Structure-based design, synthesis, and biological evaluation of potent and selective macrocyclic checkpoint kinase 1 inhibitors

Article abstract of DOI:10.1021/jm061247v

Based on the crystallographic analysis of a urea-checkpoint kinase 1 (Chk1) complex and molecular modeling, a class of macrocyclic Chk1 inhibitors were designed and their biological activities were evaluated. An efficient synthetic methodology for macrocyclic ureas was developed with Grubbs metathesis macrocyclization as the key step. The structure-activity relationship studies demonstrated that the macrocyclization retains full Chk1 inhibition activity and that the 4-position of the phenyl ring can tolerate a wide variety of substituents. These novel Chk1 inhibitors exhibit excellent selectivity over a panel of more than 70 kinases. Compounds 5b, 5c, 5f, 15, 16d, 17g, 17h, 17k, 18d, and 22 were identified as ideal Chk1 inhibitors, which showed little or no single-agent activity but significantly potentiate the cytotoxicities of the DNA-damaging antitumor agents doxorubicin and camptothecin. These novel Chk1 inhibitors abrogate the doxorubicin-induced G2 and camptothecin-induced S checkpoint arrests, confirming that their potent biological activities are mechanism-based through Chk1 inhibition.

Full text of DOI:10.1021/jm061247v

1514
J. Med. Chem. 2007, 50, 1514-1527
Structure-Based Design, Synthesis, and Biological Evaluation of Potent and Selective
Macrocyclic Checkpoint Kinase 1 Inhibitors
Zhi-Fu Tao,* Le Wang, Kent D. Stewart, Zehan Chen, Wendy Gu, Mai-Ha Bui, Philip Merta, Haiying Zhang, Peter Kovar,
Eric Johnson, Chang Park, Russell Judge, Saul Rosenberg, Thomas Sowin, and Nan-Horng Lin
Cancer Research, Global Pharmaceutical Research and DeVelopment, Abbott Laboratories, Abbott Park, Illinois 60064
ReceiVed October 23, 2006
Based on the crystallographic analysis of a urea-checkpoint kinase 1 (Chk1) complex and molecular
modeling, a class of macrocyclic Chk1 inhibitors were designed and their biological activities were evaluated.
An efficient synthetic methodology for macrocyclic ureas was developed with Grubbs metathesis
macrocyclization as the key step. The structure-activity relationship studies demonstrated that the
macrocyclization retains full Chk1 inhibition activity and that the 4-position of the phenyl ring can tolerate
a wide variety of substituents. These novel Chk1 inhibitors exhibit excellent selectivity over a panel of
more than 70 kinases. Compounds 5b, 5c, 5f, 15, 16d, 17g, 17h, 17k, 18d, and 22 were identified as ideal
Chk1 inhibitors, which showed little or no single-agent activity but significantly potentiate the cytotoxicities
of the DNA-damaging antitumor agents doxorubicin and camptothecin. These novel Chk1 inhibitors abrogate
the doxorubicin-induced G2 and camptothecin-induced S checkpoint arrests, confirming that their potent
biological activities are mechanism-based through Chk1 inhibition.
Introduction
phatases (Cdc25A, B, and C) and upregulating Wee1, thereby
inhibiting progression into mitosis.10 The inhibition of Chk 1
abrogates the S and G2 checkpoints, thereby preferentially
sensitizing tumor cells, especially p53-null cells, to various DNA
damaging agents. Downregulation of Chk1 by siRNA or
antisense abrogates DNA-damaging agent-induced G2/S arrest
and sensitizes tumor cells to topoisomerase inhibitors, anti-
metabolite anticancer agents, and radiation.10-14 Consequently,
Chk1 has emerged as an attractive chemosensitization target,
and the inhibition of Chk1 may significantly improve the
efficacy and selectivity of DNA-damaging agents in the
clinic.3,15-18
DNA-damaging agents constitute a major class of anticancer
therapeutics and have significantly contributed to the survival
increases of cancer patients.1 However, the clinical use of DNA-
damaging anticancer agents is limited by their severe toxicity
and by resistance from tumor cells, especially p53-deficient
tumor cells. While there has been much interest in identifying
more selective and effective agents that target DNA and its
associated processes,1 the development of adjuvant therapeutics
that improve the efficacy and selectivity of DNA-damaging
agents in the clinic has recently attracted particular attention.
Such treatments may either sensitize tumor tissue or protect
normal tissue from DNA damage. A promising approach to these
treatments is the targeting of specific differential biological
pathways in tumors such as DNA-damage-response pathways.2-7
With DNA damage, cells are arrested (G1, S, G2) to initiate
the DNA repair process.2-4 If cells progress into mitosis with
damaged DNA, they will undergo mitotic catastrophe and
eventually apoptosis. Most tumor cells distinguish themselves
from normal cells by lacking the G1 checkpoint due to loss of
p53, and therefore they are selectively arrested at the S or G2
checkpoint after DNA damage. If the S and G2 checkpoints
are abrogated, G1-deficient cancer cells will not arrest to repair
damaged DNA and will enter mitosis, which results in premature
chromosome condensation and leads to cell death. In contrast,
normal cells are still arrested in the G1 phase and are less
affected by S and G2 checkpoint abrogation, suggesting that a
favorable therapeutic window may be achieved for G2 and/or
S abrogators.
An ideal Chk1 inhibitor should show no single-agent activity
and should significantly potentiate DNA-damaging antitumor
agents. To date, several classes of Chk1 inhibitors have been
reported,18 including indolocarbazoles such as 7-hydroxystau-
rosporine (UCN-01),19 isogranulatimide,20 debromohymenial-
disine,21 aminopyrimidine,22 pyrrolopyridines,23 indolinones,24
and benzimidazole-quinolinones.25 UCN-01 is a potent Chk1
inhibitor and has been extensively studied. It abrogates both
the S and G2 checkpoints and sensitizes tumor cells to a wide
Checkpoint kinase 1 (Chk 1)a is a serine/threonine protein
kinase and a key mediator in the DNA damage-induced
checkpoint network.5-7 In response to DNA damage, ATM and
ATR kinases activate Chk1 through phosphorylation at Ser317
and Ser345 in the SQ/TQ motif,8,9 and Chk1 in turn inactivates
Cdc2/cyclinB by downregulating the Cdc25 family of phos-
a Abbreviations: APCI, atmospheric pressure chemical ionization; ATM,
ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3 related;
BSA, bovine serum albumin; Chk1, checkpoint kinase 1; CPT, camptoth-
ecin; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; Dox,
doxorubicin; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid;
FACS, fluorescence-activated cell sorting; Hepes, N-(2-hydroxyethyl)-
piperazine-N′-2-ethanesulfonic acid; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium; SAR, structure-
activity relationship; siRNA, small interfering ribonucleic acid; THF,
tetrahydrofuran; UCN-01, 7-hydroxystaurosporine.
* Corresponding author: AP10, R47B, Cancer Research, Abbott Labo-
ratories, Abbott Park, IL 60064; tel (847)-938-6772; fax (847)-935-5165;
e-mail Zhi-Fu.Tao@abbott.com.
10.1021/jm061247v CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/13/2007

Macrocyclic Checkpoint Kinase 1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1515
Figure 1. X-ray crystal structure of compound 4 (green carbon atoms)
bound to Chk1 kinase (brown carbon atoms with brown protein surface).
Hydrogen bonds are indicated with black dotted lines. Glu85 and Cys87
of the hinge and Glu91 of the ribose region are indicated. The side
chain of hinge residue Tyr86 is not displayed.
Figure 2. X-ray crystal structure of macrocyclic urea 5a bound to
Chk1 kinase. Hydrogen bonds are indicated with black dotted lines.
Glu85 and Cys87 of the hinge and Glu91 of the ribose region are
indicated. The side chain of hinge residue Tyr86 is not displayed.
Scheme 1a
spectrum of DNA-damaging agents.26 However, UCN-01 also
potently inhibits a number of other kinases and exhibits strong
single-agent activity, and it has high human plasma protein
binding affinity,27 which could limit its use in the clinic. We
recently reported two potent and selective urea-based Chk1
inhibitors, 1 and 2.28,29 Both compounds abrogate the cell cycle
checkpoints and significantly potentiate the cytotoxicity of
topoisomerase inhibitors. More importantly, these urea-based
Chk1 inhibitors preferentially sensitize p53-deficient tumor cells
to DNA-damaging agents.28 The urea pharmacophore has been
a very popular scaffold for kinase inhibitors30 and has been well
covered in the patent literature, and diaryl ureas have been
specifically claimed as Chk1 inhibitors in several patents.31-34
The work presented here further explores the urea class of Chk1
kinase inhibitors in the context of a macrocyclic ring system.35,36
a Reaction conditions: (a) NaH, 1-dimethylaminopropan-2-ol, dioxane,
100 °C, overnight, 83%; (b) 1-chloro-3-isocyanatobenzenes, toluene, 120
°C, 50-70%.
urea 5a (IC50 ) 10 nM) was fully retained compared with 4
and dramatically improved, over 440-fold, compared with its
synthetic acyclic urea precursor 6. The X-ray cocrystal structure
of 5a (Figure 2) confirmed that the side chain linking the C2-
position of phenyl ring to the C6′ substituent of the pyrazine
ring lies in the vicinity of the ribose pocket as predicted from
modeling studies.
Design
Chemistry
Previous SAR studies of urea-based Chk1 inhibitors disclosed
that a variety of substituents are well tolerated at the C2-position
of the phenyl ring.37,38 X-ray crystallographic analysis and
molecular modeling showed that both the 2-position of the
phenyl ring and the C6′-position of the pyrazinyl ring point
toward the vicinity of ribose pocket, indicating the C6′-position
is an ideal site for further improvement.38 This was confirmed
by the synthesis of C6′-substituted ureas 3 and 4. Both 3 (IC50
) 26 nM) and 4 (IC50 ) 22 nM) possess potent inhibitory
activity against Chk1. An X-ray cocrystal structure of a Chk1-4
complex showed that the urea backbone forms complementary
H-bonds with Cys87 and Glu85 in the hinge region (Figure 1;
PDF ID: 2E9P). The substituents at the C6′- and C2-positions
stretch into the ribose pocket, and their ends are essentially
proximal. Importantly, the bound urea conformation showed that
the urea amide bonds were in one cis and one trans orientation
(Figure 1). This places vectors from positions 2 and 6′ pointing
toward one another. We reasoned that potency might be
improved by connecting these ribose pocket substituents in a
macrocycle, as it has been well documented that restriction of
conformation through macrocyclization can produce potent
inhibitors.39 Indeed, the Chk1 inhibition activity of macrocyclic
The synthesis of the acyclic ureas is outlined in Scheme 1.
The nucleophilic displacement of chloride from 7 smoothly gave
the aminopyrazinyl ether 8, which was coupled with chlorophen-
yl isocyanates to give compounds 3 and 4 in moderate to good
yield.
The synthesis of macrocycle 5a is shown in Scheme 2.
Alkylation of the commercially available aminophenol 9 by allyl
bromide gave 10, which was treated with phosgene in toluene
to generate isocyanate 11 in very good yield. Aminopyrazinyl
ether 13, prepared through the nucleophilic displacement of
chloride from 12 by 3-butenol, was coupled to 11 to give the
key intermediate 6 in moderate yield. The macrocylization of
6 was achieved under a condition of Grubbs olefin metathesis
ring-closure. Saturation of the cyclic olefin 14 provided 5a.
Macrocyclic ureas 5b-f (Table 1) were prepared by synthetic
procedures similar to that described for 5a.
Scheme 3 outlines the synthesis of macrocyclic ureas in which
a substituent is attached to the phenyl ring by an ether linkage.
Removal of the SEM group from the 4-position of 5e released
the hydroxyl group in quantitative yield. Alkylation of the
hydroxyl group by Mitsunobu reaction under various conditions

1516 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Tao et al.
Scheme 2a
Table 2. Structure-Activity Relationship of Ether Analogues at the
C4-Position
a Reaction conditions: (a) allyl bromide, K2CO3, acetone, rt, 65%; (b)
phosgene, toluene, 110 °C, quantitative yield; (c) 3-buten-1-ol, NaH,
dioxane, 100 °C, 31%; (d) 11, toluene, 110 °C, 52%; (e) Grubbs’ catalyst
(second generation), CH2Cl2, reflux, 75%; (f) H2, Pt/C (10%), MeOH-
THF (3:1), 80%.
Table 1. Ring Size-Activity Relationship of Macrocyclic Ureas
acid, and the resulting imines were reduced to 17 (Table 3) in
situ by NaBH4 in an aqueous THF system. Additionally,
acylation of 5f with acyl chlorides in the presence of pyridine
in methylene chloride afforded the amide analogues 18a-c, and
sulfonylation of 5f with sulfonyl chloride at 0 °C in the presence
of pyridine in methylene chloride afforded sulfonamide analogue
18d (Table 3).
Chk1
Chk1
inhibition
inhibition
(IC50, nM)
compd ring size
X
(IC50, nM) compd ring size
X
5a
5b
n ) 2
H
10
7
5c
n ) 1
CN
6
15-member
n ) 2
14-member
n ) 3
CN
5d
CN
28
15-member
16-member
The synthesis of macrocyclic urea analogues bearing carbon-
linked substituents on the phenyl ring is shown in Scheme 5.
Macrocyclic urea 15 was converted to the triflate 20 in 82%
yield. Various alkynes were coupled with 20 under Sonogashira
reaction conditions to provide 21a-d. It is worth noting that
the coupling yield was significantly improved by addition of
tetrabutylammonium iodide as reported in recent literature.41
Complete saturation of the triple bond of 21e followed by the
removal of the THP group provided 22. Coupling of the triflate
20 with tributylallyltin was effected in a system of PdCl2(PPh3)2-
Ph3P-LiCl in DMF at 110 °C to provide 23 in 65% yield. Olefin
23 was oxidized by osmium tetraoxide under Upjohn condi-
tions42 to provide the diol 24 in good yield.
gave only poor yields. Fortunately, direct alkylation of the
phenol with organic bromides was achieved in the presence of
Cs2CO3 in DMF in 20-90% yield without affecting the urea
NH. This simple alkylation expedited the synthesis of ether
analogues 16 (Table 2) and greatly facilitated SAR studies,
showing the utility of the parallel synthesis.
Scheme 4 outlines the synthesis for macrocyclic ureas in
which a substituent is linked to the phenyl ring by a nitrogen
linker. Although alkylation of an inactive aniline through
reductive amination has historically proven difficult, we were
able to successfully perform the reaction by employing a
modification of a recently published procedure.40 Thus, various
aldehydes were coupled to aniline 5f in the presence of sulfuric
Scheme 3a
a Reaction conditions: (a) HCl, dioxane-CH2Cl2-EtOH, 96%; (b) RBr, Cs2CO3, DMF, 40 °C, 20-90%.

Macrocyclic Checkpoint Kinase 1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1517
Scheme 4a
a Reaction conditions: (a) RCHO, H2SO4, THF, then NaBH4, 0 °C; (b) RCOCl, pyridine, CH2Cl2, rt; (c) CH3SO2Cl, pyridine, CH2Cl2, 0 °C.
Table 3. Structure-Activity Relationship of Amino Analogues at the
C4-Position
positions 2 and 6′ would be sufficient to span the required
distance, yet not induce strain from too small a ring or clash
with Chk1 protein from too large a ring. Thus, 14-16-member
macrocyclic ureas were prepared and tested against Chk1. As
shown in Table 1, the 15-member macrocyclic ureas 5a and 5b
both exhibit potent activity for the inhibition of Chk1 in vitro.
Although the enzymatic inhibition potencies of these two
compounds are comparable, 5b showed superior cellular activity
to 5a (Vide infra), indicating an important role for the CN group
at the 5′-position of the pyrazinyl ring of 5b. For this reason,
we focused on CN analogues in the following SAR studies.
The 15-member macrocycle 5b and 14-member 5c are both very
potent, with the same IC50 (6 and 7 nM, respectively). In
contrast, the 16-member macrocycle 5d is less potent against
Chk1, but its Chk1 inhibition activity is still fully retained
compared with the acyclic analogue 4. The reduced activity of
5d may be due to the additional conformational flexibility
resulting from the presence of more rotatable bonds in the carbon
chain linker. This is consistent with the idea that the restriction
of conformation produces more potent inhibitors.39
Structure-Activity Relationship on the C4-Position of the
Phenyl Ring. Examination of the X-ray cocrystal structure of
5a-Chk1 complex (Figure 2; PDB ID: 2E9U) shows that the
substituent (R4) at the 4-position of the phenyl ring is pointing
into in a solvent-accessible region, which should be able to
accommodate a variety of groups. To further improve the
potency and physicochemical properties of the macrocyclic
Chk1 inhibitors, we performed extensive SAR studies on the
4-position based on the 15-member-ring macrocyclic urea 5b.
Table 2 shows the SAR results of ether analogues. As predicted,
most of the analogues exhibited potent activity for Chk1
inhibition. The introduction of a polar group such as an amino
group at the terminal position of various long chain substituents
resulted in a substantial increase in potency (e.g., 16g vs 16h).
This may be attributed to the hydration of the polar group in
the solvent-exposed region. Conversely, the introduction of a
hydrophobic group results in lessened activity, and the activity
decreases the further the hydrophobic group extends into the
solvent-exposed region (16k vs 16h vs 16i). Furthermore, the
nitrogen position in the pyridyl group of compounds 16l-n has
a remarkable impact on the potency. The 4′′-pyridyl (16l) and
3′′-pyridyl (16m) analogues possess much better activity than
the 2′′-pyridyl analogue 16n.
Results and Discussion
Ring Size-Activity Relationship. Molecular modeling stud-
ies suggested that a linker length of 6-8 atoms between ring
Tables 3 and 4 show the SAR results of analogues having
substituents connected to the C4-position by a nitrogen atom.

1518 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Tao et al.
Scheme 5a
a Reaction conditions: (a) trifluoromethanesulfonyl chloride, TEA, DMF, 0 °C, 82%; (b) alkynes, TRA, (PPh3)4Pd, CuI, n-Bu4NI, DMF, 70 °C; (c) Pt/C
(5%), H2, THF, 80%; (d) HOAc, THF, H2O, 45 °C, 85%; (e) PdCl2(PPh3)2, Ph3P, LiCl, tributylallyltin, DMF, 110 °C, 65%; (f) N-methylmorpholine N-oxide,
2.5% (wt %) OsO4 in 2-methyl-2-propanol, THF, H2O, rt, 85%.
Table 4. Structure-Activity Relationship of Dialkylamino Analogues at
the C4-Position
Table 5. Structure-Activity Relationship at the C4-Position with
Carbon Linkage
The amine analogues in Table 3 showed SAR similar to the
ether analogues in Table 2. Again, the 3- and 4-pyridyl amine
analogues 17g and 17h showed potent activity, and removal of
the nitrogen atom (i.e., benzyl analogue 17i) caused a consider-
able reduction in potency. The importance of 3′-endocyclic
nitrogen for potency was also observed for the five-membered
ring heteroaromatic analogues. For example, 17k and 17l are
much more potent than the thiophene analogue 17m. It is also
interesting to note that methylation of 17j results in a >100-
fold increase in potency (17n), a result not well understood at
this stage. The amide (18a) and the corresponding primary
amine-substituted analogues (17b) show comparable potency,
but the sulfonamide analogue (18d) is slightly less potent. Table
4 shows that analogues with dialkylamino substituents at the
C4-position are less active than their monoalkyl amino coun-
terparts (19b vs 17b).
that the connecting atom at the 4-position has little impact on
potency (16d vs 17d vs 22). The rigid alkynyl spacer showed
no negative impact on potency (e.g., 21d), and the terminal
group on the spacer is again the determinant for potency.
X-ray Crystallographic Analysis of Macrocyclic Urea-
Chk1 Complex. To better understand the SAR results and the
binding mode of macrocylic Chk1 inhibitors, we performed an
X-ray crystallographic analysis of 17h-Chk1 complex at a
resolution of 2.0 Å (Figure 3; PDB ID: 2E9V). Compound 17h
binds to the ATP-binding pocket of Chk1 in the same mode as
compound 5a. The urea amino group at the 2′-position of the
pyrazinyl ring forms a hydrogen bond (3.1 Å) to the backbone
carbonyl oxygen of Glu85, while the urea carbonyl oxygen
accepts a hydrogen bond (2.7 Å) from the amide nitrogen of
Cys87 in the hinge region. This complementary hydrogen-
bonding pattern is the key interaction for most potent ATP-
Analogues with carbon-connected substituents at the C4-
position are depicted in Table 5. These results further confirm

Macrocyclic Checkpoint Kinase 1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1519
Figure 3. (A) X-ray crystal structure of 17h bound to Chk1 kinase. Hydrogen bonds are indicated as in Figure 2 with the additional H-bond shown
between the inhibitor nitrile group and Lys38 (N‚‚‚N distance ) 2.8 Å, C-N‚‚‚N bond angle ) 140°). The three water molecules of the “water
pocket”18a,46 are shown with oxygen atoms as red balls. (B) Surface of entire active site, showing 4-pyridyl unit of 17h exposed to solvent.
Table 6. Kinase Selectivity of Macrocyclic Chk1 Inhibitors
competitive kinase inhibitors. One face of the diaryl urea core
Ki,a µM
Ki,a µM
makes many favorable van der Waals contacts with the
backbone and/or side chains of residues from the N-terminal
domain, including Leu15, Val23, Ala36, and Leu84. The other
face of the core makes van der Waals interactions with the side
chains of Val68 and Leu137. The carbon chain connecting the
two aryl rings sits in the ribose pocket and makes favorable
van der Waals contacts with Glu91, Asn135, Leu137, Glu134,
and Ser147. The 5′-CN is situated in the polar region surrounded
by Ser147, Lys38, and Asp148 and forms a hydrogen bond with
the side chain of Lys38 (3.2 Å). The 4′-N of the pyrazinyl ring
points to the water pocket lined by Asn59, Leu84, and Phe149.
The water pocket, occupied by three water molecules, is unique
for Chk1 kinase. It has been suggested that this pocket is an
attractive target for gaining both potency and selectivity for
Chk1 since it might be entropically favorable to displace the
pocket’s water molecules into the bulk solvent.18a,43 A related
water pocket has been reported for Wee1 kinase.44 In this kinase,
two specific waters were observed that correspond to the two
waters in Figure 3 that are closest to Lys38. A His residue in
Wee1 replaces Asn 59 of Chk1, and its His NE2 atom
corresponds to the third specific water reported here for Chk1.
The water structure in these two kinases is potentially an area
of interest in future inhibitor design. Other aspects of the crystal
structure of 17h include (a) the extension of its 4-pyridinylm-
ethylamino group of the phenyl ring into the solvent-exposed
region (Figure 3b) and (b) an intramolecular hydrogen bond
(2.9 Å) between a urea amino group and the 1′-pyrazinyl
nitrogen.
Kinase Selectivity Profiles of Macrocyclic Urea Chk1
Inhibitors. The selectivity profiles of three macrocyclic urea
Chk1 inhibitors (16a, 18d, and 22) against other kinases were
tested (Table 6). Under our assay conditions, remarkable
selectivity for all three compounds over a panel of more than
70 kinases was observed. Especially, 18d exhibited no inhibitory
activity toward other kinases in this panel at the highest
concentration tested except Plk1, but with a 170-fold selectivity
window.
Potentiation of Cytotoxicity of DNA-Damaging Agents by
Chk1 Inhibitors. Compounds identified as potent (IC50 < 20
nM) in an enzymatic assay were routinely screened for anti-
proliferative activity in HeLa cells, a p53-null human cervical
cancer cell line. The EC50 values for the compounds were
determined either alone or in the presence of 150 nM doxoru-
bicin (Dox), a clinical topoisomerase II inhibitor known to arrest
the G2/M checkpoint at this concentration in HeLa cells. The
EC50 values for Chk1 inhibitors in combination with Dox were
calculated from the percentage of inhibition by Chk1 compounds
kinase
Chk1
Abl
Akt1
Akt2
Akt3
Aurora1
Aurora2
Blk
Cdc2
Cdc42BPA >10
Cdk2
Cdk5/p25
Chk2
Ck2
Camk4
Ck1delt
cKit
18d
16a
22
kinase
18d
16a
22
0.0137 0.0047 0.0043 InsR
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.07
1.12
4.89
>10
>10
>10
>10
6.63
>10
>10
5.67
>10
>10
>10
>10
>10
>10
>10
>10
8.21
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.34
>10
3.15
3.97
>10
>10
>10
>10
>10
4.44
5.76
>10
>10
>10
>10
>10
>10
>10
>10
>10
7.35
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
0.38
IRAK4 >10
JAK2
JAK3
>10
>10
Jnk1a1 >10
Jnk2a2 >10
KDR
LCK
>8.21 >8.21 >8.21
>10
>10
>10
>10
0.88
>10
>10
>10
>10
>10
>10
2.07
>10
>10
>10
>8
1.10
>10
>10
1.28
0.82
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
1.57
>10
>10
>10
>10
>10
>10
0.74
>10
>10
>10
>8
0.28
>10
>10
0.94
0.56
>10
>10
>10
>10
>10
>10
>10
>10
>10
Limk1 >10
Lyn
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
Mk2
MK3
Nek2
p38d
p38g
PAK1 >10
PAK4 >10
PDK1 >10
cMet
Dyrk1A
EGFR
EMK
Erk2
EphA2
ErbB2
ErbB4
FGFR1
FGFR3
Flt1
Flt3
Flt4
Fyn
GSK3a
IGF1R
ITK
IKKa
IKKb
PKA
>10
PKCd >10
PKCg >10
PKCz >8
Pim1
Pim2
Pkd2
Plk1
Plk3
Plk4
>10
>10
>10
2.33
>10
>10
Rock1 >10
Rock2 >10
Sgk
>10
>10
>10
>10
>10
>10
Src
TrkA
TrkB
Tyk2
Zipk
a Inhibition constant (Ki) values are calculated from the Cheng-Prusoff
equation, Ki ) IC50/(1 + ([ATP]/Km).
at various concentrations above the background inhibition by
150 nM Dox. The ability of Chk1 inhibitors to potentiate Dox
is represented by the ratio of the EC50 values of the inhibitor
alone and the inhibitor with Dox. As shown in Table 7,
compounds 5b, 5c, 5f, 15, 16d, 17g, 17h, 17k, 18d, and 22
stood out as ideal Chk1 inhibitors, showing little or no
antiproliferative activity alone but significant activity in the
presence of Dox. It is noteworthy that the CN group at the C5′-
position of the pyrazynyl ring improved the cellular potency
(5a vs 5b). In addition, the substituents at the C4-position have
significant impact on the cellular activity, and amino analogues
are usually more potent in the cellular assays than the carbon-

1520 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Table 7. Cellular Activities of Macrocyclic Chk1 Inhibitors
Tao et al.
rocyclic ureas was developed with Grubbs metathesis macro-
cyclization as the key step. Structure-activity relationship
studies demonstrated that macrocyclic ring size had only modest
impact on the Chk1 inhibition activity and that the 4-position
of the phenyl ring can tolerate a wide variety of substituents.
These novel Chk1 inhibitors exhibited remarkable selectivity
over a panel of more than 70 kinases. Compounds 5b, 5c, 5f,
15, 16d, 17g, 17h, 17k, 18d, and 22 were identified as ideal
Chk1 inhibitors, which exhibited little or no antiproliferative
activity alone but significantly potentiated the cytotoxicities of
the DNA-damaging agents Dox and CPT. FACS analysis of
cell cycle profiles confirmed that these compounds express their
potent biological activities through the inhibition of Chk1. To
our knowledge, these are the first examples of macrocyclic
kinase inhibitors based on the urea scaffold. The efficient
synthetic methodology developed here should facilitate the
utilization of these macrocylic compounds as medically useful
molecules, especially in the field of kinase inhibitors.
MTS EC50, µM
compd
FACS EC50, µM
MTS
potentiation
of CPT
compd +
compd +
Dox
compd
alone
compd
Dox
alone
5a
5b
5c
5f
37.8
3.04
3.08
0.43
1.9
3.01
1.6
1.4
>59
57.6
57.6
>59
>59
>59
>59
>59
>59
>59
28.7
NAa
NA
NA
NA
NA
6-fold
8-fold
20-fold
NA
5-fold
NA
7.5
2.7
1.3
0.24
NA
0.7
0.33
1.2
>10
>10
>10
7.6
15
NA
16d
17g
17h
17k
18d
22
>10
>10
>10
>10
>10
>10
1.2
2.7
0.86
0.11
1.6
0.19
a NA, data not available.
connected and ether analogues. The lack of a perfect correlation
between Chk1 enzymatic inhibition potency and cellular anti-
proliferative activity may be due to other physicochemical
properties such as variation in cellular penetration, as has been
well documented in the literature.45
Experimental Section
General Information. All reactions were carried out under N2
atmosphere unless otherwise specified. Reagents and solvents were
obtained from commercial suppliers and were used without further
purification. 1H NMR spectra were obtained on a Varian Unity or
Inova (500 MHz), Varian Unity (400 MHz), or Varian Unity plus
or Mercury (300 MHz) instrument. Chemical shifts are reported as
δ values (ppm) downfield relative to tetramethylsilane (TMS) as
an internal standard, with multiplicities reported in the usual manner.
Mass spectral analyses were performed on a Finnigan SSQ7000
GC/MS mass spectrometer using different techniques, including
electrospray ionization (ESI) and atmospheric pressure chemical
ionization (APCI), as specified for individual compounds. Exact
mass measurement was performed on a Finnigan FTMS Newstar
T70 mass spectrometer. The compound is determined to be
“consistent” with the chemical formula if the exact mass measure-
ment is within 5.0 ppm relative mass error (RME) of the exact
monoisotopic mass. Elemental analyses were performed by Quan-
titative Technologies, Inc., Whitehouse, NJ. Column chromatog-
raphy was carried out on a Horizon Pioneer system (Biotage, Inc.).
Preparative reverse-phase HPLC was performed on an automated
Gilson HPLC system, with a SymmetryPrep Shield RP18 prep
cartridge, 250 mm × 21.20 mm i.d., 10 um, and a flow rate of 25
mL/min; λ ) 214, 245 nm; mobile phase A, 0.1% trifluoroacetic
acid (TFA) in H2O; mobile phase B, CH3CN; linear gradient
0-70% B in 40 min.
Analytical liquid chromatography/mass spectrometry (LC/MS)
was performed on a Finnigan Navigator mass spectrometer and
Agilent 1100 HPLC system running Xcalibur 1.2 and Open-Access
1.3 software. The mass spectrometer was operated under positive
APCI ionization conditions. The HPLC system comprised an
Agilent Quaternary pump, degasser, column compartment, au-
tosampler, and diode-array detector, with a Sedere Sedex 75
evaporative light-scattering detector. The column used was a
Phenomenex Luna Combi-HTS C8(2) 5 µm 100 Å (2.1 mm × 30
mm). TFA method (method A): A gradient of 10-100% aceto-
nitrile (solvent 1) and 0.1% trifluoroacetic acid in water (solvent
2) was used, at a flow rate of 2 mL/min (0-0.1 min, 10% solvent
1; 0.1-2.6 min, 10-100% solvent 1; 2.6-2.9 min, 100-10%
solvent 1; 2.9-3.0 min, 100-10% solvent 1). Ammonium method
(method B): A gradient of 10-100% acetonitrile (solvent 1) and
10 mM NH4OAc in water (solvent 2) was used, at a flow rate of
1.5 mL/min (0-0.1 min, 10% solvent 1; 0.1-3.1 min, 10-100%
solvent 1; 3.1-3.9 min, 100-10% solvent 1; 3.9-4.0 min, 100-
10% solvent 1).
The ability of selected compounds to sensitize tumor cells
was further evaluated by measuring the antiproliferative EC50
of camptothecin (CPT) in the presence of a fixed concentration
of Chk 1 inhibitors in a MTS assay. CPT is a major anticancer
agent in clinical use. It damages DNA through the inhibition
of topoisomerase I and is known to induce S-phase arrest.46
Thus, SW620 cells (a p53-deficient colon carcinoma cell line)
were treated with increasing doses of CPT in the presence and
absence of Chk1 inhibitors. The ratio of EC50 value of CPT
alone and EC50 value of CPT in combination with Chk1 inhibitor
was defined as the potentiation ratio. As shown in Table 7, Chk1
compounds potentiate the cytotoxity of CPT 5-20-fold at a
concentration of 10 µM. Reduced but still significant potentia-
tion was also achieved at a Chk1 inhibitor concentration of 3
µM, indicating that the potentiation was dose-dependent on the
Chk1 inhibitors (data not shown).
FACS Analysis of Cell Cycle Profiles Treated with Chk1
Inhibitors. To establish that the macrocyclic Chk1 inhibitors
are truly abrogating the checkpoints induced by DNA-damaging
agents and not simply altering cell populations by affecting cell
cycle progression, a detailed cell cycle kinetic analysis was
carried out by use of fluorescence-activated cell sorting (FACS).
The analysis was undertaken with the Chk1 inhibitors in H1299
cells. As expected, Dox alone conferred a prominent G2/M-
phase arrest. Table 7 shows the EC50 values for abrogating G2/M
caused by Dox in the presence of various concentrations of Chk1
inhibitors. The regular cell cycle profile was not altered by Chk1
inhibitors even at the highest concentration (10 µM) since the
FACS profile was indistinguishable from that of the DMSO
control (data not shown). This observation is consistent with
the fact that Chk1 knockout through Chk1 siRNA did not change
the cell-cycle distributions.47 Chk1 inhibitors abrogated the Dox-
induced-G2/M checkpoint with EC50 values of submicromolar
to low (single-digit) micromolar. An increasing amount of cells
accumulated in the sub-G0/G1 phase was noticed, indicating
that the checkpoint abrogation results in apoptosis, again
demonstrating the potentiation effect. In addition, we confirmed
that the macrocyclic Chk1 inhibitors efficiently abrogated CPT-
induced S arrest. As shown in Figure 4, both 17h and 18d
abrogated CPT-induced S arrest at 3 and 10 µM.
6-[1-(Dimethylamino)propan-2-yloxy]pyrazin-2-amine (8). To
a suspension of NaH (60%, 123.5 mg, 3.08 mmol) in dioxane was
added dropwise dimethylaminopropanol (0.37 mL, 3.08 mmol).
After the mixture was stirred for 30 min, 6-chloropyrazin-2-amine
(200 mg, 1.54 mmol) was added. The resulting mixture was heated
Conclusions
We have discovered macrocyclic ureas as a novel class of
Chk1 inhibitors. An efficient synthetic methodology for mac-

Macrocyclic Checkpoint Kinase 1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1521
Figure 4. FACS profiles of SW620 cells treated with CPT in the presence and absence of macrocyclic Chk1 inhibitors 17h and 18d. The cells
were stained with propidium iodide for DNA contents. G0/G1 cells contain 2N DNA while cells in G2/M phase have 4N DNA. S phase cells have
DNA content between 2N and 4N. Apoptotic cells contain less than 2N DNA.
at 100 °C overnight and concentrated. The residue was purified by
flash chromatography, eluted with a mixture of ethyl acetate/
methanol/ammonium hydroxide (100:6:1), to provide 250 mg of 8
in 83% yield. MS (DCI/NH3) m/z 197.11 (M + H)+.
added slowly a solution of 10 (1 g, 5.44 mmol) in toluene (10 mL)
with stirring under reflux. The resulting reaction mixture was
refluxed at 110 °C for 20 h and then cooled. Evaporation of the
solvent gave the desired product as an oil in quantitative yield. 1H
NMR (500 MHz, CD2Cl2) δ 4.62 (d, J ) 5.30 Hz, 2H), 5.33 (dd,
J ) 10.76, 1.40 Hz, 1H), 5.46 (dd, J ) 17.31, 1.40 Hz, 1H), 6.07
(m, 1H), 6.84 (d, J ) 8.73 Hz, 1H), 6.99 (d, J ) 2.50 Hz, 1H),
7.10 (dd, J ) 8.74, 2.50 Hz, 1H).
6-(But-3-enyloxy)pyrazin-2-amine (13). To a suspension of
NaH (60%, 618 mg, 15.45 mmol) in dioxane (30 mL) was injected
3-buten-1-ol (1.33 mL, 15.45 mmol) at 0 °C. After the reaction
mixture was stirred for 2 h, 2-amino-6-chloropyrazine (1 g, 7.72
mmol) was added. The reaction mixture was further stirred at 100
°C for 2.5 days, cooled, and diluted with ethyl acetate. The resulting
mixture was washed with water and dried over MgSO4. Evaporation
of ethyl acetate gave a residue, which was purified by flash
chromatography, eluted with hexane/ethyl acetate (2:1). The title
compound (390 mg, 31%) was obtained. MS (DCI/NH3) m/z 166.12
(M + H)+. 1H NMR (500 MHz, benzene-d6) δ 2.66 (m, 2H), 4.42
(t, J ) 6.87 Hz, 2H), 5.24 (dd, J ) 10.22, 1.98 Hz, 1H), 5.30 (m,
1H), 6.04 (m, 1H), 7.64 (s, 1H), 7.65 (s, 1H).
1-(3-Chlorophenyl)-3-[6-[1-(dimethylamino)propan-2-yloxy]-
pyrazin-2-yl]urea (3). A mixture of 8 (50 mg, 0.25 mmol) and
1-chloro-3-isocyanatobenzene (0.03 mL, 0.25 mmol) in toluene was
refluxed for 4 h and concentrated. The residue was triturated with
a mixture of ethyl acetate and hexane to provide 61.2 mg of 3 in
1
70% yield. MS (DCI/NH3) m/z 350.09 (M + H)+. H NMR (300
MHz, DMSO-d6) δ 1.29 (d, J ) 6.44 Hz, 3H), 2.35-2.42 (m, 1H),
2.52-2.61 (m, 1H), 5.15-5.28 (m, J ) 11.70, 6.61 Hz, 1H), 7.06-
7.11 (m, 1H), 7.25-7.38 (m, 2H), 7.72 (t, J ) 1.86 Hz, 1H), 7.85
(s, 1H), 8.67 (s, 1H), 9.23 (s, 1H), 9.35 (s, 1H). 13C NMR (101
MHz, DMSO-d6) δ 18.1, 45.7, 63.6, 69.9, 116.9, 117.8, 122.2,
125.0, 127.4, 130.5, 133.2, 140.3, 146.1, 151.3, 157.5, 183.1. HRMS
(ESI-TOF) calcd for C16H21ClN5O2 (M + H)+ 350.1384; found
350.1391.
1-(5-Chloro-2-methoxyphenyl)-3-[6-[1-(dimethylamino)pro-
pan-2-yloxy]pyrazin-2-yl]urea (4). Compound 4 was synthesized
by a synthetic procedure similar to that described for 3, in 55%
yield. MS (DCI/NH3) m/z 380.10 (M + H)+. 1H NMR (300 MHz,
DMSO-d6) δ 1.30 (s, 3H), 2.18 (s, 6H), 2.34-2.43 (m, 1H), 2.52-
2.61 (m, 1H), 3.89 (s, 3H), 5.19-5.29 (m, 1H), 7.05 (s, 2H), 7.86
(s, 1H), 8.23 (s, 1H), 8.68 (s, 1H), 9.16 (s, 1H), 9.95 (s, 1H). 13C
NMR (101 MHz, DMSO-d6) δ 18.1, 45.7, 56.2, 63.7, 69.9, 112.1,
118.0, 121.7, 124.2, 125.1, 127.2, 129.2, 146.2, 146.8, 151.3, 157.4,
182.6. HRMS (ESI-TOF) calcd for C17H23ClN5O3 (M + H)+
380.1489; found 380.1497.
1-[2-(Allyloxy)-5-chlorophenyl]-3-[6-(but-3-enyloxy)pyrazin-
2-yl]urea (6). A mixture of 11 (201 mg, 0.96 mmol) and 13 (158
mg, 0.96 mmol) in toluene (15 mL) was stirred at 110 °C for 15 h.
Evaporation of solvent gave a residue, which was purified by flash
chromatography, eluted with hexane/ethyl acetate (1:1). The title
compound (185 mg, 52%) was obtained. MS (DCI/NH3) m/z
375.12(M + H)+. 1H NMR (500 MHz, DMSO-d6) δ 2.52 (m, 2H),
4.33 (t, J ) 6.55 Hz, 2H), 4.70 (d, J ) 5.30 Hz, 2H), 5.09 (dd, J
) 10.29, 1.25 Hz, 1H), 5.16 (dd, J ) 17.31, 1.40 Hz, 1H), 5.31 (d,
J ) 10.61 Hz, 1H), 5.43 (dd, J ) 17.16, 1.25 Hz, 1H), 5.88 (m,
1H), 6.08 (m, 1H), 7.02 (d, J ) 8.75 Hz, 1H), 7.06 (dd, J ) 8.75,
2.5 Hz, 1H), 7.89 (s, 1H), 8.22 (d, J ) 2.50 Hz, 1H), 8.69 (s, 1H),
9.07 (s, 1H), 10.08 (s, 1H).
2-(Allyloxy)-5-chloroaniline (10). To a mixture of 2-amino-4-
chlorophenol (10 g, 69.65 mmol) and K2CO3 (14.53 g, 105 mmol)
in acetone (160 mL) was added allyl bromide (9.03 mL, 104 mmol).
The reaction mixture was stirred at room temperature overnight.
Inorganic salts were filtered off and washed with acetone. The
combined filtrate was concentrated. The residue was purified by
flash chromatography, eluted with hexane/EtOAc (10:1), to give
8.31 g (65%) of the desired compound as a light-brown oil. MS
Macrocyclic Diarylurea 14. Compound 6 (60 mg, 0.16 mmol)
in CH2Cl2 (66 mL) was treated with the second-generation Grubbs’
catalyst (20 mg, 0.024 mmol). The reaction mixture was stirred at
50 °C overnight. Solvent was removed under reduced pressure. The
residue was purified by flash chromatography, eluted with hexane/
ethyl acetate (1:1). The title compound (22 mg, 75%) was obtained.
MS (DCI/NH3) m/z 347.11 (M + H)+. 1H NMR (500 MHz, CD2-
Cl2) δ 2.70 (d, J ) 7.25 Hz, 2H), 4.63 (m, 4H), 6.05 (m, 2H), 6.90
1
(DCI/NH3) m/z 184.02 (M + H)+. H NMR (500 MHz, DMSO-
d6) δ 4.51 (m, 2H), 5.02 (s, 2H), 5.24 (dd, J ) 10.53, 1.68 Hz,
1H), 5.42 (m, 1H), 6.04 (m, 1H), 6.47 (dd, J ) 8.54, 2.44 Hz, 1H),
6.66 (d, J ) 2.75 Hz, 1H), 6.75 (d, J ) 8.54 Hz, 1H).
1-(Allyloxy)-4-chloro-2-isocyanatobenzene (11). To a solution
of 5 mL (47.3 mmol) of 20% phosgene in toluene (6 mL) was

1522 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Tao et al.
(d, J ) 8.73 Hz, 1H), 7.02 (dd, J ) 8.73, 2.57 Hz, 1H), 7.15 (s,
1H), 7.72 (s, 1H), 7.84 (s, 1H), 8.25 (d, J ) 2.57 Hz, 1H), 10.55
(s, 1H).
2H), 4.56-4.61 (m, 3H), 6.92 (s, 1H), 7.99 (s, 1H), 8.11 (s, 1H),
9.79 (s, 1H), 10.88 (s, 1H). Anal. (C20H22ClN5O5‚0.4H2O) C, H,
N.
Macrocyclic Diarylurea 5a. To a suspension of platinum-
carbon (10%, 2 mg) in a mixture of methanol-THF (3:1, 3 mL)
was added 14 (17 mg, 0.049 mmol). The reaction mixture was
bubbled with hydrogen for 10 min. Platinum-carbon was removed
by filtration through Celite. The filtrate was concentrated and the
residue was purified by recrystallization from ethyl acetate. The
title compound (13.7 mg, 80%) was obtained. MS (DCI/NH3) m/z
Macrocyclic Diarylurea 16b. A mixture of 15 (25 mg, 0.064
mmol), 2-(3-bromopropyl)isoindoline-1,3-dione (25.7 mg, 0.096
mmol), and Cs2CO3 (41.8 mg, 0.128 mmol) in DMF (2 mL) was
heated at 40 °C overnight. Inorganic salts were filtered off, and
the filtrate was concentrated. The residue was treated with hydrazine
monohydrate (0.016 mL, 0.32 mmol) in a mixture of THF (2 mL)
and methanol (0.2 mL). The reaction mixture was stirred for 4 h
and concentrated. The residue was purified by HPLC to provide
16b as the TFA salt in 55% yield (for two steps). MS (APCI) m/z
1
349.11 (M + H)+. H NMR (500 MHz, DMSO-d6) δ 1.64 (m,
2H), 1.84 (m, 2H), 1.89 (m, 2H), 4.14 (t, J ) 5.16 Hz, 2H), 4.48
(t, J ) 7.80 Hz, 2H), 7.08 (d, J ) 2.50 Hz, 1H), 7.09 (s, 1H), 7.89
(s, 1H), 7.95 (s, 1H), 8.23 (d, J ) 2.50 Hz, 1H), 10.26 (s, 1H),
10.32 (s, 1H). Anal. (C16H17ClN4O3) C, H, N.
Compounds 5b-f were prepared by synthetic procedures similar
to those described for 5a.
1
447.36 (M + H)+. H NMR (500 MHz, DMSO-d6) δ 1.55-1.65
(m, 2H), 1.79-1.87 (m, 2H), 1.92-2.07 (m, 4H), 2.94-3.05 (m,
2H), 4.18 (t, J ) 5.95 Hz, 2H), 4.24 (t, J ) 5.03 Hz, 2H), 4.52-
4.65 (m, 2H), 6.93 (s, 1H), 7.73 (s, 3H), 8.00 (s, 1H), 8.13 (s, 1H),
9.81 (s, 1H), 10.90 (s, 1H). Anal. (C20H23ClN6O4‚0.2H2O‚TFA)
C, H, N.
Macrocyclic Diarylurea 5b. MS (DCI/NH3) m/z 391.3 (M +
1
NH4)+. H NMR (500 MHz, DMSO-d6) δ 1.61 (dd, J ) 12.16,
Macrocyclic Diarylurea 16c. A mixture of 15 (52 mg, 0.13
mmol), allyl bromide (0.0138 mL, 0.16 mmol), and K2CO3 (36.8
mg, 0.267 mmol) in DMF (5 mL) was stirred at room temperature
overnight. Inorganic salts were filtered off, and the filtrate was
concentrated. The residue was dissolved in a mixture of THF (20
mL) and water (2 mL), and the solution was treated with
N-methylmorpholine N-oxide (36 mg, 0.31 mmol), followed by the
addition of 2.5% (wt %) OsO4 in 2-methyl-2-propanol (0.19 mL)
at 0 °C. The reaction mixture was stirred overnight and concen-
trated. The residue was purified by HPLC, eluted with a gradient
of 0-70% acetonitrile in 0.1% TFA aqueous solution. The desired
product (22 mg, 36%) was obtained as a white solid. Analytical
LC/MS with two different solvent systems indicated >95% purity.
MS (ESI) m/z 462.20 (M - H)-. 1H NMR (500 MHz, DMSO-d6)
δ 1.57-1.62 (m, 2H), 1.80-1.85 (m, 2H), 1.92-1.99 (m, 2H),
3.44-3.52 (m, 2H), 3.82 (m, 1H), 4.00 (m, 1H), 4.08 (m, 1H),
4.23 (t, J ) 5.19 Hz, 2H), 4.58 (t, J ) 8.24 Hz, 2H), 4.69 (t, J )
5.65 Hz, 1H), 4.98 (d, J ) 4.88 Hz, 1H), 6.93 (s, 1H), 7.98 (s,
1H), 8.12 (s, 1H), 9.79 (s, 1H), 10.87 (s, 1H).
6.24 Hz, 2H), 1.82 (m, 2H), 1.95 (m, 2H), 4.19 (t, J ) 5.15 Hz,
2H), 4.62 (t, J ) 8.45 Hz, 2H), 7.13 (m, 2H), 7.99 (s, 1H), 8.20 (d,
J ) 2.18 Hz, 1H), 9.95 (s, 1H), 10.94 (s, 1H). Anal. (C17H16ClN5O3)
C, H, N.
Macrocyclic Diarylurea 5c. MS (ESI) m/z 357.91 (M - H)-.
1H NMR (500 MHz, DMSO-d6) δ 1.85-1.91 (m, 2H), 2.00-2.06
(m, 2H), 4.17 (t, J ) 5.49 Hz, 2H), 4.75 (t, J ) 7.32 Hz, 2H), 7.09
(dd, J ) 8.85, 2.14 Hz, 1H), 7.18 (d, J ) 8.54 Hz, 1H), 7.94 (s,
1H), 8.34 (s, 1H), 10.83 (s, 1H), 11.05 (s, 1H). Anal. (C16H14-
ClN5O3) C, H, N.
Macrocyclic Diarylurea 5d. MS (ESI) m/z 386.08 (M - H)-.
1H NMR (400 MHz, DMSO-d6) δ 1.43-1.59 (m, 4H), 1.74-1.92
(m, 4H), 4.15 (s, 2H), 4.52 (t, J ) 6.90 Hz, 2H), 7.15 (s, 2H), 7.96
(s, 1H), 8.00 (s, 1H), 9.61 (s, 1H), 10.91 (s, 1H). Anal. (C18H18-
ClN5O3) C, H, N.
Macrocyclic Diarylurea 5e. MS (DCI/NH3) m/z 537.20 (M +
NH4)+. 1H NMR (500 MHz, DMSO-d6) δ 0.00 (s, 9H), 0.90-0.94
(m, 2H), 1.59-1.65 (m, 2H), 1.83-1.87 (m, 2H), 1.94-2.01 (m,
2H), 3.77-3.80 (m, 2H), 4.19-4.21 (m, 2H), 4.60-4.63 (m, 2H),
5.35 (s, 2H), 7.05 (s, 1H), 8.00 (s, 1H), 8.17 (s, 1H), 9.84 (s, 1H),
10.90 (s, 1H).
Macrocyclic Diarylurea 16d. A mixture of 15 (30 mg, 0.077
mmol), 2-(2-bromoethoxy)tetrahydropyran (32 mg, 0.154 mmol),
and Cs2CO3 (33.4 mg, 0.102 mmol) in DMF (2 mL) was heated at
40 °C overnight. Inorganic salts were filtered off, and the filtrate
was concentrated. The residue was treated with HOAc/THF/H2O
(4:2:1, 8 mL) at 45 °C overnight. The solvent was removed and
the residue was purified by HPLC, eluted with a gradient of 0-70%
acetonitrile in 0.1% TFA aqueous solution. The desired product
Macrocyclic Diarylurea 5f. MS (DCI/NH3) m/z 406.11 (M +
1
NH4)+. H NMR (500 MHz, DMSO-d6) δ 1.54-1.59 (m, 2H),
1.76-1.81 (m, 2H), 1.90-1.96 (m, 2H), 4.05 (d, J ) 5.49 Hz,
2H), 4.55 (t, J ) 8.24 Hz, 2H), 5.19 (s, 2H), 6.56 (s, 1H), 7.87 (s,
1H), 7.95 (s, 1H), 9.64 (s, 1H), 10.76 (s, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 22.7, 25.6, 26.8, 68.3, 69.0, 99.0, 106.5, 107.5, 115.5,
116.1, 122.2, 128.1, 141.4, 148.2, 149.4, 150.5, 160.3. Anal.
(C17H17ClN6O3) C, H, N.
1
(25 mg, 76%) was obtained. MS (ESI) m/z 432.04 (M - H)-. H
NMR (400 MHz, DMSO-d6) δ 1.57-1.63 (m, 2H), 1.80-1.85 (m,
2H), 1.92-2.00 (m, 2H), 3.74 (q, J ) 5.22 Hz, 2H), 4.11 (t, J )
5.06 Hz, 2H), 4.23 (t, J ) 5.06 Hz, 2H), 4.59 (t, J ) 8.29 Hz, 2H),
4.88 (t, J ) 5.37 Hz, 1H), 6.94 (s, 1H), 7.99 (s, 1H), 8.12 (s, 1H),
9.80 (s, 1H), 10.86 (s, 1H). Anal. (C19H20ClN5O5) C, H, N.
Compounds 16e-o were prepared by synthetic procedures
similar to that described for 16a, substituting 3-bromopropanol with
corresponding organic chlorides or bromides.
Macrocyclic Diarylurea 15. To a solution of 5e (1.8 g,
3.46mmol) in a mixture of dichloromethane (100 mL) and ethanol
(300 mL) was added 9 mL of 4 N HCl in 1,4-dioxane dropwise.
The reaction mixture was stirred overnight, and the white precipitate
was collected by filtration and dried. The desired product (1.3 g,
96%) was obtained as a white solid. MS (DCI/NH3) m/z 407.08
1
Macrocyclic Diarylurea 16e. MS (ESI) 446.01 (M - H)-. H
1
(M + NH4)+. H NMR (500 MHz, DMSO-d6) δ 1.60-1.65 (m,
NMR (400 MHz, DMSO-d6) δ 1.57-1.63 (m, 2H), 1.80-1.85 (m,
2H), 1.92-1.99 (m, 2H), 3.34 (s, 3H), 3.68-3.70 (m, 2H), 4.20-
4.25 (m, 4H), 4.59 (d, J ) 7.98 Hz, 2H), 6.93 (s, 1H), 7.99 (s,
1H), 8.13 (s, 1H), 9.80 (s, 1H), 10.86 (s, 1H). Anal. (C20H22ClN5O5‚
0.1H2O) C, H, N.
2H), 1.81-1.85 (m, 2H), 1.94-2.00 (m, 2H), 4.11 (t, J ) 5.15
Hz, 2H), 4.59 (t, J ) 8.11 Hz, 2H), 6.72 (s, 1H), 7.99 (s, 1H), 8.06
(s, 1H), 9.75 (s, 1H), 10.78 (s, 1H). 13C NMR (75 MHz, DMSO-
d6) δ 22.8, 25.6, 26.8, 68.4, 69.5, 100.8, 106.8, 109.7, 115.4, 118.7,
122.4, 128.1, 147.9, 149.4, 149.4, 150.6, 160.3. HRMS (ESI-TOF)
calcd for C17H17ClN5O4 (M + H)+ 390.0964; found 390.0960.
Macrocyclic Diarylurea 16a. A mixture of 15 (20 mg, 0.051
mmol), 3-bromopropanol (11 mg, 0.077 mmol), and Cs2CO3 (33.4
mg, 0.102 mmol) in DMF (2 mL) was heated at 40 °C overnight.
Inorganic salts were filtered off, and the filtrate was concentrated.
The residue was purified by HPLC, eluted with a gradient of
0-70% acetonitrile in 0.1% TFA aqueous solution. The desired
product (8.2 mg, 36%) was obtained. MS (ESI) m/z 445.98 (M -
H)-. 1H NMR (500 MHz, DMSO-d6) δ 1.57-1.62 (m, 2H), 1.80-
1.84 (m, 2H), 1.85-1.90 (m, 2H), 1.93-1.99 (m, 2H), 3.58 (q, J
) 5.90 Hz, 2H), 4.15 (t, J ) 6.26 Hz, 2H), 4.24 (t, J ) 5.03 Hz,
Macrocyclic Diarylurea 16f. MS (ESI) 490.07 (M - H)-,
1
492.07 (M + H)+. H NMR (400 MHz, DMSO-d6) δ 1.57-1.63
(m, 2H), 1.80-1.85 (m, 2H), 1.92-1.99 (m, 2H), 3.25 (s, 3H),
3.45-3.48 (m, 2H), 3.61-3.63 (m, 2H), 3.76 (d, J ) 4.60 Hz,
2H), 4.21-4.25 (m, 4H), 4.60 (t, J ) 8.29 Hz, 2H), 6.95 (s, 1H),
7.99 (s, 1H), 8.13 (s, 1H), 9.80 (s, 1H), 10.86 (s, 1H). Anal. (C22H26-
ClN5O6‚0.5H2O‚0.1TFA) C, H, N.
Macrocyclic Diarylurea 16g. MS (ESI) 459.14 (M - H)-,
1
461.07 (M + H)+. H NMR (400 MHz, DMSO-d6) δ 1.57-1.64
(m, 2H), 1.80-1.88 (m, 2H), 1.93-2.00 (m, 2H), 2.93 (s, 3H),
3.17 (s, 3H), 3.55-3.58 (m, 2H), 4.24-4.27 (m, 2H), 4.44 (t, J )

Macrocyclic Checkpoint Kinase 1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1523
4.91 Hz, 2H), 4.60 (t, J ) 8.29 Hz, 2H), 7.01 (s, 1H), 8.00 (s, 1H),
8.18 (s, 1H), 9.84 (s, 1H), 10.90 (s, 1H). Anal. (C21H25ClN6O4‚
0.1H2O‚TFA) C, H, N.
Hz, 2H), 4.56 (t, J ) 8.29 Hz, 2H), 5.02 (t, J ) 5.68 Hz, 1H), 6.39
(s, 1H), 7.93 (s, 1H), 7.96 (s, 1H), 9.66 (s, 1H), 10.74 (s, 1H).
HRMS (ESI-TOF) calcd for C19H22ClN6O3 (M + H)+ 417.1442;
found 417.1441.
Macrocyclic Diarylurea 16h. MS (APCI) m/z 460.17 (M + H)+.
1H NMR (400 MHz, DMSO-d6) δ 1.55-1.71 (m, 4H), 1.76-1.89
(m, 3H), 1.89-2.05 (m, 2H), 4.10 (t, J ) 6.60 Hz, 2H), 4.23 (d, J
) 4.60 Hz, 2H), 4.40-4.76 (m, 2H), 6.91 (s, 1H), 7.98 (s, 1H),
8.11 (s, 1H), 9.79 (s, 1H), 10.85 (s, 1H). Anal. (C22H26ClN5O4) C,
H, N.
Macrocyclic Diarylurea 17c. The desired product was prepared
by a procedure similar to that described for 17g by replacing
3-pyridinecarboxaldehyde with butyraldehyde. Analytical LC/MS
with two different solvent systems indicated > 95% purity. MS
1
(APCI) m/z 445.64 (M + H)+. H NMR (400 MHz, DMSO-d6) δ
Macrocyclic Diarylurea 16i. MS (APCI) m/z 474.16 (M + H)+.
1H NMR (500 MHz, DMSO-d6) δ 0.91 (s, 3H), 0.93 (s, 3H), 1.31-
1.39 (m, 2H), 1.57-1.67 (m, 3H), 1.71-1.79 (m, 2H), 1.80-1.89
(m, 2H), 1.93-2.03 (m, 2H), 4.08 (t, J ) 6.41 Hz, 2H), 4.25 (t, J
) 5.03 Hz, 2H), 4.49-4.74 (m, 2H), 6.91 (s, 1H), 8.00 (s, 1H),
8.14 (s, 1H), 9.80 (s, 1H), 10.90 (s, 1H). HRMS (ESI-TOF) calcd
for C23H29ClN5O4 (M + H)+ 474.1908; found 474.1908.
Macrocyclic Diarylurea 16j. MS (APCI) m/z 417.98 (M + H)+.
1H NMR (500 MHz, DMSO-d6) δ 1.35 (t, J ) 7.02 Hz, 3H), 1.53-
1.66 (m, 2H), 1.79-1.86 (m, 2H), 1.91-2.00 (m, 2H), 4.14 (q, J
) 6.92 Hz, 2H), 4.23 (t, J ) 5.03 Hz, 2H), 4.50-4.65 (m, 2H),
6.89 (s, 1H), 7.98 (s, 1H), 8.12 (s, 1H), 9.79 (s, 1H), 10.88 (s, 1H).
Macrocyclic Diarylurea 16k. MS (ESI) 429.97 (M - H)-,
432.00 (M + H)+. 1H NMR (500 MHz, DMSO-d6) δ 1.28 (d, J )
6.10 Hz, 6H), 1.56-1.62 (m, 2H), 1.79-1.84 (m, 2H), 1.92-1.99
(m, 2H), 4.22 (t, J ) 5.19 Hz, 2H), 4.59 (t, J ) 8.24 Hz, 2H),
4.66-4.71 (m, 1H), 6.92 (s, 1H), 7.99 (s, 1H), 8.12 (s, 1H), 9.80
(s, 1H), 10.89 (s, 1H). Anal. (C20H22ClN5O5) C, H, N.
0.92 (t, J ) 7.36 Hz, 3H), 1.31-1.42 (m, 2H), 1.51-1.63 (m, 4H),
1.78-1.84 (m, 2H), 1.92-2.00 (m, 2H), 3.15 (q, J ) 6.44 Hz,
2H), 4.19 (t, J ) 5.22 Hz, 2H), 4.58 (t, J ) 7.67 Hz, 2H), 5.01 (t,
J ) 5.83 Hz, 1H), 6.40 (s, 1H), 7.94 (s, 1H), 7.97 (s, 1H), 9.68 (s,
1H), 10.77 (s, 1H). HRMS (ESI-TOF) calcd for C21H26ClN6O3 (M
+ H)+ 445.1755; found 445.1753.
Macrocyclic Diarylurea 17d. The desired product was prepared
by a procedure similar to that described for 17g. Analytical LC/
MS with two different solvent systems indicated >95% purity. MS
1
(APCI) m/z 433.34 (M + H)+. H NMR (500 MHz, DMSO-d6) δ
1.52-1.64 (m, 2H), 1.76-1.85 (m, 2H), 1.90-2.01 (m, 2H), 3.22
(t, J ) 5.80 Hz, 2H), 3.60 (t, J ) 5.65 Hz, 2H), 4.19 (t, J ) 5.19
Hz, 2H), 4.45-4.70 (m, 2H), 5.04 (br s, 1H), 6.47 (s, 1H), 7.95 (s,
1H), 7.97 (s, 1H), 9.68 (s, 1H), 10.79 (s, 1H).
Macrocyclic Diarylurea 17e. The desired product was prepared
by a procedure similar to that described for 17g. Analytical LC/
MS with two different solvent systems indicated >95% purity. MS
1
(APCI) m/z 431.26 (M + H)+. H NMR (500 MHz, DMSO-d6) δ
Macrocyclic Diarylurea 16l. MS (ESI) 479.02 (M - H)-,
1.19 (d, J ) 6.10 Hz, 6H), 1.52-1.62 (m, 2H), 1.73-1.86 (m,
2H), 1.90-2.02 (m, 2H), 3.70-3.81 (m, 1H), 4.19 (t, J ) 5.03
Hz, 2H), 4.52-4.62 (m, 2H), 6.45 (s, 1H), 7.94 (s, 1H), 7.97 (s,
1H), 9.68 (s, 1H), 10.79 (s, 1H).
1
481.05 (M + H)+. H NMR (400 MHz, DMSO-d6) δ 1.55-1.62
(m, 2H), 1.78-1.84 (m, 2H), 1.91-1.99 (m, 2H), 4.20-4.23 (m,
2H), 4.57-4.61 (m, 2H), 5.41 (s, 2H), 7.03 (s, 1H), 7.66 (d, J )
6.14 Hz, 2H), 7.99 (s, 1H), 8.18 (s, 1H), 8.72 (d, J ) 6.44 Hz,
2H), 9.81 (s, 1H), 10.88 (s, 1H). Anal. (C23H21ClN6O4‚0.6TFA) C,
H, N.
Macrocyclic Diarylurea 17f. The desired product was prepared
by a procedure similar to that described for 17g by replacing
3-pyridinecarboxaldehyde with isobutylaldehyde. MS (APCI) m/z
445.42 (M + H)+. 1H NMR (500 MHz, DMSO-d6) δ 0.92 (d, J )
6.41 Hz, 6H), 1.54-1.61 (m, 2H), 1.78-1.83 (m, 2H), 1.87-1.99
(m, 3H), 2.99 (t, J ) 6.56 Hz, 2H), 4.18 (t, J ) 5.19 Hz, 2H), 4.57
(t, J ) 8.24 Hz, 2H), 5.08 (t, J ) 5.95 Hz, 1H), 6.39 (s, 1H), 7.93
(s, 1H), 7.97 (s, 1H), 9.68 (s, 1H), 10.78 (s, 1H).
Macrocyclic Diarylurea 16m. MS (ESI) 481.05 (M - H)-,
1
479.07 (M + H)+. H NMR (400 MHz, DMSO-d6) δ 1.56-1.63
(m, 2H), 1.79-1.85 (m, 2H), 1.91-2.00 (m, 2H), 4.24 (t, J ) 5.06
Hz, 2H), 4.59 (t, J ) 7.67 Hz, 2H), 5.29 (s, 2H), 7.08 (s, 1H), 7.45
(dd, J ) 7.83, 4.14 Hz, 1H), 7.88 (m, 1H), 7.97 (s, 1H), 8.15 (s,
1H), 8.56 (dd, J ) 4.76, 1.69 Hz, 1H), 8.69 (d, J ) 2.15 Hz, 1H),
9.81 (s, 1H), 10.86 (s, 1H). Anal. (C23H21ClN6O4‚0.7TFA) C, H,
N.
Macrocyclic Diarylurea 17g. The desired product was prepared
by a procedure similar to that described for 17g. To a solution of
5f (20 mg, 0.0514 mmol) in THF (3 mL) at 0 °C was added a
fresh mixture (mixture A) of 3-pyridinecarboxaldehyde (0.022 mL),
3 M H2SO4 (0.203 mL), and methanol (1 mL), followed by the
addition of NaBH4 (4 mg, 0.11 mmol). The reaction mixture was
stirred at 0 °C for 30 min and monitored by LC/MS. The addition
of mixture A and NaBH4 was repeated until the reaction was
complete. Saturated NaHCO3 was added to adjust the pH > 7. The
precipitates were collected, washed with water thoroughly, and
purified by reverse-phase HPLC, eluted with a gradient of 0-70%
of acetonitrile in 0.1% TFA aqueous solution. The desired product
was obtained in 90% yield. MS (DCI/NH3) m/z 480.11 (M + H)+.
1H NMR (500 MHz, DMSO-d6) δ 1.45-1.50 (m, 2H), 1.64-1.69
(m, 2H), 1.86-1.92 (m, 2H), 4.01 (t, J ) 5.30 Hz, 2H), 4.47 (d, J
) 6.24 Hz, 2H), 4.50 (t, J ) 8.11 Hz, 2H), 6.01 (t, J ) 6.40 Hz,
1H), 6.33 (s, 1H), 7.33 (dd, J ) 7.49, 4.37 Hz, 1H), 7.75-7.77
(m, 1H), 7.84 (s, 1H), 7.97 (s, 1H), 8.43 (dd, J ) 4.68, 1.56 Hz,
1H), 8.61 (d, J ) 1.87 Hz, 1H), 9.60 (s, 1H), 10.75 (s, 1H). Anal.
(C23H22ClN7O3‚0.1H2O‚0.9TFA) C, H, N.
Macrocyclic Diarylurea 16n. MS (ESI) 481.03 (M - H)-,
1
479.03 (M + H)+. H NMR (500 MHz, DMSO-d6) δ 1.59-1.64
(m, 2H), 1.83-1.87 (m, 2H), 1.96-2.02 (m, 2H), 4.26 (t, J ) 5.19
Hz, 2H), 4.63 (t, J ) 8.24 Hz, 2H), 5.36 (s, 2H), 7.12 (s, 1H), 7.42
(dd, J ) 7.17, 5.03 Hz, 1H), 7.64 (d, J ) 7.63 Hz, 1H), 7.92-7.95
(m, 1H), 8.03 (s, 1H), 8.20 (s, 1H), 8.64 (d, J ) 4.58 Hz, 1H),
9.85 (s, 1H), 10.94 (s, 1H). Anal. (C23H21ClN6O4‚0.6TFA) C, H,
N.
Macrocyclic Diarylurea 16o. MS (ESI) 482.03 (M - H)-,
1
484.00 (M + H)+. H NMR (400 MHz, DMSO-d6) δ 1.53-1.65
(m, 2H), 1.79-1.87 (m, 2H), 1.90-2.00 (m, 2H), 4.23 (t, J ) 4.60
Hz, 2H), 4.47 (t, J ) 5.06 Hz, 2H), 4.57-4.64 (m, 4H), 6.94 (s,
1H), 7.61 (s, 1H), 7.73 (s, 1H), 7.99 (s, 1H), 8.12 (s, 1H), 8.96 (s,
1H), 9.81 (s, 1H), 10.88 (s, 1H). Anal. (C22H22ClN7O4‚0.1H2O‚
0.8TFA) C, H, N.
Macrocyclic Diarylurea 17a. The desired product (7.7 mg, 25%)
was prepared by a procedure similar to that described for 17g. MS
1
(APCI) m/z 403.24 (M + H)+. H NMR (400 MHz, DMSO-d6) δ
Macrocyclic Diarylurea 17h. The desired product was prepared
by a procedure similar to that described for 17g by substituting
3-pyridinecarboxaldehyde with 4-pyridinecarboxaldehyde. MS (ESI)
1.54-1.62 (m, 2H), 1.78-1.85 (m, 2H), 1.92-2.00 (m, 2H), 2.78
(s, 3H), 4.21 (t, J ) 5.22 Hz, 2H), 4.58 (t, J ) 8.29 Hz, 2H), 5.33
(br s, 1H), 6.35 (s, 1H), 7.94 (s, 1H), 7.97 (s, 1H), 9.69 (s, 1H),
10.77 (s, 1H).
1
m/z 480.12 (M + H)+, 478.15(M - H)-. H NMR (300 MHz,
DMSO-d6) δ 1.42-1.53 (m, 2H), 1.58-1.70 (m, 2H), 1.84-1.95
(m, 2H), 3.96 (t, J ) 4.75 Hz, 2H), 4.53 (t, J ) 8.81 Hz, 2H), 4.60
(d, J ) 4.07 Hz, 2H), 6.23 (s, 1H), 6.28 (br s, 1H), 7.60 (s, 1H),
7.61 (s, 1H), 7.96 (s, 2H), 8.62 (s, 1H), 8.64 (s, 1H), 9.63 (s, 1H),
10.79 (s, 1H). Anal. (C23H22ClN7O3‚0.1H2O‚TFA) C, H, N.
Macrocyclic Diarylurea 17i. The desired product was prepared
by a procedure similar to that described for 17g. MS (APCI) m/z
Macrocyclic Diarylurea 17b. The desired product (9.6 mg, 30%)
was prepared by a procedure similar to that described for 17g by
replacing 3-pyridinecarboxaldehyde with acetylaldehyde. Analytical
LC/MS with two different solvent systems indicated >95% purity.
1
MS (APCI) m/z 417.29 (M + H)+. H NMR (400 MHz, DMSO-
d6) δ 1.17 (t, J ) 7.06 Hz, 3H), 1.54-1.60 (m, 2H), 1.76-1.83
(m, 2H), 1.90-1.98 (m, 2H), 3.14-3.22 (m, 2H), 4.18 (t, J ) 5.06
1
478.83 (M + H)+. H NMR (400 MHz, DMSO-d6) δ 1.38-1.57

1524 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Tao et al.
(m, 2H), 1.58-1.73 (m, 2H), 1.81-1.97 (m, 2H), 3.97 (t, J ) 5.06
Hz, 2H), 4.43 (d, J ) 5.83 Hz, 2H), 4.48-4.59 (m, 2H), 5.99 (t, J
) 5.98 Hz, 1H), 6.30 (s, 1H), 7.22 (t, J ) 7.06 Hz, 1H), 7.32 (t,
J ) 7.67 Hz, 2H), 7.35-7.41 (m, 2H), 7.92 (s, 1H), 7.96 (s, 1H),
9.62 (s, 1H), 10.76 (s, 1H). Anal. (C24H23ClN6O3‚0.1H2O‚0.1TFA)
C, H, N.
Macrocyclic Diarylurea 18c. To a mixture of 5f (50 mg, 0.13
mmol) and pyridine (5 mL) in dichloromethane (25 mL) was added
3-chloropropionyl chloride (0.037 mL, 0.39 mmol) dropwise at 0
°C. The reaction mixture was stirred at 0 °C until 5f was consumed
completely. Careful removal of solvent at low temperature (<10
°C) provided a residue. To this residue was added cyclopentylamine
(3 mL), and the mixture was heated at 80 °C for 1 h and
concentrated. The residue was purified by reverse-phase HPLC,
eluted with a gradient of 0-70% acetonitrile in 0.1% TFA aqueous
solution to provide the desired product (TFA salt, 66.8 mg, 80%
for two steps). Analytical LC/MS with two different solvent systems
indicated >95% purity. MS (ESI) m/z 528.07 (M + H)+, 526.16
(M - H)-. 1H NMR (400 MHz, DMSO-d6) δ 1.50-1.66 (m, 8H),
1.66-1.74 (m, 2H), 1.79-1.87 (m, 2H), 1.92-2.03 (m, 4H), 2.82-
2.85 (m, 2H), 3.21 (m, 1H), 3.52 (m, 1H), 4.13 (t, J ) 4.30 Hz,
2H), 4.61 (t, J ) 7.98 Hz, 2H), 7.53 (s, 1H), 8.01 (s, 1H), 8.26 (s,
1H), 8.40 (br s, 1H), 9.76 (s, 1H), 9.93 (s, 1H), 10.94 (s, 1H). HRMS
(ESI-TOF) calcd for C25H31ClN7O4 (M + H)+ 528.2126; found
528.2130.
Macrocyclic Diarylurea 17j. The desired product was prepared
by a procedure similar to that described for 17g. MS (APCI) m/z
1
468.87 (M + H)+. H NMR (500 MHz, DMSO-d6) δ 1.48-1.62
(m, 2H), 1.70-1.83 (m, 2H), 1.87-2.02 (m, 2H), 4.13 (t, J ) 5.03
Hz, 2H), 4.50 (d, J ) 4.58 Hz, 2H), 4.53-4.61 (m, 2H), 5.83 (s,
1H), 6.47 (s, 1H), 7.58 (s, 1H), 7.97 (s, 2H), 8.93 (s, 1H), 9.68 (s,
1H), 10.81 (s, 1H), 14.21 (br s, 2H). Anal. (C21H21ClN8O3‚0.2H2O‚
TFA) C, H, N.
Macrocyclic Diarylurea 17k. The desired product was prepared
by a procedure similar to that described for 17g. MS (APCI) m/z
1
485.97 (M + H)+. H NMR (500 MHz, DMSO-d6) δ 1.36-1.60
(m, 2H), 1.63-1.81 (m, 2H), 1.84-2.04 (m, 2H), 4.10 (t, J ) 5.19
Hz, 2H), 4.50-4.60 (m, 2H), 4.69 (s, 2H), 6.06 (br s, 1H), 6.51 (s,
1H), 7.89 (s, 1H), 7.93 (s, 1H), 7.96 (s, 1H), 8.94 (s, 1H), 9.66 (s,
1H), 10.79 (s, 1H). Anal. (C21H20ClN7O3S‚0.4H2O‚0.1TFA) C, H,
N.
Macrocyclic Diarylurea 18d. To a solution of 5f (20 mg, 0.051
mmol) and pyridine (3 mL) in dichloromethane (10 mL) was added
methanesulfonyl chloride (0.016 mL, 0.24 mmol) at 0 °C. The
reaction mixture was stirred for 6 h, and saturated NaHCO3 was
added to adjust pH value to 9. The precipitate was collected by
filtration, washed with water thoroughly, and recrystallized from
methanol to provide the desired product (20.2 mg, 85%). MS (DCI/
NH3) m/z 467 (M + H)+. 1H NMR (400 MHz, DMSO-d6) δ 1.66-
1.73 (m, 2H), 1.87-1.93 (m, 2H), 1.98-2.06 (m, 2H), 3.10 (s,
3H), 4.24 (t, J ) 5.22 Hz, 2H), 4.68 (t, J ) 7.98 Hz, 2H), 7.19 (s,
1H), 8.07 (s, 1H), 8.35 (s, 1H), 9.48 (s, 1H), 10.01 (s, 1H), 11.02
(s, 1H). HRMS (ESI-TOF) calcd for C18H18ClN6O5S (M - H)-
465.0753; found 465.0753. Anal. (C18H19ClN6O5S) C, H, N.
Macrocyclic Diarylurea 19a. To a solution of 5f (36 mg, 0.0925
mmol) in THF (6 mL) at 0 °C was added a fresh mixture of
formaldehyde (0.0345 mL, 0.463 mmol), 3 M H2SO4 (0.05 mL),
and THF (0.5 mL), followed by the addition of NaBH4 (11 mg,
0.28 mmol). The reaction mixture was stirred at 0 °C for 3 h.
Saturated NaHCO3 was added to adjust the pH > 7. The precipitates
were collected, washed with water thoroughly, and purified by
reverse-phase HPLC, eluted with a gradient of 0-70% acetonitrile
in 0.1% TFA aqueous solution. The desired product (33 mg, 86%)
was obtained as a yellow solid. Analytical LC/MS with two different
solvent systems indicated >95% purity. MS (ESI) m/z 415.35 (M
Macrocyclic Diarylurea 17l. The desired product was prepared
by a procedure similar to that described for 17g. MS (APCI) m/z
1
470.1 (M + H)+. H NMR (500 MHz, DMSO-d6) δ 1.49-1.61
(m, 2H), 1.71-1.85 (m, 2H), 1.86-2.04 (m, 2H), 4.11-4.21 (m,
2H), 4.52 (d, J ) 6.41 Hz, 2H), 4.54-4.60 (m, 2H), 5.87 (t, J )
6.10 Hz, 1H), 6.54 (s, 1H), 7.09 (s, 1H), 7.94 (s, 1H), 7.97 (s, 1H),
8.27 (s, 1H), 9.67 (s, 1H), 10.79 (s, 1H). Anal. (C21H20ClN7O4‚
0.5H2O) C, H, N.
Macrocyclic Diarylurea 17m. The desired product was prepared
by a procedure similar to that described for 17g. Analytical LC/
MS with two different solvent systems indicated >95% purity. MS
1
(APCI) m/z 485.22 (M + H)+. H NMR (500 MHz, DMSO-d6) δ
1.44-1.58 (m, 2H), 1.63-1.77 (m, 2H), 1.85-1.98 (m, 2H), 4.05
(t, J ) 5.19 Hz, 2H), 4.40 (d, J ) 6.10 Hz, 2H), 4.49-4.63 (m,
2H), 5.86 (t, J ) 6.10 Hz, 1H), 6.41 (s, 1H), 7.11 (d, J ) 4.88 Hz,
1H), 7.39 (d, J ) 2.14 Hz, 1H), 7.45-7.49 (m, 2H), 7.91 (s, 1H),
7.96 (s, 1H), 9.63 (s, 1H), 10.78 (s, 1H). HRMS (ESI-TOF) calcd
for C22H22ClN6O3S (M + H)+ 485.1163; found 485.1160.
Macrocyclic Diarylurea 17n. The desired product was prepared
by a procedure similar to that described for 17g. Analytical LC/
MS with two different solvent systems indicated >98% purity. MS
1
- H)-. H NMR (400 MHz, DMSO-d6) δ 1.34-1.41 (m, 2H),
1
(APCI) m/z 483.28 (M + H)+. H NMR (400 MHz, DMSO-d6) δ
1.55-1.61 (m, 2H), 1.66-1.76 (m, 2H), 2.49 (s, 6H), 3.97 (t, J )
5.06 Hz, 2H), 4.37 (t, J ) 7.98 Hz, 2H), 6.62 (s, 1H), 7.74 (s, 1H),
7.88 (s, 1H), 9.59 (s, 1H), 10.62 (s, 1H). HRMS (ESI-TOF) calcd
for C19H22ClN6O3 (M + H)+ 417.1442; found 417.1442.
1.52-1.61 (m, 2H), 1.73-1.82 (m, 2H), 1.86-2.02 (m, 2H), 3.87
(s, 3H), 4.16 (t, J ) 5.06 Hz, 2H), 4.48-4.66 (m, 4H), 5.95 (br s,
1H), 6.51 (s, 1H), 7.61 (s, 1H), 7.98 (s, 1H), 7.98 (s, 1H), 9.00 (s,
1H), 9.69 (s, 1H), 10.79 (s, 1H), 14.12 (br s, 1H).
Macrocyclic Diarylurea 19b. To a solution of 5f (20 mg, 0.051
mmol) in THF (5 mL) at 0 °C was added a fresh mixture of
acetylaldehyde (0.014 mL, 0.255 mmol), 3 M H2SO4 (0.034 mL),
and THF (0.5 mL), followed by the addition of NaBH4 (7.7 mg,
0.20 mmol). The reaction mixture was stirred at 0 °C for 1 h.
Saturated NaHCO3 was added to adjust the pH > 7. The mixture
was concentrated, and the precipitates were collected, washed with
water thoroughly, and dried to provide the desired product (22 mg,
97%) as a yellow solid. Analytical LC/MS with two different
solvent systems indicated >95% purity. MS (DCI/NH3) m/z 445.16
Macrocyclic Diarylurea 18a. To a mixture of 5f (10 mg, 0.026
mmol) and pyridine (1 mL) in dichloromethane (10 mL) was added
acetyl chloride (0.011 mL, 0.154 mmol) dropwise. The reaction
mixture was stirred for 3 h, and methanol (1 mL) was added to
quench the reaction. The resulting mixture was concentrated, and
the residue was triturated with a mixture of dichloromethane and
methanol to provide the desired product (9.8 mg, 89%) as an off-
white solid. MS (DCI/NH3) m/z 430.96 (M + H)+, 429.01(M -
H)-. 1H NMR (400 MHz, DMSO-d6) δ 1.58-1.64 (m, 2H), 1.78-
1.84 (m, 2H), 1.90-1.99 (m, 2H), 2.08 (s, 3H), 4.11-4.14 (m,
2H), 4.60 (t, J ) 8.29 Hz, 2H), 7.50 (s, 1H), 7.99 (s, 1H), 8.22 (s,
1H), 9.45 (s, 1H), 9.91 (s, 1H), 10.91 (s, 1H). Anal. (C19H19ClN6O4)
C, H, N.
1
(M + H)+. H NMR (500 MHz, DMSO-d6) δ 0.96 (t, J ) 7.02
Hz, 6H), 1.57-1.64 (m, 2H), 1.79-1.84 (m, 2H), 1.92-1.99 (m,
2H), 3.06 (q, J ) 7.02 Hz, 4H), 4.20 (t, J ) 4.88 Hz, 2H), 4.61 (t,
J ) 7.93 Hz, 2H), 6.89 (s, 1H), 7.98 (s, 1H), 8.15 (s, 1H), 9.83 (s,
1H), 10.90 (s, 1H). HRMS (ESI-TOF) calcd for C21H26ClN6O3 (M
+ H)+ 445.1755; found 445.1756.
Macrocyclic Diarylurea 18b. The desired product was prepared
by a procedure similar to that described for 18a by substituting
acetyl chloride with 2-dimethylacetyl chloride. Analytical LC/MS
with two different solvent systems indicated >95% purity. MS (ESI)
Macrocyclic Diarylurea 19c. MS (APCI) m/z 477.27 (M + H)+.
1H NMR (500 MHz, DMSO-d6) δ 1.53-1.65 (m, 2H), 1.72-1.89
(m, 2H), 1.90-2.05 (m, 2H), 3.20 (t, J ) 6.56 Hz, 4H), 3.40-
3.58 (m, 4H), 4.19 (t, J ) 5.19 Hz, 2H), 4.53-4.65 (m, 2H), 7.04
(s, 1H), 7.99 (s, 1H), 8.12 (s, 1H), 9.84 (s, 1H), 10.90 (s, 1H).
HRMS (ESI-TOF) calcd for C21H26ClN6O5 (M + H)+ 477.1653;
found 477.1656.
1
m/z 474.10 (M + H)+, 472.02 (M - H)-. H NMR (400 MHz,
DMSO-d6) δ 1.60-1.66 (m, 2H), 1.80-1.87 (m, 2H), 1.93-2.00
(m, 2H), 2.87 (s, 6H), 4.15-4.17 (m, 4H), 4.62 (t, J ) 7.98 Hz,
2H), 7.47 (s, 1H), 8.01 (s, 1H), 8.29 (s, 1H), 9.81 (s, 1H), 9.96 (s,
1H), 10.24 (s, 1H), 10.97 (s, 1H). HRMS (ESI-TOF) calcd for
C21H25ClN7O4 (M + H)+ 474.1657; found 474.1661.

Macrocyclic Checkpoint Kinase 1 Inhibitors
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7 1525
Macrocyclic Diarylurea 19d. The desired product (22 mg, 94%)
was prepared by a procedure similar to that described for 19b by
replacing acetylaldehyde with glutaraldehyde. MS (DCI/NH3) m/z
substituting 2-prop-2-ynloxytetrahydropyran with diethylamino-2-
propyne. Analytical LC/MS with two different solvent systems
indicated >95% purity. MS (ESI) m/z 482.50 (M + H)+, 481.1
(M - H)-. 1H NMR (400 MHz, DMF-d7) δ 1.40 (t, J ) 7.21 Hz,
5H), 1.72-1.79 (m, 2H), 1.90-1.96 (m, 2H), 2.00-2.09 (m, 2H),
3.48 (q, J ) 7.21 Hz, 4H), 4.31 (t, J ) 4.91 Hz, 2H), 4.53 (s, 2H),
4.75 (t, J ) 7.98 Hz, 2H), 7.44 (s, 1H), 8.18 (s, 1H), 8.50 (s, 1H),
10.16 (s, 1H), 11.00 (s, 1H). HRMS (ESI-TOF) calcd for C24H28-
ClN6O3 (M + H)+ 483.1911; found 483.1925.
1
457.18 (M + H)+. H NMR (500 MHz, DMSO-d6) δ 1.50-1.56
(m, 2H), 1.58-1.63 (m, 2H), 1.63-1.68 (m, 4H), 1.79-1.84 (m,
2H), 1.92-1.98 (m, 2H), 2.92 (t, J ) 4.68 Hz, 4H), 4.21 (t, J )
4.99 Hz, 2H), 4.61 (t, J ) 8.11 Hz, 2H), 6.82 (s, 1H), 7.98 (s, 1H),
8.13 (s, 1H), 9.82 (s, 1H), 10.86 (s, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 22.8, 23.7, 25.6, 25.8, 26.8, 28.8, 52.3, 68.4, 69.5,
104.8, 106.9, 115.4, 118.1, 121.7, 122.5, 128.2, 146.2, 147.5, 149.3,
150.6, 160.3. HRMS (ESI-TOF) calcd for C22H26ClN6O3 (M + H)+
457.1755; found 457.1754.
Macrocyclic Diarylurea 20. To a mixture of 15 (100 mg, 0.26
mmol) and triethylamine (0.039 mL, 0.28 mmol) in DMF (5 mL)
at 0 °C was added trifluoromethanesulfonyl chloride (0.03 mL, 0.28
mmol). The reaction mixture was stirred at 0 °C for 30 min, and
ice-water was added to quench the reaction. The resulting mixture
was concentrated, and the residue was purified by flash chroma-
tography, eluted with 9% ethyl acetate in dichloromethane. The
desired product (110 mg, 82%) was obtained as a white solid. MS
(ESI) 520.58 (M - H)-. 1H NMR (400 MHz, DMSO-d6) δ 1.61-
1.67 (m, 2H), 1.81-1.87 (m, 2H), 1.92-1.99 (m, 2H), 4.24 (t, J )
5.22 Hz, 2H), 4.61 (t, J ) 7.98 Hz, 2H), 7.38 (s, 1H), 8.01 (s, 1H),
8.46 (s, 1H), 10.03 (s, 1H), 11.06 (s, 1H).
Macrocyclic Diarylurea 21e. To a mixture of 20 (270 mg, 0.52
mmol), 2-prop-2-ynloxytetrahydropyran (0.436 mL, 3.10 mmol),
triethylamine (0.217 mL, 1.56 mmol), and (PPh3)4Pd (180 mg, 0.156
mmol) in DMF was added CuI (20 mg, 0.10 mmol), followed by
the addition of n-Bu4NI (288 mg, 0.78 mmol). The reaction mixture
was heated at 70 °C overnight, cooled, and concentrated. The
residue was purified by flash chromatography, eluted with 9% ethyl
acetate in dichloromethane, to provide the desired product (160
mg, 60%) as an off-white solid. MS (ESI) m/z 510.02 (M - H)-.
1H NMR (500 MHz, DMSO-d6) δ 1.44-1.55 (m, 4H), 1.59-1.65
(m, 2H), 1.66-1.76 (m, 2H), 1.78-1.83 (m, 2H), 1.90-1.96 (m,
2H), 3.48 (m, 1H), 3.76 (m, 1H), 4.21 (t, J ) 4.99 Hz, 2H), 4.43-
4.54 (m, 2H), 4.60 (d, J ) 8.11 Hz, 2H), 4.85 (t, J ) 2.96 Hz,
1H), 7.26 (s, 1H), 7.98 (s, 1H), 8.31 (s, 1H), 9.99 (s, 1H), 10.98 (s,
1H).
Macrocyclic Diarylurea 21a. A mixture of 21e (6 mg, 0.014
mmol), HOAc (2 mL), THF (1 mL), and water (0.5 mL) was heated
at 45 °C for 3 h and concentrated. The residue was suspended in
methanol, and the precipitates were collected by filtration. The
desired product was obtained as a white solid in quantitative yield.
MS (ESI) m/z 425.95 (M - H)-. 1H NMR (400 MHz, DMSO-d6)
δ 1.54-1.67 (m, 2H), 1.74-1.83 (m, 2H), 1.85-1.98 (m, 2H), 4.19
(br s, 2H), 4.35 (d, J ) 5.83 Hz, 2H), 4.58 (br s, 2H), 5.37 (t, J )
5.98 Hz, 1H), 7.26 (s, 1H), 7.94 (s, 1H), 8.31 (s, 1H), 10.01 (s,
1H), 10.99 (s, 1H). Anal. (C20H18ClN5O4) C, H, N.
Macrocyclic Diarylurea 21b. The desired product (25 mg, 60%)
was prepared by a procedure similar to that described for 21e by
substituting 2-prop-2-ynloxytetrahydropyran with but-3-yn-2-ol.
Analytical LC/MS with two different solvent systems indicated
>95% purity. MS (ESI) m/z 439.97 (M - H)-. 1H NMR (500 MHz,
DMSO-d6) δ 1.41 (d, J ) 6.71 Hz, 3H), 1.60-1.66 (m, 2H), 1.79-
1.84 (m, 2H), 1.91-1.97 (m, 2H), 4.21 (t, J ) 5.19 Hz, 2H), 4.59-
4.66 (m, 3H), 5.51 (d, J ) 5.19 Hz, 1H), 7.21 (s, 1H), 8.00 (s,
1H), 8.31 (s, 1H), 10.00 (s, 1H), 11.01 (s, 1H). HRMS (ESI-TOF)
calcd for C21H21ClN5O4 (M + H)+ 442.1282; found 442.1283.
Macrocyclic Diarylurea 21c. The desired product (22 mg, 50%)
was prepared by a procedure similar to that described for 21e by
substituting 2-prop-2-ynloxytetrahydropyran with 2-methyl-but-3-
yn-2-ol. Analytical LC/MS with two different solvent systems
indicated >95% purity. MS (ESI) m/z 454.03 (M - H)-. 1H NMR
(500 MHz, DMSO-d6) δ 1.49 (s, 6H), 1.60-1.66 (m, 2H), 1.79-
1.84 (m, 2H), 1.91-1.97 (m, 2H), 4.21 (t, J ) 4.88 Hz, 2H), 4.61
(t, J ) 7.93 Hz, 2H), 5.50 (s, 1H), 7.17 (s, 1H), 8.00 (s, 1H), 8.31
(s, 1H), 9.99 (s, 1H), 11.01 (s, 1H). HRMS (ESI-TOF) calcd for
C22H23ClN5O4 (M + H)+ 456.1439; found 350.1442.
Macrocyclic Diarylurea 22. A mixture of 21e (45 mg, 0.088
mmol) and Pt/C (5%, 20 mg) in THF (5 mL) was stirred under
hydrogen atmosphere (40 psi) for 16 h. The insoluble materials
were filtered off, and the filtrate was concentrated. The residue
was purified by flash chromatography, eluted with 9% ethyl acetate
in dichloromethane, to provide the THP-protected 22 (36 mg, 80%).
MS (ESI) m/z 513.93 (M - H)-. 1H NMR (400 MHz, DMSO-d6)
δ 1.38-1.53 (m, 4H), 1.56-1.65 (m, 3H), 1.71 (m, 1H), 1.77-
1.86 (m, 4H), 1.88-1.98 (m, 2H), 2.65-2.77 (m, 2H), 3.31-3.45
(m, 2H), 3.62-3.78 (m, 2H), 4.17-4.21 (m, 2H), 4.54 (m, 1H),
4.60 (t, J ) 8.29 Hz, 2H), 7.08 (s, 1H), 7.97 (s, 1H), 8.17 (s, 1H),
9.88 (s, 1H), 10.87 (s, 1H). A mixture of this intermediate (10 mg,
0.019 mmol), HOAc (4 mL), THF (2 mL), and water (1 mL) was
heated at 45 °C overnight and concentrated. The residue was
purified by reverse-phase HPLC to provide 22 (7 mg, 85%). MS
1
(ESI) m/z 430.07 (M - H)-. H NMR (400 MHz, DMSO-d6) δ
1.59-1.65 (m, 2H), 1.67-1.75 (m, 2H), 1.79-1.86 (m, 2H), 1.91-
1.99 (m, 2H), 2.68 (t, J ) 7.98 Hz, 2H), 3.42-3.47 (m, 2H), 4.19
(t, J ) 4.91 Hz, 2H), 4.50 (t, J ) 5.22 Hz, 1H), 4.61 (t, J ) 7.98
Hz, 2H), 7.07 (s, 1H), 7.99 (s, 1H), 8.16 (s, 1H), 9.89 (s, 1H),
10.90 (s, 1H). Anal. (C20H22ClN5O4) C, H, N.
Macrocyclic Diarylurea 23. To a mixture of 20 (50 mg, 0.096
mmol), PdCl2(PPh3)2 (8.1 mg, 0.0115 mmol), Ph3P (15.1 mg, 0.058
mmol), and LiCl (32.6 mg, 0.77 mmol) in DMF (2 mL) was added
tributylallyltin (0.059 mL, 0.192 mmol). The reaction mixture was
heated at 110 °C for 1 h and cooled. Saturated potassium fluoride
aqueous solution (1 mL) was added. The resulting mixture was
stirred for 30 min and concentrated. The residue was purified by
flash chromatography, eluted with 9% ethyl acetate in dichlo-
romethane, to provide the desired product (26 mg, 65%) as an off-
white solid. MS (ESI) m/z 411.98 (M - H)-. 1H NMR (400 MHz,
DMSO-d6) δ 1.56-1.65 (m, 2H), 1.75-1.84 (m, 2H), 1.90-1.98
(m, 2H), 3.42 (d, J ) 6.14 Hz, 2H), 4.16 (t, J ) 4.30 Hz, 2H),
4.60 (t, J ) 7.98 Hz, 2H), 5.05-5.09 (m, 2H), 5.83-6.01 (m, 1H),
7.07 (s, 1H), 7.98 (s, 1H), 8.19 (s, 1H), 9.90 (s, 1H), 10.90 (s, 1H).
Macrocyclic Diarylurea 24. A mixture of 23 (10 mg, 0.024
mmol), THF (5 mL), and water (0.55 mL) was treated with
N-methylmorpholine N-oxide (8.4 mg, 0.072 mmol), followed by
the addition of 2.5% (wt %) OsO4 in 2-methyl-2-propanol (0.04
mL) at 0 °C. The reaction mixture was stirred at room temperature
overnight and concentrated. The residue was purified by HPLC,
eluted with a gradient of 0-70% acetonitrile in 0.1% TFA aqueous
solution. The desired product (9.3 mg, 85%) was obtained as a
white solid. Analytical LC/MS with two different solvent systems
indicated >95% purity. MS (ESI) m/z 445.99 (M - H)-. 1H NMR
(400 MHz, DMSO-d6) δ 1.56-1.66 (m, 2H), 1.78-1.86 (m, 2H),
1.90-2.00 (m, 2H), 2.54-2.62 (m, 2H), 2.89 (dd, J ) 13.96, 4.76
Hz, 2H), 3.70 (m, 1H), 4.17 (t, J ) 4.91 Hz, 2H), 4.61 (t, J ) 8.29
Hz, 2H), 7.10 (s, 1H), 8.00 (s, 1H), 8.16 (s, 1H), 9.90 (s, 1H),
10.90 (s, 1H). HRMS (ESI-TOF) calcd for C20H23ClN5O5 (M +
H)+ 448.1388; found 448.1389.
Chk1 Enzymatic Inhibition Assay. The Chk1 enzymatic assay
was carried out with recombinant Chk1 kinase domain protein
covering amino acid residues 1-289 and a polyhistidine tag at the
C-terminal end. Human Cdc25c peptide substrate contained a
sequence of amino acid residues 204-225. The reaction mixture
contained 25 mM Hepes at pH 7.4, 10 mM MgCl2, 0.08 mM Triton
X-100, 0.5 mM DTT, 5 µM ATP, 4 nM [33P]-ATP, 5 µM Cdc25c
peptide substrate, and 6.3 nM recombinant Chk1 protein. Compound
vehicle DMSO was maintained at 2% in the final reaction. After
30 min at room temperature, the reaction was stopped by addition
of an equal volume of 4 M NaCl and 0.1 M EDTA, pH 8. A 40 µL
Macrocyclic Diarylurea 21d. The desired product (66% yield)
was prepared by a procedure similar to that described for 21e by

1526 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 7
Tao et al.
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roles from yeasts to mammals. DNA Repair 2004, 3, 1025-1032.
(7) Liu, Q.; Guntuku, S.; Cui, X.; Matsuoka, S.; Cortez, D.; Tamai, K.;
Luo, G.; Carattini-Rivera, S.; DeMayo, F.; Bradley, A.; Donehower,
L. A.; Elledge, S. J. Chk1 is an essential kinase that is regulated by
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aliquot of the reaction was added to a well in a Flash Plate (NEN
Life Science Products, Boston, MA) containing 160 µL of
phosphate-buffered saline (PBS) without calcium chloride and
magnesium chloride, and the plate was incubated at room temper-
ature for 10 min. The plate was then washed three times in PBS
containing 0.05% Tween-20 and counted in a Packard TopCount
counter (Packard BioScience Company, Meriden, CT).
(8) Zhao, H.; Piwnica-Worms, H. ATR-mediated checkpoint pathways
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Kinase Selectivity Assays. Kinase assays were conducted in 24
µL volumes on 384-well microplates via Tecan liquid-handling
automation. Ser/Thr-kinase assays were performed with a radio-
active FlashPlate-based assay platform. In this format, biotinylated
substrate peptide (2 µM), [γ-33P]-ATP (5 µM, 2 mCi/µmol), Chk1
inhibitors (3-10 000 nM in 2% DMSO), and enzyme were
incubated for 1 h in buffer containing 25 mM Hepes, pH 7.5, 1
mM DTT, 10 mM MgCl2, 100 µM Na3VO4, and 0.075 mg/mL
Triton X-100. The reaction was stopped with 80 µL of stop buffer
containing 100 mM EDTA and 4 M NaCl, and samples were
transferred to streptavidin-coated 384-well FlashPlates (Perkin-
Elmer, Boston, MA), which were then washed 3 times and read
with a TopCount microplate reader (Perkin-Elmer). Tyrosine kinase
reactions were performed with a time-resolved fluorescence (HTRF)
platform. In this format, biotinylated substrate peptide (0.5 µM),
ATP (10 µM-1 mM), inhibitors (3-10 000 nM in 2% DMSO),
and enzyme were incubated for 1 h in buffer containing 50 mM
Hepes, pH 7.4, 1 mM DTT, 10 mM MgCl2, 2 mM MnCl2, 100µM
Na3VO4, and 0.01% BSA. Reactions were stopped with 50 µL of
revelation buffer containing (final concentrations) Eu-conjugated
anti-pY-PT66 antibody (0.05 µg/mL) (CisBio), PycolLink strepta-
vidin-allophycocyanin conjugate (0.001 µg/mL) (Prozyme), and
60 mM EDTA in a buffer containing 25 mM Hepes, pH 7.4, 250
mM KF, 0.005% Tween-20, and 0.05% BSA. Following a 60 min
incubation with revelation buffer, the reactions were read with an
Envision fluorescence microplate reader (Perkin-Elmer) with 615
nm excitation and 665 nm emission.
Cell Proliferation Assay (MTS Assay). p53-deficient cancer
cells (HeLa or SW620 cells) were seeded in 96-well plates and
treated with the indicated doses of CPT or Dox with or without
the indicated doses of Chk1 inhibitor for 48 h. MTS reagents that
measure the amount of live cells (Promega, Madison, WI) were
then added to the cells, and the reactions were allowed to develop
for 20 min-2 h. Colorimetric measurements were taken at 490 nm
on a Spectra MAX 190 instrument from Molecular Devices
(Sunnyvale, CA).
Cell Cycle Analysis. Cell cycle analysis was performed as
described.47 Briefly, after the indicated treatments, cells were
washed one time in PBS and fixed in 70% ethanol. The fixed cells
were washed twice with PBS and treated with RNase A at 37 °C
for 30 min. The cells were then stained with propidium iodide and
incubated in the dark for 60 min or overnight before analysis. The
samples were analyzed through flow cytometry on a fluorescence-
activated cell sorter manufactured by BD Bioscience (San Jose,
CA) by use of the Cell-Quest program.
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telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation
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Iliakis, G.; Wang, Y. Ku affects the ataxia and rad 3-related/CHK1-
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Cancer Res. 2002, 62, 2483-2487. (b) Xiao, Z.; Chen, Z.; Gunasek-
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Acknowledgment. We thank the Department of Structural
Chemistry for measuring NMR and MS spectra, the High-
Through-Put Purification Group for analytical LC/MS, and the
Protein Biochemistry Group for the purification of Chk1 protein
used for X-ray crystallographic analysis.
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