Discovery of Novel and Orally Bioavailable Inhibitors of PI3 Kinase Based on Indazole Substituted Morpholino-Triazines

Article abstract of DOI:10.1021/acsmedchemlett.5b00322

A new class of potent PI3Kα inhibitors is identified based on aryl substituted morpholino-triazine scaffold. The identified compounds showed not only a high level of enzymatic and cellular potency in nanomolar range but also high oral bioavailability. The three lead molecules (based on their in vitro potency) when evaluated further for in vitro metabolic stability as well as pharmacokinetic profile led to the identification of 26, as a candidate for further development. The IC₅₀ and EC₅₀ value of 26 is 60 and 500 nM, respectively, for PI3Kα enzyme inhibitory activity and ovarian cancer (A2780) cell line. The identified lead also showed a high level of microsomal stability and minimal inhibition activity for CYP3A4, CYP2C19, and CYP2D6 at 10 μM concentrations. The lead compound 26, demonstrated excellent oral bioavailability with an AUC of 5.2 μM at a dose of 3 mpk in mice and found to be well tolerated in mice when dosed at 30 mpk BID for 5 days.

Full text of DOI:10.1021/acsmedchemlett.5b00322

Letter
pubs.acs.org/acsmedchemlett
Discovery of Novel and Orally Bioavailable Inhibitors of PI3 Kinase
Based on Indazole Substituted Morpholino-Triazines
Sundeep Dugar,† Frank P. Hollinger,† Dinesh Mahajan,*,‡ Somdutta Sen,‡ Bilash Kuila,‡ Reena Arora,‡
Yogesh Pawar,‡ Vaibhav Shinde,‡ Mahesh Rahinj,‡ Kamal K. Kapoor,‡ Rahul Bhumkar,‡ Santosh Rai,‡
and Rakesh Kulkarni‡
†Sphaera Pharma Pte. Ltd., 038988, Singapore
‡Sphaera Pharma Pvt. Ltd., Plot 32, Sec 5, IMT Manesar, Harayana 122051, India
S
* Supporting Information
ABSTRACT: A new class of potent PI3Kα inhibitors is identified based on aryl substituted
morpholino-triazine scaffold. The identified compounds showed not only a high level of
enzymatic and cellular potency in nanomolar range but also high oral bioavailability. The three
lead molecules (based on their in vitro potency) when evaluated further for in vitro metabolic
stability as well as pharmacokinetic profile led to the identification of 26, as a candidate for
further development. The IC50 and EC50 value of 26 is 60 and 500 nM, respectively, for PI3Kα
enzyme inhibitory activity and ovarian cancer (A2780) cell line. The identified lead also
showed a high level of microsomal stability and minimal inhibition activity for CYP3A4,
CYP2C19, and CYP2D6 at 10 μM concentrations. The lead compound 26, demonstrated
excellent oral bioavailability with an AUC of 5.2 μM at a dose of 3 mpk in mice and found to
be well tolerated in mice when dosed at 30 mpk BID for 5 days.
KEYWORDS: Cancer, PI3K inhibitors, indazole, morpholinotriazine
hosphatidyinsoitol-3-kinase (PI3K) is a lipid kinase and an
Pimportant component in the PI3K/AKT/mTOR signaling
pathway that plays a key role in cell proliferation, growth,
survival, and apoptosis as well as protein and glucose
metabolism.1−3 Class 1 PI3K protein consists of four isoforms
known as PI3K α, β, γ, and δ. This classification is based on
their sequence homology and substrate preferences. The
deregulated activation of PI3Kα and its downstream effectors
including AKT and mTOR have been linked to tumor initiation
and maintenance. Class 1 PI3K protein in general and PI3Kα
isoform specifically plays a key role in human cancer
biology.4−7 Aberrantly activated PI3Kα protein has been
implicated in poor prognosis and survival in patients with
Figure 1. PI3Kα IC50 of identified hit 3, compared to literature leads 1
and 2.
various lymphatic tumors, as well as breast, prostate, lung, brain
(glioblastoma), skin (melanoma), colon, and ovarian cancers.4
Therefore, inhibiting PI3Kα is an attractive strategy for cancer
as it interacts with a hinge region backbone NH (Val882) of
PI3K protein.16−18 Therefore, the morpholino group retained
in all subsequent molecules for this study.
Synthesis of the various morpholino−triazines reported in
Table 1 was achieved by the general synthetic route outlined in
Scheme 1 involving a combination of Suzuki coupling and
nucleophilic substitution on morpholine substituted triazine
ring.19
therapy. Clinical interest in PI3K inhibitors has been
intensifying. In recent years, a large number of compounds
have been reported in clinical development phase having strong
antitumor activity in animal models.8−11
During our efforts toward the identification and development
of class 1 PI3K inhibitors, we identified hit 3 based on the
triazine morpholine core.12−14 This hit was inspired by the
leads reported (Figure 1, compounds 1 and 2) by Hayakawa et
al.15 It was planned to develop a quick structure−activity
relationship (SAR) around identified hit 3. Based on data
reported by others and our internal data, we had ascertained
that, the morpholine moiety is critical for activity and potency
Received: August 10, 2015
Accepted: November 2, 2015
© XXXX American Chemical Society
A
DOI: 10.1021/acsmedchemlett.5b00322
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coupled with different amines to produce their corresponding
amides (entry 9−12, Table 1) and evaluated for their in vitro
PI3Kα inhibition activity. The PI3Kα enzyme inhibitory activity
of the compounds was determined using a homogeneous time
resolved fluorescence (HTRF) assay. The percent inhibition
was calculated and plotted against the concentration of the
inhibitor to calculate IC50.19 Interestingly, and as illustrated by
compounds 7 through 13, variations of the glycine moiety of 3
does not result in any significant change in activity. Compound
13 even suggests that the glycine moiety may not be interacting
with the protein and may potentially be oriented outside the
protein into the solvent. This is complementary to literature
findings.20 The activity of compound 8 suggests that this loss of
activity may be due to steric interaction between the ligand and
the protein or the lack of hydrogen bonding interaction
between the protein and the ligand. The marginal increase in
activity of compound 13 over compound 3 may be due to the
either less steric hindrance or less encumbered ability for the
formation of the hydrogen bond between the ligand and the
kinase.
Table 1
From this series of molecules, compound 11 was selected for
further optimization, primarily based on its potency against
PI3Kα. Though, we had initially established the importance of
the phenolic group, we needed to find an alternate of phenol.
This is due to the generally accepted observation of the poor
PK/ADME characteristics imparted to a compound with the
presence of a phenol moiety. A small set of new molecules were
synthesized as analogues of 11 to find a replacement for phenol
(Table 2). A two/three step synthesis, involving a Suzuki
coupling with different boronic acids as a key step for
diversification was exploited to generate these analogues
(Scheme 2).19 The data from this new set of molecules led
to the selection of compound 16 over compound 11 for further
optimizations, even though it was 2-fold lower in in vitro
Table 2
*
The coefficient of variation (CV %) was found to be <10% for IC50.
a
Scheme 1
a
Reagents and conditions: (a) morpholine, acetone/water (4:1), 0 °C,
3 h; (b) amino acid/ester, acetone/NaHCO3 aq. (1:1), RT, 16 h; (c)
aryl borate, Pd(PPh3)4, Na2CO3, DME/water (4:1), 120 °C, 20 min,
MW.
Compounds 4, 5, and 6 illustrate the importance of the
phenolic group, as protection, removal, or replacement results
in complete loss of activity. This finding is in line with literature
reports17,18 that highlighted the importance of hydrogen bond
donor moieties for potency in PI3K inhibitors having aryl
morpholine based structural motifs. Further, compound 3 was
*
The coefficient of variation (CV %) was found to be <10% for IC50.
B
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a
Scheme 2
Table 3
a
Reagents and conditions: (b) glycine amide, acetone/NaHCO3 (aq.)
(1:1), RT, 16 h; (c) aryl borate, Pd(PPh3)4, Na2CO3, DME/water
(4:1), 120 °C, 20 min, MW.
potency, primarily due to the anticipated issues with the
phenol.
Further optimization was planned targeting enhancement of
the in vitro enzymatic potency by varying the substituents on
the glycine amide side chain on compound 16 (Table 2).
Scheme 3 illustrates the synthetic route deployed for the
a
Scheme 3
a
Reagents and conditions: (d) benzyl mercaptan, DIPEA, morpholine,
THF, 0 °C, 5 h; (e) aryl borate, Pd(PPh3)4, Na2CO3, DME/water
(4:1), 90 °C, 18 h; (f) oxone, THF/water (1:1), 0 °C−RT, 25 h; (g)
RX, DMF, K2CO3, RT, 24 h; (h) methanesulfonic acid, methanol/
water (2:1), 55 °C, 1 h.
synthesis of this series of molecules based on the indazole-
morpholino-triazine scaffold. The new molecules in this series
were screened for their cellular potency on the human ovarian
cell line (A2780) along with enzymatic activity. The cellular
potency was determined by the MTT assay using human
ovarian cancer cell line (A2780). The results of this screening
are documented in Table 3. Compound 18, synthesized from
homoglycine amide, was found to be slightly more potent than
the original lead 16. Intriguingly, conformationally restricted
analogues of glycine amide, i.e., 6- and 7-membered cyclic
amides (compound 19 and 20) as a replacement, were found to
be more potent compared to original lead 16 and compound
18. The absence of a potential hydrogen bonding interaction by
the amino group attached to the triazine and the steric size of
the cyclic lactams negates our explanation for the loss of activity
of compound 8 over compound 3. The addition of methyl on
N-linker may have conformational impact leading to steric
clashes. The small but favorable shift in in vitro potency for
compound 18 to 20, relative to the original lead 16, highlights
*
The coefficient of variation (CV %) was found to be <10% for IC50
and <5% for EC50.
C
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Table 4
the role of the spacer and the spatial orientation of the terminal
amide. To explore this further, several other analogues were
synthesized using an aromatic ring spacer between the triazine
and terminal amide (compounds 21−24). All of the new
analogues demonstrated improved enzymatic and cellular
potency. Replacement of the bridging nitrogen (compound
22) with oxygen (compound 25) led to a 4-fold loss in
enzymatic as well as cellular potency, but this loss was
recovered by moving the amide group from the meta- to the
para-position on the phenyl ring (compound 26) resulting in
the best compound in this series. Replacing the aromatic ring
spacer of 26 with a cycohexyl ring (compound 28 and 29)
resulted in compounds equipotent to 26. Hence, compounds
26, 28, and 29 were identified as the optimized leads based on
their enzymatic and cellular potencies. These three leads were
further evaluated for their in vitro metabolic stability with
human liver microsome (HLM), dog liver microsome (DLM),
and mouse liver microsomes (MLM). The three leads were
incubated with isolated liver microsomes of the three species
for 2 h. The concentration of the parent compound was
monitored by HPLC at four time points (0, 0.5, 1, and 2 h,
respectively). All three molecules (26, 28, and 29) showed
encouraging level of microsomal stability particularly in HLM.
The data of 2 h time point is shown in Table 4 and a detailed
study report is provided as Supporting Information.
Compounds 26, 28, and 29 were screened further for their
cytochrome P450 inhibition activity. All three leads showed no
or minimal inhibitions for activity of CYP3A4, CYP2C19, and
CYP2D6 at 10 μM concentrations. All three molecules were
also evaluated for oral bioavailability in mice. Each was dosed
orally at 3 mpk to Swiss mice to evaluate their pharmacokinetic
profile and determine the key parameters such as Cmax, AUC,
tmax, and t1/2. The results are summarized in Table 4.
Compounds 26 and 28 showed better PK profile compared
to 29 with higher plasma exposure of >5 μM at 3 mpk dosing.
On the basis of its potency and pharmacokinetic profile, 26 was
selected as the molecule of choice for further evaluation
Compound 26 was evaluated in vivo for dose ranging
pharmacokinetic study in mice at three different doses of 1, 3,
and 10 mpk. A summary of the pharmacokinetic parameters are
shown in Table 5. The plasma exposure of 26 demonstrated a
reasonable dose response exposure with escalating dose from 1
to 3 to 10 mpk.
Table 5. Dose Ranging Pharmacokinetics Study of 26 by
Oral Administration in Mice
dose (oral)
PK parameters
tmax (h)
1 mpk
3 mpk
10 mpk
1.00
360
1.00
1243
5246
4.86
2.33
1798
9791
6.87
Cmax (nM)
AUC (nM·h)
elimination t1/2 (h)
2117
6.93
The lead 26 was also screened for hERG activity using the
HERG-lite assay from Chantest Corporation.18 The objective
of the study was to estimate in vitro effects of 26 on surface
expression of the hERG potassium channel. Both wild type
(WT) and the single-point mutant (SM) channel G601S were
examined. Predicted hERG liability was indicated as channel
block and/or trafficking inhibition. On the basis of the hERG
lite assay data, the IC50 of the hERG liability determined by the
patch clamp method was predicted. Overall, the predicted
hERG liability of 26 was found to be >10 μM implying that 26
is not expected to have hERG liability.
The in vivo multiday dose tolerability of 26 was evaluated in
Swiss mice.19 The lead 26 was dosed at 30 mpk BID for 5
consecutive days and monitored for effect and changes in body
weight, body temperature, body coat, and level of physical
activity. The animals did not show any significant body weight
loss (Figure 2), hyper- or hypothermia, change in body coat or
lowered physical activity. This clearly indicates that 26 is well
tolerated by mice.
D
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Figure 2. Effect of 26 on mice bodyweight when dosed at 30mpk BID
(13) Dugar, S.; Hollinger, F. P.; Kuila, B.; Arora, R.; Sen, S.; Mahajan,
D. Synthesis and evaluation of pyrrolotriazine based molecules as PI3
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includes chemical synthesis, characterization, and in vitro and in vivo
screening of all compounds.
oral for 5 days.
In conclusion, our efforts toward development of class I PI3K
inhibitors led to identification of a small molecule 26 as a
potent and orally bioavailable inhibitor of PI3Kα protein. The
lead 26 was found to have excellent metabolic stability, no
hERG liability with appreciable oral exposure, and tolerability.
Efforts for the development of 26 are in progress and will be
summarized in a future publication.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsmedchem-
lett.5b00322.
■
S
Experimental details of chemical synthesis as well as
characterization and in vitro and in vivo screening of all
compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: dinesh.m@sphaerapharma.com; chemidinesh@gmail.
com.
■
Notes
The authors declare no competing financial interest.
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