Microwave assisted synthesis of some new heterocyclic spiro-derivatives with potential antimicrobial and antioxidant activity

Article abstract of DOI:10.3390/molecules15128827

Homophthalic anhydride reacts with different aromatic amines to produce Nsubstituted homophthalimides. Bromination of the latter produces 4,4-dibromohomophthalimide derivatives that can be used as precursors for spiro-derivatives. The dibromo derivatives

Full text of DOI:10.3390/molecules15128827

Molecules 2010, 15, 8827-8840; doi:10.3390/molecules15128827
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Microwave Assisted Synthesis of Some New Heterocyclic
Spiro-Derivatives with Potential Antimicrobial and
Antioxidant Activity
Mohamed Mohamed Youssef 1,†,* and Mahmoud Ahmed Amin 2,†
1
Chemistry Department, Faculty of Science, Cairo University, Egypt
2
Chemistry Department, Faculty of Science, Suez Canal University, Egypt
†
Current Address: Chemistry Department, Faculty of Science, Taif University, Saudi Arabia.
* Author to whom correspondence should be addressed; E-Mail: mmmyousef@yahoo.com;
Tel.: +966-596072593.
Received: 31 October 2010; in revised form: 17 November 2010/ Accepted: 30 November 2010/
Published: 3 December 2010
Abstract: Homophthalic anhydride reacts with different aromatic amines to produce N-
substituted homophthalimides. Bromination of the latter produces 4,4-dibromo-
homophthalimide derivatives that can be used as precursors for spiro-derivatives. The
dibromo derivatives react with different binucleophilic reagents to produce several spiro-
isoquinoline derivatives. Reaction of the dibromo derivatives with malononitrile produces
dicyanomethylene derivatives which react with different binucleophiles to produce new
spiro-derivatives. Structures of the newly synthesized compounds are proved using
1
spectroscopic methods such as IR, H-NMR and 13C-NMR. The newly synthesized
compounds were tested for their antimicrobial and antioxidant activities, showing weak or
no antimicrobial activity. On the other hand select compounds showed promising
antioxidant activities.
Keywords: microwave; spiro-compounds; heterocycles; spiro-benzoxazoleisoquinoline;
spiro-benzimidazoleisoquinoline; spiro-isoquinolinetriazole; spiro-isoquinolinepyrazole;
spiro-isoquinolinepyrimidine; antimicrobial activity; antioxidant activity

Molecules 2010, 15
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1. Introduction
Spiro-compounds form a group of generally less investigated compounds. However, recently
growing efforts have been made to synthesize and characterize these compounds. Many spiro-
compounds possess very promising biological activities as anticancer agents [1,2], antibacterial
agents [3,4], anticonvulsant agents [5-7], anti-tuberculosis agents [8], anti-Alzheimer’s agents [9],
pain-relief agents [10,11], anti-dermatitis agents [12] and antimicrobial agents [13,14]. In addition to
their medical uses, some spiro-compounds have found other uses in the agricultural and industrial fields.
For example, they are used as antifungal agents [15], pesticides [16], laser dyes [17] and
electroluminescent devices [18]. Spiro compounds have also been recently used as antioxidants [19,20].
We were prompted by these findings to try to synthesize new spiro-compounds with potential
antimicrobial or antioxidant activities.
The microwave technique has several advantages over traditional methods of synthesis. Reduced
reaction times [21-24], less effects on the environment and better reaction yields are some of the common
advantages of using microwaves. In the present research project, we used both the microwave technique as
well as conventional methods to prepare some spiro-compounds with expected biological activity.
2. Results and Discussion
2.1. Chemistry
Homophthalic anhydride (1) was reacted with aromatic amines, namely aniline and p-anisidine, to
afford N-arylhomophthalimide derivatives 2a,b, respectively, which were used as precursors for
preparing new spiro-isoquinolines (Scheme 1). Compounds 2a,b, having an active methylene group,
reacted with two equivalents of bromine in acetic acid to produce 2-aryl-4,4-dibromoisoquinoline-1,3-
(2H,4H)dione derivatives 3a,b. The mass spectrum of compound 3a displayed the expected molecular
ion peaks at m/z 394 (3.2%), 395 (6.7%), 396 (3.0%). Compound 3b gave molecular ion peaks at 424
(7.3%), 425 (15.1%), 426 (6.8%).
Compound 3a underwent direct cyclocondensation when treated with each of o-phenylenediamine
or o-aminophenol to produce 2'-phenyl-1,3-dihydro-1'H-spiro-[benzimidazole-2,4'-isoquinoline]-1',3'
(2'H)-dione (4a) and 2'-phenyl-1'H,3H-spiro[1,3-benzoxazole-2,4'-isoquinoline]-1',3'(2'H)-dione (4b),
respectively. Compounds 4a and 4b are new ring systems. Synthesis of compounds 4a,b was carried
out under two different reaction conditions, namely by conventional heating and using microwave
irradiation conditions. Thus, when the reaction was carried out in a refluxing ethanolic sodium
ethoxide solution for 5 h under TLC monitoring, the product 4a was obtained in 42% yield, while 4b
was obtained in 48% yield. However, when the same reaction was carried out by heating at 140 °C in a
microwave oven for 15 min, the yields of 4a and 4b were 81% and 85%, respectively. It was then
concluded that using microwave as a source of heat not only improves the reaction yield, but also
1
significantly reduces the reaction time. The IR and H-NMR spectra of compounds 4a and 4b agreed
with the proposed structures. Their mass spectra showed the molecular ion peaks at m/z 341 (12.3%)
and 342 (8.6%), respectively.

Molecules 2010, 15
8829
Scheme 1. Reactions of dibromohomophthalimides with binucleophilic reagents; synthesis
of 4 and 5.
O
O
ArNH2
a, Ar = C6H5
b, Ar = C6H4-OCH3-p
O
N
Ar
1
2
O
O
Br2/AcOH, 2h reflux
Br
Br
O
N
a, Ar = C6H5, 65% yield
b, Ar = C6H4-OCH3-p, 58% yield
Ar
3
O
S
NH2
HN
H2N
C2H5ONa
C2H5OH
C2H5ONa
C2H5OH
NH2
XH
S
HN
X
NH
N
NH
O
HN
O
a, Ar = C6H5
Method A:55%
Method B:88%
b, Ar = C6H4-OCH3-p
Method A:48%
Method B:79%
a, X = NH
Method A:42%
Method B:81%
b, X = O
N
Ar
Ph
Method A:48%
Method B:85%
5
O
O
4
Similarly, compounds 3a,b reacted with thiosemicarbazide under both conventional and microwave
reaction conditions and produced 2-phenyl-5'-thioxo-1H-spiro[isoquinoline-4,3'-[1,2,4]triazolidine]-
1,3(2H)-dione (5a) and 2-(4-methoxyphenyl)-5'-thioxo-1H-spiro-[isoquinoline-4,3'-[1,2,4]-triazol-
idine]-1,3(2H)-dione (5b), respectively. Compounds 5 also have a new ring system. Again, comparing
the reaction times and overall yields for both traditional and microwave methods showed that
traditional methods took 4 h, to give 55% and 48% yields of 5a and 5b, respectively, while the
microwave method needed only 15 min to give 88% and 79% yields of 5a and 5b, respectively. The
comparison again proves the advantages of using the microwave technique. Analytical and spectral
data of 5a and 5b were in agreement with the proposed structures.
On the other hand, compounds 3a,b reacted with malononitrile, as an activated methylene
compound, by refluxing under TLC monitoring in absolute ethanol containing a catalytic amount of
piperidine for 3 h, to produce (1,3-dioxo-2-phenyl-2,3-dihydroisoquinolin-4(1H)-ylidene)-malononitrile
(6a) and [2-(4-methoxyphenyl)-1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene]-malononitrile (6b),
respectively (Scheme 2). Attempts to carry out the same reaction under microwave conditions failed
however, due to the formation of a complex mixture of products that could not be separated.
Compounds 6a and 6b gave acceptable analytical and spectral data.

Molecules 2010, 15
Scheme 2. Reactions of isoquinolinylidenemalononitriles with hydrazine hydrate and
8830
thiourea; synthesis of 7 and 8.
3
CH2(CN)2
C2H5OH/Piperidine
NC
CN
O
a, Ar = C6H5, 60% yield
b, Ar = C6H4-OCH3-p, 67% yield
N
Ar
O
6
S
NH2-NH2
C2H5OH/Piperidine
S
C2H5OH/Piperidine
H2N
NH2
H2N
NC
H2N
N
N
NC
H
H
NH
O
NH
O
N
N
Ar
Ar
O
O
8
7
Method A:59%
Method B:86%
Method A:53%
Method B:84%
Compound 6a could be used as a precursor for some spiro-isoquinolines containing five and six-
membered heterocycles. Thus, compound 6a reacted with each of hydrazine hydrate and thiourea to
produce
5'-amino-1,3-dioxo-2-phenyl-2,2',3,4'-tetrahydro-1H-spiro[isoquinoline-4,3'-pyrazole]-4'-
(7) and 6'-amino-1,3-dioxo-2-phenyl-2'-thioxo-2,2',3,5'-tetrahydro-1H,3'H-spiro-
carbonitrile
[isoquinoline-4,4'-pyrimidine]-5'-carbonitrile (8), respectively. Compound 8 has a new ring system.
Synthesis of compounds 7 and 8 was performed under TLC monitoring in refluxing ethanol solution
containing catalytic amounts of piperidine for 4 h and the yields were 53% and 59%, respectively.
Carrying out the same reaction under microwave assisted conditions reduced the reaction time to
15 min and increased the yields to 84% and 86%, respectively, for 7 and 8. Acceptable analytical data
were obtained for the new compounds 7 and 8.
2.2. Biological Evaluation
2.2.1. Antimicrobial evaluation
The newly synthesized heterocyclic compounds listed in Table 1 were tested for their antimicrobial
activity against the following microorganisms: Escherichia coli, Pseudomonas putida, Bacillus
subtilis, Streptococcus lactis, Aspergillus niger, Penicillium sp. and Candida albicans. The

Molecules 2010, 15
8831
preliminary screening of the investigated compounds was performed using the filter paper disc-
diffusion method. The most active compounds were 4a, 5a, 5b and 8, which were slightly inhibitory to
the microorganisms. The rest of compounds showed no sensitivity at all to the tested organisms, and
the results are summarized in Table 1.
Table 1. Antimicrobial activities of the newly synthesized compounds.
Inhibition zone (mm)
Comp.
Gram-negative
Gram-positive
Fungi
Yeast
C. albicans
No.
E. coli P. putida B. subtilis S. lactis
A. niger
P. sp.
4a
4b
5a
5b
6a
6b
7
8
0
5
10
0
0
0
10
22
4
0
3
9
0
0
0
8
21
4
0
5
10
0
0
0
8
18
6
0
5
8
0
0
0
7
19
3
0
4
6
0
0
0
8
20
2
0
3
5
0
0
0
5
12
0
0
0
0
0
0
0
0
0
8
Chloram-
phenicol®
Ampicilin®
24
20
19
22
24
14
14
E. coli = Escherichia coli; P. putida = Pseudomonas putida; B. subtilis = Bacillus subtilis;
S. lactis = Streptococcus lactis; A. niger = Aspergillus niger; P. sp. = Penicillium sp;
C. albicans = Candida albicans. The sensitivity of microorganisms to the tested compounds is
identified in the following manner *: Highly sensitive = Inhibition zone 15-20 mm; Moderately
sensitive = Inhibition zone: 10-15 mm; Slightly sensitive = Inhibition zone: 5-10 mm; Not sensitive
= Inhibition zone: 0 mm;* Each result represents the average of triplicate readings.
2.2.2. Anti-oxidant activity screening
The newly synthesized compounds were tested for anti-oxidant activity as reflected in the ability to
inhibit lipid peroxidation in rat brain and kidney homogenates and rat erythrocyte hemolysis. The
pro-oxidant activities of the aforementioned compounds were assessed by their effects on bleomycin-
induced DNA damage. Table 2 shows the anti-oxidant assays by erythrocyte hemolysis, which reveals
that compounds 7 and 8 manifested potent anti-oxidative activity in the lipid peroxidation assay and
considerable inhibitory activity in the hemolysis assay. Table 3 shows the anti-oxidant assays by the
ABTS method. Again, compounds 7 and 8 showed interesting anti-oxidant activity. Tables 2 and 3
also show that compounds 4a and 4b have moderate anti-oxidant properties. All compounds have been
tested on bleomycin-dependent DNA damage. The results indicate that some compounds, namely 4a,
4b, 7 and 8, may have some protective activity towards DNA from the damage induced by bleomycin
(Table 4).

Molecules 2010, 15
Table 2. Anti-oxidant assays by erythrocyte hemolysis (A/B × 100).
8832
Absorbance of
samples (A)
Compounds
Hemolysis (%)
Complete hemolysis with
0.660
–
distilled water (B)
Ascorbic acid
0.026
0.048
0.052
0.213
0.187
0.062
0.068
0.030
0.033
3.93
7.27
7.87
32.37
28.33
9.39
10.30
4.54
4a
4b
5a
5b
6a
6b
7
8
5.00
Table 3. Anti-oxidant assays by ABTS method [Abs. (control) − Abs. (test)/Abs. (control) × 100].
Absorbance of
Compounds
Inhibition (%)
samples
0.54
0.06
0.18
0.20
0.40
0.42
0.30
0.32
0.11
0.10
ABTS control
Ascorbic acid
0
88.9
66.7
63.0
25.9
22.2
44.4
40.7
79.6
81.5
4a
4b
5a
5b
6a
6b
7
8
Table 4. Assays for bleomycin-dependent DNA damage.
Absorbance of
Compound
samples
Ascorbic acid
0.020
0.038
0.040
0.024
0.026
4a
4b
7
8
3. Experimental
3.1. General
Melting points were determined in open glass capillaries on a Gallenkamp melting point apparatus
and are uncorrected. IR spectra (KBr discs) were recorded on a Shimadzu FTIR-8201PC
spectrophotometer. 1H-NMR and 13C-NMR spectra were recorded on a Varian Mercury 300 MHz, and
a Varian Gemini 200 MHz. spectrometers using TMS as an internal standard and DMSO-d6 as solvent.

Molecules 2010, 15
8833
Chemical shifts were expressed as δ (ppm) units. Mass spectra were recorded at 70 eV on a Shimadzu
GCMS-QP1000EX using an inlet type injector. All reactions were followed by TLC (silica gel,
aluminum sheets 60 F254, Merck). The Microanalytical Center of Cairo University performed the
microanalyses. Microwave reactions were performed with a Millstone Organic Synthesis Unit
(MicroSYNTH with touch control terminal) with a continuous focused microwave power delivery
system in a pressure glass vessel (10 mL) sealed with a septum under magnetic stirring. The
temperature of the reaction mixture was monitored using a calibrated infrared temperature control
under the reaction vessel, and control of the pressure was performed with a pressure sensor connected
to the septum of the vessel.
3.1.1. 2-aryl-4,4-dibromoisoquinoline-1,3-(2H,4H)dione derivatives 3a,b
A solution of either of 2a (2.37 g, 0.01 mol) or 2b (2.76 g, 0.01 mol) in glacial acetic acid (20 mL)
was heated under reflux with bromine (1.1 mL, 3.0 g, 0.02 mole) for 2 h. After cooling, the reaction
mixture was poured onto ice-water and the solid that precipitated was filtered off, dried and
crystallized from the proper solvent.
4,4-Dibromo-2-phenylisoquinoline-1,3-(2H,4H)dione (3a): crystallized from ethanol in 65% yield as
white crystals, m.p. 236-237 ºC; 1H-NMR: 7.10-8.30 (m, 9H, Ar-H); 13C-NMR: 70.2 (sp3 C-4), 120.5,
123.2, 124.3, 126.1, 127.4, 130.0, 131.5, 134.6, 135.2, 136.4 (aromatic C), 158.4, 167.6 (2 CO); IRν:
3066 cm−1 (aromatic CH), 1645 (broad, 2C=O), 1605, 1500 (aromatic C=C); MS: M+ m/z 394 (3.2%),
395 (6.7%), 396 (3.0%); Anal. Calcd. for C15H9Br2NO2 (395.04): C (45.60%), H (2.30%), Br
(40.45%), N (3.55%); Found: C (45.3%), H (2.4%), Br (45.5%), N (3.7%).
4,4-Dibromo-2-(4-methoxyphenyl)isoquinoline-1,3-(2H,4H)dione (3b): crystallized from dilute dioxane
1
in 58% yield as white crystals, m.p. 255-256 ºC; H-NMR: 3.60 (s, 3H, OCH3), δ 6.80-7.90 ppm (m,
13
8H, Ar-H); C-NMR: 57.1 (OCH3), 71.0 (sp3 C-4), 115.3, 118.2, 124.2, 126.0, 127.2, 130.3, 131.6,
134.4, 135.1, 152.2 (aromatic C), 155.4, 163.6 (2 CO); IRν: 3060 cm−1 (aromatic CH), 1640 (broad,
2C=O), 1605, 1500 (aromatic C=C); MS: M+ m/z 424 (7.3%), 425 (15.1%), 426 (6.8%); Anal. Calcd.
for C16H11Br2NO3 (425.07): C (45.21%), H (2.61%), Br (37.60%), N (3.30%); Found: C (45.1%), H
(2.5%), Br (45.0%), N (3.6%).
3.1.2. Cyclocondensation of 3a with o-phenylenediamine and o-aminophenol; formation of 4a,b.
Method A: Compound 3a (3.95 g, 0.01 mole) was heated under reflux with either of o-phenylenediamine
(1.08 g, 0.01 mol) or o-aminophenol (1.09 g, 0.01 mol) in ethanolic sodium ethoxide solution
[prepared by dissolving metallic sodium (0.27 g, 0.012 mol) in absolute ethanol (25 mL)] for 5 h,
under TLC monitoring. The reaction mixture was then cooled, acidified with few drops of conc.
hydrochloric acid and the solid that precipitated was filtered at the pump and crystallized from the
appropriate solvent.
Method B: The same reactants of method A were heated in microwave at 500 W and 140 ºC for 15
min. The reaction mixture was treated in a similar manner to method A to obtain compounds 4a,b.

Molecules 2010, 15
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2'-Phenyl-1,3-dihydro-1'H-spiro[benzimidazole-2,4'-isoquinoline]-1',3'(2'H)-dione (4a): Crystallized from
dimethylformamide as grey crystals, m.p. 218-219 ºC, in 42% yield (Method A) or 81% yield (Method
1
13
B); H-NMR: 3.80 (s, 2H, 2NH, D2O exchangeable), 6.40-7.70 (m, 13H, Ar-H); C-NMR: 83.4 (sp3
spiro C), 113.0, 115.9, 120.2, 122.9, 127.3, 127.8, 128.3, 128.9, 131.2, 133.7, 135.1, 135.9, 136.8
(aromatic C), 155.4, 163.6 (2 CO); IRν: 3180 cm−1 (broad, NH), 3065 (aromatic CH), 1645,1655
(2C=O), 1605, 1500 (aromatic C=C); MS: M+ m/z 341 (12.3%); Anal. Calcd. for C21H15N3O2 (341.36):
C (73.89%), H (4.43%), N (12.31%); Found: C (74.1%), H (4.4%), N (12.1%).
2'-Phenyl-1'H,3H-spiro[1,3-benzoxazole-2,4'-isoquinoline]-1',3'(2'H)-dione (4b): Crystallized from
dioxane as white crystals, m.p. 237-238 ºC, in 48% yield (Method A) and 85% yield (Method B); 1H-
NMR: 4.30 (s, 1H, NH, D2O exchangeable), 6.60-7.80 (m, 13H, Ar-H); 13C-NMR: 83.4 (sp3 spiro C),
116.0, 117.9, 118.2, 121.2, 124.9, 125.8, 127.5, 128.1, 128.5, 129.9, 132.1, 134.5, 135.4, 136.3, 138.8,
144.9 (aromatic C), 160.0, 168.9 (2 CO); IRν: 3210 cm−1 (NH), 3065 (aromatic CH), 1655,1670
(2C=O), 1605, 1500 (aromatic C=C); MS: M+ m/z 342 (8.6%); Anal. Calcd. for C21H14N2O3 (342.35):
C (73.68%), H (4.12%), N (8.18%); Found: C (73.3%), H (4.2%), N (7.9%).
3.1.3. Cyclocondensation of 3a,b with thiosemicarbazide; formation of 5a,b
Method A: Each of compounds 3a (3.95 g, 0.01 mol) and 3b (4.25 g, 0.01 mol) was heated under
reflux with thiosemicarbazide (0.91 g, 0.01 mol) in ethanolic sodium ethoxide solution [prepared by
dissolving metallic sodium (0.27 g, 0.012 mol), in absolute ethanol (25 mL)] for 4 h, under TLC
monitoring. The reaction mixture was then cooled, acidified with few drops of conc. hydrochloric acid
and the solid that precipitated was filtered at the pump and crystallized from the appropriate solvent.
Method B: The same reactants of method A were heated in microwave at 500 W and 140 ºC for 15
min. The reaction mixture was treated in a similar manner to method A to give compounds 5a,b.
2-Phenyl-5'-thioxo-1H-spiro[isoquinoline-4,3'-[1,2,4]triazolidine]-1,3(2H)-dione (5a): Crystallized from
1
ethanol as white crystals, m.p. 188-189 ºC, in 55% yield (Method A) and 88% yield (Method B); H-
NMR: 2.10 (s,1H, NH, D2O exchangeable), 2.30 (s,1H, NH, D2O exchangeable), 2.40 (s, 1H, NH, D2O
13
exchangeable), 7.10-8.30 (m, 9H, Ar-H); C-NMR: 77.7 (sp3 spiro C), 121.1, 123.4, 127.6, 128.4,
128.8, 129.8, 132.1, 134.0, 135.2, 136.8 (aromatic C), 154.9 (CS), 159.5, 166.9 (2 CO); IRν: 3220,
3185, 3150 cm−1 (NH), 3060 (aromatic CH), 1645,1670 (2C=O), 1600, 1490 (aromatic C=C); MS (70 eV):
M+ m/z 324 (11.3%); Anal. Calcd. for C16H12N4O2S (324.36): C (59.25%), H (3.73%), N (17.27%), S
(9.89%); Found: C (59.5%), H (3.9%), N (16.9%), S (10.0%).
2-(4-Methoxyphenyl)-5'-thioxo-1H-spiro[isoquinoline-4,3'-[1,2,4]-triazolidine]-1,3(2H)-dione
(5b):
Crystallized from dilute dioxane as white crystals, m.p. 202-203 ºC, in 48% yield (Method A) and 79%
1
yield (Method B); H-NMR: 2.10 (s, 1H, NH, D2O exchangeable), 2.30 (s, 1H, NH, D2O
exchangeable), 2.40 (s, 1H, NH, D2O exchangeable), 3.70 (s, 3H, OCH3), 7.10-8.30 (m, 9H, Ar-H); IR
ν: 3220, 3185, 3150 cm−1 (NH), 3060 (aromatic CH), 1640, 1660 (2C=O), 1600, 1490 (aromatic C=C);
MS: M+ m/z 354 (13.8%). Anal. Calcd. for C17H14N4O3S (354.38): C (57.62%), H (3.98%), N
(15.81%), S (9.05%); Found: C (57.3%), H (3.9%), N (16.1%), S (9.3%).

Molecules 2010, 15
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3.1.4. Reaction of 3a,b with malononitrile: formation of 6a,b
To a solution of each of compounds 3a (3.95 g, 0.01 mol) and 3b (4.25 g, 0.01 mol) in absolute
ethanol (30 mL) containing a catalytic amount of piperidine was added malononitrile (0.66 g, 0.01 mol).
The reaction mixture was heated under reflux for 3 h, under TLC monitoring, then cooled and poured
onto ice-cold water. The solid product that separated was filtered off, dried and crystallized
from ethanol.
(1,3-Dioxo-2-phenyl-2,3-dihydroisoquinolin-4(1H)-ylidene)-malononitrile (6a): Obtained in 60% yield
1
as pale yellow crystals, m.p. 224-225 ºC; H-NMR: 7.00-8.10 (m, 9H, Ar-H); 13C-NMR: 83.1
(ethylenic C), 112.0 (CN), 122.4, 124.7, 127.1, 127.8, 128.2, 128.6, 129.9, 131.0, 133.1, 133.9
(aromatic C), 147.3 (C3), 158.1, 163.0 (2 CO); IRν: 3060 cm−1 (aromatic CH), 2185 (CN), 1710, 1660
(2C=O), 1600, 1490 (aromatic C=C); MS: M+ m/z 299 (28.2%); Anal. Calcd. for C18H9N3O2 (299.28):
C (72.24%), H (3.03%), N (14.04%); Found: C (72.0%), H (3.3%), N (14.3%).
[2-(4-Methoxyphenyl)-1,3-dioxo-2,3-dihydroisoquinolin-4(1H)-ylidene]malononitrile (6b): Obtained as
yellow crystals in 67% yield, m.p. 241-242 ºC; 1H-NMR: 3.70 (s, 3H, OCH3), 6.70-8.00 (m, 8H, Ar-H);
13C-NMR: 57.3 (OCH3), 83.0 (ethylenic C), 113.1 (CN), 115.2, 124.1, 126.4, 127.2, 127.9, 128.3,
129.5, 131.0, 133.1, 144.2 (aromatic C), 146.9 (C3), 158.4, 163.2 (2 CO; IRν: 3060 cm−1 (aromatic
CH), 2195 (CN), 1710, 1670 (2C=O), 1600, 1490 (aromatic C=C); MS: M+ m/z 329 (24.8%); Anal.
Calcd. for C19H11N3O3 (329.31): C (69.30%), H (3.37%), N (12.76%); Found: C (69.5%), H (3.3%), N
(12.4%).
3.1.5. 5'-Amino-1,3-dioxo-2-phenyl-2,2',3,4'-tetrahydro-1H-spiro-[isoquinoline-4,3'-pyrazole]-4'-
carbonitrile (7).
Method A: A solution of 6a (2.99 g, 0.01 mol) in absolute ethanol (30 mL) containing a catalytic
amount of piperidine was heated under reflux with hydrazine hydrate (1.1 mL, 0.02 mol) for 4 h under
TLC monitoring. The reaction mixture was then cooled, poured onto ice-cold water. The solid that
separated was filtered off, dried and crystallized from dilute dimethylformamide.
Method B: The same reactants of method A were heated in microwave at 500 W and 140 ºC for 15
min. The reaction mixture was treated in a similar manner to method A to obtain compound 7.
Compound 7 was obtained as white crystals, m.p. 254-255 ºC, in 53% yield (Method A) and 84% yield
1
(Method B); H-NMR: 2.10 (s, 2H, NH2, D2O exchangeable), 3.50 (s,1H, pyrazole H4), 6.10 (s, 1H,
13
NH, D2O exchangeable) 6.90-8.10 (m, 9H, Ar-H); C-NMR: 44.1 (pyrazole C4), 55.3 (sp3 spiro C),
116.1 (CN), 120.2, 123.1, 126.9, 128.2, 128.9, 129.1, 129.7, 132.0, 135.1, 144.4 (aromatic C), 150.9
(pyrazole C3), 155.3, 169.2 (2 CO); IR ν: 3340, 3315, 3260 cm−1 (NH2 and NH), 3080 (aromatic CH),
2180 (CN), 1670,1640 (2C=O), 1600, 1490 (aromatic C=C); MS: M+ m/z 331 (7.8%); Anal. Calcd. for
C18H13N5O2 (331.33): C (65.25%), H (3.95%), N (21.14%); Found: C (65.5%), H (4.1%), N (21.5%).
3.1.6. 6'-Amino-1,3-dioxo-2-phenyl-2'-thioxo-2,2',3,5'-tetrahydro-1H,3'H-spiro[isoquinoline-4,4'-
pyrimidine]-5'-carbonitrile (8)

Molecules 2010, 15
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Method A: A solution of 6a (2.99 g, 0.01 mol) in absolute ethanol (30 mL) containing a catalytic
amount of piperidine was heated under reflux with thiourea (0.76 g, 0.01 mol) for 4 h under TLC
monitoring. The reaction mixture was then cooled, poured onto ice-cold water. The solid that separated
was filtered off, dried and crystallized from dilute dimethylformamide.
Method B: The same reactants of method A were heated in microwave at 500 W and 140 ºC for 15
min. The reaction mixture was treated in a similar manner to method A to obtain compound 8.
Compound 8 was obtained as white crystals, m.p. 243-244ºC, in 59% yield (Method A) and 86% yield
1
(Method B); H-NMR: 2.10 (s, 2H, NH2, D2O exchangeable), 2.70 (s, 1H, NH, D2O exchangeable),
3.30 (s, 1H, pyrimidine H5), 6.90-7.90 (m, 9H, Ar-H); 13C-NMR: 39.3 (pyrimidine C5), 54.9 (sp3 spiro
C), 118.4 (CN), 121.1, 124.6, 126.3, 128.8, 129.4, 129.8, 130.76, 132.7, 135.9, 143.6 (aromatic C),
150.9 (pyrimidine C4), 158.1 (CS), 161.3, 169.2 (2 CO); IR ν: 3300, 3285, 3180 cm−1 (NH2 and NH),
3070 (aromatic CH), 2150 (CN), 1670,1645 (2C=O), 1600, 1490 (aromatic C=C); MS: M+ m/z 375
(5.8%); Anal. Calcd. for C19H13N5O2S (375.40): C (60.79%), H (3.49%), N (18.66%), S (8.54%);
Found: C (61.0%), H (3.6%), N (18.5%), S (8.6%).
3.2. Antimicrobial Screening
The newly synthesized heterocyclic compounds were tested for their antimicrobial activity against
the following microorganisms: (a) Gram-negative: Escherichia coli and Pseudomonas putide; (b)
Gram-positive: Bacillus subtilis and Streptococcus lactis; (c) Fungi: Aspergillus niger and Penicillium
sp.; (d) Yeast: Candida albicans. Media: Three types of specific media were used in this study:
Medium 1: For bacteria (Nutrient Medium), consisting of (g/L distilled water): peptone, 5 and meat
extract, 3. pH was adjusted to 7.0.
Medium 2: For fungi (Potato Dextrose Medium), consisting of (g/L distilled water): Infusion from
potatoes, 4 and D(+)glucose, 20. pH was adjusted to 5.5.
Medium 3: For yeast (Universal Medium), consisting of (g/L distilled water): yeast extract, 3; malt
extract, 3; peptone, 5 and glucose, 10. pH was adjusted to 5.5.
For solid media, 2% agar was added. All media were sterilized at 121 °C for 20 min.
3.2.1. Procedure (Filter paper diffusion method) [25]
Proper concentrations of microbial suspensions were prepared from 1 (for bacteria) to 3 (for yeast
and fungi)-day-old liquid stock cultures incubated on a rotary shaker (100 rpm). In the case of fungi,
five sterile glass beads were added to each culture flask. The mycelia were then subdivided by
mechanical stirring at speed No. 1 for 30 min. Turbidity of microorganisms was adjusted with a
spectrophotometer at 350 nm to give an optical density of 1.0. Appropriate agar plates were aseptically
surface inoculated uniformly by a standard volume (ca. 1 mL) of the microbial broth culture of the tested
microorganism, namely E. coli, P. putida, B. subtilis, S. lactis, A. niger, Penicillium sp. and C. albicans.
Whatman No. 3 filter paper discs of 10 mm diameter were sterilized by autoclaving for 15 min at
121 °C. Test compounds were dissolved in 80% ethyl alcohol to give final concentration of 5 μg/mL.
The sterile discs were impregnated with the test compounds (5 μg/disc). After the impregnated discs

Molecules 2010, 15
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have been air dried, they were placed on the agar surface previously seeded with the organism to be
tested. Discs were gently pressed with forceps to insure thorough contact with the media. Three discs
were arranged per dish, suitably spaced apart, i.e. the discs should be separated by a distance that is
equal to or slightly greater than the sum of the diameters of inhibition produced by each disc alone.
Each test compound was conducted in triplicate. Plates were kept in the refrigerator at 5 °C for 1 h to
permit good diffusion before transferring them to an incubator at 37 °C for 24 h for bacteria and at 30 °C
for 72 h for yeast and fungi.
3.3. Anti-Oxidant Screening
3.3.1 Assay for erythrocyte hemolysis
Blood was obtained from rats by cardiac puncture and collected in heparinized tubes. Erythrocytes
were separated from plasma and the buffy coat and washed three times with 10 volumes of 0.15 M
NaCl. During the last washing, the erythrocytes were centrifuged at 2,500 rpm for 10 min to obtain a
constantly packed cell preparation. Erythrocyte hemolysis was mediated by peroxyl radicals in this
assay system [26]. A 10% suspension of erythrocytes in pH 7.4 phosphate-buffered saline (PBS) was
added to the same volume of 200 mM 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) solution
(in PBS) containing samples to be tested at different concentrations. The reaction mixture was shaken
gently while being incubated at 37 ºC for ~h. The reaction mixture was then removed, diluted with
eight volumes of PBS and centrifuged at 2,500 rpm for 10 min. The absorbance A of the supernatant
was read at 540 nm. Similarly, the reaction mixture was treated with eight volumes of distilled water to
achieve complete hemolysis, and the absorbance B of the supernatant obtained after centrifugation was
measured at 540 nm. The percentage hemolysis was calculated by equation (1 − A/B) × 100%. The data
were expressed as mean standard deviation. L-Ascorbic was used as a positive control.
3.3.2. Anti-oxidant activity screening assay-ABTS method
For each of the investigated compounds, 2 mL of ABTS [2,2'-azino-bis(3-ethylbenzthiazoline-6-
sulphonic acid)] solution (60 mM) was added to MnO2 suspension, all prepared in phosphate buffer
(pH 7, 0.1 M). The mixture was shaken, centrifuged, filtered to remove excess MnO2, and the
absorbance (Acontrol) of the resulting green-blue solution (ABTS radical solution) was adjusted at ca.
0.5 at λ 734 nm. Then, 50 μL of (2 mM) solution of the test compound in spectroscopic grade MeOH/
phosphate buffer (1:1) was added. The absorbance (Atest) was measured and the reduction in color
intensity was expressed as % inhibition. The % inhibition for each compound is calculated from the
following equation [27]:
Ascorbic acid (vitamin C) was used as standard anti-oxidant (positive control). Blank sample was
run without ABTS and using MeOH/phosphate buffer (1:1) instead of sample. Negative control sample
was run with MeOH/phosphate buffer (1:1) instead of tested compound.
3.3.3. Bleomycin-dependent DNA damage

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The assay was done according to Aeschlach et al. [28] with minor modifications. The reaction
mixture (0.5 mL) contained DNA (0.5 mg/mL), bleomycin sulfate (0.05 mg/mL), MgCl2 (5 mM),
FeCl3 (50 μM) and samples to be tested at different concentrations. L-Ascorbic acid was used as a
positive control. The mixture was incubated at 37 ºC for 1 h. The reaction was terminated by addition
of 0.05 mL EDTA (0.1 M). The color was developed by adding 0.5 mL thiobarbituric acid (TBA) (1%,
w/v) and 0.5 mL HCl (25%, v/v) followed by heating at 80 ºC for 10 min. After centrifugation, the
extent of DNA damage was measured by increase in absorbance at 532 nm.
4. Conclusions
New spiro-compounds have been synthesized using both traditional methods and microwave-
assisted conditions. The latter methods proved much more efficient in reducing reaction times as well
as increasing the overall yield of the reactions. The newly synthesized compounds were tested for their
antimicrobial and antioxidant activities. Some compounds showed moderate or weak antimicrobial
activity, whereas compounds 7 and 8 showed promising antioxidant activity.
Acknowledgements
This research is financed by Al-Taif University, Al-Taif, Kingdom of Saudi Arabia,
Project # 1-430-456.
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Sample Availability: Samples of the compounds are available from the authors.
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