Discovery of the first known small-molecule inhibitors of heme-regulated eukaryotic initiation factor 2α (HRI) kinase

Article abstract of DOI:10.1016/j.bmcl.2009.10.033

A series of indeno[1,2-c]pyrazoles were discovered to be the first known inhibitors of heme-regulated eukaryotic initiation factor 2α (HRI) kinase. The synthesis, structure-activity relationship profile, and in-vitro pharmacological characterization of th

Full text of DOI:10.1016/j.bmcl.2009.10.033

Bioorganic & Medicinal Chemistry Letters 19 (2009) 6548–6551
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of the first known small-molecule inhibitors of heme-regulated
eukaryotic initiation factor 2a (HRI) kinase
*
Mark D. Rosen , Craig R. Woods, Steven D. Goldberg, Michael D. Hack, A. Dawn Bounds, Young Yang,
Pamela C. Wagaman, Victor K. Phuong, Angela P. Ameriks, Terrance D. Barrett, Kimon C. Kanelakis,
Jui Chang, Nigel P. Shankley, Michael H. Rabinowitz
Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 3210 Merryfield Row, San Diego, CA 92121, 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 indeno[1,2-c]pyrazoles were discovered to be the first known inhibitors of heme-regulated
Received 23 September 2009
Revised 6 October 2009
Accepted 8 October 2009
Available online 13 October 2009
eukaryotic initiation factor 2
in-vitro pharmacological characterization of this inaugural series of HRI kinase inhibitors are detailed.
a (HRI) kinase. The synthesis, structure–activity relationship profile, and
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Anemia
Kinase inhibitors
Hemoglobin
HRI kinase
Blood
Oxygen homeostasis is critical to life processes, and multicellu-
lar organisms have developed complex mechanisms for the uptake,
distribution, and maintenance of oxygen levels. An example of
such mechanisms is transport of oxygen from the lungs to various
tissues by hemoglobin, which is the primary protein in red blood
cells.1 The role of hemoglobin is to transport oxygen from the lungs
to tissues, a function that is facilitated by reversible binding of oxy-
gen to the iron-containing heme moiety of hemoglobin.2
condition for which the only currently available treatment options
are direct blood transfusion, erythropoietin (EPO), and dietary iron
and vitamin supplementation.14
With the above model of therapeutic intervention in mind, we
initiated an exploratory program to discover and characterize
small-molecule inhibitors of HRI kinase. An HRI kinase assay was
developed and validated using recombinant full-length human
HRI kinase, and subsequent screening led to the discovery of pyr-
azolindane 1 (Fig. 1).15,16
Compound 1 was found to display potent, ATP-competitive HRI
kinase inhibition (pIC50 = 7.5). Preliminary in-vitro ADME predic-
tors and in-vivo pharmacokinetic parameters showed that this ini-
tial screening hit had good stability toward human, dog, and rat
liver microsomes (t1/2 > 60 min), with human and rat plasma pro-
tein binding levels of 87% and 90%, respectively (Table 1). Unfortu-
nately, 1 showed low bioavailability in the rat (ca. 18%) and rapid
clearance, with an IV half-life of 1.6 h.
Synthesis of hemoglobin is a complex process thatinvolves trans-
lation and modifications of globin proteins, heme synthesis, and
incorporation of an iron atom into heme.3,4 Eukaryotic initiation
factor 2
ulators of translation in eukaryotic cells.5–13 Heme-regulated eIF-2
(HRI) kinase is expressed primarily in reticulocytes and in erythroid
cells in bone marrow. HRI kinase phosphorylates eIF-2 and initiates
a reduction in globin synthesis, thereby regulating the availability of
and b globin chains relative to the amount of heme available for
a (eIF-2a) kinases are important and well characterized reg-
a
a
a
production of hemoglobin. It logically follows that a beneficial level
of small-molecule inhibition of HRI kinase should be expected to
promote an increased rate of globin production and hemoglobin lev-
els, thereby increasing the oxygen carrying capacity of red blood
cells.9 The discovery of such an HRI kinase inhibitor could hold great
promise in the treatment of anemia, a widespread and debilitating
H
N
N
MeO
Cl
N
H
HO
O
Cl
OH
1
* Corresponding author.
E-mail address: mrosen1@its.jnj.com (M.D. Rosen).
Figure 1. Aminopyrazolindane HRI inhibitor from HTS screening.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.10.033

M. D. Rosen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6548–6551
6549
Table 1
Preliminary in-vitro and rat pharmacokinetic profiling of 1
Pharmacokinetic parameters (rat)b
a
pIC50
Plasma protein binding (%)
Liver microsomal stability (t1/2, min)
Human
87
Rat
90
Human
>60
Rat
t1/2 (h)
%F
18
CL (L/kg/h)
1.9 0.2
7.5
>60
1.6 0.1
6
a
Negative logarithm of the concentration required to achieve 50% enzyme inhibition; value is 0.3 log units.
Values calculated from three animals.
b
Given the preliminary profiling data, compound 1 was deemed
activity relative to 7, suggesting that such conformational restric-
tion is detrimental in the tested compounds. With respect to com-
pounds 10–13, replacement of the R2 methoxy group with alkyl,
amino, halo, and thioether groups all resulted in a substantial loss
in activity (e.g., 10–13).
Using the same synthesis route outlined in Scheme 1, next we
examined structural changes to the benzylamine moiety (Table
3). Substitution with a chloride or a methyl group at the R1 and
R4 in compounds 19–22 provided high levels of enzyme inhibitory
activity, with the dimethyl analog 21 emerging as the most active
analog with a pIC50 value of 8.1 (IC50 = 7.9 nM). An important fea-
ture of this series is demonstrated by analogs 17 and 18, which
possess chloride atoms at the para (R3) position. Both these analogs
incur a substantial loss in activity relative to their direct compara-
tors 14 and 15. Methoxy analog 23 also suffers a significant loss in
activity.
to be a reasonable starting point from which to undertake a medic-
inal chemistry program. The general synthesis route for an initial
set of analogs is outlined in Scheme 1.
Known or commercially-available indanones were treated with
LiHMDS and dimethyl trithiocarbonate to provide thioesters,
which were then allowed to react with a variety of amines to pro-
vide intermediate thioamides. Alkylation of sulfur with methyl io-
dide, followed by exposure to excess hydrazine in refluxing
ethanol, afforded the final aminopyrazolindane products. We ini-
tially evaluated the effects of certain substituents on the indane
phenyl ring for specific compounds, as shown in Table 2.
With respect to compound 2, the removal of all substitution
from the indane phenyl ring results in a dramatic loss in activity,
and reintroduction of a single methoxy group in the R1 position
of 3 leads to the complete abolition of enzyme inhibition. This lost
activity is largely restored by installation of a methoxy group at the
R2 position of 4, and optimal activity was achieved with methoxy
groups at both the R2 and R3 positions of 7. Interestingly, the meth-
ylenedioxy analog 8 suffered a 10-fold loss of enzyme inhibitory
Having discovered favorable substitution elements for both the
phenyl portion of the tricyclic ring and the benzylamine side chain
in the tested compounds, we next examined modifications to core
ring system that included replacement, homologation, and elimi-
nation of the indane methylene group. The synthesis of these com-
pounds is described below.
O
O
The synthesis of ketone analog 27 began from known indanedi-
one 24, which reacts with carbon disulfide and methyl iodide in
the presence of potassium fluoride to provide dithioketene acetal
25.17 Exposure of 25 to 2,6-dichlorobenzylamine affords vinylo-
gous amide 26, which is converted to 27 by treatment with hydra-
zine and subsequent cyclization in refluxing iPrOH.
Ether, thioether, and sulfone analogs 31–33 were prepared from
known benzofuran-3-one 28 and benzothiophen-3-one 29
(Scheme 3).18 Compounds 28 and 29 were elaborated as described
in Scheme 1 to provide benzofuropyrazole 31 and benzothienylpy-
razole 32, respectively. Alternatively, 29 could oxidized using
mCPBA to afford sulfoxide 30, which was then processed in a man-
ner analogous to that described in Scheme 2 in order to afford ben-
zothienylpyrazole-S,S-dioxide 33.
S
a
b
R
R
R
SMe
H
N
O
N
c, d
S
R
N
H
n
HN
n
R
R
Scheme 1. General synthesis route for aminopyrazolindanes. Reagents and condi-
tions: (a) LiHMDS, (MeS)2CS, THF, 0 °C to rt; (b) amine, EtOH, reflux; (c) MeI,
iPr2NEt, THF; (d) hydrazine, EtOH, reflux.
Table 2
Indane phenyl ring substituent effects
R1
H
Table 3
N
R2
R3
N
Benzylamine substituent effects
Me
H
N
N
N
R1
MeO
H
R4
R2
R3
Cl
N
MeO
H
R4
a
Compd
R1
R2
R3
R4
pIC50
2
3
4
5
6
7
8
9
10
11
12
13
H
OMe
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
OMe
H
H
H
H
H
OMe
H
H
OMe
H
H
5.8
<5
a
Compd
R1
R2
R3
R4
pIC50
14
15
16
17
18
19
20
21
22
23
H
Cl
H
H
H
Cl
H
H
H
H
H
H
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
6.2
6.7
6.0
5.6
5.9
7.4
7.7
8.1
7.2
5.5
7.3
6.3
6.1
7.7
6.6
6.9
6.5
6.6
6.1
6.0
H
OMe
OMe
Cl
Cl
Cl
Me
Me
H
–OCH2O–
Cl
Me
Me
H
OMe
Et
NMe2
Br
H
H
H
H
H
H
H
H
H
H
OMe
H
SMe
a
a
Negative logarithm of the concentration required to achieve 50% enzyme
inhibition. All values are 0.3 log units.
Negative logarithm of the concentration required to achieve 50% enzyme
inhibition. All values are 0.3 log units.

6550
M. D. Rosen et al. / Bioorg. Med. Chem. Lett. 19 (2009) 6548–6551
Table 4
O
O
MeO
Central ring structure–activity relationship
MeO
MeO
SMe
SMe
a
b
H
N
MeO
N
MeO
MeO
R
O
O
O
24
25
N
H
Cl
X
H
N
N
MeO
MeO
MeO
MeO
Cl
SMe
HN
Cl
c
a
Compd
X
R
pIC50
N
H
Cl
Cl
27
31
32
33
37
38
39
C@O
O
S
SO2
–H,H–
(CH2)2
(CH2)3
Cl
Cl
Me
Cl
Cl
Cl
8.2
7.2
6.3
7.1
5.6
<5
O
O
26
27
Scheme 2. Synthesis of 33. Reagents and conditions: (a) CS2, MeI, CsF, DMSO, 46%;
(b) 2,6-dichlorobenzylamine, iPrOH, reflux, 95%; (c) hydrazine, iPrOH, reflux, 53%.
Cl
<5
a
Negative logarithm of the concentration required to achieve 50% enzyme
O
inhibition. All values are 0.3 log units.
MeO
Scheme 1
X
MeO
H
N
28 (X = O)
29 (X = S)
N
MeO
MeO
R
N
H
a
X
Cl
O
MeO
MeO
31 (X = O, R = Cl)
32 (X = S, R = Me)
33 (X = SO2, R = Cl)
Scheme 2
S
O
O
30
Scheme 3. Synthesis of ether 31, sulfide 32, and sulfone 33. Reagents and
conditions: (a) mCPBA, CH2Cl2, 74%.
Figure 2. Overlay of energy-minimized structures of 19 (magenta), 38 (cyan), and
39 (green).
Compounds 37, 38, and 39 encompass excision of the central
ring, one carbon homologation, and two carbon homologation,
respectively (Scheme 4). These compounds were prepared from
commercially-available acetophenone 34, tetralone 35, or benzo-
cycloheptanone 36 in a manner analogous to that described in
Scheme 1.
Figure 2, where the overlaid, energy-minimized structures of 19,
38, and 39 demonstrate the repositioning of the methoxyphenyl
portion of these particular molecules.20 Unfortunately, despite pro-
tracted efforts we were unable to obtain a X-ray crystal structure of
full-length HRI kinase in either apo or holo form to shed further
light on this aspect of our observations. Additional crystallographic
efforts using truncated forms of the protein will be reported in due
course.
In summary, we have discovered a series of aminopyrazoloind-
anes that are, to the best of our knowledge, the first known small-
molecule inhibitors of HRI kinase. Furthermore, the early in-vitro
data for this series was demonstrated to be robust, tractable, and
amenable to optimization. Modifications to the central ring of
the indane core were examined in detail, ultimately leading to
compounds such as 27 that display inhibitory activity against the
full-length protein at low nanomolar concentrations. Additional
studies of this inaugural series of HRI kinase inhibitors, including
selectivity profiling as well as other second-generation studies, will
be reported in the near future.
As shown in Table 4, replacement of the methylene unit of 19
with a ketone group in 27 results in an almost 10-fold increase
in enzyme inhibition. Similar replacement of the methylene group
with oxygen gives ether 31, which is essentially isoactive with 19,
while substitution with divalent sulfur incurs a greater than 10-
fold loss in activity (i.e., 20 to 32). This loss of activity is largely re-
gained in sulfoxide analog 33. In order to examine the purely geo-
metrical aspects of these data, we turn to carbon homologs 38 and
39. Insertion of either one or two additional methylene units re-
sulted in complete loss of activity, likely without incurring signifi-
cant electronic perturbation. The total removal of this carbon
bridge in seco analog 37 restores a portion of this lost activity.
**
B3LYP/6-31G optimizations on the cores of 38 and 39 have pre-
dicted tautomer-averaged dihedral angles of 13.7° and 20.4°,
respectively, between the phenyl and pyrazole rings, while for 19
the two rings are coplanar to within 0.3°.19 This lack of planarity
in 38 and 39 and the concomitant positional shift of the phenyl
ring relative to the pyrazole ring is illustrated graphically in
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O
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N
N
MeO
MeO
MeO
MeO
Cl
Scheme 1
N
H
Cl
n
n
34 (n = 0, seco)
35 (n = 2)
37 (n = 0, seco)
38 (n = 2)
39 (n = 3)
36 (n = 3)
Scheme 4. Seco and homologated analogs 37–39.

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