Isoquinoline-pyridine-based protein kinase B/Akt antagonists: SAR and in vivo antitumor activity

Article abstract of DOI:10.1016/j.bmcl.2006.03.041

The structure-activity relationships of a series of isoquinoline-pyridine-based protein kinase B/Akt antagonists have been investigated in an effort to improve the major short-comings of the lead compound 3, including poor pharmacokinetic profiles in several species (e.g., mouse iv t_1/2 = 0.3 h, po F = 0%). Chlorination at C-1 position of the isoquinoline improved its pharmacokinetic property in mice (iv t_1/2 = 5.0 h, po F = 51%) but resulted in >500-fold drop in potency. In a mouse MiaPaCa-2 xenograft model, an amino analog 10y significantly slowed the tumor growth, however was accompanied by toxicity.

Full text of DOI:10.1016/j.bmcl.2006.03.041

Bioorganic & Medicinal Chemistry Letters 16 (2006) 3150–3155
Isoquinoline–pyridine-based protein kinase B/Akt antagonists:
SAR and in vivo antitumor activity
Gui-Dong Zhu,a,* Jianchun Gong,a Akiyo Claiborne,a Keith W. Woods,a Viraj B. Gandhi,a
Sheela Thomas,a Yan Luo,a Xuesong Liu,a Yan Shi,a Ran Guan,a Shayna R. Magnone,a
Vered Klinghofer,a Eric F. Johnson,a Jennifer Bouska,a Alexander Shoemaker,a
Anatol Oleksijew,a Vincent S. Stoll,b Ron De Jong,c Tilman Oltersdorf,c
Qun Li,a Saul H. Rosenberga and Vincent L. Girandaa
aCancer Research, GPRD, Abbott Laboratories, Abbott Park, IL 60064, USA
bStructural Biology, GPRD, Abbott Laboratories, Abbott Park, IL 60064, USA
cIDUN Pharmaceuticals Inc., 9380 Judicial Drive, San Diego, CA 92121, USA
Received 22 February 2006; accepted 17 March 2006
Available online 5 April 2006
Abstract—The structure–activity relationships of a series of isoquinoline–pyridine-based protein kinase B/Akt antagonists have been
investigated in an effort to improve the major short-comings of the lead compound 3, including poor pharmacokinetic profiles in
several species (e.g., mouse iv t1/2 = 0.3 h, po F = 0%). Chlorination at C-1 position of the isoquinoline improved its pharmacoki-
netic property in mice (iv t1/2 = 5.0 h, po F = 51%) but resulted in >500-fold drop in potency. In a mouse MiaPaCa-2 xenograft mod-
el, an amino analog 10y significantly slowed the tumor growth, however was accompanied by toxicity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Protein kinase B (also known as Akt) is a 57 kDa serine/
threonine kinase that in mammals is comprised of three
highly homologous isoforms, namely PKBa (Akt1),
PKBb (Akt2), and PKBc (Akt3).1 The cellular activa-
tion of PKB/Akt triggers a cascade of responses, from
cell growth and proliferation to survival and motility,
driving tumor progression. Overexpression of Akt as a
result of, for example, inactivation of tumor suppressor
PTEN has been correlated with an increasing number of
human cancers.2 Therefore, inhibition of Akt alone, or
in combination with other standard cancer chemothera-
peutics, is widely considered as a practical strategy for
the treatment of cancers.3 While initial development of
small Akt inhibitors has been summarized in two review
articles,3 Lindsley et al. recently reported a series of
selective allosteric and non-ATP competitive diphenyl-
quinoxaline- and diphenylpyridine-based inhibitors of
Akt that target the pleckstrin homology (PH) domain
of the protein kinase.4 Some of these Akt inhibitors
(e.g., 1) are selective over other members of the closely
related AGC family of protein kinases and displayed
good selectivity among the individual Akt isozymes.
An optimized dual Akt1/Akt2 inhibitor 2 was demon-
strated to sensitize tumor cells to apoptotic stimuli and
inhibit the phosphorylation of both Akt1 and Akt2
in vitro.5
R
R
CH3
O
N
N
N
N
N
H
N
H
2
1
N
O
N
NH
R=
8
1
NH
N
NH2
4
O
Keywords: Akt inhibitor; Protein kinase B; PKB; GSK3; FL5.12-Akt;
Anticancer.
2
6
N
*
Corresponding author. Tel.: +1 847 935 1305; fax: +1 847 935
5165; e-mail: gui-dong.zhu@abbott.com
3
0960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.03.041

G.-D. Zhu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3150–3155
3151
We have reported a novel, potent (IC50 = 2.0 nM), and
selective Akt inhibitor 3 that showed statistically signif-
icant efficacy in several mouse xenograft models.6 Major
short-comings of this Akt inhibitor as a clinically useful
agent include short half-life in mice, rat, and monkey,
and a narrow therapeutic window. In an effort to im-
prove the pharmacokinetic profile and retain potency
against Akt, the structure–activity relationships of this
class of Akt inhibitors have been investigated under
the guidance of an X-ray structure of 3 bound to analo-
gous protein kinase A (PKA).
89% yield (Scheme 2). The Stille coupling of 11 with 2-
tributylstannylfuran went smoothly with Cy-MAP as
ligand, giving the coupled product 13b in 75% yield.
However, a similar reaction with tributylvinyltin provid-
ed only 26% of desired product 13a. Likewise, the
reaction of 11 with tributyl(ethynyl)tin in the presence
of Pd2(dba)3 and Cy-MAP furnished 13c in 20% yield.
When the tributylstannylethynyl C–H was substituted
with a phenyl group, reaction with 11 under the same
conditions afforded 13d in 96% yield.
Incorporation of a chlorine at the C-6 position of the
pyridine is described in Scheme 3. 5-Bromo-3-benzyl-
oxypyridine (15), prepared from dibromopyridine 14 in
75% yield, was oxidized with m-CPBA to afford N-oxide
16. Chlorination of 16 with POCl3 provided a mixture of
17 and 18 with the desired chloride 18 predominating.
The regio-chemistry of the two isomers was determined
by comparison of de-benzylated material (30% HBr in
HOAc) with an authentic sample of 4 (R1 = Cl,
R2 = R3 = H).7 The chloride 19 was converted to 10g
by the standard protocol as shown in Scheme 1 in 30%
overall yield.
Outlined in Scheme 1 is a general synthesis of the
pyridine–isoquinoline-based Akt inhibitors. Mitsunobu
coupling of 5-bromopyridin-3-ol 4 with N-Boc-tryp-
tophanol (5) provided aryl ether 6. Stille coupling with
trimethylstannyl-isoquinoline 8 and Boc-deprotection
afforded Akt inhibitors 10. Alternately, the aryl bromide
6 was converted to the corresponding trimethylaryltin 7
by treatment with hexamethylditin in refluxing toluene
under the catalysis of Pd(PPh3)4. Compounds 7, under
typical Stille conditions [Pd2(dba)3/(o-tol)3P/DMF],
smoothly coupled with bromide 9 (X = Br). The reaction
of 7 with commercially available 8-methyl-6-chloroiso-
quinoline 9 (X = Cl, R4 = R5 = R6 = H), however,
required the catalyst dicyclohexylphosphino-20-(N,N-dim-
ethylamino)-biphenyl (Cy-MAP). Boc-deprotection under
acidic conditions provided Akt inhibitors 10.
BOC
NH
N
NH
i or ii
O
The C-2 position of the pyridine in 10 was modified
through an intermediate chloride 11, that was in turn
prepared according to the general protocol described
in Scheme 1 from 6-chloro-5-bromopyridin-3-ol 4
(R1 = Cl, R2 = R3 = H).7 The cyanation of 11 under typ-
ical conditions [Zn(CN)2/Pd(Ph3)4/DMF] required more
than 3 days heating at 90 ꢁC, providing cyanide 12 in
Cl
N
N
11
BOC
O
NH
NH
iii
10
R1
Method (i) 12: R1= CN
Method (ii) 13a: R1= Vinyl
13b: R1= 2-Furyl
13c: R1=
N
BOC
NH
NH
R3
HO
BOC
O
R3
N
NH
NH
Br
R1
OH
13d: R1=
5
R
R1
iii
i
N
R2
R2
Scheme 2. Reagents: (i) Zn(CN)2, Pd(PPh3)4, DMF, 89%; (ii)
Pd2(dba)3, dicyclohexylphosphino-20-(N,N-dimethylamino)biphenyl
(Cy-MAP), Et3N, DMF; (iii) CF3CO2H, CH2Cl2.
4
6: R= Br
7: R= SnMe3
R4 R6
Br
OBn
Br
i
R
N
ii, iv
X=Br, Cl
iii
ii
R5
X
N+
O-
R4 R6
N
R7
9
N
R4 R6
14: R=Br
15: R=OBn
16
R5
SnMe3
N
R3
4
NH2
NH
8
O
R5
Br
Cl
OBn Br
OBn
Cl
Br
OH
Cl
v
iv
(via 7)
ii
+
6
10g
R7
R1
2
N
R2
N
N
N
1:5
10
17
18
19
Scheme 1. Reagents and conditions: (i) DEAD, Ph3P, THF. (ii) a—
Pd2(dba)3, P(o-tol)3, DMF; b—CF3CO2H, CH2Cl2; (iii) Pd(PPh3)4,
Scheme 3. Reagents: (i) BnOH, NaH, DMF, 75%; (ii) m-CPBA,
CH2Cl2, 100%; (iii) POCl3, CH2Cl2, 61%; (iv) HBr, HOAc, 84%; (v)
Me3SnSnMe3,
toluene,
110 ꢁC,
3 h,
85%;
(iv)
a—2-
a—Tryptophanol,
DEAD,
Ph3P,
THF,
70%;
b—8
Dicyclohexylphosphino-20-(N,N-dimethylamino)biphenyl, Pd2(dba)3,
Et3N, DMF; b—CF3CO2H, CH2Cl2.
(R4 = R5 = R6 = H), Pd2(dba)3, P(o-tol)3, DMF; c—CF3CO2H,
CH2Cl2, 44% over two steps.

3152
G.-D. Zhu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3150–3155
O
R
The C-4 position of the pyridine was alkylated or acyl-
ated in good to excellent yields through lithiation of
chloride 20b with LDA followed by treatment with
iodomethane or methyl formate (Scheme 4). A similar
reaction with bromide 15 failed to provide any product.
Wittig olefination of the aldehyde 21b with diethylphos-
phonoacetonitrile afforded 21c. The para-methoxyben-
zyl protecting group in 21c was cleaved in refluxing
trifluoroacetic acid to give 22. Conversion to 10h and
10i was carried out by standard procedures as described
in Scheme 1.
i
HN
N
Br
Br
23
24: R= Cl
25a: R= NH2
25b: R= NHBOC
ii
iii
iv or v
vi
R
N
vi
10
Modification at C-1 position of the isoquinoline is de-
scribed in Scheme 5, starting from 6-bromo-isoquinoli-
none 23.8 Treatment of 23 with POCl3 at reflux
provided chloride 24 which was converted to the amine
25. O-methylation of isoquinolinone 23 was conducted
under Mitsunobu conditions. 1-Fluoroisoquinoline 27
was obtained in an average of 15% yield in several at-
tempts through heating 23 with commercially available
perfluoro-2-methyl-2-pentene and triethylamine in ace-
tonitrile in a sealed tube. Stille coupling of 24–27 with
trimethylstannyl 7, followed by Boc-deprotection, gave
the Akt inhibitors 10j–10n.
Br
26: R= OMe
27: R= F
Scheme 5. Reagents and conditions: (i) POCl3, 100 ꢁC, 4 h., 62%; (ii)
Acetamide, K2CO3, 180 ꢁC, 5 h., 65%; (iii) (Boc)2O, DMAP, Et3N,
MeCN; (iv) DEAD, PPh3, THF, 62%; (v) Perfluoro-2-methyl-2-
pentene, Et3N, MeCN, 15%; (vi) a—7, Pd2(dba)3, P(o-tol)3, DMF;
b—CF3CO2H, CH2Cl2.
BOC
NH
N
NH
N
iii
O
R5
R
Br
Syntheses of 3-substituted isoquinoline analogs 10p–
10y are depicted in Scheme 6. 3-Chloro-6-bromoiso-
quinoline 28 and 3-amino-6-bromoisoquinoline 29 were
prepared according to literature procedures.9 Sandmey-
er reaction of 3-aminoisoquinoline 29, followed by
treating with 70% hydrogen fluoride in pyridine, fur-
nished 3-fluoro-analog 30. Stille coupling of 28, 29,
and 30 with trimethylstannyl 7 gave compounds 31,
32, and 33.
N
31: R5= Cl
32: R5= NH2
33: R5= F
28: R= Cl
29: R= NH2
30: R= F
i
ii
iv
vi
34: R5= CN
35: R5= Et
v
36a: R5= Vinyl
36b: R5= 2-Furyl
36c: R5= Phenyl
36d: R5= 4-Pyridyl
36e: R5=
The 3-chloro-functionality in compound 31 was readily
replaced with cyano to give 34 under the same cyanation
condition as detailed in Scheme 2. The Stille reaction of
36f: R5=
vii
10
Scheme 6. Reagents: (i) a—NaH, BnNH2, DMF, 42%, b—CF3CO2H,
reflux, 66%; (ii) NaNO2, HF/pyridine, 92%; (iii) 7, Pd2(dba)3, (o-tol)3P,
Et3N, DMF, 75%; (iv) Zn(CN)2, Pd(PPh3)4, DMF, 71%; (v) H2, 10%
Pd/C, 30%; (vi) Pd2(dba)3, dicyclohexylphosphino-20-(N,N-dimethyl-
amino)biphenyl (Cy-MAP1), Et3N, DMF; (vii) CF3CO2H, CH2Cl2.
OMe
R
Cl
OR
ii or iii
Cl
O
N
N
20a: R= H
20b: R= p-Methoxybenzyl
21a: R= CH3
21b: R= CHO
21c: R=
31 with a variety of tin reagents, under the catalysis of
Cy-MAP, provided 36a–36f. Hydrogenation of 36a in
the presence of 10% palladium on carbon afforded 35.
Boc-deprotection of 31–36f under acidic conditions pro-
vided 10o–10y.
i
iv
CN
R
vi
v
Cl
OH
10h
10i
Nitration of 6-bromoisoquinoline 4 (R4 = R5 = R6 =
R7 = H) with concentrated nitric acid in concentrated
sulfuric acid regio-specifically afforded 5-nitroisoquino-
line 37 (Scheme 7). No other isomer was detected by
either HPLC analysis or 1H NMR of the crude product.
Reduction of the nitro-compound with iron dust in ace-
tic acid provided amino analog 38 which was converted
to 10z through Stille coupling and Boc-deprotection.
N
22a: R= CH3
22b: R=
CN
Scheme 4. Reagents: (i) DEAD, Ph3P, CH2Cl2, 87%; (ii) LDA, MeI,
THF, 65%; (iii) LDA, HCO2Me, THF, 78%; (iv) (EtO)2P(O)CH2CN,
LiHMDS, THF, 95%; (v) CF3CO2H, 95%; (vi) a—N-Boc-tryptopha-
nol, DEAD, Ph3P, THF, 70% for 22a, 60% for 22b; b—8
(R4 = R5 = R6 = H), Pd2(dba)3, 1,3-bis(2,6-di-i-propylphenyl)imidazo-
lium chloride, DMF, 32% for 22a, 48% for 22b; c—CF3CO2H,
CH2Cl2, 81% for 22a, 80% for 22b.
Illustrated in Figure 1 is a depiction of the X-ray struc-
ture of 3 in PKA, a closely related protein kinase of Akt
in the same AGC family.3,6 Corresponding residues for

G.-D. Zhu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3150–3155
3153
Table 1. Enzyme assay results for compounds 3 and 10a–10i against
Akt
N
ii
N
7
i
10z
Br
iii
Br
NH
N
R3
4
NH2
R
4
O
37: R= NO2
38: R= NH2
2
N
6
R1
R2
Scheme 7. Reagents: (i) 63% HNO3/96% H2SO4, 88% yield; (ii) Fe,
HOAc/H2O, 92%; (iii) a—Pd2(dba)3, P(o-tol)3, Et3N, DMF, 60%;
b—CF3CO2H, CH2Cl2, 66%.
10
R2 R3
Compound R1
a
Akt1 IC50 (nM)
3
H
Cl
H
H
H
H
H
H
H
H
2.0
1.8
4.6
2.0
10a
10b
10c
CN
Glu91
Lys72
Val104
Akt1:Thr-211
Akt1:Met
O
H3N
N
O
10d
H
H
1.5
O
Leu173
Thr183
50'S Loop
10e
10f
10g
10h
10i
H
H
Cl
H
H
H
H
H
0.8
1240
12
OH
O
Ph
O
H
H
H
Glu121
NH3 +
O
HN
O
NH
CH3
84
N
O
Phe5
4
H
CN
NH2
O
697
N
O
Akt1:Ala
Val123
a Values are means of two or more experiments. Unless otherwise
Side Chain
specified, all compounds were tested at 5 lM ATP.
Docking Motif
Phe327
300' Strand
tion of a methyl (10h) or acrylonitrile (10i) group at
the C-4 position of the pyridine (IC50 = 84 and
697 nM, respectively).
Figure 1. An illustration of the X-ray structure of 3 bound to PKA.
Akt in the ATP-binding site are shown on the left. As
shown in Figure 1, three sets of hydrogen bonds of 3
to Val-123, Lys-72, and Asn-171/Asp-184 are critical
for its high potency against PKA (IC50 = 2.1 nM). The
hydrophobic indole moiety in 3 fits nicely underneath
the glycine-rich loop. Our initial goal was to optimize
the physico-properties of 3 through substitution on the
pyridine and isoquinoline rings, keeping the remainder
of molecule unchanged. The appropriate substituents
may also pick up additional interactions (e.g., with
Thr-211 in Akt) and lead to more potent and Akt-selec-
tive analogs. In addition, 10k has been characterized as
a major metabolite of 3 in most species and this metab-
olism of 3 was attributed to its short half-life (e.g., 0.3 h
in mice). Therefore, substitution at the C-1 position of
the isoquinoline could block this metabolism. From
the X-ray structure, the space between Glu-121 and
the isoquinoline is rather limited, so the group intro-
duced at the C-1 position required to be relatively small.
As revealed in Table 2, any modification at the C-1 po-
sition of the isoquinoline led to more than two orders of
magnitude loss in potency against Akt. This reduced
potency seems to correlate with the size of the substitu-
ents, less for fluorine analog (10j: IC50 = 227 nM) and
more for chlorine (10l: IC50 = 778 nM) and methoxy
group (10m: IC50 = 39,500 nM). Possible interaction of
an amino group with Val-123 backbone carbonyl is pos-
sibly responsible for the modest potency of amino ana-
log 10n (IC50 = 160 nM).
Substituents are significantly better tolerated at the C-3
position of the isoquinoline scaffold. No obvious corre-
lation was observed though between activity and either
sterics or electronics of the substituents (10o–10y). It is
likely that a combination of factors involved in the pro-
tein–ligand binding, including inhibitor-induced confor-
mational change of the protein (i.e., 10x: IC50 = 56 nM)
and formation of additional interaction with the protein
(i.e., 10y: IC50 = 3.4 nM).
SAR studies of analogs of 3 modified at the ortho- and
para-positions of the central pyridine are summarized
in Table 1. Illustrated by examples 10a–10e, relatively
small groups are tolerated at the C-2 position of the pyr-
idine. No significant impact from a weakly electron-
withdrawing group (e.g., chlorine) was observed at this
position (10a), while introduction of a strongly elec-
tron-withdrawing functionality such as a cyano group
is detrimental to its activity (10b vs 10e). Relatively larg-
er groups, even with a linear linker (10f), led to a much
less potent Akt inhibitor. Introduction of a chlorine at
the C-6 position of the pyridine (10g) resulted in 6-fold
drop in Akt activity (IC50 = 12 nM). An even more dra-
matic reduction in potency was observed by introduc-
Introduction of an amino group at the 5-position of the
isoquinoline resulted in a nearly 10-fold drop in its Akt
activity (10z). In an effort to reduce the metabolic
liability at the C-1 position of the isoquinoline through
a 1,8-steric interaction, a methyl group was installed at
the C-8 position. However, the 500-fold drop in the
potency of 10aa suggests a limited space between the
isoquinoline and the hinge protein backbone.
Summarized in Table 3 is the selectivity profile of selec-
ted Akt inhibitors with representative substituents on
the pyridine and isoquinoline rings. Like compound 3,
all inhibitors, except for 10y, displayed from modest to

3154
G.-D. Zhu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3150–3155
Table 2. Enzyme assay results for compounds 3 and 10j–10aa against
Akt
Table 4. Cellular activity of selected Akt inhibitors in comparison to 3
(IC50a, lM)
R4 R6
Cell
3
10a
10e
10o
10y
GSK3-P
FL5.12-Akt (MTT)
MiaPaCa-2 (MTT)
1.5
3.0
5.7
ndb
1.7
nd
0.93
0.20
0.1
NH
N
NH2
0.42
0.59
2.3
3.18
O
R5
13.3
10.8
R7
R5
a Values are means of two or more experiments.
b Not determined.
N
Compound R4
R6
R7
Akt1
a
IC50 (nM)
inhibitors were evaluated in MiaPaCa-2 human pancre-
atic cancer cells and FL5.12-Akt murine prolymphocytic
cells that overexpress Akt1. As shown in Table 4, signif-
icantly less cellular activity was observed for compounds
10a, 10e, and 10o. 3-Amino analog 10y was statistically
more active in both FL5.12-Akt and MiaPaCa-2 cells,
correlating to its broader spectrum of kinase activity.
3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
2.0
227
10j
10k
10l
10m
10n
10o
10p
10q
10r
10s
F
H
OH
Cl
H
1240
778
H
OMe
NH2
H
H
39,500
160
H
F
3.5
H
Cl
CN
Et
407
640
H
One of the major issues for use of compound 3 as an
antitumor agent is its poor pharmacokinetic profile,
including short half-life in most species (e.g., 0.3 h in
mice), high plasma clearance, and lack of oral bioavail-
ability. The C-1 position was identified as a major site of
metabolism. As summarized in Table 5, substitution at
the metabolically labile C-1 position with chlorine
(10l) significantly increases half-life in mice (5 h). When
administered orally in mice, this compound achieved
respectable plasma exposure with a 3.9 lg-h/mL AUC
and 51% bioavailability (10 mg/kg dose). Introduction
of a chlorine at C-3 position of the isoquinoline (10p)
also improved the pharmacokinetic profile with an in-
creased half-life in mice (2.0 h) and modest oral bio-
availability (27%). No statistically significant difference
was observed for a 3-amino group at the same position
(10y). As we envisioned, the 1,8-steric influence from a
C8-methyl group (10aa) indeed inhibited the metabolism
of the isoquinoline, leading to a prolonged half-life
(4.5 h vs 0.3 h for 3) and 35% oral bioavailability.
Unfortunately, all of these compounds with improved
PK profiles suffered from diminished Akt activity.
H
144
22
H
10t
H
H
H
57
O
10u
10v
H
H
Ph
H
H
H
H
305
122
N
10w
H
CH3
Ph
H
H
180
10x
10y
10z
H
H
H
H
H
H
56
3.4
15
NH2
H
H
H
H
CH3
NH2
H
10aa
H
995
a Values are means of two or more experiments. All compounds were
tested at 5 lM ATP.
Table 3. Selectivity profile of selected Akt inhibitors in comparison to
3 (IC50a, nM)
Kinase
3
10a
10e
10o
10y
Akt1
Akt2
Akt3
PKA
PKCc
PKCn
CDK1
ERK2
CK2
2.0
6.8
1.2
0.8
3.5
3.4
21
5
73
5
39
ndb
nd
35
2.1
76
Due to its overall profile in enzyme- and cellular assays
as well as its PK property, 10y was evaluated in a Mia-
PaCa-2 human pancreatic cell xenograft model. Mia-
PaCa-2 is a human pancreatic carcinoma line and it
has been reported that the Akt signaling pathway is
important in the pathogenesis of this tumor. As illustrat-
ed in Figure 2, at its maximally tolerated dose (75 mg/
kg/day) for subcutaneous administration, this com-
pound significantly slowed tumor growth. However,
toxicity developed, including lethargy, weight loss and
skin irritation at the site of injection after administration
of the Akt inhibitor. All animals were humanely eutha-
nized on day 27 due to the development of these
toxicities.
5
540
1
260
48
nd
8
31
270
6,100
100
910
11,800
2000
>5000
870
8900
4900
27,200
>50,000
>50,000
nd
8300
22
3400
nd
37,000
12,700
12,100
1200
620
480
nd
18,000
SRC
a All compounds were tested at 5 lM ATP.
b Not determined.
excellent selectivity against different families of protein
kinases. Installation of small groups at the C-2 position
of the pyridine as exemplified by 10a and 10e improves
selectivity over some of the kinases such as CDK1, while
3-amino analog 10y was generally less selective.
In summary, substitution at the C-2 position of the pyr-
idine in 3 could improve the selectivity against certain
protein kinases. Chlorination at the C-1 site of metabo-
lism or methylation at C-8 position of the isoquinoline
resulted in compounds with improved pharmacokinetic
properties, but diminished potency against Akt. 3-Ami-
no analog 10y was identified as a potent Akt inhibitor
Phosphorylation of the Akt downstream targets such as
GSK a/b was measured in the presence of inhibitors as
an indication of their Akt inhibitory activity in cells.
Anti-proliferative activities of the majority of our Akt

G.-D. Zhu et al. / Bioorg. Med. Chem. Lett. 16 (2006) 3150–3155
Table 5. Mouse PK summary of representative Akt inhibitorsa
3155
Compound
iv t1/2 (h)
Vd (L/kg)
CL (L/h)
po F (%)
po AUC (lg-h/mL)
sc AUC (lg-h/mL)
3
0.3
5
7.0
9.5
19.5
1.3
0
51
27
0
0
7.6
ndb
nd
10l
10p
10y
10aa
3.9
0.91
0
2.0
0.7
4.5
15.5
11.8
16.4
5.4
12.1
2.5
6.9
nd
35
1.5
a All pharmacokinetic studies shown in this table were carried out in CD1 mice with 3 mg/kg intravenous, 10 mg oral, and 30 mg subcutaneous
dosing. Plasma samples were diluted with 2 volumes of acidified methanol, centrifuged at 11,000g for 5 min, and directly analyzed by UV–LC.
b Not determined.
3. For review, see: (a) Li, Q.; Zhu, G.-D. Curr. Top. Med.
Chem. 2002, 2, 939; (b) Barnett, S.; Bilodeau, M.;
2500
A-423795 at 75 mg/kg/day, b.i.d., s.c., d1-6
Lindsley, C. Curr. Top. Med. Chem. 2005, 5, 109.
4. (a) Lindsley, C.; Zhao, Z.; Leister, W. H.; Robinson, R.;
Barnett, S.; Defeo-Jones, D.; Jones, R.; Hartman, G.;
Huff, J.; Huber, H.; Duggan, M. Bioorg. Med. Chem. Lett
2005, 15, 761; (b) Barnett, S.; Defeo-Jones, D.; Fu, S.;
Hancock, P.; Haskell, K.; Jones, R.; Kahana, A.; Kral, A.;
Leander, K.; Lee, L.; Malinowski, J.; McAvoy, E.; Nahas,
D.; Robinson, R.; Huber, H. Biochem. J. 2005, 385(Pt. 2),
399.
5. Zhao, Z.; Leister, W.; Robinson, R.; Barnett, S.; Defeo-
Jones, D.; Jones, R.; Hartman, G.; Huff, J.; Huber, H.;
Duggan, M.; Lindsley, C. Bioorg. Med. Chem. Lett. 2005,
15, 905.
Vehicle
2000
1500
1000
500
0
Untreated
0
5
10
15
20
25
30
35
6. (a) Luo, Y.; Shomaker, A.; Liu, X.; Woods, K.; Thomas,
S.; de Jong, R.; Han, E.; Li, T.; Stoll, V.; Powlas, J.;
Oleksijew, A.; Mitten, M.; Shi, S.; Guan, R.; McGonigal,
T.; Klinghofer, V.; Johnson, E.; Leverson, J.; Bouska, J.;
Mamo, M.; Smith, R.; Gramling-Evans, E.; Zinker, B.;
Mika, A.; Nguyen, P.; Oltersdorf, T.; Rosenberg, S.; Li,
Q.; Giranda, V. Mol. Cancer Ther. 2005, 4, 977; (b) 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, S.; Liu, X.; Klinghofer, V.; de Jong,
R.; Oltersdorf, T.; Stoll, V.; Jakob, C.; Rosenberg, S.;
Giranda, V. Bioorg. Med. Chem. Lett. 2006, 16, 2000.
7. Koch, V.; Schnatterer, S. Synthesis 1990, 499.
8. Becker, M. R.; Ewing, W. R.; Davis, R. S.; Pauls, H. W.;
Ly, C.; Li, A.; Mason, H. J.; Choi-Sledeski, Y. M.; Spada,
A. P.; Chu, V.; Brown, K. D. Bioorg. Med. Chem. Lett.
1999, 9, 2753.
Days post inoculation
Figure 2. Analysis of 10y (A-423795) in the MiaPaCa-2 xenograft
pancreatic cancer model.10 10y was subcutaneously dosed at 75 mg/kg
twice daily from day 1 to day 6 after inoculation. Tumor size was
evaluated by twice weekly measurements with digital calipers. 10y
significantly inhibited tumor growth but also resulted in significant
toxicity.
both in enzymatic and cellular assays. This compound
significantly slowed tumor growth in vivo, but was
accompanied by toxicity. The improved in vitro cytotox-
icity of 10y over 3 in a number of cell lines, as well as
in vivo efficacy, seemed to correlate to its broader spec-
trum of activity against other protein kinases such as
CDKs. Further profiling studies for this series of Akt
inhibitors are underway and will be published in due
course.
9. Li, Q.; Woods, K.; Zhu, G.-D.; Fischer, J.; Gong, J.; Li,
T.; Gandhi, V.; Thomas, S.; Packard, G.; Song, X.;
Abrams, J.; Diebold, R.; Dinges, J.; Hutchins, C.; Stoll,
V.; Rosenberg, S.; Giranda, V.; Colussi, D. J.; Leadley, R.
J.; Bentley, R.; Bostwick, J.; Kasiewski, C.; Morgan, S.
WO Patent 2003051366, 2005.
Acknowledgments
10. Animal studies were conducted following the guidelines of
the internal Institutional Animal Care and Use Commit-
tee. Immunocompromised male scid mice (C.B-17-
Prkdcscid) were randomly assigned to treatment groups
and therapy was initiated the day after inoculation. Ten
animals were assigned to each group, including controls.
MiaPaCa-2 cells were obtained from the American Type
Culture Collection (Manassa, VA). MiaPaCa-2 cells
(2 · 106) in 50% Matrigel (BD Biosciences, Bedford,
MA) were inoculated subcutaneously into the flank.
Tumor size was evaluated by twice weekly measurements
with digital calipers. Tumor volume was estimated using
the formula: V = L · W2/2.
We thank Drs. Thomas Penning and Milan Bruncko for
proofreading this manuscript and valuable suggestions.
References and notes
1. For a review, see: Nicholson, K. M.; Anderson, N. G.
Cell. Signalling 2002, 14, 381.
2. For reviews, see: (a) Vivanco, I.; Sawyers, C. Nat. Rev.
2002, 2, 489; (b) Gills, J.; Dennis, P. Expert Opin. Investig.
Drugs 2004, 13, 787.