Synthesis of quinazolinone conjugated shorter analogues of bactenecin7 as potent antimicrobials

Article abstract of DOI:10.2174/092986613804725235

A series of shorter peptide analogues of Bactenecin7 (RP, PRP, GPRP and RPRP) were synthesized and conjugated to 3-(4-oxo-3,4-dihydroquinazolin-2-yl) propanoic acid to study the effect of conjugation. All the peptides and their conjugates were characteriz

Full text of DOI:10.2174/092986613804725235

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146
Protein & Peptide Letters, 2013, 20, 146-155
Synthesis of Quinazolinone Conjugated Shorter Analogues of Bactenecin7
as Potent Antimicrobials
R. Suhas, S. Chandrashekar, S.M. Anil and D. Channe Gowda*
Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, India
Abstract: A series of shorter peptide analogues of Bactenecin7 (RP, PRP, GPRP and RPRP) were synthesized and conju-
gated to 3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoic acid to study the effect of conjugation. All the peptides and their
conjugates were characterized by analytical and spectroscopic techniques. The synthesized compounds viz., peptides, het-
erocyclic conjugates and the hydrogenolyzed products were evaluated for antimicrobial activity against a panel of patho-
gens. The results revealed that all the conjugates have shown enhanced activity than their counterparts. Further, hydro-
genolyzed tetrapeptide conjugates (10 and 13) have exerted highly potent activity nearly 3-4 times than the standard drugs
used.
Keywords: Quinazolinone. bactenecin7. conjugation. potent antimicrobials.
INTRODUCTION
that Gly and Arg containing tetrapeptide fragments showed
more potent activity compared to rest of the analogues syn-
thesized [7]. Therefore, in the current investigation the two
potent analogues GPRP and RPRP were chosen for the con-
jugation.
Despite a number of antibiotics available for the treat-
ment of microbial infections, emergence of multi-drug resis-
tant organisms has posed a great challenge to the scientists.
There appears to be a need for the development of new drugs
with improved efficacy and better toxicity [1]. Researchers
however are continuing their efforts to develop new classes
of antimicrobials. One of these research lines aims to exploit
the therapeutic potential of a variety of natural cationic an-
timicrobial peptides and their synthetic analogues, for the
development of antimicrobials with a mechanism of action
different from those of conventional antibiotics [2].
Bactenecin7 (Bac7), a cathelicidin-derived antimicrobial
peptide of bovine neutrophil granules [3], exhibits antibacte-
rial activity primarily against gram negative bacteria and has
been found to represent a new family of Pro/Arg-rich antibi-
otics [4]. Bac7 comprises 59 amino acid residues and in-
cludes three tandem repeats of a tetradecamer characterized
by several Pro-Arg-Pro triplets spaced by single hydrophobic
amino acid residues. Proline is known as a strong ꢀ-helix
breaker, which can induce kinks in ꢀ-helix of proteins, sug-
gesting that the repeating triplet may play an important role
in antibacterial activity and/or in maintaining the peptide
conformation [5]. In this connection, two peptide fragments
were synthesized corresponding to residues 1-17 (an Arg-
clustered region) and 46-59 (one of three tandem repeats)
sequences of Bac7 [6]. While the fragment 1-17 showed
weak antimicrobial activity against several bacteria, the frag-
ment 46-59 was almost inactive. Therefore, a detailed study
on the property and function of the repeating regions that
extends from the near N-terminal to the C-terminal portions
of the peptides were of interest. Hence, in our laboratory, a
series of tetrapeptide fragments (X-Pro-Arg-Pro, where X =
Gly, Arg, Leu, Ile and Phe) were synthesized and found
On the other hand, the pharmacodynamic versatility of
quinazolinone moiety has been documented not only in
many of its synthetic derivatives but also in several naturally
occurring alkaloids isolated from animals [8], families of
plant kingdoms [9] and from microorganisms [10]. The
quinazolinone derivatives are found to have wide range of
biological properties including anti-cancer [11], antimicro-
bial [12], antihyperlipidemic [13] and anti-inflammatory [14]
activities. In the light of the above and in continuation of our
work [12, 15-19] the present work aims at the synthesis and
biological studies of the quinazolinone conjugated shorter
active analogues of Bac7.
MATERIALS AND METHODS
Reagents
All the amino acids used except glycine were of L-
configuration unless otherwise mentioned. All Boc-amino
acids, EDCI and HOBt were purchased from Advanced
Chem. Tech. (Louisville, Kentucky, USA). NMM was pur-
chased from Sigma Chemical Co. (St. Louis, MO). All sol-
vents and reagents used for the synthesis were of analytical
grade. The progress of the reaction was monitored by TLC
using silica gel coated on glass plates with the solvent sys-
tem comprising chloroform/methanol/acetic acid in the ratio
98:2:3. IR spectra were recorded on Shimadzu FTIR-8300
1
Spectrometer (USA). H NMR spectra were obtained on
VARIAN 400 MHz instrument (Palo Alto, USA) using
DMSO and the chemical shifts are reported as parts per mil-
lion (ꢁ ppm) using TMS as an internal standard. Mass spec-
tra were obtained on LCMSD-Trap-XCT instrument. Ele-
mental analysis was performed by using VARIO EL III
CHNS Elementar (Germany). The chemical/reagents used
*Address correspondence to this author at the DOS in Chemistry, University
of Mysore, Manasagangotri, Mysore – 570 006, India; Tel: 0821 – 2419664
(O); E-mail: dchannegowda@yahoo.co.in
1875-5305/13 $58.00+.00
© 2013 Bentham Science Publishers

Quinazolinone conjugated shorter analogues of Bac7…
Protein & Peptide Letters, 2013, Vol. 20, No. 2 147
for the microbial studies were of bacteriological grade and
purchased from Himedia (Mumbai, India). All the pathogens
used for the assay were obtained from a local hospital.
DMF (120 mL) was added slowly. After 20 minutes, pH of
the solution was adjusted to 8 by the addition of NMM and
the reaction mixture was stirred overnight at room tempera-
ture. Acetonitrile was removed under reduced pressure and
the residual DMF solution was poured into about 2000 mL
ice-cold 90% saturated KHCO3 solution and stirred for 30
minutes. The precipitated peptide was filtered, washed with
water, 1N HCl, water and dried. The crude peptide was re-
crystallized from ether and petroleum ether to obtain 2.
Yield: 88.0%, M.P. 44-46 °C (Lit. 45-46 °C [7]).
Chemistry
The 3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoic acid
1 (QZN) was synthesized as previously reported using an-
thranilamide and succinic anhydride [20, 21]. The peptides 2,
5, 8 and 11 were synthesized by stepwise solution phase
method using Boc chemistry as shown in scheme 1. The Boc
group used for temporary Nꢀ protection was removed with
4N HCl in dioxane. Peptides were coupled to quinazolinone
moiety 1 using EDCI/HOBt as coupling agent and NMM as
base to get heterocyclic conjugated peptides 3, 6, 9 and 12.
The C-terminus carboxyl group protected by the benzyl ester
and the guanidine function of arginine was masked by nitro
group, and their removal was effected by hydrogenolysis
using HCOONH4 as hydrogen donor and 10% Pd on carbon
as catalyst [22] to get the hydrolysed products 4, 7, 10 and 13
respectively. The procedure followed for the synthesis is as
shown in the Scheme 2.
Boc-Pro-Arg(NO2)-Pro-OBzl (5)
Compound 2 (20.25 g) was deblocked with 4N
HCl/dioxane (200 mL) for 1.5 hr. Excess HCl and dioxane
were removed under reduced pressure, triturated with ether,
filtered, washed with ether and dried (yield: 100%). HCl.H-
Arg(NO2)-Pro-OBzl (17.72 g, 0.04 mol) in DMF (180 mL)
was neutralized with NMM (4.4 mL, 0.04 mol) and coupled
to Boc-Pro-OH (8.2 g, 0.04 mol) in acetonitrile (80 mL) and
NMM (4.4 mL) using IBCF (5.5 mL, 0.04 mol) and worked
up the same as 2 to obtain 5. The sample was recrystallized
from ether/petroleum ether. M.P. 46-48 °C (Lit. 47.24 °C
[7]).
Pro
Gly/Arg
Arg
Pro
Boc-X-Pro-Arg(NO2)-Pro-OBzl (X is Gly for 8 and Arg
(NO2) for 11)
NO2
OH
Boc
Boc
H
H
OBzl
OBzl
OBzl
OBzl
Compound 5 (9.9 g) was deblocked with 4N HCl/dioxane
(100 mL) for 1.5 hr. Excess HCl and dioxane were removed
under reduced pressure, triturated with ether, filtered,
washed with ether and dried (yield: 100%). HCl.H-Pro-
Arg(NO2)-Pro-OBzl was divided in to two equal parts and
each part in DMF was neutralized with NMM (0.76 mL,
0.007 mol) and coupled to Boc-X-OH (0.007 mol) in aceto-
nitrile and NMM (0.77 mL, 0.007 mol) using IBCF (0.95
mL, 0.007 mol) and worked up the same as 2 to obtain 8 and
11. The samples were recrystallized from ether/petroleum
ether. Compound 8: M.P. 47-49 °C (Lit. 48.97 °C [7]) and
Compound 11: M.P. 58-60 °C (Lit. 59-61 °C [7]).
NO2
NO2
NO2
i
ii
iii
ii
Boc
Boc
OH
NO2
NO2
NO2
Boc
OBzl
OH
H
iii
ii
Boc
H
OBzl
OBzl
General Procedure for the Coupling of Benzyl Esters of
Peptides 2, 5, 8 and 11 with 3-(4-Oxo-3,4-dihydro-
quinazolin-2-yl)Propanoic Acid 1
To 3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoic acid
(0.436 g, 2 mmol) dissolved in DMF (5 mL) and cooled to 0
°C was added NMM (0.21 mL, 2 mmol) and EDCI (0.380 g,
2 mmol) under stirring while maintaining the temperature at
0 °C. The reaction mixture was stirred for 10 minutes and
HOBt (0.765 g, 2 mmol) in DMF (8 mL) was added and
stirred the reaction mixture for further 10 minutes. To this a
pre-cooled solution of HCl.H-Zcc-OBzl [Zcc = RP (2), PRP
(5), GPRP (8) and RPRP (11), 2 mmol] and NMM (0.21 mL,
2 mmol) in DMF was added slowly. After 20 minutes, pH of
the solution was adjusted to 8 by the addition of NMM and
the reaction mixture was stirred over night at room tempera-
ture. DMF was removed under reduced pressure and the
residue was poured into about 100 mL ice-cold 90% satu-
rated KHCO3 solution and stirred for 30 minutes. The pre-
cipitated product was dissolved in CHCl3 (15 mL) and the
organic layer was washed with 0.1N HCl (2 x 20 mL), water
(2 x 20 mL), 5% NaHCO3 solution (2 x 20 mL) and finally
with brine solution (2 x 20 mL). The CHCl3 layer was dried
over anhydrous Na2SO4 and the solvent was removed under
Scheme 1. Schematic representation of the synthesis of tetrapep-
tides. HCl.H.Gly/Arg-Pro-Arg-Pro-OBzl by stepwise approach
i. IBCF/HOBt, NMM
ii. HCl/dioxane
iii. IBCF, NMM
Peptide Synthesis
Boc-Arg(NO2)-Pro-OBzl (2)
To Boc-Arg(NO2)-OH (15.95 g, 0.05 mol) dissolved in
acetonitrile (160 mL) and cooled to 0 °C was added NMM
(5.5 mL, 0.05 mol). The solution was cooled to -15 °C ± 1°C
and IBCF (6.85 mL, 0.05 mol) was added under stirring
while maintaining the temperature at -15 °C. After stirring
the reaction mixture for 10 minutes at this temperature, a
pre-cooled solution of HOBt (6.80 g, 0.05 mol) in DMF (70
mL) was added. The reaction mixture was stirred for an addi-
tional 10 minutes and a pre-cooled solution of HCl.H-Pro-
OBzl (12.10 g, 0.05 mol) and NMM (5.5 mL, 0.05 mol) in

148 Protein & Peptide Letters, 2013, Vol. 20, No. 2
Suhas et al.
Scheme 2. Synthesis of quinazolinone conjugated peptides. Reagents and conditions: a: EDCI/HOBt/NMM, 0 °C
b: HCOONH4/10% Pd-C, 30-45 min, rt
reduced pressure. The product obtained was triturated with
ether/petroleum ether to obtain QZN-Zcc-OBzl [Zcc = RP
Agar Well Diffusion Method
(3), PRP (6), GPRP (9) and RPRP (12)] as hygroscopic
foam.
The microorganisms were inoculated in to the sterilized
nutrient broth and maintained at 37 °C for 24 hours. On the
day of testing, bacteria were subcultured separately into 25
mL of sterilized nutrient broth. Inoculated subcultured broths
were kept at room temperature for the growth of inoculums.
Each test compounds (1-13) and standard drug (ciproflox-
acin) of 10 mg was dissolved in 10 mL of DMSO to get a
concentration of 1 mg/mL and further diluted to get a final
concentration of 30 μg/mL. About 15-20 mL of molten nu-
trient agar was poured into each of the sterile plates. With
the help of cork borer of 6 mm diameter, the cups were
punched and scooped out of the set agar and the plates were
inoculated with the suspension of particular organism by
spread plate technique. The cups of inoculated plates were
then filled with 0.1 mL of the test solution, ciprofloxacin
solution and DMSO (negative control). The plates were al-
lowed to stay for 24 hours at 37 °C and zone of inhibition
(mm) was then measured.
General Procedure for the Hydrogenolysis of Benzyl Ester
and Nitro Group of Quinazolinone Conjugated Peptides
Each conjugate QZN-Zcc-OBzl [Zcc = R(NO2)P (3),
PR(NO2)P (6), GPR(NO2)P (9) and R(NO2)PR(NO2)P (12),
1 mmol] was hydrogenolysed in methanol (10 mL/g of com-
pound) using ammonium formate (2.0 equi.) and 10% Pd-C
(0.1g/g of peptide) for 30-45 minutes at room temperature.
The completion of the reaction was monitored by TLC. The
catalyst was filtered and washed with methanol. The com-
bined washings and filtrate were evaporated in vacuo and the
residue taken in to CHCl3, washed with water and dried over
anhydrous Na2SO4. The solvent was removed under reduced
pressure and triturated with ether, filtered, washed with ether
and dried to obtain final products QZN-Zcc-OH [Zcc = RP
(4), PRP (7), GPRP (10) and RPRP (13)]. Yield of the com-
pounds obtained were 85.2%, 87.6%, 88.5% and 83.0% for
4, 7, 10 and 13 respectively.
Microdilution Method
All the microorganisms were grown in Muller-Hinton
broth. After cultivation for 16-18 hours at 37 °C, the bacteria
were harvested and their density was determined by measur-
ing O.D at A600. MIC of the compounds was determined by
agar dilution method. Suspension of each microorganism
was prepared to contain approximately (1X104 – 2X104
CFU/mL) and applied to the plates with serially diluted
compounds (dissolved in DMSO) to be tested and also refer-
Antibacterial Activity
In vitro antibacterial activity was evaluated against hu-
man pathogens of both gram positive organisms namely K.
pneumoniae and P. putida and gram negative organisms
namely S. faecalis, S. pyogenes and E. coli by agar well dif-
fusion method [23] (Fig. 1) as well as microdilution method
[23] (Fig. 2).

Quinazolinone conjugated shorter analogues of Bac7…
Protein & Peptide Letters, 2013, Vol. 20, No. 2 149
Figure 1. Diagramatic representation of antibacterial activity of the synthesized compounds by agar well diffusion method.
Figure 2. Diagramatic representation of antibacterial activity of the synthesized compounds by microdilution method.
ence drug and incubated at 37 °C overnight. The minimum
inhibitory concentration was considered to be the lowest
concentration that completely inhibited the growth of micro-
organisms on the plates. Zone of inhibition (mm) was meas-
ured after 24 hours and MIC values were determined.
verticellartar and A. alternata by agar well diffusion method
(Fig. 3) as well as microdilution method (Fig. 4).
Agar Well Diffusion Method [24]
The fungal strains were subcultured separately into 25
mL of sterilized nutrient broth and incubated for one day to
obtain the inoculums. Each test compounds (1-13) and stan-
dard drug (griseofulvin) of 10 mg was dissolved in 10 mL of
DMSO to get a concentration of 1 mg/mL and further diluted
to get a final concentration of 30 μg/mL. Molten media of
Antifungal Activity
In vitro antifungal activity was evaluated against five
fungal species namely A. flavus, C. capsisi, F. oxysporum, F.

150 Protein & Peptide Letters, 2013, Vol. 20, No. 2
Suhas et al.
Figure 3. Diagramatic representation of antifungal activity of the synthesized compounds by agar well diffusion method.
Figure 4. Diagramatic representation of antifungal activity of the synthesized compounds by microdilution method.
Sabouraud agar of 10-15 mL was poured into the petriplates
and allowed to solidify. Fungal subculture was inoculated on
Microdilution Method [24]
the solidified media. With the help of 6 mm cork borer, the
cups were punched and scooped out of the set agar. The cups
of inoculated plates were then filled with 0.1 mL of the test
solution, griseofulvin solution and DMSO (negative control).
The plates were allowed to stay for 3 days at room tempera-
ture and zone of inhibition (mm) was then measured.
Sabouraud agar was used for the preparation of plates.
Suspension of each microorganism was prepared to contain
105 CFU/mL. The agar plates were inoculated with fungal
strains and serially diluted test compounds and reference
drug dissolved in DMSO. The plates were incubated at 25 °C
for 48-72 hours. The minimum inhibitory concentration was

Quinazolinone conjugated shorter analogues of Bac7…
Protein & Peptide Letters, 2013, Vol. 20, No. 2 151
considered to be the lowest concentration that completely
inhibited the growth of microorganisms on the plates. Zone
of inhibition (mm) was measured after 72 hours and MIC
values were determined.
peptides were conjugated with the precursor 1. The protected
peptides and heterocycle conjugated peptides were character-
1
ized by TLC, IR, H NMR, mass spectroscopic techniques
1
and also elemental analysis. The H NMR; analytical and
physical data of the synthesized compounds are provided in
1
Table 1 and 2 respectively. The H NMR, mass and elemen-
RESULTS AND DISCUSSION
tal analysis data were found to be in good agreement with the
structures assigned.
Previous work has shown that it is possible to improve
the activities, or reduce the toxicities of naturally occurring
peptide antibiotics by producing synthetic analogues with
modified primary and/or secondary structures [25]. The pre-
sent work was primarily aimed to synthesize some hetero-
cyclic conjugated shorter active analogues of Bac7 to obtain
some information on the importance of conjugation. The
type of modification included attachment of bioactive quina-
zolinone moiety at the N-terminal of the peptides to study the
change in the biological activity.
Biology
The efficacy of the synthesized compounds 1-13 were
evaluated for antibacterial activities against different strains
of gram negative bacteria like S. faecalis, S. pyogenes and E.
coli and gram positive bacteria like K. pneumoniae and P.
putida and antifungal activities against A. flavus, C. capsisi,
F. oxysporum, F. verticellartar and A. alternata. The results
obtained as zone of inhibition (mm) and MIC values
(ꢀg/mL) are presented in Table 3 and 4 respectively.
Chemistry
The peptide synthesis was performed by step-wise solu-
tion phase method using Boc chemistry. The synthesized
Table 1. 1H NMR Data of the Synthesized Compounds
Compounds
Boc-RP-OBzl
Component
1H NMR (DMSO-d6, ꢀ ppm)
Boc
1.42 (s, 9H, Me3)
Arg1
Pro2
8.41 (s, 1H, NH), 4.53 (m, 1H, ꢁCH), 1.53 (m, 2H, ꢂCH2), 1.45 (m, 2H, ꢃCH2), 3.07 (m, 2H, ꢄCH2)
4.35 (m, 1H, ꢁCH), 3.06 (m, 2H, ꢂCH2), 2.20-2.25 (m, 2H, ꢃCH2), 3.52-3.64(t, 2H, ꢄCH2)
5.07 (m, 2H, CH2), 7.30-7.34 (m, 5H, Ar-H)
OBzl
Boc-PRP-OBzl
Boc
1.36 (s, 9H, Me3)
Pro1
Arg2
Pro3
OBzl
4.10 (m, 1H, ꢁCH), 1.82-1.90 (m, 2H, ꢂCH2), 1.80-1.81 (m, 2H, ꢃCH2), 3.67-3.76 (t, 2H, ꢄCH2)
8.00 (s, 1H, NH), 4.48 (m, 1H, ꢁCH), 1.55 (m, 2H, ꢂCH2), 1.47 (m, 2H, ꢃCH2), 3.09 (m, 2H, ꢄCH2)
4.46 (m, 1H, ꢁCH), 3.09 (m, 2H, ꢂCH2), 2.15-2.30 (m, 2H, ꢃCH2), 3.52-3.67 (t, 2H, ꢄCH2)
5.08 (m, 2H, CH2), 7.29-7.34 (m, 5H, Ar-H)
Boc-GPRP-OBzl
Boc
1.40 (s, 9H, Me3)
Gly1
Pro2
Arg3
Pro4
OBzl
8.10 (s, 1H, NH), 4.13 (s, 2H, ꢁCH2)
4.29 (m, 1H, ꢁCH), 2.31-2.48 (m, 2H, ꢂCH2), 2.16-2.31 (m, 2H, ꢃCH2), 3.77-3.84 (t, 2H, ꢄCH2)
7.95 (s, 1H, NH), 4.50 (m, 1H, ꢁCH), 1.52 (m, 2H, ꢂCH2), 1.47 (m, 2H, ꢃCH2), 3.10 (m, 2H, ꢄCH2)
4.44 (m, 1H, ꢁCH), 3.12 (m, 2H, ꢂCH2), 2.16-2.30 (m, 2H, ꢃCH2), 3.53-3.64 (t, 2H, ꢄCH2)
5.08 (m, 2H, CH2), 7.29-7.34 (m, 5H, Ar-H)
Boc-RPRP-OBzl
Boc
1.39 (s, 9H, Me3)
Arg1
Pro2
Arg3
Pro4
OBzl
8.22 (s, 1H, NH), 4.60 (m, 1H, ꢁCH), 1.61 (m, 2H, ꢂCH2), 1.52 (m, 2H, ꢃCH2), 3.15 (m, 2H, ꢄCH2)
4.30 (m, 1H, ꢁCH), 2.35-2.51 (m, 2H, ꢂCH2), 2.19-2.33 (m, 2H, ꢃCH2), 3.78-3.81 (t, 2H, ꢄCH2)
8.34 (s, 1H, NH), 4.51 (m, 1H, ꢁCH), 1.59 (m, 2H, ꢂCH2), 1.50 (m, 2H, ꢃCH2), 3.13 (m, 2H, ꢄCH2)
4.46 (m, 1H, ꢁCH), 3.03 (m, 2H, ꢂCH2), 2.21-2.24 (m, 2H, ꢃCH2), 3.53-3.66 (t, 2H, ꢄCH2)
5.11 (m, 2H, CH2), 7.30-7.35 (m, 5H, Ar-H)
QZN
-
12.11 (s, 1H, NH), 11.69 (s, 1H, COOH), 7.06-8.43 (m, 4H, Ar-H), 2.48-2.53 (t, 4H, (CH2)2)
QZN-RP-OBzl
QZN
Arg1
Pro2
12.11 (s, 1H, NH), 7.07-8.45 (m, 4H, Ar-H), 2.46-2.55 (t, 4H, (CH2)2)
7.88 (s, 1H, NH), 4.40 (m, 1H, ꢁCH), 1.53 (m, 2H, ꢂCH2), 1.45 (m, 2H, ꢃCH2), 3.07 (m, 2H, ꢄCH2)
4.37 (m, 1H, ꢁCH), 3.07 (m, 2H, ꢂCH2), 2.20-2.25 (m, 2H, ꢃCH2), 3.52-3.64(t, 2H, ꢄCH2)
5.08 (m, 2H, CH2), 7.30-7.34 (m, 5H, Ar-H)
OBzl

152 Protein & Peptide Letters, 2013, Vol. 20, No. 2
Suhas et al.
(Table 1) Contd….
Compounds
Component
QZN
1H NMR (DMSO-d6, ꢀ ppm)
QZN-PRP-OBzl
12.11 (s, 1H, NH), 7.07-8.45 (m, 4H, Ar-H), 2.46-2.55 (t, 4H, (CH2)2)
Pro1
Arg2
Pro3
OBzl
4.26 (m, 1H, ꢀCH), 1.84-1.91 (m, 2H, ꢁCH2), 1.80-1.83 (m, 2H, ꢂCH2), 3.66-3.76 (t, 2H, ꢃCH2)
8.21 (s, 1H, NH), 4.49 (m, 1H, ꢀCH), 1.55 (m, 2H, ꢁCH2), 1.49 (m, 2H, ꢂCH2), 3.08 (m, 2H, ꢃCH2)
4.48 (m, 1H, ꢀCH), 3.10 (m, 2H, ꢁCH2), 2.15-2.30 (m, 2H, ꢂCH2), 3.52-3.67 (t, 2H, ꢃCH2)
5.08 (m, 2H, CH2), 7.29-7.33 (m, 5H, Ar-H)
QZN-GPRP-OBzl
QZN
Gly1
Pro2
Arg3
Pro4
12.11 (s, 1H, NH), 7.10-8.44 (m, 4H, Ar-H), 2.45-2.57 (t, 4H, (CH2)2)
8.12 (s, 1H, NH), 4.15 (s, 2H, ꢀCH2)
4.30 (m, 1H, ꢀCH), 2.31-2.48 (m, 2H, ꢁCH2), 2.16-2.32 (m, 2H, ꢂCH2), 3.77-3.86 (t, 2H, ꢃCH2)
7.97 (s, 1H, NH), 4.50 (m, 1H, ꢀCH), 1.52 (m, 2H, ꢁCH2), 1.46 (m, 2H, ꢂCH2), 3.09 (m, 2H, ꢃCH2)
4.43 (m, 1H, ꢀCH), 3.13 (m, 2H, ꢁCH2), 2.17-2.31 (m, 2H, ꢂCH2), 3.14-3.53 (t, 2H, ꢃCH2)
5.10 (m, 2H, CH2), 7.30-7.32 (m, 5H, Ar-H)
OBzl
QZN-RPRP-OBzl
QZN
Arg1
Pro2
Arg3
Pro4
12.11 (s, 1H, NH), 7.11-8.43 (m, 4H, Ar-H), 2.44-2.56 (t, 4H, (CH2)2)
8.23 (s, 1H, NH), 4.62 (m, 1H, ꢀCH), 1.61 (m, 2H, ꢁCH2), 1.52 (m, 2H, ꢂCH2), 3.15 (m, 2H, ꢃCH2)
4.31 (m, 1H, ꢀCH), 2.35-2.51 (m, 2H, ꢁCH2), 2.19-2.33 (m, 2H, ꢂCH2), 3.78-3.81 (t, 2H, ꢃCH2)
8.3 (s, 1H, NH), 4.52 (m, 1H, ꢀCH), 1.60 (m, 2H, ꢁCH2), 1.51 (m, 2H, ꢂCH2), 3.13 (m, 2H, ꢃCH2)
4.48 (m, 1H, ꢀCH), 3.04 (m, 2H, ꢁCH2), 2.21-2.24 (m, 2H, ꢂCH2), 3.53-3.67 (t, 2H, ꢃCH2)
5.09 (m, 2H, CH2), 7.30-7.33 (m, 5H, Ar-H)
OBzl
Table 2. Physical and Analytical Data of the Synthesized Compounds
Elemental analysisb
IR
Molecular For-
mula
Theoretical
Mol.Wt.
Actual MS Values
(M+)
Yield
(%)
Entrya
cm-1 (KBr)
% C
54.51
% H
6.74
% N
16.60
1655 (C=O), 3176
(NH)
Boc-RP-OBzl
QZN-RP-OBzl
Boc-PRP-OBzl
C23H34N6O7
C29H34N8O7
C28H41N7O8
C34H41N9O8
C30H44N8O9
C36H44N10O9
C34H52N12O11
C40H52N14O11
506
606
603
703
660
760
804
904
507
607
604
704
661
761
805
905
88.3
85.0
88.0
84.2
89.6
85.8
89.1
86.7
(54.53) (6.77) (16.59)
1689 (C=O),
3125 (NH)
57.40
5.66
18.49
(57.42) (5.65) (18.47)
1642 (C=O),
3120 (NH)
55.68
6.84
16.26
(55.71) (6.85) (16.24)
1712 (C=O),
3182 (NH)
58.06
5.88
17.93
QZN-PRP-
OBzl
(58.03) (5.87) (17.91)
1676 (C=O),
3136 (NH)
54.50
6.70
16.98
Boc-GPRP-
OBzl
(54.43) (6.71) (16.96)
1646 (C=O),
3193 (NH)
56.81
5.85
18.44
QZN-GPRP-
OBzl
(56.83) (5.83) (18.41)
1710 (C=O),
3148 (NH)
50.72
6.52
20.86
Boc-RPRP-
OBzl
(50.74) (6.51) (20.88)
1688 (C=O),
3175 (NH)
53.11
5.82
21.69
QZN-RPRP-
OBzl
(53.09) (5.79) (21.67)
aG-Glycine, P-Proline, R-Arginine
bValues given in the parentheses are the calculated for elemental analysis
It is observed from the results that all the heterocyclic
conjugated peptides (3, 6, 9 and 12) have shown enhanced
activity than either heterocycle (1) or peptides (2, 5, 8 and
11) which are moderately active. Among the conjugated
compounds, tetrapeptide conjugates 9 and 12 (5 and 4 ꢄg/mL
respectively) have exerted high activity which is attributed to
the increase in charge, hydrophobicity as well as the length
of the peptide chain [15]. Whereas the dipeptide conjugate 3

Quinazolinone conjugated shorter analogues of Bac7…
Protein & Peptide Letters, 2013, Vol. 20, No. 2 153
Table 3. Inhibitory Zone (diameter) mm of the Synthesized Conjugates (1-13) Against Tested Bacterial and Fungal Strains by
Agar Well Diffusion Method
Antibacterial Activity
Antifungal Activity
Zone of Inhibitiona (mm) ± SD (n = 3)
Entry
A. alternata
S. pyo-
genes
F. ox-
F. verticel-
S. faecalis
E. coli
K. pneumoniae
P. putida
A. flavus
C. capsisi
ysporum
lartar
QZN (1)
04 ± 0.31
03 ± 0.46
16 ± 0.49
20 ± 0.78
06 ± 0.36
19 ± 0.49
23 ± 0.35
08 ± 0.35
26 ± 0.32
34 ± 0.53
09 ± 0.36
29 ± 0.10
37 ± 0.56
20 ± 0.59
-
03 ± 0.26
02 ± 0.42
07 ± 0.42
09 ± 0.50
03 ± 0.40
10 ± 0.57
13 ± 0.61
05 ± 0.40
24 ± 0.51
37 ± 0.23
05 ± 0.42
27 ± 0.46
39 ± 0.59
22 ± 0.42
-
00 ± 0.10
00 ± 0.10
09 ± 0.26
13 ± 0.45
01 ± 0.25
10 ± 0.36
14 ± 0.38
03 ± 0.35
16 ± 0.31
23 ± 0.62
04 ± 0.30
18 ± 0.36
26 ± 0.32
16 ± 0.42
-
01 ± 0.30
03 ± 0.35
14 ± 0.35
22 ± 0.31
05 ± 0.58
17 ± 0.56
24 ± 0.52
06 ± 0.40
25 ± 0.75
31 ± 0.38
08 ± 0.30
27 ± 0.66
34 ± 0.47
18 ± 0.46
-
03 ± 0.15
02 ± 0.20
11 ± 0.57
16 ± 0.82
04 ± 0.20
14 ± 0.35
18 ± 0.25
05 ± 0.29
24 ± 0.50
33 ± 0.47
07 ± 0.35
27 ± 0.67
35 ± 0.70
18 ± 0.38
-
03 ± 0.20
00 ± 0.06
05 ± 0.25
07 ± 0.20
01 ± 0.35
07 ± 0.47
09 ± 0.21
01 ± 0.38
12 ± 0.45
27 ± 0.38
03 ± 0.49
14 ± 0.23
30 ± 0.42
-
02 ± 0.15
02 ± 0.38
13 ± 0.32
18 ± 0.35
03 ± 0.31
16 ± 0.25
21 ± 0.40
05 ± 0.45
26 ± 0.38
35 ± 0.21
08 ± 0.40
29 ± 0.40
37 ± 0.55
-
02 ± 0.25
01 ± 0.17
11 ± 0.36
16 ± 0.36
03 ± 0.32
13 ± 0.46
18 ± 0.35
03 ± 0.30
21 ± 0.30
29 ± 0.23
05 ± 0.25
23 ± 0.42
31 ± 0.55
-
04 ± 0.35
03 ± 0.20
17 ± 0.17
22 ± 0.56
05 ± 0.26
20 ± 0.47
24 ± 0.56
06 ± 0.29
28 ± 0.50
35 ± 0.64
09 ± 0.36
31 ± 0.32
38 ± 0.61
-
03 ± 0.26
00 ± 0.12
13 ± 0.36
18 ± 0.46
02 ± 0.20
15 ± 0.55
22 ± 0.61
04 ± 0.35
24 ± 0.36
32 ± 0.57
05 ± 0.21
26 ± 0.50
33 ± 0.57
-
Boc-RP-OBzl (2)
QZN-RP-OBzl (3)
QZN-RP-OH (4)
Boc-PRP-OBzl (5)
QZN-PRP-OBzl (6)
QZN-PRP-OH (7)
Boc-GPRP-OBzl (8)
QZN-GPRP-OBzl (9)
QZN-GPRP-OH (10)
Boc-RPRP-OBzl (11)
QZN-RPRP-OBzl (12)
QZN-RPRP-OH (13)
Ciprofloxacin
Griseofulvin
14 ± 0.17
18 ± 0.35
15 ± 0.60
21 ± 0.30
17 ± 0.51
aValues are mean of three determinations, the ranges of which are <5% of the mean in all cases
and tripeptide conjugate 6 have shown comparatively mod-
erate activity (12 and 9 ꢀg/mL respectively). Thus, as the
length of the peptide chain increases the activity also in-
creases [12]. All the quinazolinone conjugated analogues are
more effective in arresting or killing the growth of gram
negative microorganisms when compared to gram positive
microorganisms since the cationic antimicrobial peptides are
likely to first be attracted to the net negative charges that
exist on the outer envelope of gram negative bacteria [12].
One exception to this is E. coli and hence it may be inferred
that compounds exhibit strain specificity particularly against
E. coli.
over GPRP conjugate. This may be due to the presence of
more bulky and charged groups in the former which interacts
more with the anionic phosphate groups of the microbial cell
membranes.
CONCLUSION
In the present study, novel potent antimicrobial agents
were synthesized by conjugating quinazolinone motif to
shorter active analogues of Bac7. Our studies revealed that
the heterocyclic conjugated peptides showed enhanced activ-
ity against all the pathogens tested compared to peptides and
quinazolinone tested alone. Further, the hydrogenolysed
compounds have shown high potency than their counterparts
in arresting the growth of microbes. Also, as the length of the
peptide chain increases, activity also increases. Thus, it can
be inferred that conjugation plays a vital role in biological
studies.
When the C-terminal benzyl ester and nitro group of the
conjugates were removed, all the free acid containing com-
pounds (4, 7, 10 and 13) have exerted high potency in arrest-
ing the growth of microorganisms which is nearly 3-4 times
greater than the antibiotics used. This fact is due to the in-
crease in the polarity of these compounds [12, 16] which
would help the molecules to interact or penetrate more
through the cell membranes of microbes and there by inacti-
vating them [6]. This indicates that increase in the polarity
has an impact on the activity.
In conclusion, the shorter quinazolinone conjugated pep-
tides with small size, easy to synthesize, simple amino acid
composition, high antimicrobial activity and low toxicity,
makes them highly potent as novel templates for the design
of pharmaceutical compounds and peptidomimetics for topi-
cal and systemic treatment of the antibiotic resistant strains
of bacteria and fungi.
Among the tetrapeptide conjugates, RPRP containing
analogue has exhibited slight enhancement in the activity

154 Protein & Peptide Letters, 2013, Vol. 20, No. 2
Suhas et al.
Table 4.
Minimum Inhibitory Concentration (MIC) in ꢀg/mL of the Synthesized Conjugates (1-13) Against Tested Bacterial and
Fungal Strains by Microdilution Method
Antibacterial activity
Antifungal activity
Minimum Inhibitory Concentration (MIC) in ꢀg/mLa
Entry
S. pyo-
genes
K. pneumo-
niae
F. ox-
F. verticel-
A. alter-
nata
S. faecalis
E. coli
P. putida
A. flavus
C. capsisi
ysporum
lartar
QZN (1)
>30
18
12
10
14
09
07
13
05
03
11
04
03
10
-
>30
22
18
10
20
15
09
16
06
04
14
05
03
12
-
>30
29
22
16
28
18
14
26
08
10
24
10
08
08
-
>30
22
14
10
19
13
08
18
08
06
16
07
04
07
-
>30
26
17
12
24
15
10
20
08
07
19
06
04
11
-
>30
32
24
16
29
20
13
28
12
10
26
10
09
-
>30
28
19
18
26
18
09
23
08
06
20
07
04
-
>30
26
20
10
24
17
09
23
08
06
21
06
05
-
>30
22
15
08
19
13
06
17
05
04
15
04
03
-
>30
23
17
09
21
15
08
19
07
06
19
05
03
-
Boc-RP-OBzl (2)
QZN-RP-OBzl (3)
QZN-RP-OH (4)
Boc-PRP-OBzl (5)
QZN-PRP-OBzl (6)
QZN-PRP-OH (7)
Boc-GPRP-OBzl (8)
QZN-GPRP-OBzl (9)
QZN-GPRP-OH (10)
Boc-RPRP-OBzl (11)
QZN-RPRP-OBzl (12)
QZN-RPRP-OH (13)
Ciprofloxacin
Griseofulvin
08
12
14
07
10
aValues are mean of three determinations, the ranges of which are <5% of the mean in all cases
[2]
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CONFLICT OF INTEREST
The authors confirm that this article content has no con-
flicts of interest.
[4]
ACKNOWLEDGEMENTS
[5]
[6]
Chou, P.Y.; Fasman, G.D. ꢀ-turns in proteins. J. Mol. Biol., 1977,
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One of the authors RS gratefully acknowledges Lady
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[7]
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ABBREVIATIONS
AMP
Boc
=
=
=
Antimicrobial peptide
[8]
[9]
t-Butoxycarbonyl
EDCI
1-(3-Dimethylaminopropyl)-3-ethyl-
carbodiimide.HCl
[10]
[11]
Michael, J.P. Quinoline, quinazoline and acridone alkaloids. Nat.
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apy. Mol. Cancer Ther., 2006, 5, 3001-3013.
HOBt
IBCF
NMM
=
=
=
1-Hydroxybenzotriazole
Isobutyl chloroformate
N-Methylmorpholine
[12]
Suresha, G.P.; Prakasha, K.C.; Shivakumara, K.N.; Wethroe, K.;
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Received: April 24, 2012
Revised: June 22, 2012
Accepted: June 25, 2012