-
etamide (46c). The title compound was prepared in a similar
manner as described in the preparation of 46b in 80% yield. MS
(ESI) 580 (M + H)+; 1H NMR (DMSO-d6) δ 9.90 (s, 1H), 9.87 (s,
1H), 8.01 (d, J ) 8.4 Hz, 1H), 7.96 (s, 1H), 7.80 (d, J ) 8.1 Hz,
1H), 7.52 (d, J ) 1.2 Hz, 1H), 7.44 (d, J ) 9.0 Hz, 2H), 7.35 (m,
2H), 7.30 (dd, J ) 8.3, 1.7 Hz, 1H), 6.95 (m, 3H), 6.88 (d, J ) 9.0
Hz, 2H), 4.03 (s, 3H), 3.72 (m, 4H), 3.48 (s, 2H), 3.03 (m, 4H).
Anal. (C32H29N5O6‚1.4 HCl‚2.75H2O) C, H, N.
2-[3-(3-Methoxy-4-nitrophenyl)-11-oxo-10,11-dihydro-5H-
dibenzo[b,e][1,4]diazepin-8-yl]-2-methyl-N-(4-morpholin-4-ylphe-
nyl)propanamide (46d). The title compound was prepared in a
similar manner as described in the preparation of 46b from
compound 42c. MS (DCI) m/e 608 (M + H)+; 1H NMR (300 MHz,
DMSO-d6) δ 9.87 (s, 1H), 8.79 (s, 1H), 8.01 (m, 2H), 7.80 (d, J )
8.1 Hz, 1H), 7.52 (s, 1H), 7.42 (d, J ) 8.7 Hz, 2H), 7.30-7.35 (m,
3H), 7.06 (s, 1H), 7.00 (m, 1H), 6.95 (m, 1H), 6.84 (d, J ) 8.7 Hz,
2H), 4.03 (s, 3H), 3.71 (m, 4H), 3.00 (m, 4H), 1.49 (s, 6H). Anal.
(C34H33N5O6‚0.9H2O) C, H, N.
Methyl 2-(4-Fluoro-3-nitrophenyl)acetate (51). 4-Fluoro-3-
nitrobenzoic acid (50; 35.0 g, 189 mmol) was dissolved in SOCl2
(300 mL) and heated under reflux for 2 h. The reaction was cooled
and concentrated, then the last of the SOCl2 was removed using a
toluene azeotrope. The resultant brown oil (40.4 g) was dissolved
in Et2O (100 mL) and that solution was added dropwise to 2.0 M
(TMS)CHN2 in hexanes. After stirring the reaction at room
temperature for 45 min, it was concentrated and the crude was
purified by flash chromatography using 7:3 hexanes/ethyl acetate.
The diazoketone intermediate (24.5 g, 62%) was recovered as
orange solids. The diazoketone (20.0 g, 96 mmol) was dissolved
in MeOH (100 mL) and heated to 70 °C, and to that was added in
portions over 1 h a solution of silver benzoate (0.4 g, 1.7 mmol) in
triethylamine (4.0 mL). After heating for an additional 1 h, the
reaction was cooled, filtered, and concentrated, and the crude was
purified by flash chromatography using 78:22 hexanes/ethyl acetate.
The homologated ester (8.8 g, 43%) was recovered as a yellow
3-[3-(3-Methoxy-4-nitrophenyl)-11-oxo-10,11-dihydro-5H-
dibenzo[b,e][1,4]diazepin-8-yl]-N,N-dimethylpropanamide (46e).
The title compound was prepared in a similar manner as described
in the preparation of 46b from compound 42b. MS (ESI) m/e 461
1
(M + H)+; H NMR (300 MHz, DMSO-d6) δ 9.83 (s, 1H), 8.01
(d, J ) 8.14 Hz, 1H)), 7.93 (s, 1H), 7.79 (d, J ) 8.14 Hz, 1H), 7.5
(d, J ) 1.7 Hz, 1H), 7.3 (m, 3H), 6.9 (m, 1H), 6.83 (d, J ) 5.43
Hz, 2H), 4.03 (s, 3H), 2.92 (s, 3H), 2.80 (s, 3H), 2.67 (t, J ) 7.46,
2H), 2.50-2.52 (m, 2H). Anal. (C25H24N4O5‚0.42TFA‚0.4CH3OH)
C, H, N.
3-[3-(3-Methoxy-4-nitrophenyl)-11-oxo-10,11-dihydro-5H-
dibenzo[b,e][1,4]diazepin-8-yl]-N-(4-morpholin-4-ylphenyl)pro-
panamide (46f). The title compound was prepared in a similar
manner as described in the preparation of 46b from compound 42b.
1
oil. MS (DCI/NH3) m/e 231 (M + H + NH3)+; H NMR (300
MHz, CDCl3) δ 8.00 (dd, J ) 6.8, 2.4 Hz, 1H), 7.57 (m, 1H), 7.26
1
MS (ESI) m/e 594 (M + H)+; H NMR (300 MHz, DMSO-d6) δ
(m, 1H), 3.73 (s, 3H), 3.69 (s, 2H).
9.78 (s, 1H), 9.7 (s, 1H), 7.93 (d, J ) 8.42 Hz, 1H), 7.85 (s, 1H),
7.72 (d, J ) 8.11 Hz, 1H), 7.45 (d, J ) 1.56 Hz, 1H), 7.34 (d, J )
9.04 Hz, 2H), 7.27 (m, 2H), 7.22 (dd, J ) 8.26, 1.72 Hz, 1H), 6.86
(d, J ) 8.11 Hz, 1H), 6.75-6.79 (m, 4H), 3.96 (s, 3H), 3.64 (br,
m, 4H), 2.95 (br, m, 4H), 2.65-2.68 (m, 2H), 2.43-2.45 (m, 2H).
Anal. (C33H31N5O6‚0.7TFA) C, H, N.
Methyl 4-Chloro-2-(4-(2-methoxy-2-oxoethyl)-2-nitrophenoxy)-
benzoate (52). Compound 51 (1.0 g, 4.7 mmol) and methyl
4-chloro-2-hydroxybenzoate (1.0 g, 5.4 mmol) were dissolved in
DMA (2.0 mL), K2CO3 (0.73 g, 5.3 mmol) was added, and the
reaction was heated at 80 °C under nitrogen overnight. The reaction
was diluted with water and extracted with ethyl acetate. The organic
layer was washed with brine and dried over Na2SO4. The crude
was purified by flash chromatography using 4:1 hexanes/ethyl
acetate. The above diaryl ether (0.89 g, 50%) was recovered as an
oil that slowly crystallized. MS (DCI/NH3) m/e 399, 397 (M + H
+ NH3)+; 1H NMR (300 MHz, DMSO-d6) δ 8.04 (d, J ) 2.2 Hz,
1H), 7.95 (d, J ) 8.4 Hz, 1H), 7.57 (dd, J ) 8.4, 2.2 Hz, 1H), 7.49
(dd, J ) 8.8, 1.8 Hz, 1H), 7.37 (d, J ) 1.8 Hz, 1H), 7.00 (d, J )
8.8 Hz, 1H), 3.83 (s, 2H), 3.70 (s, 3H), 3.65 (s, 3H).
Methyl 2-(3-Chloro-11-oxo-10,11-dihydrodibenzo[b,f][1,4]-
oxazepin-8-yl)acetate (53). The title compound was prepared using
general method C from compound 52 in 70% yield. MS (DCI/
NH3) m/e 320, 318 (M + H)+; 1H NMR (300 MHz, DMSO-d6) δ
10.80 (s, 1H), 7.77 (d, J ) 8.5 Hz, 1H), 7.53 (d, J ) 2.0 Hz, 1H),
7.40 (dd, J ) 8.5, 2.0 Hz, 1H), 7.49 (d, J ) 7.8 Hz, 1H), 7.04 (m,
2H), 3.65 (s, 2H), 3.59 (s, 3H).
2-[3-(3-Methoxy-4-nitrophenyl)-11-oxo-10,11-dihydro-5H-
dibenzo[b,e][1,4]diazepin-7-yl]-N,N-dimethylacetamide (46g).
The title compound was prepared in a similar manner as described
in the preparation of 46b from compound 42d. MS (DCI) m/e 447
1
(M + H)+, H NMR (500 MHz, DMSO-d6) δ 9.85 (s, 1H), 8.00
(m, 2H), 7.80 (d, J ) 8.11 Hz, 1H), 7.52 (d, J ) 1.56 Hz, 1H),
7.37 (d, J ) 1.56 Hz, 1H), 7.34 (dd, J ) 8.42, 1.56 Hz, 1H), 7.30
(dd, J ) 8.11 Hz, 8.42, 1.56 Hz, 1H), 6.89-6.91 (m, 2H), 6.78
(dd, J ) 7.95, 1.72 Hz, 1H), 4.03 (s, 3H), 3.56 (s, 2H), 2.98 (s,
3H), 2.82 (s, 3H). Anal. (C24H22N4O5‚0.9H2O) C, H, N.
2-[3-(3-Methoxy-4-nitrophenyl)-11-oxo-10,11-dihydro-5H-
dibenzo[b,e][1,4]diazepin-7-yl]-2-methyl-N-(4-morpholin-4-ylphe-
nyl)propanamide (46h). The title compound was prepared in a
similar manner as described in the preparation of 46b from
compound 42e. MS (ESI) m/e 608 (M + H)+; 1H NMR (300 MHz,
DMSO-d6) δ 9.89 (s, 1H), 8.85 (s, 1H), 8.06 (s, 1H), 8.01 (d, J )
8.5 Hz, 1H), 7.79 (d, J ) 8.1 Hz, 1H), 7.52 (d, J ) 1.7 Hz, 1H),
7.42 (d, J ) 9.2 Hz, 2H), 7.29-7.38 (m, 3H), 7.04 (d, J ) 1.7 Hz,
1H), 6.91 (m, 2H), 6.83 (d, J ) 9.2 Hz, 2H), 4.03 (s, 3H), 3.71 (m,
4H), 3.00 (m, 4H), 1.49 (s, 6H). Anal. (C34H33N5O6‚0.1CH2Cl2)
C, H, N.
Methyl
2-(3-(3-Methoxy-4-nitrophenyl)-11-oxo-10,11-
dihydrodibenzo[b,f][1,4]oxazepin-8-yl)acetate (54). The title com-
pound was prepared using general method D from compound 53
1
in 70% yield. MS (DCI/NH3) m/e 435 (M + H)+; H NMR (300
MHz, DMSO-d6) δ 10.62 (s, 1H), 8.00 (d, J ) 8.5 Hz, 1H), 7.89
(d, J ) 8.1 Hz, 1H), 7.82 (d, J ) 1.7 Hz, 1H), 7.75 (dd, J ) 8.1,

Design, Synthesis, and ActiVity of Chk-1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 17 4175
2.0 Hz, 1H), 7.64 (d, J ) 1.4 Hz, 1H), 7.48 (dd, J ) 8.5, 1.4 Hz,
1H), 7.34 (d, J ) 8.1 Hz, 1H), 7.08 (d, J ) 1.7 Hz, 1H), 7.05 (dd,
J ) 8.1, 2.0 Hz, 1H), 4.06 (s, 3H), 3.65 (s, 2H), 3.60 (s, 3H). Anal.
(C23H18N2O7‚0.55 H2O) C, H, N.
incubate for 48 h. Doxorubicin (DOX) and CPT were purchased
from Sigma (St. Louis, MO). After the treatment, MTS reagents
that measured the amount of live cells (Promega, Madison, WI)
were added to the incubated cells and allowed to develop for 20
min to 2 h. Colorimetric measurements were taken at 490 nm on
a Spectra MAX 190 plate reader from Molecular Device (Sunny-
vale, CA).
Chk1 Assay. The Chk1 enzymatic assay was carried out using
a recombinant Chk1 kinase domain. A human Cdc25C peptide was
used as the substrate in the assay. The reaction mixture contained
25 mM of HEPES at pH 7.4, 10 mM MgCl2, 0.08 mM Triton
X-100, 0.5 mM DTT, 5 µM ATP, 4nM 33P ATP, 5 µM Cdc25C
peptide substrate, and 5 nM of the recombinant Chk1 protein. For
potent compounds with Ki below 1 nM, 0.5 nM of the recombinant
Chk1 protein and 8 nM of 33P were used in the assays. The
compound vehicle DMSO was maintained at 2% in the final
reaction. After 30 min at room temperature, the reaction was stopped
by the addition of equal volumes of 4 M NaCl and 0.1 M EDTA,
pH 8. A 40 µL aliquot of the reaction mixture was added to a well
in a FlashPlate (NEN Life Science Products, Boston, MA) contain-
ing 160 µL of phosphate-buffered saline (PBS) without calcium
chloride and magnesium chloride and incubated at room temperature
for 10 min. The plate was then washed 3 times in PBS with 0.05%
of Tween-20 and counted in a Packard TopCount counter (Packard
BioScience Company, Meriden, CT).
In Vitro Kinase Assay for Selectivity. Recombinant Cdc2,
PKCγ, and PKCδ (Calbiochem, San Diego, California); PKA
(Panvera, Madison, Wisconsin); Src, SGK and full-length cTak1
(Upstate Biotechnology, Charlottesville, Virginia); ERK2 (New
England Biolabs, Beverly, Massachusetts); and cKit(544-976;
ProQinase, Freiburg, Germany) were commercially obtained. His-
tagged AKT1[S378A, S381A, T450D, S473D](139-480), Chk1(1-
269), KDR(789-1354), Flt-1(786-1338), and PDK1(1-396), Lck(62-
509), Lyn, Fyn-catalytic domain, HCK, Tie2, FGR(60-529), and
Flt4 were expressed using the FastBac bacculovirus expression
system (GIBCO BRL, Gaithersburg, MD) and purified using either
nickel (his-tag) or glutathione (GST) affinity S5 chromatography.
His-tagged Flt3 (569-993) was expressed in E. coli. Peptide
substrates had the general structure biotin-Ahx-peptide with
sequences: Chk1 and Chk2, AKVSRSGLYRSPSMPENLNRPR;
AKT, EELSPFRGRSRSAPPNLWAAQR; Cdc2, PKTPKKAKKL;
ERK2, KRELVEPLTPSGEAPNQALLR; SGK, RPRAATF; PKA,
LRRASLG; PKCγ and PKCδ, ERMRPRKRQGSVRRRV; cTak1,
AKVSRSGLYRSPSMPENLNRPR; KDR, Flt-1, Flt-3, Flt-4, and
cKit, AEEEYFFLFA-amide; Tie2, biotin-poly(Glu-Tyr); FGR, Lck,
Lyn, Fyn, and HCK, GAEEEIYAAFFA. For Src assays, the
biotinylated substrate PTK-2 (Promega, Madison, Wisconsin) was
used.
For S/T kinases and Src, a radioactivity-based assay was utilized.
In this format, compounds were incubated with gamma-[33P]-ATP
(5 µM), enzyme, and biotinylated peptide substrates (2 µM) in
kinase assay buffer for 30 min at room temperature and then the
reaction was stopped by the addition of 100 mM EDTA. The
quenched reactions were transferred to streptavidin-coated Flash-
Plates (Perkin-Elmer), the plates were washed, and peptide phos-
phorylation was quantified on a TopCount scintillation plate reader.
For all TKs (except Src and Tie2), an HTRF assay format was
employed. In this format, compounds were incubated with enzymes,
biotinylated-peptide substrates (0.5 µM), and ATP (1 mM) for 1 h
at room temperature before stopping with 100 mM EDTA. To the
stopped reaction, revelation buffer containing Eu-labled anti-pY-
Ab (Cis Bio HTRF) and streptavidin-APC (Prozyme) were added,
and the mixture was allowed to incubate for 1 h. Plates were
subsequently read on a Discovery HTRF plate reader monitoring
the time-resolved fluorescence ratio (665 nm/615 nm). Tie-2 activity
was measured using an Elisa-type assay.
Cell Culture. SW620 cells were grown in RPMI 1640 supple-
mented with 10% fetal bovine serum (FBS) at 37 °C in a 5% CO2
incubator, H1299 cells were grown in RPMI 1640 S6 supplemented
with 10% FBS, 1 mM sodium pyruvate, and 0.45% glucose, and
Hela cells were grown in MEM supplemented with 10% FBS.
MTS Assay. Exponential growing cells were seeded in 96-well
plates and allowed to attach an incubator. After 24 h, the cells
received various amounts of the compounds and continued to
FACS Analysis. Cells were treated with compounds at the
indicated concentrations for the indicated time. The medium and
the washing solution, phosphate-buffered saline (PBS), were
collected to include the floating cells. The cells were then
trypsinized and added to the tubes with the medium and PBS. After
spinning at 1000 × g for 5 min, the cells were washed in PBS and
fixed in 70% ethanol. The washed cells were treated with RNase
A at 37 °C for 30 min. Finally, the cells were stained with propidium
iodide (PI) and analyzed by fluorescence-activated cell sorting
(FACS) on a FACSCalibur (Becton Dickinson, San Jose, CA). Cells
were counted and analyzed using the CellQuest Pro program
provided by the vendor. Thymidine and paclitaxel were used as
controls for positioning the S and G2/M phases, respectively.
Soft Agar Assay. The soft agar experiments were performed in
12-well plates. The bottom layer contained 0.5 mL of 0.7% agar
with the growth medium. The top layer contained 0.5 mL of 0.35%
agar with the medium mixed with 5000 cells/well of SW620 cells.
Chk1 compounds were diluted in 0.5 mL of the medium and added
to each well. After 14 days, colonies were stained with iodoni-
trotetrazolium at 0.5 µg/mL overnight and counted using a Sony
CCD camera linked to the Image-Pro Plus software (MediaCyber-
netics, Silver Spring, MD).
Acknowledgment. We gratefully acknowledge the Structural
Chemistry Department of Abbott Pharmaceutical Discovery
(Dept. 0418) for collecting NMR and MS data. For crystal
structure analysis, data were collected at beamline 17-ID in the
facilities of the Industrial Macromolecular Crystallography
Association Collaborative Access Team (IMCA-CAT) at the
Advanced Photon Source. These facilities are supported by the
companies of the Industrial Macromolecular Crystallography
Association.
Supporting Information Available: FACS analysis of 46d with
doxorubicin and camptothecin, soft agar assay of 46d with
doxorubicin, and elemental analysis data. This material is available
free of charge via the Internet at http://pubs.acs.org.
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