Design, synthesis, and anticancer evaluation of acetamide and hydrazine analogues of pyrimidine

Article abstract of DOI:10.1002/jhet.3867

A library of acetamide and hydrazine analogues were generated on the pyrimidine ring through a multistep reaction starting from 5-nitro-pyrimidine-4,6-diol and pyrimidine-4,6-diol, respectively. The synthesized analogues were screened for in vitro cytotoxic activity against various human cancer cell lines like HCT-1 and HT-15 (colon), MCF-7(breast), PC-3 (prostrate), SF268 (CNS) using MTT method. From the bioassay results, it was observed that even though many of the synthesized derivatives exhibited a good potency against various screened cancer cell lines, compound 14a from the acetamide series was found to show potent anticancer activity on all the tested cancer cell lines with IC₅₀ value of 0.36μM on CNS cell line and 1.6μM on HT-21 cell line, and compound 19xxi from hydrazine series of pyrimidine showed potent activity against three tested cancer cell lines with IC₅₀ value of 0.76μM on HT-29 cell line, 2.6μM on HCT-15, and 3.2μM on MCF-7 cell line.

Full text of DOI:10.1002/jhet.3867

Received: 27 October 2019
DOI: 10.1002/jhet.3867
Revised: 8 December 2019
Accepted: 11 December 2019
A R T I C L E
Design, synthesis, and anticancer evaluation of acetamide
and hydrazine analogues of pyrimidine
Jabeena Khazir1,5
| Bilal Ahmad Mir2 | Gousia Chashoo3 | Tariq Maqbool4
|
Darren Riley5 | Lynne Pilcher5
1Department of Chemistry, Government
Abstract
Degree College (Boys), Pulwama,
Pulwama, Jammu and Kashmir, India
A library of acetamide and hydrazine analogues were generated on the
pyrimidine ring through a multistep reaction starting from 5-nitro-pyrimidine-
4,6-diol and pyrimidine-4,6-diol, respectively. The synthesized analogues were
screened for in vitro cytotoxic activity against various human cancer cell lines
like HCT-1 and HT-15 (colon), MCF-7(breast), PC-3 (prostrate), SF268 (CNS)
using MTT method. From the bioassay results, it was observed that even
though many of the synthesized derivatives exhibited a good potency against
various screened cancer cell lines, compound 14a from the acetamide series
was found to show potent anticancer activity on all the tested cancer cell lines
with IC50 value of 0.36μM on CNS cell line and 1.6μM on HT-21 cell line, and
compound 19xxi from hydrazine series of pyrimidine showed potent activity
against three tested cancer cell lines with IC50 value of 0.76μM on HT-29 cell
line, 2.6μM on HCT-15, and 3.2μM on MCF-7 cell line.
2Department of Botany, Satellite Campus
Kargil University of Kashmir, Kargil
(Ladakh), Jammu and Kashmir, India
3Cancer Pharmacology Division,
CSIR-Indian Institute of Integrative
Medicine, Jammu, Jammu and Kashmir,
India
4Department of Nanotechnology,
University of Kashmir, Srinagar, Jammu
and Kashmir, India
5Department of Chemistry, University of
Pretoria, Pretoria, South Africa
Correspondence
Jabeena Khazir, Department of
Chemistry, Government Degree College
(Boys) Pulwama, Jammu and Kashmir
192301, India.
Email: jabina.khazir@gmail.com
1 | INTRODUCTION
entities with proven biological properties, nitrogen-
containing heterocyclic compounds are of great impor-
With the continuous increase in mortality rate due to
cancer, it has gradually become one of the most compli-
cated diseases that threatens the human life and is
responsible for an estimated 9.6 million deaths in 2018.[1]
Globally, about one in six deaths is due to cancer.
Although there are many effective therapeutic methods
in clinical use for the control of cancer,[2] chemotherapy
alone or in combination with surgery is commonly the
most efficient method for the treatment of cancer. How-
ever, the use of currently available chemotherapeutic
drugs is limited because of their toxic side effects and
drug resistance.[3] Therefore, over the years, the main
effort in the field of medicinal chemistry has been the
identification of new chemical entities with special char-
acteristics as effective anticancer molecules from both
natural and synthetic sources. Out of diverse chemical
tance as their structural subunits exist in many natural
products such as vitamins, hormones, and antibiotics
because of which they have engrossed significant atten-
tion in the field of medicinal chemistry.[4–6]
Heterocyclic compound pyrimidine occupies a signifi-
cant position in the medicinal world because of its diverse
biological properties. It has been identified as an important
pharmacophore interacting with the synthesis and func-
tions of nucleic acids.[7] Pyrimidine ring is the building
component of DNA and RNA because derivatives of
pyrimidine exhibit diverse pharmacological activities
such as antiviral, antimalarial, antimicrobial, anti-inflam-
matory, and anticancer.[8–13] Among its diverse medicinal
attributes, anticancer activity is most extensively reported.
From the last few decades, several chemotherapeutic
agents have been developed from the reprivatization of the
J Heterocyclic Chem. 2020;1–13.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

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KHAZIR ET AL.
pyrimidine ring and have found wide clinical applications.
Various nucleoside drugs containing pyrimidine nucleus
were synthesized and developed as highly effective anti-
cancer agents. These drugs like cytarabine (1), gemcitabine
(2) are analogues of cytosine and drugs like fluorouracil
(3), floxuridine (4) are analogues of uracil (Figure 1) and
these have been found to demonstrate a range of antineo-
plastic activity in cell and animal models.[14–19] These
agents induce cytotoxicity by acting as antimetabolites,
competing with physiologic pyrimidine nucleosides and,
consequently, interacting with a large number of intracel-
lular targets.[20,21] Their antiproliferative activities is
achieved through incorporation into DNA which leads to
chain termination and finally inhibits the synthesis of
DNA. They can also interfere with enzymes involved in
nucleic acid synthesis, such as DNA polymerases and ribo-
nucleotide reductase.
Because of the wide clinical applications, interest in
pyrimidine derivatives has led to the preparation and eval-
uation of hundreds of such type of molecules as potent
anticancer agents revealing that pyrimidine is an impor-
tant pharmacophore in the discovery of novel active mole-
cules. In view of the above facts and in continuation of
our previous efforts in the field of synthesis of novel anti-
cancer therapeutics,[26,27] the target in this study was to
synthesize analogues having pyrimidine moiety conju-
gated through acetamide and hydrazinyl linkage with dif-
ferent aromatic/heterocyclic groups of documented
cytotoxic potency against several cancer cell lines.
2 | RESULTS AND DISCUSSION
2.1 | Chemistry
Other drugs containing fused pyrimidine ring in the
moiety include epidermal growth factor receptor—
tyrosine kinase inhibitors Gefitinib (5) and Erlotinib (6)
as shown in Figure 1, which are approved for the treat-
ment of nonsmall-cell lung cancer.[22,23] Protein tyrosine
kinase inhibitor like Imatinib (7) (Figure 1) is approved
for the treatment of chronic myeloid leukemia, gastroin-
testinal stromal tumors, and a number of other malignan-
cies.[24] Tegafur (8; Figure 1) is an oral anticancer drug
containing 5-fluorouracil widely used for adjuvant che-
motherapy of colorectal cancer.[25]
The acetamide derivatives of pyrimidine given in Table 1
are synthesized as described in Scheme 1. Synthesis was
started with the regioselective chlorination of hydroxyl
group at C-6 of 5-nitro-pyrimidine-4,6-diol (9) with pho-
sphoxoxyl chloride leading to the formation of 6-chloro-
5-nitro-pyrimidin-4-ol (10). The nitro group of (10) was
reduced to amino group forming 5-amino-6-chloro-
pyrimidin-4-ol (11).The SNAr on C-6 of (11) was effi-
ciently achieved using benzylamine as a nucleophile,
1-butanol as solvent and disopropylethylamine as base
O
O
N
HO
O
HO
N
N
O
O
F
N
NH2
NH2
NH
F
F
HO
N
O
H
HO
1. Cytarabine
OH
3. 5-Fluoro uracil
2. Gemcitabine
F
O
O
HN
Cl
NH
O
HO
N
O
O
N
O
N
HO
4. Floxiuridine
N
F
6. Geftinib
O
HN
N
O
O
O
O
N
NH
O
F
N
O
5. Erlotinib
8. Tegafur
N
O
N
N
N
H
O
H
N
FIGURE 1 Structures of anticancer
drugs containing pyrimidine nucleus
N
7. Imatinib

KHAZIR ET AL.
3
1
followed by refluxing for 12 hours to form 5-amino-
6-benzylamino-pyrimidin-4-ol (12), which was treated
with chloroacetylchloride leading to the substitution of
amino group with chloro acetyl group and the hydroxyl
group at position C-2 was also replaced with a chloro
group to form the N-(4-benzylamino-6-chloro-pyrimidin-
5-yl)-2-chloro-acetamide (13). Finally, the SNAr on C-2 of
(13) was achieved using the cyclic secondary amine in
the presence of 1-butanol as solvent to form the required
acetamide compounds 14(a-g). The compounds were
identified by HNMR, 13C, and mass spectroscopy (MS).
Figure 2 represents the X-ray crystallographic structure
of compound (14a).
The hydrazinyl derivatives of pyrimidine given in
Table 2 are synthesized as described in Scheme 2.
Pyrimidine-4,6-diol (15) was used as a starting material. The
chlorination of two hydroxyl groups located at C-4 and C-6
of starting substrate (15) was done with chlorinating agent
phosphoxoxyl chloride forming 4,6-dichloro-pyrimidine
(16). The SNAr on C-6 of (16) was regioselectively achieved
using hydrazine hydrochloride as a nucleophile and
1-Butanol as solvent followed by refluxing for 4 hours to
form 6-chloro-(pyrimidin-4-yl)-hydrazine (17). Condensa-
tion reaction on the amino group at C-6 of (17) was carried
out with different substituted aldehydes to form analogues
of the type (18). Finally, the SNAr on C-2 of (18) was
achieved using the different cyclic secondary amine in the
presence of triethylamine and THF as solvent to form the
required compounds 19(i-xxviii). The compounds were
identified by 1HNMR, 13C, and MS.
TABLE 1 Acetamide analogues of pyrimidine
R
R
NH
NH
N
O
N
S.NO
R
Yield
14a
85%
N
N
2.2 | Biological activity
14b
14c
90%
92%
N
Acetamide analogues (14a-g) and hydrazine analogues
(19i-xxviii) of pyrimidine were evaluated for in vitro
anticancer activity against a panel of human cancer cell
O
NH
14d
14e
80%
90%
HO
O
N
Et
14f
87%
85%
14g
FIGURE 2 X-ray crystal structure of compound 14a
O
OH
OH
Cl
N
OH
OH
NH2
NH2
Cl
HN
NO2
a
Cl
NO2
N
c
d
N
N
N
N
b
SCHEME 1 Synthesis of
target compounds 14(a-g).
Reagents and conditions:
N
NH
NH
N
N
OH
N
Cl
11
13
95%
9
10
12
90%
85%
O
R
(A) POCl3 (B) Zn, HCOOH, rt,
2 hours; (C) benzyl amine, N-
BuOH, DIPEA, reflux 6 hours;
85%
HN
R
N
e
N
NH
(D) chloroacetyl chloride, DMF,
reflux, 8 hours; (E) substituted
amine, Et3N, reflux, 12 hours
80-95% (gradient)
14(a-g)

4
KHAZIR ET AL.
TABLE 2 Hydrazinyl analogues of pyrimidine
lines such as HCT-1 and HT-15 (colon), MCF-7(breast),
PC-3(prostrate), and SF268 (CNS).Tables 3 and 4 summa-
rizes the inhibitory activity (IC50) values of the analogues
synthesized and the well-known anticancer drugs pacli-
taxel, doxorubicin, and 5-fluorouracil were used as stan-
dard drugs. It was observed from the results of screening
that some of the acetamide and hydrazine analogues of
pyrimidine showed good anticancer activities in lower
μM range against all the human cancer cell lines tested.
Further, it was also observed that while many of the syn-
thesized analogues exhibited potency against colon can-
cer cell lines, compound 14a from acetamide series was
found to show potent anticancer activity on all the tested
cancer cell lines with IC50 value of 0.36μM on CNS cell
line and 1.6μM on HT-21 cell line (Table 3) and com-
pound 19xxi from hydrazine series of pyrimidine showed
potent activity against three tested cancer cell lines with
IC50 value of 0.76μM on HT-29 cell line, 2.6μM on HCT-
15, and 3.2μM on MCF-7 cell line (Table 4).
R'
N
N
NH
N
R
S.No
19i
R
R0
Yield
90%
Piperidine
H
19ii
”
2-OMe
4-OMe
92%
19iii
”
95%
19iv
”
3,4 dimethoxy 90%
19v
”
2 Br
98%
97%
95%
96%
93%
90%
19vi
”
4 Br
19vii
19viii
19ix
”
2 OH
H
Pyrolidine
”
2-OMe
4-OMe
19x
”
19xi
”
3,4 dimethoxy 94%
3 | CONCLUSION
19xii
19xiii
19xiv
19xv
”
2 Br
95%
92%
90%
85%
88%
90%
”
4 Br
Commercially available 5-nitro-pyrimidine-4,6-diol was
used as starting substrate for the synthesis of acetamide
analogues of pyrimidine and pyrimidine-4,6-diol was
used as starting substrate for the synthesis of hydrazine
analogues of pyrimidine. C-4 position of pyrimidine was
substituted with cyclic secondary amines like pyrolidine,
piperidine, and morpholine and (piperidine-4-yl)-pro-
pane-1-one. All the analogues were tested for their cyto-
toxic activity against a panel of four human cancer cell
lines. Many of the synthesized derivatives exhibited
potent cytotoxicity. However, compound 14a from the
acetamide series was found to show potent anticancer
activity on all the tested cancer cell lines with IC50 value
of 0.36μM on CNS cell line and 1.6μM on HT-21 cell line
while compound 19xxi from the hydrazine series of
pyrimidine showed potent activity against three tested
cancer cell lines with IC50 value of 0.76μM on HT-29 cell
line, 2.6μM on HCT-15, and 3.2 μM on MCF-7 cell line.
”
2 OH
H
Morpholine
19xvi
19xvii
19xviii
19xix
19xx
”
”
”
”
”
”
2-OMe
4-OMe
3,4 dimethoxy 95%
2 Br
91%
90%
87%
86%
98%
90%
4 Br
19xxi
19xxii
19xxiii
19xxiv
19xxv
19xxvi
19xxvii
19xxviii
2 OH
H
(Piperidine-4-yl)-propan-l-one
”
”
”
”
”
”
2-OMe
4-OMe
3,4 dimethoxy 95%
2 Br
4 Br
2 OH
92%
87%
90%
Cl
Cl
OH
Cl
N
N
c
N
N
a
b
N
R
N
NH
N
N
NHNH2
N
15
OH
Cl
90%
80%
16
17
18
R'
N
85-95%
SCHEME 2 Synthesis of compounds 19(i-
xxviii). Reagents and conditions: (A) POCl3, rt,
2 hours; (B) NH2NH2.HCl, n-BuOH, reflux at
90ꢀC, 2 hours; (c) substituted aromatic
N
d
R
N
NH
aldehydes, EtOH, THF, reflux at 90ꢀC, 4 hours;
19(i-xxviii) 85-95%
(D) cyclic amines, Et3N, THF, reflux, 12 hours

KHAZIR ET AL.
5
TABLE 3 IC50 values of
acetamide analogues of pyrimidine
against panel of human cancer cell lines
Cancer cell lines
Breast
Colon
Prostrate
PC-3
CNS
SF268
Compound No.
[HCT-15
HT-29]
MCF-7
12.9 0.3
15.6 0.3
41.0 0.25
26.0 0.22
21.2 0.2
>100
14a
14b
14c
14d
14e
14f
10.2 0.25
65.0 0.2
>23.5 0.25
>34.9 0.22
66.7 0.2
>100
1.6 0.2
13.9 0.22
12.0 0.25
19.0 0.30
56.0 0.25
24.5 0.30
54.0 0.23
>100
0.36 0.25
10.5 0.22
85.0 0.30
>100
44.0 0.25
35.6 0.30
54.5 0.36
32.4 0.3
>100
69.8 0.25
>100
14g
10.2 0.25
18.0 0.25
34.5 0.2
88.8 0.22
Note: Results are Mean SD of three independent experiments.
Finally, it was observed that analogues from the acet-
amide series containing the piperidine and pyrolidine
secondary amine base showed better results compared
with the analogues with other amine bases. Analogues
from the hydrazine series with piperidine, pyrolidine,
and morpholine amine bases and with 2-OH, 2-OMe, and
4-OMe substitutions on the benzene ring showed much
better activity on all cancer cell lines than compounds
with other substitution.
concentrated on rotavapor. The residue was purified by
flash chromatography eluted with hexane/ethyl acetate
(8:2 v/v), and compound (10) with (90%) yield was
obtained as yellowish crystals; 1H NMR (400 MHz,
CDCl3): δ 8.81 (s, 1H), 5.02 (s, 1H); 13C NMR (100 MHz,
CDCl3): 139.5, 150.0, 153.2, 170.0; ESI MS: 174.0.
4.2 | Procedure for synthesis of
compound 5-Amino-6-chloro-pyrimidin-
4-ol (11)
4 | EXPERIMENTAL
6-Chloro-5-nitro-pyrimidin-4-ol (1.6 g), taken in a round
bottom flask, was added formic acid in excess as solvent
and then zinc (1 equiv, 4.4 g) was added to it in small por-
tions with continuous stirring. The reaction mixture was
left for stirring at rt for 2 hours. The progress of the reaction
was monitored with TLC and after the completion of reac-
tion, the reaction mixture was quenched with saturated
solution of Na2CO3 and transferred to a separatory funnel
and finally extracted with diethyl ether. The combined
organic layers were dried with anhydrous MgSO4, filtered
and concentrated on rotavapor. The residue was purified by
flash chromatography eluted with hexane/ethyl acetate,
and compound (11) with (85%) yield was obtained as white
crystals; 1H NMR (400 MHz, CDCl3): δ 8.21 (s, 1H), 5.02 (s,
1H), 4.0(s, 2H); 13C NMR (100 MHz,CDCl3): 139.5, 149.0.0,
150.2, 163.0; ESI MS: 145.0.
Buchi melting point apparatus D-545 was used to record
the melting points of the synthesized compounds. Bruker
DPX400 instrument was used to record the NMR spectra
with samples taken in CDCl3. δ (ppm) was used to report
chemical shift values with coupling constants taken in
Hertz. Electrospray ionization MS (ESI-MS) was used to
record mass spectra. Monitoring of reactions was done by
thin-layer chromatography (TLC) on a 2- to 5-cm perco-
lated silica gel 60F254 plates of thickness 0.25 mm
(Merck). Ultraviolet (UV) 254 to 366 nm and iodine were
used to visualize the chromatograms.
4.1 | Procedure for synthesis of
6-chloro-5-nitro-pyrimidin-4-ol (10)
5-Nitro-pyrimidine-4,6-diol (2 g) was taken in a round
bottom flask and DMF was added as a solvent, to it was
added phosphoxychloride (1 equiv, 2.07 g) and kept for
stirring at room temperature (rt) for 2 hours. The progress
of reaction was checked with TLC and after the comple-
tion of reaction, the reaction mixture was quenched with
water and transferred to a separatory funnel and finally
extracted with diethyl ether. The combined organic
layers were dried with anhydrous MgSO4, filtered and
4.3 | Procedure for synthesis of
compound 5-Amino-6-benzylamino-
pyrimidin-4-ol (12)
5-Amino-6-chloro-pyrimidin-4-ol (9) (0.5 g) was taken in a two-
necked, round bottom flask, to which n-Butanol (10 mL) was
added. To the above solution, benzyl amine (1.2 equiv, 0.56 g)
and diisopropylamine (1.5 equiv, 0.37 g) was added, and

6
KHAZIR ET AL.
TABLE 4 IC50 values of hydrazine
analogues of pyrimidine against panel
of human cancer cell lines
Cancer cell lines
Colon
Breast
MCF-7
Prostrate
PC-3
CNS
SF268
Compound No.
19i
[HCT-15
53.2 0.3
85.0 0.25
15.2 0.23
>100
HT-29]
>100
>100
>100
97.5 0.3
>100
19ii
68.3 0.2
>100
>100
>100
19iii
65.0 0.3
37.5 0.2
>100
76 0.20
45.6 0.35
>100
25.5 0.25
>100
19iv
24.0 0.3
>100
19v
>100
>100
19vi
19.8 0.23
16.5 0.26
8.8 0.4
3.9 0.2
2.5 0.2
>100
38.6 0.3
5.6 0.3
68.6 0.2
5.4 0.18
8.4 0.3
>100
22.1 0.2
16.5 0.2
>100
49.7 0.75
14.2 0.3
>100
23.0 0.35
12.0 0.3
>100
19vii
19viii
19ix
11.9 0.11
12.4 0.23
>100
14.6 0.75
>100
21.3 0.21
>100
19x
19xi
>100
>100
19xii
35.0 0.2
>100
26.0 0.3
>!00
28.0 0.30
>100
56.0 0.30
>100
67.0 0.3
>100
19xiii
18xiv
>100
>!00
>100
>100
>100
19xvv
13.2 0.23
37.6 0.4
47.1 0.1
>100
25.6 0.24
37.5 0.22
18 0.5
4.6 0.4
32 0.3
12.3 0.2
0.76 0.75
>100
43.5 0.23
87.8 0.21
18.6 0.4
>100
>100
10.9 0.3
23.4 0.2
12.6 0.2
>100
19xvi
63.7 0.3
19.3 0.33
>100
19xvii
19xviii
19xix
50.6
1
>100
>100
>100
19xx
8.2 0.23
2.6 0.13
>100
15.3 0.23
3.2 0.2
>100
>100
>100
19xxi
7.3 0.13
>100
11.8 0.2
>100
19xxii
19xxiii
19xxiv
19xxv
>100
>100
>!00
>100
>100
12.6 0.3
>100
13.2 0.2
>100
12.5 0.25
>100
49.5 0.32
>100
>100
58.5 0.3
>100
19xxvi
19xxvii
19xxviii
Paclitaxel(1μM)
Doxorubicin(1μM)
5-FU (20μM)
80.7 0.2
>100
61.4 0.3
>100
>100
44.8 0.2
>100
>100
>100
67.5 0.2
—
45.3 0.75
—
>100
>100
>100
—
72
—
—
—
62
—
—
—
58
—
—
—
the reaction mixture was allowed to reflux at 90ꢀC for
12 hours. The progress of the reaction was monitored with
TLC and after the completion of reaction; the reaction
mixture was quenched with water, transferred to a
separatory funnel and extracted with EtOAc. The com-
bined organic layers were dried with anhydrous MgSO4,
filtered and concentrated on rotavapor. The residue was
purified by flash chromatography eluted with hexane/
ethyl acetate, and compound (12) was obtained with 95%
NMR (100 MHz,CDCl3): 57.6, 123.4, 126.0, 127.2, 127.3,
128.3, 128.5, 137.5, 142.4, 152.0, 163.9; ESI MS: 216.0.
4.4 | Procedure for synthesis of N-
(4-Benzylamino-6-chloro-pyrimidin-5-yl)-
2-chloro-acetamide (13)
5-Amino-6-benzylamino-pyrimidin-4-ol (0.5 g) was taken
in a round bottom flask and dissolved in DMF, chloro
acetamide (1.5 equiv, 0.77 g) was added to it in small
1
yield; H NMR (400 MHz, CDCl3): δ 7.1(s, 1H), 7.05 (d,
2H), 7.00 (d, 2H), 6.96(t, 1H), 5.00 (s, 1H), 4.31(s, 2H); 13
C

KHAZIR ET AL.
7
portions at 0ꢀC. The reaction was then left overnight at
rt. The progress of reaction was monitored with TLC.
After completion of reaction, water was added to the
reaction mixture, transferred to a separatory funnel, and
extracted with EtOAc. The combined organic layers were
dried with anhydrous MgSO4, filtered and concentrated
on rotavapor. The residue was purified by flash chroma-
tography eluted with hexane/ethyl acetate, and com-
pound (13) was obtained with 85% yield; 1H NMR
(400 MHz, CDCl3): δ 8.43 (s, 1H), 7.15 (dd, 2H), 7.10 (t,
1H), 7.00 (d, 2H), 4.31(s, 2H), 4.25(s, 2H); 13C NMR
(100 MHz,CDCl3): 46.7, 57.6, 114.5, 126.5127.1, 127.1,
129.3, 129.3, 142.4, 152.0, 163.0,163.9; ESI MS: 310.0.
4.7 | Procedure for synthesis of
compound 6-Chloro-pyrimidin-4-yl)-
hydrazine (17)
4, 6-dichloro-pyrimidine (16) (1.5 g) was taken in a
100-mL round bottom flask to which n-BuOH was
added as a solvent. To this solution, hydrazine hydro-
chloride (1 equiv) was added at 0 to 5ꢀC and the reac-
tion mixture was allowed to reflux at 90ꢀC for 2 hours.
The progress of reaction was checked with TLC and
after the completion of reaction, the reaction mixture
was quenched with water and transferred to
a
separatory funnel and finally extracted with diethyl
ether. The combined organic layers were dried with
anhydrous MgSO4, filtered and concentrated on
rotavapor. The residue was purified by flash chromatog-
raphy eluted with hexane/ethyl acetate, and compound
(17) with (80%) yield was obtained as crystalline solid;
1H NMR (400 MHz, CDCl3): δ 8.32 (s, 1H), 6.78 (s, 1H);
13C NMR (100 MHz, CDCl3): 105.0, 159.0, 161.0, 173.2;
ESI MS: 144.0.
4.5 | General reaction procedure for
synthesis of compounds 14(a-g)
N-(4-benzylamino-6-chloro-pyrimidin-5-yl)-2-chloro-acetamide
(0.5 g) was taken in a 100 mL two-necked RBF. THF
(5 mL) was added to it under inert atmosphere as solvent.
Triethylamine (0.5 equiv, 3.06 g) was added to the above
solution, followed by the addition of cyclic secondary
amine piperidine/pyrolidine (2 equiv) and the reaction mix-
ture was allowed to reflux at 90ꢀC for 12 hours. The
progress of reaction was monitored with TLC. After com-
pletion of reaction, the reaction mixture was concentrated
on rotavapor; water was added to the reaction mixture,
transferred to a separatory funnel and extracted with
EtOAc. The combined organic layers were dried with anhy-
drous MgSO4, filtered and concentrated on rotavapor. The
residue was purified by flash chromatography eluted with
hexane/ethyl acetate, and compound 14(a-g) were obtained
with varying yield.
4.8 | General procedure for synthesis
of compounds of type 18
6-Chloro-pyrimidin-4-yl-hydrazine (1.5 g) was taken in a
100-mL round bottom flask to which EtOH was added as
a solvent. To the solution, various substituted aromatic
aldehydes were added, and the reaction mixture was
allowed to reflux at 90ꢀC for 4 hours. The progress of
reaction was checked with TLC and after the completion
of reaction, the reaction mixture was concentrated on
rotavapor and transferred to a separatory funnel and
finally extracted with ethyl acetate. The combined
organic layers were dried with anhydrous MgSO4, filtered
and concentrated on rotavapor. The residue was purified
by flash chromatography eluted with hexane/ethyl ace-
tate, and compounds of type (18) were obtained with
varying yields.
4.6 | Procedure for synthesis of
compound 4,6-Dichloro-pyrimidine (16)
Pyrimidine-4,6-diol (2 g) was taken in a round bottom
flask and DMF was added as a solvent. Phosphoxychloride
(1 equiv) was added to it and kept for stirring at rt for
2 hours. The progress of reaction was checked with TLC
and after the completion of reaction, the reaction mixture
was quenched with water and transferred to a separatory
funnel and finally extracted with diethyl ether. The com-
bined organic layers were dried with anhydrous MgSO4,
filtered and concentrated on rotavapor. The residue was
purified by flash chromatography eluted with hexane/
ethyl acetate, and compound (16) with (90%) yield was
obtained as crystalline solid; 1H NMR (400 MHz, CDCl3):
δ 9.31 (s, 1H), 8.02 (s, 1H); 13C NMR (100 MHz, CDCl3):
122.0, 159.0, 163.0, 163.2; ESI MS: 147.0.
4.9 | General procedure for synthesis
of hydrazine analogues of pyrimidine
(19i-xxviii)
Compounds (18) (1.2 g) was taken in a 100-mL two-
necked RBF. THF (5 mL) was added to it under inert
atmosphere as solvent. Triethylamine (0.5 equiv) was
added to the above solution, followed by the addition of
cyclic secondary amine piperidine/pyrolidine (2 equiv),
and the reaction mixture was allowed to reflux at 90ꢀC
for 12 hours. The progress of reaction was monitored

8
KHAZIR ET AL.
with TLC. After completion of reaction, the reaction
mixture was concentrated on rotavapor; water was
added to it, transferred to a separatory funnel, and
extracted with EtOAc. The combined organic layers
were dried with anhydrous MgSO4, filtered and concen-
trated on rotavapor. The residue was purified by flash
chromatography eluted with hexane/ethyl acetate, and
compound 19(i-xxviii) were obtained with varying
yields.
5 | COMPOUND
CHARACTERIZATION
5.1 | N-(4-Benzylamino-6-piperidin-1-yl-
pyrimidin-5-yl)-2-piperidin-1-yl-
acetamide (14a)
Crystalline white solid; mp: 149-151ꢀC; 1HNMR
(400 MHz, CDCl3): δ 7.36 (s, 1H), 7.15 (dd, 2H,
J = 6.7 Hz, 7.3 Hz), 7.05-7.00 (m, 3H), 4.30 (s, 2H), 3.25(s,
2H), 2.7-2.65(m, 4H), 2.24-2.21(m, 4H), 1.60-1.53(m,
12H);13C (100 MHz, CDCl3): 23.1,24.9, 25.0, 25.2, 26.2,
26.5, 51.5, 51.9, 54.3, 57.1, 97.0, 126.5, 126.8, 127.5, 128.5,
128.7, 143.2, 151.5, 162.0, 162.5, 169.0; ESI MS: 409 (M+);
Anal. Calcd for C23H32N6O: C,67.65; H, 7.58; Found: C,
66.45; H, 5.54.
4.10 | Cell culture and growth
conditions
A panel of human cancer cell lines—HCT-116 (colon),
HT-29 (colon), MCF-7 (breast), PC-3 (prostate), SF-268
(CNS)—were procured from the US National Cancer
Institute (NCI). Cell lines were grown in tissue culture
flasks in complete growth medium (RPMI-1640) sup-
plemented with 10% fetal bovine serum, 100 μg/mL strep-
tomycin, and 100 units/mL penicillin in carbon dioxide
incubator (New Brunswick, Galaxy 170R, Eppendorf) at
37ꢀC, 5% CO2, and 98% RH.
5.2 | N-(4-Benzylamino-6-pyrrolidin-1-yl-
pyrimidin-5-yl)-2-pyrrolidin-1-yl-
acetamide (14b)
1
White solid; mp: 143-145ꢀC; HNMR (400 MHz, CDCl3):
δ 7.36 (s, 1H), 7.15 (dd, 2H, J = 6.7 Hz, 7.3 Hz), 7.05-7.00
(m, 3H), 4.30 (s, 2H), 3.25(s, 2H), 2.8-2.75(m, 4H),
2.24-2.21(m, 4H), 1.55 (m, 8H);13C (100 MHz, CDCl3):
22.0, 23.1, 24.0, 51.0, 53.9, 57.1, 57.5, 97.0, 126.5, 126.8,
127.5, 128.0, 128.3, 142.2, 150.5, 162.0, 162.5, 169.0; ESI
MS: 381 (M+);Anal. Calcd for C21H28N6O: C, 66.25; H,
7.42; Found: C, 64.25; H, 6.24.
4.11 | Method for sulforhodamine B
assay
For cytotoxicity evaluation, cell suspension of optimum
cell density was seeded in 96 well flat bottom plates
(NUNC). Cell lines were seeded at respective inocula-
tion densities per well as HCT-116 (7500), HT-29 (7500),
MCF-7 (8000), Prostate (7500), SF-268 (10000). Cell sus-
pension of 100 μL was plated. The cells were then
exposed to different concentrations of test materials con-
taining complete growth medium for 24 hours. The
plates were again incubated under the same conditions
for another 48 hours at 37ꢀC. Further, cells were fixed
with ice cold TCA (trichloroacetic acid) for 1 hour at
4ꢀC. After 1 hour, the plates were rinsed three times
with water and allowed to air dry. After drying, 100 μL
of 0.4% sulforhodamine B assay (SRB) dye was added
for half an hour at room temperature. Plates were then
washed three times with 1% vol/vol acetic acid to
remove the unbound SRB. After drying at room temper-
ature, the bound dye was solubilized by adding 100 μL
of 10 mM TRIS (tris [hydroxymethyl]aminomethane)
buffer (pH −10.4) to each well. The plates were kept on
the shaker for 5 minutes to solubilize the protein-bound
dye. Finally, OD was taken at 540 nm in a microplate
reader (Thermo Scientific). IC50 was determined by plot-
ting OD against concentration using graph pad prism
software.
5.3 | N-(4-Benzylamino-6-morpholin-
4-yl-pyrimidin-5-yl)-2-morpholin-4-yl-
acetamide (14c)
1
White solid; mp: 145-146ꢀC; HNMR (400 MHz, CDCl3):
δ 7.36 (s, 1H), 7.15 (dd, 2H, J = 6.7 Hz, 7.3 Hz), 7.05-7.00
(m, 3H), 4.32 (s, 2H), 3.65-3.50 (m, 8H), 3.25 (s, 2H),
2.9-2.85 (m, 4H), 2.34-2.29 (m, 4H);13C (100 MHz,
CDCl3): 55.9, 57.1, 57.5, 60.0, 70.1, 71.0, 71.5, 97.0, 126.5,
127.5, 128.0, 142.2, 150.5, 162.0, 162.5, 169.0; ESI MS:
413 (M+);Anal. Calcd for C21H28N6O3: C, 61.25; H, 6.82;
Found: C, 60.25; H, 6.24.
5.4 | N-[4-Benzylamino-6-(2-hydroxy-
ethylamino)-pyrimidin-5-yl]-2-(2-hydroxy-
ethylamino)-acetamide (14d)
1
White solid; mp: 152-154ꢀC; HNMR (400 MHz, CDCl3):
δ 7.36 (s, 1H), 7.15 (dd, 2H, J = 6.7 Hz, 7.3 Hz),
7.05-7.00 (m, 3H), 4.32 (s, 2H), 3.70 (t, 2H), 3.65(t, 2H),

KHAZIR ET AL.
9
3.25(t, 2H), 2.70 (t, 2H); 13C (100 MHz, CDCl3): 51.0,
53.5, 54.0, 57.1, 64.0, 64.5, 70.1, 97.0, 126.5, 127.0, 128.0,
142.2, 150.5, 162.0, 162.5; ESI MS: 361 (M+);Anal.
Calcd for C17H24N6O3: C,56.25; H, 6.72; Found: C,
55.25; H, 6.24.
5.8 | N-Benzylidene-N0-(6-piperidin-1-yl-
pyrimidin-4-yl)-hydrazine (19i)
1
White solid; mp: 126-127ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.60 (d, 2H, J = 6.8 Hz), 7.55 (s, 1H),
7.32-7.30(m, 3H), 5.55(s, 1H), 2.80-2.75(m, 4H), 1.50-1.47
(m, 6H); 13C (100 MHz, CDCl3): 25.0, 25.2, 25.7, 53.7,
54.5, 92.0, 127.9, 128.2, 129.0, 130.3, 131.2, 154.5, 155.9,
170.0; ESI MS: 282.0 (M+);Anal. Calcd for C16H19N5: C,
68.30; H, 6.81; Found: C, 67.25; H, 6.34.
5.5 | N-[4-Benzylamino-6-(4-propionyl-
piperidin-1-yl)-pyrimidin-5-yl]-
2-(4-propionyl-piperidin-1-yl)-
acetamide (14e)
White solid; mp: 155-156ꢀC; 1HNMR (400 MHz,
CDCl3): δ 7.36 (s, 1H), 7.15 (dd, 2H, J = 6.7 Hz,
7.3 Hz), 7.05-7.00 (m, 3H), 4.32 (s, 2H), 3.25 (s, 2H),
2.70 (m, 4H), 2.55-2.49 (m, 4H), 2.40-2.35 (m, 2H), 2.26
(m, 4H), 1.80-1.75 (m, 8H), 1.06 (t, 6H);13C (100 MHz,
CDCl3): 8.0, 8.5, 23.0, 23.5, 23.9, 30.9, 31.5, 45.3, 45.5,
48.5, 48.9, 52.0, 55.0, 57.1, 97.0, 126.5, 127.0, 128.0,
128.3, 150.5, 162.0, 162.5. 168.2, 210.5; ESI MS:
521 (M+);Anal. Calcd for C29H40N6O3: C,66.90; H, 7.74;
Found: C, 65.25; H, 7.24.
5.9 | N-(2-methoxy-benzylidene)-N0-
(6-piperidin-1-yl-pyrimidin-4-yl)-
hydrazine (19ii)
1
White solid; mp: 116-118ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.60 (d, 2H, J = 7.8 Hz), 7.55 (s, 1H), 7.50
(d, 1H, J = 7.7 Hz), 7.2 (dd, 2H, J = 6.7 Hz, 5.4 Hz), 6.80
(m, 2H), 5.55 (s, 1H), 3.75(s, 3H), 2.80-2.75(m, 4H),
1.50-1.47 (m, 6H); 13C (100 MHz, CDCl3): 25.0, 25.2, 25.7,
53.7, 54.5, 56.0, 92.0, 114.5, 122.0, 130.3, 131.2, 154.5,
155.9, 162.5, 170.0; ESI MS: 313.0 (M+);Anal. Calcd for
C17H21N5O: C,65.57; H, 6.81; Found: C, 65.25; H, 6.34.
5.6 | N-[4-Benzylamino-6-(1-phenyl-
ethylamino)-pyrimidin-5-yl]-2-(1-phenyl-
ethylamino)-acetamide (14f)
5.10 | N-(4-methoxy-benzylidene)-N0-
(6-piperidin-1-yl-pyrimidin-4-yl)-hydrazine
(19iii)
1
White solid; mp: 143-145ꢀC; HNMR (400 MHz, CDCl3):
δ 7.36 (s, 1H), 7.20-7.17 (m, 4H), 7.15 (dd, 2H,
J = 6.7 Hz, 7.3 Hz), 7.12 (m, 4H), 7.08 (t, 2H), 7.07 (t,
2H), 7.06 (d, 2H), 4.32 (s, 2H), 4.00 (m, 2H), 1.35 (d,
6H); 13C (100 MHz, CDCl3): 23.0, 23.5,52.0, 56.0, 57.1,
97.0, 126.0, 126.5, 127.0, 127.5, 128.0, 128.3, 137.5, 142.6,
150.5, 162.0, 162.5. 168.2, 210.5; ESI MS: 482.0 (M+);
Anal. Calcd for C29H32N6O: C,72.47; H, 6.74; Found: C,
70.25; H, 5.34.
1
White solid; mp: 122-124ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.55 (s, 1H), 7.50 (d, 2H, J = 7.7 Hz), 6.80
(d, 2H, J = 5.7 Hz), 5.55 (s, 1H), 3.75 (s, 3H), 2.80-2.75
(m, 4H), 1.50-1.47 (m, 6H); 13C (100 MHz, CDCl3): 25.0,
25.2, 25.7, 53.7, 54.5, 56.0, 92.0, 114.5, 122.0, 130.3, 131.2,
154.5, 155.9, 164.5, 170.0; ESI MS: 313.0 (M+);Anal. Calcd
for C17H21N5O: C, 65.57; H, 6.81; Found: C, 65.25;
H, 6.34.
5.7 | N-[4-Benzylamino-6-(1-phenyl-
ethylamino)-pyrimidin-5-yl]-2-(1-phenyl-
ethylamino)-acetamide (14g)
5.11 | N-(3,4-Dimethoxy-benzylidene)-N0-
(6-piperidin-1-yl-pyrimidin-4-yl)-
hydrazine (19iv)
1
White solid; mp: 148-149ꢀC; HNMR (400 MHz, CDCl3):
1
δ 7.36 (s, 1H), 7.20-7.17 (m, 4H), 7.15 (dd, 2H, J = 6.7 Hz,
7.3 Hz), 7.12 (m, 4H), 7.08 (t, 2H), 7.07 (t, 2H), 7.06 (d,
2H, J = 6.4 Hz), 4.32 (s, 2H), 4.00 (m, 2H), 1.35 (d, 6H,
J = 3.4 Hz); 13C (100 MHz, CDCl3): 23.0, 23.5, 52.0, 56.0,
57.1, 97.0, 126.0, 126.5, 127.0, 127.5, 128.0, 128.3, 137.5,
142.6, 150.5, 162.0, 162.5. 168.2, 210.5; ESI MS: 482.0
(M+);Anal. Calcd for C29H32N6O: C, 72.47; H, 6.74;
Found: C, 70.25; H, 5.34.
White solid; mp: 118-120ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.55 (s, 1H), 7.10 (d, 1H, J = 7.1 Hz), 7.00
(s, 1H), 6.80 (d, 1H, J = 5.7 Hz), 5.55 (s, 1H), 3.73(s, 6H),
2.80-2.75 (m, 4H), 1.50-1.47 (m, 6H); 13C (100 MHz,
CDCl3): 25.0, 25.2, 25.7, 53.7, 54.5, 56.0, 56.1, 92.0, 115.5,
122.0, 124.5, 147.5, 149.0, 154.5, 170.0; ESI MS: 342.0
(M+);Anal. Calcd for C18H23N5O2: C, 63.57; H, 6.79;
Found: C, 62.25; H, 6.14.

10
KHAZIR ET AL.
5.12 | N-(2-Bromo-benzylidene)-N0-
(6-piperidin-1-yl-pyrimidin-4-yl)-
hydrazine (19v)
1H), 6.8 (d, 1H J = 5.7 Hz), 5.52 (s, 1H), 3.73(s, 3H),
2.80-2.75(t, 4H), 1.56(t, 4H); 13C (100 MHz, CDCl3): 23.0,
52.5, 56.5, 92.0, 113.5, 117.5, 120.0,130.0, 131.5,
154.7156.0, 162.5, 170.0; ESI MS: 298.0 (M+);Anal. Calcd
for C16H19N5O: C, 64.63; H, 6.42; Found: C, 62.76;
H, 5.92.
1
White solid; mp: 120-122ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.55 (s, 1H), 7.50 (d, 2H, J = 7.8 Hz),
7.20 (d, 2H, J = 7.0 Hz), 5.55 (s, 1H), 2.80-2.75 (m, 4H),
1.50-1.47 (m, 8H); 13C (100 MHz, CDCl3): 25.0, 25.2,
25.7, 53.7, 54.5, 92.0, 123.0, 127.6131.2, 131.9133.0, 134.5,
154.5156.0, 170.0; ESI MS: 360.0 (M+);Anal. Calcd for
C16H18BrN5: C, 53.34; H, 5.04; Found: C, 52.25; H, 4.54.
5.17 | N-(4-Methoxy-benzylidene)-N0-
(6-pyrrolidin-1-yl-pyrimidin-4-yl)-
hydrazine (19x)
1
White solid; mp: 114-115ꢀC; HNMR (400 MHz, CDCl3):
5.13 | N-(4-Bromo-benzylidene)-N0-(6-
piperidin-1-yl-pyrimidin-4-yl)-hydrazine (19vi)
δ 8.10 (s, 1H), 7.52 (s,1H), 7.50 (d, 2H, J = 7.8 Hz), 6.8(d,
2H, J = 5.7 Hz), 5.52 (s, 1H), 3.73(s, 3H), 2.80-2.75 (t,
4H), 1.56 (t, 4H); 13C (100 MHz, CDCl3): 23.0, 52.5, 56.5,
92.0, 113.5, 123.5, 130.0, 154.7156.0, 164.5, 170.0; ESI MS:
298.0 (M+); Anal. Calcd for C16H19N5O: C, 64.63; H, 6.42;
Found: C, 62.76; H, 5.92.
1
White solid; mp: 123-124ꢀC; HNMR (400 MHz, CDCl3): δ
8.10 (s, 1H), 7.55 (s, 1H), 7.50-7.45 (m, 4H), 5.55 (s, 1H),
2.80-2.75(m, 4H), 1.50-1.47 (m, 8H); 13C (100 MHz, CDCl3):
25.0, 25.2, 25.7, 53.7, 54.5, 92.0, 123.0,130.2, 131.5, 131.7,
131.9, 156.2, 170.0; ESI MS: 360.0 (M+);Anal. Calcd for
C16H18BrN5: C, 53.34; H, 5.04; Found: C, 52.25; H, 4.54.
5.18 | N-(3,4-dimethoxy-benzylidene)-N0-
(6-pyrrolidin-1-yl-pyrimidin-4-yl)-
hydrazine (19xi)
5.14 | 2-[(6-Piperidin-1-yl-pyrimidin-
4-yl)-hydrazonomethyl]-phenol (19vii)
1
White solid; mp: 131-133ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.52 (s,1H), 7.15 (d, 1H, J = 6.7 Hz), 7.00
(s, 1H), 6.73(d, 1H, J = 5.7 Hz), 5.52 (s, 1H), 3.73 (s, 6H),
2.80-2.75 (t, 4H), 1.56 (t, 4H); 13C (100 MHz, CDCl3):
23.0, 52.5, 56.5, 92.0, 115.5, 115.8, 121.5, 124.5,
154.7156.0, 170.0; ESI MS: 328.0 (M+);Anal. Calcd for
C17H21N5O2: C,62.37; H, 6.47; Found: C, 62.06; H, 5.63.
1
White solid; mp: 128-129ꢀC; HNMR (400 MHz, CDCl3): δ
8.10 (s, 1H), 7.55 (s, 1H), 7.40 (d, 1H, J = 7.7 Hz), 7.10 (dd,
1H, J = 7.5 Hz, 6.8 Hz), 6.80 (m, 2H), 5.55 (s, 1H), 2.80-2.75
(m, 4H), 1.50-1.47 (m, 8H); 13C (100 MHz, CDCl3): 25.0, 25.2,
25.7, 53.7, 54.5, 92.0, 115.5, 117.9, 121.5, 130.2, 133.0,
154.5156.0, 157.8, 170.0; ESI MS: 298.0 (M+);Anal. Calcd for
C16H19N5O: C, 64.63; H, 6.44; Found: C, 62.25; H, 5.74.
5.19 | N-(2-bromo-benzylidene)-N0-
(6-pyrrolidin-1-yl-pyrimidin-4-yl)-hydrazine
(19xii)
5.15 | N-Benzylidene-N0-(6-pyrrolidin-
1-yl-pyrimidin-4-yl)-hydrazine (19viii)
1
White solid; mp: 113-115ꢀC; HNMR (400 MHz, CDCl3):
1
White solid; mp: 124-125ꢀC; HNMR (400 MHz, CDCl3): δ
δ 8.10 (s, 1H), 7.52 (s,1H), 7.50 (d, 2H, J = 7.7 Hz), 7.20
(dd, 2H, J = 5.4 Hz, 6.2 Hz), 5.52 (s, 1H), 2.80-2.75 (t,
4H), 1.56 (t, 4H); 13C (100 MHz, CDCl3): 23.0, 52.5, 92.0,
123.5, 126.0, 130.0, 131.9, 133.2, 134.5, 154.7156.0, 170.0;
ESI MS: 347(M+); Anal. Calcd for C15H16BrN5:
C,52.05; H, 4.67; Found: C, 51.06; H, 4.63.
8.10 (s, 1H), 7.60 (d, 2H, J = 6.7 Hz), 7.52 (s,1H), 7.3 (m, 3H),
5.52 (s, 1H), 2.80-2.75 (t, 4H), 1.56 (t, 4H); 13C (100 MHz,
CDCl3): 23.0, 52.5, 92.0, 127.5, 129.0, 130.0, 131.0, 154.7156.0,
169.5, 170.0; ESI MS: 268.0 (M+);Anal. Calcd for
C15H17N5: C, 67.39; H, 6.42; Found: C, 65.12; H, 5.74.
5.16 | N-(2-Methoxy-benzylidene)-N0-(6-
pyrrolidin-1-yl-pyrimidin-4-yl)-
hydrazine (19ix)
5.20 | N-(4-bromo-benzylidene)-N0-
(6-pyrrolidin-1-yl-pyrimidin-4-yl)-hydrazine
(19xiii)
1
1
White solid; mp: 128-130ꢀC; HNMR (400 MHz, CDCl3):
White solid; mp: 128-129ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.52 (s,1H), 7.50 (d, 1H, J = 6.7 Hz), 7.25(t,
δ 8.10 (s, 1H), 7.52 (m, 4H), 5.52 (s, 1H), 2.80-2.75 (t, 4H),

KHAZIR ET AL.
11
1.56 (t, 4H); 13C (100 MHz, CDCl3): 23.0, 52.5, 92.0,
125.5, 130.0, 131.2, 131.9, 133.2, 134.5, 154.7156.0, 170.0;
ESI MS: 347 (M+);Anal. Calcd for C15H16BrN5: C,
52.05; H, 4.67; Found: C, 51.06; H, 4.63.
114.5, 116.0, 123.0, 130.0, 131.5, 154.5, 156.5, 162.0, 170.0;
ESI MS: 315.0 (M+);Anal. Calcd for C16H19N5O2: C,
61.33; H, 6.11; Found: C, 60.12; H, 6.04.
5.25 | N-(3,4-Dimethoxy-benzylidene)-N0-
(6-morpholin-4-yl-pyrimidin-4-yl)-
hydrazine (19xviii)
5.21 | 2-[(6-Pyrrolidin-1-yl-pyrimidin-
4-yl)-hydrazonomethyl]-phenol (19xiv)
1
1
White solid; mp: 126-127ꢀC; HNMR (400 MHz, CDCl3):
White solid; mp: 116-118ꢀC; HNMR (400 MHz, CDCl3): δ
δ 8.10 (s, 1H), 7.52 (s,1H), 7.40 (d, 1H, J = 6.7 Hz), 6.75
(dd, 2H, J = 6.7 Hz, 5.3 Hz), 7.10 (dd, 1H, J = 6.8 Hz,
6.0 Hz), 5.52 (s, 1H), 2.80-2.75(t, 4H), 1.56 (t, 4H); 13C
(100 MHz, CDCl3): 23.0, 52.5, 92.0, 115.5, 118.5121.0,
130.0, 132.2, 131.9, 154.7156.0, 170.0; ESI MS: 284(M+);
Anal. Calcd for C15H17N5O: C,63.59; H, 6.05; Found: C,
62.15; H, 5.73.
8.10 (s, 1H), 7.52 (s,1H), 7.10 (d, 1H, J = 6.3 Hz), 7.00 (s,
1H), 6.80 (d, 1H, J = 5.7 Hz), 5.52 (s, 1H), 3.70 (s, 6H), 3.67
(m, 4H), 2.80-2.75 (m, 4H); 13C (100 MHz, CDCl3): 55.5,
58.9, 71.0, 92.0, 114.5, 116.0, 123.0, 124.5, 147.5, 149.0,
154.5, 156.5170.0; ESI MS: 344.0 (M+); Anal. Calcd for
C17H21N5O3: C, 59.96; H, 6.16; Found: C, 57.12; H, 6.04.
5.26 | N-(2-Bromo-benzylidene)-N0-
(6-morpholin-4-yl-pyrimidin-4-yl)-
hydrazine (19xix)
5.22 | N-Benzylidene-N0-(6-morpholin-
4-yl-pyrimidin-4-yl)-hydrazine (19xv)
1
1
White solid; mp: 132-133ꢀC; HNMR (400 MHz, CDCl3):
White solid; mp: 122-123ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.65 (d, 2H, J = 5.7 Hz), 7.52 (s, 1H), 7.30
(m, 3H), 5.55 (s, 1H), 3.67(m, 4H), 2.80-2.75(m, 4H); 13C
(100 MHz, CDCl3): 38.4, 57.6, 71.0, 109.0, 112.5, 118.5,
121.7, 126.0, 127.0, 128.0, 129.3, 134.5, 138.5, 162.0, 170.0;
ESI MS: 280.0 (M+);Anal. Calcd for C19H21NO: C,
81.68; H, 7.58; Found: C, 80.25; H, 7.04.
δ 8.10 (s, 1H), 7.52 (s,1H), 7.50 (d, 2H, J = 6.3 Hz), 7.20
(dd, 2H, J = 5.7 H z, 7.8 Hz), 5.52 (s, 1H), 3.67 (m, 4H),
2.80-2.75(m, 4H); 13C (100 MHz, CDCl3): 58.9, 71.0, 92.0,
123.0, 127.5, 131.5, 133.0, 134.5, 154.5, 156.5170.0; ESI
MS: 362.0 (M+);Anal. Calcd for C15H16BrN5O: C,
49.74; H, 4.45; Found: C, 47.72; H, 4.04.
5.23 | N-(2-Methoxy-benzylidene)-N0-
(6-morpholin-4-yl-pyrimidin-4-yl)-
hydrazine (19xvi)
5.27 | N-(4-Bromo-benzylidene)-N0-
(6-morpholin-4-yl-pyrimidin-4-yl)-
hydrazine (19xx)
1
1
White solid; mp: 121-123ꢀC; HNMR (400 MHz, CDCl3):
White solid; mp: 116-118ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.52 (s,1H), 7.50 (d, 1H, J = 6.3 Hz), 7.20
(dd, 1H, J = 5.6 Hz, 6.4 Hz), 6.80 (m, 2H), 5.52 (s, 1H),
3.67(m, 4H),2.80-2.75 (m, 4H); 13C (100 MHz, CDCl3):
55.5, 58.9, 71.0, 92.0, 114.5, 116.0, 120.0, 130.0, 131.5,
154.5, 156.5, 162.0, 170.0; ESI MS: 315.0 (M+);Anal. Calcd
for C16H19N5O2: C, 61.33; H, 6.11; Found: C,
60.12; H, 6.04.
δ 8.10 (s, 1H), 7.52 (s,1H), 7.50 (m, 4H), 5.52 (s, 1H), 3.67
(m, 4H),2.80-2.75 (m, 4H); 13C (100 MHz, CDCl3): 58.9,
71.0, 92.0, 125.0, 130.0, 131.5, 154.5, 156.5170.0; ESI MS:
362.0 (M+);Anal. Calcd for C15H16BrN5O: C, 49.74; H,
4.45; Found: C, 47.72; H, 4.04.
5.28 | 2-[(6-Morpholin-4-yl-pyrimidin-
4-yl)-hydrazonomethyl]-phenol (19xxi)
5.24 | N-(4-Methoxy-benzylidene)-N0-
(6-morpholin-4-yl-pyrimidin-4-yl)-
hydrazine (19xvii)
White solid; mp: 110-111ꢀC; 1HNMR (400 MHz, CDCl3): δ
8.10 (s, 1H), 7.52 (s,1H), 7.40 (d, 1H, J = 7.8 Hz), 7.1(t,
1H), 6.8 (dd, 1H, J = 3.4 Hz, 5.2 Hz), 5.52 (s, 1H), 3.67(m,
4H), 2.80-2.75(m, 4H); 13C (100 MHz, CDCl3): 58.9, 71.0,
92.0, 115.8, 118.0, 121.0, 130.0, 132.2, 154.5, 156.5, 157.5,
170.0; ESI MS: 300.0 (M+);Anal. Calcd for C15H17N5O2: C,
60.19; H, 5.72; Found: C, 58.42; H, 5.04.
1
White solid; mp: 118-120ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.52 (s,1H), 7.50 (d, 1H, J = 7.3 Hz), 6.80
(d, 2H, J = 5.4 Hz), 5.52 (s, 1H), 3.67 (m, 4H), 2.80-2.75
(m, 4H); 13C (100 MHz, CDCl3): 55.5, 58.9, 71.0, 92.0,

12
KHAZIR ET AL.
5.29 | 1-{1-[6-(N0-Benzylidene-
hydrazino)-pyrimidin-4-yl]-piperidin-4-yl}-
propan-1-one (19xxii)
1H), 1.00 (t, 3H); 13C (100 MHz, CDCl3): 7.1, 23.0, 23.5,
30.5, 45.0, 52.1, 56.5, 92.0, 114.5, 115.0, 122.0, 124.5,
147.0, 148.0, 154.0, 156.0, 170.0, 170.3, 211.0; ESI MS: 398
(M+);Anal. Calcd for C21H27N5O3: C, 63.47; H, 6.87;
Found: C, 62.09; H, 6.83.
White crystalline solid; mp: 112-114ꢀC; 1HNMR
(400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.60 (d, 2H,
J = 6.3 Hz), 7.52 (s,1H), 7.30 (m, 3H), 5.52 (s, 1H), 2.70 (t,
4H), 2.45 (q, 2H), 2.36-2.30 (m, 1H), 1.00 (t, 3H); 13C
(100 MHz, CDCl3): 7.1, 23.0, 23.5, 30.5, 45.0, 52.1, 52.7,
92.0, 128.0, 128.3, 129.0, 130.5, 131.2, 154.0, 156.0, 170.0,
170.3, 211.0; ESI MS: 338(M+);Anal. Calcd for
C19H23N5O: C, 67.63; H, 6.87; Found: C, 65.15; H, 6.13.
5.33 | 1-(1-{6-[N0-(2-Bromo-benzylidene)-
hydrazino]-pyrimidin-4-yl}-piperidin-4-yl)-
propan-1-one (19xxvi)
White crystalline solid; mp: 108-110ꢀC; 1HNMR
(400 MHz, CDCl3): δ 8.10 (s, 1H), 7.52 (s,1H), 7.50 (d, 2H,
J = 7.7 Hz), 7.20 (dd, 2H, J = 4.5 Hz, 5.8 Hz), 5.52 (s, 1H),
2.70 (t, 4H), 2.45 (q, 2H), 2.36-2.30 (m, 1H), 1.00 (t, 3H);
13C (100 MHz, CDCl3): 7.1, 23.0, 23.5, 30.5, 45.0, 52.1,
92.0, 123.5, 127.0, 130.5, 131.5, 133.0, 154.0, 156.0, 170.0,
170.3, 211.0; ESI MS: 416(M+);Anal. Calcd for
C19H22BrN5O: C, 54.82; H, 5.33; Found: C, 53.09; H, 5.13.
5.30 | 1-(1-{6-[N0-(2-Methoxy-
benzylidene)-hydrazino]-pyrimidin-4-yl}-
piperidin-4-yl)-propan-1-one (19xxiii)
Yellow solid; mp: 124-126ꢀC; 1HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.55 (d, 1H, J = 5.7 Hz), 7.52 (s,1H), 7.20
(dd, 1H, J = 5.4 Hz, 6.0 Hz), 5.52 (s, 1H), 3.72 (s, 3H),
2.70 (t, 4H), 2.45 (q, 2H), 2.36-2.30 (m, 1H), 1.00 (t, 3H);
13C (100 MHz, CDCl3): 7.1, 23.0, 23.5, 30.5, 45.0, 52.1,
52.7, 56.5, 92.0, 114.5, 115.9, 120.5, 130.5, 131.2, 154.0,
156.0, 162.5, 170.0, 170.3, 211.0; ESI MS: 368(M+);Anal.
Calcd for C20H25N5O2: C, 65.37; H, 6.87; Found: C,
65.09; H, 5.73.
5.34 | 1-(1-{6-[N0-(4-Bromo-benzylidene)-
hydrazino]-pyrimidin-4-yl}-piperidin-4-yl)-
propan-1-one (19xxvii)
1
White solid; mp: 114-115ꢀC; HNMR (400 MHz, CDCl3):
δ 8.10 (s, 1H), 7.52 (s,1H), 7.50-7.43 (m, 4H), 5.52 (s, 1H),
2.70 (t, 4H), 2.45 (q, 2H), 2.36-2.30 (m, 1H), 1.00 (t, 3H);
13C (100 MHz, CDCl3): 7.1, 23.0, 23.5, 30.5, 45.0, 52.1,
92.0, 123.5, 127.0, 130.5, 131.5, 131.9, 133.0, 154.0, 156.0,
170.0, 170.3, 211.0; ESI MS: 416(M+); Anal. Calcd for
C19H22BrN5O: C, 54.82; H, 5.33; Found: C, 53.09; H, 5.13.
5.31 | 1-(1-{6-[N0-(4-Methoxy-
benzylidene)-hydrazino]-pyrimidin-4-yl}-
piperidin-4-yl)-propan-1-one (19xxiv)
Pale yellow solid; mp: 118-120ꢀC; 1HNMR (400 MHz,
CDCl3): δ 8.10 (s, 1H), 7.55 (d, 1H, J = 6.3 Hz), 7.52
(s,1H), 6.75 (d, 2H, J = 5.8 Hz), 5.52 (s, 1H), 3.72(s, 3H),
2.70 (t, 4H), 2.45 (q, 2H), 2.36-2.30 (m, 1H), 1.00 (t, 3H);
13C (100 MHz, CDCl3): 7.1, 23.0, 23.5, 30.5, 45.0, 52.1,
56.5, 92.0, 114.5, 124.5, 130.5, 154.0, 156.0, 170.0, 170.3,
211.0; ESI MS: 368(M+);Anal. Calcd for C20H25N5O2: C,
65.37; H, 6.87; Found: C, 65.09; H, 5.73.
5.35 | 1-(1-{6-[N0-(2-Hydroxy-
benzylidene)-hydrazino]-pyrimidin-4-yl}-
piperidin-4-yl)-propan-1-one (19xxviii)
White crystalline solid; mp: 123-124ꢀC; 1HNMR
(400 MHz, CDCl3): δ 8.10 (s, 1H), 7.52 (s,1H), 7.43 (d, 1H,
J = 8.3 Hz), 7.10 (dd, 1H, 6.8 Hz, 5.3 Hz), 6.73(dd, 2H,
7.8 Hz, 6.3 Hz), 5.52 (s, 1H), 2.70 (t, 4H), 2.45 (q, 2H),
2.36-2.30 (m, 1H), 1.00 (t, 3H); 13C (100 MHz, CDCl3):
7.1, 23.0, 23.5, 45.0, 52.0, 52.1, 92.0, 115.0, 117.5, 120.5,
130.5, 131.5, 132.9, 154.0, 156.0, 170.0, 170.3, 211.0; ESI
MS: 354(M+);Anal. Calcd for C19H23N5O2: C, 64.82; H,
6.56; Found: C, 63.39; H, 6.19.
5.32 | 1-(1-{6-[N0-(3,4-Dimethoxy-
benzylidene)-hydrazino]-pyrimidin-4-yl}-
piperidin-4-yl)-propan-1-one (19xxv)
Pale yellow solid; mp: 123-124ꢀC; 1HNMR (400 MHz,
CDCl3): δ 8.10 (s, 1H), 7.52 (s,1H), 7.10 (d, 1H,
J = 6.8 Hz), 7.00 (s, 1H), 6.75 (d, 1H, J = 5.7 Hz), 5.52 (s,
1H), 3.72 (s, 6H), 2.70 (t, 4H), 2.45 (q, 2H), 2.36-2.30 (m,
ORCID
Jabeena Khazir https://orcid.org/0000-0002-6578-1157
Lynne Pilcher https://orcid.org/0000-0003-3382-8536

KHAZIR ET AL.
13
[17] U. N. Desai, K. P. Shah, S. H. Mirza, D. K. Panchal,
S. K. Parikh, R. M. Rawal, J Cancer Res Ther 2015, 11, 352.
[18] S. Kimura, K. Kuramoto, J. Homan, H. Naruoka, T. Ego,
M. Nogawa, S. Sugahara, H. Naito, Anticancer Res. 2012, 32, 795.
[19] E. Jabbour, I. Jean-Pierre, G. Garcia-Manero, H Kantarjian.
Cancer. 2008, 112, 2341.
[20] W. B. Parker, Chem Rev 2009, 109, 2880.
[21] Z. Nikolova, B. Peng, M. Hubert, Cancer Chem Pharmacol.
2004, 53, 433.
REFERENCES
[1] https://www.who.int/news-room/fact-sheets/detail/cancer
[2] M. Arruebo, N. Vilaboa, B. S. Gutierrez, J. Lambea, A. Tres,
ꢀ
M. Valladares, A. G. Fernández, Cancers (Basel) 2011, 3, 3279.
[3] C. Chun. 2019 MPH What you need to know about chemo-
therapy
https://www.medical
newstoday.com/articles/
158401.php
[4] T. Atul, IJTAS. 2016, 8(1), 53–4.
[5] P. Arora, V. Arora, H. S. Lamba, D. Wadhwa, IJPSR. 2012, 3,
[22] G Pizzorno, D Pharm, B. R Diasio, Y. C Cheng. Holland-Frei
Cancer Medicine. 6th edition. Hamilton (ON): BC Decker;
2003. ISBN-10: 1–55009–213-8
2947.
[6] A. Al-Mulla, Der Pharma Chemica 2017, 9, 141.
[23] M. Wolf, H. Swaisland, S. Averbuch, Clin Cancer Res. 2004, 10,
[7] S. B. Patil, IJPSR. 2018, 9, 44.
4607.
[8] De. E Clercq. Targets for the Design of Antiviral Agents.1984,
73, 203–230.
[9] K. Singh, H. Kaur, P. Smith, D. C. Kock, K. Chibale,
[24] P. Pandey, H. Dureja, Cancer Ther Rev. 2017, 13, 3.
[25] S. Kobuchi, Y. Ito, D. Takamatsu, T. Sakaeda, Eur J Pharm Sci.
2018, 123, 452.
J. Balzarini, J Med Chem 2014, 57, 435.
[26] J. Khazir, I. Hyder, J. L. Gayatri, L. P. Gayatri, L. P. Yandrati,
N. Nalla, G. Chashoo, A. Mahajan, A. K. Saxena, M. S. Alam,
G. N. Qazi, H. M. S. Kumar, Eur J Med Chem 2014, 82, 255–62.
[27] J. Khazir, P. P. Singh, D. M. Reddy, I. Hyder, S. Shafi,
S. D. Sawant, G. Chashoo, A. Mahajan, M. S. Alam,
A. K. Saxena, S. Aravinda, B. D. Gupta, H. M. S. Kumar, Eur J
Med Chem 2013, 83, 279–89.
[10] B. Chandrasekaran, S. Cherukupalli, S. Karunanidhi,
A. Kajee, R. R. Aleti, N. Sayyad, B. Kushwaha, S. R. Merugu,
K. P. Mlisana, J Mol Structure 2019, 1183, 246.
[11] M. Amir, S. A. Javed, H. Kumar, Ind J Pharm Scie 2007,
69, 337.
[12] C. M. Galmarini, J. R. Mackey, C. Dumontet, Lancet Oncol
2002, 3, 415–24.
[13] C. M. Galmarini, L. Jordheim, C. Dumontet, D. Charles, Exp
Rev Anticancer Therapy 2003, 3, 717.
How to cite this article: Khazir J, Mir BA,
Chashoo G, Maqbool T, Riley D, Pilcher L. Design,
synthesis, and anticancer evaluation of acetamide
and hydrazine analogues of pyrimidine.
J Heterocyclic Chem. 2020;1–13. https://doi.org/10.
1002/jhet.3867
[14] D. B. Longley, D. P. Harkin, P. G. Johnston, Nat Rev Cancer
2003, 3, 330–8.
[15] D. G. Power, N. E. Kemeny, Mol Cancer Ther 2009, 8, 17.
[16] R. Okita, D. Wolf, K. Yasuda, A. Maeda, T. Yukawa, S. Saisho,
K. Shimizu, Y. Yamaguchi, M. Oka, E. Nakayama,
A. Lundqvist, R. Kiessling, B. Seliger, M. Nakata, PLoS One.
2015, 10, e0139809.