Discovery and Optimization of 2-Amino-4-methylquinazoline Derivatives as Highly Potent Phosphatidylinositol 3-Kinase Inhibitors for Cancer Treatment

Article abstract of DOI:10.1021/acs.jmedchem.8b00416

Increased phosphatidylinositol 3-kinase (PI3K) signaling is among the most common alterations in cancer, spurring intensive efforts to develop new cancer therapeutics that target this pathway. In this work, we discovered a series of novel 2-amino-4-methylquinazoline derivatives through a hybridization and subsequent scaffold hopping approach that were highly potent class I PI3K inhibitors. Lead optimization resulted in several promising compounds (e.g., 19, 20, 37, and 43) with nanomolar PI3K potencies, prominent antiproliferative activities, favorable PK profiles, and robust in vivo antitumor efficacies. More interestingly, compared with 19 and 20, 37 and 43 demonstrated improved brain penetration and in vivo efficacy in an orthotopic glioblastoma xenograft model. Furthermore, preliminary safety assessments including hERG channel inhibition, AMES, CYP450 inhibition, and single-dose toxicity were performed to characterize their toxicological properties.

Full text of DOI:10.1021/acs.jmedchem.8b00416

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Article
Discovery and Optimization of 2-Amino-4-methylquinazoline Derivatives as
Highly Potent Phosphatidylinositol 3-kinase Inhibitors for Cancer Treatment
Songwen Lin, Chunyang Wang, Ming Ji, Deyu Wu, Yuanhao Lv, Kehui Zhang, Yi Dong,
Jing Jin, Jiajing Chen, Jingbo Zhang, Li Sheng, Yan Li, Xiaoguang Chen, and Heng Xu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00416 • Publication Date (Web): 21 Jun 2018
Downloaded from http://pubs.acs.org on June 21, 2018
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Discovery and Optimization of 2-Amino-4-
methylquinazoline Derivatives as Highly Potent
Phosphatidylinositol 3-kinase Inhibitors for Cancer
Treatment
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†
,‡,∇
†,∇
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†,‡,
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†,‡
Songwen Lin,
Chunyang Wang, Ming Ji, Deyu Wu, Yuanhao Lv, Kehui Zhang, Yi
†
,‡
†
†,‡
†,‡
#
#
, †
Dong, Jing Jin, Jiajing Chen, Jingbo Zhang, Li Sheng, Yan Li, Xiaoguang Chen,*
,
†,‡
Heng Xu*
†
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, China.
‡
Beijing Key Laboratory of Active Substances Discovery and Drugability Evaluation, Institute of
Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College,
Beijing 100050, China.
#
Beijing Key Laboratory of Non-Clinical Drug Metabolism and PK/PD study, Chinese Academy
of Medical Sciences and Peking Union Medical College, Beijing 100050, China
KEYWORDS (Word Style “BG_Keywords”). Phosphoinositide 3-kinase, hybridization,
scaffold-hopping, brain penetration, in vivo anti-tumor efficacy
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ABSTRACT
Increased phosphatidylinositol 3-kinase (PI3K) signaling is among the most common alterations
in cancer, spurring intensive efforts to develop new cancer therapeutics that target this pathway.
In this work, we discovered a series of novel 2-amino-4-methylquinazoline derivatives through a
hybridization and subsequent scaffold hopping approach that were highly potent class I PI3K
inhibitors. Lead optimization resulted in several promising compounds (e.g. 19, 20, 37, and 43)
with nanomolar PI3K potencies, prominent anti-proliferative activities, favorable PK profiles and
robust in vivo antitumor efficacies. More interestingly, compared with 19 and 20, 37 and 43
demonstrated improved brain penetration and in vivo efficacy in an orthotopic glioblastoma
xenograft model. Furthermore, preliminary safety assessments including hERG channel
inhibition, AMES, CYP450 inhibition and single-dose toxicity were performed to characterize
their toxicological properties.
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INTRODUCTION
Phosphoinositide 3-kinases (PI3Ks) are a family of signaling enzymes that play crucial roles in
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-3
cell growth and proliferation, survival, metabolism, and migration. PI3Ks are subdivided into
three classes (I-III) according to their subunit composition and substrate specificity for
4, 5
phosphoinositides. Class I PI3Ks are the best characterized and consist of four subtypes (alpha,
beta, gamma and delta). PI3Ks phosphorylate phosphatidylinositol 4,5-diphosphate (PIP2) at the
3ʹ position on its inositol ring, converting PIP2 to the second messenger phosphatidylinositol
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,4,5-triphosphate (PIP3) at the plasma membrane, an activity that is downstream of receptor
tyrosine kinases (RTKs) or G-protein coupled receptors (GPCRs). PIP3 levels are negatively
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regulated by phosphatase and tensin homologue (PTEN). Subsequently, PIP3 recruits AKT
(
also known as protein kinase B) to the membrane through its pleckstrin homology (PH)
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domain. AKT activation involves the phosphorylation of two residues: threonine 308 (Thr308)
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in the activation loop and serine 473 (Ser473) in the C-terminal hydrophobic motif.
Once
activated, signaling through AKT can be propagated to a diverse array of substrates, including
the mammalian target of rapamycin (mTOR), a key regulator of protein translation. mTOR is an
atypical serine/threonine protein kinase that interacts with several proteins, forming two distinct
complexes named mTOR complex 1 (mTORC1) and 2 (mTORC2), which regulate different
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cellular processes including metabolism, growth, proliferation and survival.
It is well documented that PI3K pathway dysregulation is involved in human cancers.
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, 3, 12
The
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oncogenic potential of the PI3K pathway is largely associated with its molecular aberrations.
Through molecular profiling of 19784 consecutive diverse solid tumor samples submitted by
clinicians from more than 60 countries, Millis et al. recently demonstrated that PI3K pathway
aberrations are among the most common in cancer; overall 38% of patients had an alteration in
one or more PI3K pathway components, including PTEN loss (30%) and mutations in PIK3CA
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(
13%), PTEN (6%), and AKT1 (1%). The PI3K pathway has been extensively studied over the
past two decades for new cancer therapeutics, and quite a few PI3K inhibitors have entered into
15-19
clinical trials.
Based on their target specificity, these PI3K inhibitors can be characterized
into three classes (Figure 1), including dual PI3K/mTOR inhibitors (e.g. PF-04691502,
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GSK2126458, GDC-0084 and PQR309),
pan-class I PI3K inhibitors (e.g. BKM-120 and
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copanlisib),
and isoform selective PI3K inhibitors (e.g. alpha-selective BYL-719, beta-
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selective GSK2636771 and delta-selective idelalisib).
Among them, the PI3K delta-selective
inhibitor idelalisib was the first PI3K inhibitor approved by the FDA for the treatment of three
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types of blood cancers (chronic lymphocytic leukemia, follicular lymphoma, and small
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lymphocytic lymphoma) in 2014, while copanlisib was the second. Of particular note,
copanlisib, a pan-PI3K inhibitor with alpha and delta isoform potencies slightly higher than beta
and gamma isoforms, obtained an accelerated FDA approval in 2017 for relapsed FL based on its
phase II clinical trial that included only 104 patients. While these two drugs validated PI3K as a
target for blood cancers, the clinical progress regarding solid tumor treatment was not significant
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until recent Phase III trials demonstrated that BKM-120 prolonged progression-free survival
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(PFS) from 4 to 6 months in metastatic breast cancer patients with activating PI3K mutations.
Significant efforts are currently in clinical development by pharmaceutical companies and
academic institutes to realize the full potential of PI3K inhibitors for solid tumor treatment,
including exploring effective combinations, predictive biomarkers, target patient populations as
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well as underlying resistance mechanisms.
In addition to the range of solid tumor types that
would benefit from PI3K inhibitors, including breast cancer, head and neck squamous cell
carcinoma (HNSCC) and non-small cell lung carcinoma (NSCLC), these inhibitors might offer
an effective treatment option for brain tumors, such as primary brain tumors or brain metastases,
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as these tumors also show a high frequency of PI3K pathway alterations.
For example,
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PI3K signaling is implicated in more than 80% of glioblastoma multiforme (GBM) cases. This
is of great value, particularly considering the limited progress that has been made in treating
brain tumors with kinase inhibitors. However, the molecular design of such PI3K inhibitors
requires additional consideration of the brain-blood barrier, which limits penetration of the
inhibitor into the brain. Few PI3K inhibitors (e.g. GDC-0084 and PQR309, Figure 1) have been
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reported to be brain penetrant or demonstrated to be effective in orthotopic brain models,
suggesting there is rationale for them to be indicated to treat brain tumors or brain metastases.
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Therefore, the discovery of structurally distinct PI3K inhibitors, in particular those capable of
penetrating into the brain, would facilitate drug discovery for cancer therapeutics.
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Figure 1. Selected examples of PI3K inhibitors: the dual PI3K/mTOR inhibitors 1-4, pan-PI3K
inhibitors 5-6, a PI3K alpha-selective inhibitor 7, a PI3K beta-selective inhibitor 8, a PI3K delta-
selective inhibitor 9.
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RESULTS AND DISCUSSION
Our initial design was inspired from two known PI3K inhibitors PF-04691502 and
GSK2126458. Using insight from their co-crystal structures with PI3Kγ, we noticed that both
compounds established hydrogen bonding interactions with Val882 and a conserved water
molecule bridge with Tyr867 and Asp841, while they had one additional hydrophobic interaction
within the ribose pocket and one charged interaction between the deprotonated sulfonamide and
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Lys833, respectively.
Based on this analysis, we intended to exploit the hybrid drug design
approach to access all four aforementioned interactions with the goal of identifying novel
chemical scaffolds with better drug-like properties.
Figure 2. Design strategy of quinazoline-based PI3K inhibitors.
Compound 10 was synthesized to validate the hybrid design. In a PI3K enzymatic assay,
compound 10 potently inhibited PI3Kα with an IC of 0.87 nM, displaying approximately 4-fold
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increased potency compared with PF-04691502. However, the translation of inhibited enzymatic
activity into cellular potency was suboptimal. Compound 10 was approximately 2-fold less
potent than PF-04691502 against human NCI-H460 cells. We attributed this drop in cellular
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potency to its unfavorable physicochemical properties. In particular, its molecular weight (M.W.
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4, 35
=
616.64) and polar surface area (PSA = 168.3) were far from the desired range.
This
prompted us to reduce its M.W. and PSA through a scaffold hopping approach. As seen from the
docking study, the cyclic amide functionality in the pyrido[2,3-d]pyrimidin-7(8H)-one core of
compound 10 was not directly involved in the binding to the kinase, and thus, provided a
reasonable place to make structural changes (Figure 3A). The quinazoline scaffold, which is
capable of allowing similar orientations for both aminopyrimidine and methoxyl pyridine
moieties, was then chosen to replace the pyrido[2,3-d]pyrimidin-7(8H)-one core (Figure 3B).
Additionally, the synthetic feasibility of attaching various functionalities to the 8-position of the
quinazoline to access hydrophobic interactions within the ribose pocket of the protein made this
scaffold more attractive for exploration.
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Figure 3. Predicted binding mode for 10 (A) and 11 (B) with PI3Kα (PDB ID: 4JPS). Hydrogen
bonds are shown as yellow dashed lines to Val851 in PI3Kα (Val882 in PI3Kγ) in the hinge
region, Lys802 in PI3Kα (Lys 833 in PI3Kγ), and the conserved water molecule bridge between
Tyr 836 and Asp810 in PI3Kα (Tyr867 and Asp841 in PI3Kγ). Images generated by PyMol.
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Table 1. SAR studies of the ether functionalities
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PI3Kα
NCI-H460
a
a
Compd.
R1
--
R2
--
R3
--
cLogP
1.2
2.7
4.6
5.5
5.6
4.5
5.6
6.1
6.6
6.1
4.3
PSA
b
c
(nM)
(μM)
1
122.1 3.3±0.92
168.3 0.87±0.19
127.7 1.2±0.06
127.7 0.70±0.13
127.7 1.4±0.49
137.0 1.6±0.01
127.7 0.95±0.21
127.7 1.1±0.07
127.7 0.95±0.49
127.7 1.6±1.0
0.20±0.03
1
1
1
1
1
0
1
2
3
4
--
--
--
0.36±0.09
CH
O
O
O
O
O
O
O
O
O
0.28±0.06
3
3
3
3
3
3
3
3
3
CH
CH
CH
CH
CH
CH
CH
CH
0.14±0.02
0.20±0.09
0.20±0.04
15
16
0.063±0.015
0.094±0.069
0.096±0.064
0.083±0.007
0.029±0.013
1
1
1
7
8
9
137.0 0.80±0.01
2
0
CH
3
O
4.4
137.0 1.0±0.24
0.093±0.028
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CH
3
3
O
O
4.4
4.5
137.0 0.83±0.16
0.094±0.048
0.063±0.030
2
2
2
3
CH
137.0 1.2±0.28
137.0 1.0±0.04
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
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4
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0
1
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0
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0
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9
0
1
2
3
4
5
6
7
8
9
0
CH
O
5.3
0.041±0.029
3
2
2
2
2
2
4
5
6
7
8
CH
CH
CH
CH
CH
3
3
3
3
O
O
O
O
O
4.9
5.0
4.5
4.8
2.3
137.0 0.73±0.16
137.0 1.1±0.34
131.0 4.8±0.99
0.021±0.016
0.24±0.10
0.66±0.33
1.1±0.24
140.2 3.3±1.1
137.0 2.6±0.21
137.0 2.5±0.28
0.86±0.19
3
2
3
3
9
0
1
CH
CH
CH
3
O
O
O
2.8
2.8
2.8
0.93±0.24
1.1±0.15
0.98±0.03
F
90.8
90.7
16±7.8
31±1.4
3
3
H
3
3
2
3
CH
3
O
4.5
137.0 1.1±0.55
0.017±0.003
CH
CH
O
O
4.1
4.4
137.0 1.2±0.14
137.0 1.1±0.47
0.096±0.014
0.025±0.017
3
34
3
3
3
5
6
CH
Cl
3
3.4
3.5
127.7 1.4±0.35
127.7 1.7±0.92
2.1±0.41
1.2±0.51
a
b
c
Calculated from ChemBioDraw Ultra 14.0. Mean of at least two separate experiments. Mean
of at least three separate experiments.
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5
5
5
5
6
First, simple methyl ether 11 was synthesized and its IC values against PI3Kα and NCI-H460
5
0
cells were 1.2 nM and 0.28 μM respectively, showing comparable enzymatic and cellular
potencies to PF-04691502 (Table 1). Isopropyl ether 12 displayed approximately two-fold
increased potency for PI3Kα and NCI-H460 cells relative to 11. We attribute this increased
potency to better hydrophobic interactions brought by the two methyl group of the isopropyl
moiety. The cyclopropyl methyl ether 13 and 1-methoxyethyl ether 14 exhibited cellular
potencies similar to PF-04691502. When cycloalkyl groups were incorporated through the ether
linkage, the generated compounds 15–18 had IC values against PI3Kα within a range of 0.95–
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
0
1
.6 nM. More impressively, they inhibited NCI-H460 cell growth with IC values below 100
50
nM. It has been noted that the cellular activities of these cycloalkyl compounds incrementally
increase as their cLogPs decrease. While these cycloalkyl compounds give excellent cellular
potency, all their cLogP values were greater than 5.5. Such high cLogPs often give rise to poor
3
6
oral absorption and low metabolic stability. With this in mind, we further explored
heterocycloalkyl analogues to decrease the cLogP. Among O-containing heterocycloalkyls,
tetrahydropyrane 19 and tetrahydrofuran 20 were very potent against both PI3Kα (IC ≤ 1 nM)
5
0
and NCI-H460 cells (IC < 0.1 μM). Notably, the chiral tetrahydrofuran 20 displayed equal in
50
vitro potencies with its enantiomer 21. When the tetrahydrofuran moiety was one carbon away
from the oxygen atom, the racemic compound 22 had an incremental improvement in cellular
potency over 20 and 21. In particular, tetrahydropyrane 19 showed a remarkable IC value of
50
0.029 μM against NCI-H460 cells, over 3-fold more potent than the cyclohexyl compound 17. Its
close analogues of 2,2-dimethyl tetrahydropyrane 23 and 4-methyl tetrahydropyrane 24 also
showed excellent cellular potencies (IC50 < 0.05 μM). More importantly, relative to the
cycloalkyl compounds (e.g. 15–18), cLogP values of these O-containing heterocycloalkyl
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compounds (e.g. 19–22, 24) fell into a range that could potentially be translated into better oral
absorption and metabolic stability. In addition to O-containing heterocyclic alkyls,
methylpiperidine 26 and morpholine 27 were also examined. However, the introduction of the
polar nitrogen resulted in a noticeable decrease in enzymatic potency, indicating that the
piperidine and morpholine moieties formed unfavorable interactions with the lipophilic side
chains of the residues in its ribose binding pocket. The exploration of various ether analogues
revealed that the tetrahydropyrane moiety yielded excellent enzymatic and cellular potencies as
well as reasonable cLogPs, and thus were retained for further structure-activity relationship
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(SAR) studies. Sulfonamide functionality was then targeted for the next round of modifications.
Consistent with earlier findings, alkyl sulfonamide (e.g. 28) and cycloalkyl sulfonamide (e.g. 29)
were less potent than phenyl sulfonamides (e.g. 19), which was largely attributed to the
resonance effect of phenyl sulfonamides that rendered the more acidic sulfonamide NH proton to
2
1
have a stronger charged interaction with Lys802 in PI3Kα (Lys833 in PI3Kγ). As expected,
removal of the sulfonamide moiety (e.g. 30 and 31) significantly decreased both PI3Kα and
cellular potencies. Small substitution changes to the phenyl ring could also make an impact on
cellular activity. For example, compared with 2,4-difluoro-phenyl sulfonamide 19, 2-chloro-4-
floro-phenyl sulfonamide 32 was slightly more potent (NCI-H460 IC : 0.017 vs 0.029 μM),
5
0
whereas 4-fluoro-phenyl sulfonamide 33 was slightly less potent (NCI-H460 IC : 0.096 vs
5
0
0
.029 μM). In addition to phenyl sulfonamide, 5-chlorothiophene-2-sulfonamide 34 was also
demonstrated to be a favorable moiety for in vitro potencies. Notably, when the methoxyl group
adjacent to the pyridine nitrogen was replaced by methyl (e.g. 35) or chloride (e.g. 36), the
cellular potencies dropped over 10-fold.
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 2. SAR Studies of substitutions on the pyrimidylamine.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
a
Compd.
R R N-
R1
R4
cLogP
5.1
PSA
PI3Kα
(nM)
NCI-H460
(μM)
2
3
b
c
37
CH
3
NH-
123.0
123.0
123.0
114.2
140.0
1.4±0.35
1.5±0.21
5.4±2.3
125±16
1.4±0.35
11±0.71
0.059±0.023
0.24±0.12
0.41±0.02
4.3±0.64
3
3
8
9
C
2
H
5
NH-
5.6
6.1
40
(CH
3
)
2
N-
5.2
4
1
AcNH-
AcNH-
3.8
0.053±0.027
3.2±1. 8
4
4
2
3
CH
3
1.8
5.2
140.0
123.0
CH
CH
CH
NH-
NH-
NH-
0.96±0.20 0.10±0.01
3
3
3
4
4
4
5
5.2
5.3
123.0
123.0
1.4±0.61
2.0±0.42
0.10±0.003
0.31±0.06
46
CH
3
3
NH-
NH-
6.1
5.7
123.0
123.0
1.8±0.71
1.8±0.64
0.25±0.01
0.40±0.03
4
7
CH
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CH
CH
CH
3
3
3
NH-
NH-
NH-
6.4
6.9
7.5
113.7
4.1±1.3
6.6±2.8
9.8±7.4
1.4±0.26
2.0±0.31
113.7
113.7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
1.7±0.22
a
b
c
Calculated from ChemBioDraw Ultra 14.0. Mean of at least two separate experiments. Mean
of at least three separate experiments.
Finally, our SAR exploration focused on derivatizations on the pyrimidine amine (Table 2).
When a methyl, ethyl or cyclopropylmethyl group was attached, methyl amine 37 had the highest
enzymatic and cellular potencies, whereas the cyclopropylmethyl amine 39 had the lowest. We
found that the in vitro potencies of above alkylated compounds were inversely correlated with
the size of the alkyl group. Of particular note, the cellular potency of methylated
tetrahydropyrane 37 was comparable to its corresponding amine 19. For tertiary amine 40, its
PI3K potency dropped >100-fold compared with 19, indicating that the NH proton was critical
for protein binding. Besides the methyl group, acetyl 41 was also favorable for in vitro potency,
3
7
consistent with the earlier findings from another PI3K series. Similar to tetrahydropyrane 37,
tetrahydrofuran 43 and 44 retained their potencies after methylation. Methylation reactions were
also performed with other O-containing heterocyclic compounds. Compared with their
corresponding pyrimidine amines 21–24, methylated compounds 44–47 generally displayed
decreased PI3K potencies (>1.5-fold) and cellular activities (>4-fold), respectively. A more
pronounced decrease in potency was observed for methylated compounds 48–50 that featured a
cycloalkyl moiety versus their corresponding amines (>4-fold decrease in PI3Kα potency; >10-
fold decrease in cellular potency). Compared to the cycloalkyls, the O-atom in a tetrahydropyran
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
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or tetrahydrofuran moiety (e.g., compounds 37 and 43) could be aligned with Gln859 in PI3Kα
to form one hydrogen bond (Figure S1), which may explain the more pronounced potency
decrease for compounds 48-50 that do not have such an interaction.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 3. Enzymatic and cellular potencies of compounds 19, 20, 37 and 43
Enzyme/Cell
line
1
9
20
37
43
Enzyme
Inhibition
PI3K alpha
PI3K beta
PI3K gamma
PI3K delta
AKT1
0.80±0.01
0.53±0.09
1.3±0.05
0.18±0.004
>10000
1.0±0.24
0.40±0.08
1.2±0.25
0.23±0.04
>10000
1.4±0.35
1.2±0.27
1.7±0.16
0.35±0.06
>10000
0.96±0.20
1.1±0.39
0.93±0.41
0.37±0.02
>10000
a
IC (nM)
5
0
mTOR
7.9±0.77
9.9±0.48
43±14
56±8.4
Cell
proliferation
NCI-H460
A549
0.029±0.013 0.093±0.028 0.059±0.023 0.10±0.01
0.053±0.020 0.084±0.014 0.11±0.02
0.18±0.02 0.30±0.05 0.26±0.07
0.12±0.01
0.48±0.24
b
IC (μM)
5
0
MDA-MB-231
HGC-27
DU145
0.025±0.001 0.056±0.007 0.016±0.002 0.027±0.001
1.7±0.25
2.1±0.51
5.2±0.77
6.5±0.24
0.24±0.03
0.40±0.15
1.3±0.51
0.46±0.36
0.41±0.10
PC3
0.15±0.02
0.12±0.05
0.28±0.06
0.32±0.12
0.42±0.09
0.28±0.16
0.26±0.20
0.95±0.40
0.14±0.05
0.28±0.08
0.38±0.03
0.25±0.02
0.51±0.08
0.70±0.10
0.39±0.20
U87MG
SH-SY-5Y
U251
T98G
a
b
Mean of at least two separate experiments. Mean of at least three separate experiments.
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0
1
2
3
4
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8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Kinase selectivity profiling of compound 19 at a concentration of 1 μM
TM
TM
(
Kinomescan , DiscoveRx). A) TREEspot interaction maps for 19 among 468 kinase targets.
B) 9 non-mutant kinases were hit with <35% control binding remaining. Results for primary
screen binding interactions are reported as % control, where lower numbers indicate stronger hits
in the matrix. S-score is a quantitative measure of compound selectivity and it is calculated by
dividing the number of kinases that compounds bind to by the total number of distinct kinases
tested, excluding mutant variants.
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Based on their superior enzymatic and cellular potencies as well as their favorable
physicochemical properties, compounds 19, 20, 37 and 43 were selected for further profiling. As
illustrated in Table 3, these compounds potently inhibited all four class I PI3K isoforms with
sub- to low-nanomolar IC s. While the PI3K delta isoform was most potently inhibited, these
0
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0
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0
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4
5
6
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9
0
50
compounds did not show much isoform selectivity. Compounds 19, 20, 37 and 43 also had
potent inhibitory activities against mTOR. In contrast with compounds 19 and 20, their
methylated counterparts 37 and 43 displayed more than 20-fold increased selectivity for mTOR
over class I PI3Ks, which can be explained by the unfavorable interaction between the methyl on
2
0
the pyrimidine amino group and the side chain of TRP-80 that is unique to mTOR. Moreover,
these compounds did not inhibit AKT1 (IC >10 μM), a key downstream effector of PI3K. To
5
0
further understand their kinase selectivity, compound 19 was chosen as a representative for this
quinazoline chemotype to examine its kinome-wide selectivity profile using DiscoverX’s
Kinomescan technology. As depicted in Figure 4, 19 possessed a high selectivity, as indicated by
the selectivity S score (1) of 0.015 against 468 kinases and mutants tested at a 1 μM
concentration. 19 did not show any binding activity to this broad panel of kinases except for the
PI3Ks and mTOR, illustrating excellent kinase selectivity for this quinanoline chemotype.
Compounds 19, 20, 37 and 43 were also tested against a panel of human cancer cell lines (Table
3
). Among them, the most sensitive (with IC s in double-digit nanomolar concentration ranges)
50
was HGC-27 gastric cancer cells, which harbor both a PIK3CA mutation and PTEN loss, while
the least sensitive was DU145 prostate cancer cells with wild-type PTEN. Notably, they also
potently inhibited the proliferation of brain tumor cells such as U87MG, U251, SH-SY-5Y and
T98G with sub-micromolar IC s.
5
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Compounds 19, 20, 37 and 43 were further progressed into in vivo pharmacokinetic studies
Table 4). When intravenously administered at a dose of 3 mg/kg in mice, compounds 19, 20, 37
(
and 43 had moderate clearances with CL values of 7.58, 54.9, 14.3 and 34.5 mL/min/kg,
respectively. Compounds 19 and 37 featuring a tetrahydropyrane moiety displayed a slower
clearance rate than compounds 20 and 43 that bear a tetrahydrofuran moiety. When orally
administered at a dose of 30 mg/kg, compounds 19, 20, 37 and 43 were quickly absorbed with
Tmax values of <30 min. These compounds gave reasonable oral exposures with area under the
curve (AUC) values of >9000 h*ng/mL. Among them, the oral exposure of compound 19 was
the highest (normalized AUC(0-∞) = 1205 h*ng/mL). The oral bioavailability of compounds 19,
20, 37 and 43 were determined to be 33.2%, 96.2%, 44.4% and 78.4% respectively, supporting a
further evaluation of their in vivo efficacy.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 4. Murine pharmacokinetic profiles of compounds 19, 20, 37 and 43
CL
Tmax
h
Cmax
AUC(0-t) AUC(0-∞) T1/2
Vss
Dose
mg/kg
Cmpd Route
F%
mL/min/k
g
ng/mL h*ng/mL h*ng/mL
h
L/kg
a,c
19
20
37
iv
3
30
3
0.03 10283
7192
23877
904
7238
36160
911
1.48 0.853
11.1
0.54 2.57
4.68
0.62 0.763
4.25
0.53 1.56
16.2
7.58
/
33.2
/
b,d
po
0.5
4287
1583
8827
7723
/
/
a,c
iv
0.03
0.08
0.05
54.9
b,d
po
30
3
8694
3484
15478
1453
11396
9040
3501
16109
1462
17097
/
/
14.3
/
96.2
/
a,c
iv
b,d
po
30
3
0.18 12844
/
44.4
/
a,c
43
iv
0.03
0.25
2623
5390
34.5
/
b,d
po
30
/
78.4
a
b
c
Three mice per study for IV; three to five mice per study for PO; iv formulation: 10%
d
DMSO/saline; po formulation: 0.1% PEG400/ 0.5% CMC/water
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5
5
6
Next, we evaluated the brain penetration properties of these compounds. Compounds 19, 20,
7 and 43 were orally administered at a dose of 30 mg/kg, and mouse brain tissues were
3
analyzed to determine their brain pharmacokinetic profiles. As shown in Figure 5, compounds 19,
0
1
2
3
4
5
6
7
8
9
0
1
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3
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9
0
1
2
3
4
5
6
7
8
9
0
2
0, 37 and 43 were capable of crossing the brain-blood-barrier with brain Cmax values of 93,
1
61, 723 and 433 ng/g and brain AUC values of 234, 28.6, 895, 742 ng/g*h, respectively. It
0
-t
should be noted that compared with 19 and 20, compounds 37 and 43 demonstrated significantly
higher maximum brain concentrations and brain exposures. Based on our rational design,
compounds 37 and 43 have one less hydrogen-bond donor than their corresponding non-
methylated forms 19 and 20. Lower hydrogen-bond formation potential in a molecule generally
3
8
favors brain penetration. Compounds 37 and 43, with significantly higher brain penetration
compared with 19 and 20, provide solid evidence that the number of hydrogen bonding donors in
the molecule is inversely correlated with its capability of crossing the brain-blood-barrier.
1
0 0 0
1
2
3
9 (3 0 m g /kg , p o )
0
7
(3 0 m g /kg , p o )
(3 0 m g /kg , p o )
8
6
4
2
0 0
0 0
0 0
0 0
0
4
3
(3 0 m g /kg , p o )
0
1
2
3
4
T im e (h )
Figure 5. Brain concentration versus time curves for compounds 19, 20, 37 and 43. Results are
expressed as the mean brain concentration and standard deviation (SD) (n = 3 for each time
point).
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Next, compounds 19, 20, 37 and 43 were tested for their in vivo antitumor activities in lung
and gastric cancer xenograft models (Figures 6 and 7). In an NCI-H460 NSCLC xenograft model,
compound 19 orally administered at 5 and 10 mg/kg daily demonstrated robust in vivo antitumor
activity, and both dosage groups showed tumor growth inhibition (TGI) of >70%. Compound 19
showed greater efficacy than PF-04691502 (PF-04691502: TGI= 67.4% at 10 mg/kg vs 19:
TGI=79.8% at 10 mg/kg). In a separate NCI-H460 xenograft model, compound 20 orally
administrated at 5, 10 and 20 mg/kg daily effectively inhibited tumor growth in a dose-dependent
manner, with the high-dose group of 20 mg/kg achieving TGI of 75.9%. We also evaluated
compound 19 in a SCLC NCI-H209 xenograft model, in which compound 19 orally administered
at doses of 2.5 and 5 mg/kg daily for 19 consecutive days produced a dose-dependent effect on
TGI. In a MGC-803 gastric cancer xenograft model, compounds 19 and 20 dose-dependently
suppressed tumor growth. It was found that both 19 and 20 had significant exposures in the
tumor tissue and the exposure correlated with the dose as well as the anti-tumor efficacy (Figure
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C). In a HGC-27 gastric cancer xenograft model, compounds 19, 37 and 43 achieved significant
tumor regression at oral daily doses of 2.5, 2.5 and 10 mg/kg respectively, demonstrating that
HGC-27 cells harboring both PIK3CA mutation and PTEN deletion were more sensitive to these
compounds (Figure 7D). Their plasma concentrations at 2h after the final dose were also
determined to show the correlation between exposure and antitumor efficacy (Figure 7F). It was
also noted that in these lung and gastric cancer xenograft models, compounds 19, 20, 37 and 43
induced body weight loss and that their antitumor efficacy was correlated with the severity of
body weight loss.
Compounds 37 and 43 (with reasonable brain exposures) were further evaluated in a mouse
U87MG/Luc orthotopic xenograft model (Figure 8 and Figure S2). In this model, the
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U87MG/Luc cell suspension was stereotaxically injected into the mouse brain to grow orthotopic
tumors, and then compounds 37 and 43 were orally administered to test whether they could
inhibit growth of the orthotopic brain tumor. This model demonstrated that both 37 and 43
exhibited remarkable efficacy in suppressing brain tumor growth, comparable to the positive
control trimetazidine (TMZ), which is the only oral anticancer agent approved by the FDA for
GBM treatment. Of particular note, at the same dose level (5 mg/kg), compound 37 showed
better efficacy than 43, which was correlated with their absolute brain exposures.
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Figure 6. In vivo antitumor efficacy of compounds 19 and 20 in lung cancer xenograft models.
Non-small cell lung cancer NCI-H460 cells and small cell lung cancer NCI-H209 cells were
subcutaneously implanted into nude mice. A) Tumor volume changes following treatment with
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9 in the NCI-H460 xenograft model; B) body weight changes after treatment with 19 in the
NCI-H460 xenograft model; C) tumor volume changes following treatment with 20 in the NCI-
H460 xenograft model; D) body weight changes after treatment with 20 in the NCI-H460
xenograft model; E) tumor volume changes after treatment with 19 in the NCI-H209 xenograft
model; F) body weight changes after treatment with 19 in the NCI-H209 xenograft model.
Results are expressed as the mean ± SD (n = 5–7 for each group); **p <0.01 and ***p <0.001 vs
vehicle.
Figure 7. In vivo antitumor efficacy of compounds 19, 20, 37 and 43 in gastric cancer xenograft
models. Gastric cancer MGC-803 and HGC-27 cells were subcutaneously implanted into nude
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mice. A) Tumor volume changes after treatment with 19 and 20 in the MGC-803 xenograft
model; B) body weight changes after treatment with 19 and 20 in the MGC-803 xenograft model;
C) concentrations of 19 and 20 in tumor tissue at 24h after the final dose in the MGC-803
xenograft model; D) tumor volume changes after treatment with 19, 20, 37 and 43 in the HGC-
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7 xenograft model; E) body weight changes after treatment with 19, 20, 37 and 43 in the HGC-
7 xenograft model; F) concentrations of 19, 20, 37 and 43 in plasma at 2h after the final dose in
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the HGC-27 xenograft model. Results are expressed as the mean ± SD (n = 6 for each group);
*p <0.01 and ***p <0.001 vs vehicle.
*
Figure 8. In vivo antitumor efficacy of 37 and 43 in the U87MG/Luc glioblastoma orthotopic
xenograft model. A) Tumor volume in the mouse brain at the end of treatment; B) body weight
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changes; C) representative NMR images of U87MG/Luc tumors in the mouse brain at the end of
treatment. Results are expressed as the mean ± SD (n = 5–7 for each group). **p <0.01 and ***p
<0.001 vs vehicle. Temozolomide (TMZ) was used as the positive control.
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To measure the magnitude and duration of the pharmacodynamic response, phosphorylated
(p)AKT levels in tumor tissues from the NCI-H460 xenograft model following a single oral dose
of 10 mg/kg were determined over a time course. As shown in Figure 9, compounds 19 and 37
significantly reduced pAKT levels, and the duration of the pharmacodynamic response was
greater than 24 h. Their plasma and tumor tissue concentrations at each time point greatly
exceeded their PI3K IC s and NCI-H460 antiproliferative IC s. Considering protein binding
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(human 95.78%, mouse 91.77%, for 19; human 95.46%, mouse 91.58%, for 37), their theoretical
free drug concentrations at each time point are still higher than the PI3K IC s, rationalizing the
90
PD effect over the time course. Of particular note, the ratios of AUC(0-24h) in tumor to AUC(0-24h)
in plasma of compounds 19 and 37 were calculated to be 0.71 (Tumor AUC(0-24h) = 8456 ng/g*h
vs plasma AUC(0-24h) = 11831 ng/mL*h) and 1.02 (Tumor AUC(0-24h) = 5576 ng/g*h vs plasma
AUC(0-24h) = 5455 ng/mL*h), respectively, illustrating their high tumor uptakes.
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Figure 9. Pharmacodynamic response of 19 and 37 in the NCI-H460 xenograft model following
a single oral dose of 10 mg/kg. pAKT(S473) levels were determined at 2, 4, 8, 12 and 24 h post-
administration (A and B). Compound concentrations in plasma and tumor tissues were measured
at the same time point and the dashed lines represent the PI3Kα IC value (green), the PI3Kα
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are expressed as the mean ± SD (n = 3 for each time point).
We further evaluated the safety profiles of these compounds. In the hERG inhibition assay, all
tested compounds showed low hERG channel activity with IC values of >10 μM, indicating
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that they had low cardiotoxicity potential. We also studied their mutagenicity by the bacterial
reverse mutation (AMES) test. Compounds 19, 20 and 43 were non-mutagenic at the tested
concentrations (0.5–500 μg/plate) with or without metabolic activation (using the five tester
strains: TA97, TA98, TA100, TA102 and TA1535). For compound 37, however, we found that
its AMES test was positive at the concentration of 500 μg/plate in strains TA97, TA98, TA100,
and TA1535; it was also positive in strain T1535 at a concentration of 50 μg/plate with the S9
mix. Compounds 19, 20, 37 and 43 were also tested in cytochrome P450 (CYP450) inhibition
assays for a range of isoforms including CYP1A2, CYP2D6, CYP2C9, CYP2C19, CYP2E1 and
CYP3A4, and their IC values against all these CYP450 isoforms were greater than 10 μM,
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confirming their low liability for drug-drug interactions. Single-dose toxicity studies were also
performed for compounds 19, 20, 37 and 43, and their maximum tolerated doses were
determined to be >40 mg/kg.
Table 5. The safety profiles of compounds 19, 20, 37 and 43
b
Compd.
hERG
AMES
cytochrome P450
Single-dose Toxicity
a
c
d
(
IC , μM)
(IC , μM)
(MTD, mg/kg)
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>10
>10
>10
Negative
Negative
Positive
Negative
>10
>10
>10
>10
>40
>50
>40
>40
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hERG assays were performed using wide-type Chinese hamster ovary cells expressing hERG
b
potassium channels; compounds were tested at concentrations from 0.5–500 μg/plate with or
without metabolic activation using the five tester strains TA97, TA98, TA100, TA102 and
c
TA1535; the cytochrome P450 isoforms included CYP1A2, CYP2D6, CYP2C9, CYP2C19,
d
CYP2E1 and CYP3A4; mice were observed for 14 d after a single-dose oral administration.
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Chemistry
As shown in scheme 1, compound 10 was synthesized by Suzuki coupling of compound 51
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1, 37
and 52a, which were prepared according to the reported methods.
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Scheme 1. Synthesis of compound 10
a
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Reagents and conditions: (a) PdCl (dppf), 2M K CO , dioxane, 100 C, Ar, 4h, 53%.
2
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The preparation of compounds 11-36 are illustrated in scheme 2. The starting material 53 was
brominated by NBS to afford compound 54, which was then reacted with triphosgene to give 55.
Aminolysis of 55 by N,O-dimethylhydroxylamine hydrochloride afforded compound 56, which
was converted to compound 57. The 2-amino-4-methylquinazoline core structure was
constructed by reaction of compound 57 with cyanamide and concentrated hydrochloric acid,
and the resulting 58 was then reacted with 2,5-hexanedione to yield 59, which was further
demethylated to afford the general intermediate 60. 60 was converted to 61-76 via nucleophilic
substitution reaction or Mitsunobu reaction, followed by removing the amino-protective group to
give compound 77-92. Finally, compounds 58 and 77-92 were coupled with various aryl boronic
esters to afford the final products 11-36.
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a
Scheme 2. Synthesis of compound 11-36.
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Reagents and conditions: (a) NBS, DMF, rt, 2 h, 95%; (b) triphosgene, THF, reflux, 3 h, 80%;
c) N,O-dimethylhydroxylamine hydrochloride, TEA, dioxane, reflux, overnight, 91%; (d)
CH MgBr, THF, -20 C, 30 min, 26%; (e) conc. HCl, 50% cyanamide in water, 120 °C, 15 min,
98%; (f) 2,5-hexanedione, p-toluenesulfonic acid, NMP, toluene, 160 °C, 6 h, 87%; (g) AlCl3,
DCE, 80 °C, 1.5 h, 77%; (h) alkyl bromide, K CO , acetonitrile, sealed tube, reflux, overnight,
(
o
3
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5
8-98%; (i) ROH, DEAD, PPh , THF, rt, overnight, 42-94%; (j) hydroxylamine hydrochloride,
EtOH, H O, reflux, overnight, 27-85%; (k) aryl boronic ester, PdCl (dppf), 2M K CO , dioxane,
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00 C, Ar, 4h, 25-95%.
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The synthetic procedure of compound 37-50 is described in scheme 3. Compound 80-82 and
4-89 were alkylated or acylated to afford compound 93-105, which was then coupled with aryl
8
boronic esters to afford the final products 37-50.
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Scheme 3. Synthesis of compound 37-50.
a
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pyridine, DMF, rt, 4 h, 76%; (c) aryl boronic ester, PdCl (dppf), 2M K CO , dioxane, 100 C, Ar,
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h, 30-87%.
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CONCLUSION
In summary, we identified a class of novel quinazoline derivatives as highly potent PI3K
inhibitors through a hybridization followed by scaffold hopping strategy. Systematic SAR
studies led to the discovery of the promising anticancer compounds 19, 20, 37 and 43 with
favorable drug-like properties. These promising compounds displayed nanomolar enzymatic
potencies as well as sub-micromolar anti-proliferative effects on a broad panel of human cancer
cell lines. Compound 19, an exemplar of these compounds, showed excellent kinase selectivity
with an S(1) score of 0.015 against 468 kinases and mutants using DiscoverX’s Kinomescan
assay. The PK profiles of compounds 19, 20, 37 and 43 were also favorable. Compared with 19
and 20, 37 and 43 exhibited higher brain penetration, with AUC values of 895 and 742 ng/g*h at
the oral dose of 30 mg/kg, respectively. Several lung cancer and gastric cancer xenograft models
were performed to demonstrate the robust in vivo efficacies of these compounds. Of particular
note, the HGC-27 gastric xenograft model was the most sensitive to these compounds, showing
significant tumor regression. In the orthotopic U87-MG brain xenograft model, compounds 37
and 43 (with reasonable brain penetration properties) were highly effective, with TGIs of >90%,
and thus, were comparable to TMZ. Furthermore, preliminary safety assessments including
hERG channel inhibition assays, AMES, CYP450 inhibition assays and single-dose toxicity
studies demonstrated an acceptable safety profile. Overall, these pharmacological and safety data
have compelled us to advance 19 and 43 into formal preclinical evaluations, the results of which
will be reported in due course.
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