Investigation of novel 7,8-disubstituted-5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-ones as potent Chk1 inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.02.080

The synthesis and structure-activity relationships (SAR) of Chk1 inhibitors based on a 5,10-dihydro-dibenzo[b,e][1,4]diazepin-11-one core are described. Specifically, an exploration of the 7 and 8 positions on this previously disclosed core afforded compounds with improved enzymatic and cellular potency.

Full text of DOI:10.1016/j.bmcl.2008.02.080

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2311–2315
Investigation of novel 7,8-disubstituted-5,10-dihydro-dibenzo-
[b,e][1,4]diazepin-11-ones as potent Chk1 inhibitors
Lisa A. Hasvold,* Le Wang,* Magdalena Przytulinska, Zhan Xiao, Zehan Chen,
Wen-Zhen Gu, Philip J. Merta, John Xue, Peter Kovar, Haiying Zhang,
Chang Park, Thomas J. Sowin, Saul H. Rosenberg and Nan-Horng Lin
Abbott Laboratories, Cancer Research, 100 Abbott Park Road, Dept. R4N6 AP10-307, Abbott Park, IL 60064-6101, USA
Received 24 January 2008; revised 28 February 2008; accepted 29 February 2008
Available online 6 March 2008
Abstract—The synthesis and structure–activity relationships (SAR) of Chk1 inhibitors based on a 5,10-dihydro-dibenzo[b,e][1,4]dia-
zepin-11-one core are described. Specifically, an exploration of the 7 and 8 positions on this previously disclosed core afforded com-
pounds with improved enzymatic and cellular potency.
Ó 2008 Elsevier Ltd. All rights reserved.
Progression of cells through the cell-cycle is mediated by
checkpoints where the integrity of the cell is monitored.1
Specifically, checkpoint kinase 1 (Chk1 kinase) arrests p-
53 deficient tumor cells that have sustained DNA dam-
age due to radiation or chemotherapeutic agents at the
G2/M checkpoint.2,3 This prevents them from entering
mitosis and allows time for repair.4 It is thought that
inhibition of Chk1 kinase will lead to abrogation of
the G2/M checkpoint, therefore allowing DNA-dam-
aged cells to enter mitosis. This would result in cell
death, thus preventing the replication of aberrant cells
and the progression of cancer.3 Since normal cells arrest
at the G1/S checkpoint and should remain unaffected,
small molecule Chk1 inhibitors have consequently been
targeted as possible adjuvant therapies to traditional
cancer treatments.2,5
matic activity, however, compound 2 did not significantly
potentiate the activity of the chemotherapeutic agent
doxorubicin during co-administration in an MTS assay
measuring antiproliferative ability.6,7 A survey of the
remaining sites of the molecule demonstrated that, with
the exception of positions 7 and 8, the Chk1 enzyme was
intolerant to further substitution of the benzodiazepinone
core.6 Herein we disclose the SAR of 7,8-disubstituted
benzodiazepinones and detail the identification of a com-
pound with good enzymatic and cellular profiles.
O
1
NH
O
8
NH
Cl
N
H
7
3
N
H
O2N
1
2
O
In a previous report from our laboratories, we disclosed
the identification and subsequent optimization of benzo-
diazepinone-based HTS hit 1. Inthat effort, position 3was
targeted. The X-ray structure of 1 bound to the Chk1 en-
zyme showed a deep cleft that could accommodate
additional functionality at that site. A variety of 3-substi-
tuted compounds were thus investigated, leading to the
identification of compound 2 as the most potent Chk1
inhibitor within this class.6 Despite the favorable enzy-
IC50 = 10 nM
MTS EC50 = 47.7 μM
MTS EC50 with Doxorubicin = 33.8 μM
We began our investigation by substituting position 7
with a number of ether- and carbon-linked moieties
while holding position 8 constant as a methoxy group.
Substitution at position 8 with a methoxy group had
previously given a compound with reasonable enzymatic
activity (IC50 = 14 nM).6
Keyword: Chk1.
*
Synthesis of the 7-position ether-substituted benzodiaz-
epinones is shown in Scheme 1. SEM-protection of the
Corresponding authors. Tel.: +1 847 937 7217; fax: +1 847 935 5165
(L.A.H.); e-mail: lisa.hasvold@abbott.com
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.02.080

2312
L. A. Hasvold et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2311–2315
OMe
d, e
O
CO2Me
NH
OMe
OH
a, b, c
SEM
AcHN
Cl
O2N
OMe
Br
a, b, c, d
e, f, g
NO2
3
4
H2N
CO2Me
NH
Br
12
OMe
13
O2N
H2N
OMe
CO2Me
I
f
Cl
+
SEM
NO2
O
Cl
O
5
6
SEM
O
O
NH
NH
OMe
OMe
7
OMe
Br
h, i, j
N
N
H
Cl
H
O
O
B
N
NH
14
15
O2N
OMe
OMe
SEM
O
g, h,
i
j, k
+
N
k, i
O2N
Cl
O
H
O
OMe
NH
O
8
9
OMe
R
NH
OMe
CO2Me
l, j
N
O
O
H
NH
NH
N
Cl
H
OMe
OH
OMe
l
O2N
16
j
17
N
N
OMe
R
O
H
H
O2N
O2N
O
OMe
OMe
10
11
NH
OMe
CO2Me
Scheme 1. Reagents and conditions: (a) SEMCl (Me3SiCH2-
CH2OCH2Cl), DIPEA, CH2Cl2, rt; (b) 5% Pt/C, H2, EtOH, rt; (c)
Ac2O, DMAP, DCE, rt, 81% over 3 steps; (d) Ac2O, conc. HNO3,
À20 °C, 70%; (e) K2CO3, MeOH, rt, 91%; (f) Cu, K2CO3, PhCl, reflux,
84%; (g) LiOHÆH2O, THF, H2O, 65 °C; (h) Et3N, SnCl2Æ2H2O, 65 °C;
(i) HATU, Et3N, DMF, rt, 89% over 3 steps; (j) Pd(PPh3)4, 1 M
Na2CO3, 7:2:3 DME/EtOH/H2O, 160 °C microwave, 62%; (k) 4 N
HCl/dioxane, MeOH, CH2Cl2, 100%; (l) RX, K2CO3, DMF, 100 °C.
N
H
18
m,n
O2N
CO2Me
CONMe2
OMe
Scheme 2. Reagents and conditions: (a) Ac2O, Et3N, CH2Cl2, 0 °C,
95%; (b) Ac2O, conc. HNO3, À10 °C, 96%; (c) MeOH, H2SO4, reflux,
95%; (d) 6, Cu, K2CO3, PhCl, reflux, 88%; (e) 5% Pt/C, H2, MeOH, rt,
73%; (f) LiOHÆH2O, THF, H2O; (g) HATU, Et3N, DMF, 97% over
two steps; (h) 4-pyridyl-CH@CH2, Pd(dppf)Cl2, Et3N, DMF, 110 °C;
(i) H2, 5% Pt/C, MeOH, rt, 17% over 2 steps toward 15, 70% for 16; (j)
9, Pd(OAc)2, Cy-Map, CsF, DMF, 85 °C, 29% for 15, 73% for 18; (k)
MeO2C–CH@CH2, Pd(dppf)Cl2, Et3N, DMF, 110 °C, 82%; (l) LAH
(for 17a, 80%) or MeMgBr (for 17b, 78%), THF; (m) LiOHÆH2O,
DMF, THF, H2O, 100 °C, 48%; (n) Me2NHÆHCl, HATU, Et3N,
DMF, rt, 91%.
hydroxyl group of 2-methoxy-5-nitrophenol (3) fol-
lowed by nitro reduction and acetylation of the resulting
aniline gave intermediate 4. Nitration followed by
deprotection of the amine afforded 5. Copper-assisted
coupling of compound 5 with methyl ester 6 (obtained
by esterification of the commercially available acid)
yielded amine 7. The benzodiazepinone core 8 was
formed in a 3-step, one-pot reaction through ester
hydrolysis, tin-mediated nitro-group reduction and
cyclization via HATU-activated amide formation. Suzu-
ki coupling of intermediates 8 and 9 (prepared from 5-
chloro-2-nitroanisole) followed by removal of the SEM
protecting group provided 10. Alkylation of the 7-hy-
droxy with various alkyl halides afforded the final ether
products (11a–j).
hydride reduction of ester 16 followed by Suzuki cou-
pling with 9 gave 17a. Grignard reaction with methyl-
magnesium bromide and subsequent Suzuki coupling
produced 17b. Arylation of 16 with pinacol borate 9
provided 18a which was converted to 18b via ester
hydrolysis and HATU-assisted amidation.
Carbon-linked analogs 24 and 25 were synthesized as
shown in Scheme 3. Nitration of (2-hydroxyphenyl)ace-
tic acid (19) was followed by treatment with TMS-diazo-
methane resulting in methylation of both the hydroxyl
and carboxyl groups. Catalytic hydrogenation of the ni-
tro group afforded aniline 20. Acetyl protection of the
aniline followed by nitration and then deprotection gave
21. Copper-mediated coupling of 21 with 6 resulted in
intermediate 22. Reduction of the nitro group with sub-
sequent ester hydrolysis and intramolecular amide for-
mation gave benzodiazepinone 23. Treatment of 23
with methylmagnesium bromide then Suzuki coupling
with 9 produced compound 24. Alternatively, lithium
aluminum hydride reduction of the ester of 23 followed
by Suzuki coupling provided alcohol 25.
The 7-position carbon-substituted analogs 15, 17, and
18 were prepared as shown in Scheme 2. Acetylation
of 3-bromo-4-methoxyaniline (12) followed by nitration,
deacetylation, and copper-mediated coupling with 6 re-
sulted in intermediate 13. Platinum-catalyzed nitro-
group reduction, ester hydrolysis, and intramolecular
amide formation gave benzodiazepinone intermediate
14. The pyridylethyl analog 15 was prepared by a Heck
coupling with 4-vinylpyridine and subsequent hydroge-
nation of the double bond, then attachment of the 3-
methoxy-4-nitrophenyl ring via Suzuki coupling.
Alternatively, Heck coupling of intermediate 14 with
methyl acrylate followed by hydrogenation of the result-
ing olefin afforded intermediate 16. Lithium aluminum

L. A. Hasvold et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2311–2315
2313
ation. Similar transformations to those described previ-
ously for Schemes 1–3 allowed for efficient preparation
of the carbon-substituted compounds (Scheme 4).
OH
CO2H
OMe
CO2Me
a, b, c
d, e, f
H2N
19
20
CO2Me
NH
A limited number of compounds with small substitu-
ents at position 8 are presented in Table 2. As with
our findings for position 7, a hydroxyalkyl substituent
proved to be optimal for maximizing inhibitor potency
(compound 32). Methyl ester, dimethylamide, and ethyl
substitution (29a, 29b and 31, respectively) were not
well tolerated in comparison to the hydroxypropyl moi-
ety, conferring diminished activity by 5- to 8-fold. It
was not surprising that polar substitutions were supe-
rior to non-polar substitutions at both positions 7
and 8 since the X-ray structure of our HTS hit com-
pound 1 bound to the Chk1 enzyme had shown that
these positions point toward the hydrophilic solvent
front.6
Cl
MeO2C
O2N
H2N
OMe
CO2Me
h, i, j
g
NO2
21
OMe
22
O
NH
O
OMe
OH
NH
k, l
OMe
CO2Me
N
H
N
Cl
H
24
O2N
23
OMe
m, l
O
Compounds with enzymatic activities less than 50 nM
were taken forward into three cellular assays, MTS (a
measure of antiproliferative ability),6,7 FACS (a mea-
NH
OMe
OH
N
H
CO2Me
O2N
25
OMe
X
Cl
NH
a, b, c, d
e, f, g
NO2
27
Scheme 3. Reagents and conditions: (a) conc. HNO3, H2O, 0 °C; (b)
TMS–CHN2, CH2Cl2, MeOH, 45% over two steps; (c) Pd/C, H2,
MeOH; (d) Ac2O, Et3N, CH2Cl2, 0 °C, 88% over two steps; (e) Ac2O,
conc. HNO3, À10 °C, 88%; (f) MeOH, H2SO4, reflux, 95%; (g) 6, Cu,
K2CO3, PhCl, reflux, 80%; (h) 5% Pt/C, H2, MeOH, rt, 93%; (i)
LiOHÆH2O, THF, H2O; (j) HATU, Et3N, DMF, 75% over two steps;
(k) MeMgBr, THF, 75%; (l) 9, Pd(OAc)2, Cy-Map, CsF, DMF, 85 °C,
68% for 24, 75% for 25; (m) LAH, THF, 80%.
H2N
OMe
MeO
26
X
X = Br or CO2Me
O
O
NH
NH
CO2Me
X
h
X = CO2Me
N
N
H
OMe
OMe
H
Cl
28
29
O2N
We found that incorporation of polar groups at position
7 provided compounds with greater activity against
Chk1 than did hydrophobic groups. Specifically, com-
pounds containing hydroxy and methoxy groups, as well
as straight-chain hydroxyalkyl or aminoalkyl ethers had
IC50 values of <1 nM (Compounds 10, 11a–d). Varying
the length of the substitution from 4–6 atoms did not al-
ter the activity. The hydrophobic 2-trifluorobenzyl ether
compound (11f) was over 1000-fold less active than the
straight-chain ethers. Heteroaryl and aliphatic heterocy-
cles were tolerated, however, they were typically 5- to
10-fold less active than the straight-chain ether-contain-
ing compounds (11g–j). The 7-position carbon-linked
analogs were generally less active than the correspond-
ing ether analogs (11b vs 17a and 11h vs 15). The addi-
tion of geminal dimethyl groups on the terminal carbons
of the straight-chain carbon-linked compounds was well
tolerated (17b vs 17a and 24 vs 25). The presence of lar-
ger or more hydrophobic substituents conferred deleteri-
ous effects on the enzymatic potency (data in Table 1
and data not shown).
OMe
CO2Me
i, j
k, l
CONMe2
X = Br
O
O
NH
NH
h
R
N
H
OMe
R = H
N
OMe
Cl
H
31
O2N
30
OMe
m, h
R = CO2Et
O
NH
OR
N
H
OMe
32
O2N
OMe
Scheme 4. Reagents and conditions: (a) Ac2O, Et3N, CH2Cl2, 0 °C,
100% for X@Br; (b) Ac2O, conc. HNO3, À10 °C; (c) K2CO3, MeOH,
67% over two steps for X@Br, 91% for X@CO2Me; (d) 6, Cu, K2CO3,
PhCl, reflux, 67% for X@Br, 57% for X@CO2Me; (e) LiOHÆH2O,
THF, MeOH, H2O, 50 °C; (f) SnCl2Æ2H2O, 70 °C; (g) HATU, Et3N,
DMF, rt, 65% over three steps for X@Br, 20% over three steps for
X@CO2Me; (h) 9, Pd(OAc)2, Cy-Map, CsF, DMF, 85 °C, 95% for 29,
47% for 31, 60% for 32; (i) LiOHÆH2O, DMF, THF, H2O, 100 °C,
27%; (j) Me2NHÆHCl, HATU, Et3N, DMF, rt, 27%; (k) R–CH@CH2,
Pd(dppf)Cl2, Et3N, DMF, 110 °C, 66%; (l) H2, 5% Pt/C, MeOH, 95%
for R@H, 94% for R@CO2Et; (m) LAH, THF, 0 °C, 99%.
Having surveyed substitutions at position 7 of the ben-
zodiazepinone, we proceeded with the analysis of posi-
tion 8 while position 7 was held constant as methoxy.
Our survey of position 8 was limited to carbon substitu-
tions as attempts to produce ethers via either alkylation
or Mitsunobu reaction resulted in preferential N-alkyl-

2314
L. A. Hasvold et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2311–2315
Table 1. Inhibition of benzodiazepinones against the Chk1 enzyme
O
NH
OMe
N
R
H
O2N
O
Compound
R
IC50 (nM)7
MTS (lM) EC50
/
Facs (lM) EC50
/
Cdc25A EC50 (lM)8
EC50 with doxorubicin7
EC50 with doxorubicin7
10
OH
OCH3
OCH2CH2OH
0.9
0.6
>59/2.5
>59/2.7
>59/1.2
>59/1.0
17.3/2.1
52/0.8
>10/2.3
>10/0.4
>10/1.4
>10/1.6
>10/>10
>10/1.4
0.07
0.28
3.1
2
11a
11b
11c
11d
11e
0.58
0.7
OCH2CH2CH2CH2OH
OCH2CH2CH2NH2
OCH2CH2OCH2CH2OCH3
0.98
3.1
2.6
3.1
O
11f
11g
11h
11i
11j
15
1050
4.0
>59/2.4
>59/>5.9
>59/1.7
2.0/0.2
>10/2.4
>10/>10
>10/1.6
2.4/0.4
NA
>10
0.05
0.2
CF3
S
O
O
N
N
6.8
O
4.8
O
O
N
8.1
2.0/0.2
2.4/0.4
0.2
O
N
16.2
9.6/0.6
>10/0.4
2.1
H2C
17a
17b
18a
18b
24
CH2CH2CH2OH
CH2CH2C(CH3)2OH
CH2CH2CO2CH3
CH2CH2CON(CH3)2
CH2C(CH3)2OH
CH2CH2OH
2.6
4.0
22
>59/1.8
>59/0.6
>59/>5.9
>59/2.6
>59/1.2
>59/3.0
>10/0.45
>10/0.55
>10/>10
>10/0.9
1.8/0.6
1.5
0.6
>10
0.05
0.04
0.9
7.9
3.4
3.5
25
>10/0.44
sure of specificity for Chk1 through G2/M checkpoint
abrogation)6,7 and Cdc25A (a measure of Chk1 inhibi-
tion through S checkpoint abrogation).8 We defined an
ideal Chk1 inhibitor as having no single agent activity
in the MTS and FACS assays (MTS EC50 > 59 lM
and FACS EC50 > 10 lM), but significant combination
activity when co-administered with a DNA-damaging
agent (EC50 < 1 lM). The ideal EC50 value for the
Cdc25A assay was also defined to be <1 lM.
with no single agent cellular activity in either the
MTS or FACS assays, but had combination activities
of 1.0–1.6 lM, just above the upper limit for an ideal
compound. The similar carbon-linked compounds
(17a and 25) also showed no single agent activity,
but were better G2/M abrogators with ideal combina-
tion FACS EC50 values of 0.45 and 0.44 lM, respec-
tively. They failed to meet the requirements of the
MTS assay, however, with compound 17a having
slightly diminished antiproliferative activity (MTS
According to the parameters set forth for an ideal
Chk1 inhibitor, only compound 17b performed as de-
sired in all three cellular assays. Compounds with un-
branched hydroxyalkyl substituents (11b–c, 17a, and
25) typically possessed adequate cellular activity.
These compounds often showed ideal or near ideal
activity in one or more of the cellular assays. Com-
pounds 11b and 11c showed similar cellular profiles
EC50 = 1.8 lM)
and
compound
25
(MTS
EC50 = 3.0 lM) showing an even greater loss. In con-
trast, 8-substituted hydroxypropyl analog 32 demon-
strated significant single agent activity in both the
MTS (single agent EC50 = 22 lM) and FACS (single
agent EC50 = 4.3 lM) assays. Ester substituents,
whether directly attached to the benzodiazepinone
core as in compound 29a or attached through a two-

L. A. Hasvold et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2311–2315
2315
Table 2. Inhibition of 7-oxygen,8-carbon disubstituted benzodiazepinones against the Chk1 enzyme
O
NH
R
N
OMe
H
O2N
O
Compound
R
IC50 (nM)7
MTS (lM) EC50
/
Facs (lM) EC50
/
Cdc25A EC50 (lM)8
EC50with doxorubicin7
EC50 with doxorubicin7
29a
29b
31
CO2CH3
11.2
15.5
9.0
>59/>5.9
45/4.5
>10/>10
>10/>10
>10/1.2
4.3/0.7
>10
3.5
CON(CH3)2
CH2CH3
CH2CH2CH2OH
4.5/2.6
22/1.2
0.35
0.22
32
1.9
carbon linker as in compound 18a, showed no activity
in any of the three assays. The same results were seen
for the 4-pyridylmethoxy substituted compound 11h.
Interestingly, the corresponding carbon analog (15)
showed significant single agent activity in the MTS as-
say, ideal activity in the FACS assay and moderate
activity in the Cdc25A assay.
References and notes
1. Walworth, N. C. Curr. Opin. Cell Biol. 2000, 12(24), 697–704.
2. Powell, S. N.; DeFrank, J. S.; Connell, P.; Eogan, M.;
Preffer, F.; Dombkowski, D.; Tang, W.; Friend, S. Cancer
Res. 1995, 55, 1643.
3. Kawabe, T. Mol. Cancer Ther. 2004, 3, 513.
4. Zhou, B. B.; Elledge, S. J. Nature 2000, 408, 433.
5. Li, Q.; Zhu, G.-D. Curr. Top. Med. Chem. 2002, 2, 939.
6. Wang, L.; Sullivan, G. M.; Hexamer, L. A.; Hasvold, L. A.;
Thalji, R.; Przytulinska, M.; Tao, Z. F.; Li, G.; Chen, Z.;
Xiao, Z.; Gu, W. Z.; Xue, J.; Bui, M. H.; Merta, P.; Kovar,
P.; Bouska, J. J.; Zhang, H.; Park, C.; Stewart, K.; Sham,
H. L.; Sowin, T. J.; Rosenberg, S. H.; Lin, N. H. J. Med.
Chem. 2007, 50(17), 4162.
7. Tong, Y.; Claiborne, A.; Stewart, K. D.; Park, C.; Kovar,
P.; Chen, Z.; Credo, R. B.; Gu, W.-Z.; Gwaltney, S. L., II;
Judge, R. A.; Zhang, H.; Rosenberg, S. H.; Sham, H. L.;
Sowin, T. J.; Lin, N.-H. Bioorg. Med. Chem. 2007, 15, 2759.
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Herein we have described the synthesis and SAR of a
variety of 7,8-disubstituted-5,10-dihydro-dibenzo[b,e]
[1,4]diazepin-11-one compounds. Several of these com-
pounds showed an improved in vitro profile relative to
parent compound 2. Specifically, compound 17b
met all of our in vitro requirements having an IC50 of
4.0 nM, combination EC50 values with doxorubicin of
0.6 and 0.55 lM in the MTS and FACS assays, respec-
tively, and an EC50 value of 0.6 lM in the Cdc25A as-
say. Thus, substitution of positions 7 and 8 provided
improved cellular activity and progress toward obtain-
ing the desired profile.