Design and synthesis of pyridine-pyrazolopyridine-based inhibitors of protein kinase B/Akt

Article abstract of DOI:10.1016/j.bmc.2007.01.010

Thr-211 is one of three different amino acid residues in the kinase domain of protein kinase B/Akt as compared to protein kinase A (PKA), a closely related analog in the same AGC family. In an attempt to improve the potency and selectivity of our indazole-pyridine series of Akt inhibitors over PKA, efforts have focused on the incorporation of a chemical functionality to interact with the hydroxy group of Thr-211. Several substituents including an oxygen anion, amino, and nitro groups have been introduced at the C-6 position of the indazole scaffold, leading to a significant drop in Akt potency. Incorporation of a nitrogen atom into the phenyl ring at the same position (i.e., 9f) maintained the Akt activity and, in some cases, improved the selectivity over PKA. The structure-activity relationships of the new pyridine-pyrazolopyridine series of Akt inhibitors and their structural features when bound to PKA are also discussed.

Full text of DOI:10.1016/j.bmc.2007.01.010

Bioorganic & Medicinal Chemistry 15 (2007) 2441–2452
Design and synthesis of pyridine–pyrazolopyridine-based
inhibitors of protein kinase B/Akt
Gui-Dong Zhu,a,* Jianchun Gong,a Viraj B. Gandhi,a Keith Woods,a Yan Luo,a
Xuesong Liu,a Ran Guan,a Vered Klinghofer,a Eric F. Johnson,a Vincent S. Stoll,b
Mulugeta Mamo,b Qun Li,a Saul H. Rosenberga and Vincent L. Girandaa
aCancer Research, GPRD, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064-6101, USA
bStructural Biology, GPRD, Abbott Laboratories, 100 Abbott Park Road, Abbott Park, IL 60064, USA
Received 2 November 2006; revised 10 January 2007; accepted 11 January 2007
Available online 17 January 2007
Abstract—Thr-211 is one of three different amino acid residues in the kinase domain of protein kinase B/Akt as compared to protein
kinase A (PKA), a closely related analog in the same AGC family. In an attempt to improve the potency and selectivity of our inda-
zole–pyridine series of Akt inhibitors over PKA, efforts have focused on the incorporation of a chemical functionality to interact
with the hydroxy group of Thr-211. Several substituents including an oxygen anion, amino, and nitro groups have been introduced
at the C-6 position of the indazole scaffold, leading to a significant drop in Akt potency. Incorporation of a nitrogen atom into the
phenyl ring at the same position (i.e., 9f) maintained the Akt activity and, in some cases, improved the selectivity over PKA. The
structure–activity relationships of the new pyridine–pyrazolopyridine series of Akt inhibitors and their structural features when
bound to PKA are also discussed.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
As summarized in two review articles,5,6 a significant
number of small molecule inhibitors of Akt have been
developed and shown to sensitize tumor cells to apopto-
tic stimuli. Some of these Akt inhibitors slowed the
tumor growth in animal models. We have reported
several series of potent and ATP competitive inhibitors
of Akt.7 These single-digit nanomolar inhibitors have
demonstrated very good selectivity over more distinct
members of protein kinase family but are less selective
against some of the highly homologous kinases, espe-
cially the closely related protein kinase A (PKA). By
using structure-based approaches, Breitenlechner et al.
discovered several nanomolar Akt selective inhibitors
over PKA.8 Herein, to further improve the potency
and selectivity of our indazole–pyridine series of inhibi-
tors, we have incorporated a variety of substituents or
chemical moieties at the C-6 position of the indazole
scaffold to interact with the hydroxy group of Thr-211,
one of the three different amino acid residues in the
kinase domain of Akt as compared to PKA. These
efforts have resulted in the discovery of a new series of
potent and selective pyrazolopyridine inhibitors of
Akt. An X-ray structure of our reference inhibitor 1
bound to PKA provided guidance in these efforts.
Protein kinase B, also known as Akt, is a 57-kDa serine/
threonine kinase that plays critical roles in anti-apopto-
tic processes.1 Overexpression of Akt can result from
inactivation of tumor suppressor PTEN and has been
correlated with an increasing number of human can-
cers.2 Akt is also responsible for promoting survival sig-
nals that downregulate apoptotic pathways and
contribute to cancer progression.3 Correlation between
resistance to chemotherapy and Akt activation has also
been observed in prostate cancer cell lines and in human
tumor tissue.4 Inhibition of Akt alone or in combination
with other standard cancer chemotherapeutics results in
increased programmed death of cancer cells, leading to
decreased tumor growth and tumor resistance to
chemotherapy.
Keywords: Protein kinase B; Akt inhibitor; Serine/threonine kinase;
Pyrazolopyridine.
*
Corresponding author. Tel.: +1 847 935 1305; fax: +1 847 935
5165; e-mail: gui-dong.zhu@abbott.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.01.010

2442
G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
H
N
R
Br
NH
Br
Br
NH2
i
ii
R
R
N
N
O
N
N
N
N
O2N
H2N
H
R
N
12a: R= Me
12b: R= H
11a: R= Me
11b: R= H
10a: R= Me
10b: R= H
1: R= H
2: R= Cl
3: R= CF
iv
R= H
iii
3
Cl
N
Br
iii
4j, 4g or 4f
N
N
H
13
2. Chemical synthesis
Br
Br
Br
ii
v
N
A general synthesis of the indazole–pyridine series of
Akt inhibitors and pyrazolo[3,4-c]pyridine–pyridine-
based inhibitors is depicted in Scheme 1. Stille reactions
of either an arylbromide 4 or arylchloride 5 with
trimethylstannane 7,9 under the catalysis of tri-o-tolyl-
phosphine and Pd2(dba)3 (for bromide 4) or bis(tri-
tert-butylphosphine)palladium (0) (for chloride 5),
provided compound 8. While the typical yields for the
coupling of bromoindazoles 4a and 4b with 7 were
around 70%, the reactions of 4c–4j, under the same con-
ditions, were less efficient, affording less than 35% of
desired products. In the case of 4g, the best catalysis
was found to be either bis(tri-tert-butylphosphine)palla-
dium (0) or Pd2(dba)3, leading to desired product in 20%
yield. Other ligands including Nolan’s,10 BINAP, and
tri-2-furylphosphine did not furnish any desired prod-
uct. To improve the coupling reaction, we also explored
reversal of the Stille coupling partners. However, we
were not able to synthesize trimethylstannane 6 by either
the standard metal-exchange/Me3SnCl procedure or a
N
H
NO2
H2N
H2N
NO2
14
15
16
iv
iii
Cl
N
Br
iii
4e or 4c
N
H
NO2
17
Scheme 2. Reagents and conditions: (i) H2, Raney Ni, THF, yield:
85–100%; (ii) NaNO2, HOAc, H2O, rt, 3 days, yield: 63–86%;
(iii) (Boc)2O, Et3N, DMAP, CH3CN, rt, overnight, yield: 90–100%;
(iv) 10–13% NaClO, H2O, NaOH, 0 ꢁC, 5 h, yield: 77–95%; (v) KNO3,
H2SO4, rt, overnight, 95%.
CF3
N
NH2
i
ii
CF3
N
H
BOC
NH
18
19
Me3Sn
O
R2
CF3
CF3
N
CF3
N
Br
Br
NHR3
R2
N
iii
from 20
N
+
7
R1
O
Ar-Br (4)
Ar-Cl (5)
N
N
N
H
H
Boc
i or ii
Br
N
20
21
4b
8: R3 =BOC
9: R3 =H
R2 = 3-Indole or Phenyl
iii
Scheme 3. Reagents and conditions: (i) NaNO2, HOAc, H2O, rt,
3 days, yield: 49% (as a 1:1 mixture with an uncharacterized side-
product); (ii) NBS, CH3CN, rt, 2 days, yields: 35% of 20 and 6% of 21;
(iii) (Boc)2O, Et3N, DMAP, CH3CN, rt, yield: 86%.
X
Cl
N
Br
Y
N
N
N
N
Boc
Boc
5
6
R
BOC
O
4a: X= Cl, Y= CH
4b: X= CF3, Y= CH
4c: X= H, Y= C-NO2
4d: X= H, Y= C-NH2
4e: X= Cl, Y= C-NO2
4f: X= H, Y= N
NH
N
NH
Cl
i
N
SnMe3
O2N
N
N
N
N
22
23: R= NO2
24: R= NH2
ii
Boc
4g:X= CH3, Y= N
4j: X= Cl, Y= N
iii
4k: X= CH3, Y= N+- O-
9f
Scheme 1. Reagents and conditions for Ar–Br: (i) Pd2(dba)3, P(o-tol)3,
Et3N, DMF, 80 ꢁC, 6 h; for Ar–Cl: (ii) bis(tri-tert-butyl-
phosphine)palladium (0), CsF, dioxane, 90 ꢁC, overnight, 30%; (iii)
CF3CO2H, CH2Cl2, yield: 40–90%.
Scheme 4. Reagents and conditions: (i) compound 7 (R2 = 3-indole),
Pd2(dba)3, P(2-Fur)3, Et3N, DMF, 75 ꢁC, 8 h, yield: 64%; (ii) H2,
Raney Ni, THF, rt, 3 h, yield: 89%; (iii) a—NaNO2, HOAc, H2O, rt, 3
days, yield: 30%; b—CF3CO2H, CH2Cl2, yield: 40%.

G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
2443
palladium-catalyzed reaction [Pd(PPh3)4/Me3SnSnMe3/
H
Boc
O
N
N
NH
toluene/100 ꢁC]. The Stille reaction of arylchloride 5
with 7, using tri-o-tolylphosphine, tri-2-furylphosphine,
Xantphos, and 2-dicyclohexylphosphino-20-(N,N-dim-
ethylamino)-biphenyl (Cy-MAP) as ligands, failed to
provide any coupled product as well. Boc-deprotection
of compound 8 with trifluoroacetic acid afforded 9.
N
i, ii
Cl
N
8j
H
N
N
NH2
N
O
As shown in Scheme 2, aryl bromides 4c–j were
synthesized by oxidative cyclization of appropriately
substituted 3-amino-4-methylpyridine (e.g., 11) or
ortho-aminotoluene (e.g., 15). Intermediates 11a and
11b were in turn prepared by reduction of nitro-precur-
sor 10. Nitration of commercially available 14 with
one equivalent of KNO3 in concentrated H2SO4
afforded intermediate 15 in nearly quantitative yield.
Treatment of 12 or 16 with aqueous sodium hypochlo-
rite provided 3-chloropyrazolopyridine 13 or indazole
17. Boc-protection of 12, 13, 16, and 17 then furnished
compound 4.
R
N
25a: R= Vinyl
25b: R= 2-Furyl
25c: R=
N
25d: R= Ph
Scheme 5. Reagents and conditions: (i) a—tributylvinyltin, 2-dic-
yclohexylphosphino-20-(N,N-dimethylamino)biphenyl, Pd2(dba)3, Et3N,
DMF, 85 ꢁC, 3 h, Yield: 64%; b—tributylvinyltin was replaced with
2-tributylstannylfuran, yield: 70%; c—replaced with N-methyl-2-tribu-
tylstannylpyrrole, yield: 37%, d—replaced with tributylphenyltin, yield:
52%; (ii) CF3CO2H, CH2Cl2, yield: 60–80%.
The synthesis of 3-trifluoromethylindazole 4b is
described in Scheme 3. Starting material 18 was
prepared as a 1:4 mixture with aniline according to a
literature procedure.11 Treatment of the mixture with
sodium nitrite in acetic acid as described in Scheme 2
provided indazole 19 in 49% yield as a 1:1 mixture with
an uncharacterized side product. Bromination of the
impure 19 with N-bromosuccinimide afforded 35% of
5-bromo derivative 20 and 6% of 7-bromo analog 21.
Boc-protection of 20 provided 4b, which was converted
to 3 as described in Scheme 1.
were also introduced at the same position of the indazole,
in an effort to block the potentially labile site of
metabolism.
To structurally understand the binding mode of our
indazole–pyridine series of Akt inhibitors and to find
potential sites of modification to improve selectivity
over other closely related protein kinases, we crystallized
our reference compound 1 with PKA, a highly homolo-
gous protein kinase of Akt in the same AGC family. The
Due to the poor yields of the Stille coupling of 7 with
both chloride 5 and bromide 4f, an alternative synthetic
route as shown in Scheme 4 was also investigated. The
commercially available chloride 22 was first coupled
with trimethylstannane 7 (R2 = 3-indole), under the
catalysis of Pd2(dba)3 and tri-2-furylphosphine, afford-
ing 23 in 64% yield. Reduction of 23, followed by oxida-
tive cyclization of 24 with sodium nitrite, gave, after
Boc-deprotection, 9f in 12% overall yield from 23.
˚
structure was resolved at 2.2 A and is illustrated in
Figure 1. Corresponding residues for Akt in the ATP
binding site are shown on left. As shown in Figure 1,
four key hydrogen bonding interactions of 1 (Val-123,
Glu-121, Lys-72, and Asn-171/Asp-184) are important
for its high-affinity to PKA (IC50 = 18 nM). The hydro-
phobic indole moiety in 1 fits nicely underneath the
glycine-rich loop. As indicated on the left, Akt has three
different amino residues, namely Thr-211, Met-173, and
Ala-123, in the ligand-binding domain. It was thought
that introduction of an appropriate functionality at
Syntheses of 3-substituted pyrazolopyridine analogs
25a–d are described in Scheme 5. The 3-chloro-function-
ality in 8j, prepared according to the general protocol
described in Scheme 1, readily underwent Stille reaction
with a variety of tin reagents in modest to good yields,
using Cy-MAP as ligand. Boc-deprotection of the cou-
pled products afforded 25a–d.
Site of Modification
Glu91
Lys72
Akt1: Thr211
Akt1: Met
Val104
O
H3N
N
O
Lys Binding Motif
Leu173
Thr183
OH
3. Results and discussion
O
50'S Loop
NH
O
H
Glu121
N
HN
Phe
54
Hinge Binding
Motif
+
N
We previously reported our indazole–pyridine series of
compounds as exemplified by 1 as very potent Akt inhib-
itors (Ki = 1 nM for 1).7g Major drawbacks of this series
of Akt inhibitors as clinically useful agents include short
half-life in animals and poor oral bioavailability. Substi-
tution at C-3 position of the indazole scaffold by alkyl,
aryl, and amino groups failed to provide Akt inhibitors
with improved pharmacokinetic properties. Herein,
relatively more inert chloro- and trifluoromethyl groups
N
-
O
H
O
O
N
O
NH2
O
Akt1: Ala
Val123
Side Chain
300 strand
Figure 1. An illustration of the X-ray structure of 1 bound to protein
kinase A. Shown on left are the corresponding amino acid residues in
Akt.

2444
G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
Table 1. Enzyme and cellular assay results for compounds 1–3 and 9
NH2
R1
O
R2
N
9
R1
R2
MTT (MiaPaCa) EC50 (lM)
a
Akt1 IC50 (nM)
a
PKA IC50 (nM)
a
Compound
H
N
1
3-Indole
1.5
18
0.82
0.38
N
H
N
N
2
3
3-Indole
3-Indole
1.0
1.8
6.8
Cl
H
N
N
16
1.29
CF3
H
N
NO2
9c
9d
3-Indole
3-Indole
953
51
ndb
nd
2.94
nd
N
H
N
NH2
NH2
N
H
N
N
9e
9f
9g
9h
9i
3-Indole
3-Indole
3-Indole
Ph
7.1
0.6
0.34
223
25
58
0.33
1.16
0.041
1.69
0.68
Cl
H
N
N
N
110
15
N
H
N
N
H
N
N
N
nd
32
N
H
N
Ph
N
H
N
N
N
9j
Ph
Ph
59
46
nd
nd
Cl
H
N
O-
N+
9k
104
nd
N
a Values are means of two or more experiments. All compounds were tested under 5 lM ATP.
b Not determined.
the C-6 position of the indazole scaffold would interact
with the hydroxyl group of Thr-211 and would improve
the binding affinity to Akt.
indazole. Since it appeared that a hydrogen bond
accepting group was preferred and due to space con-
straints, we next incorporated a nitrogen atom into the
phenyl ring of the indazole at the same position. The
resulting pyrazolopyridine analog 9f demonstrated a
sub-nanomolar IC50 to Akt and was more than 100-fold
selective over PKA. A 3-methyl analog 9g showed excel-
lent potency against Akt as well (IC50 = 0.34 nM).
As shown in Table 1, both chloro-analog 2 and trifluo-
romethyl-analog 3 displayed a similar profile of activity
as compound 1. Installation of a nitro group at the C-6
position of the indazole moiety resulted in nearly three
orders of magnitude loss of potency against Akt, sug-
gesting a limited space between the indazole and protein.
Reduction of the nitro to an amino group partially
restored the potency as exemplified by both 9d and 9e,
the latter with a chlorine at the C-3 position of the
In a previous investigation, the pharmacokinetic profile
of the indazole–pyridine series of Akt inhibitors was
successfully improved through a replacement of the
indole side-chain moiety with
a
phenyl group.7f

G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
Table 2. Enzyme and cellular assay results for compounds 9h–j and 25
2445
H
N
N
NH2
N
O
R
N
10
a
Akt1 IC50 (nM)
a
PKA IC50 (nM)
a
Compound
R
MTT (MiaPaCa) EC50 (lM)
9h
9i
H
223
25
ndb
32
1.69
0.68
ndb
0.66
1.2
CH3
Cl
9j
59
13
46
32
25a
25b
Vinyl
2-Furyl
6.1
274
N
25c
25d
228
8.3
ndb
nd
Ph
371
0.584
a Values are means of two or more experiments. All compounds were tested under 5 lM ATP.
b Not determined.
Table 3. Fold-selectivity of Akt inhibitors 9f and 1 for Akt1 over
selected kinases
Therefore, analogs of the current pyrazolopyridine ser-
ies with a phenyl-containing side chain were synthesized
and the results are summarized in Tables 1 and 2. Unlike
analogs with an indole side chain, compounds 9h–j with
a phenyl group are much less potent. There was no
selectivity against PKA observed as well. In addition,
the 3-methylpyrazolopyridine analog 9i was 9-fold more
potent than the un-substituted 9h. An N-oxide analog
9k, where the oxygen anion serves as a hydrogen bond
acceptor, is 4-fold less potent than compound 9i.
Kinase family
AGC
Kinase
IC50 (fold selectivity)
Compound 9f Compound 1
Akt1
PKA
1
180
1
18
PKCc
PKCd
CDK1
CDK2
ERK2
CK2
130
100
180
220
250
42
110
10
>8750
3310
240
630
5300
780
8100
>920
5000
Table 2 summarizes our preliminary investigation at the
C-3 position of the pyrazolopyridine scaffold containing
a phenyl side chain. Surprisingly, 2-furyl (25b) and
phenyl (25d) analogs were 20-fold more potent than
the sterically similar N-methyl-pyrrol-2-yl derivative
(25c). Smaller substituents such as vinyl- (25a), chloro-
(9j), and methyl- (9i) only showed slightly reduced
potency against Akt as compared to 25b or 25d. Com-
pounds 25b and 25d were 10-fold more selective versus
PKA than compounds 9i, 9j, and 25a. The best selectiv-
ity was observed for compound 9f with an indole side-
chain. Also included in Tables 1 and 2 are the MTT data
of our Akt inhibitors in MiaPaCa-2 human pancreatic
cancer cells as an indication of their anti-proliferative
activity. The majority of these compounds showed cor-
relations between Akt activity and cytotoxicity. A com-
bination of factors including cell penetration and
selectivity would complicate the direct comparison of
enzyme and cellular activity.
SGK
CAMK
TK
MAPK
KDR
SRC
1250
>12,500
>10,000
the case of compound 1, four hydrogen bond interac-
tions of 9f to the ligand-binding domain of PKA were
observed, with the hydrophobic indole positioned
underneath the glycine-rich loop. Overlap of the two
Akt inhibitors in PKA complex as shown in the right,
however, shows visible differences between the two
ligands. Due to the structural difference between the
pyrazolopyridine scaffold (in purple) versus indazole (in
green), the middle pyridine of 9f orients slightly forward
as compared to compound 1, leading to more significant
change in the orientation of the side-chain indole. Precise
positioning of the indole underneath the hydrophobic
glycine-rich loop is known to be critical to binding affin-
ity.7a,f Overall, these changes must be detrimental for
compound 9f on the binding affinity to PKA and resulted
in a 6-fold loss in potency. For Akt, however, the
hydroxyl group of Thr-211 may interact with the N-6
of the pyrazolopyridine scaffold in 9f, leading to an
improved affinity to Akt. Because of the formation of
an extra hydrogen bond interaction, or perhaps due to
the size difference between the pyrazolopyridine and inda-
zole, compound 9f also repositioned in Akt complex and
compromised the predicted boost in potency against Akt.
Displayed in Table 3 is a head-to-head comparison of
the selectivity profile between 9f and 1. Compound 9f
demonstrates 10-fold higher selectivity than compound
1 against PKA. The Akt selectivity of 9f over other
selected protein kinases in AGC, CAMK, and TK fam-
ilies is similar to that of compound 1, varying from mod-
est to excellent.
The X-ray structure of 9f in complex with PKA was
˚
resolved at 2.7 A and is shown in Figure 2 (left). As for

2446
G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
˚
Figure 2. (Left) X-ray structure of 9f in protein kinase A at 2.7 A. (Right) Overlap of 9f (shown in purple) with the crystal structure of 1 (shown in
green) in PKA.12
4. Conclusion
coupling constants (J) in Hertz (Hz). When peak multi-
plicities are given the following abbreviations are used:
s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet;
br, broadened. Mass spectra were obtained as follows:
ESI (electrospray ionization) was performed on a Finn-
igan SSQ7000 MS run as a flow injection acquisition;
DCI (desorption chemical ionization) was performed
on a Finnigan SSQ7000 MS using a direct exposure
probe with ammonia gas; APCI (atmospheric pressure
chemical ionization) was performed on a Finnigan Nav-
igator MS run as flow injection acquisition. Elemental
analyses were performed by Quantitative Technologies
Inc. Whitehouse, New Jersey. All manipulations were
performed under nitrogen atmosphere unless otherwise
mentioned. All solvents and other reagents were
obtained from commercial sources and used without
further purification, except where noted. Flash column
chromatography was performed on silica gel 60 (Merck,
230–400 mesh) using the indicated solvent. For routine
aqueous workup, the reaction mixture was partitioned
between brine and EtOAc, and the organic layer was
washed with brine, and dried over MgSO4.
In an effort to search for additional interactions with
Thr-211 of Akt, we have explored the SAR at the C-6
position of the indazole scaffold of compound 1. Intro-
duction of an oxygen anion, amino and nitro groups
at this position led to a significant drop in potency
against Akt. Incorporation of a nitrogen atom into the
phenyl ring provided a new series of potent and selective
pyridine–pyrazolopyridine-based inhibitors. The Akt
selectivity of this series of compounds over PKA is sig-
nificantly dependent on the C-3 substitution of the pyr-
azolopyridine scaffold, as well as the side-chain
structures. Compound 9f was 180-fold selective over
the closely related protein kinase A, which is 10-fold
higher than the corresponding indazole–pyridine-based
inhibitor 1 against PKA. However, the boosted potency
against Akt was less than predicted and the deleterious
effect on PKA is not conclusive from their X-ray struc-
tures. Compound 9f showed a similar selectivity profile
as compound 1 against other selected AGC, CAMK,
and TK families of protein kinases. It is interesting that
the SAR at the C-3 position of the pyrazolopyridine
scaffold is significantly different from the corresponding
indazole series. Better potency and selectivity were
obtained with larger groups such as phenyl and 2-furyl
(25d and 25b) while less tolerance was observed with
the same changes in the indazole series.7g Despite lack
of structural evidence, it is reasonable to assume the
existence of an additional hydrogen bond interaction
with Thr-211 for compounds 9d–f. However, formation
of the extra hydrogen bond may change the conforma-
tion and/or orientation of the inhibitor in protein and
compromise the overall binding affinity. In vivo evalua-
tions, including pharmacokinetics and mouse MiaPaCa
xenograft models, will be published in due course.
5.2. Chemistry
5.2.1. tert-Butyl 5-bromo-3-chloro-1H-indazole-1-carbox-
ylate (4a). 5-Bromo-1H-indazole7g (10.0 g, 50.8 mmol)
was suspended in
a solution of NaOH (8.13 g,
0.203 mol) in water (250 mL). This suspension was heat-
ed until a transparent solution formed. After cooling with
an ice-water bath to 5 ꢁC, sodium hypochlorite solution
(10–13% chlorine, 43.26 g, 60.7 mmol) was added. The
reaction mixture was stirred at 0–5 ꢁC for 5 h and was
neutralized with 1 M aq HCl. The reaction mixture was
extracted with ethyl acetate and the combined organic
phases were washed with water and concentrated. The
residual solid was dissolved in 400 mL of acetonitrile.
Triethylamine (24 mL, 172.7 mmol), DMAP (620 mg,
5.08 mmol), and BOC anhydride (16.63 g, 76.2 mmol)
were then added. The formed dark-red solution was stir-
red at rt for 5 h and was concentrated. The residue was
partitioned between ethyl acetate and brine. The organic
phase was washed with water and concentrated. The
residual solid was purified by flash chromatography (sil-
ica gel, 5% EtOAc in hexane) to give 4a (11.3 g, 67%). 1H
NMR (300 MHz, CDCl3) d 1.71 (s, 9H), 7.67 (dd,
J = 8.99, 1.87 Hz, 1H), 7.85 (d, J = 1.70 Hz, 1H), 8.06
(d, J = 8.82 Hz, 1H). MS (DCI) m/z: 332 (M+H)+.
5. Experimental
5.1. General procedure
The NMR spectra were obtained on Varian M-300,
Bruker AMX-400, and Varian U-400 magnetic
1
resonance spectrometers (300/400 MHz for H and 75/
100 MHz for 13C) with deuteriochloroform as solvent
and internal standard unless otherwise indicated. The
chemical shifts are given in delta (d) values and the

G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
2447
5.2.2. 3-(Trifluoromethyl)-1H-indazole (19). A solution
of 2-(20,20,20-trifluoroethyl)aniline (18) as a 1:4 mixture
with aniline (16.5 g)10 in glacial acetic acid (990 mL)
was treated with a solution of sodium nitrite (11.1 g,
0.161 mol) in water (25 mL). The reaction mixture
was stirred for 15 min and allowed to stand at rt for
3 days. Volatiles were removed and the residual oil
was partitioned between EtOAc and aq NaHCO3 solu-
tion. The organic layer was washed with aq NaHCO3
solution and concentrated. The residue was separated
by flash chromatography (silica gel, 15–30% gradient
EtOAc in hexane) to give a 1:1 mixture of 19 with an
uncharacterized side-product (3.40 g). This material
was directly used in the next step without further
separation.
5.2.6. 5-Bromo-6-nitro-1H-indazole (16). To a solution of
15 (10.9 g, 47.17 mmol) in glacial acetic acid (1 L) was
added a solution of sodium nitrite (3.25 g, 47.17 mmol)
in water (10 mL) at rt. The solution was stirred for
15 min and allowed to stand at rt for 3 days. Volatiles
were removed under vacuum and the residue was stirred
with 100 mL of water. The formed solid was collected by
filtration, washed with water, and dried. The crude
product was recrystallized from a mixture of ethanol
and water to give 16 (7.22 g, 63%). 1H NMR
(300 MHz, DMSO-d6) d 8.26 (s, 1H), 8.33 (s, 1H), 8.35
(s, 1H), 13.84 (br s, 1H). MS(DCI) m/z: 241, 243
(M+1)+.
5.2.7. tert-Butyl 5-bromo-6-nitro-1H-indazole-1-carbox-
ylate (4c). To a suspension of 16 (1.26 g, 5.21 mmol) in
acetonitrile (50 mL) were added triethylamine (2.47 mL,
17.75 mmol), DMAP (64 mg, 0.521 mmol), and di-t-butyl
dicarbonate (1.71 g, 7.81 mmol). The formed dark-red
solution was stirred at rt for 15 h and was concentrated.
The residue was partitioned between ethyl acetate and
brine. The organic phase was concentrated and the
residual solid was purified by flash chromatography
(silica gel, 20–50% gradient EtOAc in hexane) to give
5.2.3. 5-Bromo-3-(trifluoromethyl)-1H-indazole (20) and
7-bromo-3-(trifluoromethyl)-1H-indazole (21). The mix-
ture of compound 19 (3.3 g) was dissolved in anhyd
CH3CN (50 mL). N-Bromosuccinimide (15.8 g, 88.64
mmol) was then added and the reaction mixture was
stirred at rt for 2 days. Ethyl acetate (200 mL) was
added and the mixture was washed with 5% aq NaHSO3
solution (200 mL). The organic phase was washed
with brine and concentrated. The residue was separated
by flash chromatography (silica gel, 10–40% gradient
EtOAc in hexane) to give 1.67 g of compound 20
(35%) and 302 mg of 21 (6%). Compound 20: 1H
NMR (300 MHz, CDCl3) d 7.46 (d, J = 8.50 Hz, 1H),
7.60 (dd, J = 1.70, 8.48 Hz, 1H), 8.03 (s, 1H), 10.58 (br
s, 1H); MS(DCI) m/z: 264, 266 (M+H)+. Compound
21: 1H NMR (300 MHz, CDCl3) d 7.21 (t, J =
8.14 Hz, 1H), 7.65 (d, J = 7.46 Hz, 1H), 7.83 (d, J =
8.48 Hz, 1H), 10.42 (br s, 1H). MS(DCI) m/z: 264, 266
(M+H)+.
1
4c (1.64 g, 92%). H NMR (300 MHz, CDCl3) d 1.73
(s, 9H), 8.11 (s, 1H), 8.22 (s, 1H), 8.67 (s, 1H). MS(DCI)
m/z: 359, 361 (M+NH4)+.
5.2.8. tert-Butyl 5-bromo-6-amino-1H-indazole-1-carbox-
ylate (4d). To a solution of 4c (0.5 g, 1.46 mmol) in THF
(10 mL) was added Raney 2800 nickel (0.2 g, slurry in
water) at rt. This suspension was purged with hydrogen
and stirred under hydrogen (balloon) for 6 h. Solid
material was filtered off and the filtrate was concentrat-
ed. The residual solid was purified by flash chromatog-
raphy (silica gel, 20–50% gradient EtOAc in hexane) to
1
5.2.4. tert-Butyl 5-bromo-3-trifluoromethyl-1H-indazole-
1-carboxylate (4b). To a solution of 20 (1.66 g,
6.26 mmol) in anhyd acetonitrile (50 mL) were added
triethylamine (3.0 mL, 21.28 mmol), DMAP (153 mg,
1.25 mmol), and BOC anhydride (2.05 g, 9.40 mmol).
The dark-red solution was stirred at rt for 5 h and was
partitioned between ethyl acetate and brine. The organic
phase was washed with brine and concentrated. The
residual solid was purified by flash chromatography (sil-
ica gel, 5–10% gradient EtOAc in hexane) to give 4b
give 4d (446 mg, 97%). H NMR (300 MHz, CDCl3) d
1.64 (s, 9H), 7.83 (s, 1H), 7.96 (s, 1H), 8.16 (s, 1H),
8.52 (s, 1H), 8.86 (d, J = 1.70 Hz, 1H). MS(DCI) m/z:
312, 314 (M+1)+, 328, 330 (M+NH4)+.
5.2.9. 5-Bromo-3-chloro-6-nitro-1H-indazole (17). Two
grams of compound 16 was dissolved in a hot solution
of NaOH (1.32 g) in water (40 mL). The formed solu-
tion was cooled in an ice-water bath and sodium
hypochlorite solution (10–13% chlorine, 7.04 g,
9.92 mmol) was added. The reaction mixture was stir-
red at 0 ꢁC for 5 h and neutralized with 1 M aq HCl
solution. This mixture was extracted with ethyl acetate
and the combined organic phases were washed with
water and concentrated. The residual solid was puri-
fied by flash chromatography (silica gel, 20–50% gra-
dient EtOAc in hexane) to afford 17 (2.19 g, 95%).
1H NMR (300 MHz, DMSO-d6) d 8.25 (s, 1H), 8.39
(s, 1H), 14.09 (br s, 1H). MS(DCI) m/z: 275, 277,
279 (M+1)+.
1
(1.97 g, 86%). H NMR (300 MHz, CDCl3) d 1.74 (s, 9
H), 7.70 (dd, J = 8.99, 1.87 Hz, 1H), 7.99 (s, 1H), 8.12
(d, J = 9.16 Hz, 1H). MS(DCI) m/z: 382, 384
(M+NH4)+.
5.2.5. 4-Bromo-2-methyl-5-nitroaniline (15). A solution
of 4-bromo-2-methylaniline hydrochloride (11.12 g,
50 mmol) in 40 mL of concn H2SO4 was added in one por-
tion to an ice-cold solution of KNO3 (5.05 g, 50 mmol) in
100 mL of concentrated H2SO4. The resulting solution
was stirred overnight at rt before being poured over
1.25 L of crushed ice and neutralized with concentrated
ammonium hydroxide (350 mL). The formed yellow solid
was collected by filtration, washed with water, and dried
to give 11.05 g of 15 (95%). 1H NMR (300 MHz, CDCl3)
d 2.19 (s, 3H), 3.92 (br s, 2H), 7.21 (s, 1H), 7.36 (s, 1H).
MS(DCI) m/z: 248, 250 (M+NH4)+.
5.2.10. tert-Butyl 6-amino-5-bromo-3-chloro-1H-inda-
zole-1-carboxylate (4e). To a solution of 17 (2.10 g,
7.59 mmol) in acetonitrile (55 mL) were added triethyl-
amine
0.759 mmol), and di-t-butyl dicarbonate (2.48 g,
(3.6 mL,
25.8 mmol),
DMAP
(93 mg,
11.38 mmol). The yellow solution was stirred at rt for

2448
G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
1
15 h and concentrated. The residue was partitioned
between ethyl acetate and brine. The organic phase
was washed with brine and concentrated. The residue
was purified by flash chromatography (silica gel,
5–20% gradient EtOAc in hexane) to give 4e (2.9 g,
100%). 1H NMR (300 MHz, CDCl3) d 1.72 (s, 9H),
8.07 (s, 1H), 8.63 (s, 1H). MS(DCI) m/z: 393, 395
(M+1)+.
(7.5 g, 80%). H NMR (300 MHz, CD3OD) d 1.72 (s,
9H), 2.59 (s, 3H), 8.05 (s, 1H), 9.15 (d, J = 1.02 Hz,
1H). MS (DCI) m/z: 313 (M+H)+.
5.2.14. 5-Bromo-1H-pyrazolo[3,4-c]pyridine (12b).
A
solution of 11b (3.76 g, 20.11 mmol), which was pre-
pared according to the procedure for 11a substituting
10b for 10a, in glacial acetic acid (300 ml) was treated
with a solution of sodium nitrite (1.39 g, 20.11 mmol)
in water (2.5 ml). The reaction mixture was stirred at
rt for 15 min and allowed to stand at ambient tempera-
ture for 3 days. The acetic acid was removed under re-
duced pressure and the residue was partitioned
between EtOAc and aq sodium bicarbonate solution.
The organic phase was washed with water and concen-
trated. The residual solid was purified by flash chroma-
5.2.11. 2-Bromo-4-ethyl-5-nitropyridine (10a). To a flask
containing 41.2 mL of 48% HBr was added 4-ethyl-5-
nitropyridin-2-amine (10.0 g, 59.8 mmol) in small por-
tions with vigorous stirring. The formed thick paste
was cooled in a dry-ice/isopropanol bath to ꢀꢁ30 ꢁC.
A cooled solution of Br2 (8.58 mL, 167.5 mmol) was
added maintaining the temperature below ꢁ10 ꢁC and
stirred for 1.5 h. A solution of sodium nitrite (11.14 g,
161.5 mmol) in 23 mL of water was then added slowly,
and the reaction mixture was warmed to +15 ꢁC over
a period of 1 h and stirred for an additional 45 min.
The reaction mixture was cooled to ꢁ20 ꢁC and an
aqueous solution of NaOH (43.2 g in 193 mL water)
was added maintaining the temperature below 0 ꢁC.
The mixture was allowed to warm to room temperature
and stirred for 1 h. The aqueous layer was extracted
with ethyl acetate and the combined organic phases were
concentrated on a rotary evaporator. The residual oil
was purified by flash chromatography eluting with 6%
EtOAc in hexanes to provide 10a (7.5 g, 54%). 1H
NMR (300 MHz, DMSO-d6) d 1.23 (t, J = 7.46 Hz,
3H), 2.89 (q, J = 7.23 Hz, 2H), 7.92 (s, 1H), 8.95 (s,
1H). MS (DCI) m/z: 232 (M+H)+.
1
tography to give 12b (3.9 g, 99%). H NMR (300 MHz,
CD3OD) d 7.99 (s, 1H), 8.15 (s, 1H), 8.80 (s, 1H). MS
(DCI) m/z: 199 (M+H)+.
5.2.15. tert-Butyl 5-bromo-1H-pyrazolo[3,4-c]pyridine-1-
carboxylate (4f). Compound 4f was prepared according
to the procedure for 4g by substituting 12b for 12a. H
NMR (300 MHz, CDCl3) d 1.70 (s, 9H), 7.70 (s, 1H),
8.20 (s, 1H), 9.40 (s, 1H). MS (DCI) m/z: 298, 300
(M+H)+.
1
5.2.16. 5-Bromo-3-chloro-1H-pyrazolo[3,4-c]pyridine (13).
Compound 12b (4.0 g, 20.2 mmol) was suspended in a
solution of sodium hydroxide (3.23 g, 80.8 mmol) in
water (120 ml). The suspension was heated until a trans-
parent solution formed. The solution was cooled in an
ice-water bath for 15 min before sodium hypochloride
solution (10–13% chlorine, 13.6 mL) was added. Some
solid material precipitated after the addition of sodium
hypochloride and re-dissolved into solution within 1 h
of stirring. The reaction mixture was stirred at 0 ꢁC
for 5 h before being neutralized with diluted HCl. The
mixture was extracted with ethyl acetate and the
combined organic phases were washed with water and
concentrated. The residual solid was purified by flash
chromatography to give 13 (3.6 g, 78%). 1H NMR
(300 MHz, CDCl3) d 7.85 (s, 1H), 8.81 (s, 1H). MS
(DCI) m/z: 233 (M+H)+.
5.2.12. 6-Bromo-4-ethylpyridin-3-amine (11a). A solution
of 10b (2.5 g, 10.8 mmol) in THF (50 mL) was treated
with Raney Ni (1.25 g) under hydrogen (balloon) for
18 h. The catalyst was filtered off and the filtrate concen-
trated. The residual solid was purified by flash chroma-
tography on silica gel eluting with 50% EtOAc in
hexanes to provide 11a (1.97 g, 90%). 1H NMR
(300 MHz, CD3OD) d 1.22 (t, J = 7.46 Hz, 3H), 2.52
(q, J = 7.46 Hz, 2H), 7.16 (s, 1H), 7.69 (s, 1H). MS
(DCI) m/z: 202 (M+H)+.
5.2.13. tert-Butyl 5-bromo-3-methyl-1H-pyrazolo[3,4-
c]pyridine-1-carboxylate (4g). To a solution of 11a
(6.06 g, 30.15 mmol) in glacial acetic acid (450 mL)
was added a solution of sodium nitrite (2.08 g) in water
(3.8 mL). This solution was stirred for 15 min and al-
lowed to stand at rt for 1 day. Volatiles were removed
on Rotavap and the residue was partitioned between
ethyl acetate (200 mL) and saturated sodium bicarbon-
ate solution (200 mL). The organic phase was washed
with brine and concentrated. The crude solid 12a was
dissolved in 250 mL of anhyd acetonitrile. Di-t-butyl
dicarbonate (9.87 g), triethylamine (12.61 mL), and
DMAP (369 mg) were then added. The reaction mixture
was stirred at room temperature overnight. The solvent
was evaporated on a rotary evaporator and the residue
diluted with ethyl acetate (200 mL) and washed with
water (200 mL). The organics were concentrated and
the residue was purified by flash chromatography (silica
gel, 5–10% gradient EtOAc in hexanes) to provide 4g
5.2.17. tert-Butyl 5-bromo-3-chloro-1H-pyrazolo[3,4-c]pyr-
idine-1-carboxylate (4j). To a suspension of 13 (1.4 g,
6.07 mmol) in acetonitrile (50 ml) were added triethyl-
amine (2.5 ml), DMAP (73 mg, 0.6 mmol), and di-t-butyl
dicarbonate (1.98 g, 9.1 mmol). The reaction mixture was
stirred at room temperature for 0.5 h and concentrated.
The residue was partitioned between ethyl acetate and
brine. The organic phase was washed with water and
concentrated. The residual solid was purified by flash
chromatography to give 4j (1.49 g, 75%). 1H NMR
(300 MHz, CDCl3) d 1.73 (s, 9H), 7.83 (s, 1H), 9.32 (s,
1H). MS (DCI) m/z: 333 (M+H)+.
5.2.18. 5-Bromo-1-(tert-butoxycarbonyl)-3-methyl-1H-pyr-
azolo[3,4- c]pyridine 6-oxide (4k). To a solution of com-
pound 4g (500 mg, 1.6 mmol) in dichloromethane
(25 mL) was added m-CPBA (861 mg, 3.85 mmol). This
solution was stirred at rt for 16 h and partitioned

G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
2449
between ethyl acetate and aq NaHCO3 solution. The
organic phase was washed with water and concentrated.
The residual oil was purified by flash chromatography
(EtOAc) to provide 4k (150 mg, 28%). 1H NMR
(300 MHz, CD3OD) d 1.63 (s, 9H), 3.31 (s, 3H), 8.55
(d, J = 0.68 Hz, 1H), 8.93 (d, J = 0.68 Hz, 1H). MS
(DCI) m/z: 329 (M+H)+.
CD3OD) d 3.27–3.35 (m, 2H), 3.97–4.05 (m, 1H), 4.31
(dd, J = 6.00, 9.00 Hz, 1H), 4.46 (dd, J = 3.00, 9.00 Hz,
1H), 7.04 (t, J = 7.46 Hz, 1H), 7.13 (t, J = 7.63 Hz, 1H),
7.25 (s, 1H), 7.38 (d, J = 8.14 Hz, 1H), 7.61 (d,
J = 7.80 Hz, 1H), 7.67 (d, J = 9.00 Hz, 1H), 7.75 (d,
J = 9.00 Hz, 1H), 7.91 (d, J = 2.03 Hz, 1H), 7.97 (s, 1H),
8.37 (d, J = 2.37 Hz, 1H), 8.64 (s, 1H). MS (APCI) m/z:
418 (M+H)+. Anal. Calcd for C23H20ClN5OÆ2.7TFA: C,
47.00; H, 3.15; N, 9.65. Found: C, 47.24, H, 3.08; N,
9.87.
5.2.19. tert-Butyl 5-chloro-1H-pyrazolo[3,4-c]pyridine-1-
carboxylate (5). Compound 5 was prepared according to
the procedure for 4g by substituting 2-chloro-4-methyl-
5-nitropyridine for 2-bromo-4-ethyl-5-nitropyridine.
1H NMR (300 MHz, CDCl3) d 1.74 (s, 9H), 7.69 (s,
1H), 8.19 (s, 1H), 9.34 (s, 1H). MS (DCI) m/z: 254
(M+H)+.
5.2.23. (S)-1-(5-(3-Trifluoromethyl-1H-indazol-5-yl)pyri-
din-3-yloxy)-3-(1H-indol-3-yl)propan-2-amine (3). Com-
pound
3
was synthesized according to general
1
procedure Method A followed by Boc-deprotection. H
NMR (300 MHz, CD3OD) d 3.32–3.36 (m, 2H), 3.97–
4.04 (m, 1H), 4.31 (dd, J = 10.51, 5.76 Hz, 1H), 4.45
(dd, J = 11.00, 2.50 Hz, 1H), 7.03 (t, J = 6.95 Hz, 1H),
7.13 (d, J = 7.00 Hz, 1H), 7.24 (s, 1H), 7.38 (d,
J = 8.14 Hz, 1H), 7.60 (d, J = 7.80 Hz, 1H), 7.78–7.80
(m, 2H), 7.86–7.88 (m, 1H), 8.06 (s, 1H), 8.38 (d,
J = 2.71 Hz, 1H), 8.62 (d, J = 1.36 Hz, 1H). MS (APCI)
m/z: 452 (M+H)+. Anal. Calcd for C24H20F3N5OÆ3TFA:
C, 45.41; H, 2.92; N, 8.83. Found: C, 45.65, H, 3.44; N,
8.89.
5.2.20. General procedure for Stille reaction of trimeth-
ylstannane 7 with an aryl bromide 4 or aryl chloride 5
5.2.20.1. Method A.
equipped with a septum was charged with bromide 4
(0.395 mmol), trimethylstannane (0.395 mmol),
A
round-bottomed flask
7
Pd2(dba)3 (36 mg, 0.0395 mmol), and (o-tol)3P (36 mg,
0.118 mmol), and was purged with N2. Anhydrous
DMF (10 mL) and Et3N (165 lL, 1.18 mmol) were add-
ed via syringe. The solution was purged with N2 again
and heated at 70 ꢁC overnight. After cooling, the reac-
tion mixture was partitioned between ethyl acetate and
brine. The organic phase was washed with brine and
concentrated. The residue was separated by flash chro-
matography (silica gel) to afford the title compound 8.
5.2.24. (S)-1-(1H-indol-3-yl)-3-(5-(6-nitro-1H-indazol-5-
yl)pyridin-3-yloxy)propan-2-amine (9c). Compound 9c
was synthesized as 3· TFA salt according to general
procedure Method A followed by Boc-deprotection.
1H NMR (300 MHz, CD3OD) d 3.26–3.32 (m, 2H),
3.93–4.00 (m, 1H), 4.24 (dd, J = 5.76, 9.00 Hz, 1H),
4.37 (dd, J = 7.46, 3.05 Hz, 1H), 7.01 (t, J = 7.46 Hz,
1H), 7.10 (t, J = 6.95 Hz, 1H), 7.21 (s, 1H), 7.35 (d,
J = 7.80 Hz, 1H), 7.54 (t, J = 2.71 Hz, 1H), 7.57 (d,
J = 8.14 Hz, 1H), 7.90 (s, 1H), 8.26–8.29 (m, 2H), 8.34
(s, 1H), 8.38 (d, J = 2.71 Hz, 1H). MS (APCI) m/z: 429
(M+H)+.
5.2.20.2. Method B. A round-bottomed flask was
charged with the bromide 4 or chloride 5 (0.16 mmol),
trimethylstannane 7 (0.24 mmol), bis(tri-t-butylphos-
phine)palladium (0) (20 mg, 0.039 mmol), and cesium
fluoride (130 mg, 0.855 mmol), and was purged with
N2. Anhydrous dioxane (5 mL) was added via syringe.
The solution was purged with N2 again and heated at
80 ꢁC for 16 h. After cooling, the reaction mixture was
partitioned between ethyl acetate and brine. The organic
phase was washed with brine and concentrated. The res-
idue was separated by flash chromatography (silica gel)
to provide the title compound 8.
5.2.25. (S)-1-(1H-indol-3-yl)-3-(5-(6-amino-1H-indazol-
5-yl)pyridin-3-yloxy)propan-2-amine (9d). Compound
9d was synthesized as 3· TFA salt according to gen-
eral procedure Method A followed by Boc-deprotec-
tion. 1H NMR (300 MHz, CD3OD) d 3.28–3.33 (m,
2H), 3.96–4.03 (m, 1H), 4.27 (dd, J = 5.76, 9.00 Hz,
1H), 4.42 (dd, J = 3.05, 7.46 Hz, 1H), 6.96 (s, 1H),
7.03 (d, J = 7.46 Hz, 1H), 7.13 (d, J = 6.95 Hz,
1H), 7.23 (s, 1H), 7.38 (d, J = 8.14 Hz, 1H), 7.53
(s, 1H), 7.58 (d, J = 7.80 Hz, 1H), 7.84 (d,
J = 1.70 Hz, 1H), 7.95 (s, 1H), 8.41 (d, J = 2.71 Hz,
1H), 8.44 (d, J = 1.36 Hz, 1H). MS (APCI) m/z:
399 (M+H)+.
5.2.21. General procedure for acidic Boc-deprotection of
compound 8. To a solution of 8 (0.2 mmol) in CH2Cl2
(5 mL) was added trifluoroacetic acid (1 mL) at 0 ꢁC.
The solution was stirred at 0 ꢁC for 5 min and allowed
to warm up to rt for 1 h. After cooling to 0 ꢁC again,
CH3CN (10 mL) was added, and the solution was
concentrated. The residue was purified by HPLC
(Zorbax, C-18, 250 · 2.54 column, Mobile phase A,
0.1% TFA in H2O; B, 0.1% TFA in CH3CN; 0–100%
gradient) to provide compound 9 as TFA salt. A HCl salt
of compound 9 was obtained by dissolving the TFA salt in
a mixture of methylene chloride and methanol, and
precipitating with 1 M HCl solution in ether. Removal
of the volatiles afforded 9 as HCl salt.
5.2.26. (S)-5-(5-(2-Amino-3-(1H-indol-3-yl)propoxy)pyri-
din-3-yl)- 3-chloro-1H-indazol-6-amine (9e)
5.2.26.1. Step 1. A 100-mL RBF equipped with a sep-
tum was charged with 4e (376 mg, 1.0 mmol), 7 (R2 = 3-
indole, 530 mg, 1.0 mmol), Pd2(dba)3 (183 mg,
0.2 mmol), and (o-tol)3P (183 mg, 0.6 mmol), and was
purged with N2. Anhydrous DMF (20 mL) and Et3N
(418 lL, 3.0 mmol) were added via syringe. The solution
was purged with N2 again and heated at 70 ꢁC for 60 h.
After cooling, the reaction mixture was partitioned
5.2.22. (S)-1-(5-(3-Chloro-1H-indazol-5-yl)pyridin-3-yloxy)-
3-(1H-indol-3-yl)propan-2-amine (2). Compound 2 was
synthesized according to general procedure Method A
followed by Boc-deprotection. 1H NMR (300 MHz,

2450
G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
between ethyl acetate and brine. The organic phase was
washed with brine and concentrated. The residual oil
was separated by flash chromatography (silica gel, 20–
80% gradient EtOAc in hexane) to provide the coupled
product (232 mg, 35%) of sufficient purity to carry on
to the next step.
2H), 4.35 (br s, 1H), 4.94 (br s, 1H), 7.04 (br s, 1H),
7.08 (t, J = 8.14 Hz, 1H), 7.19 (t, J = 7.63 Hz, 1H),
7.36 (d, J = 8.14 Hz, 1H), 7.65 (d, J = 7.80 Hz, 1H),
7.76 (s, 1H), 7.96 (s, 1H), 8.09 (s, 1H), 8.43 (d,
J = 2.71 Hz, 1H), 8.86 (s, 1H), 9.25 (s, 1H). MS (APCI)
m/z: 504 (M+H)+.
5.2.26.2. Step 2. 150 mg of the nitro compound from
step 1 was dissolved in 10 mL THF. Raney 2800 nickel
(0.2 g, Aldrich, slurry in water) was added and the sus-
pension was stirred at rt under hydrogen (balloon) for
16 h. Solid material was filtered off and the filtrate was
concentrated. The residual solid was purified by flash
chromatography (silica gel, 0–15% gradient MeOH in
2:1 EtOAc/hexane) to give (S)-tert-butyl 6-amino-5-(5-
(2-(tert-butoxycarbonylamino)-3-(1H-indol-3-yl)prop-
oxy)pyridin-3-yl)-3-chloro-1H-indazole-1-carboxylate
(88 mg, 62%). Boc-deprotection of this compound as de-
scribed in general procedure afforded 9e. 1H NMR
(300 MHz, CD3OD) d 3.28–3.32 (m, 2H), 3.97–4.04
(m, 1H), 4.32 (dd, J = 5.76, 10.00 Hz, 1H), 4.46 (dd,
J = 3.39, 10.55 Hz, 1H), 6.84 (s, 1H), 7.02 (t,
J = 6.95 Hz, 1H), 7.12 (t, J = 7.12 Hz, 1H), 7.24 (s,
1H), 7.37 (d, J = 8.00, 1H), 7.38 (s, 1H), 7.59 (d,
J = 7.80 Hz, 1H), 7.91–7.93 (m, 1H), 8.45 (d,
J = 2.71 Hz, 1H), 8.49 (d, J = 1.70 Hz, 1H). MS (APCI)
m/z: 433 (M+H)+. Anal. Calcd for C23H21ClN6OÆ3.1T-
FA: C, 44.60; H, 3.09; N, 10.69. Found: C, 44.60, H,
2.60; N, 10.54.
5.2.28.2. Step 2: (S)-tert-Butyl 1-(1H-indol-3-yl)-3-(5-
amino-4-methyl-2,30-bipyridin-50-yloxy)propan-2-ylcar-
bamate (24). To a solution of 23 (1.85 g, 3.67 mmol) in
THF (50 mL) was added Raney 2800 nickel (1.0 g, slur-
ry in water) at rt. This suspension was purged with
hydrogen and stirred under hydrogen (balloon) for
3 days. Solid material was filtered off and the filtrate
was concentrated. The residual solid was purified by
flash chromatography (0–15% gradient MeOH in 2:1
EtOAc/hexane) to afford 24. Yield: 1.56 g (89%). 1H
NMR (400 MHz, CDCl3) d 1.45 (s, 9H), 2.19 (s, 3H),
3.12 (m, 2H), 3.90 (br s, 1H), 3.92–3.96 (m, 2H), 4.30
(br s, 1H), 5.11 (d, J = 7.98 Hz, 1H), 6.94 (d,
J = 1.53 Hz, 1H), 7.04 (t, J = 7.36 Hz, 1H), 7.13 (t,
J = 7.52 Hz, 1H), 7.30 (d, J = 7.98 Hz, 1H), 7.39 (s,
1H), 7.64 (d, J = 7.67 Hz, 1H), 7.73 (s, 1H), 8.04 (s,
1H), 8.23 (d, J = 2.76 Hz, 1H), 8.68 (d, J = 1.53 Hz,
1H), 8.70 (s, 1H). 13C NMR (101 MHz, CDCl3) d
16.92, 27.02, 28.34, 31.50, 50.20, 68.18, 79.49, 111.19,
118.11, 118.81, 119.40, 121.91, 122.35, 123.04, 127.55,
130.89, 135.89, 136.22, 136.50, 136.86, 139.68, 141.21,
144.32, 155.08, 155.40. MS (DCI) m/z: 474 (M+H)+.
5.2.27. (S)-1-(5-(1H-pyrazolo[3,4-c]pyridin-5-yl)pyridin-
3-yloxy)-3-(1H-indol-3-yl)propan-2-amine (9f). Compound
9f was synthesized from chloride 5 and trimethylstannane
7 (R2 = 3-indole) according to general procedure Method
5.2.28.3. Step 3. To a solution of 24 (440 mg,
0.93 mmol) in glacial acetic acid (70 mL) was added
a solution of sodium nitrite (64 mg, 0.93 mmol) in
water (1.0 mL). The solution was stirred at rt for
1 h and aged at ambient temperature overnight. Sol-
vent was removed under reduced pressure. The resid-
ual oil was separated by HPLC (Zorbax, C-18,
250 · 2.54 column, Mobile phase A: 0.1% TFA in
H2O; B: 0.1% TFA in CH3CN; 0–100% gradient) to
provide the coupled product (8f). Yield: 197 mg
(30%). This product was dissolved in CH2Cl2 (5 mL)
and treated with trifluoroacetic acid (1 mL) as de-
scribed in Section 5.2.21 to give 9f as 3· TFA salt.
Yield: 80 mg (40%).
1
B followed by Boc-deprotection. H NMR (300 MHz,
CD3OD) d 3.31–3.36 (m, 2H), 4.05 (dd, J = 5.76,
3.05 Hz, 1H), 4.37 (dd, J = 5.76, 10 Hz, 1H), 4.52 (dd,
J = 3.39, 11.00 Hz, 1H), 7.03 (t, J = 6.95 Hz, 1H), 7.12
(t, J = 7.12 Hz, 1H), 7.26 (s, 1H), 7.38 (d, J = 8.14 Hz,
1H), 7.61 (d, J = 7.80 Hz, 1H), 8.33 (s, 1H), 8.44–8.47
(m, 2H), 8.46 (s, 1H), 9.05 (s, 1H), 9.18 (s, 1H). MS
(APCI) m/z: 385 (M+H)+. Anal. Calcd for
C22H20N6OÆ3.7TFA: C, 43.79; H, 2.96; N, 10.42. Found:
C, 43.97, H, 2.58; N, 10.41.
5.2.28. An alternative approach to compound 9f (Scheme
4)
5.2.29. (S)-1-(1H-indol-3-yl)-3-(5-(3-methyl-1H-pyrazolo-
[3,4-c]pyridin-5-yl)pyridin-3-yloxy)-propan-2-amine (9g).
Compound 9g was synthesized from bromide 4g and tri-
methylstannane 7 (R2 = 3-indole) according to general
procedure Method B followed by Boc-deprotection.
1H NMR (300 MHz, CD3OD) d 2.68 (s, 3H), 3.32–
3.37 (m, 2H), 4.01–4.09 (m, 1H), 4.38 (dd, J = 5.76,
9.00 Hz, 1H), 4.52 (dd, J = 3.05, 9.00 Hz, 1H), 7.03 (t,
J = 7.46 Hz, 1H), 7.12 (t, J = 7.63 Hz, 1H), 7.26 (s,
1H), 7.38 (d, J = 8.14 Hz, 1H), 7.61 (d, J = 7.80 Hz,
1H), 8.43–8.46 (m, 2H), 8.45 (s, 1H), 9.05 (s, 1H), 9.10
(s, 1H). MS (APCI) m/z: 399 (M+H)+. Anal. Calcd for
C23H22N6OÆ3.1TFA: C, 46.64; H, 3.36; N, 11.18.
Found: C, 46.59, H, 2.72; N, 10.89.
5.2.28.1. Step 1: (S)-tert-butyl 1-(1H-indol-3-yl)-3-(4-
methyl-5-nitro-2,30-bipyridin-50-yloxy)propan-2-ylcarbamate
(23). A 250-mL RBF was charged with 2-chloro-4-meth-
yl-5-nitropyridine (22b) (990 mg, 5.73 mmol), 7 (R2 = 3-
indole, 3.04 g, 5.73 mmol), Pd2(dba)3 (525 mg, 0.573
mmol), and tri-2-furylphosphine (399 mg, 1.72 mmol),
and was purged with N2. Anhydrous DMF (60 mL)
and triethylamine (2.40 mL) were added via syringe.
The solution was purged with N2 again and heated at
75 ꢁC for 8 h. After cooling, the reaction mixture was
partitioned between EtOAc and brine. The organic
phase was washed with brine and concentrated. The
residue was separated by flash chromatography (40–
80% gradient EtOAc in hexane) to afford 23. Yield:
5.2.30. (S)-1-(5-(1H-Pyrazolo[3,4-c]pyridin-5-yl)pyridin-
3-yloxy)-3-phenylpropan-2-amine (9h). Compound 9h
(36 mg) as 3· TFA salt was synthesized from bromide
1
1.86 g (64%). H NMR (300 MHz, CDCl3) d 1.46 (s,
9H), 2.75 (s, 3H), 3.16–3.21 (m, 2H), 4.04–4.13 (m,

G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
2451
4f (51 mg, 0.17 mmol) and trimethylstannane 7 (R2 = Ph,
100 mg, 0.2 mmol) according to general procedure Meth-
od A followed by Boc-deprotection. 1H NMR (300 MHz,
CD3OD) d 3.17 (d, J = 7.46 Hz, 2H), 3.95–4.03 (m,
1H), 4.28 (dd, J = 5.43, 10.50 Hz, 1H), 4.43 (dd,
J = 3.05, 10.51 Hz, 1H), 7.30–7.41 (m, 5H), 8.31 (s, 1H),
8.35–8.38 (m, 1H), 8.42–8.47 (m, 2H), 9.01 (d,
J = 1.36 Hz, 1H), 9.17 (s, 1H). MS (DCI) m/z: 455
(M+H)+.
3.35 (s, 9H), 3.85–4.02 (m, 1H), 4.19–4.29 (m, 2H),
4.40 (dd, J = 10.85, 3.05 Hz, 2H), 7.22–7.47 (m, 5H),
8.24–8.26 (m, 1H), 8.28 (d, J = 1.36 Hz, 1H), 8.40 (d,
J = 2.37 Hz, 1H), 9.00 (s, 1H), 9.12 (s, 1H). MS (DCI)
m/z: 581 (M+H)+.
5.2.34. (S)-1-(5-(3-Chloro-1H-pyrazolo[3,4-c]pyridin-5-yl)-
pyridin-3-yloxy)-3-phenylpropan-2-amine (9j). Compound
9j (30 mg) as 3· TFA salt was prepared from 8j
(31 mg) according to the general procedure for Boc-de-
protection. 1H NMR (300 MHz, CD3OD) d 3.16 (t,
J = 7.29 Hz, 2H), 3.85–4.05 (m, 1H), 4.11–4.31 (m, 1H),
4.32–4.52 (m, 1H), 7.17–7.46 (m, 5H), 8.32 (s, 1H),
8.40–8.48 (m, 1H), 8.54 (s, 1H), 9.05 (s, 1H), 9.13 (s,
1H). MS (DCI) m/z: 380 (M+H)+.
5.2.31. (S)-1-(5-(3-Methyl-1H-pyrazolo[3,4-c]pyridin-5-
yl)pyridin-3-yloxy)-3-phenylpropan-2-amine (9i). Com-
pound 9i (26 mg) was synthesized from bromide 4g
(150 mg, 0.48 mmol) and trimethylstannane 7 (R2
=
Ph, 236 mg, 0.48 mmol) according to general procedure
Method B followed by Boc-deprotection. Yield: 15%.
1H NMR (400 MHz, CD3OD) d 2.63 (s, 3H), 3.13 (d,
J = 7.67 Hz, 2H), 3.91–3.98 (m, 3H), 4.26 (dd,
J = 10.43, 5.52 Hz, 1H), 4.37–4.45 (m, 1H), 7.24–7.29
(m, 1H), 7.29–7.33 (m, 3H), 7.33–7.36 (m, 1H), 8.39–
8.43 (m, 3H), 9.01 (s, 1H), 9.05 (d, J = 1.23 Hz, 1H).
MS (DCI) m/z: 360 (M+H)+. Anal. Calcd for
C21H21N5OÆ2.9 TFA: C, 46.64; H, 3.49; N, 10.15.
Found: C, 46.68; H, 3.19; N, 10.12.
5.2.35. (S)-1-Phenyl-3-(5-(3-vinyl-1H-pyrazolo[3,4-c]pyri-
din-5-yl)pyridin-3-yloxy)propan-2-amine (25a). A 25-ml
RBF was charged with 8j (28 mg), tributylvinyltin
(32 mg), 2-dicyclohexylphosphino-2-(N,N-dimethylami-
no)biphenyl (4 mg), and Pd2(dba)3 (5 mg), and was
purged with nitrogen. Anhydrous DMF (4 ml) was added
via syringe. The solution was purged with nitrogen again
and heated at 85 ꢁC for 3 h. After cooling, the reaction
mixture was partitioned between ethyl acetate and brine.
The organic phase was washed with water and concen-
trated. The residue was separated by flash chromatogra-
phy to give the Boc-protected coupling product which
was deprotected according to the general procedure for
5.2.32. (S)-5-(5-(2-Amino-3-phenylpropoxy)pyridin-3-yl)-
3-methyl-1H-pyrazolo[3,4-c]pyridine 6-oxide (9k). A sus-
pension of 4k (50 mg, 0.15 mmol), trimethylstannane 7
(R2 = Ph, 75 mg, 0.15 mmol), and Pd(PPh3)4 (17 mg,
0.015 mmol) in anhyd xylene (10 mL) was heated at
120 ꢁC under nitrogen for 3 h. After cooling to rt, ethyl
acetate (50 mL) was added. The mixture was washed
with brine (50 mL) and water (50 ml), dried (MgSO4),
filtered, and concentrated. The residual oil was purified
by flash column chromatography (EtOAc) to provide
the corresponding coupling product. This product was
dissolved in dichloromethane (6 mL) and treated with
TFA (3 mL) at rt for 1 h. After concentration, the resid-
ual oil was purified by HPLC (Zorbax, C-18, 250 · 2.54
column, Mobile phase A: 0.1% TFA in H2O; B: 0.1%
TFA in CH3CN; 0–100% gradient) to provide 9k
1
Boc-deprotection to provide 25a. H NMR (300 MHz,
CD3OD) d 3.17 (d, J = 7.12 Hz, 2H), 3.91–4.05 (m,
1H), 4.21–4.33 (m, 1H), 4.37–4.47 (m, 1H), 5.65 (d,
J = 11.53 Hz, 1H), 6.30 (d, J = 16.95 Hz, 1H), 7.13 (dd,
J = 17.97, 11.53 Hz, 1H), 7.23–7.47 (m, 5H), 8.35–8.42
(m, 1H), 8.44 (t, J = 2.71 Hz, 1H), 8.56 (d, J = 1.36 Hz,
1H), 9.02 (s, 1H), 9.10 (s, 1H). MS (DCI) m/z: 295
(M+H)+.
5.2.36. (S)-1-(5-(3-(furan-2-yl)-1H-pyrazolo[3,4-c]pyridin-
5-yl)pyridin-3-yloxy)-3-phenylpropan-2-amine (25b). Com-
pound 25b as 3· TFA salt (20 mg) was synthesized from
8j (50 mg) according to procedure for 25a substituting
2-tributylstannylfuran for tributylvinyltin. 1H NMR
(300 MHz, CD3OD) d 3.11–3.23 (m, 2H), 3.93–4.07 (m,
1H), 4.28 (dd, J = 10.51, 5.43 Hz, 1H), 4.44 (dd,
J = 10.68, 3.22 Hz, 1H), 6.68 (dd, J = 3.56, 1.86 Hz,
1H), 7.14 (d, J = 3.56 Hz, 1H), 7.25–7.44 (m, 5H), 7.77
(d, J = 1.86 Hz, 1H), 8.34–8.38 (m, 1H), 8.44 (d,
J = 2.71 Hz, 1H), 8.64 (s, 1H), 9.04 (s, 1H), 9.16 (s,
1H). MS (DCI) m/z: 335 (M+H)+.
1
(5 mg, 6%). H NMR (400 MHz, CD3OD) d 2.62 (s,
3H), 3.11–3.17 (m, 2H), 3.89–3.97 (m, 1H), 4.16 (dd,
J = 10.59, 5.68 Hz, 1H), 4.29–4.36 (m, 1H), 7.28–7.31
(m, 2H), 7.32– 7.36 (m, 2H), 7.36–7.39 (m, 1H), 7.87–
7.90 (m, 1H), 8.08 (s, 1H), 8.42 (s, 1H), 8.51 (s, 1H),
8.87 (s, 1H). MS (DCI) m/z: 376 (M+H)+.
5.2.33. (S)-tert-Butyl 5-(5-(2-(tert-butoxycarbonylami-
no)-3-phenylpropoxy)pyridin-3-yl)-3-chloro-1H-pyrazolo-
[3,4-c]pyridine-1-carboxylate (8j: R2 = Ph). A 100-ml
RBF was charged with 4j (1.2 g, 3.61 mmol), trimeth-
ylstannane 7 (R2 = Ph, 1.77 g, 3.61 mmol), bis(tri-t-
butylphosphine)palladium (0) (184 mg, 0.36 mmol),
and cesium fluoride (1.37 g, 9.02 mmol), and was purged
with nitrogen. Anhydrous dioxane (60 ml) was added
via syringe. The suspension was purged with nitrogen
again and was heated at 85 ꢁC for 5 h. After cooling,
the reaction mixture was partitioned between ethyl ace-
tate and brine. The organic phase was washed with
water and concentrated. The residual solid was separat-
ed by flash chromatography to give 8j (R2 = Ph, 0.4 g,
20%). 1H NMR (300 MHz, CD3OD) d 3.13 (s, 9H),
5.2.37. (2S)-1-(5-(3-(1-Methyl-1H-pyrrol-2-yl)-1H-pyraz-
olo[3,4-c]pyridin-5-yl)pyridin-3-yloxy)-3-phenylpropan-2-
amine (25c). Compound 25c as 3· TFA salt (20 mg) was
synthesized from 8j (50 mg) according to procedure for
25a substituting N-methyl-2-tributylstannylpyrrole for
1
tributylvinyltin. H NMR (300 MHz, CD3OD) d 3.11–
3.23 (m, 2H), 3.93–4.07 (m, 1H), 4.28 (dd, J = 10.51,
5.43 Hz, 1H), 4.44 (dd, J = 10.68, 3.22 Hz, 1H), 6.68
(dd, J = 3.56, 1.86 Hz, 1H), 7.14 (d, J = 3.56 Hz, 1H),
7.25–7.44 (m, 5H), 7.77 (d, J = 1.86 Hz, 1H), 8.34–8.38
(m, 1H), 8.44 (d, J = 2.71 Hz, 1H), 8.64 (s, 1H), 9.04 (s,
1H), 9.16 (s, 1H). MS (DCI) m/z: 348 (M+H)+.

2452
G.-D. Zhu et al. / Bioorg. Med. Chem. 15 (2007) 2441–2452
Mamo, M.; Smith, R.; Gramling-Evans, E.; Zinker, B.;
Mika, A.; Nguyen, P.; Oltersdorf, T.; Rosenberg, S.; Li,
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Li, T.; Zhu, G.-D.; Gong, J.; Claiborne, A.; Dalton, C.;
Luo, Y.; Johnson, E.; Shi, S.; Liu, X.; Klinghofer, V.;
Bauch, J.; March, K.; Bouska, J.; Arries, S.; de Jong, R.;
Oltersdorf, T.; Stoll, V.; Jakob, C.; Rosenberg, S.;
Giranda, V. Bioor. Med. Chem. Lett. 2006, 16, 1679; (c)
Li, Q.; Woods, K.; Thomas, S.; Zhu, G.-D.; Packard, G.;
Fisher, J.; Li, T.; Gong, J.; Dinges, J.; Song, X.; Abrams,
J.; Luo, Y.; Johnson, E.; Shi, Y.; Liu, X.; Klinghofer, V.;
de Jong, R.; Oltersdorf, T.; Stoll, V.; Jakob, C.; Rosen-
berg, S.; Giranda, V. Bioor. Med. Chem. Lett. 2006, 16,
2000; (d) Zhu, G.-D.; Gong, J.; Claiborne, A.; Woods, K.;
Gandhi, V.; Thomas, S.; Luo, Y.; Liu, X.; Shi, S.; Guan,
R.; Magnone, S.; Klinghofer, V.; Johnson, E.; Bouska, J.;
Shoemaker, A.; Oleksijew, A.; Stoll, V.; de Jong, R.;
Oltersdorf, T.; Li, Q.; Rosenberg, S.; Giranda, V. Bioorg.
Med. Chem. Lett. 2006, 16, 3150; (e) Zhu, G.-D.; Gandhi,
V.; Gong, J.; Luo, Y.; Liu, X.; Shi, S.; Guan, R.;
Magnone, S.; Klinghofer, V.; Johnson, E.; Bouska, J.;
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5.2.38. (S)-1-Phenyl-3-(5-(3-phenyl-1H-pyrazolo[3,4-c]pyri-
din-5-yl)pyridin-3-yloxy)propan-2-amine (25d). Compound
25d as 3· TFA salt (28 mg) was synthesized from 8j
(50 mg) according to procedure for 25a substituting phe-
nyltributyltin for tributylvinyltin. 1H NMR (300 MHz,
CD3OD) d 3.08–3.20 (m, 2H), 3.74–4.09 (m, 1H),
4.12–4.31 (m, 1H), 4.41 (dd, J = 10.68, 3.22 Hz, 1H),
7.14–7.42 (m, 5H), 7.45–7.51 (m, 1H), 7.51–7.64 (m, 2H),
7.90–8.13 (m, 2H), 8.23–8.35 (m, 1H), 8.41 (s, 1H), 8.55
(d, J = 1.36 Hz, 1H), 9.02 (s, 1H), 9.17 (s, 1H). MS
(DCI) m/z: 348 (M+H)+.
Acknowledgment
We thank Dr. Thomas Penning for proofreading this
manuscript and valuable suggestions.
References and notes
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12. Crystallization and X-ray analysis. PKA was purified,
concentrated to 20 mg/mL, and complexed with PKI
peptide for 1 h and then complexed with Akt inhibitors.
Crystals were transferred to cryo-solutions that contained
well solution plus increasing amounts of glycerol, soaking
for 1 min in 5%, 15%, and 25% glycerol. Crystals were
then frozen in a stream of 100 K nitrogen using an Oxford
Cryo-stream cooling device. Diffraction data were record-
ed using a MAR-165 CCD detector system on a Rigaku
RU-2000 rotating anode X-ray generator operating at
100 mA and 50 kV. Diffraction data were reduced using
DENZO and the protein model (Accession No. 1YDT)
with the inhibitor (H89) and the phosphorylation sites
omitted from the Protein Data Bank entry 1YDT was
used for initial phasing. Generation of initial electron
density maps and structure refinement were achieved using
CNX program package. Electron density maps were
inspected on a Silicon Graphics Inc. workstation using
the program QUANTA 97/2001 (Molecular Simulations
Inc., San Diego, CA). Crystallographic data described in
this paper have been deposited with PDB (ID for 1:
2OHO; ID for 9f: 2OJF).
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