Discovery of spirocyclic sulfonamides as potent Akt inhibitors with exquisite selectivity against PKA

Article abstract of DOI:10.1016/j.bmcl.2011.02.098

We describe the design and synthesis of novel bicyclic spiro sulfonamides as potent Akt inhibitors. Through structure-based rational design, we have successfully improved PKA selectivity of previously disclosed spirochromanes. Representative compounds sho

Full text of DOI:10.1016/j.bmcl.2011.02.098

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2335–2340
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of spirocyclic sulfonamides as potent Akt inhibitors
with exquisite selectivity against PKA
Rui Xu a, , Anna Banka a, James F. Blake a, Ian S. Mitchell a, Eli M. Wallace a, Josef R. Bencsik a,
⇑
Nicholas C. Kallan a, Keith L. Spencer a, Susan L. Gloor a, Matthew Martinson a,
Tyler Risom a, Stefan D. Gross a, Tony H. Morales a, Wen-I Wu a, Guy P. A. Vigers a,
Barbara J. Brandhuber a, Nicholas J. Skelton b
a Array BioPharma Inc., 3200 Walnut Street, Boulder, CO 80301, USA
b Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080-4990, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe the design and synthesis of novel bicyclic spiro sulfonamides as potent Akt inhibitors.
Through structure-based rational design, we have successfully improved PKA selectivity of previously
disclosed spirochromanes. Representative compounds showed favorable Akt potency while exhibiting
up to 1000-fold selectivity against PKA.
Received 21 December 2010
Revised 17 February 2011
Accepted 22 February 2011
Available online 26 February 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Akt
PKA
Selectivity
Spiro
Kinase
Akt, also known as protein kinase B (PKB), is a serine/threonine
kinase belonging to the AGC family of kinases. Three isoforms of
Akt are known to exist, namely Akt1, Akt2 and Akt3, which exhibit
an overall homology of 80%.1–3 The Akt isoforms share a common
domain organization that consists of a pleckstrin homology do-
main at the N-terminus, followed by a kinase catalytic domain,
and a short regulatory region at the C-terminus. Akt plays a central
role in the PI3K-Akt-mTOR pathway as a key effector of receptor
tyrosine kinase signaling, regulating diverse cellular functions
including cell growth, proliferation, apoptosis and metabolism.
The upstream receptor tyrosine kinases, PI3K and Akt itself, are
commonly mis-regulated or amplified in a variety of human
tumors. For example, the tumor suppressor PTEN, a negative regu-
lator of PI3K, is deleted or mutated in many cancers.4–6 In addition,
activating mutations in PIKC3A, one of the class IA PI3K isoforms,
are found in a high proportion of breast, endometrial, colon and
other cancers.7 These abnormalities result in activation of the
PI3K–Akt pathway which leads to cell proliferation and survival,
as well as confers resistance to various types of cancer therapy,
such as chemotherapy, radiation, and EGFR inhibition. Accordingly,
Akt inhibition has attracted considerable attention as an oncology
target.8–11
Due to the high degree of homology between the ATP binding
sites of protein kinases, the development of selective ATP compet-
itive Akt inhibitors presents a substantial challenge. In a previous
report,12 we reported the discovery of a novel series of Akt inhibi-
tors that contain the spirochromane scaffold resulting from modi-
fications of a HTS hit 1 (Fig. 1). Of particular interest were
compounds with a phenol hinge binder that exhibit profound
selectivity (>250-fold) against the closely homologous AGC family
SO2
SO2
N
OEt
N
OEt
HO
N
HO
N
HO
HN
N
O
O
O
1
2
Akt1 IC50: 9 nM
PKA IC50: 30 nM
pPRAS40 IC50: 44 nM
µ
Akt1 IC50: 3.4
PKA IC50 > 50
M
M
µ
µ
pPRAS40 IC50 >10
M
⇑
Corresponding author.
E-mail address: rxu@arraybiopharma.com (R. Xu).
Figure 1. Spirochromanone Akt inhibitors.12
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.02.098

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R. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2335–2340
O
HO
N
SO2
N
N
c
SO2
N
a
b
O2S
1
H
HO
O2S
N
O2S
OEt
N
OEt
HO
N
H
2
HN
N
HO
N
( )m
HN
N
21
18
19
20
O
N
N
( )n
3
2
SO2
N
SO2
N
Potent, but not selective against PKA
Polycyclic ring, flat structure
d
O
O
Figure 2. Design of spirobicyclic analogs 3.
22b
22a
e
e
O
S
O
S
O
( )m
HN
O
( )m
N
Boc
Boc
b
N
Ar Cl
a
N
Ar
N
N
HO
N
H2N
N
( )n
HO
N
( )n
H2N
N
8
7
N
N
m = 0, 1;
n = 0, 1
N
N
23b
23a
O
O
SO2
O
S
N
Scheme 3. Reagents and conditions: (a) Et3N, DCM, 2,6-dimethylbenzene-1-
sulfonyl chloride, rt, 2 h, 67%; (b) sulfur trioxide pyridine complex, Et3N, DMSO,
DCM, 0 °C, 1 h; (c) methyltriphenylphosphonium bromide, NaH, DMSO, rt, 16 h, 36%
for two steps; (d) m-CPBA, DCM, reflux, 48 h, 64% for 22a, 19% for 22b; (e) 17, TEA,
EtOH, 55 °C.
OEt
N
10
c
OEt
HO
N
( )m
N
( )m
N
Ar
NH
2HCl
Ar
( )n
3-6
( )n
9
indazole compound 2 was highly potent in a mechanistic cellular
assay, with an IC50 of 44 nM, but selectivity over PKA dropped to
ca. 3-fold.
N
HN
N
HN
Ar =
N
N
N
In this Letter, we report our continued efforts to optimize this
series with emphasis on improving PKA selectivity while retaining
favorable Akt activity. As shown in Figure 2, our SAR studies initi-
ated by breaking the polycyclic indazole spirochromanone scaffold
in compound 2 in order to determine whether the chromane ring
could be replaced by an azabicyclic spiro system. Initial proof of
concept efforts focused on preparation of compounds with the gen-
eral structure 3. Based on molecular modeling, compound 3 was
postulated to maintain relative arrangement of the key pharmaco-
phore elements.12 The pyrrolopyrimidine ring in 3 should bind
similarly to the hinge as the indozole ring in compound 2, and
the spiro ring-system should maintain the desired conformation
to project the 2,6-dimethyl phenyl ring to the P-loop lipophilic
pocket observed in crystal structures of the original chromane
series.12
N
N
N
N
Scheme 1. Reagents and conditions: (a) DIEA, NMP, 70 °C, 16 h, 63–95%; (b) HCl/
dioxane, 61–97%; (c) TEA, EtOH, 55 °C, 16 h, 51–87%.
kinase PKA, which we used as a surrogate of general kinase selec-
tivity. Replacement of the phenol ring with a lactam or an indazole
resulted in compounds with excellent Akt enzyme and cell po-
tency, but with reduced selectivity over PKA. For example, the
Bn
HN
N
Cl
Cl
N
b
Bn
13
N
N
N
N
N
Preparation of compounds with different hinge binding cores
and various azabicyclic spiro linkers are depicted in Scheme 1. SNAr
reaction between readily available mono Boc protected spirobicyclic
a
Cl
14
12
Ph
Ph
H2N
N
d
N
N
c
Bn
O
H
N
N
Bn
N
N
N
Boc
b
N
HO
N
Boc
N
N
H
H2N
N
H2N
N
16
24
15
N
NH
a
N
O
N
N
S
O
25
17
N
OEt
HO
N
H2N
N
H2N
N
10
R
HO
N
N
HO
N
H2N
N
N
NH
e
NH2
H2N
N
H
N
c
N
N
11
N
N
17
N
N
3HCl
27
Scheme 2. Reagents and conditions: (a) DIEA, NMP, 80 °C, 1 h, 98%; (b) benzophe-
none imine, Pd(OAc)2, rac-BINAP, NaOBut, 95 °C, 7 h, 80%; (c) hydroxylamine
hydrochloride, NaOAc, MeOH, rt, 16 h, 91%; (d) ammonium formate, 10% Pd/C,
MeOH, reflux, 16 h, 78%; (e) TEA, EtOH, 55 °C, 16 h, 53%.
26
Scheme 4. Reagents and conditions: (a) TEA, EtOH, 55 °C 16 h, 91%; (b) HCl,
dioxane, 88%; (c) acylation or reductive amination.

R. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2335–2340
2337
Table 1
Azabicyclic spiro linker SARa
SO2
N
OEt
HO
N
HN
N
( )m
N
N
( )n
Compd
m
n
Akt1 IC50
(lM)
PKA IC50
(lM)
PKA/Akt (fold)
pPRAS40 IC50 (lM)
3a
3b
3c
1
0
0
1
1
0
0.041
0.002
0.017
0.21
0.067
0.71
5
28
42
1.2b
0.18
3.1
a
Values are means of at least three experiments unless otherwise stated.
Values are means of two experiments.
b
diamine 7 and aryl chloride gave intermediate 8. Removal of the Boc
group followed by reaction with epoxide 1012 afforded compounds
3–6.
(PKA/Akt1 = 5–42). Despite this, we remained encouraged by these
early results, since the pyrrolopyrimidine core had relatively low
PKA selectivity.15,17
Synthesis of compound 11 with the amino pyrimidine core
was carried out as outlined in Scheme 2. SNAr reaction between
4,6-dichloro-5-cyclopropylpyrimidine (12) and (S)-7-benzyl-2,
7-diazaspiro[4.5]decane (13)13 afforded intermediate 14. Conver-
sion of 14 to amino pyrimidine intermediate 16 was effected
through Buchwald coupling with benzophenone imine followed
by deprotection with hydroxylamine. Removal of the benzyl
protecting group under transfer hydrogenation conditions gave
the amino intermediate 17 in good yield. Subsequent reaction with
the epoxide intermediate 10 provided the desired compound 11.
Racemic 11 was prepared following a similar sequence except that
racemic 7-Boc-2,7-diazaspiro[4.5]decane was used in the first step.
We further extended our SAR to encompass conformationally
constrained pyrrolidine analogues as described in Scheme 3.
Commercially available (R)-prolinol (18) was reacted with
2,6-dimethylbenzenesulfonyl chloride to give sulfonamide 19. Oxi-
dation to the aldehyde 20 was carried out with sulfur trioxide
pyridine complex in DMSO at 0 °C. Aldehyde 20 was converted to
olefin 21 by Wittig reaction with methyltriphenylphosphonium
bromide in good yield. Epoxidation of 21 with mCPBA produced
oxiranes 22a (erythro) and 22b (threo) in a combined 83% total
yield with an erythro/threo ratio of 3:1. The mixture of 22a and
22b was readily separated by silica gel column chromatography.
Subsequent reaction of both intermediates with 17 gave com-
pounds 23a and 23b.
We then turned our attention to evaluating a series of heterocy-
clic ring hinge binders (Table 2). The pyrazolopyrimidine 4 was
found to be moderately selective against PKA (27-fold). However,
PKA selectivity was greatly improved in compounds 5, 6 and ( )-
11 with the quinazoline and pyrimidine cores. The vastly different
levels of PKA selectivity for these cores could be explained by our
observations from other series of inhibitors that PKA selectivity can
be modulated by modifying the hinge interactions and increasing
the size of substituents near Met227, the gatekeeper residue in
Akt1.12,15,16 Improved hinge interactions tend to greatly benefit
Table 2
Core SARa
O
S
O
N
OEt
HO
N
R
N
R
Akt1 IC50
M)
PKA IC50
M)
PKA/Akt
(fold)
pPRAS40
IC50 (lM)
(
l
(
l
Scheme 4 summarizes the synthesis of compounds of general
structure 27. Reaction of amine 17 and epoxide 2414 gave alcohol
25 in good yield. Subsequent deprotection of the Boc group with
HCl gave intermediate 26, which was then converted to 27 by acyl-
ation or reductive amination.
N
HN
N
4
5
6
0.021
0.038
0.012
0.56
4.7
27
120
280
0.31
N
N
N
As described previously,15,16 the resulting compounds were
evaluated for their ability to inhibit Akt1 activity in an enzyme as-
say (IMAP format) and phosphorylation of PRAS40 in LNCaP cells
(PRAS40 is a direct substrate of Akt). Selectivity was assessed by
their ability to inhibit PKA activity in a biochemical assay (IMAP
format). We initially prepared compounds 3a–c with various aza-
bicyclic spiro linkers while keeping the pyrrolopyrimidine core
and the sulfonamide moiety constant. The enzyme and cellular
activities are summarized in Table 1. To our delight, all three com-
pounds had good potency (<100 nM) in the Akt1 enzyme assay.
Compound 3b with the 2,7-diazaspiro[4.5]decane linker showed
the best potency, with an IC50 of 2 nM in the Akt1 enzyme assay,
2.4
N
N
3.4
0.74
H2N
N
( )-11
0.012
2.2
180
0.47
N
and an IC50 of 0.18
lM in the pPRAS40 assay. However, all three
a
Values are means of at least three experiments.
compounds only exhibited moderate selectivity against PKA

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R. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2335–2340
Table 3
Constrained analogsa
H2N
N
R
N
N
N
R
Akt1 IC50
M)
PKA IC50
PKA/Akt
(fold)
pPRAS40 IC50
(
l
(l
M)
(lM)
O
S
O
O
O
23a
23b
23c
0.009
3.9
430
>40
250
0.69
N
HO
HO
HO
O
Figure 3. X-ray structure of 5 bound to Akt1 (PDB code: 3QKM).18
S
0.25
>10
13b
N
PKA inhibition while having modest effects on Akt potency.12 For
example, both 3b and 4 are capable of forming a pair of hydrogen
bonds with the backbone structure of Ala230 and Glu228 (Akt1),
while the quinazoline and pyrimidine cores in 5 and 6 can only
act as hydrogen bond acceptors. Clearly, loss of the H-bond donor
interaction from the core affects potency at PKA more than Akt1. In
addition, as we noted in previous studies,15,16 there are several key
differences between the active site residues of Akt1 and PKA that
could be exploited to influence the selectivity of various inhibitors.
In particular, the substitutions of Akt1-Ala230 to PKA-Val, Akt1-
Thr211 to PKA-Val, Akt1-Met281 to PKA-Leu gives rise to a nar-
rower cavity in PKA. The narrower cavity of PKA, relative to Akt1
was found to be less forgiving of larger substituents, leading to in-
creased selectivity. This seems to be borne out in the pyrimidine
series, where the larger cyclopropyl substitution of compound 6
leads to 280-fold selectivity relative to PKA. Interestingly, although
the amino pyrimidine compound ( )-11 can also interact with the
hinge via a pair of hydrogen bonds, it retained 180-fold selectivity
against PKA, again illustrating the effects of increasing size as a
means of improving the selectivity profile. It is worthwhile to note
that selectivity against p70S6K, another closely related protein ki-
nase in the AGC family, generally parallels the PKA selectivity. For
example, compounds 3a, 3b, 3c, 4 and 5 were about 3-, 25-, 43-,
4- and 230-fold selective against p70S6K, respectively.
O
S
0.019
4.7
2.4
N
a
Values are means of at least three experiments unless otherwise stated.
Values are means of two experiments.
b
propyl linker and ethoxy ethyl side chain of 3 via a pyrrolidine
ring.19 Among the three compounds examined, 23a had the best
potency, comparable to 11. The corresponding (R)-alcohol 23b
was found to be 28-fold less potent than 23a. A modest difference
in potency was observed between the two stereoisomers of the pyr-
rolidine ring, with the (R)-isomer slightly preferred (23a vs 23c).
Further SAR of the sulfonamide region attempted to replace the
sulfonamide group with an amide, carbamate, urea or benzyl
amine. All modifications led to substantial losses in potency (data
not shown). Interestingly, removal of the ethoxy ethyl side chain in
(R)-11 retained Akt1 activity (Table 4, compound 27a, Akt1
IC50 = 4 nM and pPRAS40 IC50 = 0.90 lM), and maintained excellent
The X-ray structure of 5 in complex with Akt1 has been deter-
mined (Fig. 3) and revealed that the (R)-enantiomer of the 2,7-
diazaspiro[4.5]decane linker binds preferentially.18 The crystal
structure also confirmed that the quinazoline nitrogen acts as a
hydrogen bond acceptor in binding to the backbone NH of
Ala230 (N–N distance 2.82 Å). The piperidine nitrogen and the hy-
droxyl group form bifurcated interaction with Asp292, while the
2,6-dimethyl phenyl ring occupies a lipophilic pocket underneath
the glycine-rich loop.
selectivity against PKA (850-fold). To better understand the hydro-
phobic interactions of the benzene ring with the lipophilic pocket
under the nucleotide binding loop, we prepared a series of sulfon-
amide analogs (27b–i). The 2-methyl compound 27b had compara-
ble potency to 27a, while the 2-F compound 27c was about 20-fold
less potent. Increasing the size of the ortho-substituent recovered
Akt1 activity (27d and 27e). Fluorine substitution at the para posi-
tion (27f) was tolerated, while substitution with a larger methyl
group resulted in 40-fold drop in activity (27g). Potency also de-
creased sharply by replacing the 2-methyl benzene group with a
4-methyl-3-pyridyl moiety (27h), which is consistent with the
highly lipophilic nature of the binding pocket. We also evaluated
replacement of the phenyl group with five-membered heterocy-
cles. The isoxazole analog 27i was found to be the most potent
compound in this group, with an Akt1 enzyme IC50 of 3 nM, cell po-
Encouraged by the selectivity and potency of ( )-11, we pre-
pared the active (R)-enantiomer. Consistent with the SAR in the spi-
rochromane series,12 (R)-11 was about 2-fold more potent than the
racemate (Akt1 IC50 = 6 nM and pPRAS40 IC50 = 0.38 lM). This
compound also exhibited good selectivity against PKA, with a
PKA/Akt1 ratio of 180-fold. Although compound (R)-11 exhibited
acceptable potency, we were concerned about the impact of high
molecular weight (ca. 587), high C log P (4.2) and large number of
rotatable bonds (14 total) on ADME. In order to improve the
drug-like properties of compound (R)-11, conformationally re-
stricted analogs 23a–c were prepared (Table 3). These were de-
signed to restrain the rotation of sp3–sp3 bonds by connecting the
tency of 0.49 lM, and greater than 600-fold selectivity against PKA.
In summary, we have described the syntheses and biological
activities of several novel and potent bicyclic spiro sulfonamide
Akt inhibitors. We have successfully improved the selectivity
against PKA of previously published spirochromanes.12 Future
studies will probe other aspects of these compounds to evaluate

R. Xu et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2335–2340
2339
Table 4
Sulfonamide SARa
R
HO
N
H
H2N
N
N
N
N
R
Akt1 IC50
0.004
(lM)
PKA IC50
3.4
(lM)
PKA/Akt (fold)
850
pPRAS40 IC50
0.90
(lM)
27a
S
O2
27b
27c
0.009
0.081
5.0
560
1.2
14
S
O2
F
>10
>120
S
O2
Cl
Br
27d
27e
0.009
0.017
8.3
9.4
920
550
1.9
2.0
S
O2
S
O2
27f
27g
27h
0.008
0.16
1.1
7.8
>10
970
>63
>9
2.2
F
S
O2
18b
S
O2
>10
ND
S
O2
N
N
O
27i
0.003
1.9
630
0.49
S
O2
ND = not determined.
a
Values are means of at least three experiments unless otherwise stated.
Values are means of two experiments.
b
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We would like to thank James Graham and Dr. Francis Sullivan
for helpful discussions and careful proofreading in the preparation
of this manuscript. The authors also thank Dr. Lingyang Zhu for
NMR support.
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4615.
(3) Boc deprotection with 4N HCl in dioxane; (4) reductive amination of the
resulting amine with benzaldehyde, NaBH(OAc)3, THF, HOAc, rt, 16 h, 91% yield
for two steps; (5) amide hydrolysis with 9N HCl, dioxane, reflux, 36 h, 73%.
14. Kitchin, J.; Bethell, R. C.; Cammack, N.; Dolan, S.; Evans, D. N.; Holman, S.;
Holmes, D. S.; McMeekin, P.; Mo, C. L.; Nieland, N.; Orr, D. C.; Saunders, J.;
Shenoy, B. E. V.; Starkey, I. D.; Storert, R. J. Med. Chem. 1994, 37, 3707.
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L.; Mitchell, I. S.; Woessner, R.; Gloor, S. L.; Risom, T.; Gross, S. D.; Martinson,
M.; Morales, T.; Vigers, G. P. A.; Brandhuber, B. J. Bioorg. Med. Chem. Lett. 2010,
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R.; Gloor, S. L.; Martinson, M.; Risom, T.; Woessner, R.; Dizon, F.; Wu, W.;
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17. For pyrrolo[2,3-d]pyrimidine Akt inhibitors with various PKA selectivity
profiles see: (a) Caldwell, J. J.; Davies, T. G.; Donald, A.; McHardy, T.;
Rowlands, M. G.; Aherne, G. W.; Hunter, L. K.; Taylor, K.; Ruddle, R.;
18. Atomic co-ordinates of the structure 5 bound to Akt1 were deposited with the
RCSB Protein Data Bank (PDB) under the accession code 3QKM.
19. Such a linkage was compatible with the conformation of compound 5 observed
in the co-crystal structure with Akt1 (Fig. 3).