Synthesis and biological evaluation of N-dehydrodipeptidyl-N,N′- dicyclohexylurea analogs

Article abstract of DOI:10.1016/j.ejmech.2014.03.037

Some N-[(N-benzoyldehydrophenylalalnyl)glycinyl/cysteinyl]-N,N′- dicyclohexylurea analogs (3a-3o) were synthesized by conjugating different substituted N-benzoyldehydrophenylalanyl glycines/cysteines (differing substitutions on benzylidene ring) and dicyclohexyl carbodiimide (DCC) using base as a catalyst. The synthesized compounds were characterized and evaluated for biological activities. Compounds 3a and 3h with unsubstituted dehydrophenylalanyl glycinyl/cysteinyl moiety exhibited moderate anti-inflammatory and analgesic activities. Compound 3j bearing 4-hydroxy substitution on benzylidene ring of dehydrophenylalanyl cysteinyl moiety displayed potent antimicrobial and antioxidant activities. The results obtained from docking studies on compound 3j with penicillin binding protein and protease supported the results.

Full text of DOI:10.1016/j.ejmech.2014.03.037

European Journal of Medicinal Chemistry 78 (2014) 72e85
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of N-dehydrodipeptidyl-N,N0-
dicyclohexylurea analogsq
*
B. Haseena banu , K.V.S.R.G. Prasad, K. Bharathi
Institute of Pharmaceutical Technology, Sri Padmavathi Women’s University, Tirupati 517502, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Some N-[(N-benzoyldehydrophenylalalnyl)glycinyl/cysteinyl]-N,N0-dicyclohexylurea analogs (3ae3o)
were synthesized by conjugating different substituted N-benzoyldehydrophenylalanyl glycines/cysteines
(differing substitutions on benzylidene ring) and dicyclohexyl carbodiimide (DCC) using base as a
catalyst. The synthesized compounds were characterized and evaluated for biological activities. Com-
pounds 3a and 3h with unsubstituted dehydrophenylalanyl glycinyl/cysteinyl moiety exhibited moderate
anti-inflammatory and analgesic activities. Compound 3j bearing 4-hydroxy substitution on benzylidene
ring of dehydrophenylalanyl cysteinyl moiety displayed potent antimicrobial and antioxidant activities.
The results obtained from docking studies on compound 3j with penicillin binding protein and protease
supported the results.
Article history:
Received 31 May 2013
Received in revised form
14 January 2014
Accepted 12 March 2014
Available online 13 March 2014
Keywords:
Dehydrophenylalanyl
Dicyclohexylurea
Antimicrobial
Ó 2014 Elsevier Masson SAS. All rights reserved.
Antioxidant
Penicillin binding protein
Protease
1. Introduction
Dicyclohexylurea (DCU) analogs are of considerable interest with
diverse bioactivities (Fig. 1). Various amino acid conjugates of DCU,
Peptides that incorporate urea as substructure unit either in
open or cyclic form have been associated with several bioactivities
such as anti-infective, antitumor, anticancer and for various
metabolic disorders [1]. TAN-1057A, urea containing dipeptide
isolated from Flexibacter sp was found to exhibit potent bioactivity
against methicillin-resistant Staphylococcus aureus (MRSA) [2].
Novel leucine ureido derivatives [3] and l-lysine ureido derivatives
were reported as inhibitors of aminopeptidase [4]. Interestingly,
some ureides and ureido-based peptidomimetic inhibitors such as
2-[((2-3-benzoyl-1, 3-dicyclohexylureido)-2-oxoethyl) amino]-3-
(4-substituted phenyl) propanoic acids were synthesized and re-
ported to possess good antioxidant and anti-inflammatory activ-
ities [5]. In addition,
a series of novel 1-substituted-1,3-
dicyclohexylurea analogs of long chain carboxylic acids have been
synthesized were reported as potent antimicrobial agents [6].
Furthermore, the synthesized DCU analogs such as Benzoyl-N,N0-
dicyclo hexyl urea, N,N0-dicyclohexyl-N-3,5-dinitro benzoyl urea
and N,N0-dicyclohexyl-N-[(E)-3-phenylacryloyl]urea were found to
possess free radical scavenging activity [8].
Dehydropeptides containing a-dehydroamino acid residues are
important constituents in an increasing number of antibiotic and
phytotoxic peptides [9,10] and among them dehydrophenylalanine
Abbreviations: BHT, butylated hydroxyl toluene; CUPRAC, cupric ion reducing
antioxidant capacity; CPCSEA, committee for the purpose of control and supervi-
sion on experiments on animals; DCC, dicyclohexyl carbodiimide; DMF, dimethyl
formamide; DPPH, 2, 2-diphenyl-1-picryl hydrazyl; GPCR, G-protein coupled re-
ceptor; HOBT, hydroxy benzotriazole; IR, infra-red; MP, melting point; OECD,
organisation for economic co-operation and development; PASS, prediction of ac-
tivity spectra of substances; PBP, penicillin binding protein; PDB, protein data bank;
Rf, retention factor; SEM, standard error of mean; TBA, thiobarbituric acid; TBARS,
thiobarbituric acid reactive substances; TCA, trichloro acetic acid; TEA, triethyl-
amine; TEAC, trolox equivalent antioxidant capacity; THF, tetrahydrofuran; TLC,
thin layer chromatography; TPSA, topological polar surface area; UV, ultra violet;
WHO, World Health Organization.
Part of the work was presented as research paper at the 71st International
Congress of FIP, World Congress of Pharmacy, held at Hyderabad, India, September
(5e8) 2011.
* Corresponding author.
E-mail address: luckyaftab2002@gmail.com (B. Haseena banu).
(D
Phe) residue may be of potential use in designing peptides with
preferred secondary structures. Phe containing undecapeptides
exhibited antibacterial activities [10] and new endomorphin-2
analogs incorporating Phe in place of native phenylalanine
showed higher opiod receptor agonistic activity and selectivity
[11]. A number of other Phe analogs were found to possess
D
D
m
D
analgesic, anti-inflammatory, antimicrobial and bradykinin antag-
onist activities [12e15].
Thus, in our search for new lead compounds, we have come up
with a rational design of target hybrid molecules constituting of
dehydrophenylalanyl residue and dicyclohexylurea (DCU) group
q
http://dx.doi.org/10.1016/j.ejmech.2014.03.037
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
73
Fig. 1. Some reported DCU analogs.
whichmight contributeto interestingbiologicalactivities. Thereforea
series of N-dehyrodipeptidyl-N,N0-dicyclohexylurea analogs 3(aeo)
were synthesized by a facile one pot reaction of Dicyclohexyl Carbo-
diimide (DCC) with N-benzoyl(substituted dehydrophenylalalnyl)
glycines/cysteines 2(aeo) in the presence of triethylamine. These
compounds were evaluated for anti-inflammatory, analgesic, in vitro
antioxidant and antimicrobial activities. The study was further
extended to prediction of in silico parameters like some molecular
properties, bioactivity scores for predicting oral bioavailability and
docking studies for active compounds to investigate the nature of
interactions contributing to the biological activities.
oxazolones 1(aeo) and corresponding dehydrodipeptides, N-
benzoyl (substituted dehydrophenylalalnyl) glycines/cysteines,
2(aeo) were synthesized in our earlier work [14] following the
previous reported methods [16,17]. Conjugation of 2(aeo) with
DCC afforded the title compounds N-[(N-benzoyldehy-
drophenylalalnyl) glycinyl/cysteinyl]-N,N0-dicyclohexylurea ana-
logs (3ae3o). Such type of reactions of carboxylic acids and amino
acids with DCC to form urea analogs were previously reported
[5,7]. Similar method was followed with some modifications using
equal quantities of dehydrodipeptides 2(aeo), DCC and a base as
catalyst. Initially, heterogeneous reaction of dehydrodipeptides in
dry toluene in the presence of sodium carbonate was carried out
at room temperature to generate carboxylate anion as a nucleo-
phile. The reaction was monitored by TLC chromatogram and
found unchanged of the starting materials even at refluxing con-
ditions. However, the carboxylate anion of dehydrodipeptide was
successfully generated in the presence of triethylamine (TEA)
instead of using carbonate base (Fig. 3). The formed carboxylate
2. Results and discussion
2.1. Synthesis of compounds
Synthesis of title compounds was carried according to the
scheme described in Fig. 2. 4-Arylidene-2-phenyl-5(4H)

74
B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
Fig. 2. Synthetic scheme for preparation of N-dehydrodipeptidyl N,N0-dicyclohexylurea analogs, 3ae3o.
anion underwent nucleophilic addition to DCC to give O-aroyl urea
intermediate, which on further rearrangement afforded title
compounds in good yields.
2.2. Biological evaluation
Acute toxicity studies showed that the compounds were safe up
to 1500 mg/kg p.o dose and no mortality or gross behavioral
changes were observed in the animals used.
The chemical structures of title compounds were elucidated
using spectroscopic evidences. The IR spectra of synthesized com-
pounds, 3(aeo) showed NeH stretching at 3400e3277 cmꢀ1
,
aliphatic CH2 stretching at 2900e2700 cmꢀ1, C]O of amide bond at
1610e1669 cmꢀ1, band due to C]O of urea at 1550e1600 cmꢀ1 and
600e900 cmꢀ1 band for CH-deformation of benzene. The disap-
pearance of OeH band of free carboxylic group, appearance of
strong peaks due to aliphatic CeH stretch (cyclohexyl) and a band
due to urea carbonyl observed in the IR spectra of the title com-
pounds indicated the successful conjugation of carboxyl group of
dehydrodipeptides with DCC to yield urea analogs. 1H NMR spectra
2.2.1. Anti-inflammatory activity
Anti-inflammatory activity of all the synthesized compounds
3(aeo) was evaluated by carrageenan induced rat paw edema
method (Table 1). The test compounds and standard drug, diclo-
fenac were evaluated at 100 mg/kg p.o and their effects were
measured at 180 min. Compound 3a with unsubstituted dehy-
drophenylalanine and glycinyl moiety exhibited 35.5% edema in-
hibition whereas the standard drug reduced the inflammation by
66.75% at an equidose. The anti-inflammatory activity of these di-
peptides analogs was influenced by the nature of substitutions on
benzylidene ring. Compounds 3be3g with substitutions such as
methyl, methoxy, chloro, fluoro, and isopropyl at position 4 of
benzylidene ring showed a decrease in activity and among them
only compound 3c with phenolic functionality exhibited 31.5%
activity.
of title compounds showed two methine protons of
hexane rings bonded to nitrogen atoms which appeared as quintet
at 3.5e4.5 ppm. The methylene protons of both cyclohexyl rings
and aromatic protons were observed as multiplet at 1e2 and 7e
8 respectively. Doublet at 6e7 appeared due to styryl hydrogen,
NH of urea as singlet at 6.0e6.5 and amide, NeH protons appeared
a-C of cyclo-
d
d
d
d
as doublet at
d 8.0e8.5. Mass spectra of compounds exhibited the
molecular ion as (M þ H)þ at their characteristic m/z values, other
daughter fragmented ions at [M þ He(cyclohexene)], [M þ He
another cyclohexene], [M þ H-dicyclohexyl carbodiimide], [m/z of
benzoyl] and [m/z of benzene] etc. Detailed spectral data is given in
experimental protocols.
Compound 3h bearing cysteinyl moiety in place of glycinyl
moiety attached to unsubstituted benzylidene ring of dehy-
drophenylalanine moiety was found to exhibit 43.4% inhibition of
inflammation. It is in accordance with the earlier reports on
cysteine analogs as suppressors of prostaglandin production in

B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
75
Fig. 3. Plausible mechanism of reaction in formation of title compounds.
monocytes and enhances the action of some anti-inflammatory
drugs [18]. N-acetyl cysteine is well known for its potent antioxi-
dant, anti-inflammatory and analgesic activities [19]. Compounds
3he3o can be considered as derivatives resembling N-acetyl
cysteine where acetyl is replaced by N-benzoyl dehy-
drophenylalanyl residue and free carboxyl group is conjugated with
DCC. Further, N-benzoyl dehydrophenylalanyl moiety has similar
inflammatory drugs possess free carboxylic group as an impor-
tant structural feature. Hence, it is probable that the conjugation of
free carboxylic group in the dipeptidyl urea analogs of the present
study might be responsible for the decreased activity.
2.2.2. Analgesic activity by acetic acid induced writhing method
The title compounds were tested for analgesic activity by acetic
acid induced writhing assay at 100 mg/kg p.o (Table 1). Among all
the compounds, compound 3n which consists of cystienyl moiety
and methoxy and hydroxyl substitutions at position 3 and 4 on
benzylidene ring of dehydrophenylalanyl moiety was found to
possess significant analgesic activity (58.5%) which is comparable
to standard drug, aspirin (63.15%) and compound 3h with unsub-
stituted dehydrophenylalanyl moiety showed 56.8% of activity.
Compound 3h also exhibited good anti-inflammatory activity, thus
indicating the importance of presence of unsubstituted dehy-
drophenylalanyl nucleus. Compound 3a possessing glycinyl moiety
exhibited 54% of activity whereas substitutions on the benzylidene
moiety did not enhance the activity. The replacement of glycinyl
moiety with cysteinyl moiety resulted in a slight increase in ac-
tivity. Sulfur containing amino acid derivatives was reported to
possess analgesic and related antioxidant activities [20]. Thus, the
presence of dipeptidyl dehydrophenylalanyl feature in addition to
structural features as a-benzoyl amino cinnamoyl moiety and many
cinnamic acid derivatives are found versatile and have featured in
various drugs. Substitutions on benzylidene ring resulted in
decreased activity as observed with glycinyl analogs 3be3g.
Compound 3j with phenolic substitution at position 4 of ben-
zylidene ring and compound 3o possessing methoxy at position 3
and hydroxy at position 4 exhibited good activities of 41.3% and
38.4% respectively. Similar result was observed with 2-[((2-3-
benzoyl-1,
3-dicyclohexylureido)-2-oxoethyl)
amino]-3-(4-
hydroxyphenyl) propanoic acid where hydroxy substitution
contributed to the antiinflammatory activity [5]. In our study,
introduction of cysteinyl moiety attached to dehydrophenylalanine
ring did not result in significant increase in activity compared to
corresponding glycinyl analogs. This could be due steric hindrance
imposed by bulkier groups at N-terminal and C-terminal positions
of cysteine or glycine. Majority of the therapeutically used anti-

76
B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
Table 1
negative bacteria such as Escherichia coli (ESS2231) and fungi such
as Aspergillus niger (MTCC292) and Candida albicans (MTCC227).
Anti-inflammatory and analgesic activities of N-dehydrodipeptidyl N,N0-dicyclo-
hexylurea analogs 3(aeo).
Compound
3j
possessing
phenolic
substituted
dehy-
Compd code Carrageenan
induced rat
% Inhibition of Acetic acid
% Analgesic
activity
drophenylalanine nucleus and cysteinyl moiety was found to be
active against gram þve, gram ꢀve bacteria and also fungi
compared to the standard drugs amoxicillin and fluconazole
(Table 2). The result is analogous to the previous reports on the
antibacterial, antifungal, and antiviral activities of natural and
synthetic sulphydryl and phenolic containing compounds [21].
Literature also revealed that peptides that incorporate urea as
substructure also possess potent antimicrobial activities [2]. Com-
pound 3a possessing unsubstituted benzylidene and glycinyl moi-
ety exhibited good activity against all the microorganisms tested.
Substitutions on position-4 of benzylidene ring gave varied results
such as, methyl substitution reduced the activity against gram þve,
gram ꢀve bacteria and also fungi, phenolic substitution retained
the activity against gram þve and gram ꢀve bacteria but decreased
the activity against fungi and methoxy substitution was found to
decrease the activity against gram þve, gram ꢀve bacteria but
retained the antifungal activity. Among the halogen substitutions,
4-chloro did not display significant activity, but 4-fluoro retained
the activity against all microorganisms tested and substitution with
isopropyl group retained the activity against bacteria but decreased
against fungi.
inflammation induced
paw edema
method
writhing methodb
Edema volume
after 3hr
Number of
writhings
(ml ꢁ SEM)a
Mean ꢁ SEMb
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
Control
Standard
0.62 ꢁ 0.02
0.70 ꢁ 0.12
0.63 ꢁ 0.20
0.81 ꢁ 0.15
0.78 ꢁ 0.33
0.73 ꢁ 0.21
0.68 ꢁ 0.17
0.52 ꢁ 0.02
0.62 ꢁ 0.45
0.54 ꢁ 0.14
0.63 ꢁ 0.22
0.71 ꢁ 0.16
0.69 ꢁ 0.10
0.66 ꢁ 0.11
0.57 ꢁ 0.22
0.92 ꢁ 0.10
0.47 ꢁ 0.01
(Diclofenac
sodium)
35.5***
23.9*
31.5
12.0
15.2
20.6
26.0
43.4***
35.5
41.3*
31.5
22.8
25.0*
28.2
38.4
e
11.5 ꢁ 0.02
12.8 ꢁ 0.12
18.1 ꢁ 0.01
15.1 ꢁ 0.11
18.1 ꢁ 0.11
19.5 ꢁ 0.11
12.0 ꢁ 0.20
10.8 ꢁ 0.07
14.7 ꢁ 0.12
14.2 ꢁ 0.32
16 ꢁ 0.021
14.8 ꢁ 0.02
14.5 ꢁ 0.02
10.4 ꢁ 0.08
13.0 ꢁ 0.02
25.0 ꢁ 0.10
8.4 ꢁ 0.02
(Aspirin)
54.0***
48.8*
27.6**
39.6*
27.6*
22.0*
47.36*
56.8**
41.2
43.2
36.0
40.8**
42.0**
58.5*
48.0***
e
66.75***
63.15***
2.2.4. Brine shrimp lethality test
Significance levels *p < 0.5, **p < 0.01 and ***p < 0.001 by Dunnets t-test.
a
Compounds 3a, 3c, 3h, 3j, 3l and 3n which were found active in
other models were tested for cytotoxic activities by brine shrimp
lethality test. The results were given as ED50 values and are
compared with standard drug, podophyllotoxin (3.77
(Table 2). Among the compounds tested the lower ED50 of 3.53
At 100 mg/kg (p.o) edema volume was measured 3 h after carrageenan injection
and each value represents as the mean ꢁ SEM (n ¼ 5), activity presented as %in-
hibition of inflammation.
b
Number of writhings in 15 min beginning 5 min after acetic acid injection and
m
g/ml)
each value represents as the mean ꢁ SEM (n ¼ 5).
mg/
ml was observed with compound 3h which is comparable to the
standard. The next comparable activity was observed with com-
the cysteinyl moiety in compounds 3h and 3n might have
contributed to the activity.
pounds 3j ED50 of 4.38
chloro derivative 3l and 4-isopropyl derivative 3n were 10.01 and
16.68 g/ml respectively.
mg/ml. The ED50 values obtained with 4-
m
2.2.3. Antimicrobial activity
Among the activities tested above these urea analogs exhibited
potent antimicrobial activity, good analgesic, cytotoxic activities
and moderate anti-inflammatory activity which are in compliance
with the earlier reports on urea containing peptides.
The title compounds were screened for antimicrobial activity
against gram positive bacteria such as Bacillus subtilis (MTCC441), S.
aureus (MTCC96), Pseudomonas aeruginosa (MTCC1688), gram
Table 2
Antimicrobial and cytotoxic activities of N-dehydrodipeptidyl N,N-dicyclohexylurea analogs 3(aeo).
Compd code
Antimicrobial activity by cup plate method
Brine shrimp lethality test
Diameter of zone of inhibition (mm) Mean ꢁ SDa
ED50
(
mg/ml)
b
B. subtilis
S. aureus
P. aeuroginsa
19.4 ꢁ 0.32
17.6 ꢁ 0.23
18.5 ꢁ 0.18
10.3 ꢁ 0.22
9.2 ꢁ 0.75
17.6 ꢁ 0.43
17.3 ꢁ 0.19
20.6 ꢁ 0.06
18.4 ꢁ 0.12
20.7 ꢁ 0.19
17.7 ꢁ 0.36
17.4 ꢁ 0.27
16.6 ꢁ 0.16
15.4 ꢁ 0.51
20.8 ꢁ 0.12
21.4 ꢁ 0.09
e
E. coli
C. albicans
20.7 ꢁ 0.19
13.4 ꢁ 0.21
13.2 ꢁ 0.13
19.6 ꢁ 0.42
12.7 ꢁ 0.09
19.2 ꢁ 0.16
13.1 ꢁ 0.23
17.4 ꢁ 0.17
14.2 ꢁ 0.77
26.6 ꢁ 0.07
19.8 ꢁ 0.11
13.5 ꢁ 0.43
17.1 ꢁ 0.17
12.2 ꢁ 0.08
20.4 ꢁ 0.14
e
A. niger
3a
3b
19.2 ꢁ 0.21
13.2 ꢁ 0.09
22.4 ꢁ 0.03
11.3 ꢁ 0.42
13.5 ꢁ 0.26
20.1 ꢁ 0.11
18.4 ꢁ 0.19
23.2 ꢁ 0.06
15.8 ꢁ 0.23
24.4 ꢁ 0.31
20.1 ꢁ 0.07
18.4 ꢁ 0.11
14.5 ꢁ 0.22
15.2 ꢁ 0.34
20.2 ꢁ 0.12
21.2 ꢁ 0.07
e
18.1 ꢁ 0.47
15.5 ꢁ 0.11
13.4 ꢁ 1.01
14.3 ꢁ 0.34
11.8 ꢁ 0.39
19.3 ꢁ 0.47
16.4 ꢁ 0.08
24.4 ꢁ 0.19
16.8 ꢁ 0.16
24.8 ꢁ 0.07
19.2 ꢁ 0.18
16.5 ꢁ 0.24
11.4 ꢁ 0.47
12.9 ꢁ 0.31
22.1 ꢁ 0.11
22 ꢁ 0.05
e
20.2 ꢁ 0.27
13.3 ꢁ 0.18
19.8 ꢁ 0.44
15.8 ꢁ 0.51
16.3 ꢁ 0.43
16.5 ꢁ 0.32
18.1 ꢁ 0.11
25.8 ꢁ 0.07
22.7 ꢁ 0.62
23.5 ꢁ 0.12
16.4 ꢁ 0.41
18.3 ꢁ 0.72
14.3 ꢁ 0.81
11.8 ꢁ 0.32
24.6 ꢁ 0.33
22.6 ꢁ 0.12
e
19.1 ꢁ 0.08
14.5 ꢁ 0.14
12.4 ꢁ 1.02
18.8 ꢁ 0.06
13.5 ꢁ 0.11
20.8 ꢁ 0.08
21.2 ꢁ 0.42
18.3 ꢁ 0.35
15.4 ꢁ 0.48
24.5 ꢁ 0.14
20.3 ꢁ 0.25
21.2 ꢁ 0.08
18.2 ꢁ 0.40
14.4 ꢁ 0.12
19.3 ꢁ 0.13
e
6.02
NT
3c
9.1
3d
NT
3e
NT
3f
NT
3g
NT
3h
3i
3.53
NT
3j
3k
4.38
NT
3l
3m
10.01
NT
3n
3o
16.68
NT
Amoxicillin
Fluconazole
Podophyllotoxin
e
23.2 ꢁ 0.08
e
24 ꢁ 0.11
e
e
e
e
e
3.77
a
Test compounds (0.02 ml of 1 mg/ml), Amoxicillin and Fluconazole (200 and 500 mg/ml respectively) and DMSO as a control. Activity is measured as zone of inhibition
obtained in triplicate and is expressed as mm (Mean ꢁ SD).
b
ED50 was calculated based on the % of larvae survived at different concentrations of test and standard drugs.NT-not tested.

B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
77
Table 4
2.3. In vitro antioxidant studies
Prediction of molecular properties descriptors and drug likeness scores of N-dehy-
drodipeptidyl N,N-dicyclohexylurea analogs 3(aeo).
All the synthesized compounds 3ae3o were evaluated for free
radical scavenging activities at different concentrations (25, 50,100,
LogPa TPSAb SASAc NONd NOHNHe Ntrobf Drug-likeness score
125 mM) of test compounds and standard. IC50 was calculated for
each compound as well as standard graphically and summarized in
Table 3.
3a
3b
3c
3d
3e
3f
3g
3h 5.0
3i
3j
4.44
4.96
4.14
4.28
5.0
107.6 796.5
107.6 829.3
127.8 808.0
116.8 845.1
107.6 812.5
107.6 804.5
107.6 891.4
107.6 838.4
107.6 875.2
127.8 851.2 10
116.8 889.0 10
107.6 857.5 10
107.6 848.0 10
8
8
9
9
9
9
8
8
8
3
3
4
3
3
3
3
3
3
5
4
4
4
4
4
8
8
8
0.55
0.21
0.53
0.42
0.33
0.23
0.25
0.91
0.49
0.89
0.31
0.66
0.75
0.53
0.44
9
10
10
8
9
9
4.58
5.69
2.3.1. Reduction of DPPH
Among the test compounds the lowest IC50 of 44.44
exhibited by compound 3h which is comparable to standard drug,
tocopherol (42.28 M). The potentiality of this compound may be
mM was
5.57
4.76
9
m
3k 4.90
3l 5.66
3m 5.20
3n 6.30
10
10
10
10
10
attributed to the presence of cysteine moiety which may be syn-
ergizing the activity due to its capability of donating the proton of
SH group [22]. Compounds 3a and 3j also exhibited good activity
107.6 933.0
137.0 898.9 10
9
with IC50 values of 49.75 and 47.16
mM respectively. Similar result
3o
4.60
was observed by Quin Fan et al. in his work on N-[3-(3,4-
a
Log P-Partition coefficient.
b
c
d
e
f
TPSA-Topological polar surface area (A2).
SASA-solvent accessible surface area (A2).
NON-No of hydrogen bond acceptors.
NOHNH-no. of hydrogen bond donors.
Ntrob-No.of rotatable bonds.
dihydroxyphenyl)-1-oxo-2-prope-1-yl]-phenylalaninate
found it as potent antioxidant [23].
and
2.3.2. Hydroxyl radical scavenging activity
Compound 3m with fluoro substitution on position 4 of ben-
zylidene ring and cysteinyl moiety exhibited good activity with IC50
2.3.4. CUPRAC (cupric reducing antioxidant capacity) assay
The highest activity was exhibited by compound 3j and 3h
possessing cysteine moiety which showed absorbance of 0.68 and
0.64 and TEAC (Trolox Equivalent Antioxidant Capacity) of 3.91 and
3.84 respectively while ascorbic acid showed TEAC of 2.63. This
result can be supported by the previous reports on the potent ac-
tivity of thiol containing phenolic compounds by CUPRAC assay
[25]. Compounds with glycinyl moiety 3ae3g exhibited lower ac-
tivity compared to 3he3o with cysteinyl moiety.
of 50.95
mM which is comparable to standard (Ascorbic acid) with
IC50 49.89
mM. Sulphydryl group (eSH) and highly electronegative
atom fluorine in the molecule. Compound 3j exhibited good IC50 of
54.02 and compound 3a and 3g also exhibited IC50 of 57.13 and
60.37 respectively. Similar results were obtained with N,N0-dicy-
clohexyl-N-[(E)-3-phenylacryloyl] urea which displayed radical
scavenging
activity
[7]
and
2-[((2-3-benzoyl-1,
3-
dicyclohexylureido)-2-oxoethyl)
amino]-3-(4-hydroxyphenyl)
propanoic acid exhibited good antioxidant activity [5].
2.4. In silico evaluation
2.3.3. Inhibition of lipid peroxidation
Compounds 3j, 3a and 3h were found to possess potent activity
2.4.1. Molecular descriptors and drug likeness
by exhibiting lower IC50 of 54.02, 58.81 and 59.53
m
M respectively
Some physicochemical parameters of the synthesized com-
pounds were predicted and listed in Table 4. The predicted values
revealed that the compounds obeyed Lipinski rule of five by pos-
sessing not more than 5 hydrogen bond donors (OH and NH
groups), not more than 10 hydrogen bond acceptors (notably N and
O) and not more than 15 rotatable bonds (rotb), a partition coeffi-
cient log P was found to be not more than 5 for some compounds.
comparable to the standard drug, BHT (57.81 M). The ability to
m
scavenge hydroxyl radicals probably plays the most important role
in the inhibition of brain lipid peroxidation [24]. This property at-
tracts attention to the study of these compounds as medicinal
agents for the treatment of pathologies related to lipid
peroxidation.
Table 3
In vitro antioxidant activities of N-dehydrodipeptidyl eN,N0-dicyclohexylurea analogs 3(aeo).
Compd code
Reduction of
Hydroxyl radical
Inhibition of lipid peroxidation IC50
(
m
M)a
CUPRAC assay (Absorbance ꢁ SEM)
TEACb
DPPH IC50
(
m
M)a
scavenging IC50 (m
M)a
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
3l
3m
3n
3o
49.75
71.91
50.95
92.71
83.66
88.66
61.02
44.44
83.99
47.16
88.66
57.13
58.81
74.7
57.13
101.11
74.69
92.71
83.66
88.66
60.37
64.44
66.28
54.02
97.2
58.81
127.36
65.57
108.36
96.78
97.2
60.37
59.53
113.05
54.44
126.63
60.72
74.69
92.82
81.62
0.15 ꢁ 0.06
0.89
1.24
2.91
1.24
0.93
1.22
1.19
3.84
2.36
3.91
2.91
2.65
3.60
2.36
3.60
2.63 (Trolox)
0.20 ꢁ 0.004
0.48 ꢁ 0.08
0.20 ꢁ 0.04
0.15 ꢁ 0.44
0.21 ꢁ 0.0045
0.20 ꢁ 0.015
0.64 ꢁ 0.0478
0.39 ꢁ 0.0031
0.68 ꢁ 0.006
0.48 ꢁ 0.06
66.28
50.95
82.24
92.82
0.44 ꢁ 0.007
0.61 ꢁ 0.0076
0.39 ꢁ 0.0031
0.61 ꢁ 0.0185
0.44 ꢁ 0.0007 (Ascorbic acid)
82.24
42.28 (Tocopherol)
Standard
49.89 (Ascorbic acid)
57.81 (BHT)
a
IC 50 values were calculated graphically by using the data obtained in each test for % scavenging at different concentrations of (25, 50, 75, 100 and 125 mM).
Trolox Equivalent Antioxidant Capacity (TEAC); TEAC test ¼ ε test/ε TR.
b

78
B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
However, descriptors such as Log P and solvent accessible surface
area contributed towards the activity of the molecules, TPSA is
another key property that has been linked to bioavailability. It was
found that passively absorbed molecules with a TPSA more than
140 are thought to have low oral availability [26]. TPSA obtained for
the tested compounds were below 140 predict good oral
bioavailability.
Drug likeness scores of the synthesized compounds were pre-
dicted using molsoft website. Good drug likeness scores of 0.91 and
0.89 were obtained for cysteine containing compounds 3h and 3j
and glycinyl analogs 3a and 3c with 0.55 and 0.53. This result is
adding support to the biological activities observed for these
compounds. Ability to predict the percent oral absorption is pri-
mary goal in the design, optimization, and selection of potential
candidates in the development of oral drugs. HIA% of the com-
pounds 3a, 3c, 3h and 3j were found to be 43.1, 48.7, 33.2 and 59.7%
respectively.
dock software on compound 3j with HIV-1 protease (PDB ID: 1A30)
PBP, Penicillin binding protein (PDB ID: 1CEF) and compared with
docking studies on ritonavir for protease and amoxicillin for PBP.
For better understanding of interactions of the docked compounds
3j, amoxicillin and ritonavir, their chemical structures with
numbering on oxygen, nitrogen and sulfur atoms is given in Fig. 4.
The hypothetical binding model for interactions of compound 3j
with these target proteins are represented in Figs. 5 and 6 and
Table 7 indicates the results obtained.
Probable interactions observed for ritonavir with protease in its
best fit score showed full fitness energy of ꢀ1088.81 Kcal/mol and
binding energy (DG) ꢀ9.63 Kcal/mol. The binding interactions
observed due to oxygen atoms were, O-1 with Arg 8B (w4.14 Ǻ), O-
2 with Asp 29A (w4.14 Ǻ) and O-3 with Ala 28A (w6.14 Ǻ). In-
teractions observed with nitrogen atoms were N-2 with Asp 507C
(w6.14 Ǻ), N-3 with Leu 508C (w5.64 Ǻ), N-4 with Asp 25A
(w7.04 Ǻ) and N-5 with Asp 25B (w5.09 Ǻ). Interactions contrib-
uted by sulfur atoms were S-1 with Asp 25B (w6.44 Ǻ) and S-2 with
Ile 47A (w4.66 Ǻ). Benzene rings showed hydrophobic and Van der
Waals interactions with Asp 30A, Arg 8B and Glu 506C at distances
of 5.66 Ǻ, 4.01 Ǻ and 4.22 Ǻ respectively. Aliphatic hydrogens
showed Van der Waals interactions with Asp 25B, Asp 25A and Leu
508C. Hydrogen bonds were observed with NH of Arg 8B with ar-
omatic hydrogens (w2.06 Ǻ) and N-5 with OH of Asp 25B
(w3.14 Ǻ).
2.4.2. Bioactivity score prediction
The bioactivity scores of the compounds 3a, 3c, 3h and 3j were
calculated for their GPCR ligand, kinase inhibitor, protease inhibitor
and enzyme inhibitor activities and compared to standard drugs
such as ritonavir and amoxicillin (Table 5). For average organic
molecules the probability is that if the bioactivity score is more
than 0 then it is active, if ꢀ0.5 to 0 then moderately active [27].
Kinase inhibitor and enzyme inhibition scores of 0.01 and 0.08
obtained for compound 3j tested supported the presence of mod-
erate anti-inflammatory and analgesic activity [28]. The protease
inhibitor score of compounds 3h was predicted as 0.35 and for
compound 3j, 0.45 that is comparable with score of standard pro-
tease inhibitor, ritonavir (0.47). Good protease inhibitor scores
obtained predicted the efficiency of these compounds as good an-
timicrobials and might possess protease inhibitor activities.
Compound 3j when docked with the same target protease, full
fitness energy of ꢀ1027.66 Kcal/mol and binding energy
(DG) ꢀ8.43Kcal/mol were obtained. The strong interactions
observed due to oxygen atoms were of O-1 with Ala 28A (w1.14 Ǻ),
O-3 with Val 82B (w3.14 Ǻ) and O-5 with Asp 30A (w4.01 Ǻ). In-
teractions observed with nitrogen atoms were Nꢀ1 with Leu 508C
(w4.33 Ǻ), N-3 with Asp 25B (w3.40 Ǻ) and N-4 with Asp 25A
(w2.04 Ǻ). An interaction contributed by sulfur atom was S-1 with
Asp 25B (w1.44 Ǻ). Benzene rings showed hydrophobic and Van der
Waals interactions with Asp 30A, Arg 8B and Glu 506C at distances
of 6.06 Ǻ, 4.11 Ǻ and 3.22 Ǻ respectively. Aliphatic hydrogens and
dicyclohexyl hydrogens showed Van der Waals interactions with
Asp 25B, Asp 25A and Leu 508C. Hydrogen bonds were observed
with NH of Arg 8B with aromatic hydrogens (w0.88 Ǻ) and SH with
OH of Asp 25B (w1.44 Ǻ), OH on benzylidene ring with OH of Asp
30A (w1.72 Ǻ). Interestingly it was observed that both compound
3j and ritonavir showed similar type of interactions and sulphydryl
group and hydroxyl substitution on benzylidene ring of compound
3j contributed towards the hydrogen bonding in the active site of
protease. This result is substantial with the report on ureido based
peptidomimetics with antiviral activity [30].
2.4.3. Docking studies
PharmMapper server, a web server was used for potential drug
target identification using pharmacophore mapping approach for
compounds 3h and 3j which exhibited good biological activities.
According to the computed fit scores and z0-scores for compounds
3h and 3j, Penicillin binding protein (PBP) and Gal pol protein were
found to be the best fit target proteins. Standard drugs binding with
these proteins such as amoxicillin and ritonavir were also mapped.
The fit scores and z0-scores computed for all tested molecules are
given in Table 6.
Protease and reverse transcriptase are the subunits of Gal pol
protein, Protease inhibitors (PIs) are a class of antiviral drugs that
are widely used to treat HIV/AIDS and hepatitis caused by hepatitis
C virus. Protease inhibitors prevent viral replication by selectively
binding to viral proteases (e.g. HIV-1 protease) and blocking pro-
teolytic cleavage of protein precursors that are necessary for the
production of infectious viral particles [29]. Fit scores and z-scores
obtained for compound 3j were found comparable to that of
standard drugs, thus docking studies were performed using SWISS
Drug-resistant bacteria have caused serious medical problems
in recent years, and the need for new antibacterial agents is un-
disputed. The protein transpeptidase is also known as Novel
penicillin-binding protein, which is necessary for bacterial cell wall
peptidoglycan formation [31]. Standard antibacterial drugs such as
b-Lactam antibiotics bind at active site of PBP. Compounds 3j and
amoxicillin were docked with target PBP and it was found that both
Table 5
Table 6
Prediction of bioactivity scores of compounds 3a, 3c, 3h and 3j.
Scores obtained in Pharmmapper server for compounds 3h & 3j.
Compound
GPCR-L
score
Kinase
inhibitor score
Protease
inhibitor score
Enzyme
inhibitor score
Compound
Gal pol protein
(PDB ID: 1kzk)
Penicillin binding protein
(PDB ID: 3FWL)
3a
3c
3h
3j
0.04
0.06
0.09
ꢀ0.03
ꢀ0.02
ꢀ0.01
0.01
0.12
0.14
0.35
0.45
0.47
0.80
ꢀ0.10
ꢀ0.10
ꢀ0.02
0.08
Fit score
Z0-score
Fit score
Z0-score
3h
3j
2.841
3.449
4.162
e
0.352
0.613
0.909
e
3.167
4.572
e
0.533
0.848
e
0.08
Ritonavir
Amoxycillin
ꢀ0.33
ꢀ1.02
ꢀ0.05
Ritonavir
Amoxicillin
0.07
ꢀ0.65
0.30
4.076
0.772

B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
79
Fig. 4. Chemical structures of A-ritonavir, B-amoxycillin and C-compound 3j.
of them exhibited similar interactions. Docking interactions of
amoxicillin with PBP were observed as O-1, O-2, O-3 and O-5
showed interactions with [Val] 302, [Thr] 123 [Asn] 37 and [Ser]
160 (w3.02, 4.89, 3.74 and 4.64 Ǻ) respectively. N-1 and N-2
interacted with [Thr] 301 and [Ser] 261 (w3.94 Ǻ and 4.22 Ǻ)
whereas S-1 showed interaction with [Thr] 309 (w5.42 Ǻ) and S-2
with [Ser] 261 (w7.2 Ǻ). Tertiary butyl hydrogens showed van der
Waals interactions with [Val] 302, [Asn] 237 and [Ser] 160 (w3.8,
4.2 and 5.72 Ǻ) respectively. Aromatic phenyl group showed hy-
drophobic interactions with [Tyr] 159 and [Val] 329.
Interactions observed with compound 3j on PBP were found as
O-1, O-2, O-3 and O-5 showed interactions with [Thr] 301, [Thr]
123, [Ser] 160 and [Asn] 327 (w3.52, 4.20, 6.22 and 5.44 Ǻ)
respectively. N-1, N-2 and N-4 interacted with [Thr] 123, [Tyr] 159
and [Thr] 301 (w5.86 Ǻ, 5.26 and 5.12 Ǻ) whereas S-1 showed
interaction with [Thr] 309 (w4.99 Ǻ). Dicyclohexyl hydrogens
showed van der Waals interactions with [Val] 302 and [Ser] (w4.72
and 4.83 Ǻ). Aromatic phenyl group showed hydrophobic in-
teractions with [Tyr] 159 and benzylidene ring showed interactions
with [Lys] 65. It was interesting to note that compound 3j was
found to interact with the same amino acids of PBP as that of
amoxicillin.
dehydropeptidyl nature and dicyclohexylurea moiety for biolog-
ical activities.
4. Experimental protocols
4.1. Synthesis of compounds
4.1.1. Materials
All the chemicals and solvents used in the present study were
purchased from Merck, HIMEDIA, S.D. Fine Chemicals limited,
Mumbai and Sigma Aldrich, USA. Clean and sterile necessary lab-
oratory equipment was used. Carousel 6 plus station from Radley’s
UK was used for parallel synthesis. Melting points were determined
in an open capillary tube in Sigma melting point apparatus and are
uncorrected. Silica gel G coated on laboratory micro slides prepared
by dipping method or precoated TLC sheets were used. Eluant was
the mixture of different polar and non-polar solvents in varying
proportions and detection of spots was done by observing in UV
light. Absorbance was measured in Systronic UVeVIS-Spectro-
photometer-117, Infrared Spectra of compounds were obtained on a
Bruker FTIR spectrophotometer, SPMVV, Tirupati, 1H NMR Spectra
were recorded on Bruker Avance at IICT, Hyderabad, mass spectra
were scanned on Jeol GCmate Mass spectrophotometer at SAIF
(Sophisticated Analytical Instrumentation Facility), Elemental
analysis was performed on Perkin Elmer C, H and N elemental
analyzer, Indian Institiute of Technology, Chennai.
Thus, results observed with in silico studies were found to
support the observed potent antimicrobial activities of com-
pound 3j.
3. Conclusion
4.1.2. Synthesis of N-benzoyl(substituted dehydrophenylalanyl)
glycines/cysteines (2ae2o)
In our earlier work, different substituted oxazolones, 1(aeo)
were condensed with glycine/cysteine in presence of THF: water
(3:1) solvent system to obtain 2a-2o [14].
In the present study some N-[(N-benzoyldehydro
phenylalalnyl) glycinyl/cysteinyl]eN,N0-dicyclohexylurea analogs
(3ae3o) were synthesized and screened for varied biological ac-
tivities. Of these compounds, compounds 3a and 3h exhibited
moderate anti-inflammatory and analgesic activities and com-
pound 3j displayed potent antimicrobial and antioxidant activ-
ities. Compound 3j showed similar binding interactions as that of
standard drugs, amoxicillin and ritonavir at the binding sites of
PBP and protease. Thus, the study emphasizes the importance of
4.1.3. General method for synthesis of title compounds 3(aeo)
4.1.3.1. N-{2-[cyclohexyl(cyclohexylcarbamoyl)amino]-2-oxoethyl}-
3-phenyl-2-(phenyl formamido) prop-2-enamide (3a). To a solution
of N-benzoyl-dehydrophenylalanyl glycine, 2a (1 mmol) and TEA

80
B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
Fig. 5. Docking studies on compound 3j with protease (1A30); 5a-interactions of compound 3j at active site, 5b-superimposing of compound 3j (purple colored stick model) on
ritonavir (yellow colored ball and stick model) and interaction with similar amino acids at the active site, 5c-compound 3j (purple colored stick model) and ritonavir (blue colored
ball and stick model) inside the hydrophobic pockets of active site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of
this article.)
(1 mmol) in dry toluene (20 ml), N,N0-dicyclohexyl carbodiimide
(1 mmol) in toluene (10 ml) was added and the reaction stirred for
30e40 min. Further the reaction mixture was heated up to reflux
overnight. The crude precipitate, after removal of all the solvents,
was recrystallized from a mixture of methylene chloride and hex-
ane. Yellow crystals; yield: 64%; M.P: 217e218 ꢂC; Rf ¼ 0.63; UV
Similar procedure was followed for the synthesis of other title
compounds, 3be3o by reacting N-benzoyl (substituted dehy-
drophenylalanyl)glycines, (2be2g) and N-benzoyl(substituted
dehydrophenylalanyl) cysteines (2he2o) with DCC in presence of
TEA.
lmax (nm): 402.5; 1H NMR (300 MHz, CDCl3,
d
) 1.0e1.7 (m, 20H, CH2
4.1.3.2. N-{2-[cyclohexyl(cyclohexylcarbamoyl)amino]-2-oxoethyl}-
of cyclohexyl rings), 3.1e3.4 (m, 2H, CH of cyclohexyl rings), 3.6e
3.7 (d, J ¼ 4 Hz, 2H, CH2), 6.2 (s, 1H, NH of Dicylclohexyl urea), 6.7e
6.8 (d, J ¼ 6 Hz, 1H, C]CH), 6.9e7.4 (m, 10H, AreH), 7.6 (s, 1H, NH),
3-(4-methylphenyl)-2-(phenylformamido)prop-2-enamide
(3b).
Orange crystals; yield: 66%; M.P: 196.5e197.3 ꢂC; Rf ¼ 0.61; UV lmax
(nm): 422; 1H NMR (300 MHz, CDCl3,
d) 1.2e2.2 (m, 23H, CH2 of
7.9 (t, J ¼ 3.7 Hz, NH); IR (KBr)
n
: 3328.45 (NH), 2927.43 (AreH),
cyclohexyl & CH3), 3.6e3.8 (m, 2H, CH of cyclohexyl), 3.8e3.9 (d,
J ¼ 3 Hz, 2H, CH2), 6.2 (s, 1H, NH of Dicylclohexyl urea), 7.0 (d,
J ¼ 2.6 Hz, 1H, C]CH), 7.2e7.9 (m, 9H, AreH), 8.0e8.2 (t, J ¼ 6.7 Hz,
2851.78 (aliphatic CeH), 1626.96 (C]O), 1534.36 (CH]C), 694.35
(ArCeH). MS (EIþ): m/z (%) 531.8 (33) [M þ Hþ], 90 (100) [m/z of
PheMe], 105 (45) [m/z of CNeC6H5], 77 (24) [m/z of benzene], Anal.
Calc. for C31H38N4O4: C, 70.16; H, 7.22; N, 10.56. Found: C, 69.98; H,
7.26; N, 10.62.
2H, NH); IR (KBr) n: 3284.5 (NH), 2974.3 (AreH), 2817.8 (aliphatic
CeH), 1669.6 (C]O), 1533.6 (CH]C), 743.5 (ArCeH). MS (EIþ): m/z
(%)401.03 (33) [M þ Hþ], 375 (100) [M-dicyclohexyl urea], 90 (68)

B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
81
Fig. 6. Docking studies on compound 3j with penicilling binding protein (1CEF); 6a-superimposition of compound 3j (purple colored stick model) on amoxicillin (blue colored ball
and stick model) interacting with at the similar binding site, 6b-compound 3j (purple colored stick model) and amoxicillin (light blue colored stick model) inside the hydrophobic
pockets of active site. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
[m/z of PheMe], 381 (43) [M-dicyclohexene], 105 (68) [m/z of CNe
C6H5], 77 (34) [m/z of benzene], 464 (15) [M-cyclohexene]. Anal.
Calc. for C32H40N4O4: C, 70.56; H, 7.4; N, 10.28. Found: C, 70.76; H,
7.52; N, 10.16.
(CH]C), 845.4 (ArCeH). MS (EIþ): m/z (%) 561.7 (45) [M þ Hþ], 104
(100) [m/z of CNeC6H5] 91 (54) [m/z of PheMe], 78 (35) [m/z of
benzene], Anal. Calc. for C32H40N4O5: C, 68.38; H, 7.21; N, 10.1.
Found: C, 68.55; H, 7.19; N, 9.99.
4.1.3.3. N-{2-[cyclohexyl(cyclohexylcarbamoyl)amino]-2-oxoethyl}-
3-(4-hydroxyphenyl)-2-(phenyl formamido) prop-2-enamide(3c).
Pale orange crystals; yield: 45.6%; M.P: 198.6e199.4 ꢂC; Rf ¼ 0.81;
4.1.3.5. N-{2-[cyclohexyl(cyclohexylcarbamoyl)amino]-2-oxoethyl}-
3-(4-chlorophenyl)-2-(phenylformamido)
prop-2-enamide
(3e).
Cream colored amorphous solid; yield: 66%; M.P: 212.3e213.7 ꢂC;
UV lmax (nm): 432; 1H NMR (300 MHz, CDCl3,
d
) 1.2e2.2 (m, 20H,
Rf ¼ 0.88; UV lmax (nm): 371; 1H NMR (300 MHz, CDCl3,
d) 1.1e1.9
CH2 of cyclohexyl), 3.8e4.0 (m, 2H, CH of cyclohexyl), 4.1e4.2 (d,
J ¼ 3.9 Hz, 2H, CH2), 5.9 (s, 1H, OH), 6.2 (s, 1H, NH of Dicylclohexyl
urea), 7.0 (d, J ¼ 5.6 Hz, 1H, C]CH), 7.2e7.9 (m, 9H, AreH), 8.0e8.2
(m, 20H, CH2 of cyclohexyl), 3.8e4.0 (m, 2H, CH of cyclohexyl), 4.3
(d, J ¼ 7.9 Hz, 2H, CH2), 6.0 (s, 1H, NH of Dicylclohexyl urea), 7.3e7.4
(d, J ¼ 5.6 Hz, 1H, C]CH), 7.6e8.2 (m, 9H, AreH), 8.3e8.4 (t,
(t, J ¼ 6.7 Hz, 2H, NH); IR (KBr)
n
: 3309.76 (OH), 3198.93 (NH),
J ¼ 6.7 Hz, 2H, NH); IR (KBr)
n: 3289.46 (NH), 2973.20 (AreH),
3055.66 (AreH), 2924.69 (aliphatic CeH), 1648.80 (C]O), 1548.21
(CH]C), 696.81 (ArCeH). MS (EIþ): m/z (%) 547.2 (33) [M þ Hþ],
104 (100) [m/z of CNeC6H5] 90 (70) [m/z of PheMe], 77 (54) [m/z of
benzene], Anal. Calc. for C31H38N4O5 C, 68.11; H, 7.01; N, 10.25.
Found: C, 68.39; H, 7.04; N, 10.29.
2901.52 (aliphatic CeH), 1609.32 (C]O), 1526.39 (CH]C), 812.22
(ArCeH), 712.39 (CeCl). MS (EIþ): m/z (%) 566.7 (25) [M þ Hþ], 90
(100) [m/z of CNeC6H5] 104 (54) [m/z of PheMe], 78 (45) [m/z of
benzene], Anal. Calc. for C31H37ClN4O4: C, 65.89; H, 6.6; N, 9.91.
Found: C, 66.16; H, 6.62; N, 9.94.
4.1.3.6. N-{2-[cyclohexyl(cyclohexylcarbamoyl)amino]-2-oxoethyl}-
4.1.3.4. N-{2-[cyclohexyl(cyclohexylcarbamoyl)amino]-2-oxoethyl}-
3-(4-methoxyphenyl)-2-(phenyl formamido) prop-2-enamide(3d).
Chrome yellow amorphous solid; yield: 56%; M.P: 195 ꢂC; Rf ¼ 0.68;
3-(4-fluorophenyl)-2-(phenylformamido)
prop-2-enamide
(3f).
White amorphous solid; yield: 82%; M.P: 192.3e193 ꢂC; Rf ¼ 0.80;
UV lmax (nm): 312; 1H NMR (300 MHz, CDCl3,
d) 1.0e1.7 (m, 20H,
UV lmax (nm): 417; 1H NMR (300 MHz, CDCl3,
d) 1.3e2.0 (m, 20H,
CH2 of cyclohexyl), 3.1e3.4 (m, 2H, CH of cyclohexyl), 3.6e3.7 (d,
J ¼ 4 Hz, 2H, CH2), 6.3 (s, 1H, NH of Dicylclohexyl urea), 6.7e6.8 (d,
J ¼ 6 Hz, 1H, C]CH), 6.9e7.4 (m, 10H, AreH), 7.6 (s, 1H, NH), 7.9 (t,
CH2 of cyclohexyl), 2.3e2.5 (s, 3H, OCH3) 3.8e4.0 (q, 2H, CH of
cyclohexyl), 4.2e4.3 (d, J ¼ 5.2 Hz, 2H, CH2), 6.0 (s, 1H, NH of
Dicylclohexyl urea), 7.2 (d, J ¼ 5.6 Hz, 1H, C]CH), 7.4e8.0 (m, 9H,
J ¼ 3.7 Hz, NH); IR (KBr)
n: 3225.6 (NH), 2943.8 (AreH), 2817.2
AreH), 8.3e8.4 (t, J ¼ 6.7 Hz, 2H, NH); IR (KBr)
n: 3312.12 (NH),
(aliphatic CeH), 1681.3 (C]O), 1522.1 (CH]C), 1010.3 (CeF), 804.2
(ArCeH); MS (EIþ): m/z (%) 550.7 (34) [M þ Hþ], 90 (100) [m/z of
PheMe], 105 (45) [m/z of CNeC6H5], 77 (24) [m/z of benzene], Anal.
Calc. for C31H37FN4O4: C, 67.86; H, 6.81; N, 10.21. Found: C, 67.56; H,
6.82; N, 10.25.
3074.38 (AreH), 2932.77 (aliphatic CeH), 1646.81 (C]O), 1502.5
Table 7
Results of docking studies on compound 3j with protease and Penicillin binding
protein.
Compound
Target
PDB ID Full fitness
energy (Kcal/mol)
Estimated
G (Kcal/mol)a
4.1.3.7. N-{2-[cyclohexyl(cyclohexylcarbamoyl)amino]-2-oxoethyl}-
2-(phenylformamido)-3-[4-(propan-2-yl) phenyl]prop-2-enamide
(3g). White amorphous solid; yield: 72%; M.P: 199.6e201 ꢂC;
D
3j
Protease
Protease
Penicillin
binding protein
1A30
1A30
1CEF
ꢀ1027.66
ꢀ8.43
ꢀ9.63
ꢀ9.22
Ritonavir
3j
ꢀ1088.81
ꢀ1213.70
Rf ¼ 0.80; UV lmax (nm): 337; 1H NMR (300 MHz, CDCl3,
d) 1.3 (d,
J ¼ 2.5 Hz, 6H, 2CH3) 1.4e1.8 (m, 20H, CH2 of cyclohexyl), 3.1 (m, 1H,
CH), 3.5e3.6 (m, 2H, CH of cyclohexyl), 4.2e4.3 (d, J ¼ 5.2 Hz, 2H,
CH2), 6.0 (s, 1H, NH of Dicylclohexyl urea), 6.8 (d, J ¼ 4.6 Hz, 1H, C]
Amoxicillin Penicillin
binding protein
1CEF
ꢀ1088.60
ꢀ8.00
a
DG ¼ binding energy.
CH), 7.4e8.2 (m, 9H, AreH), 8.3 (t, J ¼ 6.7 Hz, 2H, NH); IR (KBr)
n:

82
B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
3356.4 (NH), 3043.8 (AreH), 2972.3 (aliphatic CeH), 1643.3 (C]O),
1512.8 (CH]C), 842.9 (ArCeH); MS (EIþ): m/z (%) 573 (24)
[M þ Hþ], 121 (100) [m/z of Ph-CH(Me)2], 352 (80) [M-dicyclohexyl
urea], 77 (53) [m/z of benzene], 105 (52) [m/z of CNeC6H5], 410 (38)
[M-dicyclohexene], 492 (30) [M-cyclohexene]. Anal. Calc. for
607 (5) [M þ Hþ], 385 (100) [M-dicyclohexyl urea], 109 (58) [m/z of
Ph-OCH3], 442 (44) [M-dicyclohexene], 104 (41) [m/z of CNeC6H5],
78 (28) [m/z of benzene], 524 (20) [M-cyclohexene], 90 (12) [m/z of
PheOH], 237 (28), Anal. Calc. for C33H42N4O5S: C, 65.32; H, 6.98; N,
9.23. Found: C, 66.66; H, 4.62; N, 12.96.
C
34H44N4O4: C, 71.3; H, 7.74; N, 9.78. Found: C, 71.1; H, 7.77; N, 9.75.
4.1.3.12. N-{1-[cyclohexyl(cyclohexylcarbamoyl)amino]-1-oxo-3-
sulfanylpropan-2-yl}-3-(4-chlorophenyl)-2-(phenylformamido)prop-
2-enamide (3l). Cream colored amorphous solid; yield: 64%; M.P:
207.5 ꢂC; Rf ¼ 0.731; UV lmax (nm): 402; 1H NMR (300 MHz, CDCl3,
4.1.3.8. N-{1-[cyclohexyl(cyclohexylcarbamoyl)amino]-1-oxo-3-
sulfanylpropan-2-yl}-3-phenyl-2-(phenylformamido)prop-2-
enamide (3h). Yellow amorphous solid; yield: 72%; M.P: 206 ꢂC;
Rf ¼ 0.73; UV lmax (nm): 402; 1H NMR (300 MHz, CDCl3,
d) 1.7e2.0
d
) 1.0e2.0 (m, 21H, CH2 of cyclohexyl & SH), 3.4e3.7 (m, 2H, CH of
(m, 13H, CH2 of cyclohexyl), 2.0e2.2 (t, J ¼ 10.6 Hz 1H, SH), 2.3e2.5
(m, 8H, CH2 of cyclohexyl), 4.3e4.5 (m, 2H, CH of cyclohexyl), 4.6e
4.8 (t, J ¼ 3.5 Hz, 3H, CH, CH2), 6.4 (s, 1H, NH of Dicylclohexy lurea),
7.3 (d, J ¼ 4.6 Hz, 1H, C]CH), 7.4e7.9 (m, 10H, AreH), 7.9e8.0 (d,
cyclohexyl), 3.8e4.0 (d, J ¼ 5.3 Hz, 3H, CH, CH2), 3.8e4.0 (t, J ¼ 5.5,
1H, CH), 6.3 (s, 1H, NH of Dicylclohexyl urea), 7.0 (d, J ¼ 1.8 Hz, 1H,
C]CH), 7.1e7.5 (m, 9H, AreH), 7.8e7.9 (d, J ¼ 7.2 Hz, 2H, NH); IR
(KBr)
n: 3325.31 (NH), 2991.1 (AreH), 2883.23 (aliphatic CeH),
J ¼ 5.7 Hz, 2H, NH); IR (KBr)
n: 3285.33 (NH), 3074.31 (AreH),
2536.8 (SH), 1660.7 (C]O), 1542.5 (CH]C), 843.4 (ArCeH). MS
(EIþ): m/z (%) 612 (32) [M þ Hþ], 390 (100) [M-dicyclohexyl urea],
77 (44) [m/z of benzene], 105 (32) [m/z of CNeC6H5], 446 (38) [M-
dicyclohexene], 530 (30) [M-cyclohexene]; Anal. Calc. for
2978.4 (aliphatic CeH), 1660.6 (C]O), 1513.6 (CH]C), 735.22
(ArCeH); MS (EIþ): m/z (%) 577 (44) [M þ Hþ], 121 (100) [m/z of Ph-
CH], 355 (80) [M-dicyclohexyl urea], 77 (44) [m/z of benzene], 105
(32) [m/z of CNeC6H5], 413 (38) [M-dicyclohexene], 495 (30) [M-
cyclohexene]; Anal. Calc. for C32H40N4O4S: C, 66.64; H, 6.99; N,
9.71. Found: C, 66.90; H, 7.01; N, 9.65.
C32H39ClN4O4S: C, 62.88; H, 6.43; N, 9.17. Found: C, 62.63; H, 6.40;
N, 9.20.
4.1.3.9. N-{1-[cyclohexyl(cyclohexylcarbamoyl)amino]-1-oxo-3-
sulfanylpropan-2-yl}-3-(4-methylphenyl)-2-(phenylformamido)prop-
2-enamide (3i). Light yellow amorphous solid; yield: 62%; M.P:
158e160 ꢂC; Rf ¼ 0.60; UV lmax (nm): 352; 1H NMR (300 MHz,
4.1.3.13. N-{1-[cyclohexyl(cyclohexylcarbamoyl)amino]-1-oxo-3-
sulfanylpropan-2-yl}-3-(4-fluorophenyl)-2-(phenylformamido)prop-
2-enamide (3m). White amorphous solid; yield: 64%; M.P:
182.7 ꢂC; Rf ¼ 0.63; UV lmax (nm): 392; 1H NMR (300 MHz, CDCl3,
d)
CDCl3, d) 1.1e2.2 (m, 24H, CH2 of cyclohexyl, CH3 & SH), 3.7e4.0 (m,
1.3e2.0 (m, 21H, CH2 of cyclohexyl & SH), 3.6e3.8 (m, 2H, CH of
cyclohexyl), 4.0 (d, J ¼ 8.3 Hz, 3H, CH, CH2), 4.1e4.2 (t, J ¼ 7.5, 1H,
CH), 6.3 (s, 1H, NH of dicylclohexyl urea), 7.0 (d, J ¼ 3.8 Hz, 1H, C]
CH), 7.1e7.5 (m, 9H, AreH), 7.8e7.9 (d, J ¼ 8.2 Hz, 2H, NH); IR (KBr)
4H, CH of cyclohexyl, CH2), 6.4 (s, 1H, NH of Dicylclohexyl urea), 6.8
(d, J ¼ 3.6 Hz, 1H, C]CH), 7.0e7.8 (m, 10H, AreH), 8.0 (d, J ¼ 3.7 Hz,
2H, NH); IR (KBr)
n: 3256.11 (NH), 3020.76 (AreH), 2972.75
(aliphatic CeH), 2511.06 (SH), 1641.3 (C]O), 1510.06 (CH]C), 774.2
(ArCeH). MS (EIþ): m/z (%) 591 (35) [M þ Hþ], 105 (100) [m/z of
CNeC6H5], 93 (100) [m/z of Ph-CH3], 78 (64) [m/z of benzene], 369
(60) [M-dicyclohexyl urea], 273 (48) [M-dicyclohexene], 509 (40)
[M-cyclo hexene]; Anal. Calc. for C33H42N4O4S: C, 67.09; H, 7.17; N,
9.48. Found: C, 67.3; H, 7.14; N, 9.43.
n: 3318.9 (NH), 3027.5 (AreH), 2854.81 (aliphatic CeH), 2503.2
(SH), 1633.34 (C]O), 1522.28 (CH]C), 825.11 (ArCeH). MS (EIþ):
m/z (%) 612 (32) [M þ Hþ], Anal. Calc. for C32H39 FN4 O4S: C, 64.62;
H, 6.61; N, 9.42. Found: C, 64.88; H, 6.64; N, 9.40.
4.1.3.14. N-{1-[cyclohexyl(cyclohexylcarbamoyl)amino]-1-oxo-3-
sulfanylpropan-2-yl}-2-(phenylformamido)-3-[4-(propan-2-yl)
phenyl]prop-2-enamide (3n). Orange crystalline solid; yield: 64%;
M.P: 177.5e180 ꢂC; Rf ¼ 0.51; UV lmax (nm): 382; 1H NMR
4.1.3.10. N-{1-[cyclohexyl(cyclohexylcarbamoyl)amino]-1-oxo-3-
sulfanylpropan-2-yl}-3-(4-hydroxyphenyl)-2-(phenylformamido)
prop-2-enamide (3j). Deep yellow amorphous solid; yield: 70%;
M.P: 206.5e207.3 ꢂC; Rf ¼ 0.731; UV lmax (nm): 382; 1H NMR
(300 MHz, CDCl3,
d
) 1.3 (d, J ¼ 2.5 Hz, 6H, 2CH3) 1.4e1.8 (m, 21H,
(300 MHz, CDCl3, d) 1.0e2.0 (m, 20H, CH2 of cyclohexyl & SH), 3.5e
CH2 of cyclohexyl & SH), 3.1 (m, 1H, CH), 3.5e3.6 (m, 3H, CH), 4.2e
4.3 (d, J ¼ 5.2 Hz, 2H, CH2), 6.0 (s, 1H, NH of dicylclohexyl urea), 6.8
(d, J ¼ 4.6 Hz, 1H, C]CH), 7.4e8.2 (m, 9H, AreH), 8.3 (d, J ¼ 6.7 Hz,
3.7 (m, 3H, CH), 3.8e4.0 (d, J ¼ 3 Hz, 2H, CH2), 5.3 (S, 1H, OH), 6.2 (s,
1H, NH of Dicylclohexyl urea), 7.0 (d, J ¼ 4.5 Hz, 1H, C]CH), 7.1e7.6
(m, 9H, AreH), 7.6e7.8 (d, J ¼ 6.3 Hz, 2H, NH); IR (KBr)
n: 3449.65
2H, NH); IR (KBr)
n: 3289.3 (NH), 3010.29 (AreH), 2881.54
(OH), 3331.05 (NH), 2927.41 (AreH), 2851.33 (aliphatic CeH),
2311.36 (SH), 1626.96 (C]O), 1576.54 (CH]C), 875.34 (ArCeH). MS
(EIþ): m/z (%) 592 (35) [M þ Hþ], 371 (100) [M-dicyclohexyl urea],
426 (83) [M-dicyclohexene], 105 (54) [m/z of CNeC6H5], 77 (50) [m/
z of benzene], 510 (25) [M-cyclohexene], 90 (12) [m/z of Ph-OH],
237 (28), Anal. Calc. for C32H40N4O5S: C, 64.84; H, 6.8; N, 9.45.
Found: C, 64.59; H, 6.82; N, 9.48.
(aliphatic CeH), 2502.12 (SH), 1633.43 (C]O), 1508.11 (CH]C),
729.1 (ArCeH). MS (EIþ): m/z (%) 620 (33.2) [M þ Hþ] Anal. Calc. for
C35H46 N4O4S: C, 67.93; H, 7.49; N, 9.05. Found: C, 67.66; H, 7.52; N,
9.08.
4.1.3.15. N-{1-[cyclohexyl(cyclohexylcarbamoyl)amino]-1-oxo-3-
sulfanylpropan-2-yl}-3-(4-hydroxy-3-methoxyphenyl)-2-(phenyl-
formamido)prop-2-enamide (3o). Yellow solid; yield: 54%; M.P:
280e282 ꢂC; Rf ¼ 0.71; UV lmax (nm): 412; 1H NMR (300 MHz,
4.1.3.11. N-{1-[cyclohexyl(cyclohexylcarbamoyl)amino]-1-oxo-3-
sulfanylpropan-2-yl}-3-(4-methoxyphenyl)-2-(phenylformamido)
prop-2-enamide (3k). Deep yellow amorphous solid; yield: 64%;
M.P: 207.5 ꢂC; Rf ¼ 0.731; UV lmax (nm): 402; 1H NMR (300 MHz,
CDCl3, d) 1.2e2.2 (m, 21H, CH2 of cyclohexyl & SH), 2.8 (s, 3H, OCH3),
3.8e4.0 (m, 2H, CH of cyclohexyl), 4.1e4.2 (d, J ¼ 3.9 Hz, 2H, CH2),
5.9 (s, 1H, OH), 6.2 (s, 1H, NH of Dicylclohexyl urea), 7.0 (d,
J ¼ 5.6 Hz, 1H, C]CH), 7.2e7.9 (m, 9H, AreH), 8.0e8.2 (d, J ¼ 6.7 Hz,
CDCl3, d) 1.0e1.3 (m, 12H, CH2 of cyclohexyl), 1.4e1.7 (m, 9H, SH &
CH2 of cyclohexyl), 2.2 (s, 3H, OCH3), 3.1e3.3 (m, 2H, CH of cyclo-
hexyl), 3.6e3.7 (d, J ¼ 5.3 Hz, 2H, CH2), 3.8e4.0 (t, J ¼ 5.5.1H, CH),
5.6 (s, 1H, NH of Dicylclohexyl urea), 6.7 (d, J ¼ 1.8 Hz, 1H, C]CH),
2H, NH); IR (KBr) n: 3327.93 (OH), 3197.58 (NH), 3062.58 (AreH),
2918.17 (aliphatic CeH), 2606.25 (SH), 1640.87 (C]O), 1540.51
(CH]C), 830.57 (ArCeH). MS (EIþ): m/z (%) 623 (40.2) [M þ Hþ]
Anal. Calc. for C33H42N4O6S: C, 63.64; H, 6.8; N, 9.0 Found: C, 63.92;
H, 6.82; N, 9.03.
6.9e7.5 (m, 9H, AreH), 7.4e7.5 (d, J ¼ 7.3 Hz, 2H, NH); IR (KBr)
n:
3310.5 (NH), 2974.1 (AreH), 2853.3 (aliphatic CeH), 2513.6 (SH),
1661.0 (C]O), 1554.2 (CH]C), 834.3 (ArCeH). MS (EIþ): m/z (%)

B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
83
4.2. Pharmacological studies
jar for observation after 5 min of injection. The onset of writhing
was noted. The numbers of abdominal constrictions or total writhes
were recorded during a period of 15 min. The mean value, SEM was
calculated for all groups and % inhibition was calculated using the
formula. Percentage decrease in number of writhing is calculated
by the formula, %analgesic activity ¼ 100 (1ꢀWt/Wc), where Wc is
average number of writhes observed in control group and Wt is
average number of writhes observed in test group. Statistical sig-
nificance of the results was tested by Anova and Dunnet’s t-test.
4.2.1. Materials
The chemicals used in the studies were procured from Merck,
HIMEDIA, Mumbai. Animal care and handling was carried out ac-
cording to the ethical guidelines set by WHO. Swiss albino mice
aged 6e8 weeks, of weight 25e30 g, male and female adult Wister
rats (150e220 g) were procured from an inbred colony from Gold
Mohur, Bangalore. The animals were maintained under control
conditions of temperature and light. They were provided standard
mouse feed/rat feed and water ad libitum. Paw edema was
measured by UGO BASILE 7140 Plethysmometer. Analgesic activity
was observed using Eddy’s hot plate. The experiment protocols
were approved by the institutional ethical committee (CPCSEA
registration no.1016/a/06/CPCSEA). All the biological activities
including in vitro antioxidant studies by standard methods were
carefully conducted in IPT, SPMVV. Prescreening of the cytotoxicity
studies were conducted in Laila Implex Research Laboratories,
Vijayawada.
Acute toxicity studies for the synthesized compounds were
tested in mice (500 mg/kg, 1000 mg/kg, 1500 mg/kg, p.o.) as per
OECD guidelines [32]. Any significant changes in the body weight,
food, water intake and other behavioral patterns and mortality was
observed in the control and compound treated groups. Prior to the
evaluation of biological activities, the animals were kept starved of
solid food before 4 h but fed with water ad libitum.
4.2.4. Antimicrobial activity
Cup plate method using Mueller-Hinton agar medium was
employed to study the antibacterial activity against, B. subtilis,
P. aeruginosa, S. aureus, E. coli and antifungal activity against A. niger
and C. albicans. Preparation of nutrient broth, subculture, base layer
medium, agar medium and peptone water was done as per the
standard procedure [35]. Each test compound of 5 mg was dissolved
in DMSO (5 ml). Nutrient agar was used as culture medium & DMSO
was used as solvent control. Laminar airflow bench was swapped
with 70% alcohol and UV lamp was switched on. After 30 min, the
UV lamp was switched off. The reagents, media, inoculums and
glassware were placed in laminar airflow bench observing all
aseptic conditions. Plates were inoculated within minutes of the
preparation of suspension, so that the density does not change. A
sterile cotton swab was dipped into the suspension and the medium
was inoculated by even streaking of the swab over the entire surface
of the plate in three directions. After the inoculums had dried, cups
each of 6 mm diameter were made by scooping out the medium
with a sterile cork borer. From the prepared solutions of each
compound 0.02 ml were added separately in the cups with a
micropipette and the plates were then incubated at 37 ꢂC for 24 h.
Amoxicillin and Fluconazole were used as standard reference drugs
4.2.2. Acute inflammatory model e carrageenan induced paw
edema assay
The rats were divided into 17 groups, one group consisting of six
animals served as control, while the other groups of five animals
each received the test compounds and standard drug. The rats were
administered orally with test compounds (100 mg/kg), 100 mg/kg
Diclofenac sodium (positive control) or 10 ml/kg 0.5% sodium car-
boxy methyl cellulose (vehicle control) 1 h before injection of
0.05 ml of 1% suspension of Carrageenan into the sub plantar region
of the rat hind paw [33]. A mark was made at the lateral maleolous
of the right paw and the foot was dipped to the same distance of the
mark into the arm of plethysmograph. The volume of the injected
paw was measured by water displacement in a digital plethysmo-
graph immediately after carrageenan injection. The paw volume
was again measured after 30, 60, 120 and 180 min. Average edema
volumes for test compound treated and positive control rats were
compared statistically with those of the vehicle control animals and
expressed as percent edema inhibition, which is calculated using
the formula.
(200 and 500
mg/ml respectively) and DMSO as a control which
did not reveal any inhibition. Zone of inhibition produced by each
compound was measured in mm. The experiment values were
obtained in triplicate and expressed as Mean ꢁ SD values.
4.2.5. Brine shrimp lethality test
Sea water was prepared (38 g sea salt per liter of water), filtered
and is taken in small tank and shrimp eggs were added to one side
of the divided tank and other side was covered. The shrimps were
allowed to hatch and mature as nauplii (in warmer climates,
hatching may take place sooner). After two days (when the shrimp
larvae are ready), 4 ml of sea water is added to each vial, 10 shrimp
per vial (30 shrimp per dilution) were counted, placed in the vial
and volume adjusted with sea water to 5 ml/vial. The vials were
uncovered and placed under the lamp. Twenty-four hours later the
number of survivors were counted and recorded. The data was
analyzed with the Finney computer program for probit analysis to
determine ED50 values and 95% confidence intervals [36].
Percentage edema inhibition
¼
100 (1ꢀVt/Vc) Where,
Vt ¼ volume of edema in treated group and Vc ¼ volume of the
edema in the control group. Statistical significance of the results
was tested by Anova and Dunnet’s t-test.
4.2.3. Analgesic activity by writhing method
4.2.6. In vitro antioxidant studies
Analgesic effect of the synthesized drugs was also determined
by the chemical method namely acetic acid induced writhing. The
injected chemical produces pain reaction which is characterized as
a writhing response. Constriction of abdomen, trunking of the
trunk and extension of hind limbs are considered as reactions due
to chemically induced pain. Both narcotic and nonnarcotic anal-
gesics inhibit writhing response [34]. Swiss albino mice weighing
25e30 g were chosen. The animals were divided into thirteen
groups, each group containing five animals. The animals were
administered with the test compounds and standard drug aspirin
100 mg/kg p.o for a group as positive control and SCMC as negative
control for a group. After 1 h, 10 ml/kg dose of 0.6% acetic acid was
injected intraperitonially. Each mouse was placed under the glass
4.2.6.1. Interaction with stable free radical DPPH. One of the quick
methods to evaluate antioxidant capacity is the scavenging activity
on DPPH, a stable free radical. 2 ml of standard drug, tocopherol and
title compounds at 25, 50, 75, 100 and 125
mM were added to 2 ml
of 100 M DPPH ethanolic solution. The tubes were kept at an
m
ambient temperature for 20e30 min protected from light by
covering with aluminum foil and absorbance was measured at
517 nm using ethanol as blank. The experiment values were ob-
tained in triplicate, SEM (Standard Error Mean) and % reduction
were calculated for test samples and standard drug. (Note: DPPH
solution should be made freshly and should be kept in dark.)%
scavenging/Reduction ¼ [Absorbance of controlꢀAbsorbance of
test sample/Absorbance of control] ꢃ 100. Concentration of drug

84
B. Haseena banu et al. / European Journal of Medicinal Chemistry 78 (2014) 72e85
showing 50% radical inhibition activity (IC50) was calculated from
the graph of % free radical scavenging activity against the drug
concentration [37].
online tool [40]. Using these parameters the compounds were
checked for their compliance with Lipinski rule of five [41]. Drug-
likeness score were computed using molsoft website [42]. The
drug-likeness scores were analyzed by comparing with the earlier
reports [43]. Bioactivity score predictions were also done using Mol
inspiration online tool. Pharmmapper serves as a valuable tool for
identifying targets for a novel synthetic compound, a newly iso-
lated natural product, a compound with known biological activity
or an existing drug whose mechanism action is unknown [44].
Suitable targets were predicted by Pharmmapper and docking
studies were conducted using Swiss Dock software [45].
4.2.6.2. Hydroxyl radical scavenging activity. 1 ml 2-deoxy-2-ribose
(2.8 mM); KH2PO4eKOH buffer (20 mM, pH 7.4); FeCl3 (100
EDTA (100 M); H2O2 (1.0 mM); ascorbic acid (100 M) and 1 ml of
test compounds dissolved in methanol (25e100 g/ml). After in-
mM);
m
m
m
cubation for 1 h at 37 ꢂC, 0.5 ml of the reaction mixture was added
to 1 ml of 2.8% TCA, then 1 ml of 1% aqueous TBA was added and the
mixture was incubated at 90 ꢂC for 15 min to develop the pink
chromogen. After cooling, the absorbance was measured at 532 nm.
Percentage inhibition was evaluated by comparing the test and
blank solutions. The experiment values were obtained in triplicate,
SEM, % scavenging and IC50 was calculated for test samples and
standard drug.
Acknowledgments
We are thankful UGC-New Delhi, India for granting the Research
Fellowship in Sciences for Meritorious Students.
4.2.6.3. Inhibition of iron induced lipid peroxidation. Adult Wister
rats of either sex and of approximately the same age weighing
about 200e220 g were used. Rats were fasted overnight and
sacrificed by cervical dislocation, dissected and the whole brain
except cerebellum was removed quickly. The brain issue was
weighed and homogenate (10% w/v) was prepared in 0.15 M KCl
and centrifuged at 800 rpm for 10 min using homogenizer. The
supernatant liquid was used immediately for the study and used as
a source of polyunsaturated fatty acids for determining the extent
of lipid peroxidation. The extent of lipid peroxidation in rat brain
homogenate was measured in vitro in terms of formation of thio-
barbituric acid reactive substances (TBARS). 1 ml of 25, 50, 100 and
Appendix A. Supplementary data
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2014.03.
037. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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