Discovery of chiral dihydropyridopyrimidinones as potent, selective and orally bioavailable inhibitors of AKT

Article abstract of DOI:10.1016/j.bmcl.2018.03.092

During the course of our research efforts to develop potent and selective AKT inhibitors, we discovered enatiomerically pure substituted dihydropyridopyrimidinones (DHP) as potent inhibitors of protein kinase B/AKT with excellent selectivity against ROCK₂. A key challenge in this program was the poor physicochemical properties of the initial lead compound 5. Integration of structure-based drug design and physical properties-based design resulted in replacement of a highly hydrophobic poly fluorinated aryl ring by a simple trifluoromethyl that led to identification of compound 6 with much improved physicochemical properties. Subsequent SAR studies led to the synthesis of new pyran analog 7 with improved cell potency. Further optimization of pharmacokintetics properties by increasing permeability with appropriate fluorinated alkyl led to compound 8 as a potent, selective AKT inhibitors that blocks the phosphorylation of GSK3β in vivo and had robust, dose and concentration dependent efficacy in the U87MG tumor xenograft model.

Full text of DOI:10.1016/j.bmcl.2018.03.092

Accepted Manuscript
Discovery of Chiral Dihydropyridopyrimidinones as Potent, Selective and Or-
ally Bioavailable Inhibitors of AKT
Saravanan Parthasarathy, Kenneth Henry, Huaxing Pei, Josh Clayton, Mark
Rempala, Deidre Johns, Oscar De Frutos, Pablo Garcia, Carlos Mateos, Sehila
Pleite, Yong Wang, Stephanie Stout, Bradley Condon, Sheela Ashok, Zhohai
Lu, William Ehlhardt, Tom Raub, Mei Lai, Sandaruwan Geeganage, Timothy
P. Burkholder
PII:
S0960-894X(18)30297-X
https://doi.org/10.1016/j.bmcl.2018.03.092
BMCL 25746
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
9 February 2018
30 March 2018
31 March 2018
Please cite this article as: Parthasarathy, S., Henry, K., Pei, H., Clayton, J., Rempala, M., Johns, D., De Frutos, O.,
Garcia, P., Mateos, C., Pleite, S., Wang, Y., Stout, S., Condon, B., Ashok, S., Lu, Z., Ehlhardt, W., Raub, T., Lai,
M., Geeganage, S., Burkholder, T.P., Discovery of Chiral Dihydropyridopyrimidinones as Potent, Selective and
Orally Bioavailable Inhibitors of AKT, Bioorganic & Medicinal Chemistry Letters (2018), doi: https://doi.org/
10.1016/j.bmcl.2018.03.092
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Bioorganic & Medicinal Chemistry Letters
Discovery of Chiral Dihydropyridopyrimidinones as Potent, Selective and
Orally Bioavailable Inhibitors of AKT
Saravanan Parthasarathya,*, Kenneth Henrya, Huaxing Peia, Josh Claytona, Mark Rempalaa, Deidre Johnsa,
Oscar De Frutosb, Pablo Garciab, Carlos Mateosb, Sehila Pleiteb, Yong Wanga, Stephanie Stouta, Bradley
Condonc, Sheela Ashokc, Zhohai Lua, William Ehlhardta, Tom Rauba, Mei Laia, Sandaruwan Geeganagea,
and Timothy P. Burkholdera
aLilly Research Laboratories, Lilly Corporate Center, Eli Lilly and Company, Indianapolis, IN-46285-0150
bLilly S.A., Avda.de la Industria 30, 28108-Alcobendas, Madrid, Spain
cLilly Research Laboratories, Lilly Biotechnology Center, Eli Lilly and Company, San Diego, CA-92121
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
During the course of our research efforts to develop potent and selective AKT
inhibitors, we discovered enatiomerically pure substituted dihydropyridopyrimidinones
(DHP) as potent inhibitors of protein kinase B/AKT with excellent selectivity against
ROCK2. A key challenge in this program was the poor physicochemical properties of
the initial lead compound 5. Integration of structure-based drug design and physical
properties-based design resulted in replacement of a highly hydrophobic poly
fluorinated aryl ring by a simple trifluoromethyl that led to identification of compound
6 with much improved physicochemical properties. Subsequent SAR studies led to the
synthesis of new pyran analog 7 with improved cell potency. Further optimization of
pharmacokintetics properties by increasing permeability with appropriate fluorinated
alkyl led to compound 8 as a potent, selective AKT inhibitors that blocks the
phosphorylation of GSK3 in vivo and had robust, dose and concentration dependent
efficacy in the U87MG tumor xenograft model
Dedicated to Professor E. J. Corey on the occasion
of his 90th birthday
Keywords:
AKT Inhibitor
GSK3
ATP competitive
Orally Bioavailable
© 2018 Elsevier Ltd. All rights reserved.
AKT, also known as protein kinase B (PKB), is a serine-
threonine protein kinase that plays key role in the
sequence identity (100% similarity) when the 15 residues within
3 Å of ATP pocket are considered. Several compounds with
varying levels of AKT selectivity have been identified, but only a
limited number of chemotypes have been reported to have
entered early phase clinical trials,7 including the orally
bioavailable dihyrocyclopentapyrimidine 1 (GDC-0068)8 and
triazolopyrimidine an allosteric AKT inhibitor 2 (MK-2206)9
(Figure 1).
a
phosphotidylinositol-3-kinase (PI3K)/AKT/mammalian target of
rapamycin (mTOR) pathway.1 Increased AKT activation has
been implicated in a wide variety of cancers. AKT is identified
in three forms with distinct biological relevance: AKT1 is
ubiquitously expressed and is the isoform that is believed to play
a key role in cancer; AKT2 is highly expressed in muscle, liver
and adipocytes and contributes to cell motility and invasion;
AKT3 is predominately overexpressed in certain breast, glioma
and prostate tumors.2-4 Based on these factors, many efforts to
identify AKT inhibitors with acceptable pharmaceutical
properties have been pursued.5
However, the discovery of AKT inhibitors having AGC kinase
family selectivity has posed a significant challenge due to high
homology in the adenosine triphosphate (ATP) binding pocket
among the AGC kinase family members. The kinase selectivity
over ROCK2 is important since it’s another member of the AGC
kinase family and is involved in regulation of vascular tone and
thus control of blood pressure.6 There is high homology within
the AGC kinase family, with AKT1 and ROCK2 sharing 86%
Figure 1. ATP competitive type I inhibitor of AKT (1), allosteric
inhibitor of inactive AKT (2), type 1 inhibitor of p70S6K (3), and
type 1 dual inhibitor of AKT-p70 S6K (4)
* Corresponding author: Tel. +1 317 655 6132. Fax.: + 1 317 655 0422
E-mail address: shakthisarav@gmail.com

We recently described the development of selective p70S6K
During this Hit optimization, through several scaffold morphing
steps we have discovered chiral DHP 5 as potent and selective
AKT inhibitor. A crystal structure of 5 in complex with AKT1
confirmed the presence of key hydrogen bonds from the amine
group of the ligand and to Glu-234 of the protein back bone that
is necessary for the AKT1 biological activity. Among the
hydrogen bond donors we explored in this region N-ethyl
pyrollidine provided the best potency and improved metabolic
stability profile. In addition, the dramatic improvement of cell
potency with R enantiomer of the lactam could be rationalized by
chiral methyl group nicely accommodating the lipophilic pocket
near the hinge binder region, whereas the S enantiomer had
sterically hindered effect with protein backbone with loss
biological activity. To characterize the kinase selectivity of 5 and
its analogs, we profiled their ability to inhibit a panel of ~100 in
vitro expressed human protein kinases assays and many of them
with IC50 values listed in Table 1.
inhibitor 3 (LY-2584702) and AKT-p70S6K dual inhibitor 4
11
(LY-2780301) for treatment of advanced solid tumors,10, we
also have pursued development of selective AKT inhibitors for
this indication. Here we report for the first time the identification
of novel chiral DHP as potent AKT inhibitors with high
selectivity for AGC family kinases. Our initial approach was
focused on optimizing the hinge binding bicyclic moiety of the
inhibitor for selectivity and potency optimization, and then
turned our attention to the N-alkyl substitution of the imidazole
towards optimizing the physicochemical property of our hit series
by replacing the aryl-heteroaryl bond with less lipophilic
aliphatic substitutions that led to improved aqueous solubility and
druglike properties as shown in Figure 2.
Table 1. In vitro characterization and Kinase Selectivitya
Parameter
AKT1
5
6
7
8
0.003
0.035
>20
0.011
0.876
>20
0.004
1.90
>20
0.005
0.399
>20
P70S6K
ROCK2
PKA
0.653
0.036
0.020
0.039
0.124
16.3
0.412
20
0.084
3.98
PKC2
RSK1
0.272
2.46
0.699
0.230
0.568
0.092
Parameter
LogPa
Solubilityb pH7.4
hERGc
5
6
7
0.95
>2
8
pGSK3
4.84
1.66
2.39
0.99
18.5
aThe remaining kinases (>90) have IC50 values of >20 uM.
Where bMeasured at the apparent ATP Km, bEach value
represents the average of ≥2 independent experiments, where
each experiments considered of a single determination.
0.035 1.86
1.74 >100
>100
Figure 2. Chiral Dihydropyridopyrimidinone AKT Inhibitors (5-
8), aLogP in, Data in mg/mL, cData for hERG dofetilide binding
b
assay in uM.
In general, the DHP war head demonstrated high AKT
selectivity in enzyme and low activity against ROCK2 and many
other kinases (90 Kinases had <80% inhibition at 1 uM, >1000-
fold selectivity over ROCK2). The key difference between
AKT1 and ROCK2 in the ATP binding site is the gate keeper
pocket where there is a favorable hydrogen bond interaction from
the carbonyl moiety of the hinge binder with Thr-211 hydroxyl
group of AKT1, however in ROCK2 the gatekeeper residue is
Val-153. A summary of this structure based drug design, which
led to the discovery of potent and selective carbonyl containing
chiral dihydropyridopyrimidinone hinge binder 5 as AKT
inhibitors, is shown in Figure 3.12 While 5 and related analogs
had attractive cell potency, a major challenge in optimization of
this series was a struggle with poor physicochemical properties.
Lipophilic compound 5 (LogP/D = 4.84) suffered from relatively
high plasma protein binding, poor aqueous solubility (0.035
mg/mL at pH = 7.4) and hERG activity.13 While the addition of
lipophilicity improved potency, clearance and solubility further
deteriorated. Alternately, if polar functional groups were simply
appended to the periphery of the hinge binder, then potency was
compromised and passive permeability decreased.
To guide our attempts at achieving AKT potency and selectivity
against p70S6K and other kinases from 3 (p70 S6K IC50: 0.004
uM) and 4, we took advantage of exploiting the subtle sequence
differences in the active sites of the AGC family kinases, we
utilized the information around crystal structure of AKT1 at the
hinge region with analogs of 3-4.
Thus, the focus of further work was to determine whether
analogues could be generated with better solubility and
permeability while maintaining the kinase selectivity. With this
goal in mind, our medicinal chemistry strategy was therefore to
investigate the liphophillic P-loop pocket of lead compound 5
where the fluorinated aryl ring occupies. We reasoned that if we
could reduce the molecular weight and co-planarity of 5 on the
aryl- heteroaryl junction, then we might improve
physicochemical characteristics and in vivo ADME properties.14
Figure 3. X-ray crystal structure of 5 bound to AKT1.
Resolution = 2.2 Å, PDB code 6CCY.

With the aim of deconstructing the aryl ring, we made the
rational design decision of introducing smaller hydrophobic
trifluoromethyl substitutions at the imidazole represented by
compound 6. Replacement of the aryl ring with an aliphatic
substitutions lowered logP by >2 units and led to increased
aqueous solubility, lack of hERG binding and decreased unbound
intrinsic clearance (Fig 2).
absorption of compound 7 might be impacted by P-gp mediated
efflux.
Therefore, to confirm oral absorption impacted by increased P-
gp efflux, we examined oral exposure of compound 7 in P-gp and
breast cancer resistance protein (Bcrp) KO mice.17 Compound 7
had 5 fold greater plasma exposure in P-gp KO mice compared to
normal mice at the same dose (Table 3). Bcrp alone and together
with P-gp was not a factor, which was consistent with in vitro
results showing that compound 7 is not a Bcrp substrate.
Although compound 6 and analogues was sufficiently potent in
enzyme with >10 fold selectivity for many of the AGC kinase
family member, cell potency remained less attractive. We
hypothesized that reducing the ring size followed by significant
changes to planarity at the P-loop pocket resulted in a dramatic
loss of cell activity. On the basis of structural guidance, our
attention was directed towards identifying a similarly sized
saturated aliphatic ring at the P-loop pocket that might mimic the
aryl ring and spatially suitable enough for accommodating the
hydrophobic pocket.15 With this goal in mind we rationalized that
symmetrical 4-pyran could be an appropriate saturated ring that
could fit well without significantly increased hydrophobicity.
Replacing the trifluromethyl substitution with six-membered 4-
pyran as compound 7 that resulted in 10 fold boost in both cell
and enzyme potency.
Table 3. P-gp Properties of Lead 7-8a
Parameter
7
8
Units
ng.h/mL
ng.h/mL
ng/mL
AUC0-24h, PO,wt
AUC0-24h, PO, TKO
Cmaxwt
108
501
33
290
359
76
CmaxTKO
189
88
109
79
ng/mL
TPSAb
HBA Strengthc
13.9
12.2
aAUC
Area under the plasma concentration time curve from
0-24
0-24 hours post-dosing, All data generated in normal or wild-type
and P-gp/Bcrp knockout or mdr1a,b(-/-, -/-) and abcg2 (-/-) triple
genotype mice, Values are an average of n= x animals, Data
reported using a 2.5 mg/kg PO dose. bPredicted Topological polar
surface area. cTotal strength of hydrogen bond acceptors
predicted using.
Gratifyingly, compound
7
also demonstrated improved
physicochemical properties with significant improvement in
aqueous solubility. Compound 7 was sufficiently potent to
determine its pharmacokinetic (PK) and pharmacodynamics
(PD); data is summarized in Table 2.
Table 2. ADME Properties of Lead 6-8a
Both passive permeability and P-gp substrate recognition
appeared to change with relative constancy of hydrogen bond
(HB) count and topological polar surface area (TPSA). So, based
upon a negative relationship between Ppass and total HB strength
and a positive relationship between P-gp efflux ratio and total HB
strength (Table 2 and 3). We focused on reducing total HB
strength especially with regard to imidazole substitutions. So, we
decided to revisit the fluorinated alkyl substituent that could
maintain the potency with improved permeability.18 We reasoned
that if we could extend the alkyl chain farther (from simple -CF3
substitution 6) while reaching into the enzyme p-loop pocket
deep enough, then that should maintain its activity similar to the
aryl (5) or cyclic ring (7) system.15 In addition, such a fluorinated
alkyl group with reduced polarity and hydrogen bond accepting
strength should improve passive permeability and decrease the P-
gp effect. To validate our design hypothesis, we synthesized
trifluoropropyl substituted compound 8 as shown in scheme 3.
Parameter
Dose (IV/PO)
Bioavailability
AUC0-24h, PO
CL
6
10, 5
64
7
8
Units
mg/kg
10, 5 10, 5
14
55
%
24100 173
638
74
ng.h/mL
mL/min/kg
mL/min/kg
L/kg
2.18
66
64
Intrinsic CLub
85
43
Vdss
0.65
3
13.8
8.6
60
11.6
2.3
200
54
T1/2
h
MDCK Ppassc
410
nm/sec
P-gp Efflux Ratio
aMeasured data for compound fraction unbound to human plasma
29
82
protein, AUC
Area under the plasma concentration time
0-24
curve from 0-24 hours post-dosing, All data generated in SD rats,
Values are an average of n= 3 animals, Data reported using a 5
b
mg/kg PO dose and 10 mg/kg IV dose. Scaled intrinsic unbound
clearance in mL/min/Kg. cPassive permeability coefficient
(Ppass) was measure bidirectionally in duplicate across confluent
monolayers of MDCK-MDR1 cells with inhibition of P-gp.
In vivo PK parameters obtained in rat after intravenous
administration at 10 mg/kg and oral dosing at 5 mg/kg are
highlighted in Table 2. Replacing the fluorinated aryl ring 5 and
hydrophilic 4-pyranyl in 7 with an appropriate fluorinated alkyl
led to compound 8 displaying improved permeability and oral
bioavailability in rat as expected. PK attributes were also
favorable in mouse and dog (data not shown in manuscript
preparation). In addition, compound 8 demonstrated enhanced
cell potency and ideal physicochemical properties. Based on
these encouraging results compound 8 was tested in vivo for
efficacy in a 4 weeks U87MG rat xenograft model. Oral doses
were chosen to be 25, 50 mg/kg once or 25 mg/kg twice, daily as
crystalline suspensions in Hydroxyethyl cellulose (HEC). Tumor
volume was measured relative to controls animal treated with
HEC vehicle only. Compound 8 induced dose-dependent growth
inhibition and was well tolerated with no body-weight loss
observed at all doses and dose regimens tested.
Table 2 shows ADME characteristics for compound 6 and 7 as a
result of reduced hydrophobicity. Indeed, while the introduction
of 4-pyran in the place of CF3- enhances the cell potency, and
maintained its selectivity. A key issue at this stage was poor oral
exposure, low bioavailability and reduced passive permeability
with increased plasma clearance.16 This presented us with
conundrum in that although the pyran ring was the most efficient
replacement for the aryl ring in terms of hydrophobicity/potency
(LogP = 0.95), it suffered from poor permeability and low oral
exposure. Compound 6 had been shown to be a substrate for P-
glycoprotein (P-gp) in vitro (Table 2), but this did not appear to
impact its oral absorption. With the decrease in Ppass of
compound 7 and increase in efflux ratio, we suspected that oral

Scheme 2. Imidazole Synthesisa
Figure 5. In vivo efficacy of compound 8 in a 28 day mouse
U87MG xenograft model
The synthesis of final compounds 5-8 is outlined in Scheme 1-3.
The synthesis of enantiomerically pure (R)- and (S)-4-chloro-5-
R1-6,8-dihydro-5H-pyrido[2,3-d]pyrimidin-7-one started from
commercially available propanedioic acid dimethyl ester 9
subjected for Michael addition with methyl crotonate ((E)-methyl
but-2-enoate) followed by an in situ ring formation to give 10.
Compound 10 was prepared by chlorination of the hydroxyl
group with POCl3 in the presence of base such as N,N-
diethylaniline. The racemic compound 12 was obtained by
treating excess amount of aqueous ammonium hydroxide. In
order to improve the throughput of the chiral resolution 12 was
further protected as N-Boc derivative by treating with Boc
anhydride in the presence of DMAP to provide 13. The chiral
lactam 14a was prepared by chiral chromatography and
subsequent removal of the protecting group under acidic
condition furnished the final compound with good optical purity.
a Reagents and Conditions: (a) (i) SeO2, AcOH, Dioxane-H2O, 90
°C, (b) NaOAc, H2O, 90 °C; (c) Aq. NH4OH, MeOH, 30 °C.
Shown in Scheme 3, is a convergent synthetic route to final
compounds 5-8. The key intermediates 21a-21d were made by
reacting imidazole 19a-19c and 19d with 1-chloro ethyl
pyrrolidine HCl salt in the presence of base resulted in alkylated
imidazole derivatives 20a-20d, followed by trifluoroacetic acid
mediated deprotection gave the corresponding piperidines. With
the requisite piperidine amine and chiral Core 14a in hand,
preparation of the desired compounds was accomplished by base
promoted SNAr reaction to give 5-8.
Scheme 3. Convergent Synthesisa
Scheme 1. War Head Synthesisa
a
Reagents and Conditions: (a) (i) NaOMe, ethyl crotonate,
a
Reagents and Conditions: (a) (i) KOH, DMSO, 25 °C; (b)
MeOH, 70 °C, (ii) Formamidine acetate, -20 °C-RT; (b) POCl3,
MeCN, Et2NPh, 85 °C; (c) NH3 in iPrOH, 65 °C; (d) (Boc)2O,
DMAP, CH2Cl2, room temp. ; (e) chiral HPLC chromatography,
4M HCl in Dioxane, room temp.
Aq.HCl, iPrOH, 50 °C; (c) Et3N, N-methylpyrrolidinone, 110 °C.
In summary, we have outlined the optimization of 5 to advanced
molecules such as 8 using a combination of both structure- and
physical-property-based design parameters.19 We have identified
chiral dihyropyridopyrimidinone as a unique kinase inhibitor
scaffold of AKT with ROCK2 selectivity, the pharmacological
activity resides almost exclusively in the (R)-enantiomer. In
addition, the cyclic amine pyrrolidine provided optimum AKT
potency and metabolic stability. Furthermore, the aliphatic
substituent in place of the aryl ring of the imidazole lead 6 served
Scheme 2 shows the synthesis of imidazole intermediates 19a,
19b, 19c and 19d. The key precursor keto aldehyde 18a-18d was
prepared in situ by SeO2 mediated oxidation of corresponding
carbonyl compounds 16 and 17 or hydrolysis of dibromo ketone
15. Subsequent treatment with N-Boc piperidine-4-carbaldehyde
in the presence of aqueous NH4OH afforded the respective
imidazoles in good yield.

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investigated elaborately for preclinical development and
represents a broadly selective, potent, ATP-competitive AKT
inhibitor.
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Supplementary data
Supplementary data (Experimentals for the synthesis of
compound 7 and procedures for AKT1 and ROCK2 binding and
GSK3 cell assays.) associated with this article can be found, in
the online version, at http://.
Acknowledgement
We thank Gerri Sawada, Karen Huss and Laura Bloem for their
help in in vitro evaluations of compound. We acknowledge the
cloning work for the crystallography construct done by Aiping
Zhang and the scientific leadership by Devon Thompson in the
early crystallography enablement. This research used resources
of the Advanced Photon Source, a U.S. Department of Energy
(DOE) Office of Science User Facility operated for the DOE
Office of Science by Argonne National Laboratory under
Contract No. DE-AC02-06CH11357. Use of the Lilly Research
Laboratories Collaborative Access Team (LRL-CAT) beamline
at Sector 31 of the Advanced Photon Source was provided by Eli
Lilly Company, which operates the facility.
19. See supplementary material for crystallography details of 5.
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Discovery of Chiral
Dihydropyridopyrimidinones as Potent,
Selective and Orally Bioavailable
Inhibitors of AKT
Saravanan Parthasarathya,*, Kenneth Henrya, Huaxing
Peia, Josh Claytona, Mark Rempalaa, Deidre Johnsa, Oscar
De Frutosb, Pablo Garciab, Carlos Mateosb, Sehila Pleiteb,
Yong Wanga, Stephanie Stouta, Bradley Condonc, Sheela
Ashokc, Zhohai Lua, William Ehlhardta, Tom Rauba, Mei
Laia, Sandaruwan Geeganagea, and Timothy P.
Burkholdera
AKT1 IC50: 0.003 M
pGSK3 IC50: 0.039
Fonts or abstract dimensions should not be changed or altered.

Highlights of the Paper:
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Chiral DHP as hinge binder provided high AKT1
selectivity over ROCK2/AGC kinases
Emphasis on physicochemical properties over
potency led to developable compounds
Reducing the HB acceptor strength of
compounds with high P-gp efflux improved
Ppass