Macrocyclic ureas as potent and selective Chk1 inhibitors: An improved synthesis, kinome profiling, structure-activity relationships, and preliminary pharmacokinetics

Article abstract of DOI:10.1016/j.bmcl.2007.09.063

A new series of potent macrocyclic urea-based Chk1 inhibitors are described. A detailed SAR study on the 4-position of the phenyl ring of the 14-member macrocyclic ureas 1a and d led to the identification of the potent Chk1 inhibitors 2, 5-7, 10, 13, 14,

Full text of DOI:10.1016/j.bmcl.2007.09.063

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 17 (2007) 6593–6601
Macrocyclic ureas as potent and selective Chk1 inhibitors:
An improved synthesis, kinome profiling, structure–activity
relationships, and preliminary pharmacokinetics
Zhi-Fu Tao,* Zehan Chen, Mai-Ha Bui, Peter Kovar, Eric Johnson, Jennifer Bouska,
Haiying Zhang, Saul Rosenberg, Thomas Sowin and Nan-Horng Lin
Cancer Research, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064, USA
Received 27 August 2007; revised 14 September 2007; accepted 18 September 2007
Available online 22 September 2007
Abstract—A new series of potent macrocyclic urea-based Chk1 inhibitors are described. A detailed SAR study on the 4-position of
the phenyl ring of the 14-member macrocyclic ureas 1a and d led to the identification of the potent Chk1 inhibitors 2, 5–7, 10, 13, 14,
19–21, 25, 27, and 31–34. These compounds significantly sensitize tumor cells to the DNA-damaging antitumor agent doxorubicin in
a cell-based assay and efficiently abrogate the doxorubicin-induced G2/M and camptothecin-induced S checkpoints, indicating that
the potent biological activities of these compounds are mechanism-based through Chk1 inhibition. Kinome profiling analysis of a
representative macrocyclic urea 25 against a panel of 120 kinases indicates that these novel macrocyclic ureas are highly selective
Chk1 inhibitors. Preliminary PK studies of 1a and b suggest that the 14-member macrocyclic inhibitors may possess better PK prop-
erties than their 15-member counterparts. An improved synthesis of 2 and 20 by using 2-(trimethylsilyl)ethoxycarbonyl (Teoc) to
protect the amino group not only readily provided the desired compounds in pure form but also facilitated the scale up of potent
compounds for various biological studies.
Ó 2007 Elsevier Ltd. All rights reserved.
Since nitrogen mustards were developed for cancer che-
motherapy more than six decades ago,1 DNA-damaging
agents have been a mainstay of cancer treatment. How-
ever, their clinical use is increasingly limited by their
severe toxicity to normal cells and resistance by tumor
cells.2 To overcome this limitation, the development of
adjuvant therapeutics that improve the efficacy and
selectivity of DNA-damaging agents in the clinic has re-
cently attracted much attention.3,4 Such treatments may
either sensitize tumor tissue or protect normal tissue
from DNA damage.
tiate the DNA repair process.7,8 Since p53-deficent
tumor cells lack the G1 checkpoint, they are selectively
arrested at the S or G2 checkpoint after DNA damage.
The inhibition of Chk1 abrogates the S and G2 check-
points and disrupts the DNA repair process, resulting
in premature chromosome condensation, leading to cell
death. Tumor cells, especially p53-null cells, are thereby
preferentially sensitized to various DNA-damaging
agents by inhibition of Chk 1.9,10 In contrast, normal
cells can still arrest 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.11
Checkpoint kinase 1 (Chk1) is a serine/threonine protein
kinase which plays a critical role in DNA damage-in-
duced checkpoints, and Chk1 has emerged as an attrac-
tive chemosensitization target.5,6 In response to DNA
damage, ATM and ATR kinases activate Chk1 through
phosphorylation in the SQ/TQ domain to arrest cells at
various DNA-damaging checkpoints (G1, S, G2) to ini-
Several classes of Chk1 inhibitors have been reported.6
However, it still remains a great challenge to identify
ideal Chk1 inhibitors that significantly potentiate
DNA-damaging antitumor agents without showing sin-
gle agent activity. Based on the crystallographic analysis
of a urea–Chk1 complex and molecular modeling, we
have recently discovered a class of macrocyclic Chk1
inhibitors (1).12–14 SAR studies based on 1b, a 15-mem-
ber macrocycle, have led to the identification of several
compounds that not only potently inhibit Chk1 in an
enzymatic assay, but also significantly potentiate the
Keywords: Checkpoint kinase 1; Chk1; Ureas; Kinase inhibitors;
Macrocycles; Ring-closure metathesis; DNA damage.
*
Corresponding author. Tel.: +1 847 938 6772; fax: +1 847 935
5165; e-mail: Zhi-Fu.Tao@abbott.com
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.09.063

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Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6593–6601
cytotoxicity of DNA-damaging agents to tumor cells.12
Preliminary SAR on the ring size revealed that the enzy-
matic potency of 1a–c decreased in the order of 1a
(IC50 = 6 nM, 14-member) P1b (IC50 = 7 nM, 15-mem-
ber) > 1c (IC50 = 28 nM, 16-member). This indicates
that the additional conformational flexibility resulting
from the presence of more rotatable bonds in the carbon
chain linker is detrimental. This is consistent with other
literature examples that show that the restriction of con-
formation produces more potent inhibitors.15 We there-
fore further elaborated the more constrained macrocycle
1a. Described herein are: (1) SAR based on the 14-mem-
ber macrocycle 1a and its olefin counterpart 1d; (2) the
kinome profiling of a representative compound; (3) pre-
liminary pharmacokinetics (PK); (4) an improved syn-
thesis of macrocyclic ureas.
Table 2 summarizes the SAR results at the 4-position of
the phenyl ring based on macrocyclic urea 1a. These
compounds (19–36) were also found to be potent
Chk1 inhibitors, and the SAR based on 1a is very simi-
lar to that based on 1d (Table 1); for the same substitu-
ent at the 4-position of the phenyl ring, analogues
derived from the saturated core 1a exhibit the same or
very comparable potency as those derived from the ole-
fin core 1d (3 vs 21, 4 vs 22, 7 vs 25, 18 vs 36, etc.). Nota-
bly, the diethylamino analogue 23 is 11-fold less potent
than the dimethylamino analogue 21, indicating that the
larger ethyl groups may clash with the Chk1 protein.
Compound 31 is a phosphate derivative of 30. Although
the enzymatic activity of 31 is weaker than that of its
parent compound 30, the cellular potency of 31 shows
dramatic improvement. Furthermore, the phosphate
group of 31 should improve the aqueous solubility.
Cl
O
H
H
Cl
O
H
H
N
All the potent Chk1 inhibitors identified in the enzy-
matic assay were further evaluated in an MTS assay
using HeLa cells, a p53-null human cervical cancer cell
line. The EC50 values for the compounds were deter-
mined either alone or in the presence of 150 nM of
doxorubicin (Dox), a clinical topoisomerase II inhibitor
known to arrest the G2/M checkpoint at this concentra-
tion in HeLa cells. The EC50 values for Chk1 inhibitors
in combination with Dox were calculated from the per-
centage of inhibition by Chk1 inhibitors at various con-
centrations above the background inhibition by 150 nM
Dox. The ability of Chk1 inhibitors to potentiate Dox is
represented by the ratio of the EC50 value of the inhib-
itor with Dox to that of the inhibitor alone. As shown in
Tables 1 and 2, these compounds fit into three catego-
ries: the first class of compounds show no cellular activ-
ity at all (4, 8, 11, 12, 15, 22–24, and 28–30) or show
little or no potentiation of the cytotoxicity of Dox (9,
16–18, 26, 35, and 36); the second class of compounds
show significant potentiation but also have single agent
activity (20); the third class of compounds show little or
no antiproliferative activity alone but significantly
potentiate Dox (2, 5–7, 10, 13, 14, 19, 21, 25, 27, and
31–34). This latter class of compounds fit the definition
of ideal Chk1 inhibitors. The lack of a good correlation
between Chk1 enzymatic inhibition potency and cellular
antiproliferative activity may be due to variation in
physicochemical properties such as cellular permeability
and potential off-target activity.
N
N
4
N
N
4
CN
N
N
1
CN
N
1
2
2
O
O
O
O
1a: n=1, 14-member ring, IC50=6 nM
1b: n=2, 15-member ring, IC50=7 nM
1c: n=3, 16-member ring, IC50=28 nM
1d
Examination of an X-ray co-crystal structure of a mac-
rocyclic urea–Chk1 complex12 reveals that the 4-posi-
tion of the phenyl ring is very open to a solvent
accessible region, which should be able to accommo-
date a variety of groups for further optimization. Thus,
an extensive SAR on the 4-position of the phenyl ring
was performed. The substituents at the 4-position were
selected based on our previous SAR results of the mac-
rocyclic urea-based Chk1 inhibitors.12 These groups
may improve the cellular activity or physical proper-
ties. Table 1 shows the analogues (2–18) derived from
1d. As predicted, all the analogues exhibited potent
activity for Chk1 inhibition.16 Compounds 2–12 are
macrocyclic ureas in which a substituent is linked to
the phenyl ring by a nitrogen linker. The amino ana-
logue 2 exhibits a subnanomolar IC50 against Chk1
(Table 1). The dimethylamino (3) and mono-methyla-
mino analogues (4) are equally potent to one another.
Analogues 5 and 6, with extended alkyl tails, not only
fully retain low single digit nanomolar potency but also
potentially have better solubility due to the presence of
the terminal polar hydroxyl groups. The introduction
of an aromatic group such as a thiazolyl group (7) to
the terminal position of the substituent gave Chk1 inhi-
bition activity comparable to 4. Amides (8–10) and car-
bamates (11 and 12) are also very potent but are
slightly weaker than the alkyl amino analogues. Com-
pounds 13–18 are macrocyclic ureas in which a substi-
tuent is linked to the phenyl ring by an oxygen atom.
All these compounds have polar moieties on the termi-
nus of the substituent that are intended to point into
the solvent exposed area. The polar moieties may be
solvated and therefore may boost the potency as previ-
ously observed.12 Indeed, compounds 13–18 are very
potent Chk1 inhibitors with IC50 values ranging from
subnanomolar to low single digital nanomolar.
To confirm the cellular activity obtained in the above-
described MTS assay, and to make sure the biological
activity of the Chk1 inhibitors is truly expressed through
the abrogation of DNA damage-induced checkpoints,
fluorescence-activated cell sorting (FACS) analysis of
cell cycle profiles was performed. H1299 cells were trea-
ted with Chk1 inhibitors in the presence and absence of
Dox and their detailed cell cycle kinetics were assessed
by FACS analysis. As predicted, Dox itself induced a
remarkable G2/M-phase arrest at a concentration of
500 nM. The EC50 values for abrogation of the G2/M
checkpoint caused by Dox in the presence of various
concentrations of Chk1 inhibitors are shown in Tables
1 and 2. FACS analysis charts of H1299 cells treated
with Dox in the presence or absence of Chk1 inhibitor

Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6593–6601
Table 1. SAR and cellular activities of unsaturated macrocyclic Chk1 inhibitors
6595
H
N
O
Cl
N
N
N
H
R
CN
O
O
Compound
R=
Chk1 inhibition
(IC50, nM)
MTS EC50 (lM) compound
+ Dox/ (compound alone)
Potentiation
ratio
FACS EC50 (lM) compound
+ Dox/ (compound alone)
2
3
NH2
0.3
1.2/>59.3
>49
<0.03/>10
N
2
1.9/18.7
10
1.7/>10
HN
HO
4
5
6
2
1
2
>5.9/>59.3
1.5/34.1
NA
22
>10/>10
N
NA
HO
HO
N
H
2.3/>59.3
>26
1.3/>10
NH
7
8
9
4
6
6
0.59/13.6
>5.9/>59.3
2.9/5.4
25
NA
2
0.2/>10
>10/>10
>10/>10
N
S
O
NH
O
N
NH
Cl
N
H2N
O
10
11
12
3
1.9/>59.3
>31
NA
NA
0.09/>10
>10/>10
NA
N
H
O
O
N
H
3
>5.9/>59.3
>5.9/>59.3
N
O
O
10
O
N
13
14
OH
0.3
1
0.93/45.7
2.3/>59.3
49
0.7/>10
1.9/>10
>26
O
HO
O
15
16
17
2
>5.9/>59.3
4.2/14.1
1.1/1.9
NA
3
NA
HO
O
O
1
>10/>10
>10/>10
N
O
0.3
2
N
O
O
N
O
18
1
0.7/1.2
2
>10/>10
N

6596
Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6593–6601
Table 2. SAR and cellular activities of saturated macrocyclic Chk1 inhibitors
H
N
O
Cl
N
N
N
H
R
CN
O
O
Compound
R=
Chk1 inhibition
(IC50, nM)
MTS EC50 (lM) compound
+ Dox/ (compound alone)
Potentiation
ratio
FACS EC50 (lM) compound
+ Dox/ (compound alone)
19
20
H
6
3.08/57.6
0.2/3.4
19
28
1.3/>10
NH2
1
2
2
<0.03/2.6
N
21
22
1.8/54.5
30
0.54/>10
>10/>10
HN
>5.9/>59.3
NA
23
24
N
23
3
>5.9/>59.3
>5.9/>59.3
NA
NA
>10/>10
NA
N
H
NH
25
4
1.0/59.3
>59
NA
N
S
O
NH
O
26
27
4
4
5.1/24.3
5
NA
NA
N
NH
2.6/>59.3
23
Cl
N
O
O
N
H
28
29
3
6
>5.9/>59.3
>5.9/>59.3
NA
NA
>10/>10
N
O
O
NA
O
N
O
30
31
OH
0.3
8
>5.9/>59.3
0.23/17.4
NA
78
NA
NA
HO P
HO
32
33
34
8
1
3
2.0/>59.3
1.5/>59.3
0.9/13.9
>30
>40
18
3.2/>10
2.5/>10
NA
O
HO
O
HO
O
H3CO
O
35
36
1
1
2.1/5.0
1.2/2.8
2
2
>10/>10
>10/>10
N
O
O
N
O
N

Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6593–6601
6597
2 are shown in Figure 1a. Ideal Chk1 inhibitors (2, 6, 7,
10, 13, 14, 19, 21, 32, and 33) did not affect the regular
cell cycle profile even at high concentrations (up to
10 lM), but efficiently abrogated the Dox-induced-G2/M
checkpoint with subnanomolar to low single digital
micromolar EC50 values. Notably, compounds that do
not show cellular activity at all (4, 8, 11, 22, 23, and
28) or only show little or no potentiation of the cytotox-
icity of Dox (9, 16–18, 35, and 36) do not abrogate the
Dox-induced-G2/M checkpoint even at 10 lM concentration.
Figure 1. FACS profiles of cancer cells treated with DNA-damaging agents in the presence or absence of Chk1 inhibitor 2. 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. (a) H1299 cells treated with Dox. (b) SW620 cells treated with CPT.
Table 3. Kinase selectivity of macrocyclic Chk1 inhibitor 25
Kinase
Kia (lM)
Kinase
Kia (lM)
Kinase
Kia (lM)
Kinase
Kia (lM)
CHK1
GSK3a
PLK3
SRPK1
AUR1
CDK2
IRAK1
AKT1
MK2
0.004
0.911
1.085
2.307
2.025
3.088
3.011
3.698
3.886
3.401
0.949
4.266
4.800
1.539
5.195
1.911
6.560
7.157
8.176
3.482
1.119
6.846
6.812
9.182
>1.503
>0.706
>1.453
>9.524
>9.275
>1.549
CDK7
CDK9
CHK2
CLK2
>1.525
>1.644
>5.578
>8.753
>8.951
>6.667
>1.677
>8.571
>5.833
>6.916
>7.826
>7.166
>9.570
>5.804
>1.803
>8.000
>1.438
>3.750
>8.750
>1.863
>4.737
>0.440
>2.481
>0.809
>7.059
>3.338
>7.664
>0.991
>9.921
>1.525
IKKa
IKKb
INSR
>3.75
PKCc
PKCf
>8.750
>8.333
>1.777
>1.683
>8.195
>1.525
>1.488
>1.424
>8.980
>9.217
>9.105
>7.500
>7.368
>1.442
>1.638
>1.936
>1.606
>9.334
>1.525
>1.596
>1.526
>1.936
>2.481
>4.536
>1.518
>8.573
>1.391
>2.857
>8.913
>7.500
>2.338
>5.238
>1.453
>1.722
>3.056
>4.118
>8.876
>2.701
>9.546
>1.667
>8.493
>9.234
>1.667
>9.350
>1.892
>1.736
>1.650
>6.516
>1.611
>1.549
>9.242
>1.667
>6.711
>6.668
>9.727
>5.454
>4.902
>7.500
>8.889
PKG1A
PKG1B
PKN2
PRAK
PRKX
PIM2
JAK2
JAK3
CLK4
CTAK-1
CAMK1
CAMK4
CK2
JNK1a1
JNK2a2
KDR
LCK
LIMK1
LYN
PKD2
PRKCN
ROCK1
ROCK2
RSK2
DYRK1A
MSK2
EMK
CK1a 1
CK1d
CK1c2
DCAMKL2
DYRK3
EGFR
EPHA2
ERBB2
ERBB4
ERK2
MINK1
MK3
AKT2
AMPK
PBK
MLK1
MNK2
MSK1
MSSK1
MUSK
MAP4K2
NLK
STK31
STK33
SRC
AUR2
PLK1
AKT3
SGK
TAK1-TAB1
TAOK2
TBK1
PDK1
CKIT
FLT4
FGFR3
FGFR
FLT1
TSSK1
TSSK2
TYK2
TRKA
TRKB
WNK2
ZAK
NEK11
NEK2
NEK3
NEK4
P70S6K
PAK1
PAK4
PHKk2
PKA
P38d
PIM1
FLT3
FYN
ARK5
ABL
BLK
GSK3b
HIPK2
HIPK4
IGF1R
IRAK4
ITK
ZIPk
CDC42BPA
CDK5
CDK6
CMET
CDC2
p38c
PKCd
a The inhibition constant (Ki) values are calculated from the Cheng-Prusoff equation, Ki = IC50/1 + ([ATP]/Km).

6598
Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6593–6601
The results obtained from the MTS assays are very con-
sistent with those derived from FACS analysis. In addi-
tion, we found that the Chk1 inhibitors efficiently
abrogate camptothecin (CPT)-induced S arrest. CPT
damages DNA through the inhibition of topoisomerase
I and is known to cause S phase arrest. As shown in Figure
1b, compound 2 alone did not alter the regular cell cycle
profile, but it efficiently abrogated CPT-induced S arrest
and forced the cells further into the G2/M phase. The
subG0/G1 population indicates the resulting apoptosis.
transition. Recently it has been reported that selectivity
for Chk1 over Cdk7 is essential for the abrogation of
DNA damage-induced checkpoint arrests.17
To investigate how the ring size affects PK, compounds 1a
and b were intraperitoneally administered to mice with a
dosage of 10 mg/kg. As shown in Figure 2, both com-
pounds show moderate plasma exposure with AUC val-
ues of 3.8 lmol h/L for 1a and 1.5 lmol h/L for 1b. It is
very interesting to note that the 14-member macrocycle
1a exhibits over 2-fold more exposure than the 15-mem-
ber 1b.
The macrocyclic urea Chk1 inhibitor 25 was tested
against a panel of 120 kinases to obtain a selectivity pro-
file, and excellent selectivity was observed. As shown in
Table 3, the compound exhibited no inhibitory activity
toward a majority of kinases in this panel even at the
highest concentration tested. Moreover, compound 25
does not inhibit Chk2, an important DNA damage-
involving checkpoint kinase, or Cdk7, a cyclin-depen-
dent kinase that is critical in the regulation of cell cycle
Scheme 1 outlines the syntheses of 2 and 20, which are
the key intermediates toward macrocyclic ureas in which
a nitrogen atom links the substituent to the phenyl ring.
Compound 39 was prepared in high yield by the cou-
pling of 37 and 38, both of which were prepared as pre-
viously described,18 followed by reduction of the nitro
group. The macrocyclization of 39 was first attempted
with Grubbs catalysts (1st and 2nd generation) in
DCM under reflux. LCMS indicated a 40–60% conver-
sion of 39 to 2. Hoveyda-Grubbs catalyst (2nd) was then
tried and gave better conversion (70–90%). However, it
was very difficult to obtain 2 in pure form due to con-
taminants of dark green color originating from the cat-
alysts. Attempts to remove the color by using reported
methods19–21 were not successful. In addition, the con-
version of 2 to 20 by hydrogenation also proved to be
problematic. We therefore decided to protect the amino
group of 39. After screening several protecting groups,
2-(trimethylsilyl)ethoxycarbonyl (Teoc)22 stood out as
the best. Thus, 39 was coupled with Teoc-Cl in the pres-
ence of pyridine to give 40 in quantitative yield. Cycliza-
tion of 40 in the presence of Hoveyda-Grubbs catalyst
(2nd) provides 41 in 90% isolated yield. Deprotection
of 41 in TFA at 0 °C quantitatively produced 2. Satura-
tion of olefin 41 in the presence of Wilkinson’s catalyst
smoothly gave 42. Compound 20 was obtained by the
deprotection of 42 in quantitative yield. This improved
10
Plasma levels in CD-1 mouse
Compound 1a
1
0.1
Compound 1b
0.01
0
1
2
3
4
5
6
7
8
Time (hrs)
Figure 2. Plasma levels of compounds 1a and b with intraperitoneal
(IP) dosing in CD-1 mice.
Cl
O
Cl
O
N
Cl
H2N
NH
N
O
N
O2N
HN
O
Ph
N
H
H2N
b
N
H
NH
a
N
+
NH2
O
90%
O
N
O
O
CN
O
N
O
CN
37
38
39
CN
2
e
100%
Cl
Cl
H
N
H
O
N
Cl
N
O
NH
N
R'
d
R
O
N
c
HN
R
42: R'=R
e
100%
N
NH
39
b
N
H
H
N
NH
99%
H
O
90%
O
O
N
92%
O
CN
N
O
N
20: R'=H
O
O
CN
41
Si
40
CN
O
R=
Scheme 1. Reagents and conditions: (a) i—DMF, 50 °C; ii—iron powder, NH4Cl, EtOH, H2O, 80 °C; (b) Hoveyda-Grubbs catalyst, CH2Cl2, reflux;
(c) RCl, THF, pyridine; (d) H2, Wilkinson’s catalyst, THF, rt; (e) TFA, 0 °C.

Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6593–6601
6599
synthetic sequence not only readily provided 2 and 20 in
pure form, but also facilitated the scale up of potent
compounds for various biological studies.
bromoethyl chloroformate, was treated with morpholine
to provide 11 and 12 concurrently. Amidation of 39 gave
44, which was readily cyclized to provide 45. Removal of
the Fmoc group in piperidine produced 10. Compounds
21–29 were prepared using routes similar to those in
Scheme 2.
Scheme 2 depicts the synthesis of compounds 3–12. Com-
pounds 3–7 were prepared by the reductive amination of 2
in moderate to good yield. The coupling of 2 with the cor-
responding acyl chlorides gave amides 8 and 9. Com-
pound 43, obtained through the coupling of 2 with
Scheme 3 shows the synthetic routes to macrocyclic ureas
in which an oxygen atom links the substituent to the
Cl
3 - 7
H
N
O
N
O
a
30% - 90%
c
O
N
H
NH
d
2
11
12
+
80%
O
50%-60%
b
Br
N
O
8 - 9
CN
Cl
43
O
N
H
Cl
N
O
NH
O
f
HN
g
N
H
NH
e
O
10
H
N
O
N
85%
FmocHN
39
90%
43%
O
O
N
CN
FmocHN
N
44
O
CN
45
Scheme 2. Reagents and conditions: (a) RCHO, H2SO4, THF, then NaBH4, 0 °C; (b) RCOCl, pyridine, CH2Cl2, rt; (c) bromoethyl chloroformate,
pyridine, DMF–THF; (d) morpholine, DMF–THF; (e) Fmoc-L-Ala-Cl, aqueous Na2CO3 (1 M), DCM; (f) Hoveyda-Grubbs catalyst (2nd), DCM,
rt; (g) piperidine, DMF.
Cl
O
N
Cl
O
N
Cl
SEMO
NH
N
O
N
SEMO
HN
O
Ph
SEMO
b
N
H
NH
H
a
N
+
NH2
90%
O
89%
O
N
O
O
CN
O
48
N
O
CN
46
38
47
CN
c
95%
O
Cl
Cl
O
Cl
HO
SEMO
O
HO
O
N
N
H
NH
N
NH
N
NH
d
c
95%
48
O
H
H
O
N
85%
N
N
N
N
CN
O
O
O
CN
30
CN
49
13
Cl
O
O
N
g
( )n
13
e
16 - 17
N
H
NH
O
60-70%
f
90-100%
O
14, 15
70-80%
h
THP
86%
N
O
50
18
CN
Scheme 3. Reagents and conditions: (a) DMF, 50 °C; (b) Hoveyda-Grubbs catalyst, CH2Cl2, reflux; (c) HCl, dioxane–CH2Cl2–EtOH; (d) H2,
Wilkinson’s catalyst, THF, rt; (e) RBr, Cs2CO3, DMF; (f) HOAc, THF–H2O; (g) ROH, polymer-supported PPh3, DBAD, THF; (h) 1,40-
bipiperidine-10-carbonyl chloride, pyridine, DMF–THF.

6600
Z.-F. Tao et al. / Bioorg. Med. Chem. Lett. 17 (2007) 6593–6601
O
N
O
Cl
Cl
O
N
Cl
NH
N
NH
N
O
P
NH
N
N
H
O
HO
SEMO
BnO
a
b
H
N
N
H
N
OBn
O
91%
90%
O
O
O
CN
O
CN
O
CN
51
52
47
Cl
O
O
Cl
O
O
P
O
O
N
P
O
O
HO
BnO
OH
N
H
NH
c
d
OBn
N
H
NH
85%
83%
N
N
31
O
N
53
O
CN
CN
Scheme 4. Reagents and conditions: (a) HCl, dioxane–CH2Cl2–EtOH; (b) P(H)(O)(OBn)2, CCl4, DIEA, DMAP, CH3CN; (c) Hoveyda-Grubbs
catalyst, CH2Cl2, reflux; (d) H2, Wilkinson’s catalyst, THF, rt.
4-position of the phenyl ring. Compound 46 was prepared
according to a literature procedure.18 Macrocyclic pre-
cursor 48 was synthesized by the olefin metathesis of 47
in the presence of Hoveyda-Grubbs catalyst (2nd).
Deprotection of 48 under acidic conditions gave 13 in
excellent yield. Hydrogenation of 48 smoothly produced
49, which was deprotected to provide 30. Alkylation of
13 with organic bromides gave ethers 50, which were trea-
ted with acetic acid to provide alcohols 14 and 15. Alter-
natively, alkylation of 13 by alcohols under
Mitsunobu’s conditions gave 16 and 17 in good yield.
Compound 18 was obtained by the pyridine-catalyzed
coupling of 13 with 1,40-bipiperidine-10-carbonyl chlo-
ride. Compounds 32–36 were prepared using routes simi-
lar to those in Scheme 3.
ber counterparts. The improved synthesis of 2 and 20 by
using Teoc as an amine protecting group not only readily
provided the desired compounds in pure form but also
facilitated the scale up of potent compounds for various
biological studies.
Acknowledgments
The authors thank Drs. Zhenping Tian and Daniel Plata
for scaling up a pyrazine-containing intermediate, Drs. Le
Wang, Kent D. Stewart, and Chang Park for helpful dis-
cussion on the design of macrocyclic kinase inhibitors.
The authors also thank the Department of Structural
Chemistry for measuring NMR and MS spectra, the
High-Through-Put Purification Group for analytical
LC–MS.
The synthesis of 31 is shown in Scheme 4. Compound
51, obtained by the deprotection of 47, was phosphory-
lated with dibenzyl phosphate using a recently reported
procedure23 to give 52 in excellent yield. The ring-clo-
sure olefin metathesis of 52 was effected by Hoveyda-
Grubbs catalyst to provide 53. The debenzylation and
olefin saturation of 53 were effected simultaneously in
the presence of Wilkinson’s catalyst under a hydrogen
atmosphere to give the final product 31.
References and notes
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In summary, a new series of potent macrocyclic urea-
based Chk1 inhibitors are described. A detailed SAR
study on the 4-position of the phenyl ring of 1a and d
led to the identification of the potent Chk1 inhibitors 2,
5–7, 10, 13, 14, 19, 21, 25, 27, and 31–34. These com-
pounds significantly sensitize tumor cells to the DNA-
damaging antitumor agent Dox in a cell-based assay
and efficiently abrogate the Dox-induced G2/M and
CPT-induced S checkpoints, indicating that their potent
biological activities are mechanism-based through Chk1
inhibition. Kinome profiling analysis of a representative
macrocyclic urea 25 against a panel of 120 kinases indi-
cates that these novel macrocyclic ureas are highly selec-
tive Chk1 inhibitors. The preliminary PK (IP) studies of
1a and b suggest that the 14-member macrocyclic inhibi-
tors may possess better PK properties than their 15-mem-
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Li, G.; Wang, G.; Tao, Z.-F.; Tong, Y.; Lin, N.-H.; Sham,

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5 lM ATP, 4 nM 33P ATP, 5 lM Cdc25c peptide sub-
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more details, see Refs. 11 and 12.
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