Indole RSK inhibitors. Part 1: Discovery and initial SAR

Article abstract of DOI:10.1016/j.bmcl.2011.10.030

A series of inhibitors for the 90 kDa ribosomal S6 kinase (RSK) based on an 1-oxo-2,3,4,5-tetrahydro-1H-[1,4]diazepino[1,2-a]indole-8-carboxamide scaffold were identified through high throughput screening. An RSK crystal structure and exploratory SAR were

Full text of DOI:10.1016/j.bmcl.2011.10.030

Bioorganic & Medicinal Chemistry Letters 22 (2012) 733–737
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Indole RSK inhibitors. Part 1: Discovery and initial SAR
Stephen J. Boyer a, , Jennifer Burke a, Xin Guo a, Thomas M. Kirrane a, Roger J. Snow a, Yunlong Zhang a,
⇑
Chris Sarko a, Lida Soleymanzadeh a, Alan Swinamer a, John Westbrook a, Frank DiCapua a, Anil Padyana a,
Derek Cogan a, Amy Gao a, Zhaoming Xiong a, Jeffrey B. Madwed b, , Mohammed Kashem a,
Stanley Kugler a, Margaret M. O’Neill b
a Department of Medicinal Chemistry, Boehringer-Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, PO Box 368, Ridgefield, CT 06877, United States
b Department of CardioMetabolic Diseases Research, Boehringer-Ingelheim Pharmaceuticals, Inc., 900 Ridgebury Road, PO Box 368, Ridgefield, CT 06877, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of inhibitors for the 90 kDa ribosomal S6 kinase (RSK) based on an 1-oxo-2,3,4,5-tetrahydro-1H
-[1,4]diazepino[1,2-a]indole-8-carboxamide scaffold were identified through high throughput screening.
An RSK crystal structure and exploratory SAR were used to define the series pharmacophore. Compounds
with good cell potency, such as compounds 43, 44, and 55 were identified, and form the basis for subse-
quent kinase selectivity optimization.
Received 28 July 2011
Revised 5 October 2011
Accepted 7 October 2011
Available online 14 October 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
RSK
MAPKAP-K1
Kinase inhibitor
Myocardial ischemia rapidly sets in motion a series of patholog-
ical processes, most significantly intracellular acidosis that arises
from anaerobic glycolytic metabolism. The sodium–hydrogen ex-
changer (NHE) plays a critical role in regulating cellular pH post-
MI by removing protons while concomitantly internalizing Na+.1
However, what begins as a protective response later transforms
into a maladaptive process; elevated cellular Na+ decreases Ca2+ ef-
flux, ultimately leading to Ca2+ overload and cardiac hypertrophy.2
Inhibitors of NHE have been shown to reverse this sequence of
events, and have demonstrated efficacy in preclinical models of
ischemia-reperfusion injury and heart failure.3
K1]) has been shown to be one such NHE activating factor and a
potential point for therapeutic intervention.6 A member of the
ACG kinase subfamily, the RSK family consists of four human iso-
forms, RSK 1–4.7 The RSK 1–3 isoforms have a high degree of
homology, are ubiquitously expressed, and have all been shown
to stimulate NHE1 via phosphorylation in vitro8; RSK4 appears to
demonstrate different pharmacology.9 A feature atypical for most
kinases, RSK possesses two functionally distinct kinase domains:
an N-terminal domain homologous to other ACG kinases, and a
C-terminal autophosphorylation domain homologous to the cal-
cium/calmodulin-dependent protein kinases.10
Unfortunately, NHE inhibitors have also been associated with
adverse clinical events.4 Moreover, the NHE inhibitor zoniporide
was found to be neurotoxic.5 One possible explanation for the unde-
sirable effects of NHE inhibitors is that the compounds not only
block NHE during times of stress, but also completely inhibit the
protein’s housekeeping function. An alternative approach for a car-
dioprotective agent could be to reduce the activation of NHE, while
retaining the basal activity necessary for cellular pH maintenance.
The 90 kDa ribosomal S6 kinase (RSK, also known as mitogen-
activated protein kinase-activated protein kinase-1 [MAPKAP-
Few small molecule RSK inhibitors have been documented in
the literature, most notably fmk, SL0101, and BI-D1870.11 Addi-
tionally, RSK has appeared in the selectivity profiles for other ki-
nase inhibitors.12 As part of an ongoing program directed at the
NHE pathway,13 we undertook a high-throughput screen (HTS) to
identify novel scaffolds for inhibitors of the RSK N-terminal kinase
domain. Indole 1 (Fig. 1) is representative of an attractive cluster
from the hitset. Interestingly, selected members were found previ-
ously to be MAPKAP-K2 (MK2) inhibitors.14
O
NH
⇑
Corresponding author at present address: Global Regulatory Sciences,
H
Bristol-Myers Squibb,
States.
5 Research Parkway, Wallingford, CT 06492, United
N
N
N
O
1
E-mail address: stephen.boyer.b@gmail.com (S.J. Boyer).
Present address: Department of Cardiovascular Diseases, Merck
126 E. Lincoln Avenue, PO Box 2000, Rahway, NJ 07065, United States.
&
Co. Inc.,
Figure 1. An HTS hit from the indole series.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.10.030

734
S. J. Boyer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 733–737
O
O
O
NBoc
( )n
S
3
a, b, c
R
R
( )n
4
5
NHBoc
HO
d, e, f
O
O
H
N
n, o
CO2Et
N
R
N
R
1, 9-26
N
H
TsO
OTs
EtO2C
m
NH
N
NH
EtO2C
g, h, i, j
F
F
( )n
( )n
O
2
8
k
CO2Et
N
R
6 R = H
EtO2C
l
CN
7 R = 1,1-diMe
Scheme 1. Reagents and conditions: (a) NaH, DMF, 0 °C—rt, 48–72 h; (b) TFA, CH2Cl2, 1 h, rt; (c) K2CO3, EtOH, reflux, 1–4 h, 20–66% (three steps); (d) DIAD, Ph3P, THF, 16–
60 h, rt; (e) TFA, CH2Cl2, 1–2 h, rt; (f) K2CO3, Et3N, EtOH, reflux, 1–4 h, 15–66% (three steps); (g) K2CO3, DMF, 135 °C, microwave, 1 h, 38%; (h) NaN3, DMF, 95 °C, 40 h; (i) H2,
10% Pd/C, MeOH/CH2Cl2 (1:1 v/v), 3.5 h; (j) K2CO3, Et3N, EtOH, 80 °C, 16 h, 15–66% (three steps); (k) BrCH2CN, K2CO3, DMF, 60–80 °C, 4–6 h, 84–96%; (l) NaHMDS, MeI, THF,
0 °C—rt, 24 h, 61%; (m) H2, PtO2/EtOH or Raney Ni/water, 85–95%; (n) 1 M NaOH, EtOH, 80 °C, 2 h, 58–96%; (o) 3-aminopyridine, PyBOP, Et3N, DMF, 30–40% or 3-
aminopyridine, TBTU, Et3N, DMF, 40–75%.
Although we postulated the binding modes would be similar,15
early SAR for RSK and MK2 appeared divergent, and thus, the in-
dole series offered an attractive starting point.
The synthesis of indole series analogs proceeded through the in-
dole lactam intermediate 8 (Scheme 1). Beginning with indole dies-
ter 2,14a the lactam was assembled through one of four alternative
routes that allowed access to a broad range of ring substituents.
The cyclic sulfamidate16 3 (n = 1 or 2) was used to synthesize the cor-
responding intermediate 8 en route to compounds 9, 12, 13, 21, and
23–25 (Table 1) through a ring opening, deprotection and cycliza-
tion sequence. Alternatively, Mitsunobu reaction of the Boc-pro-
tected amino alcohol 4 (n = 1) with indole 2, deprotection, and
subsequent cyclization furnished intermediate 8 toward com-
pounds 10, 14, and 16–19. Mono displacement of ditosylate 5,17
installation of the lactam nitrogen, and cyclization provided the
difluorinated intermediate 8 leading to compound 15. In another ap-
proach, indole 2 was alkylated with bromoacetonitrile. The resultant
nitrile 6 could either be directly carried on toward compound 20 via
a one step hydrogenation/cyclization, or alkylated to afford the gem-
inal di-methyl intermediate 7 necessary for compound 22. Once in
hand, the substituted indole lactam 8 was readily converted to final
products following hydrolysis and coupling with the 3-aminopyri-
dine left-hand side (LHS). The syntheses of compounds 1, 11, and
26 have been described previously.14b
With the high degree of homology between the RSK family
members and the general lack of RSK isoform selectivity observed
during early optimization of several other lead series, we chose to
develop SAR primarily against RSK2.18 Additionally, we used a pro-
prietary X-ray crystal structure of RSK2 for novel analog design.
Affinity for other RSK isoforms was periodically confirmed in a
broad selectivity panel for key compounds, and binding interac-
tions were corroborated with homology models of RSK1 and RSK3.
The structure of compound 1 docked into RSK2 is shown in
Figure 2a. Members of the indole series are thought to form key
hydrogen bonds with RSK2; the three within 2.2 Å are shown as
yellow lines. These were corroborated by SAR developed during
hit evaluation. The amide carbonyl anchors the molecule to the
backbone N-H of Leu150 in the hinge region. An additional strong
hydrogen-bonding interaction appears between the lactam car-
bonyl and the catalytic Lys100 side chain nitrogen. A third, out of
plane hydrogen bond connects the lactam nitrogen with the side
chain carboxylate of Asp211. Analogs lacking any of these three
critical elements of the indole series pharmacophore were found
to be poor inhibitors of RSK2. Additionally, the LHS amide substitu-
ent binds in a lipophilic pocket bounded in part by Phe149, and en-
Table 1
Piperazinone and diazepinone SAR
O
O
H
N
H
N
NH
3
N
1
N
N
N
NH
O
O
1
2
2
Core A
Core B
a
19
Compound Core Substituent
RSK2 IC50 (nM) HLR-CREB IC50 (nM)
1
9
A
A
A
A
A
A
A
A
A
A
A
A
B
B
B
B
B
B
B
—
1-Me
2-Me
2-CH2NH2
1,2-cis Di Me
1,2-trans Di Me
2,2-Di Me
2,2-Di F
2-spiro c-Pr
2-spiro c-Bu
2-spiro (4-THP) >10000
3-Me
—
1-Me
1,1-Di Me
1,2-cis Di Me
1,2-trans Di Me
2-Me
240
41
1200
1800
240
1100
4600
1700
410
1940
890
4890
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
3300
2500
730
170
210
45
871
350
970
>10000
1630
1550
840
gages in p-stacking with Phe357.
2-CH2NH2
Bound to RSK2 in this manner, the seven-membered indole
lactam fits tightly into the ATP pocket with limited room for sub-
stitution (Fig. 2b). The majority of ring substituents led to a loss
a
Values are means of at least two experiments, standard deviation is typically
50% of the reported value.

S. J. Boyer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 733–737
735
erate potency increase. This is likely due to a different orientation of
the smaller ring within the pocket, which avoids the negative clash
with the wall, and also allows the methyl to interact constructively
with the lipophilic roof indentation. Similar to the 7-membered lac-
tam, the b-aminomethyl derivative 26 did not provide an expected
potency boost through a bridge to the bound water.
Having explored the lactam ring SAR, we then turned toward
amide LHS optimization (Table 2). Throughout, we held the more
potent seven-membered lactam constant while seeking to define
interactions within the lipophilic pocket necessary for RSK inhibi-
tion. It was quickly established that a
p-stacking interaction with
Phe357 is another critical element of the indole series pharmaco-
phore, as demonstrated by the poor activity of unsubstituted
amide 27 and cyclohexyl analog 28. Moreover, the pyridyl nitrogen
(1) does not appear to be critical for binding, as the corresponding
phenyl derivative 29 is equipotent.
Probing space around the aromatic LHS with a chlorine, the
para- (30) and meta- (31) derivatives were found to be tolerated,
while the ortho- analog 32 significantly lost potency. This effect ap-
Table 2
SAR of amide substituent—six membered rings
O
NH
H
N
N
R
O
Compound
27
R
RSK2 IC50 (nM)
9100
HLR-CREB IC50 (nM)
H
Figure 2. Docking of 1 into a crystal structure of RSK2 [30-350] (2.9 Å resolution,
Boehringer-Ingelheim unpublished results). The binding mode is based on an X-ray
structure for an analogous compound bound to MK2.15
28
29
>10000
290
1930
in potency (Table 1); however, introduction of a methyl alpha- to
the indole provided a six-fold improvement in RSK2 IC50 (9). This
30
31
180
270
can be understood by the allylic 1,3-strain between the
a-methyl
Cl
Cl
and the indole 7-position C–H, which forces the -methyl to adopt
a
an axial conformation and places it into a lipophilic indentation in
the roof of the pocket. Substitution at the adjacent b-position sub-
stantially decreased potency (10). In this region, the roof of the
pocket appears to be less accommodating toward an equatorial
substituent. Alternatively, axial orientation of the b-substituent
either displaces the nearby bound water or clashes with the roof
in the ring conformation not shown in Figure 2b. Attempts to di-
rectly address the bound water through hydrogen-bonding substit-
uents were unsuccessful, as demonstrated by compound 11.
Cl
32
33
6000
400
OMe
Interestingly, a,b-vicinal disubstitution couldcompensate for the
H2N
O
potency loss due to b-monosubstitution, with the cis-diastereomer
12 favored over the corresponding trans- (13). This diastereomeric
preference reinforces the binding hypothesis: compound 12 allows
for the preferred axial methyl orientation without bound water dis-
placement, while compound 13 can only adopt conformations with
disfavored interactions. Geminal disubstitution of the central meth-
ylene also reduced potency (14–18). Reduced steric demand at this
position has a less detrimental effect (compare 14 with 16).
Substituents next to the lactam nitrogen were also not tolerated
(compound 19), consistent with the proposed binding mode which
places this methylene in close proximity to the wall of the binding
pocket.
34
21
560
35
36
34
620
N
220
2630
N
N
N
37
38
>10000
>10000
The SAR trends in six-membered lactam analogs were in part
consistent with those observed for the seven-membered counter-
parts: methyl substituents alpha- to the indole improved potency
>10000
N
N
39
52
(compounds 20–22), and cis-a,b disubstitution was preferred over
trans- (compare 23 and 24). However, in contrast to the
N
H
seven-membered lactams, a b-methyl substituent in 25 led to mod-

736
S. J. Boyer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 733–737
Table 3
pears to be driven by a steric clash between the ortho- substituent
and the amide that forces the LHS to twist out of plane with the in-
dole. As seen in Figure 2b, the lipophilic pocket is narrow and
would be unable to accommodate more demanding functionality.
In contrast, an ortho-methoxy substituent capable of enforcing pla-
narity via an intramolecular hydrogen bond to the amide had lim-
ited impact on potency (33). A boost in potency was observed for
the ortho-amide 34, which may also provide an interaction beyond
the intramolecular hydrogen bond by reaching out to Asp154 on
the lip of the ATP pocket.
Similar conformational effects on IC50 were observed for other
heterocycles, in this case driven by electrostatic repulsion between
Leu150 of RSK and the LHS rather than sterics. Although the
4-pyridyl regioisomer 35 gains potency relative to the HTS hit 1
and the 2-pyridine regioisomer 36 is equipotent, the 2-pyrimidine
analog 37 surprisingly loses affinity for RSK. We reasoned that a
repulsive interaction between the two pyrimidine nitrogens and
the Leu150 backbone carbonyl would either lead 37 to adopt a
twisted conformation upon binding analogous to the unbound con-
formation of 32, or significantly disfavor binding. In this context,
pyridines 1 and 36 are free to adopt an orientation that points the
ring nitrogen away from the backbone, while 35 has no repulsive
interaction with RSK.
SAR of amide substituent—five membered rings
O
H
N
NH
N
R
O
Compound
40
R
RSK2 IC50 (nM)
2200
HLR-CREB IC50 (nM)
N
N
N
S
41
42
26
290
NH
N
2200
43
44
45
19
11
43
200
790
O
N
N
N
HN
N
>10000
We also targeted the space in the lipophilic pocket off of the
phenyl meta-position. Chain extension to better position the pyri-
dyl deeper into the pocket led to a complete loss in potency (38). It
N
46
540
appears that maintaining aromatic LHS
p-stacking while expand-
N
N
ing into the lipophilic pocket is critical. Thus, we carried out a li-
brary directed at validating the hypothesis. The imidazole
derivative 39 is a representative example that provided a five-fold
boost in IC50 relative to the parent phenyl; however, it came at the
expense of a modest reduction in ligand efficiency.
47
48
93
N
H
Five membered heterocycle LHS offered a potential path to more
ligand efficient compounds (Table 3). But, thiazole 40, a bioisostere
of pyridine 36, lost 10-fold RSK potency. We hypothesized that elec-
trostatic repulsion impacts thiazole 40 binding, similar to that of 37,
and then chose to introduce a heterocycle that could constructively
interact with the Leu150 backbone carbonyl. Gratifyingly, 41 of-
fered a 10-fold boost in potency, and an additional hydrogen bond-
ing opportunity from the imidazole N–H. The methylated analog 42
prevents the hydrogen bond formation with corresponding loss of
potency. Complicating the interpretation, it also introduces a sub-
stituent adjacent to the amide that leads to a conformational
change and analogous to ortho-chlorophenyl 32. Interestingly, a
bidentate interaction is not necessary to increase affinity for RSK,
as demonstrated by isoxazole 43 and pyrazole 44.
In general, the rank order for potency was the same for both the
primary soluble kinase and for cellular mechanistic phosphorylation
assays, although physicochemical properties were found to impact
cell potency in some cases. While pyrazoles 44 and 45 are equipo-
tent in the kinase assay, the methyl of pyrazole 44 appears to allow
better cell penetration. Specifically, permeability as measured in the
PAMPA assay was found to be 10-fold lower for 45 than 44.
The interplay of electronic and steric factors on potency is well
illustrated by the 3-aminopyrazole regioisomers 46–48. Electro-
static repulsion orients the pyrazole LHS of 46 such that the trajec-
tory for the methyl substituent differs from that of 43 and 44.
Ultimately, this leads to a clash with the lipophilic pocket near
the entrance and loss in potency. Removal of the methyl (47) re-
solves the conflict, while the other regioisomer (48) further con-
firms that adjacent LHS substitution is disfavored.
Fused ring systems provided further insight into the LHS SAR
(Table 4). We observed a potency boost through increased lipophil-
icity with quinoline 49, and found that the lipophilic pocket could
accommodate relatively large LHS such as the pyrazolopyridine 50.
Compounds 51 and 52, direct analogs of the monocycles 36 and 40,
>1000
N
N
Table 4
SAR of amide substituent—fused ring systems
O
H
N
NH
N
R
O
Compound
49
R
RSK2 IC50 (nM)
34
HLR-CREB IC50 (nM)
340
N
N
50
53
840
N
N
N
51
52
53
68
>10000
367
1130
N
S
N
>10000
>10000
O
N
54
55
4
9
N
H
N
200
N

S. J. Boyer et al. / Bioorg. Med. Chem. Lett. 22 (2012) 733–737
737
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deleterious effect on potency. It is noteworthy that although ring
fusion significantly improved the potency of quinoline 51 it was
not sufficient to overcome the negative conformational effects for
benzothiazole 52.
With the LHS SAR apparently well established, we were sur-
prised to find that the benzoxazole 53 demonstrated modest RSK
inhibition. This counterintuitive result can be explained through
the imino–amino tautomeric equilibrium of the LHS. In the case
of 53, the aminobenzoxazole spends a portion of its time in the
imino tautomer, effectively replacing a ligand-receptor repulsive
interaction with a hydrogen bonding opportunity. The hypothesis
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behave differently than their unfused counterparts. While 54
showed the expected increase in potency compared to 41 that
can be attributed to ring fusion, the methyl analog 55 maintained
excellent potency, in stark contrast to 42. In this case, it appears
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same hydrogen bonding interaction as the unalkylated analog.
N-methylation also provides 55 with better cell penetration mea-
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We were also interested in the kinome selectivity of the indole
series, particularly given the chronic disease indication and the po-
tential for off-target kinase inhibition that could lead to safety con-
cerns. During the early optimization phase described in this report,
we found most compounds to be highly selective against a limited
panel of kinases including Aurora A, CHEK1, GSK3b, MEK5, and
STK33. Having defined critical elements of the indole series SAR
and also identified compounds with good cell potency, such as
isoxazole 43, pyrazole 44, and benzimidazole 55, we turned toward
optimization of selectivity against a broader panel of kinases.
These results will be reported in a subsequent communication.20
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Acknowledgments
17. Binsch, G.; Eliel, E. L.; Mager, S. J. Org. Chem. 1973, 38, 4079.
The authors would like to thank I. Mugge for discussions, J. Wo-
lak and S. Marques for help providing HTS data, and D. Brennan for
RSK crystals
18. Inhibition of soluble RSK2 phosphorylation activity was determined with a
luminescent assay format similar to that described in: Zegzouti, H.;
Zdanovskaia, M.; Hsiao, K.; Goueli, S. A. Assay Drug Dev. Technol. 2009, 7, 560.
19. Inhibition of the phosphorylation of the transcription factor CREB (cAMP
Response Element Binding) by RSK2 in cells was determined according to the
method described in: Boyer, S.; Gao, A.; Guo, X.; Kirrane, T.; Sarko, C.; Snow, R.;
Soleymanzadeh, F.; Zhang, Y. WO2011071716.
20. Kirrane, T. K.; Boyer, S. J.; Burke, J.; Guo, X.; Snow, R. J.; Soleymanzadeh, L.;
Swinamer, A.; Zhang, Y.; Madwed, J. B.; Kashem, M.; Kugler, S.; O’Neill, M. M.
Biorg. Med. Chem. Lett. 2011. doi:10.1016/j.bmcl.2011.10.029.
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