Efficient synthesis and molecular docking studies of new pyrimidine-chromeno hybrid derivatives as potential antiproliferative agents

Article abstract of DOI:10.1080/00397911.2021.1922920

Various novel heterocyclic compounds containing pyrimidine nuclei 5H-chromeno[4,3-d]pyrimidine (4a–c, e–h, l–r, t) and pyrimidine-5-yl-(2-hydroxyphenyl)methanone (5a, c, d, f–k, m–o, r, s, u) were synthesized from the reaction of guanylhydrazones (2a–u) a

Full text of DOI:10.1080/00397911.2021.1922920

Synthetic Communications
An International Journal for Rapid Communication of Synthetic Organic
Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/lsyc20
Efficient synthesis and molecular docking studies
of new pyrimidine-chromeno hybrid derivatives as
potential antiproliferative agents
Sevtap Çağlar Yavuz, Senem Akkoç, Burak Tüzün, Onur Şahin & Emin
Saripinar
To cite this article: Sevtap Çağlar Yavuz, Senem Akkoç, Burak Tüzün, Onur Şahin & Emin
Saripinar (2021) Efficient synthesis and molecular docking studies of new pyrimidine-chromeno
hybrid derivatives as potential antiproliferative agents, Synthetic Communications, 51:14,
2135-2159, DOI: 10.1080/00397911.2021.1922920
To link to this article: https://doi.org/10.1080/00397911.2021.1922920
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SYNTHETIC COMMUNICATIONSV
2021, VOL. 51, NO. 14, 2135–2159
https://doi.org/10.1080/00397911.2021.1922920
Efficient synthesis and molecular docking studies of new
pyrimidine-chromeno hybrid derivatives as potential
antiproliferative agents
d
e
Sevtap C¸aglar Yavuza,b, Senem Akkoc¸c , Burak Tuzun , Onur S¸ahin , and
ꢀ
€ €
Emin Saripinara
b
aDepartment of Chemistry, Faculty of Science, Erciyes University, Kayseri, Turkey; Department of
c
Veterinary Science, S¸efaatli Vocational School, Yozgat Bozok University, Yozgat, Turkey; Department of
€
Basic Pharmaceutical Sciences, Faculty of Pharmacy, Suleyman Demirel University, Isparta, Turkey;
e
dDepartment of Chemistry, Faculty of Science, Sivas Cumhuriyet University, Sivas, Turkey; Scientific and
Technological Research Application and Research Center, Sinop University, Sinop, Turkey
ABSTRACT
ARTICLE HISTORY
Received 28 February 2021
Various novel heterocyclic compounds containing pyrimidine nuclei
5H-chromeno[4,3-d]pyrimidine (4a–c, e–h, l–r, t) and pyrimidine-5-yl-(2-
hydroxyphenyl)methanone (5a, c, d, f–k, m–o, r, s, u) were synthesized
from the reaction of guanylhydrazones (2a–u) and 3-formylchromone
(3). These compounds were tested against human liver hepatocellular
carcinoma cell line (HepG2) and human breast adenocarcinoma cell
line (MDA-MB-231) using the MTT assay method. Furthermore, molecu-
lar docking calculations were performed to compare the biological
activities of various novel heterocyclic compounds against cancer pro-
teins. In these calculations, the protein used are crystal structure of the
BRCT repeat region from the breast cancer associated protein, 1JNX,
crystal structure of VEGFR kinase (liver cancer) protein, 3WZE, and crys-
tal structure of an allosteric Eya2 phosphates inhibitor (lung cancer)
protein, 5ZMA, respectively. After molecular docking calculations,
absorption, distribution, metabolism, and excretion/toxicity analysis was
performed to examine the properties of various novel heterocyclic
compounds for their future use as drugs.
KEYWORDS
Antiproliferative activity;
HepG2; MDA-MB-231;
pyrimidine; molecu-
lar docking
GRAPHICAL ABSTRACT
CONTACT Emin Saripinar
emin@erciyes.edu.tr
Department of Chemistry, Faculty of Science, Erciyes University,
Kayseri, Turkey.
Supplemental data for this article can be accessed on the publisher’s website.
ß 2021 Taylor & Francis Group, LLC

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S. Ç. YAVUZ ET AL.
Introduction
Although newly introduced drugs today have significantly increased the success of treat-
ment in many cancer types, nevertheless, there is still a need to develop more effective
drugs for the treatment of breast, colon, liver, and many types of cancer. Heterocyclic
compounds containing electron-rich nitrogen atoms are of great importance in this
medicinal field.[1]
Synthetic drugs containing the pyrimidine nucleus, which is a structural compo-
nent of critical drugs, are currently used in the clinics for treatments. These drugs
have a very important role to play in many diseases.[2–5] Heterocyclic chemistry
includes at least half of all organic chemistry research in the world. In particular,
heterocyclic structures are the basis of many pharmaceutical, agricultural, and veter-
inary products forms. Heterocyclic compounds exhibit a high degree of structural
diversity and wide therapeutic agents and play an effective role economically.
Pyrimidine pharmacophore is an important and integral part of DNA and RNA[6]
and plays an important role in biological processes.Due to its high activity, it has an
outstanding recognition in organic and medicinal chemistry. Pyrimidine is the
nucleus structural component[7] of critical drugs such as fluorouracil, etravirine, ris-
peridone, iclaprim, avanafil, and rosuvastatin. Because of the wide range of biological
activities of pyrimidines (anti-inflammatory, COX inhibitor, anticancer, antiallergic,
analgesic, etc.) attention is drawn to the wide range broad class of compounds.[8]
Because of their biological activities, they form an important group in the chemical
compounds. Pyrimidine derivatives are known to be used in epilepsy,[9] measles dis-
ease treatments,[10] germs,[11] parasitic killers,[12] cancer chemotherapy,[13] and hepa-
titis B virus inhibitor.[14]
Chromones, heterocyclic compounds containing benzene ring condensed to the pyran
ring is a large family of compounds, some of which are used as medicaments.
Chromones are an important class of natural and synthetic compounds with a broad
spectrum of pharmacological activity such as anti-inflammatory, antibacterial, antitu-
mor, anti-HIV, antioxidant, antiallergic, antiviral,[15–19] antifungal, antiulcer, immunosti-
mulator, biological killer, wound curative, inflammation preventive, and
antimicrobial.[20–33] 3-Formylchromone is a very active reagent and owing to the active
centers in its structure, it has been reported to interact easily with the molecule.
Chemical of substituted chromones reactivity depends on the nature and reaction con-
ditions of the functional group present in the third position.
Among three-functionalized chromones, 3-formyl derivatives are widely used in het-
erocyclic synthesis. 3-Formylchromones provide a new pharmacophore for the treat-
ment of diabetes (type II) and obesity as well as several tumor cells.[34] Biological
activities could be as a result of the presence of an acting recipient of Michael a,
b-unsaturated reactive aldehyde.[35–37]
3-Formylchromone (I) has three electrophilic centers. The first of these is carbonyl
center C-4, the second is the formyl group in C, and the third is carbon with high
reactive electrophilic properties of unsaturated C-2 carbon atoms.[38] In this case, the
reaction of (I) with some reagents results in different yields leading to the formation of
product mixtures.[39] As reported by Lowe, the reaction of 3-formylchromone with

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SYNTHETIC COMMUNICATIONSV
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formamidine, 5-(2-hydroxy benzoyl) pyrimidine (R ¼ H, 13%) and 5-hydroxy-5H-chro-
meno[4,3-d]pyrimidine (R ¼ H, 31%) mixtures were given with different C form substi-
tuted formamidine (R is alkyl, aryl, hetaryl, NH2, NHCN, SH, SMe, OH, OMe, 1-
pyrrolidinyl, or 1-piperidinyl, 4-morpholinyl) reaction with 26–90% yielded chromeno-
pyrimidine.[40–42] Thus, 3-formylchromones can be used with 1,3-bifunctional nucleo-
philes which are amidine derivatives as a good precursor for the synthesis of pyrimidine
derivatives. Based on the experiment of Bruno et al., Vilsmeier-Haack[43,44] started from
the use of hydroxyacetophenone with (POCl3 in DMF) to produce 3-formylchromone,
2-cycloamino-5-hydroxy-5H-[1]benzopyrano[4,3-d]pyrimidine intermediates to give pyr-
rolidine-1-carboxamidine hydrochloride, piperidine-1-carboxamidine sulfate or morpho-
line-4-carboxamidine hydrochloride (S-starting from methylisothioure sulfate and
suitable amines) were used for the experiment. Subsequently, the hemiacetalic hydroxy
at position 5 of the benzopyrano[4,3-d] pyrimidine system group was replaced with
[42].
appropriate amines in the presence of TiCl4.
When many studies conducted in recent years are examined, the results of experi-
mental and theoretical studies are seen together.[45–48] It has been observed that the
work done in this way is of higher quality and realistic. Because it has been observed
that the results obtained support each other, thus increasing the accuracy of the results.
In these studies, it was observed that the numerical values of the results obtained from
experimental and theoretical studies were quite close to each other. Therefore, when
theoretical calculations are made before experimental studies, it has been seen that time
will be an important guide for experimental studies. In this direction, it is possible to
synthesize more effective and active molecules with theoretical calculations.
In theoretical calculations, molecular docking is the best method to compare the bio-
logical activities of molecules against enzymes.[49] In the calculations made by molecular
docking method, theoretical biological activity values of molecules against enzymes are
found. It is possible to compare the biological activities of other molecules with the
numerical values obtained because of the calculation. Many parameters are obtained in
calculations made new pyrimidine-chromeno hybrid derivatives against enzymes.
It provides important information about the biological activities of molecules with
the parameters obtained because of these calculations. After these calculations, new pyr-
imidine-chromeno hybrid derivatives absorption, distribution, metabolism, excretion
/Toxicity(ADME/T) analysis was performed.[50–53] With the ADME/T analysis, the
effects and reactions of drug molecules in human metabolism in cells and tissues are
tried to be predicted theoretically. These effects and responses are tried to be predicted
by numerical values of parameters found by ADME/T calculations in molecular docking
calculations. The numerical value of each parameter obtained gives important informa-
tion about the action and reaction of molecules in different organs or tissues. These
results give the properties of the molecule to be used as a drug in the future.
Guanylhydrazones known as amidinohydrazones, with oxo compounds (aldehyde/
ketone), were obtained from the reaction of aminoguanidine. These compounds are antipro-
tozoal, antibacterial, antimalarial, tripanosidal, antisecretory, antidiuretic, anticoagulant,
antihypertensive, antiviral, and possess anticancer activities. Other than medical importance,
it could also be used for the synthesis of important heterocyclic compounds. The importance
of both 3-formylchromone and guanylhydrazones was also considered in our reactions. In a

2138
S. Ç. YAVUZ ET AL.
O
R1
NH
NH
. HNO3
H2N
+
R1
NH2
R
(1a-u)
R
N
N
HN
H2O, NaOH
N
R1
O
O
N
NH2
NH2
N
H
O
OH
+
EtOH/reflux
O
4a-c, e-h, l-r, t
R
O
(2a-u)
3
N
N
OH
N
N
H
R1
R
5a, c, d, f, g-k, m-o, r, s, u
Scheme 1. A synthesis scheme of compound 5H-chromeno[4,3, d]pyrimidine (4a–c, e–h, 1–r, t) and
pyrimidine-5-yl-(2-hydroxyphenyl)methanone (5a, c, d, f, g–k, m–o, r, s, u) derivatives.
literature review, some pyrimidine-chromeno hybrid heterocyclic compounds containing
nuclei have been shown to exhibit biological activity. Due to the active centers in the 3-for-
mylchromone structure, it is known that the reactivity against nucleophilic substances is
quite high and it interacts easily with many molecules. These compounds are versatile sub-
strates that can be used as raw materials for synthetic drug synthesis. Due to above men-
tioned properties of these compounds, new molecules having pyrimidine-chromeno
structure were synthesized and characterized. The antiproliferative activities of these synthe-
sized compounds were studied as in vitro.
Results and discussion
Although numerous pyrimidine-chromeno compound derivatives have been introduced
to the literature up to today, there is still a need to synthesize novel compound deriva-
tives containing the pyrimidine nucleus in view of the broad biological properties of
these compounds.
In many structure-activity studies, it has been found that substitution with donor/
electron-withdrawing groups on aryl rings or binding of aryl/heteroaryl groups to the
functional structure has a significant effect on anticancer activity. Designed molecules
were synthesized, as shown in Scheme 1.
Pyrimidine-5-yl-(2-hydroxyphenyl)methanone and 5H-chromeno[4,3-d]pyrimidine-
derived compounds were synthesized. A product mixture was obtained from the reac-
tion of 3 and 2a-u derivatives. Because of compound 3 having three electrophilic cen-
ters, it mainly produces two products after synthesis. This may result in different yields
in a reaction when mixed with different reagents. When the reaction scheme is exam-
ined, it will be seen that 3 and 2a–u reaction will result in the formation of a product

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Table 1. Some characteristic data and properties of synthesized compounds.
Melting point
(oC)
1H NMR(OH)
(d, ppm)
IR(C¼O)
Yield
(%)
Entry
Compound
R
R1
(cmꢀ1
)
Color
1
2
3
4
5
6
7
8
4a
5a
4b
4c
5c
5d
4e
4f
4-CH3
4-CH3
H
2-Cl
2-Cl
4-CH3O
H
4-NO2
4-NO2
4-Cl
H
H
H
H
H
H
CH3
H
H
H
H
H
H
H
H
H
CH3
H
H
H
H
CH3
CH3
H
H
H
251–253
213–215
256–258
260–262
221–223
243–245
217–218
269–271
260–262
243–245
208–210
221–223
231–233
209–211
168–170
233–234
234–245
198–201
212–213
238–240
245–247
233–235
219–221
226–228
249–251
235–237
184–186
185–187
225–227
11.37
11.89
11.45
11.70
12.15
11.84
10.27
11.83
11.27
11.56
12.01
11.11
11.67
11.90
11.81
12.12
10.14
11.25
11.77
11.67
12.14
11.33
10.98
11.61
11.78
12.06
12.01
11.26
11.99
Yellow
Yellow
Cream
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Cream
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Cream
Yellow
Yellow
11
82
70
68
26
71
85
14
70
21
65
12
80
83
91
69
94
13
82
13
67
10
78
72
66
13
77
59
73
1600.8
1590.9
1594.3
9
5f
1614.2
1615.0
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
4g
5g
4h
5h
5i
5j
5k
4l
4m
5m
4n
5n
4o
5o
4p
4r
4-Cl
4-N(CH3)2
4-N(CH3)2
4-CH(CH3)2
4-CH3CH2O
2-Cl-6-F
4-CH3O
2,4-diCH3O
2,4-diCH3O
4-CF3
4-CF3
4-Cl
4-Cl
2,6-diF
2,4-diCl
2,4-diCl
4-F
3,4-diCH3O
3-Br
1655.2
1582.6
1597.5
1574.3
1599.7
1579.6
1600.8
5r
5s
4t
1599.6
1602.0
H
H
H
5u
1568.1
or product mixture. In this type of experiment, normally two types of products are
formed. But in some syntheses, only one product is formed, and second products are
not formed or could not be fully purified. The resulting products have been represented
in the form of “4” and “5”. They were represented as 4 and 5 because of the change in
the original skeletal structure of the compound. Products marked with “4” refers to pyr-
imidine compound derivatives 5H-chromeno[4,3,d], while products designated with “5”
refers to compound derivatives of pyrimidine-5-yl-(2-hydroxyphenyl)methanone. 4a–c,
e–h, l–r, t and 5a, c, d, f, g–k, m–o, r, s, u was obtained in yields of 11–94%. All spec-
tral data, with the structure of compounds 4a–c, e–h, l–r, t and 5a, c, d, f, g–k, m–o, r,
s, u is consistent. Selected properties of compounds 4a–c, e–h, l–r, t and 5a, c, d, f,
g–k, m–o, r, s, u is given in Table 1.
Compounds represented by “4” contain chromene, while compounds represented
1
by “5” contain C¼O. Thus, the H NMR and 13C NMR spectra of the synthesized
compounds show different chemical shift values. The exception of alkyl groups dem-
onstrated similar structures and characteristic signals that are common. As shown in
Scheme 1, guanylhydrazone derivatives (2a–u) obtained by reaction of oxo com-
pounds (aldehyde/ketone; 1a–u) with aminoguanidine reacted with 3. The diversity
in the series results from the presence of electron donor-withdrawing groups of sub-
stituents attached to aromatic rings present in 1a–u. In general, the yields were sat-
isfactory. A substituent electron donor group attached to the ring was observed to
be more efficient in reactions. First, the reaction product obtained as a single

2140
S. Ç. YAVUZ ET AL.
product and that of two different products 4l and 5d base on the structure of the
compounds is explained in detail based on spectroscopic data. After obtaining a sin-
gle crystal of 4l and 5d compounds after the synthesis, X-ray single-crystal studies
were also carried out.
When the Fourier transform- infrared (FT-IR) spectrum of compound 4 l was exam-
ined, NH, C–H, C¼N, and C–O groups with typical frequency values of 3305.3,
1
3053.4–2842.5, 1602.8–1581.2, and 1551.8–1510.5 cmꢀ1 were observed. When the H
NMR spectra of compound 4I were examined, whereas the proton of the OH group
located on the fifth position carbon of the chromeno ring showed a characteristic singlet
signal of d ¼ 10.14 ppm, the signal of H which is bound to the same position on the car-
bon showed d ¼ 6.46–6.47 ppm due to geminal interaction of protons which showed a
doublet signal. This peak was seen specifically in the compound derivatives represented
by “4”. The proton on the -NH group showed a signal of d ¼ 7.61 ppm. In the presence
of –OH and –NH protons after the addition of deuterium oxide (D2O), the intensity of
the peaks of their protons was seen to decrease or disappear because of displacement
1
with deuterium. When the displacement of H NMR D2O spectrum was observed, the
peaks of the protons of OH and NH of 4b and 4e (d4b: 11.45 and 7.63 ppm; d4e: 10.27
and 7.63 ppm) were seen to disappear (in Figure S8 and Figure S22). Protons bound to
carbons in the aromatic ring (Ar-H), showed a signal between d: 8.51–6.98 ppm. Singlet
peaks of CH3O– and –CH3 protons are obtained at d¼ 3.80 ppm and d¼ 2.39 ppm,
respectively. When the 13C NMR spectrum of compound 4l was examined, the carbon
(OCHOHC) on the fifth position of the chromene ring was seen to have a signal of
d¼ 91.27 ppm by giving a characteristic signal of the structure illumination. The pres-
ence of carbon atoms (Ar-C) in the aromatic region was seen with signals d:
161.36–114.13 ppm. The peaks of the CH3O– and –CH3 carbons are obtained at
d¼ 55.64 ppm and d¼ 14.04 ppm, respectively.
When the FT-IR spectrum of compound 5d was examined, NH, C-H, C¼O, and the
C¼N groups were observed at 3227.8, 3111.7–2972.7, 1594.3, and 1532.6–1512.6 cmꢀ1
.
It was observed that the frequency displayed absorption bands showing vibration peaks
1
confirming the structure of the substance. When the H NMR spectra of the compound
5d were examined, the –OH group bound to the aromatic ring showed a characteristic
singlet signal at d ¼ 11.84 ppm, while the –NH nitrogen-dependent proton showed the
spectrum of the group at a characteristic singlet signal at d ¼ 10.43 ppm. Equivalent pro-
tons bound to carbons in the pyrimidine ring (NCHCCHNC) showed d ¼ 8.70 ppm at
very close chemical shift values as a singlet signal. The proton of the imine
(CCHNNHC) group showed a characteristic sharp singlet peak around 8.19 ppm. The
protons bonded to carbons in the aromatic ring (Ar-H) were signaled in the range of
6.94–7.66 ppm. The methoxy (CH3Oꢀ) group connected to the aromatic ring showed a
singlet peak at 3.79 ppm.
1
When the H NMR D2O change spectrum was examined, ꢀOH and ꢀNH proton
peaks (d5d: 11.84 and 10.43 ppm) after the addition of D2O proton it was observed
that the intensity of the peaks decreased because of displacement with deuterium
(Fig. S18). Thus, the chemical shift value of –OH and –NH proton peaks were clari-
fied. When the 13C NMR spectra of compound 5d were examined, the guanylhydra-
zone derivative of compound 3, C ¼ O group, formed by ring-opening during the

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Scheme 2. Possible reaction mechanism.
reaction was observed to be d ¼ 192.87 ppm. The carbon groups present in the pyr-
imidine ring derived from the guanylhydrazone derivative imine (CCHNNHC) and
the presence of carbon atoms (Ar-C) in the aromatic region, showed peaks between
d ¼ 161.02 and 114.78 ppm. The signal of the carbon in the CH3O- group was
observed at d ¼ 55.73 ppm.
In the literature, while the proton at the fifth position of the benzopyran[4,3-d] pyr-
imidine system gives a peak between d¼ 6.39 and 6.55 ppm, the chemical shift value was
observed to be between d¼ 6.43 and 6.51 ppm. While the signal of the fourth position
proton on the structure was showing 8.10–8.55 ppm from experimental results however
the signal observed was between d ¼ 8.26 and 8.55 ppm. Characterization data were
found to be in harmony with the product symbolized as “4” in our study. Likewise, the
characteristic spectra of 5-(2-hydroxybenzoyl)pyrimidine derivatives, which are repre-
sented as 5 are consistent with the data in the literature.[40]
3-Formylchromone, which has three electrophilic centers, particularly high electro-
philic unsaturated C-2 carbon atom opens the ring by attacking the nucleophilic guanyl-
hydrazone amine groups. The known Michael addition reaction mechanism observed
on the a,b-unsaturated carbonyl compounds resulting in the splitting of the ring can be
carried out in two ways. This condition, however, has led to the formation of product
mixtures. The possible reaction mechanism is given in Scheme 2.
Cytotoxic activity studies
The synthesized compounds containing pyrimidine-chromeno nucleus were tested
toward two different cancer cell lines using MTT assay method. The results obtained
are given in Table 2.
Two series of heterocyclic organic compounds were tested against human breast
adenocarcinoma cell line (MDA-MB-231) and human liver hepatocellular carcinoma
cell line (HepG2) cell lines at concentrations ranging from 5–200 mM for 48 h. The
structure-activity relationship (SAR) studies of the 5H-chromeno[4,3-d]pyrimidin-5-ol
(4) and pyrimidine-5-yl-(2-hydroxyphenyl)methanone (5) demonstrate that cytotoxic
activities of these compounds are mostly dependent on the position of R group, which

2142
S. Ç. YAVUZ ET AL.
Table 2. IC50 results of compounds against MDA-MB-231and
HepG2 cells.
IC50 (mM)
Compounds
MDA-MB-231
HepG2
4a
5a
4b
4c
5c
5d
4e
4f
5f
4g
5g
4h
5h
5i
5j
5k
4l
4m
5m
4n
5n
4o
5o
4p
4r
5r
5s
4t
>200
>200
>200
65.21
>200
88.54
119.30
>200
>200
>200
155.20
152.00
198.00
96.66
163.80
>200
>200
N.T.
>200
>200
199.40
>200
N.T.
>200
>200
>200
186.55
>200
>200
2.78
48.61
95.59
66.38
67.03
>200
103.40
99.59
>200
>200
71.22
199.90
51.99
51.56
80.88
35.87
154.70
53.27
13.56
82.37
97.67
44.23
65.68
194.80
>200
146.90
22.76
83.30
70.88
183.30
31.68
187.70
22.25
146.90
5u
Cisplatin
Melphalen
Ploxal-S
Fluorouracil
25.88
4.30
N.T.
N.T.: Not tested.
is dependent on the benzylidenehydrazin, namely: –F, –Cl, –Br, –CH3, –CF3, –OCH3,
–CH(CH3)2, –OCH2CH3, –NO2, or –N(CH3)2. For instance, the most potent anticancer
drug candidate 4c against the MDA-MB-231 cell line has –Cl group on meta position
of benzylidene ring. Compounds 4a, 5a, 4b, 5c, 5f, 4 g, 5h, 5k, 4l, 5 m, 4n, 5n, 4o, 4p,
4r, 5r, 4t, and 5u were found not to have cytotoxic activity toward breast cancer cell
line. On the other hand, compound 4f having an electron-withdrawing group (–NO2),
located in the para position of benzylidene ring, was also seen not to have cytotoxic
activity in the same cell line. Compounds 4c, 5d, and 5i were more potent than other
compounds in MDA-MB-231 cell line, with the half maximal inhibitory concentration
(IC50) values 65.21, 88.54, and 96.66 mM, respectively.
The effects of all compounds were also screened in the HepG2 cell line. Compounds,
except 5c, 4f, 5f, 5 g, 5o, and 4p, exhibited antiproliferative activities against HepG2 cell
line. Compounds 5n, 5j, 4a, and 5r were especially found to be more effective potential
anticancer drug candidates than the commonly used positive control drugs cisplatin,
melphalen, and fluorouracil in HepG2 cells as they showed IC50 values of 44.23, 35.87,
48.61, and 22.76 mM, respectively. Furthermore, compound 4m showed better antiproli-
ferative activities than ploxal-S and other positive control drugs with following IC50

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value of 13.56 mM in HepG2. This is a remarkable result indicating that compound 4m
may be a possible drug candidate for liver cancer.
As shown in Table 2, compounds 5c, 4f, 5f, and 4p displayed a similar trend with
IC50 values greater than 200 mM toward both HepG2 and MDA-MB-231. It is note-
worthy that although compounds 4a, 4b, 4c, 4g, 4h, 5h, 5j, 4 l, 4m, 5n, 4o, 5r, and 4t
showed measurable antiproliferative activities in HepG2, it was found to have no cyto-
toxic activity in MDA-MB-231. Therefore, these compounds have more suitability for
further research on liver cancer rather than on breast cancer.
Such as the substituent depending on designed compounds, the drug concentration
of compounds has a significant role on screened cell lines. The live-cell percentages
were observed to increase with decreasing concentrations of tested heterocyclic organic
compounds toward MDA-MB-231 and HepG2 (Figs. S91 and S92). The most cell viabil-
ity ratio was obtained at 5 mM of tested compounds in HepG2 and MDA-MB-231.
Molecular docking studies
As a result of molecular docking calculations, many parameters of new pyrimidine-
chromeno hybrid derivatives against enzymes were found. These parameters are given
in the Table 3. As a result of the calculations, the most important parameter among the
parameters found is the docking score. It should be well known that the molecule with
the most negative numerical value of the docking score parameter obtained because of
the interaction between molecules and enzymes has higher biological activity than other
molecules.[54] As the interaction between new pyrimidine-chromeno hybrid derivatives
and enzyme increases, the biological activity value of new pyrimidine-chromeno hybrid
derivatives increases, showing both experimental and theoretical results.
After the docking calculations made to compare the biological activities of molecules,
the drug-ability properties of new pyrimidine-chromeno hybrid derivatives were exam-
ined. As a result of the ADME/T analysis, many parameters were found for the mol-
ecule. These calculated parameters give the effects and responses of new pyrimidine-
chromeno hybrid derivatives in human metabolism of tissues or organs.[55] As a result
of this analysis, each parameter is numerical value in different organs or tissues. All the
calculated parameters are detailed in Table 4. Molecules marked with “–” in Table 4 did
not show any interaction with cancer proteins.
Many other parameters were found in the calculations. These parameters are used to
explain the interaction between the new pyrimidine-chromeno hybrid derivatives and
the enzyme. Other parameters found because of the calculations are Glide hbond, Glide
evdw, and Glide ecoul parameters. These parameters give numerical values of hydrogen
bonding, Van der Waals, and Coulomb interactions, which are many interactions
between molecules and enzymes. These interactions have many interactions such as
hydrogen bonds, polar and hydrophobic interactions, p-p and halogen.[56] These are
given in Figures 1–6. On the other hand, the last remaining parameters are Glide emo-
del, Glide energy, Glide einternal, and Glide posenum parameters, which gives numer-
ical value about the interaction exposure between new pyrimidine-chromeno hybrid
derivatives and enzyme.[57]

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The biological activities of the studied molecules against cancer proteins were com-
pared. The molecules with the highest activity are 4t and 4g molecules against breast
cancer protein, 4n molecules against liver cancer protein, and 5r molecules against lung
cancer protein.
Among all ADME/T parameters, another important two parameters are Rule of five
and Rule of three. The Rule of five[58,59] and Rule of three[60] parameters are more
important than any other parameter. In the analysis for molecules, the numerical value
of these two parameters is expected to be zero. The Rule of five parameter, also known
as Lipinski’s Pfizer’s fifth rule. The rules are: mol MW <500, QPlogP o/w < 5, donorHB
ꢁ5, and accptHB ꢁ10. However, the Rule of three parameter is known as the three of
Jorgensen’s rule. The three rules are: QPlogS> ꢀ5.7, QP PCaco> 22 nm / s, and
#Primary Metabolites <7. If the numerical value of the Rule of three parameter is zero,
this molecule can be used orally as a drug. These two parameters are the two parame-
ters that best explain the drug properties of molecules.
X-ray diffraction analysis for 4l
The molecular structure of 4l, with the atom numbering scheme, is shown in Figure 7.
The N4 ¼ C12 bond distance [1.282(5) Å] is a typical double bond character. The dihe-
dral angles between pyrimidine and phenyl rings are 8.59(13) and 10.28(17)ꢂ. The dihe-
dral angle of the phenyl rings is 6.54(18)ꢂ, respectively. The pyrimidine ring plane is
approximately planar, with a maximum deviation from the least-squares plane being
0.0474(27) Å for atom C11. The values of the O1–C5/C9 ring puckering parameters:
Q ¼ 0.319 (2) Å, h¼ 61.65(2)ꢂ and u¼ 315 (2)ꢂ. The C14-C12 ¼ N4–N3 angle is
178.6(3)ꢂ, respectively. The molecules of 4l are connected by O–HꢃꢃꢃN hydrogen bonds.
Atom O2 acts as a hydrogen-bond donor, via atom H2A, to atom N2i, forming a C(6)
chain running which is parallel to the direction [x þ 1/2, y, ꢀz þ 1/2].
X-ray diffraction analysis for 5d
The molecular structure of 5d, with the atom numbering scheme, is shown in Figure 8.
The C7¼O2 [1.234(5) Å] and N4¼C12 [1.273(6) Å] bond distances are a typical double
bond character. The C1–C7–C8 and C10–N3–N4¼C12 angles are 122.0(4) and
ꢀ172.8(4)ꢂ, respectively. The pyrimidine ring makes dihedral angles of 44.38(13) and
7.50(20)ꢂ with the two phenyl rings. The dihedral angle of the phenyl rings is
41.08(13)ꢂ, respectively. The pyrimidine ring plane is approximately planar, with a max-
imum deviation from the least-squares plane being 0.0292(24) Å for atom C10. The
molecules of 5d are connected by N–HꢃꢃꢃN and C–HꢃꢃꢃO hydrogen bonds. Atom N3
atom acts as a hydrogen-bond donor, via atom H3A, to atom N1ii, forming a centro-
2
symmetric R2 (8) ring centered at (1, 1/2, 1). Similarly, atom C11 in the molecule at (x,
y, and z) acts as a hydrogen-bond donor, via atom H11, to atom O3i, forming a centro-
2
symmetric R2 (24) ring centered at (1/2, 1/2, 1/2). The combination of the hydrogen
2
2
bonds generates a chain of edge fused R2 (8) and R2 (24) rings running parallel to
the direction.

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Figure 1. Representation of interactions between molecule 4c and breast cancer protein.
Figure 2. Representation of interactions between molecule 4c and breast cancer protein.

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Figure 3. Representation of interactions between molecule 4m and liver cancer protein.
Figure 4. Representation of interactions between molecule 4m and liver cancer protein.

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Figure 5. Representation of interactions between molecule 5r and lung cancer protein.
Figure 6. Representation of interactions between molecule 5r and lung cancer protein.

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Figure 7. The molecular structure of 4l showing the atom numbering scheme.
Figure 8. The molecular structure of 5d showing the atom numbering scheme.
Conclusion
Novel 5H-chromeno[4,3-d]pyrimidine (4a–c, e–h, l–r, t) and pyrimidine-5-yl-(2-
hydroxyphenyl)methanone (5a, c, d, f–k, m–o, r, s, u) derivatives were synthesized and
characterized. Furthermore, the structures of compounds 4 l and 5d were completely
verified by X-ray crystal structural analysis. All compounds were screened against two
human cancer cell lines using MTT assay. Most of the compounds were found to have
more cytotoxic effects toward liver cancer cells than breast cancer cells. Particularly,
compounds 4a, 5a, 4b, 4g, 4h, 5h, 5j, 4 l, 5m, 4n, 5n, 4o, 5r, 5s, and 4t exhibited anti-
proliferative effects at lower micromolar concentrations in HepG2 cells rather than
MDA-MB-231 cells. On the other hand, molecule 4m exhibited the best activity with
IC50 value of 13.56 lM against HepG2.
Biological activities of new pyrimidine-chromeno hybrid derivatives against enzymes
were compared. As a result of these docking calculations, the molecules with the highest
activity are 4t and 4g molecules against breast cancer protein, 4n molecules against liver
cancer protein, and 5r molecules against lung cancer protein. The theoretical

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calculations made are generally similar to the experimental results. However, the main
reason for the differences occurred is that the theoretical calculations were made in a
pure and isolated environment, but there are many experimental inputs in experimental
processes. After examining the interaction of these new pyrimidine-chromeno hybrid
derivatives against enzymes, a theoretical ADME/T analysis was made. Considering the
interaction of the molecule with enzymes because of docking calculations, it is seen that
it has a high interaction. Accordingly, since ADME/T parameters meet the necessary
conditions, it is safe to use it as an advanced drug. The numerical values of the parame-
ters obtained from this study are used in future in vivo and in vitro studies, providing a
great deal for new drug candidate discovery.
Experimental section
For each of the series, parameters such as temperature, type of solvent, time, and cata-
lyst were considered for the determination of the optimum reaction conditions of the
experiment. During the synthesis, thin layer chromatography (TLC) application was
used. With specific time intervals, the material from the reaction balloon and the start-
ing reactant material are put in an already prepared solution with silica gel selected as
adsorbent and the help of 60/F-254 coated aluminum applied to the plates with the aid
of capillary tubes which aid dragging in moving phases. Reaction times were determined
according to the TLC results. For each part of the experiment, TLC mobile phases were
tested by using different solvent mixtures for the control of the synthesis. The impurity
controls were first performed by TLC and the compounds that contained impurity were
purified by washing, crystallization, and purification by column chromatography. 9200
melting point apparatus was used to define the melting points (m.p.) of each of the
compounds. The structures of the products obtained were confirmed by using nuclear
1
magnetic resonance (NMR, H and 13C NMR), FT-IR, elemental analysis, and X-ray
diffraction.
A general method for the synthesis of compounds 5H-chromeno[4,3-d]pyrimidine
(4a) and pyrimidine-5-yl-(2-hydroxyphenyl) methanone (5) derivatives
Guanylhydrazone derivatives (2a-u; 1 mmol) and 3-formylchromone (3; 1 mmol) were
added to a 100 ml flask. The mixture was stirred under reflux for 3–8 hours in 10 cm3
of ethyl alcohol. The precipitates were filtered, and the impurity was checked by TLC.
In column chromatography (4a, c, f–h, m–o, r, t and 5a, c, d, f–k, m–o, r, s, uin solv-
ent mixtures of specified percentages) and/or usage of crystallization was used in the
purification method (4b, e, l, or p). It was then dried in a 60 ꢂC vacuum oven or vac-
uum desiccator with the aid of phosphorus pentoxide (Scheme 1). The structures of
1
compounds were verified by H NMR, 13C NMR, IR, elemental, and X-ray diffrac-
tion analysis.
Spectral data of synthesized compounds
(4-methylphenyl)methylidenehydrazino-5H-chromeno[4,3-d]pyrimidine-5-ol, 4a. Color:
yellow, yield: 11%, m.p.: 251–253 oC. IR (cm-1): 3212.6 (NH), 2926.3 (C–H),

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1
1570.9–1590.9 (C¼N), 1532.1–1438.0 (C–O). H NMR (400 MHz, DMSO–d6) d: 11.37
(s, 1H, OH), 8.49 (s, 1H, NCHC), 8.22–7.08 (m, 9H, Ar-H), 7.61 (s, 1H, CNHN),
6.44–6.46 (d, 1H, OCHOHC), 2.34 (s, 3H, CH3). 13C NMR (100 MHz, DMSO–d6) d:
161.29, 156.82, 154.86, 154.31, 147.79, 139.20, 133.54, 128.97, 128.77, 126.39, 124.89,
122.25, 120.10, 118.46, 116.71, 91.21 (OCHOHC), and 14.11 (CH3). Anal. Calcd. for
C19H15N4O2 (331.346 g/mol): C: 68.87; H: 4.56; and N: 16.91. Found: C: 68.62; H: 4.51;
and N: 16.87.
(4-methylphenyl)methylidenehydrazinopyrimidine-5-yl)(2-hydroxyphenyl) methanone, 5a.
Color: yellow, yield: 82%, m.p.: 213–215oC. IR (cm-1): 3222.2 (NH), 3049.7–2871.8 (C-
1
H), 1600.8 (C¼O), 1579.6 (C¼N). H NMR (400 MHz, DMSO-d6) d: 11.89 (s, 1H,
OH), 10.41 (s, 1H, NH), 8.72 (ss, 2H, NCHCCHNC), 8.22 (s, 1H, CCHNNHC),
6.94–7.61 (m, 8H, Ar-H), 2.33 (s, 3H, CH3). 13C NMR (100 MHz, DMSO-d6) d: 192.89
(C¼O), 160.89, 160.74, 156.78, 145.59, 139.93, 133.91, 132.24, 130.85, 129.90, 127.27,
124.96, 122.96, 119.90, 117.18, and 21.47 (CH3). Anal. Calcd. for C19H16N4O2
(332.354 g/mol): C: 68.66; H: 4.85; and N: 16.86. Found: C: 68.68; H: 4.81; and N: 16.82.
In vitro cytotoxic activity studies of compounds
Cytotoxic activity studies were done according to the literature procedures.[61] HepG2
and MDA-MB-231, which were purchased from the American Type Culture Collection
(ATCC, Manassas, VA), were maintained in DMEM medium containing 10% foetal
bovine serum and 1% glutaMAX. The cells were seeded into sterile 96-well plates at a
density of 4 ꢅ 103 cells/well. After 24 h, cells were exposed to drug candidates at concen-
trations ranging from 200 to 5 mM (200, 100, 50, 20, 10, and 5 mM) for 48 h. 50 mL of
MTT stock solution (5 mg/mL) was added to each well after 48 h and the plates were
incubated for another 3 h in an incubator. Finally, DMSO (200 mL) was put to each
well. Absorbance values were read with the Promega reader device at 560 nm.
Docking studies
An important method used to calculate the theoretical biological activities of molecules
against enzymes is molecular docking. In the calculations made by molecular docking
method, many parameters are obtained to compare the biological activities of mole-
cules.[62,63] These parameters are very important parameters to explain the theoretical
biological activities of molecules. Many enzymes were used to calculate the biological
activity of new pyrimidine-chromeno hybrid derivatives. Enzymes used for new pyrimi-
dine-chromeno hybrid derivatives in this study crystal structure of the BRCT repeat
region from the breast cancer associated protein, 1JNX,[64] crystal structure of VEGFR
kinase (liver cancer) protein, 3WZE,[65] and crystal structure of an allosteric Eya2 phos-
phatase inhibitor (lung cancer) protein, 5ZMA,[66] was used.
Molecular docking calculations to calculate the biological activity of new pyrimidine-
chromeno hybrid derivatives were performed using the Maestro Molecular modeling
€
platform (version 12.2) by Schrodinger. Proteins and new pyrimidine-chromeno hybrid
derivatives molecule should be prepared for calculations using the Maestro Molecular

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€
modeling platform (version 12.2) by Schrodinger program. In docking calculations, each
stage is completed by different processes for molecules and enzymes. Firstly, it was used
from Gaussian software program[67] to obtain optimized structures of molecules. Using
these optimized structures, all calculations were made with the Maestro Molecular mod-
[68]
€
eling platform (version 12.2) by Schrodinger, LLC.
The Maestro Molecular modeling
€
platform (version 12.2) by Schrodinger comes together from many modules. In the first
[69,70]
module between these modules, the protein preparation module
is used to prepare
the enzymes formed by proteins for calculations. In the next module, the LigPrep mod-
ule[71,72] was used to prepare the new pyrimidine-chromeno hybrid derivatives for
calculations.
In the next module, the Glide ligand docking module[73] was used to interact with
the molecule and enzymes. OPLS3e method was used in all calculations for docking cal-
culations of molecules and proteins in all modules used. After the docking calculations,
ADME/T analysis was performed to examine the future drug properties of the molecule.
The Qik-prop module[74] of the Schrodinger software was used for ADME/T analysis.
€
X-ray diffraction analysis studies
Suitable crystals of 4l and 5d were selected for data collection which was performed on
a D8-QUEST diffractometer equipped with graphite-monochromatic Mo-Ka radiation at
296 K. The structure was solved by direct methods using SHELXS-2013[75] and refined
by full-matrix least-squares methods on F2 using SHELXL-2013.[76] All nonhydrogen
atoms were refined with anisotropic parameters. For 4l compound; the hydroxyl H
atom was in a difference map refined freely. The other H atoms were located from dif-
ferent maps and then treated as riding atoms with C-H distances of 0.93–0.96 Å and N-
H distances of 0.86 Å. For 5d compound; the H atoms were located from different
maps and then treated as riding atoms with C-H distances of 0.93–0.96 Å, N-H distance
of 0.86 Å, and O-H distance of 0.82 Å. The following procedures were implemented in
our analysis: data collection: Bruker APEX2;[77] a program used for molecular graphics:
MERCURY programs;[78] the software used to prepare material for publication:
WinGX.[79] Details of data collection and crystal structure determinations are given in
Tables 3 and 4, respectively.
Crystallographic data for the structural analyses have been deposited with the
Cambridge Crystallographic Data Center, CCDC No. 1878421 for compound 4 l and
CCDC No. 1900837 for compound 5d. Copies of this information may be obtained free
of charge from the Director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax:
þ44-1223-336033; e-mail: deposit@ccdc.cam.ac.uk or www: http://www.ccdc.cam.ac.uk).
1H NMR, 13C NMR, IR spectra, X-ray data and full characterization data of all com-
pounds can be found via the “Supplementary File” section of this article’s webpage.’
Acknowledgments
€
This work was financially supported by the Research Fund of Erciyes Universitesi [FDK-2016-
6664]. The authors acknowledge the Scientific and Technological Research Application and
Research Center, Sinop University, Turkey, for the use of the Bruker D8 QUEST diffractometer.

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S. Ç. YAVUZ ET AL.
This research was made possible by TUBITAK ULAKBIM, High Performance and Grid
Computing Center (TR-Grid e-Infrastructure).
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was financially supported by Erciyes University Research Fund [FDK-2016-6664].
ORCID
Senem Akkoc¸
http://orcid.org/0000-0002-1260-9425
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