Enaminonitrile in heterocyclic synthesis: Synthesis and antimicrobial evaluation of some new pyrazole, isoxazole and pyrimidine derivatives incorporating a benzothiazole moiety

Article abstract of DOI:10.1016/j.ejmech.2009.07.024

Enaminonitrile 2 was used as key intermediate for the synthesis of polyfunctionally substituted heterocycles (e.g. pyrazoles, isoxazole, pyrimidines, thiazolo[3,2-a]pyrimidine, tetrazolo[1,5-a]pyrimidine, pyrido[1,2-a]pyrimidine, 1,5-benzodiazepine, and p

Full text of DOI:10.1016/j.ejmech.2009.07.024

European Journal of Medicinal Chemistry 44 (2009) 4813–4818
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Enaminonitrile in heterocyclic synthesis: Synthesis and antimicrobial evaluation
of some new pyrazole, isoxazole and pyrimidine derivatives incorporating
a benzothiazole moiety
*
Samir Bondock , Walid Fadaly, Mohamed A. Metwally
Department of Chemistry, Faculty of Science, Mansoura University, ET-35516 Mansoura, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Enaminonitrile 2 was used as key intermediate for the synthesis of polyfunctionally substituted
heterocycles (e.g. pyrazoles, isoxazole, pyrimidines, thiazolo[3,2-a]pyrimidine, tetrazolo[1,5-a]pyrimi-
dine, pyrido[1,2-a]pyrimidine, 1,5-benzodiazepine, and pyrazolo[1,5-a]pyrimidine) incorporating ben-
zothiazole moiety via its reactions with some N-nucleophiles. The newly synthesized compounds were
characterized by IR, 1H NMR and mass spectral studies. Representative compounds of the synthesized
products were tested and evaluated as antimicrobial agents.
Received 25 May 2009
Received in revised form
19 July 2009
Accepted 23 July 2009
Available online 30 July 2009
Ó 2009 Elsevier Masson SAS. All rights reserved.
Keywords:
Benzothiazoles
Enaminonitriles
Pyrimidines
Antimicrobial activity
1. Introduction
compound, N-(benzothiazol-2-yl)-2-cyanoacetamide (1) was
prepared according to the previously reported procedure [21].
Enaminonitriles are versatile reagents and their chemistry has
recently received a considerable attention as precursors to, otherwise
not readily obtainable heteroaromatics [1–6]. Elnagdi et al. have
reported several novel syntheses of azoles, azines, and azoloazines
utilizing enaminonitriles as starting components [7–9]. On the other
hand, the considerable biological and medicinal activities of benzo-
thiazoles initiated considerable recent interest in the development of
syntheses of these molecules [10–18]. In continuation of our interest
in the synthesis of heterocycles containing a benzothiazole moiety
[19,20], to identify new candidates that may be value in designing
new, potent, selective and lesstoxic antimicrobial agents, we reported
here a facial synthesis of some new pyrazole, isoxazole and pyrimi-
dine derivatives pendant to a benzothiazole via the reactions of
enaminonitrile with some nitrogen nucleophiles.
Thus, treatment of compound 1 with dimethylformamide dime-
thylacetal (DMF-DMA) in dry dioxane under reflux afforded
a bright red crystalline product that was identified as N-(benzo-
thiazol-2-yl)-2-cyano-3-(dimethylamino)acrylamide (2) in high
yield. The structure of the latter product was established on the
basis of its elemental analysis and spectral data. For example, its 1H
NMR spectrum displayed four singlet signals at
d 3.25, 3.32, 8.10,
and 11.60 ppm due to magnetically nonequivalent N(CH3)2 group,
methine, and NH protons, respectively, in addition to an aromatic
multiplet in the region
d 7.26–7.90 ppm. To study the structure–
activity relationship with respect to antimicrobial properties, the
reactivity of enaminonitrile 2 towards some nitrogen nucleophiles
was investigated. Thus, when enaminonitrile 2 was treated with
ammonia, the transamination adduct 3 was formed in good yield.
Cyclization of 3-amino-N-(benzothiazol-2-yl)-2-cyanoacrylamide
(3) with triethyl orthoformate and acetic anhydride afforded
a yellow crystalline product that was identified as 1-(benzothiazol-
2-yl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile (4). The struc-
ture of compound 4 was established on the basis of its elemental
analysis and spectral data. For example, its 1H NMR spectrum
2. Results and discussion
2.1. Chemistry
The synthetic strategies adopted to obtain the target
compounds are depicted in Schemes 1 and 2. The starting
showed two singlet signals at d 8.63 and 9.86 ppm characteristic for
H-4 and H-2 of the pyrimidine moiety. The mass spectrum of
compound 4 showed the molecular ion peak at m/z 254 (Mþ).
* Corresponding author. Tel.: +20 50 2369267; fax: +20 50 2246781.
E-mail address: bondock@mans.edu.eg (S. Bondock).
Treatment of enaminonitrile
2 with hydrazine hydrate and
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.024

4814
S. Bondock et al. / European Journal of Medicinal Chemistry 44 (2009) 4813–4818
N
O
Me2NCH(OMe)2
NH3
-Me2NH
N
O
CN
S
N
H
CN
S
N
H
1
HC(OEt)3
Ac2O
3
H2N
NH
NH2
EtONa
N
O
N
N
O
H2N
RNHNH2
EtOH
CN
S
CN
S
N
H
4
N
NMe2
2
HONH2.HCl
Pyridine
N
O
N
O
H
N
O
H
CN
H
CN
NH
S
N
CN
S
N
H
S
N
H
H
NHR
Me2N
N
H
OH
Me2N
N
H
NH2
Me2N
N
H
-Me2NH
-Me2NH
-Me2NH
N
O
NH2
N
O
N
O
CN
NH2
S
N
H
N
S
N
S
N
H
H
NHR
O
N
NH2
N
H
8
5a-b
N
7
Pyridine
O
N
NH2
R
S
N
H
N
N
6a-b
6a R = H
6b R = Ph
Scheme 1.
phenylhydrazine in refluxing ethanol furnished the isolated inter-
mediates 5a and b which could be cyclized to pyrazole derivatives
6a and b upon boiling in pyridine. Structures of compounds 6a and
b were established on the basis of elemental analysis and spectral
data. For example, the IR spectra of pyrazoles 6a and b were free of
nitrile function and showed absorption bands for NH2 in the region
3450–3325 cmꢀ1, pyrazole 6a showed also an absorption band due
Similarly, the enaminonitrile 2 reacts with hydroxylamine
hydrochloride in refluxing pyridine to afford one isolable product
identified as 5-amino-N-(benzothiazol-2-yl)isoxazole-4-carbox-
amide (7). The IR spectrum of isoxazole 7 showed absorption bands
at 3379, 3271, 3138, 1648 cmꢀ1 due to the amino, amide NH and
carbonyl groups, respectively. We have also investigated the reac-
tivity of enaminonitrile
2 towards guanidine. Thus, when
to NH at 3125 cmꢀ1
.
compound 2 was treated with guanidine nitrate in the presence of
NH2
N
N
O
NH
H N
N
O
2
H2N
S
S
N
H
N
NH2
EtOH/Pip.
N
S
N
H
EtOH/Pip.
N
S
9
N
12
H
H
N
N
N
N
NH2
H2N
N
H
N
O
NH2
N
N
O
NH2
N
N
2
EtOH/Pip.
EtOH/Pip.
S
N
N
N
S
N
H
H
N
N
N
N
10
13
NH2
N
N
N
O
NH2
N
N
H
N
O
NH
N
H2N
EtOH/Pip.
S
N
N
H
EtOH/Pip.
S
N
H
N
N
N
14
N
11
Scheme 2.

S. Bondock et al. / European Journal of Medicinal Chemistry 44 (2009) 4813–4818
4815
sodium ethoxide, it afforded an excellent yield of a product which
was identified as 2,4-diamino-N-(benzothiazol-2-yl)pyrimidine-5-
carboxamide (8) on the basis of its spectral data. Compounds 3–8
are assumed to be formed via addition of N-nucleophiles to the
activated ethylenic double bond of enaminonitrile 2 followed by
cyclization and elimination of a dimethylamine molecule as
depicted in Scheme 1.
pyrazolo[3,4-b]pyridine [22] under the same experimental
condition furnished only one isolable product, which was
identified as 4-amino-N-(benzothiazol-2-yl)-8,10-dimethylpyr-
ido[20,30:3,4]pyrazolo[1,5-a]pyrimidine-3-carboxamide (14). The
spectral data of the isolated product were in complete agreement
with the assigned structure. For example, the IR spectrum of the
reaction product 14 revealed no bands due to nitrile function and
showed characteristic absorption bands at 3430, 3301, 3179,
1678 cmꢀ1 due to the amino, amidic-NH, and amidic carbonyl
groups, respectively. The mass spectrum of compound 14 showed
a molecular ion peak at m/z 389 (Mþ) corresponding to a molecular
formula C19H15N7OS.
Formation of 2-heterarylbenzothiazoles 9–14 from enaminoni-
trile 2 and N,N-binucleophiles proceeds by initial substitution of
the dimethylamino group followed by cyclization. It is worthwhile
to mention that the intermediate substitution products 9–14 could
not be isolated. The regioselectivity for the formation of
compounds 9–14 is in line with the reported results of the reaction
of enaminonitriles with heterocyclic amines [23].
The foregoing results prompted us to investigate the behavior of
enaminonitrile 2 towards some bis-N-nucleophiles as well as
heterocyclic amines as potential precursors for fused heterocyclic
systems. Therefore, it was of interest to explore the scope, limita-
tions and generality of enaminonitrile 2 as a precursor for the
synthesis of some intricate to access polyfunctionally substituted
fused pyrimidine derivatives for which we might expect, a wide
spectrum of bioresponses. Thus, reaction of enaminonitrile 2 with
equimolar amounts of heterocyclic amines namely 2-amino-
thiazole, 5-aminotetrazole and 2-aminopyridine upon reflux in
ethanol containing a catalytic amount of piperidine, afforded the
corresponding thiazolo[3,2-a]pyrimidine (9), tetrazolo[1,5-
a]pyrimidine (10) and pyrido[1,2-a]pyrimidine (11) derivatives,
respectively, in reasonable yields. The identity of the products was
established on the basis of elemental analyses and spectral back-
ground in each case. o-Phenylene diamine has been reported as
useful precursors for the synthesis of diazepine which possesses
tranquilizers activity. Thus, treatment of enaminonitrile 2 with
o-phenylene diamine under the same experimental condition
afforded a yellow product identified as 4-amino-N-(benzothiazol-
2-yl)-1H-benzo[b][1,4]diazepine-3-carboxamide (12). The IR spec-
trum of compound 12 showed two characteristic absorption bands
at 3466, 3350, 3725, 3150 cmꢀ1 due to amino group and two NH
groups and no band due to the nitrile function. The mass spectrum
of compound 12 showed a molecular ion peak at m/z 335 (Mþ).
In the same manner, the enaminonitrile 2 reacts regiose-
lectively with 2-aminobenzimidazole in refluxing ethanolic
piperidine solution to give pyrimido[1,2-a]benzimidazole deriva-
tive (13). The structure of compound 13 was established on the
basis of elemental analysis and spectral data of the isolated reac-
tion product. The presence of an amino group in structure 13 was
evidenced by the appearance of two absorption bands at 3452 and
3340 cmꢀ1 in the IR spectrum of the reaction product. Similarly,
the reaction of enaminonitrile 2 with 3-amino-4,6-dimethyl-1H-
3. Pharmacology
3.1. Antimicrobial evaluation
Thirteen of the newly synthesized target compounds were
evaluated for their in vitro antibacterial activity against Bacillus
subtilis and Bacillus thuringiensis as examples of Gram positive
bacteria and Escherichia coli and Pseudomonas aeruginosa as
examples of Gram negative bacteria. They were also evaluated for
their in vitro antifungal potential against Botrytis fabae and Fusa-
rium oxysporum fungal strains.
Agar-diffusion method was used for the determination of the
preliminary antibacterial and antifungal activities. Streptomycin,
Chloroamphenicol and Treflucan were used as reference drugs. The
results were recorded for each tested compound as the average
diameter of inhibition zones (IZ) of bacterial or fungal growth
around the disks in mm. The minimum inhibitory concentration
(MIC) measurement was determined for compounds showed
significant growth inhibition zones (>10 mm) using twofold serial
dilution method [24]. The MIC (
m
g/mL) and inhibition zone diam-
eter values are recorded in Table 1.
Table 1
Minimal inhibitory concentrations (MIC,
m
g/mL) and inhibition zone (mm) of some new synthesized compounds.
Compound no.
MIC in
Bacteria
mg/mL, and zone of inhibition (mm)
Fungi
Gram positive bacteria
Gram negative bacteria
B. subtilis
B. thuringiensis
E. coli
P. aeruginosa
B. fabae
25 (25–30)
F. oxysporum
2
3
4
5a
5b
6a
6b
7
8
9
10
11
12
100 (13–18)
100 (13–18)
12.5 (31–36)
100 (13–18)
100 (13–18)
12.5 (31–36)
12.5 (31–36)
50 (19–24)
12.5 (31–36)
12.5 (31–36)
3.125 (43–48)
6.25 (37–42)
6.25 (37–42)
25 (25–30)
50 (19–24)
50 (19–24)
50 (19–24)
50 (19–24)
50 (19–24)
50 (19–24)
25 (25–30)
25 (25–30)
25 (25–30)
12.5 (31–36)
12.5 (31–36)
12.5 (31–36)
100 (13–18)
25 (25–30)
50 (19–24)
25 (25–30)
12.5 (31–36)
12.5 (31–36)
50 (19–24)
50 (19–24)
25 (25–30)
25 (25–30)
50 (19–24)
12.5 (31–36)
25 (25–30)
50 (19–24)
100 (13–18)
50 (19–24)
25 (25–30)
25 (25–30)
12.5 (31–36)
50 (19–24)
50 (19–24)
50 (19–24)
6.25 (37–42)
50 (19–24)
50 (19–24)
6.25 (37–42)
25 (25–30)
12.5 (31–36)
50 (19–24)
50 (19–24)
12.5 (31–36)
6.25 (37–42)
50 (19–24)
25 (25–30)
6.25 (37–42)
25 (25–30)
6.25 (37–42)
25 (25–30)
25 (25–30)
6.25 (37–42)
12.5 (31–36)
6.25 (37–42)
6.25 (37–42)
3.125 (43–48)
12.5 (31–36)
6.25 (37–42)
3.125 (43–48)
12.5 (31–36)
3.125 (43–48)
6.25 (37–42)
12.5 (31–36)
Reference drugs
Streptomycin
Chloramphenicol
Treflucan
3.125 (43–48)
6.25 (37–42)
–
6.25 (37–42)
6.25 (37–42)
–
6.25 (37–42)
6.25 (37–42)
–
6.25 (37–42)
6.25 (37–42)
–
–
–
–
–
3.125 (43–48)
3.125 (43–48)

4816
S. Bondock et al. / European Journal of Medicinal Chemistry 44 (2009) 4813–4818
The results depicted in Table 1 revealed that most of tested
4.1. Synthesis of N-(benzothiazol-2-yl)-2-cyano-3-
(dimethylamino)acrylamide (2)
compounds displayed variable inhibitory effects on the growth of
the tested Gram positive and Gram negative bacterial strains, and
also against antifungal strains.
A
mixture of compound 1 (2.17 g, 10 mmol) and dime-
In general, most of the tested compounds revealed better
activity against the Gram positive rather than the Gram negative
bacteria. It would be also noticed that compounds belonging to the
cyclized pyrazole series exhibited better antibacterial potentials
than the transamination adduct.
Regarding the activity of the pyrimidines against Gram positive
bacteria, the results revealed that compounds 4, 8–11 exhibited
broad spectrum antibacterial profile against the tested organisms.
In this view, compound 10 was equipotent to streptomycin in
thylformamide dimethylacetal (1.2 g, 10 mmol) in dioxane (30 mL)
was refluxed for 4 h. After cooling to room temperature, the solid
precipitate that formed was filtered off, washed with light petro-
leum ether (bp 40–60 ꢁC) and dried. Recrystallization from dioxane
afforded compound 2.
Bright red crystals; yield 81%; mp 231–232 ꢁC (Lit. [9] 232 ꢁC); IR
(KBr): ymax/cmꢀ1 ¼3400 (NH), 2186 (CN), 1678 (CO). 1H NMR
(CDCl3): dppm ¼ 3.25 (s, 3H, CH3), 3.32 (s, 3H, CH3), 7.26 (t, J ¼ 8.1 Hz,
1H, Ar–H), 7.40 (t, J ¼ 7.8 Hz, 1H, Ar–H), 7.63 (d, J ¼ 8.1 Hz, 1H, Ar–
H), 7.90 (d, J ¼ 7.8 Hz, 1H, Ar–H), 8.10 (s, 1H, CH]), 11.6 (s, 1H, NH).
MS: m/z (%) ¼ 272 (Mþ, 25), 177 (10), 149 (13), 123 (100), 122 (3.4),
80 (13). Anal. for C13H12N4OS (272.33): Calcd.: C 57.34; H 4.44; N
20.57%. Found: C 57.37; H 4.51; N 20.53%.
inhibiting the growth of B. subtilis (MIC 3.125 mg/mL), while its
activity was 50% lower than streptomycin against B. thuringiensis.
Compounds 11 and 12 showed 50% of the activity of streptomycin
(MIC 6.25
mg/mL) but they were equipotent to Chloroamphenicol in
inhibiting the growth of B. subtilis (MIC 6.25
mg/mL).
On the other hand, compounds 4, 5a, b, 6a, b and 7 exhibited
4.2. 3-Amino-N-(benzothiazol-2-yl)-2-cyanoacrylamide (3)
weak to moderate growth inhibitory activity against Gram positive
bacteria as revealed from their MIC values (25–100
these compounds 6a and b showed relatively good growth inhibi-
tory profiles against B. subtilis (MIC 12.5 g/mL) which were about
25% of the activity of streptomycin and 50% of Chloroamphenicol
against the same organism.
m
g/mL). Among
To a solution of the enaminonitrile 2 (0.217 g, 1 mmol) in
ethanol (20 mL), concentrated aqueous ammonia (0.7 mL, sp. gr.
0.90) was added. The reaction mixture was refluxed for 6 h, then
cooled. The solid product so formed was filtered off, washed with
dry ether, dried and recrystallized from ethanol afforded
compound 3.
m
Concerning the antibacterial activity of the compounds 4, 6b, 7,
and 10 revealed weak growth inhibitory against the tested Gram
Yellow crystals; yield 73%; mp 226–227 ꢁC; IR (KBr): ymax
/
negative bacteria (MIC 50
mg/mL).
cmꢀ1 ¼3379, 3274 (NH2), 3166 (NH), 2195 (CN), 1655 (CO). 1H NMR
(CDCl3): dppm ¼ 7.20–7.94 (m, 4H, Ar–H), 8.21 (br s, 2H, NH2), 8.32
(s, 1H, CH]), 9.86 (s, 1H, NH). Anal. for C11H8N4OS (244.27): Calcd.:
C 54.09, H 3.30, N 22.94%; Found: C 54.12, H 3.26, N 22.89%.
Regarding the activity of pyrazoles, pyrimidines and azolopyr-
imidines incorporating benzothiazole moiety, against antifungal
strains, the results revealed that compounds 6b, 8, and 10 were
equipotent to Treflucan in inhibiting the growth of B. fabae (MIC
3.125
m
g/mL), while their reactivity was 50% lower than Treflucan
4.2.1. 1-(Benzothiazol-2-yl)-6-oxo-1,6-dihydropyrimidine-5-
carbonitrile (4)
against F. oxysporum (MIC 6.25
mg/mL).
It is worth mentioning that incorporation of benzothiazole to
pyrimidine nucleus at position 5 via a carboxamide linker produced
a high antimicrobial activity. Conversion of 5a and b to pyrazoles 6a
and b also enhanced the antimicrobial activity. On the other hand,
incorporation of benzothiazole nucleus to isoxazole at position 4 in
compound 7 unfortunately produced weak antimicrobial activity.
In conclusion, the objective of the present study was to
synthesize and investigate the antimicrobial activities of some new
heterocycles incorporating benzothiazole moiety with the hope of
discovering new structure leads serving as potent antimicrobial
agents. Our aim has been verified by the synthesis of two different
groups of structure hybrids comprising basically the benzothiazole
moiety attached to either polysubstituted pyrazole or pyrimidine
counter parts through various linkages of synergistic purpose. The
obtained results clearly revealed that compounds derived from
pyrimidines exhibited better antimicrobial activity than their pyr-
azole structure variants.
A solution of compound 3 (0.26 g, 1 mmol) in a mixture of
triethyl orthoformate (2.5 mL) and acetic anhydride (2.5 mL) was
heated under reflux for 3 h during which the product partially
separated. The reaction mixture was allowed to cool to room
temperature and the separated product was filtered off, washed
with ethanol, dried, and recrystallized from acetic acid.
Yellow crystals; yield 53%; mp 282–283 ꢁC; IR (KBr): ymax
/
cmꢀ1 ¼ 2198 (CN), 1695 (CO), 1625 (C]N). 1H NMR (DMSO-d6):
dppm ¼ 7.22–7.78 (m, 4H, Ar–H), 8.63 (s, 1H, pyrimidine–H4), 9.25 (s,
1H, pyrimidine–H2). MS m/z (%): 254 (Mþ, 24), 227 (30), 203 (20),
150 (100), 149 (80), 122 (39), 95 (28), 68 (40), 51 (28). Anal. for
C12H6N4OS (254.27): Calcd.: C 56.68, H 2.38, N 22.03%; Found: C
56.61, H 2.33, N 22.08%.
4.3. General procedure for the reaction of enaminonitrile 2 with
hydrazines
To a solution of the enaminonitrile 2 (0.45 g, 2 mmol) in ethanol
(20 mL), hydrazine hydrate (80%, 0.2 mL) or phenylhydrazine
(0.2 mL, 2 mmol) was added. The reaction mixture was refluxed for
4 h, and then cooled. The solid product so formed was filtered off,
washed with ethanol, dried and recrystallized from a mixture of
DMF/EtOH (1:2) to give compounds 5a and b.
4. Experimental
All melting points were measured on a Gallenkamp electro-
thermal melting point apparatus. IR spectra were recorded for KBr
disc on a Mattson 5000 FTIR spectrophotometer. 1H NMR spectra
were measured on a Bruker AC 300 (300 MHz) in CDCl3 or DMSO-d6
as solvent, using TMS as an internal standard, and chemical shifts
are expressed as dppm. Mass spectra were determined on Finnigan
Incos 500 (70 eV). Elemental analyses were carried out in the
Microanalytical Unit of the Faculty of Science, Cairo University. N-
(Benzothiazol-2-yl)-2-cyanoacetamide (1) [21] and 4,6-dimethyl-
1H-pyrazolo[3,4-b]pyridin-3-amine [22] were prepared following
literature procedure.
4.3.1. N-(Benzothiazol-2-yl)-2-cyano-3-hydrazinylacrylamide (5a)
Pale yellow crystals; yield 83%; mp 303–304 ꢁC; IR (KBr): ymax
/
cmꢀ1 ¼3436, 3328 (NH2), 3273, 3135 (2NH), 2194 (CN), 1667 (CO).
1H NMR (DMSO-d6): dppm ¼ 5.13 (br s, 2H, NH2), 7.26 (t, J ¼ 7.6 Hz,
1H, Ar–H), 7.41 (t, J ¼ 7.6 Hz, 1H, Ar–H), 7.60 (d, J ¼ 5.4 Hz, 1H, Ar–
H), 7.81 (d, J ¼ 6 Hz,1H, Ar–H), 7.86 (s,1H, olefinic CH]), 8.30 (s,1H,
NH–hydrazine), 10.6 (s, 1H, NH). MS m/z (%): 259 (Mþ, 36%), 226

S. Bondock et al. / European Journal of Medicinal Chemistry 44 (2009) 4813–4818
4817
(7.5), 185 (6.6), 177 (6.8), 150 (96), 110 (100), 83 (22), 69 (27.5). Anal.
for C11H9N5OS (259.29): Calcd.: C 50.95, H 3.50, N 27.01%; Found: C
50.90, H 3.45, N 27.13%.
dissolving 0.23 g of sodium in 20 mL absolute ethanol) was refluxed
for 8 h. The reaction mixture was allowed to cool to room
temperature and diluted with ice-cold water (30 mL) containing
few drops with HCl. The solid product so formed was collected by
filtration, washed with water, dried, and recrystallized from
a mixture of EtOH/DMF (2:1) to give compound 8.
4.3.2. 3-(2-Phenylhydrazinyl)-N-(benzothiazol-2-yl)-2-
cyanoacrylamide (5b)
Yellow crystals; yield 67%; mp 135–136 ꢁC; IR (KBr): ymax
/
Buff powder; yield 86%; mp 333–334 ꢁC; IR (KBr): ymax
/
cmꢀ1 ¼3429, 3325, 3175 (3NH), 2216 (CN), 1660 (C]O). 1H NMR
(DMSO-d6): dppm ¼ 7.26–7.90 (m, 4H, Ar–H), 8.11 (s, 1H, olefinic
CH]), 8.52 (s, 1H, NH), 10.2 (s, 1H, Ph–NH), 10.6 (s, 1H, NH). MS m/z
(%): 335 (Mþ, 14.6), 272 (20), 186 (68), 123 (100), 77 (39). Anal. for
C17H13N5OS (335.38): Calcd.: C 60.88, H 3.91, N 20.88%; Found: C
60.79, H 3.87, N 20.78%.
cmꢀ1 ¼3360, 3256 (NH2), 3144 (NH), 1660 (C]O). 1H NMR (DMSO-
d6): dppm ¼ 7.33 (t, J ¼ 7.2 Hz,1H, Ar–H), 7.46 (t, J ¼ 7.2 Hz,1H, Ar–H),
7.65 (br s, 2H, NH2), 7.64 (d, J ¼ 8.0 Hz, 1H, Ar–H), 7.95 (d, J ¼ 7.8 Hz,
1H, Ar–H), 8.72 (s, 1H, pyrimidine–H6), 8.89 (br s, 2H, NH2), 12.80
(s, 1H, NH). MS m/z (%): 286 (Mþ, 13%), 137 (98), 136 (100), 95 (53),
94 (53), 68 (19), 67 (18). Anal. for C12H10N6OS (286.31): Calcd.:
C 50.34, H 3.52, N 29.35%; Found: C 50.31, H 3.43, N 29.31%.
4.4. General procedure for synthesis of pyrazole derivatives
(6a and b)
A solution of compounds 5a and b (1 mmol) in pyridine (20 mL)
was refluxed for 8 h. The solution was evaporated under vacuum
and triturated with ethanol. The precipitated product was filtered
off, washed with ethanol and recrystallized from a mixture of EtOH/
DMF (1:1) to give pyrazoles 6a and b.
4.7. General procedure for the reaction of enaminonitrile 2 with
heterocyclic amines and o-phenylene diamine
A mixture of the enaminonitrile 2 (0.45 g, 2 mmol) and an equi-
molar amount of the appropriate heterocyclic amines (2-amino-
thiazole, 5-aminotetrazole, 2-aminopyridine, 2-aminobenzimidazole
or 4,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-3-amine) or o-phenylene
diamine in 30 mL ethanol containing few drops of piperidine
(3 drops) was refluxed for 8 h, then left to cool to room temperature.
The solid deposited was collected by filtration and recrystallized from
a mixture of EtOH/DMF (3:1) to give compounds 9–14.
4.4.1. 5-Amino-N-(benzothiazol-2-yl)-1H-pyrazole-4-carboxamide
(6a)
Yellow powder; yield 88%; mp 284–285 ꢁC (Lit. [9] 286 ꢁC); IR
(KBr): ymax/cmꢀ1 ¼3450, 3369 (NH2), 3275, 3125 (2NH), 1660
(CO), 1607 (C]N). 1H NMR (DMSO-d6): dppm ¼ 6.12 (br s, 2H,
NH2), 7.27 (t, J ¼ 7.8 Hz, 1H, Ar–H), 7.40 (t, J ¼ 8.1 Hz, 1H, Ar–H),
7.72 (d, J ¼ 8.1 Hz, 1H, Ar–H), 7.93 (d, J ¼ 7.8 Hz, 1H, Ar–H), 8.16
(s, 1H, pyrazole–H5), 11.93 (br s, 2H, 2NH). Anal. for C11H9N5OS
(259.29): Calcd.: C 50.95, H 3.50, N 27.01%; Found: C 50.87,
H 3.42, N 27.12%.
4.7.1. N-(Benzothiazol-2-yl)-5-imino-5H-[1,3]thiazolo[3,2-
a]pyrimidine-6-carboxamide (9)
Yellow crystals; yield 74%; mp 283–284 ꢁC; IR (KBr): ymax
/
cmꢀ1 ¼3345, 3169, (2NH), 1665 (CO), 1625(C]N). 1H NMR (DMSO-
d6): dppm ¼ 6.98, 7.22 (2d, J ¼ 4.5 Hz, thiazole–H), 7.25–7.86 (m, 4H,
Ar–H), 8.96 (s, 1H, pyrimidine–H4), 9.21 (s, 1H, NH), 9.65 (s, 1H, NH).
Anal. for C14H9N5OS2 (327.38): Calcd.: C 51.36, H 2.77, N 21.39%;
Found: C 51.21, H 2.67, N 21.42%.
4.4.2. 5-Amino-N-(benzothiazol-2-yl)-1-phenyl-1H-pyrazole-4-
carboxamide (6b)
Yellow powder; yield 65%; mp 246–247 ꢁC; IR (KBr): ymax
/
cmꢀ1 ¼3441, 3328 (NH2), 3219 (NH), 1666 (C]O), 1637 (C]N). 1H
NMR (DMSO-d6): dppm ¼ 6.70 (s, 2H, NH2), 7.29–7.94 (m, 9H, Ar–H),
8.41 (s, 1H, pyrazole–H5), 12.15 (s, 1H, NH). Anal. for C17H13N5OS
(335.38): Calcd.: C 60.88, H 3.91, N 20.88%; Found: C 60.69, H 3.85, N
20.77%.
4.7.2. 7-Amino-N-(benzothiazol-2-yl)-tetrazolo[1,5-a]pyrimidine-
6-carboxamide (10)
Yellow crystals; yield 54%; mp 272–273 ꢁC; IR (KBr): ymax
/
cmꢀ1 ¼3434, 3329 (NH2), 3169 (NH), 1672 (CO). 1H NMR (DMSO-
d6): dppm ¼ 6.98 (s, 2H, NH2), 7.23–7.98 (m, 4H, Ar–H), 8.23 (s, 1H,
pyrimidine–H4), 9.87 (s, 1H, NH). MS m/z (%): 312 (39), 225 (33),
258 (39), 177 (28), 135 (21), 123 (37), 77 (10). Anal. for C12H8N8OS
(312.31): Calcd.: C 46.15, H 2.51, N 35.88%; Found: C 46.11, H 2.47, N
35.76%.
4.5. Synthesis of 5-amino-N-(benzothiazol-2-yl)isoxazole-4-
carboxamide (7)
A mixture of enaminonitrile 2 (0.45 g, 2 mmol) and hydroxyl-
amine hydrochloride (2.3 mmol) in 30 mL pyridine was refluxed for
8 h and then allowed to cool to room temperature and diluted with
ice-cold water (20 mL). The solid product so formed was collected
by filtration, washed with water, dried, and recrystallized from
ethanol to give compound 7.
4.7.3. N-(Benzothiazol-2-yl)-4-imino-4H-pyrido[1,2-a]pyrimidine-
3-carboxamide (11)
Yellow crystals; yield 74%; mp 288–289 ꢁC; IR (KBr): ymax
/
cmꢀ1 ¼3320, 3195 (2NH), 1660 (CO), 1618 (C]N). 1H NMR (DMSO-
d6): dppm ¼ 7.21–7.85 (m, 8H, Ar–H), 8.16 (s, 1H, pyrimidine–H4),
8.98 (s, 1H, NH), 9.95 (s, 1H, NH). Anal. for C16H11N5OS (321.36):
Calcd.: C 59.80, H 3.45, N 21.79%; Found: C 59.69, H 3.35, N 21.61%.
Yellow crystals; yield 73%; mp 260–261 ꢁC; IR (KBr): ymax
/
cmꢀ1 ¼3379, 3271 (NH2), 3138 (NH), 1648 (CO). 1H NMR (DMSO-
d6): dppm ¼ 7.20–7.92 (m, 4H, Ar–H), 8.02 (br s, 2H, NH2), 8.31 (s, 1H,
isoxazole–H3), 9.86 (s, 1H, NH). MS m/z (%): 260 (Mþ, 34%), 259 (30),
217 (7), 177 (29), 150 (77), 149 (81), 123 (19), 122 (24.5), 77 (24), 68
(100), 67 (70). Anal. for C11H8N4O2S (260.27): Calcd.: C 50.76, H 3.10,
N 21.53%; Found: C 50.66, H 3.05, N 21.43%.
4.7.4. 4-Amino-N-(benzothiazol-2-yl)-1H-1,5-benzodiazepine-3-
carboxamide (12)
Yellow powder; yield 68%; mp 206–207 ꢁC; IR (KBr): ymax
/
cmꢀ1 ¼3466, 3350 (NH2), 3275, 3150 (2NH), 1678 (CO). 1H NMR
(DMSO-d6): dppm ¼ 6.82 (s, 2H, NH2), 7.25–8.36 (m, 8H, Ar–H), 8.62
(s, 1H, CH]), 9.87 (s, 1H, NH), 12.22 (s, 1H, NH). MS m/z (%): 335
(Mþ, 100), 267 (55), 178 (14), 150 (67), 135 (12), 133 (33), 123 (31),
108 (31), 77 (47). Anal. for C17H13N5OS (335.38): Calcd.: C 60.88, H
3.91, N 20.88%; Found: C 60.73, H 3.89, N 20.72%.
4.6. Synthesis of 2,4-diamino-N-(benzothiazol-2-yl)pyrimidine-5-
carboxamide (8)
A mixture of enaminonitrile 2 (0.45 g, 2 mmol) and guanidine
nitrate (0.28 g, 2.3 mmol) in sodium ethoxide solution (prepared by

4818
S. Bondock et al. / European Journal of Medicinal Chemistry 44 (2009) 4813–4818
4.7.5. 4-Amino-N-(benzothiazol-2-yl)-pyrimido[1,2-a]benzimidazole-
3-carboxamide (13)
suspensions at 106 CFU/mL (Colony Forming Unit/mL) concentra-
tion were inoculated to the corresponding wells. Plates were
incubated at 36 ꢁC for 24–48 h and the minimal inhibitory
concentrations (MICs) were determined. Control experiments were
also done.
Yellow powder; yield 49%; mp 285–286 ꢁC; IR (KBr): ymax
/
cmꢀ1 ¼3452, 3340 (NH2), 3218 (NH), 1645 (CO). 1H NMR (DMSO-
d6): dppm ¼ 7.19–8.12 (m, 8H, Ar–H), 8.51 (s, 2H, NH2), 8.89 (s, 1H,
pyrimidine–H6), 12.82 (s, 1H, NH). MS m/z (%): 360 (Mþ, 8), 343
(100), 212 (36), 177 (2), 150 (16), 134 (7), 123 (5), 90 (15), 77 (9), 63
(17). Anal. for C18H12N6OS (360.39): Calcd.: C 59.99, H 3.36, N
23.32%; Found: C 59.83, H 3.32, N 23.24%.
Acknowledgments
The authors are very grateful to Dr. Ashraf Nouval and
Dr. Ahmed Abduo, Depatment of Botany, Faculty of Science, Man-
soura University, for performing the antimicrobial evaluation.
4.7.6. 4-Amino-N-(benzothiazol-2-yl)-8,10-dimethylpyrido
[20,30:3,4]pyrazolo[1,5-a] pyrimidine-3-carboxamide (14)
Deep red powder; yield 54%; mp 296–297 ꢁC; IR (KBr): ymax
/
References
cmꢀ1 ¼3430, 3301 (NH2), 3179 (NH),1678 (CO).1H NMR (DMSO-d6):
dppm ¼ 2.70 (s, 3H, CH3), 3.11 (s, 3H, CH3), 6.98 (s, 1H, pyridine–H5),
7.36–7.68 (m, 4H, Ar–H), 7.88 (s, 2H, NH2), 8.27 (s, 1H, pyrimidine–
H4),10.24 (s, H, NH). MS m/z (%): 389 (Mþ, 61), 356 (17), 240 (100),173
(25),150 (23), 77 (6.5). Anal. for C19H15N7OS (389.43): Calcd.: C 58.60,
H 3.88, N 25.18%; Found: C 58.45, H 3.61, N 25.07%.
[1] N. Oukli, S. Comesse, N. Chafi, H. Oulyadi, A. Daich, Tetrahedron Lett. 50 (2009)
1459–1462.
[2] M.V. Pilipecz, T.R. Varga, Z. Mucsi, P. Scheiber, P. Nemes, Tetrahedron 64 (2008)
5545–5550.
[3] M.R. Shaaban, Heterocycles 75 (2008) 3005–3014.
[4] M.A. Radwan, M. El-Sherbiny, Bioorg. Med. Chem. 15 (2007) 1206–1211.
[5] F. Al-Qalaf, M.M. Abdelkhalik, A. El-Enezi, J.R. Al-Ajimi, Heterocycles 75 (2008)
145–156.
[6] A.M. Salaheldin, M.K. Alphy, J. Heterocycl. Chem. 45 (2008) 307–310.
[7] S.M. Riyadh, I.A. Abdelhamid, H.M. Al-Matar, N.M. Hilmy, M.H. Elnagdi,
Heterocycles 75 (2008) 1849–1905.
5. Antimicrobial evaluation
Standard sterilized filter paper disks (5 mm diameter) impreg-
nated with a solution of the test compound in DMF (1 mg/mL) were
placed on an agar plate seeded with the appropriate test organism
in triplicates. The utilized test organisms were: B. subtilis and
B. thuringiensis as examples of Gram positive bacteria and E. coli and
P. aeruginosa as examples of Gram negative bacteria. They were also
evaluated for their in vitro antifungal potential against B. fabae and
F. oxysporum fungal strains. Streptomycin, Chloroamphenicol and
Treflucan were used as standard antibacterial and antifungal
agents, respectively. DMF alone was used as control at the same
above-mentioned concentration. The plates were incubated at
37 ꢁC for 24 h for bacteria and for 48 days for fungi. Compounds
that showed significant growth inhibition zones (>10 mm) using
the twofold serial dilution technique, were further evaluated for
their minimal inhibitory concentrations (MICs).
[8] R.M. Abdel-Motaleb, A.M.A.S. Makhloof, H.M. Ibrahim, M.H. Elnagdi, J. Heter-
ocycl. Chem. 44 (2007) 109–114.
[9] H.M. Ibrahim, S. Makhseed, R.M. Abdel-Motaleb, A.M.A.S. Makhlouf,
M.H. Elnagdi, Heterocycles 71 (2007) 1951–1966.
[10] K.S. Lin, M.L. Debnath, C.A. Mathis, W. Klunk, Bioorg. Med. Chem. Lett. 19
(2009) 2258–2262.
[11] K. Serdons, T. Verduyckt, D. Vanderghinste, P. Borghgraef, J. Cleynhens, F. Van
Leuven, H. Kung, G. Bormans, A. Verbruggen, Eur. J. Med. Chem. 44 (2009)
1415–1426.
¨
[12] G. Turan-Zitouni, S. Demirayak, A. Ozdemir, Z.A. Kaplancikli, M.T. Yildiz, Eur. J.
Med. Chem. 39 (2003) 267–272.
[13] S.T. Huang, I.J. Hsei, C. Chen, Bioorg. Med. Chem. 14 (2006) 6106–6119.
[14] C.J. Lion, C.S. Matthews, G. Wells, T.D. Bradshaw, M.F.G. Stevens, A.D. Westwell,
Bioorg. Med. Chem. Lett. 16 (2006) 5005–5008.
[15] N. Siddiqui, S.N. Pandeya, S.A. Khau, J. Stables, A. Rana, M. Alam, Md. F. Arshad,
M.A. Bhat, Bioorg. Med. Chem. 17 (2007) 255–259.
[16] F. Russo, G. Romeo, N.A. Santagati, A. Caruso, V. Cutuli, D. Amore, Eur. J. Med.
Chem. 29 (1994) 569–578.
[17] F. Russo, M. Santagati, Farmaco Ed. Sci. 31 (1976) 41–48.
[18] J.H. Musser, R.E. Brown, B. Love, K. Baily, H. Jones, R. Kahen, F. Haung,
A. Khandwala, M. Leibowitz, J. Med. Chem. 27 (1984) 121–125.
[19] S. Bondock, W. Fadaly, M.A. Metwally, J. Sulfur Chem. 30 (2009) 74–107.
[20] M.A. Metwally, E.M. Keshk, A. Fekry, H.A. Etman, J. Chem. Res. 9 (2004)
602–604.
[21] A.M. Hussein, F.A. Abu-Shanab, M.A.M. Abdel-Raheim, M.S.A. El-Gaby, Phos-
phorus, Sulfur Silicon Relat. Elem. 183 (2008) 1722–1734.
[22] A.M. El-Dean, A.A. Geies, T.A. Mohamed, A.A. Atalla, Bull. Fac. Sci., Assiut Univ.
20 (1991) 15–21: Chem. Abstr. 116 (1992) 106161g.
5.1. Minimal inhibitory concentration (MIC) measurement
The microdilution susceptibility test in Mu¨ller–Hinton Broth
(Oxoid) and Sabouraud Liquid Medium (Oxoid) was used for the
determination of antibacterial and antifungal activities, respec-
tively. Stock solutions of the tested compounds, Streptomycin,
Chloroamphenicol and Treflucan were prepared in DMF at
[23] M.A.A. Radwan, E.A. Ragab, M.R. Shaaban, A.O.H. El-Nezhawy, Arkivoc vii
(2009) 281–291.
[24] A.H. Shamroukh, M.E.A. Zaki, E.M.H. Morsy, F.M. Abdel-Motti, F.M.E. Abdel-
Megeid, Arch. Pharm. Chem. Life Sci. 340 (2007) 345–351.
concentration of 1000
mg/mL followed by twofold dilution at
concentrations of (500, 250,., 3.125
mg/mL). The microorganism