Novel antiproliferative agents bearing morpholinopyrimidine scaffold as PI3K inhibitors and apoptosis inducers; design, synthesis and molecular docking

Article abstract of DOI:10.1016/j.bioorg.2020.104051

Two series of novel morpholinopyrimidine derivatives were synthesized and screened for their in-vitro cytotoxic activity against 60 tumor cell line by the National Cancer Institute, USA. The in-vitro cytotoxic IC₅₀ values for the most active compounds 6e, 6g, and 6l against the most sensitive cell line leukemia SR were estimated (IC₅₀ = 0.76, 13.59, and 4.37 uM, respectively). To investigate their PI3K enzyme inhibition activity, the assay was done on Class IA (α, β, & δ) isoforms. The IC₅₀ values were very promising: compound [6e = 11.73 (α), 6.09 (β), 11.18 (δ)], compound [6g = 8.43 (α), 15.84 (β), 30.62 (δ)], and compound [6l = 13.98 (α), 7.22 (β), 10.94 (δ)], compared to the reference compound LY294002 = 6.28 (α), 4.51 (β), 4.60 (δ) uM, respectively. Moreover, cell cycle analysis and annexin V-FITC staining were done on Leukemia SR, there was arrest at G2/M phase and apoptosis was induced. Finally, docking study was performed to analyze the interactive mode of these derivatives in PI3Kα ATP-binding site. These outcomes proved that compounds 6e, 6g, and 6l are potential leads for further optimization as antileukemic agents.

Full text of DOI:10.1016/j.bioorg.2020.104051

BioorganicChemistry102(2020)104051
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Novel antiproliferative agents bearing morpholinopyrimidine scaffold as
PI3K inhibitors and apoptosis inducers; design, synthesis and molecular
docking
⁎
Amira A. Helwaa, , Nehad M. El-Dydamonyb, Rasha A. Radwanc, Sahar M. Abdelraoufd,
Rana M. Abdelnabye
a Pharmaceutical Organic Chemistry Department, College of Pharmaceutical Sciences and Drug Manufacturing, Misr University for Science and Technology, 6th of October
City, Egypt
b Pharmaceutical Chemistry Department, College of Pharmaceutical Sciences and Drug Manufacturing, Misr University for Science and Technology, 6th of October City,
Egypt
c Biochemistry Department, Faculty of Pharmacy and Pharmaceutical Industries, Sinai University- Kantara Branch, New City, El Ismailia, Egypt
d Biochemistry Department, Faculty of Pharmacy, Misr International University, Cairo, Egypt
e Pharmaceutical Chemistry Department, Faculty of Pharmacy, Heliopolis University, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two series of novel morpholinopyrimidine derivatives were synthesized and screened for their in-vitro cytotoxic
activity against 60 tumor cell line by the National Cancer Institute, USA. The in-vitro cytotoxic IC50 values for the
most active compounds 6e, 6g, and 6l against the most sensitive cell line leukemia SR were estimated
(IC50 = 0.76, 13.59, and 4.37 uM, respectively). To investigate their PI3K enzyme inhibition activity, the assay
was done on Class IA (α, β, & δ) isoforms. The IC50 values were very promising: compound [6e = 11.73 (α),
6.09 (β), 11.18 (δ)], compound [6g = 8.43 (α), 15.84 (β), 30.62 (δ)], and compound [6l = 13.98 (α), 7.22
(β), 10.94 (δ)], compared to the reference compound LY294002 = 6.28 (α), 4.51 (β), 4.60 (δ) uM, respectively.
Moreover, cell cycle analysis and annexin V-FITC staining were done on Leukemia SR, there was arrest at G2/M
phase and apoptosis was induced. Finally, docking study was performed to analyze the interactive mode of these
derivatives in PI3Kα ATP-binding site. These outcomes proved that compounds 6e, 6g, and 6l are potential leads
for further optimization as antileukemic agents.
Morpholinopyrimidine
Cytotoxicity
Leukemia
PI3K inhibition
Apoptosis
Molecular modeling
1. Introduction
outcomes in cancer patients. The phosphoinositide 3-kinase (PI3K)/
AKT signaling pathway (PI3K/AKT) which is activated by receptor
The dream of all researchers in the field of oncology is to find an
agent that can reverse the uncontrolled proliferation process occurring
in neoplastic cells. Thus, lot of efforts and money are spent to find that
effective cure for cancer which is the second leading cause of death
world wide [1]. Conventional chemotherapy as much as it is effective,
causes a lot of serious side effects such as myelopenia, mucositis, gen-
eral weakness, nausea, vomiting, susceptibility to infection, and hair
fall [2] and at the same time, the cancer cell itself can develop re-
sistance towards these types of medications [3]. Thus, targeted therapy
era has begun to find novel selective tyrosine kinases inhibitors and
scientists continuously think of attractive, fewer side effects scaffolds to
combine in producing new drugs against this demanding disease [3,4].
Targeting tyrosine Kinase Inhibitors (TKIs), vastly improve clinical
tyrosine kinase (RTKs), is a critical signal transduction system corre-
lated with essential cellular functions, such as cell survival, prolifera-
tion, differentiation, and contributes to tumor genesis in different types
of cancer [5–7]. The PI3Ks is a lipid kinase family, divided into three
classes (class I, II& III) according to their sequence homology, mode of
action and substrate preferences. Class I PI3Ks is subdivided into class
IA (PI3Kα, β and δ) isoforms and class IB (PI3Kγ) isoforms. The PI3K
signaling pathway regulates the cellular responses and plays a critical
role in maintaining the balance between cellular survival (proliferation,
differentiation, and lipid metabolism) and apoptosis. Consequently,
activation of PI3K signaling can result in uncontrolled cell proliferation
and may enhance malignant tumor formation [8,9]. The PI3K catalytic
subunit is one of the most highly mutated/amplified oncogenes in
⁎ Corresponding author at: Department of Pharmaceutical Organic Chemistry, College of Pharmaceutical Sciences and Drug Manufacturing, Misr University for
Science and Technology, Al-Motamayez District, P.O. Box: 77, 6th of October City, Egypt.
E-mail address: amira.helwa@must.edu.eg (A.A. Helwa).
https://doi.org/10.1016/j.bioorg.2020.104051
Received 27 January 2020; Received in revised form 10 May 2020; Accepted 27 June 2020
Availableonline30June2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Fig. 1. Pyrimidine derivatives with anticancer activity.
several cancer types such as breast, kidney, colorectal, liver, brain,
leukemia, and others and that has prompted the advancement of in-
hibitors of several segments of this pathway, including the PI3K, AKT,
mTOR kinases, and rapamycin analogs that hinder mTORC1 [10–17].
Therefore, selective inhibition of PIK3 enzyme provides a potential
target to develop promising anticancer agents.
compound IV [22] in Fig. 1. Furthermore, pyrimidine containing
compounds V-X in Fig. 2 [14,15,23–25] especially containing mor-
pholine moiety known to act through PI3K ATP-competitive inhibition.
The reported structural features of morpholinopyimidine hybrids are: a)
The morpholine ring is crucial for binding to Val amino acid at hinge
region. b) The central core can be any heterocycle (either fused or
single) having an aryl substituent at meta position to morpholine
moiety. c) Hydrogen bond donor/acceptor group should be present on
the aryl substituent preferably at 3 position. 3-OH showed best activity,
but it is metabolically labile due to glucuronidation. Therefore, it can be
replaced by 3-hydroxypyridyl, indazoleheterocycle or 2-aminopyr-
imidinyl to maintain the same hydrogen bonds. d) Some structures
show extension toward solvent exposed area in PI3K binding site. This
extension may be reported to form additional hydrogen bond
Pyrimidine is an interesting heterocyclic ring with great importance
as it constitutes the building block of a variety of compounds playing
critical role in the biological processes [18,19]. Many literature re-
ported that 59 patents were published on pyrimidine-based derivatives
as anticancer agents between the years 2009–2014 across the globe
[20,21] through different mechanism of action as the FDA approved
compound Imatinib (III) [21] which is reported to inhibit many kinases
like EGFR, c-Kit, PDGFR, and also, through induction of apoptosis as
Fig. 2. PI3K inhibitors with pyrimidine and morpholinopyrimidine scaffolds.
2

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Fig. 3. The design of the target compounds passed on PI3K inhibitor PI-103.
interaction with Lys and Ala or improve the pharmacokinetics of the
designed compounds [26–27].
NH peak which proved the success of chlorination. Refluxing compound
3 with double mole of morpholine in dry benzene resulted in the for-
mation of morpholinopyrimidine-5-carbonitrile 4 as reported proce-
dure [27].
From these findings, our research team was encouraged to design
and synthesize novel 6-morpholoinopyrimidines substituted at position
two with different aryl moieties (6a-l) or isatin moieties (7a-c) spaced
by hydrazono linker and at C4 p-methoxyphenyl was attached directly
to pyrimidine scaffold participating in hydrogen bond interaction as
shown in Fig. 3. The cytotoxic activity of these newly synthesized
compounds was assayed against NCI 60 cell lines panel. To discover the
The 2-hydrazinyl-4-(4-methoxyphenyl)-6-morpholinopyrimidine-5-
carbonitrile 5 was obtained through reacting compound 4 with hy-
drazine hydrate in absolute ethanol (Scheme 1). The 1H NMR spectrum
of compound 5 verified exchangeable singlet peak around δ 4.42 and
8.81 ppm due to NH2 and NH protons, respectively. On the other hand,
condensing the key intermediate hydrazinyl compound 5 with appro-
priate aldehydes and isatins in absolute ethanol in presence of catalytic
amount of glacial acetic acid yielded the corresponding 2-(2-ar-
ylidenehydrazinyl)-4-(4-methoxyphenyl)-6-morpholinopyrimidine-5-
carbonitriles 6a-l and 2-(2-(5-substitutedindolin-2-oxo-3-ylidene)hy-
drazinyl)-4-(4-methoxyphenyl)-6-orpholinopyrimidine-5-carbonitriles
7a-c, respectively.
underlying molecular mechanism of the most active derivatives, IC50
,
in-vitro PI3K class 1A enzyme inhibition (PI3Kα, β and δ), cell cycle
analysis, and apoptosis determination were done on the most suscep-
tible cell line Leukemia SR.
2. Results and discussion
2.1. Chemistry
The IR spectra of compounds 6a-l and 7a-c revealed the dis-
appearance of forked peak of NH2 group. The 1H NMR spectra of
compounds 6a-l showed the appearance of singlet signal of one proton
around δ 8.07–8.58 ppm pointing to the presence of CH=N and dis-
appearance of NH2 signal at 4.42 ppm. The 1H NMR spectra of com-
pounds 6a-l revealed two exchangeable singlet signals around δ
11.22–11.81 ppm due to NH protons. In addition, the hydroxyl groups
were detected in compounds 6g, h, and i as exchangeable singlet signal
of one proton in the range of δ 9.24 to 9.59 ppm. Meanwhile, the IR
spectra of compounds 7a-c disclosed the appearance of one absorption
band around 1685–1714 cm−1 pointing to the presence of one carbonyl
group. The 1H NMR spectra of compounds 7a-c showed two ex-
changeable singlet signals around δ 11.06–12.91 ppm due to NH pro-
tons. (Scheme 2)
The synthesis of the target compounds is depicted in Scheme 1 and
2. The structures of the newly synthesized compounds were confirmed
by elemental analysis and spectral data (IR, 1H NMR, 13C NMR, and
MS). The starting pyrimidinone-5-carbonitrile 1 was synthesized in a
one-pot three component reaction according to the reported procedure
of Bigenilli [28]. Furthermore, the synthesis of 2-metylthio-4-(4-meth-
oxyphenyl)-6-oxo-1,6-dihydropyrimidine-5-carbonitrile
2 has been
done by the reaction of 1 with methyl iodide in dimethylformamide
[28,29]. Compound 4-chloro-6-(4-methoxyphenyl)-2-(methylthio)pyr-
imidine-5-carbonitrile (3) was prepared on refluxing 2 with phosphorus
oxychloride. The IR spectrum of compound 3 was free of NH and C]O
amide bands. Also, the 1H NMR spectrum revealed the disappearance of
3

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Scheme 1. Reagents and Conditions: i) CH3I/anhydrous K2CO3/DMF/stirring 3 h 0–5 °C; ii) POCl3/reflux 5 h; iii) morpholine/dry benzene/reflux 8 h; iv) NH2NH2/
absolute ethanol/ stirring 3 h and then reflux.
2.2. Biological activity
[30–33]. The data was reported as mean graph of the percent growth of
the treated cells and presented as percentage growth inhibition (GI%) in
Table 1.
2.2.1. In-vitro cytotoxicity using sulforhodamine b assay
In the present work, all the synthesized compounds (1–5, 6a-l & 7a-
c) were chosen by the National Cancer Institute (NCI), Bethesda, MD,
U.S.A., for in-vitro screening at a single dose of 10 μM against full NCI
60 cell lines panel representing leukemia, melanoma and cancers of
lung, colon, central nervous system, ovary, kidney, prostate and breast
From the NCI growth inhibition percentage (GI%) results in Table 1,
it was found that the newly synthesized compounds showed moderate
to potent cytotoxic activity. Regarding the activity towards individual
cell lines; 4-chloropyrimidine 3 showed high potency against leukemia
SR cell line with GI value 83.25%. Introduction of morpholine at C-6
Scheme 2. Reagents and Conditions: i) aryl aldehydes/glacial acetic acid/absolute ethanol/reflux 6 h; ii) isatin derivatives/glacial acetic acid/ absolute ethanol/
reflux 6 h.
4

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Table 1
Percentage growth inhibition (GI%) of in-vitro subpanel tumor cell lines at 10 μM concentration of compounds 1–5, 6a-l and 7a-c.
Subpanel tumor cell lines
Compounds
1
2
3
4
5
6a
6b
6c
6d
Leukemia
CCRF-CEM
HL-60(TB)
K-562
–
–
–
–
–
–
–
–
–
–
–
–
46.53
30.62
42.39
40.30
–
11.31
–
–
–
–
–
–
10.60
11.81
–
–
–
–
–
15.18
11.19
12.89
–
–
–
–
–
–
MOLT-4
RPMI-8226
SR
12.38
–
13.95
–
22.85
–
11.65
–
83.25
21.11
39.97
55.51
27.16
28.35
Non-small cell lung cancer
A549/ATCC
EKVX
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10.14
12.39
HOP-62
–
–
11.52
–
14.19
20.29
HOP-92
–
–
28.79
–
16.27
10.82
NCI-H226
NCI-H23
13.94
15.82
–
11.20
–
–
–
–
–
–
–
–
–
–
–
–
–
–
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
–
–
–
–
–
–
–
–
–
–
16.01
8.77
10.53
13.85
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12.23
24.22
–
–
–
–
–
–
–
–
–
–
10.16
10.60
16.74
20.57
17.67
–
16.15
HCT-15
13.91
–
–
–
–
HT29
–
–
–
KM12
SW-620
–
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12.20
–
–
–
–
13.54
–
–
22.28
–
–
–
–
–
–
–
10.49
28.89
–
11.44
–
16.09
–
39.55
–
19.51
–
23.75
–
16.9
–
Melanoma
LOX IMVI
MALME-3M
M14
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12.80
13.47
–
–
–
–
–
–
–
–
–
–
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGORV1
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
10.06
16.55
13.52
–
–
–
–
–
–
–
–
–
15.01
11.64
20.60
34.98
30.83
13.12
29.12
–
11.05
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
–
–
–
–
–
–
–
–
–
–
12.88
–
–
–
–
–
–
–
–
–
10.12
–
–
–
–
–
–
–
–
–
–
–
18.96
15.37
26.24
–
–
–
–
10.64
–
10.92
12.60
10.59
13.44
Renal cancer
786–0
–
–
–
–
–
–
–
–
–
23.23
36.93
–
–
32.68
64.58
–
21.34
56.15
13.39
16.47
–
–
26.52
A498
–
–
–
–
58.65
ACHN
–
–
–
14.04
30.74
–
–
CAKI-1
15.73
20.2
18.65
–
–
13.48
–
–
SN12C
–
–
–
–
TK-10
–
–
–
–
–
–
–
–
UO-31
10.71
10.04
32.57
13.51
37.03
50.45
27.55
29.51
Prostate cancer
PC-3
–
–
–
–
–
–
18.88
–
–
–
–
–
16.36
–
20.58
10.14
–
–
DU-145
Breast cancer
MCF7
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
13.05
14.64
14.01
11.98
15.97
–
13.4
MDA-MB-231/ATCC
HS 578T
–
18.28
–
–
–
–
–
–
18.41
–
–
–
–
–
–
–
–
–
–
–
–
–
BT-549
16.29
–
T-47D
27.14
14.57
–
MDA-MB-468
Mean
–
–
–
–
11.75
11.26
(continued on next page)
5

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Table 1 (continued)
Subpanel tumor cell lines
Compounds
6e
6f
6g
6h
6i
6j
6k
6l
7a
7b
7c
Leukemia
CCRF-CEM
HL-60(TB)
K-562
64.44
89.32
96.86
53.1
64.18
L
–
–
–
–
–
–
73.73
50.78
41.66
60.25
67.91
L
20.80
27.27
–
19.50
–
25.76
–
33.45
48.69
24.96
42.83
36.97
79.24
43.07
52.12
62.04
44.4
28.06
L
24.49
–
–
–
–
–
–
–
–
–
–
12.43
37.84
36.00
39.76
25.00
19.25
39.78
58.26
–
MOLT-4
31.98
23.58
54.55
29.83
22.34
32.27
–
RPMI-8226
SR
–
14.24
Non-small cell lung cancer
A549/ATCC
EKVX
37.79
–
–
–
–
–
–
–
–
–
–
18.29
11.29
–
14.14
–
18.13
17.67
31.93
50.76
15.20
19.00
12.91
33.64
23.07
14.41
–
48.35
25.50
32.01
80.37
61.60
18.09
22.86
53.48
27.94
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
16.20
–
HOP-62
20.89
18.02
24.25
–
–
–
–
–
HOP-92
52.31
–
25.34
24.06
34.5
30.93
NCI-H226
NCI-H23
–
–
–
–
–
–
–
–
–
–
–
–
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
11.53
68.19
29.38
–
–
–
–
16.23
20.86
–
–
17.21
–
10.17
26.39
59.16
24.56
67.07
92.7
–
–
–
–
–
–
–
25.94
–
–
–
–
–
–
–
–
–
14.33
–
20.85
–
21.5
32.22
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
18.15
–
40.87
53.07
27.43
27.82
41.11
14.08
10.93
11.66
–
17.67
20.49
25.19
–
40.21
55.89
48.55
16.05
27.38
70.33
30.08
50.81
26.55
33.17
19.04
HT29
78.45
55.83
86.01
–
–
–
KM12
13.34
10.16
SW-620
11.62
CNS cancer
SF-268
18.60
45.81
44.05
–
–
21.19
30.34
45.47
14.09
76.41
41.10
–
–
19.92
–
10.36
–
–
37.61
13.62
L
–
–
SF-295
–
–
–
31.75
32.38
–
–
–
SF-539
–
29.93
–
–
25.77
11.09
84.42
–
27.86
–
–
–
SNB-19
–
–
27.40
56.03
62.23
–
–
SNB-75
L
17.88
–
21.21
20.75
38.21
–
21.98
18.54
38.39
41.28
22.55
–
17.61
–
U251
47.60
Melanoma
LOX IMVI
MALME-3M
M14
–
–
–
–
–
–
–
–
–
–
20.43
34.13
42.49
–
12.64
–
–
–
–
–
–
–
–
–
11.74
40.53
14.67
–
–
–
–
–
–
–
–
–
–
–
33.16
49.03
39.08
20.9
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
92.69
75.21
12.13
19.87
L
–
19.72
–
11.54
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
–
–
14.45
66.06
19.93
42.14
–
–
–
–
22.13
27.84
42.54
26.38
49.71
–
19.15
19.57
–
58.66
27.98
L
14.45
–
–
–
78.58
15.58
24.42
–
Ovarian cancer
IGORV1
10.86
25.38
18.80
–
–
–
–
–
–
–
–
28.81
24.2
10.75
–
38.63
–
–
–
–
–
–
–
–
62.71
29.75
22.02
10.36
11.42
13.63
35.41
50.57
19.15
12.23
32.58
40.50
18.13
55.81
40.28
33.19
–
–
–
–
–
–
–
10.48
OVCAR-3
14.7
–
–
–
–
–
–
–
–
–
–
–
–
OVCAR-4
15.95
–
21.23
OVCAR-5
–
–
–
–
OVCAR-8
30.14
16.72
–
31.18
25.25
–
17.36
16.44
–
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
64.37
54.91
45.37
40.37
10.56
–
–
55.94
14.34
42.17
22.99
21.70
11.55
65.55
–
28.72
45.31
93.33
21.69
40.96
19.83
10.22
71.31
40.38
22.77
12.71
31.13
24.82
14.62
42.34
54.12
48.77
23.91
–
20.27
32.65
74.97
56.66
26.73
28.54
15.16
–
–
A498
20.29
–
17.4
–
–
ACHN
–
12.55
25.27
20.40
–
–
–
–
CAKI-1
SN12C
TK-10
–
–
18.74
–
–
–
–
–
–
–
–
–
–
–
UO-31
56.52
25.78
40.65
29.48
36.33
23.71
22.12
Prostate cancer
PC-3
52.18
50.23
–
–
43.50
43.97
22.52
–
–
–
40.50
–
22.12
–
25.71
20.27
27.27
36.33
–
–
–
–
DU-145
Breast cancer
MCF7
40.74
27.83
32.02
–
–
–
–
–
–
–
–
38.30
25.11
30.67
15.37
28.05
12.91
30.67
21.60
12.51
13.24
–
18.21
64.76
13.92
19.37
21.54
45.85
64.04
25.98
24.31
10.56
–
19.75
39.35
57.39
52.46
37.21
34.59
29.03
38.15
13.68
–
MDA-MB-231/ATCC
HS 578T
15.65
27.59
–
–
–
–
–
–
–
–
–
–
BT-549
–
–
–
–
T-47D
22.99
26.60
43.34
17.98
–
26.34
14.19
16.72
–
–
10.02
–
MDA-MB-468
Mean
–
–
13.52
19.49
0.99
0.52
a Only GI% higher than 10% are shown.
6

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Table 2
unsubstituted isatin ring was promising derivative in this series with
lethal activity on CNS (SF-539) cell line and remarkable potency to-
wards non-small lung cancer (Hop-92) and colon cancer (HCT-116)
with GI values 80.37 and 70.33%, respectively. It also exhibited
moderate cytotoxic activity on other types of cancer cell lines.
Based on these findings, compounds 6e, 6g, and 6l were the most
promising lead compounds with lethal activity on leukemia SR cell line
which are known for their PI3K over-activity [15]. Thus, they were
chosen for further investigations to gain insight into their mechanism of
antiproliferative action.
IC50 values of the most cytotoxic compounds against Leukemia SR cell line and
non-tumorigenic cell line and their selectivity indices.
Compounds
IC50 (uM)
IC50 (uM)
Selectivity Index (S.I.)
L.SR
PCS-800–011
PCS-800–011/L.SR
6e
0.76
13.59
4.37
3.38
1.72
7.38
–
0.05
0.72
40.44
22.29
24.05
17.73
–
1.85
53.21
1.64
5.50
–
6g
1.27
1.63
0.79
6l
0.26
Etoposide
Doxorubicin
Levofloxacin
Erlotinib
0.18
0.08
0.04
–
–
17.73
0.79
2.2.2. In-vitro antiproliferative activity against leukemia SR cell line
The most active compounds 6e, 6g, and 6l were assessed for their
cytotoxic efficacy against the most sensitive cell line leukemia SR (L.SR)
using the 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bro-
mide (MTT) colorimetric assay [34,35]. Etoposide, Doxorubicin, and
Levofloxacin were used as the reference compounds as shown in
Table 2. The cytotoxic efficacy of these compounds is expressed as IC50
values, which represent the concentration of the compound required to
produce a 50% inhibition of cell growth after 48 h of incubation.
The IC50 results showed that compounds 6e, 6l, and 6g possess
potent antiproliferative activity with values of 0.76, 4.37, and 13.59
uM, respectively compared to the reference compounds etoposide (3.38
uM), doxorubicin (1.72 uM), and levofloxacin (7.38 uM) as represented
graphically in Fig. 4. Compound 6e was found to be the most potent
among the novel compounds.
aIC50 values are the mean
SD of three separate experiments.
position produced inactive compound 4, also the replacement of the
thiomethyl group in compound 4 with hydrazinyl group in 5 showed
the same activity. With regard to broad-spectrum antitumor activity;
elaborating 6-morpholinopyrimidine-5-carbonitrile scaffold with aryl
hydrazone moieties in 6a-l and 7a-c conferred the most active members
in this study, displaying effectiveness against numerous cell lines that
belong to different tumor subpanels. The potency of hydrazone deri-
vatives 6a-l and 7a-c was found to be influenced by the nature and type
of substituent on arylidine moiety.
Grafting 2-flurobenzylidene 6a showed moderate activity against
renal cancer cell line (A 498) with GI value 64.58%. By changing the
position of fluorine from ortho to meta position in compound 6b re-
sulted in expanded activity against both leukemia SR cell line with GI
value 55.51% and renal cancer (A498 and UO-31) cell lines with GI
values 56.15 and 50.45%, respectively. On the other hand, 4-flur-
obenzylidene 6c exhibited weak antitumor activity. Also, compound 6d
with 2-bromobenzylidene moiety gave the same selectivity with mod-
erate activity against renal cancer cell line (A498) with GI value
58.65%. Likewise, incorporation of 3-bromobenzylidene 6e enhanced
broad-spectrum and potent antitumor activity.
Furthermore, the cytotoxicity of these compounds was assayed
against non-tumorigenic cell line (Primary Peripheral Blood Monocytes,
(ATCC® PCS-800-010™) to investigate their safety taking Erlotinib as
the reference compound. The results were given as IC50 values and
selectivity index (SI) [36] in Table 2. Compound 6e with SI = 53.21
provided high safety profile as anticancer agent, while compounds 6l
with SI = 5.50 showed moderate safety levels on healthy cells. Com-
pound 6g with SI < 2 indicates certain level of toxicity to normal cells.
Further insights of their mechanisms of action were conducted.
Compound 6e exhibited lethal activity to leukemia SR, CNS cancer
(SNB-75) and melanoma cell lines (SK-MEL-28 and UACC-257). It
worth mentioning that compound 6e showed potency against leukemia
cell lines (HL-60(TB) and K-562) with GI values 89.32 and 96.86%,
respectively, Colon cancer (HCT-15 and SW-620) GI values 92.70 and
86.01%, respectively and melanoma cell line (MALME-3M) with GI
value 92.69%. In contrast, the introduction of 4-bromobenzylidene
produced inactive compound 6f. While, compound 6g (3-hydro-
xyphenyl derivative) showed lethal activity on leukemia SR cell line
and high growth inhibition activity (GI%) on the other leukemia cell
2.2.3. PI3K enzyme inhibition assay
The PI3K inhibition assay of the most active compounds 6e, 6g, and
6l was done on Class IA isoforms. The three compounds displayed po-
tent inhibitory activity on different isoforms in comparable values to
the reference compound LY294002 as shown in Table 3 which indicates
that they are pan class IA inhibitors. Compound 6e was found to be
more selective to PI3K-β isoform and nearly has the same activity on
PI3K-α and δ isoforms. While compound 6g was found to be selective to
PI3K-α in half the amount required to inhibit the other two isoforms.
Compound 6l was more selective to PI3K-β isoform and with compar-
able values against PI3K-α and δ isoforms.
lines (CCRF-CEM
= 73.73%, RPMI-8226 = 67.91%, & MOLT-
4 = 60.25%). It displayed moderate activity on other cell lines as in
colon cell lines (HCT-15 = 53.17%), CNS (SNB-75 = 76.41%), mel-
anoma (SK-MEL-28 = 66.06%), renal cancer (786–0 = 55.94 & UO-
31 = 65.55%). Incorporation of 3-hydroxy-4-methoxybenzylidene in
6 h and 4-hydroxy-3-methoxybenzylidene in 6i exhibited the same
selectivity with weak activity against numerous cell lines.
2.2.4. Cell cycle analysis and apoptosis induction
In cell cycle, there are two significant stages which are the G2 phase
to the M phase and the G1 phase to the S phase. The G2 phase is the
Moreover, compound 6j (furo derivative) showed potent activity
with GI% on Renal cancer (A498
= 93.33%) and CNS (SNB-
75
(ICORV1
31 71.31%), and breast (MCF-7
=
84.42%) cell lines and moderate GI% on Ovarian
62.71%), renal cancer (A498 93.33%), (UO-
64.76% MDA-MB-
=
=
=
=
&
468 = 64.04%) cell lines, while replacement of (furan-2-yl) in 6j with
(1H-pyrrol-2-yl) produced compound 6k with enhanced antitumor ac-
tivity against leukemia cell lines and decreased antitumor activity to-
wards the other different cell lines. Compound 6l (indolo derivative)
showed lethal activity on leukemia SR and showed moderate activity on
other leukemia cell lines (HL-60 = 52.12% & K-562 = 62.04%), colon
(HCT-15 = 55.89%), on melanoma (SK-MEL-28 = 58.66% & UACC-
257 = 78.58%) and on renal (786–0 = 54.12%) cell lines.
Fig. 4. The IC50 values in uM of the most active compounds against Leukemia
In the isatin derivative series (7a-c), only compound 7a with
SR cell line.
7

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Table 3
Table 5
IC50 values of PI3K Class 1A Inhibition of compounds 6e, 6g, & 6l.
Percentage of apoptotic cells in the annexin V-FITC experiment.
Compounds
IC50 (uM)
PI3K-α
11.73
Compounds
Apoptosis %
Total
Necrosis
PI3K-β
PI3K-δ
Early
Late
6e
0.71
0.61
6.09
15.84
7.22
4.51
0.24
11.18
30.62
10.94
4.60
0.81
1.25
0.77
6e
14.07
25.21
18.55
2.01
6.99
4.72
8.26
0.81
5.14
18.11
8.72
0.28
1.94
2.38
1.57
0.92
6g
8.43
13.98
6.28
0.31
0.92
6g
6l
0.25
0.13
6l
LY294002
0.23
0.14
Control LSR
Table 4
cells in the G1 phase following incubation with 6e, 6g and 6l p < 0.01
as shown in Table 4 and represented graphically in Fig. 5. Compounds
6e, 6g, and 6l were found to inhibit proliferation through cell cycle
arrest at G2-M phase which will induce apoptotic cell death.
Cell cycle analysis of Leukemia SR cells after treatment with the tested com-
pounds.
DNA content %
Inducing apoptosis is one of the promising fields for cancer.
Accordingly, the proapoptotic activity of the new active compounds
was assayed. The assay depends on that the cells translocate the
membrane phosphatidylserine (PS), hall mark of apoptosis from the
inner surface of the plasma membrane to the outer cell surface during
early apoptosis which can be detected using AnnexinV-FITC/PI assay.
Cells that were propidium iodide (PI) negative and Annexin V negative
are considered healthy cells, while PI negative and Annexin V positive
cells are considered apoptotic and cells that are positive to both PI and
Annexin V are considered necrotic [39,40].
Compound
%G0-G1 %S
%G2-M %Pre G1 Comment
6e
6g
6l
39.23
26.81
32.72
30.47 30.3
14.07
25.21
18.55
2.01
Cell growth arrest at G2/
M
27.44 45.75
25.67 41.61
33.56 19.52
Cell growth arrest at G2/
M
Cell growth arrest at G2/
M
Control LSR 46.92
—
From the data shown in Table 5 and represented graphically in
Fig. 6, it is obvious that treating Leukemia SR cells for 48 h with the
tested compounds resulted in increased percentage of annexin V posi-
tive apoptotic cells both in the early and latest age which represents
total significant 9 folds increase for compound 6l, 12 folds increase for
compound 6g, and 7 folds increase for compound 6e compared to the
control with p < 0.0001. These results prove that the cytotoxic ac-
tivity in LSR cell line of these new 6-morpholinopyrimidine compounds
is through inducing apoptosis. Regarding 6g, there is increased per-
centage of late apoptotic cells and increased percentage of necrotic cell
than 6e and 6l and this indicates some toxicity together with its low
selectivity index (SI < 2) in Table 2.
time of the cell cycle following DNA amplification, only before cells
entering mitosis. In the event that the cell cycle is captured during the
G2 stage, development is restrained. Thus, to examine the mechanism
responsible for induced inhibition of cell growth in leukemia SR (LSR),
cell cycle distribution was assessed by flow cytometric analysis as re-
ported [37,38]. The flow cytometric analysis indicated a disruption in
the cell cycle of LSR cells following incubation with 6e, 6g and 6l. LSR
cells incubated with vehicle had normal cell cycle phases, where
46.92%, 33.56% and 19.52% of the cells were in the G1, S and G2
phase, respectively. However, cells incubated with 6e, 6g and 6l were
significantly shifted toward the G2 phase (30.3, 45.75, 41.61, respec-
tively with p < 0.001 (There was a significant reduction in the % of
Fig. 5. The distribution of cells in cell cycle phases after treatment with the tested compounds (6e, 6g, & 6l).
8

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Fig. 6. Effect of active compounds 6e, 6g, & 6l on inducing apoptosis in Leukemia SR cell line.
2.3. In-silico studies
inhibitory activity.
Insights into PI3Kα ATP-binding site (Fig. 7) revealed that 6e and 6l
interact similarly to PI-103 with the morpholine directed to the hinge
region with the oxygen accepting one hydrogen bond to the amide of
the hinge Val851 [41,43]. A second hydrogen bond was formed be-
tween the oxygen atom of methoxy group and Lys802 residue near the
solvent accessible area. Compound 6e showed an additional hydrogen
bond between the nitrogen atom of cyano group and Tyr836 residue
which is considered a key amino acid in the interaction [14,41]. In
addition, the following interactions were formed similar to reported
binding of reference inhibitors: (i) The morpholine ring formed two
Alkyl interactions with residue Val850 and Met922 and carbon hy-
drogen interactions with Ser854. (ii) The pyrimidine ring formed two
pi-Alkyl interactions with residue Ile932 in the two compounds and
with Ile800 in 6e only. (iii) The methoxyphenyl was more fitted by pi-
Alkyl interaction with Ile848 and pi-anion interaction with Asp933.
Also, two carbon hydrogen interactions were observed between
methoxy hydrogens and Asp933 and Leu807 in 6l and with Asp933 and
Asp810 in 6e. (iv) Finally, the indole ring of 6l exhibited Pi-Pi T-Shaped
2.3.1. Molecular docking
Zhao et al. [41] reported that the ATP binding site is characterized
by the presence of key region: the hinge region (Val) that is crucial for
activity may accommodate substituents containing hydrogen bond
donor/acceptor such as morpholine, piperazine, indole, or methoxy
group [14,15,26].
Accordingly, docking studies were performed using Discovery
Studio 4.1 (Accelyrs, Inc. San Diego, USA) to gain insightful under-
standing for the binding pattern of the newly synthesized compounds
6e, 6g and 6l inside the PI3K ATP-catalytic site. The human PI3Kα
isoform (PDB code: 4L23) [41] was selected and the co-crystalized li-
gand (PI-103) was taken as the reference structure. The alpha isoform
was selected to perform the docking procedures over PI3Kβ and PI3Kγ
isoforms as their selective inhibitors possess immune and cardiovas-
cular toxicities [42]. The docking studies revealed that the compounds
under investigation displayed comparable binding modes (Fig. 7) like
the reference compound (PI-103) matched along with their PI3K
9

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Fig. 7. The 2D & 3D interactions of PI-103, 6e, 6g, and 6l in the ATP binding site of PI3Kα: a) PI-103 with docking score = -26.75 Kcal/mol; b) 6e, score = −20.95
Kcal/mol; c) 6g, score = –22.25 Kcal/mol; d) 6l, score = –23.71 Kcal/mol.
10

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Fig. 7. (continued)
interaction with Trp780 residue.
examine the possibility of the synthesized molecules to become a drug
(the molecule can reach its target and stay there long enough to give
their therapeutic effect) their ADMET and pharmacokinetic properties
were calculated using SWISSADME online tool (http://www.
swissadme.ch.) to predict their drug ability [44,45]. The results are
listed in Table 6.
Considering the binding pose of 6g with different orientation from
the others, the methoxy oxygen accepted one hydrogen bond from
hinge Val851 residue instead of the morpholine ring. The 5-phenyl ring
was involved in pi-Alkyl interaction with Ile932 and pi-Anion interac-
tion with Met922 representing a key amino acid for anchoring in the
hinge region [41]. The nitrogen atom of the cyano group interacts with
Gln859 through hydrogen bond formation. Also, an intramolecular
hydrogen bond was observed between the morpholine hydrogen and
the cyano nitrogen leading to an extra stability for this pose. The 3-
hydroxyphenyl at C2 of pyrimidine ring was directed towards solvent
accessible area forming a hydrogen bond with Lys802 and was also
involved in pi-Alkyl interaction with Ile848 and pi-Anion interaction
with Asp933. Finally, Pi-Alkyl interactions were also observed between
the pyrimidine ring and Met772.
Our compounds obeyed Lipiski's rule which assesses qualitatively
the chance for a molecule to become an oral drug with respect to
bioavailability. Also, they didn't match with CYP1A2 inhibition which
is one major cause of pharmacokinetics-related drug-drug interactions.
Zero alert in PAINS filter confirmed freedom from instability or re-
activity that may interfere with biological assays.
3. Conclusion
The present study reports the design and synthesis of novel series of
6-morpholinopyrimidine derivatives as PI3K inhibitors and apoptosis
inducers. The newly synthesized compounds were first evaluated for
their cytotoxic activity in the NCI 60-cell lines program. Most of com-
pounds showed moderate to high growth inhibitory activity of which
three compounds 6e and 6g with m-substitution (electron withdrawing
or electron donating groups) on the phenyl ring, and 6l unsubstituted
indole derivative gave high growth inhibitory activity at dose of 10 uM
on leukemia cell lines and especially on leukemia SR cell line with IC50
values of 0.76, 13.59, &4.37, respectively. The IC50 values of PI3K in-
hibition assay were in the micromolar range with very promising ac-
tivity against the three isoforms of class IA. Flow cytometry was used to
analyze the DNA content of the cells after inhibition with the three
selected 6-morpholinopyrimidine derivatives. The study showed that
the cell cycle was arrested at the G2/M phase that lead to decreased
DNA synthesis and induced apoptosis. Thus, the data obtained indicates
that the PI3K inhibitory effect of the three selected derivatives may
correlate with changes in the cell cycle, and this data is also in ac-
cordance with the results of apoptosis assay. Through Molecular mod-
eling studies, the binding modes of 6e, 6g, and 6l were similar to that of
the reference compound supporting their mode of action. By ADME in-
silico studies predicted that they are promising orally available com-
pounds with low toxicity profile and suitable for further optimization as
selective PI3Kα inhibitors in treatment of leukemia.
2.3.2. ADME studies
It is obvious from the docking studies that our compounds have
affinity for the protein target with promising inhibitory activity. To
Table 6
Physicochemical descriptors, ADME parameters, pharmacokinetic properties,
and drug like nature of compounds 6e, 6g, 6l.
Compound
6e
6g
6l
Molecular weight
Num. rotatable bonds
Num. H-bond donors
Num. H-bond acceptors
TPSA
493.09 g/mol
430.28
453.32
6
6
6
1
2
2
6
7
6
95.66 Å2
High
115.89 Å2
High
107.69 Å2
High
GI absorption
BBB permeant
No
No
No
P-gp substrate
No
No
Yes
CYP1A2 inhibitor
Log Kp (skin permeation)
Lipinski
No
No
No
−6.27 cm/s
Yes; 0 violation
0 alert
3.61
−6.63 cm/s
Yes; 0 violation
0 alert
3.59
−6.80 cm/s
Yes; 0 violation
0 alert
5.05
PAINS
Synthetic accessibility
Topological polar surface area (TPSA), gastrointestinal absorption (GI absorp-
tion), blood brain barrier (BBB) permeant, P-glycoprotein substrate (P-gp sub-
strate), cytochrome P50 1A2 inhibitor (CYP1A2 inhibitor), pan-assay inter-
ference structure (PAINS).
11

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
4. Experimental
4H, CH2-O- CH2), 7.10 (d, 2H, J = 8.8, ArH), 7.20–7.24 (m, 1H, ArH),
7.45–7.53 (m, 3H, ArH), 7.88 (d, 2H, J = 8.0, ArH), 8.20 (s, 1H,
N]CH), 11.69 (s, 1H, NH, exchangeable by D2O); MS (m/z): 432.36
(M+, 100%), 433.35 (M+H, 35.07%); Anal. Calcd. For C23H21FN6O2
(432.45): C:63.88; H: 4.89; N: 19.43. Found: C: 64.09; H: 5.01; N:
19.68.
4.1. Chemistry
All reagents were commercially available and were used without
further purification. Melting points were determined on Stuart appa-
ratus and the values given are uncorrected. The IR spectra were de-
termined using potassium bromide discs on Shimadzu IR 435 spectro-
photometer, Faculty of Pharmacy, Cairo University, Egypt and the
values were represented in cm−1. The 1H NMR and 13C NMR spectra
were carried out on Bruker 400 MHz (Bruker Corp., MA, USA), 13C
NMR (100 MHz) spectrometer, Microanalytical Unit, Faculty of
Pharmacy, Cairo University, Egypt, using TMS as internal standard and
chemical shift values were recorded in ppm on δ scale. The mass spectra
were recorded on Hewlett Packard 5988 spectrometer, Microanalytical
Center, Cairo University, Egypt. The elemental analyses were carried
out at the Regional Center for Mycology and Biotechnology, Al-Azahr
University, Egypt.
4.1.2.3. 2-(2-(4-Fluorobenzylidene)hydrazinyl)-4-(4-methoxyphenyl)-6-
morpholinopyrimidine-5-carbonitrile 6c. Pale yellow crystals, Yield: %;
m.p. 254–256 °C; IR (KBr, cm−1): 3251 (NH), 3064 (N]CH), 3045
(ArH), 2960, 2864 (CH aliphatic), 2198 (CN). 1H NMR (DMSO‑d6-
400 MHz, δ ppm): 3.75 (s, 4H, CH2-N-CH2), 3.85 (s, 4H, CH2-O- CH2),
3.92 (s, 3H, OCH3), 7.09 (d, 2H, J = 8.8, ArH), 7.25–7.29 (m, 2H, ArH),
7.73–7.76 (m, 2H, ArH), 7.87 (d, 2H, J = 8.0, ArH), 8.21 (s, 1H,
N]CH), 11.60 (s, 1H, NH, exchangeable by D2O); 13C NMR (DMSO‑d6-
100 MHz, δ ppm): 47.54 (2C), 55.85, 66.44 (2C), 80.00, 114.08,
116.18, 116.40, 119.81, 129.13, 129.21, 129.48, 131.32, 131.82,
131.85, 141.13, 158.94, 161.92, 161.97, 164.43, 164.91, 171.69; MS
(m/z): 432.33 (M+, 100.00%), 433.32 (M + H, 32.38%), 434.28 (M
+2, 5.45%); Anal. Calcd. For C23H21FN6O2 (432.45): C:63.88; H: 4.89;
N: 19.43. Found: C: 64.12; H: 4.97; N: 19.71.
The progress of the reaction was monitored using TLC aluminum
sheets precoated with UV fluorescent silica gel (Merck 60F 254) and
were visualized using UV lamp using eluents, chloroform-methanol (9:1
v/v) and benzene-acetone (8:2 v/v).
Compounds 1 [28], 2 [28,29], 3 and 4 [27] were prepared ac-
cording to the reported procedure.
4.1.2.4. 2-(2-(2-Bromobenzylidene)hydrazinyl)-4-(4-methoxyphenyl)-6-
morpholinopyrimidine-5-carbonitrile 6d. White powder, Yield: 95%; m.p.
249–250 °C; IR (KBr, cm−1): 3221 (NH), 3088 (N]CH), 3059 (ArH),
2976, 2879 (CH aliphatic), 2198 (CN). 1H NMR (DMSO‑d6-400 MHz, δ
ppm): 3.75 (s, 4H, CH2-N-CH2), 3.85 (s, 4H, CH2-O- CH2), 3.91 (s, 3H,
OCH3), 7.09 (d, 2H, J = 8.0, ArH), 7.32 (t, 1H, ArH), 7.44 (t, 1H, ArH),
7.66 (d, 1H, J = 8.0, ArH), 7.88 (d, 2H, J = 8.0, ArH), 8.00 (d, 1H,
J = 8.0, ArH) 8.58 (s, 1H, N]CH), 11.81 (s, 1H, NH, exchangeable by
D2O); MS (m/z): 310.19 (100%), 493.60 (M+, 11.72%); Anal. Calcd.
For C23H21BrN6O2 (493.37): C:55.99; H: 4.29; N: 17.03. Found: C:
56.21; H: 4.53; N: 17.19.
4.1.1. 2-Hydrazineyl-4-(4-methoxyphenyl)-6-morpholinopyrimidine-5-
carbonitrile (5)
A suspension of 4 (0.003 mmol) in 20 mL of absolute ethanol was
stirred with hydrazine hydrate (99–100%) (2 mL) for 3 h and then re-
fluxed until the odor of hydrogen sulfide ceased. The precipitate formed
was filtered, dried and crystallized from methanol.
Yellow crystals, yield: 65%; m.p. 200–202 °C; IR (KBr, cm−1):3471,
3311 (NH, NH2), 3008 (ArH), 2978, 2899 (CH aliphatic), 2198 (CN); 1H
NMR (DMSO‑d6-400 MHz, δ ppm): 3.71 (s, 3H, OCH3), 3.83 (s, 4H,
CH2-N-CH2), 3.87 (s, 4H, CH2-O-CH2), 4.42 (s, 2H, NH2, exchangeable
by D2O), 7.04–7.06 (m, 2H, ArH), 7.77–7.93 (m, 2H, ArH), 8.81 (s, 1H,
NH, exchangeable by D2O). 13C NMR (DMSO‑d6-100 MHz, δ ppm):
47.59, 55.82 (2C), 66.45 (2C), 76.20, 113.97, 120.30 (2C), 129.74 (2C),
131.09, 161.73, 162.82, 165.24, 171.11; MS (m/z): 326.31 (M+,
100%), 327.33 (M+H, 19.72); Anal. Calcd. For C16H18N6O2 (326.35):
C: 58.88; H: 5.56; N: 25.75. Found: C: 59.17; H: 5.70; N: 26.03.
4.1.2.5. 2-(2-(3-Bromobenzylidene)hydrazinyl)-4-(4-methoxyphenyl)-6-
morpholinopyrimidine-5-carbonitrile 6e. White powder, Yield: 75%; m.p.
234–236 °C; IR (KBr, cm−1): 3313 (NH), 3078 (N]CH), 3061 (ArH),
2964, 2873 (CH aliphatic), 2191 (CN). 1H NMR (DMSO‑d6-400 MHz, δ
ppm): 3.75 (s, 4H, CH2-N-CH2), 3.85 (s, 4H, CH2-O- CH2), 3.92 (s, 3H,
OCH3), 7.10 (d, 2H, J = 8.0, ArH), 7.40 (t, 1H, ArH), 7.58 (d, 1H,
J = 8.0, ArH), 7.70 (d, 1H, J = 8.0, ArH), 7.85–7.88 (m, 3H, ArH), 8.17
(s, 1H, N]CH), 11.69 (s, 1H, NH, exchangeable by D2O); MS (m/z):
310.26 (100%), 493.09 (M+, 6.12%); Anal. Calcd. For C23H21BrN6O2
(493.37): C:55.99; H: 4.29; N: 17.03. Found: C: 55.87; H: 4.48; N:
17.32.
4.1.2. General procedure for the preparation of 2-(2-arylidenehydrazinyl)-
4-(4-fluorophenyl)-6-morpholinopyrimidine-5-carbonitriles (6a-l)
A mixture of compound 5 (0.25 g, 0.0007 mol), the appropriate
aldehyde (0.0007 mol) and the glacial acetic acid (1 mL) in absolute
ethanol (15 mL) was heated under reflux for 6–8 h. The obtained pre-
cipitate was filtered, dried and crystallized from ethanol.
4.1.2.6. 2-(2-(4-Bromobenzylidene)hydrazinyl)-4-(4-methoxyphenyl)-6-
morpholinopyrimidine-5-carbonitrile 6f. White powder, Yield: 90%; m.p.
270–271 °C; IR (KBr, cm−1): 3226 (NH), 3093 (N]CH), 3066 (ArH),
2966, 2866 (CH aliphatic), 2193 (CN). 1H NMR (DMSO‑d6-400 MHz, δ
ppm): 3.74 (s, 4H, CH2-N-CH2), 3.85 (s, 4H, CH2-O- CH2), 3.91 (s, 3H,
OCH3), 7.09 (d, 2H, J = 8.0, ArH), 7.63 (s (br), 4H, ArH), 7.87 (d, 2H,
J = 8.0, ArH), 8.17 (s, 1H, N]CH), 11.65 (s, 1H, NH, exchangeable by
D2O), 13C NMR (DMSO‑d6-100 MHz, δ ppm): 47.54 (2C), 55.86, 66.44
(2C), 79.00, 114.09 (2C), 119.77, 123.10, 128.94 (2C), 129.46 (2C),
131.33 (2C), 132.24, 134.50, 143.01, 158.88, 161.93, 164.87, 171.10;
MS (m/z): 492.26 (M−1, 100%), 493.23 (M+, 74.98%); Anal. Calcd.
For C23H21BrN6O2 (493.37): C:55.99; H: 4.29; N: 17.03. Found: C:
56.12; H: 4.50; N: 17.28.
4.1.2.1. 2-(2-(2-Fluorobenzylidene)hydrazinyl)-4-(4-methoxyphenyl)-6-
morpholinopyrimidine-5-carbonitrile 6a. Pale yellow powder, Yield: 75%;
m.p. 245–246 °C; IR (KBr, cm−1): 3251 (NH), 3064 (N]CH), 3045
(ArH), 2995, 2864 (CH aliphatic), 2198 (CN). 1H NMR (DMSO‑d6-
400 MHz, δ ppm): 3.75 (s, 3H, OCH3), 3.85 (s, 4H, CH2-N-CH2), 3.92 (s,
4H, CH2-O- CH2), 7.09 (d, 2H, J = 8.0, ArH), 7.25–7.30 (m, 2H, ArH),
7.42–7.47 (m, 1H, ArH), 7.87–7.97(m, 3H, ArH), 8.44 (s, 1H, N]CH),
11.72 (s, 1H, NH, exchangeable by D2O), MS (m/z): 432.36 (M+
,
100%), 433.35 (M+H, 49.20%); Anal. Calcd. For
C23H21FN6O2
(432.45): C:63.88; H: 4.89; N: 19.43. Found: C: 64.16; H: 5.03; N:
19.39.
4.1.2.7. 2-(2-(3-Hydroxybenzylidene)hydrazinyl)-4-(4-methoxyphenyl)-
6-morpholinopyrimidine-5-carbonitrile 6g. Pale yellow crystals, Yield:
50%; m.p. 260–262 °C; IR (KBr, cm−1):3315, 3280 (NH, OH), 3099
(N]CH), 3057 (ArH), 2993, 2873 (CH aliphatic), 2191 (CN). 1H NMR
(DMSO‑d6-400 MHz, δ ppm): 3.75 (s, 4H, CH2-N-CH2), 3.85 (s, 4H, CH2-
O- CH2), 3.92 (s, 3H, OCH3), 6.80 (d, 1H, ArH), 7.06–7.10 (m, 3H,
4.1.2.2. 2-(2-(3-Fluorobenzylidene)hydrazinyl)-4-(4-methoxyphenyl)-6-
morpholinopyrimidine-5-carbonitrile 6b. Pale yellow powder, Yield: 80%;
m.p. 250–252 °C; IR (KBr, cm−1): 3242 (NH), 3066 (N]CH), 3056
(ArH), 2962, 2864 (CH aliphatic), 2198 (CN). 1H NMR (DMSO‑d6-
400 MHz, δ ppm): 3.75 (s, 3H, OCH3), 3.85 (s, 4H, CH2-N-CH2), 3.92 (s,
12

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
ArH), 7.14 (s, 1H, ArH), 7.23 (t, 1H, ArH), 7.86–7.88 (d, 2H, ArH), 8.12
(d, 2H, ArH) 8.39 (s, 1H, N]CH), 8.46 (d, 1H, ArH), 11.45 (s, 2H, 2NH,
exchangeable by D2O); MS (m/z): 310.30 (100%), 453.32 (M+,
7.80%),454.60 (M+H, 1.30%) 455.41 (M+2, 1.29%); Anal. Calcd. For
(s, 1H, N]CH), 9.59 (s, 1H, OH, exchangeable by D2O), 11.53 (s, 1H,
NH, exchangeable by D2O); MS (m/z): 326.28 (100%), 430.28 (M+
,
53.55%), 431.17 (M+H, 15.92%); Anal. Calcd. For
C
23H22N6O3
C25H23N7O2 (453.50): C: 66.21; H: 5.11; N: 21.62. Found: C: 66.47; H: 5.28;
(430.47): C:64.18; H: 5.15; N: 19.52. Found: C: 64.34; H: 5.37; N:
19.38.
N: 21.89.
4.1.3. General procedure for the preparation of 2-(2-(5-substitutedindolin-
2-oxo-3-ylidene)hydrazinyl)-4-(4-methoyphenyl)-6-morpholinopyrimidine-
5-carbonitriles (7a-c)
4.1.2.8. 2-(2-(3-Hydroxy-4-methoxybenzylidene)hydrazinyl)-4-(4-
methoxyphenyl)-6-morpholinopyrimidine-5-carbonitrile 6h. Yellow powder,
Yield: 75%; m.p. 262–264 °C; IR (KBr, cm−1): 3529, 3244 (NH, OH),
3116 (N]CH), 3066 (ArH), 2969, 2850 (CH aliphatic), 2194 (CN);1H
NMR (DMSO‑d6-400 MHz, δ ppm): 3.75 (s, 4H, CH2-N-CH2), 3.80 (s, 3H,
OCH3), 3.85 (s, 4H, CH2-O-CH2), 3.93 (s, 3H, OCH3), 6.95 (d, 2H, ArH),
7.09 (d, 2H, ArH), 7.23 (s, 1H, ArH), 7.86 (d, 2H, ArH), 8.07 (s, 1H,
N]CH), 9.24 (s, 1H, OH, exchangeable by D2O), 11.44 (s, 1H, NH,
exchangeable by D2O); MS (m/z): 310.24 (100%), 460.94 (M+, 13.15%),
461.62 (M+H, 4.17%); Anal. Calcd. For C24H24N6O4 (460.49): C: 62.60;
H: 5.25; N: 18.25. Found: C: 62.56; H: 5.39; N: 18.47.
A mixture of compound 5 (0.25 g, 0.0007 mol), substituted isatin
(0.0007 mol) and the glacial acetic acid (1 mL) in absolute ethanol
(15 mL) was heated under reflux for 5–7 h. The obtained precipitate
was filtered, dried and crystallized from ethanol.
4.1.3.1. 2-(2-(Indolin-2-oxo-3-ylidene)hydrazinyl)-4-(4-methoxyphenyl)-
6-morpholinopyrimidine-5-carbonitrile 7a. Yellow solid, Yield: 85%; m.p.
˃300 °C; IR (KBr, cm−1): 3502, 3460 (2NH), 3080 (N]CH), 3040
(ArH), 2960, 2840 (CH aliphatic), 2204 (CN), 1714 (C]O). 1H NMR
(DMSO‑d6-400 MHz, δ ppm): 3.76 (s (br), 4H, CH2-N-CH2), 3.87 (s, 3H,
OCH3), 3.98 (s (br), 4H, CH2-O- CH2), 6.95 (d, 1H, ArH), 7.05–7.13 (m,
3H, ArH), 7.37 (d, 1H, ArH), 7.91–8.02 (m, 3H, ArH), 11.06 (s, 1H, NH,
exchangeable by D2O), 12.91 (s, 1H, NH, exchangeable by D2O); MS
(m/z): 320.26 (100%), 455.32 (M+, 7.86%), 456.19 (M+2, 2.62%);
Anal. Calcd. For C24H21N7O3 (455.48): C: 63.29, H: 4.65; N: 21.53.
Found: C: 63.15; H: 4.82; N: 21.80.
4.1.2.9. 2-(2-(4-Hydroxy-3-methoxybenzylidene)hydrazinyl)-4-(4-
methoxyphenyl)-6-morpholinopyrimidine-5-carbonitril 6i. Yellow solid,
Yield: 80%; m.p. 270–271 °C; IR (KBr, cm−1): 3523, 3259 (NH, OH),
3080 (N]CH), 3047 (ArH), 2974, 2862 (CH aliphatic), 2193 (CN);1H
NMR (DMSO‑d6-400 MHz, δ ppm): 3.74 (s, 4H, CH2-N-CH2), 3.83 (s,
3H, OCH3), 3.84 (s, 4H, CH2-O-CH2), 3.92 (s, 3H, OCH3), 6.84 (d, 1H,
ArH), 7.07–7.11 (m 3H, ArH), 7.30 (s, 1H, ArH), 7.87 (s (br), 2H, ArH),
8.10 (s, 1H, N]CH), 9.45 (s, 1H, OH, exchangeable by D2O), 11.48 (s,
1H, NH, exchangeable by D2O); 13C NMR (DMSO‑d6-100 MHz, δ ppm):
47.54 (2C), 55.83 (2C), 66.45 (2C),78.50, 109.87, 114.05 (2C), 116.03,
119.95, 121.33, 126.77, 129.56 (2C), 131.28, 144.63, 148.36, 148.87,
158.91, 161.86, 164.95, 171.54; MS (m/z): 310.22 (100%), 460.35
(M+, 59.16%), 461.31 (M+H, 11.51%); (Anal. Calcd. For C24H24N6O4
(460.49): C: 62.60; H: 5.25; N: 18.25. Found: C: 62.49; H: 5.43; N:
18.59.
4.1.3.2. 2-(-2-(5-Chloroindolin-2-oxo-3-ylidene)hydrazinyl)-4-(4-
methoxyphenyl)-6-morpholinopyrimidine-5-carbonitrile 7b. Dark yellow
powder, Yield: 75%; m.p. ˃300 °C; IR (KBr, cm−1): 3419, 3226
(2NH), 3078 (N]CH), 3064 (ArH), 2972, 2862 (CH aliphatic), 2206
(CN), 1685 (C]O). 1H NMR (DMSO‑d6-400 MHz, δ ppm): 3.75 (s (br),
4H, CH2-N-CH2), 3.86 (s, 3H, OCH3), 3.95 (s (br), 4H, CH2-O- CH2),
6.94 (d, 1H, ArH), 7.11 (d, 2H, ArH), 7.36 (d, 1H, ArH), 7.51 (s, 1H,
ArH), 7.91 (d, 2H, ArH), 11.35 (s, 1H, NH exchangeable by D2O), 12.86
(s, 1H, NH exchangeable by D2O); MS (m/z): 354.23 (100%), 489.29
(M+, 5.71%), 491.30 (M+2, 1.94%); Anal. Calcd. For C24H20ClN7O3
(489.92): C: 58.84, H: 4.11; N: 20.01. Found: C: 59.12; H: 4.18; N:
20.23.
4.1.2.10. 2-(2-((furan-2-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)-
6-morpholinopyrimidine-5-carbonitrile 6j. Green crystals, Yield: 50%;
m.p. 254–255 °C; IR (KBr, cm−1): 3259 (NH), 3074 (N]CH), 3045
(ArH), 2958, 2854 (CH aliphatic), 2194 (CN); 1H NMR (DMSO‑d6-
400 MHz, δ ppm): 3.74 (s (s, 4H, CH2-N-CH2), 3.85 (s, 4H, CH2-O-CH2),
3.89 (s, 3H, OCH3),6.61–6.62 (m, 1H, ArH), 6.84 (d, 1H, ArH), 7.09 (d,
2H, ArH), 7.82–7.86 (m, 3H, ArH), 8.10 (s, 1H, N]CH), 11.52 (s, 1H,
NH, exchangeable by D2O); MS (m/z): 404.30 (M+, 100.00%), 405.32
(M+H, 44.57%); Anal. Calcd. For C21H20N6O3 (404.42): C: 62.37; H:
4.98; N: 20.78. Found: C: 62.53; H: 5.12; N: 21.04.
4.1.3.3. 2-(2-(5-Bromoindolin-2-oxo-3-ylidene)hydrazinyl)-4-(4-
methoxyphenyl)-6-morpholinopyrimidine-5-carbonitrile 7c. yellow powder,
Yield: 95%; m.p. ˃300 °C; IR (KBr, cm−1): 3419, 3244 (2NH), 3055
(N]CH), 3008 (ArH), 2970, 2860 (CH aliphatic), 2204 (CN), 1685(C]
O). 1H NMR (DMSO‑d6-400 MHz, δ ppm): 3.75 (s (br), 4H, CH2-N-CH2),
3.86 (s, 3H, OCH3), 3.94 (s (br), 4H, CH2-O- CH2), 6.88 (d, 1H, ArH),
7.10 (d, 2H, ArH), 7.48 (d, 1H, ArH), 7.61 (s, 1H, ArH), 7.91 (d, 2H,
ArH), 11.35 (s, 1H, NH exchangeable by D2O), 12.85 (s, 1H, NH
4.1.2.11. 2-(2-((1H-pyrrol-2-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)-
6-morpholinopyrimidine-5-carbonitrile 6k. Brown crystals, Yield: 60%; m.p.
265–267 °C; IR (KBr, cm−1): 3460, 3271 (2NH), 3097 (N]CH), 3041
(ArH), 2962, 2873 (CH aliphatic), 2193 (CN);1H NMR (DMSO‑d6-400 MHz,
δ ppm): 3.74 (s, 4H, CH2-N-CH2), 3.85 (s, 4H, CH2-O-CH2), 3.93 (s, 3H,
OCH3), 6.14 (s, 1H, ArH), 6.43 (s, 1H, ArH), 6.90 (s, 1H, ArH), 7.07 (d, 2H,
ArH), 7.86 (s (br), 2H, ArH), 8.08 (s, 1H, N]CH), 11.22 (s, 1H, NH,
exchangeable by D2O), 11.31 (s, 1H, NH, exchangeable by D2O); 13C NMR
(DMSO‑d6-100 MHz, δ ppm): 47.49 (2C), 55.84, 66.51 (2C),78.00, 109.69,
112.58 (2C), 114.02, 120.07, 122.32, 127.97, 129.63 (2C), 131.30, 137.97,
158.73, 161.82, 164.84, 171.51; MS (m/z): 403.37 (M+, 100%), 404.33 (M
+H, 31.34%); Anal. Calcd. For C21H21N7O2 (403.44): C: 62.52; H: 5.25; N:
24.30. Found: C: 62.75; H: 5.13; N: 24.38.
exchangeable by D2O); MS (m/z): 398.21 (100.00%); 534.26 (M+
,
37.85%); Anal. Calcd. For C24H20BrN7O3 (534.37): C: 53.94; H: 3.77;
N: 18.35. Found: C: 53.79; H: 3.93; N: 18.51.
4.2. Biological activity
4.2.1. Measurement of potential cytotoxic activity
These compounds were subjected to the NCI’s disease-oriented
human cell lines screening assay to be evaluated for their in-vitro an-
titumor activity. A single dose (10 µM) of the test compounds were used
in the full NCI 59 cell lines panel assay which includes nine tumor
subpanels namely; leukemia, non-small cell lung, colon, CNS, mela-
noma, ovarian, renal, prostate, and breast cancer cells [30–33]. The
data reported as mean graph of the percent growth of the treated cells
and presented as percentage growth inhibition (GI%).
4.1.2.12. 2-(2-((1H-indol-3-yl)methylene)hydrazinyl)-4-(4-methoxyphenyl)-6-
morpholinopyrimidine-5-carbonitrile 6l. Yellow powder, Yield: 85%; m.p.
270–272 °C; IR (KBr, cm−1): 3423, 3261 (2NH), 3109 (N]CH), 3049
(ArH), 2956, 2864 (CH aliphatic), 2198 (CN). 1H NMR (DMSO‑d6-400 MHz,
δ ppm): 3.81 (s, 4H, CH2-N-CH2), 3.85 (s, 4H, CH2-O-CH2), 4.02 (s, 3H,
OCH3), 7.08–7.21 (m, 4H, ArH), 7.43 (d, 1H, ArH), 7.75 (d, 1H, ArH), 7.85
4.2.2. Cytotoxicity using MTT assay
Cell Line cells were obtained from American Type Culture
Collection, cells were cultured using DMEM (Invitrogen/Life
13

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Technologies) supplemented with 10% FBS (Hyclone), 10 ug/ml of
insulin (Sigma), and 1% penicillin-streptomycin. All the other chemi-
cals and reagents were from Sigma, or Invitrogen. Plate cells (cells
density 1.2–1.8 × 10,000 cells/well) in a volume of 100 µL complete
growth medium + 100 ul of the tested compound per well in a 96-well
plate for 24 h before the MTT assay. Leukemia SR cells were incubated
with the serial concentrations of the target compound to be tested for
48 h at 37 °C, then the plates are to be examined under the inverted
microscope and proceed for the MTT assay as reported [34,35].
4.3. Molecular modeling
Molecular docking simulation studies were carried out by Discovery
studio software 4.1 (Accelrys, Inc, San Diego, USA) in drug design lab,
College of pharmacy, Misr University for Science and Technology. The
target compounds were drawn on Chem Draw Professional 16.0 and
subjected to energy minimization using CHARMm-Force Field. The X-
ray crystal structure of PI3Kα in complex with PI-103 (PDB ID: 4L23)
was downloaded from the protein data bank (http://www.rscb.org/
pdb) [23,41,43]. The protein-ligand complex was prepared for docking
as follows: 1- The unwanted chains and water molecules were deleted.
2- The enzyme was 3D protonated and missing amino acids were fixed.
Constraints were applied to heavy atoms to keep its 3D structure before
the energy minimization step. 3-The binding pocket was identified.
Validation of the lead compound, docking, and selection of proper
binding pose was performed. 4- Docking of the prepared ligands into
the 3D structure of the protein was carried out adopting flexible ligand-
rigid receptor docking using CDOCKER protocol. 5- Ten of the most
stable docking poses for each ligand were obtained and arranged ac-
cording to their docking scores (CDOCKER energy). The ideal pose
which fulfills the reported key binding features was selected.
4.2.3. PI3K enzyme inhibition assay
The inhibitory rate against PI3 Kinase of selected 3 compounds were
assayed in-vitro using The PI3 Kinase Activity/Inhibitor Assay (ELISA
Kit Merck Millipore) according to the manufacturer’s instructions. First,
setup the PI3 Kinase reaction in the Glutathione-coated strips/plate for
inhibitor reaction: Preincubate the kinase and inhibitor for 10 min prior
to adding PIP2 substrate then add 5 μL/well of 5X Kinase reaction
buffer and 5 μL/well of PIP2 substrate. After that add distilled H2O to
each well based on Table 1 below to make up to a final 25 μL/well.
Incubate at RT for 1 h then add 25 μL/well of Biotinylated-PIP3/EDTA
working solution excluding the buffer control wells and add 25 μL/well
1XTBS to the buffer control wells. Add 50 μL/well of GRP1 working
solution to all wells and incubate at RT for 1 h. Wash the wells 4 times
with 200 μL/well 1XTBST. Add 50 μL/well SA-HRP working solution,
incubate at RT for 1 h. Allow the Substrate TMB and Stop Solution to
warm up at RT. Wash the wells 3 times with 200 μL of 1X TBST per
well, then 2 times with 200 μL of 1X TBS per well. 11. Add 100 μL of the
Substrate TMB per well, develop in the dark for 5–20 min. Monitor the
appearance of the blue color to avoid over-development. Stop the re-
action by adding 100 μL of the Stop Solution per well. Read at 450 nm.
The lower the signal, the higher the PI3 Kinase activity. The relative %
to B-PIP3 is calculated as:
4.4. Statistical analysis
Data are reported as Means
SD. Statistical analysis was per-
formed using GraphPad Prism version. A p value less than 0.05 was
considered statistically significant.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
A450 ofsamplesincludebuffer, kinase&inhibitorsx100
A450 ofBiotinylated PIP3average
References
4.2.4. Cell cycle analysis and apoptosis
[1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer sta-
tistics, CA-CANCER J. CLIN. 61 (2011) 69–90.
Apoptosis study was performed at Cell Culture Unit, VACSERA,
Cairo, Egypt. These studies were employed against the most potent
compound 6e, 6g, & 6l to investigate their cytotoxicity in the most
sensitive cell line, Leukemia SR.
[2] (a) C. Lindley, J.S. McCune, T.E. Thomason, D. Lauder, A. Sauls, S. Adkins,
W.T. Sawyer, Perception of chemotherapy side effects cancer versus noncancer
patients, Cancer Pract. 7 (1999) 59–65;
(b) L. Galluzzi, L. Senovilla, L. Zitvogel, G. Kroemer, The secret ally: im-
munostimulation by anticancer drugs, Nat. Rev. Drug Discov. 11 (2012) 215–219.
[3] S. Raguz, E. Yagüe, Resistance to chemotherapy: new treatments and novel insights
into an old problem, Br. J. Cancer 99 (2008) 387–439.
4.2.4.1. Cell cycle analysis. The effect on cell cycle progression was
analyzed by flow cytometric analysis. The data represents means
SD
[4] M. Murahari, K.V. Prakash, G.J. Peters, Y.C. Mayur, Acridone-pyrimidine hybrids-
design, synthesis, cytotoxicity studies in resistant and sensitive cancer cells and
molecular docking studies, Eur. J. Med. Chem. 139 (2017) 961–981.
[5] W. Jiang, M. Ji, Receptor tyrosine kinases in PI3K signaling: the therapeutic targets
in cancer, Semin. Cancer Bio. 59 (2019) 3–22.
of three independent experiments performed in triplicate. The
Leukemia SR cells were treated with the active compounds. After
treatment, the cells were washed twice with ice-cold phosphate buffer
saline (PBS), collected by centrifugation, and fixed in ice-cold 70% (v/
v) ethanol, washed with PBS, re-suspended with 0.1 mg/mL RNase,
stained with 40 mg/mL Propidium iodide (PI), and analyzed by flow
cytometry using FACSCalibur (Becton Dickinson). The cell cycle
distributions were calculated using Cell Quest software (Becton
Dickinson) [37,38]. Further evaluation of pro-apoptotic effect was
carried out using Annexin V-FITC and propidium iodide.
[6] S. Jia, T.M. Roberts, J.J. Zhao, Should individual PI3 kinase isoforms be targeted in
cancer, Curr. Opin. Cell Biol. 21 (2009) 199–208.
[7] B. Vanhaesebroeck, J. Guillermet-Guibert, M. Graupera, B. Bilanges, Theemerging
mechanisms of isoform-specific PI3K signaling, Nat. Rev. Mol. Cell Biol. 11 (2010)
329–341.
[8] R. Marone, V. Cmiljanovic, B. Giese, M.P. Wymann, Targeting phosphoinositide 3-
kinase: moving towards therapy, BBA, Proteins Proteom 1784 (2008) 159–185.
[9] P.T. Cherian, L.N. Koikov, M.D. Wortman, J.J. Knittel, Exploring the PI3Kα and γ
binding sites with 2,6-disubstituted isonicotinic derivatives, Bioorg. Med. Chem.
Lett. 19 (2009) 2215–2219.
[10] P. Workman, P.A. Clarke, S. Guillard, F.I. Raynaud, Drugging the PI3 kinome, Nat.
Biotechnol. 24 (2006) 794–796.
4.2.4.2. Annexin V-FITC apoptosis assay. Apoptosis was determined by
staining the cells with annexin V (fluorescein isothiocyanate) FITC and
counterstaining with propidium iodide (PI) using the Annexin-V-FITC/
PI apoptosis detection kit (BD Biosciences, San Diego, CA) according to
the manufacturer’s instructions. Cells were exposed to the selected
compounds for 48 h. The cells were collected by trypsinization and
0.5 × 106 cells were washed twice with phosphate-buffered saline
(PBS) and stained with 5 μL Annexin-V-FITC and 5 μL PI in 1 × binding
buffer for 15 min at room temperature in the dark. Analyses were
performed using FACSCaliber flow cytometer (BD Biosciences, San
Diego, CA) [39,40].
[11] R.J. Shaw, L.C. Cantley, Ras, PI(3)K and mTOR signaling controls tumour cell
growth, Nature 441 (2006) 424–430.
[12] S. Jamieson, J.U. Flanagan, S. Kolekar, C. Buchanan, J.D. Kendall, W. Lee,
G.W. Rewcastle, W.A. Denny, R. Singh, J. Dickson, B.C. Baguley, P.R. Shepherd, A
drug targeting only p110alpha can block phosphoinositide 3-kinase signaling and
tumor growth in certain cell types, Biochem. J. 438 (2011) 53–62.
[13] W.C. Hon, A. Berndt, R.L. Williams, Regulation of lipid binding underlies the ac-
tivation mechanism of class IA PI3-kinases, Oncogene 31 (2012) 3655–3666.
[14] Y. Liu, W. Wan, Y. Li, G. Zhou, X. Liu, Recent development of ATP-competitive
small molecule phosphatidylinostitol-3-kinase inhibitors as anticancer agents,
Oncotarget 82 (2017) 7181–7200.
[15] L. Liu, B. Shi, X. Li, X. Wang, X. Lu, X. Cai, A. Huang, G. Luo, Q. You, H. Xiang,
14

A.A. Helwa, et al.
BioorganicChemistry102(2020)104051
Design and synthesis of benzofuro[3,2-b]pyridin-2(1H)-one derivatives as anti-
thiopyrimidine analogues, Chem. Pharm. Bull. 60 (2012) 521–530.
leukemia agents by inhibiting Btk and PI3Kδ, Bioorg. Med. Chem. J. 26 (2018)
4537–4543.
[30] M.R. Grever, S.A. Schepartz, B.A. Chabner, The National Cancer Institute: cancer
drug discovery and development program, Semin. Oncol. 19 (1992) 622–638.
[31] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose,
J. Langley, P. Cronise, A. Vaigro-Wolff, M. Gray-Goodrich, H. Campbell, J. Mayo,
M. Boyd, Feasibility of a high-flux anticancer drug screen using a diverse panel of
cultured human tumor cell lines, J. Natl. Cancer. Inst. 83 (1991) 757–766.
[32] M.R. Boyd, K.D. Paull, Some practical considerations and applications of the
National Cancer Institute in vitro anticancer drug discovery screen, Drug Dev. Res.
34 (1995) 91–109.
[16] V.H. Vangapandu, N. Jain, V. Gandhi, Duvelisib: a phosphoinositide-3 kinase δ/γ
inhibitor for chronic lymphocytic leukemia, Expert Opin. Investig. Drugs 26 (2017)
625–632.
[17] N. Zhang, Z. Yu, X. Yang, Y. Zhou, J. Wang, S.-L. Zhang, M.-W. Wang, Y. He,
“Synthesis, biological evaluation and structure-activity relationship of a novel class
of PI3Kα H1047Rmutant inhibitors, Eur. J. Med. Chem. 158 (2018) 707–719.
[18] T.P. Selvam, C.R. James, P.V. Dniandev, S.K. Valzita, A mini review of pyrimidine
and fused pyrimidine marketed drugs, J. Res. Pharm. 2 (2012) 01–09.
[19] R.B. Ajmal, Biological activity of pyrimidine derivatives: a review, Org. Med. Chem.
I J. 2 (2017).
[33] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J.T. Warren,
H. Bokesch, S. Kenney, M.R. Boyd, New colorimetric cytotoxicity assay for antic-
ancer-drug screening, J. Natl. Cancer Inst. 82 (1990) 1107–1112.
[34] J. Carmichael, W.G. DeGraff, A.F. Gazdar, J.D. Minna, J.B. Mitchell, Evaluation of
atetrazolium-based semi-automated colorimetric assay: assessment of chemosensi-
tivity testing, Cancer Res. 47 (1987) 936–942.
[20] R. Kaur, P. Kaur, S. Sharma, G. Singh, S. Mehndiratta, P.M. Bedi, K.l. Nepali, Anti-
cancer pyrimidines in diverse scaffolds: a review of patent literature, Rec. Patents
on Anti-Cancer Drug Disov. 10 (2015) 23–71.
[21] M. Radi, E. Dreassi, C. Brullo, E. Crespan, C. Tintori, V. Bernardo, M. Valoti,
C. Zamperini, H. Daigl, F. Musumeci, F. Carraro, A. Naldini, I. Filippi, G. Maga,
S. Schenone, M. Botta, Design, synthesis, biological activity, and ADME properties
of pyrazolo[3,4-d]pyrimidines active in hypoxic human leukemia cells: a lead op-
timization study, J. Med. Chem. 54 (2011) 2610–2626.
[35] H. Takeuchi, M. Baba, S. Shigeta, An application of tetrazolium (MTT) colorimetric
assay for the screening of anti-herpes simplex virus compounds, J. Virol. Methods
33 (1991) 61–71.
[36] R.B. Badisa, S.F. Darling-Reed, P. Joseph, J.S. Cooperwood, L.M. Latinwo,
C.B. Goodman, Selective cytotoxic activities of two novel synthetic drugs on human
breast carcinoma MCF-7 cells, Anticancer Res. 29 (2009) 2993–2996.
[37] P. Pozarowski, Z. Darzynkiewicz, Analysis of cell cycle by flow cytometry, Methods
Mol. Biol. 281 (2004) 301–311.
[22] K.P. Shao, X.Y. Zhang, P.J. Chen, D.Q. Xue, P. He, L.Y. Ma, J.X. Zheng, Q.R. Zhang,
H.M. Liu, Synthesis and biological evaluation of novel pyrimidine–benzimidazol
hybrids as potential anticancer agents, Bioorg. Med. Chem. Lett. 24 (2014)
3877–3881.
[38] Z. Darzynkiewicz, E. Bedner, P. Smolewski, Flow cytometry in analysis of cell cycle
and apoptosis, Semin. Hematol. 38 (2001) 179–193.
[23] A.K. Al-Ashmawy, F.A. Ragab, K.M. Elokely, M.M. Anwar, O. Perez-Leal, M.C. Rico,
J. Gordon, E. Bichenkov, G. Mateo, E.M. Kassem, G.H. Hegazy, M. Abou-Gharbia,
W. Childers, Design, synthesis and SAR of new-di-substituted pyridopyrimidines as
ATP-competitive dual PI3Ka/mTOR inhibitors, Bioorg. Med. Chem. Lett. 27 (2017)
3117–3122.
[39] I. Vermes, C. Haanen, H. Steffens-Nakken, C.J.A. Reutelngsperger, A novel assay for
apoptosis Flow cytometric detection of phosphatidylserine early apoptotic cells
using fluorescein labelled expression on Annexin V, J. Immunol. Meth. 184 (1995)
39–51.
[24] T. Saurat, F. Buron, N. Rodrigues, M. Tauzia, L. Colliandre, S. Bourg, P. Bonne,
G. Guillaumet, M. Akssira, A. Corlu, C. Guillouzo, P. Berthier, P. Rio, M. Jourdan,
H. Bénédetti, S. Routier, Design, synthesis, and biological activity of pyridopyr-
imidine scaffolds as novel PI3K/mTOR dual inhibitors, J. Med. Chem. 57 (2014)
613–631.
[40] I. Lakshmanan, S.K. Batra, Protocol for apoptosis assay by flow cytometry using
Annexin V Staining method, Bio. Protoc. 3 (2013) 374.
[41] Y. Zhao, X. Zhang, Y. Chen, S. Lu, Y. Peng, X. Wang, C. Guo, A. Zhou, J. Zhang,
Y. Luo, Q. Shen, J. Ding, L. Meng, J. Zhang, Crystal structures of PI3Kα complexed
with PI103 and its derivatives: new directions for inhibitors design, ACS Med.
Chem. Lett. 5 (2014) 138–142.
[25] D. Sarker, J.E. Ang, R. Baird, R. Kristeleit, K. Shah, V. Moreno, P.A. Clarke,
F.I. Raynaud, G. Levy, J.A. Ware, K. Mazina, R. Lin, J. Wu, J. Fredrickson,
J.M. Spoerke, M.R. Lackner, Y. Yan, L.S. Friedman, S.B. Kaye, M.K. Derynck,
P. Workman, J.S. de Bono, First-in-human Phase I study of Pictilisib (GDC-0941), a
Potent Pan-Class I Phosphatidylinositol-3-Kinase (PI3K) inhibitor, in patients with
advanced solid tumors, Clin. Cancer Res. 21 (2015) 77–86.
[42] D.A. Sabbah, M.G. Brattain, H. Zhong, Dual inhibitors of PI3K/mTOR or mTOR-
selective inhibitors: which way shall we go, Curr. Med. Chem. 18 (2011)
5528–5544.
[43] M.A. Ibrahim, S.M. Abou-Seri, M.M. Hanna, M.M. Abdalla, N.A. El Sayed, “Design,
synthesis and biological evaluation of novel condensed pyrrolo[1,2-c]pyrimidines
featuring morpholine moiety as PI3Kα inhibitors, Eur. J. Med. Chem. 99 (2015)
1–13.
[26] F.M. Elmenier, D.S. Lasheen, K.A.M. Abouzid, Phosphatidylinositol 3 kinase (PI3K)
inhibitors as new weapon to combat cancer, Eur. J. Med. Chem. 183 (2019)
111718–111748.
[44] A. Daina, O. Michielin, V. Zoete, iLOGP: a simple, robust, and efficient description
of n-octanol/water partition coefficient for drug design using the GB/SA approach,
J. Chem. Inf. Model. 54 (2014) 3284–3301.
[27] A.A. Helwa, E.M. Gedawy, A.T. Taher, A.K. El-Ansary, S.M. Abou-Seri, Synthesis
and biological evaluation of novel pyrimidine-5-carbonitriles featuring morpholine
moiety as antitumor agents, Future Med. Chem. 12 (5) (2020) 403–421.
[28] V.J. Ram, Chemotherapeutic agents, XVIII: Synthesis of pi-deficient pyrimidines
and fused pyrimidines as leishmanicidal agents, Arch. Pharm. 323 (1990) 895–899.
[29] A.T. Taher, A.A. Helwa, Synthesis, antitumor and antimicrobial testing of some new
[45] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate phar-
macokinetics, drug-likeness and medicinal chemistry friendliness of small mole-
cules, Sci. Rep. 7 (2017) 1–13.
15