Solvent-free Synthesis of Halogenated Furo[2,3-d] pyrimidines and Their Cytotoxic Activity on the T47D Breast Cancer Cell Line

Full text of DOI:10.1080/00304948.2020.1771961

Organic Preparations and Procedures International
The New Journal for Organic Synthesis
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Solvent-free Synthesis of Halogenated Furo[2,3-d]
pyrimidines and Their Cytotoxic Activity on the
T47D Breast Cancer Cell Line
Afsaneh Zonouzi, Mehran Habibi Rezaei, Roghieh Mirzazadeh & Maryam
Rezaei Arjomand
To cite this article: Afsaneh Zonouzi, Mehran Habibi Rezaei, Roghieh Mirzazadeh & Maryam
Rezaei Arjomand (2020): Solvent-free Synthesis of Halogenated Furo[2,3-d] pyrimidines and Their
Cytotoxic Activity on the T47D Breast Cancer Cell Line, Organic Preparations and Procedures
International, DOI: 10.1080/00304948.2020.1771961
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ORGANIC PREPARATIONS AND PROCEDURES INTERNATIONAL
https://doi.org/10.1080/00304948.2020.1771961
OPPI BRIEF
Solvent-free Synthesis of Halogenated Furo[2,3-d]
pyrimidines and Their Cytotoxic Activity on the T47D
Breast Cancer Cell Line
Afsaneh Zonouzia,b , Mehran Habibi Rezaeic , Roghieh Mirzazadeha,d, and
Maryam Rezaei Arjomandc
b
aSchool of Chemistry, College of Science, University of Tehran, Tehran, Iran; Pharmaceutical and
c
Cosmetic Research Center (PCRC), University of Tehran, Tehran, Iran; School of Biology, University
d
College of Science, University of Tehran, Tehran, Iran; Biochemistry Department, Pasteur Institute of
Iran, Tehran, Iran
ARTICLE HISTORY Received 30 September 2019; Accepted 24 February 2020
Motivated by the important biological activities displayed by furanopyrimidines and
their congeners,1–11 we have been investigating their synthesis using easily available pre-
cursors under mild and environmentally friendly conditions.12–22 We now describe a
high- yielding preparation of halogenated furo[2,3-d]pyrimidine-2,4(1H,3H)-diones
2a-f23–24 using a solvent-free method (Scheme 1). In brief, heating a mixture of N,N’
-dimethyl barbituric acid, cyclohexyl isocyanide and aldehydes 1 at 70 ꢀC for 20-
35 minutes produced compounds 2a-f in 85-95% yields. To the best of our knowledge,
this is the first report of the use of this reactant combination in a solvent-free system.
Significantly, we found that some of these compounds destroyed breast cancer cells.
The half maximal inhibitory concentrations (IC50) were determined as a measure of
the effectiveness of compounds 2a-e on the T47D cell line. The IC50 values at 95% con-
fidence intervals (95% CI) were determined from least-squares non-linear regression
analyses of log (inhibitor) versus normalized dose-response plots. In vitro data were
analyzed using the computer program Graphpad PRISM 5 from which the IC50 values
(mM) were calculated. The reported IC50 values are the mean (SEM) from experiments
run in triplicate with 8 or 10 concentrations of test compounds. Figure 1 shows the
model micrographs of the T47D cell line cultures at three living, half inhibited and
inhibited states that represent three concentration ranges of toxicants including: less
than IC50, IC50 and much more than IC50, respectively.
Calculated IC50 parameters of each compound are presented in Table 1. The results
suggest that dichloro-substitutions on the aryl ring are more toxic than mono-substitu-
tions against the cancerous cell line under the defined conditions used in this report.
In summary, we have demonstrated a solvent-free procedure for the synthesis of a
series of halogenated furo[2,3-d]pyrimidine-2,4(1H,3H)-diones 2a-e. The products have
been obtained in high yields (90-95%). The simplicity of this procedure makes it an
attractive method for the preparation of the target compounds. Some of these
CONTACT Afsaneh Zonouzi
zonouzi70@gmail.com
School of Chemistry, College of Science, University of Tehran,
14155-6455, Tehran, Iran.
ß 2020 Taylor & Francis Group, LLC

2
A. ZONOUZI ET AL.
N
Scheme 1. Preparation of furanopyrimidines 2.
Figure 1. Representative model micrographs of the T47D cell line cultures at three concentration
ranges of toxicants.
Table 1. Cytotoxic activity (IC50, in mM) of compounds 2a-e on the
cancer human cell line T47D comparing with Etoposide.
Compounds
LogIC50
IC50 (mM)
2a
2b
2c
2d
2.26
2.38
2.18
1.58
1.58
1.61
183.30
237.70
150.40
39.53
38.11
41.21
2e
Etoposide
compounds destroyed breast cancer cells. Further investigation of this method is cur-
rently in progress to establish its scope and utility.
Experimental section
Chemicals and solvents were obtained from Merck (Germany) and Fluka (Switzerland)
and were used without further purification. Column chromatography was performed on
silica gel (0.015-0.04 mm, mesh-size) and TLC on pre-coated plastic sheets (25 DCUV-
254) respectively. Melting points were measured on Barnstead Electrothermal melting
point apparatus and are not corrected. Elemental analysis for C, H and N were per-
formed using a Thermo Finnigan Flash EA1112 instrument. IR spectra were measured
1
on a Bruker EQUINOX 55 spectrophotometer in the ATR method. H NMR and 13C
NMR spectra were determined in CDCl3 on a Bruker 500 spectrophotometer and chem-
ical shifts were expressed in ppm downfield from tetramethylsilane. Mass spectra were
recorded on a Finnigan-MAT 8430 spectrometer at an ionization potential of 70 ev.

ORGANIC PREPARATIONS AND PROCEDURES INTERNATIONAL
3
Table 2. Formulas, mps, yields, and Elemental Analysis of white compounds 2a-e.
Elemental analysis (Calc.)
Cmpd
Formula
mp (ꢀC)
Yield%
C
H
N
2a
2b
2c
2d
2e
C20H22BrN3O3
C20H22ClN3O3
164-165
125-126
187-188
155-156
153-154
95
90
92
95
90
55.56 (55.57)
61.90 (61.93)
56.85 (56.88)
56.84 (56.88)
56.87 (56.88)
5.14 (5.13)
5.75 (5.72)
5.04 (5.01)
5.00 (5.01)
5.02 (5.01)
9.74 (9.72)
10.86 (10.83)
9.98 (9.95)
10.01 (9.95)
9.97 (9.95)
C20H21Cl2N3O3
C20H21Cl2N3O3
C20H21Cl2N3O3
Cytotoxicities of compounds 2 on the T47D cell line were assessed using the MTT [3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay. RPMI-1640 (Roswell
Park Memorial Institute) medium was obtained from PAA Laboratories GmbH,
Germany. Fetal bovine serum (FBS) inactivated (EU approved origin) was purchased
from Gibco Invitrogen (Life Technologies, Paisley, UK). Penicillin and streptomycin
and other chemicals were purchased from Sigma Chemical Company (St. Louis, MO,
USA). The pictures in Figure 1 have been taken on a model Axiovert 25 microscope
made by the Zeiss Company (Germany).
General procedure for the synthesis of furo[2,3-d]pyrimidine-2,4(1H,3H)-diones (2)
To a magnetically stirred mixture of N,N’-dimethyl barbituric acid (1 mmol) and alde-
hyde 1 (1 mmol), cyclohexylisocyanide (1.1 mmol) was added dropwise. The reaction
mixture was stirred for 20-35 min at 70 ꢀC. The progress of reaction was monitored by
TLC. When the spot for the aldehyde derivatives (Rf ꢁ 0.8-0.9) (silica gel, n-
hexane:EtOAc 4:1) disappeared and the spot for the products (Rf ꢁ 0.5-0.6) was
formed, the solid products 2 were washed with hot n-hexane and were recrystallized
from EtOAc:n-hexane 3:1. The white to light yellow crystals of compounds 2a-f were
thus obtained in high yields (85-95%). Characterization data for compound 2a are given
below, and the data for 2a-e are summarized in Tables 2 and 3.
5-(2-Bromophenyl)-6-(cyclohexylamino)1,3-dimethylfuro[2,3-d]pyrimidine-
2,4(1H,3H)-dione (2a)
White powder, yield 95%; mp 164-165 ꢀC; IR (cmꢂ1): 3330 (N–H), 1671, 1632 (C¼O),
1
1195, 1079, 1026 (C–O), 783, 741(C–Br). H NMR (500 MHz, CDCl3): d 1.06-1.27,
1.56-1.99 (m, 10H, CH2 of cyclohexyl) 3.13-3.14 (m, 1H, CH of cyclohexyl), 3.36, 3.58
(2s, 6H, 2CH3N), 3.41 (br s, NH) 7.23-7.37, 7.65-7.66 (4H, Ar protons) ppm. 13C NMR
(125 MHz, CDCl3): 24.26, 24.71, 25.51, 33.86, 55.21 (cyclohexyl carbons), 28.13, 29.44
(CH3N), 96.96, 103.04, 124.71, 127.16, 129.43, 131.48, 132.53, 132.75, 148.76, 150.25
(unsaturated carbons), 150.51, 157.80 (C¼O) ppm. MS: m/z (%) 431 (Mþ, 80), 433
(Mþ2, 79), 351 (13), 349 (13), 270 (100), 243 (37), 220 (43), 222 (43), 211 (37), 127
(20), 83 (20), 55 (87), 41 (67).
Anal. Calcd for C20H22BrN3O3: C, 55.57; H, 5.13; N, 9.72. Found: C, 55.56; H, 5.14;
N, 9.74.

4
A. ZONOUZI ET AL.
Table 3. Spectroscopic Data of 2a-e.
1H NMR (d)
13C NMR (d)
MS (m/z)
IR (cm-1)
2a
2b
2c
1.06-1.27, 1.56-1.99 (5m,
10H, CH2 of cyclohexy)
l3.13-3.14 (m, 1H, CH of
cyclohexyl), 3.36, 3.58
(2s, 6H, 2CH3N), 3.41 (br
s, NH) 7.23-7.37 ,7.65-
7.66 (4H, Ar protons)
24.26, 24.71, 25.51, 33.86,
55.21 (cyclohexyl carbons),
28.13, 29.44 (CH3N), 96.96,
103.04, 124.71, 127.16,
129.43, 131.48, 132.53,
132.75, 148.76, 150.25
(unsaturated carbons),
150.51, 157.80 (C¼O)
25.1,1, 25.94, 34.34, 55.91
(cyclohexyl carbons), 28.67,
29.85 (2CH3N), 96.21,
104.52, 128.77, 129.43,
130.97, 133.14, 149.22,
150.76 (unsaturated
3330 (N–H), 1671,
1632 (C¼O), 1195,
1079, 1026 (C–O),
783, 741 (C–Br)
431, 433 (Mþ, Mþþ2),
334, 336 (Mþ, Mþþ2 –
C6H11N), 276 (Mþ-
C6H4Br), 177
(C8H5N2O3þ), 85, 83, 55
1.15-1.32, 1.61-1.95 (5m,
10H, CH2 of cyclohexyl),
3.15-3.17 (m, 1H, CH of
cyclohexyl), 3.40, 3.57
(2s, 6H, 2CH3N), 3.53 (br
s, NH), 7.33-7.31, 7.36-
7.38, 7.54-7.56, (4H,
Ar protons)
1.07-1.92 (5m, 10H, CH2
of cyclohexyl), 3.16-
3.17(m, 1H, CH of
cyclohexyl), 3.34, 3.57
(2s, 6H, 2CH3N), 3.42 (br
s, NH), 7.24-7.46 (3H,
Ar protons)
3287 (N–H), 1687,
1634 (C¼O), 1182,
1094 (C–O),
387, 389 (Mþ, Mþþ2),
304, 306 (Mþ, Mþþ2 –
C6H11N), 276 (Mþ-
C6H4Cl), 177
746 (C–Cl)
(C8H5N2O3þ), 97, 83, 55
carbons), 151.29, 158.
57 (C¼O)
24.64, 24.70, 25.41, 30.88,
33.82, 54.99 (cyclohexyl
carbons), 28.09, 29.40,
(2CH3N), 96.76, 99.98,
126.89, 129.80, 130.68,
131.67, 132.48, 133.25,
148.96, 150.21 (unsaturated
carbons), 150.33,
3227 (N–H), 1673,
163 (C¼O), 1194,
1041 (C–O),
421, 423 (Mþ, Mþþ2),
324, 326 (Mþ, Mþþ2 –
C6H11N), 276 (Mþ-
C6H3Cl2), 177
793,741 (C–Cl)
(C8H5N2O3þ), 97, 83,
55, 43
157.72 (C¼O)
2d
2e
1.13-2.03 (5m, 10H, CH2
of cyclohexyl), 3.13-3.14
(m, 1H, CH of
cyclohexyl), 3.32, 3.55
(2s, 6H, 2CH3N), 3.47 (br
s, NH), 7.27- 7.45 (3H,
Ar protons)
1.11-1.90 (5m, 10H, CH2
of cyclohexyl), 3.12 (m,
1H, CH of cyclohexyl),
3.28 (br s, NH), 3.35,
3.58 (2s, 6H, 2CH3N),
7.26-7.42 (3H,
24.54, 25.35, 33.71, 54.89
(cyclohexyl carbons), 27.98,
29.31 (2CH3N), 96.58, 99.20,
126.78, 127.97, 129.22,
133.19, 134.03, 148.89
(unsaturated carbons),
150.21, 157.64(C¼O)
3277(N–H), 1674,
1637(C¼O), 1175,
1097(C–O),
421, 423 (Mþ, Mþþ2),
324, 326 C6H11N), 276
(Mþ-C6H3Cl2), 177
(C8H5N2O3þ), 97, 85, 83,
55, 43, 41
811, 739(C–Cl)
24.69, 25.51, 33.87, 55.04
(cyclohexyl carbons), 28.12,
29.48 (2NCH3), 97.05, 98.66,
127.93, 128.79, 129.84,
136.66, 148.99, 150.32
(unsaturated carbons),
150.52, 157.69 (C¼O)
3312 (N–H), 1671,
1636 (C¼O), 1191,
1049 (C–O), 783, 725
(C-Cl)
421, 423 (Mþ, Mþþ2),
324, 326 C6H11N), 276
(Mþ-C6H3Cl2), 177
(C8H5N2O3þ), 97, 55
Ar protons)
6-(Cyclohexylamino)-1,3-dimethyl-5-(pyridin-4-yl)furo[2,3-d]pyrimidine-2,4(1H,3H)-
dione (2f)
White powder, yield 85%; mp 176-177 ꢀC, lit mp25 [sic] 151-164 ꢀC; this is a known
1
compound, which was characterized by matching IR and H NMR spectral data with
the literature.
General procedure for the cytotoxicity assay of compounds 2a-e
The human breast cancer T47D cell line (ATCC, HTB-133, USA) came from the Cell
Bank of the Pasteur Institute in Tehran, Iran. The growth medium used was RPMI1640
medium supplemented with 10% v/v FBS, 2% NaHCO3, penicillin (100 units/ml) and
streptomycin (100 mg/ml). All of the cultures were maintained in 25 cm2 culture flasks
at 37 ꢀC, 95% air humidity, and 5% CO2. The suspended cells were seeded into 96-well
flat bottom cell culture plates (Nest), at a density designed to keep the cultures in

ORGANIC PREPARATIONS AND PROCEDURES INTERNATIONAL
5
Figure 2. IC50 values for the compound 2a-e.
logarithmic growth phase during the whole experiment. We used 7 ꢃ 103 cells per well
in 100 ml of medium. After seeding, the plates were incubated 24 h prior to
the experiment.
The chemicals were dissolved in 100% dimethyl sulfoxide (DMSO) as 100 mM stock
solutions then were diluted to appropriate concentrations in RPMI. Cells were treated

6
A. ZONOUZI ET AL.
with at least 8 concentrations in the range of 9-450 mM and all samples were re-incu-
bated for the next 24 h (all treatments were repeated six times). As control wells, cells
were treated with DMSO dissolved in medium (drug-free media) and the final concen-
tration of DMSO in each well did not exceed 0.45%. Then, 100 ml MTT solution
(0.5 mg.mlꢂ1 in PBS), was added to every well and incubated for 3 h at 37 ꢀC in a
humidified atmosphere with 5% CO2. After that, the MTT-containing medium was
removed gently and 100 ml DMSO was added to each well to dissolve blue formazan
crystals. The plates were read on a microtiter plate reader at 570 nm using an ELISA
TM
reader (Gen5TM ELISA reader, PowerWave
XS2, BioTek).
Among the compounds listed in Table 1, compounds 2a-e showed better results in
primary tests. The IC50 values for the compounds 2a-e are present at the mean (SEM)
from experiments run in triplicate with 8 or 10 concentrations of test compounds.
(Figure 2)
Acknowledgments
We thank the Research Council of the University of Tehran and Pharmaceutical and Cosmetic
Research Center (PCRC) for financial support.
ORCID
Afsaneh Zonouzi
http://orcid.org/0000-0001-8352-1976
Mehran Habibi Rezaei
http://orcid.org/0000-0002-4819-8613
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