Synthesis and in vitro investigation of novel cytotoxic pyrimidine and pyrazolopyrimidne derivatives showing apoptotic effect

Article abstract of DOI:10.1016/j.bioorg.2020.103621

A series of novel derivatives of hydrazinylpyrimidines, pyrazolylpyrimidines and 3-amino[3,4-d]pyrazolopyrimidines have been synthesized and tested for their in vitro cytotoxic activity against 60 tumor cell lines by NCI. The in vitro cytotoxic IC₅₀ values for the most active compounds were determined against the colon-KM12 cell line (5d, 7c and 7d), breast-MCF-7 (6a) and melanoma-MDA-MB-435 (6h) using 5-fluorouracil (5-FU) as a positive control. Derivatives 5d and 7c were found to be the most potent derivatives against KM12 cell line (IC₅₀ = 1.73 and 1.21 μM, respectively) with a high selectivity index (SI) (18.82 and 35.49, respectively) compared to 5-FU (IC₅₀ = 12.26 μM, SI = 1.93). Compounds 5d and 7c were further investigated for their apoptotic behavior in KM12 cell line. The investigations showed the up-regulation of caspase 3/9 and the pro-apoptotic factor Bax. On the other hand, the expression of the anti-apoptotic factor Bcl-2, was down-regulated, as well as its inhibition at a nanomolar concentration. Furthermore, the apoptotic effect for derivatives 5d and 7c in KM12 cells was detected using annexin V-FITC staining method.

Full text of DOI:10.1016/j.bioorg.2020.103621

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Synthesis and in vitro investigation of novel cytotoxic pyrimidine and pyrazo-
lopyrimidne derivatives showing apoptotic effect
Fatma A. Ragab, Yassin M. Nissan, Emad M. Seif, Ahmed Maher, Reem K.
Arafa
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DOI:
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Please cite this article as: F.A. Ragab, Y.M. Nissan, E.M. Seif, A. Maher, R.K. Arafa, Synthesis and in vitro
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Synthesis and in vitro investigation of novel cytotoxic pyrimidine and
pyrazolopyrimidne derivatives showing apoptotic effect
Fatma A. Ragab a*, Yassin M. Nissan a,b, Emad M. Seif b*, Ahmed Maher c and Reem
K. Arafa d
aPharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Elini
St., Cairo 11562, Egypt
bPharmaceutical Chemistry Department, Faculty of Pharmacy, October University for Modern
Sciences and Arts (MSA), Giza, Egypt
cBiochemistry Department, Faculty of Pharmacy, October University for Modern Sciences
and Arts (MSA), Giza, Egypt
dBiomedical Sciences Program, University of Science and Technology, Zewail City of
Science and Technology, 12578, Cairo, Egypt
*Corresponding authors:
Emad M. Seif, Pharmaceutical Chemistry Department, Faculty of Pharmacy, October
University for Modern Sciences and Arts (MSA), Giza, Egypt, Mobile +2-01001734143, E-mail:
ph.emad.seif@gmail.com
Fatma A. Ragab, Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo
University, Kasr Elini St., Cairo 11562, Egypt
Abstract
A series of novel derivatives of hydrazinylpyrimidines, pyrazolylpyrimidines and 3-
amino[3,4-d]pyrazolopyrimidines have been synthesized and tested for their in vitro cytotoxic
activity against 60 tumor cell lines by NCI. The in vitro cytotoxic IC50 values for the most active
compounds were determined against the colon-KM12 cell line (5d, 7c and 7d), breast-MCF-7 (6a)
and melanoma-MDA-MB-435 (6h) using 5-fluorouracil (5-FU) as a positive control. Derivatives
5d and 7c were found to be the most potent derivatives against KM12 cell line (IC50 =1.73 and
1.21 µM, respectively) with a high selectivity index (SI) (18.82 and 35.49, respectively) compared
to 5-FU (IC50 = 12.26 µM, SI= 1.93). Compounds 5d and 7c were further investigated for their
apoptotic behavior in KM12 cell line. The investigations showed the up-regulation of caspase 3/9
and the pro-apoptotic factor Bax. On the other hand, the expression of the anti-apoptotic factor
Bcl-2, was down-regulated, as well as its inhibition at a nanomolar concentration. Furthermore,
the apoptotic effect for derivatives 5d and 7c in KM12 cells was detected using annexin V-FITC
staining method.
Keywords: pyrimidine based hybrids; pyrazolopyrimidine hybrids; cytotoxicity; apoptosis
1. INTRODUCTION
Inhibition of cell cycle progression and induction of apoptosis represent a key strategy for
prevention and treatment of cancer [1]. Apoptosis or programmed cell death is considered a natural
1

process in elimination of any unneeded or abnormal cells [1,2]. Two major pathways of apoptosis
have been identified; the intrinsic (mitochondrial mediated) pathway which is regulated by Bcl-2
family and extrinsic (death receptor mediated) pathway which is regulated by a subgroup of tumor
necrosis factor receptors superfamily (TNFR) [3]. Instantly as apoptosis process is initiated,
caspases, a family of cysteine proteases, are activated. Caspases are subdivided into initiator
caspases as caspases 2, 8 and 9, effector caspases as caspases 3, 6 and 7 and inflammatory caspases
as caspases 1, 4, 5, 11 and 12 [3]. Caspase 9 is the initiator of the intrinsic apoptosis while caspase
8 is the initiator of the extrinsic apoptosis. Caspases 8 and 9 activate the effector caspases 3 and 7
which trigger apoptosis. Diverse anticancer drugs act by induction of apoptosis [4–8]. Cancer cells
can avoid apoptosis by hindering caspase function, decreasing Bax expression level or increasing
gene expression level of anti-apoptotic Bcl-2 [2]. Apoptosis process could be induced by death
receptors agonists, DNA damage, mitotic spindle dysfunction and cell cycle progression
disturbance[9,10].
One of the most important building blocks of nucleic acids is the pyrimidine ring,
subsequently its derivatives possess a diverse chemotherapeutic profile as antimicrobial, antiviral
or anticancer activity [11,12]. Among these reported pharmacological activities of pyrimidine
based scaffolds, antitumor activity is the most widely reported [11–19]. The antiproliferating
activity of the pyrimidine based scaffold is owing to their ability to bind with several enzymes,
receptors and target proteins (Fig. 1) [20]. 5-Fluoruracil is a dihydropyrimidine derivative used in
the treatment of various types of cancer as metastatic colorectal, colon, nasopharyngeal and breast
cancer and acts through irreversible inhibition of thymidylate synthase [21,22]. 5-FU induce
varying degrees of apoptosis in colorectal cancer cells by the activation of caspase-9 [23] and is
known to affect the cell cycle [24]. Monastrol is another dihydropyrimidine derivative inhibits the
motility of the mitotic kinesin Eg5 and arrest the cell cycle at G2/M phase [25]. In addition, several
pyrimidin-5-carbonitrile derivatives as I and II exhibit cytotoxic activity against wide range of
cancer cell lines [26–34]. Moreover, several bicyclic condensed pyrimidines possess antitumor
activity by inhibiting different protein kinases. On the other hand, pyrazoles demonstrate a great
potential in the design of anticancer agents and also stimulate activity of proapoptotic proteins
[35–37]. Furthermore, dihydropyrazoles act as selective inhibitors for EGFR and/or Her-2 and
telomerase enzyme which are useful targets for cancer treatment [38–40]. Some pyrazoline
derivatives as III and IV inhibit also the proliferation of some types of cancer through inhibition
of cell cycle progression and induction of apoptosis through upregulation of caspase 3 and Bax
expression level [41,42]. Pyrazolopyrimidines as Roscovitine, CYC065 and V, bioisosteres of
adenine, act as apoptotic inducers through elevation of caspase 3 level and cell cycle arrest at G1,
S and G2/M phases (Fig. 1) [43–46].
2

O
O
N
F
N
HN
HN
O
HN
OH
O
N
O
S
N
N
H
H
O
N
H
F
5-Fluorouracil
Monastrol
I
O
S
O
N
R
N
O
S
O
O
H2N
O
O
F
S
N
H
N
N
N
N
N
NH2
N
N
R'
S
II
III
IV
O
N
NH2
N
N
HN
NH
N
N
HO
N
N
N
N
N
N
N
N
N
N
N
H
N
H
OH
O
Roscovitine
V
CYC065
Fig. 1. Biologically active pyrimidine, pyrazoline and fused pyrimidine derivatives with anticancer activity.
Consequently, the present investigation describes the synthesis of new pyrimidine derivatives
(5a-d) and pyrimidine based hybrids molecules containing fused (7a-d and 8a-f) and non-fused
(6a-h) pyrazole moiety to be tested as anticancer agents (Fig. 2).
R
O
H2N
O
CN
NH
O
HN
N
S
HN
N
CN
N
N
N
R1
X
NH2
X
N
N
N
NH2
X
R
X
N
N
S
N
R
S
S
N
6a-h
5a-d
7a-d
8a-f
Fig. 2. The targeted compounds 5a-d, 6a-h, 7a-d and 8a-f
3

2. RESULTS AND DISCUSSION
2.1. Chemistry
The key starting dihydropyrimidines 1a and 1b have been synthesized according to the
reported method involving cyclocondensation of thiourea, furan-2-carboxaldehyde or 2-
thiophenecarboxaldehyde, respectively with ethylcyanoacetate in anhydrous K2CO3 / ethanol [47].
Hydrazinolysis of 1a and 1b afforded 2-hydrazino derivatives 2a and 2b, respectively [48,49].
Regioselective methylation of 1a and 1b with methyl iodide in DMF at 0°C in presence of K2CO3
yielded the S-methyl derivatives 3a and 3b, respectively [50]. Reaction of 1a, 1b, 3a and 3b with
a mixture of phosphorous oxychloride and phosphorous pentachloride gave the corresponding
chloro derivatives 4a-d [50,51]. The structure of 4b was confirmed by IR, which showed bands at
3440 cm-1 (NH) and 2228 cm-1 (CN). 1H NMR of 4b showed three signals attributed to thiophene
protons at δ 7.30, 7.98, 8.08 ppm and a singlet at δ 11.81 ppm (NH-exchangeable with D2O). The
chloro derivatives 4a-d were reacted with hydrazine hydrate at room temperature to give the
hydrazino derivatives 5a-d (Scheme 1). IR spectra of derivatives 5a-d revealed bands at 3350-
3309 cm-1 (NH, NH2).
4

O
H2N
NC
NH2
Cl
N
CN
CN
HN
S
d
X
a
HN
+
CHO
X
S
N
H
X
S
O
1a:
1b:
X=O
X=S
OEt
4a
:X=O
4b
:X=S
b
c
e
H2N
NH
N
CN
O
N
O
HN
S
CN
CN
X
HN
HN
X
H2N
X
S
N
N
H
5a
:X=O
3a
:X=O
5b
:X=S
2a
2b
:X=O
:X=S
3b
:X=S
d
H2N
NH
N
Cl
N
CN
CN
N
S
N
e
X
X
S
5c
:X=O
4c
:X=O
5d
4d
:X=S
:X=S
Scheme 1.
Reagets and conditions: (a) K2CO3, abs. ethanol, reflux 12 h: (b) NH2NH2, methanol, reflux 12
h: (c) MeI, DMF, Ice bath, 3 h: (d) POCl3, PCl5, reflux 30 h: (e) NH2NH2, Methanol, rt, 5 h.
Reaction of the hydrazino derivatives 2a and 2b with various chalcones yielded the cyclic
pyrazoline derivatives 6a-h (Scheme 2). The IR spectra of 6a-h derivatives exhibited bands at
3436-3363 cm-1 (NH of pyrimidine) and at 1658-1644 cm-1 (C=O). 1H NMR spectra showed the
characteristic AMX prominent pattern of pyrazoline protons where C4-HA, HM and C5-HX
appeared as doublet of doublet signals at δ 3.17-3.40, 3.95-4.01 and 5.74-5.79 ppm, respectively.
13
Besides exchangeable NH pyrimidine singlet signals appeared at δ 12.27-13.43 ppm. C NMR
spectra elucidated C4 and C5 of pyrazoline at δ 40.42-40.62 and δ 55.64-62.34 ppm, respectively
(Scheme 2).
5

R
O
O
N
CN
CN
HN
a
HN
H2N
X
X
N
N
N
H
2a, b
N
6a
:R= H; X= O
6b
:R= Cl; X= O
:R= F; X= O
6c
6d
:R= OMe; X= O
6e
6f
:R= H; X= S
:R= Cl; X= S
6g
:R= F; X= S
6h
:R= OMe; X= S
Scheme 2.
Reagets and conditions: (a) chalocones, abs ethanol, NaOH, reflux, 70 h.
The pyrazolopyrimidine derivatives 7a-d were obtained through intramolecular cyclization of
hydrazino derivatives 5a-d. Reaction of 7c and 7d with p-substituted arylsulfonyl chlorides yielded
the phenyl sulfonyl pyrazolopyrimidine derivatives 8a-f.
IR of 7a-d was characterized by absence of CN band. 1H NMR of 7a-d showed a singlet signal
at δ 5.35-5.92 ppm (NH2) in addition to a singlet signals at δ 12.27-12.77 (NH pyrazole
1
exchangeable with D2O). IR spectra of 8a-f showed bands at 3495-3286 (NH2) while H NMR
spectra showed singlet signals at δ 2.65-2.66 (SCH3) and at δ 6.00-6.60 (NH2) (Scheme 3).
6

H2N
NH
HN
N
CN
a
HN
HN
S
NH2
X
S
N
X
N
5a, b
7a
:X=O
7b
:X=S
H2N
O
NH
O
S
N
HN
CN
N
N
R1
N
S
b
a
NH2
X
N
NH2
X
X
N
N
S
N
S
N
5c, d
7c
:X=O
7d
:X=S
8a
:R1= H; X= O
8b
:R1= Me; X= O
8c
:R1= Cl; X= O
8d
8e
8f
:R1= H; X=S
:R1= Me; X= S
:R1= Cl; X= S
Scheme 3.
Reagets and conditions: (a) abs ethanol, conc. HCl, reflux, 12 h: (b) arylsulfonyl chloride derivatives,
dry pyridine, reflux, 8h.
2.2. Biological evaluation
2.2.1. Antiproliferative activity
The synthesized compounds were chosen and tested for their in vitro antiproliferative activity
by the National Cancer Institute (NCI), Germantown MD, USA, under the Development
Therapeutic Program (DTP) on a panel of 60 tumor cell lines representing breast, CNS, colon,
leukemia, melanoma, non-small cell lung, ovarian, prostate and renal cancers. The compounds
were screened in an initial one dose at conc. 10 µM and results are presented as percent growth
inhibition of the treated cells (GI %) (As seen in supplementary data).
With regards to the cytotoxicity against the breast cancer cell line (MCF-7), the unsubstituted
derivative 6a was the most active (GI % = 59.97) while the para fluoro derivative 6c and para
methoxy derivative 6d showed a little less cytotoxic activity (GI % of 47.43 and 33.97,
respectively). The thiophene derivatives 6e, 6g and 6h displayed significant less cytotoxic activity
(GI % = 23.63, 22.50 and 23.82).
With respect to the colon cancer cell line (KM12), three derivatives 5d, 7c and 7d exhibited
good potency with GI % = 57.94, 55.93 and 51.22, respectively. In the series of the hydrazino
derivatives 5a-d, it was noticed that the thiomethyl derivatives 5c (GI % = 33.16) and 5d (GI % =
57.94) were more potent against colon cancer cell line (KM12) than thioxo derivatives 5a (GI %
= 0.27) and 5b (GI % = 6.51), respectively.
7

Furthermore, the fused pyrazolopyrimidine derivative 7c and 7d exhibited high potency (GI%
= 55.93 and 51.22, respectively) when compared to the desmethyl analogs 7a and 7b (GI% less
than 20)
Referring to melanoma (MDA-MB-435) cancer cell line, the only active derivative was 6h
(GI % = 55.30) (Fig. 3).
70
60
50
40
30
20
10
0
55.3
5c
5d
6a
6c
6d
6e
6g
6h
7c
7d
8a
8c
Breast cancer MCF-7
Colon cancer KM12
Melanoma MDA-MB-435
Fig. 3. Growth inhibition percentage of the tested compounds exceeding 20% at 10 µM concentration in MCF-7,
KM12 and MDA-MB-435 cell lines.
2.2.2. In vitro IC50 evaluation
The in vitro cytotoxic IC50 values of the most active derivatives in the NCI growth inhibition
assay 5d, 6a, 6h, 7c and 7d were determined against the most sensitive cancer cell lines viz. colon
(KM12), breast (MCF-7) and melanoma (MDA-MB-435) and against the corresponding normal
colon (FHC), breast (MCF10a) and skin (HFB4) cell lines using 5-fluorouracil as a positive control
(Table 1).
Table 1. IC50 values of the MTT assayed compounds against the most sensitive cell lines.
IC50 results (µM)*
Cancer cell lines
MDA-MB-435
Normal cell lines
HFB4
Compound #
MCF7
NT**
KM12
MCF10a
NT
FHC
5d
6a
6h
7c
NT
1.73±0.06
SI=18.82***
NT
NT
32.57±1.97
4.22±0.31
SI= 21.50
NT
NT
90.74 ±6.8
NT
NT
NT
NT
5.25±0.24
SI= 8.42
NT
NT
44.25±2.1
NT
NT
1.21±0.04
NT
42.95±2.55
8

SI= 35.49
7d
NT
NT
5.55±0.31
SI= 2.49
12.26±1.4
SI= 1.93
NT
NT
13.87±1.74
23.78 ±1.5
5-FU
2.44±0.23
SI=43.21
2.52±0.12
SI=14.08
105.45±7.22
35.5±1.9
* IC50 values are the average of 3 independent runs ±SD
** NT = not tested
*** SI = selectivity index
Compounds 5d, 7c and 7d were more potent against KM12 cell line (IC50 =1.73, 1.21 and
5.55 µM, respectively) with higher selectivity index (SI) (18.82, 35.49 and 2.49, respectively) than
5-FU (IC50 = 12.26 µM, SI= 1.93). Compound 6a showed potent activity against MCF-7 cell line
(IC50= 4.22 µM) with high SI (21.50) although lower than 5-FU (IC50= 2.44 µM and SI= 43.21).
Compound 6h also exhibited remarkable activity against MDA-MB-435 cells (IC50= 5.25 µM, SI=
8.42) but still lower than 5-FU (IC50= 2.52 µM, SI= 14.08).
2.2.3. Caspase 3/9 assay
Compounds 5d and 7c were selected for further evaluation of their caspase activation ability
in colon cancer cell line (KM12). They showed significant increase in the cellular levels of both
caspase 3 (0.5217 and 0.5951 ng/mL, respectively) and caspase 9 (14.77 and 18.45 ng/mL,
respectively) compared to untreated cells (caspase 3 = 0.04316 ng/mL and caspase 9 = 0.91 ng/mL)
(Table 2).
Table 2. Caspase 3/9 concentrations in treated KM12 cells with 5d and 7c.
Compound #
Caspase 3 (ng/mL)*
0.5217±21.1
Caspase 9 (ng/mL)*
14.77±0.83
5d
7c
0.5951±18.3
18.45±1.21
Control
0.04316±3.42
0.91±0.04
* Data are presented as the mean of 3 independent runs ±SD
2.2.4. Evaluation of Bax and Bcl-2 expressions
Bax and Bcl-2 cellular levels were assayed for 5d and 7c in colon (KM12) cells. The gene
expression fold values were presented in Table 3. Results showed an increase of the pro-apoptotic
factor Bax by 6.50 and 6.07 folds, respectively while anti-apoptotic protein Bcl-2 demonstrated a
concentration decrease by 0.29 and 0.35 folds, respectively.
Table 3. Bax and Bcl-2 expression levels in treated KM12 cells with 5d and 7c.
Compound #
Pro-apoptotic Bax (fold)*
Anti-apoptotic Bcl-2 (fold)
5d
7c
Control
6.50
6.07
1.00
0.29
0.35
1.00
2.2.5. In vitro Bcl-2 inhibition assay
Bcl-2 anti-apoptotic protein, a member of Bcl-2 family, is an attractive target in cancer therapy
where the ability of the cancer cells to survive and resist apoptosis is closely related to the high
expression level of Bcl-2 protein [52,53]. In a trial for further investigation of the apoptotic
9

induction mechanism exhibited by 5d and 7c, in vitro Bcl-2 inhibition assay was performed using
venetoclax as a reference. Venetoclax is a selective Bcl-2 inhibitor used in the treatment of chronic
lymphocytic leukeamia (CLL). It restores the normal apoptosis process in malignant cells through
liberating the pro-apoptotic factors from Bcl-2[54].
Fortunately, both compounds showed inhibitory action at nanomolar level (IC50= 852.78 and
449.42 nM, respectively) towards the evaluated protein. These results may add an extra mechanism
of action for the newly synthesized compounds and contribute to their apoptotic behavior in cancer
cells (Table 4).
Table 4. Bcl-2 inihibtion assay of 5d and 7c
Compound #
Bcl-2 IC50 (nM)
852.78±24.5
449.42±13.01
202.01±7.32
5d
7c
venetoclax
2.2.6. Cell cycle analysis
To further investigate the apoptotic capacity of 5d and 7c, flowcytometric analysis was
performed on colon (KM12) cancer cell line treated with the tested compounds at their IC50
concentrations using untreated colon (KM12) cancer cells as a negative control. There was an
elevation of apoptotic cells at pre-G1 phase (14.36% and 22.81%, respectively) compared to
control KM12 cells (2.16%) as well as an accumulation of cells at the G2-M phase (34.28% and
42.08%, respectively) versus control KM12 cells (11.19%). On the contrary, the percentage of
treated cells in G0/G1 phase was reduced (44.15% and 35.28%, respectively) compared to
untreated cells (55.48%) also in S phase the percentage of treated cells was reduced (21.57% and
22.64%, respectively) compared to untreated cells (33.33%) (Fig. 4 and Table 5).
Control / KM12
7c / KM12
5d / KM12
Fig. 4. Effect of 5d and 7c on the cell cycle of KM12 cells.
Table 5. Cell cycle analysis in treated KM12 cells with 5d and 7c.
Compound #
% G0-G1*
%S*
%G2-M*
%Pre-G1
apoptosis*
14.36±1.1
5d
44.15±2.82
21.57±1.92
34.28±1.86
10

7c
Control
35.28±2.36
55.48±3.71
22.64±1.73
33.33±2.36
42.08±3.17
11.19±1.24
22.81±2.5
2.16±0.22
* Data are presented as the mean of 3 independent runs ±SD
2.2.7. Apoptotic cells sub-population determination
To support the fact that derivatives 5d and 7c induce apoptosis in colon (KM12) cancer cell
line, annexin V-FITC and propidium iodide double staining method was used. Table 6 displays
the percentage of early and late apoptotic populations as well as the necrotic population relative to
the total apoptotic cells. When KM12 cells were treated with the IC50 dose of compounds 5d and
7c, the percentage of early and late apoptotic populations increased from 1.31% and 0.26%,
respectively in control untreated cells to 6.77% and 6.05%, respectively in 5d treated cells and to
5.92% and 13.6%, respectively in 7c treated cells. This proves that the antiproliferative effect is
associated with a pro-apoptotic activity of the tested compounds (Fig. 5).
Table 6. Apoptotic cells sub-population percentage in treated KM12 cells with 5d and 7c.
Compound #
Total*
Early*
Late*
Necrosis*
1.54±0.65
3.29±0.19
0.59±0.03
5d
7c
Control
14.36±0.88
22.81±2.11
2.16±0.13
6.77±0.34
5.92±0.29
1.31±0.07
6.05±0.33
13.6±1.21
0.26±0.04
* Data are presented as the mean of 3 independent runs ±SD
5d / KM12
7c / KM12
Control / KM12
Fig. 5. Effect of 5d and 7c on the percentage of annexin V-FITC-positive staining in KM12 cells
Molecular docking
2.3.
Docking studies of 5d and 7c in the BH3 groove of Bcl-2 were performed to predict the
binding mode of interactions between the synthesized derivatives and the active site. Both the co-
crystallized ligand 398 and the reference drug venetoclax were also docked using the same
protocol of 5d and 7c docking process for validation (Table 7). The docked pose of 398 (RMSD <
1) and venetoclax showed interaction with Tyr 67 (1 hydrogen bond; 1.97 Å) and Arg 105 (2
hydrogen bonds; 1.98 and 2.67 Å), respectively (Fig. 6).
11

Table 7. Docking scores of 5d and 7c in the active site of Bcl-2 [PDB ID: 4AQ3].
Compound #
Docking score (Kcal/mol)
5d
7c
-5.4
-5.6
Co-crystallized ligand (398)
venetoclax
-11.1
-9.4
A
B
Fig. 6. The docked pose of the co-crystallized ligand 398 A (orange) and venetoclax B (orange) in the active site of
Bcl-2 [PDB ID: 4AQ3]
Investigational analysis of the predicted binding modes of 5d and 7c helps their assumed direct
inhibitory role where 5d form two hydrogen bonds with Arg 105 residue (2.13 and 2.38 Å) and 7c
form single hydrogen bond with Tyr 161 (2.35 Å) residue in the active site (Fig. 7)
12

A
B
Fig. 7. The proposed binding pose for the interaction of the 5d (A; blue) and 7c (B; blue) in the active site of Bcl-2
[PDB ID: 4AQ3]
3. CONCLUSIONS
Three series of hydrazinopyrimidines 2a, b and 5a-d, 2-pyrazolylpyrimidines 6a-h, and 3-
amino[3,4-d]pyrazolopyrimidines 7a-d and 8a-f have been synthesized and tested for their in vitro
cytotoxic activity against 60 tumor cell lines by NCI. In vitro IC50 determination of the best five
derivatives were determined against cancer and normal cell lines. Compounds 5d and 7c were
found to be the most potent and selective derivatives against KM12 cell line. Caspase activation
assay was performed for both 5d and 7c derivatives in colon (KM12) cancer cell line. The results
showed significant increase of caspase 3 (0.5217 and 0.5951 ng/mL, respectively) and caspase 9
(14.77 and 18.45 ng/mL, respectively) compared to untreated KM12 (0.04316 and 0.91 ng/mL,
respectively). Bax and Bcl-2 concentration levels were also evaluated and showed a titer increase
of the pro-apoptotic protein Bax (6.50 and 6.07 fold, respectively) and a decrease of the anti-
apoptotic protein Bcl-2 (0.29 and 0.35 fold, respectively) compared to untreated KM12 cells, thus
confirming their involvement in apoptosis induction. In vitro Bcl-2 inhibition assay was performed
for compounds 5d and 7c to confirm their apoptotic activity. Compound 7c showed Bcl-2
inhibition with IC50= 449.42 nM. Furthermore, flow cytometric assay results showed that the
apoptotic cells percentage was significantly elevated at the pre-G1 phase to 14.36% and 22.81%,
respectively compared to untreated control KM12 2.16%. Derivatives 5d and 7c which revealed
selective cytotoxic activity against KM12 cells in addition to apoptotic effect confirmed by
annexin V-FITC staining method may serve as promising leads as apoptotic inducers in colon
cancer treatment.
13

4. EXPERIMENTAL SECTION
4.1.
Chemistry
Melting points were determined with Stuart SMP3 version 5.0 apparatus and were
uncorrected. FT-IR spectra were recorded on Shimadzu-FTIR spectrophotometer using KBr discs
1
and expressed in wave number (cm-1). H and 13C NMR spectra were recorded with Bruker
Advance 400 spectrophotometer operating at 400 MHz and 100 MHz, respectively and a varian
mercury VXR-300 spectrophotometer (300 MHz for 1H) and the chemical shifts were given in δ
as parts per million (ppm) downfield from tetramethylsilane (TMS) as internal standard. Elemental
microanalysis was performed at the Regional Center for Mycology and Biotechnology, AL-Azhar
University, Egypt. The reactions were monitored by TLC (Merck, Germany), the spots were
detected by exposure to UV lamp at λ 254 nm. All reagents and solvents were purified and dried
by standard techniques.
4.1.1. Compounds 1a, 1b, 2a, 2b, 3a, 3b, 4a, 4c and 4d
Have been prepared according to the reported methods [47–51,55].
4.1.2. Synthesis of 6-Chloro-4-(thiophen-2-yl)-2-thioxo-1,2-dihydropyrimidin-5-carbonitrile
(4b)
4-Oxo-6-(thiophen-2-yl)-2-thioxo-1,2,3,4-tetrahydropyrimidin-5-carbonitrile
(1b)
(8.5
mmol, 2 g) in a mixture of POCl3 (10 mL) and PCl5 (8.5 mmol, 1.76 g) was refluxed for 30 h. The
reaction mixture was poured on ice/water and the residue was filtered off and crystallized from
methanol.
Yield: 84%; Melting point: 280-282 ºC; IR υmax/cm-1: 3440 (NH), 3113 (CH aromatic), 3000
(CH aliphatic) 2228 (CN). 1H NMR (300 MHz, DMSO-d6) δ 7.30 (dd, 1H, thiophene C4-H, J =
4.2, 5.1 Hz), 7.98 (d, 1H, thiophene C3-H, J = 4.2 Hz), 8.08 (d, 1H, thiophene C5-H, J = 5.1 Hz),
11.81 (s, 1H, NH, exchangeable with D2O). Anal. Calcd for C9H4ClN3S2 (253.73): C, 42.60; H,
1.59; N, 16.56. Found: C, 42.76; H, 1.83; N, 16.82.
4.1.3. Synthesis of compounds 5a-d
To a solution of 4a-d (1 mmol) in methanol (10 mL), hydrazine hydrate 99% (3 mmol) was
added dropwise. The reaction mixture was stirred for 5 h at room temperature then the precipitate
was filtered, washed and crystallized from methanol.
4.1.3.1.
4-(Furan-2-yl)-6-hydrazinyl-2-thioxo-1,2-dihydropyrimidin-5-carbonitrile (5a)
Yield: 82%; Melting point: >300 ºC; IR υmax/cm-1: 3350-3194 (NH, NH2), 3140 (CH
aromatic), 2193 (CN). 1H NMR (400 MHz, DMSO-d6) δ 6.73 (dd, 1H, furan C4-H, J = 3.56, 1.72
Hz), 7.19-7.39 (s, br., 4H, NH pyrimidine and NHNH2, exchangeable with D2O), 7.43 (d, 1H,
furan C3-H, J = 3.56 Hz), 7.98 (d, 1H, furan C5-H, J = 1.0 Hz). Anal. Calcd for C9H7N5OS
(233.25): C, 46.34; H, 3.02; N, 30.03. Found: C, 46.13; H, 3.25; N, 30.21.
14

4.1.3.2.
6-Hydrazinyl-4-(thiophen-2-yl)-2-thioxo-1,2-dihydropyrimidin-5-carbonitrile (5b)
Yield: 76%; Melting point: 169-171 ºC; IR υmax/cm-1: 3332-3146 (NH, NH2), 3099 (CH
aromatic), 2209 (CN). 1H NMR (400 MHz, DMSO-d6) δ 5.19-6.76 (s, br., 4H, NH pyrimidine and
NHNH2, exchangeable with D2O), 7.04-7.97 (m, 3H, aromatic-H). 13C NMR (100 MHz, DMSO-
d6) δ 75.30, 119.03, 129.71, 132.27, 132.56, 141.84, 154.99, 155.62, 185.78. Anal. Calcd for
C9H7N5S2 (249.32): C, 43.36; H, 2.83; N, 28.09. Found: C, 43.59; H, 3.01; N, 28.41.
4.1.3.3.
4-(Furan-2-yl)-6-hydrazinyl-2-(methylthio)pyrimidin-5-carbonitrile (5c)
Yield: 86%; Melting point: 181-183 ºC; IR υmax/cm-1: 3315-3265 (NH, NH2), 3133 (CH
aromatic), 2921 (CH aliphatic), 2208 (CN). 1H NMR (400 MHz, DMSO-d6) δ 2.54 (s, 3H, SCH3),
3.42-3.50 (s, br., 3H, NHNH2, exchangeable with D2O), 6.76 (dd, 1H, furan C4-H, J = 3.52, 1.72
Hz), 7.43 (d, 1H, furan C3-H, J = 3.4 Hz), 8.04 (d, 1H, furan C5-H, J = 0.88 Hz). 13C NMR (100
MHz, DMSO-d6) δ 14.12, 77.54, 112.71, 113.19, 115.90, 116.44, 147.69,150.04, 161.89, 162.95,
174.08. Anal. Calcd for C10H9N5OS (247.28): C, 48.57; H, 3.67; N, 28.32. Found: C, 48.34; H,
3.80; N, 28.65.
4.1.3.4.
4-Hydrazinyl-2-(methylthio)-6-(thiophen-2-yl)pyrimidin-5-carbonitrile (5d)
Yield: 87%; Melting point: 260-262 ºC; IR υmax/cm-1: 3309-3170 (NH, NH2), 3093 (CH
aromatic), 2970 (CH aliphatic), 2210 (CN). 1H NMR (400 MHz, DMSO-d6) δ 2.54 (s, 3H, SCH3),
4.76 (s, 2H, NH2, exchangeable with D2O), 7.23 (m, 1H, thiophene C4-H), 7.90 (d, 1H, thiophene
C3-H, J = 4.84 Hz), 8.18 (d, 1H, thiophene C5-H, J = 2.52 Hz), 9.39 (s, 1H, NH, exchangeable
with D2O). 13C NMR (100 MHz, DMSO-d6) δ 14.09, 77.64, 116.92, 129.28, 130.68, 133.09,
140.71, 162.10, 173.84. Anal. Calcd for C10H9N5S2 (263.34): C, 45.61; H, 3.44; N, 26.59. Found:
C, 45.89; H, 3.61; N, 26.78.
4.1.4. Synthesis of compounds 6a-h
A mixture of 2a, b (5mmol), the appropriate chalcone (5mmol) and sodium hydroxide (5
mmol, 0.2 g) in absolute ethanol (30 mL) was refluxed for 70 h. The reaction mixture was filtered
on hot then the filtrate was poured on ice\water, neutralized with acetic acid and left for 24h at
room temperature. The residue was filtered off and the crude product was crystallized from
methanol.
4.1.4.1.
2-(3,5-Diphenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-(furan-2-yl)-6-oxo-1,6-
dihydropyrimidin-5-carbonitrile (6a)
Yield: 61%; Melting point: 267-269 ºC; IR υmax/cm-1: 3367 (NH), 3032 (CH aromatic), 2993
(CH aliphatic), 2218 (CN), 1658 (C=O). 1H NMR (400 MHz, DMSO-d6) δ 3.40 (dd, 1H, C4-HA
pyrazoline, JAX =4.96 Hz, JAM =18.28 Hz), 3.98 (dd, 1H, C4-HM pyrazoline, JMX = 11.52 Hz, JMA
= 18.28 Hz), 5.77 (dd, 1H, C5-HX pyrazoline, JXA = 4.96, JXM = 11.52), 6.71-8.18 (m, 13H, aromatic
H), 12.27 (s, 1H, NH, exchangeable with D2O). 13C NMR (100 MHz, DMSO-d6) δ 40.20, 62.34,
100.08, 113.05, 113.18, 116.25, 125.56, 125.85, 126.36, 127.04, 127.79, 128.14, 128.59, 129.01,
129.10, 129.14, 129.28, 130.40, 130.95, 134.67, 147.00, 156.94, 157.97, 172.50. Anal. Calcd for
C24H17N5O2 (407.42): C, 70.75; H, 4.21; N, 17.19. Found: C, 70.62; H, 4.36; N, 17.32.
15

4.1.4.2.
2-(5-(4-Chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-(furan-2-yl)-6-oxo-
1,6-dihydropyrimidin-5-carbonitrile (6b)
Yield: 72%; Melting point: >300 ºC; IR υmax/cm-1: 3371 (NH), 3024 (CH aromatic), 2985 (CH
1
aliphatic), 2218 (CN), 1651 (C=O). H NMR (400 MHz, DMSO-d6) δ 3.33 (dd, 1H, C4-HA
pyrazoline, JAX =4.72 Hz, JAM =17.48 Hz), 3.97 (dd, 1H, C4-HM pyrazoline, JMX = 11.52 Hz, JMA
= 17.48 Hz), 5.79 (dd, 1H, C5-HX pyrazoline, JXA = 4.72, JXM = 11.52), 6.73-8.16 (m, 12H, aromatic
H), 12.35 (s, 1H, NH, exchangeable with D2O). 13C NMR (100 MHz, DMSO-d6) δ 21.58, 40.41,
61.55, 82.68, 112.88, 115.31, 127.26, 127.51 , 128.35, 129.01, 129.08, 129.57, 130.64, 131.70,
132.15, 142.41, 146.46, 151.10, 156.92, 172.51. Anal. Calcd for C24H16ClN5O2 (441.87): C, 65.24;
H, 3.65; N, 15.85. Found: C, 64.95; H, 3.56; N, 16.04.
4.1.4.3.
2-(5-(4-Fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-4-(furan-2-yl)-6-oxo-
1,6-dihydropyrimidin-5-carbonitrile (6c)
Yield: 74%; Melting point: >300 ºC; IR υmax/cm-1: 3367 (NH), 3055 (CH aromatic), 2908 (CH
1
aliphatic), 2218 (CN), 1658 (C=O). H NMR (400 MHz, DMSO-d6) δ 3.37 (dd, 1H, C4-HA
pyrazoline, JAX =5.04 Hz, JAM =18.32 Hz), 3.98 (dd, 1H, C4-HM pyrazoline, JMX = 11.68 Hz, JMA
= 18.32 Hz), 5.79 (dd, 1H, C5-HX pyrazoline, JXA = 5.04 Hz, JXM = 11.68 Hz), 6.80-8.17 (m, 12H,
aromatic H), 12.36 (s, 1H, NH, exchangeable with D2O). 13C NMR (100 MHz, DMSO-d6) δ 42.83,
61.85, 82.65, 100.07, 113.39, 115.86, 116.07, 116.50, 117.17, 127.95, 128.21, 128.57, 129.10,
130.92, 131.43, 138.49, 146.11, 150.25, 150.77, 154.06, 156.93, 160.72, 162.15, 163.14. Anal.
Calcd for C24H16FN5O2 (425.41): C, 67.76; H, 3.79; N, 16.46. Found: C, 67.89; H, 3.50; N, 16.25.
4.1.4.4.
4-(Furan-2-yl)-2-(5-(4-methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-6-
oxo-1,6-dihydropyrimidin-5-carbonitrile (6d)
Yield: 68%; Melting point: 236-238 ºC; IR υmax/cm-1: 3363 (NH), 3032 (CH aromatic), 2954
(CH aliphatic), 2214 (CN), 1658 (C=O). 1H NMR (400 MHz, DMSO-d6) δ 3.35 (dd, 1H, C4-HA
pyrazoline, JAX =4.64 Hz, JAM =18.28 Hz), 3.7 (s, 3H, OCH3), 3.95 (dd, 1H, C4-HM pyrazoline,
JMX = 11.36 Hz, JMA = 18.28 Hz), 5.74 (dd, 1H, C5-HX pyrazoline, JXA = 4.64, JXM = 11.36), 6.75-
13
8.18 (m, 12H, aromatic H), 12.31 (s, 1H, NH, exchangeable with D2O). C NMR (100 MHz,
DMSO-d6) δ 40.21, 55.54, 61.99, 82.37, 113.39, 114.51, 114.51, 114.67, 116.53, 126.80, 126.92,
127.66, 127.95, 128.19, 128.56, 130.27, 131.41, 134.21, 147.40, 150.67, 153.91, 156.99, 158.23,
159.16, 162.13. Anal. Calcd for C25H19N5O3 (437.45): C, 68.64; H, 4.38; N, 16.01. Found: C,
68.77; H, 4.20; N, 16.28.
4.1.4.5.
2-(3,5-Diphenyl-4,5-dihydro-1H-pyrazol-1-yl)-6-oxo-4-(thiophen-2-yl)-1,6-
dihydropyrimidin-5-carbonitrile (6e)
Yield: 64%; Melting point: 253-255 ºC; IR υmax/cm-1: 3436 (NH), 3025 (CH aromatic), 2929
(CH aliphatic), 2209 (CN), 1644 (C=O). 1H NMR (400 MHz, DMSO-d6) δ 3.40 (dd, 1H, C4-HA
pyrazoline, JAX =4.44 Hz, JAM =18.36 Hz), 4.01 (dd, 1H, C4-HM pyrazoline, JMX = 11.68 Hz, JMA
= 18.36 Hz), 5.77 (dd, 1H, C5-HX pyrazoline, JXA = 4.44, JXM = 11.68), 7.22-8.47 (m, 13H, aromatic
H), 12.44 (s, 1H, NH, exchangeable with D2O). 13C NMR (100 MHz, DMSO-d6) δ 40.21, 62.85,
82.60, 100.09, 117.78, 125.56, 126.33, 127.69, 128.15, 128.26, 129.13, 129.27, 130.88, 131.18,
131.52, 134.32, 135.62, 140.71, 142.04, 150.28, 158.50, 161.63. Anal. Calcd for C24H17N5OS
(423.49): C, 68.07; H, 4.05; N, 16.54. Found: C, 67.84; H, 3.90; N, 16.33.
16

4.1.4.6.
2-(5-(4-Chlorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-6-oxo-4-(thiophen-2-
yl)-1,6-dihydropyrimidin-5-carbonitrile (6f)
Yield: 73%; Melting point: 232-234 ºC; IR υmax/cm-1: 3433 (NH), 3066 (CH aromatic), 2924
(CH aliphatic), 2210 (CN), 1651 (C=O). 1H NMR (400 MHz, DMSO-d6) δ 3.35 (dd, 1H, C4-HA
pyrazoline, JAX =4.8 Hz, JAM =18.28 Hz), 4.00 (dd, 1H, C4-HM pyrazoline, JMX = 11.52 Hz, JMA
=
18.28 Hz), 5.78 (dd, 1H, C5-HX pyrazoline, JXA = 4.8, JXM = 11.52), 7.22-8.12 (m, 12H, aromatic
H), 13.43 (s, 1H, NH, exchangeable with D2O). 13C NMR (100 MHz, DMSO-d6) δ 40.20, 56.28,
87.29, 100.36, 116.84, 125.57, 127.24, 128.39, 129.27, 129.33, 129.81, 131.97, 135.07, 139.89,
158.91, 161.08, 162.55. Anal. Calcd for C24H16ClN5OS (457.93): C, 62.95; H, 3.52; N, 15.29.
Found: C, 62.74; H, 3.39; N, 15.48.
4.1.4.7.
2-(5-(4-Fluorophenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-6-oxo-4-(thiophen-2-
yl)-1,6-dihydropyrimidin-5-carbonitrile (6g)
Yield: 71%; Melting point: 234-236 ºC; IR υmax/cm-1: 3394 (NH), 3032 (CH aromatic), 2931
(CH aliphatic), 2210 (CN), 1651 (C=O). 1H NMR (400 MHz, DMSO-d6) δ 3.33 (dd, 1H, C4-HA
pyrazoline, JAX =4.72 Hz, JAM =18.32 Hz), 3.99 (dd, 1H, C4-HM pyrazoline, JMX = 11.52 Hz, JMA
= 18.32 Hz), 5.78 (dd, 1H, C5-HX pyrazoline, JXA = 4.72, JXM = 11.52), 7.16-8.12 (m, 12H, aromatic
H), 12.45 (s, 1H, NH, exchangeable with D2O). 13C NMR (100 MHz, DMSO-d6) δ 40.62, 56.28,
87.29, 100.07, 116.03, 116.25, 116.83, 125.55, 127.52, 127.60, 128.33, 129.30, 129.80, 130.06,
131.97, 135.06, 139.89, 158.89, 161.07, 162.54. Calcd for C24H16FN5OS (441.48): C, 65.29; H,
3.65; N, 15.86. Found: C, 65.08; H, 3.50; N, 15.99.
4.1.4.8.
2-(5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-6-oxo-4-(thiophen-
2-yl)-1,6-dihydropyrimidin-5-carbonitrile (6h)
Yield: 64%; Melting point: 263-265 ºC; IR υmax/cm-1: 3421 (NH), 3032 (CH aromatic), 2958
(CH aliphatic), 2210 (CN), 1651 (C=O). 1H NMR (400 MHz, DMSO-d6) δ 3.32 (dd, 1H, C4-HA
pyrazoline, JAX = 4.24 Hz, JAM =18.32 Hz), 3.70 (s, 3H, OCH3), 3.97 (dd, 1H, C4-HM pyrazoline,
JMX = 11.32 Hz, JMA = 18.32 Hz), 5.74 (dd, 1H, C5-HX pyrazoline, JXA = 4.24, JXM = 11.32), 6.89-
13
8.14 (m, 12H, aromatic H), 12.39 (s, 1H, NH, exchangeable with D2O). C NMR (100 MHz,
DMSO-d6) δ 40.62, 55.64, 65.73, 87.10, 114.55, 116.91, 125.53, 126.92, 127.77, 128.11, 128.24,
129.22, 129.84, 130.07, 131.87, 132.01, 135.00, 135.64, 140.03, 152.86, 158.43, 158.59, 159.46,
161.12, 162.58. Anal. Calcd for C25H19N5O2S (453.52): C, 66.21; H, 4.22; N, 15.44. Found: C,
66.37; H, 4.48; N, 15.71.
4.1.5. Synthesis of compounds 7a-d
A solution of 5a-d (4 mmol) in absolute ethanol (20 mL) and Conc. HCl (1 mL) was was
heated under reflux for 12 h. The reaction mixture was cooled, neutralized with conc. Ammonia
solution. The precipitate was filtered and washed with ethanol and crystallized from ethanol.
4.1.5.1.
3-Amino-4-(furan-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6(7H)-thione (7a)
Yield: 78%; Melting point: >300 ºC; IR υmax/cm-1: 3460-3243 (NH, NH2), 3100 (CH
aromatic). 1H NMR (400 MHz, DMSO-d6) δ 5.92 (s, 2H, NH2, exchangeable with D2O), 6.68-8.32
(m, 4H, aromatic 3H and NH pyrimidine, exchangeable with D2O), 12.27 (s, 1H, NH pyrazole,
17

exchangeable with D2O). Anal. Calcd for C9H7N5OS (233.25): C, 46.34; H, 3.02; N, 30.03. Found:
C, 46.50; H, 3.17; N, 30.26.
4.1.5.2.
3-Amino-4-(thiophen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-6(7H)-thione (7b)
Yield: 82%; Melting point: 265-267 ºC; IR υmax/cm-1: 3401-3205 (NH, NH2), 3103 (CH
aromatic). 1H NMR (400 MHz, DMSO-d6) δ 5.38 (s, 2H, NH2, exchangeable with D2O), 6.39 (s,
1H, NH pyrimidine, exchangeable with D2O), 7.27 (m, 1H, thiophene C4-H), 7.87 (dd, 1H,
thiophene C3-H, J = 1.2, 5.1 Hz), 8.34 (d, 1H, thiophene C5-H, J = 3.6 Hz), 12.77 (s, 1H, NH
pyrazole, exchangeable with D2O). Anal. Calcd for C9H7N5S2 (249.32): C, 43.36; H, 2.83; N,
28.09. Found: C, 43.2; H, 3.09; N, 28.41.
4.1.5.3.
4-(Furan-2-yl)-6-(methylthio)-1H-pyrazolo[3,4-d]pyrimidin-3-amine (7c)
Yield: 89%; Melting point: 233-235 ºC; IR υmax/cm-1: 3373-3288 (NH, NH2), 3190 (CH
aromatic), 2924 (CH aliphatic). 1H NMR (400 MHz, DMSO-d6) δ 2.72 (s, 3H, SCH3), 5.84 (s, 2H,
NH2, exchangeable with D2O), 6.80 (dd, 1H, furan C4-H, J = 1.72, 3.52 Hz), 7.49 (m, 1H, furan
C3-H), 8.09 (dd, 1H, furan C5-H, J =0.84, 25 Hz), 12.48 (s, 1H, NH, exchangeable with D2O). 13C
NMR (100 MHz, DMSO-d6) δ 14.10, 98.59, 129.40, 132.30, 132.84, 141.50, 148.51, 154.35,
156.43, 167.97. Anal. Calcd for C10H9N5OS (247.28): C, 48.57; H, 3.67; N, 28.32. Found: C,
48.79; H, 3.81; N, 28.04.
4.1.5.4.
6-(Methylthio)-4-(thiophen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-3-amine (7d)
Yield: 92%; Melting point: 253-255 ºC; IR υmax/cm-1: 3371-3190 (NH, NH2), 3082 (CH
aromatic), 2997 (CH aliphatic). 1H NMR (400 MHz, DMSO-d6) δ 2.56 (s, 3H, SCH3), 5.35 (s, 2H,
NH2, exchangeable with D2O), 7.30 (s, 1H, thiophene C4-H), 7.89 (d, 1H, thiophene C3-H, J =
13
3.52 Hz), 8.34 (s, 1H, thiophene C5-H), 12.63 (s, 1H, NH, exchangeable with D2O). C NMR
(100 MHz, DMSO-d6) δ 14.11, 98.58, 129.39, 132.29, 132.84, 141.50, 154.37, 156.45, 163.19,
167.98. Anal. Calcd for C10H9N5S2 (263.34): C, 45.61; H, 3.44; N, 26.59. Found: C, C, 45.68; H,
3.58; N, 26.87.
4.1.6. Synthesis of compounds 8a-f
To a solution of 7c or 7d (4 mmol) in dry pyridine (10 mL), the appropriate aryl sulfonyl
chloride (4 mmol) was added and heated under reflux for 12 h. The reaction mixture was cooled,
poured into 10% ice cooled HCl. The precipitate was filtered and washed with methanol and
crystallized from methanol.
4.1.6.1.
4-(Furan-2-yl)-6-(methylthio)-1-(phenylsulfonyl)-1H-pyrazolo[3,4-d]pyrimidin-3-
amine (8a)
Yield: 72%; Melting point: 229-231 ºC; IR υmax/cm-1: 3495, 3413 (NH2), 3082 (CH aromatic),
2923 (CH aliphatic), 1383, 1190 (SO2). 1H NMR (300 MHz, DMSO-d6) δ 2.65 (s, 3H, SCH3), 6.60
(s, 2H, NH2, exchangeable with D2O), 6.79-8.15 (m, 8H, aromatic H). 13C NMR (100 MHz,
DMSO-d6) δ 14.40, 100.87, 113.54, 117.75, 127.72, 128.78, 130.15, 135.23, 137.23, 148.89,
149.87, 150.71, 151.50, 158.77, 171.26. Anal. Calcd for C16H13N5O3S2 (387.44): C, 49.60; H,
3.38; N, 18.08. Found: C, 49.51; H, 3.47; N, 18.25.
18

4.1.6.2.
4-(Furan-2-yl)-6-(methylthio)-1-tosyl-1H-pyrazolo[3,4-d]pyrimidin-3-amine (8b)
Yield: 79%; Melting point: 193-195 ºC; IR υmax/cm-1: 3472, 3390 (NH2), 3052 (CH aromatic),
2927 (CH aliphatic), 1387, 1188 (SO2). 1H NMR (300 MHz, DMSO-d6) δ 2.34 (s, 3H, CH3), 2.65
(s, 3H, SCH3), 6.58 (s, 2H, NH2, exchangeable with D2O), 6.79 (dd, 1H, furan C4-H, J = 1.5, 3.3
Hz), 7.40 (d, 2H, aromatic-H, J = 7.8 Hz), 7.55 (dd, 1H, furan C3-H, J = 0.9, 3.6 Hz), 7.78 (d, 2H,
aromatic-H, J = 8.4 Hz), 8.14 (dd, 1H, furan C5-H, J = 1.5, 0.6 Hz). 13C NMR (100 MHz, DMSO-
d6) δ 14.39, 21.58, 100.84, 113.54, 117.73, 127.40, 127.77, 129.79, 130.56, 134.32, 146.08,
148.88, 149.83, 150.72, 151.41, 158.70, 171.18. Anal. Calcd for C17H15N5O3S2 (401.46): C, 50.86;
H, 3.77; N, 17.44. Found: C, 51.18; H, 3.89; N, 17.68.
4.1.6.3.
1-((4-Chlorophenyl)sulfonyl)-4-(furan-2-yl)-6-(methylthio)-1H-pyrazolo[3,4-
d]pyrimidin-3-amine (8c)
Yield: 69%; Melting point: 209-211 ºC; IR υmax/cm-1: 3480-3298 (NH2), 3110 (CH aromatic),
2927 (CH aliphatic), 1388, 1184 (SO2). 1H NMR (300 MHz, DMSO-d6) δ 2.66 (s, 3H, SCH3), 6.60
(s, 2H, NH2, exchangeable with D2O), 6.80-8.16 (m, 7H, aromatic H). 13C NMR (100 MHz,
DMSO-d6) δ 14.21, 101.00, 113.38, 113.56, 117.82, 129.54, 129.90, 135.90, 140.27, 148.94,
149.91, 150.71, 151.72, 156.43, 169.19, 171.38. Anal. Calcd for C16H12ClN5O3S2 (421.88): C,
45.55; H, 2.87; N, 16.60. Found: C, 45.31; H, 3.04; N, 16.89.
4.1.6.4.
6-(Methylthio)-1-(phenylsulfonyl)-4-(thiophen-2-yl)-1H-pyrazolo[3,4-d]pyrimidin-
3-amine (8d)
Yield: 75%; Melting point: 177-179 ºC; IR υmax/cm-1: 3443, 3294 (NH2), 3092 (CH aromatic),
2925 (CH aliphatic), 1373, 1189 (SO2). 1H NMR (300 MHz, DMSO-d6) δ 2.65 (s, 3H, SCH3), 6.0
(s, 2H, NH2, exchangeable with D2O), 7.25-8.11 (m, 8H, aromatic H). 13C NMR (100 MHz,
DMSO-d6) δ 14.30, 102.36, 127.51, 127.81, 129.44, 129.50, 129.71, 130.22, 133.80, 135.34,
137.24, 139.74, 152.24, 155.47, 158.48, 171.14. Anal. Calcd for C16H13N5O2S3 (403.50): C, 47.
63; H, 3.25; N, 17.36. Found: C, 47.44; H, 3.42; N, 17.58.
4.1.6.5.
6-(Methylthio)-4-(thiophen-2-yl)-1-tosyl-1H-pyrazolo[3,4-d]pyrimidin-3-amine (8e)
Yield: 87%; Melting point: 185-187 ºC; IR υmax/cm-1: 3436, 3288 (NH2), 3207 (CH aromatic),
2923 (CH aliphatic), 1383, 1190 (SO2). 1H NMR (300 MHz, DMSO-d6) δ 2.35 (s, 3H, CH3), 2.65
(s, 3H, SCH3), 6.08 (s, 2H, NH2, exchangeable with D2O), 7.26 (dd, 1H, thiophene C4-H, J = 3.9,
4.8 Hz), 7.41 (d, 2H, aromatic-H, J = 8.4 Hz), 7.82 (d, 2H, aromatic-H, J = 8.4 Hz), 7.93 (dd, 1H,
thiophene C3-H, J = 0.9, 4.8 Hz), 8.10 (d, 1H, thiophene C5-H, J = 0.9 Hz). 13C NMR (100 MHz,
DMSO-d6) δ 14.40, 21.60, 102.32, 127.86, 129.39, 129.71, 129.85, 130.62, 133.60, 134.33,
139.76, 141.50, 148.42, 152.13, 155.43, 167.99, 171.06. Anal. Calcd for C17H15N5O2S3 (417.53):
C, 48.90; H, 3.62; N, 16.77. Found: C, 49.13; H, 3.78; N, 17.01.
4.1.6.6.
1-((4-Chlorophenyl)sulfonyl)-6-(methylthio)-4-(thiophen-2-yl)-1H-pyrazolo[3,4-
d]pyrimidin-3-amine (8f)
Yield: 77%; Melting point: 174-176 ºC; IR υmax/cm-1: 3390, 3286 (NH2), 3087 (CH aromatic),
2926 (CH aliphatic), 1384, 1184 (SO2). 1H NMR (300 MHz, DMSO-d6) δ 2.65 (s, 3H, SCH3), 6.1
(s, 2H, NH2, exchangeable with D2O), 7.27-8.11 (m, 7H, aromatic H). 13C NMR (100 MHz,
DMSO-d6) δ 14.26, 102.47, 129.73, 129.90, 130.46, 130.48, 133.67, 133.83, 138.36, 139.70,
19

140.39, 152.46, 155.53, 158.56, 171.26. Anal. Calcd for C16H12ClN5O2S3 (437.95): C, 43.88; H,
2.76; N, 15.99. Found: C, 44.20; H, 2.89; N, 16.24.
4.2.
Biological screening
4.2.1. Preliminary in vitro anticancer screening
The cytotoxicity assays for the synthesized compounds were performed by the National
Cancer Institute (NCI), Germantown MD, USA, under the Development Therapeutic Program
(DTP) on a panel of 60 tumor cell lines at a single high dose of 10 µM. Data were reported as %
growth inhibition of treated cells.
4.2.2. MTT cytotoxicity assay
To determine the IC50 of the tested compounds, MTT assay was adopted. The basic element
is 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT). The assay depends on
mitochondrial dehydrogenase enzyme produced from the viable cells and its ability to open
tetrazolium ring leading to change in color of MTT solution from yellow to purple due to formazan
crystals formation [56]. Cells were seeded at a density of 6000 per well in 96-well microtiter plates
and incubated for 72 h. Cells were treated with five dilutions of the tested compounds (100, 10,
1.0, 0.1 and 0.01 µM) for 72 h. After that, cells were stained with MTT solution (0.5 mg/mL) at
37 ºC. DMSO was added to each well to solubilize the intracellular formazan crystals. Absorbance
was measured using a spectrophotometer at a wavelength of 570 nm using a back ground
absorbance at 690 nm. The cell viability curves were plotted and the IC50 (concentration required
to inhibit 50% of cell growth) values were determined.
4.2.3. Caspase 3/9 assay
Colon (KM12) cancer cells were treated with 5d (1.73 µM) or 7c (1.21 µM). The cells were
trypsinized, rinsed with PBS and centrifuged. Subsequently, the cells were harvested, suspended
in 1mL PBS, frozen at < -20 °C and thawed with gentle mixing. This cycle of freeze/thraw was
repeated for three times and centrifuged for 10 min to purify from cell debris. Caspase 3/9 human
kit is a solid phase sandwich Enzyme Linked Immuno-Sorbent Assay (ELISA). The wells of the
microtiter strips provided were coated with a monoclonal specific antibody for human target
caspases. Samples and unknowns were pipetted into these wells and then a rabbit antibody specific
for human target caspases were added to the wells. Through the first incubation, the human target
caspases 3/9 bind to the immobilized (capture) antibody and the specific active caspase 3/9
antibody served as a detection of antibody by binding to the immobilized active caspase 3/9
proteins. After the first incubation and washing to remove excess protein, a horseradish
peroxidase-labeled anti-Rabbit IgG (anti-rabbit IgG HRP) was added. After a third incubation and
washing to remove all excess anti-rabbit IgG HRP, a substrate solution was added to produce a
colored product as a result of interaction with the bound enzyme. The intensity of the color is
directly proportional to the concentration of human active caspase 3/9 in the original specimen
[57].
20

4.2.4. Evaluation of Bax and Bcl-2 expressions
Total RNA was extracted from at least 1×106 cells using RNeasy Mini-spin column kit
RNeasy RNA extraction kit (Qiagen). A reaction mixture (50 µL) was prepared according to the
following recipe: 2X SYBR® Green RT-PCR Reaction Mix (25 µL); Forward primer (10 μM)
(1.5 µL); Reverse primer (10 μM) (1.5 µL); Nuclease-free H2O; RNA template (1 pg to 100 ng
total RNA); iScript Reverse Transcriptase for One-Step RT-PCR (1 µL).
Amplification was performed using a real-time thermal detection system (Rotorgene) as
follows: cDNA synthesis: 10 min at 50°C; iScript Reverse transcriptase inactivation: 95 °C (5
min); PCR cycling and detection 45 cycles: 95 °C (10 sec); 55 °C (30 sec) then data collection step
and Melt curve analysis [58].
The gene expression fold values were calculated using 2ˆ(-delta delta CT) method for data
analysis of quantitative real time polymerase chain reaction [59]
4.2.5. In vitro Bcl-2 inhibition assay
The Bcl-2 inhibition assay was performed at Confirmatory diagnostic unit, VACSERA, Egypt
using Bcl-2 TR-FRET Assay Kit- BPS Bioscience (Catalog # 50222) and protocol [60]. All
samples and controls were tested in duplicate. 1x BCL TR-FRET Assay Buffer was prepared by
dilution of 3x BCL TR-FRET Assay Buffer (one part) with distilled water (2 parts). Anti-His Tb
labeled donor and Dye labeled acceptor were 100-fold diluted in 1x BCL TR-FRET Assay Buffer.
In each well, 5 μL of both Anti-His Tb labeled donor and Dye labeled acceptor were added. In
“Test inhibitor” labeled wells, 2 μL of inhibitor solution was added while in “negative control”
and “positive control” wells 2 μL of the inhibitor buffer was added.
Bcl-2 peptide ligand was 40 fold diluted using 1x BCL TR-FRET Assay Buffer. 5 μL of the
diluted Bcl-2 peptide ligand was added in positive control and test inhibitor wells while 5 μL of
1x BCL TR-FRET Assay Buffer was added to the negative control wells.
The reaction was initiated by adding 3 μL of diluted Bcl-2 protein to negative control, positive
control and test inhibitor wells. Wells were incubated for 3 hours at room temperature and the
fluorescence intensity was read using microtiter plate reader of TR-FRET.
The activity percentage was calculated as follow:
% Activity = (FRETs - FRETneg / FRETp - FRETneg) ×100%
Where, FRETs = Sample FRET; FRETneg = negative control FRET and FRETp = positive
control FRET.
4.2.6. Cell cycle analysis
The colon (KM12) cancer cell line was exposed to IC50 dose of 5d (1.73 µM) and 7c (1.21
µM) or to DMSO (0.002%) as a control for 24 h. Treated cells then were suspended in 0.5 mL of
PBS, centrifuged, collected and fixed in ice/cold ethanol (70% v/v) for 2h at 4 °C then, they washed
with PBS, suspended using 0.1 mg/mL RNase and stained by 40 mg/mL PI. Flow cytometry
analysis was performed using FACScalibur (Becton Dickinson) and Phoenix Flow Systems and
Verity Software House was used in the cell cycle distributions calculations [61].
4.2.7. Apoptotic cells sub-population determination
The colon (KM12) cancer cells were exposed to IC50 dose of 5d (1.73 µM) and 7c (1.21 µM)
and DMSO (0.002%) as a control for 24h. Treated cells then were suspended in 0.5 mL of PBS,
21

°
centrifuged, collected and fixed in ice/cold ethanol (70% v/v) for 2h at 4 C. Subsequently, the
cells were washed with PBS and centrifuged. The cells were stained using a mixture of fluorescein
isothiocyanate (FITC), annexin V (component no. 51-65875X) and propidium iodide (PI) and
suspended in dark at 37 °C for 30 min. Flow cytometry analysis was performed using FACScalibur
(Becton Dickinson) and Phoenix Flow Systems and Verity Software House was used in the cell
cycle distributions calculations [62].
4.2.8. Molecular docking
AutoDock Vina [63] was used to perform docking while UCSF Chimera [64] was used in the
protein crystal structure preparation [PDB ID: 4AQ3]. Gasteiger charges were applied for the
protein and all ligands. The dimension of the grid box were 20 x 20 x 20 grid points and was
centered on the co-crystallized ligand 398 spacing 0.375.
ACKNOWLEDGEMENTS
The authors would like to acknowledge the gratitude of the National Cancer Institute at
Germantown, US for its assistance in biological screening of chemical compounds.
CONFLICT OF INTEREST
The authors declare that there are no conflicts of interests.
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26

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
27

Synthesis and in vitro investigation of novel cytotoxic pyrimidine and
pyrazolopyrimidne derivatives showing apoptotic effect
Fatma A. Ragab a, Yassin M. Nissan a,b, Emad M. Seif b*, Ahmed Maher c and Reem
K. Arafa d
28

Synthesis and in vitro investigation of novel cytotoxic pyrimidine and
pyrazolopyrimidne derivatives showing apoptotic effect
 A series of novel pyrimidine based compounds have been synthesized and tested for their
in vitro cytotoxic activity against 60 tumor cell lines by NCI.
 5d and 7c (IC50 = 1.73±0.06 & 1.21±0.04 µM, respectively) derivatives were found to be
the most potent derivatives against KM12 cell line with a high selectivity index.
 Derivatives 5d and 7c showed the up-regulation of caspase-3/9 and the pro-apoptotic
factor Bax in KM12 cells.
 The expression of the anti-apoptotic factor Bcl-2, was down-regulated, as well as its
inhibition at a nanomolar concentration.
 The apoptotic effect for derivatives 5d and 7c in KM12 cells was detected using annexin
V-FITC staining method.
29