Synthesis, characterization, and biological study of phenylalanine amide derivatives

Article abstract of DOI:10.1007/s00706-016-1700-3

In the present study, a series of amide derivatives of 4-nitro-l-phenylalanine were synthesized with good yield from 4-nitro-l-phenylalanine and substituted anilines using propylphosphonic anhydride (T3P) as coupling reagent and were characterized through the IR, LC–MS, ¹H and ¹³C NMR spectral studies. The isolated products were screened for antimicrobial and antioxidant activities. Some of the compounds showed Microsporum gypsuem activity or were active against Candida albicans, also a few of compounds showed antibacterial activities. The resultant compounds screened for anti-oxidant activity, which showed good activity for DPPH scavenging and ABTS assay methods and others had moderately activity. Some amide compounds act as metal chelating ligands with Fe²⁺ ions. Graphical abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00706-016-1700-3

Monatsh Chem
DOI 10.1007/s00706-016-1700-3
ORIGINAL PAPER
Synthesis, characterization, and biological study of phenylalanine
amide derivatives
Mahesh Bhat1 S. L. Belagali1 P. Rajesh Shastry2 V. Ravishankar Rai2
•
•
•
Received: 8 October 2015 / Accepted: 9 February 2016
Ó Springer-Verlag Wien 2016
Abstract In the present study, a series of amide deriva-
tives of 4-nitro-L-phenylalanine were synthesized with
good yield from 4-nitro-L-phenylalanine and substituted
anilines using propylphosphonic anhydride (T3PÒ) as
coupling reagent and were characterized through the IR,
Graphical abstract
1
LC–MS, H and 13C NMR spectral studies. The isolated
products were screened for antimicrobial and antioxidant
activities. Some of the compounds showed Microsporum
gypsuem activity or were active against Candida albicans,
also a few of compounds showed antibacterial activities.
The resultant compounds screened for anti-oxidant activity,
which showed good activity for DPPH scavenging and
ABTS assay methods and others had moderately activity.
Some amide compounds act as metal chelating ligands with
Fe2? ions.
Keywords T3PÒ Á Amide Á DPPH Á Antimicrobial Á
Antioxidant Á Chelating activity
Introduction
Phenylalanine is an amino acid and one of the ‘‘building
block’’ of proteins. Major dietary sources of L-phenylalanine
include meat, fish, eggs, cheese, milk, etc. The synthetic and
biological interest on phenylalanine began from 1960
onwards. Esters of certain L-phenylalanine and substituted
phenylalanine derivatives showed very good antimicrobial
activity [1]. Some hydroxyethylpiperazine derivatives of
phenylalanine showed the antimalarial activity towards
Plasmodium falciparum [2] and short peptides of phenylala-
nine were antimalarial active [3]. Some of the 4-amino-N-
acetylphenylalanine amide ATP phosphate ester derivatives
are potent protein kinaseinhibitors [4]. N-Tetrahydrofuroyl-L-
phenylalanine and 1-[(3,5-dichlorophenyl)sulfonyl]pyrro-
lidine-2-carboxamido-3-phenylpropanoic acid derivatives act
as potent antigen-4 antagonists and studied for inflammatory
autoimmune diseases [5].
Electronic supplementary material The online version of this
article (doi:10.1007/s00706-016-1700-3) contains supplementary
material, which is available to authorized users.
& Mahesh Bhat
Maheshbhat08@gmail.com
1
Environmental Chemistry Laboratory, Department of Studies
in Environmental Science, University of Mysore, Mysore,
Karnataka 570006, India
2
Department of Studies in Microbiology, University of
Mysore, Mysore, Karnataka 570006, India
123

M. Bhat et al.
Cyclic amide derivatives of L-phenylalanine showed
significant potential inhibition and toxicity profile for anti-
HIV test [6]. Different a-amino-b-napthylamides of pheny-
lalanine derivatives are the sensitive fluorogenic substrates
for selective in vitro and in vivo assay on leukotriene A4
hydrolase activity [7]. So, phenylalanine derivatives exhibit
a variety of applications and are biologically important.
Antimicrobial agents are considered one of the most sig-
nificant milestones in modern medicinal chemistry, the
recent advance studies were carried out day by day to
develop the pathogen resistant medicine [8–11]. All the
amino acids occur in proteins are in L form and participate
in all bio-physiological process. L-Phenylalanine is an
essential amino acid and needed for functioning of the
central nervous system. So it will be an advantage, if
structural modified L-phenylalanine compound can be uni-
versalized with less toxic effect. Because of this we planned
the synthesis of 4-nitro-L-phenylalanine amide derivatives
and evaluate their antimicrobial and antioxidant activities.
synthesized 12 compounds with different substitution on
phenyl group and the compound structure, their percentage
yield is shown in the Table 1. The synthesized products
were confirmed by [M?1]? molecular ion peak by
recording LC–MS. The appearance of singlet broad peak at
1
10.0 ppm in H NMR spectrum clearly indicates the for-
mation of the amide product and it was characterized
through FT-IR, shows the peak at 1670 cm-1 correspond-
ing to the amide C=O stretching frequency indicates the
formation of critical amide bond, also characterized by 13
C
NMR spectrum. From the spectroscopic study, we can
conclude that synthesized products were complying with
the assigned structure, in very high purity.
Biology
The characterized fine products were screened for their
in vitro antimicrobial activity against Staphylococcus epi-
dermidis, Streptococcus pyogens, and Salmonella typhi and
in vitro anti fungal activity against Candida albicans and
Microsporum gypsum and their zone of inhibition was
shown in Table 2.
Results and discussion
Chemistry
Compounds 4 and 13 (methyl group at para position)
are active against S. epidermidis, 4 and 10 (p-methyl and
3,5-dichloro on phenyl side chain, respectively) show
activity against S. pyogens, compound 4 also active against
S. typhi, it is shown in Table 2 with its of zone of inhibi-
tion. The MIC of the synthesized compound was
experimentally determined and it is shown in Table 3. The
compounds 2, 5, 7, 9, and 11 did not show antimicrobial
activity against selected bacteria/fungi. Recent studies
reported that L-phenylalanine-derived C5-substituted rho-
danine showed significant antibacterial activity against
Gram-positive bacteria [14]. Sulfonamide phenyl-amides
of N-(4-nitrobenzoyl)phenylalanine has activity against
Gram-positive and Gram-negative bacteria strains [15].
Compounds 3, 10, 12, and 13 (p- and m-substituents on
phenyl side chain) are active against M. gypsum and shows
better activities compare to the standard. 6 and 8 (p-hy-
droxy and p-chloro substituents) show C. albicans anti
fungal activity, this is may be the resultant delocalization
of the electron because of the electron donating and
withdrawing groups present in the aromatic substitution.
In the literature L-phenylalanine derivatives of 40-OMe-
asperphenamate showed activity against Aspergillus ele-
gans fungus [16]. In general, strongly electron donating
group present at para position has less active, has large
MIC values. Weakly electron donating group (methyl) at
para position and chloro substituted compounds have
remarkable antimicrobial activities.
Amides are wide spread in compounds of biological and
pharmaceutical importance [12]. Here 4-nitro-L-pheny-
lalanine amide compounds were synthesized by the
coupling of N-Boc protected 4-L-nitrophenylalanine and
aromatic amines, whereby amide bond was generated by
one pot synthesis in presence of propylphosponic anhy-
dride and base (triethylamine). The synthetic strategies for
the targeted compounds were shown in the Scheme 1.
Propylphosphonic anhydride is very good amide coupling
reagent compared to DCC, HOBt, HATU, EDC etc.,
because of less epimerization, easy water work up and
purification techniques [13]. From the Scheme 1 we have
Scheme 1
(a) Boc anhydride, NaOH, tert-butyl alcohol; (b) R1NH2, T3P®, EtOAc, trimethylamine;
The phenylalanine amide derivatives were also screened
for antioxidant and chelating activity. Compounds 2, 3, 4,
5, and 6 (hydrogen, ethyl ester, p-methyl, p-methoxy, and
(c) TFA, dichloromethane
123

Synthesis, characterization, and biological study of phenylalanine amide derivatives
DPPH scavenging method. In ABTS assay method 6 and 7
Table 1 Chemical structures and yields of the synthesized com-
pounds (Scheme 1)
show excellent activity and the IC50 values 11.38 and
11.16 lg/cm3, respectively. The compounds containing
easily replaceable H? or radical stabilizing group show
better antioxidant activities [17]. In the synthesized com-
pound 6 has OH group which is mainly responsible for the
activity and 7 has five membered oxygen containing ring
(furan), which is attached to amide bond through CH2
group, also shows the remarkable activity.
Product
R1
Yield/%a
2
C6H5–
93
86
90
89
90
82
95
91
85
91
83
90
3
4-(EtO-CO)–C6H4–
4-CH3–C6H4–
4-CH3O–C6H4–
4-OH–C6H4–
4
5
6
7
2-Furanylmethyl
4-Cl–C6H4–
In human and animal systems, transition metal ions such
as ferrous ion (Fe2?) can facilitate the production of
reactive oxygen species leading to oxidative damage of the
cells. The powerful ferrous ion chelators afford protection
against oxidative damage by removing iron [18]. The fer-
rous ion metal chelating activity was determined by Lim
et al. method [23]. The present study reveals that 3 (93.29),
5 (58.88), 6 (59.78), 13 (114.15) act as ligands for Fe2?
ions. This indicates that some of the synthesized com-
pounds are good chelating ligands, because of the presence
of NH2 group present in the all the synthesized compounds
and also some of the moiety contains OH, OMe, CO2Et
which act as chelating sites for Fe2?. The IC50 values of
8
9
1,4-Phenylenebis–
3,5-Cl2–C6H3–
4-F–C6H4–
10
11
12
13
a
3,5-(CH3)2–C6H3–
3,4-(CH3)2–C6H3–
Yields are calculated over the last two steps (acid amine coupling
and Boc deprotection)
p-hydroxy substituents on phenyl side chain) are good
antioxidants, both in DPPH and ABTS assay method, in
which 3 and 6 have IC50 value 37.81, 78.50 lg/cm3 in
Table 2 Antimicrobial activity of 4-nitro-L-phenylalanine amide derivatives
Compounds
Zone of inhibition/mmb
Staphylococcus
epidermidis
Streptococcus
pyogens
Salmonella
typhi
Candida
albicans
Microsporum
gypsum
3
–
–
–
–
14 0.4a
4
18 0.6a
10 0.5a
8
–
–
–
–
–
0.7a
NA
–
6
–
–
–
8
0.5a
–
8
–
11 0.6a
–
10
–
9
–
–
0.5
–
26 0.4a
12
–
–
9
0.7a
13
Chloramphenicolc (30 lg/disc)
Nystatinc (30 lg/disc)
13 0.7a
26 0.5a
ND
–
19 0.3a
16 0.6a
18 0.7a
ND
10 0.5a
ND
ND
ND
8
0.6a
a
significant at P \ 0.05 (one way ANOVA followed by Tukey’s test)
The zone of inhibition was expressed as an average from three experiments standard deviation
b
c
Standard drug, – = no activity, ND = not done
Table 3 Minimum inhibitory concentrations of 4-nitro-L-phenylalanine amide derivatives
Microorganisms
MIC/lg/cm3
3
4
6
8
10
12
13
Chloramphenicol
Nystatin
Staphylococcus epidermidis
Streptococcus pyogens
Salmonella typhi
–
15.6
62.5
125
–
–
–
–
–
15.6
–
15.6
7.8
ND
ND
ND
125
125
–
–
–
125
–
–
–
–
–
–
–
7.8
Candida albicans
–
250
–
250
–
–
–
–
ND
ND
Microsporum gypsum
125
–
15.6
125
62.5
ND not done, – no activity
123

M. Bhat et al.
amides from acid amine coupling and the byproduct can be
removed by simple water work up, hence the synthesis of
4-nitro-L-phenylalanine amides were achieved in very effi-
cient method. Here, starting material is amino acid
derivative, so needs amino functional group protection, by
Boc anhydride. After synthesis of the amides, purification
was done by acid base work up. Once amides are obtained,
the protecting group was removed by TFA. The purity of the
compounds was checked by LCMS and structures of all the
derivatives were assigned by IR, 1H and 13C NMR spectro-
scopic data and they comply with all the proposed structures.
Reactions were performed in two necked 100 cm3 round
bottomed flask. The glass wares were previously rinsed
with acetone and dried in hot air oven. The raw materials
were purchased from Chem-Impex International (USA)
and T3PÒ from Aldrich with purity [99 %. The monitor-
ing of reaction was carried out by thin layer
chromatography (TLC), using aluminium plate, coated
with silica, which were obtained from Merck, by using
appropriate solvent mixtures. The melting points were
determined by open capillary method. The products were
confirmed by LCMS and 1H and 13C NMR spectral studies.
NMR spectrum was recorded by an Agilent 400 MHz
instrument and LC–MS were recorded by positive and
negative mode, using solvents 0.1 % formic acid in Ace-
tonitrile. Antioxidant activity was found out by DPPH and
ABTS assay method, using 96-well micro titer plate and
readings were taken from Varioskan Flash instruments.
Table 4 Antioxidant IC50 values of the synthesized amide
compounds
Comp
IC50a/lg cm-3
DPPH method
ABTS assay
Chelating activity
2
325.05 0.23
37.81 0.36
198.67 0.15
98.90 0.33
78.50 0.30
[500
175.82 0.29
92.55 0.23
488.44 0.46
79.12 0.32
11.38 0.27
11.16 0.22
442.65 0.39
[500
218.49 0.28
93.29 0.20
[500
3
4
5
[500
6
58.88 0.30
59.78 0.26
[500
7
8
477.69 0.56
[500
9
[500
10
11
12
13
AA
EDTA
a
478.99 0.57
428.00 0.36
[500
455.01 0.39
310.35 0.78
[500
333.62 0.48
169.62 0.78
[500
[500
326.13 0.63
14.64 0.46
–
114.15 0.62
–
14.64 0.46
–
1.10 0.02
The IC50 value was expressed as an average from three experiments
Standard deviation
antioxidant activity of the synthesized compounds are
shown in Table 4.
Conclusions
Boc-4-nitro-L-phenylalanine (1)
A series of phenylalanine amide derivatives were synthe-
sized with very good yield and characterized through FT-
To the stirred solution of 4.0 g 4-nitro-L-phenylalanine
(0.019 mol) in 30 cm3 tert-butyl alcohol and 0.8 g NaOH
(0.0209 mol, in 5 cm3 water), 5 cm3 BOC anhydride
(0.0228 mol in 5 cm3 tert-butyl alcohol) was added drop-
wise at 0 °C. The resulting solution was stirred for 10 h at
room temperature and after completion of the reaction
(confirmed by TLC), solvent was evaporated, quenched
into water, extracted with EtOAc (2 9 50 cm3), washed
with 50 cm3 citric acid (10 % solution), 50 cm3 brine
solution, dried over sodium sulfate, and concentrated to get
title compound as white solid [4]. Yield 5.0 g (86 %); m.p.:
105–106 °C (Ref. [24] 105–107 °C).
1
IR, LC–MS, H NMR, and 13C NMR spectra. From the
activity studies we can conclude that phenylalanine amide
derivatives having methyl, dimethyl, dichloro substituents
on phenyl group revealed as active compounds against
tested bacteria and fungi. (R)-2-Amino-3-(4-nitrophenyl)-
N-(p-tolyl)propanamide has showed significant activity
(P \ 0.05) against tested three bacteria suggesting the
importance of electron donating methyl group in para
position. Furthermore, compounds having substituent like
chloro and hydroxy are significantly active against C.
albicans and amide derivatives with hydroxyl, methoxy,
ethyl ester, and furan substituent were revealed as most
potent antioxidants.
General procedure for the synthesis of amide
derivatives
Experimental
Chemistry
To the solution of Boc-4-nitro-L-phenylalanine (1.0 mmol)
and substituted aniline (1.1 mmol) in 10 cm3 ethyl acetate,
T3PÒ (1.5 mmol) was added drop-wise followed by tri-
ethylamine (3.0 mmol) and stirred at room temperature for
6 h. After completion of the reaction (confirmed by TLC),
it was quenched to water, extracted with ethyl acetate
(3 9 15 cm3). The combined organic layer was washed
The reaction sequence for the synthesis of amide derivatives
of titled compounds is outlined in Scheme 1. T3PÒ (propyl
phosphonic anhydride) is a green reagent for preparation of
123

Synthesis, characterization, and biological study of phenylalanine amide derivatives
with 10 cm3 aqueous sodium bicarbonate solution (10 %),
10 cm3 citric acid solution (10 %), and finally 10 cm3
brine solution. The organic layer was dried and evaporated
to get title compounds. The crude material was purified by
recrystallisation with ethanol [19–21].
143.2, 136.7, 135.8, 130.2, 129.8, 124.6, 122.5, 54.2, 39.3,
21.4 ppm; LC–MS: m/z = 300.2 ([M ? 1]?).
(R)-2-Amino-N-(4-methoxyphenyl)-3-(4-nitro-
phenyl)propanamide (5, C16H17N3O4)
Off white solid; 88 % yield; m.p.: 120–121 °C; FT-IR (KBr):
vꢀ = 3388 (–NH2), 3267 (–NH), 2938 (Ar–CH), 1670 (C=O,
amide), 1513 (Ar–C=C), 1597, 1344 (NO2), 1242 (C–N),
1026 (O–CH3) cm-1; 1H NMR (400 MHz, CDCl3): d = 9.24
(s, 1H), 8.19–8.17 (d, J = 7.6 Hz, 2H), 7.48–7.41 (m, 4H),
6.88–6.86 (d, J = 7.6 Hz, 2H), 3.79 (s, 3H), 3.61–3.59 (m,
1H), 3.44–3.42 (d, J = 12.0 Hz, 1H), 3.03–2.98(m, 1H)ppm;
13C NMR (100 MHz, CDCl3): d = 171.0, 156.4, 147.1,
145.6,130.5,130.2,123.9,121.1,114.1,56.3, 55.5, 40.6 ppm;
LC–MS: m/z = 315.9 ([M ? 1]?).
General procedure for BOC group deprotection (com-
pounds 2–13)
To the solution of Boc-amide compound (1.0 mmol) in 5 cm3
dichloromethane, 5 cm3 trifluoroacetic acid was added drop-
wiseat0 °C and the reaction mixturewas stirred atRTfor 5 h.
After completion of the reaction, it was quenched to 10 %
aqueous bicarbonate solution and extracted with dichloro-
methane, washed with brine solution (pH = 7.0), dried over
sodium sulfate, and evaporated to get final compound [22].
(R)-2-Amino-N-(4-hydroxyphenyl)-3-(4-nitro-
(R)-2-Amino-3-(4-nitrophenyl)-N-phenylpropanamide
(2, C15H15N3O3)
phenyl)propanamide (6, C15H15N3O4)
Brown solid; 89 % yield; m.p.: 171–172 °C; FT-IR (KBr):
vꢀ = 3395 (–OH), 3264 (–NH2), 3107 (–NH), 3957 (Ar–
CH), 1669 (C=O, amide), 1517 (Ar–C=C), 1606, 1340
Yellowish solid; 93 % yield; m.p.: 139–140 °C; FT-IR
(KBr): vꢀ = 3345 (–NH2), 3215 (–NH), 3021 (Ar–CH), 1672
(C=O, amide), 1521 (Ar–C=C), 1603 1341 (NO2), 1266 (C–
1
(NO2), 1224 (C–N) cm-1; H NMR (400 MHz, DMSO-
1
N) cm-1; H NMR (400 MHz, CDCl3): d = 9.3 (s, 1H),
d6): d = 9.8 (bs, 1H), 9.2 (bs, 1H), 8.13–8.11 (m, 2H),
7.51–7.49 (t, J = 2.0, 8.8 Hz, 2H), 7.34–7.32 (m, 2H),
6.67–65 (m, 2H), 3.56–3.52 (m, 1H), 3.09–3.05 (m, 1H),
2.84–2.80 (m,1H) ppm; 13C NMR (100 MHz, DMSO-d6):
d = 172.8, 153.8, 147.9, 146.5, 131.1, 130.8, 123.5, 121.5,
115.5, 57.1, 41.2 ppm; LC–MS: m/z = 302.0 ([M ? 1]?).
8.18–8.16 (d, J = 8.4 Hz, 2H), 7.58–7.56 (d, J = 8.0 Hz,
2H), 7.43–7.41 (d, J = 8.0 Hz, 2H), 7.35–7.31 (t, J = 8.0,
15.6 Hz, 2H), 7.14–7.10 (t, J = 7.2, 14.8 Hz, 1H),
3.80–3.78 (t, J = 4.8, 8.4 Hz, 1H), 3.45–3.41 (m, 1H),
3.03–2.97 (m, 1H) ppm; 13C NMR (100 MHz, CDCl3):
d = 171.3, 147.1, 145.5, 137.4, 130.2, 129.1, 124.4, 123.9,
119.5, 56.4, 40.5 ppm; LC–MS: m/z = 286.1 ([M ? 1]?).
(R)-2-Amino-N-(furan-2-ylmethyl)-3-(4-nitro-
phenyl)propanamide (7, C14H15N3O4)
(S)-Ethyl 4-[[2-amino-3-(4-nitrophenyl)-1-oxo-
Pale orange solid; 82 % yield; m.p.: 83–84 °C; FT-IR
(KBr): vꢀ = 3329 (–NH2), 3231 (–NH), 3012 (Ar–CH),
2917 (alkyl-CH), 1664 (C=O, amide), 15,145 (Ar–C=C),
propyl]-amino]benzoate (3, C18H19N3O5)
Orange solid; 86 % yield; m.p.: 157–158 °C; FT-IR (KBr):
vꢀ = 3418 (–NH2), 3252 (–NH), 2985 (Ar–CH), 1694 (C=O,
ester), 1607 (C=O, amide), 1513 (Ar–C=C), 1607, 1349
1
1603, 1342 (NO2), 1239 (C–N), 1230 (C–O) cm-1; H
1
(NO2), 1285 (C–N) cm-1; H NMR (400 MHz, CDCl3):
NMR (400 MHz, CDCl3): d = 8.15–8.13 (d, J = 8.0 Hz,
2H), 7.51 (s, 1H), 7.35–7.27 (d, J = 30.0 Hz, 3H),
6.32–6.30 (d, J = 8.2 Hz, 2H), 4.48–4.36 (m, 2H),
3.17–3.68 (m, 1H), 3.33–3.29 (m, 1H), 3.01–2.95 (m,
1H) ppm; 13C NMR (100 MHz, CDCl3): d = 173.0, 151.1,
146.9, 145.5, 142.3, 130.2, 123.8, 110.4, 107.5, 55.9, 40.7,
36.1 ppm; LC–MS: m/z = 290.2 ([M ? 1]?).
d = 9.65 (s, 1H), 8.20–8.18 (d, J = 8.4 Hz, 2H), 8.04–8.01
(t, J = 1.6, 8.8 Hz, 2H),7.67–7.65 (d, J = 8.8 Hz, 2H),
7.44–7.42 (d, J = 8.8 Hz, 2H), 4.39–4.36 (m, 1H),
4.39–4.34 (m, 2H), 3.85–3.81 (m, 1H), 3.03–2.98 (m, 1H),
1.67–1.36 (t, 3H) ppm; 13C NMR (100 MHz, CDCl3):
d = 171.3, 147.1, 145.5, 137.4, 130.2, 129.1, 124.4, 123.9,
119.5, 56.4, 40.5 ppm; LC–MS: m/z = 358.0 ([M ? 1]?).
(R)-2-Amino-N-(4-chlorophenyl)-3-(4-nitro-
phenyl)propanamide (8, C15H14ClN3O3)
(R)-2-Amino-3-(4-nitrophenyl)-N-(p-tolyl)propanamide
(4, C16H17N3O3)
Off white solid; 95 % yield; m.p.: 142–143 °C; FT-IR
(KBr): vꢀ = 3324 (–NH2), 3184 (–NH), 3073 (Ar–CH),
1663 (C=O, amide), 1511 (Ar–C=C), 1608, 1351 (NO2),
;
1257 (C–N), 690 (C–Cl) cm-1 1H NMR (400 MHz,
Pale orange solid; 89 % yield; m.p.: 137–138 °C; FT-IR
(KBr): vꢀ = 3312 (–NH2), 3194 (–NH), 3061 (Ar–CH), 2844
(alkyl-CH), 1668 (C=O, amide), 1514 (Ar–C=C), 1605 1346
1
(NO2), 1250 (C–N) cm-1; H NMR (400 MHz, CDCl3):
CDCl3): d = 9.45 (s, 1H), 8.19–8.17 (d, J = 8.0 Hz, 2H),
7.55–7.53 (d, J = 8.8 Hz, 2H), 7.43–7.41 (d, J = 8.0 Hz,
2H), 7.31–7.29 (d, J = 8.4 Hz, 2H), 3.81–3.72 (m, 1H),
3.46–3.42 (m, 1H), 3.03–2.98 (m, 1H) ppm; 13C NMR
(100 MHz, CDCl3): d = 171.4, 147.1, 145.4, 135.9, 130.2,
d = 9.28 (bs, 1H), 8.15–8.12 (m, 2H), 7.40–7.38 (t, J = 4.0,
8.8 Hz, 2H), 7.34–7.32 (m, 2H), 6.67–65 (m, 2H), 3.84–3.81
(m, 1H), 3.39–3.25 (m, 1H), 3.07–3.04 (m, 1H), 2.30 (s, 3H)
ppm; 13C NMR (100 MHz, CDCl3): d = 174.2, 146.5,
123

M. Bhat et al.
129.3, 129.1, 56.3, 40.5 ppm; LC–MS: m/z = 320.1
([M ? 1]?).
(R)-2-Amino-N-(3,4-dimethylphenyl)-3-(4-nitro-
phenyl)propanamide (13, C17H19N3O3)
Pale yellow solid; 80 % yield; m.p.: 100–101 °C; FT-IR
(KBr): vꢀ = 3378 (–NH2), 3280 (–NH), 2920 (Ar–CH),
2859 (alkyl C–H), 1667 (C=O amide), 1516 (Ar–C=C),
(2S,20S)-N,N0-(1,4-Phenylene)bis[2-amino-3-(4-nitro-
phenyl)propanamide] (9, C24H24N6O6)
White solid; 91 % yield; m.p.: 194–196 °C; FT-IR (KBr):
vꢀ = 3317 (–NH2), 3184 (–NH), 3061 (Ar–CH), 3045 (Ar–
CH), 1667 (C=O, amide), 1516 (Ar–C=C), 1607, 1341
1606, 1346 (NO2) cm-1 1H NMR (400 MHz, CDCl3):
;
d = 9.23 (s, 1H), 8.18–8.16 (d, J = 8.8 Hz, 2H),
7.41–7.38 (d, J = 12.0 Hz, 2H), 7.27–7.26 (d,
J = 4.0 Hz, 1H), 7.3 (s, 1H), 7.09–7.07 (d, J = 8.4 Hz,
1H), 3.79–3.77 (m, 1H), 3.44–3.39 (m, 1H), 3.04–3.00 (m,
1H), 2.25 (s, 3 H), 2.23 (s, 3H) ppm; 13C NMR (100 MHz,
CDCl3): d = 171.1, 147.0, 145.6, 137.3, 135.1, 132.7,
130.2, 129.9, 123.9, 120.8, 116.9, 56.4, 40.6, 19.9,
19.2 ppm; LC–MS: m/z = 314.1 ([M ? 1]?).
1
(NO2), 1252 (C–N) cm-1; H NMR (400 MHz, DMSO-
d6): d = 10.13 (bs 2H), 8.18–8.16 (d, J = 8.0 Hz, 4H),
7.57–7.53 (t, J = 8.4, 15.2, 8H), 3.83–3.79 (t, J = 6.4,
12.4 Hz, 2H), 3.21–3.16 (m, 2H), 2.99–2.94 (m, 2H) ppm;
13C NMR (100 MHz, DMSO-d6): d = 171.4, 146.7, 146.6,
134.6, 131.2, 123.6, 120.3, 56.3, 40.6 ppm; LC–MS:
m/z = 493.1 ([M ? 1]?).
(S)-2-Amino-N-(3,5-dichlorophenyl)-3-(4-nitro-
Antimicrobial activity
phenyl)propanamide (10, C15H13Cl2N3O3)
Off white solid; 85 % yield; m.p.: 134–135 °C; FT-IR
(KBr): vꢀ = 3388 (–NH2), 3233 (–NH), 2932 (Ar–CH),
1678 (C=O amide), 1511 (Ar–C=C), 1586, 1346 (NO2),
Microbial cultures used in the study were collected from
Microbial Type Culture Collection (MTCC), Institute of
Microbial Technology (IMTECH), Chandigarh, India. The
cultures used were S. epidermidis (MTCC-435), S. pyogens
(MTCC-1925), S. typhi (MTCC-733), Escherichia coli
(MTCC-40), C. albicans (MTCC-183), and Microsporum
gypseum (MTCC-2830). Stock cultures of bacteria were
maintained on nutrient agar (Himedia, Mumbai) slants at
4 °C with periodical sub-culturing. The fungal cultures
were maintained on Sabouraud’s dextrose agar (Himedia,
Mumbai) slants at 4 °C with periodic sub-culturing.
1281 (C–N), 844 (C–Cl) cm-1 1H NMR (400 MHz,
;
CDCl3): d = 9.57 (s, 1H), 8.21–8.18 (m, 2H), 7.56–7.55
(d, J = 2.0 Hz, 2H), 7.43–7.40 (m, 2H), 7.10–7.09 (t,
J = 2.0, 4.0 Hz, 1H), 3.81–3.78 (m 1H), 3.45–3.41 (m,
1H), 3.03–2.98 (m, 1H) ppm; 13C NMR (100 MHz,
CDCl3): d = 171.6, 147.2, 145.1, 139.2, 135.3, 130.2,
124.3, 117.6, 56.2, 40.3 ppm; LC–MS: m/z = 354.1
([M ? 1]?).
The antimicrobial activities of compounds were
screened by disc diffusion method [23]. Briefly, the sterile
paper discs loaded with extract (500 lg/disc) were placed
on the pre-seeded agar plates. 100 mm3 of 24 h culture of
test microorganisms in broth was used for the seeding.
Chloramphenicol discs (6 mm, 30 lg/disc) and nystatin
discs (Himedia, 6 mm diameter, 30 lg/disc) were used as
positive controls for bacteria and fungi respectively, while,
the discs with only the solvent (30 mm3/disc) were used as
negative control. The plates were pre-incubated for one h at
4 °C. The bacterial plates were incubated at 37 °C for 24 h
and fungal plates for 5 days at 30 °C temperatures. The
diameters of the inhibition zones (in mm) were measured
after the incubation and values are shown in Table 2.
(R)-2-Amino-N-(4-fluorophenyl)-3-(4-nitro-
phenyl)propanamide (11, C15H14FN3O3)
Off white solid; 90 % yield; m.p.: 109–110 °C; FT-IR
(KBr): vꢀ = 3346 (–NH2), 3236 (–NH), 2937 (Ar–CH),
1690 (C=O, amide), 1516 (Ar–C=C), 1605 1343 (NO2),
;
1208 (C–N), 1090 (C–F) cm-1 1H NMR (400 MHz,
CDCl3): d = 9.39 (s, 1H), 8.19–8.17 (m, 2H), 7.55–7.52
(m, 2H), 7.44–7.41 (m, 2H), 7.05–7.01 (m, 2H), 3.82–3.78
(m, 1H), 3.47–3.42 (m, 1H), 3.04–2.99 (m, 1H) ppm; 13C
NMR (100 MHz, CDCl3): d = 171.3, 160.6, 158.2, 147.1,
145.5, 133.5, 130.2, 123.9, 121.2, 121.1, 115.8, 115.6,
56.2, 40.5 ppm; LC–MS: m/z = 304.1 ([M ? 1]?).
(S)-2-Amino-N-(3,5-dimethylphenyl)-3-(4-nitro-
phenyl)propanamide (12, C15H14FN3O3)
White solid; 82 % yield; m.p.: 160–162 °C; FT-IR (KBr):
vꢀ = 3400 (–NH2), 3297 (–NH), 3016 (Ar–CH), 2922 (alkyl
C–H), 1673 (C=O amide), 1515 (Ar–C=C), 1604, 1343
Determination of minimum inhibitory concentration
(MIC)
1
(NO2) cm-1; H NMR (400 MHz, DMSO-d6): d = 9.94
A broth micro dilution method was used to determine the
MIC of the isolated compounds [24, 25]. A serial twofold
dilution of the compounds were prepared in a 96-well
micro titer plate (Tarsons, F bottom) over the range
500–0.24 lg/cm3. Each of the wells was inoculated with
10 mm3 of the turbidity adjusted inoculums. The bacterial
plates were incubated at 37 °C for 24 h and those of fungi
(bs, 1H), 8.15–8.13 (d, J = 8.8 Hz, 2H), 7.53–7.50 (d,
J = 8.4 Hz, 2H), 7.16 (s, 2H), 6.68 (s, 1H), 3.78 (s, 1H),
3.17–3.12 (m, 1H), 2.96–2.90 (m, 1H), 2.19 (s, 6H) ppm;
13C NMR (100 MHz, DMSO-d6): d = 171.3, 146.8, 146.5,
1387.7, 138.2, 131.2, 125.6, 123.7, 117.6, 56.3, 39.3,
21.5 ppm; LC–MS: m/z = 314.1 ([M ? 1]?).
123

Synthesis, characterization, and biological study of phenylalanine amide derivatives
at 28 °C for 72 h. MIC for bacteria was determined by
color change in the medium to red color after addition of
10 mm3 of 2,3,5-triphenyltetrazolium chloride (Himedia,
Mumbai, India) and MIC of fungi were determined as the
lowest concentrations without visible growth under optical
microscope and MIC values are shown in Table 3.
Free radical scavenging ability by DPPH radical
assay (1,1-diphenyl-2-picrylhydrazyl)
DPPH radical scavenging activity of purified compound was
determined by the method of Raj et al. [24]. Various con-
centrations of test sample in an aliquot of 100 mm3 were
mixed with 100 mm3 of 40 lM methanolic solution of
DPPH (Himedia, Mumbai, India) in a 96-well micro titer
plate. The decrease in absorbance at 517 nm was recorded
after the incubation of 15 min at room temperature. The
absorbance of the DPPH solution with only methanol and
Fig. 1 Antioxidant activity of the 4-nitro-L-phenylalanine amide
compounds at 500 lg/cm3
without sample was used as the control. The ascorbic acid
(AA, Himedia, Mumbai) was used as a standard to compare
the activity. Appropriate blank readings at 517 nm were
recorded for each tested dilution. The assay was carried out
in triplicate. The percentage inhibition of the DPPH radical
by the samples was calculated according to the formula:
the compound at 500 lg/cm3 were shown in the Fig. 1. The
IC50 value was calculated graphically based on capacity of
compound concentration to scavenge 50 % of free radicals
and it is shown in Table 4.
Percentage of inhibition ¼ ½ðAC À ASÞ=ACފ  100
ð1Þ
Ferrous ion chelating activity
where AC is the absorbance of the control and AS is the
absorbance of the sample/standard at 15 min. The IC50
value was calculated graphically based on capacity of
compound concentration to scavenge 50 % of free radicals.
The ferrous ion metal chelating activity was determined by
Lim et al. method [27]. Briefly, equal volumes of
0.125 mM FeSO4 and 0.3125 mM of ferrozine (Himedia,
Mumbai) were mixed with different dilutions of test
compound. The reaction mixture was equilibrated for
10 min and absorbance measured at 562 nm. The mixture
of iron and ferrozine solutions without the chelators was
used as the control and EDTA (Himedia, Mumbai) was
used as a standard to compare. The chelating capacity of
the compound was calculated based on following formula:
ABTS (2,20-azinobis(3-ethylbenzthiazoline-6-
sulfonic acid) radical scavenging activity
ABTS assay was performed with modification of the
Samaga et al. method [26]. The ABTS stock solution was
prepared by mixing equal volumes of 7.4 mM ABTS
(Sigma-Aldrich, USA) solution and 2.6 mM potassi-
umpersulfate solution followed by incubation for 12 h at
room temperature in the dark. The reaction mixture con-
sisted of 50 mm3 of the purified compound at different
concentrations (0.5–500 lg/cm3 in respective solvents) and
150 mm3 standardized ABTS solution. The decrease in
absorbance was measured at 734 nm after 15 min of
incubation. Data for each assay was recorded in triplicate.
Ascorbic acid (NICE Chemicals Pvt. Ltd., Cochin, India)
was used as positive control. The scavenging activity was
estimated based on the percentage of ABTS radicals
scavenged by the following formula:
ÂÀ
Á
Ã
Percentage of iron chelation ¼ Acontrol À Asample =Acontrol
 100
ð3Þ
Statistical analysis
The values of antimicrobial activity were expressed as
mean standard deviation. The inhibition zone was
measured from the antimicrobial activity of compound and
analysed using one way analysis of variance (ANOVA)
followed by Tukey’s test at P \ 0.05. The software Origin
Pro 9.0 was employed for the statistical analysis.
Percentage of scavenging ¼ ½ðAC À ASÞ=ACŠ  100
where AC is absorbance of control, AS is absorbance of
ð2Þ
Acknowledgments One of the authors, Mr. Mahesh Bhat is
thankful to Department of Science and Technology, New Delhi, India,
for providing INSPIRE Fellowship [IF130464] and Institute of
Excellence, University of Mysore, for providing NMR facilities.
tested sample/standard solution, percentage absorbance of
123

M. Bhat et al.
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