Synthesis of 6-unsubstituted 2-oxo, 2-thioxo, and 2-amino-3,4-dihydropyrimidines and their antiproliferative effect on HL-60 cells

Article abstract of DOI:10.1016/j.tetlet.2020.151967

A general and efficient synthetic method for 6-unsubstituted 3,4-dihydropyrimidin-2(1H)-thiones 6 and -ones 7 has been developed. In three-component reactions, the reactivity of reagents serving as the C5–C6 fragment of the dihydropyrimidine ring was comp

Full text of DOI:10.1016/j.tetlet.2020.151967

Journal Pre-proofs
Synthesis of 6-unsubstituted 2-oxo, 2-thioxo, and 2-amino-3,4-dihydropyrimi‐
dines and their antiproliferative effect on HL-60 cells
Yoshio Nishimura, Hidetomo Kikuchi, Takanori Kubo, Yuki Gokurakuji,
Yuri Nakamura, Rie Arai, Bo Yuan, Katsuyoshi Sunaga, Hidetsura Cho
PII:
DOI:
Reference:
S0040-4039(20)30410-X
https://doi.org/10.1016/j.tetlet.2020.151967
TETL 151967
To appear in:
Tetrahedron Letters
Received Date:
Revised Date:
Accepted Date:
21 March 2020
17 April 2020
21 April 2020
Please cite this article as: Nishimura, Y., Kikuchi, H., Kubo, T., Gokurakuji, Y., Nakamura, Y., Arai, R., Yuan,
B., Sunaga, K., Cho, H., Synthesis of 6-unsubstituted 2-oxo, 2-thioxo, and 2-amino-3,4-dihydropyrimidines and
their antiproliferative effect on HL-60 cells, Tetrahedron Letters (2020), doi: https://doi.org/10.1016/j.tetlet.
2020.151967
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
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Highlights
・6-Unsubstituted 3,4-dihydropyrimidin-2(1H)-thiones and -ones were synthesized.
・The three-component reaction was mediated by a catalytic amount of Lewis acid.
・The reaction had broad scope for substrates and proceeded in high yields.
・Novel 6-unsubstituted 2-aminodihydropyridimidines were synthesized.
・The antiproliferative effect of the compounds on HL-60 cells was explored.
Tetrahedron Letters
journal homepage: www.elsevier.com
Synthesis of 6-unsubstituted 2-oxo, 2-thioxo, and 2-amino-3,4-
dihydropyrimidines and their antiproliferative effect on HL-60 cells
Yoshio Nishimuraa,*, Hidetomo Kikuchib, Takanori Kuboa, Yuki Gokurakujia, Yuri Nakamuraa,
Rie Araib, Bo Yuanb, Katsuyoshi Sunagab, Hidetsura Choc
a Faculty of Pharmacy, Yasuda Women’s University, 6-13-1, Yasuhigashi, Asaminami-ku, Hiroshima, 731-0153, Japan
b Faculty of Pharmacy and Pharmaceutical Sciences, Josai University, Keyakidai, Sakado, Saitama 350-0295, Japan
c Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3 Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan

ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
A general and efficient synthetic method for 6-unsubstituted 3,4-dihydropyrimidin-2(1H)-
thiones 6 and -ones 7 has been developed. In three-component reactions, the reactivity of
reagents serving as the C5–C6 fragment of the dihydropyrimidine ring was compared. The
reaction of thiourea or urea 12, aldehydes 13, and ethyl 3-dimethylaminoacrylate 9 in the
presence of a catalytic amount of AlCl3 by heating smoothly proceeds to give 6 and 7 in high
yields. A synthetic novelty of our protocol is as follows: 1) Lewis acid-mediated reaction, 2)
good to high yields, 3) broad scope as for aldehydes, and ureas. Hitherto unavailable 6-
unsubstituted 2-aminodihydropyridimidine 8 has been obtained from the 2-thioxo derivative 6
by a stepwise method involving a substitution reaction with the amine at the 2-position. These
6-unsubstituted compounds 6, 7, and some 6-methyl derivatives 16 were assessed for their
antiproliferative effect on the human promyelocytic leukemia cell line, HL-60. The 4-propyl-
6-methyl derivative 16b showed relatively strong activity with the IC50 value of 952 nM.
Available online
Keywords:
dihydropyrimidine
three-component reactions
AlCl3
antiproliferative effect
HL-60 cells
2009 Elsevier Ltd. All rights reserved.

A three-component reaction called Biginelli reaction involving
using AlCl3 for the synthesis of 2-thioxo derivatives 6 and 2-oxo
derivatives 7 (Figure 1). General and catalytic methods for
synthesis of 6 and 7 have not been reported thus far. Compared
with synthetic methods for 6 and 7 mentioned above,4,5 a synthetic
novelty of our protocol is as follows: 1) Lewis acid-mediated
reaction, 2) good to high yields, 3) broad scope as for aldehydes
and ureas. We also transformed the 2-thioxo group of 6 to an
amino group by a stepwise method involving a substitution
reaction with an amine. This protocol enables the synthesis of
hitherto unavailable 6-unsubstituted 2-aminodihydropyrimidines
8. Thus, we established a synthetic method for a series of 2-
heteroatom- (S, O, and N-) substituted 6-unsubstituted derivatives
6–8 in this study. Their in vitro antiproliferative effect on HL-60
(human promyelocytic leukemia) cell lines was also evaluated.
the condensation of ureas 1, aldehydes 2, and -dicarbonyl
compounds
3 gives 3,4-dihydropyrimidin-2(1H)-ones and
thiones (DHPMs) 4. The heterocycles show a wide range of
biological activities for medicinal applications (Scheme 1).1 They
display calcium channel inhibition, anticancer, antiviral,
antibacterial, antifungal, antimicrobial, anti-HIV, antimalarial,
anti-inflammatory, antihypertensive, and antioxidation activities.
Recent additional studies on the synthesis and biological
activities of DHPMs suggest their great and significant potential
as leading compounds for developing medicines.2
R1
R1
4
O
R1
O
O
H
O
O
3
2
HN
R3
HN
R3
R3
5
Initially, the reactivity of three reagents serving as the source of
the C5–C6 fragment of the dihydropyrimidine ring was compared.
NH2
NH2
1
R2
N
N
H
2
X
X
6
6
R2
X
H
Thus,
ethyl
3-dimethylaminoacrylate
9,
ethyl
3,3-
3
1
4
5
diethoxypropanoate 10, and ethyl propiolate 11 were subjected to
a three-component reaction involving thiourea 12a and 4-
chlorobenzaldehyde 13a in the presence of 20 mol% AlCl3
hexahydrate in DMF at 90 ºC for 6 h (Scheme 2). Among these
three compounds, 9 showed relatively higher reactivity, and the
corresponding 6-unsubstituted DHPM 6a was obtained in 40%
yield. Therefore, 9 was employed as a suitable C5–C6 source.
X = O, S
R3 = alkyl, aryl, OR, NR2
R1, R2 = alkyl, aryl
Scheme 1. Synthesis of 3,4-dihydropyrimidin-2(1H)-ones and -
thiones 4 (DHPMs) and 6-unsubstituted DHPMs 5
Cl
* Corresponding author. Tel.: +81 82 878 9498; fax: +81 82 878 9540. E-mail
address: nishimura-y@yasuda-u.ac.jp (Y. Nishimura).
Cl
13a
(1.5 eq)
AlCl3 6 H2O
(20 mol%)
In most Biginelli reactions, -dicarbonyl compounds 3 such as -
ketoesters and 1,3-diketones have long been employed as the
source of C5–C6 fragment, which predetermined the substituents
at the 6-position (R2 of 4 in Scheme 1). DHPMs 5 having no
substituent at the 6-position were rarely accessible by the
classical reaction, probably because of lack of availability of -
formylcarbonyl compounds 3 (R2 = H) and difficulty in
controling the reactivity of the compounds. Indeed, it has been
reported in the literature that the classical Biginelli reactions
using 3 (R2 = H) failed.3 Therefore, alternative methods to
synthesize 5 have been developed, in which a reactant serving as
an equivalent to -formylcarbonyl compounds was used. For
example, alkyl 3,3-dialkoxypropanoate was used for this
purpose.4 Namely, Kappe reported a synthetic method using a
Yb(OTf)3 catalyst to give 5 in moderate yields.4a Other reactions
using methyl 3,3-dimethoxypropanoate to synthesize 5 as a
synthetic intermediate either for 1A receptor antagonists4b or
marine alkaloid batzeladine D3c were also reported. Electron-
deficient alkynes, enaminones, and enaminoates also served as
the source of the C5–C6 fragment of 5.5 Wan carried out studies
o n t h r e e - c o m p o n e n t r e a c t i o n s u s i n g a l k y n e s a n d
piperazine/TsOH/TMSCl systems to give 5 with diverse
substituents.5a Other reactions involving alkynes or enaminoates
using excess acid5b, or using enaminones to yield 5-aroyl
derivatives were also reported.5c–5d Related three-component
reactions involving two aldehydes, and urea to give 6-
unsubstituted 4,5-dialkyl derivatives were also reported.6 As a
result of our studies on the synthesis of less substituted
dihydropyrimidines,7 we realized the three-component reactions
O
H
CO2Et
NH2
NH2
HN
5
6
9 10
, or
11
,
S
DMF, 90 ºC, 6 h
S
N
H
(1.0 eq)
CO2Et
12a
(1.5 eq)
6a
CO2Et
CO2Et
Me2N
EtO
OEt
9
10
27%
11
5%
Cl
6a
Yield of
:
40%
Cl
O
H
Acid
CO2Et
13a
(1.0 eq)
CO2Et
(20 mol%)
HN
NH2
S
N
H
S
NH2
Me2N
DMF, 90 ºC, 3 h
6a
12a
(1.5 eq)
9
(1.0 eq)
Entry
Acid (20 mol%)
Yield (%)
1
2
none
8
2 M HCl
CF3CO2H
MsOH
13
38
34
38
33
55
40
39
19
39
43
38
31
10
23
52
61
88
59
3
4
5
BF3 Et2O
TMSOTf
Sc(OTf)3
Y(OTf)3
Yb(OTf)3
La(OTf)3
AlCl3 6 H2O
InCl3
6
7
8
9
R
R
R
10
11
12
13
14
15
16
17
18
19a
20a
CO2Et
CO2Et
CO2Et
HN
HN
N
N
H
N
H
N
H
S
O
R'NH
CeCl3
6
7
8
FeCl3
MgCl2
ZnCl2
Figure 1. A series of 2-heteroatom (S, O, and N-) substituted 6-
unsubstituted dihydropyrimidines 6–8
TiCl4
AlCl3
AlCl3
AlCl3 6 H2O
12a 13a 9
: ) is 1:1.5:1.5.
a) Reaction temperature was 110 ºC, and molar ratio (
:

R
O
H
13
(1.5 eq)
R
CO2Et
CO2Et
AlCl3 (20 mol%)
DMF, 110 ºC, 3 h
HN
NH2
NH2
Scheme 2. The reactivity and optimization of C5-C6 source
X
N
H
X
Me2N
9
(1.5 eq)
12a
12b
6
7
(X = S)
(X = O)
(X = S)
(X = O)
Next, we examined the reaction conditions, and revealed the
effect of the acid. The results are summarized in Table 1. All
protonic acids and Lewis acids improved the yield of 6a. The
reactions using some Lewis acids [Sc(OTf)3, TiCl4, AlCl3] gave
6a in good yields of >50% (entries 7, 17, and 18) at 90 ºC, and
the highest yield of 61% was obtained in the reaction using
anhydrous AlCl3 (entry 18). Subsequently, other reaction
conditions including solvents (DMA, NMP, DMSO, EtOH, THF,
1,4-dioxane, toluene, and CH3CN), reaction temperature, catalyst
loading, and molar ratio of the three reaction components were
examined and optimized (see Table S1 in Supplementary
entry
X
R
Yield (%)
6a
6b
6c
6d
6e
6f
1
2
S
S
S
S
S
S
S
S
S
S
S
S
S
O
O
O
4-ClC6H4
88
95
83
84
95
85
84
81
58
75
88
73
38
91
94
71
3-ClC6H4
2-ClC6H4
4-BrC6H4
4-CF3C6H4
4-NO2C6H4
C6H5
3
4
5
6
6g
6h
6i
7
8
3-OHC6H4
4-CH3OC6H4
4-CH3C6H4
cyclo-C6H11
n-C3H7
9
6j
10
11
12
13a
14
15
16
6k
6l
Table 1. Screening of acid catalyst, and optimization of other
reaction conditions
6m
7a
7b
7c
H
4-ClC6H4
4-CF3C6H4
n-C3H7
Material). As a result, the optimal conditions were those for entry
19; namely, 20 mol% anhydrous AlCl3 and 1:1.5:1.5 molar ratio
of 12a:13a:9 at 110 ºC in DMF, which produced 6a in a high yield
of 88%. The reaction using AlCl3 hexahydrate yielded 6a in
moderate yield of 59% (entry 20); use of anhydrous AlCl3
promoted the reaction more effectively.
12a 13 9
a) Paraformaldehyde was used, and molar ratio ( : ) is 1.5:1.5:1.
:
aminodihydropyrimidine 8a (R = n-C3H7) having no Boc group
was obtained in 74% yield. To our surprise, this result is in
contrast to that of our previous study, in which no ring opening
product 15a (R = n-C3H7) was obtained.11 In the reaction of 14b
(R = Ph), the major product was 8b (R = Ph) accompanied by
15b (R = Ph) in 67% and 25% yields, respectively. We again
confirmed our previous finding that the reaction of the 4-H
Under the optimized reaction conditions, various aryl- and
alkylaldehydes were subjected to the three-component reaction to
obtain 6-unsubstituted DHPMs 6 and 7; the results are summarized
in Table 2.8 Arylaldehydes having electron-withdrawing groups
showed especially high reactivity, and the corresponding 4-aryl
products 6a–6f were obtained in good to high yields (entries 1–6).
Although 3-hydroxybenzaldehyde, anisaldehyde, and 4-
tolaldehyde showed relatively low reactivity, the reactions gave
the desired products 6h–6j in acceptable yields (entries 8–10). The
reaction using alkylaldehydes proceeded without incident to afford
the products 6k and 6l in good yields (entries 11 and 12). In entry
13, the reaction using paraformaldehyde in a modified 1.5:1.5:1
molar ratio of 12a:13:9 gave the simple 4,6-unsubstituted 6m in
38% yield. The yield in the reaction using formalin solution was
<20%, and paraformaldehyde showed higher reactivity. The result
for entry 13 demonstrated the synthesis of 6m from commercially
available reagents in one step, whereas only multistep synthetic
methods to obtain 6m have been reported thus far.9 The reactions
using urea 12b also proceeded smoothly to give 2-oxo products
7a–7c in high yields (entries 14–16). Unfortunately, the use of a
ketone, heptan-4-one, in the reaction failed to produce the desired
4,4-dialkyl product.
MeO
NH2
R
R
CO2Et
CO2Et
1. MeI, Et3N
PPTS
CH2Cl2, reflux
N
HN
6
1
N
MeS
MeO
S
N
2. NaH, Boc2O
H
Boc
6l
14a
14b
(R = n-C3H7)
(R = n-C3H7)
6m
(R = Ph)
(R = Ph)
14c
from
(R = H)
Ref. 11
Scheme 3. Attempt to synthesize 6-unsubstituted 2-
aminodihydropyrimidine 8
derivative 14c (R = H) with 4-methoxyaniline in the presence of
0.1 eq of PPTS exclusively gave 15c (R = H) in 76% yield with a
minute amount (6%) of the 2-substituted product 8c (R = H). These
results indicate that different 4-substituents (H, n-C3H7, and Ph)
have a crucial effect on the reactivity. Our research is underway to
clarify the details.
Table 2. Synthesis of 6-unsubstituted 3,4-dihydropyrimidin-2-
thiones 6 and 2-ones 7
In addition to 6 and 7, 6-methyl DHPMs 16a and 16b were also
prepared in order to clarify the 6-substituent on their biological
activities (Scheme 4). To explore the antiproliferative effect of
fourteen compounds of 6 and 7 (excluding 6m and 7c), 16a, and
16b in HL-60 cells, the viability of the cells was determined by
6-Unsubstituted 2-aminodihydropyrimidines, such as 8 in
Scheme 3, have been hitherto unavailable; thus, we next
attempted to transform the 2-thioxo group of 6 into an amino
group. The 4-propyl derivative 6l was S-methylated, and the
corresponding 2-methylthio derivative was obtained as a mixture
of tautomers (Scheme 3). In our previous report, a Boc group on
the nitrogen atom of dihydropyrimidines increased the
electrophilicity in the substitution reaction with amines, and the
group was introduced.10 The reaction occurred selectively at the
nitrogen atom in the 1-position to give 14a (R = n-C3H7); the
structure of 14a was determined by HSQC (heteronuclear single
quantum correlation) and HMBC (heteronuclear multiple bond
correlation) experiments. A significant HMBC was observed
between the 6-proton and the carbonyl carbon of the Boc group at
the 1-position (see Supplementary Material). Subsequently, 14a
was reacted with 4-methoxyaniline in the presence of 1.2 eq of
P P T S ( p y r i d i n i u m p - t o l u e n e s u l f o n a t e ) , a n d 2 -
XTT
[2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-
hydroxide] assay
{(phenylamino)carbonyl}-2H-tetrazolium
following treatment for 96 h with 1,000 nM ATRA (all-trans
retinoic acid, a positive control reagent).12 As shown in Figure 2,
treatment with ATRA and 16b showed relatively strong activity (p
< 0.01).
R
O
H
R
13
(1.0 eq)
AlCl3 6 H2O
(20 mol%)
CO2Et
Me
CO2Et
HN
NH2
S
N
H
6
EtOH, reflux, 6 h
S
NH2
O
Me
12a
16a
16b
(1.3 eq)
(R = cyclo-C6H11) 26%
(R = n-C3H7) 38%
17
(1.0 eq)

following treatment for 96 h with various concentrations (100, 300,
700, and 1,000 nM) of 16b.13–15 As shown in Figure 3 (left), a dose-
dependent decrease of TB-negative cells (viable cells) was
observed in 16b-treated HL-60 cells. The IC50 value was 952.2 nM
(95% confidence interval, 658.4−2428; R2 = 0.6679). Moreover,
the ratio of the number of TB-positive cells (dead cells) to that of
TB-negative cells in HL-60 cells at each concentration of 16b
resulted in an increase in dead cells rate in a dose-dependent
manner [Figure 3 (right)]. The detail types of cell death, such as
apoptosis, necrosis, autophagy, and so on, may become clear in
future studies. These results suggest that the 16b-induced
antiproliferative effect was derived in large part from cell death;
thus, 16b inducing cell death at 100 nano-order concentrations
might be a leading compound for the development of novel
anticancer agents.
Scheme 4. Preparation of 16
In summary, a three-component reaction involving ureas,
aldehydes, and ethyl 3-dimethylaminoacrylate using anhydrous
AlCl3 to give 6 and 7 has been developed. General, efficient and
catalytic methods for synthesis of 6 and 7 have not been reported
thus far.4,5 A synthetic novelty of our protocol is as follows: 1)
Lewis acid-mediated reaction, 2) good to high yields, 3) broad
scope as for aldehydes, and ureas. Hitherto unavailable 2-amino
derivatives 8 have been obtained by transformation of the 2-thioxo
group of 6 via the substitution reaction of 14 with amines.
Although 6 and 7 showed a weak antiproliferative effect on HL-
60 cells, related 6-methyl derivative 16b showed relatively strong
activity with 100 nano-order IC50 value.
Figure 2. Antiproliferative effect of 6, 7, and 16 in HL-60 cells.
Following treatment for 96 h with 1,000 nM ATRA, 6, 7, and 16,
respectively, the cell viability of HL-60 cells was determined by
XTT assay as described in “Supplementary Material”. Relative
cell viability was calculated as the ratio of the absorbance at 450
nm of each treatment group against those of the corresponding
untreated control group. Data are shown as the means and SD
†
(standard deviation) from three independent experiments. p <
0.01 vs. control.
Acknowledgments
This work was financially supported by JSPS KAKENHI Grant
Number 16K08335, 16K01939, and 19K05703.
Supplementary Material
Supplementary Material (synthesis and characterization of
compounds, spectroscopic data of IR, NMR, MS) associated with
the article can be found, in the online version, at doi:
******/j.tetlet. *********.
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Figure 3. 16b induces cell death in a dose-dependent manner.
Following treatment with various concentrations of 16b for 96 h,
the cell viability of HL-60 cells was determined by TB exclusion
test as described in “Supplementary Material”. (Left) Relative cell
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the ratio of TB-positive cells to TB-negative cells in HL-60 cells
treated with each concentration of 16b was calculated. Data are
shown as the means and SD from three independent experiments.
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(5 mL), dried over anhydrous MgSO4, and concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography [n-hexane-EtOAc (4:1 to 2:1)] to give 6a (78.9 mg,
0.266 mmol, 88%) as pale yellow crystals.
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Synthesis of 6-unsubstituted 2-oxo, 2-thioxo, and 2-
amino-3,4-dihydropyrimidines and their
antiproliferative effect on HL-60 cells
Leave this area blank for abstract info.
Yoshio Nishimuraa,*, Hidetomo Kikuchib, Takanori Kuboa, Yuki Gokurakujia, Yuri Nakamuraa, Rie Araib, Bo Yuanb,
Katsuyoshi Sunagab, Hidetsura Choc
R
R
R
O
H
CO2Et
CO2Et
CO2Et
AlCl3
HN
HN
NH2
2
6
6
heating
2
R' N
X
N
N
X
NH2
Me2N
H
H
2-(N, O, and S-) substituted derivatives
X = S, O
1) Lewis acid-mediated reaction
2) good to high yields
3) broad scope for aldehydes and ureas