Discovery and Development of SPR519 as a Potent, Selective, and Orally Bioavailable Inhibitor of PI3Kα and mTOR Kinases for the Treatment of Solid Tumors

Article abstract of DOI:10.1021/acs.jmedchem.0c01061

Herein, we report the identification and preclinical profile of a lead compound 10, (SPR519) as an equally potent dual inhibitor of PI3Kα and mTOR kinases. SPR519 exhibits an EC50 of low sub-micromolar range among various tested cancer cell lines such as A2780 (0.23 μM), PC3 (0.48 μM), and SKOV3 (0.50 μM). When administrated orally, it shows a considerably high plasma exposure (area under curve: 26,858 nM/h at 1 mg/kg) in mice. Moreover, it is found to be safe in animals with a dose of 30 mg/kg BID for 12 days in the dose tolerance study. SPR519 did not show any CYP or hERG liability. The identified lead compound demonstrates significant efficacy and bioavailability in ovarian and colon cancer xenograft models when evaluated for dose-ranging efficacy studies, at a dose as low as 2.5 mg/kg.

Full text of DOI:10.1021/acs.jmedchem.0c01061

Subscriber access provided by CORNELL UNIVERSITY LIBRARY
Article
Discovery and Development of SPR519 as a Potent,
Selective and Orally Bioavailable Inhibitor of PI3Kalpha
and mTOR Kinases for the Treatment of Solid Tumours
Dinesh Mahajan, Somdutta Sen, Bilash Kuila, Amit Sharma, Reena Arora, Milind
Sagar, Amal Ray Mahapatra, Lalita Babasaheb Gawade, and Sundeep Dugar
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.0c01061 • Publication Date (Web): 08 Sep 2020
Downloaded from pubs.acs.org on September 8, 2020
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 12
Journal of Medicinal Chemistry
Discovery and Development of SPR519 as a Potent, Selective and Orally Bioavailable Inhibitor of
PI3Kα and mTOR Kinases for the Treatment of Solid Tumors
1
2
Dinesh Mahajan,1*† Somdutta Sen,1 Bilash Kuila,1 Amit Sharma,1 Reena Arora,1 Milind Sagar,1 Amal Ray Mahapatra,1 Lalita Babasaheb
Gawade,1 Sundeep Dugar,2*
3
4
5
6
7
1Sphaera Pharma Pvt. Ltd., Plot 32, Sec 5, IMT Manesar, Haryana 122051, India.
2Sphaera Pharma Pte. Ltd., 038988, Singapore.
8
KEYWORDS PI3K, mTOR, dual inhibitor, cancer, triazine
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
ABSTRACT: Herein we report, the identification and pre-clinical profile of a lead compound 10, (SPR519) as an equally potent
dual inhibitor of PI3Kα and mTOR kinases. SPR519 exhibits an EC50 of low sub micro molar range among various tested cancer
cell lines such as A2780 (0.23 µM), PC3 (0.48 µM), and SKOV3 (0.50 µM) respectively. When administrated orally, it shows a
considerably high plasma exposure (AUC: 26,858 nM/h at 1 mg/Kg) in mice. Moreover, it is found to be safe in animals with a
dose of 30 mg/Kg BID for 12 days in dose tolerance study. SPR519 did not show any CYP or hERG liability. The identified lead
demonstrates significant efficacy and bioavailability in ovarian and colon cancer xenograft models when evaluated for dose-ranging
efficacy studies, at a dose as low as 2.5 mg/Kg.
INTRODUCTION
pharmacologically well characterized advance leads in this
structure class are mentioned in Figure 1. Compound
Phosphatidyinsoitol-3-kinases (PI3Ks) play a key role in cell
proliferation, growth and survival.1-2 Dysregulated activity of
PI3Ks has been implicated in poor prognosis and survival in
patients with various solid as well as liquid tumors.3 There are
four classes of PI3Ks reported in literature.4 Ambiguity over
the involvement and role of individual PI3K isoforms of class
I PI3Ks in various types of tumors leads to an argument on
isoform specific over pan PI3K inhibitors for therapeutic
efficacy against cancer.5 However, literature suggests a
prominent role of PI3K α and PI3K isoforms of class I PI3Ks
in human cancer biology. The dysregulated activation of PI3K
α or other isoforms of class I PI3K group and its downstream
effectors including AKT and mTOR have been linked to
tumor initiation and maintenance.6 So, an effective inhibitor of
the PI3K/AKT/mTOR pathway could both prevent cancer cell
proliferation and induce programmed cell death (apoptosis).
Significant efforts have been made in drug discovery to
develop therapeutic interventions for cancer by inhibiting the
functional roles of PI3K, AKT, or mTOR in this signaling
pathway.6-7 The selective inhibition of mTOR kinase faces a
challenge of drug resistance due to over activation of
oncogene PI3K by a feedback mechanism involving S6
kinase.8 Recent literature reports suggest that single molecule
based dual inhibitors of PI3K/mTOR may have better
therapeutic advantages over stand-alone PI3K or mTOR
inhibitors, but this certainly requires clinical validation.9
ZSTK474 was the first published clinical lead based on
morpholine substituted triazine core having pan PI3 kinase
activity.11 PKI-587 was a second inhibitor disclosed in this
class possessing PI3K/mTOR dual activity.12 PKI-587 is an
experimental drug under phase 2 evaluation at present.
PQR309 is a PI3K/mTOR dual inhibitor in phase 2 trials.13
Wymann et al. has published an ingenious SAR study to
understand PI3K/mTOR differential activity leading to
discovery of PQR309 and other follow-up analogues i.e.,
PQR514 and PQR530.13-15 It is interesting to see how minor
structural changes can lead to improve in potency as well as
mTOR activity. In all these reported inhibitors, oxygen atom
of morpholine found to have a critical hydrogen bond
interaction with NH of the amino acid residue Val851 (PI3Kα)
and Val2240 (mTOR) in the hinge region.11-16 The aryl group
of these reported inhibitors is understood to occupy affinity
pocket of ATP binding site of the PI3K. The presence of two
morpholine units put these reported leads under a common
genus.
In our ongoing endeavor for the development of
cancer therapies based on the targeted inhibition/modulation
of PI3K/AKT/mTOR signaling pathway, we reported the
discovery of an early lead compound 26.17 Compound 26 was
found to be an orally bioavailable, well-tolerated potent
inhibitor of PI3K α. Herein, we report further optimization and
development studies around this initial hit leading to discovery
of a more potent as well as efficacious analogue compound 10,
Stocks et al. discussed and described different
structure classes of PI3K inhibitors and their design strategies
from medicinal chemistry prospectus in an elegantly collated
review.10 There are quite a few PI3K inhibitors known in
literature based on morpholine substituted triazine core. Few
an equipotent dual inhibitor of PI3K
(Figure 1).
α and mTOR proteins
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 12
A) Literature reported leads
1
2
3
4
5
6
7
8
O
N
O
N
O
N
O
N
O
N
N
N
N
N
F
N
N
O
N
N
F
N
N
F
N
N
N
(S)
N
N
F
F
N
N
N
O
N
N
N
F
N
N
N
F
N
O
N
F
O
O
O
O
NH2
N
N
N
NH2
N
NH2
H
H
F
ZSTK474
PQR514
PKI-587
PQR309
PQR530
9
B) This work
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
O
N
O
N
O
O
N
N
HN
N
N
N
N
N
N
O
N
O
N
S
SPR519
26
PI3K= 60nM
mTOR=1350nM
AUC= 2.1uM/hr@1mg/Kg in mice
H2N
Compound 10
PI3K= 30nM
mTOR=10nM
AUC= 26.8uM/hr@1mg/Kg in mice
Figure 1: PI3Kα inhibitors reported in literature based on morpholine substituted triazine scaffold.
determined using
a
Homogeneous Time Resolved
RESULTS AND DISCUSSION
Fluorescence (HTRF) assay. The percentage of inhibition was
calculated and plotted against the concentration of the
inhibitor to calculate the IC50. The newly synthesized
analogues in this series were also screened for their cellular
potency. The cellular potency was determined by the MTT
assay using human ovarian cancer cell line (A2780).
In the course of our efforts towards development of a dual
PI3K/mTOR inhibitor, we designed and synthesized new
analogues around 26 and evaluated them for PI3Kα enzyme
inhibitory activity as well as cellular potency against human
ovarian cancer cell line (A2780). The new analogues were
designed with an aim to enhance in vitro therapeutic potency.
The variations on indazole heterocycle was selected as a
preferred alteration on initial lead 26 based on the previously
reported SAR and understanding by us17-18 and others,19-20
where it is understood to be a critical pharmacophore for
potency. The new series of compounds were designed and
synthesized (Table 1), as replacements of indazole. All new
analogues 1-12 were synthesized using Scheme 1 as reported
before. 21 To facilitate rapid analogue synthesis, we utilized 13
as a late stage key intermediate for Suzuki coupling reactions
with various aryl boronates, exploiting Pd(PPh3)4 as catalyst in
biphasic reaction conditions. The desired core 13 was
synthesized in multi gram scale exploiting nucleophilic
addition of morpholine followed by addition of 4-hydroxy
benzamide on commercially available cyanuric chloride. 21
Compound 1 was found to be the most potent analogue
with an IC50 of 10 nM against PI3K and EC50 of 0.09 µM in
A2780 cell line. Compared to 26, compound 1 exhibited 6-
fold higher potency (both for enzymatic as well as cellular
activity) and was further considered for pharmacokinetic
study. Disappointingly, the pharmacokinetic analysis indicated
no plasma exposure for compound 1 when administrated
orally to a group of mice at a dose of 10 mg/Kg. The absence
of plasma levels is probably related to the potentially rapid
Phase II conjugation and clearance associated with the
phenolic group of 1. Interestingly, none of other analogues
synthesized had better affinity for PI3K protein except 10.
But compounds 2, 3, and 8 (Table 1) showed improved or
equal cellular potency compared to 26. Replacement of
indazole with similar bicyclic moieties (i.e., compounds 7, 8,
and 9) provided interesting insights. The change from indazole
(26) to indole (7) resulted in a significant drop in potency,
which regained significantly in the azaindole analog (8; IC50 =
0.08 µM). This possibly suggested the involvement of pKa of
the NH of the hetroaryl, in this series of compounds. However,
replacement with the benzotriazole (9) resulted in a total loss
of potency (IC50 = > 10 µM). Replacement of indazole with
amino-pyridine (3; IC50 = 0.21 µM) or amino-pyrimidine (2;
IC50 = 0.27 µM) led to a 3 to 4-fold loss in kinase activity.
Introduction of steric constraints in compound 3, through
methyl group on the pyridine ring (compound 4, 5 and 6
compared to compound 3) found to have dramatic effect on
IC50 value. Methyl substitution was found to be more tolerable
at 5-position of pyridine ring (i.e. compound 4). Compound 10
with a five member heterocycle as a new replacement of
indazole led to 2-fold increase in activity. Here again, an
additional substitution of methyl on this five-member
heterocycle resulting in compound 11 proved detrimental for
O
O
Cl
N
O
c
a,b
N
O
N
N
N
N
N
N
N
N
Cl
N
Cl
Cl
N
O
Ar
N
O
1-12
13
Scheme 1: Synthesis of compounds 1-12 for SAR study.
Reagents and conditions: (a) Morpholine (1 equiv), anhyd.
THF, DIPEA (1 equiv.), 0 °C, 2 h, 69%; (b) 4-Hydroxy
benzamide, anhyd. DMF, K2CO3 (2.5 equiv.), 18-Crown-6
(catalytic), 60 °C, 4 h, 88%; (c) Aryl borate (1.5 equiv.),
Pd(PPh3)4 (0.1 equiv.), Na2CO3 (3 equiv.), DME: water (4:1),
100 °C or microwave irradiation, 16-20 h, HCl in dioxane
(4N), RT, 30-60 min, 60-85%.
All new compounds synthesized using this synthetic route
were evaluated for their in vitro PI3Kα inhibition activity. The
PI3Kα enzyme inhibitory activity of the new compounds was
ACS Paragon Plus Environment

Page 3 of 12
Journal of Medicinal Chemistry
ligand protein interaction. Interestingly, but with no obvious
explanation available, compound 2, 3,
with PI3K as well as cellular potency. Two different cancer
1
2
3
4
5
6
7
8
cell lines were used to evaluate their EC50. The results are
tabulated in table 2. As is evident from the data, these
compounds demonstrated a good affinity for mTOR along
with PI3K. The compounds 26, 2, and 8 showed higher
affinity for PI3K (0.06 µM, 0.27 µM and 0.08 µM)
compared to mTOR (1.35 µM, 0.96 µM and 0.22 µM) while
compounds 4 and 10 showed equal affinities for PI3K and
mTOR proteins. The new compounds 2, 3, 8, and 10 are also
found to be more potent in prostate cancer cell line (PC3) as
well as ovarian cancer cells (A2780) compared to initial hit
26.
Table 1: Medicinal chemistry optimization and SAR around
initial hit 26.
Compound
Number
Ar
Cell line A2780
*EC50 (µM)
PI3K
*IC50 (µM)
N
HN
26
0.06
0.52
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
HO
1
2
0.01
0.27
0.09
0.56
Table 2: Potency evaluation of identified leads.
N
H2N
N
*IC50 and *EC50 (µM)
Compoun
d
Number
Ar
p110

mTO
R
PC3
SKO
V3
logP/tPS
A#
3
4
0.21
0.46
0.20
1.5
H2N
H3C
N
H2N
N
N
HN
CH3
3.78/103.
5
26
0.06
1.35
1.97
0.52
5
>10
>10
H2N
N
N
2
3
0.27
0.21
0.96
0.20
0.80
0.28
0.22
0.21
2.4/129.8
H2
N
N
6
7
8
3.40
0.56
0.08
>10
0.90
0.37
H2N
N
CH3
3.21/117.
5
HN
H2N
N
3.58/103.
5
HN
8
0.08
0.03
0.22
0.01
0.30
0.48
0.37
0.50
N
H
N
S
N
N
HN
N
N
10
3.6/117.5
9
>10
>10
H2N
* All the experiments were done in triplicates. The coefficient of variation
(CV %) was found to be <10% for IC50 and <5% for EC50. Values are
calculated using ChemOffice 2015.
#
N
10
0.03
0.23
S
S
H2N
H3C
Based on the dual activity of compounds 3, 8,
and 10 and high cellular efficacy, these three leads were
further evaluated for their in vitro metabolic stability using
human liver microsome (HLM), dog-liver microsome (DLM),
and mouse liver microsomes (MLM). The three leads were
incubated individually, with liver microsomes of three
different species for 2 h. The concentration of the parent
compound was monitored by HPLC at four-time points. All
three leads (3, 8, and 10) showed a high level of microsomal
stability, particularly in HLM (Table 3), just like original
lead 26. These three molecules were also evaluated for oral
bioavailability in mice. The results are summarized in table 3.
All new compounds demonstrated higher plasma exposure
than the original lead 26 when administrated orally to a group
of mice (3 mg/Kg). Compound 10 showed considerably better
pharmacokinetic (PK) profile compared to others leads (AUC
>32 µM.h). This study concluded that replacement of indazole
(26) with 2-aminothiazole (10) provided higher potency for
mTOR and higher plasma exposure.
N
H2N
N
11
0.14
1.1
12
0.90
0.04
2.36
0.29
NH
H2N
-
GDC0940
*All the experiments were done in triplicates. The coefficient of variation
(CV %) was found to be <10% for IC50 and <5% for EC50.
and 8 demonstrated comparable or higher cellular potency
with respect to 26 even though they have lower IC50. A
possible explanation for this could be enhanced cellular
permeability of these compounds or perhaps involvement of
an additional complementary target leading to enhanced
cellular potency. We envisaged that enhanced cellular
permeability, however, might be less likely to be the reason
for this increased potency as these compounds are structurally
similar. We next investigated the possibility of the
involvement of a complementary target. It is reported that
PI3K and mTOR share a high degree of sequence homology.
Keeping this understanding in mind we screened some
selected molecules for their mTOR binding affinities along
The lead compounds 3, 8, and 10 were further
evaluated for possible hERG liability. Compounds were
screened for hERG activity using the HERG-lite assay from
Chantest Corporation. The objective of the study was to
estimate the in vitro effects of these leads on surface
expression of the hERG potassium channel. Both wild type
(WT) and the single-point mutant (SM) channel G601S were
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 12
examined. Predicted hERG liability was indicated as channel This screening demonstrated that the lead 10 has similar
1
2
3
4
5
6
7
8
block and/or trafficking inhibition. While none of the lead
showed trafficking inhibition, but all of these demonstrated
channel blocking. On the basis of the hERG lite assay data, the
IC50 of the hERG liability was predicted by the patch-clamp
method. The anticipated hERG liability of 3 was found to be
>1 μ M, while those of compound 8 and 10 were >10 μ M
(Table 4), which was very encouraging.
inhibitory activity against PI3Kδ along with PI3Kα and mTOR
kinases. With encouraging level of selectivity across a limited
panel of kinases, it was planned to profile 10 in vivo in mice to
evaluate pharmacological parameters and optimum dosing
regimen for efficacy studies. We performed a dose-ranging in
pharmacokinetics and multi-day repeat dose toxicity studies to
understand dose linearity and tolerable dose in a rodent model.
N
All three leads showed no or minimal inhibitions for
activity against CYP3A4, CYP2C19, and CYP2D6 at 10 µM
concentration, as mentioned in table 4. Based on its potency
and DMPK profile, compound 10 was selected for further
(n-Bu3)Sn
NHBoc
S
14
9
O
N
O
N
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
14
a) , Pd(PPh3)2Cl2
O
O
DIPEA, DMF, 90oC, 16 h
evaluation.
To
evaluate
off-target
selectivity,
N
N
N
N
N
N
b) HCl in Dioxane, RT
compound 10 was screened for its binding affinity against a
panel of kinases. A panel of 23 kinases was selected based on
their close sequence homology with PI3Kα and/or mTOR
protein. The lead 10 was screened at a single concentration of
1 µM across this panel. The results are mentioned in table 5.
N
O
Cl
N
O
N
S
13
10
ClH.H2N
Scheme 2: Multigram synthesis of 10
Table 3: Metabolic stability and pharmacokinetic analysis of leads.
Metabolic stability*
Mouse Pharmacokinetic**
Compound
Number
Ar
DLM
83
MLM
HLM
100
AUC
(nM/h)
Cmax
(nM)
Tmax
(h)
t1/2
(h)
N
HN
26
45
5246
1243
1.0
4.9
3
8
93
77
102
68
93
92
10940
16770
2010
3076
1.4
1.0
2.7
2.8
H2N
N
N
N
H
N
10
105
103
98
32146
6903
1.5
2.7
S
H2N
*@10 µM: %remaining after 2 h; ** n=3/group; PO @ 3mpk; Cmax: maximal concentration, Tmax: time of maximal, concentration in hours; t1/2: half-life
elimination; AUC: area under the curve.
Table 4: Off-target screening of the leads.
hERG binding
CYP inhibition IC50 (M)
Compound
Number
Ar
CYP
3A4
>10
CYP
2C19
>10
CYP
2D6
>10
Channel Blocking
Yes
Trafficking Inhibition
Predicted IC50 (M)
No
No
>1.0
3
H2N
N
>10
>10
>10
>10
>10
>10
Yes
Yes
>10.0
>10.0
8
N
N
H
No
N
10
S
H2N
Table 5: Screening of lead 10 across a small panel of human
kinases at 1 µM concentration.
ACS Paragon Plus Environment

Page 5 of 12
Journal of Medicinal Chemistry
Sr
No
1
2
3
4
5
6
7
8
9
10
Kinase
%
Sr
No
13
14
15
16
17
18
19
20
21
22
Kinase
%
and distribution in different vital organs. After 30 mg/Kg
inhibition
inhibition
1
2
3
4
5
6
7
8
BID/five day dosing, animals were sacrificed, and their liver,
kidney, lung, uterus, and brain were extracted and observed
for any physical color change as well as test drug
concentration. No change in physical morphology or
discoloration was observed in these vital organs except very
faint color change in the liver. The LCMS analysis of the
homogenized extracted organs showed maximum drug
concentration (Figure 2) in kidneys (160 ng/g) followed by
liver (85 ng/g), lungs (50 ng/g), and uterus (10 ng/g). No
significant amount of test drug was observed in the brain of
the animals.
AKT1
AKT2
AKT3
ERBB2
FLT3
GSK3B
IKBKB
IKBKE
MEK1
-1
0
-1
-1
9
-1
-3
-1
0
PDK1
PI3K
PI3K
PI3K
PI4K
PRKCZ
SPHK1
TIE2
-1
86
42
88
-3
0
-1
1
3
TRKA
CDK7
MEK2
1
-10
9
(MAP2K2)
P70S6K1
(RPS6KB1)
RPS6KB2
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
11
12
-1
-3
23
mTOR
89
Tissue concentration of 10
In dose-ranging pharmacokinetic study, compound
10 was evaluated for various parameters at three incremental
oral doses of 1, 3, and 10 mg/Kg in Swiss mice (Table 6). A
dose-dependent increase in plasma exposure (AUC) was
observed without any significant change in Tmax and T1/2 at
three different doses. However, Cmax did not show any dose
linearity at three tested doses.
160
140
120
100
80
60
40
Table 6: Detailed pharmacokinetic analysis of 10
20
0
Swiss Mouse PK (Oral)
Control
Liver
Liver A
Kidney
A
Lung A
Uterus
A
Brain A
Compound
10
dose
AUC
(nM/h)
26858
32146
76365
Cmax
(nM)
5014
6903
12558
Tmax
(h)
1.33
1.50
1.67
T1/2
(h)
2.66
2.74
2.52
Figure 2: Distribution/accumulation of 10 in tissues after 5 days repeat
dosing with 30 mg/Kg BID oral in Swiss female mice.
1 mg/Kg
3 mg/Kg
10 mg/Kg
Based on the encouraging in vitro and in vivo profile
of the lead 10, we planned to determine in vivo efficacy
against two different solid tumors. An efficacy study in two
individual xenograft models with CD1 nude female mice was
performed using SKOV-3 (ovarian cancer) and HCT116
(colon cancer) cells. In each xenograft study, tumor was
allowed to grow and sustain for a few days to a specific pre-
defined tumor volume before start of treatment with
compound 10. Animals were randomized and divided in
different groups (3 or 4 doses and one control) with five
animals per group. The average body weight of animals and
tumor volume was within the same range across all groups.
The three dosing groups in the case of SKOV-3 model were
treated with three different incremental oral doses of 10 (2.5,
5, and 10 mg/Kg; once-a-day) for 27 days. Similarly, four
dosing groups of HCT116 model were treated with four
different doses of 10 (2.5, 5, 10, and 20 mg/Kg once-a-day)
for 21 days. Tumor volume was determined every third day to
monitor the study progress for each dosing group (Figures 3
and 4). It was encouraging to see a considerable inhibition of
tumor growth in two different tumors after first 3 days at dose
as low as 2.5 mg/Kg. This reduction in tumor volume was
dose-dependent, with all treated animals responding in the
treated groups compared to the untreated group. The study was
terminated on day 28 for SKOV-3 and day 22 in the case of
HCT116 due to animal ethics reasons as untreated arms in two
cases had to be sacrificed because of large tumor volume. It
was exciting to see a considerable inhibition in tumor growth
in both studies within the initial 3 days at a dose as low as 2.5
mg/Kg to 20 mg/Kg QD, which was consistent with the
progress of the study.
Cmax: maximal concentration, Tmax: time of maximal, concentration in
hours; t1/2: half-life elimination; AUC: area under the curve
For the planned dose tolerance and efficacy studies in
rodents, multi-gram quantity of compound 10 was required.
Attempts to get gram-scale synthesis of 10 using Scheme 1
through Suzuki coupling of corresponding boronic acid was
not efficient due to practical limitations involving poor yields
and/or need of a microwave reactor. Most of the gram-scale
attempts for Suzuki coupling of boronic acid led to undesired
dehalogenation of 13 as a major product. To solve this
problem, a Stille coupling based synthesis was developed as
presented in Scheme 2. An organo-tin reagent (14) was
synthesized in lab.21 The core 13 was subjected to a Stille
coupling with 14, followed by a treatment with a premixed
solution of HCl in dioxane lead to formation of compound 10
in high yield.21 This reaction was performed on 10 gm scale to
afford sufficient amount of 10 to support all necessary animal
experiments.
A repeat dose tolerance study was performed at two
different doses of 10 mg/Kg and 30 mg/Kg, twice daily (BID),
oral for 5 days, and 12 days respectively. For this, three study
groups (two doses and one vehicle control group) of mice
were selected. All three groups were observed daily for
bodyweight loss as well as other vital clinical signs such as
loss of appetite, body coat, eyes, feces, and general body
conditions. In the 10 mg/Kg group after 5 days of treatment,
no significant average weight loss was observed compared to
vehicle control. There was no change in clinical or vital signs
seen in the 10 mg/Kg BID dosing group. However, slight
piloerection was observed on the 9th day in 30 mg/Kg BID
dosing arm. A tissue distribution analysis was done in Swiss
female mice. Mice were dosed orally at 30 mg/Kg, twice a day
for five consecutive days to determine the drug accumulation
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 12
quoted in parts per million (ppm) relative to internal solvent
1
reference (CDCl3 δ = 7.26 for H NMR and δ = 77.0 for 13C
Tumor Volume after dosing of 10 (2.5, 5 and 10mpk) vs control
1
2
3
4
5
6
7
8
9
NMR and DMSO-d6 δ = 2.50 for 1H NMR and δ = 39.9 for 13C
NMR). Coupling constants are given in Hz. Data is reported as
followed: chemical shift, multiplicity (s = singlet, bs = broad
singlet, d = doublet, t = triplet, dd = double doublet, dt =
double triplet, m = multiplet), coupling constants (Hz), and
integration. Purity of all new compounds was > 95% and
confirmed using high performance liquid (HPLC)
chromatography. The actual purity of individual compounds is
mentioned in experimental detail. The detailed HPLC method
used for purity determination is provided below. Mass spectra
were recorded in ESI mode on an Agilent 6120 Quadrupole
LC/MS instrument. HRMS spectra were recorded on Agilent
HRMS-6540-UHD machine at IIIM Jammu, INDIA. Authors
confirm that all animal experiments performed in the
manuscript were conducted in compliance with institutional
guidelines following ethical guidelines and necessary
approvals.
990.0
890.0
790.0
690.0
590.0
490.0
390.0
290.0
190.0
90.0
Control
SPR519@ 10 MPK
SPR519@ 5 MPK
SPR519@ 2.5 MPK
*
**
**
*
**
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
Day 0 Day 3 Day 6 Day 9 Day 12Day 15Day 18Day 21Day 24Day 27
Figure 3: Tumor growth inhibition study after oral dosing of 10 in mice
xenograft model, inoculated with SKOV-3 cell line. The tumor volume
was determined at each time point. Statistics analysis was done using two-
way ANOVA with Dunnett's multiple comparisons test: *p<0.05;
**p<0.01; n=5
HPLC method for chemical purity determination: The
chemical purity of the final compounds and/or intermediates
1190.0
Tumor Volume after dosing of 10 (2.5, 5, 10, 20mpk) vs control
1090.0
were
characterized
by
high
performance
liquid
990.0
chromatography (HPLC) using
a
Waters Millenium
Control
890.0
790.0
690.0
590.0
490.0
390.0
290.0
190.0
90.0
chromatography system with a 2695 Separation Module
(Milford, MA). The analytical columns used for this analysis
was Gemini C-8 4.6 x 150 mm 5 micron with flow rate 1
mL/min of mobile phase consist of a mixture of water and
acetonitrile. A gradient elution was used, typically starting
with 5% acetonitrile/ 95% water contained 0.1%
trifluoroacetic acid (TFA) with a progression to 100%
acetonitrile over a period of 20 min. Compounds were
detected by ultraviolet light (UV) absorption at either 220 or
240 nm. HPLC grade solvents were procured from Merck and
Fisher Scientific.
SPR519 @ 20 MPK
SPR519 @ 10 MPK
SPR519@ 5 MPK
SPR519@ 2.5 MPK
*
**
*
**
*
Day 0
Day 3
Day 6
Day 9
Day 12
Day 15
Day 18
Day 21
Figure 4: Tumor growth inhibition study after oral dosing of 10 in mice
xenograft model, inoculated with colon cancer (HCT116). The tumor
volume was determined at each time point. Statistics analysis was done
using two-way ANOVA with Dunnett's multiple comparisons test:
*p<0.05; **p<0.01; n=5
Synthesis of compound 26, 13 and 14: Compounds was
synthesized as per previously reported procedures.17,21
General procedure for synthesis of compounds 1-12:21 To a
solution of compound 13 (1 mmol) in DME: H2O (4:1; 10 mL)
was added corresponding aryl boronic acid or boronate ester
(1.5 mmol) and Na2CO3 (3 mmol). Degassing of the reaction
was done for 5 min (using nitrogen and vacuum in repeated
cycles) followed by addition of Pd(PPh3)4 (0.1 mmol) under
inert atmosphere. The reaction mixture was heated at 100-110
°C for 16-20 h. Alternatively, reaction can be performed using
microwave irradiation for 1-2 h. The progress of reaction was
monitored using TLC (1-5% MeOH in DCM). After
completion, excess of solvent was removed under vacuum, the
reaction mass was diluted with water and extracted with of
DCM (3 x 100 mL). The organic layer was separated, dried
over Na2SO4, filtered, and concentrated under reduced
pressure to obtain crude product. Further purification was
done by silica gel chromatography to obtain pure product. In
case of compounds 3, 8, 10 and 12, since N-Boc protected aryl
boronate esters were used, obtained products were further
treated with HCl in dioxane solution at RT for 30-60 min and
dried under vacuum to obtain corresponding hydrochloride
salt.
CONCLUSION
In conclusion, our efforts towards development of
drug like molecules for the treatment of solid tumor led to the
identification of a drug lead 10 (SPR519). SPR519 is a potent
inhibitor of PI3K and mTOR kinases. The identified lead
was evaluated in vivo and found to be orally bioavailable, and
well-tolerated up to a high dose of 30 mg/Kg. SPR519 was
found to be efficacious in animal models of ovarian and colon
cancer. It was encouraging to see noticeable efficacy in
ovarian cancer xenograft at lower oral dose (2.5 mg/Kg).
Further pre-clinical investigations are in progress to develop
SPR519 as a candidate for investigational new drug studies.
EXPERIMENTAL SECTION
General information: Unless otherwise noted, commercial
available materials were used without further purification. Air
sensitive reactions were carried out under argon atmosphere.
Anhydrous solvents were obtained from Sigma Aldrich,
Merck. Thin layer chromatography (TLC) was carried out
using 0.2 mm Kieselgel F254 (Merck) silica plates and
compounds viewed under an ultraviolet lamp. NMR spectra
were recorded on a Bruker 300 MHz spectrometer operating at
4-((4-(1H-indazol-4-yl)-6-morpholino-1,3,5-triazin-2-
yl)oxy)-N,N ’ -dimethylbenzamide hydrochloride salt (26):
Yellow solid; Yield: 83%; HPLC purity: 98.2%; mp: 150-155
300 MHz for H and 75 MHz for 13C. Chemical shifts (δ) are
1
ACS Paragon Plus Environment

Page 7 of 12
Journal of Medicinal Chemistry
1
°C; H NMR (300 MHz, DMSO-d6): δ 8.21 (d, J = 8.1 Hz,
1H), 8.07 (s, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.54-7.57 (m, 2H),
7.44 (t, J = 7.5 Hz, 1H), 7.36-7.41 (d, J = 10.8 Hz, 2H), 4.01-
4.02 (m, 2H), 3.70-3.75 (m, 6H ), 3.03 (brs, 6H); 13C NMR (75
MHz, DMSO-d6): δ 35.4 (2CH3), 43.9, 44.4, 66.2 (2CH2),
115.2, 115.4, 121.5, 122.4, 122.9, 125.9, 128.3, 129.0, 129.5,
134.2, 134.8, 141.0, 153.1, 166.2, 170.0, 171.5, 172.6;
ESIMS: 446.2 [M+1]+. HRMS calcd. for C23H24N7O3:
446.1941, found: 446.1924 [M+H]+.
2H), 3.66 (s, 6H), 2.94-2.99 (brs, 6H), 2.40 (s, 3H); ESIMS:
436.2 [M+1]+.
1
2
3
4
5
6
7
8
4-(4-(1H-indol-4-yl)-6-morpholino-1,3,5-triazin-2-yloxy)-
N,N ’ -dimethylbenzamide (7): Off-white solid; Yield 64%;
1
HPLC purity: 87.3%; mp: 120-130 °C; H NMR (300 MHz,
DMSO-d6): δ 11.32-11.34 (m, 1H), 8.10-8.13 (m, 1H), 7.58-
7.61 (m, 1H), 7.52-7.55 (m, 2H), 7.35-7.37 (m, 2H), 7.28-7.32
(m, 1H), 7.13-7.22 (m, 1H), 6.81-6.83 (m, 1H), 3.99 (s, 2H),
3.69-3.73 (m, 6H), 3.01 (s, 6H); ESIMS: 445.1 [M+1]+.
9
4-(4-(3-hydroxyphenyl)-6-morpholino-1,3,5-triazin-2-
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
N,N
’
-dimethyl-4-((4-morpholino-6-(1H-pyrrolo[2,3-
yloxy)-N,N’-dimethylbenzamide (1): Off-white solid; Yield:
83%; HPLC purity: 99.7%; mp: 208-215 °C; H NMR (300
1
c]pyridin-4-yl)-1,3,5-triazin-2-yl)oxy)benzamide
hydrochloride salt (8) : Off-white solid; Yield: 66 %; HPLC
purity: 97.8%; mp: 206-215 °C; H NMR (300 MHz, DMSO-
d6): δ 12.14 (s, 1H), 9.09 (d, J = 1.74 Hz, 1H), 8.85 (d, J =
1.62 Hz, 1H), 7.58-7.59 (m, 1H), 7.51 (d, J = 8.4 Hz, 2H),
7.35 (d, J = 8.4 Hz, 2H), 6.60-6.61 (m, 1H), 3.99 (bs, 2H),
3.63-3.70 (m, 6H), 2.97-2.99 (brs, 6H); ESIMS: 446.5
[M+1]+.HRMS calcd. for C23H24N7O3: 445.1941, found:
446.1919 [M+1]+.
MHz, DMSO-d6): δ 9.67 (s, 1H), 7.66-7.70 (m, 2H), 7.50 (d, J
= 9.0 Hz, 2H), 7.33 (d, J = 9.0 Hz, 2H), 7.27 (m, 1H), 6.95 (d,
J = 9.15 Hz, 1H), 3.94 (s, 2H), 3.62-3.69 (brs, 6H), 2.95-2.99
(m, 6H); ESIMS: 422.2 [M+1]+. HRMS calcd. for
C23H24N7O3: 421.1828, found: 422.1852 [M+1]+.
1
4-(4-(2-aminopyrimidin-5-yl)-6-morpholino-1,3,5-triazin-2-
yloxy)-N,N ’ -dimethylbenzamide hydrochloride salt (2):
Off-white solid; Yield: 74%; HPLC purity: 97.5%; mp: 245-
1
4-(4-(1H-benzo[d][1,2,3]triazol-4-yl)-6-morpholino-1,3,5-
triazin-2-yloxy)-N,N ’ -dimethylbenzamide (9): Off-white
solid; Yield 55%; HPLC purity: 86.7%; mp: 142-150 °C; H
NMR (300 MHz, DMSO-d6): δ 8.18-8.21 (m, 1H), 7.87-7.90
(m, 1H), 7.36-7.56 (m, 5H), 3.79-3.85 (m, 2H), 3.60-3.66 (m,
6H), 3.59 (m, 1H), 2.99-3.01 (brs, 6H); ESIMS: 447.5
[M+1]+.
255 °C; H NMR (300 MHz, DMSO-d6): δ 8.95 (s, 2H), 7.46-
7.50 (m, 4H), 7.30-7.33 (m, 2H), 3.91-3.92 (m, 2H), 3.64-3.66
(m, 2H), 3.60 (s, 4H), 2.94-2.98 (brs, 6H); 13C NMR (75 MHz,
MeOD): δ 34.8, 43.2, 43.7, 65.6, 117.2, 121.6, 128.2, 133.3,
152.3, 158.8, 164.9, 165.1, 169.4, 169.7, 170.2; ESIMS:
423.2 [M+1]+. HRMS calcd. for C20H23N8O3: 422.1893, found:
423.1890 [M+1]+.
1
4-((4-(2-aminothiazol-5-yl)-6-morpholino-1,3,5-triazin-2-
yl)oxy)-N,N ’ -dimethylbenzamide hydrochloride salt (10):
Off-white solid. Yield: 85%; mp: 276-283 °C; HPLC purity:
99.6%; 1H NMR (300 MHz, DMSO-d6): δ 8.39 (bs, 2H), 7.94
(s, 1H), 7.46 (d, J = 8.4 Hz, 2H), 7.28 (d, J = 8.4 Hz, 2H), 3.80
(s, 2H), 3.57-3.64 (m, 6H), 2.93-2.98 (brs, 6H); 13C NMR (75
MHz, MeOD): δ 34.3, 43.5, 44.0, 65.9, 66.0, 121.6, 123.4,
128.2, 129.7, 133.2, 153.0, 165.1, 166.2, 170.4, 171.3, 171.5;
ESI-MS: 428.2 [M+1]+. HRMS calcd. for C19H22N7O3S:
427.1505, found: 428.1488 [M+1]+.
4-((4-(6-aminopyridin-3-yl)-6-morpholino-1,3,5-triazin-2-
yl)oxy)-N,N ’ -dimethylbenzamide hydrochloride salt (3):
White solid; Yield: 81%; mp: 250-259 °C; HPLC purity:
1
96.4%; H NMR (300 MHz, DMSO-d6): δ 8.71 (s, 1H), 8.49-
8.52 (m, 2H), 7.49 (d, J = 8.6 Hz, 2H), 7.32 (d, J = 8.6 Hz,
2H), 7.01 (d, J = 9.2 Hz, 1H), 3.94 (bs, 2H), 3.67 (m, 2H),
3.61 (s, 4H), 2.95-2.98 (brs, 6H); 13C NMR (75 MHz, MeOD):
δ 34.7, 43.3, 43.7, 65.7, 113.1, 120.0, 121.4, 128.4, 133.4,
138.4, 141.2, 152.2, 155.5, 165.0, 168.8, 169.4, 170.3;
ESIMS: 422.2 [M+1]+. HRMS calcd. for C21H24N7O3:
421.1941, found: 422.1922 [M+1]+.
4-((4-(2-amino-4-methylthiazol-5-yl)-6-morpholino-1,3,5-
triazin-2-yl)oxy)-N,N ’ -dimethylbenzamide hydrochloride
salt (11): White solid; Yield 70%; HPLC purity: 95.8%; mp:
4-((4-(6-amino-5-methylpyridin-3-yl)-6-morpholino-1,3,5-
triazin-2-yl)oxy)-N,N-dimethylbenzamide (4): Sticky brown
1
1
225-232 °C; H NMR (300 MHz, DMSO-d6): δ 7.65 (s, 2H),
oil; Yield: 66%; HPLC purity: 99.0%; H NMR (DMSO-d6,
7.44-7.47 (m, 2H), 7.25-7.28 (m, 2H), 3.75 (m, 2H), 3.61-3.65
(m, 6H), 2.92-2.98 (brs, 6H), 2.30 (s, 3H); ESIMS: 442.3
[M+1]+.
300 MHz): δ 8.59 (s, 1H), 8.37 (s, 1H), 8.03 (bs, 2H), 7.49 (d,
J = 8.7 Hz, 2H), 7.32 (d, J = 8.7 Hz, 2H), 3.95 (bs, 2H), 3.68
(bs, 2H), 3.60 (s, 4H), 2.95-2.99 (m, 6H), 2.21 (s, 3H);
ESIMS: 436.3 [M+1]+.
4-((4-(2-amino-1H-imidazol-5-yl)-6-morpholino-1,3,5-
triazin-2-yl)oxy)-N,N ’ -dimethylbenzamide hydrochloride
salt (12): Sticky yellow solid; Yield 62%; HPLC purity:
4-(4-(6-amino-4-methylpyridin-3-yl)-6-morpholino-1,3,5-
triazin-2-yloxy)-N,N ’ -dimethylbenzamide (5): Light green
1
1
97.9%; H NMR (300 MHz, DMSO-d6): δ 10.97 (bs, 1H),
solid; Yield: 69%; HPLC purity: 92.1%; mp: 118-125 °C; H
7.44-7.47 (m, 2H), 7.25-7.32 (m, 2H), 5.81 (m, 2H), 3.87 (s,
2H), 3.52-3.64 (m, 6H), 2.94-2.98 (brs, 6H); ESIMS: 411.7
[M+1]+.
NMR (DMSO-d6, 300 MHz): δ 8.27-8.30 (m, 1H), 7.50-7.53
(m, 3H), 7.33 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 5.7 Hz, 1H),
3.79 (s, 3H), 3.63-3.67 (m, 6H), 2.95-2.99 (brs, 6H), 2.40 (s,
3H); ESIMS: 436.3 [M+1]+.
Gram scale synthesis of 4-((4-(2-aminothiazol-5-yl)-6-
morpholino-1,3,5-triazin-2-yl)oxy)-N,N
’
-
4-(4-(6-amino-2-methylpyridin-3-yl)-6-morpholino-1,3,5-
dimethylbenzamide hydrochloride salt (10): To a mixture of
compound 13 (10 g, 25.0 mmol) in anhydrous DMF (20 mL)
was added tert-butyl 5-(tributylstannyl)thiazol-2-ylcarbamate
14 (14.7 g, 30.0 mmol). The resulting mixture was degassed
(repeated nitrogen and vacuum cycles) for 15 min followed by
triazin-2-yloxy)-N,N’-dimethylbenzamide (6): Pale yellow
1
solid; Yield: 69%; HPLC purity: 95.6%; mp: 220-230 °C; H
NMR (DMSO-d6, 300 MHz): δ 7.77-7.93 (m, 2H), 7.49-7.58
(m, 3H), 7.32 (d, J = 8.5 Hz, 2H), 7.12-7.15 (m, 1H), 3.76 (s,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 12
addition of Pd(PPh3)4 (2.85 g, 2.50 mmol) under inert RT before a 10 µL preparation of kinase quench buffer and
1
2
3
4
5
6
7
8
atmosphere. The reaction mixture was heated to reflux under
inert atmosphere for 4 h. Progress of the reaction was
monitored by TLC. After consumption of starting material, the
excess of DMF was removed under reduced pressure and
reaction mixture was extracted with DCM (3 x 500 mL). The
organic layer was washed with brine, seperated and dried over
anhydrous Na2SO4, filtered and evaporated under reduced
pressure to obtain viscous dark brown material which was
further purified by silica gel chromatography (Eluent: 0-35%
EtOAC/ Hexane mixture) to obtain product as light yellow
Tb–labeled Antibody in TR-FRET dilution buffer was added.
Plate was incubated at RT for 1 h. FRET signal was read in
Envision Multilabel Reader. TR FRET Ratio (Fluorescence
520 nm/Fluorescence 486 nm) was plotted against the
concentration of inhibitor and data was fit to Non Linear
Regression curve fit (sigmoidal dose response curve with
variable slope-four parameters) using Graph pad prism 5.
In vitro Anti-proliferative activity by MTT assay:
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
A2780: A2780 cells (human ovarian cancer) were cultured in
DMEM (Sigma) supplemented with 10% FBS, L-glutamine
and antibiotics. Cells were plated in the same medium at the
density of 10000 cells/ well into 96 well tissue culture plates
and allowed to adhere overnight. Cells were treated with
compounds in the concentration range 20 µM - 0.001 µM for
48 h. A 10 µL solution of MTT (3-(4,5-dimethythiazol-2-yl)-
2,5-diphenyl tetrazolium bromide) 5 mg/ mL in PBS was
added to the wells. The plates were incubated for 4 h. The
plates were then centrifuged at 1000 rpm for 5 min,
supernatant was removed and 100 µL DMSO was added and
mixed on shaker for 30 min. Absorbance was measured at 550
nm in an Envision Multilabel Plate Reader (Perkin Elmer).
The percentage of inhibition was calculated and a dose
response curve was plotted using Graph pad Prism with a non-
linear regression fit (Log inhibitor vs. variable slope, four
parameters).
sticky
solid,
i.e.,
tert-butyl
(5-(4-(4-
(dimethylcarbamoyl)phenoxy)-6-morpholino-1,3,5-triazin-2-
yl)thiazol-2-yl)carbamate, [N-Boc protected 10 (9.43 g, 65%).
ESIMS: 528.1 [M+1]+. This obtained product (9.4 g, 17.8
mmol) was dissolved in anhydrous DCM (100 mL). To this
mixture, 4N HCl in dioxane (100 mL) was added drop wise at
0 °C and resulting mixture was then stirred at RT for 6 h. After
completion (TLC), reaction mixture was evaporated under
vacuum and residue so obtained was treated with diethyl ether
and dried under vacuum to obtain compound 10 as off-white
solid. Yield: (7.02g, 85%).
PI3Kα enzyme assays: Kinase activity was measured in a
homogeneous assay in low-binding Corning 384 well white
plates. Detection was performed by HTRF using ADP
detection mixture comprising of ADP HiLyte 647 tracer bound
to an ADP2 antibody-Tb conjugate. (Transcreener® ADP2
TR-FRET Red Assay, Bell Brook Labs, Madison, WI) 2.5 µL
of compound ranging from 10 µM - 0.001 µM were added to a
384 well plate. 2.5 µL of diluted PI3K alpha 150 ng/mL
(Invitrogen Life Sciences) in a reaction buffer consisting of
50mM HEPES, pH 7.4, 3mM MgCl2, 2 mM DTT, 0.05%
CHAPS, 50 mM NaCl, and 1mM EGTA was added and the
plates were incubated for 15 min with shaking. The reaction
was initiated by addition of PIP2 & ATP (50 µM PIP2 & 1
µM ATP in final assay). The samples were incubated for 60
minutes. 10 µL of ADP Detection mix was added and
incubated for 1 h. The plate was read in an Envision
Multilabel Plate Reader. Fluorescence was measured at 665
nm & 615 nm. The ratio between the signal at 665 and 615 nm
was calculated. The percentage of inhibition was calculated
and plotted against the concentration of inhibitor. IC50 was
calculated using Graph Pad prism Non-linear regression fit
(Log inhibitor vs variable slope using four parameters).
PC3: PC3 human prostate cancer cells (American Type
Culture Collection, Manassas, VA) were cultured in RPMI
1640 (Invitrogen, Carlsbad, CA) supplemented with 10% fetal
bovine serum and 1% penicillin and streptomycin in a 5% CO2
atmosphere at 37 °C. Cells were plated in the same medium at
the density of 10000 cells/ well into 96 well tissue culture
plates. Cells were treated with compounds in the concentration
range 20 µM - 0.001 µM for 48 h. A 10 µL solution of MTT
(3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide) 5 mg/ ml in PBS was added to the wells. The plates
were incubated for 4 h. The plates were then centrifuged at
1000 rpm for 5 min, supernatant was removed and 100 µL
DMSO was added and mixed on shaker for 30 min.
Absorbance was measured at 550 nm in an Envision
Multilabel Plate Reader (Perkin Elmer). The percentage of
inhibition was calculated and a dose response curve was
plotted using Graph pad Prism with a non-linear regression fit
(Log inhibitor vs. variable slope, four parameters).
mTOR Assay: Compounds were evaluated for mTOR activity
using invitro kinase assay. mTOR activity is measured in vitro
by determining the level of phosphorylation of the protein
substrate 4EBP-1. The phosphorylation of GFP-4E-BP1 at
threonine residue is recognized by Ab Tb-Anti-p4EBP1,
which results in time resolved fluorescence energy transfer
(TR-FRET) between GFP and Terbium in the Ab-Protein
complex. Recombinant mTOR (FRAP1), Kinase reaction
buffer, GFP-4E-BP1 and LanthaScreen Tb-anti-4E-BP1
(pThr46), TR FRET dilution buffer, Kinase Quench Buffer
were obtained from Invitrogen. Kinase reactions were
performed in 10 µL volume in 384 well plates. Serially diluted
compounds (10 µM-0.0005 µM) prepared in DMSO were
incubated with mTOR in the assay buffer for 15 min. Reaction
was initiated by the addition of substrate (GFP-4E-BP1) and
ATP at final conc. of 400 nM and 8 µM ATP in the reaction
respectively. Kinase reaction was allowed to proceed for 1 h at
SKOV3: SK-OV-3 human ovarian cells was grown in
McCoy's 5a medium supplemented with 10% fetal bovine
serum, 100 U/mL penicillin and 100 μg/mL streptomycin at
37ºC in an atmosphere of 5% CO2 in air. Cells were plated in
the same medium at the density of 10000 cells/ well into 96
well tissue culture plates. Cells were treated with compounds
in the concentration range 20 µM - 0.001 µM for 48 h. A 10
µL solution of MTT (3-(4,5-dimethythiazol-2-yl)-2,5-diphenyl
tetrazolium bromide) 5 mg/mL in PBS was added to the wells.
The plates were incubated for 4 h. The plates were then
centrifuged at 1000 rpm for 5 min, supernatant was removed
and 100 µL DMSO was added and mixed on shaker for 30
min. Absorbance was measured at 550 nm in an Envision
Multilabel Plate Reader (Perkin Elmer). The percentage of
inhibition was calculated and a dose response curve was
ACS Paragon Plus Environment

Page 9 of 12
Journal of Medicinal Chemistry
plotted using Graph pad Prism with a non-linear regression fit µM and 1 nM, respectively. At the end of the incubation, the
1
2
3
4
5
6
7
8
(Log inhibitor vs. variable slope, four parameters).
reaction was quenched by the addition of an EDTA-containing
buffer. Substrate and product (i.e., phosphorylated substrate)
were separated electrophoretically using the microfluidic-
based LabChip 3000 Drug Discovery System from Caliper
Life Sciences and both were quantitated by fluorescence
intensity.
Microsomal stability assay: 10 µM Compound was
incubated with 0.5 mg/mL liver microsomes (MLM, DLM,
HLM purchased from BD Gentest). NADPH was maintained
at 1 mM in the final assay in 700 µL reaction volume. The
reaction was evaluated at 0, 15, 30, 60 & 120 min and was
terminated by the addition of acetonitrile. Samples were
centrifuged for 10 min at 10000 rpm and supernatant analyzed
using HPLC. Percentage parent remaining was calculated
considering percent parent area at 0 min as 100%.
Cyp P450 inhibition assay: The in vitro inhibition of
cytochrome P450 (CYP) 2C9, 2C19, 2D6, and 3A4 isozymes
by SPR519 was evaluated in human liver microsomes. The
inhibition of each isozyme by the test items was monitored by
following the production of a selected metabolite of an FDA-
recommended probe substrate of the respective isozyme using
HPLC and LC-MS/MS technique. For each isozyme, the
incubations were performed in duplicate in a 96-well plate.
Microsomes-buffer-substrate mixtures (MBS mix) were
prepared by pre-mixing appropriate volumes of buffer, human
liver microsomes and CYP-specific probe substrates. This
MBS mixture (179 µL) was transferred to a 96-well plate and
1 µL of inhibitor stock solutions were spiked into it such that
the final target inhibitor concentrations were achieved. The
plate was incubated at 37 °C for 5 min following which the
reaction was initiated by the addition of 20 µL of 10 mM
NADPH. The reaction plate was incubated at 37 °C for a pre-
determined time and subsequently it was quenched with 200
µL of respective stop solutions. The formation of each
substrate metabolite was monitored using HPLC and LCMS.
The effect of increasing the concentration of test items on the
production of the metabolite was evaluated, and the
concentration of inhibitor required for a 50% reduction in the
measured enzyme activity was determined.
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
Experimental details for pharmacokinetic evaluations of
the compounds: Female Swiss mice, (3 animals per group)
after overnight fasting were dosed orally (via gavage) with test
compound in 0.5% CMC/0.5% Tween 80 (10 mL/kg) at dose
levels of 1, 3, 10 mg/kg. Blood was collected by serial
bleeding at 0.16 h, 0.5 h, 1 h, 2 h, 4 h, 6 h, 8 h, respectively in
heparinized tubes. Blood samples were centrifuged at 10,000
rpm for 5 min at 4 °C to obtain the plasma, which were
aspirated into separate labeled tubes and stored at -80 °C. 400
ng/mL of Verapmil in acetonitrile was used as the drug
extraction solvent for extracting drug from plasma. Extraction
solvent was added to plasma was vortexed and shaken using
an auto-shaker for 10 min, centrifuged at 10000 rpm for 10
min at 40 °C. Supernatant was retained for analysis.
Acetonitrile and plasma calibration curves were generated and
percentage of drug recovery from plasma determined by a
quantitative analysis using LCMS. Quantitative analysis was
done by liquid chromatography tandem mass spectrometer
(API3000 LC-MS/MS). Cmax, Tmax, AUC and t1/2 were
calculated using Graph Pad PRISM version 5.04.
Experimental details for multiple day dose tolerance
study: Female swiss mice were dosed 10mpk for 5 days BID
and 30mpk for 12 days BID to evaluate drug related toxicity in
mice on repeat dosing. 3 Female Swiss mice, were
administered 10mpk of SPR519 formulated in CMC 0.5% and
Tween 80, by oral gavage twice a day (BID) for a period of 5
days. 2 mice were kept as control. Parameters like body
weight, body temperature, body coat, feed intake and general
activities were monitored on a daily basis. 4 Female Swiss
mice, were administered 30mpk of test compound (SPR519)
formulated in CMC 0.5% and Tween 80, by oral gavage twice
a day (BID) for a period of 12 days. 2 mice were kept as
control. Parameters like body weight, body temperature, body
coat, feed intake and general activities were monitored on a
daily basis.
Experimental detail for hERG activity analysis: HERG-
Lite monitors the expression of hERG (human ether-à-go-go)-
related gene at the cell surface in two different stable
mammalian cell lines. One cell line acts as a biosensor for
drugs that inhibit hERG trafficking, while the other predicts
hERG blockers based on their ability to act as
pharmacological chaperones. The in vitro effects of the
selected compounds were evaluated for effecting changes in
relative surface expression (RSE) of the hERG potassium
channel. Trafficking inhibition was assessed by monitoring
surface expression of the wild-type (WT) channel, while
channel block was predicted based on the rescue of surface
expression of a hERG single mutant (SM). Compounds were
tested at 1, 10, 30 µM concentrations. Geldanamycin at 1 µM
was used as inhibitor of hERG trafficking and Astemizole was
used as a standard hERG binder.
Experimental details for tissue distribution study of
SPR519 after repeat dosing: Three female Swiss mice, were
administered 30mpk of SPR519 formulated in CMC 0.5% and
Tween 80, by oral gavage twice a day (BID) for a period of 5
days. Two mice were kept as control. Parameters like body
weight, body temperature, body coat, feed intake and general
activities were monitored on a daily basis. On the last day,
mice were anesthetized by isofluorane and euthanized via
cardiac puncture. Liver, brain, uterus and kidneys were
removed from each animal by gross dissection. After flushing
with saline, tissues were stored. Tissues were rinsed gently,
but thoroughly with water to remove remaining traces of blood
before storage. Dissecting instruments were also washed
between tissue procurements to avoid cross-contamination.
Kinase biochemical assay for 23 kinases by microfluidic
mobility shift detection: SPR519 was tested against 23
kinases at a single test concentration of 1 µM in duplicate
wells. Test compound was diluted in DMSO to 100x assay
concentration and then further diluted in assay buffer. The
final concentration of DMSO in all assays was kept at 1%.
Reference compounds, Staurosporine and Wortmannin, were
tested in 8-point IC50 format using 3-fold dilution steps.
Test compound, enzyme, fluorescently labeled substrate, and
cofactors (ATP and Mg2+) were combined in a well of a
microtiter plate and incubated typically for 3 h at 25 ºC. ATP
was used at
1 x Km concentration, while typical
concentrations of the peptide substrate and the kinase are 1
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Aqueous homogenates of the tissue were prepared. Extraction PI3K, phosphoinositide 3-kinase; Akt, protein kinase B;
Page 10 of 12
1
2
3
4
5
6
7
8
solvent was added to tissues vortexed and shaken using an
auto-shaker for 10 min, centrifuged at 10000 rpm for 10 min at
40 °C. Supernatant was retained for analysis. Acetonitrile and
plasma calibration curves were generated and percentage of
drug recovery from tissue was determined by a quantitative
analysis using LCMS. Tissue levels of the drug were evaluated
by liquid chromatography tandem mass spectrometer
(API3000 LC-MS/MS).
mTOR, mechanistic or mammalian target of rapamycin; S6K,
S6 kinase; mTOR, mammalian target of rapamycin; TR-
FRET, time-resolved Forster resonance energy transfer;
DIPEA, N,N-diisopropylethylamine; DMF,
Dimethylformamide; PK, pharmacokinetic; AUC, Area Under
Curve; DTT, Dithiothreitol; CHAPS, 3-[(3-
Cholamidopropyl)dimethylammonio]-1-propanesulfonate;
EGTA, Ethylene-bis(oxyethylenenitrilo)tetraacetic acid
9
Experimental detail for in vivo efficacy study: The
xenograft was generated by subcutaneous injection of
HCT116 and SKOV3 in CD1 nude mice. Animals were
randomized in 5 groups (Vehicle control, Standard Drug, test
Compound in multiple doses) and treatment was started when
average tumor size was 120-130 mm3. The body weight was
checked prior to dosing or alternate day as required. The size
of the tumor was measured every alternate day. The tumor size
was measured by taking longest length and shortest width of
the tumor with digital caliper. The tumor volume was
calculated with the formula (width2 x length)/2. On the last
day of study, CO2 overdosing was done to euthanize the
animals to complete the experiment. Tumor isolation was done
and snap frozen in liquid nitrogen followed by storage at -80
°C for further studies. Complete liver and lung tissue was
isolated and snap frozen.
REFERENCES
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
1. Cantley, L. C. The phosphoinositide 3-kinase pathway.
Science 2002, 296, 1655−1657.
2. Toker, A.; Cantley, L. C. Signalling through the lipid
products of phosphoinositide-3-OH kinase. Nature 1997,
387, 673−676.
3. Fruman, D. A.; Rommel, C. PI3K and cancer: Lessons,
challenges and opportunities. Nat. Rev. Drug Discov. 2014,
13, 140−156.
4. Jean, S.; Kiger, A. A. Classes of phosphoinositide 3-
kinases at a glance. J. Cell Sci. 2014 127, 923–928.
5. Thorpe, L. M.; Yuzugullu, H.; Zhao, J. J. PI3K in cancer:
Divergent roles of isoforms, modes of activation and
therapeutic targeting. Nat. Rev. Cancer 2015, 15, 7−24.
6. Janku, F. Phosphoinositide 3-kinase (PI3K) pathway
inhibitors in solid tumors: From laboratory to patients.
Cancer Treat. Rev. 2017, 59, 93−101.
7. Janku, F.; Yap, T. A.; Bernstam, M. F. Targeting the PI3K
pathway in cancer: Are we making headway? Nat. Rev.
Clin. Oncol. 2018, 15, 273-291.
AUTHOR INFORMATION
Corresponding Authors
8. Enrique Rozengurt, E.; Soares, H.P.; Smith, J. S.
Suppression of feedback loops mediated by PI3K/mTOR
induces multiple over-activation of compensatory
pathways: An unintended consequence leading to drug
resistance. Mol. Cancer Ther. 2014, 13, 2477–2488.
9. Liu Y. N.; Wan R. Z.; Liu Z. P. Recent developments of
small molecule PI3K/mTOR dual inhibitors. Mini Rev.
Med. Chem. 2013, 13, 2047−2059.
*†dinesh.mahajan@thsti.res.in; chemidinesh@gmail.com
*dugar.s@sphaerapharma.com
Present Addresses
† Principal Scientist-II, Translational Health Science and
Technology Institute (THSTI), NCR Biotech Cluster, Faridabad,
INDIA 121001.
10. Garces, A.E.; Stocks, M.J. Class I PI3K clinical candidates
and recent inhibitor design strategies:
chemistry perspective. J. Med. Chem., 2019, 62, 4815-
4850.
A medicinal
Notes
11. Yaguchi, S.I.; Fukui, Y.; Koshimizu, I.; Yoshimi, H.;
Matsuno, T.; Gouda, H. ; Hirono, S.; Yamazaki, K.;
Yamori, T. Antitumor activity of ZSTK474, a new
phosphatidylinositol 3-kinase inhibitor. J. Natl. Cancer
Inst. 2006, 98, 545-556.
12. Venkatesan, A.M.; Dehnhardt, C.; Santos, E.D.; Chen, Z.;
Santos, O.D.; Kaloustian, S.A.; Khafizova, G.;
Brooijmans, N.; Mallon, R.; Hollander, I.; Feldberg, L.;
Lucas, J.; Yu, K.; Gibbons, J.; Abraham, R.T.; Chaudhary,
All experimental in vivo work related to animals were
performed after due approvals and performed in accordance
with institutional guidelines as defined by Institutional Animal
ethics committee.
The authors declare the following competing financial interest
(s): All the authors are current or past employee of Sphaera
Pharma. SD is shareholder of Sphaera Pharma.
I.;
derivatives: Potent adenosine 50-triphosphate competitive
phosphatidylinositol-3-kinase/mammalian target of
Mansour,
T.S.
Bis(morpholino-1,3,5-triazine)
ACKNOWLEDGMENT
Authors wish to acknowledge Dr Sandip Bharate -IIIM Jammu
for kind help on HRMS experiments and Mr. Ashish Dugar at
Sphaera Pharma for his untiring efforts for extraction of old
data files.
rapamycin inhibitors: Discovery of compound 26 (PKI-
587), a highly efficacious dual inhibitor. J. Med. Chem.
2010, 53, 2636–2645.
13. Beaufils, F.; Cmiljanovic, N.; Cmiljanovic, V.; Bohnacker,
T.; Anna Melone, A.; Marone, R.; Jackson, E.; Zhang, X.;
Sele, A.; Borsari, C.; Mestan, J.; Hebeisen, P.; Hillmann,
P.; Giese, B.; Zvelebil, M.; Fabbro, D.; Williams, R.L.;
Rageot, D.; Wymann, M.P. 5 ‑ (4,6-Dimorpholino-1,3,5-
triazin-2-yl)-4-(trifluoromethyl)pyridin-2-amine (PQR309),
a potent, brain-penetrant, orally bioavailable, Pan-Class I
PI3K/mTOR inhibitor as clinical candidate in oncology. J.
Med. Chem. 2017, 60, 7524−7538.
ASSOCIATED CONTENT
Supporting Information
Images of NMR (1H and 13C) as well as MS/HRMS spectra,
HPLC chromatograms and molecular formula strings for all new
compounds described in this paper are available as supporting
information at http://pubs.acs.org.
14. Borsari, C.; Rageot, D.; Beaufils, F.; Bohnacker, T.; Keles,
E.; Buslov, I.; Melone, A.; Sele, A.M.; Hebeisen, P.;
Fabbro, D.; Hillmann, P.; Wymann, M.P. Preclinical
ABBREVIATIONS
ACS Paragon Plus Environment

Page 11 of 12
Journal of Medicinal Chemistry
development of PQR514, a highly potent PI3K inhibitor
bearing difluoromethyl−pyrimidine moiety. ACS Med.
Chem. Lett. 2019, 10, 1473−1479.
based molecules as PI3Kinase inhibitors. Bioorg. Med.
Chem. Lett. 2015, 25, 3142−3146.
1
2
3
4
5
6
7
8
19. Folkes, A. J.; Ahmadi, K.; Alderton, W. K.; Alix, S.;
Baker, S. J.; Box, G.; Chuckowree, I. S.; Clarke, P. A.;
Depledge, P.; Eccles, S. A.; Friedman, L. S.; Hayes, A.;
Hancox, T. C.; Kugendradas, A.; Lensun, L.; Moore, P.;
Olivero, A. G.; Pang, J.; Patel, S.; Pergl-Wilson, G. H.;
Raynaud, F. I.; Robson, A.; Saghir, N.; Salphati, L.; Sohal,
S.; Ultsch, M. H.; Valenti, M.; allweber, H. J. A.; Wan, N.
C.; Wiesmann, C.; Workman, P.; Zhyvoloup, A.; Zvelebil,
M. J.; Shuttleworth, S. J. The identification of 2-(1H -
Indazol-4-yl)-6-(4-methanesulfonyl-piperazin-1-ylmethyl)-
4-morpholin-4-yl-thieno[3,2-d]pyrimidine (GDC-0941) as
a potent, selective, orally bioavailable inhibitor of class I
PI3 kinase for the treatment of cancer†. J. Med. Chem.
2008, 51, 5522−5532.
20. Berndt, A.; Miller, S.; Williams, O.; Le, D. D.; Houseman,
B. T.; Pacold, J. I.; Gorrec, F.; Hon, W.-C.; Ren, P.; Liu,
Y.; Rommel, C.; Gaillard, P.; Rückle, T.; Schwarz, M. K.;
Shokat, K. M.; Shaw, J. P.; Williams, R. L. The P110δ
structure: Mechanisms for selectivity and potency of new
PI(3)K inhibitors. Nat. Chem. Biol. 2010, 6, 117−124.
21. Dugar, S.; Mahajan, D.; Hollinger, F. P.; Sharma, A; Kuila,
B. Novel triazine compounds. US 2015/0197515 A1.
15. Rageot, D.; Bohnacker, T.; Keles, E.; McPhail, J.A.;
Hoffmann, R.M.; Melone, A.; Borsari,C.; Sriramaratnam,
R.; Sele, M.A.; Beaufils, F.; Hebeisen, P.; Fabbro, D.;
Hillmann, P.; Burke, J.E.; Wymann, M.P. (S)
(Difluoromethyl)-5-(4-(3-methylmorpholino)-6-
‑4-
morpholino-1,3,5-triazin-2-yl)pyridin-2-amine (PQR530), a
potent, orally bioavailable, and brain-penetrable dual
inhibitor of class I PI3K and mTOR Kinase. J. Med. Chem.
2019, 62, 6241−6261.
9
16. Andrs, M.; Korabecny, J.; Jun, D.; Hodny, Z.; Bartek, J.;
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
Kuca, K. Phosphatidylinositol 3
‑kinase (PI3K) and
phosphatidylinositol 3 ‑kinase-related kinase (PIKK)
inhibitors: Importance of the morpholine ring. J. Med.
Chem. 2015, 58, 41−71.
17. Dugar, S.; Hollinger, F. P.; Mahajan, D.; Sen, S.; Kuila, B.;
Arora R.; Pawar, Y.; Shinde, V.; Rahinj, M.; Kapoor, K.
K.; Bhumkar, R.; Rai, S.; Kulkarni, R. Discovery of novel
and orally bioavailable inhibitors of PI3Kinase based on
indazole substituted morpholino-triazines. ACS Med.
Chem. Lett. 2015, 6, 1190−1194.
18. Dugar, S.; Hollinger, F. P.; Kuila, B.; Arora, R.; Sen, S.;
Mahajan, D. Synthesis and evaluation of pyrrolotriazine
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 12
Graphical Abstract
1
2
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
ACS Paragon Plus Environment