Structure-Based Optimization of Novel Azepane Derivatives as PKB Inhibitors

Article abstract of DOI:10.1021/jm0310479

Novel azepane derivatives were prepared and evaluated for protein kinase B (PKB-α) and protein kinase A (PKA) inhibition. The original (-)-balanol-derived lead structure (4R)-4-(2-fluoro-6-hydroxy-3-methoxy-benzoyl)-benzoic acid (3R)-3-[(pyridine-4-carbon

Full text of DOI:10.1021/jm0310479

J . Med. Chem. 2004, 47, 1375-1390
1375
Str u ctu r e-Ba sed Op tim iza tion of Novel Azep a n e Der iva tives a s P KB In h ibitor s
Christine B. Breitenlechner,‡ Thomas Wegge,† Laurent Berillon,† Klaus Graul,† Klaus Marzenell,†
Walter-Gunar Friebe,† Ulrike Thomas,† Ralf Schumacher,† Robert Huber,‡ Richard A. Engh,†,‡ and
Birgit Masjost*,†
Pharma Research, Roche Diagnostics GmbH, Werk Penzberg, Nonnenwald 2, D-82372 Penzberg, Germany, and
Abteilung fu¨r Strukturforschung, MPI fu¨r Biochemie, Am Klopferspitz 18a, D-82152 Martinsried, Germany
Received October 1, 2003
Novel azepane derivatives were prepared and evaluated for protein kinase B (PKB-R) and
protein kinase A (PKA) inhibition. The original (-)-balanol-derived lead structure (4R)-4-(2-
fluoro-6-hydroxy-3-methoxy-benzoyl)-benzoic acid (3R)-3-[(pyridine-4-carbonyl)amino]-azepan-
4-yl ester (1) (IC50 (PKB-R) ) 5 nM) which contains an ester moiety was found to be plasma
unstable and therefore unsuitable as a drug. Based upon molecular modeling studies using
the crystal structure of the complex between PKA and 1, the five compounds N-{(3R,4R)-4-
[4-(2-fluoro-6-hydroxy-3-methoxy-benzoyl)-benzoylamino]-azepan-3-yl}-isonicotinamide (4), (3R,4R)-
N-{4-[4-(2-fluoro-6-hydroxy-3-methoxy-benzoyl)-benzyloxy]-azepan-3-yl}-isonicotinamide (5),
N-{(3R,4S)-4-[4-(2-fluoro-6-hydroxy-3-methoxy-benzoyl)-phenylamino]-methyl}-azepan-3-yl)-
isonicotinamide (6), N-{(3R,4R)-4-[4-(2-fluoro-6-hydroxy-3-methoxy-benzoyl)-benzylamino]-
azepan-3-yl}-isonicotinamide (7), and N-{(3R,4S)-4-(4-{trans-2-[4-(2-fluoro-6-hydroxy-3-methoxy-
benzoyl)-phenyl]-vinyl}-azepan-3-yl)-isonicotinamide (8) with linkers isosteric to the ester were
designed, synthesized, and tested for in vitro inhibitory activity against PKA and PKB-R and
for plasma stability in mouse plasma.1 Compound 4 was found to be plasma stable and highly
active (IC50 (PKB-R) ) 4 nM). Cocrystals with PKA were obtained for 4, 5, and 8 and analyzed
for binding interactions and conformational changes in the ligands and protein in order to
rationalize the different activities of the molecules.
In tr od u ction
(PKB-R, PKB-â, and PKB-γ). The kinase domain of
PKB-R has a sequence homology identity with PKB-â
of 91% and with PKB-γ of 88%. PKB-R also exhibits a
sequence homology identity to PKC-η of 53% and PKA-
CR of 47%.16 Recently, a crystal structure of PKB-â with
GSK3-peptide and AMP-PNP has been reported.17,18
Several structural classes of PKC and PKA inhibitors
are known. One class of PKC inhibitors with pharma-
ceutical potential are bisindolylmaleimides, derived
from the natural product staurosporin.19-22 Another
important class of inhibitors is derived from the natural
product (-)-balanol which was first isolated as a me-
tabolite of the fungus Verticillium balanoides at Sphinx
Pharmaceuticals (Eli Lilly) in 199323 and from a species
of Fusarium at Nippon Roche in 1994.24 (-)-Balanol
inhibits PKC in the low nanomolar range. Cocrystalli-
zation of (-)-balanol with PKA was achieved in 1999.25,26
Balanol derivatives with PKC inhibitory activity have
been reported in the literature.27-35
Cell signaling pathways regulate cell growth, prolif-
eration, and apoptosis. Kinases are the key players in
cell signaling. They transduct signals from growth factor
receptors for cell growth or apoptosis by phosphorylation
of their substrates which are mostly downstream ki-
nases involved in cell signaling processes themselves.
The activity of kinases is regulated by phosphorylation
and dephosphorylation effecting conformational changes
in the kinases. Overexpression or constitutive activation
of kinases involved in anti-apoptotic or proliferation
signaling pathways are one typical feature of tumor
cells.2
PKB is located downstream in the PI-3 kinase path-
way3,4 and phosphorylates a growing list of substrates
involved in several pathways for cell survival and
inhibition of apoptosis as has been shown recently.5-9
Constitutive activation of PKB-R is frequently found in
human prostate,10,11 breast,12 and ovarian carcinomas.
It is due to a complete loss of the lipid phosphatase
PTEN gene, a negative regulator of the PI-3 kinase
pathway.13 These data indicate that PKB-R is a central
player in tumorigenesis and a potential target for cancer
intervention.14 Inhibitors of PKB-R are therefore prom-
ising drugs for cancer therapy as effective sensitizers
or inducers of apoptosis.15
In sum, these research results show PKB-R to be an
attractive target in oncology. We found that certain
novel azepane derivatives such as 1 are potent inhibitors
of PKB-R in vitro and in various tumor cell lines (see
Table 2). They also possess anti-cell-proliferation prop-
erties, and induce apoptosis. For the use as potential
drugs with an in vivo effect on solid tumors, the
compounds need to be stable in blood plasma. However,
first in vitro experiments indicated that these com-
pounds were not stable in mouse plasma. For compound
1 we measured a half-life time t1/2 < 1 min. Therefore,
the first optimization step to render these molecules
potential drugs was to improve plasma stability. The
PKB-R belongs to the AGC-family of serine/threo-
nine protein kinases. There are three isoforms known
* To whom correspondence should be addressed. Phone: +49(0)-
8856/60 4749. Fax +49(0)8856/60-4445. E-mail: birgit.masjost@
roche.com.
† Roche Diagnostics GmbH, Penzberg.
‡ Max-Planck-Institut, Martinsried.
10.1021/jm0310479 CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/17/2004

1376 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Breitenlechner et al.
F igu r e 1. Overlay of X-ray crystal structures of inhibitor 1 (orange/green) and AMP-PNP and two Mn2+ (magenta/blue) in
complex with PKA and PKI(5-24). The backbone of the kinase domain of PKA is shown in R-helix and â-sheet ribbon presentation.
instability is presumably caused by a rapid cleavage of
the ester bond by ester hydrolases; LC-MS results show
that cleavage products 2 and 3 appear after incubation
of 1 at 37 °C in mouse plasma (Table 1). Therefore, we
set out to find a replacement of the ester group which
would not adversely affect the inhibitory activities of
the molecules.
Because it crystallizes readily, PKA has been used as
a surrogate kinase to study structural features of closely
related enzymes such as Rho-kinase.36 For similar
reasons, we used PKA for cocrystallization with PKB-R
inhibitors for rapid access to structural information on
inhibitor binding, enabling structure-based guidance of
inhibitor design issues.
Cocrystallization of 1 with PKA revealed the inter-
molecular bonding contacts between the amino acids of
the enzyme and compound 1 as well as its binding
conformation. Here we describe the rational design,37-40
synthesis, and in vitro inhibitory activities of analogues
of compound 1 with plasma stable linkers replacing the
ester moiety.1 These new inhibitors were also cocrys-
tallized with PKA, and the X-ray structures are thor-
oughly analyzed for binding contacts and conformation
of bound ligands to the enzyme.
This space is occupied by the adenine in the complex of
AMP-PNP and PKA41 as shown in Figure 2b. Both
aromatic systems are in the same plane. One of the
amino acids involved in bonding interactions with these
moieties is Val123 which makes an OCNH‚‚‚N hydrogen
bond to the pyridine via its main chain amide. This
H-bond to Val123 or homologue is nearly universal
among protein kinase inhibitor complexes and is ap-
parently critical for tight binding inhibitors.36 Further-
more, the amide makes hydrogen bonds with two water
molecules, one of which is an OCNH‚‚‚OH2 H-bond to a
water molecule that in turn hydrogen-bonds to the side
chain carboxylate of Glu127 and to the carbonyl of the
amide of Leu49; the other one is a HNCO‚‚‚HO H-bond
to a water molecule that is further hydrogen-bonded to
the side chain amine of Lys72 and the side chain
carboxylate of Asp184. Leu49, Val57, Ala70, Met120,
Tyr122, Val123, Glu127, Leu173, Thr183, and Phe327
are involved in van der Waals contacts to the pyridine
and amide portion of compound 1. The second pocket,
stretching over the ribose subsite, contains the azepane
and the ester part of the molecule and will be referred
to as the azepane pocket. It consists of three amino acids
+
making hydrogen bonds to the NH2 of the azepane
ring: namely, Glu170 which makes a NHCO‚‚‚HN
Resu lts a n d Discu ssion
+
hydrogen bond with its main chain CO to the NH2 of
the azepane ring, Asn171 which makes a hydrogen bond
with its side chain CO to the NH2+ of the azepane ring,
and Asp184 which makes a bifurcated hydrogen bond
with its side chain COO to the NH2+of the azepane ring.
Moreover, Asp184 makes a COO‚‚‚HO H-bond to the
side chain of Thr183 and a COO‚‚‚HO H-bond to a water
molecule. Furthermore, Gly50, Thr51, Gly52, Val57,
Lys72, Glu170, Asn171, and Asp184 make van der
Waals contacts to the azepane and ester moiety. The
third binding pocket contains the benzophenone part of
the ligand and it will be called the benzophenone pocket.
This pocket is covered by the flexible so-called glycine
loop which is displaced compared to the AMP-PNP
PKA complex (see Figure 1). Phe54 from the tip of the
glycine loop has an OCNH‚‚‚OC H-bond via its main
Design a n d P r ed iction . We cocrystallized com-
pound 1 with PKA and PKI(5-24) in a ternary complex
and solved the X-ray structure at 2.5 Å resolution under
equal conditions as with AMP-PNP and other inhibi-
tors before.25,41-44 Figure 1 shows the overall structure
of this complex superimposed with the AMP-PNP
complex.41 PKA consists of an N-terminal lobe and a
C-terminal lobe. The ATP-binding site which the inhibi-
tor molecules occupy is located inbetween these two
lobes.
Figures 2a,b show the intermolecular bonding con-
tacts in the complex between PKA and compound 1.
Three binding pockets were defined: the first contains
the pyridine moiety and the amide connecting it to the
azepane ring and will be referred to as pyridine pocket.

Novel Azepane Derivatives as PKB Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1377
F igu r e 2. (a) X-ray crystal structure of inhibitor 1 situated in the active site of PKA with the final 2Fo - Fc electron density map
contoured at 1σ. (b) Overlay of X-ray crystal structures of inhibitor 1 and AMP-PNP situated in the active site of PKA. The
hydrogen bonds between the inhibitor 1 and the amino acids of PKA and water molecules are indicated.
chain amide to the carbonyl oxygen of the benzophe-
none. Glu91 which is essential for catalysis45 forms a
salt bridge to the NH3+ of Lys72 with its side chain COO
in the active state as well as in the inhibitor complex
with 1. In this complex Glu91 also forms a COO‚‚‚HO
hydrogen bond with its side chain to the inhibitor
hydroxyl group of the benzophenone. In addition, Ser53,
Phe54, Gly55, Arg56, Leu74, Gln84, His87, Thr88,
Glu91, Gly186, and Phe187 make van der Waals con-
tacts with the benzophenone moiety. The crystal struc-
ture shows that the plasma unstable ester moiety of
compound 1 is not hydrogen-bonded to the enzyme. Also
there are no strong attractive van der Waals interac-
tions with the amino acids of PKA which are likely to
make a significant contribution to the enthalpy of
binding. On the other hand, its trans conformation
rigidly orients the azepane and the benzophenone of 1
toward their binding pockets. We therefore sought to
replace the ester moiety by an isosteric chemical linker
to achieve plasma stability without losing potent inhibi-
tion. The design and estimation of relative binding
affinities for the inhibitors 4, 5, 6, 7, and 8 was
performed by computational methods, using the pro-
grams MOLOC46 and Insight II.47 Molecular modeling
predicted that the double amide 4 and the compound
with the vinyl linker 8 should have the highest binding
affinities, mostly because these molecules have linkers
that are preorganized in the trans conformation favored
for binding (see Figure 2a). The inhibitors with the ether
linkage 5 and the benzylamino linker 7 would have to
rearrange to adopt the right conformation for binding
which costs free energy. For the molecule with the
phenylaminomethyl linker 6 we expected a significantly
lower binding energy, because the molecule cannot
adopt the conformation preferred for binding due to
steric hindrance between the methylene protons and the
protons of the neighboring azepane ring.
Syn th esis. For the synthesis of the various enan-
tiopure balanol analogues (structurally related to (-)-
balanol) such as 4, 5, 6, 7, and 8, in which the carboxylic
ester of the lead is replaced by different linkers, we
employed five different synthetic routes. The synthesis
of the double amide 4 started with cis-amino alcohol
(9)48 as chiral building block (Scheme 1). Treatment of
starting material 9 with isonicotinic acid 10/DCC/DMAP
in methylene chloride yielded the isonicotinic amide 11,
which was transformed to the corresponding mesylate
12. SN2-type reaction of 12 by treatment with sodium
azide afforded the trans-azide 13 which was selectively
hydrogenated to the trans amine 14. The second amide
moiety was introduced by reacting amine 14 with acid
1535 in methylene chloride using DCC/DMAP as cou-
pling reagents. Removal of the protecting groups from
carbamate 16 led to the desired target compound 4
which could be isolated in 94% yield.
For the synthesis of the aniline 6, a SN2-type reaction
of 12 with sodium cyanide yielded the trans cyanide 17,
which could not be isolated from elimination byproducts
(Scheme 2). Hydrogenation of the raw material gener-
ated the unstable amine 18. The key step in this
synthetic pathway was the palladium-catalyzed Buch-
wald49 coupling of aryl iodide 19 (synthesized from
lithiated 20 and 21 as reaction partner and subsequent
oxidation of the resulting carbinol 22) with amine 18.
The desired coupling product 23 was deprotected using
HCl in dioxane.
The synthesis of ether 5 was performed by protection
of carbinol 2435 using TIPS-Cl (Scheme 3). Reduction
with LiAlH4 yielded the hydroxymethyl compound 26
which could be transformed to the corresponding chlo-

1378 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Breitenlechner et al.
Sch em e 1a
a
Reagents and conditions: (a) isonicotinic acid 10, DCC, DMAP, CH2Cl2, r. t., 24 h, 98%; (b) MeSO2Cl, pyridine, rt, 24 h, 79%; (c)
NaN3, DMF, 90 °C, 3 h, 91%; (d) Ra-Ni, 1 bar H2, CH3OH, 95%; (e) 4-(2-fluoro-3-methoxy-6-methoxymethoxy-benzoyl)-benzoic acid 15,35
DMAP, DCC, CH2Cl2, rt, 6 h, 76%; (f) 4 M HCl in dioxane, rt, 15 h, 94%.
romethyl compound 27 by treatment with PPh3/CCl4.50
The ether was introduced by reacting chloromethyl
compound 27 with amino alcohol 935,48 and NaH to give
28. Reaction of compound 28 with isonicotinic acid/DCC/
DMAP in methylene chloride led to isonicotinic amide
29. The carbinol function of the resulting ether 29 was
selectively deprotected with TBAF and oxidized to the
corresponding benzophenone 30 using activated MnO2.
Deprotection with HCl in dioxane afforded the desired
ether 5 in good yield.
to vinyl compound 38 by Wittig reaction (Ph3P+CH3I-,
n-BuLi, THF). At this stage the desired trans-vinyl
compound could be isolated by preparative HPLC with
chiral column OD 40 and 2-propanol/heptane ) 1:4 as
eluent. The aryl iodide 19 was coupled with the vinyl
compound 38 using a Heck protocol52 ((o-Tol)3P, Et3N,
Pd(OAc)2, DMF) to yield the trans-vinyl compound 39.
Removal of the protecting groups with 2 N HCl in ether
afforded the desired product 8.
Biologica l Resu lts. The plasma stability was deter-
mined for the inhibitors 1, 4, 5, and 7. Table 1 lists the
half-lives of the molecules in mice blood plasma under
the conditions mentioned above. All molecules except
for the ester are plasma stable with half-lives t1/2 > 29
h (compound 5) up to 161 h (compound 7). These
findings confirm our hypothesis that the plasma insta-
bility of lead compound 1 is caused by cleavage of the
ester bond. The biological activities of the compounds
in the in vitro kinase assay are listed in Table 2. The
most active compound is the double amide 4 (IC50(PKB-
R) ) 4 nM) as was predicted by molecular modeling,
because the conformation of the amide is trans, and that
is the preferred conformation of the linker in the bound
state according to the crystal structure of 1 in PKA. The
compound with the trans-vinyl linker 8 is considerably
less active (IC50(PKB-R) ) 160 nM) than the ester which
was not predicted from the modeling.
The synthetic pathway for benzylamine 7 was as
follows: carbinol 2435 was converted to aldehyde 32
in two steps. Reduction (LiAlH4) of 24 afforded alcohol
31 and was followed by oxidation (activated MnO2) to
give aldehyde 32 (Scheme 4). Reductive amination
(NaCNBH3) of aldehyde 32 with amine 14 resulted in
amine 34. Subsequent deprotection with HCl in dioxane
led to the desired balanol analogue 7.
For the preparation of compound
8 with the
trans-vinyl linker Swern oxidation51 of 11 to ketone 35
and subsequent Wittig reaction (Ph3P+CH2OCH3Cl-,
KOtBu, ether) afforded enol ether 36 as an aldehyde
synthon (Scheme 5). The enol ether 36 in CHCl3 was
thermodynamically hydrolyzed using Cl3CCOOH with-
out deprotection of the carbamate group to the resulting
aldehyde 37 which was a diastereomeric mixture (trans:
cis ) 3:1). The desired trans-aldehyde 37a could not be
separated from the cis-aldehyde 37b. Therefore the
diastereomeric mixture of aldehyde 37 was transformed
The ether 5 (IC50(PKB-R) ) 355 nM) is less active
than double amide 4, as predicted by modeling, presum-

Novel Azepane Derivatives as PKB Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1379
Sch em e 2a
a
Reagents and conditions: (a) NaCN, DMSO, 100 °C, 48 h, 57%; (b) Ra-Ni, 50 atm H2, CH3OH, 22%; (c) 4-iodobenzaldehyde 21,
t
n-BuLi, THF, -78 °C, 14 h, 50%; (d) MnO2, CH2Cl2, rt, 24 h, 95%; (e) 19, 18-crown-6, BuOK, Pd2(dba)3, BINAP, THF, 15 h, reflux, 10%;
(f) 4 M HCl in dioxane, rt, 15 h, 70%.
ably because the ether bond has to rearrange in order
to adopt the trans binding conformation. A NOESY
spectrum of 5 in D2O was obtained to analyze its
conformation in water (Figure 3). The NOE between the
pyridine protons H-C(3,5) and the benzoyl protons
H-C(3’,5’) as well as the NOE between the pyridine
protons H-C(2,6) and the benzoyl protons H-C(2′,6′)
indicate that the unbound state of the molecule is
stabilized by hydrophobic collapse. Hydrophobic collapse
could stabilize the ligand in two ways. On one hand, it
is favorable enthalpically, because the two terminal
chromophores, pyridine and benzophenone, are brought
into proximity for attractive π-π interactions. On the
other hand, this phenomenon is accompanied by desol-
vation of the molecule resulting in an entropy gain. In
the collapsed conformation that molecule 5 adopts in
solution, the pyridine is not in the right conformation
to bind to the active site of PKB-R and, therefore, upon
binding, significant reorganizational energy would be
required, which is unfavorable for the binding free
enthalpy of the ligand.
be required, which is unfavorable for the binding free
enthalpy of the ligand as well. According to molecular
modeling a protonated amine would result in steric
hindrance between the ammonium protons and the ring
protons of the azepane ring. Compound 6 is inactive
(IC50 ) 25.8 µM) which was predicted by modeling and
is mainly due to steric hindrance between the methylene
protons and the ring protons of the azepane ring which
do not allow the linker to adopt the right conformation
for binding of the different moieties of the molecule to
their binding pockets.
Cr ysta llogr a p h ic Resu lts. The inhibitors 4, 5, and
8 could be cocrystallized with PKA and were overlayed
with the ester 1 as shown in Figure 4. The crystal
structures reveal the conformations of the aforemen-
tioned inhibitors which differ only in the chemical entity
of the linker between the azepane and benzophenone
moieties in the bound state. It can be seen that inhibi-
tors 4, 5, and 8 bind practically in the same conforma-
tion as the ester 1. The conformation of the linker for
which the torsional angle is antiperiplanar in all
molecules is trans with respect to azepane and ben-
zophenone. This confirms our modeling hypothesis that
this is the favorable binding confirmation, because it
orients the azepane and the benzophenone toward their
binding pockets. Therefore, molecules which are preor-
ganized in aqueous solution in the trans conformations
have higher binding affinities which is in accordance
with the biochemical data (see Table 2). In the complex
between the double amide 4 and PKA (Figure 5a) there
is a rearrangement of the side chains of Asp184, Thr183,
Molecule 7 (IC50(PKB-R) ) 2.8 µM) is much less active
than double amide 4 and ether 5. A hypothetical
interpretation of this result might be that the benzyl-
amino linker in molecule 7 is more flexible and presum-
ably not preorganized in the trans conformation in
aqueous solution, and therefore a loss in entropy would
occur upon binding. Furthermore, the amine is hydro-
philic and basic and therefore protonated and hydrated
in aqueous solution, and upon binding, dehydration
(depends on protein) and deprotonation energy would

1380 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Breitenlechner et al.
Sch em e 3a
a
Reagents and conditions: (a) imidazole, TIPS-Cl, DMF, rt, 15 h, then TIPSOSO2CF3, 100 °C, 36 h, 90%; (b) LiAlH4, THF, rt, 3.5 h,
70%; (c) PPh3, CCl4, rt, 3.5 h, 74%; (d) (3 R, 4 S)-3-amino-4-hydroxy-azepane-1-carboxylic acid tert-butyl ester 9, NaH, THF, reflux, 2 h,
43%; (e) isonicotinic acid 10, DCC, DMAP, CH2Cl2, rt, 15 h, 92%; (f) Bu4NF, THF, rt, 15 h, then MnO2, CH2Cl2, rt, 24 h, 72%; (g) 4 M HCl
in dioxane, rt, 15 h, 91%.
and Lys72 compared to the complex of the ester with
PKA as can be seen in Figure 4 and Figure 5a. In the
complex of the double amide with PKA, the NH of the
amide moiety (next to the benzophenone) of molecule 4
forms an OCNH‚‚‚OOC hydrogen bond with the side
chain of Asp184 (Table 3). Furthermore, Asp184 makes
the double amide 4. All other intermolecular bonding
contacts are practically the same as described for the
complex between PKA and the ester 1 (see Figure 2b).
Analysis of the crystal structure of the complex of the
compound with the vinyl linker 8 with PKA (Figure 5c)
reveals no significant changes in intermolecular binding
contacts compared to the ester 1 except for an additional
+
a COO‚‚‚HN H-bond to the NH2 of the azepane, and
+
+
two more hydrogen bonds to the NH3 of Lys72 and a
NH‚‚‚OH H-bond between Lys72 with its NH3 to the
water molecule. Thr183 makes an OH‚‚‚OCNH H-bond
with its side chain OH to the carbonyl oxygen of the
amide next to pyridine in the inhibitor 4. Lys72 forms
inhibitor hydroxyl group of the benzophenone. However,
analysis of the crystal structure does not provide an
explanation of its lower binding affinity of IC50(PKA) )
360 nM (Table 2).
+
an additional NH‚‚‚OH H-bond with its NH3 to the
inhibitor hydroxyl group of the benzophenone as com-
pared to the complex of the ester 1 and PKA. These
extra hydrogen bonding contacts between inhibitor and
protein detected in the crystal structure of the complex
of double amide 4 with PKA might contribute to its
slightly higher binding affinity of IC50 (PKA) ) 2 nM
compared to the ester (IC50 (PKA) ) 4 nM). In the
cocrystal structure of the ether 5 with PKA (Figure 5b)
Lys72 forms an additional NH‚‚‚OH H-bond with its
Con clu sion s. Based upon molecular modeling stud-
ies on the X-ray crystal structure of the complex
between the lead molecule 1 and PKA, new linkers
isosteric to the plasma unstable ester were designed and
introduced into 1 to give the compounds 4, 5, 6, 7, and
8. The in vitro PKA and PKB-R inhibitory activities of
these molecules as inhibitors were measured and evalu-
ated with respect to the modeling predictions. No
significant differences in binding affinities for PKA and
PKB-R could be observed. The binding affinities are in
agreement with the modeling predictions for inhibitors
4, 5, 6, and 7. This confirms our hypothesis that PKA
is a suitable surrogate kinase for PKB, and crystal
structures of inhibitors cocrystallized with the kinase
domain of PKA can be used for molecular modeling
+
NH3 to the inhibitor hydroxyl group of the benzophe-
none as compared to the complex of the ester 1 and
PKA. Thr183 adopts two conformations in the same
crystal: one as observed in the crystal structure of the
complex of PKA with the ester 1, the other one as
observed in the crystal structure of the complex with

Novel Azepane Derivatives as PKB Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1381
Sch em e 4a
a
Reagents and conditions: (a) LiAlH4, THF, rt, 3 h, 63%; (b) MnO2, CH2Cl2, rt, 22 h, 69%; (c) 14, C2H5OH, reflux, 15 h, 95%; (d)
NaBH3CN, CH3OH, rt, 1 h, 57%; (e) 4 M HCl in dioxane, rt, 15 h, 57%.
to the mouse plasma (t ) 0, 0.5, 1, 2, 4 h), equal portions were
isolated from the plasma, separated with HPLC and analyzed
by mass spectrometry. During all these steps, the temperature
was kept constant at 37 °C.
studies for a design of active PKB-R inhibitors. This
opens the possibility for further homology modeling
studies with PKA as a model for PKB. It also allows
the design of inhibitors which are selective for PKB-R
against PKA, because there are different amino acids
in PKB-R in a deeper pocket close to the benzophenone
binding site in PKA which is not occupied by the
aforementioned inhibitors. These studies also confirm
the validity of selectivity predictions based on the X-ray
structures of inhibitors cocrystallized with PKA/B mu-
tant protein constructs and will be published sepa-
rately.36,53a Based upon the available structures, it is
also possible to design molecules that belong to com-
pletely different chemical classes as PKB-R inhibitors.
Results of this approach will be published separately.53b
It was also shown that inhibitors 4, 5, and 7 are
plasma stable analogues of the original lead 1.1 Inhibitor
4 had an inhibitory activity in the in vitro kinase assay
on PKB-R of IC50 ) 4 nM and did not have the liability
of plasma instability as the original lead. It was used
as the new lead structure for further lead optimization
of selectivity between PKB and PKA, bioavailability,
and tolerability.
In Vitr o Kin a se Assa ys. To study PKB-R inhibitory
activity of the compounds, an ELISA-based assay has been
developed for the serine/threonine kinase, PKB-R. The assay
design utilizes an N-terminally biotinylated substrate peptide.
When this substrate is phosphorylated by the PKB-R kinase,
the product is recognized by a substrate/sequence-specific
antibody known to bind to particular phosphorylated serine
residues. For this assay a Biotin-SGRARTSSFAEPG peptide
and anti-phospho-GSK-3R Ser21 antibody (rabbit) from Cell
Signaling Technology/New England Biolabs have been used.
The enzymatic reactions were carried out with PKB-R
expressed in Sf9 cells (7.7 ng/reaction), substrate peptide (200
nM), and ATP (5 µM) in the absence or presence of different
concentrations of compounds dissolved in DMSO. The final
concentration of DMSO was 4%. The mixtures were reacted
for 30 min at room temperature in assay buffer containing 50
mM Tris-HCl, 10 mM MgCl2, 1.0 mM DTT, 0.2 mM Na3VO4,
pH 7.5, in a final volume of 50 µL. The reaction was stopped
by addition of 10 µL 0.12 M EDTA/0.12 M EGTA. The reaction
mixture was transferred to a SA-coated microtiter plate. After
1 h incubation, the plate was washed with PBS using a 384-
well Embla plate washer. Anti-phospho-GSK-3R Ser21 anti-
body was added. After 1 h incubation, the plate was washed
with PBS and bound antibody was detected by addition of
polyclonal<rabbit>S-IgG-POD conjugate from Roche Diag-
nostics GmbH. After 1 h incubation, the plate was washed with
PBS. The amount of phosphorylated peptide was measured
by the enzyme-catalyzed ABTS (ABTS is a trademark of a
Exp er im en ta l Section
P la sm a Sta bility Assa y. To test the plasma stability of
the inhibitors, mouse plasma tests have been used as follows:
Samples of mouse plasma containing each compound in a
standard concentration (10 µmol/L) were prepared. After
defined periods of time after the addition of said compounds

1382 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Breitenlechner et al.
Sch em e 5a
a
Reagents and conditions: (a) ClCOCOCl, DMSO, CH2Cl2, Et3N, -78 °C, 30 min, 85%; (b) Ph3P+CH2OCH3Cl-, KOtBu, Et2O, -30 °C
to 0 °C, 59%; (c) Cl3CCOOH, CHCl3, reflux, 1 h, 68%; (d) n-BuLi, Ph3P+CH3I-, THF, -78 °C to rt, 14 h, 50%; (e) 19, Pd(OAc)2, (o-Tol)3P,
N,N-diisopropylamine, DMF, 105 °C, 14 h, 20%; (f) 2 M HCl in diethyl ether, rt, 15 h, 97%.
member of the Roche group) conversion and photometrical
measurement at 405 nm.
To determine IC50 values, the compounds were tested in the
concentration range from 100 µM to 20 pM. Calculation of IC50
values were performed with ActivityBase.
ature. The kinase reaction was started subsequently by adding
ATP (5.76 µM final concentration) and incubated for 90 min
at room temperature. Finally, the reaction was stopped by
adding the same volume of a 1:1 mixture of quenching buffer
and phosphoserine antibody (both from PanVera). After an-
other 30 min of incubation, enzymatic activity was measured
by detection of fluorescence polarity in a Victor 2 reader
(Wallac, Finland).
To study PKA inhibitory activity of the compounds, a
fluorescence polarization-based assay (Protein Kinase C Assay
Kit, Green, PanVera), which is also suitable for the serine/
threonine kinase PKA, was used. In this assay, a fluorescent
phosphopeptide tracer and the nonfluorescent phosphopeptides
generated during a PKA reaction compete for binding to an
antiphosphoserine antibody. In a reaction mixture containing
no phosphopeptide product, the fluorescent tracer is bound by
the antibody, and the emission signal is polarized. However,
in a reaction mixture containing phosphopeptide product, the
fluorescent tracer is displaced from the antibody and the
emission signal becomes depolarized. Different concentrations
of compounds dissolved in DMSO (max. final DMSO concen-
tration 2.5%) were incubated with PKA-CR enzyme (recombi-
nant from E. coli, 10 ng/reaction) together with PKC buffer,
PKC peptide substrate (2 µM), and PKC tracer (all from
PanVera) as well as 9 mM Hepes-buffer pH 7.2, 0.05%
Nonidet-P40, and 50 µM Na3VO4 for 30 min at room temper-
To determine IC50 values, the compounds were tested in the
concentration range from 100 µM to 20 pM. Calculation of IC50
values were performed with ActivityBase.
P r otein Exp r ession a n d P u r ifica tion . Recombinant
bovine CR catalytic subunit of cAMP dependent protein kinase
(PKA) (which differs from the human protein at two posi-
tions: N32S and M63K) was solubly expressed in E. coli BL21-
(DE3) cells and then purified via affinity chromatography and
ion exchange chromatography as previously described.42,43
Heterogeneously phosphorylated PKA was used for the kinase
assay. Three fold phosphorylated protein was used for crystal-
lization of all inhibitors.
Cr ysta lliza tion . The inhibitors 1, 4, 5, and 8 were cocrys-
tallized with PKA and PKI(5-24) at 75 mM LiCl, 25 mM
MesBisTris, pH 6.4. Hanging drop vapor diffusion method

Novel Azepane Derivatives as PKB Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1383
Ta ble 1. Half-Lives of Inhibitors in Mouse Plasma at 37 °C
minimized by conjugate gradients to a final value of the sum
of the squares of the components of the gradient of less than
the accuracy (0.1 or relative value of 1).
(3R,4S)-4-H yd r oxy-3-[(p yr id in e-4-ca r b on yl)-a m in o]-
a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (11). A solu-
tion of compound 948 (4.60 g, 20.0 mmol), isonicotinic acid (2.46
g, 20.0 mmol) and DMAP (1.22 g, 10.0 mmol) in anhydrous
CH2Cl2 (100 mL) was cooled to 0 °C. A solution of DCC (4.13
g, 20.0 mmol) in anhydrous CH2Cl2 (50 mL) was added
dropwise. The resulting mixture was stirred for 24 h at room
temperature. Water (30 mL) was added, and stirring was
continued for 30 min. After filtration and separation, the
organic layer was washed with water (2 × 20 mL), and the
aqueous layers were re-extracted with CH2Cl2 (2 × 20 mL).
The combined organic layers were washed with brine and dried
over Na2SO4. After filtration and evaporation, the residue was
purified by column chromatography on silica gel (column, 3
cm × 25 cm; eluent, ethyl acetate: CH3OH ) 97:3). A yellow
oil was obtained (6.6 g, 98%). [R]20 14.2° (c 0.6, CHCl3). IR:
D
ν (cm-1) ) 1666, 1415, 1165. 1H NMR (250 MHz, CDCl3): δ
1.44 (s, 9H), 1.50-1.95 (m, 4H), 2.83-3.12 (m, 2H), 3.67-3.87
(m, 1H), 3.89-4.15 (m, 2H), 4.37 (br s, 1H), 4.65 (br s, 1H),
7.51-7.72 (m, 2H), 8.40 (br s, 1H), 8.62-8.73 (m, 2H). 13C NMR
(62.5 MHz, CDCl3): δ 20.79, 27.36, 46.63, 47.29, 56.20, 59.40,
72.98, 80.01, 120.09, 139.50, 149.52, 157.20, 166.54. MS (70
eV), m/z (%): 336 (30, M+ + 1), 123 (100); HR-MS (ESI): calcd
for C17H26N3O4 [M + H]: 336.1923; found: 336.1968.
inhibitor
T1/2
1
4
5
7
<1 min
69 h
29 h
161 h
(3R,4S)-4-Meth an esu lfon yloxy-3-[(pyr idin e-4-car bon yl)-
a m in o]-a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (12).
A solution of 11 (6.60 g, 19.7 mmol) in anhydrous pyridine (100
mL) was cooled to 0 °C. Methanesulfonic acid chloride (6.76
g, 59.0 mmol) was added during 1 h. The mixture was stirred
for 24 h at room temperature, and the volatile components
were removed in vacuo. The residue was dissolved in ethyl
acetate/water (200 mL, 1:1), and the aqueous layer was
extracted with ethyl acetate (3 × 30 mL). The combined
organic layers were washed with brine and dried over Na2-
SO4. After filtration and evaporation, the residue was purified
by column chromatography on silica gel (column, 5 cm × 35
cm; eluent, (1) ethyl acetate, (2) ethyl acetate/CH3OH ) 99:1,
(3) ethyl acetate/CH3OH ) 97:3). A yellow oil was obtained
(6.4 g, 79%). 1H NMR (250 MHz, CDCl3): δ 1.39 (s, 9H), 1.55-
2.35 (m, 4H), 2.78-2.95 (br s, 3H), 2.90-3.82 (m, 4H), 4.35-
4.55 (m, 1H), 5.12-5.19 (m, 1H), 6.82 (br d, 0.3H), 7.58-7.65
(m, 2H), 7.90-8.05 (br d, 0.7H), 8.62-8.76 (m, 2H). 13C NMR
(62.5 MHz, CDCl3): δ 21.09, 27.36, 28.11, 36.99, 47.12, 51.30,
52.06, 59.39, 79.40, 119.93, 139.83, 149.65, 156.20, 163.56. MS
(70 eV), m/z (%): 414 (100, M+ + 1); HR-MS (ESI): calcd for
C18H28N3O6S [M + H]: 414.1699; found: 414.1726.
Ta ble 2. IC50 Values Measured for Binding of Inhibitors to
PKB-R and PKA
inhibitor
IC50 (PKB-R)
IC50 (PKA)
1
4
5
6
7
8
5 nM
4 nM
5 nM
2 nM
39 nM
45 µM
800 nM
360 nM
355 nM
25 µM
3 µM
160 nM
against 15% methanol as precipitant was used to obtain ca.
100 × 100 × 500 µm crystals.
Da ta Collection a n d Str u ctu r e Deter m in a tion . Diffrac-
tion data were measured at 4 °C in a sealed capillary on an
image plate detector (Mar research) or Bruker ×1000 area
detector using a copper target Rigaku Rotaflex X-ray generator
and graphite crystal monochromator. In each case one crystal
was sufficient to obtain a complete data set. In case of
compound 8, the crystals were flash frozen in liquid nitrogen
and measured at the synchrotron beamline BW-6 at the DESY
(Hamburg, Germany). The data were processed with the
program MOSFLM and SCALA or ASTRO and SAINT. All
crystals have orthorhombic symmetry (P212121) with nearly
the same cell constants.
(3R,4R)-4-Azid o-3-[(p yr id in e-4-ca r b on yl)-a m in o]-a ze-
p a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (13). Compound
12 (21.5 g, 52.0 mmol) was dissolved in DMF (500 mL), and
sodium azide (16.9 g, 26.0 mmol) was added. The mixture was
stirred for 3 h at 90 °C. Another portion of sodium azide (16.9
g, 26.0 mmol) was added, the mixture was stirred for 3 h at
90 °C and for 24 h at room temperature, and the solvent was
evaporated. The residue was dissolved in ethyl acetate/water
(200 mL, 1:1), and the aqueous layer was extracted with ethyl
acetate (3 × 30 mL). The combined organic layers were washed
with brine and dried over Na2SO4. After filtration and evapo-
ration, the residue was purified by column chromatography
on silica gel (column, 5 cm × 35 cm; eluent, ethyl acetate/
heptane ) 9:1). A yellow oil was obtained (17.0 g, 91%). 1H
NMR (250 MHz, CDCl3): δ 1.39 (s, 9H), 1.55-2.01 (m, 4H),
2.82-2.93 (m, 1H), 3.12-3.24 (m, 1H), 3.72-3.85 (m, 1H),
The structures were determined by molecular replacement
using the CCP4 program package (www.ccp4.ac.uk/main/
html). As a starting model, we chose a PKA structure in a
closed conformation (to be published). Refmac 5.1.24 was used
for refinement, while MOLOC46 (www.moloc.ch) was used for
graphical evaluation and model building. The structures are
depicted in the Figures 1, 2a,b, 4, and 5a-c with graphics from
programs MOLSCRIPT,54 BOBSCRIPT,54-56 and Raster3d.57,58
Da ta Dep osition . Atomic coordinates have been deposited in
the Protein Data Bank.
Molecu la r Mod elin g. The design of target molecules with
different linkers between the azepane and benzophenone part
of lead molecule 1 was carried out on a Silicon Graphics Octane
workstation. The starting geometries for the molecular me-
chanics studies were constructed with the program MOLOC,
from crystallographic data and standard molecular fragments.
The proposed inhibitor was minimized separately and docked
manually into its expected binding site. The coordinates of
PKA were constrained. The inhibitors were minimized inside
the enzyme. Energy minimizations were performed in vacuo
by MOLOC with the MAB force field. The energy was
3
3.94-4.24 (m, 3H), 7.63 (d, J ) 5.2 Hz, 2H), 8.41 (br s, 1H),
8.62 (d, 3J ) 5.2 Hz, 2H). 13C NMR (62.5 MHz, CDCl3): δ 26.17,
28.76, 30.01, 36.87, 48.19, 55.59, 62.56, 81.48, 121.32, 141.35,
150.94, 158.65, 162.91. MS (70 eV), m/z (%): 359 (22, M+
1), 259 (100).
-
(3R,4R)-4-Am in o-3-[(p yr id in e-4-ca r bon yl)-a m in o]-a ze-
p a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (14). Compound
13 (17.0 g, 47.2 mmol) dissolved in CH3OH (850 mL) was
hydrogenated (anhydrous Ra-Ni (7 g), 1 bar). The mixture

1384 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Breitenlechner et al.
F igu r e 3. 1H NMR (D2O, 500 MHz) NOESY spectrum of 5. The NOE between the pyridine protons H-(C3,5) and the benzoyl
protons H-C(3′,5′) as well as the NOE between the pyridine protons H-C(2,6) and the benzoyl protons H-C(2′,6′) indicate that the
unbound state of the molecule is stabilized by hydrophobic collapse.
F igu r e 4. Overlay of X-ray crystal structures of inhibitors 1, 4, 5, and 8 situated in the active site of PKA. The protein backbone
ribbon with conformation of side chains relevant for binding interactions with the inhibitors is shown.
was filtered carefully, and the volatile components were
removed in vacuo. The residue was solved in ethyl acetate (200
mL), dried over Na2SO4, filtered, and evaporated to yield a
light brown amorphous compound (16.5 g, 95%). [R]20D -27.1°
(c 0.6, CHCl3). IR: ν (cm-1) ) 1665, 1417, 1163. 1H NMR (400
MHz, DMSO): δ 1.37 (s, 6H), 1.42 (s, 3H), 1.45-1.74 (m, 2H),
1.71-1.92 (m, 2H), 2.72-2.88 (m, 1H), 3.05-3.30 (m, 2H),
3.28-3.44 (br s, 2H), 3.45-3.65 (m, 2H), 3.73 (br s, 1H), 7.74
(d, J ) 5.0 Hz, 0.6H), 7.78 (d, J ) 5.8 Hz, 1.4H), 8.29-8.38
(br s, 1H), 8.70-8.76 (m, 2H). 13C NMR (62.5 MHz, CDCl3): δ
25.44, 27.40, 31.67, 45.00, 49.01, 56.25, 57.89, 79.71, 119.99,
140.36, 149.53, 156.71, 164.16. MS (70 eV), m/z (%): 334 (100,
M).
purified by column chromatography on silica gel (column, 2
cm × 20 cm; eluent, ethyl acetate). An amorphous colorless
1
compound was obtained (240 mg, 76%, mp: 105-107 °C). H
NMR (400 MHz, DMSO): δ 1.41 (s, 6H), 1.45 (s, 3H), 1.55-
2.05 (m, 4H), 3.05 (s, 3H), 3.05-3.26 (m, 2H), 3.45-3.70 (m,
3
2H), 3.84 (s, 3H), 4.09-4.23 (m, 2H), 5.02 (s, 2H), 7.03 (d, J
3
3
) 7.8 Hz, 1 H), 7.28 (t, J ) 8.0 Hz, 1H), 7.55 (d, J ) 5.4 Hz,
3
1H) 7.59 (d, J ) 5.5 Hz, 1H), 7.74-7.83 (m, 4H), 8.53-8.71
3
3
(m, 4H). MS (70 eV), m/z (%): 650 (80, M+), 649 (100); HR-
MS (ESI): calcd for C34H40N4O8F [M + H]: 651.2830; found:
651.2841.
(3R,4R)-N-{4-[4-(2-F lu or o-6-h yd r oxy-3-m et h oxy-b en -
zoyl)-ben zoyla m in o]-a zep a n -3-yl}-ison icotin a m id e Hy-
d r och lor id e (4). HCl in dioxane (4 M, 2 mL, 8.0 mmol) was
added to a solution of 16 (260 mg, 0.42 mmol) in anhydrous
dioxane (2 mL). The mixture was stirred for 15 h at room
temperature, and the resulting light yellow crystals were
separated (185 mg, 78%, mp: 200 °C dec). The purity of the
(3R,4R)-4-[4-(2-F lu or o-3-m eth oxy-6-m eth oxym eth oxy-
ben zoyl)-ben zoylam in o]-3-[(pyr idin e-4-car bon yl)-am in o]-
a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (16). 4-(2-
Fluoro-3-methoxy-6-methoxymethoxy-benzoyl)-benzoic acid
(15)35 (176 mg, 0.50 mmol), DMAP (31 mg, 0.30 mmol), and
DCC (113 mg, 0.55 mmol) were added to a solution of 14 (167
mg, 0.50 mmol) in CH2Cl2 (5 mL). The mixture was stirred
for 6 h at room temperature and filtered. The filter cake was
washed with CH2Cl2 (3 × 4 mL), and the combined organic
layers were washed with water (2 × 3 mL). After drying over
Na2SO4, filtration, and concentration in vacuo, the residue was
1
compound was determined as 99.3% by analytical HPLC. H
NMR (500 MHz, DMSO): δ 1.85-2.10 (m, 4H), 3.07 (br s, 1H),
3.24 (br s, 1H), 3.32-3.45 (m, 2H), 3.80 (s, 3H), 4.32-4.40 (m,
3
3
1H), 4.47-5.56 (m, 1H), 6.74 (d, J ) 7.8 Hz, 1H), 7.18 (t, J
3
3
) 7.9 Hz, 1H), 7.76 (d, J ) 8.3 Hz, 2H), 7.84 (d, J ) 5.3 Hz,
2H), 8.05 (d, 3J ) 8.3 Hz, 2H), 8.85-8.92 (m, 3H), 9.31 (m,

Novel Azepane Derivatives as PKB Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1385
F igu r e 5. X-ray crystal structures of PKA with inhibitor 4 (a), 5 (b), and 8 (c), respectively, were superimposed with the AMP-
PNP complex (1CDK). The figures show the binding mode in the active site of PKA. The hydrogen bonds between the inhibitors
and the amino acids of PKA and water molecules are indicated.
1H), 9.40 (br s, 1H), 9.68 (br s, 1H). 13C NMR (125 MHz,
DMSO): δ 22.52, 30.09, 46.60, 47.31, 52.20, 54.13, 95.88,
111.56, 116.78, 123.93, 128.39, 129.36, 138.82, 139.73, 140.19,
140.28, 146.35, 148.06, 149.24, 150.01, 163.57, 165.98, 191.68.
MS (70 eV), m/z (%): 507 (100, M+ + 1). HR-MS (ESI): calcd
for C27H28N4O5F [M + H]: 507.2044; found: 507.2067.
(3R,4R)-4-Cya n o-3-[(p yr id in e-4-ca r bon yl)-a m in o]-a ze-
p a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (17). (3R,4S)-4-
Methanesulfonyloxy-3-[(pyridine-4-carbonyl)-amino]-azepane-
1-carboxylic acid tert-butyl ester (12) (103 mg, 0.25 mmol) and
NaCN (122.5 mg, 2.50 mmol) in anhydrous DMSO (2 mL) were
stirred for 48 h at 100 °C under argon. Water (20 mL) was
added, and the suspension was extracted with ethyl acetate
(3 × 20 mL). The combined organic layers were washed with
brine and dried over Na2SO4. After filtration and evaporation,
the residue was purified by flash chromatography on silica gel
(column, 2 cm × 15 cm; eluent, (1) ethyl acetate/heptane, (2)
ethyl acetate to (3) ethyl acetate/CH3OH ) 95:5). A yellow gum
was obtained (102 mg, 57%). 1H NMR (250 MHz, CDCl3): δ
1.35 (s, 3H), 1.42 (s, 6H), 1.55-2.00 (m, 4H), 2.95-3.07 (m,
1H), 3.15-3.25 (m, 1H), 3.55-4.35 (m, 4H), 7.55-7.68 (m, 2H),
8.60-8.72 (m, 3H). 13C NMR (62.5 MHz, CDCl3): δ 22.86,
27.33, 29.83, 30.98, 45.90, 48.59, 52.50, 80.54, 119.84, 139.32,
149.62, 164.18. MS (70 eV), m/z (%): 345 (100, M+ + 1).
(3R ,4S )-4-Am in o m e t h y l-3-[(p y r id in e -4-c a r b o n y l)-
a m in o]-a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (18).
Cyanide 17 (445 mg, 1.29 mmol) in CH3OH (25 mL) and liquid
ammonia (25 mL) was hydrogenated (anhydrous Ra-Ni (100
mg), 50 bar). The mixture was filtered carefully, and the
volatile components were removed in vacuo. The residue was
purified by flash chromatography on silica gel (column, 2 cm
× 15 cm; eluent, (1) CH2Cl2/2N NH3 in CH3OH ) 99:1 to CH2-
Cl2/2 N NH3 in CH3OH ) 90:10) to yield a light brown oil (100
1
mg, 22%). H NMR (250 MHz, CDCl3): δ 1.40 (s, 9H), 1.45-
1.95 (m, 5H), 2.65-2.83 (m, 1H), 3.02-3.63 (m, 5H), 3.73-
3
3
3.85 (m, 1H), 7.59 (d, J ) 5.3 Hz, 2H), 8.67 (d, J ) 5.3 Hz,
2H). 13C NMR (62.5 MHz, CDCl3): δ 22.98, 26.40, 29.57, 30.88,
43.44, 45.63, 46.62, 47.26, 78.95, 119.99, 140.85, 149.39,
155.38, 163.74. MS (70 eV), m/z (%): 349 (100, M+ + 1).
(2-F lu or o-3-m et h oxy-6-m et h oxym et h oxy-p h en yl)-(4-
iod o-p h en yl)-m eth a n ol (22). BuLi solution (1.6 M in hexane,
12.1 mL, 19.3 mmol) was added to a solution of 2-fluoro-1-
methoxy-4-methoxymethoxybenzene 20 (2.4 g, 12.9 mmol) in
anhydrous THF (30 mL) at -78 °C. The solution was stirred
for 40 min at -70 °C and then added dropwise in a period of
20 min to a solution of 4-iodo-benzaldehyde 21 (4.49 g, 19.3
mmol) in anhydrous THF (25 mL) at -78 °C. The mixture was
warmed to room temperature in a period of 14 h. Water (20
mL) and ethyl acetate (20 mL) were added, and the separated
aqueous layer was re-extracted with ethyl acetate (2 × 40 mL).
The combined organic layers were washed with brine and dried
over Na2SO4. After filtration and evaporation, the residue was

1386 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Ta ble 3. H-Bonds
Breitenlechner et al.
H-bonds between
protein atom
PKA Ester 1
PKA amide 4
PKA ether 5
PKA vinyl 8
inhibitor atom
distance (Å)
distance (Å)
distance (Å)
distance (Å)
N11
O22
N33
N33
N33
N33
X41
X41
O62
O92
O92
Val123 (N)
3.3
-
3.2
2.8
2.7
3.4
2.9
2.9
3.0
3.4
3.0
2.6
3.1
3.1
[O¨ ]
2.5
3.3
3.1
3.2
-
3.1
-
Thr183 (OG1)
Asp184 (OD1)
Asp184 (OD2)
Glu170 (O)
Asn171 (OD1)
Asp184 (OD2)
Asp184 (OD1)
Phe54 (N)
2.9
3.4
2.9
3.1
-
2.9
2.9
3.1
-
-
-
-
3.1
2.5
-
3.3
2.7
3.1
2.0
2.7
3.2
Glu91 (OE1)
Lys72 (NZ)
inhibitor atom
water atom
distance (Å)
distance (Å)
distance (Å)
distance (Å)
(connects to protein)
O22
X54-Lys72 (NZ)
-Asp184 (OD1)
-Asp184 (OD1)
X22-Glu127 (OE2)
-Leu49 (O)
2.9-2.6
-2.5
2.9-3.4
-2.7
2.8-2.8
-3.2
3.1-2.9
-2.9
-3.1
-2.9
-2.9
N23
2.9-2.6
-3.0
2.9-2.7
-2.9
3.1-2.7
-3.0
3.1-2.6
-2.9
purified by flash chromatography on silica gel (column, 4 cm
(m, 2H), 7.01-7.20 (m, 2H), 7.50-7.60 (m, 2H), 7.65-7.80 (m,
2H), 8.60-8.75 (s, 2H). 13C NMR (250 MHz, CDCl3): δ 21.45,
28.83, 29.75, 30.09, 41.50, 45.17, 47.76, 49.50, 56.52, 57.41,
60.81, 95.71, 111.07, 112.38, 112.80, 114.69, 121.24, 127.00,
133.07, 143.14, 143.32, 148.48, 148.58, 150.85, 152.50, 156.80.
MS (70 eV), m/z (%): 637 (100, M+ + 1).
× 25 cm; eluent, ethyl acetate/heptane ) 1:1) to yield a light-
1
yellow oil (4.2 g, 50%). H NMR (500 MHz, CDCl3): δ 3.27 (s,
3H), 3.80 (d, 1H), 3.87 (s, 3H), 4.98 (d, 1H), 5.07 (d, 1H), 6.18
(d, 1H), 6.85 (m, 2H), 7.12 (d, 2H), 7.64 (d, 2H). 13C NMR (100
MHz, CDCl3): δ 56.25, 56.80, 67.79, 92.38, 95.04, 109.95,
112.85, 120.86, 127.54, 137.15, 143.07, 143.29, 148.77, 151.30.
(3R,4S)-N-(4-{[4-(2-F lu or o-6-h yd r oxy-3-m et h oxy-b en -
zoyl)-p h en yla m in o]-m et h yl}-a zep a n -3-yl)-ison icot in a -
m id e h yd r och lor id e (6). HCl in dioxane (4 M, 1 mL, 4.0
mmol) was added to a solution of 23 (11.5 mg, 0.02 mmol) in
2 mL anhydrous dioxane. The mixture was stirred for 15 h at
room temperature, and the volatile components were evapo-
rated. The residue was dissolved in a minimum of MeOH, and
anhydrous ether was added to precipitate the product. The
resulting light yellow amorphous powder was separated and
washed with anhydrous ether (9.4 mg, 95%). 1H NMR (250
MHz, CD3OD): δ 1.45-2.50 (m, 5H), 3.05-3.85 (m, 7H), 4.05
(s, 3H), 6.65-6.95 (m, 4H), 7.20-7.80 (m, 2H), 7.90-8.30 (m,
2H), 8.70-9.0 (m, 2H). 13C NMR (250 MHz, CD3OD): δ 24.73,
29.41, 30.19, 43.43, 44.32, 60.83, 63.55, 73.06, 111.55, 114.50,
117.88, 128.67, 128.85, 129.55, 130.37, 134.10, 134.24, 137.55,
146.46, 151.55, 152.50, 164.00, 164.50. MS (70 eV), m/z (%):
493 (100, M+ + 1); HR-MS (ESI): calcd for C27H30N4O4F [M +
H]: 493.2251; found: 493.2233.
HR-MS (EI): calcd for C16H16FIO4 [M]+: 418.0072; found:
+
418.0073.
(2-F lu or o-3-m et h oxy-6-m et h oxym et h oxy-p h en yl)-(4-
iod o-p h en yl)-m eth a n on e (19). Carbinol 22 (300 mg, 0.72
mmol) was dissolved in anhydrous CH2Cl2 (2 mL). Activated
MnO2 (350 mg) was added, and the mixture was stirred for
24 h. After filtration through a plug of Celite and evaporation,
the residue was purified by flash chromatography on silica gel
(column, 2 cm × 10 cm; eluent, ethyl acetate/heptane ) 4:1)
to yield a colorless oil (290 mg, 95%). 1H NMR (500 MHz,
CDCl3): δ 3.32 (s, 3H), 3.90 (s, 3H), 5.03 (s, 2H), 6.95 (d, 1H),
7.02 (t, 1H), 7.59 (d, 2H), 7.84 (d, 2H). 13C NMR (100 MHz,
CDCl3): δ 56.22, 57.09, 95.32, 102.19, 110.67, 115.29, 119.42,
130.88, 136.40, 137.98, 142.98, 148.22, 150.49, 190.70.
HR-MS (EI): calcd for C16H14FIO4 [M]+: 415.9915; found:
+
415.9926.
(3R,4S)-4-{[4-(2-F lu or o-3-m eth oxy-6-m eth oxym eth oxy-
ben zoyl)-ph en ylam in o]-m eth yl}-3-[(pyr idin e-4-car bon yl)-
a m in o]-a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (23).
18-crown-6 (84.0 mg, 0.32 mmol), aryl iodide 19 (60.0 mg, 0.15
4-[(2-F lu or o-3-m et h oxy-6-m et h oxym et h oxyp h en yl)-
(1,1,1-tr iisopr opylsilan yloxy)-m eth yl]-ben zoic Acid Meth -
yl Ester (25). Carbinol 2434 (500 mg, 1.43 mmol), imidazole
(244 mg, 3.58 mmol), TIPS-Cl (359 mg, 1.86 mmol) in anhy-
drous DMF were stirred for 15 h at room temperature.
TIPSOSO2CF3 (0.7 equiv) was added, and the mixture was
stirred for 36 h at 100 °C. Water (5 mL) was added, and the
suspension was extracted with ethyl acetate (3 × 10 mL). The
combined organic layers were washed with brine and dried
over Na2SO4. After filtration and evaporation, the residue was
purified by flash chromatography on silica gel (column, 2 cm
× 15 cm; eluent, (1) ethyl acetate/heptane ) 1:9). A colorless
t
mmol), amine 18 (45.0 mg, 0.15 mmol), BuOK (31.0 mg, 0.32
mmol), Pd2(dba)3 (5.2 mg), and BINAP (10.8 mg) were added
to anhydrous THF (0.5 mL), and the mixture was stirred for
15 h at reflux. The volatile components were evaporated in
vacuo, and the residue was purified by flash chromatography
on silica gel (column, 1.5 cm × 15 cm; eluent, ethyl acetate/
MeOH ) 19:1) to yield a dense colorless oil (12 mg, 12.5%).
1H NMR (250 MHz, CDCl3): δ 1.25 (s, 9H), 1.55-2.01 (m, 5H),
2.05 (s, 2H), 2.95-3.21 (m, 2H), 3.28 (s, 3H), 3.30-3.82 (m,
3H), 3.85 (s, 3H), 5.00 (s, 2H), 6.65-6.85 (m, 2H), 6.88-7.00
1
oil was obtained (650 mg, 90%). H NMR (250 MHz, CDCl3):

Novel Azepane Derivatives as PKB Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1387
δ 0.85-1.19 (m, 18H), 3.32 (s, 3H), 3.72 (s, 3H), 3.79 (s, 3H),
4.98-5.08 (m, 2H), 6.42 (s, 1H), 6.67-6.78 (m, 2H), 7.50 (d, 3J
the suspension was extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were washed with brine and dried
over Na2SO4. After filtration and evaporation, a colorless oil
was obtained (114 mg, 92%). 1H NMR (250 MHz, CDCl3): δ
0.85-1.05 (m, 18H), 1.41 (s, 9H), 1.48-2.02 (m, 4H), 2.91-
3.05 (m, 1H), 3.15-3.29 (m, 1H), 3.36 (s, 3H), 3.70 (s, 3H),
3.90-4.07 (m, 3H), 4.18-4.25 (m, 1H), 4.52-4.60 (s, 2H), 5.00-
5.10 (m, 2H), 6.38 (s, 1H), 6.62-6.78 (m, 2H), 7.19 (d, 3J ) 8.3
3
) 8.3 Hz, 2H), 7.87 (d, J ) 8.3 Hz, 2H). 13C NMR (62.5 MHz,
CDCl3): δ 12.67, 18.17, 52.34, 56.37, 57.21, 67.96, 95.61,
109.52, 113.26, 123.00, 125.69, 128.68, 129.54, 143.45, 149.00,
149.998, 151.40 (d, 2J ) 250 Hz), 167.58. MS (70 eV), m/z (%):
507 (100, M+ + 1).
{4-[(2-F lu or o-3-m eth oxy-6-m eth oxym eth oxy-p h en yl)-
(1,1,1-t r iisop r op ylsila n yloxy)-m et h yl]-p h en yl}-m et h a -
n ol (26). Compound 25 (650 mg, 1.28 mmol) was dissolved in
anhydrous THF (8 mL). LiAlH4 (106 mg, 2.80 mmol) was added
portionwise, and the mixture was stirred for 3 h at room
temperature. Water (5 mL) was added, and the suspension
was extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were washed with brine and dried over Na2-
SO4. After filtration and evaporation, the residue was purified
by flash chromatography on silica gel (column, 2 cm × 15 cm;
eluent, (1) ethyl acetate/heptane ) 1:3). A colorless oil was
obtained (430 mg, 70%). 1H NMR (250 MHz, CDCl3): δ 0.97-
1.22 (m, 18H), 3.51 (s, 3H), 3.86 (s, 3H), 4.70 (s, 2H), 5.19 (s,
2H), 6.53 (s, 1H), 6.78-6.90 (m, 2H), 7.35 (d, 3J ) 8.3 Hz, 2H),
7.57 (d, 3J ) 8.3 Hz, 2H). 13C NMR (250 MHz, CDCl3): δ 12.68,
18.22, 56.38, 57.19, 65.75, 68.08, 95.70, 109.52, 112.98, 123.70,
126.18, 126.96, 139.30, 143.63, 144.31, 149.04, 153.10. MS (70
eV), m/z (%): 479 (100, M+ + 1).
3
Hz, 2H), 7.37 (d, J ) 8.3 Hz, 2H), 7.62 (br d, 2H), 8.29 (br s,
1H), 8.57-8.69 (m, 2H). MS (70 eV), m/z (%): 797 (100, M+
1).
+
(3R,4R)-4-[4-(2-F lu or o-3-m eth oxy-6-m eth oxym eth oxy-
b en zoyl)-b en zyloxy]-3-[(p yr id in e-4-ca r b on yl)-a m in o]-
a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (30). Com-
pound 29 (114 mg, 0.14 mmol) and Bu4NF‚4 H2O (183 mg, 0.70
mmol) in THF (2 mL) were stirred for 15 h at room temper-
ature. Water (5 mL) was added, and the suspension was
extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were washed with brine and dried over Na2-
SO4. After filtration and evaporation, the residue was purified
by flash chromatography on silica gel (column, 2 cm × 10 cm;
eluent, (1) ethyl acetate) to yield a colorless crude oil (70 mg,
76%) which was dissolved in anhydrous CH2Cl2 (2 mL).
Activated MnO2 (50 mg) was added, and the mixture was
stirred for 24 h. After filtration through a plug of Celite and
evaporation, the residue was purified by flash chromatography
on silica gel (column, 2 cm × 10 cm; eluent, ethyl acetate) to
[(4-Ch lor om eth yl-p h en yl)-(2-flu or o-3-m eth oxy-6-m eth -
oxym et h oxy-p h en yl)-m et h oxy]-(1,1,1-t r iisop r op yl)-si-
la n e (27). Compound 26 (210 mg, 0.44 mmol) and PPh3 (115
mg, 0.44 mmol) were dissolved in anhydrous CCl4 (5 mL). The
mixture was stirred for 3 h at room temperature. Water (5
mL) was added, and the suspension was extracted with ethyl
acetate (3 × 10 mL). The combined organic layers were washed
with brine and dried over Na2SO4. After filtration and evapo-
ration, the residue was purified by flash chromatography on
silica gel (column, 2 cm × 10 cm; eluent, (1) ethyl acetate/
1
yield a colorless crude oil (50 mg, 72%). H NMR (250 MHz,
CDCl3): δ 1.48 (s, 9H), 1.52-2.08 (m, 4H), 2.97-3.09 (1H),
3.24-3.36 (m, 1H), 3.27 (s, 3H), 3.72-3.82 (m, 1H), 3.86 (s,
3H), 4.02-4.13 (m, 2H), 4.74 (s, 2H), 4.99 (s, 2H), 6.89-7.0
3
3
(m, 2H), 7.43 (d, J ) 9.1 Hz, 2H), 7.67 (d, J ) 6.8 Hz, 2H),
3
3
7.83 (d, J ) 9.1 Hz, 2H), 8.43 (br d, 1H), 8.72 (d, J ) 6.8 Hz,
2H). 13C NMR (250 MHz, CDCl3): δ 21.19, 25.27, 28.77, 33.42,
47.19, 47.93, 55.21, 56.56, 57.42, 70.92, 81.27, 95.66, 110.99,
115.26, 121.29, 127.56, 130.18, 136.64, 141.60, 143.15, 145.39,
148.55, 149.63, 150.93, 159.06, 165.45, 191.39. MS (70 eV), m/z
(%): 638 (100, M+ + 1).
1
heptane ) 1:9) to yield a colorless oil (162 mg, 74%). H NMR
(250 MHz, CDCl3): δ 0.85-1.19 (m, 18H), 3.32 (s, 3H), 3.70
(s, 3H), 4.48 (s, 2H), 5.04 (q, 3J ) 5.6 Hz, 2H), 6.39 (s, 1H),
3
3
6.63-6.78 (m, 2H), 7.20 (d, J ) 8.3 Hz, 2H), 7.43 (d, J ) 8.3
Hz, 2H). 13C NMR (250 MHz, CDCl3): δ 12.56, 18.20, 56.36,
57.18, 67.08, 67.96, 95.51, 109.35, 113.02, 123.35, 126.34,
128.88, 132.57, 136.09, 143.37, 145.14, 148.94. MS (70 eV), m/z
(%): 498 (100, M+ + 1).
(3R,4R)-N-{4-[4-(2-F lu or o-6-h yd r oxy-3-m et h oxy-b en -
zoyl)-b en zyloxy]-a zep a n -3-yl}-ison icot in a m id e h yd r o-
ch lor id e (5). HCl in dioxane (4 M, 2 mL, 8.0 mmol) was added
to a solution of 30 (45.0 mg, 0.07 mmol) in anhydrous MeOH
(2 mL). The mixture was stirred for 15 h at room temperature,
and the volatile components were evaporated. The residue was
dissolved in a minimum of MeOH, and anhydrous diethyl ether
was added to precipitate the product. The resulting light yellow
amorphous powder was separated and washed with anhydrous
diethyl ether (40 mg, 91%). 1H NMR (250 MHz, D2O): δ 1.89-
2.38 (m, 3H), 2.32-2.35 (m, 1H), 3.27-3.48 (m, 3H), 3.85-
3.92 (m, 2H), 3.92 (s, 3H), 4.24-4.32 (m, 1H), 4.53 (d, 3J )
(3R,4R)-4-{4-[(2-F lu or o-3-m eth oxy-6-m eth oxym eth oxy-
ph en yl)-(1,1,1-tr iisopr opylsilan yloxy)-m eth yl]-ben zyloxy}-
a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (28). NaH (8.0
mg, 0.3 mmol) was added to a solution of (3R,4S)-3-amino-4-
hydroxy-azepane-1-carboxylic acid tert-butyl ester 2848 (83 mg,
0.36 mmol) in anhydrous THF (2 mL). The mixture was stirred
for 30 min at reflux, a solution of 27 (160 mg, 0.32 mmol) in
anhydrous THF (1 mL) was added and stirring was continued
for 2 h at reflux. Water (5 mL) was added, and the suspension
was extracted with ethyl acetate (3 × 10 mL). The combined
organic layers were washed with brine and dried over Na2-
SO4. After filtration and evaporation, the residue was purified
by flash chromatography on silica gel (column, 2 cm × 10 cm;
eluent, ethyl acetate/2N NH3 in MeOH ) 98:2) to yield a
colorless oil (102 mg, 43%). 1H NMR (250 MHz, CDCl3): δ 0.85
(s, 18H), 1.35 (s, 9H), 1.40-1.55 (m, 4H), 2.75-3.20 (m, 4H),
3.35 (s, 3H), 3.35-3.57 (m, 2H), 3.67 (s, 3H), 4.21-4.58 (m,
2H), 5.01-5.09 (m, 2H), 6.39 (s, 1H), 6.62-6.78 (m, 2H), 7.11-
7.23 (m, 2H), 7.39-7.48 (m, 2H). 13C NMR (250 MHz, CDCl3):
δ 12.28, 17.84, 22.52, 27.31, 28.83, 46.00, 47.11, 47.63, 56.53,
57.16, 68.09, 71.35, 79.98, 84.88, 95.68, 109.39, 112.91, 123.67,
126.04, 127.57, 136.72, 143.43, 144.21, 148.91, 156.04, 175.14.
MS (70 eV), m/z (%): 692 (100, M+ + 1).
3
3
14.1 Hz, 1H), 4.83 (d, J ) 14.1 Hz, 1H), 6.83 (d, J ) 9.1 Hz,
3
3
1H), 7.27 (d, J ) 9.1 Hz, 1H), 7.42 (d, J ) 8.3 Hz, 2H), 7.60
3
3
3
(d, J ) 8.3 Hz, 2H), 7.96 (d, J ) 6.7 Hz, 2H), 8.85 (d, J )
6.7 Hz, 2H). 13C NMR (62.5 MHz, D2O): δ 19.30, 27.59, 45.79,
47.20, 52.82, 57.02, 69.99, 79.55, 111.55, 117.56, 124.16,
124.23, 128.93, 129.00, 129.74, 135.59, 140.03, 140.85, 143.35,
144.40, 147.06, 164.13, 194.54. MS (70 eV), m/z (%): 567 (100,
M+ + 1); HR-MS (ESI): calcd for C27H29N3O5F [M + H]:
494.2091; found: 494.2101.
(2-F lu or o-3-m et h oxy-6-m et h oxym et h oxy-p h en yl)-(4-
h yd r oxym eth yl-p h en yl)-m eth a n ol (31). A suspension of
LiAlH4 (0.68 g, 17.8 mmol) in anhydrous THF (10 mL) was
added during 15 min to a solution of 4-[(2-fluoro-3-methoxy-
6-methoxymethoxy-phenyl)-hydroxy-methyl]-benzoic acid meth-
yl ester 24 (3.0 g, 8.9 mmol) in anhydrous THF (25 mL) at
room temperature. The mixture was stirred for 3 h at room
temperature. Water (5 mL) was added dropwise at 0 °C. After
stirring for 30 min water (15 mL) was added and the mixture
was filtered. The filter cake was washed with ethyl acetate (3
× 4 mL) and the aqueous layer re-extracted with ethyl acetate
(2 × 10 mL). The combined organic layers were washed with
brine (2 × 3 mL), dried over Na2SO4, filtered, and concentrated
(3R,4R)-4-{4-[(1,1,1-Tr iisop r op ylsila n yloxy)-(2-flu o-
r o-3-m eth oxy-6-m eth oxym eth oxy-ph en yl)-m eth yl]-ben zyl-
oxy}-3-[(pyridine-4-carbonyl)-amino]-azepane-1-carboxylic
Acid ter t-Bu tyl Ester (29). Compound 28 (102 mg, 0.15
mmol) was dissolved in anhydrous CH2Cl2 (2 mL). Isonicotinic
acid (21 mg, 0.17 mmol), DMAP (10 mg, 0.1 mmol), and DCC
(36 mg, 0.17 mmol) were added to this solution, and the
mixture was stirred for 15 h. Water (5 mL) was added, and
1
in vacuo to yield a colorless viscous oil (1.8 g, 63%). H NMR
(500 MHz, CDCl3): δ 1.70 (br, 2H), 3.28 (s, 3H), 3.87 (s, 3H),

1388 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6
Breitenlechner et al.
4.67 (s, 2H), 4.98 (d, 1H), 5.06 (d, 1H), 6.25 (s, 1H), 6.82-6.87
(m, 2H), 7.31 (d, 2H), 7.37 (d, 2H); 13C NMR (100 MHz,
CDCl3): δ 56.22, 56.82, 65.11, 68.18, 95.08, 109.97, 112. 67,
121.38, 125.72, 126.86, 139.66, 142.92, 143.08, 148.85, 148.91.
HR-MS (EI): calcd for C17H19O5F+ [M]+: 322.1211; found:
322.1223.
mmol) in CH2Cl2 (10 mL) was added during 5 min at -78 °C,
and the mixture was stirred for 20 min at this temperature.
Triethylamine (8.3 mL, 60 mmol) was added, and the mixture
was stirred for 5 min and slowly warmed to room temperature.
Water (5 mL) was added, and the suspension was extracted
with CH2Cl2 (3 × 10 mL). The combined organic layers were
washed with brine and dried over Na2SO4. After filtration and
evaporation, the residue was purified by flash chromatography
on silica gel (column, 4 cm × 25 cm; eluent, ethyl acetate/
MeOH-2N NH3 ) 95:5) to yield a light yellow gum (3.18 g,
85%). 1H NMR (250 MHz, CDCl3): δ 1.38 (s, 2H), 1.44 (s, 7H),
1.65-2.09 (m, 2H), 2.55-2.90 (m, 2H), 2.92-3.05 (m, 0.75H),
3.18-3.40 (0.25H), 3.60-4.25 (m, 3H), 4.85-4.96 (m, 0.25H),
4.96-5.14 (m, 0.75H), 7.72 (d, 3J ) 5.4 Hz, 2H), 8.20 (br d,
4-(2-F lu or o-3-m et h oxy-6-m et h oxym et h oxy-b en zoyl)-
ben za ld eh yd e (32). Activated MnO2 (0.78 g, 9.00 mmol) was
added to a solution of 31 (1.8 g, 5.6 mmol) in CH2Cl2 (150 mL).
Two further portions of activated MnO2 (2 × 0.78 g, 9.00 mmol)
were added after 2 and 4 h of vigorous stirring. The stirring
was continued for 15 h. The filter cake was washed with CH2-
Cl2 (3 × 30 mL), and the combined organic layers were
concentrated in vacuo to yield light yellow crystals (1.22 g,
69%, mp. 104-106 °C). 1H NMR (500 MHz, CDCl3): δ 3.31 (s,
3H), 3.92 (s, 3H), 5.03 (s, 2H), 6.98 (d, 1H), 7.05 (t, 1H), 7.99
(d, 2H), 8.04 (d, 2H), 10.14 (s, 1H); 13C NMR (100 MHz,
CDCl3): δ 56.24, 57.09, 95.33, 110.69, 115.59, 119.11, 129.77,
130.00, 139.43, 141.26, 143.02, 148.30, 150.59, 190.76, 191.60.
HR-MS (EI): calcd for C17H15O5F+ [M]+: 318.0898; found:
318.0903.
3
1H), 8.88 (d, J ) 5.4 Hz, 2H). 13C NMR (62.5 MHz, CDCl3):
δ two rotamers: A: 27.29, 28.61, 40.03, 49.28, 51.09, 62.21,
81.61, 121.46, 141.42, 150.90, 156.81, 165.43, 206.36; B: 25.13,
28.62, 40.15, 48.67, 48.88, 61.03, 81.08, 121.29, 141.00, 150.90,
155.11, 165.06, 207.36. MS (70 eV), m/z (%): 334 (100, M+
1); HR-MS (ESI): calcd for C17H23N3O4 [M + H]: 334.1767;
found: 334.1782.
+
(3R)-4-Met h oxym et h ylen e-3-[(p yr id in e-4-ca r b on yl)-
a m in o]-a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (36).
Potassium tert-butoxide (1.60 g, 13.9 mmol) was added over a
period of 0.5 h via a sidearm addition funnel under argon
atmosphere to a suspension of (methoxymethyl)-triphenylphos-
phonium chloride (4.90 g, 13.9 mmol) in anhydrous diethyl
ether (60 mL) at -15 °C. The reaction mixture was stirred at
-15 °C for 1 h, and then a solution of ketone 35 (1.65 g, 4.95
mmol) in anhydrous diethyl ether (20 mL) was added over a
period of 20 min at this temperature. Stirring at room
temperature was continued for 24 h. The reaction mixture was
hydrolyzed with water (30 mL), and the organic and aqueous
phases were separated. The aqueous phase was re-extracted
with diethyl ether (2 × 20 mL), and the combined organic
layers were dried over Na2SO4. Cooling of the organic phase
to -30 °C resulted in crystallization of triphenylphosphine
oxide, which was separated by decantation. After filtration and
evaporation, the residue was purified by flash chromatography
on silica gel (column, 4 cm × 25 cm; eluent, ethyl acetate/
MeOH-2 N NH3 ) 99:1) to yield a light yellow gum (1.05 g,
59%). 1H NMR (400 MHz, CDCl3): δ 1.45 (s, 2H), 1.51 (s, 7H),
1.62-2.19 (m, 4H), 2.82-2.96 (m, 2H), 3.55-3.70 (m, 3H),
3.80-3.91 (m, 1H), 4.18-4.30 (m, 1H), 5.20 (br s, 1H), 6.00 (s,
1H), 7.66 (d, 3J ) 5.2 Hz, 2H), 8.20 (br d, 1H), 8.70 (d, 3J )
5.2 Hz, 2H). 13C NMR (62.5 MHz, CDCl3): d 21.05, 28.41,
47.63, 50.29, 52.53, 60.10, 71.12, 80.44, 114.07, 120.91, 131.74,
145.75, 150.47, 157.99, 164.33. MS (70 eV), m/z (%): 362 (100,
M+ + 1); HR-MS (ESI): calcd for C19H28N3O4 [M + H]:
362.2080; found: 362.2071.
(3R ,4S )-4-F or m yl-3-[(p yr id in e -4-ca r b on yl)-a m in o]-
a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (37). Enol
ether 36 (200 mg, 0.55 mmol) was stirred in CHCl3 (30 mL)
at reflux. Trichloroacetic acid (200 mg, 2.77 mmol) was added
via a sidearm addition funnel. The mixture was stirred for 60
min at reflux and then cooled to room temperature. The
reaction mixture was washed with saturated aqueous NaHCO3
solution (10 mL), and after separation the aqueous phase was
re-extracted with CH2Cl2 (2 × 5 mL). The combined organic
layers were washed with brine and dried over Na2SO4. After
filtration and evaporation the residue was purified by flash
chromatography on silica gel (column, 4 cm × 25 cm; eluent,
ethyl acetate/MeOH-2 N NH3 ) 99:1) to yield a light yellow
oil (130 mg, 68%). The product contains 20% (3R,4S)-4-formyl-
3-[(pyridine-4-carbonyl)-amino]-azepane-1-carboxylic acid tert-
butyl ester. 1H NMR (400 MHz, CDCl3): δ 1.53 (s, 9H), 1.51-
2.19 (m, 4H), 2.55-2.94 (m, 2H), 3.14-3.24 (m, 1H), 3.82-
4.25 (m, 2H), 4.18-4.30 (m, 1H), 4.51-4.84 (m, 1H), 7.57-
7.78 (m, 2H), 8.70-8.85 (m, 2H), 9.81 (s, 1H). MS (70 eV), m/z
(%): 348 (100, M+ + 1); HR-MS (ESI): calcd for C18H26N3O4
[M + H]: 348.1923; found: 364.1889.
(3R,4R)-4-{[4-(2-F lu or o-3-m eth oxy-6-m eth oxym eth oxy-
ben zoyl)-ben zylid en e]-a m in o}-3-[(p yr id in e-4-ca r bon yl)-
a m in o]-a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (33).
Compound 32 (0.78 g, 9.00 mmol) was added to a solution of
amine 14 (1.8 g, 5.6 mmol) in C2H5OH (150 mL). The solution
was heated at reflux under argon for 15 h and stirred for 48
h at room temperature. The volatile components were evapo-
1
rated to yield an off-white foam (302 mg, 95%). H NMR (500
MHz, CDCl3): δ 1.57 (s, 9Η), 1.83-1.99 (m, 4H), 3.20 (d, 1H),
3.31 (s, 3H), 3.54 (d, 1H), 3.90 (s, 3H), 3.96 (d, 1H), 3.97 (m,
1H), 4.13 (m, 1H), 4.16 (d, 1H), 5.02 (s, 2H), 6.95 (t, 1H), 7.02
(t, 1H), 7.70 (s, 2H), 7.83 (d, 2H), 7.92 (d, 2H), 8.47 (s, 1H),
8.57 (s, 1H), 8.74 (s, 2H). 13C NMR (100 MHz, CDCl3): δ 22.92,
28.07, 28.46, 48.43, 48.90, 56.22, 57.10, 58.67, 61.23, 70.46,
80.75, 95.34, 110.68, 115.24, 115.56, 120.94, 128.40, 129.87,
138.58, 140.67, 141.26, 142.88, 148.29, 149.66, 150.53, 165.89,
190.96, 191.61.
(3R,4R)-4-[4-(2-F lu or o-3-m eth oxy-6-m eth oxym eth oxy-
ben zoyl)-ben zyla m in o]-3-[(p yr id in e-4-ca r bon yl)-a m in o]-
a zep a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (34). NaBH3-
CN (35.0 mg, 0.56 mmol) was added to a solution of 14 (180
mg, 0.28 mmol) in CH3OH (3 mL), and the resulting mixture
was stirred for 1 h at room temperature. Water (25 mL) was
added, and the suspension was extracted with ethyl acetate
(3 × 20 mL). The combined organic layers were washed with
brine and dried over Na2SO4. After filtration and evaporation,
the residue was purified by flash chromatography on silica gel
(column, 2 cm × 15 cm; eluent, (1) ethyl acetate/heptane, (2)
ethyl acetate to (3) ethyl acetate/CH3OH ) 95:5). A yellow gum
was obtained as raw material (102 mg, 57%). HR-MS (ESI):
calcd for C34H42N4O7F [M + H]: 637.3038; found: 637.3082.
(3R,4R)-N-{4-[4-(2-F lu or o-6-h yd r oxy-3-m et h oxy-b en -
zoyl)-b en zyla m in o]-a zep a n -3-yl}-ison icot in a m id e H y-
d r och lor id e (7). HCl in dioxane (4 M, 3 mL, 12.0 mmol) was
added to a solution of 34 (45.0 mg, 0.07 mmol) in anhydrous
dioxane (2 mL). The mixture was stirred for 15 h at room
temperature, and the resulting light yellow crystals were
separated and washed with anhydrous diethyl ether (24 mg,
1
57%, mp 196 °C dec). H NMR (500 MHz, DMSO): δ 1.93 (m,
1H), 2.08 (m, 1H), 2.19 (m, 1H), 2.35 (m, 1H), 3.05 (m, 1H),
3.25 (m, 1H), 3.40 (s, 2H), 3.79 (s, 3H), 4.25 (m, 1H), 4.37 (m,
1H), 4.92 (m, 1H), 6.76 (d, 1H), 7.15 (t, 1H), 7.72 (d, 2H), 7.75
(d, 2H), 7.97 (d, 2H), 8.77 (d, 2H), 9.61-10.01 (m, 5H). 13C
NMR (100 MHz, DMSO): δ 22.5, 25.7, 46.9, 47.0, 48.0, 48.3,
57.3, 61.2, 111.3, 116.4, 116.8, 122.5, 129.5, 131.2, 136.9, 138.3,
140.3, 141.5, 149.6, 149.8, 151.0, 165.4, 191.7. HR-MS (ESI):
calcd for C27H30N4O4F [M + H]: 493.2251; found: 493.2250.
(3R)-4-Oxo-3-[(p yr id in e-4-ca r b on yl)-a m in o]-a zep a n e-
1-ca r boxylic Acid ter t-Bu tyl Ester (35). Oxalyl chloride
(1.07 mL, 12.3 mmol) was dissolved in anhydrous CH2Cl2 (25
mL) and cooled to -78 °C. DMSO (1.93 g, 24.7 mmol) in CH2-
Cl2 (3 mL) was added to this solution at -78 °C, and the
mixture was stirred for 5 min. Compound 11 (3.76 g, 11.2
(3R,4S)-3-[(P yr id in e-4-ca r b on yl)-a m in o]-4-vin yl-a ze-
p a n e-1-ca r boxylic Acid ter t-Bu tyl Ester (38). n-BuLi solu-
tion (1.6 M in hexane, 605 µL, 0.97 mmol) was added to a

Novel Azepane Derivatives as PKB Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 6 1389
suspension of methyltriphenylphosphonium iodide (390 mg,
0.97 mmol) in anhydrous THF (10 mL). The mixture was
stirred for 40 min at -10 °C and then cooled to -78 °C.
Aldehyde 37 (240 mg, 0.69 mmol) in THF (5 mL) was added,
and the mixture was warmed to room temperature in a period
of 14 h. Water (5 mL) and ethyl acetate (5 mL) were added,
and the separated aqueous layer was re-extracted with ethyl
acetate (2 × 5 mL). The combined organic layers were washed
with brine and dried over Na2SO4. After filtration and evapo-
ration, the residue was purified by preparative HPLC (Chiracel
OD CSP 20 µm from Daicel, 2-propanol/heptane ) 1:4 as
eluent) to yield a light yellow oil (120 mg, 50%). 1H NMR (400
MHz, CDCl3): δ 1.53 (s, 9H), 1.51-1.78 (m, 4H), 2.10-2.19
(m, 1H), 2.72-2.83 (m, 1H), 3.29 (dd, 3J ) 15.3 Hz, 5.6 Hz,
1H), 3.92-4.05 (m, 2H), 4.21-4.32 (m, 1H), 4.93-5.08 (m, 2H),
5.80-5.94 (m, 1H), 7.50 (m, 2H), 8.21 (d, 3J ) 7.3 Hz, 1H),
8.73 (d, 3J ) 4.2 Hz, 2H). 13C NMR (100 MHz, CDCl3): δ 27.02,
27.41, 28.16, 48.02, 49.18, 51.27, 53.65, 79.76, 113.68, 119.94,
139.53, 140.89, 149.46, 157.21, 163.15. MS (70 eV), m/z (%):
346 (100, M+ + 1); HR-MS (ESI): calcd for C19H28N3O3 [M +
H]: 346.2131; found: 346.2131.
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(3R,4S)-4-{tr a n s-2-[4-(2-F lu or o-3-m eth oxy-6-m eth oxy-
m eth oxy-ben zoyl)-p h en yl]-vin yl}-3-[(p yr id in e-4-ca r bon -
yl)-a m in o]-a zep a n e-1-ca r b oxylic Acid ter t-Bu t yl E st er
(39). Alkene 38 (60 mg, 0.17 mmol), iodide 19 (65 mg, 0.16
mmol), Pd(II) acetate (3.90 mg, 0.017 mmol), tri-o-tolylphos-
phine (10.9 mg, 0.034 mmol), and N,N-diisopropylamine (60.1
µL, 0.34 mmol) were dissolved in anhydrous DMF (1 mL) and
stirred for 14 h at 105 °C. The volatile components were
evaporated in vacuo and the residue was purified by flash
chromatography on silica gel (column, 1.5 cm × 15 cm; eluent,
ethyl acetate/MeOH ) 19:1) to yield a dense colorless oil (20
1
mg, 18%). H NMR (400 MHz, CDCl3): δ 1.54 (s, 9H), 1.51-
1.92 (m, 4H), 2.33-2.42 (m, 1H), 2.72-2.88 (m, 1H), 3.25-
3.39 (m, 1H), 3.29 (s, 3H), 3.87 (s, 3H), 3.95-4.09 (m, 2H),
4.32-4.42 (m, 1H), 5.00 (s, 2H), 6.32-6.45 (m, 2H), 6.87-7.02
3
3
(m, 2H), 7.35 (d, J ) 8.3 Hz, 2H), 7.58 (d, J ) 5.3 Hz, 2H),
3
3
3
7.77 (d, J ) 8.2 Hz, 2H), 8.28 (d, J ) 7.8 Hz, 1H), 8.68 (d, J
) 5.4 Hz, 2H). MS (70 eV), m/z (%): 634 (100, M+ + 1).
(3R,4S)-N-(4-{tr a n s-2-[4-(2-Flu or o-6-h ydr oxy-3-m eth oxy-
ben zoyl)-ph en yl]-vin yl}-azepan -3-yl)-ison icotin am ide Hy-
d r och lor id e (8). HCl in diethyl ether (2 M, 180 µL, 0.36
mmol) was added to a solution of 39 (8.00 mg, 0.0126 mmol)
in anhydrous MeOH (0.2 mL) and anhydrous diethyl ether (0.5
mL). The mixture was stirred for 15 h at room temperature,
and the volatile components were evaporated. The residue was
dissolved in a minimum of MeOH, and anhydrous diethyl ether
was added to precipitate the product. The resulting light yellow
amorphous powder was separated and washed with anhydrous
diethyl ether (7 mg, 98%). 1H NMR (500 MHz, CD3OD): δ
1.82-2.20 (m, 4H), 2.86-2.96 (m, 1H), 3.12-3.75 (m, 4H), 3.84
(s, 3H), 4.39 (br s, 1H), 6.38-6.48 (m, 1H), 6.58-6.76 (m, 3H),
3
3
7.06-7.17 (m, 2H), 7.47 (d, J ) 8.0 Hz, 2H), 7.73 (d, J ) 7.8
Hz, 2H), 8.36 (br s, 8.36, 2H), 8.97 (br s, 2H). MS (70 eV), m/z
(%): 563 (100, M+ + 1); HR-MS (ESI): calcd for C28H29N3O4F
[M + H]: 490.2142; found: 490.2165.
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