Synthesis and biological activity of peptide derivatives of iodoquinazolinones/nitroimidazoles

Article abstract of DOI:10.3390/molecules13040958

Two substituted quinazolinyl/imidazolyl-salicylic acids 5, 6 were synthesized by the reaction of 6-iodo-2-methylbenzoxazin-4-one/5-nitroimidazole with 5-aminosalicylic acid (5-ASA). Coupling of compounds 5 and 6 with different amino acid ester hydrochlorides, dipeptide and tripeptide methyl esters yielded novel quinazolino/imidazolopeptide derivatives 5a-f and 6a-g. The chemical structures of all newly synthesized compounds were confirmed by means of FT-IR, ¹H- and ¹³C-NMR, MS and elemental analysis. Selected peptide ester derivatives were further hydrolyzed by using lithium hydroxide (LiOH) to afford the corresponding acid derivatives 5b_a-d_a and 6e_a-g_a. All peptide derivatives were assayed for antimicrobial and anthelmintic activities against eight pathogenic microbes and three earthworm species. Among the tested compounds, 5e, 5d, 6e and their hydrolyzed analogs 5d_a and 6e_a exhibited higher antimicrobial activity against Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans, and 5a, 6g and 6g_a displayed better antifungal activity against the dermatophytes Trichophyton mentagrophytes and Microsporum audouinii. Moreover, 6f and its hydrolyzed derivative 6f_a showed good anthelmintic activity against Megascoplex konkanensis, Pontoscotex corethruses and Eudrilus eugeniea at dose of 2 mg mL^-1.

Full text of DOI:10.3390/molecules13040958

Molecules 2008, 13, 958-976
molecules
ISSN 1420-3049
© 2008 by MDPI
www.mdpi.org/molecules
Full Paper
Synthesis and Biological Activity of Peptide Derivatives of
Iodoquinazolinones/Nitroimidazoles
Rajiv Dahiya*, Anil Kumar and Rakesh Yadav
Department of Pharmaceutical Chemistry, Rajiv Academy for Pharmacy, Mathura-281001 (UP), India
*Author to whom correspondence should be addressed; Tel.: +91-9897417450; +91-565-6531680;
E-mail: rajivdahiya04@yahoo.co.in or rajivdahiya77@rediffmail.com
Received: 3 April 2008; in revised form: 18 April 2008 / Accepted: 19 April 2008 / Published: 24 April
2008
Abstract: Two substituted quinazolinyl/imidazolyl-salicylic acids 5, 6 were synthesized by
the reaction of 6-iodo-2-methylbenzoxazin-4-one/5-nitroimidazole with 5-aminosalicylic
acid (5-ASA). Coupling of compounds 5 and 6 with different amino acid ester
hydrochlorides,
dipeptide
and
tripeptide
methyl
esters
yielded
novel
quinazolino/imidazolopeptide derivatives 5a-f and 6a-g. The chemical structures of all
newly synthesized compounds were confirmed by means of FT-IR, 1H- and 13C-NMR, MS
and elemental analysis. Selected peptide ester derivatives were further hydrolyzed by using
lithium hydroxide (LiOH) to afford the corresponding acid derivatives 5ba-da and 6ea-ga.
All peptide derivatives were assayed for antimicrobial and anthelmintic activities against
eight pathogenic microbes and three earthworm species. Among the tested compounds, 5e,
5d, 6e and their hydrolyzed analogs 5da and 6ea exhibited higher antimicrobial activity
against Pseudomonas aeruginosa, Klebsiella pneumoniae and Candida albicans, and 5a,
6g and 6ga displayed better antifungal activity against the dermatophytes Trichophyton
mentagrophytes and Microsporum audouinii. Moreover, 6f and its hydrolyzed derivative
6fa showed good anthelmintic activity against Megascoplex konkanensis, Pontoscotex
corethruses and Eudrilus eugeniea at dose of 2 mg mL–1.
Keywords: Quinazolinone; imidazole; 5-aminosalicylic acid; peptides; antimicrobial
activity; anthelmintic activity.

Molecules 2008, 13
959
Introduction
During past decades, compounds bearing heterocyclic nuclei have received much attention due to
their chemotherapeutic value in the development of novel antimicrobials and anthelmintics.
Quinazolinone and imidazole analogs are associated with a variety of pharmacological activities
including antibacterial and antifungal [1-3], anti-inflammatory and analgesic [4-6], antitubercular
[7,8], cytotoxic [9-11], antiviral [12, 13], anticonvulsant [14], antimuscarinic [15], insecticidal [16],
farnesyltransferase, gastric H+/K+-ATPase and MAP kinase p38 inhibitory properties [17-19].
Furthermore, the literature is enriched with several findings indicating antimicrobial potential of
salicylic acid and its analogs [20-22].
Prompted by the chemotherapeutic importance of quinazolinones/imidazoles and salicylic acid
derivatives, these two vital moieties were combined together into a single molecule by varying the
substitution pattern on heterocyclic moieties to yield 2-hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-
dihydro-3-quinazolinyl)benzoic acid (5) and 2-hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoic acid (6).
The literature contains several reports on the incorporation of amino acids and peptides into the
aromatic and heterocyclic congeners resulting in compounds with potent bioactivities [23-27]. Thus,
keeping in mind the pharmacological potential of quinazolinones/imidazoles/salicylic acids as well as
taking advantage of biodegradability and biocompatibility of amino acids/peptides and further, in
continuation of our earlier work on synthesis of bioactive peptide analogs of aroylbenzoic acids,
aryloxyacetic acids, benzimidazoles and furoic acid [28], an attempt was made towards the synthesis
of two novel series of peptidyl derivatives of the iodoquinazolinones/nitroimidazoles – 2-hydroxy-5-
(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoyl amino acids/peptides 5a-f and 2-hydroxy-
5-(5-nitro-1H-imidazol-2-yl)benzoyl amino acids/peptides 6a-g. Selected peptide derivatives were
further hydrolyzed to get corresponding acid derivatives 5ba-da and 6ea-ga. The potential antibacterial,
antifungal and anthelmintic activities of the synthesized compounds were also evaluated.
Results and Discussion
6-Iodo-2-methylbenzoxazin-4-one (1a) was prepared in good yield according to a literature
procedure [16] by refluxing of 5-iodoanthranilic acid and acetic anhydride with stirring. Imidazole was
nitrated using nitrating mixture by the standard procedure [29] to afford 5-nitroimidazole (1b).
Dipeptides Boc-Pro-Val-OMe (2a), Boc-Leu-Phe-OMe (2b), Boc-His-Phe-OMe (2c), Boc-Gly-Gly-
OMe (2d), Boc-Ile-Tyr-OMe (2e), Boc-Phe-Pro-OMe (2f) were prepared by coupling Boc-amino acids
with the respective amino acid methyl ester hydrochlorides using dicyclohexylcarbodiimide (DCC) as
coupling agent and triethylamine (TEA) as base, according to the Bodanzsky and Bodanzsky
procedure with suitable modifications [30]. Similarly, tripeptides Boc-Ala-Pro-Try-OMe (3a),
Boc-Gly-Leu-His-OMe (3b) and tetrapeptide Boc-Val-Tyr-Phe-Gly-OMe (4a) were synthesized by
coupling Boc-dipeptides with respective amino acid methyl ester hydrochlorides/dipeptide methyl
esters under alkaline conditions. Prior to coupling, all di-/tri- and tetrapeptides were deprotected at the
amino end using trifluoroacetic acid (TFA). Compound 5 was prepared by the reaction of 1a and
5-ASA in ethanol, whereas compound 6 was synthesized by stirring 1b with a solution of diazotized
5-ASA. Finally, compound 5 was coupled with different amino acid methyl ester hydrochlorides and

Molecules 2008, 13
960
peptide methyl esters using DCC and N-methylmorpholine (NMM) in THF to afford peptide
derivatives 5a-f. Similarly, coupling of compound 6 with different peptide methyl esters in the
presence of DCC and TEA in DMF afforded amino acid/peptide conjugates 6a-g. Furthermore, amino
acid/peptide derivatives 5b-d and 6e-g were hydrolyzed by stirring with LiOH to yield corresponding
acid derivatives 5ba-da and 6ea-ga (Scheme 1).
Scheme 1. Synthesis of peptide analogs of iodoquinazolinones/nitroimidazoles 5a-6ga.
O
O
O
H2N
H2N
I
N
OH
OH
O
+
+
NO2
N
H
OH
OH
N
CH3
1b
5-ASA
5-ASA
1a
Diazotization
Stirring
NaNO2
CuCl2, 0-10 oC
170-180 oC
Heating
O
OH
OH
N
O
O
I
NO2
N
H
OH
N
OH
N
CH3
5
6
DCC, TEA
Coupling
Coupling
O
DCC, NMM
O
Me
X
NH
Me
OH
N
O
O
O
X
NO2
N
H
N
H
I
N
OH
N
CH3
6a-g
5a-f
LiOH Hydrolysis
LiOH Hydrolysis
OH
X
NH
O
N
OH
O
OH
O
X
NO2
N
H
N
H
I
N
OH
N
CH3
6ea-ga
5ba-da

Molecules 2008, 13
961
NO
2
NH
HO
O
H
N
O
O
HN
N
H
N
H
N
X =
O
O
O
5a
5e
5b, 5ba
5d, 5da
5c, 5ca
O
O
H
N
O
N
H
O
NH
N
H
N
5f
N
O
O
O
O
O
NH
6d
N
NH
6c
O
HO
OH
6b
O
6a
H
N
O
O
O
N
O
O
HN
O
NH
H
N
O
HN
O
6e, 6ea
O
N
HO
NH
6f, 6fa
6g, 6ga
All peptide derivatives 5a-6ga were synthesized in good yields using DCC as coupling agent and
TEA/NMM as bases. Presence of bands at 3365-3362, 3305-2505, 1702-1697, 1542, 1349, 590 cm–1 in
the IR spectra of compounds 5 and 6 clearly indicated presence of functional groups like -COOH,
-OH, -NO2 and -I and the absence of a free -NH2 group which was present in the starting material
5-ASA. Furthermore, the IR spectra of peptide derivatives 5a-6g showed amide I and amide II bands at
1660-1637 cm–1 and 1538-1525 cm–1, indicating formation of peptide bonds and confirming the
success of the coupling reactions. This fact was further supported by the appearance of broad singlets
for imino proton of the CO–NH moiety at 8.62-6.50 ppm in the 1H-NMR spectra and singlets at 177.3-
13
166.2 ppm (for the carbonyl carbon of the CO–NH moiety) in the C-NMR spectra of compounds
5a−6g. Moreover, the presence of a NO2 group in the peptide analogs of compound 6 was indicated by
appearance of medium bands at 1544-1540 cm–1 and 1349-1345 cm–1 (asymmetric and symmetric NO2
stretching) in the IR spectra, whereas presence of a iodo group in the peptide derivatives of compound
5 was indicated by the appearance of medium intensity bands at 592-587 cm–1 (C–Istr) in the IR spectra.
The mass spectra of peptide ester derivatives showed molecular ion peaks along with isotopic peaks at
m/z values consistent with their respective molecular formulas. All peptide ester derivatives showed
easily distinguishable R–C≡O+ ion peaks at M+– 31, along with characteristic fragmentation patterns
on both sides of the carbonyl moiety in their respective structures. Furthermore, [CH3O+] and
[CH3OCO+] fragment ion peaks appeared at m/z values 31 and 59 in the mass spectra of the
synthesized peptide derivatives. Structures of hydrolyzed derivatives 5ba-6ga were confirmed by the
appearance of strong bands at 1713-1710 cm–1 (C=Ostr, COOH) in the IR spectra, broad singlets at
1
8.17-7.42 ppm (for hydroxyl proton of COOH) in the H-NMR spectra and singlets at 177.2-175.4
ppm (for carbonyl carbon of COOH) in their 13C-NMR spectra. This fact was further supported by the
disappearance of the medium to strong bands at 1752-1742 cm–1 (C=Ostr, ester) and 1272-1268 cm–1
(C−Ostr, ester) in the IR spectra and the singlets at 53.9-52.1 ppm (for the carbonyl carbon of OCH3) in
the 13C-NMR spectra of compounds 5ba-6ga.

Molecules 2008, 13
962
All hydrolyzed peptide derivatives showed peaks at M+ – 17 and M+ – 45 in their mass spectra
corresponding to the loss of OH and COOH, respectively. Moreover, a [COOH+] fragment ion peak
appeared at m/z value 45 in the mass spectra of compounds 6fa and 6ga, along with characteristic
fragmentation patterns on both sides of the carbonyl moiety. The newly synthesized compounds were
also analyzed for C, H and N content and the results revealed deviations of ± 0.04 from calculated
values.
Almost all the synthesized compounds were found to exhibit moderate to good bioactivity against
Gram negative bacteria, dermatophytes and C. albicans. However, 5b-6ga displayed mild to moderate
activity against Gram positive bacteria and A. niger. Comparison of the antimicrobial activity data
suggested that the hydrolyzed peptide derivatives 5ba-da and 6ea-ga are more potent antimicrobial
agents than their corresponding methyl ester derivatives 5b-6g, but the methyl ester analogs were
found to be more potent than the corresponding acid derivatives against dermatophytes.
Compounds 5a, 5d, 5da, 5e, 6e, 6g, 6ga and 6ea were found to be the most active compounds, with
higher antimicrobial activity against P. aeruginosa, K. pneumoniae, C. albicans and dermatophytes.
Against Gram positive bacteria and A. niger, only compounds 5a, 5f and 6c exhibited significant
activity in comparison to reference drugs - ciprofloxacin and griseofulvin respectively (Table 1, Figure
1).
Table 1. Antimicrobial activity of compounds 5a-6ea.
Diameter of zone of inhibition (mm)
Bacterial strains
S. aur. P. aeru.
16(12.5) 22(6)
Fungal strains
Compound
B. sub.
K. pneu. C. alb.
M. audo. A. niger T. menta.
5a
5b
5c
5d
5e
5f
14(6)†
11(25)
10(12.5)
13(25)
9(12.5)
16(6)
17(12.5) 14(12.5) 19(6)
14(12.5) 22(6)
9(25)
9(50)
10(25)
8(25)
17(12.5) 13(25)
15(6)
14(12.5) 18(6)
20(12.5) 22(6)
22(12.5) 24(6)
12(6)
13(6)
12(6)
9(12.5)
15(25)
16(50)
16(50)
7(12.5) 13(12.5)
15(12.5) 9(25)
15(6)
17(6)
18(6)
20(6)
27(6)
26(6)
17(12.5) 20(6)
11(25)
18(12.5) 11(12.5) 10(6)
5ba
5ca
5da
6a
13(25)
21(12.5) 19(25)
17(6)
17(12.5) 19(6)
24(12.5) 25(6)
10(6)
11(6)
10(6)
16(25)
17(50)
17(50)
14(6)
14(6)
16(6)
12(25)
12(6)
12(12.5) 12(50)
23(6)
29(6)
24(6)
23(6)
14(25)
16(25)
14(50)
17(6)
17(25)
14(25)
15(50)
16(25)
16(25)
16(50)
17(25)
–
14(25)
10(50)
12(25)
18(25)
15(12.5) 13(12.5) 9(6)
8(25)
15(12.5) 10(12.5) 14(25)
11(12.5) 11(12.5) 14(25)
6b
19(25)
6c
6d
6e
6f
16(12.5) 23(12.5) 16(12.5) 12(25)
14(12.5) 10(6)
12(25)
11(50)
12(25)
12(50)
13(50)
13(25)
14(50)
–
22(6)
26(6)
20(6)
18(25)
21(12.5) 23(6)
17(12.5) 15(6)
12(12.5)
13(6)
17(6)
23(6)
11(6)
15(6)
25(6)
–
11(6)
12(6)
21(6)
10(6)
11(6)
23(6)
–
13(25)
15(50)
14(50)
14(25)
16(50)
15(50)
–
6g
22(12.5) 16(25)
16(6)
6ea
6fa
6ga
27(6)
23(6)
24(12.5) 19(25)
23(12.5) 25(6)
18(12.5) 16(6)
17(6)
–
Control*
Ciprofloxacin
Griseofulvin
–
–
20(6)
–
20(12.5) 25(6)
19(12.5)
–
–
–
–
–
–
–
20(6)
17(6)
18(12.5) 20(6)
† Values in bracket are MIC values (μg mL–1).
* Dimethylformamide (DMF) / Dimethylsulfoxide (DMSO).

Molecules 2008, 13
Figure 1. Comparison of antimicrobial activity of quinazolino/imidazolopeptide derivatives.
963
35
30
25
20
15
10
5
0
P. aeruginosa
K. pneumoniae
B. subtilis
S. aureus
Compounds
Table 2. Anthelmintic activity of compounds 5a-6ea
Earthworm species
M. konkanensis
P. corethruses
E. eugeniea
Mean
Mean
Mean
Mean
Mean
Mean
Compound
paralyzing
time (min)‡
death
time (min)‡
paralyzing
time (min)
death
paralyzing
time (min)
death
time (min)
time (min)
5a
40.38 ± 0.52 52.58 ± 0.59 42.57 ± 0.26 54.26 ± 0.42 39.25 ± 0.23 49.34 ± 1.62
21.56 ± 0.28 31.56 ± 0.45 26.65 ± 0.44 38.22 ± 0.87 25.67 ± 0.82 37.21 ± 0.82
42.34 ± 0.59 55.40 ± 0.84 41.17 ± 0.88 55.40 ± 0.43 40.73 ± 0.49 51.54 ± 0.93
44.68 ± 0.12 52.59 ± 0.72 44.55 ± 0.23 54.18 ± 0.17 41.49 ± 0.32 51.28 ± 0.44
24.22 ± 0.21 35.48 ± 0.16 29.35 ± 0.65 37.24 ± 0.54 25.45 ± 0.58 34.34 ± 0.62
43.25 ± 0.44 50.52 ± 0.43 42.22 ± 0.24 57.50 ± 0.42 43.59 ± 0.41 55.10 ± 0.60
20.15 ± 0.52 29.29 ± 0.76 26.04 ± 0.12 37.96 ± 0.54 24.38 ± 0.69 35.12 ± 0.71
40.29 ± 0.89 53.27 ± 0.92 39.61 ± 0.21 50.39 ± 0.20 39.80 ± 0.85 49.24 ± 0.55
40.54 ± 0.83 52.18 ± 0.28 40.47 ± 0.47 52.02 ± 0.18 37.44 ± 0.89 47.08 ± 0.38
24.28 ± 0.67 36.46 ± 0.28 29.14 ± 0.23 38.82 ± 0.58 26.34 ± 0.56 37.30 ± 0.49
26.04 ± 0.53 38.28 ± 0.71 30.10 ± 0.38 38.29 ± 0.30 27.50 ± 0.51 36.02 ± 0.66
27.23 ± 0.56 38.23 ± 0.38 28.60 ± 0.33 40.32 ± 0.37 30.22 ± 0.75 38.45 ± 0.48
15.33 ± 0.40 25.04 ± 0.77 19.57 ± 0.56 31.42 ± 0.86 16.43 ± 0.60 27.02 ± 0.53
21.73 ± 0.64 31.73 ± 0.49 26.50 ± 0.23 36.58 ± 0.63 24.48 ± 0.59 31.08 ± 0.37
11.31 ± 0.43 21.10 ± 0.44 15.19 ± 0.31 24.24 ± 0.45 13.39 ± 0.33 23.45 ± 0.12
15.55 ± 0.21 25.56 ± 0.62 20.02 ± 0.50 32.32 ± 0.34 16.27 ± 0.38 27.15 ± 0.46
17.25 ± 0.52 27.48 ± 0.83 22.38 ± 0.33 34.33 ± 0.28 18.37 ± 0.44 29.06 ± 0.21
09.21 ± 0.22 18.34 ± 0.30 12.41 ± 0.15 21.55 ± 0.26 12.45 ± 0.33 23.09 ± 0.10
14.05 ± 0.46 23.52 ± 0.66 18.19 ± 0.36 31.03 ± 0.44 14.02 ± 0.43 25.34 ± 0.22
5b
5c
5d
5e
5f
5ba
5ca
5da
6a
6b
6c
6d
6e
6f
6g
6ea
6fa
6ga
Control#
–
–
–
–
–
–
Mebendazole 13.85 ± 0.64 22.85 ± 0.53 17.82 ± 0.43 29.60 ± 0.22 13.54 ± 0.45 24.05 ± 0.62
‡ Data are given as mean ± S.D. (n = 3).
# Tween 80 (0.5 %) in distilled water.

Molecules 2008, 13
Figure 2. Comparison of anthelmintic activity of quinazolino/imidazolopeptide derivatives.
964
Mean Paralyzing Time (MPT) Mean Death Time (min)
45
40
35
30
25
20
15
10
5
0
M. konkanensis
P. corethruses
E. eugeniea
Compounds
All imidazolopeptide derivatives 6a-ga showed moderate to good anthelmintic activity at 2 mg
mL−1 concentration in Tween 80 (0.5 %) and distilled water, whereas quinazolinopeptide analogs
5a-da displayed mild to moderate activity. Comparison of anthelmintic activity data revealed that
hydrolyzed peptide derivatives 5ba-6ga are slightly more active than their corresponding ester
derivatives 5b-6g. Compound 6f and its hydrolyzed derivative 6fa were found to exhibit higher
bioactivity against all three earthworm species, in comparison to the standard drug - mebendazole.
Compounds 6d, 6g and 6ga showed good anthelmintic activity whereas compounds 6a-c, 6e and 6ea
displayed only moderate level of activity. Among quinazolinopeptides, no compound exhibited any
significant anthelmintic activity except moderate activity for compounds 5e, 5b and its hydrolyzed
derivative 5ba (Table 2, Figure 2).
Conclusions
The present study reports the successful synthesis of the title compounds in good yields via
coupling reactions. For peptide coupling, the method employing DCC/TEA in DMF solvent proved to
be better than the DCC/NMM method utilizing THF as solvent, providing 10-15 % additional yields.
Gram negative bacteria proved to be more sensitive in comparison to Gram positive bacteria towards
newly synthesized peptide derivatives. Greater anthelmintic activity was found in derivatives with
histidine and tyrosine constituents in their amino acid chain. Hydrolyzed derivatives exhibited more
antimicrobial and anthelmintic activity in comparison to their corresponding methyl ester derivatives
except against dermatophytes. Among the tested compounds, 5a, 5d, 5da, 5e, 5f, 6c, 6g, 6ga, 6e and
6ea showed good antimicrobial activity and, 6f and 6fa exhibited better anthelmintic activity. On
passing toxicity tests, these compounds may prove good candidates for clinical studies and may be
potential antimicrobial and anthelmintic agents of future.

Molecules 2008, 13
965
Experimental
General
Melting points were determined by the open capillary method and are uncorrected. L-Amino acids,
di-tert-butylpyrocarbonate (Boc2O), dicyclohexylcarbodiimide (DCC), trifluoroacetic acid (TFA),
triethylamine (TEA) and N-methylmorpholine (NMM) were procured from Spectrochem Limited
(Mumbai, India). IR spectra were recorded on a Shimadzu 8700 FTIR spectrophotometer using a thin
13
film supported on KBr pellets or chloroform solutions. 1H-NMR and C-NMR spectra were recorded
on Bruker AC NMR spectrometer (300 MHz) using CDCl3 as solvent and TMS as internal standard.
The mass spectra were recorded on a JMS-DX 303 mass spectrometer (Jeol, Tokyo, Japan) operating
at 70 eV using the electron spray ionization technique (ESI MS). Optical rotation of synthesized
peptide derivatives was measured on automatic polarimeter in a 2 dm tube at 25 °C using sodium lamp
and methanol as solvent. Elemental analyses of all compounds were performed on Vario EL III
elemental analyzer. Purity of all synthesized compounds was checked by TLC on precoated silica gel
G plates utilizing chloroform/methanol in different ratios (9:1 / 7:3 v/v) as developing solvent system
and spots were detected on exposure to iodine vapours in a tightly closed chamber.
Compounds 3a, 3b and 4a were synthesized by coupling Boc-dipeptides with respective amino acid
methyl ester hydrochlorides/dipeptide methyl esters under alkaline conditions.
tert-Butyloxycarbonyl-alanyl-prolyl-tryptophan methyl ester (3a). Semisolid mass; yield 82 %; [α]D
–55.6°; Rf - 0.81; IR (CHCl3): v 3491 (N–Hstr, ring), 3122, 3117 (N–Hstr, amide), 3077, 3053 (C–Hstr,
ring), 2994-2989 (C–Hstr, CH2, pro), 2962, 2925 (C–Hstr, asym, CH3 and CH2), 2872, 2849, (C–Hstr,
sym, CH3 and CH2), 1750 (C=Ostr, ester), 1680, 1670, 1645 (C=Ostr, 3˚ and 2˚ amide), 1585, 1482
(skeletal bands), 1538, 1534 (N–Hbend, 2˚ amide), 1390, 1372 (C–Hbend, butyl-t), 1267 (C–Ostr, ester),
1
732, 675 (C–Hbend, out-of-plane (oop), ring), 478 (C–Cbend, aliphatic) cm−1; H-NMR: δ 8.95 (1H, s,
NH, ring), 8.65 (1H, br. s, NH), 7.50 (1H, s, H-β, ring), 7.20-6.98 (4H, m, H-δ−η, ring), 6.90 (1H, br.
s, NH), 5.38-5.31 (1H, m, H-α, try), 4.52-4.43 (2H, m, H-α, ala and pro), 3.64 (2H, t, H-δ, pro), 3.55
(3H, s, OCH3), 3.21 (2H, d, J = 6.8 Hz, H-β, try), 2.72-2.66 (2H, q, H-β, pro), 1.98-1.89 (2H, m, H-γ,
pro), 1.52 (9H, s, butyl-t), 1.35 (3H, d, J = 5.2 Hz, H-β, ala) ppm; Anal. Calcd. for C25H34N4O6: C,
61.7; H, 7.1; N, 11.5. Found: C, 61.5; H, 7.3; N, 11.44 %.
tert-Butyloxycarbonyl-glycyl-leucyl-histidine methyl ester (3b). Semisolid mass; yield 71 %; [α]D
–19.2°; Rf - 0.72; IR (CHCl3): v 3495 (N–Hstr, ring), 3129 (N–Hstr, amide), 3076 (C–Hstr, ring), 2928,
2852 (C–Hstr, asym and sym, CH2), 1748 (C=Ostr, ester), 1641 (C=Ostr, amide), 1583, 1488 (skeletal
bands), 1533, 1526 (N–Hdef, amide), 1390, 1368 (C–Hdef, butyl-t), 932, 925 (CH3rocking, butyl-t and
1
propyl-i), 910 (C–Hdef, oop, ring) cm−1; H-NMR (CDCl3): δ 9.10 (1H, br. s, NH, ring), 7.73 (1H, d,
J = 7.25 Hz, H at C3, ring), 7.59 (1H, s, H at C2, ring), 7.54 (1H, br. s, NH), 7.02 (1H, br. s, NH), 6.50
(1H, br. s, NH), 4.56-4.53 (1H, m, H-α, leu), 4.14-4.09 (1H, m, H-α, his), 3.55 (3H, s, OCH3), 3.52
(2H, d, J = 4.8 Hz, CH2, gly), 3.06 (2H, d, J = 6.65 Hz, H-β, his), 1.84 (2H, t, H-β, leu), 1.55 (9H, s,
butyl-t), 1.51-1.43 (1H, m, H-γ, leu), 0.98 (6H, d, J = 6.15 Hz, H-δ, leu) ppm; Anal. Calcd. for
C20H33N5O6: C, 54.66; H, 7.57; N, 15.93. Found: C, 54.65; H, 7.58; N, 15.95 %.

Molecules 2008, 13
966
tert-Butyloxycarbonyl-valyl-tyrosinyl-phenylalanyl-glycine methyl ester (4a). Semisolid mass; Yield
79 %; [α]D +37.7°; Rf - 0.66; IR (CHCl3): v 3375 (O–Hstr, tyr), 3127-3122 (N–Hstr, amide), 3075-3069,
3035 (C–Hstr, rings), 2929-2925, 2856-2849 (C–Hstr, asym and sym, CH2), 1748 (C=Ostr, ester), 1644-
1640, 1639-1637 (C=Ostr, 2° amide), 1585-1578, 1425-1420 (skeletal bands, rings), 1532-1528
(N–Hbend, 2° amide), 1465 (C–Hbend (scissoring), CH2), 1392, 1370 (C–Hbend, butyl-t), 1383, 1359
(C–Hbend, propyl-i), 1268 (C–Ostr, ester), 930, 922 (CH3rocking, butyl-t and propyl-i), 829, 730, 695
(C–Hdef, oop) cm−1; 1H-NMR: δ 8.19 (1H, br. s, NH, phe), 7.60 (1H, br. s, NH, tyr), 7.20-7.16 (2H, tt,
H-m, phe), 7.02-6.96 (3H, m, H-m, tyr and H-p, phe), 6.90-6.82 (4H, m, H-o, tyr and phe), 6.52 (1H,
br. s, NH, gly), 6.45 (1H, br. s, NH, val), 5.95 (1H, br. s, OH, tyr), 4.52-4.48 (1H, q, H-α, tyr), 4.20
(1H, t, H-α, val), 4.15-4.11 (1H, q, H-α, phe), 4.04 (2H, d, J = 5.45 Hz, H-α, gly), 3.66 (3H, s, OCH3),
2.95-2.83 (4H, m, H-β, tyr and phe), 1.86-1.74 (1H, m, H-β, val), 1.54 (9H, s, butyl-t), 1.06 (6H, d,
J = 4.6 Hz, H-γ, val) ppm; Anal. Calcd. for C31H42N4O8: C, 62.19; H, 7.07; N, 9.36. Found: C, 62.22;
H, 7.05; N, 9.35 %.
2-Hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoic acid (5)
Equimolar amounts (0.025 mol) of 6-iodo-2-methylbenzoxazin-4-one (1a, 7.18 g) and 5-ASA
(3.83 g) were heated at 170-180 °C for 2 h in an oil bath. The separated jelly-like mass solidified upon
cooling. The crude product was finally crystallized from ethanol to give a 71 % yield of pure 5 as a
white solid; m.p. 124-125 °C; Rf - 0.52; IR (KBr): v 3365 (O−Hstr, Ar−OH), 3295-2505 (O−Hstr,
COOH), 3072-3066, 3052 (Ar−Hstr), 2967, 2875 (C−Hstr, CH3), 1702 (C=Ostr, COOH), 1669 (C=Ostr,
ring), 1589, 1575, 1425, 1417 (skeletal bands), 1405 (O−Hdef, COOH), 875, 836, 760, 752, 696
1
(C−Hdef, oop), 590 (C−Istr) cm−1; H-NMR: δ 11.43 (2H, br. s, OH and COOH), 8.42 (1H, s, H-ε,
quinazolinone moiety (qz)), 8.14 (1H, d, J = 7.15 Hz, H-η, qz), 7.89 (1H, s, H-ζ, o-hydroxybenzoic
acid moiety (hba)), 7.65 (1H, d, J = 6.9 Hz, H-θ, qz), 7.50 (1H, d, J = 6.65 Hz, H-δ, hba), 6.98 (1H, d,
13
J = 6.9 Hz, H-γ, hba), 8.42 (3H, s, CH3-β, qz) ppm; C-NMR: δ 172.3 (C=O, COOH), 163.9 (C-2,
hba), 158.7 (C=O, qz), 153.4, 148.5 (2C, C-2 and C-2′, qz), 141.7, 137.8 (2C, C-7 and C-5, qz), 135.0,
131.5 (2C, C-4 and C-5, hba), 128.4, 124.1 (2C, C-8 and C-3′, qz), 123.3, 117.5 (2C, C-6 and C-3,
hba), 114.0 (C-1, hba), 95.1 (C-6, qz), 19.8 (CH3, qz) ppm; Anal. Calcd. for C16H11IN2O4 (422): C,
45.52; H, 2.63; N, 6.64. Found: C, 45.49; H, 2.60; N, 6.65 %.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoic acid (6)
A mixture of 5-ASA (7.65 g), dilute hydrochloric acid (15%, 30 mL) and water (45 mL) was
heated to give a clear solution. After cooling to 0 °C, the solution was diazotized by addition of sodium
nitrite solution (30%, 12 mL). To the filtrate of diazotized salt solution, 5-nitroimidazole (1b, 5.65 g)
and aqueous cupric chloride solution (1.3 g in 5 mL water) were added with stirring, followed by slow
addition of water (25 mL), maintaining the temperature between 5-10 °C. Stirring was continued for
6 h and finally the reaction mixture was kept overnight in the refrigerator. The separated solid was
collected by filtration and washed with cold water. The crude product was recrystallized from acetone
to give a 77 % yield of pure 6 as a pale-yellow solid; m.p. 108-110 °C; Rf - 0.67; IR (KBr): v 3488

Molecules 2008, 13
967
(N−Hstr, ring), 3362 (O−Hstr, Ar−OH), 3305-2509 (O−Hstr, COOH), 3077, 3054 (Ar−Hstr), 1697
(C=Ostr, COOH), 1588, 1576, 1428, 1419 (skeletal bands), 1542, 1349 (NO2str), 1408 (O−Hdef, COOH),
872, 838, 822, 698 (C−Hdef, oop) cm−1; 1H-NMR: δ 10.89 (2H, br. s, OH and COOH), 10.85 (1H, br. s,
NH, imidazole moiety (imz)), 9.10 (1H, s, H-ζ, hba), 8.80 (1H, d, J = 6.5 Hz, H-δ, hba), 8.52 (1H, s,
13
H-δ, imz), 6.83 (1H, d, J = 6.75 Hz, H-γ, hba) ppm; C-NMR: δ 173.8 (C=O, COOH), 164.2 (C-2,
hba), 155.4 (C-2, imz), 148.7, 139.4 (2C, C-5 and C-4, imz), 135.1, 128.6 (2C, C-6 and C-4, hba),
125.0, 122.3 (2C, C-5 and C-3, hba), 117.6 (C-1, hba) ppm; Anal. Calcd. for C10H7N3O5 (249): C,
48.20; H, 2.83; N, 16.86. Found: C, 48.22; H, 2.84; N, 16.83 %.
General procedure for synthesis of amino acid / peptide derivatives of 2-hydroxy-5-(6-iodo-2-methyl-
4-oxo-3,4-dihydro-3-quinazolinyl)benzoic acids 5a-f
Amino acid methyl ester hydrochloride/di/tri/tetrapeptide methyl ester (0.01 mol) was dissolved in
THF (75 mL). To this, NMM (2.3 mL) was added at 0 °C and the reaction mixture was stirred for
15 min. Compound 5 (4.22 g, 0.01 mol) in THF (75 mL) and DCC (2.1 g) were added to the above
mixture with stirring. After 36 h, the reaction mixture was filtered and the residue was washed with
THF (25 mL). Then, filtrate was washed with 5% NaHCO3 and saturated NaCl solutions (15 mL). The
organic layer was dried over anhydrous Na2SO4, filtered and evaporated in vacuum. The crude product
was recrystallized from a mixture of chloroform and n-hexane followed by cooling at 0 °C.
2-Hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoyl nitro(arginine) methyl ester
(5a). Semisolid mass; yield 62 %; [α]D +34.8° (c, 0.2 in MeOH); Rf - 0.63; IR (CHCl3): v 3362
(O−Hstr, Ar−OH), 3340, 3328 (N−Hstr, amine), 3122 (N−Hstr, amide), 3075-3069, 3056 (Ar−Hstr),
2965, 2872 (C−Hstr, CH3), 2926, 2919, 2856, 2850 (C−Hstr, CH2), 1738 (C=Ostr, ester), 1672 (C=Ostr,
ring), 1643 (C=Ostr, amide), 1587, 1576, 1422, 1415 (skeletal bands), 1551 (NO2str, asym), 1532
(N−Hbend, amide), 1468, 1459 (C−Hbend (scissoring), CH2), 1370 (NO2str, sym), 1269 (C−Ostr, ester),
1156 (C–Nstr, amine), 870, 833, 763, 751, 694 (C−Hdef, oop), 588 (C−Istr) cm−1; 13C-NMR: δ 173.5
(C=O, ArCO), 171.8 (C=O, arg), 160.6 (C=NH), 158.3 (C=O, qz), 156.1 (C-2, hba), 152.8, 148.9 (2C,
C-2 and C-2′, qz), 141.9, 139.0 (2C, C-7 and C-5, qz), 137.4, 132.2 (2C, C-4 and C-5, hba), 127.7
(C-8, qz), 123.1 (C-1, hba), 122.4 (C-3′, qz), 120.7, 118.0 (2C, C-6 and C-3, hba), 96.6 (C-6, qz), 54.2
(C-α, arg), 50.9 (OCH3), 40.1 (C-δ, arg), 28.0 (C-β, arg), 24.3 (C-γ, arg), 20.6 (CH3, qz) ppm; Anal.
Calcd. for C23H24IN7O7 (637): C, 43.34; H, 3.80; N, 15.38. Found: C, 43.36; H, 3.79; N, 15.42 %.
2-Hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoyl tyrosine methyl ester (5b).
Pale-yellow solid; m.p. 163-164 °C; yield 65 %; [α]D +17.7° (c, 0.2 in MeOH); Rf - 0.81; IR (KBr): v
3364, 3359 (O−Hstr, Ar−OH), 3126 (N−Hstr, amide), 3072-3068, 3058-3053 (Ar−Hstr), 2967, 2874
(C−Hstr, CH3), 2922, 2852 (C−Hstr, CH2), 1742 (C=Ostr, ester), 1670 (C=Ostr, ring), 1640 (C=Ostr,
amide), 1587-1578, 1425-1417 (skeletal bands), 1535 (N−Hbend, amide), 1272 (C−Ostr, ester), 876,
835-824, 764-753, 698 (C−Hdef, oop), 588 (C−Istr) cm−1; 1H-NMR: δ 8.43 (1H, s, H-ε, qz), 8.12 (1H, d,
J = 7.2 Hz, H-η, qz), 7.67 (1H, d, J = 6.85 Hz, H-θ, qz), 7.39 (1H, d, J = 6.7 Hz, H-δ, hba), 7.35 (1H,
s, H-ζ, hba), 7.18 (1H, d, J = 6.95 Hz, H-γ, hba), 6.90 (2H, dd, J = 8.55, 5.25 Hz, H-o, tyr), 6.77 (2H,
dd, J = 8.6, 4.9 Hz, H-m, tyr), 6.50 (1H, br. s, NH), 5.12 (2H, br. s, OH, tyr and hba), 4.68-4.62 (1H,

Molecules 2008, 13
968
m, H-α, tyr), 3.56 (3H, s, OCH3), 2.82 (2H, d, J = 4.9 Hz, H-β, tyr), 2.58 (3H, s, CH3-β, qz) ppm; Anal.
Calcd. for C26H22IN3O6 (599): C, 52.10; H, 3.70; N, 7.01. Found: C, 52.09; H, 3.72; N, 7.05 %.
2-Hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoyl prolyl-valine methyl ester
(5c). Off-white solid; m.p. 102-103 °C; yield 69 %; [α]D −53.2° (c, 0.2 in MeOH); Rf - 0.72; IR (KBr):
v 3367 (O−Hstr, Ar−OH), 3124 (N−Hstr, amide), 3069, 3055 (Ar−Hstr), 2995-2989 (C−Hstr, CH2, pro),
2965, 2876 (C−Hstr, CH3), 1746 (C=Ostr, ester), 1668 (C=Ostr, ring), 1659, 1642 (C=Ostr, 3° and 2°
amide), 1582, 1423 (skeletal bands), 1531 (N−Hbend, amide), 1385, 1362 (C−Hbend, propyl-i), 1270
(C−Ostr, ester), 1224 (C–Ostr, phenolic), 875, 832, 767, 750, 692 (C−Hdef, oop), 589 (C−Istr) cm−1;
13C-NMR: δ 174.9 (C=O, val), 172.7 (C=O, pro), 171.9 (C=O, ArCO), 158.1 (C=O, qz), 155.7 (C-2,
hba), 152.5, 149.3 (2C, C-2 and C-2′, qz), 141.6, 137.7 (2C, C-7 and C-5, qz), 137.1, 131.9 (2C, C-4
and C-5, hba), 128.8 (C-8, qz), 122.5 (C-1, hba), 121.7 (C-3′, qz), 120.5, 117.2 (2C, C-6 and C-3, hba),
95.3 (C-6, qz), 70.7 (C-α, pro), 58.3 (C-α, val), 52.1 (OCH3), 45.5 (C-δ, pro), 31.4, 29.8 (2C, C-β, val
and pro), 28.7 (C-γ, pro), 21.2 (2C, C-γ, val), 19.9 (CH3, qz) ppm; Anal. Calcd. for C27H29IN4O6 (632):
C, 51.28; H, 4.62; N, 8.86. Found: C, 51.30; H, 4.61; N, 8.85 %.
2-Hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoyl leucyl-phenylalanine methyl
ester (5d). White crystals; m.p. 90-92 °C; yield 71 %; [α]D +4.8° (c, 0.2 in MeOH); Rf - 0.60; IR
(KBr): v 3365 (O−Hstr, Ar−OH), 3129-3125 (N−Hstr, amide), 3067, 3059-3955 (Ar−Hstr), 2966, 2874
(C−Hstr, CH3), 2928-2924, 2853 (C−Hstr, CH2), 1749 (C=Ostr, ester), 1669 (C=Ostr, ring), 1645, 1641
(C=Ostr, 2° amide), 1587-1583, 1426-1422 (skeletal bands), 1534, 1531 (N−Hbend, amide), 1387, 1363
(C−Hbend, propyl-i), 1268 (C−Ostr, ester), 1226 (C–Ostr, phenolic), 878, 830, 765-754,712-695 (C−Hdef,
oop), 587 (C−Istr) cm−1; 1H-NMR: δ 8.41 (1H, s, H-ε, qz), 8.10 (1H, d, J = 7.15 Hz, H-η, qz), 7.65 (1H,
d, J = 6.9 Hz, H-θ, qz), 7.61 (1H, br. s, NH), 7.58 (1H, s, H-ζ, hba), 7.38 (1H, d, J = 6.65 Hz, H-δ,
hba), 7.14 (1H, t, J = 6.15 Hz, H-p, phe), 7.05 (1H, br. s, NH), 6.98 (2H, m, H-m, phe), 6.95 (1H, d,
J = 6.9 Hz, H-γ, hba), 6.84 (2H, dd, J = 8.75, 4.1 Hz, H-o, phe), 4.48-4.42 (1H, m, H-α, leu), 4.27 (1H,
br. s, OH), 3.94-3.89 (1H, m, H-α, phe), 3.55 (3H, s, OCH3), 2.98 (2H, d, J = 5.6 Hz, H-β, phe), 2.60
(3H, s, CH3-β, qz), 1.72 (2H, t, J = 4.9 Hz, H-β, leu), 1.50-1.42 (1H, m, H-γ, leu), 0.98 (6H, d,
J = 6.2 Hz, H-δ, leu) ppm; Anal. Calcd. for C32H33IN4O6 (696): C, 55.18; H, 4.78; N, 8.04. Found: C,
55.19; H, 4.80; N, 8.02 %.
2-Hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoyl-histidinyl-phenylalanine
methyl ester (5e). Semisolid mass; yield 63 %; [α]D −14.9° (c, 0.2 in MeOH); Rf - 0.55; IR (CHCl3): v
3489 (N–Hstr, ring), 3368 (O−Hstr, Ar−OH), 3127-3123 (N−Hstr, amide), 3068-3063, 3058-3954
(Ar−Hstr), 2964, 2876 (C−Hstr, CH3), 2929-2923, 2852 (C−Hstr, CH2), 1751 (C=Ostr, ester), 1667
(C=Ostr, ring), 1644, 1639 (C=Ostr, 2° amide), 1589-1582, 1428-1423 (skeletal bands), 1536, 1533
(N−Hbend, amide), 1269 (C−Ostr, ester), 1224 (C–Ostr, phenolic), 878, 848-839, 765, 758, 714-698
(C−Hdef, oop), 589 (C−Istr) cm−1; 13C-NMR: δ 173.2 (C=O, his), 169.8 (C=O, phe), 170.5 (C=O,
ArCO), 157.6 (C=O, qz), 156.0 (C-2, hba), 153.3, 149.1 (2C, C-2 and C-2′, qz), 146.7 (C-2, imz),
141.5 (C-7, qz), 137.1 (C-γ, phe), 136.4 (C-5, qz), 135.1, 133.9 (2C, C-4 and C-5, hba), 129.1 (2C,
C-o, phe), 128.4 (2C, C-m, phe), 127.2 (C-p, phe), 126.4 (C-5, imz), 124.9 (C-8, qz), 122.6 (C-3′, qz),
120.2 (C-1, hba), 119.4, 117.7 (2C, C-6 and C-3, hba), 115.3 (C-γ, his), 94.9 (C-6, qz), 60.6 (C-α, his),

Molecules 2008, 13
969
55.9 (C-α, phe), 53.7 (OCH3), 38.2, 26.5 (2C, C-β, phe and his), 18.7 (CH3, qz) ppm; Anal. Calcd. for
C32H29IN6O6 (720): C, 53.34; H, 4.06; N, 11.66. Found: C, 53.35; H, 4.06; N, 11.68 %.
2-Hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoyl alanyl-prolyl-tryptophan
methyl ester (5f). Semisolid mass; yield 66 %; [α]D −102.6° (c, 0.5 in MeOH); Rf - 0.76; IR (CHCl3): v
3483 (N–Hstr, ring), 3358 (O−Hstr, Ar−OH), 3133-3126 (N−Hstr, amide), 3069-3065, 3058 (Ar−Hstr),
2996-2989 (C−Hstr, CH2, pro), 2968, 2963, 2876 (C−Hstr, CH3), 2926, 2854 (C−Hstr, CH2), 1747
(C=Ostr, ester), 1667 (C=Ostr, ring), 1658, 1643, 1639 (C=Ostr, 3° and 2° amide), 1589-1582, 1428-
1424 (skeletal bands), 1536, 1533 (N−Hbend, amide), 1274 (C−Ostr, ester), 1223 (C–Ostr, phenolic), 878,
835-829, 768, 715, 698-693 (C−Hdef, oop), 592 (C−Istr) cm−1; MS (m/z, %): 792 (2.8), 791 (3.4), 790
(M+, 5.9), 775 (9.2), 759 (26.5), 731 (8.1), 573 (100), 545 (11.4), 476 (72.7), 448 (33.1), 405 (54.9),
377 (20.6), 285 (19.3), 228 (26.6), 159 (14.7), 130 (10.3), 126 (11.5), 116 (7.7), 93 (9.7), 70 (8.2), 59
(21.8), 44 (17.4), 42 (10.5), 31 (11.9), 15 (9.2); Anal. Calcd. for C36H35IN6O7 (790): C, 54.69; H, 4.46;
N, 10.63. Found: C, 54.72; H, 4.45; N, 10.65 %.
General procedure for synthesis of amino acid/peptide derivatives of 2-hydroxy-5-(5-nitro-1H-
imidazol-2-yl)benzoic acids 6a-g
To a mixture of amino acid methyl ester hydrochloride/di/tri/tetrapeptide methyl ester (0.01 mol) in
DMF (50 mL), TEA (2.8 mL) was added at 0 °C with stirring. Compound 6 (2.5 g, 0.01 mol) in DMF
(50 mL) and DCC (2.1 g) were added to the above mixture and stirring was done for 24 h. After 24 h,
the reaction mixture was filtered. To the filtrate, water was added in equal proportions and the aqueous
layer was washed with ether (3 × 50 mL). The organic layer was separated and dried over anhydrous
Na2SO4, filtered and evaporated under vacuum. The product obtained was dissolved in chloroform,
washed with 10 % HCl, saturated NaHCO3 solution and water (25 mL each) followed by evaporation
under vacuum. The crude product was recrystallized from a mixture of ethyl acetate and petroleum
ether.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoyl threonine methyl ester (6a). Semisolid mass; yield
79 %; [α]D −22.4° (c, 0.35 in MeOH); Rf - 0.53; IR (CHCl3): v 3485 (N−Hstr, ring), 3366 (O−Hstr,
Ar−OH), 3132, 3127 (N−Hstr, amide), 3075, 3052 (Ar−Hstr), 2969, 2874 (C−Hstr, CH3), 1744 (C=Ostr,
ester), 1642 (C=Ostr, 2° amide), 1586, 1424 (skeletal bands), 1540, 1348 (NO2str), 1532 (N−Hbend
,
amide), 1422, 1329 (O−Hdef), 1271 (C−Ostr, ester), 1225 (C–Ostr, phenolic), 870, 839, 820, 696
(C−Hdef, oop) cm−1; 1H-NMR: δ 9.38 (1H, s, H-δ, imz), 8.90 (1H, s, H-ζ, hba), 8.85 (1H, d, J = 6.5 Hz,
H-δ, hba), 7.05 (1H, d, J = 6.85 Hz, H-γ, hba), 6.50 (1H, br. s, NH), 5.75 (3H, br. s, OH, hba and thr,
NH, imz), 4.65-4.57 (1H, m, H-β, thr), 4.52-4.48 (1H, m, H-α, thr), 3.60 (3H, s, OCH3), 1.32 (3H, d,
J = 4.95 Hz, H-γ, thr) ppm; Anal. Calcd. for C15H16N4O7 (364): C, 49.45; H, 4.43; N, 15.38. Found: C,
49.49; H, 4.40; N, 15.39 %.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoyl proline methyl ester (6b). Semisolid mass; yield 85 %;
[α]D +71.5° (c, 0.35 in MeOH); Rf - 0.69; IR (CHCl3): v 3482 (N−Hstr, ring), 3368 (O−Hstr, Ar−OH),
3133 (N−Hstr, amide), 3077, 3055 (Ar−Hstr), 2998-2992 (C−Hstr, CH2, pro), 1742 (C=Ostr, ester), 1660,

Molecules 2008, 13
970
1644 (C=Ostr, 3° and 2° amide), 1584, 1421 (skeletal bands), 1544, 1347 (NO2str), 1535 (N−Hbend
,
amide), 1269 (C−Ostr, ester), 1223 (C–Ostr, phenolic), 869, 835, 824, 695 (C−Hdef, oop) cm−1;
13C-NMR: δ 178.5 (C=O, ArCO), 170.3 (C=O, pro), 159.6 (C-2, hba), 153.3 (C-2, imz), 154.2, 138.0
(2C, C-5 and C-4, imz), 133.7 (C-4, hba), 130.6 (C-1, hba), 126.9, 123.7 (2C, C-6 and C-3, hba), 120.8
(C-5, hba), 65.8 (C-α, pro), 54.0 (OCH3), 46.5 (C-δ, pro), 29.2 (C-β, pro), 26.0 (C-γ, pro) ppm; Anal.
Calcd. for C16H16N4O6 (360): C, 53.33; H, 4.48; N, 15.55. Found: C, 53.32; H, 4.50; N, 15.58 %.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoyl glycyl-glycine methyl ester (6c). White solid; m.p.
155 °C; yield 80 %; [α]D −57.4° (c, 0.35 in MeOH); Rf - 0.66; 1H-NMR: δ 9.35 (1H, s, H-δ, imz), 9.15
(1H, s, H-ζ, hba), 8.88 (1H, d, J = 6.45 Hz, H-δ, hba), 8.62 (1H, br. s, NH), 8.15 (1H, br. s, NH), 6.98
(2H, br. s, OH, hba and NH, imz), 6.87 (1H, d, J = 6.8 Hz, H-γ, hba), 4.05 (2H, d, J = 5.15 Hz, H-α,
13
gly-2), 3.90 (2H, d, J = 5.2 Hz, H-α, gly-1), 3.48 (3H, s, OCH3) ppm; C-NMR: δ 177.3, 171.5 (2C,
C=O, gly-1 and gly-2), 165.7 (C=O, ArCO), 161.9 (C-2, hba), 156.0 (C-2, imz), 149.5, 139.9 (2C, C-5
and C-4, imz), 134.3 (C-4, hba), 129.2 (C-1, hba), 125.6, 122.4 (2C, C-6 and C-5, hba), 119.5 (C-3,
hba), 54.1 (OCH3), 47.2 (C-α, gly-1), 41.6 (C-α, gly-2) ppm; Anal. Calcd. for C15H15N5O7 (377): C,
47.75; H, 4.01; N, 18.56. Found: C, 47.78; H, 3.98; N, 18.55 %.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoyl isoleucyl-tyrosine methyl ester (6d). Semisolid mass;
yield 88 %; [α]D +29.4° (c, 0.35 in MeOH); Rf - 0.58; 13C-NMR: δ 172.0, 170.6 (2C, C=O, ile and tyr),
166.8 (C=O, ArCO), 157.6 (C-2, hba), 154.0 (C-p, tyr), 153.4 (C-2, imz), 146.2, 139.2 (2C, C-5 and
C-4, imz), 134.0 (C-4, hba), 133.3 (2C, C-m, tyr), 130.6 (2C, C-o, tyr), 127.9 (C-1, hba), 125.0, 123.7
(2C, C-6 and C-5, hba), 118.9 (C-3, hba), 117.5 (C-γ, tyr), 58.2 (C-α, ile), 55.7 (C-α, tyr), 53.1
(OCH3), 39.7 (C-β, tyr), 34.2 (C-β, ile), 30.1 (C-γ, ile), 14.5 (C-γ′, ile), 10.2 (C-δ, ile) ppm; 1H-NMR:
δ 9.37 (1H, s, H-δ, imz), 9.10 (1H, s, H-ζ, hba), 8.85 (1H, d, J = 6.5 Hz, H-δ, hba), 7.50 (1H, br. s,
NH), 7.16 (1H, br. s, NH), 6.92 (1H, d, J = 6.75 Hz, H-γ, hba), 6.88 (2H, dd, J = 8.6, 5.3 Hz, H-o, tyr),
6.80 (2H, dd, J = 8.55, 4.85 Hz, H-m, tyr), 6.65 (3H, br. s, OH, hba and tyr, NH, imz), 4.79-4.75 (1H,
m, H-α, ile), 3.96-3.92 (1H, m, H-α, tyr), 3.55 (3H, s, OCH3), 2.78 (2H, d, J = 4.85 Hz, H-β, tyr), 1.59-
1.47 (3H, m, H-β and H-γ, ile), 0.98 (3H, t, J = 7.8 Hz, H-δ, ile), 0.95 (3H, d, J = 5.9 Hz, H-γ′, ile)
ppm; Anal. Calcd. for C26H29N5O8 (539): C, 57.88; H, 5.42; N, 12.98. Found: C, 57.90; H, 5.40; N,
13.02 %.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoyl phenylalanyl-proline methyl ester (6e). Semisolid
mass; yield 86 %; [α]D −109.2° (c, 0.35 in MeOH); Rf - 0.71; 13C-NMR: δ 174.5, 168.1 (2C, C=O, pro
and phe), 165.4 (C=O, ArCO), 158.3 (C-2, hba), 156.6 (C-2, imz), 154.7, 139.0 (2C, C-5 and C-4,
imz), 135.4 (C-γ, phe), 132.5 (C-4, hba), 131.5 (2C, C-o, phe), 129.9 (2C, C-m, phe), 127.7 (C-1, hba),
127.0 (C-p, phe), 134.2, 124.8, 122.0 (3C, C-6, C-5, C-3, hba), 58.6 (C-α, pro), 53.9 (OCH3), 52.1
1
(C-α, phe), 45.6 (C-δ, pro), 38.5 (C-β, phe), 29.5, 23.8 (2C, C-β and C-γ, pro) ppm; H-NMR: δ 9.38
(1H, s, H-δ, imz), 9.13 (1H, s, H-ζ, hba), 8.88 (1H, d, J = 6.55 Hz, H-δ, hba), 7.86 (1H, br. s, NH),
7.22 (2H, m, H-m, phe), 7.05 (1H, t, J = 6.2 Hz, H-p, phe), 6.95 (2H, br. s, OH, hba and NH, imz),
6.90 (1H, d, J = 6.8 Hz, H-γ, hba), 6.82 (2H, dd, J = 8.8, 4.1 Hz, H-o, phe), 4.69-4.65 (1H, m, H-α,
phe), 3.90 (1H, t, J = 6.9 Hz, H-α, pro), 3.62 (3H, s, OCH3), 3.40 (2H, t, J = 7.2 Hz, H-δ, pro), 2.78

Molecules 2008, 13
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(2H, t, J = 5.6 Hz, H-β, phe), 2.05-1.97 (4H, m, H-γ and H-δ, pro) ppm; Anal. Calcd. for C25H25N5O7
(507): C, 59.17; H, 4.97; N, 13.80. Found: C, 59.20; H, 4.94; N, 13.82 %.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoyl glycyl-leucyl-histidine methyl ester (6f). Semisolid
mass; yield 83 %; [α]D +7.2° (c, 0.5 in MeOH); Rf - 0.76; IR (CHCl3): v 3493, 3489 (N−Hstr, ring),
3365 (O−Hstr, Ar−OH), 3132-3128 (N–Hstr, amide), 3076-3072, 3056 (Ar−Hstr), 2929-2923, 2854-2851
(C–Hstr, CH2), 1752 (C=Ostr, ester), 1645-1641 (C=Ostr, amide), 1589-1575, 1429-1422 (skeletal
bands), 1535-1531, 1527 (N–Hdef, amide), 1544, 1346 (NO2str), 1385, 1362 (C−Hbend, propyl-i), 1272
(C−Ostr, ester), 1227 (C–Ostr, phenolic), 912, 869, 839-828, 696 (C−Hdef, oop) cm−1; MS (m/z, %): 572
(1.9), 571 (2.7), 570 (M+, 4.1), 555 (6.9), 539 (17.7), 511 (15.1), 402 (59.3), 374 (11.3), 289 (100), 261
(38.6), 232 (31.4), 204 (15.9), 110 (13.8), 86 (17.4), 81 (10.2), 55 (6.7), 54 (4.4), 112 (16.3), 93 (14.4),
86 (12.8), 85 (7.5), 59 (17.9), 57 (10.5), 46 (7.7), 43 (11.3), 42 (3.9), 30 (6.6), 31 (12.7), 15 (3.6);
Anal. Calcd. for C25H30N8O8 (570): C, 52.63; H, 5.30; N, 19.64. Found: C, 52.65; H, 5.29; N, 19.66 %.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoyl valyl-tyrosinyl-phenylalanyl-glycine methyl ester (6g).
Semisolid mass; yield 90 %; [α]D −49.1° (c, 0.5 in MeOH); Rf - 0.85; IR (CHCl3): v 3492 (N−Hstr,
ring), 3365, 3362 (O−Hstr, Ar−OH), 3132-3125 (N−Hstr, amide), 3079-3072, 3057 (Ar−Hstr), 2966,
2874 (C−Hstr, CH3), 2925, 2922, 2857, 2852 (C−Hstr, CH2), 1748 (C=Ostr, ester), 1645-1638 (C=Ostr, 2°
amide), 1589-1575, 1429-1422 (skeletal bands), 1544, 1347 (NO2str), 1539-1532 (N−Hbend, amide),
1469, 1457 (CHbend (scissoring), CH2), 1388, 1362 (C−Hbend, propyl-i), 1268 (C−Ostr, ester), 1226
(C–Ostr, phenolic), 870, 839-831, 758-752, 695 (C−Hdef, oop) cm−1; MS (m/z, %): 730 (3.2), 729 (M+,
4.7), 714 (8.6), 698 (19.9), 670 (12.7), 641 (69.6), 613 (10.2), 494 (100), 466 (13.5), 331 (57.3), 303
(20.9), 232 (32.6), 204 (16.7), 136 (13.9), 120 (11.4), 112 (14.3), 107 (17.2), 93 (19.8), 91 (15.1), 86
(11.3), 85 (7.9), 72 (9.7), 66 (11.5), 59 (19.5), 46 (8.9), 43 (12.8), 42 (5.7), 30 (7.1), 31 (13.2), 15 (4.5);
Anal. Calcd. for C36H39N7O10 (729): C, 59.25; H, 5.39; N, 13.44. Found: C, 59.28; H, 5.40; N, 13.45
%.
General method for hydrolysis of amino acid /peptide derivatives of 2-hydroxy-5-(6-iodo-2-methyl-4-
oxo-3,4-dihydro-3-quinazolinyl)benzoic acid (5) / 2-hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoic acid
(6)
To a solution of the amino acid / peptide methyl ester (0.01 mol) in THF-H2O (1:1, 36 mL), LiOH
(0.36 g) was added at 0 °C. The mixture was stirred at RT for 1 h and then acidified to pH 3.5 with
1N H2SO4. The aqueous layer was extracted with Et2O (3 × 25 mL). The combined organic extracts
were dried over anhydrous Na2SO4 and concentrated under reduced pressure. The crude products were
recrystallized from methanol and ether to afford hydrolyzed peptide derivatives 5ba-da and 6ea-ga.
2-Hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoyl tyrosine (5ba). Yellowish
solid, m.p. 195 °C; yield 70 %; [α]D +30.3° (c, 0.25 in MeOH); Rf - 0.64; IR (KBr): v 3366, 3357
(O−Hstr, Ar−OH), 3298-2509 (O−Hstr, COOH), 3122 (N−Hstr, amide), 3075, 3069, 3055 (Ar−Hstr),
2965, 2872 (C−Hstr, CH3), 2920, 2855 (C−Hstr, CH2), 1710 (C=Ostr, COOH), 1672 (C=Ostr, ring), 1644
(C=Ostr, amide), 1589-1582, 1424-1418 (skeletal bands), 1533 (N−Hbend, amide), 1408 (O−Hdef,

Molecules 2008, 13
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1
COOH), 879, 832-826, 765-752, 695 (C−Hdef, oop), 589 (C−Istr) cm−1; H-NMR: δ 8.42 (1H, s, H-ε,
qz), 8.32 (1H, br. s, NH), 8.10 (1H, d, J = 7.15 Hz, H-η, qz), 7.61 (1H, d, J = 6.9 Hz, H-θ, qz), 7.42
(3H, br. s, OH, tyr, hba and COOH), 7.38 (1H, d, J = 6.65 Hz, H-δ, hba), 7.33 (1H, s, H-ζ, hba), 7.19
(2H, dd, J = 8.6, 5.25 Hz, H-o, tyr), 7.08 (1H, d, J = 7.0 Hz, H-γ, hba), 6.75 (2H, dd, J = 8.6, 4.85 Hz,
H-m, tyr), 4.92-4.86 (1H, m, H-α, tyr), 2.91 (2H, d, J = 4.85 Hz, H-β, tyr), 2.59 (3H, s, CH3-β, qz)
ppm; Anal. Calcd. for C25H20IN3O6 (585): C, 51.30; H, 3.44; N, 7.18. Found: C, 51.30; H, 3.47; N,
7.15 %.
2-Hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoyl prolyl-valine (5ca). Semi-
solid mass; yield 73 %; [α]D −81.9° (c, 0.25 in MeOH); Rf - 0.54; IR (CHCl3): v 3366 (O−Hstr,
Ar−OH), 3306-2512 (O−Hstr, COOH), 3126 (N−Hstr, amide), 3066, 3052 (Ar−Hstr), 2994-2988 (C−Hstr,
CH2, pro), 2962, 2874 (C−Hstr, CH3), 1712 (C=Ostr, COOH), 1669 (C=Ostr, ring), 1658, 1644 (C=Ostr,
3° and 2° amide), 1587, 1422 (skeletal bands), 1536 (N−Hbend, amide), 1405 (O−Hdef, COOH), 1386,
1360 (C−Hbend, propyl-i), 1228 (C–Ostr, phenolic), 876, 835, 765-753, 690 (C−Hdef, oop), 586 (C−Istr)
cm−1; 13C-NMR: δ 175.4 (C=O, COOH), 171.9 (C=O, pro), 170.2 (C=O, ArCO), 157.8 (C=O, qz),
156.1 (C-2, hba), 152.7, 150.1 (2C, C-2 and C-2′, qz), 141.2, 136.8 (2C, C-7 and C-5, qz), 135.3, 131.5
(2C, C-4 and C-5, hba), 127.3 (C-8, qz), 124.0 (C-1, hba), 123.5 (C-3′, qz), 120.8, 116.4 (2C, C-6 and
C-3, hba), 95.1 (C-6, qz), 71.5 (C-α, pro), 57.7 (C-α, val), 47.1 (C-δ, pro), 30.8, 28.9 (2C, C-β, val and
pro), 28.3 (C-γ, pro), 18.6 (2C, C-γ, val), 16.9 (CH3, qz) ppm; Anal. Calcd. for C26H27IN4O6 (618): C,
50.50; H, 4.40; N, 9.06. Found: C, 50.48; H, 4.43; N, 9.05 %.
2-Hydroxy-5-(6-iodo-2-methyl-4-oxo-3,4-dihydro-3-quinazolinyl)benzoyl leucyl-phenylalanine (5da).
White solid; m.p. 151-152 °C; yield 69 %; [α]D +15.8° (c, 0.25 in MeOH); Rf - 0.68; IR (CHCl3): v
3368 (O−Hstr, Ar−OH), 3296-2502 (O−Hstr, COOH), 3129-3126 (NHstr, amide), 3065, 3058-3953
(Ar−Hstr), 2968, 2876 (C−Hstr, CH3), 2929-2925, 2852 (C−Hstr, CH2), 1711 (C=Ostr, COOH), 1668
(C=Ostr, ring), 1646, 1643 (C=Ostr, 2° amide), 1589-1583, 1425-1420 (skeletal bands), 1537, 1532
(N−Hbend, amide), 1402 (O−Hdef, COOH), 1387, 1362 (C−Hbend, propyl-i), 1225 (C–Ostr, phenolic),
1
879, 833, 768-756, 711-692 (C−Hdef, oop), 588 (C−Istr) cm−1; H-NMR: δ 8.43 (1H, s, H-ε, qz), 8.17
(2H, br. s, OH, hba and COOH), 8.12 (1H, d, J = 7.2 Hz, H-η, qz), 7.65 (1H, d, J = 6.85 Hz, H-θ, qz),
7.60 (1H, br. s, NH), 7.57 (1H, s, H-ζ, hba), 7.39 (1H, d, J = 6.7 Hz, H-δ, hba), 7.29 (1H, br. s, NH),
7.10 (1H, t, J = 6.2 Hz, H-p, phe), 7.05 (2H, dd, J = 8.8, 4.15 Hz, H-o, phe), 6.98 (1H, d, J = 6.85 Hz,
H-γ, hba), 6.95 (2H, m, H-m, phe), 5.41-5.37 (1H, m, H-α, leu), 4.19-4.14 (1H, m, H-α, phe), 2.94 (2H,
d, J = 5.55 Hz, H-β, phe), 2.58 (3H, s, CH3-β, qz), 1.70 (2H, t, J = 4.85 Hz, H-β, leu), 1.51-1.44 (1H,
m, H-γ, leu), 0.99 (6H, d, J = 6.15 Hz, H-δ, leu) ppm; Anal. Calcd. for C31H31IN4O6 (682): C, 54.55; H,
4.58; N, 8.21. Found: C, 54.58; H, 4.58; N, 8.24 %.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoyl phenylalanyl-proline (6ea). White crystals; m.p.
123-125 °C; yield 68 %; [α]D −87.4° (c, 0.35 in MeOH); Rf - 0.51; 13C-NMR: δ 177.2 (C=O, COOH),
168.8 (C=O, ArCO), 166.2 (C=O, phe), 158.0 (C-2, hba), 156.2 (C-2, imz), 152.9, 140.9 (2C, C-5 and
C-4, imz), 140.4 (C-γ, phe), 134.1 (C-4, hba), 131.1 (2C, C-o, phe), 129.4 (2C, C-m, phe), 127.5 (C-1,
hba), 127.2 (C-p, phe), 126.2, 124.1, 121.8 (3C, C-6, C-5, C-3, hba), 61.2 (C-α, pro), 51.5 (C-α, phe),
45.2 (C-δ, pro), 36.6 (C-β, phe), 30.2, 24.5 (2C, C-β and C-γ, pro) ppm; 1H-NMR: δ 9.35 (1H, s, H-δ,

Molecules 2008, 13
973
imz), 9.11 (1H, s, H-ζ, hba), 8.86 (1H, d, J = 6.6 Hz, H-δ, hba), 8.15 (3H, br. s, OH, hba and COOH,
NH, imz), 7.85 (1H, br. s, NH), 7.20 (2H, m, H-m, phe), 7.02 (1H, t, J = 6.15 Hz, H-p, phe), 6.92 (1H,
d, J = 6.75 Hz, H-γ, hba), 6.84 (2H, dd, J = 8.75, 4.15 Hz, H-o, phe), 5.89-5.85 (1H, m, H-α, phe),
4.07 (1H, t, J = 6.85 Hz, H-α, pro), 3.38 (2H, t, J = 7.15 Hz, H-δ, pro), 2.80 (2H, t, J = 5.55 Hz, H-β,
phe), 2.07-1.98 (4H, m, H-γ and H-δ, pro) ppm; Anal. Calcd. for C24H23N5O7 (493): C, 58.42; H, 4.70;
N, 14.19. Found: C, 58.39; H, 4.70; N, 14.22 %.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoyl glycyl-leucyl-histidine (6fa). Semisolid mass; yield
71 %; [α]D +19.5° (c, 0.35 in MeOH); Rf - 0.84; IR (CHCl3): v 3492, 3489 (N−Hstr, ring), 3368
(O−Hstr, Ar−OH), 3298-2510 (O−Hstr, COOH), 3130-3125 (N–Hstr, amide), 3077-3073, 3055
(Ar−Hstr), 2929-2922, 2855-2851 (C–Hstr, CH2), 1712 (C=Ostr, COOH), 1644-1640 (C=Ostr, amide),
1588-1576, 1427-1423 (skeletal bands), 1538-1533, 1525 (N–Hdef, amide), 1543, 1345 (NO2str), 1409
(O−Hdef, COOH), 1385, 1360 (C−Hbend, propyl-i), 1222 (C–Ostr, phenolic), 910, 867, 838, 829, 698
(C−Hdef, oop) cm−1; MS (m/z, %): 557 (1.9), 556 (M+, 3.2), 539 (22.9), 511 (17.3), 402 (55.3), 374
(10.6), 289 (100), 261 (39.2), 232 (27.7), 204 (12.8), 110 (11.5), 86 (15.9), 81 (11.8), 55 (8.8), 54
(3.7), 112 (12.9), 93 (19.3), 86 (11.5), 85 (7.9), 57 (12.7), 46 (8.9), 45 (14.4), 43 (10.2), 42 (4.5), 30
(5.8), 17 (3.6), 15 (2.9); Anal. Calcd. for C24H28N8O8 (556): C, 51.80; H, 5.07; N, 20.13. Found: C,
51.77; H, 5.05; N, 20.15 %.
2-Hydroxy-5-(5-nitro-1H-imidazol-2-yl)benzoyl valyl-tyrosinyl-phenylalanyl-glycine (6ga). Semisolid
mass; yield 70 %; [α]D −81.9° (c, 0.35 in MeOH); Rf - 0.61; IR (CHCl3): v 3489 (N−Hstr, ring), 3362,
3359 (O−Hstr, Ar−OH), 3298-2510 (O−Hstr, COOH), 3133-3127 (N−Hstr, amide), 3077-3072, 3053
(Ar−Hstr), 2965, 2876 (C−Hstr, CH3), 2925-2921, 2856, 2850 (C−Hstr, CH2), 1713 (C=Ostr, COOH),
1643-1637 (C=Ostr, 2° amide), 1587-1574, 1427-1422 (skeletal bands), 1542, 1345 (NO2str), 1537-1532
(N−Hbend, amide), 1467, 1459 (C−Hbend (scissoring), CH2), 1407 (O−Hdef, COOH), 1389, 1360
(C−Hbend, propyl-i), 1224 (C–Ostr, phenolic), 872, 838, 830, 756-753, 698 (C−Hdef, oop) cm−1; MS
(m/z, %): 715 (M+, 3.9), 698 (25.2), 670 (18.9), 641 (63.8), 613 (8.9), 494 (100), 466 (11.2), 331
(59.4), 303 (25.4), 232 (38.2), 204 (14.6), 136 (13.2), 120 (16.1), 112 (11.8), 107 (16.7), 93 (18.9), 91
(13.6), 86 (16.7), 85 (11.6), 72 (7.9), 66 (10.7), 46 (6.7), 45 (10.9), 43 (11.2), 42 (5.9), 30 (6.8), 17
(2.8), 15 (3.6); Anal. Calcd. for C35H37N7O10 (715): C, 58.74; H, 5.21; N, 13.70. Found: C, 58.75; H,
5.20; N, 13.74 %.
Antimicrobial activity
Experimental details of the antimicrobial and anthelmintic activity test procedures are given in our
previously published reports [28]. All the newly synthesized compounds 5a-6ga were evaluated for
their antimicrobial activity against four bacterial strains: Bacillus subtilis (NCIM 2063),
Staphylococcus aureus (NCIM 2079), Pseudomonas aeruginosa (NCIM 2034) and Klebsiella
pneumoniae (NCIM 2011) and four fungal strains: Microsporum audouinii (MUCC 545),
Trichophyton mentagrophytes (MUCC 665), Candida albicans (MUCC 29) and Aspergillus niger
(MUCC 177) at 50-6 μg mL–1 concentration, according to the modified Kirby-Bauer disk diffusion
method [31]. MIC values of test compound were determined by the tube dilution technique. The

Molecules 2008, 13
974
solvents DMF/DMSO were used as negative controls and ciprofloxacin/griseofulvin were used as
standards. Diameters of the zones of inhibition (in mm) were measured and the average diameters for
test sample were calculated for triplicate sets. The diameters obtained for the test sample were
compared with that produced by the standard drug - ciprofloxacin. The antibacterial study results are
presented in Table 1.
Anthelmintic activity
Anthelmintic activity studies were carried out against three different species of earthworms:
Megascoplex konkanensis (ICARBC 211), Pontoscotex corethruses (ICARBC 117) and Eudrilus
eugeniea (ICARBC 042) at 2 mg mL−1 concentration following Garg’s method [32]. Tween 80 (0.5%)
in distilled water was used as control and mebendazole was used as a reference compound. The
paralysis and death times were noted and their mean was calculated for triplicate sets. The death time
was ascertained by placing the earthworms in warm water (50 °C) which stimulated the movement, if
the worm was alive. The anthelmintic study results are tabulated in Table 2.
Acknowledgements
We greatfully acknowledge the Head, U.S.I.C., DU, Delhi (India) and Head, R.S.I.C., I.I.T., Delhi
(India) for spectral analysis. Also, great thanks to the Head, C.P.C.R.I., Kasaragod, Kerala (India) for
providing earthworms for testing anthelmintic activity.
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evaluation of imidazo(4,5-b)pyridinylethoxypiperidones. Eur. J. Med. Chem. 2006, 41, 268-275.
3. Sengupta, A. K.; Bhattacharya, T. Synthesis and antimicrobial activity of some substituted
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