Synthesis and antiproliferative activitiy of novel diaryl ureas possessing a 4H-pyrido[1,2-a]pyrimidin-4-one group

Article abstract of DOI:10.1002/ardp.200900130

We herein disclose a series of novel diaryl urea derivatives possessing a 4H-pyrido[1,2-a]pyrimidin-4-one group as novel potent anticancer compounds. The structures were confirmed by IR, ¹H-NMR, and MS. All the compounds were screened for their antiprofilerative activity agaist the human breast cancer cell line (MDA-MB-231). The pharmacological results indicated that most of the compounds showed moderate activity. The best of this series is compound 4c (IC₅₀ = 0.7 μmol/L), with a potency 3.6-fold higher than Sorafenib (IC₅₀ = 2.5 μmol/L), which was approved in 2005.

Full text of DOI:10.1002/ardp.200900130

Arch. Pharm. Chem. Life Sci. 2010, 343, 17–23
P. Yao et al.
17
Full Paper
Synthesis and Antiproliferative Activitiy of Novel Diaryl Ureas
Possessing a 4H-Pyrido[1,2-a]pyrimidin-4-one Group
Peng Yao, Xin Zhai, Dong Liu, Bao Hui Qi, Hai Liang Tan, Yong Cai Jin, and Ping Gong
Key Lab of New Drugs Design and Discovery of Liaoning Province, School of Pharmaceutical Engineering,
Shenyang Pharmaceutical University, Shenyang, P. R. China
We herein disclose a series of novel diaryl urea derivatives possessing a 4H-pyrido[1,2-a]pyrimi-
din-4-one group as novel potent anticancer compounds. The structures were confirmed by IR, 1H-
NMR, and MS. All the compounds were screened for their antiprofilerative activity agaist the
human breast cancer cell line (MDA-MB-231). The pharmacological results indicated that most of
the compounds showed moderate activity. The best of this series is compound 4c (IC50 = 0.7
lmol/L), with a potency 3.6-fold higher than Sorafenib (IC50 = 2.5 lmol/L), which was approved in
2005.
Keywords: Antiprofilerative activity / 4H-Pyrido[1,2-a]pyrimidin-4-one / Synthesis /
Received: June 24, 2009; accepted: September 11, 2009
DOI 10.1002/ardp.200900130
Introduction
Cancer is considered to be one of the major killer diseases
worldwide. It is caused by mutations in critical genes
that alter normal cell functioning. Since kinases are
involved in many critical biological signaling pathways,
which are essential for the cell cycle regulation [1], the
development of selective protein kinase inhibitor is
widely considered a promising approach for antitumor
Figure 1. Structures of Sorafenib and target compounds.
drug development. Since the discovery of Sorafenib,
which had been approved by FDA for use in patients with
hepatocellular carcinoma and renal cell carcinoma in the selective introduction of a 4H-pyrido[1,2-a]pyrimidin-
2005 [2], derivatives with a diaryl urea framework consti- 4-one group and the modification of the aryl substituents
tuted an interesting class of compounds as multi-target on the 1-position at the diaryl urea framework should be
protein kinase inhibitors [3].
of significant importance for the antitumor activity.
In this context, many derivatives based on the essential Thus, in our efforts developing new antitumor agents
diaryl urea scaffold were synthesized [3–5], however, with improved solubility and better biological interac-
tions, 20 new diaryl urea derivatives possessing a 4H-pyr-
there are hardly any studies referring to compounds
bearing a 4H-pyrido[1,2-a]pyrimidin-4-one group so far. ido[1,2-a]pyrimidin-4-one group were designed and syn-
After reading many references, we had concluded that thesized (Fig. 1). All the prepared compounds, which are
represented by the general structure of 4a–4f, 8a–8g,
13a–13g in Tables 1 and 2, were identified as potent anti-
Correspondence: Key Lab of New Drugs Design and Discovery of
tumor agents and screened for their antiprofilerative
activity against the human breast cancer cell line (MDA-
MB-231; Sorafenib had been tested against MDA-MB-231
by Chunrong Yu et al.).
Liaoning Province, School of Pharmaceutical Engineering, Shenyang
Pharmaceutical University, Ping Gong, 103 Wenhua Road, Shenhe Dis-
trict, 110016 Shenyang, Liaoning, P. R. China.
E-mail: gongpinggp@126.com
Fax: +86 24 2398-6429
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18
P. Yao et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 17–23
Table 1. The substituents and IC50 values against the human
breast cancer cell line of compounds 4a–4f and 8a–8g.
Compound
R1
R2
IC50 (lmol/L)
MDA-MB-231
4a
4b
4c
4d
4e
4f
8a
8b
8c
8d
8e
8f
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
2,6-difluoro
2-fluoro
3-fluoro
3-trifluoromethyl
3-chloro-4-fluoro
4-fluoro
9.5
2.1
0.7
1.8
4.2
1.6
5.1
20.0
8.2
14.0
5.9
3.9
4.4
2.5
4-fluoro
2,6-difluoro
3-chloro-4-fluoro
3,5-dichloro
2-fluoro
3-fluoro
3-trifluoromethyl
Reagents and conditions: (a) Polyphosphate, 45 – 1258C, 2 h; (b) K2CO3, DMF, 4-
nitrophenol, 758C, 6 h; (c) Fe/AcOH, 95% EtOH, reflux, 4 h; (d) substituted arylisocya-
nate, DMF, 308C, 2 – 3 h.
Scheme 1. Synthesis of the compounds 4a–4f and 8a–8g.
H
H
8g
Sorafenib
Table 2. The substituents and IC50 values against the human
breast cancer cell line of compounds 13a–13g.
Compound
R
IC50 (lmol/L)
MDA-MB-231
13a
13b
13c
13e
13f
13g
Sorafenib
3-fluoro
2,6-difluoro
2-fluoro
4-fluoro
3-trifluoromethyl
3-chloro-4-fluoro
188.0
102.0
71.0
46.0
37.0
41.0
2.5
Reagents and conditions: (a) Conc. sulfuric acid (98%), EtOH, reflux, 8 h; (b) DMF/
DMA, reflux, 4 – 5 h; (c) Pyridin-2-amine, AcOH, reflux, 6 h; (d) Fe/AcOH, EtOH,
reflux, 8 h; (e) substituted arylisocyanate, DMF, 308C, 2 – 4 h.
Scheme 2. Synthesis of the compounds 13a–13g.
thesized from 3 with a variety of substituted arylisocya-
nates [9]. The new compounds 8a–8g were prepared suc-
cessfully according to the same method as described for
4a–4f when the initial material was replaced with pyri-
Results and discussion
Chemistry
The title compounds 4a–4f were synthesized via a con- din-2-amine (Scheme 1).
venient four-step method outlined in Scheme 1. The com-
Compounds 13a–13g were synthesized as shown in
mercially available 4-methylpyridin-2-amine in polyphos- Scheme 2. 2-(4-Nitrophenyl)acetic acid was refluxed in
phate was cyclizated with ethyl 4-chloro-3-oxobutanoate ethanol with 0.15 equivalents of 98% sulfuric acid to
at 1258C to provide compound 1 as a grey solid [6], which afford compound 9 in good yield [10], which was then
was then reacted with 4-nitrophenol in the presence of treated with DMF/DMA to give 10 [11]. The cyclization of
K2CO3 in DMF to afford 2 [7]. Subsequently, the intermedi- 10 with pyridine-2-amine was carried out in acetic acid to
ate 2 underwent reduction with Fe in ethanol to produce obtain the intermediate 11 [12, 13]. The intermediate 11
compound 3 [8]. The target compounds 4a–4f were syn- was subsequently reduced with powdered iron in ethanol
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Arch. Pharm. Chem. Life Sci. 2010, 343, 17–23
4H-Pyrido[1,2-a]pyrimidin-4-one
19
lmol/L and 8b, IC50 = 20 lmol/L). In addition, the bulki-
ness of the substituent can also affect the activity, a
flouro atom was superior to a trifluoromethyl group
(4c vs. 4d and 8f vs. 8g).
In conclusion, a series of diaryl urea derivatives pos-
sessing a 4H-pyrido[1,2-a]pyrimidin-4-one group were
designed and synthesized in order to find potent antitu-
mor compounds. All the compounds were screened for
their antiproliferative activity against the human breast
cancer cell line MDA-MB-231. The pharmacological
results indicated that most of the compounds showed
moderate activity, and the best one of them was com-
pound 4c (IC50 = 0.7 lmol/L), with potency a 3.6-fold
higher potency than Sorafenib (IC50 = 2.5 lmol/L). The pre-
liminary structure-activity relationship (SAR) showed
that the oxymethylene group on the 4-position of ben-
zene ring was essential for the activity, and a methyl
group was favorable on the 8-position of 4H-pyrido[1,2-a]
pyrimidin-4-one. Furthermore, the position of the substi-
tutent and its size also contributed important effects to
the activity. The SAR results of this study will provide use-
ful information for the design of novel diaryl urea antitu-
mor derivatives bearing fused heterocyclic groups.
Reagents and conditions: (a) Bis(trichloromethyl)carbonate(BTC), ethyl acetate,
reflux, 12 h.
Scheme 3. Synthesis of the compounds 14a–14g.
to give compound 12 [8], which was reacted with substi-
tuted arylisocyanate to afford the target compounds
13a–13g [9]. In addition, the intermediates 14a–14g
were prepared from substituted anilines with bis(tri-
chloromethyl)carbonate (BTC) in ethyl acetate or 1,4-
dioxane at 808C [14] (Scheme 3).
Antiproliferative activity and discussion
The antiproliferative activity of the synthesized com-
pounds 4a–4f, 8a–8g, and 13a–13g against MDA-MB-231
(human breast cancer cell lines) were determined by MTT
assay. Sorafenib was used as positive control, and the
results expressed as IC50 values are summarized in Table
1 and Table 2.
As listed in Tables 1 and 2, most of the compounds
showed moderate activity, while compounds 4b, 4c, 4d,
and 4f, having IC50 values ranging from 0.7 to 2.1 lmol/L
with the R2 moieties 2-fluoro, 3-fluoro, 3-trifluomethyl,
and 4-fluoro, respectively, exhibited an activity more
potent than that of Sorafenib (IC50 = 2.5 lmol/L).
All the compounds in Table 1 were more potent than
those in Table 2, which suggests that the oxymethylene
group on the 4-position of the benzene ring was essential
for the activity. In our opinion, the oxygen atom is able
to act as a receptor of hydrogen bonds; hydrogen bonds
are formed with some amino acid residues of a variety of
enzymes.
On the other hand, compounds 4a–4f and 8a–8g were
chosen as examples to study the effect of different sub-
stituents on the antitumor activity. The activity of 4a–4g
bearing a methyl group on the 8-position of the 4H-pyr-
ido[1,2-a]pyrimidin-4-one moiety was decreased nearly 1
to 7-times compared to 8a–8f substituted with a hydro-
gen atom (4b vs. 8e, 4c vs. 8f, and 4d vs. 8g). One can con-
clude that a methyl group is favorable in this region.
The substitution position of groups on the 1-anilino
moiety also exerts an important effect on the pharma-
cological activity. Generally, substituents on the 3-
position were optimal (4c, IC50 = 0.7 lmol/L and 8f, IC50
= 3.9 lmol/L), followed by the 4-position (4f, IC50 = 1.6
lmol/L and 8a, IC50 = 5.1 lmol/L), while the introduc-
tion of substituents both on the 2- and 6-positions will
cause a dramatically decreased activity (4a, IC50 = 9.5
Experimental
Chemistry
All melting points were obtained on a Bꢀchi Melting Point B-540
apparatus (Bꢀchi Labortechnik, Flawil, Switzerland) and are
uncorrected. Mass spectra (MS) were taken in ESI mode on Agi-
lent 1100 LC-MS (Agilent, Palo Alto, CA, USA). Proton (1H) nuclear
magnetic resonance spectroscopy was performed using Bruker
ARX-300, 300 MHz spectrometers (Bruker Bioscience, Billerica,
MA, USA) with TMS as an internal standard. IR spectra (KBr disks)
were recorded with a Bruker IFS 55 instrument (Bruker). Elemen-
tal analysis was determined on a Carlo-Erba 1106 Elemental
analysis instrument (Carlo Erba, Milan, Italy). The oxygen anal-
ysis was carried out using the Unterzaucher principle by sample
pyrolysis in a helium stream at 16008C. Unless otherwise noted,
all solvents and reagents were commercially available and used
without further purification.
2-(Chloromethyl)-8-methyl-4H-pyrido[1,2-a]pyrimidin-4-
one 1
A mixture of 4-methylpyridin-2-amine (38 g, 0.35 mol) and ethyl
4-chloro-3-oxobutanoate (55 mL) in PPA (250 g) was stirred at
1258C for 2 h and then cooled to room temperature, poured into
water, and extracted with CHCl3. The extract was washed with
water and brine, dried (MgSO4), filtered and concentrated to pro-
vide pale solid (48 g, 64.7%). M.p.: 110–1128C; MS (ESI) m/z: 210.1
[M + H+].
8-Methyl-2-((4-nitrophenoxy)methyl)-4H-pyrido[1,2-a]
pyrimidin-4-one 2
A mixture of 1 (21 g, 0.1 mol), 4-nitrophenol (15.3 g, 0.11 mol)
and K2CO3 (41.4 g, 0.3 mol) in DMF was heated at 758C for 5 h. It
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P. Yao et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 17–23
was then poured into water, filtered and washed with water,
and dried to give the title compound without further purifica-
tion (25.1 g, 80%). M.p.: 156–1598C; MS (ESI) m/z: 312.1 [M + H+].
1-(4-((8-Methyl-4-oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)
methoxy)phenyl)-3-(3-(trifluoromethyl)phenyl)urea 4d
Prepared from 3 and 14f, 73% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3332.3, 3089.7, 2660.2, 1737.0, 1656.5, 1555.3, 1512.6, 1443.7,
1319.9, 1248.8, 1184.9, 1043.1; 1H-NMR (DMSO-d6) d [ppm]: 9.21
(s, 1H), 8.94–8.97 (d, J = 7.2 Hz, 1H), 8.90 (s, 1H), 8.02 (s, 1H), 7.65
(s, 1H), 7.57–7.59 (d, J = 8.4 Hz, 1H), 7.48–7.51 (d, J = 7.8 Hz, 1H),
7.41–7.44 (d, J = 8.7 Hz, 3H), 7.26–7.29 (d, J = 7.5 Hz, 1H), 7.00–
7.03 (d, J = 8.7 Hz, 2H), 6.53 (s, 1H), 5.13 (s, 2H), 2.55 (s, 3H); MS m/
z: 469.1 [M + H+]. Anal. calcd. for C24H19F3N4O3: C, 61.54; H, 4.09; N,
11.96; O, 10.25. Found: C, 61.48; H, 4.07; N, 12.01; O, 10.19.
2-((4-Aminophenoxy)methyl)-8-methyl-4H-pyrido[1,2-a]
pyrimidin-4-one 3
A mixture of 3 (21.8 g, 0.07 mol), Fe (23.5 g, 0.42 mol), and
CH3COOH (1 mL) in 95% ethanol (300 mL) was refluxed for 4 h. The
mixture was filtered immediately and washed three times with
hot ethanol. The combined filtrates were evaporated in vacuo to
yield a light yellow solid, which was then crystallized from etha-
nol and water. Compound 3 was afforded as a light yellow solid
(15.9 g, 81%). M.p.:172–1758C. MS(ESI) m/z:282.1 [M+ H+].
1-(3-Chloro-4-fluorophenyl)-3-(4-((8-methyl-4-oxo-4H-
pyrido[1,2-a]pyrimidin-2-yl)methoxy)phenyl)urea 4e
Prepared from 3 and 14g, 74% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3279.3, 3084.8, 1736.9, 1652.6, 1603.4, 1555.0, 1500.4, 1384.4,
General procedure for preparation of compounds 4a–4f
Compound 3 (0.5 g, 1.78 mmol) was dissolved in N,N-dimethyl-
formamide (10 mL) at 308C. Then, substituted isocyanate (2.67
mmol) was added and the mixture was stirred for 3 h, poured
into water, filtered, and washed with water. The crude product
was dissolved in refluxed ethanol and chloroform, then, meth-
ane sulfonic acid (approx. 2 mmol) was added. The mixture was
refluxed for 0.5 h, filtered, and dried to afford yellow solid 4a–
4f, 8a–8g, and 13a–13g.
1
1313.4, 1207.5, 1168.6, 1047.4; H-NMR (DMSO-d6) d [ppm]: 9.02
(s, 1H), 8.95–8.97 (d, J = 7.2 Hz, 1H), 8.83 (s, 1H), 7.78–7.80 (d, J =
7.2 Hz, 1H), 7.65 (s, 1H), 7.39–7.45 (t, J1 = 9.6 Hz, J2 = 9 Hz, 3H),
7.30–7.33 (t, J1 = 6.6 Hz, J2 = 1.2 Hz, 2H), 7.00–7.03 (d, J = 9 Hz,
2H), 6.53 (s, 1H), 5.13 (s, 2H), 2.56 (s, 3H); MS m/z: 454.1 [M + H+].
Anal. calcd. for C23H18ClFN4O3: C, 61.00; H, 4.01; N, 12.37; O,
10.60. Found: C, 60.95; H, 4.07; N, 12.21; O, 10.57.
1-(4-Fluorophenyl)-3-(4-((8-methyl-4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 4f
1-(2,6-Difluorophenyl)-3-(4-((8-methyl-4-oxo-4H-
pyrido[1,2-a]pyrimidin-2-yl)methoxy)phenyl)urea 4a
Prepared from 3 and 14b, 81% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3272.6, 1707.6, 1646.6, 1605.3, 1559.2, 1508.7, 1479.6, 1332.3,
Prepared from 3 and 14e, 77% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3282.9, 1735.8, 1641.6, 1560.1, 1507.2, 1336.6, 1213.9, 1166.7,
1039.2; 1H-NMR (DMSO-d6) d [ppm]: 8.96–8.98 (d, J = 7.2 Hz, 1H),
8.80 (s, 1H), 8.70 (s, 1H), 7.67 (s, 1H), 7.39–7.46 (m, 5H), 7.07–7.13
(t, J = 9 Hz, 2H), 6.99–7.02 (d, J = 9 Hz, 2H), 6.54 (s, 1H), 5.13 (s,
2H), 2.58 (s, 3H); MS m/z: 419.0 [M + H+]. Anal. calcd. for
C23H19FN4O3: C, 66.02; H, 4.58; N, 13.39; O, 11.47. Found: C, 65.98;
H, 4.60; N, 13.41; O, 11.44.
1
1240.7, 1172.0, 1071.9, 1002.6; H-NMR (DMSO-d6) d [ppm]: 8.87
(s,1H),8.79 (s, 1H), 8.05 (s, 1H), 7.53 (s, 1H), 7.37 (s, 2H), 7.27 (s, 2H),
7.13 (s, 2H), 6.99 (s, 2H), 6.39 (s, 1H), 5.04 (s, 2H), 2.49 (s, 3H); MS
m/z: 437.1 [M + H+]. Anal. calcd. for C23H18F2N4O3: C, 63.30; H, 4.16;
N, 12.84; O, 11.00. Found: C, 63.24; H, 4.19; N, 12.91; O, 11.04.
1-(2-Fluorophenyl)-3-(4-((8-methyl-4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy) phenyl)urea 4b
2-(Chloromethyl)-4H-pyrido[1,2-a]pyrimidin-4-one 5
Prepared in a similar procedure as described for 1, prepared
from pyridin-2-amine and ethyl 4-chloro-3-oxobutanoate, 67%
yield. M.p.: 98–1018C; MS (ESI) m/z: 196.1 [M + H+].
Prepared from 3 and 14c, 80% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3277.0, 3062.4, 1736.1, 1661.4, 1601.4, 1545.7, 1510.1, 1454.6,
1335.5, 1218.5, 1185.8, 1164.3, 1034.2; 1H-NMR (DMSO-d6) d
[ppm]: 9.03 (s, 1H), 8.97–8.99 (d, J = 7.2 Hz, 1H), 8.54 (s, 1H), 8.08–
8.13 (t, J = 8.1 Hz, 1H), 7.70 (s, 1H), 7.48–7.50 (d, J = 7.2 Hz, 1H),
7.4–7.43 (d, J = 8.7 Hz, 2H), 7.18–7.25 (dd, J1 = 11.4 Hz, J2 = 8.4 Hz,
1H), 7.09–7.14 (t, J = 7.5 Hz, 1 H), 6.97–7.04 (m, J1 = 8.7 Hz, J2 = 6.3
Hz, 3H), 6.57 (s, 1H), 5.16 (s, 2H), 2.57 (s, 3H); MS m/z: 419.1 [M +
H+]. Anal. calcd. for C23H19FN4O3: C, 66.02; H, 4.58; N, 13.39; O,
11.47. Found: C, 66.00; H, 4.55; N, 13.41; O, 11.44.
2-((4-Nitrophenoxy)methyl)-4H-pyrido[1,2-a]pyrimidin-4-
one 6
Prepared in a similar procedure as described for 2, prepared
from 5 and 4-nitrophenol, 81% yield. M.p.: 148–1518C; MS (ESI)
m/z: 298.1 [M + H+].
2-((4-Aminophenoxy)methyl)-4H-pyrido[1,2-a]pyrimidin-
1-(3-Fluorophenyl)-3-(4-((8-methyl-4-oxo-4H-pyrido[1,2-a] 4-one 7
Prepared in a similar procedure as described for 3, prepared
pyrimidin-2-yl)methoxy)phenyl)urea 4c
from 6, 80% yield. M.p.: 205–2088C; MS (ESI) m/z: 268.1 [M + H+].
Prepared from 3 and 14a, 78% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3271.7, 3086.8, 1734.3, 1661.3, 1607.7, 1553.8, 1513.2, 1439.9,
1
1384.0, 1335.0, 1218.3, 1160.8, 1089.8, 1036.8; H-NMR (DMSO-
General procedure for preparation of compounds 8a–8g
Prepared in a similar procedure as described for 4a–4f.
d6) d [ppm]: 9.03 (s, 1H), 8.96–8.98 (d, J = 7.2 Hz, 1H), 8.80 (s, 1H),
7.67 (s, 1H), 7.44–7.51 (m, 2H), 7.40–7.43 (d, J = 9 Hz, 2H), 7.24–
7.32 (dd, J1 = 8.1 Hz, J2 = 15 Hz, 1H), 7.11–7.13 (d, J = 8.1 Hz, 1H),
7.0–7.03 (d, J = 9.3 Hz, 2H), 6.72–6.77 (m, 1H), 6.55 (s, 1H), 5.14 (s,
2H), 2.57 (s, 3H); MS m/z: 419.1 [M + H+]. Anal. calcd. for
C23H19FN4O3: C, 66.02; H, 4.58; N, 13.39; O, 11.47. Found: C, 65.98;
H, 4.57; N, 13.41; O, 11.51.
1-(4-Fluorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8a
Prepared from 7 and 14e, 78% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3317.5, 3077.9, 1725.6, 1658.0, 1611.0, 1563.3, 1503.7, 1446.4,
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Arch. Pharm. Chem. Life Sci. 2010, 343, 17–23
4H-Pyrido[1,2-a]pyrimidin-4-one
21
1339.1, 1313.7, 1232.1, 1205.4, 1162.3, 1038.0;1H-NMR (DMSO-d6)
d [ppm]: 9.03–9.05 (d, J = 6.9 Hz, 1H), 8.81 (s, 1H), 8.71 (s, 1H),
8.17–8.22 (t, J = 7.5 Hz, 1H), 7.82–7.85 (d, J = 8.7 Hz, 1H), 7.50–
7.55 (t, J = 6.9 Hz, 1H), 7.44–7.48 (dd, J1 = 9 Hz, J2 = 5.1 Hz, 2H),
7.39–7.42 (d, J = 8.7 Hz, 2H), 7.06–7.12 (t, J = 9 Hz, 2H), 6.99–7.02
(d, J = 8.7 Hz, 2H), 6.57 (s, 1H), 5.13 (s, 2H); MS m/z: 404.1 [M + H+].
Anal. calcd. for C22H17FN4O3: C, 65.34; H, 4.24; N, 13.85; O, 11.87.
Found: C, 65.38; H, 4.20; N, 13.91; O, 11.84.
1-(3-Fluorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8f
Prepared from 7 and 14a, 76% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3274.0, 3100.5, 1736.7, 1661.4, 1554.0, 1513.4, 1440.8, 1336.2,
1219.2, 1160.0, 1039.6; 1H-NMR (DMSO-d6) d [ppm]: 9.02–9.04 (d,
J = 8.7 Hz, 2H), 8.80 (s, 1H), 8.14–8.19 (t, J = 7.5 Hz, 1H), 7.81–7.83
(d, J = 8.7 Hz, 1H), 7.47–7.53 (m, 2H), 7.39–7.42 (d, J = 9 Hz, 2H),
7.23–7.31 (dd, J1 = 15 Hz, J2 = 7.8 Hz, 1H), 7.10–7.13 (d, J = 8.1 Hz,
1H), 7.00–7.03 (d, J = 9 Hz, 2H), 6.72–6.77 (m, 1H), 6.56 (s, 1H),
5.13 (s, 2H); MS m/z: 405.1 [M + H+]. Anal. calcd. for C22H17FN4O3: C,
65.34; H, 4.24; N, 13.85; O, 11.87. Found: C, 65.36; H, 4.21; N,
13.88; O, 11.84.
1-(2,6-Difluorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8b
Prepared from 7 and 14b, 85% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3283.2, 1712.8, 1698.2, 1645.0, 1619.7, 1604.1, 1560.9, 1510.1,
1467.0, 1383.4, 1241.6, 1074.3, 1003.1; 1H-NMR (DMSO-d6) d
[ppm]: 8.94–8.97 (d, J = 6.9 Hz,1H), 8.79 (s, 1H), 8.04 (s, 1H) 7.97–
8.02 (t, J1 = 8.4 Hz, J2 = 7.2 Hz, 1H) 7.68–7.71 (d, J = 9.0 Hz, 1H),
7.35–7.38 (t, J1 = 3 Hz, J2 = 5.7 Hz, 3H), 7.26–7.32 (t, J = 7.8 Hz, 1H),
7.11–7.16 (t, J = 8.1 Hz, 2H) , 6.97–7.0 (d, J = 9.0 Hz, 2H) , 6.45 (s,
1H), 5.01 (s, 2H). MS m/z: 415.1 [M + H+]. Anal. calcd. for
C22H16F2N4O3: C, 62.56; H, 3.82; N, 13.26; O, 11.36. Found: C,
62.58; H, 3.80; N, 13.21; O, 11.40.
1-(4-((4-Oxo-4H-pyrido[1,2-a]pyrimidin-2-yl)methoxy)
phenyl)-3-(3-(trifluoromethyl)phenyl)urea 8g
Prepared from 7 and 14f, 72% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3308.9, 3100.5, 1749.8, 1675.9, 1566.9, 1513.6, 1447.0, 1383.8,
1337.9, 1223.1, 1163.8, 1111.0, 1046.2; 1H-NMR (DMSO-d6) d
[ppm]: 9.06 (s, 1H), 8.98–9.0 (d, J = 6.9 Hz, 1H), 8.73 (s, 1H), 8.04–
8.09 (t, J = 7.5 Hz, 1H), 8.01 (s, 1H), 7.73–7.76 (d, J = 8.7 Hz, 1H),
7.55–7.58 (d, J = 8.1 Hz, 1H), 7.46–7.52 (t, J = 7.5 Hz, 1H), 7.38–
7.41 (d, J = 8.7 Hz, 2H), 7.27–7.29 (d, J = 7.2 Hz, 1H), 7.0–7.03 (d,
J = 8.7 Hz, 2H), 6.50 (s, 1H), 5.10 (s, 2H); MS: 455.0 [M + H+]. Anal.
calcd. for C23H17F3N4O3: C, 60.79; H, 3.77; N, 12.33; O, 10.56.
Found: C, 60.78; H, 3.79; N, 12.37; O, 10.54.
1-(3-Chloro-4-fluorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8c
Prepared from 7 and 14g, 74% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3363.2, 1747.1, 1661.4, 1601.7, 1552.8, 1498.2, 1384.3, 1297.9,
1206.4, 1042.5; 1H-NMR (DMSO-d6) d [ppm]: 9.03–9.05 (d, J = 3.9
Hz, 2H), 8.85 (s, 1H), 8.17–8.23 (m, 1H), 7.78–7.86 (m, 2H), 7.51–
7.55 (t, J = 6.9 Hz, 1H), 7.39–7.42 (d, J = 9 Hz, 2H), 7.30–7.33 (d, J =
7.8 Hz, 2H), 7.0–7.03 (d, J = 9 Hz, 2H), 6.57 (s, 1H), 5.14 (s, 2H); MS
m/z: 440.0 [M + H+]. Anal. calcd. for C22H16ClFN4O3: C, 60.21; H,
3.67; N, 12.77; O, 10.94. Found: C, 60.18; H, 3.70; N, 12.81; O,
10.91.
Ethyl 2-(4-nitrophenyl)acetate 9
To a solution of 2-(4-nitrophenyl)acetic acid (50 g, 0.276 mol) and
ethanol (250 mL), conc. H2SO4 (12 mL, 0.22 mol) was added drop-
wise in 30 min. The mixture was refluxed for 8 h, after that, it
was cooled to room temperature. Ethanol was evaporated in
vacuo, then poured into water, filtered, washed with water, and
dried to afford a white solid (53.4 g, 92.5%). M.p.: 64–668C; MS
m/z: 210.0 [M + H+].
Ethyl 3-(dimethylamino)-2-(4-nitrophenyl)acrylate 10
Compound 9 (50 g, 0.239 mol) was refluxed in DMF/DMA (48 mL)
and toluene (350 mL) for 4–5 h, the solvent was evaporated in
vacuo. The residue was extracted twice with 125 mL CH2Cl2. The
combined extracts were washed with brine and water, dried
(MgSO4), filtered, and evaporated in vacuum, followed by col-
umn chromatographic purification to afford the desired com-
pound 10 (43.6 g, 69.0%). M.p.: 139–1428C; MS (ESI) m/z: 265.1 [M
+ H+].
1-(3,5-Dichlorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8d
Prepared from 7 and 14d, 82% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3368.0, 3278.6, 3084.7, 1739.5, 1663.8, 1589.6, 1545.7, 1511.0,
1451.1, 1242.5, 1197.3, 1074.1, 1047.9; 1H-NMR (DMSO-d6) d
[ppm]: 9.23 (s, 1H), 9.02–9.04 (d, J = 6.6 Hz, 1H), 8.97 (s, 1H), 8.14–
8.17 (t, J1 = 7.8 Hz, J2 = 7.2 Hz, 1H), 7.80–7.83 (d, J = 8.7 Hz, 1H),
7.50–7.53 (d, J = 9.3 Hz, 3H), 7.39–7.42 (d, J = 8.7 Hz, 2H), 7.12 (s,
1H), 7.0–7.03 (d, J = 8.7 Hz, 2H), 6.56 (s, 1H), 5.13 (s, 2H); MS m/z:
456.1 [M + H+]. Anal. calcd. for C22H16Cl2N4O3: C, 58.04; H, 3.54; N,
12.31; O, 10.54. Found: C, 58.08; H, 3.50; N, 12.33; O, 10.51.
3-(4-Nitrophenyl)-4H-pyrido[1,2-a]pyrimidin-4-one 11
A solution of 10 (25 g, 94.7 mmol) and pyridin-2-amine (27.8 g,
284 mmol) in glacial acetic acid (150 mL) was refluxed for 6 h,
then cooled to 08C and filtered to give a light yellow solid which
was used without further purification (17.2 g, 68.0%). M.p.: 164–
1678C; MS (ESI) m/z: 268.1 [M + H+].
1-(2-Fluorophenyl)-3-(4-((4-oxo-4H-pyrido[1,2-a]
pyrimidin-2-yl)methoxy)phenyl)urea 8e
Prepared from 7 and 14c, 79% yield. M.p.: A3308C; IR (KBr) [cm– 1]:
3340.7, 1738.3, 1663.9, 1620.5, 1601.6, 1536.6, 1511.7, 1454.5,
1320.9, 1214.7, 1171.3, 1079.7, 1039.9; 1H-NMR (DMSO-d6) d
[ppm]: 8.96–9.01 (d, J = 6.3 Hz, 2H), 8.51 (s, 1H), 8.10–8.17 (d, J =
7.5 Hz, 2H), 7.79–7.81 (d, J = 8.7 Hz, 1H), 7.47–7.51 (t, J = 6.9 Hz,
1H), 7.38–7.41 (d, J = 8.7 Hz, 2H), 7.18–7.25 (t, J1 = 8.7 Hz, J2 = 7.8
Hz, 1H), 7.09–7.14 (t, J1 = 7.8 Hz, J2 = 7.2 Hz, 1H), 6.97–7.03 (t, J =
8.4 Hz, 3H), 6.54 (s, 1H), 5.12 (s, 2H); MS m/z: 405.1 [M + H+]. Anal.
calcd. for C22H17FN4O3: C, 65.34; H, 4.24; N, 13.85; O, 11.87.
Found: C, 65.38; H, 4.20; N, 13.87; O, 11.84.
3-(4-Aminophenyl)-4H-pyrido[1,2-a]pyrimidin-4-one 12
Prepared in a similar procedure as described for 3, prepared from
11, 58.5% yield. M.p.: 226–2298C;MS (ESI) m/z:238.1 [M + H+].
General procedure for preparation of compounds 13a–
13g
Prepared in a similar procedure as described for 4a–4f.
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P. Yao et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 17–23
Hz, 2H), 7.47–7.52 (m, 2H), 7.10–7.16 (t, J = 8.7 Hz, 2H); MS m/z:
375.1 [M + H+]. Anal. calcd. for C21H15FN4O2: C, 67.37; H, 4.04; N,
14.97; O, 8.55. Found: C, 67.35; H, 4.02; N, 14.95; O, 8.58.
1-(3-Fluorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl)urea 13a
Prepared from 12 and 14a, 73% yield. M.p.: A3308C; IR (KBr)
[cm–1]: 3273.7, 3089.7, 1725.0, 1601.0, 1534.7, 1490.5, 1384.1,
1324.3, 1220.8, 1182.0, 1043.4; 1H-NMR (DMSO-d6) d [ppm]: 9.18–
9.26 (m, 3H), 8.75 (s, 1H), 8.30–8.35 (t, J = 7.5 Hz, 1H), 7.93–7.96
(d, J = 8.7 Hz, 1H), 7.76–7.79 (d, J = 8.7 Hz, 2H), 7.66–7.71 (t, J = 6.9
Hz, 1H), 7.60–7.63 (d, J = 9 Hz, 2H), 7.50–7.54 (t, J1 = 12 Hz, J2 = 1.8
Hz, 1H), 7.27–7.35 (m, 1H), 7.16–7.18 (d, J = 8.1 Hz, 1H), 6.75–
6.82 (m, 1H); MS m/z: 375.1 [M + H+]. Anal. calcd. for C21H15FN4O2:
C, 67.37; H, 4.04; N, 14.97; O, 8.55. Found: C, 67.38; H, 4.08; N,
14.92; O, 8.54.
1-(4-(4-Oxo-4H-pyrido[1,2-a]pyrimidin-3-yl)phenyl)-3-
(3-(trifluoromethyl)phenyl)urea 13f
Prepared from 12 and 14f, 65% yield. M.p.: A3308C; IR (KBr)
[cm–1]: 3317.8, 3091.7, 1725.8, 1662.1, 1595.9, 1536.8, 1447.1,
1384.1, 1319.5, 1181.5,1119.2, 1047.2; 1H-NMR (DMSO-d6) d
[ppm]: 9.17–9.19 (d, J = 6 Hz, 2H), 9.05 (s, 1H), 8.70 (s, 1H), 8.11–
8.17 (t, J1 = 8.4 Hz, J2 = 7.2 Hz, 1H), 8.05 (s, 1H), 7.85 (s, 2H), 7.79–
7.82 (d, J = 9 Hz, 2H), 7.58–7.61 (d, J = 8.4 Hz, 2H), 7.50–7.55 (t, J1
= 7.5 Hz, J2 = 8.7 Hz, 2H), 7.31–7.33 (d, J = 7.2 Hz, 1H); MS m/z:
425.1 [M + H+]. Anal. calcd. for C22H15F3N4O2: C, 62.26; H, 3.56; N,
13.20; O, 7.54. Found: C, 62.25; H, 3.52; N, 13.25; O, 7.57.
1-(2,6-Difluorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl)urea 13b
Prepared from 12 and 14b, 68% yield. M.p.: A3308C; IR (KBr)
[cm–1]: 3261.1, 1725.0, 1649.7, 1599.1, 1535.0, 1466.6, 1384.4,
1296.6, 1191.8, 1042.0; 1H-NMR (DMSO-d6) d [ppm]: 9.28 (s, 1H),
9.23–9.26 (d, J = 6.9 Hz, 1H), 8.74 (s, 1H), 8.35 (brs, 2H), 7.94–7.97
(d, J = 9 Hz, 1H), 7.75–7.78 (d, J = 8.7 Hz, 2H), 7.67–7.72 (t, J = 6.9
Hz, 1H), 7.58–7.61 (d, J = 8.7 Hz, 2H), 7.27–7.37 (m, 1H), 7.12–
7.20 (m, 2H); MS m/z: 394.1 [M + H+]. Anal. calcd. for C21H14F2N4O2:
C, 64.28; H, 3.60; N, 14.28; O, 8.16. Found: C, 64.26; H, 3.61; N,
14.24; O, 8.14.
1-(3-Chloro-4-fluorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl) urea 13g
Prepared from 12 and 14g, 81% yield. M.p.: A3308C; IR (KBr)
[cm–1]: 3426.9, 1715.2, 1647.6, 1597.6, 1535.3, 1497.9, 1384.4,
1297.4, 1194.3, 1042.8;1H-NMR (DMSO-d6) d [ppm]: 9.19–9.21 (d, J
= 6.6 Hz, 1H), 9.08 (s, 2H), 8.70 (s, 1H), 8.17–8.19 (d, J = 7.2 Hz, 1H),
7.82–7.88 (t, J1 = 9.9 Hz, J2 = 8.4 Hz, 2H), 7.77–7.80 (d, J = 8.4 Hz,
2H), 7.57–7.60 (d, J = 8.7 Hz, 3H), 7.33–7.36 (t, J = 6.6 Hz, 2H); MS
m/z: 409.1 [M + H+]. Anal. calcd. for C21H14ClFN4O2: C, 61.70; H,
3.45; N, 13.70; O, 7.83. Found: C, 61.68; H, 3.48; N, 13.69; O, 7.86.
1-(2-Fluorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl)urea 13c
Evaluation of the biological activity
Prepared from 12 and 14c, 76% yield. M.p.: >3308C; IR (KBr)
[cm–1]: 3272.2, 1729.9, 1651.5, 1596.4, 1536.4, 1384.1, 1302.7,
1255.5, 1190.3, 1039.5; 1H-NMR (DMSO-d6) d [ppm]: 9.30 (s, 1H),
9.21–9.23 (d, J = 6.9 Hz, 1H), 8.73 (s, 1H), 8.65–8.66 (d, J = 2.1 Hz,
1H), 8.23–8.28 (t, J1 = 7.2 Hz, J2 = 8.4 Hz, 1H), 8.13–8.18 (dd, J1 =
8.1 Hz, J2 = 6.9 Hz, 1H), 7.89–7.92 (d, J = 8.7 Hz, 1H), 7.78–7.87 (d,
J = 8.7 Hz, 2H), 7.63–7.66 (d, J = 6.9 Hz, 1H), 7.57–7.60 (d, J = 8.7
Hz, 2H), 7.22–7.28 (m, 1H), 7.13–7.18 (t, J = 7.5 Hz, 1H), 6.99–
7.05 (m, 1H); MS m/z: 375.0 [M + H+]. Anal. calcd. for C21H15FN4O2:
C, 67.37; H, 4.04; N, 14.97; O, 8.55. Found: C, 67.34; H, 4.01; N,
14.99; O, 8.60.
The antiproliferative activities of compounds 4a–4f, 8a–8g, and
13a–13g were evaluated with MDA-MB-231 (a human breast can-
cer cell line) by the MTT method in vitro, with Sorafenib as posi-
tive control. The cancer cells (of MDA-MB-231) were cultured in
minimum essential medium (MEM) supplemented with 10%
fetal bovine serum (FBS).
Approximately 46103 cells, suspended in MEM medium, were
plated onto each well of a 96-well plate and incubated in 5% CO2
at 378C for 24 h. The test compounds were added to the culture
medium at the indicated final concentrations and the cell cul-
tures were continued for 72 h. Fresh MTT was added to each well
at a final concentration of 5 lg/mL and incubated with cells at
378C for 4 h. The formazan crystals were dissolved in 100 lL
DMSO per each well, and the absorbency at 492 nm (for the
absorbance of MTT formazan) and 630 nm (for the reference
wavelength) was measured with the ELISA reader. All of the com-
pounds were tested twice in each of the cell lines. The results
expressed as IC50 (inhibitory concentration of 50%) were the aver-
ages of two determinations and were calculated by using the
Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
1-(3,5-Dichlorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl)urea 13d
Prepared from 12 and 14d, 71% yield. M.p.: >3308C; IR (KBr)
[cm–1]: 3330.5, 3082.9, 1722.7, 1646.5, 1586.5, 1531.5, 1448.6,
1
1383.8, 1297.4, 1194.1; H-NMR (DMSO-d6) d [ppm]: 9.36 (s, 1H),
9.30 (s, 1H), 9.23–9.26 (d, J = 7.2 Hz, 1H), 8.75 (s, 1H), 8.29–8.34
(m, 1H), 7.91–7.94 (d, J = 9 Hz, 1H), 7.77–7.80 (d, J = 8.7 Hz, 2H),
7.65–7.70 (t, J = 6.9 Hz, 1H), 7.59–7.62 (d, J = 8.7 Hz, 2H), 7.57–
7.58(d, J = 1.5 Hz, 2H), 7.17 (s, 1H); MS m/z: 426.0 [M + H+]. Anal.
calcd. for C21H14Cl2N4O2: C, 59.31; H, 3.32; N, 13.17; O, 7.52.
Found: C, 59.28; H, 3.31; N, 13.21; O, 7.54.
The authors have declared no conflict of interest.
1-(4-Fluorophenyl)-3-(4-(4-oxo-4H-pyrido[1,2-a]
pyrimidin-3-yl)phenyl)urea 13e
References
Prepared from 12 and 14e, 70% yield. M.p.: A3308C; IR (KBr)
[cm–1]: 3326.3, 3084.4, 1714.8, 1647.5, 1603.4, 1531.3, 1505.4,
1333.8, 1300.0, 1208.1, 1157.6, 1044.3; 1H-NMR (DMSO-d6) d
[ppm]: 9.23–9.26 (d, J = 6.9 Hz, 1H), 9.03 (s, 1H), 8.93 (s, 1H), 8.74
(s, 1H), 8.28–8.34 (m, 1H), 7.92–7.94 (d, J = 8.7 Hz, 1H), 7.75–7.78
(d, J = 8.7 Hz, 2H), 7.66–7.70 (t, J = 6.9 Hz, 1H), 7.58–7.61 (d, J = 9
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