Synthesis of pyranochromene and pyranopyrimidine derivatives from substituted natural coumarin isolated from Ammi majus L. and their biological evaluation

Article abstract of DOI:10.1007/s00044-013-0724-z

A series of novel coumarin derivatives were synthesized from 6-hydroxy-7-methoxy-4-methyl coumarin which was isolated from the aerial parts of the Egyptian medicinal plant Ammi majus L. (Apiaceae). The key intermediate 3-amino-5-methoxy-1-(4-methoxyphenyl)-10-methyl-8-oxo-1,8-dihydropyrano[3,2-f] chromene-2-carbonitrile (3c) was obtained in one-pot synthesis by treating α-cyanocinnamonitrile (1-c) with the natural compound: 6-hydroxy-7-methoxy-4-methyl coumarin (2). Chemical, elemental and spectroscopic evidences confirmed the structures of the synthesized compounds. Some of the newly synthesized compounds exhibited better anti-inflammatory activities at low concentrations compared with indomethacin as positive control.

Full text of DOI:10.1007/s00044-013-0724-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:1180–1188
DOI 10.1007/s00044-013-0724-z
ORIGINAL RESEARCH
Synthesis of pyranochromene and pyranopyrimidine derivatives
from substituted natural coumarin isolated from Ammi majus L.
and their biological evaluation
•
•
•
N. H. Ouf Y. A. Selim M. I. Sakran
A. S. Badr El-din
Received: 30 April 2013 / Accepted: 14 August 2013 / Published online: 25 August 2013
Ó Springer Science+Business Media New York 2013
Abstract A series of novel coumarin derivatives were syn-
thesized from 6-hydroxy-7-methoxy-4-methyl coumarin
which was isolated from the aerial parts of the Egyptian
medicinal plant Ammi majus L. (Apiaceae). The key interme-
diate 3-amino-5-methoxy-1-(4-methoxyphenyl)-10-methyl-8-
oxo-1,8-dihydropyrano[3,2-f]chromene-2-carbonitrile (3c)
was obtained in one-pot synthesis by treating a-cyanocinna-
monitrile (1-c) with the natural compound: 6-hydroxy-7-
methoxy-4-methyl coumarin (2). Chemical, elemental and
spectroscopic evidences confirmed the structures of the syn-
thesized compounds. Some of the newly synthesized com-
pounds exhibited better anti-inflammatory activities at low
concentrations compared with indomethacin as positive
control.
Introduction
Coumarin is a plant flavonoid widely distributed in nature.
In previous studies, pyran and fused 4H-pyran derivatives
have attracted great interest owing to their antimicrobial
activity (El-Agrody et al., 2000, 2001; Bedair et al., 2000)
inhibition of influenza, virus sialidases (Taylor et al.,
1998), mutagenic activity (Hirmoto et al., 1997), antiviral
(Martinez-Grau and Marco, 1997), antiproliferaction
agents (Dell and Smith, 1993), sex-hormones (Bianchi and
Tava, 1987), antitumor (Eiden and Denk, 1991), and anti-
inflammatory agents (Shishoo et al., 1981). Moreover,
pyrane derivatives are well known for their antihistaminic
activity. Recent studies have shown the syntheses of some
pyrimidines and their fused derivatives occurring as anti-
bacterial and antitumor agents as well as in agrochemical
and veterinary products (Ismail et al., 2008; El-Gaby
et al., 2006; Prikazchikova et al., 1975; Brown et al.,
1984). The significance of pyranopyrimidine derivatives is
recognized because of their occurrence in the structure of
various natural products, their biological activity, and their
synthetic potential (Hren et al., 2009; Yu and Wang,
2005). We believed it would be of interest to combine the
above mentioned heterocyclic compounds in a molecular
framework to investigate a possible additive effect of
these rings regarding biological activity. In this study, new
coumarin, 6-hydroxy-7-methoxy-4 methyl coumarin 2, is a
natural product isolated from Ammi majus L. (Apiaceae),
and was found to have anti-inflammatory and antiviral
activity (Selim and Ouf, 2012). We have synthesized some
new heterocyclic-fused system containing coumarin
nuclei, and tested their anti-inflammatory activities in
comparison with indomethacin as positive control. Cyto-
toxicity and some biochemical parameters were also
determined.
Keywords Coumarin ꢀ Pyranochromene ꢀ
Pyranopyrimidinochromene ꢀ Anti-inflammatory
N. H. Ouf ꢀ M. I. Sakran ꢀ A. S. Badr El-din
Department of Chemistry and Biochemistry, Faculty of Science,
Tabuk University, Tabuk, Saudi Arabia Kingdom
N. H. Ouf
Department of Chemistry, Faculty of Science, Zagazig
University, Zagazig 44519, Egypt
Y. A. Selim (&)
Faculty of Specific Education, Zagazig University,
Zagazig 44519, Egypt
e-mail: y2selem@yahoo.com
M. I. Sakran ꢀ A. S. Badr El-din
Department of Chemistry, Faculty of Science, Tanta University,
Tanta 31527, Egypt
123

Med Chem Res (2014) 23:1180–1188
Experimental
1181
H6), 7.1(2H, br, NH2 cancelled by D2O), 7.5–8(5H, m, Ar–
H20, 30, 40, 50, 60). 13C NMR (DMSO-d6 100 MHz) d ppm:
19.8(CH3), 56.9(OCH3), 104(C-6),110(C-1), 112(C-2),
113(C-9), 116.3(CN), 119(C-10), 121.4(C-20), 125.3(C-40,
60), 127.6(C-30, 50), 143(C-10a), 146.2(C-6a), 147.5(C-4a,
10b), 174.1(C-3), 152(C-5,10), 162(C-8). MS: m/z [M?1]?
360.1. Anal. Calcd. for C21H16N2O4: C, 70.00; H, 4.48; N,
7.77; O 17.76. Found: C, 70.05; H, 4.37; N, 7.72; O 17.69.
Materials and instrumentation
All melting points were measured on Electrothermal IA
9000 series digital melting point apparatus. Elemental
analysis data were obtained from the micro-analytical unit,
Cairo University, Cairo, Egypt, and the results were in
favorable agreement with the calculated values. The UV
spectra were recorded on Bye-Unicam SP-1800 spec-
trometer. The IR spectra (KBr) were recorded on a Pye-
Unicam spectrophotometer. The 1H- and 13C NMR spectra
were measured in DMSO and recorded at 400 and
100 MHz on a PerkinElmer R12B Spectrometer using
TMS as an internal standard. All reactions were followed
by TLC (silica gel, aluminum sheets 60 F254, Merck), and
spots were visualized by UV Lamb.
3-amino-1-(4-chlorophenyl)-5-methoxy-10-methyl-8-oxo-
1,8-dihydropyrano[3,2-f] chromene-2-carbonitrile (3b)
It was obtained as yellow crystals from benzene mp 265 °C
yield (90 %). UV (CH3OH) kmax (log e): 275(2.81). IR
(KBr)1700(C=O of coumarin), 2200(CN), 2840(CH-
stretching), 2930, 2960, 3319(NH2), 3448 cm-1
.
1H
NMR(DMSO-d6 400 MHz) d ppm: 2.1(3H, s, CH3),
3.84(3H, s, OCH3), 5.3(1H, s, H1),6.2(1H, s, H9), 5.3(1H,
s, H1) ppm 6.7(1H, s, H6), 7.3(2H, br, NH2 cancelled by
D2O), 7.5–8(4H, m, Ar-H20, 30, 50, 60). 13C NMR(DMSO-d6
100 MHz) d ppm: 19.8(CH3), 57.9(OCH3), 104.7(C-6),
110.1(C-1), 112(C-2), 114.5(C-9), 116.1(CN), 119.2(C-10),
Reaction of 1(a–f) with 6-hydroxy-7-methoxy-4-methyl
coumarin
These compounds were prepared by a solution of 1a–f
(0.01 mol) in ethanol (30 ml), and were treated with
6-hydroxy-7-methoxy-4-methyl coumarin 2 (0.01 mol) and
piperidine (0.5 ml). The reaction mixture was heated until
complete precipitation (reaction times: 15 min for 1(a–c);
120 min for 1(d–f). The solid product that formed was
collected by filtration and recrystallized from a suitable
solvent to give new synthesized pyrano chromene deriva-
tives 3(a–f). See (Scheme 1).
121.4(C-20),
125.3(C-60),127.6(C-30,
50),
131.2(C-
40),143(C-10a), 146.2(C-6a), 147.2(C-4a, 10b), 173.1(C-3),
152.4(C-5, 10), 162.5(C-8). MS: m/z [M?1]? 394.06.
Anal. Calcd. for C21H15ClN2O4: C, 63.89; H, 3.83; Cl,
8.98; N, 7.10; O, 16.22. Found: C, 63.98; H, 3.81; Cl 8.88,
N, 7.10; O, 16.19.
3-amino-5-methoxy-1-(4-methoxyphenyl)-10-methyl-8-oxo-
1,8-dihydropyrano[3,2f] chromene-2-carbonitrile (3c)
3-amino-5-methoxy-10-methyl-8-oxo-1-phenyl-1,8-
dihydropyrano[3,2-f]chromene-2-carbonitrile (3a)
It was obtained as colorless crystals from benzene, mp 250 °C,
yield (80 %). UV (CH3OH) kmax (log e): 275(2.85). IR
(KBr)1700(C=O of coumarin), 3411, 3317(NH2), 2969,
2927,2833(CH-stretching), 2195(CN)cm-1. 1H NMR(DMSO-
d6)dppm: 2.1(3H, s, CH3), 3.85 (6H, s, 2OCH3), 5.2(1H, s, H1),
6.2(1H, s, H9), 6.7(1H, s, H6), 7.1(2H, br, NH2 cancelled by
D2O), 7.5–8(4H, m, Ar-H20, 30, 50, 60). 13C NMR(DMSO-d6
100 MHz) d ppm: 19.8(CH3), 56.9, 59.5(2OCH3), 104(C-6),
It was obtained as colorless crystals from benzene, mp
240 °C, yield (89 %). UV (CH3OH) kmax (log e):
275(2.83). IR (KBr)1700(C=O of coumarin), 2195(CN),
2833(CH-stretching), 2927, 2966, 3317(NH2), 3400, cm-1
1H NMR(DMSO-d6 400 MHz) d ppm: 2.2(3H, s, CH3), 3.8
(3H, s, OCH3), 5.3(1H, s, H1), 6.2(1H, s, H9), 6.7(1H, s,
.
Scheme 1 Synthesis of
pyranochromene derivatives
123

1182
Med Chem Res (2014) 23:1180–1188
110(C-1), 112(C-2), 114.1(C-9), 116.3(CN), 119(C-10), 121.4
(C-20), 125.3(C-60), 127.6(C-30, 50), 143(C-10a), 146.2(C-6a),
147.5(C-4a, 10b), 174.1(C-3), 152(C-5, 10, 40), 162.5(C-8).
MS: m/z [M?1]? 390.11. Anal. Calcd. for C22H18N2O5: C,
67.69; H, 4.65; N, 7.18; O, 20.46. Found: C, 68.01; H, 4.64; N,
7.18; O, 20.22.
s, OCH3), 4.15(2H, q, J = 7.2 Hz, CH2CH3), 5.3(1H, s,
H1), 6.2(1H, s, H9), 6.7(1H, s, H6), 7.11(2H, br, NH2
cancelled by D2O), 7.5–8.2(4H, m, Ar-H20, 30, 50, 60). 13C
NMR(DMSO-d6 100 MHz) d ppm: 19.8, 28.2(2CH3),
56.9,58.9 (2OCH3), 61.7(CH2CH3), 104(C-6), 109(C-1),
112.5(C-2), 114.1(C-9), 119(C-10), 121.5(C-20), 125.3(C-
60), 127.6(C-30, 50), 143(C-10a), 146.2(C-6a), 147.5(C-4a,
10b), 174.1(C-3), 152(C-5, 10, 40), 162,5(C-8), 192.7
(COOC2H5). MS: m/z [M?1]? 437.15. Anal. Calcd. for
C24H23NO7: C, 65.90; H, 5.30; N, 3.20; O, 25.60. Found:
C, 65.96; H, 5.22; N, 3.10; O, 25.24.
Ethyl-3-amino-5-methoxy-10-methyl-8-oxo-1-phenyl-1,
8-dihydropyrano [3,2-f] chromene-2-carboxylate (3d)
It was obtained as colorless needles from benzene, mp
190 °C, yield (85 %). UV (CH3OH) kmax (log e):
275(2.82). IR (KBr)1700(C=O of coumarin), 1749(C=O of
ester), 2847(CH-stretching), 2925, 2966, 3320(NH2),
Synthesis of 4(a–c)
3440 cm-1
.
1H NMR (DMSO-d6 400 MHz) d ppm :
These compounds were prepared by a mixture of 3c
(0.01 mol), benzaldhyde, p-chlorobenzaldhyde, p-anisald-
hyde (0.01 mol), DMSO (20 ml), and piperidine (0.5 ml),
and were refluxed for 4 h to give new synthesized pyrano
derivatives 4a–c (Scheme 2).
1.25(3H, t, J = 7.05 Hz, CH2CH3), 2.2(3H, s, CH3), 3.8.
(3H, s, OCH3), 4.15(2H, q, J = 7.2 Hz, CH2CH3), 5.3(1H,
s, H1), 6.2(1H, s, H9), 6.7 (1H, s, Ar-H6), 7.1(2H, br, NH2
cancelled by D2O), 7.5–8(5H, m, Ar-H20, 30, 40, 50, 60). MS:
m/z [M?1]? 407.14. Anal. Calcd. for C23H21NO6: C,
67.80; H, 3.44; N, 7.18; O, 23.56. Found: C, 67.80; H, 5.20;
N, 3.43; O, 23.42.
(E)-3-(benzylideneamino)-5-methoxy-1-(4-
methoxyphenyl)-10-methyl-8-oxo-1, 8-dihydro pyrano [3,2-
f]chromene-2-carbonitrile (4a)
Ethyl-3-amino-1-(4-chlorophenyl)-5-methoxy-10-methyl-8-
oxo-1, 8-dihydropyrano [3, 2-f] chromene-2-carboxylate
(3e)
It was obtained as yellow crystals from benzene, mp
280 °C, yield (82 %). IR (KBr)1641(C=NH), 1700(C=O of
coumarin), 2210(CN), 2833(CH-stretching), 2925,
3074 cm-1. 1H NMR(DMSO-d6 400 MHz) d ppm: 2.2(3H,
s, CH3), 3.8(6H, s, 2OCH3), 5.3(1H, s, H1), 6.2(1H, s, H9),
6.7(1H, s, H6), 7.5–8.2(9H, m, Ar-H20, 30, 50, 60, 200, 300, 400,
500, 600), 9.06(1H, s, N=CH). 13C NMR(DMSO-d6
100 MHz) d ppm: 19.8(CH3), 56.9, 59.9(2OCH3), 104(C-
6), 110.2(C-1)0, 112.9(C-2), 114.5(C-9), 116.3(CN), 119(C-
10), 121.4(C-2 , 200), 125.3(C-60, 400, 600), 127.6(C-30, 5, 300,
500), 133.7(c-100), 143(C-10a), 143.5(N=CH), 146.2(C-6a),
147.5(C-4a, 10b), 174.1(C-3), 152(C-5, 10, 40), 162(C-8).
MS: m/z [M?1]? 478.45. Anal. Calcd. for C29H22N2O5: C,
72.79; H, 4.63; N, 5.85; O, 16.72. Found: C, 71.96; H, 4.21;
N, 5.13; O, 16.21.
It was obtained as colorless needles from benzene, mp
180 °C, yield (80 %). UV (CH3OH) kmax (log e):
275(2.86). IR (KBr)1700(C=O of coumarin), 1740(C=O of
ester), 2930, 2966, 2844(CH-stretching), 3320(NH2),
1
3400 cm-1. H NMR(DMSO-d6 400 MHz) d ppm: 1.26
(3H, t, J = 7.05 Hz, CH2CH3), 2.2(3H, s, CH3), 3.8(3H, s,
OCH3), 4.11(2H, q, J = 7.1 Hz, CH2CH3), 5.3(1H, s, H1),
6.2(1H, s, H9), 6.68(1H, s, H6), 7.1(2H, br, NH2 cancelled
by D2O), 7.5–8.2(m, 4H, Ar-H20, 30, 50, 60), 4.15 (q, 7.2 Hz,
CH2). MS: m/z [M?1]? 441.11. Anal. Calcd. for
C23H20ClNO6: C, 62.52; H, 4.56; Cl, 8.03; N, 3.17; O,
21.76. Found: C, 52.83; H, 4.46; Cl, 7.93; N, 3.09; O,
21.24.
(E)-3-(4-chlorobenzylideneamino)-5-methoxy-1-(4-
methoxyphenyl)-10-methyl-8-oxo-1, 8-dihydropyrano [3,2-
f] chromene-2-carbonitrile (4b)
Ethyl-3-amino-5-methoxy-1-(4-methoxyphenyl)-10-methyl-
8-oxo-1,8-dihydropyrano [3, 2-f] chromene-2-carboxylate
(3f)
It was obtained as yellow crystals from benzene, mp
310 °C, yield (88 %). IR (KBr)1641(C=N)1700(C=O of
coumarin), 2211(CN), 2833 (CH-stretching), 2925,3074
It was obtained as colorless needles from benzene, mp
200 °C, yield (80 %). UV (CH3OH) kmax (log e):
275(2.85). IR (KBr)1700(C=O of coumarin), 1744(C=O of
ester), 2840(CH-stretching), 2925, 2966, 3320(NH2),
1
cm-1. H NMR (DMSO-d6 400 MHz) d ppm: 2.2(3H, s,
CH3), 3.83(6H, s, 2OCH3), 5.3(1H, s, H1), 6.2(1H, s, H9),
6.7 (1H, s, H6),7.5–8.1(8H, m, Ar-H20, 30, 50, 60, 200, 300, 500,
600), 9.06(1H, s, N=CH). MS: m/z [M?1]? 512.11. Anal.
1
3400 cm-1. H NMR(DMSO-d6 400 MHz) d ppm: 1.25
(3H, t, J = 7.05 Hz, CH2CH3), 2.2(3H, s, CH3), 3.85(6H,
123

Med Chem Res (2014) 23:1180–1188
1183
Scheme 2 Synthesis of
pyranochromene and
pyranopyrimidine derivatives
Synthesis of N-(2-cyano-5-methoxy-1-(4-
methoxyphenyl)-10-methyl-8-oxo-1,8-dihydro-pyrano
[3,2-f]chromen-3-yl)acetamide, (7)
Calcd. for C29H21ClN2O5: C, 67.90; H, 4.13; Cl, 6.90; N,
5.46; O, 15.60. Found: C, 67.92; H, 4.10; Cl 6.54; N, 5.47;
O, 15.21.
This new pyrano compound was prepared by a solution of 3c
(0.01 mol) in acetic anhydride (20 ml) and was heated under
reflux for 30 min. The solid product formed was filtered,
washed with cold ethanol, dried, and recrystallized from
ethanol. It was obtained as colorless needles, mp 240 °C,
yield(81 %).IR (KBr)1700(C=O of coumarin), 2206(CN),
2916(CH-stretching), 3200(NH), 3046, 1H NMR (DMSO-d6
400 MHz)d ppm: 2.2(3H, s, CH3), 3.2(3H, s, COCH3),
3.8(6H, s, 2OCH3), 5.13(1H, s, H1), 6.21(1H, s, H9),
6.17(1H, s, H6),7.5–8.2(4H, m, Ar-H20, 30, 50, 60), 11–12(1H,
br, NH). 13C NMR(DMSO-d6 100 MHz) d ppm: 17.2
(2CH3), 57.2, 59.6(2OCH3), 106.2(C-6),111.3(C-1), 112.3
(C-2), 114.6(C-9), 117.2(CN),119.2(C-10), 121.4(C-20),
126.3(C-60), 129.2(C-30, 50), 143.2(C-10a), 148.1(C-6a),
(E)-5-methoxy-3-(4-methoxybenzylideneamino)-1-(4-
methoxyphenyl)-10-methyl-8-oxo-1, 8-dihydropyrano [3,2-
f] chromene-2-carbonitrile (4c)
It was obtained as yellow crystals from benzene, mp
272 °C, yield (90 %). IR (KBr) 1641(C=N), 1710(C=O of
coumarin), 2211(CN), 2833(CH-stretching), 2929, 3174,
1
cm-1. H NMR (DMSO-d6 400 MHz) d ppm: 2.2(3H, s,
CH3), 3.86(9H, s, 3OCH3), 5.3(1H, s, H1), 6.2(1H, s, H9),
6.7(1H, s, H6), 7.5–8.2(8H, m, Ar-H20, 30, 50, 60, 200, 300,
500, 600), 9.16(1H, s, N=CH) ppm. MS: m/z [M?1]?
508.16. Anal. Calcd. for C30H24N2O6: C, 70.86; H, 4.76;
N, 5.51; O, 18.85. Found: C, 71.10; H, 4.68; N, 5.47; O,
18.42.
123

1184
Med Chem Res (2014) 23:1180–1188
149.1(C-4a, 10b), 177.1(C-3), 152.4(C-5, 10, 40), 162.5(C-
8), 169.3(COCH3). MS: m/z [M?1]? 423.13. Anal. Calcd.
for C24H20N2O6: C, 66.66; H, 4.49; N, 6.35; O, 22.25. Found:
C, 66.76; H, 4.49; N, 6.35; O, 21.99.
NMR(DMSO-d6 100 MHz)
d
ppm: 19.8(CH3),
56.1,59.2(2OCH3), 104.7(C-10), 112.5(C-4a), 113(C-5),
114.0(C-7), 119(C-10), 121.4(C-20), 125.3(C-60),126(C-
2),127.6(C-30,50), 131.2(C-40), 143(C-5b),146.2(C-9a),
147.5(C-5a, 11a, 12a), 152.0(C-6,11), 159(C-4), 162,5(C-8).
MS: m/z [M?1]? 418.23. Anal. Calcd. for C23H18N2O6: C,
66.02; H, 4.34; N, 6.70; O, 22.92. Found: C, 66.52; H, 4.25;
N, 6.66; O, 22.58.
Synthesis of 2, 6-dimethyl-4, 8-dioxo-5-(4-methoxy
phenyl)-11-methoxy-5, 8-dihydro pyrano [3, 2-f]-3,
4-dihydropyrimidino [4, 5-i] chromene (8)
Method (a)
Method (b)
This new pyrano pyrimidine compound was prepared by a
solution of 3c(0.01 mol) in acetic anhydride (20 ml) and was
heated under reflux for 3 h. The solid product formed was
filtered, washed with cold ethanol, dried, and recrystallized
from ethanol to give 8 as colorless needles, mp 170 °C, yield
(67 %).IR (KBr)1700(C=O of coumarin), 2916(CH-
This compound was prepared by a solution of 3f (0.01 mol)
in formamide (20 ml) and was heated under reflux for 6 h.
The solid product formed was filtered, washed with cold
ethanol, dried, and recrystallized from ethanol to give 9
(Yield 70 %)
stretching), 3200(NH) 3046, cm-1 1H NMR(DMSO-d6
.
Biology part
400 MHz) d ppm: 2.3(6H, s, 2CH3),3.8(6H, s, 2OCH3),
5.3(1H, s, H5), 6.2(1H, s, H10), 6.7(1H, s, H7),7.2–9.07(5H,
m, Ar-H20, 30, 50, 60 ? NH). 13C NMR (DMSO-d6 100 MHz)
d ppm: 19.8(2CH3), 55.2, 57.1(2OCH3), 108.2(C-10),
111.9(C-4a), 113.3(C-5), 115.1(C-7), 119(C-10), 123.6 (C-
20), 126.3(C-60),127.2(C-2),129.6 (C-30,50), 131.5(C-
40),143(C-5b), 146.2(C-9a), 148.3 (C-5a, 11a, 12a), 152.4(C-
6,11), 160.2(C-4), 164,5(C-8). MS: m/z [M?1]? 423.23.
Anal. Calcd. for C24H20N2O6: C, 66.61; H, 4.46; N, 6.35; O,
22.22. Found: C, 66.71; H, 4.49; N, 6.33; O, 21.89.
Experimental animals
Male albino mice weight around 150–200 g were pur-
chased from Faculty of Science, Tanta University, Egypt.
They were acclimatized to animal house conditions. Ani-
mals were provided with standard diet.
Anti-inflammatory activity
The anti-inflammatory activity was carried out following the
method of Domenjoz (1952). Rats were divided into nine
different groups each of ten animals. At the beginning, the
thickness of the left paw was measured. They were treated
orally with the tested compounds, at 40 mg/kg body weight
or indomethacin 600 mg/kg as a reference standard. After
1 h of administration, the inflammation was induced by S.C.
injection of 0.1 ml of 6 % formalin solution in normal saline.
The right hind paw was injected with unequal volume of
saline. The difference in thickness between the two paws
gave the swelling induced by formalin. The anti-inflamma-
tory efficacy was estimated by comparing the swelling of the
treated one with the control. The difference in thickness was
recorded after 0.5, 1, 1.5, and 2 h.
Method (b)
This compound was prepared by a stream of dry HCl gas
and was passed through a mixture of 3f (0.01 mol) and
acetonitrile (30 ml) for 5–8 h. The reaction mixture was
poured into ice water and basified with 10 % ammonium
hydroxide to give 8 with yield (80 %).
Synthesis of 6-methyl-4, 8-dioxo-5-(4-methoxy
phenyl)-11-methoxy-5, 8-dihydro pyrano[3, 2-f]-3,
4-dihydropyrimidino[4, 5-i] chromene (9)
Method (a)
This new pyrano pyrimidine compound was prepared by a
solution of 3c (0.01 mol) in formic acid (20 ml) was heated
under reflux for 30 min. The solid product formed was fil-
tered, washed with cold ethanol, dried, and recrystallized
from ethanol. It was obtained as needles, mp 240 °C, yiel-
d(81 %).IR (KBr)1700(C=O of coumarin), 2916(CH-
Acute toxicity studies (LD50)
Preliminary experiments were carried out on six main
groups (10 mice/each dose/each group). Compounds 7, 8,
and 9 were injected in different doses to find out the range
of doses which cause zero and 100 % mortality of animals.
A range of doses was determined for each compound, and
LD50 was determined. See (Table 2,3,4). The LD50 was
evaluated by Spearman and Karber method (Finney, 1964)
stretching), 3200(NH), 3046 cm-1 1H NMR(DMSO-d6
.
400 MHz) d ppm: 2.3(3H, s, CH3),3.8(6H, s, OCH3),5.3(1H,
s, H5), 6.22(1H, s, H10), 6.71(1H, s, H7),7.2–9.07(5H, m,
Ar-H2, 20, 30 ,50 , 60 ? NH),8.3(1H, s, H3). 13C
123

Med Chem Res (2014) 23:1180–1188
1185
Scheme 3 Mechanism of
formation of compound 5 from
4a
on groups of mice, and the number of animals that died
within 24 h was recorded.
from each animal. The separated blood was used for the
estimation of AST, ALT, and c-GT, ALP, LDH, and MDA.
The LD50 was then calculated by the application of the
following formula:
Biochemical parameters
Dm ꢁ RðZ:dÞ
Serum levels of aspartate transaminase (AST), alanine trans-
aminase(ALT) (Reitman and Frankel, 1975), alkaline phos-
phatase (ALP) (King and Armstrong, 1988), gamma
glutamyltransferase c-GT(Fiala et al., 1972), lactate dehy-
drogenase(Buhl and Jackson, 1978), and Malondiadhyde as
TBARS in serum (Uchiyama and Mihara, 1978). Blood
superoxide dismutase (SOD) (Marklundand Marklund,1974),
glutathione peroxidase (GPx) activities (Paglia and Valentine,
1967), reduced GSH levels (Hussein et al., 2001), and blood
hemoglobin concentration (Van Kampen and Zijlstra, 1961).
LD50
¼
n
where Dm is the dose by which killed all the mice in the
group; ZHalf the sum of the dead rats from two2 successive
groups; dthe difference between 2 successive doses;
Nnumber of animals in each group.
Biochemical studies
Experimental design
This experiment was carried out to examine the effect of anti-
inflammatory compounds 7, 8, and 9 on liver enzymes. A
solution of 6 g % for compounds 7, 8, and 9 in DMSO was
prepared for intragastric intubation of male albino rats.
Groups of animals each consisting of six rats in each were
treated daily for 15 days as follows: group I, control (was
given similar volume of saline), group II, Normal (was given
similar volume of DMSO); group III, was treated with
compound 7(300 mg/kg. b.w.) dissolved in DMSO orally in
a single daily dose; group IV, was treated with compound 8
(300 mg/kg. b.w.) dissolved in DMSO orally in a single daily
dose; group V, was treated with compound 9 (300 mg/kg.
b.w.) dissolved in DMSO orally in a single daily dose; and
group VI, was treated with indomethacin (600 mg/kg. b.w.)
dissolved in DMSO orally in a single daily dose (Lavergne
et al., 2005).After 10 days of treatment, animals were killed
by cervical dislocation, and blood samples were withdrawn
Statistical analysis
All the grouped data were statistically evaluated using
SPSS/7.5 software. Hypothesis testing methods included
one-way analysis of variance (ANOVA) followed by least
significant difference (LSD) test. P values of less than 0.05
were considered to indicate statistical significance. All the
results were expressed as mean SD for 10 animals in
each group.
Results and discussion
Chemistry
In continuation of previous studies (Ismail et al., 2008; El-
Gaby et al., 2006; Prikazchikova et al., 1975; Brown et al.,
123

1186
Med Chem Res (2014) 23:1180–1188
Table 1 Anti-inflammatory activity of the biologically active compounds
Formalin induced rat paw oedema thickness (mm)/min
Compound
Dose (mg/kg)
0.5 h
1 h
1.5 h
2 h
Control
9.66 0.035
6.44 0.035**
6.11 0.014**
8.67 0.071*
6.5 0.03**
9.73 0.06
9.55 0.045
6.00 0.11**
5.98 0.028**
8.10 0.096*
7.39 0.071*
10.00 0.0.08
5.90 0.026**
5.98 0.030**
7.80 0.174**
6.7 0.057**
7
30
6.17 0.054**
6.00 0.042**
8.45 0.165*
7.33 0.07*
8
30
9
30
Indomethacin#
600
# Indomethacin is used as a reference
* Significant at P \ 0.05
** Significant at P \ 0.01
*** Significant at P \ 0.005
Table 2 Determination of LD50 of compound (7) given i.p. in adult
mice
Table 3 Determination of LD50 of compound (8) given i.p. in
adult mice
Number
Dose
(mg/kg)
Animals/
group
Dead
animals
(Z)
(d)
(Z.d)
Number Dose
Animals/ Dead
animals
(Z)
(d)
(Z.d)
(mg/kg) group
1
2
3
4
5
6
80
10
10
10
10
10
10
0
0.5
1.5
2.5
6
40
20
45
1
2
3
4
5
6
75
110
10
10
0
1
40
40
130
160
200
250
295
1
30
50
2
2.5 50
125
300
560
2
125
360
1080
00
175
250
320
400
10
10
10
10
3
4
7
9
0
75
80
4
60
5
7
9
120
00
8
100 900
00 00
10
0
10
LD50 = 216.91 mg/100 g b.w
LD50 = 217.5 mg/100 g.b.w
1984; Hren et al., 2009) on the synthesis of fused pyrans
using enaminonitriles, we report here the synthesis of a
variety of new heterocyclic compounds. Thus, condensa-
tion of various substituted a-cyanocinnamonitriles (1a–f)
with the natural product 6-hydroxy-7-methoxy-4-methyl-
coumarin 2 isolated from Ammi majus L. (Selim and Ouf,
2012) in ethanolic piperidine afforded 1:1 adducts (Ismail
et al., 2008; Prikazchikova et al., 1975). Structure 3
Table 4 Determination of LD50 of compound (9) given i.p. in adult
mice
Number
Dose
(mg/kg)
Animals/
group
Dead
animals
(Z)
(d)
(Z.d)
1
2
3
4
5
6
90
10
10
10
10
10
10
0
1 .5
2.5
4
20
30
100
240
560
900
00
140
190
240
290
390
1
40
60
1
(Scheme 1) was established on the basis of the H NMR
2
spectra which showed 1-H at d 5.27–5.51 ppm (3a–f). The
increased chemical shift for this signal, compared to the
expected value d 4.0–5.0 ppm for such protons, can be
attributed to the deshielding effect of the diamagnetic
current of the coumaryl, aryl, and allylic p-electrons. The
UV spectrum of new synthesized d (3a–f) revealed a weak
shoulder (Prikazchikova et al., 1975; Yang et al., 2012),
characteristic for 4H-pyran, at kmax (CH3OH) 275 nm
(log e 2.80–2.86) (3a–f), respectively, (Scheme 1).
Interaction of 3-amino-5-methoxy-1-(4-methoxyphenyl)-
10-methyl-8-oxo-1,8-dihydro pyrano [3,2-f]-chromene-2-
carbonitrile (3c) with aromatic aldehydes in DMSO–piperi-
dine under reflux gave the corresponding 3-aryl methylene
aminonewsynthesizedderivatives(4a–c)(Scheme 2). When
(E)-3-(benzylideneamino)-5-methoxy-1-(4-methoxyphenyl)-
10-methyl-8-oxo-1,8-dihydropyrano[3,2-f]-chromene-2-
5
7
80
8
9
100
00
10
0
LD50 = 207 mg/100 g b.w
carbonitrile (4a) was treated with hydrazine hydrate or
phenyl hydrazine in ethanol at room temperature or under
reflux, an additional product formed 5,from which elim-
ination of benzaldehydehydrazone and benzaldehyde-
phenylhydrazone, respectively, gave the enaminonitrile
3c (Prikazchikova et al., 1975; Domenjoz, 1952) instead
of the pyrimidine derivative 6. Treatment of 3c with
acetic anhydride for30 min afforded the new N-acetyl
derivative 7, while heating of 3c with acetic anhydride
under reflux for 3 h afforded 2,6-dimethyl-4,8-dioxo-5-
123

Med Chem Res (2014) 23:1180–1188
1187
Table 5 Serum levels of AST, ALT, ALP, c-GT, LDH and MDA in normal and experimental groups of rats
Group
AST U/l
ALT U/l
ALP U/l
c–GT U/l
LDH U/l
MDA mmol/ml
Normal Saline
9.32 1.82
10.8 1.73
11.9 1.14
12.11 1.5
10.9 1.1
33 3.5
30.5 2.8
33.5 2.73
32.8 3.6
35.8 2.90
34.6 3.56
4.5 1.5
4.82 0.43
4.95 1.12
4.76 0.53
4.86 1.2
220.53 10.17
237.5 6.5
4.20 1.1
4.54 0.95
4.4 5 0.35
4.76 0.42
4.32 0.88
Control (DMSO)
37.5 3.7
38.4 3.5
39.5 3.9
36.75 3.8
Compound 7 (300 mg/kg.b.)
Compound 8 (300 mg/kg.b.)
Compound 9 (300 mg/kg.b.)
235.65 5.5
233.2 7.8
236.1 7.51
Compounds 7, 8, 9 and indomethacin were given orally as a single daily dose for 15 days. Control group was compared to normal group.
Experimental groups were compared to control group. Values are given as mean SD for groups of six animals each
* Significantly different from control group at p \ 0.05
Table 6 Level of reduced glutathione (GSH) and activities of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in blood of normal
and experimental groups of rats
Group
GSH (mg %)
SOD(U/g Hb)
GPx (U/g Hb)
Normal (Saline)
65.5 2.30
61.51 2.50
63.40 2.40
64.12 2.31
63.60 2.2
14.22 2.11
12.50 1.5
12.4 2.51
12.87 2.65
12.92 1.53
8.1 2.1*
185.5 4.50
180.4 5.76
179.51 8.50
181.10 5.33
180.32 4.54
135.5 9.12*
Control (DMSO)
Compound 7 (300 mg/kg. b.w)
Compound 8 (300 mg/kg. b.w)
Compound 9 (300 mg/kg. b.w)
Indomethacin (600 mg/kg. b.w)
48.50 3.50*
Compounds 7, 8, 9 and indomethacin were given orally as a single daily dose for 15 days. Control group was compared to normal group.
Experimental groups were compared to control group. Values are given as mean SD for groups of six animals each
* Significantly different from control group at p \ 0.05
(4-methoxy phenyl)-11-methoxy-5,8-dihydropyrano[3,2-f]-
3,4-dihydropyrimidino[4,5-i]-chromene 8. Structure of new
derivative 8 is supported by an independent synthesis of the
same product from 3f and acetonitrile in the presence of
HCl gas (23) (Scheme 2). Reaction of 3c with formic acid
6-methyl-4,8-dioxo-5-(4-methoxy phenyl)-11-methoxy-5,8-
dihydropyrano[3,2-f]-3,4-dihydropyrimidino[4,5-i] chro-
mene 9. The structure of new derivative 9 was supported
by an independent synthesis from 3f and formamide
(Scheme 2). Also we suppose the mechanism of how
0, 1, 2, 4, 7, and 10 mice, respectively. The dose of com-
pound 7 that killed half of the mice (LD50) was 216.91 mg/
100 g b.w. See (Table 2).The results show that i.p. injec-
tion of compound 8 in doses of 75,110, 175, 250, 320, and
400 mg/100 g b.w. resulted in mortalities of 0, 2, 3, 5, 8,
and 10, respectively. The dose of compound 8 that killed
half of the mice (LD50) was 217.5 mg/100 g b.w.
(Table 3). The results are given in (Table 4) shows that i.p.
injection of compound 9 in doses of 90, 140, 190, 240, 290,
and 390 mg/100 g b.w. resulted in mortalities of 0, 1, 2, 5,
8, and 10, respectively. The dose of compound 9 that killed
half of the mice (LD50) was 207 mg/100 g b.w.
compound 4a was converted to compound
5 in
Scheme 3, which is nucleophilic attack of RNHNH2 to
N=C, followed by proton transfer.
Biochemical studies
Biological activity
We showed that administration of compounds 7, 8, and 9
orally to the rats at dose of 300 mg/kg b.w. for 15 days
revealed non-significant differences in liver enzymes AST,
ALT, ALP, LDH, c–GT, and serum MDA compared with
the control group. See (Table 5). On the other hand, oral
administration of indomethacin showed significant increase
of serum AST, ALT, ALP, LDH, c–GT, and TBARs
compared with the control group. Table 6 shows the con-
centration of GSH, SOD, and GPx in blood of normal and
experimental groups of rats. The levels of GSH, SOD, and
GPx in treated rats were nonsignificantly changed
Anti-inflammatory activity
Data listed in Table 1 indicate that compounds 7, 8, and 9
have promising anti-inflammatory activity compared with
the reference anti-inflammatory drug, indomethacin.
Acute toxicity (LD50) studies
The i.p. injection of compound 7 in doses of 80,130, 160,
200, 250, and 295 mg/100 g b.w. resulted in mortalities of
123

1188
Med Chem Res (2014) 23:1180–1188
Finney DJ (1964) Statistical methods in biological assay. Charles
Griffen & Company Limit, London
Hiramoto K, Nasuhara A, Michiloshi K, Kato T, Kikugawa K (1997)
DNA strand-breaking activity and mutagenicity of 2,3-dihydro-
compared with the control group, but administration of
indomethacin orally at dose of 600 mg/kg.b.w. revealed
significant decrease of blood SOD, GPx, and GSH as
compared with the control group. The result of the present
study indicates that in the present case, a series of Cou-
marnyl derivatives were synthesized. The results indicate
that Compounds 7, 8, and 9 exhibited anti-inflammatory
activity, high LD50 value, and found to be more safe on
liver enzymes. Further studies are in progress to investigate
the effect of Compounds 7, 8, and 9 on different blood
parameters to ensure their anti-inflammatory mechanism.
3,5-dihydroxy-6-methyl-4H-pyran-4-one(DDMP).
A Maillard
reaction product of glucose and glycine. Mutat Res 395:47–56
Hren J, Po_zgan F, Buni CA, Parvulescu VI, Polanc S, Ko_cevar M
(2009) An expeditious synthesis of b-pyrimidyl-a, b-didehydro-
a-amino acid derivatives and pyrano[2,3-d]pyrimidines using
microwave-assisted conditions. Tetrahedron 65:8216–8221
Hussein HEMA, Jamil K, Rao M (2001) Preliminary studies on the
hypoglycemia effect of abromaaugustain alloxan diabetic rats.
Ind J clin Biochem 16(1):77–80
Ismail ZH, Ghorab MM, Mohamed EMA, Aly HM, El-Gaby MSA
(2008) Antitumor activity of some novel 1, 2, 5-thiadiazole
derivatives. Phosphorus Sulfur Silicon 183:2541–2554
King EJ, Armstrong AR (1988) Calcium, phosphorus and phosphate.
In: Varley H (ed) In practical clinical biochemistry. CBS
Publishers, New Delhi, p 458
Acknowledgments The authors gratefully acknowledge the finan-
cial support by Greece and are thankful to the Lab. of organic
chemistry (natural product synthesis), Department of Chemistry,
Faculty of science Aristotle University of Thessaloniki, Thessaloniki,
Greece for the assistance in the performance of some experiments of
1D&2D NMR & MS spectral analyses.
Lavergne N, Volkman M, Maki JE, Yoder R, Trepanier LA (2005)
Evaluation of the clinical, immunologic, and biochemical effects
of sulfamethoxazole-nitroso administration to dogs: a pilot
study. Toxicology 208:63–72
Marklund S, Marklund D (1974) Involvement of the superoxide anion
radical in the autoxidation of pyrogallol and a convenient assay
for superoxide dismutase. Eur J Biochem 47:469–476
Martinez-Grau A, Marco LJ (1997) Friedlander reaction on 2-amino-
3-cyano-4H-pyrans, synthesis of derivatives of 4H-pyran[2,3-
b]quinoline, new tacrine analogues. Bioorg Med Chem Lett
7:3165–3170
Paglia D, Valentine W (1967) Studies on the quantitative and
qualitative characterization of erythrocyte glutathione peroxi-
dase. J Lab Clin Med 70:158–164
Prikazchikova LP, Khutova BM, Vladimirtsev IF, Boldyrev IV,
Zhuravskaya NI (1975) Fiziol. Akt. Veshchestva. 7, 84. ref.
Chem. Abstr. 83,127346 m
Reitman S, Frankel A (1975) A colorimetric method for the
determination of serum glutamic oxaloacetic acid and glutamic
pyruvic transaminases. Am J Clin Pathol 28:56–62
Selim YA, Ouf NH (2012) Anti-inflammatory new coumarin from the
Ammi majus. L. Org Med Chem Lett 2(1):1–4
Shishoo CJ, Devani MB, Ullas GV, Ananthan S, Bhadti VS (1981)
Studies in the synthesis and interconversion of isomeric triaz-
olopyrimidines. J Heterocycl Chem 18:43–46
Taylor R, Cleasby NA, Singh O, Skarzynski T, Wonacott AJ, Smith
PW, Sollis SL, Howes PD, Cherry PC, Bethell R, Colman P,
Varghese J (1998) Dihydropyrancarboxamides related to za-
namivir: a new series of inhibitors of influenza virus sialidases.
2. Crystallographic and molecular modeling study of complexes
of 4-amino-4H-pyran-6-carboxamides and sialidases from influ-
enza virus types A and B. J Med Chem 41:798–807
Uchiyama M, Mihara M (1978) Determination of malonaldehyde
precursor in tissues by thiobarbituric acid. Anal Biochem 86:271
Van Kampen EJ, Zijlstra WJ (1961) Standardization of hemoglobi-
nometry II. The hemoglobin cyanide method. Clin Chim Acta
6:538–543
References
Bedair AH, Nagwa AE, Abd El-Latif MS, Fakery AH, El-Agrody AM
(2000) 4-Hydroxy coumarin in heterocy-clic synthesis. Part III:
Synthesis of some new pyrano [2,3-d] [1,2,4] traizolo [1,5-c]
pyrimidines and pyrimido [1,6-b] [1,2,4]–triazine derivatives.
lLFarmaco 55:708–714
Bianchi G, Tava A (1987) Synthesis of (2R) (?)-2, 3-dihydro-2,
6-dimethyl-4H-pyran-one, a homologue of pheromones of a
species in the hepialid family. Agric Biol Chem 51:2001–2002
Brown D, Katritzky AR, Rees CW (1984) Comprehensive heterocy-
clic chemistry 3. Pergamon Press, Oxford, p 443
Buhl SN, Jackson KY (1978) Optimal conditions and comparison of
lactate dehydrogenase catalysis of the lactate to pyruvate to
lactate reactions in human serum at 25, 30 and 37°C. Clin Chem
24:828–835
Dell CP, Smith CW (1993) Ant proliferative derivatives of 4H-
naphtho [1, 2- b] pyran and process for their preparation. Eur Pat.
537:94, 9, 21; ref Chem Abstr (1993), 119, 139102d
Domenjoz R, Schwarz (1952) Med Wochschr 82:1023
Eiden F, Denk F (1991) Synthesis and CNS activity of pyrane
derivatives: 6, 8-dioxa bicyclo (3, 2, 1)-octanes. Arch Pharm
Weinheim Ger 324:353–354
El-Agrody AM, El-Hakim MH, Abd El-Latif MS, Fakery AH, El-
Sayed EM, El-Ghareab KA (2000) Synthesis of pyrano[2,3-
d]pyrimidine and pyrano[3,2-e] [1,2,4]traizolo[2,3-c]pyrimidine
derivatives with promising antibacterial activities. Acta Pharm
50:111–120
El-Agrody AM, Abd El-Latif MS, El-Hady NA, Fakery AH, Bedair
AH (2001) Heteroaromatization with hydroxy coumarin. Part II:
Synthesis of some new pyrano[2,3-d][1,2,4]traizolo [1,5-c]
pyrimidines and pyrimido [1,6-b][1,2,4]-tri-azine derivatives.
Molecules 6:519–527
El-Gaby MSA, Abdel-Gawad SM, Ghorab MM, Heiba HI, Aly HM
(2006) Synthesis and biological activity of some novel thie-
no[2,3-b]quinoline, quinolino[30,20:4,5] thieno[3,2-d]pyrimidine
and pyrido[20,30:4,5] thieno[2,3-b]quinoline derivatives. Phos-
phorus Sulfur Silicon 181:279–297
Fiala S, Fiala AE, Dixon B (1972) Gamma glutamyl transpeptidase in
transplantable chemically induced rat hepatomas and spontane-
ous mouse hepatomas. J Natl Cancer Inst 48:1393
Yang JL, Jan MH, Chang CW, Lin JJ (2012) Effectiveness of the end-
range mobilization and scapular mobilization approach in a
subgroup of subjects with frozen shoulder syndrome: a random-
ized control trial. Man Ther 17(1):47–52
Yu J, Wang H, Hanqing (2005) Green synthesis of pyrano [2, 3-d]-
pyrimidine derivatives in ionic liquids. Synth Commun 35:
3133–3140
123