Structural characterization and antimicrobial activities of 7H-benzo[h]chromeno[2,3-d]pyrimidine and 14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c] pyrimidine Derivatives

Article abstract of DOI:10.3390/molecules21111450

Three new series of chromene molecules have been synthesized in order to explore their antimicrobial activity. The series encompass 2-substituted 14-(4-halophenyl)-12-methoxy-14Hbenzo[ h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]pyrimidines 7a-o, 9-benzylideneamino-7-(4-halophenyl)- 5-methoxy-8-imino-7H-benzo-[h]chromeno[2,3-d]pyrimidines 8a-b and 3-ethoxycarbonyl- 14-(4-halophenyl)-12-methoxy-14H-benzo-[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine-2-one derivatives 12a-b. The structure of these novel compounds were confirmed using IR, 1H- and 13C-NMR as well as MS spectroscopy. The new compounds were evaluated in vitro for their antimicrobial activity and it was demonstrated that 7H-benzochromenopyrimidine and derivatives of 14H-benzochromenotriazolopyrimidine exhibited the most promising antibacterial activities compared to the reference antimicrobial agents. The structure activity relationship (SAR) studies of the target compounds agreed with the in vitro essays and confirmed higher potent antimicrobial activity against some of the tested microorganisms.

Full text of DOI:10.3390/molecules21111450

molecules
Article
Structural Characterization and Antimicrobial
Activities of 7H-Benzo[h]chromeno[2,3-d]pyrimidine
and 14H-Benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]
pyrimidine Derivatives
Rawda M. Okasha 1,*, Fawzia F. Albalawi 1, Tarek H. Afifi 1, Ahmed M. Fouda 2,
Al-Anood M. Al-Dies 2 and Ahmed M. El-Agrody 3
1
Chemistry Department, Faculty of Science, Taibah University, Al-Madinah Al-Munawarah 30002,
Saudi Arabia; ffs_chem334@hotmail.com (F.F.A.); afifith@yahoo.com (T.H.A.)
Chemistry Department, Faculty of Science, King Khalid University, Abha 61413, P.O. Box 9004, Saudi Arabia;
2
amfouda@hotmail.com (A.M.F.); nood-mansyaa@hotmail.com (A.M.A.-D.)
Chemistry Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo 11884, Egypt;
3
elagrody_am@yahoo.com
*
Correspondence: rawdao@yahoo.com; Tel.: +966-567-947-939
Academic Editor: Philippe Belmont
Received: 30 September 2016; Accepted: 28 October 2016; Published: 1 November 2016
Abstract: Three new series of chromene molecules have been synthesized in order to explore their
antimicrobial activity. The series encompass 2-substituted 14-(4-halophenyl)-12-methoxy-14H-
benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]pyrimidines 7a
phenyl)-5-methoxy-8-imino-7H-benzo-[h]chromeno[2,3-d]pyrimidines 8a
–
o
,
9-benzylideneamino-7-(4-halo-
and 3-ethoxycarbonyl-
–b
14-(4-halophenyl)-12-methoxy-14H-benzo-[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine-2-one
1
derivatives 12a
–
b
. The structure of these novel compounds were confirmed using IR, H- and
13C-NMR as well as MS spectroscopy. The new compounds were evaluated in vitro for their
antimicrobial activity and it was demonstrated that 7H-benzochromenopyrimidine and derivatives
of 14H-benzochromenotriazolopyrimidine exhibited the most promising antibacterial activities
compared to the reference antimicrobial agents. The structure activity relationship (SAR) studies of
the target compounds agreed with the in vitro essays and confirmed higher potent antimicrobial
activity against some of the tested microorganisms.
Keywords: 4H-benzochromene; 7H-benzochromenopyrimidine; 14H-benzochromenotriazolo-
pyrimidine; antimicrobial activities; SAR
1. Introduction
The development of antimicrobial agents is an area of great activity due to the emergence
of multidrug resistance in common pathogens and the appearance of new infections. Infectious
diseases are considered the second-leading cause of death worldwide; consequently, tremendous
efforts have been made to develop new antimicrobial agents that are active especially against the drug
resistant strains.
The majority of antimicrobial agents are diverse five- and six-membered heterocyclic molecules
that play a crucial role in the metabolism of all living cells [1]. Moreover, a great deal of interest has
been directed toward condensed ring systems due to their various types of physiological activities
and the success in utilizing them as privileged medicinal scaffolds. In particular, fused chromene and
benzochromene molecules have become some of the best potential candidates for pharmacological
purposes due to their antimicrobial [2,3], antileishmanial [4,5], anticancer [6,7], antiproliferative [8],
Molecules 2016, 21, 1450; doi:10.3390/molecules21111450
www.mdpi.com/journal/molecules

Molecules 2016, 21, 1450
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antioxidant [
9
,
10], hypertensive [11], antitumor [12
–
15] effects and activities, as well as for the treatment
of Alzheimer’s disease [16] and schizophrenia disorders [17].
Fused chromene ring systems also displayed blood platelet antiaggregating [18], antihistaminic [19],
analgesic [20–22], hypolipidemic [23], DNA breaking and mutagenicity activities [24]. Furthermore,
several reports have shown that 7H-benzo[h]chromeno[2,3-d]pyrimidine and 14H-benzo[h]
chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine moieties are promising and attractive scaffolds for
the development of potent antimicrobial agents [
[2,3-d]pyrimidine derivatives exhibit anticancer activities [29–33].
In continuation of the previous works [15 25 31 34 37], it seemed interesting to synthesize some
4,25–28]. In addition, 7H-benzo[h]-chromeno
,
–
,
–
new 7H-benzo[h]chromeno[2,3-d]-pyrimidine and 14H-benzo-14H-benzo[h]chromeno[3,2-e][1,2,4]-
triazolo[1,5-c]pyrimidine derivatives and to appraise their antimicrobial activities. The structure-
activity relationships (SAR) are discussed in this work to correlate between the substituent effects and
the activities that aid in drug design.
2. Results and Discussion
2.1. Chemistry
The first series of the new chromene compounds was synthesized via multi component reactions
of 4-methoxy-1-naphthol (
1) with 4-halobenzaldehydes 2a–c and malononitrile (3) in ethanolic
piperidine solution. The solution was refluxed for 1 h and gave the corresponding 2-amino-4-(4-
halophenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitriles 4a
obtained from the same precursors using microwave irradiation for 2 min. at 140 C. These results are
depicted in Scheme 1.
–
c
. The desired compounds were also
◦
9
10
8
OH
1
10a
7
2
3
O
NH2
CN
a
h
f
EtOH / pip. 1 h. reflux
or MW / 400 W / 2 min.
6a
g
b
+
ArCHO
CH2(CN)2
+
e
d
c
4a
6
MeO
4
5
3
2a-c
1
OMe
Ar
4a-c
a: Ar = 4-FC6H4
b: Ar = 4-ClC6H4
c: Ar = 4-BrC6H4
Scheme 1. Synthesis of 2-amino-4-(4-halophenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitriles
4a–c.
Treatment of compounds 4a
the 2-ethoxymethyleneamino-4-(4-halophenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitriles 5a
Subsequent hydrazinolysis of the latter in ethanol under stirring at room temperature gave 9-amino-
–c with triethyl orthoformate in acetic anhydride at reflux gave
–c.
7-(4-halophenyl)-5-methoxy-8-imino-7H-benzo[h]chromeno[2,3-d] pyrimidines 6a–c, as illustrated in
Scheme 2.
The aminoimino compounds
of 2-substituted 14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine derivatives. Thus,
treatment of the aminoimino compounds 6a with either formic acid or methyl formate in benzene
at reflux gave 14-(4-halophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e]-[1,2,4]triazolo[1,5-c]-
pyrimidines 7a , whilst acylation of compounds 6a with acetyl chloride or acetic anhydride gave
the pentacyclic 2-methyltriazolopyrimidine derivatives 7d . On the other hand, condensation of
compounds 6a with diethyl oxalate and ethyl cyanoacetate afforded 2-ethoxycarbonyltriazolo-
and 2-cyanomethyltriazolopyrimidine derivatives 7j , respectively, as shown in
6 proved to be useful intermediates for the synthesis of a variety
–c
–
c
–c
–
f
–
c
pyrimidines 7g
–i
–l
Scheme 3.

Molecules 2016, 21, 1450
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2
5
1
3
4
11
O
NH
11a
O
N=CHOEt
CN
O
N
2
NH2NH2.H2O
EtOH
HC(OEt)3
Ac2O
10
9
c
1
b
4a
2
3
a
N
8
d
MeO
CN
7a
6a
MeO
MeO
NH
stirring for 1h
2
reflux
6
7
NH
Ar
5a-c
4a-c
Ar
Ar
6a-c
a: Ar = 4-FC6H4
b: Ar = 4-ClC6H4
c: Ar = 4-BrC6H4
Scheme 2. Synthesis of 2-ethoxymethyleneamino-4-(4-halophenyl)-6-methoxy-4H-benzo[h]chromene-
3-carbonitriles (5a ) and 9-amine-7-(4-halorophenyl)-5-methoxy-8-imino-7H-benzo[h]chromeno[2,3-
d]pyrimidines (6a–c).
–
c
a: R = H,
b: R = H,
c: R = H,
Ar = 4-FC6H4
Ar = 4-ClC6H4
Ar = 4-BrC6H4
Ar = 4-FC6H4
Ar = 4-ClC6H4
Ar = 4-BrC6H4
3
5
4
N1
6 N
N
b
a
2
c
2 R
3
5
e
4
d
7O
d: R = CH3,
e: R = CH3,
f: R = CH3,
g: R = CO2Et, Ar = 4-FC6H4
h: R = CO2Et, Ar = 4-ClC6H4
i: R = CO2Et, Ar = 4-BrC6H4
J R = CH2CN, Ar = 4-FC6H4
K: R = CH2CN, Ar = 4-ClC6H4
l: R = CH2CN, Ar = 4-BrC6H4
N
1
2
i) HCO2H or HCO2Me
ii) AcCl or Ac2O
iii) (CO2Et)2
3
1
O
N
8
14
9
Ar
N
MeO
NH2
10
13
iv) CNCH2CO2Et
v) PhCOCl
12
11
NH
6a-c Ar
OMe
7a-o
a: Ar = 4-FC6H4
b: Ar = 4-ClC6H4
c: Ar = 4-BrC6H4
m: R = Ph,
n: R = Ph,
o; R = Ph,
Ar = 4-FC6H4
Ar = 4-ClC6H4
Ar = 4-BrC6H4
PhCHO / EtOH / pip. reflux
H
N
N
N=CHPh
N
Ph
H
a
N
N
b
c
N
d
O
2
NH
Ar
O1
3
-H2
1,4-Dioxan
pip. reflux
Ar
7m-o
OMe
8a-c
OMe
Scheme 3. 2-substituted 14-(4-halophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]
pyrimidines (7a ) and 9-benzylideneamino-7-(4-halophenyl)-5-methoxy-8-imino-7H-benzo[h]chromeno
–o
[2,3-d]pyrimidines (8a–c).
Aroylation of compounds 6a
–
c
with benzoyl chloride in refluxing dry benzene proceeded
readily to give the 2-phenyltriazolopyrimidine derivatives 7m
the aminoimino compounds 6a with benzaldehyde in ethanolic piperidine solution under
reflux gave the open chain product 9-benzylideneamino-7-(4-halophenyl)-5-methoxy-8-imino-7H-
benzo[h]-chromeno[2,3-d]pyrimidines 8a , Scheme 3. Compounds 7m were also prepared by
cyclization of compounds 8a in 1,4-dioxane/piperdine solution under reflux [26] as confirmed by
the m.p., mixed m.p., and their identical IR and MS spectra, Scheme 3.
Interaction of the aminoimino compounds 6a with ethyl chloroformate in dry benzene at
reflux gave the corresponding 1:2 adducts, the 3-ethoxycarbonyl-14-(4-halophenyl)-12-methoxy-14H-
benzo[h]-chromeno[3,2-e]-[1,2,4]triazolo[1,5-c]pyrimidine-2-ones 12a instead of the 1:1 adducts, the
triazolopyrimidin-2-one derivatives 11a . These results are depicted in Scheme 4. The formation
of 12a is assumed to proceed via interaction of 6a with one mole of ethyl chloroformate
and elimination of HCl to yield the intermediates 9a , which then cyclized to the non-isolable
compounds 11a via elimination of EtOH. The intermediates 11a then reacted with another mole
of ethyl chloroformate to eliminate HCl and give 12a . Alternatively, interaction of 6a with
two moles of ethyl chloroformate could eliminate two HCl molecules and yield the intermediate
–o, Scheme 3. Condensation of
–
c
–
c
–o
–c
–
c
–c
–
c
–
c
–c
–
c
–c
–c
–
c
–c
bis-(ethoxycarbonyl) derivatives 10a
molecule (Scheme 4). The structures of
13C-NMR and MS data. The 7-position of compounds
are chiral centers, and all the reactions were monitored using the TLC technique.
–
c
, which then cyclized to 12a
–
c
with elimination of an ethanol
1
7,
8
and 12 were established on the basis of IR, H-NMR,
8
and the 14-position of compounds 7 and 12

Molecules 2016, 21, 1450
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O
N
N
MeO
NH
2
NH
6a-c Ar
ClCO Et / -HCl reflux
2ClCO Et / -2HCl reflux
2
2
N
N
N
O
O
N
N
NH
NH
MeO
MeO
MeO
CO Et
2
CO Et
2
NH
10a-c
Ar
9a-c
Ar
CO Et
2
-EtOH
-EtOH
N
O
N
O
ClCO Et
2
N
N
CO Et
NH
reflux
-HCl
2
MeO
N
O
N
11a-c Ar
N
12a-c
Ar
a: Ar = 4-FC H
b: Ar = 4-ClC H
c: Ar = 4-BrC H
O
6
4
6
4
4
6
Scheme 4. Synthesis of 3-ethoxycarbonyl-14-(4-halophenyl)-12-methoxy-14H-benzo[h]chromeno
[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine-2-ones (12a–c).
2.2. Antibacterial Evaluation
The target compounds 7a
by the agar diffusion method using Mueller-Hinton agar medium for bacteria and Sabouraud’s agar
medium for fungi [32 33]. The tested microorganisms were obtained from the Regional Center for
–o, 8a–c and 12a–c were tested in vitro for their antimicrobial activities
,
Mycology & Biotechnology (RCMP), Al-Azhar University.
The assayed collection included four Gram-positive species of pathogenic bacteria:
Staphylococcus aureus (RCMB 000106), Staphylococcus epidermidis (RCMB 000107), Bacillis subtilis (RCMB
000108), Bacillus pumilus (RCMB 000109), and two Gram-negative ones: Pseudomonas aeruginosa
(RCMB 000102), Escherichia coli (RCMB 000103), using two standard antibiotics, ampicillin, and
streptomycin (25
Candida albicans (RCMB 005002) and Saccharomyces cerevisiae (RCMB 006002), using two standard
antibiotics, mycostatine and clotrimazole (25 g/mL) as reference drugs. The mean zone of inhibition
in mm standard deviation beyond the well diameter (6 mm) was determined using a 25 g/mL
µg/mL) as reference drugs, and three fungi: Aspergillus fumigatus (RCMB 002003),
µ
±
µ
concentration of the tested compounds. The inhibitory effects of the synthetic compounds against
these organisms are given in Figures 1–3 and Table 1.
Figure 1. Antibacterial activity (Gram-positive) of the tested compounds compared to ampicillin and
streptomycin: S. aureus (blue); S. epidermidis (red); B.subtilis (green) and B. pumilus (pale purple).

Molecules 2016, 21, 1450
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Figure 2. Antibacterial activity (Gram-negative) of tested compounds compared to ampicillin and
streptomycin: P. aeruginosa (blue) and E. coli (red).
Figure 3. Antifungal activity of tested compounds compared to mycostatine and clotrimazole:
A. fumigatus (blue); C. albicans (red) and S. cerevisiae (green).

Molecules 2016, 21, 1450
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Table 1. Antimicrobial screening for compounds 7a–o, 8a–c and 12a–c (25 µg/mL).
CO2Et
N=CHPh
NH
N
N
N
N
N
N
N
N
N
R
O
N
O
O
O
Ar
Ar
Ar
7a-o OMe
8a-c
OMe
12a-c OMe
Compound
R
Ar
S. aureus (+ve)
NA
S. Epider. (+ve)
11.9 ± 0.3
20.5 ± 0.2
23.5 ± 0.1
21.1 ± 0.1
23.5 ± 0.1
24.3 ± 0.1
18.9 ± 0.1
17.2 ± 0.2
16.1 ± 0.3
25.2 ± 0.1
26.3 ± 0.3
26.9 ± 0.3
19.3 ± 0.5
20.9 ± 0.1
21.4 ± 0.1
22.9 ± 0.1
25.7 ± 0.2
28.5 ± 0.3
NA
B. Subtilis (+ve)
NA
B. pumilus (+ve)
NA
P. Aerug. (−ve)
NA
E. coli (−ve)
NA
A. fumig.
NA
C. albic.
NA
S. cerev.
NA
7a
H
4-FC6H5
4-ClC6H5
4-BrC6H5
4-FC6H5
4-ClC6H5
4-BrC6H5
4-FC6H5
4-ClC6H5
4-BrC6H5
4-FC6H5
4-ClC6H5
4-BrC6H5
4-FC6H5
4-ClC6H5
4-BrC6H5
4-FC6H5
4-ClC6H5
4-BrC6H5
4-FC6H5
4-ClC6H5
4-BrC6H5
-
7b
H
NA
NA
NA
NA
NA
NA
NA
NA
7c
H
NA
NA
NA
NA
NA
NA
NA
NA
7d
CH3
NA
NA
NA
NA
NA
NA
NA
NA
7e
CH3
NA
NA
NA
NA
NA
NA
NA
NA
7f
CH3
NA
NA
NA
NA
NA
NA
NA
NA
7g
CO2Et
NA
NA
NA
NA
NA
NA
NA
NA
7h
CO2Et
NA
NA
NA
NA
NA
NA
NA
NA
7i
CO2Et
NA
NA
NA
NA
NA
NA
NA
NA
7j
CH2CN
27.3 ± 0.5
28.8 ± 0.1
29.1 ± 0.2
NA
12.3 ± 0.3
12.2 ± 0.4
12.7 ± 0.4
NA
12.3 ± 0.3
12.2 ± 0.4
12.8 ± 0.4
NA
28.3 ± 0.07
28.2 ± 0.03
28.9 ± 0.03
NA
26.7 ± 0.1
26.8 ± 0.3
27.8 ± 0.3
NA
12.3 ± 0.3
12.2 ± 0.1
12.1 ± 0.2
NA
19.3 ± 0.1
21.2 ± 0.2
21.1 ± 0.2
NA
23.3 ± 0.1
20.2 ± 0.1
18.2 ± 0.1
NA
7k
CH2CN
7l
CH2CN
7m
Ph
7n
Ph
NA
NA
NA
NA
NA
NA
NA
NA
7o
Ph
-
NA
NA
NA
NA
NA
NA
NA
NA
8a
8b
21.8 ± 0.1
24.6 ± 0.2
26.6 ± 0.1
NA
23.1 ± 0.2
25.1 ± 0.4
25.1 ± 0.4
NA
24.0 ± 0.3
23.6 ± 0.1
21.3 ± 0.1
NA
19.3 ± 0.1
21.3 ± 0.2
23.3 ± 0.1
NA
21.9 ± 0.1
21.0 ± 0.4
20.7 ± 0.2
NA
23.8 ± 0.1
23.4 ± 0.1
22.1 ± 0.1
NA
22.9 ± 0.1
22.4 ± 0.1
21.7 ± 0.2
NA
16.8 ± 0.4
18.2 ± 0.3
19.5 ± 0.1
NA
-
8c
-
12a
-
12b
-
NA
NA
NA
NA
NA
NA
NA
NA
NA
12c
-
NA
NA
NA
NA
NA
NA
NA
NA
NA
Ampicillin
Streptomycin
Mycostatine
Clotrimazole
-
27.4 ± 0.1
25.1 ± 0.2
-
28.6 ± 0.5
29.1 ± 0.3
-
29.4 ± 0.7
30.1 ± 0.9
-
29.9 ± 0.3
30.8 ± 0.4
-
26.3 ± 0.3
24.3 ± 0.8
-
28.5 ± 0.1
25.6 ± 0.4
-
-
-
-
-
-
-
-
-
-
-
29.5 ± 0.1
26.1 ± 0.1
27.1 ± 0.1
23.1 ± 0.3
24.1 ± 0.3
18.3 ± 0.1
-
-
-
-
-
-
-
-
NA = not active; Diameter of the hole = 6 mm; Data are expressed in the form of mean ± SD.

Molecules 2016, 21, 1450
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2.3. SAR Studies
The structure activity relationship (SAR) studies of compounds 7a
–
o
,
8a
±
–
c
and 12a
–
c
revealed
0.13 and
that compounds 8a and 7j with inhibitory effects of 21.8 0.14, 24.6
–
c
±
0.21, 26.6
±
27.3
(27.4
±
0.47
0.15
µ
g/mL were 1.3, 1.1, 1.0, 1.0 times more active than the standard antibiotic ampicillin
±
µ
g/mL), while compounds 8a
, b were 1.2 and 1.0 times more active than the standard
antibiotic streptomycin (25.1
±
0.18 g/mL) against S. aureus, respectively, and other compounds
µ
showed almost equipotent activities or were inactive, implying that the 7H-benzo[h]chromeno-[2,3-
d]-pyrimidine nucleus was more active than the 14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo-[1,5-c]-
pyrimidine one and grafting a lipophilic hydrophobic group (=NH-8, –N=CHPh-9) or halogen
(
F > Cl > Br) on the 4-position of the phenyl group at the 7-position of the 7H-benzo[h]chromeno-
[2,3-d]pyrimidine moiety is more beneficial than a CH2CN group at the 2-position of a 14H-
benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]pyrimidine. Compounds 7a 8a 7c
8b 7k 8c were found to be the most potent against S. epidermidis, with inhibitory effects ranging
from 11.9 to 28.5 g/mL compared to standard antibiotics ampicillin and streptomycin (28.6 0.5 and
29.1 0.3 g/mL), suggesting that the 14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine
,i
,h,g,m
,b,n,
d,o,
,
,e,f,j,
,
,l,
µ
±
±
µ
nucleus was more effective than the 7H-benzo[h]chromeno[2,3-d]pyrimidine nucleus. Moreover,
the non-substituted triazolo ring at the 2-postion is better than a hydrophobic moiety like a CO2Et,
Ph, Me and CH2CN group. In addition, compounds 7k
B. subtilis with inhibitory effects ranging 12.2–25.1 g/mL as compared to the standard antibiotics
ampicillin and streptomycin (29.4 0.7 and 30.1 0.9 g/mL), while compounds 7k and 8c with
inhibitory effects ranging 12.2–24.0 g/mL, exhibited good activity against B. pumilus as compared to
the standard antibiotics ampicillin and streptomycin (29.9 0.3 and 30.8 0.4 g/mL). These results
,j,l and 8a–c) showed good activity against
µ
±
±
µ
,
j,
l
,b,a
µ
±
±
µ
implied that 14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine nucleus with hydrophobic
group CH2CN group at 2-position was more beneficial than 7H-benzo[h]chromeno[2,3-d]pyrimidine
nucleus with a lipophilic hydrophobic groups (=NH-8, –N=CHPh-9). Furthermore, compounds 8a
exhibited good activities against P. aeruginosa, with inhibitory effects ranging from 19.3–23.3 g/mL, as
compared to the standard antibiotics ampicillin and streptomycin (26.3 ± 0.3 and 24.3 ± 0.8 µg/mL),
while compounds 8c and 7k showed high activities against E. coli, with inhibitory effects
ranging from 20.7–27.8 g/mL as compared to the standard antibiotics ampicillin and streptomycin
(28.5 0.1 and 25.6 0.4 g/mL), suggesting that a 7H-benzo[h]chromeno[2,3-d]pyrimidine
–c
µ
,b,
a
,j,l
µ
±
±
µ
nucleus with a lipophilic hydrophobic group (=NH-8, –N=CHPh-9) was more effective than a
14H-benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]pyrimidine nucleus with a hydrophobic CH2CN
group at the 2-position. Finally, compounds 7l
from 12.1–12.3, 19.3–21.2, 22.1–23.8 and 21.7–22.9
A. fumigatus and C. albicans as compared to the standard antibiotics mycostatine (29.5
0.1 and 23.1 0.3 g/mL), respectively, implying that the
,
k
µ
,
j
,
7j
g/mL, respectively, showed high activity against
0.1 and
–l and 8c,b,a)with inhibitory effects ranging
±
27.1 ± 0.1 µg/mL) and clotrimazole (26.1
±
±
µ
14H-benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]pyrimidine nucleus with a hydrophobic CH2CN
group at the 2-position was more active than a 7H-benzo[h]chromeno[2,3-d]pyrimidine nucleus with a
lipophilic hydrophobic group (=NH-8, –N=CHPh-9), while compounds 8a
effects between 16.8–19.5 and 18.2–23.3 g/mL, respectively, are more effective against S. cerevisiae
as compared to the standard antibiotic mycostatine (24.1 0.3 g/mL) and compounds 8a and
7l (16.8–18.2 g/mL) are more active than the standard antibiotic clotrimazole (18.3 0.1 g/mL).
–c and 7l,k,j with inhibitory
µ
±
µ
,b
µ
±
µ
The rest of the compounds showed almost equipotent and moderate activities or were inactive.
3. Experimental Section
3.1. General Information
Commercial-grade solvents and reagents were purchased from Sigma-Aldrich (St. Louis, MO,
USA) and used without further purification. Melting points were measured with a Stuart Scientific
(Stone, Staffordshire, UK) apparatus and are uncorrected. IR spectra were determined as KBr pellets on

Molecules 2016, 21, 1450
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1
a Jasco FT/IR 460 plus spectrophotometer (Jasco, Tokoyo, Japan). H-NMR (500 MHz) and 13C-NMR
(125 MHz) spectra were recorded using a Bruker AV 500 MHz spectrometer (Bruker, Billerica, MA,
1
USA). Chemical shifts (
δ
) are expressed in parts per million (ppm). The H-NMR and 13C-NMR
spectra of the compounds are provided in the Supplementary Material. The MS were measured using
a Shimadzu GC/MS-QP5050A spectrometer (Shimadzu, Tokoyo, Japan). Elemental analyses were
carried out at the Regional Centre for Mycology & Biotechnology (RCMP, Al-Azhar University, Cairo,
Egypt) and the results were within
(TLC) on silica gel precoated F254 plates (Merck, Billerica, MA, USA) was used to check the purity of
the final compounds and intermediates.
±
0.3% of calculated values. Analytical thin layer chromatography
3.2. Synthesis
3.2.1. Starting Materials
2-Amino-4-(4-fluoro/chloro/bromophenyl)-6-methoxy-4H-benzo[h]chromene-3-carbonitriles
4a
–
c, 2-ethoxymethyleneamino-4-(4-fluoro/chloro/bromophenyl)-6-methoxy-4H-benzo[h]chromene-3-
carbonitriles 5a
–
c
and 9-amino-7-(4-fluoro/chloro/bromophenyl)-5-methoxy-8-imino-7H-benzo[h]-
chromeno[2,3-d]pyrimidines 6a–c were prepared by reported methods ([34–37], [29–31] and [29–31],
respectively).
3.2.2. General Procedure for the Synthesis of Compounds 7a–c
A mixture of aminoimino compound 6a (3.88, 4.04, or 4.49 g, respectively, 0.01 mol) and
–
c
formic acid or methyl formate (0.46 g or 0.6 g, 0.01 mol) in dry benzene (30 mL) was refluxed for 5 h.
The solvent was extracted and the resulting products were recrystallized from ethanol/1,4-dioxane to
give 7a–c. The physical data of the compounds 7a–c are as follows:
14-(4-Fluorophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7a). Pale
yellow crystals. Yield: 79%; m.p. 262–263 ◦C; IR (KBr, cm−1):
ν
3053, 3010 (CH-arom.), 2959, 2848
3.87 (s, 3H, OCH3), 5.34 (s, 1H, H-14), 6.68–8.22 (m,
39.02 (C-14), 55.75 (CH3), 98.75 (C-14a), 103.59 (C-13),
(CH-aliph.), 1652 (C=N); 1H-NMR (DMSO-d6)
δ
11H, Ar-H, H-2, H-5); 13C-NMR (DMSO-d6)
δ
115.22 (Ar-C), 120.66 (C-13a), 121.65 (C-8), 124.02 (C-11), 124.39 (C-10), 126.18 (C-11a), 127.37 (C-9),
129.71 (C-8a), 129.77 (Ar-C), 136.67 (C-5), 140.25 (Ar-C), 141.36, (C-8b), 146.79 (C-14b), 151.42 (C-12),
155.91 (C-2), 160.01 (Ar-C), 161.94 (C-6a); MS (m/z), 398 (M+, 11.46) with a base peak at 278 (100);
C23H15FN4O2 (398.39): calcd; % C: 69.34, % H: 3.80, % N: 14.06; found; % C: 69.40, % H: 3.84, %
N: 14.10.
14-(4-Chlorophenyl)-12-methoxy-14H-benz◦o[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7b). Pale
ν 3071, 3011 (CH-arom.), 2953, 2912, 2862,
yellow crystals. Yield: 92%; m.p. 242–243 C; IR (KBr, cm−1):
2828 (CH-aliph.), 1651 (C=N); 1H-NMR (DMSO-d6) δ 3.86 (s, 3H, OCH3), 5.34 (s, 1H, H-14), 6.68–8.22
(m, 10H, Ar-H, H-5), 8.34 (s, 1H, H-2); 13C-NMR (DMSO-d6)
δ
39.91 (C-14), 55.73 (CH3), 98.73 (C-14a),
103.51 (C-13), 120.66 (C-13a), 121.64 (C-8), 124.00 (C-11), 124.41 (C-10), 126.20 (C-11a), 127.37 (C-9),
128.28 (C-8a), 128.38 (Ar-C), 129.77 (Ar-C), 133.20 (Ar-C), 137.37 (C-5), 141.64 (Ar-C), 143.37 (C-8b),
148.77 (C-14b), 151.42 (C-12), 155.92 (C-2), 166.92 (C-6a); MS (m/z), 416 (M+ + 2, 19.25), 414 (M+, 4.67)
with a base peak at 62 (100); C23H15ClN4O2 (414.84): calcd; % C: 66.59, % H: 3.64, % N: 13.51; found; %
C: 66.64, % H: 3.70, % N: 13.56.
14-(4-Bromophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7c). Pale
yellow crystals. Yield: 83%; m.p. 232–233 ◦C; IR (KBr, cm−1):
ν
3071, 3007 (CH-arom.), 2962, 2915,
2895 (CH-aliph.), 1652 (C=N); 1H-NMR (DMSO-d6) δ 3.87 (s, 3H, OCH3), 5.32 (s, 1H, H-14), 6.67–8.22
(m, 10H, Ar-H, H-5), 8.95 (s, 1H, H-2); 13C-NMR (DMSO-d6)
40.03 (C-14), 55.75 (CH3), 96.96 (C-14a),
δ
103.63 (C-13), 120.03 (Ar-C), 120.67 (C-13a), 121.66 (C-8), 124.01 (C-11), 124.44 (C-10), 126.21 (C-11a),
127.38 (C-9), 130.16 (C-8a), 131.39 (Ar-C), 133.53 (Ar-C), 137.56 (C-5), 141.70, (C-8b), 143.24 (Ar-C),
151.44 (C-14b), 152.44 (C-12), 155.94 (C-2), 161.44 (C-6a), 163.94 (C-6a); MS (m/z), 460 (M+ + 2, 6.03), 458

Molecules 2016, 21, 1450
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(M+, 7.68) with a base peak at 279 (100); C23H15BrN4O2 (459.29): calcd; % C: 60.15, % H: 3.29, % N:
12.20; found; % C: 60.20, % H: 3.34, % N: 12.27.
3.2.3. General Procedure for the Preparation of Compounds 7d–f
A mixture of aminoimino compounds 6a
–c (3.88, 4.04, or 4.49 g, respectively, 0.01 mol) and acetyl
chloride or acetic anhydride (30 mL) was refluxed for 2 h. The solvent was extracted and the resulting
product was recrystallized from 1,4-dioxan to give 7d–f. The physical data of the compounds 7d–f are
as follows:
14-(4-Fluorophenyl)-2-methyl-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7d).
◦
Colourless crystals. Yield: 81%; m.p. 252–253 C; IR (KBr, cm−1):
ν 3068, 3035, 3006 (CH-arom.),
2967, 2938, 2848 (CH-aliph.), 1623 (C=N); MS (m/z), 412 (M+, 77.98) with a base peak at 318 (100);
C24H17FN4O2 (412.42): calcd; % C: 69.89, % H: 4.15, % N: 13.59; found; % C: 69.94, % H: 4.17, %
N: 13.61.
14-(4-Chlorophenyl)-2-methyl-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7e).
◦
Pale yellow crystals. Yield: 89%; m.p. 238–239 C; IR (KBr, cm−1):
ν
3071, 3025 (CH-arom.), 2970, 2937,
2.46 (s, 3H, CH3), 3.91 (s, 3H, OCH3), 5.71 (s,
1H, H-14), 6.68–8.35 (m, 9H, Ar-H), 9.59 (s, 1H, H-5); 13C-NMR (DMSO-d6)
14.28 (CH3), 40.05 (C-14),
2845 (CH-aliph.), 1632 (C=N); 1H-NMR (DMSO-d6)
δ
δ
55.88 (CH3), 103.76 (C-13), 117.75 (C-13a), 119.77 (C-14a), 121.75 (C-8), 124.04 (C-11), 124.59 (C-10),
126.38 (C-11a), 127.57 (C-9), 128.28 (C-8a), 129.51 (Ar-C), 130.64 (Ar-C), 135.92 (Ar-C), 136.52 (C-5),
139.47 (C-8b), 146.01 (Ar-C), 148.54 (C-14b), 152.76 (C-12), 153.91 (C-2), 165.18 (C-6a), 151.44 (C-14b),
152.38 (C-12), 155.94 (C-2), 161.94 (C-6a), 163.90 (C-6a); MS (m/z), 430 (M+ + 2, 19.45), 428 (M+, 57.92)
with a base peak at 51 (100); C24H17ClN4O2 (428.87): calcd; % C: 67.21, % H: 4.00, % N: 13.06; found; %
C: 67.30, % H: 4.09, % N: 13.10.
14-(4-Bromophenyl)-2-methyl-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7f).
Colourless crystals. Yield: 84%; m.p. 243–244 ◦C; IR (KBr, cm−1):
ν
3060, 3021, 3006 (CH-arom.), 2958,
2.41 (s, 3H, CH3), 3.97 (s, 3H, OCH3), 5.33
(s, 1H, H-14), 6.17–8.45 (m, 9H, aromatic), 9.76 (s, 1H, H-5); 13C-NMR (DMSO-d6)
14.53 (CH3), 38.58
2932, 2848 (CH-aliph.), 1637 (C=N); 1H-NMR (DMSO-d6)
δ
δ
(C-14), 55.99 (CH3), 96.80 (C-14a), 103.39 (C-13), 120.08 (Ar-C), 120.57 (C-13a), 121.73 (C-8), 124.05
(C-11), 124.56 (C-10), 128.91 (C-11a), 129.62 (C-9), 130.24 (C-8a), 131.14 (Ar-C), 134.25 (Ar-C), 137.62
(C-5), 143.90 (C-8b), 144.55 (Ar-C), 151.70 (C-14b), 156.57 (C-12), 158.48 (C-2), 163.90 (C-6a); MS (m/z),
474 (M+ + 2, 12.19), 472 (M+, 13.44) with a base peak at 75 (100); C24H17BrN4O2 (473.32): calcd; % C:
60.90, % H: 3.62, % N: 11.84; found; % C: 60.96, % H: 3.66, % N: 11.88.
3.2.4. General Procedure for the Preparation of Compounds 7g–i
A mixture of aminoimino compounds 6a
–c (3.88, 4.04, or 4.49 g, respectively, 0.01 mol) and diethyl
oxalate (1.46 g, 0.01 mol) in ethanol (30 mL) was refluxed for 2 h. The solvent was extracted and the
resulting product was recrystallized from ethanol/1,4-dioxane to give 7g–i. The physical data of the
compounds 7g–i are as follows:
2-Ethoxycarbonyl-14-(4-fluorophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]-pyrimidine
7g). Yellow crystals. Yield: 75%; m.p. 299–300 ◦C; IR (KBr, cm−1):
3067 (CH-arom.), 2995, 2987,
2939 (CH-aliph.), 1639 (C=N), 1743 (CO); 1H-NMR (DMSO-d6)
1.36 (t, 3H, CH3, J = 7.2 Hz), 3.93
(s, 3H, OCH3), 4.42 (q, 2H, CH2, J = 7.2 Hz), 5.97 (s, 1H, H-14), 6.63–8.64 (m, 9H, Ar-H), 9.82 (s, 1H,
H-5); 13C-NMR (DMSO-d6)
14.00 (CH3), 37.95 (C-14), 55.98 (CH3), 61.92 (CH2), 102.68 (C-13), 103.55
(
ν
δ
δ
(C-13a), 115.73 (Ar-C), 117.90 (C-14a), 121.79 (C-8), 123.89 (C-11), 124.62 (C-10), 126.68 (C-11a), 127.95
(C-9), 128.30 (C-8a), 129.95 (Ar-C), 138.62 (C-5), 139.95 (Ar-C), 141.32 (C-8b), 153.06 (C-14b), 154.57
(C-12), 157.43 (C-2), 159.46 (C-6a), 160.53 (Ar-C), 164.55 (CO); MS (m/z), 470 (M+, 17.39) with a base
peak at 260 (100); C26H19FN4O4 (470.45): calcd; % C: 66.38, % H: 4.07, % N: 11.91; found; % C: 66.42, %
H: 4.11, % N: 12.00.

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2-Ethoxycarbonyl-14-(4-chlorophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]-pyrimidine
(
7h). Colourless powder. Yield: 87%; m.p. 248–249 ◦C; IR (KBr, cm−1):
ν 3071 (CH-arom.), 2979, 2945,
2920 (CH-aliph.), 1625 (C=N), 1747 (CO); MS (m/z), 488 (M+ + 2, 1.15), 486 (M+, 3.52) with a base peak
at 111 (100); C26H19ClN4O4 (486.91): calcd; % C: 64.14, % H: 3.93, % N: 11.51; found; % C: 64.09, % H:
3.89, % N: 11.47.
2-Ethoxycarbonyl-14-(4-bromophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]pyri-
◦
midine (7i). Colourless needles. Yield: 80%; m.p. 288–289 C; IR (KBr, cm−1):
ν
3067, 3025, 3001
(CH-arom.), 2948, 2845 (CH-aliph.), 1626 (C=N), 1747 (CO); 1H-NMR (DMSO-d6)
δ 1.52 (t, 3H, CH3,
J = 7.2 Hz), 3.95 (s, 3H, OCH3), 4.59 (q, 2H, CH2, J = 7.2 Hz), 5.93 (s, 1H, H-14), 6.43–8.51 (m, 9H,
aromatic), 9.24 (s, 1H, H-5); 13C-NMR (DMSO-d6)
δ
14.46 (CH3), 40.84 (C-14), 56.05 (CH3), 63.02 (CH2),
102.70 (C-13), 103.49 (C-13a), 116.04 (C-14a), 121.80 (C-8), 121.47 (Ar-C), 122.47 (C-11), 125.00 (C-10),
125.99 (C-11a), 126.86 (C-9), 127.90 (C-8a), 130.50 (Ar-C), 132.12 (Ar-C), 139.20 (C-5), 139.43 (C-8b),
142.16 (Ar-C), 153.54 (C-14b), 155.06 (C-2), 153.81 (C-12), 158.78 (C-6a), 159.93 (CO); MS (m/z), 532
(
M+ + 2, 1.19), 530 (M+, 1.24) with a base peak at 76 (100); C26H19BrN4O4 (531.36): calcd; % C: 58.77, %
H: 3.60, % N: 10.54; found; % C: 58.81, % H: 3.66, % N: 10.60.
3.2.5. General Procedure for the Preparation of Compounds 7j–l
A mixture of aminoimino compounds 6a
–c (3.88, 4.04, or 4.49 g, respectively, 0.01 mol) and ethyl
cyanoacetate (1.13 g, 0.01 mol) in ethanol (30 mL) was refluxed for 2 h. (TLC monitoring). The solvent
was extracted and the resulting product was recrystallized from ethanol/1,4-dioxane to give 7j
The physical data of the compounds 7j–l are as follows:
–l.
2-Cyanomethyl-14-(4-fluorophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]-pyrimidine
7j). Pale yellow crystals. Yield: 90%; m.p. >300 ◦C; IR (KBr, cm−1):
3065, 3026 (CH-arom.), 2983,
2940 (CH-aliph.), 2260 (CN), 1631 (C=N); 1H-NMR (DMSO-d6)
3.45 (s, 2H, CH2), 3.93 (s, 3H, OCH3),
5.62 (s, 1H, H-14), 6.63–8.32 (m, 9H, aromatic), 8.55 (s, 1H, H-5); 13C-NMR (DMSO-d6)
25.07 (CH2),
(
ν
δ
δ
38.06 (C-14), 55.97 (CH3), 97.29 (C-13), 102.71 (C-13a), 116.50 (Ar-C), 117.41 (CN), 117.84 (C-14a), 120.64
(C-8), 121.79 (C-11), 123.90 (C-10), 124.63 (C-11a), 126.67 (C-9), 127.92 (C-8a), 130.00 (Ar-C), 138.65
(C-5), 140.36 (Ar-C), 151.34 (C-8b), 152.32 (C-14b), 153.37 (C-2), 159.14 (C-12), 160.80 (Ar-C), 167.38
(C-6a); MS (m/z), 437 (M+, 54.05) with a base peak at 252 (100); C25H16FN5O2 (437.43): calcd; % C:
68.64, % H: 3.69, % N: 16.01; found; % C: 68.70, % H: 3.74, % N: 16.08.
2-Cyanomethyl-14-(4-chlorophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]-pyrimidine
◦
7k). Pale yellow powder. Yield: 83%; m.p. 240–241 C; IR (KBr, cm−1):
ν 3062, 3012 (CH-arom.), 2962,
(
2905, 2890 (CH-aliph.), 2258 (CN), 1624 (C=N); MS (m/z), 455 (M+ + 2, 8.15), 453 (M+, 25.25) with a base
peak at 343 (100); C25H16ClN5O2 (453.88): calcd; % C: 66.16, % H: 3.55, % N: 15.43; found; % C: 66.55,
% H: 3.49, % N: 15.38.
2-Cyanomethyl-14-(4-bromophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]-pyrimidine
(
7l). Colourless needles. Yield: 75%; m.p. 232–233 ◦C; IR (KBr, cm−1):
ν
3077 (CH-arom.), 2959, 2925
3.45 (s, 2H, CH2), 3.93 (s, 3H, OCH3), 5.61
(s, 1H, H-14), 6.63–8.26 (m, 9H, aromatic), 8.56 (s, 1H, H-5); 13C-NMR (DMSO-d6)
25.06 (CH2), 40.04
(CH-aliph.), 2255 (CN), 1626 (C=N); 1H-NMR (DMSO-d6)
δ
δ
(C-14), 55.98 (CH3), 96.93 (C-13), 102.64 (C-13a), 117.45 (CN), 117.83 (C-14a), 120.53 (Ar-C), 120.79 (C-8),
121.79 (C-11), 123.88 (C-10), 124.62 (C-11a), 126.72 (C-9), 127.95 (C-8a), 129.88 (Ar-C), 131.76 (Ar-C),
136.96 (C-5), 141.71 (Ar-C), 151.41 (C-8b), 152.35 (C-14b), 153.38 (C-2), 159.18 (C-12), 167.38 (C-6a); MS
(m/z), 499 (M+ + 2, 32.02), 497 (M+, 33.62) with a base peak at 342; C25H16BrN5O2 (498.33): calcd; % C:
60.25, % H: 3.24, % N: 14.05; found; % C: 60.31, % H: 3.37, % N: 14.10.
3.2.6. General Procedure for the Preparation of Compounds 7m–o
Method A: A mixture of aminoimino compound 6a–c (3.88, 4.04, or 4.49 g, respectively, 0.01 mol)
and benzoyl chloride (0.01 mol) in dry benzene (30 mL) was refluxed for 5 h. The solvent was removed

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under reduced pressure and the resulting solids were recrystallized from ethanol/1,4-dioxane to give
7m–o.
Method B: A mixture of compounds 8a–c (4.76, 4.92 or 5.37 g, respectively, 0.01 mol), dioxane
(20 mL) and piperidine (0.5 mL) was refluxed for 2 h. The solvent was extracted and the resulting
product was recrystallized from ethanol/1,4-dioxane to give 7m–o in 60% yield (as verified by m.p.,
mixed m.p., identical IR and MS spectrum).
The physical data of the compounds 7m–o are as follows:
14-(4-Fluorophenyl)-2-phenyl-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]pyrimidine (7m).
Colourless needles. Yield: 67%; m.p. 248–249 ◦C; IR (KBr, cm−1):
3058, 3001 (CH-arom.), 2967, 2941
(CH-aliph.), 1625 (C=N); 1H-NMR (DMSO-d6)
3.90 (s, 3H, OCH3), 5.96 (s, 1H, H-14), 6.85–8.35 (m,
14H, aromatic), 9.69 (s, 1H, H-5); 13C-NMR (DMSO-d6)
40.03 (C-14), 55.88 (CH3), 101.24 (C-13), 103.72
ν
δ
δ
(C-13a), 115.22 (Ar-C), 117.30 (C-14a), 120.66 (C-8), 121.75 (C-11), 124.07 (C-10), 124.69 (C-11a), 126.50
(C-9), 127.64 (C-8a), 128.28 (Ar-C), 129.03 (Ar-C), 129.65 (Ar-C), 130.09 (Ar-C), 130.92 (Ar-C), 138.15
(C-5), 140.24 (Ar-C), 151.89 (C-8b), 153.13 (C-14b), 154.00 (C-12), 160.09 (C-2), 162.03 (Ar-C), 165.50
(C-6a); MS (m/z), 474 (M+, 54.05) with a base peak at 279 (100); C29H19FN4O2 (474.49): calcd; % C: 73.41,
% H: 4.04, % N: 11.81; found; % C: 73.48, % H: 4.11, % N: 11.98.
14-(4-Chlorophenyl)-2-phenyl-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo-[1,5-c]pyrimidine (7n).
Colourless needles. Yield: 66%; m.p. 295–296 ◦C; IR (KBr, cm−1):
ν 3062, 3021 (CH-arom.), 2954, 2895
(CH-aliph.), 1621 (C=N); MS (m/z), 492 (M+ + 2, 3.13), 490 (M+, 9.42) with a base peak at 104 (100);
C29H19ClN4O2 (490.94): calcd; % C: 70.95, % H: 3.90, % N: 11.41; found; % C: 71.00, % H: 3.94, %
N: 11.45.
14-(4-Bromophenyl)-2-phenyl-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo-[1,5-c]pyrimidine (7o).
◦
Colourless needles. Yield: 67%; m.p. 298–299 C; IR (KBr, cm−1):
ν 3046 (CH-arom.), 2979, 2945
(CH-aliph.), 1621 (C=N); MS (m/z), 536 (M+ + 2, 6.23), 534 (M+, 5.15) with a base peak at 433 (100);
C29H19BrN4O2 (535.39): calcd; % C: 65.06, % H: 3.58, % N: 10.46; found; % C: 65.13, % H: 3.62, %
N: 10.57.
3.2.7. General Procedure for the Preparation of Compounds 8a–c
A mixture of aminoimino compounds 6a–c (3.88, 4.04, or 4.49 g, respectively, 0.01 mol) and
benzaldehyde (1.06 g, 0.01mol) in ethanol (30 mL) and piperidine (0.5 mL) was refluxed for 2 h.
The solvent was extracted and the resulting product was recrystallized from 1,4-dioxane to give the
open chain products 8a–c. The physical data of the compounds 8a–c are as follows:
9-Benzylideneamino-7-(4-fluorophenyl)-5-methoxy-8-imino-7H-benzo[h]chromeno[2,3-d]pyrimidine (8a). Pale
◦
yellow crystals. Yield: 66%; m.p. 257–258 C; IR (KBr, cm−1):
ν
3204 (NH), 3055, 3000 (CH-arom.), 2951,
3.98 (s, 3H, OCH3), 6.13 (s, 1H, H-7), 6.80–8.26
(m, 14H, aromatic), 8.21 (s, 1H, H-10), 8.41 (N=CH), 11.03 (bs, 1H, NH); 13C-NMR (DMSO-d6)
38.52
2850 (CH-aliph.), 1631 (C=N); 1H-NMR (DMSO-d6)
δ
δ
(C-7), 56.19 (CH3), 96.78 (C-7a), 103.38 (C-6), 116.57 (Ar-C), 118.82 (C-6a), 121.74 (C-1), 124.01 (C-4),
124.54 (C-3), 126.33 (C-4a), 126.74 (C-2), 127.54 (C-1a), 128.91 (Ar-C), 129.62 (Ar-C), 130.23 (Ar-C),
132.64 (Ar-C), 134.64 (Ar-C), 137.55 (C-1b), 144.51 (C-5), 144.88 (Ar-C), 151.65 (C-11a), 156.54 (N=CH),
158.42 (C-10), 160.98 (Ar-C), 163.83 (C-8); MS (m/z), 476 (M+, 99.45) with a base peak at 380 (100);
C29H21FN4O2 (476.50): calcd; % C: 73.10, % H: 4.44, % N: 11.76; found; % C: 73.14, % H: 4.47, %
N: 11.81.
9-Benzylideneamino-7-(4-chlorophenyl)-5-methoxy-8-imino-7H-benzo[h]chromeno[2,3-d]pyrimidine
(
8b).
3214 (NH), 3065, 3010 (CH-arom.),
δ 3.97 (s, 3H, OCH3), 6.18 (s, 1H, H-7),
Colourless powder. Yield: 86%; m.p. 260–261 ◦C; IR (KBr, cm−1):
ν
1
2971, 2860 (CH-aliph.), 1629 (C=N); H-NMR (DMSO-d6)
6.82–8.29 (m, 14H, aromatic), 8.21 (s, 1H, H-10), 8.42 (N=CH), 11.04 (bs, 1H, NH); 13C-NMR (DMSO-d6)
38.54 (C-7), 55.99 (CH3), 96.79 (C-7a), 103.36 (C-6), 118.80 (C-6a), 121.76 (C-1), 124.02 (C-4), 124.52
δ

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(C-3), 126.36 (C-4a), 126.50 (Ar-C), 126.76 (C-2), 127.56 (C-1a), 128.91 (Ar-C), 129.44 (Ar-C), 129.62
(Ar-C), 131.64 (Ar-C), 131.98 (Ar-C), 132.23 (Ar-C), 137.60 (C-1b), 144.53 (C-5), 146.88 (Ar-C),151.66
(C-11a), 156.58 (N=CH), 158.44 (C-10), 163.88 (C-8); MS (m/z), 494 (M+ + 2, 2.07), 492 (M+, 5.29) with a
base peak at 385 (100); C29H21ClN4O2 (492.96): calcd; % C: 70.66, % H: 4.29, % N: 11.37; found; % C:
70.70, % H: 4.33, % N: 11.42.
9-Benzylideneamino-7-(4-bromophenyl)-5-methoxy-8-imino-7H-benzo[h]chromeno[2,3-d]pyrimidine (8c). Pale
yellow crystals. Yield: 89%; m.p. 274–275 ◦C; IR (KBr, cm−1):
ν
3216 (NH), 3040 (CH-arom.), 2937,
3.97 (s, 3H, OCH3) 6.17 (s, 1H, H-7), 6.17–8.27
(m, 14H, aromatic), 8.28 (s, 1H, H-10), 8.44 (N=CH), 11.06 (bs, 1H, NH); 13C-NMR (DMSO-d6)
38.54
2894 (CH-aliph.), 1650 (C=N); 1H-NMR (DMSO-d6)
δ
,
δ
(C-7), 55.99 (CH3), 96.77 (C-7a), 103.37 (C-6), 118.80 (C-6a), 120.57(Ar-C), 121.73 (C-1), 124.03 (C-4),
124.53 (C-3), 126.35 (C-4a), 126.77 (C-2), 127.55(C-1a), 128.50 (Ar-C), 128.91 (Ar-C), 129.44 (Ar-C), 129.62
(Ar-C), 131.64 (Ar-C), 134.23 (Ar-C), 137.59 (C-1b), 143.88 (Ar-C), 144.54 (C-5), 151.68 (C-11a), 156.57
(N=CH), 158.45 (C-10), 163.87 (C-8); MS (m/z), 538 (M+ + 2, 4.61), 536 (M+, 4.70) with a base peak at 433
(100); C29H21BrN4O2 (537.41): calcd; % C: 64.81, % H: 3.94, % N: 10.43; found; % C: 64.77, % H: 3.99, %
N: 10.48.
3.2.8. Synthesis of Compounds 12a–c
A mixture of aminoimino compounds 6a
–c (3.88, 4.04, or 4.49 g, respectively, 0.01 mol) and ethyl
chloroformate (1.08 g, 0.01 mol) in dry benzene (30 mL) was refluxed for 2 h. The solid product was
collected by filtration and recrystallized from ethanol/1,4-dioxane to give 12a
the compounds 12a–c are as follows:
–c. The physical data of
3-Ethoxycarbonyl-14-(4-fluorophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]-pyrimidine-
2-one (12a). Yellow crystals. Yield: 45%; m.p. 241–242 ◦C; IR (KBr, cm−1):
2950, 2880 (CH-aliph.), 1645 (C=N), 1721 (CO), 1770 (CO ester); H-NMR (DMSO-d6)
ν
3051 (CH-arom.), 2983,
1.35 (t, 3H,
1
δ
CH3, J = 7.2 Hz), 3.89 (s, 3H, OCH3), 4.44 (q, 2H, CH2, J = 7.2 Hz), 5.59 (s, 1H, H-14), 6.82–8.29 (m, 9H,
aromatic), 9.35 (s, 1H, H-5); 13C-NMR (DMSO-d6)
δ
13.96 (CH3), 40.03 (C-14), 55.92 (CH3), 64.47 (CH2),
98.45 (C-14a), 103.55 (C-13), 115.38 (Ar-C), 117.65 (C-13a), 120.51 (C-8), 121.79 (C-11), 123.88 (C-10),
124.63 (C-11a), 127.84 (C-9), 130.14 (C-8a), 130.19 (Ar-C), 137.46 (C-8b), 139.59 (Ar-C), 140.51 (C-5),
148.77 (C-12), 152.21 (C-6a), 155.11 (Ar-C), 155.21 (C-14b), 157.39 (CO), 160.32 (CO); MS (m/z), 486 (M+,
2.44) with a base peak at 320 (100); C26H19FN4O5 (486.45): calcd; % C: 64.20, % H: 3.94, % N: 11.52;
found; % C: 64.24, % H: 3.99, % N: 11.56.
3-Ethoxycarbonyl-14-(4-chlorophenyl)-12-methoxy-14H-benzo[h◦]chromeno[3,2-e][1,2,4]triazolo[1,5-c]-pyrimidine-
2-one (12b). Colourless needles. Yield: 43%; m.p. 200–201 C; IR (KBr, cm−1):
2970, 2860 (CH-aliph.), 1641 (C=N), 1740 (CO), 1759 (CO ester); H-NMR (DMSO-d6)
ν 3020 (CH-arom.), 2992,
1
δ 1.35 (t, 3H,
CH3, J = 7.2 Hz), 3.89 (s, 3H, OCH3), 4.44 (q, 2H, CH2, J = 7.2 Hz), 5.59 (s, 1H, H-14), 6.82–8.29 (m,
9H, aromatic), 9.35 (s, 1H, H-5); 13C-NMR (DMSO-d6)
δ
13.98 (CH3), 40.04 (C-14), 55.94 (CH3), 64.50
(CH2), 98.38 (C-14a), 103.51 (C-13), 117.33 (C-13a), 120.53 (C-8), 121.81 (C-11), 123.89 (C-10), 124.69
(C-11a), 126.70 (Ar-C), 127.88 (C-9), 128.55 (C-8a), 130.15 (Ar-C), 131.78 (Ar-C), 137.49 (C-8b), 140.59
(C-5), 142.29 (Ar-C), 148.80 (C-12), 152.25 (C-6a), 155.12 (C-14b), 155.23 (CO), 157.40 (CO); MS (m/z),
504 (M+ + 2, 5.24), 502 (M+, 16.36) with a base peak at 320 (100); C26H19ClN4O5 (502.91): calcd; % C:
62.09, % H: 3.81, % N: 11.14; found; % C: 62.03, % H: 3.77, % N: 11.17.
3-Ethoxycarbonyl-14-(4-bromophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]-triazolo[1,5-c]pyrimidine-
◦
2-one (12c). Colourless powder. Yield: 49%; m.p. 200–201 C; IR (KBr, cm−1):
ν
3068 (CH-arom.), 2975,
1.34 (t, 3H,
1
2937, 2885 (CH-aliph.), 1646 (C=N), 1736 (CO), 1773 (CO ester); H-NMR (DMSO-d6)
δ
CH3, J = 7.2 Hz), 3.89 (s, 3H, OCH3), 4.44 (q, 2H, CH2, J = 7.2 Hz), 5.57 (s, 1H, H-14), 6.81–8.28 (m,9H,
aromatic), 9.35 (s, 1H, H-5); 13C-NMR (DMSO-d6)
δ
13.98 (CH3), 40.05 (C-14), 55.93 (CH3), 64.50 (CH2),
98.31 (C-14a), 103.49 (C-13), 117.23 (C-13a), 120.53 (Ar-C), 121.81 (C-8), 123.87 (C-11), 124.68 (C-10),
126.69 (C-11a), 127.78 (C-9), 130.32 (C-8a), 130.52 (Ar-C), 131.46 (Ar-C), 137.48 (C-8b), 140.58 (C-5),

Molecules 2016, 21, 1450
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142.70 (Ar-C), 148.79 (C-12), 152.25 (C-6a), 155.11 (C-14b), 155.21 (CO), 157.37 (CO); MS (m/z), 548
M+ + 2, 10.14), 546 (M+, 10.90) with a base peak at 320 (100); C26H19BrN4O5 (547.36): calcd; % C: 57.05,
(
% H: 3.50, % N: 10.24; found; % C: 57.11, % H: 3.55, % N: 10.27.
3.3. Antimicrobial Assay
All the newly synthesized compounds 7a
–o, 8a–c and 12a–c were screened for their in vitro
antimicrobial activity at 25 g/mL to determine the zone of inhibition against four Gram-positive
µ
bacteria: Staphylococcus aureus (RCMB 000106), Staphylococcus epidermidis (RCMB 000107), Bacillis subtilis
(RCMB 000108), Bacillus pumilus (RCMB 000109), and two Gram-negative pathogenic bacteria:
Pseudomonas aeruginosa (RCMB 000102), Escherichia coli (RCMB 000103) using two standard antibiotics
(ampicillin, streptomycin) as reference drugs, and three fungi: Aspergillus fumigatus (RCMB 002003),
Candida albicans (RCMB 005002) and Saccharomyces cerevisiae (RCMB 006002) using two standard
antibiotics (mycostatine, clotrimazole) as reference drugs. The activities of these compounds were
tested by agar diffusion method using Mueller-Hinton agar medium for bacteria and Sabouraud’s
agar medium for fungi [31
(DMF) to give a solution of 1 mg
in millimeters (6 mm) at the end of an incubation period of 48 h at 28 ◦C; N,N-dimethylformamide
,32]. The tested compounds were dissolved in N,N-dimethylformamide
·
mL−1. The inhibition zones (diameter of the hole) were measured
showed no inhibition zone.
4. Conclusions
In conclusion, several 14-(4-halophenyl)-12-methoxy-14H-benzo[h]chromeno[3,2-e][1,2,4]-
triazolo[1,5-c]pyrimidine and 7H-benzo[h]chromeno[2,3-d]pyrimidine derivatives were synthesized in
good yields, starting from 4H-benzo[h]chromene derivatives. The structures of compounds 7, 8 and
12 were established on the basis of IR, 1H-NMR, 13C-NMR and MS data. A pharmacological study
has been performed in order to evaluate the effects of substituents on the antibacterial and antifungal
activities. Compounds 12a
and fungi, while compounds 7a
Staphylococcus epidermidis as compared to ampicillin and streptomycin. In contrast, compounds 7j
8a showed high activity against all tested microorganisms. The rest of compounds showed almost
equipotent and moderate activities or were inactive. The structure activity relationship (SAR) study
–
c
did not show any antimicrobial activity against any of the tested bacteria
showed high to good activities only against
and
,i,h,g,m,b,n,d,o,c,e,f
–
l
–b
-
revealed that the antimicrobial activity of 14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]-pyrimidine
nucleus was more affected by the lipophilicity of the non-substituent or 2-substituents than by lipophilic
hydrophobic groups (=NH-8, –N=CHPh-9) on the 7H-benzo[h]-chromeno[2,3-d]pyrimidine nucleus
and the 14H-benzo[h]chromeno[3,2-e][1,2,4]triazolo[1,5-c]-pyrimidine nucleus was more beneficial
than the 7H-benzo[h]chromeno[2,3-d]pyrimidine nucleus for the antimicrobial activity.
Acknowledgments: The authors profoundly thank the Regional Center for Mycology & Biotechnology (RCMP),
Al-Azhar University for carrying out the antitumor study and Elemental analyses.
Author Contributions: A.M.E., A.M.F. and R.M.O. conceived and designed the experiments; A.M.A. performed
the experiments; R.M.O., F.F.A., T.H.A., A.M.F. and A.M.E. analyzed the data, wrote the paper and edited English
language. All authors discussed the results and commented on the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds (7a–o), (8a–c) and (12a–c) are available from the authors.
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