Synthesis and fungicidal evaluation of some new anilinopyrimidine derivatives

Article abstract of DOI:10.1007/s00044-013-0535-2

New series of anilinopyrimidine and pyrimido[4,5-c]azepine derivatives were synthesized to evaluate their in vitro antifungal activities. N-acetyl anilinopyrimidine derivative 7 showed similar fungicidal activity against Aspergillus niger compared to the reference fungicidal pyrimethanil 2. In addition, it exhibits shorter bursting time (2.5 μg/ml in 9 h) than fungicidal drug 2 (2.5 μg/ml in 12 h). The brominated pyrimidine derivative 5 displayed higher fungicidal activity than those of the cyano derivative 6 and the 6-bromoalkyl analogs 4. The fused pyrimido[4,5-c]azepine derivative 10 showed lower activity toward A. niger. A new application for the pyridinium bromochromate as a selective brominating agent on the pyrimidine ring rather on the side chain methyl group was studied.

Full text of DOI:10.1007/s00044-013-0535-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0535-2
ORIGINAL RESEARCH
Synthesis and fungicidal evaluation of some new
anilinopyrimidine derivatives
•
Mohamed A. Waly Eman T. Bader-Eldien
•
•
Mohamed E. Aboudobarah El-Shahat T. Aboumosalam
Received: 28 December 2012 / Accepted: 1 February 2013
Ó Springer Science+Business Media New York 2013
Abstract New series of anilinopyrimidine and pyrimi-
do[4,5-c]azepine derivatives were synthesized to evaluate
their in vitro antifungal activities. N-acetyl anilinopyrimi-
dine derivative 7 showed similar fungicidal activity against
Aspergillus niger compared to the reference fungicidal
pyrimethanil 2. In addition, it exhibits shorter bursting time
(2.5 lg/ml in 9 h) than fungicidal drug 2 (2.5 lg/ml in
12 h). The brominated pyrimidine derivative 5 displayed
higher fungicidal activity than those of the cyano deriv-
ative 6 and the 6-bromoalkyl analogs 4. The fused
pyrimido[4,5-c]azepine derivative 10 showed lower activity
toward A. niger. A new application for the pyridinium
bromochromate as a selective brominating agent on the
pyrimidine ring rather on the side chain methyl group was
studied.
the monitoring conducted in several places of the Europe
since 1994 allowed detection of strains with highly resis-
tant to fungicidal agents (Forster and Staub, 1996; Hilber
and Hilber-Bodmer, 1998; Leroux and Greet, 1995; Leroux
et al., 1999). The development of potentially effective
antifungal compounds is one of the main objectives of the
current work. Aspergillus niger has been reported to cause
numerous chronic diseases to humans, animals, and plants,
and has a pathogenic effect called Aspergillosis (Samson
et al., 2001; Tunev et al., 1999). A. niger affect plants in
form of black molds, and targets mainly the lung and the
respiratory tract in both animals and humans (Roehrl et al.,
2007). Also, it may cause a serious damage to the ear canal
and tympanic membrane (Steinbach and Stevens, 2003).
The 2-anilinopyrimidine skeleton is a requisite structure
for a potent fungicidal activity, whatever its situation is less
clear as fungicides. It has the ability to interfere with
methionine biosynthesis (Fritz et al., 1997) and inhibit the
secretion of hydrolytic enzymes, which play an important
role in the infection process of fungi (Milling and
Richardson, 1995); however, the primary target site is
still unknown. 2-Anilino-4-methyl-6-(1-propynyl)pyrimi-
dine (KIF-3535) 1 has been given the common name me-
panipyrim (De Waard and Van Nistelrooy, 1979, 1988;
Albertson et al., 1996; Chapeland et al., 1999; Akallal
et al., 1998; Joseph-Horne et al., 1996) and showed an
excellent activity against Gray mold and no phytotoxicity.
2-Anilino-4,6-dimethylpyrimidine 2, with a common name
pyrimethanil, showed good fungicidal activity, but with
unacceptable phytotoxicity (Fritz et al., 1997; Chapeland
et al., 1999; Prasad et al., 1995; Friedrich et al., 1981;
Nagata et al., 2003).
Keywords Pyrimethanil Á
Pyridinium bromochromate (PBC) Á
Intramolecular cyclization Á Aspergillus niger Á
Antifungal activity
Introduction
Fungicides have become an integral part of efficient food
production. The loss of a fungicide to agriculture through
resistance is a problem that causes crop losses. Moreover,
M. A. Waly (&) Á E. T. Bader-Eldien Á E.-S. T. Aboumosalam
Chemistry Department, Faculty of Science, Damietta University,
Damietta, Egypt
e-mail: mohamedwaly7@yahoo.com
The main objective in this study is to synthesize of
target structures based on the anilinopyrimidine skeleton
by introducing substituents on the bridged nitrogen atom
M. E. Aboudobarah
Botany Department, Faculty of Science, Damietta University,
Damietta, Egypt
123

Med Chem Res
CH3
CH3
CH3
O
CH3
N
C
5
HN
NaO2CCH3
Fusion
O
O
N
N
N
NHPh
N
+
Ph
H2N
Ph
N
H
N
CH3
N
H
Ph
N
N
N
C
CH3
N
H
N
.
2 H2SO4
N
H
2
CH3
1
2
Bz
3
Scheme 2 Modified synthesis of pyrimethanil 2
Scheme 1 Target structures
CH3
H
C
CH3
3
Br
CN
N
CuCN
DMF
N
N
PBC
AcOH
and pyrimidine ring position 5 as shown in structure 2,
Scheme 1. In addition, the synthesis of fused pyrimido[4,5-
c]azepine derivatives 3 for their in vitro evaluation as a
fungicidal agents against A. niger will be examined in the
current study.
N
CH3
Ph
N
H
N
CH3
Ph
N
H
N
H
C
3
Ph
N
H
2
5
6
Ac2O
NBS
H
l
C
C 3
CH3
CH3
N
N
N
CH3
Ph
N
Ph
N
H
N
Br
CH2
CH3
O
7
4
Results and discussion
Scheme 3 Syntheses of different pyrimethanil substituents
Chemistry
IR spectrum for compound 6 showed the presence of cyano
group absorption signal at 2217.74 cm-1, and appeared at
d 115.30 ppm in its 13C NMR spectrum.
Refluxing of compound 2 with acetic anhydride afforded
N-acetyl anilinopyrimidine derivative 7 as in Scheme 3,
which showed amide carbonyl absorption signal at
1685.48 cm-1 in its IR spectrum. The N-acetyl (N–CO–
CH3) methyl protons signal appeared at d 2.33 ppm in its
1H NMR data and its carbonyl carbon appeared at d
172.77 ppm in its 13C NMR spectrum.
The described procedure for the synthesis of the starting
2-(N-phenylamino)-4,6-dimethylpyrimidine 2 (condensa-
tion of phenylguanidine sulfate salt with acetylacetone in
the presence of sodium carbonate) (Nagata et al., 2003)
was modified by fusion with sodium acetate instead of
refluxing with sodium carbonate in ethanol, for increasing
the reaction yield, Scheme 2 (Waly and Abou Dobara,
2009).
For syntheses of targets containing substituted pyrimi-
dine ring was achieved as was described in Scheme 3.
Attempted bromination of anilinopyrimidine derivative 2
with bromine in acetic acid afforded mixed two products
Fused pyrimido[4,5-c]azepine derivative 10 was syn-
thesized by starting with the pyrimidine nitrile derivative 6,
as described in Scheme 4. Bromination of the pyrimidine
nitrile derivative 6 with NBS gave the bromonitrile deriv-
1
difficult to separate them. The H NMR spectrum for this
reaction mixture indicated that the bromination was
exhibited at both of the pyrimidine ring position 5 and side
chain methyl group; while bromination of compound 2
with N-bromosuccinimide (NBS) afforded the side bro-
1
ative 8. The H NMR spectrum of compound 8 indicated
the presence of CH2Br protons signal at d 2.62 ppm.
Reaction of the bromopyrimidine nitrile derivative 8 with
3-(N-benzylamino)propionitrile (NBAP) in ethanol and
the presence of sodium carbonate afforded the pyrimidine
dinitrile derivative 9. The IR data for compound 9 showed
the presence of dicyano groups signals at 2210.02 and
1
momethyl derivative 4. The H NMR spectrum for com-
pound 4 indicated that it still has signal interfering with the
aromatic protons at d 7.66–7.98, characterizing the C-5
proton, in addition to the d 2.55 for CH2Br protons. On
bromination of compound 2 with pyridinium bromochro-
mate (PBC) (Patwari et al., 2003; Sarrafi et al., 2009) in
acetic acid, a new precipitated product was produced,
during 5 min. The 1H NMR spectrum for this product
showed the presence of two similar CH3 group protons at d
2.48 ppm and disappearance of the C-(5)H proton signal;
while its 13C NMR data showed the presence of signal at d
166.84 ppm for C–Br carbon. All the spectral and ele-
mental analyses data for this bromination product were
consistent with the structure 6, Scheme 3.
CH3
CH3
CN
CN
N
N
NBS
CHCl3
Ph
N
H
N
Br
CH2
Ph
N
H
N
CH3
8
6
NBAP
HN
Me
CH3
CN
CN
N
N
Bz
N
NaH THF
,
Ph
N
N
H
N
Ph
N
N
CH2
CN
Bz
H
9
10
Cyanation of the bromopyrimidine derivative 5 with
CuCN in DMF (Ellis’ and Romeny-Alexander, 1987)
afforded the pyrimidine nitrile derivative 6, Scheme 3. The
=
NBAP 3(N-benzylaminoprpionitrile
Scheme 4 Synthesis of pyrimido[4,5-c]azepine derivative 10
123

Med Chem Res
2217.11 cm-1. Cyclization of compound 9 with sodium
hydride in THF afforded the pyrimidine iminonitrile
product 10 as in Scheme 4 which showed the imino NH
Table 1 Antifungal activity of synthesized compound against
A. niger
Compounds
Zone of inhibition (mm)
1
proton at d 10.76 ppm in its H NMR spectrum and the
10 lg/ml
50 lg/ml
100 lg/ml
1000 lg/ml
imino carbon signal appeared at d 169.46 ppm in its 13C
NMR spectrum.
2 (Reference)
2.6
1.5
1.6
1.4
2.1
1.00
2.9
1.9
2.1
1.7
2.4
1.02
3.2
2.0
2.5
1.9
2.7
1.33
4
Compound 9 was synthesized by an alternative path via
the reaction of the bromopyrimidine derivative 4 with NBAP
in the presence of ethanol and sodium carbonate to produce
the nitrile derivative 11. Bromination of compound 11 with
PBC in acetic acid gave the bromopyrimidine nitrile deriv-
ative 12 that upon cyanation with CuCN in DMF produced
the same pyrimidine dinitrile derivative 9 in an independent
synthesis, Scheme 5. Compound 11 showed the cyano group
absorption signal at 2210.23 cm-1 and 11 aromatic protons
appeared at d 7.55–7.89 ppm in its 1H NMR data. Compound
12 gave the cyano group absorption at 2218.27 cm-1 in its IR
data; while the 1H NMR spectrum indicated the disappear-
ance of the pyrimidine ring C-5 proton signal.
4
2.2
2.9
2.5
3.4
2.10
5
6
7
10
Table 2 Effect of different concentrations of pyrimethanil 2 and
N-acetyl pyrimethanil 7 on bursting of A. niger
Concentrations (lg/ml)
Pyrimethanil 2 (h)
N-acetyl
pyrimethanil (h)
10
7.5
5
56
39
21
12
51
32
18
9
Fungicidal activity
2.5
Anilinopyrimidine derivatives namely; 2, 4–7, and 10 were
in vitro applied as an antifungal active against A. niger.
The pyrimethanil 2 is considered as a reference fungicidal
drug. The newly synthesized N-acetyl pyrimethanil 7 have
the highest activity compared to the others compounds; 4–6
and 10 with inhibition zone similar to the reference pyri-
methanil 2. The inhibition activities for the applied com-
pounds against A. niger and the significant inhibition zones
with different concentration are reported Table 1.
reached to 51 h for N-acetyl pyrimethanil and 56 h for
pyrimethanil 2. The previous data indicated that the time of
bursting was increased significantly as more concentrations
for the applied substances are used. This was explained
on the basis of coagulation of the polar molecules of the
N-acetyl pyrimethanil 7, which prevents the penetration of
fungous cell wall membrane.
The biological data indicated that the newly synthesized
compound 7 is considered the most active analog and it
was selected to study its bursting time of A. niger (the time
required for complete inhibition of A. niger growth)
(Toffolatti et al., 2008) in comparison with reference
pyrimethanil 2. Table 2 indicates the relation between the
concentrations of N-acetyl pyrimethanil 7 and pyrimethanil
2 on the bursting of A. niger. As the concentration of both
compounds increased, the time required to complete the
bursting of A. niger were dramatically increased till
On comparing the time required for bursting of A. niger
filaments for all the applied concentrations of N-acetyl
pyrimethanil 7 was less than of that for pyrimethanil 2
indicates that this derivative is more effective as fungicidal
agent. From the economic point of view, the N-acetyl
pyrimethanil 2 could destroy the filaments of A. niger by
concentration 2.5 lg/ml in 9 h, which reflected its high
reactivity as active ingredient for formulation of pesticides.
Conclusions
CH3
N
CH3
Several newly substituted anilinopyrimidine were prepared
and their fungicidal activities against A. niger were eval-
uated. PBC was applied as a selective brominating agent
for pyrimidine ring instead of the side chain. N-acetyl
pyrimethanil 7 showed high activity toward A. niger with
similar inhibition soon and faster bursting time with the
reference pyrimethanil fungicidal drug 2. Several pyrimi-
dine ring substituents and fused pyrimido[4,5-c]azepine
derivatives have a moderate fungicidal activity against
A. niger.
NBAP
CH2Br
N
Bz
N
N
Ph
N
H
CN
Ph
N
H
N
11
PBC
4
CH3
Br
N
Bz
CuCN
DMF
10
N
Ph
N
N
CN
12
H
Scheme 5 Independent synthesis of compound 9
123

Med Chem Res
Experimental
162.35, 166.84, 169.87. Anal. Calcd. for C12H12BrN3
(278.15): C, 51.82; H, 4.35; N, 15.11. Found: C, 52.10;
H, 4.11; N, 15.43 %.
Chemistry
Melting points were uncorrected and obtained on digital
Gallen Kamp melting point apparatus. The IR spectra were
recorded on a Jasco 4100 FTIR Spectrophotometer in KBr
disks (vmax in cm-1). 1H- and 13C NMR spectra (CDCl3) or
(DMSO-d6) were obtained using Pucker 600 MHZ spec-
trometer and chemical shift values were expressed in d
values (ppm) relative to that of the solvent. All NH and OH
protons were exchangeable with D2O. The mass spectra
were recorded on a Shimadzu GCMS-QP 1000 EX mass
spectrometer at 70 eV. Elemental analyses were recorded
on a PERKIN-ELMER 2400 C, H, N elemental analyzer,
Cairo University. Reaction progress was monitored by
analytical thin layer chromatography on pre-coated glass
plate (silica gel 60F254-plate-Merck) and the products were
visualized by UV light.
5-Bromo-4,6-dimethyl-N-phenylpyrimidin-2-amine (5)
A solution of compound 2 (4.0 g, 0.02 mol) in glacial
acetic acid (10 ml) was added to a stirred solution of PBC
(5 g, 0.02 mol) in glacial acetic acid (10 ml) in 40 °C
during 5 min. The buff precipitate was filtered off and dried
to give pure product 5 with yield (4.4 g, 79.4 %) and mp.
80–82 °C. IR (KBr, cm-1): 3400.85 (NH). 1H NMR
(CDCl3): d 2.48 (6H, s, 2CH3), 7.01(NH, br, NH,
exchangeable), 7.21–7.46 (5H, m, aromatic). 13C NMR
(CDCl3): d 25.69, 111.71, 115.17, 120.96, 132.41, 139.32,
157.95, 166.84. Anal. Calcd. for C12H12BrN3 (278.15): C,
51.82; H, 4.35; N, 15.11. Found: C, 52.11; H, 4.66; N,
15.32.
4,6-Dimethyl-2-(phenylamino)pyrimidine-5-carbonitrile
(6)
4,6-Dimethyl-2(N-phenylamino)pyrimidine (2)
A
mixture of phenylguanidine sulfate salt (3.68 g,
A mixture of the bromopyrimidine derivative 5 (2.8 g,
0.01 mol) and CuCN (0.98 g, 0.011 mol) in DMF (20 ml)
was refluxed for 4 h and poured onto a solution of FeCl3
in HCl and water at 60 °C. The product was extracted with
CH2Cl2 (50 ml 9 3), washed with water (20 ml 9 3),
dried over sodium sulfate anhydrous, and evaporated in
vacuo. Product 6 was crystallized from ethanol and has
mp. 174–176 °C and yield 1.5 g, 67 %. IR (KBr, cm-1):
3336.25 (NH) and 2217.74 (CN). 1H NMR (CDCl3): d
2.65 (6H, s, 2CH3), 7.65 (1H, br, NH, exchangeable),
7.66–7.80 (5H, m, aromatic). 13C NMR (CDCl3): d 21.61,
111.29, 115.30, 121.43, 132.37, 139.30, 157.71, 166.95,
176.61. Anal. Calcd. for C13H12N4 (224.26): C, 69.62; H,
5.39; N, 24.98. Found: C, 69.32; H, 5.50; N, 25.20 %.
0.01 mol), acetylacetone (2.0 g, 0.02 mol), and hydrated
sodium acetate (2.0 g, 0.02 mol) was heated at temperature
120 °C in open round bottom flask until all water was
removed. Then, the mixture was heated under refluxing for
further 4 h. After cooling, the residue was dissolved in
ethanol and poured onto cold water. The precipitate was
filtered off and recrystallized from aqueous ethanol to give
pure product, 3.2 g, 80 %, mp. 91–92 °C. IR (KBr, cm-1):
1
3478.23 (NH). H NMR (CDCl3): d 2.31 (6H, s, 2CH3),
6.34 (1H, br, NH, exchangeable), 7.34–7.76 {6H, m, aro-
matic and C(5)H}. 13C NMR (CDCl3): d 22.43, 97.62,
105.88, 116.75, 130.11, 150.69, 156.34, 168.22. Anal.
Calcd. for C12H13N3C (199.25): C, 72.33; H, 6.58; N,
21.09. Found: C, 72.54; H, 6.29; N, 21.11.
N-(4,6-Dimethylpyrimidin-2-yl)-N-phenylacetamide (7)
4-(Bromomethyl)-6-methyl-N-phenylpyrimidin-2-amine (4)
A mixture of anilinopyrimidine 2 (1.99 g, 0.01 mol) and
acetic anhydride (20 ml) was refluxed for 2 h and was
poured onto water (50 ml). The mixture was extracted with
water (30 ml 9 3), washed with sodium carbonate solution
(30 ml 9 3), water (20 ml 9 3), dried, and evaporated in
vacuo to give pure N-acetyl anilinopyrimidine derivative 7,
Rf 0.3 (ethyl acetate: petroleum ether 10 %) and yield
2.2 g, 83 %. IR (KBr, cm-1): 1685.48 (CO). 1H NMR
(CDCl3): d 2.33 (3H, s, COCH3), 2.54 (6H, s, 2CH3),
7.54–7.76 {6H, m, aromatic and C(5)H}. 13C NMR
(CDCl3): d 20.28, 26.31, 119.11, 126.43, 128.96, 131.03,
144.52, 164.37, 169.44, 172.77. Anal. Calcd. for
C14H15N3O (241.29): C, 69.69; H, 6.27; N, 17.41. Found:
C, 69.34; H, 6.55; N, 17.23 %.
A solution of anilinopyrimidine 2 (1.99 g, 0.01 mol),
NBS (1.77 g, 0.01 mol), and bi-benzoyl peroxide (0.1 g,
catalyst) in chloroform was irradiated using mercury
lamp 250 W for 8 h. The mixture was transferred to
separating funnel and washed with water (30 ml 9 3),
dried with Na2SO4 and evaporated in vacuo until dryness.
The solid residue was separated and crystallized from
aqueous ethanol to give pure product 4 with yield 1.8 g,
64.7 % and mp. 127–128 °C. IR (KBr, cm-1): 3554.14
1
(NH). H NMR (CDCl3): d 2.44 (3H, s, CH3), 2.55 (2H,
s, CH2Br), 7.11 (1H, br, NH, exchangeable), 7.66–7.98
{6H, m, aromatic and C(5)H}. 13C NMR (CDCl3): d
24.61, 51.08, 108.79, 118.44, 120.64, 130.18, 148.22,
123

Med Chem Res
4-(Bromomethyl)-6-methyl-2-(phenylamino)pyrimidine-5-
carbonitrile (8).
exchangeable), 7.47–7.98 (10H, m, aromatic), 10.76 (1H,
s, =NH, exchangeable). 13C NMR (DMSO-d6): d 26.22,
35.43, 60.82, 67.53, 69.66, 115.54, 118.28, 126.17, 128.22,
128.98, 129.32, 129.98, 132.41, 135.33, 139.92, 160.55,
169.46, 172.66, 177.87. Anal. Calcd. for C23H22N6
(382.46): C, 72.23; H, 5.80; N, 21.97. Found: C, 72.55; H,
6.11; N, 22.30 %.
The procedure was followed as described with compound
4, yield 88 %, mp. 137–138 °C (aqueous ethanol). IR
(KBr, cm-1): 3403.74 (NH), 2216.77 (CN). 1H NMR
(CDCl3): d 2.555 (3H, s, CH3), 2.62 (2H, d, CH2Br), 7.36
(1H, br, NH, exchangeable), 7.61–7.76 (5H, m, aromatic).
13C NMR (CDCl3): d 28.56, 35.31, 106.44, 119.03, 122.08,
126.62, 128.29, 134.22, 168.11, 169.32, 172.74. Anal.
Calcd. for C13H11BrN4 (303.16): C, 51.50; H, 3.66; N,
18.48. Found: C, 51.34; H, 3.75; N, 18.30 %.
3-[Benzyl{(6-methyl-2-(phenylamino)pyrimidin-4-
yl)methyl}amino] propanenitrile (11)
The procedure was followed as was described for the
synthesis of compound 9, yield 73 %, semi-solid, Rf = 0.3
(ethyl acetate:petroleum ether 10 %). IR (KBr, cm-1):
N-((5-Cyano-6-methyl-2-(phenylamino)pyrimidin-4-
yl)methyl)-N-(2-cyanoethyl)benzamide (9)
1
3452.21 (NH), 2216.11 (CN). H NMR (CDCl3): d 2.33
(2H, t, NCH2CH2CN), 2.40 (3H, s, CH3), 2.99 (2H, t,
NCH2CH2CN), 3.22 (2H, d, CH2NCH2CH2CN), 4.12 (2H,
s, CH2Ph), 7.00 (1H, br, NH, exchangeable), 7.55–7.89
(11H, m, aromatic and C-5). 13C NMR (CDCl3): d 18.12,
24.05, 55.83, 58.29, 60.30, 99.19, 119.32, 120.33, 122.63,
127.11, 127.88, 128.39, 129.72, 138.11, 140.53, 167.29,
168.66, 169.30. Anal. Calcd. for C22H23N5 (357.45): C,
73.92; H, 6.49; N, 19.59. Found: C, 74.23; H, 6.65; N,
19.88 %.
A mixture of the bromopyrimidine nitrile derivative 8
(3.03 g, 0.01 mol), NBAP (1.6 g, 0.01 mol), and sodium
carbonate (1.07 g, 0.01 mol) in ethanol absolute (30 ml)
was refluxed for 12 h. After cooling, the solid precipitate
was filtered off, washed with ethanol absolute (10 ml), and
the filtrate was poured onto ice water (50 ml). The pre-
cipitate was filtered off and crystallized from aqueous
ethanol to give pure product 9 (3.4 g, 85.6 %), mp.
210–212 °C. IR (KBr, cm-1): 3401.82 (NH), 2210.02
and 2217.11 (CN). 1H NMR (CDCl3): d 2.43 (2H, t,
NCH2CH2CN), 2.47 (3H, s, CH3), 3.11 (2H, d,
NCH2CH2CN), 3.29 (2H, s, CH2NCH2CH2CN), 4.21 (2H,
s, CH2Ph), 7.16 (1H, br, NH, exchangeable), 7.27–7.78
(10H, m, aromatic). 13C NMR (CDCl3): d 17.32, 22.75,
48.11, 56.30, 58.92, 102.39, 117.82, 120.17, 121.56,
123.80, 126.11, 127.59, 128.35, 129.03, 136.43, 139.83,
169.33, 170.12, 172.00. Anal. Calcd. for C23H20N6O
(396.44): C, 69.68; H, 5.08; N, 21.20. Found: C, 69.45; H,
4.88; N, 21.34 %.
3-[Benzyl{(5-bromo-6-methyl-2-(phenylamino)pyrimidin-
4-yl)methyl}amino] propanenitrile (12)
The procedure was followed as was described for the
synthesis of compound 5, yield 67 %, mp. 192–194 °C. IR
(KBr, cm-1): 3500.57 (NH), 2218.27 (CN). 1H NMR
(CDCl3): d 2.40 (2H, t, NCH2CH2CN), 2.53 (3H, s, CH3),
3.23 (2H, t, NCH2CH2CN), 3.54 (4H, d, CH2NCH2CH2CN
and CH2Ph), 7.24 (1H, br, NH, exchangeable), 7.60–8.11
(10H, m, aromatic). 13C NMR (CDCl3): d 18.84, 19.33,
49.49, 50.75, 55.27, 112.52, 117.56, 118.23, 121.06,
126.43, 127.54, 128.96, 131.03, 140.90, 148.89, 160.33,
162.63, 169.11. Anal. Calcd. for C22H22BrN5 (436.35): C,
60.56; H, 5.08; N, 16.05. Found: C, 60.34; H, 5.29; N,
16.38 %.
8-Benzyl-5-imino-4-methyl-2-(phenylamino)-6,7,8,9-
tetrahydro-5H-pyrimido [4,5-c]azepine-6-carbonitrile (10)
A solution of dinitrile 9 (1.0 g, 2.5 mmol) in THF (10 ml)
was added dropwise to a suspension of sodium hydride
(0.2 g, 40 %, 5 mmol) in THF (20 ml) at room temperature
with stirring under nitrogen. After the addition was com-
pleted, the reaction mixture was refluxed for 10 h. After
cooling, ethanol absolute (10 ml) was added gradually, was
poured onto ice water (50 ml), and acidified with dilute
HCl. The precipitate was collected by filtration and crys-
tallized from ethanol to give pure product 10 (0.6 g, 63 %),
mp. 184–186 °C. IR (KBr, cm-1): 3397.96 (NH), 2217.74
(CN) and 1560.11(=NH). 1H NMR (DMSO-d6): d 2.50
(3H, s, CH3), 2.77 (2H, m, C-7), 2.93 (1H, m, C-6), 3.34
(4H, d, C-9 and CH2Ph), 7.01 (1H, br, NHPh,
Fungicidal activity
In vitro antifungal activity
Aspergillus niger was cultured on Czapek’s-agar medium
that has the following composition: 30 g sucrose, 3 g
sodium nitrate, 1 g K2HPO4, 0.5 g MgSO4Á7H2O, 0.5 g
KCl, 0.01 g FeSO4Á7H2O, 15 g agar, and 1 l of distilled
water. The pH was then adjusted to 6.5 using 0.1 N NaOH.
In this experiment, A. niger was inoculated on solid
Czapek’s medium and poured into plate until solidification.
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After solidification, holes were made into the plate (1 cm)
using cork borer. In these holes, 200 ll of the different
synthesized compounds with different concentrations (10,
50, 100, and 1000 lg/ml) were loaded. The plates were
incubated at 30 °C for 7 days. Through the incubation
period, zone of inhibition were recorded daily.
Mechanism of extension growth in hyphae
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This experiment was carried out on S agar medium that has
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g agar, 1 l water, and pH 5.6. Plates of S agar were inoc-
ulated with a diametric streak of spores from a malt agar
slope culture of A. niger. The plates were incubated for
3 days at 25 °C.
A plate of cultured A. niger was placed on the micro-
scope stage and focused on the surface marginal hyphae
with the 910 or 920 objective. The hyphae at the margin
of the plate were avoided. The objective was raised and one
drop of dilute acetic acid (0.5 % v/v) was added to the
marginal hyphae beneath the objective and immediately the
objective was lowered and the surface marginal hyphae
was refocused. The time taken for most of the hyphae to
burst was recorded.
´
´
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The procedure was repeated, but this time one drop of a
0.2 M sucrose solution (prepared in 0.5 % v/v acetic acid)
was added to a different portion of the colony margin.
Different concentrations of acetic acid and of sucrose in
acetic acid were applied and the time taken for most of the
hyphae to burst was recorded. This technique has been
extended to estimate the osmotic equivalent of hyphae
(Park and Robinson, 1996).
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Hyphal morphogenesis in A. niger
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Plates of S agar were inoculated with a diametric streak of
spores from a malt agar slope culture of A. niger, and the
plates were incubated for 3 days at 25 °C. Drops of each of
the following concentrations were applied on separate
regions of the colony margin: 2.5, 5, 7.5, and 10 lg/ml of
the synthesized compounds. A drop of dimethyl sulphoxide
was added as a control.
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