Design, Synthesis, and Biological Evaluation of Substituted Pyrimidines as Potential Phosphatidylinositol 3-Kinase (PI3K) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.6b00235

Three series of substituted pyrimidines were designed and synthesized. All target compounds were screened for kinase inhibitory activities against PI3Kα, and most IC₅₀ values were found within the nanomolar range. Compounds 5d and 5p displayed

Full text of DOI:10.1021/acs.jmedchem.6b00235

Subscriber access provided by UNIV OF CALIFORNIA SAN DIEGO LIBRARIES
Brief Article
Design, Synthesis and Biological Evaluation of Substituted Pyrimidines
as Potential Phosphatidylinositol 3-Kinase (PI3K) Inhibitors
Ji-Quan Zhang, Yong-Jie Luo, Yan-Shi Xiong, Yang Yu, Zheng-Chao Tu, Zi-
Jie Long, Xiao-Ju Lai, Hui-Xuan Chen, Yu Luo, Jiang Weng, and Gui Lu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00235 • Publication Date (Web): 18 Jul 2016
Downloaded from http://pubs.acs.org on July 18, 2016
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 free 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 accessible to all
readers and 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.
Journal of Medicinal Chemistry 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 14
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Design, Synthesis and Biological Evaluation of Substituted Pyrim-
idines as Potential Phosphatidylinositol 3-Kinase (PI3K) Inhibitors
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
JiꢀQuan Zhang,†,‡ YongꢀJie Luo,† YanꢀShi Xiong,† Yang Yu,† ZhengꢀChao Tu,§ ZiꢀJie Long,ǁ XiaoꢀJu
Lai,⊥ HuiꢀXuan Chen,† Yu Luo,† Jiang Weng,† Gui Lu*,†,#
†Institute of Medicinal Chemsitry, School of Pharmaceutical Sciences, Sun Yatꢀsen University, Guangzhou, 510006, PR
China
‡College of Pharmacy, Guizhou Medical University, Guiyang, 550004, PR China
§Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, 510530, PR China
ǁDepartment of Hematology, The Third Affiliated Hospital, Sun Yatꢀsen University, Guangzhou, 510260, PR China
⊥State Key Laboratory of Oncology in South China, Cancer Center, Sun Yatꢀsen University, Guangzhou, 510060, PR China
#Institute of Human Virology, Sun Yatꢀsen University, Guangzhou, 510080, PR China
KEYWORDS: Substituted pyrimidine, PI3Kα, isozyme selectivity, cytotoxicity
ABSTRACT: Three series of substituted pyrimidines were designed and synthesized. All target compounds were screened for kiꢀ
nase inhibitory activities against PI3Kα, and most IC50 values were found within the nanomolar range. Compounds 5d and 5p disꢀ
played comparable activities relative to the positive control 5a, 5p also showed a significant isozyme selectivity (PI3Kβ/α). Furꢀ
thermore, the cytotoxicities of these pyrimidines against human cancer cell lines were evaluated, the in vivo anticancer effect of 5d
was also tested.
INTRODUCTION
It is well recognized that phosphatidylinositol 3ꢀkinases (PI 3ꢀ
kinases or PI3Ks) play a central role in a broad cellular funcꢀ
tions such as cell growth, proliferation, differentiation, survivꢀ
al, and intracellular trafficking.1,2 On the basis of sequence
homology and substrate preferences, PI3Ks can be categorized
Figure 1. Structures of positive controls mentioned in this study.
into class I, II, and III.3 Class I PI3Ks are further subdivided
into class IA and class IB. Class IA PI3Ks (PI3Kα, PI3Kβ, and
PI3Kδ) consist of heterodimers between a p110 catalytic subꢀ
unit (p110α, p110β, and p110δ, respectively) and a p85 regulaꢀ
tory subunit. The Class IB subtype (PI3Kγ) contains a catalytꢀ
ic p110γ and a regulatory p101 subunit.4 The main function of
class I PI3Ks in vivo is to phosphorylated phosphatidylinositol
(4,5) diphosphate (PtdIns(4,5)P2, PIP2) to phosphatidylinosiꢀ
tol triphosphate (3,4,5)P3 (PIP3) at the 3ꢀposition of the inosiꢀ
tol ring, which serves as an important second messenger in
triggering a series of downstream effectors mediating cellular
functions. This process is strictly controlled by tumor suppresꢀ
sor phosphatase and tensin homologue (PTEN), which
dephosphorylates PIP3 back to PIP2.5 Abnormalities in funcꢀ
tions of both kinase and phosphatase are commonly observed
in tumors, thus emphasizing the importance of this pathway in
cancer. A high proportion of human cancers were revealed to
rely strongly on P110α for survival and resistance to theraꢀ
py.5,6 Therefore, the targeting of PI3K pathway is one of the
most promising approaches for cancer treatment.
BKM120, a pyrimidine scaffold derivative developed by Noꢀ
vartis AG, displayed a potent and selective class I PI3K inhibiꢀ
tor over many other related kinases;7 this derivative is also
undergoing phase III trials for breast cancer treatment
(NCT01572727, NCT01610284, and NCT01633060). Similar
to the majority of reported clinical candidates, NVP-BKM120
showed comparable potency against four isoforms of class I
PI3Ks, possibly leading to offꢀtarget effects compromising
therapeutic utility. Therefore, more potent and higher isoformꢀ
selective chemical entities should be developed in clinical
trials to offer a better choice for cancer treatment. On the basis
of 2,4,6ꢀtrisubstituted pyrimidine derivatives, most previous
structureꢀactivity relationships (SAR) studies focused on the
4ꢀ and 6ꢀpositions of the pyrimidine scaffold with morpholine
substituted on the 2ꢀposition.7,8 This finding led us to design
and synthesize a series of derivatives by replacing the C2 morꢀ
pholine with various aliphatic or longꢀchain substituted aroꢀ
matic amines. Increased hydrogen bonding on the periphery of
the active site was expected to improve selective profiles (Figꢀ
ure 3), leading to the identification of compounds 5d and 5p.
Among the current clinical candidates (Figure 2), NVP-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 2 of 14
1
2
obtained via cyclization with urea, which was subjected to
3
4
5
6
7
8
9
chlorination with POCl3 in a basic environment to obtain the
key intermediate 16. Coupling with morpholine and (2S,6R)ꢀ
2,6ꢀdimethylmorpholine was then performed to produce 17a
and 17b, respectively, which underwent SuzukiꢀMiyaura
crossꢀcoupling reaction with 4 to generate the target comꢀ
pounds. The 6ꢀpyrimidineꢀsubstituted derivative 20 was preꢀ
pared from 3d and boronic ester 20 via a similar coupling reꢀ
action (Scheme 4).
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
Figure 2. Some reported structures of clinical candidates.
Figure 5. Structures of compounds12 and 13
Figure 3. Design of series derivatives based on NVP-BKM120.
RESULTS AND DISCUSSION
As outlined in Scheme 1, the desired 2,4,6ꢀtrisubstituted pyꢀ
rimidine derivatives (5aꢀp) were prepared from 2,4,6ꢀ
trichloropyrimidine (1) via amination on C4 and C2 stepwise
with various aliphatic amines or aromatic amines. Suzukiꢀ
Miyaura crossꢀcoupling reaction with 5ꢀ(4,4,5,5ꢀtetramethylꢀ
1,3,2ꢀdioxaborolanꢀ2ꢀyl)ꢀ4ꢀ(trifluoromethyl)pyridinꢀ2ꢀamine
(4) was then performed under microwave irradiation condiꢀ
tions. Optically pure compounds 6 and 7 were obtained from
5d via chiral preparative separation (Figure 4). The 2,4,6ꢀ
trisubstitutedꢀ1,3,5ꢀtriazines (11aꢀe) were prepared through a
similar procedure mentioned above (Scheme 2). Compounds
12 and 13 were also isolated from 11b via chiral separation
(Figure 5).
Scheme 3.
Scheme 4.
The detailed preparation of transꢀhexahydroꢀ2Hꢀ
[1,4]dioxino[2,3ꢀc]pyrrole (26) is shown in Scheme 5. Starting
with benzyl 2,5ꢀdihydroꢀ1Hꢀpyrroleꢀ1ꢀcarboxylate (22), inꢀ
termediate 25 was obtained via epoxidation with mꢀCPBA,
nucleophilic substitution with 2ꢀbromoethanol in the presence
of BF3·OEt2, and subsequent cyclization in EtOH. Removal of
the protecting group with 10% Pd/C produced the desired inꢀ
termediate 26.
Scheme 1.
Scheme 5.
All newly prepared compounds were assessed in vitro in
terms of biological activity against the p110α isoform of PI3K
by using the kinaseꢀGlo Plus Luminescent Assay. PI-103 and
NVP-BKM120 (5a) were assigned as positive controls. Under
these assay conditions, IC50 values were determined to be
7.4±1.3 nM and 28.0±1.2 nM, respectively (Table 1).
We first conducted an SAR study by replacing the C4 morꢀ
pholine with 2,6ꢀdimethylmorpholine and hexahydroꢀ2Hꢀ
[1,4]dioxino[2,3ꢀc]pyrrole. All tested compounds (5iꢀ5m)
showed significant decrease in activity possibly because of
increased steric hindrance, which obstructed the hydrogen
bond interaction between O (morpholine) and NH (hinge
Val882).9,10 Compounds 5bꢀ5f were characterized by replacing
Figure 4. Structures of compounds 6 and 7.
Scheme 2.
Scheme 3 shows the detailed synthetic route for the preparaꢀ
tion of quinazolineꢀscaffold derivatives 18a and 18b. Starting
with 2ꢀaminoꢀ4ꢀchlorobenzoic acid (14), intermediate 15 was
ACS Paragon Plus Environment

Page 3 of 14
Journal of Medicinal Chemistry
1
2
3
4
5
6
Table 1. Kinase inhibition against PI3Kα (IC50 values in nM).
a
a
Compound X=
R1
R2
IC50
Compound X=
R1
R2
IC50
7
8
5a
CH
28.0±1.2
5p
CH
18.0±1.9
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
5b
5c
CH
CH
CH
91.0±12.6
60.0±2.2
31.0±6.0
11a
(±)-11b
11c
N
N
N
59.0±4.5
32.0±2.4
(±)-5d
9530±1675
5e
5f
CH
CH
117.0±2.9
23.0±4.7
11d
11e
N
N
>10000
>10000
5g
CH
26.0±2.7
(-)-6
CH
61.0±5.4
5h
5i
CH
CH
568.0±28.9
224.0±6.4
(+)-7
CH
N
35.0±2.3
68.0±8.2
(-)-12
5j
CH
CH
>10000
(+)-13
N
38.0±4.5
5k
5751±1275
(±)-21
128.0±9.1
5l
CH
CH
896.0±56.7
375.0±9.9
O
N
(±)-5m
18a
>10000
CF3
N
N
5n
CH
118.0±5.6
N
N
O
H2N
18b
>10000
7.4±1.3
5o
CH
31.0±3.1
PI-103
aIn vitro lipid kinase assay. IC50 values are the mean of triplicate measurements.
the C2 morpholine of 5a with various heterocycles, and raceꢀ
mic 5d showed comparable activity relative to reference 5a.
By contrast, cisꢀform heterocycle substituted compound 5e
exhibited a fourꢀfold decrease in activity relative to 5d. To
further investigate the influence of chiral configuration of 5d
on the inhibitory activity, compounds 6 and 7, which were
separated from 5d, were evaluated. Dextroisomer 7 showed
approximately twoꢀfold higher activity than levoisomer 6.
Upon introducing an aliphatic or aromatic chain between
the pyrimidine scaffold and C2 morpholine, compound 5g was
found to be slightly more potent than 5a. By contrast, comꢀ
pound 5h showed significant loss in activity possibly because
of πꢀπ (Tꢀshaped) stacking interaction between the benzene
ring of 5g and hinge residue of Trp780. Longꢀchain substituted
compound 5p displayed the highest inhibitory activity, which
was 1.56 times higher than that of 5a. We could also deduce
that the pyrimidine scaffold carried a N atom at position 5
(11aꢀe, 12 and 13), but no activity was observed upon replacꢀ
ing the pyrimidine scaffold with quinazoline (18a and 18b).
We compared the biochemical activities of compounds 5a,
5d and 5p with other isoforms of PI3Ks. As summarized in
Table 2, compound 5d showed comparable activities with 5a
to other PI3K isoforms, whereas compound 5p showed better
selectivity over βꢀisozyme, with a selectivity of 112ꢀfold
(PI3Kβ/α) relative to 9.3ꢀfold of 5a. Interestingly, 5p also
displayed an IC50 value of 13.0±0.5 nM against δꢀisozyme,
making it a promising dual PI3Kα/δ inhibitor.
Mutations of class I PI3Ks frequently occur in various huꢀ
man cancers;11ꢀ13 thus, 19 human cancer cell lines were used in
the current experiment, Trichostatin A (TSA) and 5a were
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 4 of 14
1
2
3
Table 2. Activities of 5a, 5d and 5p against class I PI3K (IC50
valuesa in nM).
4
5
6
7
Compound
PI3Kα
28.0±1.2
31.0±6.0
PI3Kβ
259.0±13.8
175.0±2.4
PI3Kγ
129.0±9.6
158.0±14.1 61.0±7.2
80.0±7.0 13.0±0.5
PI3Kδ
62.0±7.6
5a
5d
5p
18.0±1.9 2014.0±126.9
a IC50 values are the mean of triplicate measurements.
8
employed as positive controls. The results are presented in
Tables 3 and 4. All tested compounds showed comparable
cytotoxicities with IC50 values in the micromolar range, except
for compounds 5l and 18a. Compound 5d also exhibited comꢀ
parable activities to 5a in various cell lines, and dextroisomer
7 was twice to thrice more potent than levoisomer 6. These
results were correlated with their effects on kinase. Compound
5g unexpectedly performed well on kinase but exhibited weak
cellular inhibition.
As shown in Figure 6, the suppressive effect of compound
5d on pꢀAKT473 was comparable to that of 5a, whereas 5p
showed more potent inhibition. Compounds 5d and 5p were
also shown to inhibit the PI3K/AKT/mꢀTOR pathway; thus,
5d and 5p were potential PI3K inhibitors.
9
Figure 6. Effects of compounds 5a, 5d and 5p on AKT and pꢀ
AKT473
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
.
fects of 5d.
Nude mice bearing MDAꢀMBꢀ231 xenograft tumors were
treated with 5d (30 mg/kg, every two days) by intragastric
administration for 19 days. As shown in Figures 8a and 8b, the
tumor size in the 5dꢀtreated group (402±184 mm3; P<0.001)
was smaller than that in the control group (1065±142 mm3),
indicating that the growth of xenograft tumors was significantꢀ
ly inhibited by 5d. Consistently, the tumor weight in the 5dꢀ
treated group (0.32±0.19 g; P<0.01) was significantly lower
than that in the control group (0.74±0.10 g; Figure 8c). During
the experimental period, the mice in the 5dꢀtreated group
showed no decrease in body weight (Figure 8d), indicating the
low toxicity of intragastrically administered 5d.
We have compared the effects of compounds 5a, 5d and 5p
on MDAꢀMBꢀ231 cell proliferation test.14ꢀ16 The results reꢀ
vealed that 5a (positive control) and 5d showed similar antiꢀ
cancer effects, 5d was more potent than 5p (Figure 7). So we
used a xenograft model to evaluate the in vivo anticancer efꢀ
To examine the possible binding modes for compound
,
5d
and
inside the ATPꢀbinding site of PI3Ks, docking
18a
5p
Table 3. Antiꢀproliferative activities in various cell types (IC50 valuesa in ꢁM).
Cell-lines
5a
(±)-5d
5c
5b
11a
5e
5l
(±)-5m
(±)-11b
TSA
K562
1.10±0.30 4.26±1.41 2.40±0.49 2.68±0.10
>10
1.75±0.98 >10 3.19±0.76
>10
0.12±0.03
0.02±0.00
0.10±0.01
0.06±0.00
0.07±0.01
0.05±0.04
0.06±0.00
0.04±0.00
0.06±0.00
0.03±0.00
0.07±0.00
MOLTꢀ4
0.80±0.19 1.00±0.13 1.14±0.11 0.42±0.05 1.55±0.36 2.51±0.22 NT 3.24±0.08
1.21±0.19
5.90±1.87
5.66±0.85
8.15±1.34
NT
3.30±0.60
3.05±0.14
0.77±0.18
4.46±0.98
9.57±1.18
BELꢀ402 1.92±0.43 3.37±0.55 2.79±1.45 1.64±0.36 5.90±0.48 5.82±1.23 NT 6.43±1.23
Huhꢀ7
MCFꢀ7
SKꢀBRꢀ3
DU145
U937
1.51±0.29 4.26±0.21 2.93±0.30 1.21±0.22 5.67±0.79 4.84±1.18 NT 6.29±0.84
1.50±0.29 2.42±0.56 2.54±0.55 1.41±0.37 5.37±1.80 4.95±1.45 NT 7.29±1.41
NT
NT
NT
NT
NT
NT
NT
NT
0.91±0.25 1.93±0.31 2.38±0.20 1.41±0.13 3.43±1.17 4.81±1.27 >10 7.11±0.98
0.58±0.09 0.77±0.05 0.72±0.12 0.57±0.12 2.93±0.08 1.76±0.12 NT 2.86±0.65
NCIꢀN87 0.66±0.27 1.36±0.39 1.41±0.37 0.52±0.25 1.20±0.52 1.97±0.62 NT 6.21±1.00
SGCꢀ901 1.24±0.27 3.14±0.14 2.30±0.12 1.24±0.04 5.25±0.95 3.75±1.52 NT 4.78±0.68
BGCꢀ823 1.43±0.46 3.09±0.62 2.44±0.50 1.41±0.12
>10
4.57±1.43 NT 4.33±1.10
HT1080
A431
A549
PANCꢀ1
Hela
2.08±0.35 4.73±1.00 2.72±0.84 1.98±0.88 5.51±0.02 4.84±0.11 NT 5.64±1.49 10.00±1.20 0.08±0.00
1.03±0.47 3.68±1.37 1.79±0.60 2.29±0.29 8.40±2.50 2.55±1.60 NT 3.03±0.18
1.51±0.17 3.39±1.06 3.05±1.80 1.89±0.91 6.37±0.90 4.98±0.32 NT 5.56±1.07
1.83±0.66 3.26±1.32 2.78±0.39 1.38±0.28 6.42±0.63 3.67±1.41 NT 5.97±1.63
1.17±0.36 2.66±0.77 1.76±0.21 1.81±0.83 8.33±0.85 4.31±1.40 NT 5.33±1.60
>10
0.18±0.03
0.05±0.01
0.10±0.01
0.09±0.01
4.03±0.99
6.23±1.91
>10
a IC50, the mean value of triplicate measurements. b NT, not tested.
Table 4. Antiꢀproliferative activities in various cell types (IC50 valuesa in ꢁM).
Cell-lines
5f
5g
5n
5o
5p
NTb
(-)-6
(+)-7
(-)-12
(+)-13
TSA
3.64±0.56 8.81±2.10 14.90±1.82 11.20±0.86
6.34±0.85
NT
1.96±0.35
7.47±1.04
3.47±0.50
NT
2.56±0.43
NT
0.97±0.44
3.82±0.77
1.60±0.40
NT
24.60±1.50 24.50±3.41 0.12±0.02
K562
HL60
NT
NT
4.34±1.51
4.07±0.85
0.94±0.20
1.11±0.20
4.97±1.30
1.63±0.53
5.36±1.36
6.71±1.12
7.49±1.24
1.29±0.22
2.39±1.04
NT
2.49±1.22
1.96±0.50
12.8±2.90
4.79±0.31
4.34±0.85
NT
19.50±0.08
4.22±1.05
4.74±1.35
NT
2.70±0.76
2.28±0.24
7.32±1.50
4.27±1.38
4.85±1.31
2.08±0.03
9.29±2.06
3.67±0.32
NT
MOLTꢀ4
MCFꢀ7
SKꢀBRꢀ3
MDAꢀMBꢀ231
DU145
PC3
NCIꢀN87
U937
BGCꢀ823
A431
1.11±0.22 1.77±0.52
4.39±1.10 9.41±0.83 16.50±2.54
1.59±0.17 2.67±1.28
NT
6.56±1.21
NT
0.02±0.00
0.07±0.00
0.05±0.00
NT
6.53±1.10
7.28±2.64
0.99±0.12
2.21±0.37
4.66±0.47
NT
>10
NT
NT
15.10±1.72 11.90±1.87
15.03±3.47 4.86±0.045
7.36±0.98
5.63±0.83
3.90±0.30
4.03±0.45
NT
1.08±0.12
6.24±1.95
2.77±0.46
5.53±0.46
NT
0.05±0.01
NT
0.05±0.01
0.04±0.00
0.06±0.01
NT
NT
NT
NT
NT
1.99±0.26 3.48±0.86 10.40±3.53
3.38±0.72
6.59±0.50
NT
1.51±0.50
2.79±1.20
NT
3.64±1.00 8.36±0.74
NT NT
2.95±0.35 7.79±1.48
NT NT
4.04±1.90 6.91±1.55
NT
11.50±1.47
4.57±0.96
8.52±1.37
>100
5.90±0.87
5.46±0.05
2.36±0.09
>100
10.50±2.11
9.13±0.78
3.47±0.49
>100
14.10±0.78 32.20±5.21 0.18±0.02
6.40±2.01
NT
7.53±1.48
2.49±0.36
NT
2.45±0.33
8.01±0.48
9.04±2.50
>100
9.50±1.90
7.64±1.05
>100
0.05±0.01
0.10±0.01
0.09±0.03
A549
PANCꢀ1
Hela
a IC50, the mean value of triplicate measurements. b NT, not tested.
ACS Paragon Plus Environment

Page 5 of 14
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
Surprisingly, the amino of the pyridine ring only formed a
hydrogen bond with residue Asp964 (distance: 2.2 Å), and no
interaction existed with residue Lys833, which may explain its
good selectivity over PI3Kβ. We also performed the docking
analysis of compound
binding to γ (PDB code, 4ANV)
5p
(Figure 10c) and δ isoforms (PDB code, 2WXF) (Figure 10d).
Four hydrogenꢀbond interactions with residues of PI3Kγ were
noticed: Val882 (1.8 Å), Tyr867 (2.1 Å), Asp841 (2.1 Å), and
Figure 7. Effects of compounds 5a, 5d and 5p on cell proliferaꢀ
tion test.
9
Gln893 (2.3 Å). For the binding of compound
to PI3Kδ, six
5p
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
hydrogenꢀbond interactions with the residues were indicated:
Val828 (2.0 Å), Tyr813 (1.8 Å), Asp787 (1.9 Å), Lys779 (1.8
Å), Lys708 (2.1Å), and Thr750 (2.1 Å), along with a πꢀπ (Tꢀ
shaped) stacking interaction with residue Trp760. Compound
expectedly showed no hydrogenꢀbond interaction with
18a
amino because of the replacement of pyrimidine scaffold with
quinazoline, which blocked the molecule from docking into an
active pocket (Figure 9b).
CONCLUSIONS
We have designed and synthesized three series of trisubstiꢀ
tuted pyrimidine derivatives. These compounds were evaluatꢀ
ed as potential PI3Kα inhibitors. Compound 5d showed comꢀ
parable bioactivity with NVP-BKM120, and 5p displayed the
greatest inhibitory potency. The isozymeꢀselective assay
showed that 5p exhibited higher selectivity than 5a, suggestꢀ
ing that 5p is a promising dual PI3Kα/δ inhibitor. Moreover,
the cytotoxicities of the substituted pyrimidines against human
cancer cell lines were evaluated, most of them showed compaꢀ
rable activities, the in vivo anticancer effect of 5d was also
tested. Their pharmacokinetics profiles and other biological
activities in vivo are presently being investigated.
EXPERIMENTAL SECTION
Molecular modeling. The PI3KαꢀKKR proteinꢀligand comꢀ
plex crystal structure (PDB ID: 3ZIM) was chosen as the temꢀ
plate to compare the docking mode among compounds 18a, 5d
and 5p bound to PI3Kα, as well as 5p bound to PI3Kβ (PDB
ID: 3T8M). The molecular docking procedure was per formed
in accordance with the CꢀDOCKER protocol of Discovery
Studio 2.5. For ligand preparation, the 3D structures of comꢀ
pounds 18a, 5d, and 5p were generated and minimized using
Discovery Studio 2.5. Hydrogen atoms were added for enzyme
preparation, and CHARMm force field was employed. PI3Kα
and PI3Kβ enzymes were defined as receptors, and the site
sphere was selected on the basis of the ligand binding location
of KKR. The KKR molecule was then removed, and comꢀ
pounds 18a, 5d or 5p were placed during the molecular dockꢀ
ing procedure. The types of interactions between the docked
enzyme and the ligand were analyzed at the end of the moꢀ
lecular docking. The ligandꢀprotein complex with the optimum
score was selected as the initial structure for MD simulaꢀ
tions.18ꢀ23
Figure 8. 5d attenuates xenograft tumor growth. (a) Nude mice
bearing MDAꢀMBꢀ231xenograft tumors were treated with vehicle
or 5d (30 mg/kg, intragastric administration, every two days). The
estimated tumor volume is plotted versus time. (b) Tumors were
removed from 5 mice in each group and are shown. (c) The
weights of the dissected tumors were measured. (d) The body
weights were monitored and plotted versus time. All data are preꢀ
sented as the mean+SD (n =5; **, P < 0.01, ***, P < 0.001, Stuꢀ
dent t test).
analysis was performed using CꢀDOCKER in Discovery Stuꢀ
dio 2.5. The protein crystal structure of PI3Kα (PDB code,
3ZIM)17 was used, followed by 8ꢀns molecular dynamics simꢀ
ulations to study the binding modes using the Amber12 packꢀ
age. Three hydrogen bonds interacted between
and PI3Kα
5d
(Figure 9a): the oxygen of C4 morpholine formed a key hydroꢀ
gen bond with the hinge of Val851 (distance: 1.9 Å), and the
amino of the pyridine ring formed two hydrogen bonds, which
interacted with the side chains of residues Asp993 (distance:
2.0 Å) and Lys802 (distance: 2.1 Å). Compound
also exꢀ
5p
ASSOCIATED CONTENT
Supporting information
hibited the above mentioned threeꢀhydrogenꢀbond interaction,
although it indicated additional πꢀπ (Tꢀshaped) stacking interꢀ
action between the benzene ring and hinge residue of Trp780
(Figure 10a). This finding led to a more potent activity against
Synthetic schemes and experimental procedures, characterization
of organic molecules, enzyme assays, cell proliferation assay,
western blot assay, tumor growth in xenografts. This material is
available free of charge via the Internet at http://pubs.acs.org.”
PI3Kα than . The docking analysis of
5d
with PI3Kβ (PDB
5p
code, 3T8M) (Figure 10b) showed that this compound formed
a hydrogen bond with a residue of Val882 (distance: 1.9 Å)
and a πꢀπ (Tꢀshaped) stacking interaction with residue Trp812.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 14
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
Docking modes for compounds
18a
and
into protein crystal structure of PI3Kα. (a) Compound
bound to PI3Kα. (b) Comꢀ
5d
Figure 9.
pound
5d
bound to PI3Kα (PDB ID: 3ZIM).
18a
Figure 10. Docking modes for compound 5p into protein crystal structure of PI3Ks. (a) Binding to PI3Kα (PDB ID: 3ZIM). (b) Binding to
PI3Kβ (PDB ID: 3T8M). (c) Binding to PI3Kγ (PDB ID: 4ANV). (d) Binding to PI3Kδ (PDB ID: 2WXF).
chostatin A; mꢀTOR, mammalian target of rapamycin; HEPES,
4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid; EGTA,
AUTHOR INFORMATION
ethylene
glycol
tetraacetic
acid;
CHAPS,
3ꢀ[(3ꢀ
Corresponding Author
cholamidopropyl)ꢀdimethylammonio]ꢀ1ꢀpropane
sulfonate;
* G.Lu.: phone, 86ꢀ20ꢀ39943048; Eꢀmail, lugui@mail.sysu.edu.cn.
DMEM, dulbecco minimum essential medium; CHARMm,
Chemistry at Harvard Macromolecular Mechanics; KKR, Korꢀ
ringaꢀKohnꢀRostoker; MD, molecular dynamics.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version
of the manuscript.
REFERENCES
(1) Vivanco, I.; Sawyers, C. L. The phosphatidylinositol 3ꢀ
KinaseꢀAKT pathway in human cancer. Nat. Rev. Cancer 2002, 2,
489ꢀ501.
ACKNOWLEDGMENTS
This work was financially supported by the National Highꢀ
tech R&D Program of China (863 Program, No.
2013AA092903) and the Guangdong Province Natural Sciꢀ
ence Foundation (No. 2015A030313184). We also
acknowledge Prof. RuiꢀBo Wu for help in molecular docking.
(2) Smith, A. L.; D’Angelo, N. D.; Bo, Y. Y.; Booker, S. K.; Cee,
V. J.; Herberich, B.; Hong, F.ꢀT.; Jackson, C. L. M.; Lanman, B.
A.; Liu, L.; Nishimura, N.; Pettus, L. H.; Reed, A. B.; Tadesse, S.;
Tamayo, N. A.; Wurz, R. P.; Yang, K.; Andrews, K. L.;
Whittington, D. A.; McCarter, J. D.; Miguel, T. S.; Zalameda, L.;
Jiang, J.; Subramanian, R.; Mullady, E. L.; Caenepeel, S.;
Freeman, D. J.; Wang, L.; Zhang, N.; Wu, T.; Hughes, P. E.;
Norman, M. H. StructureꢀBased Design of a Novel Series of
Potent, Selective Inhibitors of the Class I Phosphatidylinositol 3ꢀ
Kinases. J. Med. Chem. 2012, 55, 5188ꢀ5219.
ABBREVIATIONS LIST
PI3K, phosphatidylinositol 3ꢀkinase; PtdIns(4,5)P2, phosphaꢀ
tidylinositol (4,5) diphosphate; PIP3, phosphatidylinositol
triphosphate (3,4,5)P3; PTEN, phosphatase and tensin homoꢀ
logue; AKT, known as protein kinase B or PKB; TSA, triꢀ
ACS Paragon Plus Environment

Page 7 of 14
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
(3) Engelman, J. A.; Luo, J.; Cantley, L. C. The evolution of
phosphatidylinositol 3ꢀkinases as regulators of growth and
metabolism. Nat. Rev. Genet. 2006, 7, 606ꢀ619.
(4) Vanhaesebroeck, B.; Leevers, S. J.; Ahmadi, K.; Timms, J.;
Katso, R.; Driscoll, P. C.; Woscholski, R.; Parker, P. J.;
Waterfield, M. D. Synthesis and function of 3ꢀphosphorylated
insitol lipids. Annu. Rev. Biochem. 2001, 70, 535ꢀ602.
(5) Liu, P.; Cheng, H.; Roberts, T. M.; Zhao, J. J. Targeting the
phosphoinositide 3ꢀkinase pathway in cancer. Nat. Rev. Drug
Discov. 2009, 8, 627ꢀ644.
K.; Rubin, M. A.; Moch, H.; Loda, M.; Catalona, W.; Fletcher, J.;
Signoretti, S.; Kaye, F.; Anderson, K. C.; Demetri, G. D.;
Dummer, R.; Wagner, S.; Herlyn, M.; Sellers, W. R.; Meyerson,
M.; Garraway, L. A. Highꢀthroughput oncogene mutation
profiling in human cancer. Nat. Genet. 2007, 39, 347ꢀ351.
(13) Samuels, Y.; Wang, Z.; Bardelli, A.; Silliman, N.; Ptak, J.;
Szabo, S.; Yan, H.; Gazdar, A.; Powell, S. M.; Riggins, G. J.;
Willson, J. K. V.; Markowitz, S.; Kinzler, K. W.; Vogelstein, B.;
Velculescu, V. E. High frequency of mutations of the PIK3CA
gene in human cancers. Science 2004, 304, 554.
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
(6) Hollander, M. C.; Blumenthal, G. M.; Dennis, P. A. PTEN
loss in the continuum of common cancers, rare syndromes and
mouse models. Nat. Rev. Cancer 2011, 11, 289ꢀ301.
(14) Kim, K. J.; Wang, L.; Su, Y.ꢀC.; Gillespie, G. Y.; Salhotra,
A.; Lal, B.; Laterra, J. Systemic antiꢀhepatocyte growth factorꢀ
monoclonal antibody therapy induces the regression of intracraniꢀ
al glioma xenografts. Clin. Cancer Res. 2006, 12, 1292ꢀ1298.
(15) Jiang, W.; Jin, G.; Ma, D.; Wang, F.; Fu, T.; Chen, X.; Chen,
X.; Jia, K.; Marikar, F. M. M. T.; Hua, Z. Modification of cyclic
NGR tumor neovasculatureꢀhoming motif sequence to human
plasminogen kringle 5 improves inhibition of tumor growth. PLoS
ONE 2012, 7, e37132.5.
(16) Zhang, F.ꢀM.; Long, Z.ꢀJ.; Hou, Z.ꢀJ.; Luo, Y.; Xu, L.ꢀZ.;
Xia, J.ꢀL.; Lai, X.ꢀJ.; Liu, J.ꢀW.; Wang, X.; Kamran, M.; Yan, M.;
Shao, S.ꢀJ.; Lam, E. W.ꢀF.; Wang, S.ꢀW.; Lu, G.; Liu, Q. A novel
small molecule aurora kinase inhibitor attenuates breast tumorꢀ
initiating cells and overcomes drug resistance. Mol. Cancer Ther.
2014, 13, 1991ꢀ2003.
(17) Nacht, M.; Qiao, L.; Sheets, M. P.; St. Martin, T.; Labenski,
M.; Mazdiyasni, H.; Karp, R.; Zhu, Z.; Chaturvedi, P.; Bhavsar,
D.; Niu, D.; Westlin, W.; Petter, R. C.; Medikonda, A. P.; Singh, J.
Discovery of a potent and isoformꢀselective targeted covalent
inhibitor of the lipid kinase PI3Kα. J. Med. Chem. 2013, 56, 712ꢀ
721.
(18) Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.;
Zhang, W.; Yang, R.; Cieplak, P.; Luo, R.; Lee, T.; Caldwell, J.;
Wang, J.; Kollman, P. A pointꢀcharge force field for molecular
mechanics simulations of proteins based on condensedꢀphase
quantum mechanical calculations. J. Comput. Chem. 2003, 24,
1999ꢀ2012.
(19) Wang, J.; Wolf, R. M.; Caldwell, J. W.; Kollman, P. A.; Case,
D. A. Development and testing of a general amber force field. J.
Comput. Chem. 2004, 25, 1157ꢀ1174.
(20) Bayly, C. I.; Cieplak, P.; Cornell, W.; Kollman, P. A. A wellꢀ
behaved electrostatic potential based method using charge
restraints for deriving atomic charges: the RESP model. J. Phys.
Chem. 1993, 97, 10269ꢀ10280.
(21) Berendsen, H. J. C.; Postma, J. P. M.; van Gunsteren, W. F.;
DiNola, A.; Haak, J. R. Molecular dynamics with coupling to an
external bath. J. Chem. Phys. 1984, 81, 3684ꢀ3690.
(22) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R.
W.; Klein, M. L. Comparison of simple potential functions for
simulating liquid water. J. Chem. Phys. 1983, 79, 926ꢀ935.
(23) Ryckaert, J.ꢀP.; Ciccotti, G.; Berendsen, H. J. C. Numerical
integration of the cartesian equations of motion of a system with
constraints: molecular dynamics of nꢀalkanes. J. Comput. Phys.
1977, 23, 327ꢀ341.
(7) Burger, M. T.; Pecchi, S.; Wagman, A.; Ni, Z.ꢀJ.; Knapp, M.;
Hendrickson, T.; Atallah, G.; Pfister, K.; Zhang, Y.; Bartulis, S.;
Frazier, K.; Ng, S.; Smith, A.; Verhagen, J.; Haznedar, J.; Huh, K.;
Iwanowicz, E.; Xin, X.; Menezes, D.; Merritt, H.; Lee, I.;
Wiesmann, M.; Kaufman, S.; Crawford, K.; Chin, M.; Bussiere,
D.; Shoemaker, K.; Zaror, I.; Maira, S.ꢀM.; Voliva, C. F.
Identification of NVPꢀBKM120 as a potent, pelective, orally
bioavailable class I PI3 kinase inhibitor for treating cancer. ACS
Med. Chem. Lett. 2011, 2, 774ꢀ779.
(8) Burger, M. T.; Knapp, M.; Wagman, A.; Ni, Z.ꢀJ.;
Hendrickson, T.; Atallah, G.; Zhang, Y.; Frazier, K.; Verhagen, J.;
Pfister, K.; Ng, S.; Smith, A.; Bartulis, S.; Merrit, H.; Weismann,
M.; Xin, X.; Haznedar, J.; Voliva, C. F.; Iwanowicz, E.; Pecchi, S.
Synthesis and in vitro and in vivo evaluation of phosphoinositideꢀ
3ꢀkinase inhibitors. ACS Med. Chem. Lett. 2010, 2, 34ꢀ38.
(9) Walker, E. H.; Pacold, M. E.; Perisic, O.; Stephens, L.;
Hawkins, P. T.; Wymann, M. P.; Williams, R. L. Structural
determinants of phosphoinositide 3ꢀkinase inhibition by
Wortmannin, LY294002, Quercetin, Myricetin, and Staurosporine.
Mol. Cell 2000, 6, 909ꢀ919.
(10) Knight, Z. A.; Chiang, G. G.; Alaimo, P. J.; Kenski, D. M.;
Ho, C. B.; Coan, K.; Abraham, R. T.; Shokat, K. M. Isoformꢀ
specific phosphoinositide 3ꢀkinase inhibitors from an
arylmorpholine scaffold. Bioorg. Med. Chem. 2004, 12, 4749ꢀ
4759.
(11) temkeꢀHale, K.; GonzalezꢀAngulo, A. M.; Lluch, A.; Neve,
R. M.; Kuo, W.ꢀL.; Davies, M.; Carey, M.; Hu, Z.; Guan, Y.;
Sahin, A.; Symmans, W. F.; Pusztai, L.; Nolden, L. K.; Horlings,
H.; Berns, K.; Hung, M.ꢀC.; van de Vijver, M. J.; Valero, V.;
Gray, J. W.; Bernards, R.; Mills, G. B.; Hennessy, B. T. An
integrative genomic and proteomic analysis of PIK3CA, PTEN,
and AKT mutations in breast cancer. Cancer Res. 2008, 68, 6084ꢀ
6091.
(12) Thomas, R. K.; Baker, A. C.; DeBiasi, R. M.; Winckler, W.;
LaFramboise, T.; Lin, W. M.; Wang, M.; Feng, W.; Zander, T.;
MacConaill, L. E.; Lee, J. C.; Nicoletti, R.; Hatton, C.; Goyette,
M.; Girard, L.; Majmudar, K.; Ziaugra, L.; Wong, K.ꢀK.; Gabriel,
S.; Beroukhim, R.; Peyton, M.; Barretina, J.; Dutt, A.; Emery, C.;
Greulich, H.; Shah, K.; Sasaki, H.; Gazdar, A.; Minna, J.;
Armstrong, S. A.; Mellinghoff, I. K.; Hodi, F. S.; Dranoff, G.;
Mischel, P. S.; Cloughesy, T. F.; Nelson, S. F.; Liau, L. M.; Mertz,
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 8 of 14
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
8
ACS Paragon Plus Environment

Page 9 of 14
Journal of Medicinal Chemistry
1
2
3
4
Figure 1.
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
Figure 2.
Figure 3.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 10 of 14
1
2
3
4
Figure 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
Figure 5.
Figure 6.
ACS Paragon Plus Environment

Page 11 of 14
Journal of Medicinal Chemistry
1
2
3
4
Figure 7.
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
Figure 8.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 12 of 14
1
2
3
4
Figure 9.
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
Figure 10.
ACS Paragon Plus Environment

Page 13 of 14
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
Scheme 1.
Scheme 2.
Scheme 3.
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
Scheme 4.
Scheme 5.
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 14 of 14
1
2
3
4
Table of contents graphic
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