Discovery of GSK2656157: An optimized PERK inhibitor selected for preclinical development

Article abstract of DOI:10.1021/ml400228e

We recently reported the discovery of GSK2606414 (1), a selective first in class inhibitor of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), which inhibited PERK activation in cells and demonstrated tumor growth inhibition in a human tumor xenograft in mice. In continuation of our drug discovery program, we applied a strategy to decrease inhibitor lipophilicity as a means to improve physical properties and pharmacokinetics. This report describes our medicinal chemistry optimization culminating in the discovery of the PERK inhibitor GSK2656157 (6), which was selected for advancement to preclinical development.

Full text of DOI:10.1021/ml400228e

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Letter
Discovery of GSK2656157: An Optimized PERK
Inhibitor Selected for Preclinical Development
Jeffrey M Axten, Stuart P Romeril, Arthur Shu, Jeffrey M Ralph, Jesus R. Medina, Yanhong Feng, William
Hoi Hong Li, Seth W Grant, Dirk A. Heerding, Elisabeth Minthorn, Thomas Mencken, Nathan Gaul,
Aaron Goetz, Thomas B. Stanley, Annie M Hassell, Robert T Gampe, Charity Atkins, and Rakesh Kumat
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/ml400228e • Publication Date (Web): 12 Aug 2013
Downloaded from http://pubs.acs.org on August 23, 2013
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Discovery of GSK2656157: An Optimized PERK Inhibitor Selected for
Preclinical Development
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Jeffrey M. Axten,*,† Stuart P. Romeril,† Arthur Shu,† Jeffrey Ralph,† Jesús R. Medina,† Yanhong Feng,†
William Hoi Hong Li,† Seth W. Grant,† Dirk A. Heerding,† Elisabeth Minthorn,† Thomas Mencken,†
Nathan Gaul, ‡ Aaron Goetz,§ Thomas Stanley,§ Annie M. Hassell,║ Robert T. Gampe,║ Charity Atkins†
and Rakesh Kumar†
GlaxoSmithKline Research and Development,
†
Oncology Research, Protein Dynamics DPU, Collegeville, PA 19426, United States
‡
Screening and Compound Profiling, Collegeville, PA 19426, United States
§
Screening and Compound Profiling, Research Triangle Park, NC 27713, United States
║
Biomolecular Structure, Computational and Structural Chemistry, Research Triangle Park, NC 27709, United States
KEYWORDS: PERK, UPR, kinase, lead optimization, structure-activity relationship, fluorine interaction
ABSTRACT: We recently reported the discovery of GSK2606414 (1), a selective first in class inhibitor of protein kinase R (PKR)ꢀ
like endoplasmic reticulum kinase (PERK) which inhibited PERK activation in cells and demonstrated tumor growth inhibition in a
human tumor xenograft in mice. In continuation of our drug discovery program, we applied a strategy to decrease inhibitor lipoꢀ
philicity as a means to improve physical properties and pharmacokinetics. This report describes our medicinal chemistry optimizaꢀ
tion culminating in the discovery of the PERK inhibitor GSK2656157 (6), which was selected for advancement to preclinical deꢀ
velopment.
Increased endoplasmic reticulum (ER) stress resulting from nutriꢀ
ent deprivation and unfolded protein accumulation is associated
with debilitating conditions such as neurodegeneration, heart disꢀ
ease, diabetes, and cancer.1 To maintain ER homeostasis, the
unfolded folded protein response (UPR) coordinates an adaptive
cellular signaling cascade to alleviate the impact of the stress and
enhance cell survival.2 Protein kinase R (PKR)ꢀlike ER kinase
(PERK) is one of three primary effectors of the UPR.3ꢀ4 Once
activated, PERK phosphorylates eukaryotic initiation factor 2α
(eIF2α) at serine 51 which inhibits the ribosome translation initiaꢀ
tion complex and reduces overall protein synthesis.5ꢀ7 The reducꢀ
tion in translation reduces the ER burden, providing time for the
cell to process or degrade the accumulated unfolded proteins.
Although global protein synthesis is decreased, there is also speꢀ
cific increased transcription of certain messages regulated by
downstream PERK effectors, such as ATF4, which activate genes
that enhance UPR function. For example, in situations of extreme
hypoxia or nutrient starvation, UPR activation is associated with
increased vascularization. A number of studies using genetic
manipulation or siRNA knock down provide evidence that PERK
function contributes to transcriptional activation and upꢀregulation
of proꢀangiogenic genes.8ꢀ14 Therefore, inhibiting PERK in canꢀ
cer cells may limit their ability to thrive under hypoxia or nutrient
deprived conditions and lead to apoptosis or tumor growth inhibiꢀ
tion. Recently we reported the discovery and characterization of
GSK2606414 (1), a highly selective, first in class inhibitor of
PERK that demonstrated tumor growth inhibition in a human
tumor xenograft in mice.15 In this communication we disclose the
medicinal chemistry optimization leading to the discovery of the
preclinical development candidate GSK2656157 (6), which was
recently reported with extensive biological characterization.16
Although compound 1 served as an excellent, orally available tool
to elucidate the function of PERK in cells and animals, we sought
improvements to the physicochemical properties, metabolism and
pharmacokinetics. Data from in vitro metabolic studies showed
that 1 broadly inhibited cytochrome P450s in human liver microꢀ
somes, with subꢀmicromolar inhibition of CYP2C8 (IC50 = 0.89
ꢁM, Table 2). We suspected that the broad inhibitory activity
against cytochrome P450s was related to the overall lipophilicity
of the molecules and electronic nature of the arylacetamide based
on our previous work relating this part of the molecule to in vivo
rat clearance.15 Thus, our optimization strategy to increase polarꢀ
ity focused on modifying the aryl acetamide. We specifically
targeted heteroaryl acetamides to minimize molecular weight gain
and to evenly distribute polarity throughout the molecules, with
an emphasis on the design of analogs with cLogP values <3. In
early SAR investigations we prepared the pyridyl acetamide isoꢀ
mers 2ꢀ4 (Table 1), and observed a significant loss of PERK inꢀ
hibitory activity compared to the parent phenyl analog. The 4ꢀ
pyridyl analog 4 was the least potent (IC50 = 63.8 nM), consistent
with the sensitivity of the aryl acetamide to paraꢀsubstitution. We
selected the 2ꢀpyridyl isomer 2 (IC50 = 19.1 nM) for further invesꢀ
tigation, hypothesizing that substitution at the 6ꢀposition may
improve potency by filling the lipophilic back pocket observed in
Xꢀray structures. Additionally, this substitution could also serve
to impede interactions of the pyridine moiety with P450s. The
corresponding 6ꢀmethylpyridyl analog 5 (IC50 = 2.5) and trifluoꢀ
romethylpyridyl analog 7 (IC50 = 0.5 nM) offered significant (7 to
30ꢀfold) increases in potency. Xꢀray structural data confirmed the
binding mode and the interaction of the pyridyl methyl group with
the small pocket adjacent to the pyridine ring (data not shown).
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Table 1. PERK inhibition and pharmacokinetic data for compound 1 and heteroaryl acetamide analogs.
3
4
5
6
7
8
9
10
11
12
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Compound
R
X
H
cLogPa
4.1
PERK IC50 (nM)b
0.4 ± 0.32
pꢀPERK IC50 (ꢁM)c
<0.03
Rat Clb (mL minꢀ1 kgꢀ1)d
1
21.5
19.1e
23.4e
63.8e
NDf
ND
ND
ND
ND
ND
2
3
H
H
H
1.7
1.7
1.7
4
5
H
F
2.2
2.4
2.5 ± 1.0
0.1ꢀ0.3
10.5
9.5
6 (GSK2656157)
0.8 ± 0.42
0.03ꢀ0.1
7
8
H
F
2.8
2.9
0.5 ± 0.22
0.5 ± 0.15
0.03ꢀ0.1
<0.03
12.8
8.9
57.5e
16.6e
ND
ND
ND
ND
9
H
H
2.0
2.0
10
11
12
H
F
2.3
2.5
1.9 ± 1.0
0.1ꢀ0.3
2.8
1.9
2.7 ± 0.56
0.03ꢀ0.1
aCalculated using BioByte cLogP (the calculated logarithm of the 1‑octanol–water partition coefficient of the nonꢀionized molecule, see
www.biobyte.com. bInhibitory activity as measured by cytoplasmic PERK domain phosphorylation of EIF2α. Average values are reported
with standard deviation. cIC50 estimated from a Western blot 6ꢀpoint dose response, except for compounds 1 and 9a (3ꢀpoint dose reꢀ
sponse). dAverage in vivo blood clearance determined after i.v infusion in SD rats (n = 2). eData reported as the average of a single experꢀ
iment. fNot determined.
During our studies, we also found that 5ꢀmembered heteroaryl
acetamides were potent PERK inhibitors when appropriately subꢀ
stituted. In a series of pyrazoles, the 3ꢀmethylꢀpyrazolꢀ1ꢀylꢀ
acetamide 9 (IC50 = 57.5 nM) had decreased activity, yet the 5ꢀ
methylꢀpyrazolꢀ1ꢀylꢀacetamide isomer 10 (IC50 = 16.6 nM) preꢀ
served a large amount of inhibitory potency. Unlike the parent
phenyl acetamides, monoꢀsubstitution adjacent to the acetamide
linkage in the pyrazole acetamides was favored over the correꢀ
sponding 3ꢀsubtituted analog. An apparent synergistic effect was
observed when both 3ꢀ and 5ꢀpositions were substituted with meꢀ
thyl groups to give compound 11 (IC50 = 1.9 nM). The increased
potency observed with this substitution pattern is speculated to
arise from a better overall fit within the volume of the lipophilic
acetamide binding pocket.
Compounds 5, 7, and 11 were assayed in A549 cells for their
ability to inhibit thapsigargin induced PERK autophosphorylation
(Table 1). Consistent with the biochemical activity of these three
analogs, the most potent compound 7 (PERK IC50 = 0.5 nM)
demonstrated the most potent inhibition of PERK autophosphoryꢀ
lation in cells (IC50 = 0.03ꢀ0.1 ꢁM). Compounds 5 and 11 which
were 3 to 4 times weaker than 7 in the PERK biochemical assay,
showed similar weaker activity in the pPERK cellular assay (IC50
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= 0.1ꢀ0.3 ꢁM). Overall the data indicated that we could in fact
greatly decrease the lipophilicity of the PERK inhibitors (Clog P
= 2.3ꢀ2.8 compared to 4.1 for compound 1) while maintaining
biochemical and cellular potency.
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Figure 1. Crystal structure of compound 6 bound to the PERK kinase domain. (a) Closeꢀup view of 6 (green sticks) in the PERK active
site (brown cartoon and sticks).The 6ꢀmethylpyridyl group occupies the large inner lipophilic pocket while the fluoroindoline fills a narrow
channel between M887 and D954 of the inward facing DFG motif. The aminoꢀpyrimidine binds the hinge βꢀstrand with hydrogen bonds to
the back bone amide atoms of Q888 and C890. (b) Closeꢀup view of 6 bound to the PERK active site with surface rendering. Close spatial
arrangement between the indoline fluorine atom and the V606 methyl groups may account for some of the improved biochemical activity.
Atomic interactions are indicated with orange solid lines and distances reported in Angstroms. The crystal structure of compound 6 bound
to the PERK kinase domain is available with the PDB access code XXXX (will insert in accepted proof) at www.rcsb.org.
To evaluate the impact of structure and physiochemical property
changes on the pharmacokinetics of advanced inhibitors, we monꢀ
itored the in vivo rat blood clearance for the potent and cell active
compounds 5, 7, and 11. When compared to 1, each analog
showed improved pharmacokinetic profiles and low blood clearꢀ
ance in rats (Table 1), suggestive of a decrease in metabolic clearꢀ
ance. The rat blood clearance of the pyridyl acetamides 5 and 7
(Clb = 10.5 and 12.8 mL/min/kg respectively) was about 2ꢀfold
lower than that of 1 (Clb = 21.5 ml/min/kg), whereas the pyrazole
acetamide 11 exhibited very low rat blood clearance (Clb = 2.8
mL/min/kg). Overall, the data supported our strategy given that
analogs with lower Clog P values led to improved blood clearꢀ
ance, however, there was a trade off in PERK biochemical potenꢀ
cy resulting in a corresponding decrease in cell activity as measꢀ
ured by PERK autophosphorylation (vide supra).
The subtle, general trend of increased biochemical potency was
not predicted, but subsequently rationalized from crystal strucꢀ
tures of PERK with inhibitors such as 6, which binds in the kinase
ATP pocket in an identical fashion to that previously described
for 1 (Figure 1a).15 Upon close inspection, the crystal structure
revealed that the indoline fluorine atom of 6 is directed towards
the methyl groups of valine 606 in the kinase Pꢀloop (Figure 1b).
Fluorine can assume diverse roles in protein binding events, most
of which are described as polar interactions.18 However, evidence
also suggests that it is reasonable for fluorine to induce subtle
energetic gains with nonꢀpolar CꢀH bonds.19 We hypothesize that
the fluorineꢀVal606 interaction is favorable due to the lipophilic
nature of the fluorine atom, which is reflected a general trend of
increased biochemical activity we observed for 4ꢀfluoroindoline
analogs.
Fluorination, a well documented and proven strategy to improve
compound permeability and pharmacokinetics in drug discovery,
was implemented in further optimization of our PERK inhibiꢀ
tors.17 To minimize potential impact on binding affinity, we seꢀ
lected the indoline 4ꢀposition for fluorine substitution since it was
not likely to have an effect on the biaryl torsion or the critical
amide conformation observed in the Xꢀray structures. During the
course of our research we observed a trend in which the majority
of 4ꢀfluoroindolines offered small improvements to the PERK
activity relative to the indolines. We synthesized 4ꢀfluoroindoline
derivatives of the advanced analogs and found that 6ꢀ
methylpyridyl acetamide 6 had a more than 2ꢀfold improvement
in PERK biochemical activity (IC50 = 0.8 nM vs 2.5 nM for 5),
whereas the 6ꢀtrifluoromethylpyridyl acetamide 8 (IC50 = 0.5 nM
vs 0.5 nM for 7) and the 3,5ꢀdimethylpyrazolyl acetamide 12
(IC50 = 2.7 nM vs 1.9 nM for 11) maintained similar activities to
their parent compounds. A more pronounced effect of the fluoꢀ
rine substitution was observed when characterizing the intracelluꢀ
lar inhibition of PERK. The IC50 for PERK autophosphorylation
in A549 cells was uniformly improved 3 to 10ꢀfold for comꢀ
pounds 6, 8, and 12 relative to their parent 4ꢀunsubstituted inꢀ
dolines 5, 7, and 11 (Table 1).
In addition to the increased functional potency in cells, there was
also a noticeable modest improvement to in vivo rat blood clearꢀ
ance for each 4ꢀfluoroindoline compound. Concordantly, we charꢀ
acterized cytochrome P450 inhibition in human liver microsome
preparations to gauge potential for drugꢀdrug interactions and
metabolism associated with these derivatives relative to the tool
inhibitor 1 (Table 2). Unlike 1 which broadly inhibited P450s to
varying degrees with most potent inhibition observed for CYP2C8
(IC50 = 0.89 ꢁM), the 4ꢀfluoroindoline containing heteroaryl acetꢀ
amides 6, 8, and 12 generally displayed weaker P450 inhibitory
potency. The only exception was 8, which was a slightly more
potent inhibitor of the CYP3A4 enzyme than 1. Nevertheless, 8
had an improved overall profile, and compounds 6 and 12 were
uniformly less active versus the P450 panel with IC50s for all
isoforms > 19 ꢁM. The levels of P450 activity of the compounds
profiled in Table 2 align with their corresponding lipophilicity
indicated by their respective Clog P (Table 1). Taken together,
the improvements in cell potency, P450 profile, and rat blood
clearance guided our selection of the 4ꢀfluoroindolines 6, 8, and
12 for extensive selectivity screening.
In our initial assessment of selectivity, compounds 6, 8, and 12
were assayed against HRI, PKR, and GCN2 ꢀ closely related
EIF2AK family members that also phosphorylate eIF2α (Table 2).
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All three compounds showed selectivity for PERK over other
1
2
3
4
5
6
7
8
members of the EIF2AK family, however the magnitude of the
selectivity varied depending on the identity of the heteroarylaꢀ
cetamide. PERK inhibitor 6 is the least potent inhibitor of other
EIF2AK family members, and showed 500ꢀfold selectivity over
the most sensitive kinase HRI (IC50 = 460 nM). Compound 8 is
greater than 700ꢀfold selective over PKR and GCN2, but does
have significant activity against HRI (IC50 = 37 nM). The pyraꢀ
zole acetamide 12 has the poorest selectivity of the
Table 2. Human liver microsomal P450 inhibition data reported as IC50 (µM) for selected PERK inhibitorsa
9
10
11
12
13
14
15
16
17
18
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CYP1A2
phenacetin
CYP2C9
diclofenac
CYP2C19
mephenytoin
CYP2D6
bufuralol
CYP3A4
midazolam
CYP3A4
nifedipine
CYP2C8
paclitaxel
Compound
cLogPb
1
6
4.1
2.4
2.9
2.5
21
4.5
24.7
19.2
>25
7.0
>25
>25
>25
23c
>25
23.7
>25
13
10
0.89
>25
5.83
>25
>25
>25
>25
>25
9.09
>25
19.7
7.4
8
12
20.5
aCYP450s with molecular probe listed under specific isoform. For assay details, see supporting information.bCalculated using BioByte
cLogP (the calculated logarithm of the 1‑octanol–water partition coefficient of the nonꢀionized molecule, see
www.biobyte.com.cdextromethorphan was used as the probe in this experiment.
Table 3. Kinase inhibiton summary for compounds 6, 8, and 12.
EIF2AK2
(PKR)
IC50
EIF2AK4
(GCN2)
IC50
EIF2AK1
HRI IC50
(nM)a
X/300 Kinases
Inhibited >80% @
10 ꢁMc
HRI/PERK
IC50 ratio
PKR/PERK
IC50 ratio
GCN2/PERK
IC50 ratio
Compound
(nM) a
(nM) b
460
37
511
74
905
359
99
1,006
718
37
3,388
776
3,764
1,552
ND
17
39
6
8
61
23
ND
ND
12
aData from a single 10ꢀpoint dose response performed by Reaction Biology Corp. (http://www.reactionbiology.com). bValues reported
are the average of at least two experiments (see Experimental Section for details). cSee supporting information for complete kinase profile.
three analogs since it potently inhibits both HRI and PKR (IC50s =
37 and 99 nM, respectively) with < 50ꢀfold selectivity for PERK.
Compounds 6 and 8, the two most selective PERK inhibitors
within the EIF2AK family were profiled against a panel of 300
kinases. In line with the EIF2AK data, 6 continued to demonꢀ
strate superior kinase selectivity, inhibiting only 17/300 kinases
>80% at 10 ꢁM, whereas compound 8 inhibited 39/300 kinases
>80% at the same concentration.
treatment with 6 for several weeks we observed a pancreas specifꢀ
ic phenotype in mice characteristic of genetic PERK ablation.20ꢀ22
Taken together, the data strongly supported that the observed
pharmacologic effects upon treatment with 6 were associated with
PERK inhibition. The collective biological effects, exquisite
kinase selectivity, and pharmacokinetic profile supported the seꢀ
lection of 6 (GSK2656157) for progression to preclinical develꢀ
opment.
Pharmacokinetic studies were performed in mouse, rat and dog
for compound 6 (Table 4). Data collected from i.v./p.o. crossꢀ
over studies showed that compound 6 was well absorbed providꢀ
ing good exposure, high oral availability, and low to moderate
blood clearance in mouse, rat and dog. Estimated volume of disꢀ
tribution at steadyꢀstate was low in rodents and moderate to high
in the dog. Halfꢀlives were less than 2 hours in the mouse and
rat (T1/2 = 1.25 and 1.4 h, respectively), which influenced the use
of twice a day dosing in efficacy studies in mice.
Table 4. Pharmacokinetic parameters of compound 6.a
Mousec
Ratd
Doge
i.v. dose
(mg/kg)
2.0
2.2
2.9
3921.0
3128.5
3270
AUC(0ꢀinf)
ng*h/mL
We recently reported the results of extensive biological evaluation
of 6 in cell culture and in vivo.16 Chemical stress induced PERK
activation was inhibited by 6 in multiple cell lines, with correꢀ
sponding decreases in eIF2α phosphorylation and downstream
transcriptional activation. In efficacy studies, oral treatment with
6 resulted in doseꢀdependent inhibition of multiple human tumor
xenografts growth in mice. The tumor growth inhibition was
mechanistically associated with an antiꢀangiogenic affect, which
is in agreement with observations reported using genetic means to
downꢀregulate PERK in mouse tumor studies.8ꢀ10 In addition, after
(3226.8ꢀ
4615.2)
(2577.1ꢀ
3594.8)
(2817ꢀ4085)
15.5
10.5
9.5
CLb
(13.4ꢀ
18.2)
(mL/min/kg)
(8.2ꢀ11.8)
(8.0ꢀ10.9)
0.72
0.6
2.8
Vdss (L/kg)
(0.50ꢀ0.86)
(0.6)
(2.8ꢀ2.9)
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1.25
1.4
3.1
T (h)
½
1
2
3
4
5
6
7
8
9
10
(1.13ꢀ1.36)
(1.3ꢀ1.5)
(2.7ꢀ3.6)
oral dose
(mg/kg)
13.4
4.3
5.2
13378.6
8015.7
6210.0
AUC (0ꢀinf)
ng*h/mL
(13110.6ꢀ
13645.5)
(7174.5ꢀ
8856.8)
(5274.9ꢀ
7144.2)
Oral F(%)
52b
~100
~100
aData is reported as a mean, with ranges provided in parentheꢀ
ses. bBioavailability (F%) was estimated using mean AUC (0ꢀt)
values due to the nonꢀcrossover study design. cMouse i.v. (bolus,
n=3) 1% DMSO and 20% Captisol in saline, pH = 4 ; p.o. (susꢀ
pension, n = 2): 2% DMSO and 40% PEG 400 in water, pH = 4.0.
dRat i.v. (60 minute infusion, n = 2): 1%DMSO and 20% Captiꢀ
sol in saline, pH = 4; oral (solution, n = 2): 1% DMSO and
20% PEG 400 in water, pH=4. eDog i.v. (60 minute infusion, n =
3): 1% DMSO and 20% Captisol in saline, pH = 6.5; p.o. (soluꢀ
tion, n = 3) 1%DMSO and 40% PEG 400 in water, pH = 4.9.
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6. Marciniak, S. J.; GarciaꢀBonilla, L.; Hu, J.; Harding, H. P.; Ron, D.
Activationꢀdependent substrate recruitment by the eukaryotic translaꢀ
tion initiation factor 2 kinase PERK. J. Cell Biol. 2006, 172, 201ꢀ
209.
7. Ron, D.; Walter, P. Signal integration in the endoplasmic reticulum
unfolded protein response. Nat. Rev. Mol. Cell Bio. 2007, 8, 519ꢀ529.
8. Blais, J.D; Addison, C.L.; Edge, R.; Falls, T.; Zhao, H.; Wary, K.;
Koumenis, C.; Harding, H.P.; Ron, D.; Holcik, M.; Bell, J.C. Perkꢀ
Dependent Translation Regulation Promotes Tumor cell Adaptation
and Angiogenesis in response to Hypoxic Stress. Mol. Cell. Biol.
2006, 26, 9517ꢀ9532.
9. Bi, M.; Naczki, C.; Koritzinsky, M.; Fels, D.; Blais, J.; Hu, N.;
Harding, H.; Novoa, I.; Varia, M.; Raleigh, J.; Scheuner, D.; Kaufꢀ
man, R.J.; Bell, J.; Ron, D.; Wouters, B.G.; Koumenis, C. ER stressꢀ
regulated translation increases tolerance to extreme hypoxia and proꢀ
motes tumor growth. EMBO J. 2005, 24, 3470ꢀ3481.
10. Blais, J.D.; Addison, C.L.; Edge, R.; Falls, T.; Zhao, H.; Wary,
K.; Koumenis, C.; Harding, H.P.; Ron, D.; Holcik, M.; Bell, J.C.
Perkꢀdependent translational regulation promotes tumor cell adaptaꢀ
tion and angiogenesis in response to hypoxic stress. Mol. Cell Biol.
2006, 26, 9517ꢀ9532.
11. Pereira, E.R.; Liao, N.; Neale, G.A.; Hendershot, L.M. Transcripꢀ
tional and PostꢀTranscriptional Regulation of Proangiogenic Factors
by the Unfolded Protein Response. PloS One 2010, 5, e12521.
12. Ghosh, R.; Lipson, K.L.; Sargent, K.E.; Mercurio, A.M.; Hunt,
J.S.; Ron, D.; Urano, F. Transcriptional Regulation of VEGFꢀA in the
Unfolded Protein Response Pathway. PLoS One 2010, 5, e9575.
13. Pereira, E.R.; Liao, N.; Neale, G.A.; Hendershot, L.M. Transcripꢀ
tional and PostꢀTranscriptional Regulation of Proangiogenic Factors
by the Unfolded Protein Response. PloS One 2010, 5, e12521.
14. Wang, Y.; Alam, G.N.; Ning, Y.; Visioli, F.; Dong, Z.; Nör, J.E.;
Polverini, P.J. The unfolded protein response induces the angiogenic
switch in human tumor cells through the PERK/ATF4 pathway. Can-
cer Res. 2012, 72, 5396ꢀ5406.
15. Axten, J. M.; Medina, J. R.; Feng, Y.; Shu, A.; Romeril, S.P.;
Grant, S.W.; Li, W.H.H.; Heerding, D.A.; Minthorn, E.; Mencken, T.;
Atkins, C.; Liu, Q.; Rabindran, S.; Kumar, R.; Hong, X.; Goetz, A.;
Stanley, T.; Taylor, J. D.; Sigethy, S.D.; Tomberlin, G.H.; Hassell,
A.M.; Kahler, K.M.; Shewchuk, L.M.; Gampe, R.T. Discovery of 7ꢀ
methylꢀ5ꢀ(1ꢀ{[3ꢀ(trifluoromethyl)phenyl]acetyl}ꢀ2,3ꢀdihydroꢀ1Hꢀ
indolꢀ5ꢀyl)ꢀ7Hꢀpyrrolo[2,3ꢀd]pyrimidinꢀ4ꢀamine (GSK2606414), a
potent and selective firstꢀinꢀclass inhibitor of protein kinase R (PKR)ꢀ
like endoplasmic reticulum kinase (PERK) J. Med. Chem. 2012,
55(16), 7193ꢀ7207.
In summary, our PERK inhibitor lead optimization was guided by
a strategy to decrease analog lipophilicity while maintaining the
potency and exquisite kinase selectivity of tool inhibitor 1. We
focused on heteroaryl acetamide analogs to minimize molecular
weight gain and designed a series of inhibitors with low to very
low in vivo rat blood clearance. Fluorination of the indoline 4ꢀ
postion was important for the recovery of potent biochemical and
cell potency, and the optimized 4ꢀfluorindoline analogs 6, 8, and
12 had favorable pharmacokinetics and lower levels of P450 inhiꢀ
bition in human liver microsomes. Expanded profiling estabꢀ
lished the superior kinase selectivity of 6 which was selected as a
preclinical development candidate.
ASSOCIATED CONTENT
Supporting Information Available: General Synthetic scheme
and experimental procedures for the synthesis of compounds 2ꢀ12,
DMPK and biological assay descriptions, crystallographic methꢀ
ods, and kinase selectivity profile information. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
* (J.M.A.) Phone: 610ꢀ270ꢀ6368. Eꢀmail: Jefꢀ
frey.M.Axten@gsk.com
REFERENCES
1. Kim, I.; Xu, W.; Reed, J.C. Cell death and endoplasmic reticulum
stress: disease relevance and therapeutic opportunities. Nat. Rev. Drug
Disc. 2008, 7, 1013ꢀ1030.
2. Walter, P.; Ron, D. The Unfolded Protein Response: From Stress
Pathway to Homeostatic Regulation. Science 2011, 34, 1081ꢀ1086.
3. Shi, Y.; Vattem, K.M.; Sood, R.; An, J.; Liang, J.; Stramm, L.;
Wek, R.C. Identification and characterization of pancreatic eukaryotic
initiation factor 2 alphaꢀsubunit kinase, PEK, involved in translational
control. Mol. Cell Biol. 1998, 18, 7499ꢀ7509.
4. Harding, H.P.; Zhang, Y.; Ron, D. Protein translation and folding
are coupled by an endoplasmicꢀreticulumꢀresident kinase. Nature
1999, 397, 271ꢀ274.
5. Sood, R.; Porter, A.C.; Ma, K.; Quilliam, L.A.; Wek, R.C. Pancreꢀ
atic eukaryotic initiation factorꢀ2alpha kinase (PEK) homologues in
humans, Drosophila melanogaster and Caenorhabditis elegans that
mediate translational control in response to endoplasmic reticulum
stress. Biochem J. 2000, 346, 81ꢀ93 (Pt2).
16. Atkins, C.; Liu, Q.; Minthorn, E.; Zhang, S.; Figueroa, D.J.; Moss,
K.; Stanley, T.B.; Sanders, B.; Goetz, A.; Gaul, N.; Choudhry, A.E.;
Alsaid, H.; Jucker, B.M.; Axten, J.M.; Kumar, R. Characterization of
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ACS Medicinal Chemistry Letters
Page 6 of 6
a novel PERK kinase inhibitor with antiꢀtumor and antiꢀangiogenic
activity. Cancer Res. 2013, 73, 1993ꢀ2002.
1
17. Shah, P.; Westwell, A.D. The role of fluorine in medicinal chemꢀ
istry. J. Enzyme Inhib. Med. Chem. 2007, 22, 527ꢀ540.
18. Müller, K.; Faeh, C.; Diederich, F. Fluorine in Pharmaceuticals:
Looking Beyond Intuition. Science 2007, 317, 1881ꢀ1886.
19. Zhou, P.; Zou, J.; Tian, F.; Shang, Z. Fluorine Bonding – How
Does It Work In ProteinꢀLigand Interactions? J. Chem. Inf. Model.
2009, 49, 2344ꢀ2355.
20. Harding, H.P.; Zeng, H.; Zhang, Y.; Jungries, R.; Chung, P.; Plesꢀ
ken, H.; Sabatini, D.D.; Ron, D. Diabetes Mellitus and Exocrine Panꢀ
creatic Dysfunction in Perkꢀ/ꢀ Mice Reveals a Role for Translational
Control in Secretory Cell Survival. Mol. Cell 2001, 7, 1153–1163.
21. Zhang, P.; McGrath, B.; Li, S.; Frank, A.; Zambito, F.; Reinert, J.;
Gannon, M.; Ma, K.; McNaughton, K.; Cavener, D.R. Mol. Cell. Bio.
2002, 22, 3864ꢀ3874.
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3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
22. Gao, Y.; Sartori, D.J.; Li, C.; Yu, Q.; Kushner, J.A.; Simon, M.C.;
Diehl, J.A. PERK Is Required in the Adult Pancreas and Is Essential
for Maintenance of Glucose Homeostasis. Mol. Cell. Bio. 2012, 32,
5129ꢀ5139.
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