Synthesis and evaluation of debromohymenialdisine-derived Chk2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.12.054

Natural products have been the subject of interest for drug discovery and as tools for understanding the underlying cellular pathways in various diseases. We present herein the synthesis and evaluation of new analogs of the marine sponge metabolite, debro

Full text of DOI:10.1016/j.bmc.2011.12.054

Bioorganic & Medicinal Chemistry 20 (2012) 1475–1481
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of debromohymenialdisine-derived Chk2 inhibitors
⇑
Rahman Shah Zaib Saleem, Theresa A. Lansdell, Jetze J. Tepe
Department of Chemistry, Michigan State University, East Lansing, MI 48824, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Natural products have been the subject of interest for drug discovery and as tools for understanding the
underlying cellular pathways in various diseases. We present herein the synthesis and evaluation of new
analogs of the marine sponge metabolite, debromohymenialdisine, as checkpoint kinase 2 (Chk2) inhib-
itors. We illustrate herein that slight modifications to the natural product scaffold can induce strong
selectivity for Chk2 over Chk1. These Chk2 inhibitors can serve as drug templates or molecular tools to
gain insight in Chk2 mediated radioprotection.
Received 11 November 2011
Revised 20 December 2011
Accepted 23 December 2011
Available online 11 January 2012
Keywords:
Checkpoint kinases
Chk2
Ó 2012 Elsevier Ltd. All rights reserved.
Natural products
Debromohymenialdisine
Hymenialdisine
Radioprotection
1. Introduction
robust apoptotic response in those cancer types.12 Secondly, in nor-
mal cells, inhibition of Chk2 has indicated radioprotective effects by
The induction of DNA damage by chemotherapeutic agents or
ionizing radiation (IR) is one of the more effective therapy to re-
duce tumor growth. The effectiveness of the therapy lies in its
ability to damage DNA of cancer cells, resulting in an apoptotic cell
signal. Unfortunately, the DNA of normal cells is also damaged
resulting in part in the harmful side effects of these treatments.
Thus, strategies that induce DNA repair in normal cells may have
the potential to improve the overall therapeutic treatment.
inhibiting the IR-induced p53 mediated apoptosis,13 thus indirectly
inducing DNA repair. In vivo studies have validated this radiopro-
tective approach and illustrated that the Chk2 À/À mice appear
normal, fertile but resistant to IR induced apoptosis and showed in-
creased survival over the wild type mice.14
In the recent years inhibition of Chk2 has attracted great atten-
tion as a therapeutic target, however only a few Chk2 inhibitors have
been reported in the literature, which include the indolocarbazole
UCN-01,15 Gö6976,16 EXEL-9844,17 NSC109555,18,19 PV1019,12
VRX0466617,20 2-(quinazolin-2-yl)phenol (2QP) based inhibitors
like CCT241533,21,22 2-aminopyridine based inhibitors (2AP),23 the
aryl benzimidazole based inhibitors24–26 and the natural products
hymenialdisine (HMD) and debromohymenialdisine (DBH).13,27
The use of these Chk2 inhibitors for potentiating DNA-damage
agents in cancerous cells and radioprotection of normal cells has be-
come an increasingly promising strategy to improve current
chemotherapeutic regimens.
However, several studies report high variability in effectiveness
as not all inhibitors of Chk2 demonstrate similar physiological ef-
fects. For example, Chk2 inhibitor PV1019 showed potentiation of
the DNA-damaging agents, topotecan and camptothecin, in
OVCAR-4 and OVCAR-5 human tumor cells.12 However, the Chk2
inhibitor CCT241533 does not potentiate the cytotoxicity of a selec-
tion of genotoxic agents, but potentiates the selectivity of PARP
inhibitors in p53 defective cancer cell lines.21
DNA damage induced by ionizing radiation or chemotherapeu-
tics leads to the activation of DNA sensors including the ataxia tel-
angiectasia mutated (ATM) or/and ataxia telangiectasia mutated
and rad-related (ATR) kinases.1–3 These sensors activate pathways
that induce DNA repair or apoptosis. Although many cross-over
pathways are possible, activation of the ATR pathway is typically
seen following single strand DNA breaks,4 whereas ATM is activated
upon double strand breaks (DSBs),5 as seen in ionizing radiation (IR)
therapy. Following DNA DSBs, ATM is recruited to damage site,
undergoes autophosphorylation, and subsequently activates and
phosphorylates downstream effector kinases, including checkpoint
kinase 2 (Chk2).6 Chk2 plays a pivotal role in the cell cycle regula-
tion, and thus has become an attractive target in cancer therapy.
1,6–9
Inhibition of Chk2 has been postulated to significantly aug-
ment current cancer therapies in two ways.10 Firstly, some cancer-
ous cells overexpress Chk2 and require high Chk2 levels for
survival.11 Inhibition of Chk2 in those cancer types results in a
Similarly VRX046617 does not potentiate the cytotoxicity of
anticancer drugs like doxorubicin, taxol and cisplatin, but selec-
tively inhibits IR-induced Chk2-dependent degradation of Hmdx.20
⇑
Corresponding author. Tel.: +1 517 355 9715; fax: +1 517 353 1793.
E-mail address: tepe@chemistry.msu.edu (J.J. Tepe).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.054

1476
R. S. Z. Saleem et al. / Bioorg. Med. Chem. 20 (2012) 1475–1481
These studies indicate that different structural classes of Chk2
inhibitors may invoke very different physiological effects when
the cells are treated by mechanistically different classes of chemo-
therapeutics. Thus, the development of different and selective
Chk2 inhibitors can be used as tools to aid our understanding of
Chk2 mediated phenotypical responses and as potential lead
agents for pharmaceutical development.
indolic structure. The second set of modifications involved the al-
kyl substitution on the exocyclic amine of the glycocyamidine ring.
This amine is projected towards the adjacent cavity as shown in
Figure 2b. We envisioned that placing substitution at this position
will allow for additional interactions in the adjoining cavity.
2.1. Chemistry
Previously, debromohymenialdisine (DBH, 1) and our DBH-de-
rived indoloazepine (2) Chk2 inhibitor (Fig. 1) have been found
to inhibit both Chk1 and Chk2.27 We and others demonstrated that,
while DBH is a good inhibitor of Chk2, it lacked in kinase selectiv-
ity.13,27–30 However, the DBH-derived indoloazepine was found to
be more selective towards the checkpoint kinases and was found
to be a nanomolar inhibitor of Chk2 (IC50 8 nM) and Chk1 (IC50
234 nM).27 More excitingly, the DBH-derived indoloazepine in-
duced strong radioprotection of normal cells from ionizing radia-
tion, without affecting the survival of cancerous cells.13 In an
effort to elucidate the role of the Chk2 pathway involved chemo-
sensitization of cancer cells and radioprotection of normal cells,
we set out to improve the selectivity of Chk2 inhibition over
Chk1 by this potent class of radioprotecting agents. Herein, we re-
port the synthesis and evaluation of new derivatives of DBH (3–
11), with significant improvement of Chk2 over Chk1 selectivity.
Compound 12 was prepared by previously published proce-
dure.32 In order to prepare compounds 13–15 from compound 12,
we utilized the chemistry that we have reported recently (Scheme
1). Treatment of compound 12 with appropriate aryl boronic acid
yielded the respective 2-aryl aldisine 13–15, smoothly in good
yields. The aryl aldisines 13–15 were subsequently condensed with
2-(methylthio)-1H-imidazol-4(5H)-one to give aldol adducts 16–
18. Subsequent treatment of the methylthio-imidazolones 16–18
with ammonium hydroxide led to the formation of respective
DBH-analogs 3–5 in 69%, 65% and 52% yields, respectively.
The exocyclic amine on glycocyamidine ring was subsequently
modified, in order to explore the effects on Chk2 inhibition. Previ-
ous studies had already indicated that secondary amines were de-
pleted of all biological activity,13 thus only a range primary amines
were prepared in these studies. The primary exocyclic amines were
prepared by heating thio-imidazolone 16 with appropriate amine
in sealed tube, which provided the analogs 6–11 in good yields
(Scheme 2).
2. Results and discussion
The design of our modifications to the DBH-scaffold was based
on the previously reported X-ray crystal structure of DBH-bound in
the Chk2 binding pocket.31 The first set of modifications involved
the substitution of hydrogen-atom at the C2-pyrrole position of
DBH with an aryl moiety. This change will allow flexibility to the
phenyl ring to orient better inside the binding pocket (Fig. 2a), as
compared to the DBH-derived indoloazepine (Fig. 1), where phenyl
ring was fused with the pyrrole ring making the rigid and planar
2.2. Biological evaluation and structure–activity relationships
Analogs 3–11 were examined for their ability to inhibit both
Chk1 and Chk2 using a HTRF serine/threonine KinEASE assay. All
compounds were compared to their parent agent DBH and its indo-
loazepine analog 2, with the goal to improve Chk2 selectivity over
Chk1, while maintaining low nanomolar Chk2 activity. The results
H
H
N
H
N
H
N
O
N
O
OH
O
N
N
HN
H2N
NH
NH2
N
N
N
O
N
NH
HN
OCH3
NHCH3
NSC 109555
NC
Go6976
UCN-01
HO
HN
OH
NH
NH
S
NH
NH2
HN
HO
O
O
H
N
O
O
Br
N
N
N
N
H
H
HN
O
N
F
N
N
N
H
HO
CCT241533
VRX0466617
PV1019
H2N
HN
N
O
H2N
NH2
NH
N
O
H
N
O
HN
H2N
S
O
O
N
O
NH
O
NH
N
N
N
H
NH2
H
O
Cl
O
Aryl benzimidazole
2-AP based inhibitors
Debromohymenialdisine
DBH-derived
Indoloazepine, 2
1
DBH,
Figure 1. Structures of Chk2 inhibitors.

R. S. Z. Saleem et al. / Bioorg. Med. Chem. 20 (2012) 1475–1481
1477
Figure 2. Crystal structure of Chk2 with DBH in the binding pocket (pdb code: 2CN8) (a) showing the pyrrolic N-atom and C2-carbon atom (b) showing the amine of
glycocyamidine projecting into the adjacent cavity.
H N
S
2
N
N
O
S
O
HN
HN
N
O
O
O
O
HN
ArB(OH)
2
NH4OH, THF
Ar
Ar
Ar
Br
Pd(PPh3)4, Na2CO3,
EtOH, PhMe
TiCl4, py, THF
0 °C-rt
N
H
N
H
N
H
NH
NH
NH
N
H
Sealed tube,
110 °C, 24h
NH
O
O
O
H2O, 95 °C, 18h
12
13
16
3
Ar= Ph 69% ( )
Ar= Ph 72% (
)
Ar= Ph 73% (
)
Ar = 4-MeOC6H4 82% (14)
Ar= 4-MeOC6H4 49% (17)
Ar= 4-MeOC6H4 65% (4)
Ar = 3,4-(MeO)2C6H3 82% (15)
Ar= 3,4-(MeO)2C6H3 60% (18)
Ar= 3,4-(MeO)2C6H3 52% (5)
Scheme 1. Synthesis of compounds 3–5.
H
N
S
N
O
N
R
O
HN
O
HN
6
R= Bn- 78% ( )
RNH2, THF,
R= 4-MeOC6H4CH2- 62% (7)
R= Cy- 80% (8)
sealed tue
90 °C
R= EtOOCCH2CH2- 67% (9)
R= nPr- 77% (10)
N
H
NH
N
H
NH
11
)
R= Me- 74% (
O
16
Scheme 2. Synthesis of compounds 6–11.
are shown in Table 1, where the IC50 values shown are averages of a
minimum of three independent experiments, each performed in
duplicate (see Supplementary data Table SI-1 for error margins,
means and individual IC50 values). As indicated in Table 1, place-
ment of the 2-aryl-pyrole moiety led to significant gains in the
selectivity favoring Chk2, while gaining, excellent low nanomolar
potency towards Chk2. Incorporation of methoxy substituents did
not gain any significant potency over the phenyl analogue 3. The
3,4-dimethoxy substituted Chk2 inhibitor 5, achieved excellent
selectivity for Chk2 (>100-fold Chk2 over Chk1) while maintaining
excellent potency (IC50 14 nM).
Next, we evaluated the activities of various substitutions on the
exocyclic amine of the aminoimidazolone moiety. These analogs
(compounds 6–11) were subjected to the same HTRF serine/threo-
nine KinEASE assay and the results are shown in Table 2. Interest-
ingly, the alkylation on the exocyclic nitrogen (compounds 6–11)
resulted in a strong decrease of Chk1 activity compared to the free
amine (compound 3). However, the activity for Chk2 was also

1478
R. S. Z. Saleem et al. / Bioorg. Med. Chem. 20 (2012) 1475–1481
Table 1
Kinase profiling of compounds 3–5
Compound No.
Ar
IC50 in nM (std. dev.)
Fold
Chk1
Chk2
Chk1/Chk2
DBH (1)
H–
725
183
4
Indoloazepine-analog (2)
Indole
Ph–
4-MeOC6H4–
3,4-(MeO) 2C6H3–
234
8
29
66
62
111
3
4
5
1310 (475)
867 (239)
1554 (217)
20 (5)
14 (5)
14 (13)
Standard deviations of all new compounds are shown in parentheses.
Table 2
out on silica gel 60 (230–400 mesh) supplied by EM Science. Yields
refer to chromatographically and spectroscopically pure com-
pounds unless otherwise stated. Infrared spectra were recorded
on a Nicolet IR/42 spectrometer. Proton and carbon NMR spectra
were recorded on a Varian Inova-300 spectrometer or a Varian Ino-
va-500 spectrometer or Varian Inova-600 MHz. High resolution
mass spectra were obtained at the Mass Spectrometry Laboratory
Kinase profiling of compounds 6–11
Compound No.
R
IC50 in nM (std. dev.)
Fold
Chk1
Chk2
Chk1/Chk2
3
6
7
H–
Bn–
1310
20
66
32
>17
905 (38)
28 (13)
588 (72)
4-MeOC6H4CH2– >10,000
of the Michigan State University with
spectrometer.
a QTof-Ultima mass
8
9
10
11
Cy–
>10,000
>10,000
>10,000
1526 (90) >7
277 (140) >36
EtOOCCH2CH2–
nPr–
156 (46)
>64
38
Me–
6558 (1913) 171 (37)
4.2. General procedure for preparing compounds 13–15
Standard deviations of all new compounds are shown in parentheses.
Compound 12 (1 mmol) was added in a mixture of toluene and
ethanol (3:1, 40 mL) and aryl boronic acid (1.2 mmol) and tetrakis
(triphenylphosphine) palladium (5 mol %) were added to the reac-
tion mixture. Then a solution of sodium carbonate (3 mmol) in
water (3 mL) was added to the reaction mixture and the mixture
was heated to 95 °C for 18 h. Then the reaction mixture was evap-
orated and the contents were dissolved in ethyl acetate (50 mL)
and transferred to a separatory funnel. Then the contents of the
flask were washed with 10% aqueous sodium bicarbonate solution
(50 mL) and brine (50 mL). The organic layer was dried over anhy-
drous sodium sulfate (500 mg). The solvent was removed and the
crude material was purified by column chromatography (silica,
ethyl acetate) to afford product.
decreased in most cases. Simple aliphatic chains, such as seen in
compound 10, indicated good selectivity towards Chk2 over
Chk1, comparable to compound 3, yet at a lower overall potency
for Chk2.
Overall, the results indicate that unlike secondary amines, the
primary amines show a size based inhibitory ability for Chk2 and
significant depletion of Chk1 inhibitory activity. The results also
indicate that smaller substituents at this glycocyamidine amine
exhibited better IC50 values for Chk2 and as the size of the substi-
tuent increased, the IC50 values deteriorated. The compound 6
showed some deviation to the observation. Nonetheless, the fact
that these compounds show the depletion of Chk1 inhibitory activ-
ity make them attractive candidates to study the Chk2 mediated
phenotypical responses.
4.2.1. 2-Phenyl-6,7-dihydropyrrolo[2,3-c]azepine-4,8(1H,5H)-
dione (13)
1H NMR (500 MHz, DMSO-d6) d 7.90 (2H, d, J = 7.5 Hz), 7.38 (2H,
t, J = 7.5 Hz), 7.28 (1H, t, J = 7.5 Hz), 6.98 (1H, s), 3.48–3.50 (2H, m),
2.78–2.80 (2H, m); 13C NMR (125 MHz, DMSO-d6) d 194.5, 162.1,
135.3, 130.6, 128.9, 128.7, 127.6, 125.4, 124.6, 107.0, 43.7, 36.5;
IR (film): 3204, 1644, 1510, 1513, 1467, 1437, 1399, 1363; MS
(ES+) m/z: 241.1 [M+H]+; mp 222 °C; HRMS (ES+): m/z calcd for
3. Conclusion
In conclusion, we have presented the synthesis and evaluation
of a series of Chk2 inhibitors. Changing some of the substituents
on the pyrrolic region has resulted in remarkable increases in the
selectivity as compared to the natural product, debromohymeni-
aldisine (DBH), while maintaining excellent low nanomolar po-
tency. Alkylation of the exocyclic amine moiety did present good
selectivity for Chk2, but at a cost of potency. We also demonstrated
that while the secondary amines at this position lead to the loss of
the activity, the analogs with primary exocyclic amines are potent
inhibitors of Chk2. These inhibitors could also provide potential
lead to the new drug candidates, but in the mean time serve as
excellent tools to elucidate some of the complexity of the Chk1–
Chk2-mediated cell cycle regulation.
C
14H13N2O2 [M+H]+ 241.0977, found, 241.0982.
4.2.2. 2-(4-Methoxyphenyl)-6,7-dihydropyrrolo[2,3-c]azepine-
4,8(1H,5H)-dione (14)
1H NMR (500 MHz, DMSO-d6) d 7.83 (2H, d, J = 8.6 Hz), 6.94 (2H,
d, J = 8.8 Hz), 6.86 (1H, d, J = 2.4 Hz), 3.77 (3H, s), 3.35–3.38 (2H, m),
2.70–2.72 (2H, m); 13C NMR (125 MHz, DMSO-d6) d 194.5, 162.1,
158.9, 135.4, 128.3, 126.8 (s), 124.6, 123.3, 114.1 (s), 105.9 (s),
55.2 (t), 43.7 (d), 36.5 (d); IR (film): 3212, 3135, 2897, 1653,
1636, 1456, 1256 cmÀ1; MS (ES+) m/z: 271.1 [M+H]+, mp decom-
poses above 255–256 °C; HRMS (ES+) calcd for C15H15N2O3
[M+H]+ 271.1083, found 271.1085.
4. Experimental section
4.1. General methods
4.2.3. 2-(3,4-Dimethoxyphenyl)-6,7-dihydropyrrolo[2,3-
c]azepine-4,8(1H,5H)-dione (15)
1H NMR (500 MHz, DMSO-d6) d 7.57 (1H, d, J = 2.0 Hz), 7.41 (1H,
dd, J = 8.4, 2.1 Hz), 6.94 (2H, dd, J = 8.5 Hz), 6.8 (1H, d, J = 2.4 Hz),
3.84 (3H, s), 3.76 (3H, s), 3.38–3.40 (2H, m), 2.72–2.74 (2H, m);
13C NMR (125 MHz, DMSO-d6) d 194.36, 162.3, 148.8, 148.4,
All commercial reagents were used without further purification.
All solvents were reagent grade. THF was freshly distilled from so-
dium/benzophenone under nitrogen. CH2Cl2 was freshly distilled
from CaH2 under nitrogen. Column chromatography was carried

R. S. Z. Saleem et al. / Bioorg. Med. Chem. 20 (2012) 1475–1481
1479
135.7, 128.3, 124.7, 123.5, 117.9 (s), 112.0 (s), 109.2 (s), 106.2 (s),
55.7 (t), 55.5 (t), 43.7 (d), 36.6 (d); IR (film): 2923, 2849, 1653,
1644, 1636, 1491, 1468, 1256 cmÀ1; MS (ES+) m/z: 301.1 [M+H]+;
mp decomposes above 180 °C; HRMS (ES+) calcd for C16H17N2O4
[M+H]+ 301.1188, found 301.1192.
5 mL) was added to the solution. The tube was sealed and the reac-
tion mixture was heated at 90 °C for 24 h and then allowed to cool
to room temperature. Then the reaction mixture was concentrated
and the crude material was purified by column chromatography
(silica, MeOH/DCM 1:4) to afford the product.
4.3. General procedure for preparing compounds 16–18
4.4.1. (Z)-4-(2-Amino-4-oxo-1H-imidazol-5(4H)-ylidene)-2-
phenyl-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-8(1H)-one (3)
Respective compound 13–15 (1 mmol) was dissolved in THF
(25 mL) and 2-(methylthio)-1H-imidazol-4(5H)-one (2 mmol) was
added to the reaction flask. The reaction mixture was cooled to
0 °C and 1 M solution of TiCl4 in DCM (4 mmol) was added to the
reaction mixture in drop-wise manner. The reaction mixture was
stirred for 30 min and pyridine (8 mmol) was added to the reaction
mixture dropwise over 15 min. The reaction mixture was stirred for
an additional 14 h allowing it to gradually warm to room tempera-
ture. At this point saturated NH4Cl solution (40 mL) was added to
the reaction mixture and contents of the flask were transferred to
the separatory funnel. Then the crude product was extracted with
ethyl acetate (50 mL Â 3). The ethyl acetate fractions were com-
bined and dried over anhydrous Na2SO4 (500 mg). The solvent
was removed and the crude material was purified by column chro-
matography (silica, EtOAc) to afford product.
1H NMR (500 MHz, DMSO-d6 + CF3COOH)
d 7.89 (2H, d,
J = 7.4 Hz), 7.38 (2H, t, J = 7.4 Hz), 7.27 (1H, t, J = 7.4 Hz), 6.86
(1H, s), 3.29 (4H, br r); 13C NMR (125 MHz, DMSO-d6 + CF3COOH)
d 163.7, 163.5, 154.6, 135.9, 131.1, 130.2, 128.9 (s), 128.2 (s),
127.8, 125.8 (s), 121.6, 121.0, 107.6 (s), 39.2 (d), 33.2 (d); IR (film):
2915, 2857, 2444, 1697, 1683, 1650, 1634, 1621, 1607, 1578, 1560,
1542, 1509, 1470, 1456 cmÀ1; MS (ES+) m/z: 322.1 [M+H]+; mp
decomposed above 250 °C; HRMS (ES+) m/z calcd for C17H16N5O2
[M+H]+ 322.1310, found 322.1304.
4.4.2. (Z)-4-(2-Amino-4-oxo-1H-imidazol-5(4H)-ylidene)-2-(4-
methoxyphenyl)-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-8(1H)-
one (4)
1H NMR (500 MHz, DMSO-d6 + drop of CF3COOH) d 7.84 (2H, d,
J = 8.5 Hz), 6.96 (2H, d, J = 8.5 Hz), 6.76 (1H, s), 3.77 (3H, s), 3.29
(4H, br r); 13C NMR (125 MHz, DMSO-d6 + drop of CF3COOH) d
163.7, 163.6s, 154.7, 136.1, 130.6, 127.8, 127.4 (s), 126.9, 123.9,
121.7, 120.9, 114.5 (s), 106.6 (s), 55.6 (t), 39.6 (d), 33.2 (d); IR
(film): 2995, 2935, 1696, 1684, 1653, 1636, 1617, 1559, 1539,
1491, 1456, 1437, 1385, 1260, 1206, 1138 cmÀ1; MS (ES+) m/z:
352.1 [M+H]+; mp decomposes above 250 °C; HRMS (ES+) calcd
for C18H18N5O3 [M+H]+ 352.1410, found 352.1412.
4.3.1. (Z)-4-(2-(Methylthio)-4-oxo-1H-imidazol-5(4H)-ylidene)-
2-phenyl-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-8(1H)-one (16)
1H NMR (500 MHz, DMSO-d6) d 8.01 (1H, s), 7.82 (2H, d,
J = 7.5 Hz), 7.26 (1H, t, J = 7.5 Hz), 3.44–3.46 (2H, m), 3.27–3.28
(2H, m), 2.65 (3H, s); 13C NMR (125 MHz, DMSO-d6) d 170.6,
162.8, 158.3, 135.4, 134.3, 133.6, 131.3, 128.7, 128.4, 127.1 (s),
125.0 (s), 124.0, 111.8 (s), 39.1 (d), 30.3 (d), 12.1 (t); IR (film):
3184, 3046, 2929, 1691, 1658, 1623, 1605, 1508, 1470, 1436,
1411 cmÀ1; MS (ES+) m/z: 353.1 [M+H]+; mp decomposed over
250 °C; HRMS (ES+) m/z calcd for C18H17N4O2S [M+H]+ 353.1072,
found, 353.1082.
4.4.3. (Z)-4-(2-Amino-4-oxo-1H-imidazol-5(4H)-ylidene)-2-(3,4-
dimethoxyphenyl)-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-81H)
-one (5)
1H NMR (600 MHz, DMSO-d6 + drop of CF3COOH) d 7.57 (1H, d,
J = 2.2 Hz), 7.41 (1H, dd, J = 8.3, 2.0 Hz), 6.96 (1H, d, J = 8.5 Hz), 6.77
(1H, d, J = 2.4 Hz), 3.84 (3H, s), 3.77 (3H, s), 3.31 (4H, br r); 13C NMR
(150 MHz, DMSO-d6 + drop of CF3COOH) d 163.5, 163.2, 154.4,
149.1, 148.7, 136.0, 130.4, 127.4, 123.9, 121.4, 120.6, 118.2 (s),
112.4 (s), 109.7 (s), 106.4 (s), 55.8 (t), 55.7 (t), 39.2 (d), 32.9 (d);
IR (film): 2921, 2851, 1701, 1684, 1653, 1558, 1489, 1473, 1456,
1258, 1204, 1181 cmÀ1; MS (ES+) m/z: 382.2 [M+H]+; mp decom-
poses above 250 °C; HRMS (ES+) calcd for C19H20N5O4 [M+H]+
382.1515, found 382.1526.
4.3.2. (Z)-2-(4-Methoxyphenyl)-4-(2-(methylthio)-4-oxo-1H-
imidazol-5(4H)-ylidene)-4,5,6,7-tetrahydropyrrolo[2,3-
c]azepin-8(1H)-one (17)
1H NMR (500 MHz, DMSO-d6) d 7.90 (1H, s), 7.76 (2H, d,
J = 8.3 Hz,), 6.95 (2H, d, J = 8.3 Hz), 3.77 (3H, s), 3.42–3.45 (2H,
m), 3.25–3.27 (2H, m), 2.4 (3H, s); 13C NMR (125 MHz; DMSO-d6)
d 170.6, 162.8, 158.6, 135.5, 134.4, 133.5, 131.5, 127.8, 126.4 (s),
124.0, 123.9, 114.2 (s), 110.7 (s), 55.1 (t), 39.2 (d), 30.3 (d), 12.2
(t); IR (film): 2980, 2910, 1676, 1632, 1588, 1478, 1456, 1435,
1252, 1179 cmÀ1; MS (ES+) m/z: 383.1 [M+H]+; mp decomposes
4.5. General procedure for preparing compounds 6–11
above 240 °C; HRMS (ES+) calcd for
C
19H19N4O3S [M+H]+
383.1178; found 383.1185
Compound 16 (1 mmol) was added to THF (5 mL) in a sealable
tube and appropriate alkylamine (4 mmol) was added to the solu-
tion. The tube was sealed and the reaction mixture was heated at
90 °C for 24 h and then allowed to cool to room temperature. Then
the reaction mixture was concentrated and the crude material was
purified by column chromatography (silica, MeOH/DCM 1:9) to af-
ford the product.
4.3.3. (Z)-2-(3,4-Dimethoxyphenyl)-4-(2-(methylthio)-4-oxo-
1H-imidazol-5(4H)-ylidene)-4,5,6,7-tetrahydropyrrolo[2,3-c]
azepin-8(1H)-one (18)
1H NMR (500 MHz, DMSO-d6) d 7.99 (1H, d, J = 2.9 Hz), 7.51 (1H,
d, J = 2.0 Hz), 7.33 (1H, dd, J = 8.3, 2.0 Hz), 6.98 (1H, d, J = 8.6 Hz),
3.77 (3H, s), 3.83 (3H, s), 3.43–3.45 (2H, m), 3.26–3.28 (2H, m),
2.66 (3H, s); 13C NMR (125 MHz, DMSO-d6) d 170.6, 162.9, 158.1,
148.8, 148.1, 135.5, 134.6, 133.4, 127.8, 124.1, 124.0, 117.2 (s),
112.0 (s), 111.0 (s), 108.8 (s), 55.5 (t), 55.4 (t), 39.2 (d), 30.2 (d),
12.3 (t); IR (film): 2921, 2860, 1686, 1678, 1653, 1636, 1507,
1487, 1456, 1437, 1385, 1248, 1184, 1136 cmÀ1; MS (ES+) m/z:
413.2 [M+H]+; mp decomposes above 250 °C; HRMS (ES+) calcd
for C20H21N4O4S [M+H]+ 413.1284, found 413.1293.
4.5.1. (Z)-4-(2-(Benzylamino)-4-oxo-1H-imidazol-5(4H)-
ylidene)-2-phenyl-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-
8(1H)-one (6)
1H NMR (600 MHz, DMSO-d6 + drop of CF3COOH) d 7.86 (2H, d,
J = 7.8 Hz), 7.35–7.39 (5H, m), 7.25–7.30 (2H, m), 6.87 (1H, s), 4.59
(2H, br r), 3.31 (4H, br r); 13C NMR (150 MHz, DMSO-d6 + drop of
CF3COOH) d 163.8, 163.7, 154.0, 136.5, 136.0, 131.1, 130.6, 128.9
(s), 128.4, 128.1 (s), 127.9 (s), 127.7 (s), 127.6, 125.8 (s), 121.7,
121.0, 107.7 (s), 46.3 (d), 39.2 (d), 33.2 (d); IR (film): 3077, 2924,
2800, 1690, 1680, 1640, 1489, 1427, 1200, 1136 cmÀ1; MS (ES+)
m/z: 412.2 [M+H]+; mp decomposes above 220–222 °C HRMS
(ES+) calcd for C24H22N5O2 [M+H]+ 412.1774, found 412.1778.
4.4. General procedure for preparing compounds 3–5
The methylthio-imidazolone precursor 16–18 (0.32 mmol) was
added to THF (5 mL) in a sealable tube and ammonia solution (30%,

1480
R. S. Z. Saleem et al. / Bioorg. Med. Chem. 20 (2012) 1475–1481
4.5.2. (Z)-4-(2-((4-Methoxybenzyl)amino)-4-oxo-1H-imidazol-
5(4H)-ylidene)-2-phenyl-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin
-8(1H)-one (7)
120.8, 107.5 (s), 39.1 (d), 32.8 (d), 29.6 (t); IR (film): 2975, 2920,
1701, 1684, 1635, 1617, 1676, 1559, 1437, 1385, 1265, 1198,
1191, 1128 cmÀ1; MS (ES+) m/z: 336.1 [M+H]+; mp 240-242 °C;
HRMS (ES+) calcd for
336.1464.
1H NMR (600 MHz, DMSO-d6 + drop of CF3COOH) d 7.88 (2H, d,
J = 7.6 Hz), 7.36–7.40 (2H, m), 7.26–7.32 (3H, m), 6.92–6.96 (2H,
m), 6.87 (1H, s), 4.52 (2H, d, J = 5.4 Hz), 3.87 (1H, s), 3.73 (3H, s),
3.31 (4H, br r); 13C NMR (150 MHz, DMSO-d6 + drop of CF3COOH)
d 163.7, 163.4, 159.2, 153.6, 135.7, 130.9, 130.2, 129.1 (s), 129.0,
128.6 (s), 128.1, 127.7 (s), 125.6 (s), 121.4, 120.7, 114.4 (s), 107.5
(s), 55.2 (t), 45.6 (d), 39.3 (d), 32.9 (d); IR (film): 2922, 2849,
1696, 1680, 1634, 1516, 1476, 1433, 1203, 1180, 1132 cmÀ1; MS
(ES+) m/z: 442.2 [M+H]+; mp decomposes above 250 °C; HRMS
(ES+) calcd for C25H24N5O3 [M+H]+ 442.1879, found 442.1880.
C
18H18N5O2 [M+H]+ 336.1461, found
4.6. Biological methods
The HTRF KinEASE STK1 kit (from Cisbio/Millipore) was used to
evaluate the serine/threonine kinase activity of Chk1 and Chk2
according to the manufacturer’s instructions. In short, Chk1 and
Chk2 activity was analyzed in a white 96-well half volume plate
in a final reaction volume of 50 lL, human Chk1 or Chk2 (5–
10 m U) was incubated with various concentrations of test agent
or vehicle (a final DMSO vehicle concentration of 0.2%), STK1 sub-
4.5.3. (Z)-4-(2-(Cyclohexylamino)-4-oxo-1H-imidazol-5(4H)-
ylidene)-2-phenyl-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-
8(1H)-one (8)
strate (50 nM for Chk1 and 1
supplemented with 5 mM MgCl2 and 1 mM DTT. The kinase reac-
tion was initiated with the addition of 100 M ATP. After incuba-
tion for 10 min at 30 °C, the reaction was stopped by the
addition of 25 L Sa-XL665 and 25 L STK Antibody-Eu(K) in EDTA.
lM for Chk2) in 50 mM HEPES pH 7.0
1H NMR (500 MHz, CD3OD + drop of CF3COOH, 50 °C) d 7.75 (2H,
d, J = 7.6 Hz), 7.42 (2H, t, J = 7.6 Hz), 7.33 (1H, t, J = 7.6 Hz), 6.89
(1H, s), 3.46 (4H, br r), 1.98 (2H, br r), 1.80 (2H, br r r), 1.66 (1H,
d, J = 12.9 Hz), 1.41–1.47 (4H, m), 1.28 (1H, br r); 13C NMR
l
l
l
The plate was sealed and incubated for 1 h at room temperature.
The resulting TR-FRET signal was measured on a SpectaMaxM5e
plate reader. The fluorescence emission was measured at 620 nm
(cryptate) and 665 nm (XL665). A ratio was calculated (665/620)
for each well and the results were expressed as follows: specific
signal = ratio (sample)Àratio (negative control), where ra-
tio = (665 nm/620 nm) Â 104.
(125 MHz, CD3OD + drop of CF3COOH, 50 °C)
d 166.0, 164.1,
160.3, 154.0, 138.5, 132.4, 132.1, 130.0, 129.2, 128.3 (s), 126.5
(s), 124.0 (s), 105.7 (s) 54.4, 40.9 (d), 33.5 (d), 32.0 (d), 26.0 (d),
25.3 (d); IR (film): 2930, 2855, 2800, 1727, 1700, 1676, 1615,
1663, 1516, 1466, 1450, 1205, 1181, 1136 cmÀ1; MS (ES+) m/z:
404.2 [M+H]+; mp decomposes above 250 °C; HRMS (ES+) calcd
for C23H26N5O2 [M+H]+ 404.20887, found 404.2088.
Acknowledgments
4.5.4. (Z)-Ethyl 3-((4-oxo-5-(8-oxo-2-phenyl-5,6,7,8-tetra
hydropyrrolo[2,3-c]azepin-4(1H)-ylidene)-4,5-dihydro-1H-
imidazol-2-yl)amino)propanoate (9)
The authors gratefully acknowledge financial support in part
from Michigan State University and the Fulbright Scholarship Pro-
gram for funding for this work.
1H NMR (500 MHz, DMSO-d6 + drop of CF3COOH) d 7.88 (2H, d,
J = 7.6 Hz), 7.39 (2H, t, J = 7.6 Hz), 7.29 (1H, t, J = 7.6 Hz), 6.84 (1H,
br r), 4.08 (2H, q, J = 7.1 Hz), 3.59 (2H, br r), 3.31 (4H, br r), 2.71–
2.65 (2H, m), 1.18 (3H, t, J = 7.1 Hz); 13C NMR (125 MHz, DMSO-
d6 + drop of CF3COOH) d 171.0, 163.6, 163.5, 153.9, 135.8, 131.0,
130.2, 128.8 (s), 128.2, 127.8 (s), 125.7 (s), 121.4, 120.7, 107.3 (s),
60.5 (d), 39.9 (d), 39.8 (d), 33.3 (d), 33.1 (d), 14.1 (t); IR (film):
3222, 2921, 2993, 1728, 1717, 1696, 1686, 1653, 1636, 1617,
1203, 1138 cmÀ1; MS (ES+) m/z: 422.2 [M+H]+; mp 227–229 °C;
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.12.054.
References and notes
HRMS (ES+) calcd for
C
22H24N5O4 [M+H]+ 422.1828, found
1. Smith, J.; Tho, L. M.; Xu, N.; Gillespie, D. A. Adv. Cancer Res. 2010, 108, 73.
2. Kao, J.; Rosenstein, B. S.; Peters, S.; Milano, M. T.; Kron, S. J. NY Ann. Acad. Sci.
2006, 1066, 243.
422.1831.
3. Abraham, R. T. Genes Dev. 2001, 15, 2177.
4. Zou, L.; Elledge, S. J. Science 2003, 300, 1542.
5. Lee, J. H.; Paull, T. T. Science 2005, 308, 551.
6. Bartek, J.; Lukas, J. Cancer Cell 2003, 3, 421.
4.5.5. (Z)-4-(4-Oxo-2-(propylamino)-1H-imidazol-5(4H)-
ylidene)-2-phenyl-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-
8(1H)-one (10)
7. Zhou, B. S.; Bartek, J. Nat. Rev. Cancer 2004, 4, 216.
1H NMR (600 MHz, DMSO-d6 + drop of CF3COOH) d 7.88 (2H, d,
J = 7.6 Hz), 7.39 (2H, t, J = 7.7 Hz), 7.28 (1H, t, J = 7.7 Hz), 6.86 (1H,
d, J = 2.2 Hz), 3.22–3.36 (6H, m), 1.49–1.58 (2H, m), 0.88 (3H, t,
J = 7.3 Hz); 13C NMR (150 MHz, DMSO-d6 + drop of CF3COOH) d
163.7, 163.4, 153.6, 135.7, 130.9, 129.7, 128.7 (s), 128.0, 127.6
(s), 125.6 (s), 121.4, 120.7, 107.4 (s), 44.5 (d), 39.2 (d), 32.9 (d),
22.3 (d), 10.7 (t); IR (film): 2920, 1700, 1684, 1635, 1472, 1435,
1385, 1264, 1206, 1132 cmÀ1; MS (ES+) m/z: 364.2 [M+H]+; mp
decomposes above 270 °C; HRMS (ES+) calcd for C20H22N5O2
[M+H]+ 364.1774, found 364.1777.
8. Bartkova, J.; Horejsi, Z.; Koed, K.; Kramer, A.; Tort, F.; Zieger, K.; Guldberg, P.;
Sehested, M.; Nesland, J. M.; Lukas, C.; Orntoft, T.; Lukas, J.; Bartek, J. Nature
2005, 434, 864.
9. Nguyen, T. N.; Tepe, J. J. Curr. Med. Chem. 2009, 16, 3122.
10. Pommier, Y.; Weinstein, J. N.; Aladjem, M. I.; Kohn, K. W. Clin. Cancer. Res. 2006,
12, 2657.
11. Zoppoli, G.; Solier, S.; Reinhold, W. C.; Liu, H.; Connelly, J. W., Jr.; Monks, A.;
Shoemaker, R. H.; Abaan, O. D.; Davis, S. R.; Meltzer, P. S.; Doroshow, J. H.;
Pommier, Y. Oncogene 2011, 1.
12. Jobson, A. G.; Lountos, G. T.; Lorenzi, P. L.; Llamas, J.; Connelly, J.; Cerna, D.;
Tropea, J. E.; Onda, A.; Zoppoli, G.; Kondapaka, S.; Zhang, G.; Caplen, N. J.;
Cardellina, J. H., 2nd; Yoo, S. S.; Monks, A.; Self, C.; Waugh, D. S.; Shoemaker, R.
H.; Pommier, Y. J. Pharmacol. Exp. Ther. 2009, 331, 816.
13. Nguyen, T. N.; Saleem, R. S.; Luderer, M. J.; Hovde, S.; Henry, R. W.; Tepe, J. J.
ACS Chem. Biol. 2011. doi: 10.1021/cb200320c.
14. Takai, H.; Naka, K.; Okada, Y.; Watanabe, M.; Harada, N.; Saito, S.; Anderson, C.
W.; Appella, E.; Nakanishi, M.; Suzuki, H.; Nagashima, K.; Sawa, H.; Ikeda, K.;
Motoyama, N. EMBO J. 2002, 21, 5195.
15. Yu, Q.; La Rose, J.; Zhang, H.; Takemura, H.; Kohn, K. W.; Pommier, Y. Cancer
Res. 2002, 62, 5743.
16. Kohn, E. A.; Yoo, C. J.; Eastman, A. Cancer Res. 2003, 63, 31.
17. Matthews, D. J.; Yakes, F. M.; Chen, J.; Tadano, M.; Bornheim, L.; Clary, D. O.;
Tai, A.; Wagner, J. M.; Miller, N.; Kim, Y. D.; Robertson, S.; Murray, L.; Karnitz, L.
M. Cell Cycle 2007, 6, 104.
4.5.6. (Z)-4-(2-(Methylamino)-4-oxo-1H-imidazol-5(4H)-
ylidene)-2-phenyl-4,5,6,7-tetrahydropyrrolo[2,3-c]azepin-
8(1H)-one (11)
1H NMR (500 MHz, DMSO-d6 + drop of CF3COOH) d 7.89 (2H, d,
J = 7.8 Hz), 7.37–7.41 (2H, t, J = 7.8 Hz), 7.22 (1H, t, J = 7.8), 6.85
(1H, s), 3.31 (4H, br r), 2.97 (3H, d, J = 4.6 Hz); 13C NMR
(125 MHz, DMSO-d6 + drop of CF3COOH) d 163.6, 163.3, 154.3,
135.6, 130.9, 129.7, 128.7 (s), 128.1, 127.6 (s), 125.6 (s), 121.4,

R. S. Z. Saleem et al. / Bioorg. Med. Chem. 20 (2012) 1475–1481
1481
18. Lountos, G. T.; Tropea, J. E.; Zhang, D.; Jobson, A. G.; Pommier, Y.; Shoemaker, R.
H.; Waugh, D. S. Protein Sci. 2009, 18, 92.
25. McClure, K. J.; Huang, L.; Arienti, K. L.; Axe, F. U.; Brunmark, A.; Blevitt, J.;
Breitenbucher, J. G. Bioorg. Med. Chem. Lett. 2006, 16, 1924.
19. Jobson, A. G.; Cardellina, J. H., 2nd; Scudiero, D.; Kondapaka, S.; Zhang, H.; Kim,
H.; Shoemaker, R.; Pommier, Y. Mol. Pharmacol. 2007, 72, 876.
20. Carlessi, L.; Buscemi, G.; Larson, G.; Hong, Z.; Wu, J. Z.; Delia, D. Mol. Cancer
Ther. 2007, 6, 935.
21. Anderson, V. E.; Walton, M. I.; Eve, P. D.; Boxall, K. J.; Antoni, L.; Caldwell, J. J.;
Aherne, W.; Pearl, L. H.; Oliver, A. W.; Collins, I.; Garrett, M. D. Cancer Res. 2011,
71, 463.
26. Neff, D. K.; Lee-Dutra, A.; Blevitt, J. M.; Axe, F. U.; Hack, M. D.; Buma, J. C.;
Rynberg, R.; Brunmark, A.; Karlsson, L.; Breitenbucher, J. G. Bioorg. Med. Chem.
Lett. 2007, 17, 6467.
27. Sharma, V.; Tepe, J. J. Bioorg. Med. Chem. Let. 2004, 14, 4319.
28. Tasdemir, D.; Mallon, R.; Greenstein, M.; Feldberg, L. R.; Kim, S. C.; Collins, K.;
Wojciechowicz, D.; Mangalindan, G. C.; Concepcion, G. P.; Harper, M. K.;
Ireland, C. M. J. Med. Chem. 2002, 45, 529.
22. Caldwell, J. J.; Welsh, E. J.; Matijssen, C.; Anderson, V. E.; Antoni, L.; Boxall, K.;
Urban, F.; Hayes, A.; Raynaud, F. I.; Rigoreau, L. J.; Raynham, T.; Aherne, G. W.;
Pearl, L. H.; Oliver, A. W.; Garrett, M. D.; Collins, I. J. Med. Chem. 2011, 54, 580.
23. Hilton, S.; Naud, S.; Caldwell, J. J.; Boxall, K.; Burns, S.; Anderson, V. E.; Antoni,
L.; Allen, C. E.; Pearl, L. H.; Oliver, A. W.; Wynne Aherne, G.; Garrett, M. D.;
Collins, I. Bioorg. Med. Chem. 2010, 18, 707.
29. Curman, D.; Cinel, B.; Williams, D. E.; Rundle, N.; Block, W. D.; Goodarzi, A. A.;
Hutchins, J. R.; Clarke, P. R.; Zhou, B. B.; Lees-Miller, S. P.; Andersen, R. J.;
Roberge, M. J. Biol. Chem. 2001, 276, 17914.
30. Meijer, L.; Thunnissen, A. M.; White, A. W.; Garnier, M.; Nikolic, M.; Tsai, L. H.;
Walter, J.; Cleverley, K. E.; Salinas, P. C.; Wu, Y. Z.; Biernat, J.; Mandelkow, E. M.;
Kim, S. H.; Pettit, G. R. Chem. Biol. 2000, 7, 51.
24. Arienti, K. L.; Brunmark, A.; Axe, F. U.; McClure, K.; Lee, A.; Blevitt, J.; Neff, D. K.;
Huang, L.; Crawford, S.; Pandit, C. R.; Karlsson, L.; Breitenbucher, J. G. J. Med.
Chem. 2005, 48, 1873.
31. Oliver, A. W.; Paul, A.; Boxall, K. J.; Barrie, S. E.; Aherne, G. W.; Garrett, M. D.;
Mittnacht, S.; Pearl, L. H. EMBO J. 2006, 25, 3179.
32. Annoura, H.; Tatsuoka, T. Tetrahedron Lett. 1995, 36, 413.