Synthesis and structure-activity relationship of 3,4′- bispyridinylethylenes: Discovery of a potent 3-isoquinolinylpyridine inhibitor of protein kinase B (PKB/Akt) for the treatment of cancer

Article abstract of DOI:10.1016/j.bmcl.2005.12.065

Structure-based design and synthesis of the 3,4′-bispyridinylethylene series led to the discovery of 3-isoquinolinylpyridine 13a as a potent PKB/Akt inhibitor with an IC₅₀ of 1.3 nM against Akt1. Compound 13a shows excellent selectivity against distinct families of kinases such as tyrosine kinases and CAMK, and displays poor to marginal selectivity against closely related kinases in the AGC and CMGC families. Moreover, 13a demonstrates potent cellular activity comparable to staurosporine, with IC₅₀ values of 0.42 and 0.59 μM against MiaPaCa-2 and the Akt1 overexpressing FL5.12-Akt1, respectively. Inhibition of phosphorylation of the Akt downstream target GSK3 was also observed in FL5.12-Akt1 cells with an EC₅₀ of 1.5 μM. The X-ray structures of 12 and 13a in complex with PKA in the ATP-binding site were determined.

Full text of DOI:10.1016/j.bmcl.2005.12.065

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2000–2007
Synthesis and structure–activity relationship of
3,40-bispyridinylethylenes: Discovery of a potent
3-isoquinolinylpyridine inhibitor of protein kinase B
(PKB/Akt) for the treatment of cancer
Qun Li,a,* Keith W. Woods,a Sheela Thomas,a Gui-Dong Zhu,a Garrick Packard,a
John Fisher,a Tongmei Li,a Jianchun Gong,a Jurgen Dinges,a Xiaohong Song,a
Jason Abrams,a Yan Luo,a Eric F. Johnson,a Yan Shi,a Xuesong Liu,a Vered Klinghofer,a
Ron Des Jong,b Tilman Oltersdorf,b Vincent S. Stoll,a Clarissa G. Jakob,a
Saul H. Rosenberga and Vincent L. Girandaa
aCancer Research, GPRD, Abbott Laboratories, Abbott Park, IL 60064-6101, USA
bIdun Pharmaceuticals, 9380 Judicial Dr., San Diego, CA 92121, USA
Received 29 November 2005; revised 15 December 2005; accepted 19 December 2005
Available online 18 January 2006
Abstract—Structure-based design and synthesis of the 3,40-bispyridinylethylene series led to the discovery of 3-isoquinolinylpyridine
13a as a potent PKB/Akt inhibitor with an IC50 of 1.3 nM against Akt1. Compound 13a shows excellent selectivity against distinct
families of kinases such as tyrosine kinases and CAMK, and displays poor to marginal selectivity against closely related kinases in
the AGC and CMGC families. Moreover, 13a demonstrates potent cellular activity comparable to staurosporine, with IC50 values of
0.42 and 0.59 lM against MiaPaCa-2 and the Akt1 overexpressing FL5.12-Akt1, respectively. Inhibition of phosphorylation of the
Akt downstream target GSK3 was also observed in FL5.12-Akt1 cells with an EC50 of 1.5 lM. The X-ray structures of 12 and 13a in
complex with PKA in the ATP-binding site were determined.
Ó 2005 Elsevier Ltd. All rights reserved.
Kinase inhibitors have generated considerable interest
due to the remarkable success of Gleevec and other
N
inhibitors of tyrosine kinases (TKs) in clinical oncolo-
gy.1 As one of the major downstream targets of several
receptor TKs in the signal transduction pathways, Akt
(PKB) plays a central role in promoting cancer cell pro-
liferation and survival.2 Akt is a serine/threonine kinase
belonging to the AGC family of kinases.3 It is overex-
pressed and constitutively activated in a large number
of human tumors.2f Antisense inhibition of Akt has been
shown to inhibit proliferation and induce apoptosis in
several cancer cells.4 All of these findings have recently
led to an intense search for small molecule inhibitors
of Akt as anticancer agents.5 In the preceding paper,
N
Het
L
O
O
N
2-9
R
R
10a
NH2
NH2
NH2
NH2
N
N
2
3
α
X
NH
O
3'
4
4'
β
R
R
1
N
NH2
N
N
5'
11
N
N
N
O
α
O
Keywords: Inhibitors of Akt; Akt1; Akt2; Akt3; PKA; PKB; protein
kinase B; GSK3; FL5.12-Akt1; X-ray; Anticancer; Apoptosis; Anti-
tumor; Pharmacokinetics; Kinase inhibitors; Structure-based design.
5
R
R
12
N
NH2
13
*
Corresponding author. Tel.: +1 847 937 7125; fax: +1 847 936
1550; e-mail: qun.li@abbott.com
Figure 1. Modifications of 1 yielded potent Akt inhibitor (13).
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.065

Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2000–2007
2001
N
N
N
N
a
b, c
O
O
O
Br
O
HO2C
O
NH
15
14
R
Py
R
R
16
R
NHBoc
NH2
3
NHBoc
b,c
d, c
NHBoc
R=3-Indolylmethyl
N
a
N
N
N
d
O
Ar2
O
H2N
17
O
Ar1
Br
O
R
14
R
2
R
R
NH2
6-9, 13
NH2
BocNH
NHBoc
R=Ph, 3-indolyl
g, c
e, c
(for 4)
or f, c
(for 5)
Scheme 1. Reagents and conditions: (a) i—TMSCꢀCH, CuI/
Pd2Cl2(PPh3)2, Et3N, DMF, 50 °C, 15 h, 80%; ii—TBAF, THF, rt,
86%; (b) Ar1Br, CuI/Pd2Cl2(PPh3)2, Et3N, DMF, 50 °C, 20 h, 60–75%;
(c) TFA, CH2Cl2, rt, 2 h; (d) Ar2SnMe3, Pd2(dba)3/P(o-tol)3, DMF,
75 °C, 8 h, or Ar2B(OH)2, Pd(PPh3)4, DMF, 100 °C, 8–10 h or i—
Me3SnSnMe3, Pd(PPh3)4, toluene, 100 °C, 8 h; ii—Ar2Br, Pd2(dba)3/
P(o-tol)3, DMF, 75 °C, 8 h.
N
N
HN
Het
O
HN
O
R
Py
X
R
NH2
NH2
4, X=O; 5, X=2H
7
Scheme 2. Reagents and conditions: (a) CO (900 psi), Pd(dppf)Cl2,
THF/water, 100 °C, 19 h, 76%; (b) PyNH2, EDC, HOBt, DMF, rt,
overnight, 18–23%; (c) TFA, CH2Cl2, rt, 2 h; (d) i—Ph2C@NH,
Pd2(dba)3, BINAP, NaOBut, toluene, 80 °C, 4 h; ii—10% HCl, THF,
rt, 2 h, 70%; (e) PyCO2H, EDC, HOBt, DMF, rt, 50–70%; (f) PyCHO,
acetic acid, MeOH, rt, 1 h; then NaBH3CN, rt, 1 h, 70%; (g) t-BuONa,
Pd2(dba)3, dioxane, 90 °C, 3 h, 60–75%.
we reported a novel series of Akt inhibitors that contain
the 3,40-bispyridinylethylene core (1) resulting from
structural modifications of a screening lead.6 We report
herein our continued optimization efforts of the 3,40-
bispyridinylethylene series that has led to the identifica-
tion of 3-isoquinolinylpyridine 13a as a potent PKB/Akt
inhibitor with an IC50 of 1.3 nM against Akt1 (Fig. 1).
H
N
N
O
CO2H
a
Syntheses of the alkyne (2) and 3-arylsubstituted pyri-
dines 6–9 and 13 are outlined in Scheme 1. Sonogashira
coupling of 146 with TMS-acetylene followed by depro-
tection of the TMS group with TBAF produced acety-
lene 15, which underwent the Heck reaction with
selected aryl bromides to give 2. Conversion of 14 to
the 3-arylpyridines (6–9 and 13) proceeded via the Stille
or Suzuki reactions with corresponding tin or boronic
acid coupling partners.
O
+
Br
Br
OH
Br
20
O
21
19
b
c
N
R=3-Indolylmethyl
N
Py
O
Br
O
R
10
22
NH2
NH
BocNH
The analogs (3–5 and 7) with double bond replacements
were prepared as described in Scheme 2. Amide 3 was
prepared in a three-step sequence involving palladium-
catalyzed carboxylation of 14, coupling of the resulting
16 with 4-aminopyridine and deprotection with TFA.
Palladium-catalyzed amination of bromide 14 with ben-
zophenone imine following the procedure developed by
Buchwald et al.,7 and subsequent acid hydrolysis provid-
ed amine 17. Coupling of 17 with pyridinecarboxylic
acids in the presence of EDC and subsequent deprotec-
tion of the Boc group with TFA afforded amide 4. Con-
version of amine 17 to pyridinylmethylamine 5 was
achieved through reductive amination of pyridine-car-
baldehydes and subsequent TFA deprotection. Pd-cata-
lyzed amination of bromide 14 followed by deprotection
furnished compound 7.
Scheme 3. Reagents and conditions: (a) i—KOH, water, rt, 6 days,
58%; ii—nitrobenzene, reflux, 5 min, 55%; (b) N-Boc-L-tryptophanol,
DEAD/PPh3, THF, rt, 2 h, 72%; (c) i—PyB(OH)2, Pd(PPh3)4, DMF,
100 °C, 8 h, 70–76%; ii—TFA, CH2Cl2, rt, 2 h, 90%.
10 from 21 was carried out using the same three-step
protocol as described in Scheme 1.
Ullmann coupling between dibromopyridine 23a and
phenol 24a at 240 °C under microwave conditions gave
ether 25a. Palladium-catalyzed amination of aminopyri-
dine 23b with triflate 24b provided aniline 25b. Com-
pounds 25a–b were converted to amines 26 as
previously described followed by acid hydrolysis.
Reductive amination of 26 with N-Boc-tryptophanal in
the presence of NaBH3CN and Ti(i-PrO)4 and subse-
quent acidic deprotection of the Boc group provided
compounds 11a–b (Scheme 4).
Preparation of the conformationally constrained ana-
logs is illustrated in Schemes 3–5. Compound 10 with
C-b of the double bond cyclized with C-2 of the central
pyridine was synthesized as depicted in Scheme 3. Bro-
moisatin 19 reacted with bromopyruvic acid 20 in aque-
ous KOH solution to form the cinchoninic acid, which
underwent decarboxylation in refluxing nitrobenzene8
to provide bromohydroxy-quinoline 21. Synthesis of
Preparation of analogs cyclized between C-b and C-4 of
the central pyridine is shown in Scheme 5. PMB protect-
ed bromopyridine 27b (made from 27a) was converted
to 3-Boc-aminopyridine 28 via the amination protocol
as previously described followed by Boc protection in

2002
Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2000–2007
the terminal pyridine binds to the hinge region via a
N
hydrogen bond with the backbone NH of Val123, while
the nitrogen of the central pyridine hydrogen bonds to
the amino group of Lys72. Although the terminal pyri-
dine is tightly sandwiched between the isopropyl group
of Leu173 and the methyl group of Ala70, there is avail-
able space surrounding the trans-double bond linking
the two pyridines. Therefore, our initial efforts focused
on replacing the double bond with other functional
groups (Table 1). Substitution of the double bond with
a triple bond (2a) resulted in sharply lowered activity.
The lost activity in linear acetylene 2a cannot be re-
gained with an angled 3-pyridinyl group (2b). The bio-
isosteric amide analogs (3a, 4a–b) are much less active
than the corresponding double bond analogs (1a–b).
These results are consistent with the highly hydrophobic
nature of the binding pocket. All other double bond
replacements including the methylamine (5a–b), amine
(7a–d) ortho-phenyl (6a), and 2,4-thiazole (6b–c) groups
(Table 1) resulted in a significant loss in activity.
Y
N
A
Br
a (25a)
b (25b)
c
+
N
Br
X
N
A
23a: X=Br
24a: Y=OH
23b: X=NH2 24b: Y=OTf
25a: A=O
25b: A=NH
N
N
A
NH2
d, e
NH
N
N
NH2
NH
11a: A=O
11b: A=NH
26
Scheme 4. Reagents and conditions: (a) 23a + 24a, K2CO3, DMF,
microwave, 240 °C, 10 min, 23%; (b) 23b + 24b, Pd2(dba)3, BINAP,
NaOBut, DMF, 90 °C, 8 h, 18%; (c) i—Ph2C@NH, Pd2(dba)3, BINAP,
NaOBut, toluene, 80 °C, 3 h; ii—1 N HCl, THF, rt, 15 min, 44–45%;
(d) N-Boc-L-tryptophanal, Ti(i-PrO)4, CH2Cl2, 2 h; then NaBH3CN,
2 h, 24–40%; (e) TFA, CH2Cl2, rt, 2 h.
N
N
Bispyridine 8a (Table 1), which has the double bond
deleted, shows better than expected activity. Its IC50 val-
ue of 260 nM is respectable considering that the pyridine
is further away from the hinge. A series of 3-arylpyri-
dines were therefore prepared with functional groups
capable of hydrogen bonding attached to the aryl rings
(Table 2). Some of these compounds should presumably
fit in the binding pocket better than 8a and an additional
hydrogen bond with the hinge is therefore possible.
Unfortunately, none of the compounds in Table 2 shows
improved activity to 8a, except for 9m, which has an
IC50 of 227 nM. We speculate that the lack of van der
Waals interactions with Ala70 and Leu173 for these
analogs (9a–x), as seen for 1b, may contribute partially
to the poor activity.
N
c
b
BocNH
OPMB
Br
OR
BocNH
OPMB
28
27a: R=H
27b, R=PMB
O
H
a
29
N
N
d
e
f
N
OPMB
HN
OPMB
Cl
O
31
30
N
N
g
N
OPMB
N
O
N
N
NH2
NH
32
12
Having been unsuccessful in replacing the trans-double
bond, we next investigated structures resulting from
cyclizations of the trans-double bond onto the adjacent
pyridines. This approach also restricts the number of
rotatable bonds and hence has the potential to increase
potency. As shown in Figure 1, there are four possible
ways to cyclize the double bond onto either the central
or terminal pyridine. Note that in the X-ray structure
in complex with PKA, 1 adopts the conformation shown
in Figure 1. In order to cyclize C-b with C-2 (10a), the
single bond connecting C-a has to rotate 180° to a less
favorable conformation. The resulting analog 10a is
21-fold less active than 1b (Table 3). Replacing the 4-
pyridine moiety in 10a with a 3-pyridine imparts an
additional 24-fold drop in activity (10b). Activity also
decreased sharply for analogs 11a–b incorporating cycli-
zations between C-b and C-50. Cyclization between C-b
and C-4 appears to be more favorable. The resulting
naphthyridine (12) shows improved potency as com-
pared with compounds 10 and 11, but is still 14-fold less
potent than 1b. Finally, cyclization between C-a and
C-30 led to a breakthrough Akt1 inhibitor 13a, which
is 10-fold more potent than 1b with an IC50 of 1.3 nM.
Compound 13a possesses one less rotatable bond than
1b, and it contains an isoquinoline group that provides
stronger van der Waals interactions with the hydropho-
Scheme 5. Reagents and conditions: (a) PMBCl, K2CO3, Bu4NCl,
DMF, rt, 3.5 days, 50%; (b) i—Ph2C@NH, Pd2(dba)3, BINAP,
NaOBut, toluene, 80 °C, 3 h; ii—1 N HCl, THF, rt, 15 min, 71%; iii—
Boc2O, KN(TMS)2, THF, rt, 55%; (c) BuLi, HCO2CH3, ꢁ78–10 °C,
45 min, 63%; (d) i—(CF3CH2O)2POCH2CO2CH3, KN(TMS)2, THF,
ꢁ78 °C, 20 min; ii—NaN(TMS)2, rt, 30 min, 75%; (e) POCl3, DMF,
45 °C, 6 h, 79%; (f) 4-PySnBu3, Pd2(dba)3, Cy-MAP1, Et3N, DMF,
100 °C, 5 h, 49%; (g) i—1 N HCl, EtOH, 90 °C, 2 h, 74%; ii—N-Boc-L-
tryptophanol, DEAD, PPh3, THF, rt, 4 h, 83%; iii—TFA, p-meth-
oxythiobenzyl alcohol, CH2Cl2, rt, 20 min, 60%.
the presence of KN(TMS)2. Lithiation of 28 with n-BuLi
at ꢁ78 °C and subsequent quenching of the anion with
methyl formate afforded aldehyde 29. Wittig–Horner
reaction of aldehyde 29 with methyl bis(trifluoroeth-
yl)phosphonoacetate and cyclization with NaN(TMS)2
provided 30. 2-Naphthyridone 30 was converted to chlo-
ride 31 by POCl3 in DMF followed by coupling with 4-
tributylstannylpyridine to provide 32. Compound 32
was treated with HCl to remove the PMB group and
the resulting phenol was coupled to Boc-tryptophanol
via the Mitsunobu reaction. Finally, acidic deprotection
of the Boc group yielded 12.
The X-ray structure of 1b in complex with PKA6 (a ki-
nase closely related to Akt) revealed that the nitrogen of

Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2000–2007
2003
Table 1. Activity of Akt inhibitorsa
N
R2
Py
L
O
NH2
R2
a
Akt1 IC50 (nM)
Compound
Py
L
1a
690
N
1b
2a
2b
3a
4a
4b
5a
5b
6a
6b
6c
7a
7b
7c
7d
8a
14
N
NH
NH
NH
NH
210
N
N
25,200
2020
31,950
26%b
26%b
41%b
10%b
9870
9350
2860
2160
18,830
6970
260
H
N
N
O
O
N
H
N
N
O
N
H
N
H
N
N
N
H
N
NH
S
N
N
N
S
N
N
H
N
N
NH
NH
N
H
N
H
N
N
N
H
Bond
N
NH
a Against Akt1 with ATP concentration of 10 lM.6
b Inhibition at 50 lM.
bic pocket than does the vinylpyridinyl group in 1b.
Note that S-isomer 13a is 20-fold more potent than
the corresponding R-isomer 13b.
The selectivity profile and cellular activity of 13a and
1b6 are summarized in Table 4. The non-selective ki-
nase inhibitor staurosporine was used as a control.

2004
Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2000–2007
Table 2. Activity of Akt inhibitorsa
N
X
L
O
NH2
NH
a
Akt1 IC50 (nM)
Compound
X
L
9a
1340
1830
1560
13%b
6940
31,280
9310
41%b
48%b
12%b
0%b
N
O
N
Me
Me
9b
9c
9d
9e
9f
N
F
O
N
O
H2N
O
OH
O
9g
9h
9i
NH2
HN
NH2
N
HO
O
NH2
O S
9j
NH2
O
O S
9k
9l
NH2
NH2
1390
227
O
O
9m
9n
9o
9p
9q
N
H
12,030
3%b
O
O
N
H
H
N
Me
O S
O
33,010
33,160
N
H
N
N
N
N
H
O
9r
1120
N
N
N
H
H

Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2000–2007
2005
Table 2 (continued)
a
Akt1 IC50 (nM)
Compound
X
L
HN
9s
N
2710
H2N
N
HN
N
9t
14,330
3670
6230
1450
2170
N
N
HN
N
9u
9v
9w
9x
HN
N
S
O
Cl
O
H2N
NH2
a Against Akt1 with ATP concentration of 10 lM.6
b Inhibition at 50 lM.
Table 3. Activity of Akt inhibitorsa,b
Ar2
Ar1
A
NH2
NH
a
Akt1 IC50 (nM)
Compound
Ar1
Ar2
A
O
N
N
10a
300
7090
210
270
190
1.3
N
10b
O
N
N
11a
NH
NH
O
O
N
N
N
11b
HN
N
N
N
12
N
13a (S-)
13b (R-)
O
N
N
O
26
a Unless otherwise specified, all compounds are S-enantiomers.
b Against Akt1 with ATP confcentration of 10 lM.6
Overall, compound 13a demonstrates excellent selectiv-
ity against distinct families of kinases including TK
and CAMK,3 and possesses IC50 values greater than
5 lM against most of these kinases. However, selectiv-
ity against closely related kinases of the AGC3 and
CMGC3 families is poor to moderate because of the
high degree of homology in the ATP-binding pocket.
Similar to 1b, 13a largely displays no selectivity for
any of the three Akt isoforms, presumably due to very
high sequence homology, especially in the ATP-binding
pocket where the homology is nearly 100% (except one
non-crucial amino acid in Akt3). For similar reasons,
13a is non-selective against PKA (IC50 = 2.1 nM).
Selectivities versus other closely related kinases such

2006
Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2000–2007
a
Table 4. Selectivity profile and cellular activity of 1b and 13a in comparison with those of staurosporine (IC50
,
nM)
Test
Name
1b
13a
Staurosporine
Kinases
Akt1
Akt2
14
257
1.3
6.8
1.5
6.5
Akt3
PKA
354
38
35
2.1
10
3.6
PKCc
PKCf
CDK1
ERK2
MAPK-AP2
CK2
1200
930
270
6100
100
910
6.6
570
0.4
>10,000
12,850
>10,000
>50,000
16,000
>50,000b
>50,000b
>50,000b
>50,000b
370
110
3400
9.5
88b
58b
23b
1920b
>50,000
11,800
4200
Chk1
KDR
FLT4
C-KIT
SRC
>50,000b
>50,000b
26,200b
>50,000b
Cellular activity6
GSK3-Pc
FL5.12-Akt1 (MTT)d
MiaPaCa-2 (MTT)d
10,000
3000
12,000
1500
420
460
290
370
590
a Unless otherwise specified, ATP concentration at 10 lM.6
b ATP concentration at 1.0 mM.
c EC50 against GSK3 phosphorylation in the Akt1 overexpressing cell FL5.12-Akt1.
d IC50 against cell proliferation in MTT assays.
as PKC, CDK, and ERK2 are moderate, ranging from
77- to 500-fold.
1b. Compound 13a also inhibits the phosphorylation of
the Akt downstream target GSK3 in FL5.12-Akt1 cells
with an EC50 of 1.5 lM.6
Compound 13a was evaluated for its antiproliferative
activity against FL5.12-Akt1 murine prolymphocytic
cells that overexpress Akt1 and MiaPaCa-2 human pan-
creatic cancer cells.6 Its IC50 values of 0.42 and 0.59 lM
against the Akt1 overexpressing FL5.12-Akt1 and Mia-
PaCa-2, respectively, represent approximately a 10-fold
improvement over 1b, and are comparable with that of
staurosporine (Table 4). These improvements in cellular
potency for 13a are in agreement with the same compa-
rable increases in enzymatic activity observed relative to
The X-ray structures of 12 and 13a in complex with PKA
in the ATP-binding site have been determined (Fig. 2).9
The binding pattern of 13a is almost identical to that
of1b.6 The nitrogen of the isoquinoline binds to the hinge
via a hydrogen bond with the backbone NH of Val123
˚
with a distance of 3.0 A between the two nitrogens. The
pyridine of the isoquinoline is sandwiched between the
isopropyl group of Leu173 and the methyl group of
Ala70, while the benzene ring of the isoquinoline is in a
Figure 2. Stereo views of overlays of X-ray structures9 of Akt inhibitors 13a (in gray) and: (A) 1b (in purple); (B) 12 (in blue) in complex with PKA in
the ATP-binding site.

Q. Li et al. / Bioorg. Med. Chem. Lett. 16 (2006) 2000–2007
2007
hydrophobic pocket defined by amino acid residues of
Val57, Ala70, Val104, Met120, Leu173, and Thr183.
C-1 of the isoquinoline is buried deep inside the binding
of the Industrial Macromolecular Crystallography
Association.
˚
cavity with a distance of merely 3.2 A between C-1 and
the carbonyl group of Glu121, leaving little space for
any group larger than hydrogen atom. Interestingly, if
the C-1 hydrogen of the isoquinoline were capable of
hydrogen bonding, it might be a perfect fit. The nitrogen
of the central pyridine hydrogen bonds to the amino
group of Lys72 (distance between the nitrogens is
References and notes
1. (a) Druker, B. J. Adv. Cancer Res. 2004, 91, 1; (b) Krause,
D. S.; Van Etten, R. A. N. Engl. J. Med 2005, 353, 172; (c)
Ross, J. S.; Schenkein, D. P.; Pietrusko, R.; Rolfe, M.;
Linette, G. P.; Stec, J.; Stagliano, N. E.; Ginsburg, G. S.;
Symmans, W. F.; Pusztai, L.; Hortobagyi, G. N. Am. J.
Clin. Pathol. 2004, 122, 598.
2. For recent reviews on Akt see: (a) Barnett, F. S.;
Bilodeau, M. T.; Lindsley, C. W. Curr. Top. Med. Chem.
2005, 5, 109; (b) Bellacosa, A.; Kumar, C. C.; Di
Cristofano, A.; Testa, J. R. Adv. Cancer. Res. 2005, 94,
29; (c) Mitsiades, C. S.; Mitsiades, N.; Koutsilieris, M.
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˚
2.8 A). The charged primary amine of the side chain sits
tightly in the Mg-binding loop, binding to Asn171 and
Asp184 via H-bonds and ionic interactions. The indole
ring packs underneath the glycine-rich loop with an aver-
˚
age distance of 3.5 A to the loop. Note that the entrance to
the ATP-binding pocket near the hinge region is partially
blocked by residues 315–327 of the C-terminal domain.
The nearest distance from the isoquinoline to Phe327 is
˚
approximately 3.5 A, enabling the isoquinoline of 13a to
fit tightly in the hinge region.
Overlay of structures of 12 and 13a (Fig. 2b) in complex
with PKA revealed that 12 binds similarly to 13a, except
that the side chain of 12 is forced into an unfavorable
position due to the clash between H-5 of the isoquino-
line and H-a of the side chain (Fig. 1). This change leads
to weakened hydrogen bonding interactions of the pri-
mary amino group with Asn171 and Asp184. The dis-
tances of the hydrogen bonds are in sub-optimal
3. Hanks, S.; Hunter, T. FASEB 1995, 9, 576.
4. (a) Liu, X.; Shi, Y.; Han, E. K.; Chen, Z.; Rosenberg, S.
H.; Giranda, V.; Luo, Y.; Ng, S. C. Neoplasia 2001, 3, 278;
(b) Cheng, J. Q.; Ruggeri, B.; Klein, W. M.; Sonoda, G.;
Altomare, D. A.; Watson, D. K.; Testa, J. R. Proc. Natl.
Acad. Sci. U.S.A. 1996, 93, 3636.
5. (a) Zhao, Z.; Leister, W. H.; Robinson, R. G.; Barnett,
S. F.; Defeo-Jones, D.; Jones, R. E.; Hartman, G. D.;
Huff, J. R.; Huber, H. E.; Duggan, M. E.; Lindsley, C.
W. Bioorg. Med. Chem. Lett. 2005, 15, 905; (b)
Lindsley, C. W.; Zhao, Z.; Leister, W. H.; Robinson,
R. G.; Barnett, S. F.; Defeo-Jones, D.; Jones, R. E.;
Hartman, G. D.; Huff, H. E.; Duggan, M. E. Bioorg.
Med. Chem. Lett. 2005, 15, 761; (c) Breitenlechner, C.
B.; Wegge, T.; Berillon, L.; Graul, K.; Marzenell, K.;
Friebe, W.-G.; Thomas, U.; Schumacher, R.; Huber, R.;
Engh, R. A.; Masjost, B. J. Med. Chem. 2004, 47, 1375;
(d) Reuveni, H.; Livnah, N.; Geiger, T.; Klein, S.; Ohne,
O.; Cohen, I.; Benhar, M.; Gelllerman, G.; Levitzki, A.
Biochemistry 2002, 41, 10304.
6. Li, Q., Li, T.; Zhu, G-D.; Gong, J.; Claiborne, A.; Dalton,
C.; Luo, Y.; Liu, X.; Klinghofer, V.; Bauch, J. L.; March,
K. C.; Bouska, J. J.; Airies, S.; De Jong, R.; Oltersdorf, T.;
Stoll, V. S.; Jakob, C. G.; Rosenberg, S. H.; Giranda, V.
Bioorg. Med. Chem. Lett. 2005, doi:10.1016/
j.bmcl.2005.12.017.
˚
lengths of 3.4 and 4.1 A, respectively. In comparison,
˚
the primary amino group of 13a is positioned 2.9 A
equally from the above residues.
In summary, structure-based design and synthesis of the
3,40-bispyridinylethylene series of Akt/PKB inhibitors
led to the discovery of 3-isoquinolinylpyridine 13a,
which exhibits an IC50 of 1.3 nM against Akt1. Com-
pound 13a shows excellent selectivity against distinct
families of kinases such as TK and CAMK, and displays
poor to marginal selectivity against closely related ki-
nases in the AGC and CMGC families. Moreover, 13a
demonstrates potent cellular activity comparable to
staurosporine. Inhibition of the phosphorylation of the
Akt downstream target GSK3 by 13a was also observed
in FL5.12-Akt1 cells. With the available X-ray struc-
tures of the Akt inhibitors in complex with PKA, further
structural refinements of this series via structure-based
design to optimize the physiochemical and biological
properties of molecules is ongoing, the results of which
will be published in due course.10
7. Wolfe, J. P.; Ahman, J.; Sadighi, J. P.; Singer, R. A.;
Buckwald, S. L. Tetrahedron Lett. 1997, 38, 6367.
8. Cragoe, E., Jr.; Robb, C. A.; Bealor, M. D. J. Org. Chem.
1953, 18, 552.
9. PKA and Akt inhibitors (12 and 13a) were co-crystallized in
a solution containing PKA (20 mg/mL) and PKI peptide.6
The structures were determined and refined to resolutions of
˚
˚
3.0 A for 12 (Rwork = 22.97% and RFree = 31.61%) and 2.0 A
for 13a (Rwork = 28.26% and RFree = 32.28%). Crystallo-
graphic data described in this paper have been deposited
with PDB (ID 2F7Z for 12 and 2F7E for 13a).
Acknowledgments
The authors are grateful to Mr. Rick Clark, Dr. Moshe
Weitzberg, Dr. Matt Hensen, Dr. Robert Keyes, Dr. Curt
Cooper, and Dr. Chris Krueger for proofreading and
valuable suggestions. X-ray crystallographic data were
collected at beamline 17-ID in the facilities of the Indus-
trial Macromolecular Crystallography Association Col-
laborative Access Team (IMCA-CAT) at the Advanced
Photon Source, which are supported by the companies
10. Luo, Y.; Shoemaker, A.; Liu, X.; Woods, K.; Thomas, S.;
de Jong, R.; Han, E.; Li, T.; Stoll, S. S.; Powelas, J. A.;
Oleksijew, A.; Mitten, M. J.; Shi, Y.; Guan, R.; McGon-
igal, T. P.; Klinghofer, V.; Johnson, E. F.; Leverson, J. D.;
Bouska, J. J.; Mamo, M.; Smith, R. A.; Gramling-Evans,
E. E.; Zinker, B. A.; Mika, A. K.; Nguyen, P. T.;
Oltersdorf, T.; Rosenberg, S. H.; Li, Q.; Giranda, V. Mol.
Cancer Ther. 2005, 4, 977.