Microwave-assisted Synthesis and Bioevaluation of Some Semicarbazones

Article abstract of DOI:10.1111/j.1747-0285.2012.01360.x

In continuation to our efforts in finding potential therapeutic agents, a variety of biologically significant semicarbazones were synthesized by the reaction of different carbonyl compounds with phenyl semicarbazides through microwave irradiation. Initial

Full text of DOI:10.1111/j.1747-0285.2012.01360.x

ª 2012 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2012.01360.x
Chem Biol Drug Des 2012; 79: 950–959
Research Article
Microwave-assisted Synthesis and Bioevaluation
of Some Semicarbazones
Laila Jafri1, Farzana L. Ansari2, Maryam
plexes of semicarbazones have also been found to be active against
influenza, protozoa, smallpox, psoriasis, rheumatism, trypanosomiasis,
coccidiosis, malaria, and certain kinds of tumors and have been sug-
gested as possible pesticides and fungicides too (4). Present study was
Jamil1, Saima Kalsoom2, Sana Qureishi2
and Bushra Mirza1,*
1Department of Biochemistry, Qauid-i-Azam University, Islamabad
planned to synthesize a variety of semicarbazones and to screen them
for their biological properties. Initially, synthesis of 18 semicarbazones
was carried out including eight benzaldehyde semicarbazones, four
acetophenone semicarbazones, five isatin semicarbazones and a ferr-
ocenyl semicarbazone. These semicarbazones were screened against a
variety of bioassays. Antibacterial and antifungal activities were deter-
mined against four pathogenic bacterial and four fungal strains,
respectively. To evaluate the antitumor activity, potato disc antitumor
assay was performed. Antioxidant potential of the synthesized com-
pounds was determined by DPPH radical scavenging and DNA protec-
tion assay. The results of these bioassays indicated strong potential of
these compounds as antitumor and antioxidants. Later, another set of
semicarbazones was synthesized, and all these compounds were evalu-
ated for their potential as antioxidant using DPPH free radical scaveng-
ing assay. The results indicated that semicarbazones possess a great
potential to be developed into anticancerous drugs.
45320, Pakistan
2Department of Chemistry, Qauid-i-Azam University Islamabad
45320, Pakistan
*Corresponding author: Bushra Mirza, bushramirza@qau.edu.pk
In continuation to our efforts in finding potential
therapeutic agents, a variety of biologically signifi-
cant semicarbazones were synthesized by the reac-
tion of different carbonyl compounds with phenyl
semicarbazides through microwave irradiation. Ini-
tially, 18 semicarbazones were studied for their
antimicrobial, antitumor, and antioxidant poten-
tial. None of the tested compounds showed any
antibacterial activity; however, some compounds
showed significant antifungal activity. Interest-
ingly, all compounds showed antitumor activity
when tested against tumors grown on potato discs.
These compounds were also tested for their effect
on OH radical-induced oxidative DNA damage. All
the compounds showed DNA protection to varying
extent. Based on the promising results of antitu-
mor and antioxidant activities, another set of 24
semicarbazones was synthesized, and all of these
semicarbazones were evaluated for their antioxi-
dant potential. The results showed that the semic-
arbazones derived from 2-nitrobenzaldehyde and
acetophenone were the most active 2,2-diphenyl-1-
picrylhydrazyl 9 (DPPH) free radical scavengers.
The overall results have led to the identification of
some interesting compounds which seem to have
great potential to be developed into effective
anticancer drugs.
Materials and Methods
Chemicals and instruments
All chemicals and reagents were purchased from BDH Laboratory
Supplies, Poole, UK. Melting points were recorded on a MEL-Temp
apparatus. Infrared spectra were recorded on Schimadzu Infrared
Spectrophotometer model 270. Samples were taken in KBr pellets.
1H and 13C-NMR spectra were recorded on Bruker Spectrophotometer
at 300 and 75 MHz field strengths, respectively. Tetramethylsilane
was used as reference. Chemical shifts are given in d-scale (ppm).
(EI-MS) Mass Spectra were recorded on Agilent GCMS Spectrometer.
Synthesis of semicarbazones (general
procedure)
Key words: antibacterial, antifungal, antioxidant, antitumor, DNA
protection, Semicarbazones
Phenylsemicarbazones were prepared according to literature proce-
dure (10) while semicarbazones were prepared through microwave
irradiation according to the procedure reported in literature (11). Phe-
nylsemicarbazide (3 mmol), ethanol (10–20 mL), and few drops of gla-
cial acetic acid were added in equimolar quantity to an appropriate
carbonyl compound in ethanol. The reaction mixture was given pulses
in a domestic microwave oven Domestic microwave oven (LG, Model:
MS-304 A, Microwave: 1250 W, RF outt: 900 W and 2450 Mz) for
about 60–120 seconds. The completion of reaction was monitored
through TLC (Ethylacetate: n-hexane 4:1) (A). The resultant mixture
was cooled, filtered, and dried. Purification of the compounds was
Received 2 November 2010, revised 30 December 2011 and accepted
for publication 28 January 2012
Semicarbazones, also known as urea derivatives, are the product of
the addition–elimination reaction of an aldehyde (or ketone) with
semicarbazide (H2NNHCONH2). They present a wide range of pharma-
cological applications (1–3). An exhaustive literature search revealed
that semicarbazones have shown significant antifungal, antibacterial,
antitumor, anticonvulsant, and antioxidant activities (5–9). Metal com-
950

Biological Activities of Some Semicarbazones
carried out by recrystallization in hot ethanol. Semicarbazones 1–10
are already reported in literature and their physical constants agree
with the reported values (11). Following this general procedure, a vari-
ety of semicarbazones (1–42) were synthesized.
Compound (22): 1-(1-(2,4-Dihydroxyphenyl)
ethylidene)-4-phenylsemicarbazone
m.p.219 ꢀC, Rf 0.76 (A), Yield 56%. IR (KBr, m ⁄ cm): 3600, 3435,
3241, 3160, 3067, 1759, 1650, 1570, 1428, 1410, 1398. 1H NMR
(300 MHz, DMSO-d6): d1.58 (s, 3H, CH3), 6.15 (bs, 1H, Ar-NH),
6.68–7.57 (m, 8H, aromatic), 7.95 (bs, 2H, OH), 8.30 (bs, 1H, =N-
NH).13C NMR (75 MHz, DMSO-d6): d 16.5 (CH3), 110.2–154.5 (aro-
matic), 157.0 (C=N), 169.1 (C=O). m ⁄ z (%): 285 (100).
Compound (11): 1-(1-(3-Aminophenyl)
ethylidene)-4-phenylsemicarbazone
m.p.184 ꢀC, Rf0.68 (A). Yield 42%. IR (KBr, m ⁄ cm): 3321, 3242,
1760, 1656, 1580, 1420, 1409, 1398.1H NMR (300 MHz, DMSO-d6):
d1.55 (s, 3H, CH3), 5.11 (bs, 2H, NH2), 6.20 (bs, 1H, Ar-NH), 7.07–
7.66 (m, 9H, aromatic), 8.25 (bs, 1H, =N-NH).
Compound (23): 1-(1-(Anthracen-2-
yl)ethylidene)-4-phenylsemicarbazone
m.p.176 ꢀC, Rf 0.59 (A). Yield 72%. IR (KBr, m ⁄ cm): 3390, 3257,
3033, 1735, 1655, 1611, 1578, 1438.1H NMR (300 MHz, DMSO-d6):
d5.1 (s, 3H, CH3), 5.96 (bs, 1H, Ar-NH), 7.32–7.96 (m, 14H, aro-
matic), 8.10 (bs, 1H, =N-NH).13C NMR (75 MHz, DMSO-d6): d 13.1
(CH3), 121.1–135.9 (aromatic), 153.0 (C=N), 168.7 (C=O).m ⁄ z: 353
(M+•, 100), 338 (5), 261 (30), 214 (5).
13C NMR (75 MHz, DMSO-d6): d 18.9 (CH3), 116.5–151.2 (aromatic),
157.0 (C=N), 169.0 (C=O).m ⁄ z (%): 268(M+•,100).
Compound (12): 1-(1-(4-Aminophenyl)
ethylidene)-4-phenylsemicarbazone
m.p.198 ꢀC, Rf 0.57 (A). Yield 39%. IR (KBr, m⁄ cm): 3375, 3280, 1756,
1655, 1588, 1445.1H NMR (300 MHz, DMSO-d6): d1.56 (s, 3H, CH3),
5.20 (bs, 2H, NH2), 6.21 (bs, 1H, Ar-NH), 7.07–7.69 (m, 9H, aromatic),
8.18 (bs, 1H, =N-NH).13C NMR (75 MHz, DMSO-d6): d 18.9 (CH3), 116.5–
151.2 (aromatic-C), 157.0 (C=N), 169.0 (C=O).m⁄z (%): 268(M+•,100).
Compound (28): 4-Phenyl-1-(1-(thiophen-2-
yl)ethylidene)semicarbazone
m.p.198 ꢀC, Rf 0.57 (A). Yield 39%. IR (KBr, m ⁄ cm): 3343, 3225,
3018, 1732, 1630, 1600, 1560, 1428.1H NMR (300 MHz, DMSO-
d6):d 1.56 (s, 3H, CH3), 5.96 (bs, 1H, Ar-NH), 7.00–7.64 (m, 8H, aro-
matic), 8.01 (bs, 1H, =N-NH).13C NMR (75 MHz, DMSO-d6): d13.8
(CH3), 121.6–154.9 (aromatic), 155.6(C=N), 168.0 (C=O).m ⁄ z (%): 259
(M+•, 100), 244.0 (3), 167.0 (50), 135 (4).
Compound (13): 1-(1-(Ferrocen-2-y)ethylidene)-4-
phenylsemicarbazone
m.p.228 ꢀC, Rf 0.82 (A). Yield 63%. IR (KBr, m ⁄ cm): 3318, 3168,
3023, 1770, 1640, 1612, 1578.1H NMR (300 MHz, DMSO-d6): d5.96
(bs, 1H, Ar-NH), 7.00–7.64 (m, 12H, aromatic), 8.01 (bs, 1H, =N-
NH).13C NMR (75 MHz, DMSO-d6): d 11.5 (CH3), 69–80 (Ferrocenyl-
C), 121.6–135.9 (aromatic-C), 155.6 (C=N), 161.0 (C=O).m ⁄ z (%): 361
(M+•,100), 296.0 (13.5), 269 (3.29), 185 (6.1).
1-(2-Oxoindolin-3-ylidene)-4-
phenylsemicarbazone (29–33)
Semicarbazones 29–33 are already reported (Scheme 4) and their
physical constants agree with the literature values (11).
1-(2-Oxoindolin-3-ylidene)-4-
phenylsemicarbazone (14–18)
Semicarbazones derived from hydantoins
(34–35)
Isatinsemicarbazones have been synthesized according to the
reported procedure (Scheme 2). Physical constants of semicarbaz-
ones14–18 agree with the literature values (12).
Compound (34):1-(2-Oxo-5-phenylimidazolidin-4-
ylidene)-4- phenyl semicarbazone
m.p.210 ꢀC, Rf 0.69(A). Yield 72%.IR (KBr, m ⁄ cm):3255, 3198, 3032,
2980, 1760, 1645, 1590, 1457.1H NMR (300 MHz, DMSO-d6):d 2.75
(s, 2H, CH2),6.5 (bs, 1H, Ar-NH), 7.25–7.55 (m, 5H, aromatic), 7.92
(bs, 1H, NH-3), 8.0 (bs, 1H, NH-1), 8.60 (bs, 1H, =N-NH).13C NMR
(75 MHz, DMSO-d6): d 37.5 (CH2), 121.6–129.0 (aromatic), 156
(C=N), 165 (C=O).m ⁄ z (%): 233(M+•, 100), 98.04 (24), 109 (32).
Compound (18): 4-(2,4-Dinitrophenyl)-1-(2-
oxoindolin-3-ylidene) semicarbazone
m.p.187 ꢀC, Rf 0.69 (A). Yield 67%. IR (KBr, m ⁄ cm): 3350, 3218,
3025, 1710, 1665, 1635, 1575, 1560, 1455, 1342.1H NMR (300 MHz,
acetone-d6):d 5.91 (s, 1H, Ar-NH), 7.00–7.99 (m, 5H, aromatic), 8.01
(bs, 1H, C-NH), 8.56 (bs, 1H, NH, 5-membered ring), 8.56–9.10 (m,
3H, aromatic semicarbazide).13C NMR (75 MHz, acetone-d6): d
117.0–146.0 (aromatic-C), 132.8 (C=N), 159.0 (C=O amide), 167.5
(C=O, 5-membered ring).m ⁄ z(%): 370(M+•,100).
Compound (35): 1-(2-Oxo-5,5-
diphenylimidazolidin-4-ylidene)-4-phenyl
semicarbazone
m.p.276 ꢀC, Rf 0.55 (A). Yield 58%. IR (KBr, m ⁄ cm): 3300, 3278,
3135, 3045, 1750, 1730, 1635.1H NMR (300 MHz, DMSO-d6):d 5.96
(bs, 1H, Ar-NH), 7.00–7.64 (m, 15H, aromatic), 8.10 (bs, 1H, =N-NH),
8.20 (bs, 1H, NH-1), 9.10 (bs, 1H, NH-3).13C NMR (75 MHz, DMSO-
d6):d 59.0 (4eC), 121.0–135.0 (aromatic), 159.0 (C=O, ring), 162
(C=N),178.0 (C=O).m ⁄ z (%): 385(M+•,100), 386(24).
Aryl aldehyde ⁄ aryl ketone and
heteroarylsemicarbazones (19–28)
Semicarbazones 19–21 and 25–27 are already reported and their
physical constants agree with the literature values (11).
Chem Biol Drug Des 2012; 79: 950–959
951

Jafri et al.
Semicarbazones derived from miscellaneous
carbonyl compounds (36–38)
(bs, 1H, NH-1), 5.0 (bs, 1H, NH-5), 5.55 (bs, 1H, Ar-NH), 7.00–8.34
(m, 8H, aromatic), 8.29 (bs, 1H, =N-NH).13C NMR (75 MHz, DMSO-
d6): d 25.0 (CH3), 49.6 (CH2), 52.6 (CH), 114.3–135.9 (aromatic), 169.0
(C=N), 180 (C=O).m ⁄ z %): 354(M+•, 100), 355 (18).
Compound (36): 4-Phenyl-1-(5-vinyl-1H-pyrrol-2
(3H)-ylidene)semicarbazone
m.p. 210 ꢀC, Rf 0.87 (A), Yield 76%.IR (KBr, m ⁄ cm): 3210, 3148,
1760, 1686. 1420.1H NMR (300 MHz, DMSO-d6):d 2.65 (s, 1H, H-1),
2.68 (d, 2H, H-3), 5.16 (dd, 1H, H-7a), 5.27 (dd, 1H, H-7b), 5.98 (bs,
1H,Ar-NH), 6.25 (dd, 1H, H-6), 6.45 (t, 1H, H-4) 7.23–7.52 (m, 5H,
aromatic), 8.80 (bs, 1H, =N-NH).13C NMR (75 MHz, DMSO-d6) d:
36.8 (C-3), 67.5 (C-2), 95.6 (C-4), 116.4 (C-7), 124.8–136.0 (aromatic-
C), 125.0 (C-6), 140.0 (4eC), 168.0 (C=N), 171.0 (C=O).m ⁄ z (%):
242(M+•,100).
Compound (41): 1-(2-Methyl-2,3-
dihydrobenzo[b][1,4]thiazepin-4(5H)-ylidene)-4-
phenylsemicarbazone
m.p.232 ꢀC, Rf 0.67 (A). Yield 69%. IR(KBr, m ⁄ cm): 3238, 3168,
3040, 1760, 1652, 1428, 1399.1H NMR (300 MHz, DMSO-d6): d 1.23
(d, 3H, CH3), 2.00 (dd, 1H,H-3b), 2.52 (dd, 1H, H-3a), 2.89 (m, 1H, H-
4), 5.10 (bs, 1H, Ar-NH), 7.00–8.64 (m, 9H, aromatic-H), 7.96 (bs,
1H, =N-NH), 8.91 (bs, 1H, -NH).13C NMR (75 MHz, DMSO-d6): d
21.5 (CH3), 23.6 (CH2), 43.5 (CH), 123.4–144.9 (aromatic-C), 162.8
(C=N), 171.0 (C=O). m ⁄ z %): 326(M+•,100), 327 (20).
Compound (37): 1-(1H-Imidazol-2(3H)-ylidene)-4-
phenylsemicarbazone
m.p.186 ꢀC, Rf 0.59 (A),Yield 76%.IR (KBr, m ⁄ cm): 3218, 2168, 1760,
1680, 1520, 1453.1H NMR (300 MHz, DMSO-d6):d 4.18 (bs, 1H, NH),
5.90 (bs, 1H, Ar-NH), 6.18 (d, 2H, H-4,5), 7.00–7.64 (m, 9H, aromatic),
8.10 (bs, 1H, =N-NH).13C NMR (75 MHz, DMSO-d6):d 88.7 (C-2),
116.6 (C-4,5), 121.6–135.9 (aromatic), 168.0 (C=N), 172.0(C=O).m ⁄ z
%): 217.0 (M+•,100), 125.0 (80), 92 (25), 161.0 (68), 56 (10).
Compound (42): 4-(2,4-Dinitrophenyl)-1-(4-
methyl-4,5-dihydro-1H-benzo[b][1,4]diazepin-
2(3H)-ylidene)-4-phenylsemicarbazone
m.p.292 ꢀC, Rf 0.65 (A), Yield 83%. IR (KBr, m ⁄ cm): 3241, 3162,
3022, 2979, 2855, 1762, 1642, 1560, 1428, 1320, 1220.1H NMR
(300 MHz, DMSO-d6):d 1.23 (d, 3H, CH3),2.08 (dd, 1H,H-3b), 2.52
(dd, 1H, H-3a), 2.75 (m, 1H, H-4),6.16 (bs, 1H, Ar-NH), 6.52–6.77(m,
4H, aromatic), 7.96 (bs, 1H, =N-NH), 8.16 (s, N-H), 8.16–9.10 (m,
3H, aromatic-H).13C NMR (75 MHz, DMSO-d6): d 21.5 (CH3), 23.6
(CH2), 43.5 (CH), 119.0–148.3 (aromatic-C), 162.8 (C=N), 172.0
(C=O).m ⁄ z %): 415(M+•, 100).
Compound (38): 1-(4-Nitro-1 H-benzo[d]
imidazol-2(3H)-ylidene)-4-phenylsemicarbazone
m.p.256 ꢀC, Rf 0.61 (A),Yield 76%. IR (KBr, m ⁄ cm): 3320, 3244,
3018, 1712, 1686, 1445, 1350.1H NMR (300 MHz, DMSO-d6):d4.0
(bs, 1H, H-3), 4.2 (bs, 1H, H-1), 5.96 (bs, 1H, Ar-NH), 6.10–7.64 (m,
8H, aromatic), 8.01 (bs, 1H, =N-NH).13C NMR (75 MHz, DMSO-
d6):d115.2–137.0 (aromatic-C), 168.0 (C=N),175.0 (C=O amide).m ⁄ z
%): 312(M+•,100), 313(15).
Biological screening
All the synthesized compounds were screened for their antibacte-
rial, antifungal, antitumor, DNA damaging, and DPPH radical scav-
enging characteristics.
Benzoheteroazepinone semicarbazones (39–42)
Benzoheteroazepinones were synthesized according to literature
procedure (13)
Antibacterial activity
Agar-well diffusion method as reported earlier (14) was used to
check antibacterial activity of all synthesized compounds against
four bacterial strains namely: Staphylococcus aurous (ATTCC 6538),
Bacillus subtilis (ATTCC 6633), Escherichia coli (ATTCC 15224), and
Enterobacter aerogenes (ATTCC 13048). DMSO and standard anti-
bacterial drugs (Roxithromycin and Cefixim) served as negative and
positive controls respectively. The antibacterial activity was deter-
mined by measuring the diameter of zones showing complete inhibi-
tion (mm). Growth inhibition was calculated with reference to
negative control.
Compound (39): 1-(4-Methyl-4,5-dihydro-1
H-benzo[b][1,4]diazepin-2(3H)-ylidene)-4-
phenylsemicarbazone
m.p.197 ꢀC, Rf 0.78 (A),Yield 76%. IR (KBr, m ⁄ cm): 3345, 3230,
3028, 2990, 2850, 1678, 1655, 15801H NMR (300 MHz, DMSO-d6):d
1.27 (d, 3H, CH3), 2.07 (dd, 1H, H-3b), 2.32 (dd, 1H, H-3a), 3.75 (m,
1H, H-4), 4.5 (bs, 1H, NH-5), 4.8 (bs, 1H, NH-1), 5.95 (bs, 1H, Ar-
NH), 7.00–8.34 (m, 9H, aromatic), 8.19 (bs, 1H, =N-NH).13C NMR
(75 MHz, DMSO-d6): d 21.0 (CH3), 48.6 (CH2), 56.6 (CH), 114.3–
135.9 (aromatic), 162.0 (C=N), 171.0, 179.0 (C=O).m ⁄ z %): 309(M+•,
100), 310(20).
Antifungal activity
Antifungal activity against four fungal strains namely Mucor species
(0300), Aspergillus niger (0198), Aspergilus flavus (0064), and Asper-
gilus fumigatus (66) was determined by using Agar tube dilution
method (15). The medium supplemented with DMSO and Terbinafine
(200 lL ⁄ mL) was used as negative and positive control, respectively.
The growth was determined by measuring linear growth (mm), and
growth inhibition was calculated with reference to negative control.
Compound (40): 1-(4-Methyl-8-nitro-4,5-dihydro-
1H-benzo[b][1,4]diazepin-2(3H)-ylidene)-4-
phenylsemicarbazone
m.p.237 ꢀC, Rf 0.67(A), Yield 74%. IR (KBr, m ⁄ cm): 3354, 3242, 3036,
2985, 2865, 1685, 1650, 1589.1H NMR (300 MHz, DMSO-d6):d 1.37
(d, CH3), 2.27 (dd, 1H,H-3b), 2.32 (dd, 1HH-3a), 3.55 (m, 1H, 4-H), 4.6
952
Chem Biol Drug Des 2012; 79: 950–959

Biological Activities of Some Semicarbazones
Antitumor activity
DPPH free radical scavenging assay
Antitumor activity of the synthesized compounds was checked by per-
forming modified potato disc antitumor assay (16). All the synthesized
compounds were also tested against Agrobacterium tumefacians
(AT10) using the agar-well diffusion method as described in antibacte-
rial activity section. All tests were performed in replicate, and percent
tumor inhibition was calculated by the following equation
The antioxidant effect of semicarbazones was studied using stan-
dard DPPH assay (17). Test samples were examined at 100, 50, and
25 lg ⁄ mL as final concentration. Ascorbic acid was used as a posi-
tive control and DMSO as negative control. Percentage free radical
scavenging was calculated by using following formula
% Scavenging
ꢀ
ꢁ
% Age inhibition
Absorbance of control ꢀ Absorbance of test sample
¼
ꢁ 100
Average number of tumours of samples
Average number of tumours of -ve control
Absorbance of control
¼ 100 ꢀ
ꢁ 100
Results and Discussion
DNA protection activity
The determination of antioxidant or prooxidant activity of the
synthesized compounds was conducted by hydroxyl-induced DNA
damage-based assay (17).
A
variety of substituted semicarbazones with the formula
RCNNH2CONHPh were synthesized under microwave radiations, by
the reaction of quite diverse carbonyl compounds with phenyl
Scheme 1: Semicarbazones
derived from arylaldehydes and
ketones.
Scheme 2: Isatin semicarbazones derived from phenyl and dinitro phenyl semicarbazide.
Chem Biol Drug Des 2012; 79: 950–959
953

Jafri et al.
semicarbazide which in turn was synthesized by the reaction of
phenyl hydrazine with potassium cyanate. Semicarbazone 1–12
was synthesized by the reaction of commercially available aryl
aldehydes or ketones, while compound 13 was synthesized by
using acetylferrocene as the carbonyl compound (Scheme 1). Semic-
arbazones 14–17 were obtained by the reaction of various substi-
tuted isatins with phenyl semicarbazide (Schemes 2 and 3). In all
isatins, only the exocyclic carbonyl group at C-3 undergoes a con-
densation reaction as the C-2 carbonyl functionality is involved in
amide linkage with the ring nitrogen.
A few semicarbazones were also derived from heteroaryl carbonyl
derivatives (Scheme 3). Quite a few semicarbazones were synthesized
by the reaction of phenyl semicarbazide with different 5-member het-
erocycles such as hydantoins, imidazolones, and pyrrolidinone
(Scheme 3–6). Few exocyclic semicarbazones 39–42 derived from 7-
member heteroazepinones such as 1,5-benzodiazepinone and 1,5-ben-
zothiazepinones have been shown in Scheme 7. Semicarbazones 18
and 42 were derived from 2,4-dinitrophenyl semicarbazide, which in
turn was synthesized by the reaction of, 4-dinitrophenyl hydrazine with
potassium cyanate. The yields of the products vary from 40% to 80%.
Scheme 3: Semicarbazones derived from diverse carbonyl compounds.
Scheme 4: Isatin derived from phenyl semicarbazones.
954
Chem Biol Drug Des 2012; 79: 950–959

Biological Activities of Some Semicarbazones
Scheme 5: Synthesis of hydantoin semicarbazones.
Scheme 6: Semicarbazones derived from heteroazepinones.
Scheme 7: Semicarbazones derived from heteroazepinones.
Antibacterial activity
Antifungal activity
Four strains of bacteria, that is, S. aurous, E. aerogenes, E. coli and
B. subtilis, were used to assess the antibacterial activity of 18 syn-
thesized compounds by the agar-well diffusion protocol at a concen-
tration of 1 mg ⁄ mL (1000 ppm). None of the tested compounds
showed any significant antibacterial activity. These results are con-
sistent with a previous report (18).
A set of 18 semicarbazones were screened against four fungal
strains; Mucor species (0300), A. niger (0198), A. flavus (0064), and
A. fumigatus (66) using tube diffusion method. Linear growth inhibi-
tion was observed in each case. Terbinafine which were used as
standard drug showed 100% inhibition against all strains. The crite-
ria for activity were based on percent growth inhibition; more than
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955

Jafri et al.
70% growth inhibition was considered as significant, 60–70% inhibi-
tion as good, 50–60% inhibition as moderate and below 50% as
non-significant activity. Compound 7 showed quite significant activ-
ity against A. fumigatus and A. flavus (69% against both strains)
while moderate activity was seen against A. niger and Mucor. Three
compounds showed significant activity against only one fungal
strain, for example, semicarbazone 4 showed 68.4% inhibition
against A. niger while compounds 14 and 17 showed significant
antifungal activity against A. fumigatus only. Other compounds
showed varying degree of percentage inhibition (Table 1). These
results agree with the two previous reports (7,8) that semicarbaz-
ones usually have significant antifungal activity.
Antitumor activity
Potato disc antitumor assay is a reliable bench-top assay used to
detect antitumor behavior of compounds (19). All the synthesized
semicarbazones were investigated for their antitumor activity. The
Table 3: DNA protection activity of semicarbazones 1–18
Compound
Concentration
Protection
Damage
1
1000
100
10
+
++
++
++
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
+++
+++
+++
+++
–
–
–
–
–
2
3
1000
100
10
1000
100
10
++
++
++
++
++
++
++
++
++
++
+
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
++
+
++
++
+
+
++
–
–
–
–
+
++
++
++
++
–
+
++
–
Table 1: Antifungal activity of semicarbazones 1–18
Percentage growth inhibition
Sample code Aspergillus niger Aspergilus fumigatus Aspergilus flavus Mucor
4
1000
100
10
1
2
52.62
54.20
43.15
68.41
56.83
44.73
44.73
36.83
32.10
48.41
23.24
37.20
39.52
16.27
9.29
48.94
12.91
34.43
34.93
40.44
2.90
69.96
40.44
25.42
46.44
53.33
51.11
58.66
67.55
46.66
46.66
60
42.20
45.30
36.01
41.17
42.20
29.30
64.91
52.52
39.62
40.14
34.08
43.63
52.27
45.90
54.08
31.81
27.26
54.54
16.85
18.77
18.77
21.83
19.15
18.00
56.32
13.02
21.83
21.83
9.890
36.39
20.49
22.25
20.49
16.95
18.72
16.95
5
1000
100
10
3
4
5
6
1000
100
10
6
7
8
7
1000
100
10
9
10
11
12
13
14
15
16
17
18
8
1000
100
10
9
1000
100
10
13.94
9.29
2.31
10
11
12
13
14
15
16
17
18
1000
100
10
1000
100
10
1000
100
10
1000
100
10
1000
100
10
1000
100
10
1000
100
10
1000
100
10
51.11
Table 2: Antitumor activity of semicarbazones 1–18
Percentage tumor inhibition (ppm)
Organic compounds
1000
100
10
IC50 lg ⁄ mL
1
20.88
15.11
)0.88>1000
38.97
49.23
35.05
22.53
20.97
38.51
25.18
39.53
25.92
34.49
36.43
37.98
35.36
25.85
25.19
10.76
32
2
56.92
64.10
31.14
53.05
65.57
47.40
40
64.72
63.70
75.58
77.51
64.72
67.84
67.84
50.78
70.80
81.29
65.12
62.05
38.18
36.61
49.92
12.59
40
58.52
44.44
56.97
53.48
56.97
56.69
43.89
41.60
41.27
58.16
38.96
7.28
3
4
>1000
669.37
159.35
>1000
>1000
45.87
191.01
52.88
52.03
57.34
62.66
157.46
699.61
200.26
51
5
6
7
8
9
10
11
12
+++
13
14
–
+++
–
15
16
1000
100
10
++
++
17
18
–
Vincristine
100
100
100
0.003
+ Weakly effective, ++ Moderately effective, +++ Strongly effective.
Chem Biol Drug Des 2012; 79: 950–959
956

Biological Activities of Some Semicarbazones
experiment was carried out at different concentrations of semicar-
bazones to investigate whether an increase in concentration influ-
enced the tumor formation efficiency; IC50 values were calculated
later. The solvent DMSO served as a negative control for calculating
percentage inhibition as mentioned in Table 2. Results showed that
all the tested compounds had significant (p < 0.05) impact on tumor
formation at three different concentrations, that is, 10, 100, and
1000 ppm (Table 2). In our experiments, compound 3 was found to
have the highest inhibitory activity (IC50 value 7.28 lg ⁄ mL). As com-
pounds with IC50 values <10 lg ⁄ mL are usually considered highly
active antitumor agents (20), none of compounds have shown anti-
bacterial activity against A. tumefacians, which is evidence that
tumor inhibition is solely because of the intrinsic antitumor potential
of the tested compounds. Hence, they can be investigated for fur-
ther studies against different cancer cell lines.
However, the series of isatinsemicarbazone (15–17) and a ferroce-
nylsemicarbazone (18) caused cleavage of DNA at one or more
concentrations (Figure 1). Compounds 15, 17, and 18 showed
damaging effect at 1000 ppm, but protective effect at 100 and
10 ppm was observed. It has been reported in literature that sev-
eral chemopreventive agents that are antioxidant at some concen-
trations may behave as prooxidant at other concentrations (22).
The results of these two assays indicated the potential of the
tested semicarbazones for antitumor and antioxidant activities.
Therefore, another set of 24 compounds was synthesized, and all
42 compounds were then tested for their potential as free radical
scavengers.
DPPH radical scavenging assay
DPPH radical scavenge assay is a non-enzymatic method currently
used to provide basic information about the ability of compounds to
scavenge free radicals. Reduction of DPPH by an antioxidant results
in the loss of absorbance at 517 nm (23). The resulting decrease in
color from purple to yellow is observed as the radical is scavenged
by antioxidants through donation of hydrogen to form the stable
DPPH-H molecule. The antioxidant activity of the synthesized semic-
arbazones was determined spectrophotometrically by monitoring the
disappearance of DPPH at 517 nm. The results indicated that
although most of the compounds were antioxidant (except 12 and
17), however, the level of activity varied considerably. Some com-
pounds like 1, 3, 7, 9–10, 22, 25, 27, 29, 34, 39, and 41–42
were found to be strong antioxidant and showed IC50 values rang-
ing from 24 to 30 lg ⁄ mL while others showed low antioxidant
activity (Table 4). The compounds (e.g., 3 and 9) with good antitu-
mor activity were found to be strong antioxidants as well, and
these results are consistent with the previous reports of some cor-
relations found in these two activities (24). Semicarbazones 12 and
17 behaved as prooxidant, and this behavior is also reported in
DNA damaging studies
The ability of the semicarbazones to affect Fenton-mediated single-
and double-strand breaks in plasmid pBR322 DNA was assessed. In
a Fenton reaction, Fe2+ metal iron reduces hydrogen peroxide result-
ing in the production of hydroxyl radical which attack supercoiled
plasmid DNA (SC) to break it into two forms, namely, open circular
(OC) and linear form (Linear) representing single-stranded and dou-
ble-stranded breaks, respectively (17). A control run in the assay
showed changes from SC DNA to the open circular (OC) and linear
form (Linear) indicating the cleavage of DNA when treated with
H2O2 in the presence of FeSO4. DNA was also treated either with
FeSO4 or with H2O2 alone which indicated that H2O2 and FeSO4
together caused much more damage to DNA than either of them
alone, and this observation was consistent with a previously
reported results (21). It was also found that most of the semicar-
bazones like benzaldehyde semicarbazones (1–8) and acetophenone
semicarbazones (9–12) and two isatinsemicarbazones (13 and 16)
protected DNA against the attack of hydroxyl radical (Table 3).
L
P
F
H
X
1
2
3
4
5
6
Relaxed
Super
coiled
Linear
L
P
1 kb ladder
pBR322 plasmid
F
pBR322 plasmid treated with FeSO4
pBR322 plasmid treated with H2O2
(positive control) pBR322 plasmid treated with FeSO4 and H2O2
H
X
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
pBR322 plasmid treated with FeSO4 and H2O2 + compound 16(1000 ppm)
pBR322 plasmid treated with FeSO4 and H2O2 + compound 16(100 ppm)
pBR322 plasmid treated with FeSO4 and H2O2 + compound 16(10 ppm)
pBR322 plasmid treated with FeSO4 and H2O2 + compound 17(1000 ppm)
pBR322 plasmid treated with FeSO4 and H2O2 + compound 17(100 ppm)
pBR322 plasmid treated with FeSO4 and H2O2 + compound 17(10 ppm)
Figure 1: Effect of compounds 16 and 17 on pBR322 plasmid DNA.
Chem Biol Drug Des 2012; 79: 950–959
957

Jafri et al.
Table 4: DPPH free radical scavenging activity of the semicar-
bazones 1–42
against any tested bacterial strain. However, some of the com-
pounds showed variable activity against different fungi. All com-
pounds showed significant inhibition against tumor formation on
potato discs induced by AT 10. DNA protective activity was
observed for all semicarbazones. The overall results showed that
these semicarbazones have significant potential to be developed
into antioxidants and anticancer drugs.
Percentage scavenging (ppm)
Sample code
100
50
25
IC50 lg ⁄ mL Remarks
33.29 Antioxidant
1
2
82.07
11
75.47
34.40
11
4.9
>100
25.65
>100
3
86.6
17.61
15.80
11.76
91.29
7.06
91.0
51
15.99
12.39
72.11
25.90
4.2
73.38
)18.16
5.043
55.76
34.7
41.7
89
31.8
14.8
65
33
53.79
17
58.5
32.69
67
35.91
32.8
85
23.41
39.0
35.15
1.20
73.27
51.38
75.71
75.14
86.1
41.87
1.96
14.49
15.04
93
7.05
43.88
57.92
10.96
Antioxidant
4
13.55
15.46
13.29
93.27
Acknowledgments
5
>100
>100
<25
6
7
Antioxidant
The authors acknowledge the Higher Education Commission, Paki-
stan for providing financial support.
8
.136
>100
9
86.50
59
16.78
2.40
47.10
13.79
4.57
63.18
)6.62
22.61
36.30
31.7
24.82
<25
Antioxidant
Antioxidant
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
>100
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Prooxidant
Antioxidant
1.96
6.75
8.58
60.45
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76.60
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8.89
40.16
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40.12
85.76
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75.44
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959