Synthesis, and Fluorescence Properties of Coumarin and Benzocoumarin Derivatives Conjugated Pyrimidine Scaffolds for Biological Imaging Applications

Article abstract of DOI:10.1007/s10895-015-1677-z

Series of coumarin and 5,6-benzomcomarin substituted pyrimidine derivatives 11-15 and 22-25 were synthesized, aiming to develop new imaging fluorescent agents. Analogously, treatment of 4-chloropyrimidine analog 16 with coumarin 3-carbohyrazide 5 under MWI condition followed by boiling with NH₄OAc in HOAc furnished coumarin-1,2,4-triazolo-pyrimidine analog 18. The fluorescence property was investigated spectrophotometrically in MeOH with Rhodamine 6G as standard dye. All the compounds showed emission in the region between 331 and 495 nm. The quantum yield of all the compounds were found to be weak, except methyl benzocoumarin 3-carboxylate 22 which showed (Φ_F = 0.98) in comparison to Rhodamine 6G as standard (Φ_F = 0.95).

Full text of DOI:10.1007/s10895-015-1677-z

J Fluoresc
DOI 10.1007/s10895-015-1677-z
ORIGINAL ARTICLE
Synthesis, and Fluorescence Properties
of Coumarin and Benzocoumarin Derivatives Conjugated
Pyrimidine Scaffolds for Biological Imaging Applications
Najim A. Al-Masoudi1 & Niran J. Al-Salihi1 & Yossra A. Marich1 & Timo Markus2
Received: 10 June 2015 /Accepted: 28 September 2015
#
Springer Science+Business Media New York 2015
Abstract Series of coumarin and 5,6-benzomcomarin
substituted pyrimidine derivatives 11–15 and 22–25 were syn-
thesized, aiming to develop new imaging fluorescent agents.
Analogously, treatment of 4-chloropyrimidine analog 16 with
coumarin 3-carbohyrazide 5 under MWI condition followed
by boiling with NH4OAc in HOAc furnished coumarin-1,2,4-
triazolo-pyrimidine analog 18. The fluorescence property was
investigated spectrophotometrically in MeOH with
Rhodamine 6G as standard dye. All the compounds showed
emission in the region between 331 and 495 nm. The quantum
yield of all the compounds were found to be weak, except
methyl benzocoumarin 3-carboxylate 22 which showed
(ΦF=0.98) in comparison to Rhodamine 6G as standard
(ΦF=0.95).
natural products with pharmacological activity [1–6].
Coumarin compounds are known to possess a wide
range of biological activities such as antibacterial [7],
anticancer [8, 9], anticoagulants [10], anti-HIV protease
inhibitors [11], anti-HIV integrases [12, 13], serine pro-
tease inhibitors [14], inhibitors of steroid 5α-reductase
[15], and
NO synthase inhibitors [16]. Geiparvarin 1 (Fig. 1), a
naturally occurring product bearing the coumarin residue,
has been shown to possess a significant inhibitory activity
against a variety of cancer cell lines [17]. Due to efficient
light emission properties, the coumarin derivatives are of
the most importance as significant organic fluorescent ma-
terials [18] for biochemical and biological imaging appli-
cations [19]. Lee et al. [20] have reported novel coumarin-
based fluorogenic probe bearing the 2-picolyl as a fluo-
rescent chemosensor with high selectivity and suitable
affinity in biological systems toward Cu2+, meanwhile
Rajisha et al. [21] have synthesized new benzocoumarin-
oxadiazolyls as strong blue-green fluorescent brighteners
research with good bathochromic shifts. Other coumarin
derivatives are used as fluorescent imaging agents such as
7-amino-4-methyl coumarin-3-acetic acid (AMCA) 2 [22]
in fluorohistochemical examination of human kidney glo-
meruli and 3-(4-aminophenyl)-2H-chromen-2-one (CMC)
3 (Fig. 1) can be used for in situ fluorescent imaging of
myelin in the vertebrate nervous system [23].
.
.
.
Keywords Coumarins Fluorescence Pyrimidines
UV-visible absorption
Introduction
Coumarin and its derivatives are one of the important
classes of heterocyclic compounds which occur in many
Electronic supplementary material The online version of this article
(doi:10.1007/s10895-015-1677-z) contains supplementary material,
which is available to authorized users.
Fluorescence Imaging (FI) is one of the most popular im-
aging modes in biomedical sciences for the visualisation of
cells and tissues both in vitro and in vivo [24]. Despite that a
number of fluorescent imaging agents have been reported, we
synthesized here new coumarin and benzocoumarin deriva-
tives conjugated pyrimidines scaffolds with study of their
fluorescence properties, aiming to develop new imaging
agents can be used in living cells.
* Najim A. Al-Masoudi
najim.al-masoudi@gmx.de
1
Department of Chemistry, College of Science, University of Basrah,
Basrah 61001, Iraq
2
Department of Chemistry, University of Konstanz, P.O. Box 5560,
78457 Konstanz, Germany

J Fluoresc
Fig. 1 Some potential coumarin
derivatives
Experimental Details
Physical Measurements
4a), 117.0 (Ccoum.-8), 113.1 (Ccoum.-3), 102.4 (Cpyrimid.-5).
Anal. Calcd for C20H15ClN8O3 (450.84): C, 53.28; H, 3.35;
N, 24.85. Found: C, 53.01; H, 3.28; N, 24.77 %.
Melting points are uncorrected and were measured with a Büchi
melting point apparatus B‐545 (Büchi Labortechnik AG,
Switzerland). NMR data were obtained on 400 and 600 MHz
(1H) and 100 and 150.91 MHz (13C) spectrometers (Avance III,
Bruker, Germany), respectively, with TMS as internal standard
and on the δ scale in ppm. Heteronuclear assignments were
verified by 1H, 13C HMBC and 1H, 13C HSQC NMR experi-
ments. Microanalytical data were obtained with a Vario EL
(Shimadzu, Japan). Analytical silica gel TLC plates 60 F254
were purchased from Merck. Microwave-assisted reactions
were carried out in a CEM Focused Microwave Synthesis
System (100–450 W). All reagents were obtained from com-
mercial suppliers and were used without further purification.
N’-(2,6-Diamino-5-((4-bromophenyl)diazenyl)
pyrimidin-4-yl)-2-oxo-2H-chromene-3-carbohydrazide (12)
From 2,6-diamino-4-chloro-5-p-bromophenylazopyrimidine
(7) (164 mg). Yield: 153 mg (62 %), m. p. 222–223 °C (dec.).
Rf=0.72. 1H NMR (DMSO-d6): δ 11.11 (br s., 1H, NH), 9.40
(br s., 1H, NH), 9.00 (s, 1H, Hcoum.-4), 8.35 (m, 3H, NH2+H-5),
7.98 (dt, 1H, J5,7=1.5 Hz, J7,8=J6,7=7.7 Hz, Hcoum.-7), 7.68 (d,
2H, J2′,3′=7.9 Hz, Harom.-2′+Harom.-6′), 7.41 (m, 3H, Harom.-3′+
Harom.-5′+Hcoum.-6), 6.97 (m, 3H, NH2+Hcoum.-8). 13C NMR
(DMSO-d6): δ 165.1 (C9=O.), 163.2 (Cpyrimid.-4),. 161.7
(Cpyrimid.-2), 159.1 (Ccoum.-2), 156.4 (Cpyrimid.-6), 151.8
(Ccoum.-8a), 133.7 (C-Br), 132.7 (Carom.-2′+Carom.-6′), 131.3
(Ccoum.-5+Ccoum.-7), 123.8 (Ccoum.-6), 120.1 (Ccoum.-4), 119.1
General Procedure for the Preparation
(Ccoum.-4a), 118.7 (Ccoum.-8), 112.5 (Ccoum.-3), 102.7 (Cpyrimid.-
of Coumarinyl-Pyrimidine Analogues (11–15)
5′). Anal. Calcd for C20H15BrN8O3 (495.29): C, 48.50; H, 3.05;
N, 22.62. Found: C, 48.31; H, 2.98; N, 22.40 %.
To a stirred solution of coumarin-3-carbohysrazide (5)
(100 mg, 0.50 mmol) in DMF (10 mL) and substituted aryl-
azopyrimidines 6–10 (0.50 mmol) were heated in an oil bath
at 90–100 °C for 4–5 h. The mixture was evaporated to dry-
ness and the residue was co-evaporated with silica gel (1.0 g)
in MeOH then poured on a short SiO2 column (5.0 g). Elution,
in gradient, with MeOH (0–10 % v:v) and CHCl3 as eluent
afforded the desired products.
N’-(2,6-Diamino-5-((4-nitrophenyl)diazenyl)pyrimidin-4-yl)
-2-oxo-2H-chromene-3-carbohydrazide (13)
From 2,6-diamino-4-chloro-5-p-nitrophenylazopyrimidine (8)
(164 mg). Yield: 138 mg (60 %), m.p. 198–201 °C (dec.).
Rf=0.79. 1H NMR (DMSO-d6): δ 11.11 (br s., 1H, NH), 9.40
(br s., 1H, NH), 9.00 (s, 1H, Hcoum.-4), 8.48 (br s., 2H, NH2),
8.36 (d, 2H, J5′,6′=8.9 Hz, Harom.-3′+Harom.-5′), 7.98 (m, 2H,
Hcoum.-5+ Hcoum.-7), 7.69 (d, 2H, J2′,3′=8.9 Hz, Harom.-2′+
N’-(2,6-Diamino-5-((4-chlorophenyl)diazenyl)
pyrimidin-4-yl)-2-oxo-2H-chromene-3-carbohydrazide (11)
Harom.-6′), 7.40 (m, 2H, Hcoum.-6+Hcoum.-8), 6.98 (d, 2H, J=
5.9 Hz, NH2). 13C NMR (DMSO-d6): δ 163.6 (C9=O.), 161.3
(Cpyrimid.-4′),. 159.4 (Cpyrimid.-2′), 158.5 (Ccoum.-2), 156.2
From 2,6-diamino-4-chloro-5-p-chlorophenylazopyrimidine
(6) (141 mg). Yield: 120 mg (53 %), m.p. 203–210 °C Rf=
0.65. 1H NMR (DMSO-d6): δ 11.14 (br s., 1H, NH), 9.26 (br
s., 1H, NH), 9.01 (s, 1H, Hcoum.-4), 8.18 (br s., 2H, NH2), 7.82
(dd, 1H, J5,7=1.4 Hz, J5,6=7.7 Hz, H-5), 7.70 (dt, 1H, J6,7=J7,
8=7.8 Hz, H-7), 7.57 (d, 2H, J2′,3′=8.0 Hz, Harom.-2′+Harom.-6′),
7.41 (d, 2H, J5′,6′=8.0 Hz, Harom.-3′+Harom.-5′), 7.00 (m, 2H,
(Cpyrimid.-6.), 155.7 (Ccoum.-8a), 146.5 (C-NO2.), 133.3 (Carom.
-
1′), 130.7 (Carom.-2′+Carom.-6′), 126.9 (Ccoum.-5+Ccoum.-7),
124.9 (Ccoum.-6), 122.0 (Carom.-3′+Carom.-5′), 119.5 (Ccoum.-4),
118.1 (Ccoum.-4a), 116.4 (Ccoum.-8), 112.5 (Ccoum.-3), 103.0
(Cpyrimid.-5). Anal. Calcd for C20H15BrN9O5 (461.39): C,
52.06; H, 3.28; N, 27.32. Found: C, 51.89; H, 3.17; N, 27.08 %.
H
coum.-6+Hcoum.-8), 6.81 (d, 2H, J=4.5 Hz, NH2). 13C NMR
(DMSO-d6): δ 165.1 (C9=O), 163.2 (Cpyrimid.-4),. 161.7
(Cpyrimid.-2), 159.1 (Ccoum.-2), 157.0 (Cpyrimid.-6), 151.1
(Ccoum.-8a), 133.9 (C-Cl), 131.1 (Carom.-2′+Carom.-6′), 129.8
(Carom.-3′+Carom.-5′), 129.0 (Ccoum.-5+Ccoum.-7), 128.6
Methyl 4-((2,6-diamino-6-(2-oxo-2H-chromene-3-carbonyl)
hydrazinyl)pyrimidin-5-yl) diazenyl)benzoate (14)
From methyl 4-((2,6-diamino-4-chloropyrimidin-5-
(Carom.-1′), 123.5 (Ccoum.-6), 119.2 (Ccoum.-4), 118.7 (Ccoum.
-
yl)diazenyl)benzoate (9) (153 mg). Yield: 52 mg (22 %),

J Fluoresc
m.p. 212–216 °C. Rf=0.60. 1H NMR (DMSO-d6): δ 11.12 (br
s., 1H, NH), 9.00 (s, 2H, NH+H-4), 8.00 (d, 1H, J=8.7 Hz,
Methyl 3-oxo-3H-benzochromene-2-carboxylate (22)
H
arom.-2′+Harom.-6′), 7.69 (m, 2H, Hcoum.-5+ Hcoum.-7), 7.43 (d,
2H, J=8.7 Hz, Harom.-3′+Harom.-5′), 7.00-6.95 (m, 4H, NH2+
coum.-6+Hcoum.-8), 3.90 (s, 3H, CO2Me). 13C NMR (DMSO-
A mixture of 2-hydroxynaphthaldehyde (19) (500 mg,
2.91 mmol) and dimethylmalonate (20) (385 mg,
2.91 mmol) in the presence of piperidine (2.0 mL) was
heated under reflux for 1 h. After cooling, the reaction
mixture was acidified with HCl and cooled. The precipi-
tate was filtered, washed with small amount of cold water,
the product was dried and recrystallized from MeOH to
H
d6): δ 164.1 (CO2Me), 163.3 (C9=O), 162.6 (Cpyrimid.-4),.
159.6 (Cpyrimid.-2), 158.5 (Ccoum.-2), 155.5 (Cpyrimid.-6.), 152.8
(Ccoum.-8a), 133.1 (Carom.-1′), 130.7 (Carom.-4′), 129.4 (Carom.
3′+Carom.-5′), 127.8 (Ccoum.-5+Ccoum.-7+ Carom.-2′+Carom.-6′),
124.2 (Ccoum.-6), 119.5 (Ccoum.-4), 118.1 (Ccoum.-4a), 116.4
(Ccoum.-8), 113.1 (Ccoum.-3), 99.9 (Cpyrimid.-5), 51.6 (CO2Me).
Anal. Calcd for C22H18N8O5 (474.43): C, 55.70; H, 3.82; N,
23.62. Found: C, 55.49; H, 3.70; N, 23.40 %.
-
1
give 22 (593 mg, 85 %), m.p. 148 °C, Rf=0.69. H NMR
(DMSO-d6): δ 9.45 (s, 1H, H-4.), 8.63 (dd, 1H, J5,6
=
7.6 Hz, J5,7=1.5 Hz, H-5), 8.36 (dd, 1H, J7,8=7.6 Hz,
J6,8 =1.5 Hz, H-8), 8.11 (m, 1H, H-6), 7.82 (m, 1H,
H-7), 7.70 (d, 1H, J9,10=8.0 Hz, H-9), 7.64 (d, 1H, J9,
10 =8.0 Hz, H-10), 3.90 (s, 3H, CO2Me). 13C NMR
(DMSO-d6): δ 163.9 (CO2Me.), 155.8 (C-2), 145.2
(C-10a) 136.7 (C-4), 129.6 (C-9+C-5a), 129.5 (C-8),
127.0 (C-6), 122.8 (C-7+C-8a), 120.3 (C-5), 117.0
(C-4a), 115.3 (C-10), 115.3 (C-4a), 100.0 (C-3), 53.0
(OMe). Anal. Calcd for C15H10O4 (254.24): C, 70.86; H
3.96. Found: C, 70.66; H, 3.89 %.
N-(4-((2,4-Diamino-6-(2-oxo-2H-chromene-3-carbonyl)
hydrazinyl)pyrimidin-5-yl)diazenyl)phenyl)acetamide (15)
From 2,6-diamino-4-chloro-5-p-acetamidophenylazopyrimidine
(8)10 (153 mg). Yield: 88 mg (37 %), m.p. 288 °C (dec.). Rf=
0.62. 1H NMR (DMSO-d6): δ 11.11 (br s., 1H, NH), 9.00 (s, 1H,
Hcoum.-4), 7.70 (m, 6H, NH2+Harom.-2′+Harom.-6′+Hcoum.-5+
Hcoum.-7),. 7.42 (d, 2H, J3′,4′=8.5 Hz, Harom.-3′+Harom.-5′), 7.39
(m, 2H, Hcoum.-6+Hcoum.-8), 7.11 (s, 1H, NHMe), 6.97 (d, 2H,
J=6.0 Hz, NH2), 2.32 (s, 3H, NHMe). 13C NMR (DMSO-d6): δ
169.0 (CONH.), 165.0 (C9=O), 162.6 (Cpyrimid.-4), 160.1
(Cpyrimid.-2), 158.5 (Ccoum.-2), 156.5 (Cpyrimid.-6), 153.1 (Ccoum.
8a), 137.8 (Carom.4′), 133.1 (Carom.-2′+Carom.-6′), 130.7 (Carom.
1′), 127.7 (Ccoum.-5+Ccoum.-7), 123.6 (Ccoum.-6), 119.5 (Ccoum.
4+Carom.-3′+Carom.-5′), 118.1 (Ccoum.-4a), 116.4 (Ccoum.-8),
113.9 (Ccoum.-3), 99.4 (Cpyrimid.-5), 25.0 (CONHMe). Anal.
Calcd for (C22H19N9O4 (473.44): C, 55.81, H, 4.05; N, 26.63.
Found: C, 54.61; H, 3.93; N, 26.4 %.
Ethyl 3-imino-3H-benzochromene-2-carboxylate (23)
To a stirred solution of 19 (361 mg, 2.01 mmol) in EtOH
(10 ml) were added ethyl cyanoacetate (21) (227 mg,
2.01 mmol), NH4OAc (200 mg) and glacial HOAc
(1.0 ml). The mixture was heated under reflux for 1 h,
and then poured onto crushed ice. The precipitate was
filtered, dried and recrystallized from MeOH to give 23
-
-
-
1
(456 mg, 81 %), m.p. 202–205 °C (dec.), Rf=0.32. H
NMR (DMSO-d6): δ 9.29 (s, 1H, H-4.), 9.07 (s, 1H,
NH), 8.66 (d, 1H, J=8.5 Hz, H-5), 8.34 (d, 1H, J=
9.1 Hz, H-8), 8.12 (d, 1H, J6,7=8.1 Hz, H-6), 7.82 (m,
1H, H-7), 7.81 (d, 1H, J9,10=8.0 Hz, H-9), 7.68 (m, 2H,
H-9+H-10), 4.23 (q, 2H, J=7.8 Hz, CH2CH3), 1.27 (t,
3H, CH2CH3). 13C NMR (DMSO-d6): δ 167.1 (CO2Et.),
165.0 (C=NH), 154.3 (C-10a) 135.6 (C-8), 130.6 (C-5a),
129.5, 129.4 (C-4+C-6), 127.1 (C-9), 123.1 (C-5), 122.1
(C-7+C-8a), 118.8 (C-4a), 117.0 (C-10), 112.6 (C-3),
60.5 (CH2), 14.9 (CH3). Anal. Calcd for C16H13NO3
(267.28): C, 71.90; H, 4.91; N, 5.24. Found: C, 71.74;
H, 7.80; N, 5.07 %.
3-(5,7-Diamino[1,2,4]triazolo[4,3-c]pyrimidin-3-yl)
-2H-chromene-2-one (18)
A mixture of 2,6-diamino-4-chloropyrimidine (16) (200 mg,
1.39 mmol) and 5 (283 mg, 1.39 mmol) was irradiated under
microwave at 180 °C for 25 min. The crude mixture was heated
under reflux in glacial HOAc (10 ml) for 4 h. After cooling,
water was added the precipitate was filtered, washed with cold
EtOH and dried. Recrystallization from EtOH afforded 18
1
(176 mg, 43 %), m.p. 210 °C (dec.), Rf=0.17. H NMR
(DMSO-d6): δ 9.72 (br s., 2H, NH2), 8.04 (s, 1H, Hcoum.-4),
7.73 (dd, 1H, J5,6=8.3 Hz, J5,7=2.3 Hz, Hcoum.-5), 7.59-7.32
(m, 3H, Hcoum.-6+Hcoum.-7+Hcoum.-8), 6.32 (br s., 2H, NH2),
5.71 (s, 1H, Hpyrimid.-5). 13C NMR (DMSO-d6): δ 168.2
(Cpyrimid.-2′), 164.9 (Cpyrimid.-6′), 159.3 (Ccoum.-2), 152.8
(Ccoum.-8a), 149.3 Cpyrimid.-4′), 146.1 (Ctriazol-3), 144.7
3-Oxo-3H-benzochromene-2-carbohydrazide (24)
A solution of 22 (100 mg, 0.37 mmol) in EtOH (7 mL)
and hydrazine hydrate (2 mL) was heated under reflux for
3 h. After cooling, the solution was poured into crushed
ice with stirring. The separated solid was filtered, washed
with water, dried and recrystallised from EtOH to give 24
(75 mg, 80 %), as a yellow solid, m. p. 260–263 °C (Lit.
[25], m.p. 260–262 °C),
(Ccoum.-4), 130.4 (Ccoum.-3), 128.3 (Ccoum.-7), 126.5 (Ccoum.
-
5), 124.7 (Ccoum.-6), 120.4 (Ccoum.-4a), 115.8 (Ccoum.-8), 93.7
(Cpyrimid.-5). Anal. Calcd for C14H10N6O2 (294.27): C, 57.14;
H, 3.33; N, 28.56. Found: C, 56.83; H, 3.33; N, 28.29 %.

J Fluoresc
N’-(2,6-Diamino-5-((4-bromophenyl)diazenyl)
pyrimidin-4-yl)-3-oxo-3H-benzochromene-2-carbohydrazide
(25)
the carbohydrazide scaffolds. Thus, treatment of 6–10 with 5
in hot DMF for 4–5 h furnished, after chromatographic puri-
fication, 11–15 in 22–62 % yield (Scheme 1).
1
The structures of 11–15 were determined by H, 13C and
1
To a stirred solution of 24 (67 mg, 0.30 mmol) in DMF (5 mL)
was added the azopyrimidine 7 (98 mg, 0.30 mmol) and the
mixture was heated at 100 °C for 3 h. After cooling, the res-
idue was evaporated to dryness and the residue was purified
on a short SiO2 column (5.0 g). Elution, in gradient, with
MeOH (0–10 %) and CHCl3 as eluent afforded 25 (95 mg,
58 %), m.p. 279–282 °C (dec.). 1H NMR (DMSO-d6): δ 10.02
2D NMR spectroscopy. In the H NMR spectra, H-4 of the
coumarin ring were resonated as singlets at δ=9.01–9.00 ppm,
meanwhile H-5 - H-8 together with the aromatic protons con-
jugated pyrimidine ring and other substituents were fully
analysed. In the 13C NMR spectra of 11–15, the carbonyl
carbon atom (C9=O) appeared at the regions δ=165.2–
163.3 ppm, while C-4 of the pyrimidine ring resonated at
δ=163.3–161.3 ppm. The resonances at the regions δ=
161.7–159.4, 157.0–155.7 and 103–99.4 ppm were assigned
to carbon atoms 2, 6 and 5 of the pyrimidine backbone, re-
spectively, whereas the signals at the regions δ=159.1–158.5
and 155.7–151 ppm were attributed to carbon atoms 2, and 8a
of the coumarin scaffold, respectively. Other coumarin and
aromatic carbon atoms as well as the substituents were fully
analysed (c.f. Experimental section).
(br s., 2H, NH2), 9.26 (br s., 1H, NH), 8.78 (d, 1H, J9,10
8.8 Hz, Hcoum.-10), 8.00 (d, 1H, J7,8=8.0 Hz, Hcoum.-8), 7.89
(d, 1H, J5,6=8.0 Hz, H-5), 7.82 (d, 1H, J9,10=8.8 Hz, Hcoum.
9), 7.55 (d, 2H, J2′,3′=8.0 Hz, Harom.-2′+Harom.-6′), 7.50 (m,
2H, Hcoum.-6+Hcoum.-7), 7.38 (d, 2H, J5′,6′=8.0 Hz, Harom.
=
-
-
3′+Harom.-5′), 7.25 (s, 1H, Hcoum.-4), 6.97 (d, 2H, J=5.0 Hz,
NH2). 13C NMR (DMSO-d6): δ 164.9 (CONH.), 162.1
(Cpyrimid.-4), 161.2 (Cpyrimid.-2), 157.8 (Ccoum.2=O), 156.1
(Cpyrimid.-6), 137.2 (Ccoum.-3), 134.5 (C-8a), 132.8 (C-4a),
132.3, 131.0, 130.3, 129.4, 128.4, 128.0, 127.3, 127.0,
125.2, 124.3, 123.1 (Ccoum.+Carom.), 120.1 (Ccoum.-4), 102.1
(Cpyrimid.-5). Anal. Calcd for C24H17BrN8O3 (545.35): C,
52.86; H, 3.14; N, 20.55. Found: C, 52.66; H, 3.03; N,
20.36 %.
Next, treatment of 16 with carbohydrazide 5 under micro-
wave irradiation (400 Watt, 180 °C) for 20 min afforded the
crud product, 4-coumarinyl-pyrimidine carbohydrazide deriv-
ative 17 which has been used directly, without purification.
Boiling of the crude 17 with glacial HOAc for 4 h furnished ,
after chromatographic purification, the cyclized triazolo ana-
log 18 (43 %) (Scheme 2).
1
The structure of 18 was determined from its H and 13C
Results and Discussion
Chemistry
NMR spectra. In the 1H NMR spectrum, H-4 of the coumarin
ring appeared as a singlet at δ=8.04 ppm, while H-5 resonated
as a doublet of doublets at δ=7.73 ppm (J5,6=8.3 Hz, J5,7
=
2.3 Hz). H-5 of the pyrimidine backbone appeared as a singlet
δ=7.71 ppm, whereas the other coumarin protons were
analysed. The 13C NMR spectrum showed four signals at
the lower fields, δ=168.2, 164.9, 159.3 and 152.8 ppm, were
assigned to C-2′, C-6′ of the pyrimidine ring and C-2, 8a of
coumarin moiety, respectively. The resonances at δ=149.3,
146.1 and 144.7 ppm were attributed C-4′ of the pyrimidine
Treatment of methyl coumarin-3-carboxylate 4 with hydra-
zine hydrate afforded the carbohydrazide analog 5. 4-
Chloro-azopyrimidine derivatives 6–10 have been selected
as starting materials for the synthesis of the target compounds
11–15, since the electron withdrawing group, azo residue
would facilitated replacement of the chloro group at C-4 by
Scheme 1 Conditions and
reagents: (i) NH2NH2.H2O,
EtOH, reflux, 6 h; (ii) DMF, 90–
100 °C, 4–5 h

J Fluoresc
Scheme 2 Synthesis of 3-(5,7-
diamino[1,2,4-]triazolo[4,3-
c]pyrimidin-3-yl)-2H-chromene-
2-one (18)
ring, C-3 of the triazole and C-4 of coumarin moiety, respec-
tively. C-5 of the pyrimidine ring resonated at δ=93.7 ppm.
Our work was modified by selecting 19 as a precursor for
the synthesis of new benzocoumarinyl-pyrimidine, aiming to
examine its fluorescence property in comparison to the cou-
marin analogs 11–15. Thus, treatment of 19 with
dimethylmalonate 20 in the presence of piperidine afforded,
after purification, the benzocoumarin 3-methyl ester 22
(85 %). Alternatively, treatment of 19 with ethyl cyanoacetate
21 in the presence of NH4OAc and HOAc in boiling EtOH
furnished the 3-iminoethyl benzocoumarin ester 23 (81 %)
atoms resonated at δ=167.1 and 165.0 ppm, respectively.
Further, the resonances at δ=164.9 and 162.1 ppm were
assigned for CONH of coumarin and C-4′ of pyrimidine back-
bone, respectively. The rest of benzocoumarin and aromatic
carbon atoms were fully assigned (cf. Experimental section).
All the synthesized compounds were further analysed by
the HSQC [26] and HMBC [27] NMR spectroscopy.
UV–Vis Absorption and Fluorescence Spectra
The UV-visible absorption and fluorescence spectra of
the synthesized coumarin analogs 4, 5, 11–15, 18 and
22–25 in MeOH were obtained and are presented in
Table 1. The absorption and fluorescence maxima of
some of the synthesized analogs showed good
bathochromic shifts with wavelength of maximum ab-
sorption in the UV or visible region (λex.) (315–
405 nm). Wavelengths of maximum emission for the
compounds (λem.) (322–483 nm) were observed in
MeOH at room temperature. Although, compounds 4,
12, 22 and 23 showed low quantum yield values (ΦF)
of 0.02, 0.01 and 0.15 nm, respectively, but exhibited
fluorescence spectral properties with Stoke’s shift of 89,
87, 72 and 85 nm, respectively (Fig. 2). However, 22
1
which has been structurally confirmd by the H and 13C
NMR spectra. Treatment of 22 with hydrazine hydrate
afforded the carbohydrazide derivative 24 in 30 % yield.
Compound 25 was obtained in 58 % yield by condensation
of 24 with the 4-bromo-azopyrimidine derivative 7 in hot
DMF (Scheme 3).
The structures of 22–25 were assigned by their 1H and 13
C
NMR spectra, where the protons and carbon atoms of the
coumarin moiety showed almost similar pattern. The singlets
at δ=9.24, 9.29, 8.87 and 7.25 ppm were assigned for H-4 of
the coumarin ring, respectively. The other aromatic, NH and
NH2 protons were fully analysed (c.f. Experimental section).
In the 13C NMR spectrum of 23, CO2Et and C=NH carbon
Scheme 3 Conditions and
reagents: (i) 20, piperidine,
EtOH, reflux, 1 h; (ii) 21,
NH4OAc, HOAc, EtOH, reflux,
1 h; (iii) NH2NH2.H2O, EtOH,
reflux, 3 h; (iv) 7, DMF, 100 °C,
3 h

J Fluoresc
Table 1 UV–vis, fluorescence data, and quantum yields (ΦF) of
compounds some coumarin and benzocoumarin analogs
Biological Imaging Application
Organic fluorescent dyes are routinely used as contrast
agents in immunohistochemical staining protocols.
Owing to their low molecular weight, high quantum
yield, relatively low cost and the existence of well-
developed conjugation protocols, they represent an ideal
optical label for molecular imaging probes [29].
Additionally, fluorescent imaging is a relatively recent
imaging method and thus still developing in many
ways, and typically performed by exogenous delivery
of a fluorescent probe (e g, an organic dye) that inter-
acts with the target or by direct imaging of an endoge-
nously expressed fluorescent protein. Signal generation
is achieved by exciting the fluorescent probe or protein
at a given light wavelength (λ) and detecting light emis-
sion at another λ with a charge-coupled device camera
built into either a planar (fluorescence reflectance imag-
ing) or a tomographic (fluorescence molecular tomogra-
phy) imaging system [30, 31]. Most of the above imag-
ing fluorescence agents exhibited several side effects
in vivo (especially by human treatment), including the
allergy, toxicity, solubility and radioactivity. Update 125I
and sodium diatrizoate (sodium 3-(acetylamino)-2,4,6-
triiodobenzoate) are used in the coronary arterials angi-
ography [32].
Our small molecule, the benzocoumarin ester 22 has
been selected for a priminary flourescence imaging ap-
plication by using an experimental animal model (rabbit,
weight~3.2 kg) induced inrtravenasouly by 22 with a
concentration of 10−3 M in a mixture of MeOH-H2O
(1:9 v:v) solution, following Johnston et al. method
[33]. The X-ray contrast media for coronary angiogra-
phy showed some encouraging peripheral vascular an-
giographic imaging pictures. The application is under
further investigation to overcome the solubility problem
of 22 in vivo by introducing a more polar group such
hydroxyl group.
Compd.
λex[nm]
log ε [mM]−1
λem[nm]
ΦF
Stoke’s
shift
.[cm]−1
4
331
315
365
365
405
[355]
[355]
293
371
363
360
379
4.04
4.03
4.41
4.78
4.43
[4.61]
[4.42]
4.56
3.90
3.81
4.09
4.27
331
322
385
483
405
355
360
300
443
448
373
391
0.02
89
7
5
<0.01
-<0.01
0.01
11
12
13
14
15
18
22
23
24
25
20
87
10
8
<0.01-
<0.01-
<0.01-
<0.01-
0.98
5
7
72
85
13
12
0.15
0.01
0.12
Annotations: Ref.: Rh6G in EtOH (ΦF: 0.95) at room temperature;
Equipment and software: Perkin Elmer LS 50, Cary UV/vis 50;
SpekWin 1.71.3, FL WinLab. [ ]: shoulder
showed a very exciting fluorescence result in MeOH
with a quantum yield (ΦF=0.98) with stoke’s shift of
72 nm (Fig. 2), which characterized by a brightly in-
tense blue emission light at λem=443 nm, in comparison
for those of Rh6G (ΦF=0.95). These result encouraged
us to proceed further in our current fluorescence imag-
ing (FI) for the visualisation of cells and tissues both
in vitro and in vivo as well as in coronary angiographic
applications, with full biochemical study of 22.
In conclusion, the replacement of 3-ester group in the
coumarin of benzocoumarin backbone by hydrazide
group (e.g.,: 5 and 24) or hydrazide-pyrimidine moiety
(e.g.,: 11–15 and 25) would reduced the fluorescence
property unless there is an electron donating group at
the aromatic ring of the coumarin moiety. Such argu-
ment is in agreement with Mahadevan et al. results [28].
Fig. 2 UV-visible and fluorescence spectra of compounds 12 and 22 in MeOH

J Fluoresc
9. Itoigawa M, Ito C, Tan T-WT, Kuchide M, Tokuda H, Nishino H,
Furukawa H (2000) Cancer chemopreventive agents, 4-
phenylcoumarins from Calophyllum inophyllum. Cancer Lett 169:
15–19
10. Cravotto G, Nano GM, Palmisano G, Tagliapietra S (2001) An
asymmetric approach to coumarin anticoagulants via hetero-Diels-
Alder cycloaddition. Tetrahedron: Asym 12:707–709
11. Al-Soud YA, Al-Masoudi IA, Saeed B, Beifuß U, Al-Masoudi NA
(2006) Synthesis of new 1H-1,2,4-triazolylcoumarins and their an-
titumor and anti-HIV activities. Chem Heterocycl Comp 42:667–
676
12. Kirkiacharian S, Thuy DT, Sicsic S, Bakhchinian R, Kurkjian R,
Tonnaire R (2002) Structure-activity relationships of some 3-
substituted-4-hydroxycoumarins as HIV-1 protease inhibitors.
Farmaco 57:703–708
13. Yamaguchi T, Fukuda T, Ishibashi F, Iwao M (2006) The first total
synthesis of lamellarin α 20-sulfate, a selective inhibitor of HIV-1
integrase. Tetrahedron Lett 47:3755–3757
Conclusion
A new series of coumarin and 5,6-benzomcomarin substituted
pyrimidine derivatives 11–15 and 22–25 were synthesized
from treatment of coumarin and benzocoumarin 3-
carbohydrazide with 4-chloro-5-azoarylpyrimidines analogs,
Further, the coumarin-1,2,4-triazolo-pyrimidine analog 18
was prepared from 4-chloropyrimidine analog 16 with cou-
marin 3-carbohyrazide 5 under MWI condition, aiming to
develop new imaging fluorescence agents. The new analogs
were investigated spectrophotometrically in MeOH for their
fluorescence properties, which showed emission in the region
between 331 and 495 nm. Interestingly, methyl
benzocoumarin 3-carboxylate (22) showed a higher quantum
yield (ΦF=0.98) in comparison for those of Rhodamine 6G as
standard (ΦF=0.95). The priminary biological fluorescence
imaging aaplication of 22 in an experimental animal model
showed some peripheral vascular angiographic imaging pic-
tures by using a flourescence emission with some
pharmakinetic studies.
14. Pochet L, Frederic R, Masereel B (2004) Coumarin and
isocoumarin as serin protease inhibitors. Curr Pharm Design 10:
3781–3796
15. Fan G-J, Mar W, Park MK, Choi EW, Kim K, Kim S (2001) A
novel class of inhibitors for steroids 5α-reductase: synthesis and
evaluation of umbelliferone derivatives. Bioorg Med Chem Lett
11:2361–2363
16. Murakami A, Gao G, Omura M, Yano M, Ito C, Furukawa H,
Takahashi D, Koshimizu K, Ohigashi H (2000) 1,1-
Dimethylallylcoumarins potently supress both lipopoly- saccharide
and interferone-γ-induced nitric oxide generation in mouse macro-
phage RAW 264.7 cells. Bioorg Med Chem Lett 10:59–62
17. Jerris JP, Smith AB (1981) Synthesis of geiparvarin: a novel anti-
tumor agent. J Org Chem 46:577–585
Acknowledgments We thank Professor Surg. Abdul-Bari A. Alfaris of
College of Veterinary Medicine, Department of Surgery, University of
Basrah (Iraq) for the priminary biological imaging fluorescence experi-
ments. Miss A. Friemel of Chemistry Department, Konstanz University,
Germany is highly acknowledged for the 2D-NMR experiments.
18. Christie RM, Lui C-H (2000) Studies of fluorescent dyes: part 2. an
investigation of the synthesis and electronic spectral properties of
substituted 3-(2-benzimidazolyl) coumarins. Dyes Pigments 47:79–
89
References
19. Zhao Y, Zheng Q, Dakin K, Xu K, Martinez ML, Li W-H (2004)
New caged coumarin fluorophores with extraordinary uncaging
cross sections suitable for biological imaging applications. J Am
Chem Soc 126:4653–4663
1. Ito C, Itoigawa M, Katsuno S, Omura M, Tokuda H, Nishino H,
Furukawa H (2000) Chemical constituents of Clausena excavata:
isolation and structure elucidation of novel furanone-coumarins
with inhibitory effects for tumor-promotion. J Nat Prod 63:1218–
1224
2. Abyshev AZ, D’Yachuk GI, Semenov EV, Pukhov MP (1993)
Synthesis and biological activity of derivatives of benzopyran-2-
one. Pharm Chem J 27:766–770
3. Kontogiorgis CA, Hadjipavlou-Litina DJ (2004) Synthesis and bi-
ological evaluation of # novel coumarin derivatives with a 7-
azomethine linkage. Bioorg Med Chem Lett 14:611–614
4. Abyshev AZ, Alekseev AT, Platonov VG, Byrkin IA (1996)
Synthesis an antiviral activity of benzopyran-2-one derivatives.
Pharm Chem J 30:441–444
5. Teran C, Santana L, Uriarte E, Fall Y, Lena L, Unelius L, Tolf B-R
(1998) Phenyl- piperazine derivatives with strong affinity for
5HT1A, D2A and D3 receptors. Bioorg Med Chem Lett 8:3567–
3570
20. Jung HS, Kwon PS, Lee JW, Kim JI, Hong CS, Kim JW, Yan S, Lee
JY, Lee JH, Joo J, Kim JS (2009) Coumarin-derived Cu2+
−selective fluorescence sensor: synthesis, mechanisms, and appli-
cations in living cells. J Am Chem Soc 131:2008–2012
21. Rajesha G, Kumar HK, Naik HSB, Mahadevan KM (2011)
Synthesis of new benzocoumaryl oxadiazolyls as strong blue-
green fluorescent brighteners. South Afri J Chem 64:88–94
22. Khalfan H, Abuknesha R, Rand-Weaver M, Price RG, Robinson D
(1986) Aminomethyl coumarin acetic acid: a new fluorescenct la-
belling agent proteins. Histochem J 19:497–509
23. Wang C, Popescu DC, Wu C, Zhu J, Macklin W, Wang Y (2010) In
situ fluorescence imaging of myelination. J Histochem Cytochem
58:611–621
24. Wu Q, Merchant FA, Castleman KR (2008) Microscope image
processing. Academic, New York
6. Al-Soud YA, Al-Sauodoni HH, Amajaour HAS, Salih KSM,
Mubarak MS, Al-Masoudi NA, Jaber IH (2008) Synthesis, charac-
terization and anti-HIV activities of new coumarin derivatives. Z
Naturforsch 63b:83–89
7. Kayser O, Kolodziej H (1997) Antibacterial activity of extracts and
constituents of Pelargonium sidoides and Pelargonium reniforme.
Planta Med 63:508–510
8. Wang CJ, Hsieh YJ, Chu CY, Lin YL, Tseng TH (2002) Inhibition
of cell cycle progression in human leukemia HL-60 cells by escu-
letin. Cancer Lett 183:163–186
25. Bogdal D (1998) Coumarins: fast synthesis by knoevenagel
condendation under microwave inrradiation. J Chem Res (S):
468–469
26. Chiyomi M, Toshinobu M, Yasuko K, Kenichiro T, Hideyuki Y,
Hitoshi N, Masatoshi Y, Akira T (2005) Improvement of fluores-
cence characteristics of coumarins: syntheses and fluorescence
properties of 6-methoxycoumarin and benzocoumarin derivatives
as novel fluorophores emitting in the longer wavelength rRegion
and their application to analytical reagents. Chem Pharm Bull 53:
750–758

J Fluoresc
27. Davis AL, Keeler J, Laue ED, Moskau D (1992) Experiments for
recording pure- absorption heteronuclear correlation spectra using
pulsed field gradients. J Magn Reson 98:207–216
30. Hellebust A, Richards-Kotum R (2012) Advances in molecular
imaging: targeted optical contrast agents for cancer diagnostics.
Nanomedicine (Lond) 7:429–445
28. Willker W, Leibfritz D, Kerssebaum R, Bermel W (1993) Gradient
selection in inverse heteronuclear correlation spectroscopy. Magn
Reson Chem 31:287–292
29. Harishkumar HN, Mahadevan KM, Masagalli JN,
Chandrashekarappa KKH (2012) Synthesis and fluorescence
study of phenylcoumarin/cyanophenylbenzocoumarin-3- carbox-
ylates. Organ Commun 5:196–208
31. Ntziachristos V (2006) Fluorescence molecular imaging. Annu Rev
Biomed Eng 8:1–33
32. Rao J, Dragulescu-Andrasi A, Yao H (2007) Fluorescence imaging
in vivo: recent advances. Curr Opin Biotech 18:17–25
33. Hoppe JO, Archer S (1960) X-ray contrast media for cardiovascular
angiography. Angiology 11:244–254