Novel cationic quinazolin-4(3H)-one conjugated fullerene nanoparticles as antimycobacterial and antimicrobial agents

Article abstract of DOI:10.1002/ardp.201200371

A series of novel cationic fullerene derivatives bearing a substituted-quinazolin-4(3H)-one moiety as a side arm were synthesized using the 1,3-dipolar cycloaddition reaction of C₆₀ with azomethine ylides generated from the corresponding Schiff

Full text of DOI:10.1002/ardp.201200371

210
Arch. Pharm. Chem. Life Sci. 2013, 346, 210–220
Full Paper
Novel Cationic Quinazolin-4(3H)-one Conjugated
Fullerene Nanoparticles as Antimycobacterial and
Antimicrobial Agents
Manishkumar B. Patel, Uma Harikrishnan, Nikunj N. Valand, Nishith R. Modi, and
Shobhana K. Menon
Department of Chemistry, School of Sciences, Gujarat University, Navrangpura, Ahmedabad, Gujarat, India
A series of novel cationic fullerene derivatives bearing a substituted-quinazolin-4(3H)-one moiety as a
side arm were synthesized using the 1,3-dipolar cycloaddition reaction of C60 with azomethine ylides
generated from the corresponding Schiff bases of substituted quinazolinones. The synthesized
compounds 5a–f were characterized by elemental analysis, FT-IR, 1H NMR, 13C NMR, and ESI-MS
and screened for their antibacterial activity against Mycobacterium tuberculosis (H37RV) and
antimicrobial activity against selected Gram-positive (Staphylococcus aureus and S. pyogenes) and
Gram-negative (Pseudomonas aeruginosa, Klebsiella pneumonia and Escherichia coli) bacterial and fungal
strains (Candida albicans, Aspergillus clavatus, and A. niger), respectively. All the compounds exhibited
significant activity, with the most effective compounds having MIC values and zones of inhibition
comparable to those of standard drugs.
Keywords: Antibacterial study / Antifungal study / Antimycobacterial study / Cationic fulleropyrrolidines / Quinazolin-
4(3H)-one
Received: October 1, 2012; Revised: November 27, 2012; Accepted: November 30, 2012
DOI 10.1002/ardp.201200371
Introduction
It is known that the lipidic bacterial cell wall, especially of
mycobacterium, is robust and impermeable to many anti-
biotics [13]. But the high hydrophobicity of fullerene allows it
to intercalate into the bilayer lipid membrane of the cell wall
and inhibits bacterial growth by inducing membrane stress
[14]. In addition, fullerenes bearing a cationic derivative have
shown enhancement in the interaction with the negatively
charged bacterial cell surface and thereby destruct the cell
wall [14].
Since the discovery of fullerene (C60) in 1985 [1], there have
been various reports of chemically modified fullerenes
with widespread applications in chemical, biological, and
material sciences [2–4]. C60 derivatives have been well known
for their encouraging applications as antibacterial agents [5],
antimycobacterial agents [6], anti-amyloid agents [7], photo-
induced DNA cleavage agents [8]. They have also been used
for studying various photo-physical properties [9], potentio-
metric biosensing of glucose [10], and drug delivery systems
[11]. There has been a great deal of emphasis in using C60 for
diagnosis and therapeutic purposes; the main motivation
being the ability of the hydrophobic fullerene spheroid in
crossing cell membrane [12].
But bacteria are characterized by a robust central metab-
olism and cell wall, which can resist or adapt to stress stur-
dily. It can easily cause precipitation of membrane stress
inducing molecules and thus adapt to the environment.
Hence we need to design a molecule which can rupture
the cell wall (cause membrane stress) and also disturb the
central metabolism ensuring complete cell death.
Cationic fullerenes, though capable of destroying the
integrity of the bacterial cell wall, are less likely to disturb
the central metabolism of the bacteria; hence keeping both
the above points in view we have synthesized a series of
cationic fullerene derivatives having a substituted quinazo-
lin-4(3H)-one side arm.
Correspondence: Shobhana K. Menon, Department of Chemistry,
School of Sciences, Gujarat University, Navrangpura, Ahmedabad-
380009, Gujarat, India.
E-mail: shobhanamenon07@gmail.com
Fax: þ91 79 26308545
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Arch. Pharm. Chem. Life Sci. 2013, 346, 210–220
Cationic Quinazolin-4(3H)-one Conjugated Fullerene Nanoparticles
211
The quinazolin-4(3H)-one side arm was chosen due to the
versatility of the quinazolinone scaffold. Quinazolinone
derivatives are readily accessible, have diverse chemical reac-
tivity as well as a wide range of biological activities [15–17].
They are known to inhibit enzymes essential for bacterial
DNA replication and hence are very potent antibacterial
agents having immense therapeutic significance [18].
Furthermore, it is known that substitution at the 2nd, 3rd,
and 6th positions of the quinazolinone ring gives improved
bacterial activity; hence these sites were considered for deri-
vatization [17, 19].
The compounds 1a–f were synthesized by a reported pro-
cedure [17]. Schiff bases 2a–f were formed by treating 1a–f
with mono-protected terephthaldehyde in ethanol and gla-
cial acetic acid. The deprotection of the aldehyde group was
then achieved by treating with perchloric acid using dioxane
as solvent to obtain 4-(substituted-4-oxo-(3H)-quinazoline-3-yl)-
4-formyl-benzylidene hydrazide 3a–f (Scheme 1).
Compounds 3a–f, N-methyl glycine and C60 were then
allowed to reflux in toluene wherein they undergo a 1,3-
dipolar cycloaddition reaction [21] to give fulleropyrrolidines
4a–f (Scheme 2).
We have reported earlier a fullerene–isoniazid conjugate
with improved activity toward H37Rv mycobacterial strain [6].
Considering the tremendous biological importance of full-
erene and quinazolinone for the treatment of tuberculosis
and also based on our expertise in functionalization of
fullerene [8–10] and calixarene [20], we carried out the syn-
thesis of Schiff base linked cationic fullerene–quinazolinone
analogues, which would provide a synergistic enhancement
in the antimycobacterial and antibacterial activity of
both the moieties. The synthesized compounds were then
screened for their antimicrobial activity by using the
broth microdilution method and antituberculosis activity
against H37RV strain using the L. J. agar dilution method.
The IR spectra of the novel compounds 4a–f showed
characteristic bands of amide carbonyl between 1695
and 1712 cmꢀ1 and organo fullerenes between 524 and
1
527 cmꢀ1. The H NMR showed the presence of the pyrroli-
dine protons as two doublets at 3.68 and 4.23 ppm with
coupling constants between 9.3 and 10.1 Hz and a singlet
at 4.59 ppm.
The cationic derivatives 5a–f (Scheme 3) was obtained by
refluxing 4a–f with stoichiometric amount of methyl iodide
in chloroform for 2 days.
The quaternization of the nitrogen on the pyrrolidine ring
was confirmed from the spectral data. The presence of a peak
in the 1H NMR spectra of compound (5a–f) at d 10.0–10.8
attributed to the hydrazide proton and the N–H stretching
(3500–3250 cmꢀ1) in the IR spectra showed that the hydra-
Results and discussion
Chemistry
The synthetic route adopted for the synthesis of fullerene
quinazolin-4(3H)-one conjugates is shown in Schemes 1–3.
Scheme 2. Synthesis of fullerene–quinazolinone conjugates
4a–f.
Scheme 1. Synthesis of compounds 3a–f.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 210–220
zide nitrogen has not participated in salt formation. The
study of the 1H NMR and 13C NMR for the surrounding atoms
of the quinazolinone nitrogen also shows that it has not
undergone methylation. Owing to the basic nature of the
pyrrolidine nitrogen it is the most susceptible to be attacked
by methyl iodide resulting in the cationic center for com-
pounds 5a–f.
The compounds 4a–f and 5a–f have a hydrophilic cationic
center but are still highly hydrophobic and were found to be
insoluble in water and other polar solvents like DMSO. Hence
suspensions in deionized water were prepared for their bio-
logical evaluation by a similar procedure as reported earlier
[22]. The size of suspended particles of compounds 4a–f and
5a–f in water was determined by transmission electron
microscopy (TEM) which shows that the particle sizes are
50 nm (Fig. 1). The concentration of suspended aggregates
in water was determined spectrophotometrically.
The compounds 4a–f and 5a–f gave the characteristic peaks
of fullerene moiety at a UV range of 292–329 nm in chloro-
form from which a standard curve was plotted. 5 mL of
chloroform was added to 5 mL of the aqueous suspension
to extract the particles and then stirred for 1.5 h. The pro-
cedure was repeated using 2.5 mL chloroform. Both the
chloroform layers were combined and the concentration
of compounds was found by comparing the absorbance from
the standard plot.
Pharmacology
Antibacterial activity
Scheme 3. Synthesis of cationic derivatives of fullerene–quina-
zolinone conjugates 5a–f.
The antibacterial activities of compounds 3a–f, 4a–f, and 5a–f
are listed in Table 1. The results revealed that there is sig-
nificant improvement in the activity of the compounds 3a–f
Figure 1. TEM images of compounds 4a and 5a.
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Cationic Quinazolin-4(3H)-one Conjugated Fullerene Nanoparticles
213
Table 1. In vitro antibacterial activity of compounds 3a–f, 4a–f, and 5a–f.
Compd No
Gram-positive
S. pyogenes
MTCC 442
Gram-negative
S. aureus
MTCC 96
P. aeruginosa
MTCC 741
K. pneumoniae
MTCC 109
E. coli
MTCC 739
MIC
MIC
MIC
MIC
MIC
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
125
100
100
125
125
125
100
125
125
125
100
125
100
100
100
100
100
50
100
125
125
125
100
100
125
125
125
100
100
100
100
100
100
100
100
50
250
200
200
250
200
200
125
100
100
100
100
100
50
50
50
50
25
25
25
–
500
500
500
500
500
250
125
125
125
125
125
125
100
100
100
100
50
250
250
200
125
125
100
100
125
100
100
100
100
100
100
50
5d
5e
5f
25
25
25
25
50
50
–
Ciprofloxacina)
DMSO
50
–
50
–
–
MIC (mg/mL), minimum inhibitory concentration.
a)
Standard drugs.
(200 mg/mL) on conjugation with a fullerene, i.e., 4a–f
(100 mg/mL) for Gram-negative bacteria. In fact the activity
increases significantly for the cationic derivative 5a–f (50–
25 mg/mL) compared to 3a–f. On the other hand, the trend
observed for Gram-positive bacteria shows that the addition
of the fullerene to the quinazolinone moiety does not cause
significant variation in the antibacterial activity.
Additionally, an introduction of cationic charge on the com-
pound in 5a–f increases the interaction with the negatively
charged cell wall causing an increase in the activity of the
molecule (Fig. 2). On the other hand in Gram-positive bacteria
permeability is not an issue, the activities remain similar
from 3a–f to 5a–f (100 mg/mL).
This can be clearly explained on the basis of the difference
in the cell wall structure of Gram-positive and Gram-negative
bacteria. The Gram-positive bacterial cell walls are less
charged and more permeable while the Gram-negative bac-
teria have a highly lipidic and negatively charged cell wall.
The antimicrobial activity of 3a–f is majorly due to the ability
of the quinazolinone group to inhibit enzymes and disrupt
other metabolic activities inside the cell. Since the Gram-
positive bacterial cell walls are highly permeable, 3a–f are
potent antibacterials; but for Gram-negative bacteria per-
meability through the cell wall is less, consequently the con-
centration of 3a–f reaching inside the cell decreases and
therefore activity decreases relative to Gram-positive bacteria.
However, when a highly lipophilic moiety like the full-
erene spheroid is introduced, the molecules 4a–f can
intercalate into the lipid bilayers of the cell wall and
channel the accumulation of more concentration of the
quinazolinone inside the cell leading to increased potency.
Figure 2. Graphical sketch representing the attack of the com-
pounds 5a–f on the Gram-negative bacteria.
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Arch. Pharm. Chem. Life Sci. 2013, 346, 210–220
Table 2. In vitro antifungal activity of compounds 3a–f, 4a–f, and
5a–f.
Table 3. In vitro antituberculosis activity and % inhibition against
M. tuberculosis H37Rv at concentration 250 mg/mL and MIC (mg/mL)
of compounds 3a–f, 4a–f, and 5a–f.
Compound
No
C. albicans
MTCC 183
A. clavatus
MTC C 1323
A. niger
MTCC 282
Compound
No
L. J. MIC method
Inhibition (%) at 250 mg/mL
MIC
MIC
MIC
MIC (mg/mL)
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
250
500
200
200
200
200
250
250
200
200
200
200
200
200
125
200
200
125
100
–
500
250
200
200
250
125
250
250
250
200
250
125
200
200
125
250
200
125
100
–
500
200
250
200
250
200
200
200
250
200
250
200
200
200
125
250
200
125
100
–
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
4e
4f
5a
5b
5c
250
250
200
200
200
200
25
12.5
12.5
12.5
6.25
6.25
3.125
3.125
3.125
3.125
3.125
1.562
0.20
0.25
–
78.12 ꢁ 1.21
77.06 ꢁ 0.88
79.07 ꢁ 0.91
79.42 ꢁ 1.02
79.55 ꢁ 1.23
79.71 ꢁ 0. 42
80.26 ꢁ 0.31
89.02 ꢁ 1. 15
91.11 ꢁ 0.99
90.25 ꢁ 0.28
92.07 ꢁ 1. 17
93.08 ꢁ 1. 09
90.01 ꢁ 1.32
92.23 ꢁ 0.17
95.45 ꢁ 1.23
96.42 ꢁ 0.62
98.12 ꢁ 1.23
98.83 ꢁ 0.21
99.00
5d
5e
5f
5d
5e
5f
Nystatinea)
DMSO
Isoniazida)
Rifampicinea)
DMSO
99.00
–
MIC (mg/mL), minimum inhibitory concentration.
a)
Standard drugs.
MIC (mg/mL), minimum inhibitory concentration.
Mean of five replicates ꢁ standard deviation.
a)
Standard drugs.
Amongst the activities of fullerene derivatives 5a–f, it is very
obvious that the molecule having more lipophilic substi-
tution, i.e. 5f, has better activity than the other compounds
for Gram-negative bacteria.
The mycobacterium cell wall has a very high concentration of
lipids and is associated with the resistance of these bacteria to
stains and antibiotics. The waxy cell wall of these bacteria
prevents the entry of molecules 3a–f (250 mg/mL and 78%
inhibition); the only activity observed is due to the small
amount probably entering through the porin pathway like
most of the antitubercular drugs [23]. But on accessorizing
the molecule with a fullerene, 4a–f can easily seep into the
waxy cell wall, rupture it, and facilitate the entry of the
quinazolinone into the cytoplasm. Inside the cytoplasm qui-
nazolinone can easily cause cell death by inhibiting cell metab-
olism. Hence the MIC decreases to 6.25 mg/mL and inhibition
increases to 93%. The introduction of a cationic charge results
in better interaction between the carboxylic groups of mycolic
acid present in the cell envelope of mycobacterium with the
derivatives 5a–f (1.5 mg/mL and 98% inhibition), resulting in
better activity compared to 3a–f and 4a–f.
Antifungal activity
The compounds 3a–f, 4a–f, and 5a–f were tested for antifun-
gal activity against Candida albicans, Aspergillus niger, and
A. clavatus. As shown in Table 2, there is no definite trend
observed in the antifungal activities of the compounds 3a–f,
4a–f, and 5a–f suggesting that the attachment of the full-
erenes has not resulted in any significant increase in the
antifungal activities. This property can be attributed to the
cell wall structures of the fungus which are mainly composed
of proteins like chitin and contain very less amount of fats.
Here again permeability is not a concern hence the activity
observed is due to the inherent antifungal property of qui-
nazolinone compounds and the fullerene moiety does not
contribute to the potency of the molecule.
Antimycobacterial activity
Conclusion
In vitro evaluation of antimycobacterial activity of the com-
pounds 3a–f, 4a–f, and 5a–f along with the standard drugs is
summarized in Table 3. A very pronounced enhancement in
the antimycobacterial activity can be seen on attachment of
the fullerene spheroid to the quinazolinone group.
We have designed and synthesized a novel class of quinazo-
linone–fullerene conjugates as antimycobacterial/microbial
agents. The molecule 5f inhibits the growth of M. tuberculosis
effectively at MIC 1.562 mg/mL with inhibition of 98.83%,
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Arch. Pharm. Chem. Life Sci. 2013, 346, 210–220
Cationic Quinazolin-4(3H)-one Conjugated Fullerene Nanoparticles
215
3300, 3190 (–NH–), 1640 (CH N); 1H NMR (d, 400 MHz, DMSO-
d6, Me4Si) 2.45 (s, 3H, CH3), 5.82 (s, 1H, O–CH–O), 3.95–4.05 (m, 4H,
–
–
which is comparable to that of the existing front-line drug
isoniazid and rifampicin which have 99% inhibition.
We conclude that the fullerene moiety here acts as a per-
meabilizer allowing the transit of the quinazolinone group
into the cytoplasm by destroying the cell envelope of myco-
bacterium as well as for Gram-negative bacteria. Thus these
molecules constitute our important ‘‘leads’’ for further
optimization by structure–activity relationship to develop
effective antimycobacterial/microbial agents which can help
to shorten the duration of current anti-TB therapy. Further,
optimization and pharmacokinetic characterization of this
series are in progress in our laboratory.
–
–
–OCH –CH O–); 7.41–8.23 (m, 13H, aromatic H and –N CH), 9.98
2
2
(s, 1H, –NH); 13C NMR (d, 125 MHz, DMSO-d6, Me4Si) 23.33 (–CH3),
67.47 (OCH2–CH2–O), 105.49 (O–CH–O), 121.90, 121.96, 125.06,
126.35, 126.74, 126.81, 127.27, 129.56, 130.23, 130.33, 133.68,
–
–
–
–
139.44, 142.67, 147.74 (Ar C), 148.27 (–N CH), 154.04 (–C N),
–
–
–
–
–
161.47 (–C O–N ), 163.93 (–C O–NH); MS: m/z 454.50 (Mþ).
–
Analysis for C26H22N4O4, (454.48): Calcd.: % C 68.71; H 4.88;
N, 12.33. Found: % C 68.72; H 4.90; N 12.34.
4-(6-Bromo-2-methyl-4-oxo-(3H)quinazolin-3-yl)-benzoic
acid (4-[1,3]dioxolan-2-yl-benzylidene) hydrazide (2b)
Yield: 80%, mp >2408C; IR (KBr, y cmꢀ1): 1658 (–C O cyclic
–
–
tertiary amide), 1682, 1530 (–C O acyclic secondary amide),
–
–
3429, 3230 (–NH–), 1668 (CH N); 1H NMR (d, 400 MHz, DMSO-
d6, Me4Si) 2.48 (s, 3H, CH3), 5.89 (s, 1H, O–CH–O), 3.95–4.12 (m,
–
–
Experimental
–
4H, –OCH –CH O–); 7.44–8.36 (m, 12H, aromatic H and –N CH),
–
2
2
Materials and method
10.05 (1H, s, –NH); 13C NMR (d, 125 MHz, DMSO-d6, Me4Si) 23.34
(–CH3), 67.41 (–OCH2–CH2O–), 105.23 (O–CH–O), 121.92, 126.72,
127.27, 127.34, 127.37, 128.26, 130.23, 130.21, 133.36, 133.71,
All the chemicals and reagents were of analytical grade of BDH,
Aldrich, and Merck unless and otherwise specified. The solvents
used for the analysis were purified by standard methods [24].
The melting points (8C, uncorrected) were taken using MPA100
Automated Melting Point Apparatus. The FT-IR spectra were
recorded on Bruker Tensor 27 FT-IR spectrometer with KBr pel-
lets. The ESI–MS spectra were recorded on Applied Biosystems,
API 2000 LC/MS/MS System. Vario Micro cube elemental analyzer
was used as elemental analysis system; 1H NMR and 13C NMR
spectra were recorded on a Bruker Avance II 400 MHz spec-
trometer in DMSO-d6 or CDCl3 with tetramethylsilane (TMS) as
an internal standard. The nanoparticle size was determined
using transmission electron microscope (TEM) JEOL JEM 2100.
The progress of the reaction was monitored on readymade silica
gel plates (Merck) using toluene/ethyl acetate as a solvent system.
Spectral data (IR, 1H NMR, mass spectra, and elemental analysis)
confirmed the structure of the synthesized compounds and the
purity of these compounds were ascertained by microanalysis.
Elemental (C, H, N) analysis indicated that the calculated and
observed values were within the acceptable limits (ꢁ0.4%).
–
–
–
139.51, 142.62, 146.78 (Ar C), 148.22 (–N CH), 154.12 (–C N),
–
–
161.32 (–C O–N ), 163.88; (–C O–NH); MS: m/z 533.32, 535.34
–
–
–
–
–
(Mþ), (Mþ2). Analysis for C26H21N4O4Br (533.37): Calcd.: % C
58.55; H 3.97; N 10.50. Found: % C 58.56; H 3.98; N 10.52.
4-(6,8-Dibromo-2-methyl-4-oxo-(3H)quinazolin-3-yl)-
benzoic acid (4-[1,3]dioxolan-2-yl-benzylidene) hydrazide (2c)
Yield: 79%, mp >2408C; IR (KBr, y cmꢀ1): 1651 (–C O cyclic
–
–
tertiary amide), 1675, 1529 (–C O acyclic secondary amide),
–
–
3323, 3187 (–NH–), 1635 (CH N); 1H NMR (d, 400 MHz, DMSO-
d6, Me4Si) 2.39 (s, 3H, CH3), 5.79 (s, 1H, O–CH–O), 4.05–4.13 (m,
–
–
–
4H, –OCH –CH O–); 7.39–8.33 (m, 11H, aromatic H and –N CH),
–
2
2
9.93 (1H, s, –NH); 13C NMR (d, 125 MHz, DMSO-d6, Me4Si) 22.89
(–CH3), 67.44 (–OCH2–CH2O–), 105.33 (O–CH–O), 126.74, 127.21,
127.29, 130.23, 130.33, 133.68, 136.86, 139.26, 136.44, 142.67
–
–
–
–
–
–
–
–
(Ar C), 148.22 (–N CH), 152.55 (–C N), 161.42 (–C O–N ), 163.98
–
(–C O–NH); MS: m/z 612.21, 614.30, 616.10 (Mþ), (Mþ2), (Mþ4).
–
Analysis for C26H20N4O4Br2, (612.27): Calcd.: % C 51.00; H 3.29;
N 9.15. Found: % C 51.02; H 3.27; N 9.17.
Synthesis of 1a–f
Synthesis of 1a–f was carried out as reported earlier [17, 25, 26].
4-(2-Phenyl-4-oxo-(3H)quinazolin-3-yl)-benzoic acid
(4-[1,3]dioxolan-2yl-benzylidene) hydrazide (2d)
Synthesis of 4-(substituted-4-oxo-(3H)quinazolin-3-yl)-
benzoic acid ([1,3]4-dioxolan-2yl-benzylidene)hydrazide
(2a–f) [6]
Yield: 85%, mp >2408C; IR (KBr, y cmꢀ1): 1658 (–C O cyclic
–
–
tertiary amide), 1682, 1538 (–C O acyclic secondary amide),
–
–
3312, 3189 (–NH–), 1641 (CH N); 1H NMR (d, 400 MHz, DMSO-
d6, Me4Si) 4.11–4.20 (m, 4H, –OCH2–CH2O–), 5.81 (s, 1H, O–CH–O),
–
–
A mixture of 1.79 g (0.01 mol) of 4-(1,3)-dioxolan-2yl-benzal-
dehyde, 2 mL of glacial acetic acid, 2.941 (0.01 mol) of substi-
tuted quinazolinone 1a dissolved in 50 mL of ethanol was
refluxed for about half an hour. Product was isolated and puri-
fied by using column chromatography using hexane/ethyl
acetate (9:1) as the solvent system. The solvent was then distilled
under vacuum to get bright yellow product 2a. The compounds
2b–f were synthesized in an analogous manner and were charac-
terized as shown below.
–
7.51–8.29 (m, 18H, aromatic H and –N CH), 10.91 (1H, s, –NH);
–
13C NMR (d, 125 MHz, DMSO-d6, Me4Si) 67.44 (–OCH2–CH2O–),
105.52 (O–CH–O), 122.07, 122.99, 125.18, 126.29, 126.74, 127.12,
127.27, 128.07, 128.68, 130.13, 130.57, 131.41, 131.73, 133.68,
–
135.48, 139.99, 142.67, 146.02 (Ar C), 148.17 (–N CH), 152.19
–
–
(–C N), 163.11 (–C O–N ), 163.79; (–C O–NH); MS: m/z 516.50
–
–
–
–
–
–
–
(Mþ). Analysis for C31H24N4O4, (516.55): Calcd.: % C, 72.08;
H, 4.68; N, 10.85. Found: % C, 72.10; H, 4.66; N, 10.84.
4-(2-Methyl-4-oxo-(3H)quinazolin-3-yl)-benzoic acid
(4-[1,3]dioxolan-2-yl-benzylidene) hydrazide (2a)
4-(6-Bromo-2-phenyl-4-oxo-(3H)quinazolin-3-yl)-benzoic
acid (4-[1,3]dioxolan-2-yl-benzylidene) hydrazide (2e)
Yield: 84%, mp >2408C; IR (KBr, y cmꢀ1): 1659 (–C O cyclic
Yield: 83%, mp >2408C; IR (KBr, y cmꢀ1): 1655 (–C O cyclic
–
–
–
–
–
–
tertiary amide), 1680, 1535 (–C O acyclic secondary amide),
–
tertiary amide), 1684, 1535 (–C O acyclic secondary amide),
–
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216
M. B. Patel et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 210–220
1
3412, 3228 (–NH–), 1642 (CH N); H NMR (d, 400 MHz, DMSO-d ,
–
–
–
d , Me Si) 2.48 (s, 3H, CH ), 7.44–8.36 (m, 12H, Ar–H, CH N), 9.99
6 4 3
–
Me4Si) 3.97–4.15 (m, 4H, –OCH2–CH2O–), 5.81 (s, 1H, O–CH–O),
6
(s, 1 H, –CHO), 10.05 (s, 1H, –NH); 13C NMR (d, 125 MHz, DMSO-d6,
–
– –
Me Si) 23.33 (–CH ), 161.02 (–C O–N ), 163.93 (–C O–NH),
– –
4 3
–
7.43–8.41 (m, 17H, aromatic H and –N CH), 9.97 (1H, s, –NH);
–
–
13C NMR (d, 125 MHz, DMSO-d6, Me4Si), 67.57 (–OCH2–CH2O–),
105.47 (O–CH–O), 118.84, 122.07, 126.74, 127.27, 127.62, 127.90,
128.07, 128.68, 130.13, 130.57, 131.41, 131.73, 133.68, 134.20,
148.27 (CH N), 154.0 (–C N), 116.99, 121.96, 127.19, 127.34,
–
–
–
–
127.38, 128.26, 129.06, 130.23, 130.33, 131.45, 131.55, 139.44,
139.66, 146.74, 191.14 (CHO); MS: m/z 489.20, 491.20 (Mþ), (Mþ2).
Analysis for C24H17BrN4O3, (489.32): Calcd.: % C, 58.91; H, 3.50;
N, 11.63. Found: % C, 58.91; H, 3.50; N, 11.63.
–
135.48, 139.99, 142.67, 145.27 (Ar C), 148.13 (–N CH), 152.20
–
–
(–C N), 163.03 (–C O–N ), 163.83; (–C O–NH); MS: m/z 595.41,
–
–
–
–
–
–
–
597.35 (Mþ), (Mþ2). Analysis for C31H23N4O4Br, (595.44): Calcd.:
% C, 62.53; H, 3.89; N, 9.41. Found: % C, 62.54; H, 3.88; N, 9.42.
4-(2-Methyl-6,8-dibromo-4-oxo-(3H)quinazolin-3-yl)-
benzoic acid (4-formyl-benzylidene) hydrazide (3c)
Yield: 78%, mp >2408C; IR (KBr y cmꢀ1): 1647 (–C O cyclic
4-(6,8-Dibromo-2-phenyl-4-oxo-(3H)quinazolin-3-yl)-
benzoic acid (4-[1,3]dioxolan-2yl-benzylidene) hydrazide (2f)
–
–
tertiary amide), 1702, 1529 (–C O acyclic secondary amide),
–
–
Yield: 80%, mp >2408C; IR (KBr, y cmꢀ1): 1652 (–C O cyclic
3341, 3188 (–NH–), 1641 (–C N); 1H NMR (d, 400 MHz, DMSO-
–
–
–
–
tertiary amide), 1677, 1530 (–C O acyclic secondary amide),
–
–
–
d , Me Si) 2.39 (s, 3H, CH ), 7.39–8.33 (m, 11H, Ar–H, CH N), 9.93
6 4 3
–
3399, 3185 (–NH–), 1637 (CH N); 1H NMR (d, 400 MHz, DMSO-
d6, Me4Si) 4.04–4.12 (m, 4H, –OCH2–CH2O–), 5.77 (s, 1H, O–CH–O),
(s, 1 H, –CHO), 10.01 (s, 1H, –NH); 13C NMR (d, 125 MHz, DMSO-d6,
–
–
– –
Me Si) 23.33 (–CH ), 161.49 (–C O–N ), 163.93 (–C O–NH),
– –
4 3
–
–
–
–
148.27 (CH N), 152.55 (–C N), 114.75, 120.07, 121.19, 125.56,
–
–
7.41–8.36 (m, 16H, aromatic H and –N CH), 9.89 (1H, s, –NH);
–
–
13C NMR (d, 125 MHz, DMSO-d6, Me4Si), 67.57 (–OCH2–CH2O–),
105.48 (O–CH–O), 116.75, 122.07, 122.19, 125.56, 126.62, 126.73,
127.26, 128.06, 128.67, 130.56, 131.41, 131.73, 133.68,
135.47, 136.38, 137.32, 139.98, 142.67, 148.27, 152.34 (Ar C),
127.73, 127.26, 129.67, 130.56, 131.41, 131.73, 136.68, 139.47,
139.38, 139.32, 139.98 191.16 (CHO); MS: m/z 568.20, 570.20,
572.20 (Mþ), (Mþ2), (Mþ4). Analysis for C24H16Br2N4O3,
(568.22): Calcd.: % C, 50.73; H, 2.84; N, 9.86. Found: % C, 50.75;
H, 2.86; N, 9.88.
–
–
–
–
–
–
–
–
148.25 (–N CH), 152.30 (–C N), 163.63 (–C O–N ), 163.87;
–
(–C O–NH); MS: m/z 674.28, 676.29, 678.22 (Mþ), (Mþ2),
–
(Mþ4). Analysis for C31H22N4O4Br2, (674.34): Calcd.: %C, 55.21;
4-(2-Phenyl-4-oxo-(3H)quinazolin-3-yl)-benzoic acid
(4-formyl-benzylidene) hydrazide (3d)
H, 3.29; N, 8.31. Found: %C, 55.22; H, 3.31; N, 8.32.
Yield: 85%, mp >2408C; IR (KBr y cmꢀ1): 1657 (–C O cyclic
–
–
tertiary amide), 16 942, 1535 (–C O acyclic secondary amide),
General procedure for the synthesis of 4-
(substituted-4-oxo-(3H)quinazolin-3-yl)-benzoic acid
(4-formyl-benzylidene) hydrazide (3a–f) [6]
–
–
3300, 3190 (–NH–), 1640 (–C N); H NMR (d, 400 MHz, DMSO-d ,
1
–
–
Me Si) 7.42–8.29 (m, 18H, Ar–H, CH N), 9.89 (s, 1 H, –CHO), 10.91
6
–
–
4
(s, 1H, –NH); 13C NMR (d, 125 MHz, DMSO-d , Me Si), 152.19 (CH
–
4.51 g (0.01 mol) of compound 2a was mixed with a solution of
3 mL perchloric acid in 50 mL 1,4-dioxane. The mixture was
stirred at room temperature for 12 h. The solution was diluted
with chloroform (30 mL) and washed with a saturated solution of
sodium bicarbonate. The organic layer was separated and dried
over anhydrous Na2SO4. The solvent was removed in vacuum and
the light yellow solid residue was subjected to column chroma-
tography in toluene/methanol (5:1) to get a light yellow solid
product 3a. In a similar way all other products 3b–f were
obtained by the above procedure.
–
N), 163.74, (–C O–N ), 163.93 (–C O–NH), 122.07, 122.99,
6
4
–
–
–
–
–
–
125.17, 126.29, 127.19, 128.07, 128.68, 129.07, 130.57, 131.41,
131.55, 131.73, 135.48, 139.66, 139.99, 146.03. 191.18 (CHO);
MS: m/z 472.00 (Mþ). Analysis for C29H20N4O3, (472.49): Calcd.:
% C, 73.72; H, 4.27; N, 11.86. Found: % C, 73.72; H, 4.27; N, 11.85.
4-(2-Phenyl-6-bromo-4-oxo-(3H)quinazolin-3-yl)-benzoic
acid (4-formyl-benzylidene) hydrazide (3e)
Yield: 76%, mp >2408C; IR (KBr y cmꢀ1): 1655 (–C O cyclic
–
–
–
tertiary amide), 1705, 1532 (–C O acyclic secondary amide),
–
3350, 3199 (–NH–), 1640 (–C N); 1H NMR (d, 400 MHz, DMSO-
4-(2-Methyl-4-oxo-(3H)quinazolin-3-yl)-benzoic acid
(4-formyl-benzylidene) hydrazide (3a)
–
–
d , Me Si) 7.43–8.41 (m, 17H, Ar–H, CH N), 9.97 (s, 1H, –CHO),
–
–
6
4
Yield: 79%, mp >2408C; IR (KBr y cmꢀ1): 1649 (–C O cyclic
–
9.91 (s, 1H, –NH); 13C NMR (d, 125 MHz, DMSO-d6, Me4Si),
–
– – –
152.19 (CH N), 163.03 (–C O–N ), 163.93 (–C O–NH), 118.84,
– – –
–
–
tertiary amide), 1705, 1535 (–C O acyclic secondary amide),
–
–
3351, 3198 (–NH–), 1639 (–C N); 1H NMR (d, 400 MHz, DMSO-
–
122.07, 122.99, 125.17, 126.29, 127.19, 128.07, 128.68, 129.07,
130.57, 121.41, 131.55, 131.73, 135.48, 139.66, 139.99, 146.03,
192.11 (CHO); MS: m/z 551.12, 553.31 (Mþ), (Mþ2). Analysis
–
d , Me Si) 2.40 (s, 3H, CH ), 7.41–8.23 (m, 13H, Ar–H, CH N), 9.87
–
–
6
4
3
(s, 1 H, –CHO), 10.05 (s, 1H, –NH); 13C NMR (d, 125 MHz, DMSO-d6,
–
–
–
–
–
for C29H19BrN4O3, (551.39): Calcd.: % C, 63.17; H, 3.47;
Me Si) 23.38 (–CH ), 161.47 (–C O–N ), 163.93 (–C O–NH),
–
–
148.27 (CH N), 154.0 (–C N), 121.90, 121.96, 125.01, 126.35,
4
3
–
–
N, 10.16. Found: % C, 65.18; H, 3.49; N, 10.18.
–
126.81, 127.18, 127.07, 129.04, 129.56, 130.23, 130.33, 131.55,
139.44, 139.44, 147.74, 191.17 (CHO); MS: m/z 410.00 (Mþ).
Analysis for C24H18N4O3, (410.42): Calcd.: % C, 70.23; H, 4.42;
N, 13.65. Found: % C, 70.23; H, 4.44; N, 13.67.
4-(2-Phenyl-6,8-dibromo-4-oxo-(3H)quinazolin-3-yl)-
benzoic acid (4-formyl-benzylidene) hydrazide (3f)
Yield: 83%, mp >2408C; IR (KBr y cmꢀ1): 1641 (–C O cyclic
–
–
tertiary amide), 1705, 1532 (–C O acyclic secondary amide),
–
–
3347, 3189 (–NH–), 1639 (–C N); 1H NMR (d, 400 MHz, DMSO-
4-(2-Methyl-6-bromo-4-oxo-(3H)quinazolin-3-yl)-benzoic
acid (4-formyl-benzylidene) hydrazide (3b)
–
–
d6, Me4Si) 7.42–8.86 (m, 16H, J ¼ 8.5 Hz, Ar–H), 9.88 (s, 1 H,
Yield: 82%, mp >2408C; IR (KBr y cmꢀ1): 1654 (–C O cyclic
–CHO), 9.89 (s, 1H, –NH), 13C NMR (d, 125 MHz, DMSO-d6,
–
–
–
– – –
Me Si), 152.43 (CH N), 163.84 (–C O–N ), 163.93 (–C O–NH),
– – –
4
–
tertiary amide), 1699, 1531 (–C O acyclic secondary amide),
–
–
3348, 3190 (–NH–), 1638 (–C N); 1H NMR (d, 400 MHz, DMSO-
116.75, 122.07, 122.19, 125.56, 126.62, 126.73, 127.26, 128.06,
–
–
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Arch. Pharm. Chem. Life Sci. 2013, 346, 210–220
Cationic Quinazolin-4(3H)-one Conjugated Fullerene Nanoparticles
217
128.67, 130.56, 131.41, 131.41, 131.73, 133.68, 135.47, 136.38,
137.32, 139.98, 142.67, 191.11 (CHO); MS: m/z 630.14, 632.15,
634.15 (Mþ). Analysis for C29H18Br2N2O4 (630.29): Calcd.: % C,
55.26; H, 2.88; N, 8.89. Found: % C, 55.26; H, 2.88; N, 8.89.
1H, J ¼ 10.1, HHC–N of the pyrrolidine ring), 4.52 (s, 1H, CH of
the pyrrolidine ring), 10.12 (s, 1H, NH), 2.19 (s, 3H, CH3 linked to
N of pyrrolidine ring), 2.41 (s, 3H, CH3 of quinazolinone ring);
13
–
C NMR (d, 125 MHz, CDCl ) 163.62 (C O of quinazolinone
3
–
ring), 160.64 (CONH), 154.34, 151.23, 150.56, 146.95, 146.85,
145.94, 143.54, 143.36, 142.36, 142.23, 141.16, 140.86, 135.93,
134.26, 133.46, 130.46, 129.23, 128.77, 128.24, 127.64, 126.63,
125.41, 124.26, 123.54, 122.84, 118.94, 114.46, 112.62, 111.42,
28.43 (quinazolinone ring CH3), 41.48 (CH3 linked to N of the
pyrrolidine ring), 67.28 (NCH2 of pyrrolidine ring), 81.45 (NCH of
the pyrrolidine ring), 77.25, 73.50 (sp3 C– of C60), 71.91, 72.43,
67.48 (sp3 C– of C60); ESI m/z: 1315.40, 1317.40, 1319.20, (Mþ),
(Mþ2), (Mþ4). Analysis for C86H21Br2N5O2, (1315.93).
General procedure for the synthesis of fulleropyrrolidines
(4a–f) [21]
Schiff base 3a (41.0 mg, 0.1 mmol), N-methylglycine (5 mg)
and C60 (72 mg, 0.1 mmol) were refluxed in dry toluene in inert
atmosphere for 6 h. The product was first purified by column
chromatography using toluene/ethyl acetate (9:1) to get pure
product 4a. In a similar way all other products 4b–f were
obtained by the above procedure.
Compound 4d
Yield: 35%, mp >2408C; IR (KBr, y cmꢀ1) 3410, 3300 (N–H), 1687
Compound 4a
Yield: 38%, mp >2408C; IR (KBr, y cmꢀ1) 3410, 3300 (N–H), 3148
–
–
–
(C N), 1645 (–C O cyclic tertiary amide) 1695, 1535 (–C O acy-
–
–
–
clic secondary amide), 1640 (C N), 527 (organo fullerene);
–
–
–
(Ar–H), 1648 (–C O cyclic tertiary amide), 1700, 1536 (–C O
–
–
acyclic secondary amide), 1640 (C N), 527 (organo fullerene);
–
–
H NMR (d, 500 MHz, CDCl ): 7.41–8.23 (m, 18H, Ar–H, CH N),
1
–
H NMR (d, 500 MHz, CDCl ): 7.46–8.20 (m, 13H, Ar–H, CH N),
–
–
3
1
–
–
3
4.22 (dd, 1H, J ¼ 9.4, HHC–N of the pyrrolidine ring), 3.65 (dd, 1H,
4.25 (dd, 1H, J ¼ 9.3, HHC–N of the pyrrolidine ring), 3.61 (dd, 1H,
J ¼ 9.3, HHC–N of the pyrrolidine ring), 4.51 (s, 1H, CH of the
pyrrolidine ring), 9.93 (s, 1H, NH), 2.21 (s, 3H, CH3 linked to N of
J ¼ 9.4 HHC–N of the pyrrolidine ring), 4.52 (s, 1H, CH of the
13
pyrrolidine ring), 9.92 (s, 1H, NH), 2.28 (s, 3H, CH3 linked to N of
–
pyrrolidine ring); C NMR (d, 125 MHz, CDCl ) 163.24, (C O of
–
3
pyrrolidine ring), 2.45 (s, 3H, CH3 of quinazolinone ring);
13
quinazolinone ring), 162.15 (CONH), 154.12, 151.23, 150.52,
146.91, 146.81, 145.92, 143.24, 143.00, 142.32, 142.03, 141.11,
140.81, 135.93, 134.21, 133.43, 130.45, 129.23, 128.75, 128.20,
127.62, 126.65, 125.00, 124.06, 123.58, 122.80, 118.91, 114.43,
112.63, 111.40, 28.32 (quinazolinone ring CH3), 40.23 (CH3 linked
to N of the pyrrolidine ring), 67.63 (NCH2 of pyrrolidine ring),
82.94 (NCH of the pyrrolidine ring), 77.20, 73.50 (sp3 C– of C60),
72.78, 72.42, 68.6 (sp3 C– of C60); ESI m/z: 1220.10 (Mþ). Analysis
for C91H25N5O2, (1220.20).
–
C NMR (d, 125 MHz, CDCl ) 163.66, (C O of quinazolinone
3
–
ring), 160.64 (CONH), 154.32, 151.43, 150.52, 147.91, 146.81,
145.94, 143.24, 143.13, 142.35, 142.23, 141.18, 140.82, 135.94,
134.22, 133.13, 131.40, 129.23, 128.95, 128.03, 127.62, 126.63,
125.08, 124.06, 123.48, 122.85, 118.93, 114.41, 112.63, 110.40,
28.28 (quinazolinone ring CH3), 40.25 (CH3 linked to N of the
pyrrolidine ring), 69.21 (NCH2 of pyrrolidine ring), 83.31 (NCH
of the pyrrolidine ring), 77.22, 73.50 (sp3 C– of C60), 72.73,
72.43, 68.65 (sp3 C– of C60); ESI m/z: 1158.00 (Mþ). Analysis
for C86H23N5O2 (1158.14).
Compound 4e
Yield: 35%, mp >2408C; IR (KBr, y cmꢀ1) 3420, 3235 (N–H), 2958,
Compound 4b
Yield: 37%, mp >2408C; IR (KBr, y cmꢀ1) 3418, 3190 (N–H), 1652
–
–
(Ar H), 1642 (–C O cyclic tertiary amide) 1712, 1533 (–C O
–
–
acyclic secondary amide), 1677 (C N), 526 (organo fullerene);
–
–
H NMR (d, 500 MHz, CDCl ): 7.45–8.41 (m, 17H, Ar–H, CH N),
1
–
–
(–C O cyclic tertiary amide), 1698, 1535 (–C O acyclic secondary
–
–
–
–
–
3
1
amide), 1640 (C N), 526 (organo fullerene); H NMR (d, 500 MHz,
–
4.21 (dd, 1H, J ¼ 9.4, HHC–N of the pyrrolidine ring), 3.68 (dd, 1H,
–
CDCl ): 7.43–8.37 (m, 12H, Ar–H, CH N), 4.25 (dd, 1H, J ¼ 9.6,
–
J ¼ 9.4 HHC–N of the pyrrolidine ring), 4.51 (s, 1H, CH of the
13
3
HHC–N of the pyrrolidine ring), 3.62 (dd, 1H, J ¼ 9.6, HHC–N of
the pyrrolidine ring), 4.58 (s, 1H, CH of the pyrrolidine ring),
10.12 (s, 1H, NH), 2.25 (s, 3H, CH3 linked to N of pyrrolidine ring),
2.52 (s, 3H, CH3 of quinazolinone ring); 13C NMR (d, 125 MHz,
pyrrolidine ring), 9.97 (s, 1H, NH), 2.28 (s, 3H, CH3 linked to N of
–
pyrrolidine ring); C NMR (d, 125 MHz, CDCl ) 163.14, (C O of
–
3
quinazolinone ring), 161.24 (CONH), 153.92, 151.33, 150.42,
146.96, 146.80, 145.93, 143.24, 143.32, 142.31, 142.33, 141.13,
140.61, 135.73, 134.25, 133.03, 130.46, 129.53, 128.75, 128.20,
127.67, 126.65, 125.30, 124.76, 123.55, 122.82, 118.94, 114.47,
112.63, 111.49, 28.34 (quinazolinone ring CH3), 40.23 (CH3 linked
to N of the pyrrolidine ring), 66.34 (NCH2 of pyrrolidine ring),
81.26 (NCH of the pyrrolidine ring), 77.25, 73.50 (sp3 C– of C60),
72.7, 72.47, 68.67 (sp3 C– of C60); ESI m/z: 1299.10, 1301.10 (Mþ),
(Mþ2). Analysis for C91H24BrN5O2, (1299.10).
–
CDCl ) 163.64, (C O of quinazolinone ring), 161.64 (CONH),
3
–
153.82, 151.63, 150.55, 146.98, 146.86, 145.94, 143.29, 143.06,
142.22, 142.03, 141.21, 140.81, 135.93, 134.21, 133.44, 130.65,
129.22, 128.77, 128.20, 127.64, 126.68, 125.45, 124.21, 123.52,
121.81, 118.91, 114.34, 112.63, 111.42, 28.82 (quinazolinone
ring CH3), 40.58 (CH3 linked to N of the pyrrolidine ring),
67.25 (NCH2 of pyrrolidine ring), 82.23 (NCH of the pyrrolidine
ring), 77.44, 73.50 (sp3 C– of C60), 72.76, 72.44, 68.62 (sp3 C–
of C60); ESI m/z: 1237.10, 1239.20 (Mþ), (Mþ2). Analysis
for C86H22BrN5O2 (1237.03).
Compound 4f
Yield: 40%, mp >2408C; IR (KBr, y cmꢀ1) 3429, 3320 (N–H), 3068,
–
–
(Ar C–H), 1645 (–C O cyclic tertiary amide) 1702, 1535 (–C O
–
–
–
acyclic secondary amide), 1675 (C N), 524 (organo fullerene);
–
Compound 4c
Yield: 33%, mp >2408C; IR (KBr, y cmꢀ1) 3450, 3256 (N–H), 1650
1
–
H NMR (d, 500 MHz, CDCl ): 7.44–8.39 (m, 16H, Ar–H, CH N),
–
3
–
–
–
4.23 (dd, 1H, J ¼ 9.6, HHC–N of the pyrrolidine ring), 3.62 (dd, 1H,
(C N); 1645 (–C O cyclic tertiary amide) 1707, 1529 (–C O acy-
–
–
–
–
J ¼ 9.6 HHC–N of the pyrrolidine ring), 4.52 (s, 1H, CH of the
13
clic secondary amide), 1645 (C N), 528 (organo fullerene);
–
1
–
pyrrolidine ring), 9.96 (s, 1H, NH), 2.22 (s, 3H, CH3 linked to N of
–
H NMR (d, 500 MHz, CDCl ): 7.38–8.32 (m, 11H, Ar–H, CH N),
–
3
pyrrolidine ring); C NMR (d, 125 MHz, CDCl ) 163.43 (C O of
4.22 (dd, 1H, J ¼ 10.1, HHC–N of the pyrrolidine ring), 3.65 (dd,
–
3
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

218
M. B. Patel et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 210–220
quinazolinone ring), 161.65 (CONH), 154.32, 151.24, 150.54,
146.93, 146.83, 145.96, 143.44, 143.23, 142.42 142.23, 141.13,
140.81, 135.98, 134.25, 133.65 130.46, 129.27, 128.74, 128.22,
127.62, 126.64, 125.03, 124.26, 123.51, 122.85, 118.93, 114.47,
112.66, 111.44, 28.37 (quinazolinone ring CH3), 40.28 (CH3 linked
to N of the pyrrolidine ring), 69.29 (NCH2 of pyrrolidine ring),
83.23 (NCH of the pyrrolidine ring), 77.26, 73.50 (sp3 C– of C60),
72.7, 72.43, 68.65 (sp3 C– of C60); ESI m/z: 1378.00, 1380.10,
1382.30 (Mþ). Analysis for C91H23Br2N5O2, (1378.00).
lidine ring), 77.24, 73.50 (sp3 C– of C60), 72.77, 72.44, 68.62 (sp3 C–
of C60); ESI m/z: 1252.40, 1254.50 (Mþ), (Mþ2). Analysis
for C87H25BrIN5O2, (1378.97).
Compound 5c
Yield: 98%, mp 2148C; IR (KBr, y cmꢀ1) 3450, 3256 (N–H), 3116,
–
(Ar–H), 2840 (N–CH ), 1705, 1535 (–C O acyclic secondary amide),
–
–
1657 (–C O cyclic tertiary amide), 1665 (C N), 528 (organo full-
3
–
–
–
erene); 1H NMR (d, 500 MHz, CDCl3): 7.38–8.32 (m, 11H, Ar–H,
–
CH N), 4.22 (dd, 1H, J ¼ 10.1, HHC–N of the pyrrolidine ring),
–
3.65 (dd, 1H, J ¼ 10.1, HHC–N of the pyrrolidine ring), 4.52 (s, 1H,
CH of the pyrrolidine ring), 10.12 (s, 1H, NH), 2.19 (s, 3H, CH3
linked to N of pyrrolidine ring), 2.23 (s, 3H, CH3 linked to N of
General procedure for the synthesis of fullerene-N,N-
dimethylpyrrolidines iodide salt (fullerene-quinazolinone
conjugates) (5a–f)
pyrrolidine ring), 2.41 (s, 3H, CH3 of quinazolinone ring);
13
A
solution of fulleropyrrolidine derivative 4a (80 mg,
–
C NMR (d, 125 MHz, CDCl ) 161.22 (C O of quinazolinone
3
–
0.0691 mmol) in 30 mL of chloroform and iodomethane
(9.8 mg, 0.0691 mmol) was refluxed with stirring for 2 days
under argon. Then, the residue was washed with toluene (three
times) and hexane (twice). The solvent was removed under
vacuum to give fullerene-N-dimethylpyrrolidine iodide salt 5a
as brownish solid. In a similar way all other products 5b–f were
obtained by the above procedure.
ring), 162.45 (CONH), 154.22, 151.21, 150.54, 146.96, 146.83,
145.95, 143.24, 143.11, 142.35, 142.23, 141.43, 140.36, 135.65,
134.55, 133.83, 130.65, 129.29, 128.74, 128.22, 127.64, 126.66,
125.08, 124.26, 123.59, 122.80, 118.95, 114.43, 112.73, 111.45,
28.39 (quinazolinone ring CH3), 41.2, 40.5 ((CH3)2 linked to N of
the pyrrolidine ring), 67.51 (NCH2 of pyrrolidine ring), 81.42
(NCH of the pyrrolidine ring), 77.21, 73.50 (sp3 C– of C60),
72.75, 72.45, 68.66 (sp3 C– of C60); ESI m/z: 1330.50, 1332.30,
1334.50 (Mþ), (Mþ2), (M).
Compound 5a
Yield: 98%, mp 2128C; IR (KBr, y cmꢀ1) 3410, 3300 (N–H), 3148,
–
(Ar–H), 2945 (N–CH ), 1701, 1540 (–C O acyclic secondary
–
–
amide), 1643 (–C O cyclic tertiary amide), 1600 (C N), 525
3
Compound 5d
Yield: 94%, mp 2188C; IR (KBr, y cmꢀ1) 3415, 3300 (N–H), 3128,
–
–
–
(organo fullerene); 1H NMR (d, 500 MHz, CDCl3): 7.46–8.20 (m,
–
(Ar–H), 2840, 2740 (N–CH ), 1701, 1537 (–C O acyclic secondary
–
–
amide), 1653 (–C O cyclic tertiary amide), 1654 (C N), 524
3
–
13H, Ar–H, CH N), 4.25 (dd, 1H, J ¼ 9.3, HHC–N of the pyrroli-
–
–
–
–
(organo fullerene); 1H NMR (d, 500 MHz, CDCl3): 7.41–8.23 (m,
dine ring), 3.61 (dd, 1H, J ¼ 9.3, HHC–N of the pyrrolidine ring),
4.51 (s, 1H, CH of the pyrrolidine ring), 9.93 (s, 1H, NH), 2.21 (s,
3H, CH3 linked to N of pyrrolidine ring), 2.30 (s, 3H, CH3 linked to
–
18H, Ar–H, CH N), 4.22 (dd, 1H, J ¼ 9.4, HHC–N of the pyrroli-
–
dine ring), 3.65 (dd, 1H, J ¼ 9.4, HHC–N of the pyrrolidine ring),
4.52 (s, 1H, CH of the pyrrolidine ring), 9.92 (s, 1H, NH), 2.28 (s,
3H, CH3 linked to N of pyrrolidine ring), 2.35 (s, 3H, CH3 linked to
N of pyrrolidine ring), 2.45 (s, 3H, CH3 of quinazolinone ring);
13
–
C NMR (d, 125 MHz, CDCl ) 163.62 (C O of quinazolinone
3
–
N of pyrrolidine ring); 13C NMR (d, 125 MHz, CDCl ) 161.25 (C O
–
ring), 160.66 (CONH), 154.11, 151.20, 150.54, 146.96, 146.83,
145.94, 143.25, 143.00, 142.32, 142.03, 141.11, 140.81, 135.93,
134.21, 133.43, 130.45, 129.23, 128.75, 128.26, 127.67, 126.67,
125.03, 124.06, 123.56, 122.84, 118.93, 114.45, 112.67, 111.42,
28.33 (quinazolinone ring CH3), 42.54, 41.83 ((CH3)2 linked to N of
the pyrrolidine ring), 69.22 (NCH2 of pyrrolidine ring), 83.16
(NCH of the pyrrolidine ring), 77.26, 73.50 (sp3 C– of C60),
72.73, 72.45, 68.66 (sp3 C– of C60); ESI m/z: 1173.20 (Mþ).
Analysis for C87H26IN5O2, (1300.07).
–
3
of quinazolinone ring), 164.65 (CONH), 154.28, 151.28, 150.32,
146.87, 146.89, 145.94, 143.27, 143.12, 142.34, 142.13, 141.17,
140.84, 135.83, 134.25, 133.73, 130.44, 129.28, 128.73, 128.28,
127.52, 126.62, 125.26, 124.45, 123.57, 122.88, 118.71, 114.49,
112.64, 111.46, 38.29, 40.55 ((CH3)2 linked to N of the pyrrolidine
ring), 69.71 (NCH2 of pyrrolidine ring), 83.00 (NCH of the
pyrrolidine ring), 77.23, 73.50 (sp3 C– of C60), 72.71, 72.44,
68.60 (sp3 C– of C60); ESI m/z: 1235.60 (Mþ). Analysis
for C92H28IN5O2, (1362.13).
Compound 5b
Yield: 97%, mp >2408C; IR (KBr, y cmꢀ1) 3410, 3204 (N–H), 3140,
Compound 5e
Yield: 97%, mp 2128C; IR (KBr, y cmꢀ1) 3420, 3235 (N–H), 2958,
–
(Ar–H), 2945 (N–CH ) 1701, 1536 (–C O acyclic secondary amide),
–
–
1654 (–C O cyclic tertiary amide), 1660 (C N), 526 (organo full-
3
–
–
(Ar–H), 2850 (N–CH ), 1726, 1536 (–C O acyclic secondary
3
–
–
–
erene); 1H NMR (d, 500 MHz, CDCl3): 7.46–8.20 (m, 12H, Ar–H,
amide), 1658 (–C O cyclic tertiary amide), 1647 (C N), 527
–
–
–
–
CH N), 4.25 (dd, 1H, J ¼ 9.6, HHC–N of the pyrrolidine ring), 3.62
(organo fullerene); 1H NMR (d, 500 MHz, CDCl3): 7.45–8.41 (m,
–
–
–
(dd, 1H, J ¼ 9.6, HHC–N of the pyrrolidine ring), 4.58 (s, 1H, CH of
the pyrrolidine ring), 10.12 (s, 1H, NH), 2.25 (s, 3H, CH3 linked to
N of pyrrolidine ring), 2.32 (s, 3H, CH3 linked to N of pyrrolidine
ring), 2.52 (s, 3H, CH3 of quinazolinone ring); 13C NMR (d,
17H, Ar–H, CH N), 4.21 (dd, 1H, J ¼ 9.4, HHC–N of the pyrroli-
–
dine ring), 3.68 (dd, 1H, J ¼ 9.4 HHC–N of the pyrrolidine ring),
4.51 (s, 1H, CH of the pyrrolidine ring), 9.97 (s, 1H, NH), 2.28 (s,
3H, CH3 linked to N of pyrrolidine ring), 2.30 (s, 3H, CH3 linked to
125 MHz, CDCl ) 163.64 (C O of quinazolinone ring), 160.59
3
N of pyrrolidine ring); 13C NMR (d, 125 MHz, CDCl ) 161.32 (C O
–
–
–
–
3
(CONH), 154.14, 151.22, 150.54, 146.93, 146.83, 145.94, 143.26,
143.02, 142.33, 142.05, 141.13, 140.82, 135.95, 134.25, 133.45,
130.47, 129.26, 128.77, 129.20, 127.64, 126.67, 125.03, 124.05,
123.56, 122.88, 118.95, 114.42, 112.67, 111.46, 28.34 (quinazoli-
none ring CH3), 39.54, 41.48 ((CH3)2 linked to N of the pyrrolidine
ring), 67.43 (NCH2 of pyrrolidine ring), 82.45 (NCH of the pyrro-
of quinazolinone ring), 164.64 (CONH), 154.12, 151.23, 150.52,
146.91, 146.81, 145.92, 143.24, 143.00, 142.32, 142.03, 141.14,
140.88, 135.83, 134.27, 133.46, 130.55, 129.25, 128.85, 128.24,
127.63, 126.25, 125.30, 124.08, 123.52, 122.81, 118.91, 114.42,
112.66, 111.42, 39.25, 42.52 ((CH3)2 linked to N of the pyrrolidine
ring), 66.22 (NCH2 of pyrrolidine ring), 81.40 (NCH of the pyrro-
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Arch. Pharm. Chem. Life Sci. 2013, 346, 210–220
Cationic Quinazolin-4(3H)-one Conjugated Fullerene Nanoparticles
219
lidine ring), 77.22, 73.50 (sp3 C– of C60), 72.70, 72.45, 68.66 (sp3 C–
of C60); ESI m/z: 1314.40, 1316.20 (Mþ) (Mþ2). Analysis
for C92H27BrIN5O2, (1441.04).
were taken. The active synthesized drugs found in this primary
screening were further tested in a second set of dilution against
all microorganisms. The drugs found active in primary screening
were similarly diluted to obtain 100, 50, 25, 12.5, 6.250, 3.125,
and 1.5625 mg/mL concentrations. The highest dilution showing
at least 99% inhibition is taken as MIC.
Compound 5f
Yield: 98%, mp 2108C; IR (KBr, y cmꢀ1) 3429, 3320 (N–H), 3068, (Ar
The control tube, containing no antibiotic, was subcultured
(before inoculation) by spreading a loopful evenly over a quarter
of plate of medium suitable for the growth of the test organism
and put for incubation overnight at 378C. The MIC of the control
organism was read to check the accuracy of the drug concen-
trations. The lowest concentration inhibiting growth of the
organism was recorded as the MIC. All the tubes not showing
visible growth (in the same manner as control tube described
above) was subcultured and incubated overnight at 378C. The
amount of growth from the control tube before incubation
(which represents the original inoculum) was compared.
Subcultures might show similar number of colonies indicating
bacteriostatic, a reduced number of colonies indicating a partial
or slow bactericidal activity and no growth if the whole inocu-
lum has been killed. The test included a second set of the same
dilutions inoculated with an organism of known sensitivity.
The antifungal activity of the synthesized compounds 3a–f,
4a–f, and 5a–f was screened against three fungal species
(C. albicans MTCC 183, A. clavatus MTCC 1323, and A. niger
MTCC 282). Nystatine were used as standard drug. The procedure
followed was similar to as described for antibacterial studies.
The compounds were screened for the in vitro antimycobacte-
rial activity against Mycobacterium tuberculosis (H37RV), by using L.
J. (Lowenstein and Jensen) MIC method [28]. Stock solutions of
primary 1000, 500, 250, and secondary 200, 100, 62.5, 50, 25,
12.5, 6.25, 3.25, 1.562 mg/mL dilutions of each test compound in
DMSO (dimethyl sulfoxide) were added in the liquid L. J. Medium
and then media were sterilized by inspissation method. A culture
of M. tuberculosis H37Rv growing on L. J. medium was harvested in
0.85% saline in bijou bottles. These tubes were then incubated at
378C for 24 h followed by streaking of M. tuberculosis H37Rv (5 ꢂ 104
bacilli per tube). These tubes were then incubated at 37 ꢁ 18C.
Growth of bacilli was seen after 12 days, 22 days, and finally
28 days of incubation. Tubes having the compounds were com-
pared with control tubes where medium alone was incubated with
M. tuberculosis H37Rv. The concentration at which no development
of colonies occurred or <20 colonies was taken as MIC concen-
tration of test compound. The standard strain M. tuberculosis
H37Rv was tested with known drugs isoniazid and rifampicin.
–
C–H), 2921, (N–CH ), 1715, 1533 (–C O acyclic secondary amide),
–
–
1652 (–C O cyclic tertiary amide), 1667 (C N), 524 (organo full-
3
–
–
–
erene); 1H NMR (d, 500 MHz, CDCl3): 7.44–8.39 (m, 16H, Ar–H,
–
CH N), 4.23 (dd, 1H, J ¼ 9.6, HHC–N of the pyrrolidine ring), 3.62
–
(dd, 1H, J ¼ 9.6 HHC–N of the pyrrolidine ring), 4.52 (s, 1H, CH of
the pyrrolidine ring), 9.96 (s, 1H, NH), 2.22 (s, 3H, CH3 linked to N
of pyrrolidine ring), 2.28 (s, 3H, CH3 linked to N of pyrrolidine
ring); 13C NMR (d, 125 MHz, CDCl ) 162.15 (C O of quinazolinone
–
–
3
ring), 164.26 (CONH), 154.42, 151.22, 150.51, 146.98, 146.81,
145.92, 143.24, 143.11, 142.33, 142.33, 141.41, 140.85, 135.73,
134.27, 133.48, 130.35, 129.63, 128.77, 128.22, 127.52, 126.67,
125.29, 124.06, 123.63, 122.88, 118.99, 114.94, 112.63, 111.60,
28.42 (quinazolinone ring CH3), 40.4, 40.2 ((CH3)2 linked to N of
the pyrrolidine ring), 69.2 (NCH2 of pyrrolidine ring), 83.35 (NCH of
the pyrrolidine ring), 77.25, 73.50 (sp3 C– of C60), 72.71, 72.40, 68.63
(sp3 C– of C60); ESI m/z: 1392.00, 1394.40, 1396.30 (Mþ), (Mþ2),
(Mþ4) Analysis for C92H26Br2IN5O2, (1519.94).
Preparation of 4a–f and 5a–f water suspensions
The aqueous suspensions of compounds 4a–f and 5a–f were
prepared by a method reported earlier [22]. Fifty milligrams of
compound 4a in 50 mL of Milli-Q water was stirred at 408C for 2
weeks. The suspension thus obtained was then filtered through a
Whatman filter; then through a 0.45 mm Osmonics nylon mem-
brane and a 0.22 mm nylon membrane to remove aggregates
larger than 200 nm. The water suspension obtained was then
concentrated on a rotavapor to get different concentrations
ranging from 1.562 to 2000 mg/mL.
The size of suspended particles in water was determined using
TEM. TEM samples were prepared by depositing a drop of dis-
persed compound 5f particles in water on the carbon-coated
Formvar films on copper grids with electron microscope oper-
ated at 200 keV.
In vitro evaluation of antimicrobial activity
The minimum inhibitory concentrations (MIC) of the synthes-
ized quinazolinone and fullerene derivatives (3a–f) and (5a–f)
were determined by broth microdilution method as described
by Rattan [27]. Antibacterial activity was screened against two
Gram-positive bacteria (Staphylococcus aureus MTCC 96 and
Streptococcus pyogenes MTCC 442), and three Gram-negative bac-
teria (Pseudomonas aeruginosa MTCC 741, Klebsiella pneumoniae
MTCC 109 and Escherichia coli MTCC 739). Ciprofloxacin was used
as a standard drug.
M.B.P. would like to acknowledge the University Grants Commission, New
Delhi, India for a Junior Research Fellowship.
The authors have declared no conflict of interest.
All MTCC (Microbial Type Culture Collection) cultures were
provided by Institute of Microbial Technology, Chandigarh.
Mueller–Hinton broth was used as nutrient medium to grow.
Inoculum size for test strain was adjusted to 108 CFU (colony
forming unit) per milliliter by comparing the turbidity. Serial
dilutions were prepared for primary and secondary screening
and DMSO was used as diluent to get desired concentration of
drugs to test upon standard bacterial strains. A stock solution of
2000 mg/mL of each of the synthesized compounds was prepared
and diluted as and when required. In primary screening 500,
250, and 125 mg/mL concentrations of the synthesized drugs
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www.archpharm.com