Synthesis and antimicrobial activity of quinazolinone conjugated peptides

Article abstract of DOI:10.1155/2010/979675

Antimicrobial compounds were synthesized by coupling 4-(4- oxo-3,4-dihydroquinazolin-2-yl) butanoic acid with VP, GVP, VGVP and GVGVP peptides. Antimicrobial activities of the synthesized compounds were performed against various bacterial strains by disc

Full text of DOI:10.1155/2010/979675

ISSN: 0973-4945; CODEN ECJHAO
E-Journal of Chemistry
2010, 7(2), 449-456
http://www.e-journals.net
Synthesis and Antimicrobial Activity of
Quinazolinone Conjugated Peptides
G. P. SURESHA, K. C. PRAKASHA,
WETHROE KAPFO and D. CHANNE GOWDA*
Department of Chemistry,
Manasagangotri, University of Mysore, Mysore-570006, India.
dchannegowda@yahoo.co.in
Received 10 September 2009; Accepted 5 November 2009
Abstract: Antimicrobial compounds were synthesized by coupling 4-(4-
oxo-3,4-dihydroquinazolin-2-yl) butanoic acid with VP, GVP, VGVP and
GVGVP peptides. Antimicrobial activities of the synthesized compounds
were performed against various bacterial strains by disc diffusion method.
The structure activity
relationship was evaluated with respect to
hydrophobicity, polarity, chain length of peptides and alkyl chain length of
quinazolinone. Correlations of analogs with respect to their antimicrobial
activity in comparison with conventional drugs and probable mechanism for
the activity were discussed.
Keywords: Antimicrobial drugs, Quinazolinone conjugated peptides, Hydrophobicity, Polarity, Chain length.
Introduction
In recent past pharmacological applications witnessed an unprecedented rate of the
spread of microbes that are resistant to conventional antimicrobials worldwide. This
poses a threat and the rapid emergence of resistant stains involve both gram-positive
and gram-negative1. Efforts are currently underway to find new antimicrobials which act
in a different mechanism of action against microbes and can potentially evade resistance
provided by microbes. In order to achieve this in an efficient manner, it is important to
understand the mechanism of action of these agents and the reason for their selectivity
against microbes2.

450
D. CHANNE GOWDA et al.
Antimicrobial peptides are found in most living organisms and have been most
interesting compounds due to their structure and unique mode of action different from the
most conventional antimicrobials3. These peptides exhibit activity against a broad spectrum
of microbes but showed fairly low activity. Also several classes of antimicrobial peptides
have been studied and their mechanisms of action against several microbes not yet clear and
its elucidation would form a sound basis for the further development of new pharmaceutical
compounds4.
Another class of compounds of interest in this scenario is quinazolinone alkaloids and
its analogues5. These compounds extracted from natural sources were used as antimicrobials
from long time. But recently mimicking of theses compounds with synthetic routes were
found valuable route to find a new candidate as antimicrobials6.
In our earlier work, we have developed novel quinazolinone conjugated peptides by
coupling peptides with 4-(4-oxo-3, 4-dihydroquinazolin-2-yl)propanoic acid (Precursor 1)7.
The resulted compounds showed enhanced activity against both gram positive and gram
negative bacterial strains compared to conventional antimicrobial viz., Streptomycin and
tetracyclin. Further the activity depends on structural entities of the compounds; we were
interested to extend the work by introducing the analogue of precursor 1 with mentioned
peptides. The analogues were different from earlier compounds by elongating the alkyl
chain as given in structures 2a-9a. These new chemical entities were synthesized by
coupling mentioned peptides with 4-(4-oxo-3,4-dihydroquinazolin-2-yl) butanoic acid (1a)
and evaluated for their antimicrobial activities against various bacterial strains.
Experimental
All chemicals and reagents were obtained from Aldrich (USA), Spectrochem Pvt. Ltd
(India) and Rankem Pvt. Ltd. (India) and were used without further purification. The amino
1
acids used were L- series unless mentioned. The H NMR, mass and HPLC spectra were
recorded on 300 MHz Bruker FT-NMR Spectrometer, LCMSD-Trap-XCT instrument and
Agilent-1100 HPLC respectively. The bacterial strains used were obtained from department
of Studies in Biotechnology, University of Mysore, India, namely, Bacillus substilis,
Escherichia coli, Pseudomonas fluorescens, Xanthomonas campestris pvs and Xanthomonas
oryzae.
Synthesis
The 4-(4-oxo-3,4-dihydroquinazolin-2-yl)butanoic acid (1a) was synthesized as previously
reported using anthranilide and pentanedioic acid anhydride7,8. The peptides were
synthesized by stepwise solution phase method using Boc chemistry. The Boc group was
used for temporary Nα-protection and its removal was achieved with TFA. The resulting
peptides were coupled with 1a as shown in the Scheme 1. The carboxyl group was protected
by benzyl ester and its removal was effected by hydrogenolysis using HCO2NH4 as
hydrogen donor and 10% Pd-C on carbon as catalyst, described by our earlier method7. The
series of compounds 2a, 4a, 6a, 8a possess precursor 1a coupled with benzyl esters of VP,
GVP, VGVP and GVGVP peptides and compounds 3a, 5a, 7a, 9a were their hydrolyzed
products respectively.

Synthesis and Antimicrobial Activity
451
General procedure for the synthesis of 4-(4-oxo-3,4-dihydroquinazolin-2-
yl)butanoic acid conjugated peptides (2a - 9a)
i
ii
(i) EDCl/HOBt and DIEA,(ii) HCO2NH4/10% Pd-C
Scheme 1. Synthesis of heterocyclic conjugated peptides.
The coupling of 4-(4-oxo-3,4-dihydroquinazolin-2-yl)butanoic acid with benzyl
esters of peptides
To the stirred solution of 4-(4-oxo-3,4-dihydroquinazolin-2-yl)butanoic acid (0.2 g, 0.86 mmol)
0
in DMF (5.0 mL) cooled to C with crushed ice, added TFA.VP-OBzl (0.36 g, 0.86 mmol),
DIEA (0.61 mL, 3.44 mmol ), HOBt (0.17 g, 1.29 mmol ), EDCI (0.33 g, 1.72 mmol ) and THF
(5 mL). The reaction mixture was stirred over night while slowly warming to room temperature.
The reaction was quenched with H2O (2 mL) and the solvent was condensed. The residue was
dissolved in EtOAc (30 mL) and the organic layer was washed with 1 N HCl (1 x 20 mL),
saturated NaHCO3 solution (1 x 20 mL) and brine (1x 20 mL). Then the organic layer was dried
over MgSO4 and concentrated at high vacuum. Chromatography (10% EtOAc-Hexane) over
silica gel (100-200) provided compound 2a as white hygroscopic foam (Yield: 87%).
Hydrogenolysis of benzyl ester of heterocyclic conjugated peptides
To a stirred solution of compound 2a (0.15 g) and 10% Pd/C (10% w/w) in methanol (2.5 mL),
ammonium formate (2 equiv) was added. The resulting reaction mixture was stirred at room
temperature. After the completion of hydrogenolysis (monitored by TLC), the mixture
was filtered through celite and washed with methanol. The combined washings and filtrate

452
D. CHANNE GOWDA et al.
were evaporated. The residue was taken up in chloroform and washed with 50% saturated
NaC1 solution to remove ammonium formate. The solvent was removed under reduced
pressure and triturated with ether to obtain the hydrolyzed compound 3a (Yield: 90%).
Analytical data of synthesized compounds
The spectral data (H1 NMR and mass spectra) confirmed the structures of the synthesized
compounds. The analytical data of the compounds are given below.
1
Compound 2a: Yield: 87%., H NMR (DMSO-D6): δ 12.16 ( s, 1H), 8.11-8.07 (t, 2H,
J=9.0Hz), 7.77-7.75 (d, 1H, J=8.2Hz), 7.60-7.58 (d, 1H, J=8.1Hz), 7.48-7.36 (t, 1H,
J=7.8Hz), 7.34 (s, 5H ), 5.11 (s, 2H), 4.40-4.38 (m, 1H), 4.37-4.36 (m, 1H), 3.74-3.72 (m,
1H), 3.71 (m, 1H), 2.58-2.57 (m, 2H), 2.22-2.18 (m, 3H), 1.95-1.93 (m, 5H), 1.91-1.90 (m,
1H), 0.87-0.84 (d, 6H, J=11.8). MS m/z: 519.2 (M+).
Compound 3a: Yield: 90%., 1H NMR (DMSO-D6): δ 12.18 ( s, 1H), 8.11-8.07 (t, 2H, J=9.0Hz),
7.77-7.75 (d, 1H, J=8.2Hz), 7.60-7.58 (d, 1H, J=8.1Hz), 7.48-7.36 (t, 1H, J=7.8Hz), 4.40-4.38 (m,
1H), 4.37-4.35 (m, 1H), 3.75-3.72 (m, 1H), 3.71-3.70 (m, 1H), 2.58-2.57 (m, 2H), 2.20-2.18 (m, 3H),
1.95-1.94 (m, 5H), 1.91-1.90 (m, 1H), 0.86-0.84 (d, 6H, J=11.8).MS m/z: 429.3 (M+).
1
Compound 4a: Yield: 86%., H NMR (DMSO-D6): δ 12.16 ( s, 1H), 8.08-8.06 (d, 3H,
J=8.0Hz), 7.77-7.75 (d, 1H, J=8.2Hz), 7.62-7.60 (d, 1H, J=8 Hz), 7.47-7.46 (t, 1H,
J=7.4Hz), 7.34 (s, 5H ), 5.11 (s, 2H), 4.38-4.32 (m, 2H), 3.73(m, 1H), 3.72 (m, 2H), 3.71 (m,
1H), 2.61-2.50 (m, 2H), 2.21-2.19 (m, 3H), 1.95-1.93 (m, 5H), 1.91-1.89 (m, 1H), 0.87-0.82
(m, 6H). MS m/z: 576.2 (M+).
1
Compound 5a: Yield: 86%., H NMR(DMSO-D6): δ 12.17 ( s, 1H), 8.08-8.06 (d, 3H,
J=8.0Hz), 7.77-7.75 (d, 1H, J=8.2Hz), 7.62-7.60 (d, 1H, J=8 Hz), 7.47-7.46 (t, 1H, J=7.4Hz),
4.38-4.34 (m, 2H), 3.74(m, 1H), 3.72 (m, 2H), 3.70 (m, 1H), 2.62-2.50 (m, 2H), 2.20-2.19 (m,
3H), 1.96-1.92 (m, 5H), 1.91-1.89 (m, 1H), 0.86-0.82 (m, 6H). MS m/z: 484.4 (M-).
Compound 6a: Yield: 85%., 1H NMR (DMSO-D6): δ 12.17 (s, 1H), 8.23 (s, 1H), 8.09-
8.07 (d, 1H, J= 8.4Hz ), 7.93-7.91 (d, 2H, J= 8.4Hz), 7.90-7.77 (t, 1H, J=7.6Hz), 7.75-
7.73(d, 1H, J= 8.4Hz), 7.48-7.46 (t, 1H, J=7.6Hz), 7.35 (s, 5H ), 5.11 (s, 2H), 4.36 (m, 2H),
4.33(m, 1H), 3.75-3.73 (m, 3H), 3.72 (m, 1H), 2.85-2.84 (m, 2H), 2.25-2.24 (m, 3H), 1.97-
1.95 (m, 6H), 1.92-1.90 (m, 1H), 0.86-0.81 (m, 12H). MS m/z: 674.8 (M+).
1
Compound 7a: Yield: 86%., H NMR (DMSO-D6): δ 12.18 (s, 1H), 8.24 (s, 1H),
8.09-8.07 (d, 1H, J= 8.4Hz ), 7.93-7.91 (d, 2H, J= 8.4Hz), 7.90-7.77 (t, 1H, J=7.6Hz),
7.75-7.73(d, 1H, J= 8.4Hz), 7.48-7.46 (t, 1H, J=7.6Hz), 4.38 (m, 2H), 4.33(m, 1H), 3.74-
3.73 (m, 3H), 3.72 (m, 1H), 2.85-2.83 (m, 2H), 2.26-2.24 (m, 3H), 1.97-1.94 (m, 6H), 1.93-
1.90 (m, 1H), 0.86-0.80 (m, 12H). MS m/z: 585.2 (M+).
Compound 8a: Yield: 83%., 1H NMR (DMSO-D6): δ 12.19 (s, 1H), 8.24-8.22 (m, 1H), 8.11-
8.09 (m, 2H ), 7.97-7.95 (d, 1H, J=8.4Hz), 7.89-7.86 (m, 2H), 7.86-7.84 (d, 1H, J= 8.1Hz), 7.70-7.53
(t,1H, J= 7.6Hz), 7.34 (s, 5H), 5.10 (s, 2H), 4.35-4.32 (m, 2H), 4.14 (m, 1H), 3.73-3.55 (m, 6H),
2.71-2.65 (m, 2H), 2.24-2.21 (m, 3H), 1.98-1.87 (m, 7H), 0.84-0.80 (m, 12H). MS m/z: 731.8 (M+).
1
Compound 9a: Yield: 88%. H NMR (DMSO-D6): δ δ 12.20 (s, 1H), 8.25-8.22 (m, 1H),
8.11-8.08 (m, 2H ), 7.97-7.95 (d, 1H, J=8.4Hz), 7.90-7.86 (m, 2H), 7.86-7.84 (d, 1H, J= 8.1Hz),
7.70-7.53 (t,1H, J= 7.6Hz), 4.35-4.33 (m, 2H), 4.14-4.20 (m, 1H), 3.73-3.59 (m, 6H), 2.70-2.65
(m, 2H), 2.24-2.22 (m, 3H), 1.98-1.87 (m, 7H), 0.85-0.80 (m, 12H). MS m/z: 642.5 (M+)

Synthesis and Antimicrobial Activity
453
Antibacterial assay
In vitro antibacterial assays were performed against Bacillus substilis, Escherichia coli,
Pseudomonas fluorescens, Xanthomonas campestris pvs and Xanthomonas oryzae by using
the disc diffusion method9. The bacterial strains were maintained on LB agar medium at 28
οC. The bacteria were grown in LB broth, centrifuged at 10,000 rpm for 5 min; pellet was
dissolved in double distilled water and used to inoculate the plates. The paper discs
containing streptomycin and tetracyclin were used as positive control and DMSO as
negative control. Each disc contained 10 µg of standard drugs and 10 µg synthesized
compounds. Plates were first kept at 4 °C for 2 h to allow the diffusion of chemicals and
then incubated at 28 °C. All the compounds were tested in triplicate and inhibition zones
were measured in mm after 24 h of incubation.
Table 1. Heterocyclic conjugated peptides with their analytical data.
Compound
Theoretical Actual MS
HPLC
RT
R a
Formula
No.
2a
3a
4a
5a
6a
7a
8a
9a
Mol. Wt.
518.25
428.21
575.27
485.23
674.34
584.30
731.36
641.32
Values (M+)
519.2
429.3
VP-OBzl
VP-OH
GVP-OBzl
GVP-OH
C29H34N4O5 9.070
C22H28N4O5 5.397
C31H37N5O6 9.086
C24H31N5O6 5.659
C36H46N6O7 9.125
C29H40N6O7 7.038
C38H49N7O8 9.300
C31H43N7O8 7.072
576.2
484.4(M-)
674.8
585.2
VGVP-OBzl
VGVP-OH
GVGVP-OBzl
GVGVP-OH
731.8
642.5
a G= Glycine, P= Proline, V= Valine
Results and Discussion
The biological activities of the synthesized quinazolinone conjugated peptides were tested
against bacterial strains, Bacillus-substilis (gram positive) and Escherichia-coli,
Pseudomonas-fluorescens, Xanthomonas-campestris pvs and Xanthomonas-oryzae (gram
negative) and are given in Table 2. Each fragment of these compounds i.e 4-(4-oxo-3,4-
dihydroquinazolin-2-yl)butanoic acid, VP, GVP, VGVP and GVGVP peptides were tested
for antibacterial activity and found inactive10. Even though the peptides chosen were less
hydrophobic and inactive towards mentioned bacterial strains, their conjugation with
precursor 1a showed increased antimicrobial activity (Table 2).
From the Table 2, it is observed that the antimicrobial activity increases with increase in
length of peptide chain. Both benzylated and debenzylated product of quinazolinone
conjugated peptides (2a-9a) showed parallel trends towards the antimicrobial activity with
respect to peptide chain length. However, the debenzylated product of these molecules (3a,
5a, 7a, 9a) showed enhanced activity than its respective counterparts (2a, 4a, 6a, 8a). It was
observed that the quinazolinone conjugated with VGVP and GVGVP (6a, 7a, 8a, 9a)
showed increased activities in comparison with the conventional drugs (Table 2).

454
D. CHANNE GOWDA et al.
In order to correlate the appropriate structure activity relationship, the hydrophobicity of
synthesized molecules was evaluated using RP-HPLC, C18 column surface which has affinity for
hydrophobicity11, so more retained compound have higher hydrophobic nature. A plot of retention
time of compounds against its antimicrobial activity (Figure 1) showed that both benzylated
and its hydrolyzed compounds (2a - 9a) having more elongated peptide chain exhibits more
hydrophobic nature and showed higher antimicrobial activities compared to the compounds
having shorter peptide chain. But hydrolyzed compounds (3a, 5a, 7a, 9a) exhibit less hydrophobic
character compared to their counterparts (2a, 4a, 6a, 8a), still they have shown more antimicrobial
activity. This may be due to increase in polarity of these compounds and indicates that increase in
the polarity also has an impact on the activity12.
Table 2. Inhibitory Zone (diameter) mm of compounds against tested bacterial strains by
disc diffusion method.
Inhibitory zone (diameter) mm a
Bacillus- Escherichia- Pseudomonas- Xanthomonas- Xanthomonas-
Compounds
substilis
0
coli
0
fluorescens campestris pvs.
oryzae
0
1a
2a
0
5
0
4
6
7
6
3a
4a
8
10
8
9
7
8
10
11
18
20
21
28
17
-
9
6
5
5a
6a
7a
8a
10
19
23
20
24
21
-
10
18
22
24
27
23
-
13
20
23
23
28
-
11
17
21
26
30
-
9a
Streptomycin
Tetracycline
19
18
Streptomycin (10 µg/disc), Tetracycline (10 µg/disc) were used as positive control & compounds (10 µg/disc)
a Values are means of three determinations, the ranges of which are less than 5% of the mean in all cases.
Bacillus subtilis (+)
30
9
25
8
20
15
10
5
7
6
5
4
3
2
0
5
6
7
8
9
10
RT, min
Figure 1. Correlation between RT and antibacterial activity.

Synthesis and Antimicrobial Activity
455
Indeed, 4-(4-oxo-3,4-dihydroquinazolin-2-yl)butanoic acid conjugated peptides (2a-9a)
having an additional methylene group in its alkyl chain showed less hydrophobic nature and
also showed the slightly lesser antimicrobial activities compare to that of 4-(4-oxo-3,4-
dihydroquinazolin-2-yl)propanoic acid conjugated peptide (2-9) which has been discussed in
our previous study7. To evaluate this, a plot of antimicrobial activities of 4-(4-oxo-3,4-
dihydroquinazolin-2-yl)butanoic acid and 4-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoic
acid conjugated peptides against their retention times is presented in Table 2. From the
Figure 2, it is evident that the increase in the length of carbon chain in quinazolinone
precursors has little / no impact on the activity of the molecules. Both the set of molecules
(2 - 9 and 2a - 9a) showed similar and steady increase in activity as peptide chain length and
hydrophobicity (HPLC-RT) increases.
Compound
Figure 2. RT & microbial activity of B. subtilis.
Further it is evident that activity towards gram -ve bacteria is more compared to the
gram +ve. This may be due to the distance between the charged groups and the heterocyclic
ring. Another widely postulated mechanism is that of the "self-promoted uptake" of the
peptides across the outer membranes of gram negative bacteria which consists
lipopolysaccaride surface13. This suggest that the peptides interact with the negatively
charged outer membrane and subsequent channel formation in the cytoplasmic membrane
via either "barrel-stave" or a" carpet" mechanism resulting in cell death14.
Conclusion
Following our ongoing investigations on development of quinazolinone conjugated peptides
as novel antimicrobial agents, in the present study, by enhancing the alkyl chain length in

456
D. CHANNE GOWDA et al.
the quinazolinone showed similar activities but fairly less compare to quinazolinone of
shorter alkyl chain length. Thus, the increase in the length of carbon chain in quinazolinone
precursors may have little / no impact on the activity of the molecules. But the length of
peptide chain, hydrophobicity and variation in polarity of these molecules play important
role in their antimicrobial activities. It was noticed that quinazolinone conjugated VGVP and
GVGVP peptides showed increased activity by nearly two fold compared to conventional
antimicrobials.
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