Synthesis, molecular docking and biological evaluation of novel phthaloyl derivatives of 3-amino-3-aryl propionic acids as inhibitors of Trypanosoma cruzi trans-sialidase

Article abstract of DOI:10.1016/j.ejmech.2018.07.005

In the last two decades, trans-sialidase of Trypanosoma cruzi (TcTS) has been an important pharmacological target for developing new anti-Chagas agents. In a continuous effort to discover new potential TcTS inhibitors, 3-amino-3-arylpropionic acid derivatives (series A) and novel phthaloyl derivatives (series B, C and D) were synthesized and molecular docking, TcTS enzyme inhibition and determination of trypanocidal activity were carried out. From four series obtained, compound D-11 had the highest binding affinity value (?11.1 kcal/mol) compared to reference DANA (?7.8 kcal/mol), a natural ligand for TS enzyme. Furthermore, the 3D and 2D interactions analysis of compound D-11 showed a hydrogen bond, π-π stacking, π-anion, hydrophobic and Van der Waals forces with all important amino acid residues (Arg35, Arg245, Arg314, Tyr119, Trp312, Tyr342, Glu230 and Asp59) on the active site of TcTS. Additionally, D-11 showed the highest TcTS enzyme inhibition (86.9% ± 5) by high-performance ion exchange chromatography (HPAEC). Finally, D-11 showed better trypanocidal activity than the reference drugs nifurtimox and benznidazole with an equal % lysis (63 ± 4 and 65 ± 2 at 10 μg/mL) and LC₅₀ value (52.70 ± 2.70 μM and 46.19 ± 2.36 μM) on NINOA and INC-5 strains, respectively. Therefore, D-11 is a small-molecule with potent TcTS inhibition and a strong trypanocidal effect that could help in the development of new anti-Chagas agents.

Full text of DOI:10.1016/j.ejmech.2018.07.005

Accepted Manuscript
Synthesis, molecular docking and biological evaluation of novel phthaloyl derivatives
of 3-amino-3-aryl propionic acids as inhibitors of Trypanosoma cruzi trans-sialidase
Muhammad Kashif, Karla Fabiola Chacon-Vargas, Julio Cesar Lopez-Cedillo,
Benjamin Nogueda-Torres, Alma D. Paz-Gonzalez, Esther Ramírez-Moreno, Rosalia
Agusti, Maria Laura Uhrig, Alicia Reyes-Arellano, Javier Peralta-Cruz, Muhammad
Ashfaq, Gildardo Rivera
PII:
S0223-5234(18)30561-0
10.1016/j.ejmech.2018.07.005
EJMECH 10540
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 June 2018
Revised Date: 2 July 2018
Accepted Date: 3 July 2018
Please cite this article as: M. Kashif, K.F. Chacon-Vargas, J.C. Lopez-Cedillo, B. Nogueda-Torres,
A.D. Paz-Gonzalez, E. Ramírez-Moreno, R. Agusti, M.L. Uhrig, A. Reyes-Arellano, J. Peralta-Cruz, M.
Ashfaq, G. Rivera, Synthesis, molecular docking and biological evaluation of novel phthaloyl derivatives
of 3-amino-3-aryl propionic acids as inhibitors of Trypanosoma cruzi trans-sialidase, European Journal of
Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.07.005.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Synthesis, molecular docking and biological evaluation of novel phthaloyl derivatives of
3
-amino-3-aryl propionic acids as inhibitors of Trypanosoma cruzi trans-sialidase
1
2
2
Muhammad Kashif , Karla Fabiola Chacon-Vargas , Julio Cesar Lopez-Cedillo , Benjamin
2
1
3
4,5
Nogueda-Torres , Alma D. Paz-Gonzalez , Esther Ramírez-Moreno , Rosalia Agusti ,
4
,5
6
6
7
Maria Laura Uhrig , Alicia Reyes-Arellano , Javier Peralta-Cruz , Muhammad Ashfaq ,
1
*
Gildardo Rivera
1
Laboratorio de Biotecnología Farmacéutica, Centro de Biotecnología Genómica, Instituto
Politécnico Nacional, 88700, Reynosa, México
2
Departamento de Parasitología, Escuela Nacional de Ciencias Biológicas, 07320, Ciudad de
México, México
3
Escuela Nacional de Medicina y Homeopatía, Instituto Politécnico Nacional, 07320, Ciudad
de México, México
4
Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Departamento de
Química Orgánica, Pabellón 2, Ciudad Universitaria, C1428EG Buenos Aires, Argentina.
5
Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET)-UBA, Centro de
Investigaciones en Hidratos de Carbono (CIHIDECAR), Buenos Aires, Argentina
6
Departamento de Química Orgánica, Escuela Nacional de Ciencias Biológicas, 07320,
Ciudad de México, México
7
Department of Chemistry, The Islamia University of Bahawalpur, Bahawalpur, Pakistan
*
Corresponding author: Gildardo Rivera, Centro de Biotecnología Genómica, Instituto
Politécnico Nacional, Boulevard del Maestro, s/n, Esq. Elías Piña, 88710, Reynosa, México.
Phone: +52 899 9243627, Fax: +52 899 9243628, E-mail: gildardors@hotmail.com

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Abstract
In the last two decades, trans-sialidase of Trypanosoma cruzi (TcTS) has been an
important pharmacological target for developing new anti-Chagas agents. In a continuous
effort to discover new potential TcTS inhibitors, 3-amino-3-arylpropionic acid derivatives
(series A) and novel phthaloyl derivatives (series B, C and D) were synthesized and
molecular docking, TcTS enzyme inhibition and determination of trypanocidal activity were
carried out.
From four series obtained, compound D-11 had the highest binding affinity value (-
1
1.1 kcal/mol) compared to reference DANA (-7.8 kcal/mol), a natural ligand for TS enzyme.
Furthermore, the 3D and 2D interactions analysis of compound D-11 showed a hydrogen
bond, π-π stacking, π-anion, hydrophobic and Van der Waals forces with all important amino
acid residues (Arg35, Arg245, Arg314, Tyr119, Trp312, Tyr342, Glu230 and Asp59) on the
active site of TcTS. Additionally, D-11 showed the highest TcTS enzyme inhibition (86.9% ±
5
) by high-performance ion exchange chromatography (HPAEC). Finally, D-11 showed
better trypanocidal activity than the reference drugs nifurtimox and benznidazole with an
equal % lysis (63 ± 4 and 65 ± 2 at 10µg/mL) and LC value (52.70 ± 2.70 µM and 46.19 ±
5
0
2
.36 µM) on NINOA and INC-5 strains, respectively. Therefore, D-11 is a small-molecule
with potent TcTS inhibition and a strong trypanocidal effect that could help in the
development of new anti-Chagas agents.
Keywords; Trans-sialidase, Trypanosoma cruzi, molecular docking, propionic acid,
phthaloyl, inhibitors

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1
. Introduction
Chagas disease affects 8 to 10 million people worldwide, primarily in South and
Central America [1-3]. Only two approved drugs, nifurtimox (Nfx) and benznidazole (Bzn),
are currently used in the world and have been for the last four decades; however, both drugs
display low efficacy in the chronic phase of infection and are associated with severe
undesirable side effects [4- 9]. Currently, Trans-sialidase of Trypanosoma cruzi (TcTS), a
non-homologous enzyme in humans or hosts, which is mainly expressed by trypomastigotes,
has been reported by various research groups as a pharmacological target for Chagas disease.
This enzyme catalyses the transfer of sialic acid from the host glycoconjugate surface to the
Trypanosoma cruzi (T. cruzi) mucin-like glycoprotein surface and also helps T. cruzi escape
from the host immune system [10- 12].
The kinetic, structural, molecular docking, quantum mechanical/molecular
mechanical (QM/MM) molecular dynamics (MD) simulation analysis of TcTS enzyme has
provided evidence that the residues Arg35, Arg245, Arg314, Asp59, Glu230, Trp120,
Trp312, Tyr119, Tyr342, Val95 and Leu176 play a key role in transferring sialic acid, in
stabilizing the transition states and in accommodating the lactosyl moiety to the
donor/acceptor substrates. Furthermore, the aromatic residues contribute to the formation of a
hydrophobic pocket in the enzyme catalytic cavity [10,11,13,14].
In addition to rational drug design, in silico screening methodology has been adopted
to find a strong (non-sugar based) TcTS inhibitor. For example, Neres et al., in 2007 and
2
009, using an in silico screening, found some compounds with novel scaffolds (non-sugar
benzoic acid and pyridines derivatives), with submillimolar IC values (Fig. 1, 1-4) [15, 16].
5
0
Later, the virtual screening of a natural product library by Kim et al., (2009) and Arioka et
al., (2010) showed that quinolinone and flavonoid derivatives (Fig. 1, 5 and 6) are the most
potent compounds with sub-micromolar TcTS inhibition values [17, 18].

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Fig. 1. Structure and inhibition values for representative known TcTS non-sugar based
inhibitors obtained from molecular docking and virtual screening of a chemical library.
In the search for new non-sugar based inhibitors of TcTS, we propose the synthesis of
3
-amino-3-arylpropionic acid derivatives (series A) due to the presence of NH and –COOH
2
groups, which could enhance the ability to interact with amino acid residues in the catalytic
cavity of TcTS [19]. Additionally, novel phthaloyl derivatives of 3-amino-3-arylpropionic
acid (series B, C and D) were proposed from 3-amino-3-arylpropionic acid derivatives (series
A) (Scheme 1) to enhance more interaction on the active site of TcTS due to the presence of a
carbonyl group on the phthaloyl moiety. Our main focus was to propose compounds that
could bind to the TcTS residues, a) Asp59, Glu230 and Tyr342, which are involved in
stabilizing the transition state by protonation and deprotonation during sialic acid transfer;
and b) Trp312 and Tyr119, which are hydrophobic in nature and are present at the mouth of
the catalytic cleft and that control substrate entry in the catalytic pocket. Also, a
phthaloyl/phthalimide structural framework could enhance hydrogen bonding and π-π

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stacking interaction on TcTS active residues due to a carbonyl group and aromatic ring
moiety, respectively. Furthermore, phthaloyl/phthalimide derivatives have also received great
attention due to their COX-1/2 inhibition [20], anti-inflammatory [21], antihyperlipidemic
[
22], antitumor/anticancer, anxiolytic, analgesic [23-26], antimicrobial, anticonvulsant,
antitubercular [27-29], antipsychotic [30], antiproliferative [31], immunomodulatory [32],
antiangiogenic [33], schistosomicidal [34] and anti-protozoa (anti-trypanocidal) activities
[
35]. All the compounds proposed were analyzed by molecular docking on the active site of
TcTS using Auto Dock Vina 4.0. Furthermore, the TcTS enzyme inhibition assay of selected
compounds and in vitro anti-T. cruzi activity on trypomastigotes of NINOA and INC-5
strains were also evaluated.
2
. Results and Discussion
2
.1. Chemistry
Compounds from series A, B, C and D were synthesized according to scheme 1.
Series A is described as 3-amino-3-arylpropionic acid derivatives and were synthesized
following the procedure reported in the literature [36]. The phthalic anhydride (series B),
trimellitic anhydride (series C) and 4-methylphthalic anhydride (series D) phthaloyl
derivatives of 3-amino-3-arylpropionic acid were synthesized by the fused reaction as
described in the experimental section [37].
The FTIR spectra of series A, showed a broad –OH peak in the range of 3300-2900
-1
cm and the absence of a –NH peak due to an interchangeable hydrogen atom between –OH
2
-1
and –NH . Also, carbonyl (C=O) peaks in 1650-1600 (short) cm confirmed the synthesis.
2
1
Furthermore, the H-NMR showed the peak in the range of the chemical shift 4.60 – 4.40
(
singlet) ppm due to an interchangeable hydrogen atom between –OH and –NH , which also
2
confirmed the synthesis.

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The phthaloyl derivatives of 3-amino-3-arylpropionic acid (series B, C and D) were
confirmed by FTIR, phthalimide peaks in the range of C O N (phthalimido): 1770 asymmetric,
2
2
-1
-1
1
690 symmetric cm and a carbonyl (C=O) peak in the range of 1650-1610 cm . The –OH
1
proton peak of the carboxylic acid of the 3-amino-3-arylpropionic acid part in H-NMR was
found in a range of 13.20-11.90 ppm and a singlet proton peak of CH-N of phthalimide in a
range of 5.80-5.30 ppm, which also strengthened the synthesis of compounds.
R =
Scheme 1. Synthesized compounds and reaction conditions. Series A obtained by reflux for
o
2
-3 h; Series B, C and D by fused reaction in the equimolar ratio at 250 C.

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2
.2. Molecular Docking Analysis
The 3-amino-3-arylpropionic acid derivatives with different substituents were used as
-
a starting point to propose TcTS inhibitors due to the presence of –NH and –COO groups,
2
which could help to show inhibitory effects. The substituents (1-15) were categorised
according to their chemistry as electron donating substituents (-OCH , -CH , and -C H ),
3
3
2
5
electron withdrawing substituents (OH, NO , -F, -Cl, Br, and C H O -mNO ), aromatic
2
2
2
2
2
substituents (naphthyl and biphenyl) and heterocyclic moieties (furanyl and thienyl) to
understand the effects of these modifications on TcTS binding sites, enzyme inhibition and
trypanocidal activity.
To increase the interaction of 3-amino-3-arylpropionic acid derivatives (series A) on
the TcTS active site, their novel phthaloyl derivatives were proposed by coupling them with
phthalic anhydride (series B), trimellitic anhydride (benzene-1,2, 4-tricarboxylic anhydride)
(series C) and 4-methylphthalic anhydride (series D). When these phthaloyl derivatives were
sketched in 3D and their energy minimized, these compounds showed a chair-shaped
geometry, and when they were fitted in TcTS catalytic cavity, it was found the 3-aryl
propionic acid skeleton deep inside the catalytic cavity with the carboxylic group flipped near
to the active amino acid residues Arg35, Arg245, Arg314, Asp59, Glu230 and Tyr342.
On the other hand, the phthaloyl group was found at the mouth of the TcTS catalytic
pocket with a carbonyl group near the key amino acid residues Tyr119 and Trp312. To
produce a stronger interaction, the hydrophilic –COOH and hydrophobic –CH substituents at
3
4
-position on the phthaloyl ring were also considered. To understand the binding mode of the
enzyme with molecules, 3-aryl propionic acid and the phthaloyl moiety were considered
separately (Fig. 2).

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Fig. 2. The orientation of phthaloyl derivatives of 3-amino-3-arylpropionic acid inside the
TcTS catalytic cavity (ribbons showing TcTS enzyme 1MS8). The red circle shows the
phthaloyl group at the mouth of the cavity and the black circle shows 3-amino-3-
arylpropionic acid deep inside the cavity. The 3D image was produced by Discovery studio
visualizer R-2 client 2017 version.
To understand and establish the interaction of the proposed molecules with key amino
acid residues on the catalytic pocket of TcTS (PDB code 1MS8), molecular docking studies
were performed to explore the best predicted binding affinities. The TcTS natural ligand 2-
deoxy-2,3-didehydro-N-acetylneuraminic acid (DANA) was used as a reference obtaining a
predicted binding affinity value of -7.8 Kcal/mol.
From series A (Table 1), only six compounds showed predicted binding affinities that
were the same (A3 and A5) or better (A-9 to A-12) than the reference DANA (-7.8
Kcal/mol). The compounds A-3 and A-5 only bind the two amino acid residues Arg35 and
Tyr342 via conventional hydrogen bonding, while Asp96 and Glu230 showed salt bridge
interaction. Also, compound A-5 showed an unfavourable interaction with Arg53 and Asp59;
in contrast, Glu230 was bound via hydrogen bonding with NO substituents. Compound A-11
2
(Fig. 3, 3-a) showed the highest predicted binding affinity (-9.0 Kcal/mol) in this series. The
hydrogen bond was found between Tyr342, Arg53 and Arg35 and a carboxylic oxygen atom,
hydroxyl oxygen and the amino group of the 3-amino-3-arylpropionic acid chain. π-π

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stacking occurred between Val95 and the aromatic ring of naphthalene of compound A-11,
but a hydrophobic interaction was found between Tyr119 and Trp312. The rest of the
compounds A-9, A-10 and A-12, with the highest predicted binding affinity compared to the
reference DANA, only showed interaction with two key amino acids, Arg245 and Glu230,
but showed a hydrophobic interaction with other amino acids on the TcTS catalytic cavity.
Therefore, from the docking analysis of series A, we concluded that the 3-amino-3-
arylpropionic acid chain only has the ability to bind with two or three amino acid residues but
no compound showed an interaction with Tryp312 and other essential amino acid residues in
the catalytic cavity of TcTS, even if they are small molecules that can be deep inside the
catalytic cavity of TcTS.
In series B, the compound B-11 showed the highest binding affinity (-10.8 Kcal/mol)
(Table 1). The binding interaction of B-11 (Fig. 3. 3-b) on TcTS cavity, showed that the
arginine triad (Arg35, Arg245 and Arg314) was engaged by a carbonyl group of the
phthaloyl moiety via hydrogen bonding. Also, Asp96 and Asp59 were attracted via π-anion
interaction with naphthyl and phthaloyl groups of B-11 and Glu230 was found in interaction
with the naphthyl group via π-anion. Additionally, hydrogen bonding between Glu230 and
the –OH of the carboxylic acid group of B-11 was observed, while Tyr342 was bound via
hydrogen bonding with the carbonyl group of phthaloyl. These results confirm the interaction
of B-11 with three amino acid residues, Asp59, Glu230 and Tyr342, which is reported as very
important to stabilize the transition state during the catalytic mechanism of sialic acid transfer
of TcTS [38]. Moreover, Trp312, which contributes to the formation of the hydrophobic
pocket and facilitates sialic acid transfer over hydrolysis and the formation of acceptor
binding sites [10], was found in interaction with the phthaloyl ring of B-11 via the strongest
classical parallel π-π stacking. Furthermore, other residues, Tyr119, Trp120, Val95 and

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Leu176, which are involved in trans glycosylase activity and exclusion of water from the
hydrophobic pocket [39, 40] were attracted by B-11 with a hydrophobic interaction.
Compound B-2 showed the second highest predicted binding affinity (-9.9 kcal/mol)
in this series, but the binding pattern was totally different compared to B-11. Only two key
residues, Asp59 and Tyr342, which play an important role in protonation and deprotonation
of the TcTS enzyme in the transition state were bound via hydrogen bonding with the
carbonyl group of the phthaloyl and the carbonyl group of carboxylic acid, respectively. The
arginine triad only showed the weak Van der Waals forces. Val 95 and Asp96, two other
important residues showed π-sigma and π-anion interaction with the phthaloyl ring.
Compounds B-6, B7 and B-8, which have halogen substituents showed the lowest
predicted binding affinity compared to B-1, B-2 and B-3, which have electron donating
substituents. Additionally, compounds B-5 and B-13 with a –NO group at para and meta
2
position of 3-arylpropionic acid, respectively, showed a large predicted binding affinity
difference and binding pattern. The binding pattern of compound B-5 showed the three
hydrogen bonding interactions, a -NO group interaction with Trp312 and Arg245, a
2
carboxylic group interaction with Tyr342 and a carbonyl group of phthaloyl interaction with
Arg53 but in compound B-13 an unfavourable interaction of Glu230 was observed with
carboxylic acid, while, a –NO group showed hydrogen bonding with Tyr342 and Arg53,
2
which is a totally different interaction compared to compound B-5.
Compounds B-14 and B-15 which contain furanyl and thienyl heterocyclic groups
showed the same binding pattern as compound B-5 except the oxygen and sulphur atom
showed an additional hydrogen bond with Tyr342 and Arg245. When the binding pattern of
compound B-4 with a predicted binding affinity of -9.3 kcal/mol was compared with other
compounds of this B series, only Trp120 showed a hydrogen bond between the oxygen atom

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of the hydroxyl group at the aryl moiety of propionic acid and all other residues showed only
hydrophobic interaction.
The compound B-12, with a bi-phenyl ring, showed the lowest predicted binding
affinity values (-9.0 kcal/mol) compared to B-11, even though both have two aromatic rings,
because the orientation pattern of B-12 in the catalytic cavity of the enzyme was found
different compared to other compounds. The biphenyl ring did not show a strong interaction
like the naphthalene ring of compound B-11. Only hydrogen binding was found between the
carboxylic group and Tyr119.
Table 1. Predicted binding affinities of series A-D and natural ligand DANA on active site of
TcTS using Auto Dock Vina version 4.2 and MGL.
Series A
A-1
A-2
A-3
A-4
A-5
A-6
A-7
A-8
*PBA
-7.4
-7.5
-7.8
-7.2
-7.8
-7.2
-7.4
-7.5
-8.1
-8.2
-9.0
-8.2
-7.5
-6.2
-6.0
-7.8
Series B
B-1
B-2
B-3
B-4
B-5
B-6
B-7
B-8
*PBA
-9.1
-9.9
-9.4
-9.3
-9.5
-9.2
-9.3
-9.2
-9.8
-9.4
-10.8
-9.0
-9.2
-8.0
-7.0
-7.8
Series C
C-1
C-2
C-3
C-4
C-5
C-6
C-7
C-8
*PBA
-9.9
-9.9
-10.1
-9.9
-9.6
-9.8
-9.8
-9.8
-10.4
-10.6
-11.1
-9.5
-9.6
-9.2
Series D
D-1
D-2
D-3
D-4
D-5
D-6
D-7
D-8
*PBA
-9.6
-9.7
-10.0
-9.6
-9.6
-9.0
-9.6
-9.7
-10.5
-10.5
-11.1
-9.4
-9.6
-8.8
A-9
B-9
C-9
D-9
A-10
A-11
A-12
A-13
A-14
A-15
DANA
B-10
B-11
B-12
B-13
B-14
B-15
DANA
C-10
C-11
C-12
C-13
C-14
C-15
DANA
D-10
D-11
D-12
D-13
D-14
D-15
DANA
-8.0
-7.8
-8.2
-7.8
*
PBA= Predictive binding affinity in -Kcal/mol
The docking study of series C and D (Table 1), showed the same binding pattern and
orientation in the catalytic pocket of TcTS as that of the B series with some exception due to
the presence of carboxyl and methyl substituents at 4-position on the phthaloyl ring. One of
the highest ranked compounds, C-11 with a predicted binding affinity of -11.1 Kcal/mol (Fig.
3
, 3-c), showed an extra interaction due to the presence of a carboxylic acid group at 4-
position on the phthaloyl ring bound to two extra amino acid residues, Tyr364 and Glu362,

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that were not reported as essential amino acids in the TcTS catalytic process but which are
present in the catalytic pocket. Moreover, Glu230 of compound C-11 showed the π-anion
interaction with the naphthyl ring; however, Glu230 was bound by hydrogen with an –O
atom of the hydroxyl group of the carboxylic group of propionic acid of compound B-11.
Compound D-11 (Fig. 3, 3-d) with the highest predicted binding affinity (-11.1
Kcal/mol) (Table 1) possessing a methyl group at 4-position on the phthaloyl ring showed the
exact same binding pattern as that of compound C-11; only one more π-alkyl interaction was
observed on the methyl group of the phthaloyl moiety with Trp312. In the case of the
compound D-4 with a hydroxyl substituent on the aryl moiety of propionic acid, a hydrogen
bond was found between the H atom of hydroxyl and the O atom of Asp96; also, the O atom
of the hydroxyl group showed hydrogen bonding with Arg53. In a comparison of compound
D-4 with C-4, the same binding pattern was observed in the –OH group at para position of
the aryl moiety of the propionic acid chain, but the oxygen atom of the carboxyl group of the
propionic acid compound C-4 showed hydrogen bonding with Tyr119 and Arg245, while the
same carboxyl group oxygen in D-4 showed hydrogen bonding with Glu230. A hydrophobic
interaction was observed for Tyr119 and Arg245 for compound D-4. In addition, all the
remaining compounds of series C and D showed the same binding pattern as was discussed
for series B.

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Fig. 3. 3D interaction of compounds with the highest predicted binding affinities: 3-a (series
A, compound A-11), 3-b (series B, compound B-11), 3-c (series C, compound C-11) and 3-d
(series D, compound D-11) compared to the reference DANA (-7.8 Kcal/mol). The 3D image
was produced by Discovery studio visualizer R-2 client 2017 version.

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2
.3. Enzyme Inhibition
After molecular docking, twelve compounds from series A, B, C and D were selected
according to the highest, intermediate and lowest predicted binding affinity to determine their
inhibitory properties towards TcTS and to measure their capacity to inhibit the sialylation of
N-acetyllactosamine in the reaction showed in Fig. 4. A mixture of 3’-sialyllactose (1 mM)
donor, N-acetyllactosamine (1 mM) acceptor, and TcTS was prepared in the absence or
presence of the inhibitor (1 mM) and analyzed by high-performance anion exchange
chromatography with pulsed amperometric detection (HPAEC-PAD) [39, 40].
Fig. 4. Sialylation of N-acetyllactosamine catalyzed by TcTS as a target of the inhibitors
tested.
Enzyme inhibition was calculated comparing the amount of 3’sialyl-N-
acetyllactosamine in the presence or absence of selected compounds. When the equimolar
concentration of the donor substrate 3’-sialyllactose and inhibitors were used, inhibition
values ranged between 1.4 and 86.9% (Fig. 5). Compounds D-11 and D-4 exhibited the
strongest inhibition against TcTS with 86.9 and 82.5%, respectively. Furthermore, the
compound B-14, C-11, B-11 and C-4 showed 76.5, 66.9, 65.8 and 53% enzyme inhibition,
respectively. All compounds from series A showed lower inhibition values.

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Code
SD ±
A-4
1.6
A-11
1.4
A-14
4.4
B-4
0.1
B-11
4.8
B-14
5
C-4
7
C-11
5.1
C-14
6
D-4
4.6
D-11
5
D-14
6
Fig. 5. Percentage inhibition of TcTS enzymes by selected compounds of series (A-D),
determined by HPAEC-PAD. Each compound was tested in triplicate (n= 3).
Interestingly, the compound D-11 with the highest inhibition value, 86.9%, also
showed the highest predicted binding affinity -11.1 Kcal/mol compared to the reference
DANA (-7.8 Kcal/mol). However, compound C-11, which also has the same PBA value,
exhibited a lower inhibition (66.9%). Both compounds, D-11and C-11, bear the same
substituents on the aryl group of the propionic acid part but different substituents, –CH and -
3
COOH at 4-position, on the phthaloyl ring, respectively. On the other hand, compound B-11
with the same substituent on the aryl group, like compound D-11 and C-11, but with no
substituents on the phthaloyl group, showed 65.8% enzyme inhibition, which is only one
percent less than compound C-11. These results suggest that the addition of lipophilic or
electron donating substituents on the phthaloyl moiety favour enzyme inhibition and TcTS
inhibition values can be increased by the addition of these types of substituents.
2
.3.1. Correlation between molecular docking and enzyme inhibition
Compound D-11 with 86.9% inhibition showed an interaction with the most essential
amino acids on the active site of TcTS (Fig. 6) as follows: The carbonyl moiety of the

ACCEPTED MANUSCRIPT
phthaloyl group was found binding with two arginines (Arg35 and Arg314) and Tyr342 via a
hydrogen bond. A classical π-π stacking was observed between Trp312 and phthaloyl ring,
while methyl group at phthaloyl showed π-alkyl interaction. The naphthyl moiety at
propionic acid and the phthaloyl group showed π-π stacking and π-anion interaction with
Val95, Asp59 and Asp96, respectively. The amino acid Glu230 was found in hydrogen
bonding with a proton of the hydroxyl group of the carboxylic acid of the propionic acid part
and amino acid residues Tyr119, Trp120 and Arg245 were found in interaction via Van der
Waals forces. The molecular docking analysis of compound C-11 and B-11 with 66.9 and
6
1
5.8% respectively, also showed the same pattern of interaction as shown by compound D-
1, except the interaction of Glu230, which was found absent in compound C-11 (Fig. 7);
however, an additional hydrogen bond interaction of Arg245 and Tyr119 was observed in
compound C-11 which was absent in compound D-11.
Compound D-4 (Fig. 6), with the second highest inhibition value of 82.5%, in the
docking analysis showed the same binding interaction for Arg35, Arg314, Asp59, Trp312,
Glu230 and Tyr342 as compound D-11, except for the amino acid residues Asp96 and Arg53
which showed a hydrogen bond interaction with the para substituted hydroxyl group on the
phenyl ring, at the propionic acid part of compound D-4.
On the other hand, compound C-4 which has the same scaffold as D-4, except for the
–
COOH substituent at 4-position on the phthaloyl ring, showed 53% inhibition. The binding
pattern of both compound C-4 and D-4 is about the same, except Glu230, which was found in
a hydrogen bond interaction with a carboxylic group of a propionic acid part in D-4, but in
compound C-4, Glu230 was found in π-anion interaction with the phenyl ring. Compound B-
4
with the same scaffold-like compounds C-4 and D-4, but with no substituents on the
phthaloyl moiety, showed only 5.6% inhibition. The interaction pattern of compound B-4

ACCEPTED MANUSCRIPT
only showed a hydrophobic interaction with key amino acid residues in the TcTS catalytic
pocket compared to the interaction pattern of compounds C-4 and D-11.
The compound B-14 with a furanyl ring showed the third highest TcTS enzyme
inhibition, 76.5% (Fig. 6), compared to compounds C-14 and D-14, which have 40% and
1
9.2%, respectively. The interaction pattern of compound B-14 was found similar to
compounds D-11 and D-4. Herein, Trp312 showed π-π stacking with the furanyl ring and
hydrogen bonding with the carbonyl of the phthaloyl moiety. Furthermore, Tyr119 also
showed π-π stacking and hydrogen bonding with the phthaloyl moiety but in compound D-
1
1, D-4 and C-11, Tyr 119 was found in hydrophobic interaction.
In series A, compound A-11, which also has the highest predicted binding affinity (-
.0 Kcal/mol) compared to DANA, showed the lowest enzyme inhibition value (8.3 %). If we
9
compare the interaction of A-11 with compounds that have the highest enzyme inhibition
values (Fig. 6), we find that A-11 showed the lowest interaction with essential amino acids
residues and also showed unfavourable interaction bumps, compared to other compound
interactions with the highest predicted binding affinity and enzyme inhibition values.

ACCEPTED MANUSCRIPT
Fig. 6. 2D interaction of series D, B, C and A compounds with TcTS active residues, with
highest to lowest TcTS inhibition percentage. The 2D image was produced by Discovery
studio visualizer R-2 client 2017 version.
From this correlation between molecular docking and enzymatic inhibition, we
suggest that compounds with a strong interaction with Glu230, Trp312 and Tyr342 show a
strong inhibition compared to other interactions and that the presence of lipophilic
substituents (-CH ) at 4-position on the phthaloyl moiety is another key factor to enhance
3
inhibitory effects.

ACCEPTED MANUSCRIPT
2
.4. Trypanocidal activity
All synthesized compounds from series A, B, C and D were screened at a
concentration of 10 µg/mL to determine their trypanocidal effect (% lysis) on a blood sample
infected with trypomastigotes from NINOA and INC-5 strains of T. cruzi. Afterwards,
compounds with a % lysis >50 were selected to determine the lysis concentration of 50% of
the population (LC ). Furthermore, C log P values, Absorption, Distribution, Metabolism,
5
0
and Excretion (ADME) properties, pharmacokinetic properties of gastrointestinal absorption
(
GI) and blood-brain barrier (BBB) were predicted by using the online swissADME database
www.swissadme.com), and (www.cbligand.org/BBB/) respectively (Full results are shown in
(
supporting material section).
2
.4.1. 3-amino-3-arylpropion acid derivative series A
We began our investigation using 3-amino-3-arylpropionic acid derivative series A.
The modification of substituents on the aryl moiety showed a different lytic effect on both
NINOA and INC-5 strains. In series A, compounds A-1, A-2 and A-3 with electron donating
substituents such as -OCH , -CH and -C H at para position on the phenyl ring showed 83,
3
3
2
5
7
8 and 65% lysis, respectively, for NINOA strain (Table 2), values that are higher than that
of the reference drugs Bzn (46.30%) and Nfx (40.74%). This showed that the alkyl
substituent (methoxy) with an electronegative oxygen atom increases lysis. This may be due
to more polarizability compared to the methyl substituent, but a comparison of the methyl
and ethyl substituents showed that an increase in chain length decreases lysis. The electron
withdrawing substituents –OH and –NO in compounds A-4 and A-5, respectively, at para
2
position on the phenyl ring showed 50% lysis. When a substitution was made with naphthyl
(A-11) and bi-phenyl (A-12) groups, only compound A-11 showed 60% lysis, which is better
than the reference drugs, but compound A-12 showed a lower percentage of lysis (32%),

ACCEPTED MANUSCRIPT
which indicates that aromatic polarizability and conformers also have an important impact on
lysis.
The compounds A-1 to A-3 with electron donating substituents, showed the lowest
percentage of lysis, 54, 59 and 50%, respectively, for the INC-5 strain. This was about equal
to the reference drugs Bzn 50% and Nfx 56% and less than the % lysis of these compounds
for NINOA strain. This contrast in results may be due to different protein expression levels in
trypomastigotes of both strains. In INC-5 strains, the compounds A-4 and A-5 with the
electron withdrawing substituents –OH and –NO , showed 76 and 68 % lysis, respectively.
2
This is better than the lysis of these compounds for NINOA strain. These lysis results indicate
that electron withdrawing substituents favour lysis in INC-5. The compounds A-6 and A-7
with halogen atoms, such as –F and –Cl, also showed good lysis, 62 and 73%, respectively.
Interestingly, compound A-11 with a naphthyl group showed 60% lysis which is exactly the
same as with the NINOA strain. This suggests that the fused aromatic ring or the bulky
aromatic moiety is favourable for both strains.
2
.4.2. Phthaloyl derivatives of 3-amino-3-arylpropionic acids (series B)
In series B, the modification was made on the –NH group of the 3-amino-3-
2
arylpropionic acid series A derivatives by introducing a phthaloyl group. This results in an
increase of the C log P value (Table 2). The compounds B-4, B-5, B-13 and B-6 with –OH, -
NO (meta position for B-13), and –F at para position on the phenyl ring showed 77, 62, 63
2
and 56% lysis, respectively, for the NINOA strain (Table 2). This was greater than the
references drugs Bzn (46%) and Nfx (40%) and also better than compounds A-4, A-5, A-6
and A-13 for NINOA strain. The compound B-11 with a naphthyl group showed 60% lysis
for NINOA strain. This is similar to compound A-11 in series A, which showed that the
naphthyl moiety is unaffected by this phthaloyl addition.

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Addition of a phthaloyl group produced the same effect on the INC-5 strain in series
B as discussed above. A hydrophilic group (electron withdrawing), B-4, B-5, B-13 and B-7
with -OH, –NO (meta position for B-13) and –Cl, at para position showed 68, 80, 70 and
2
6
8% lysis, respectively, but the activity of B-6 with an –F group decreased, and B-7 with a –
Cl group increased compared to NINOA. This could be due to a biological difference in the
strains. In a comparison of series A, the compounds A-4 to A-7 and A-13 showed about the
same behaviour for the INC-5 strain. The compound B-11 with a naphthyl group showed a
nearly similar 62% lysis, compared to the lysis for NINOA strain of compound A-11 as
shown in series A. The rest of the compounds did not show better lysis than the reference
drugs.
2
.4.3. Carboxylic Phthaloyl derivatives (series C)
In series C, a –COOH group was added at 4-position on the phthaloyl ring of series B.
This addition caused a decrease in C log P values for all compounds compared to series B
Table 2). The compound C-2, with a –CH substituent at para position on the phenyl ring
(
3
showed the highest lysis, 85% for NINOA strain (Table 2). This was greater than all the
compounds in series C and B. The compound C-1, with a methoxy substituent at the aryl ring
showed the second highest lysis value, 72%, which was found to be about the same as
compound C-1 but greater than compound B-1. This result showed that the addition of –
COOH group at 4-position on the phthaloyl ring produced a better effect on lipophilic
substituents on the aryl ring. Compound C-11 and C-12, with a naphthyl and a biphenyl ring
at the aryl part of propionic acid, showed 42 and 17% lysis. This is less compared to
compound B-11 and B-12, which showed that the addition of an electron withdrawing group
at the phthaloyl moiety decreases lysis due to a decrease in electron density. The addition of a
–
COOH group at the phthaloyl moiety produced an appreciable effect on the lysis values of
compounds C-14 and C-15 with heterocyclic moieties, such as furanyl and thienyl at the aryl

ACCEPTED MANUSCRIPT
part of propionic acid. These compounds showed 77 and 67 % lysis, respectively, which is
much better than the B-14 and B-15 compounds.
For INC-5 strain, compounds C-4, C-5 and C-13 with –OH and –NO substituents at
2
para and meta for C-13 showed 71, 63 and 68% lysis for INC-5 strain, which is greater
compared to lysis of these compounds for NINOA strain but in comparison with the series B
compounds, B-4, B-5 and B-13, compound C-4 showed a lower lysis and compound C-5
higher lysis. Compounds C-6 to C-8 with halogen substituents such as –F, -Cl, and –Br,
respectively, showed 63, 67 and 69 % lysis, respectively, which is better than the lysis of
these compounds for NINOA and also better than series B compounds B-6 to B-8.
Compound C-11 with a naphthyl group at the aryl part of propionic acid showed 24% lysis
which was less than compound B-11 for INC-5 strain but an increase in lysis for compound
C-13 was observed for INC-5 strain compared to lysis of the same compound for NINOA
strain and B-12.
2
.4.4. Methyl Phthaloyl derivatives (series D)
In series D, the –CH group was added at 4-position on the phthaloyl moiety, which
3
showed an increase in C log P values for all compounds compared to series B and C (Table
). The compound D-1, D-2 and D-3 with a lipophilic substituent –OCH , –CH and –C H at
2
3
3
2
5
para position on the phenyl ring of propionic acid showed 78, 73 and 65% lysis, respectively,
for NINOA strain compared to the reference drugs Bzn (46%) and Nfx (40%) (Table 2).
Compounds D-1–D-3 showed better % lysis compared to compounds B-1–B-3. In
comparison with the series C compounds, C-1 and C-2, the series D compounds showed
slightly better or equal lysis, but D-3 showed a higher lysis compared to a C-3 compound;
this showed that the introduction of a lipophilic substituent on the phthaloyl group enhances
the trypanocidal effect for ethyl group at the phenyl ring of the propionic acid part.
Compounds D-6 to D-8 with halogen substituents (hydrophilic) –F, -Cl, and – Br at the para

ACCEPTED MANUSCRIPT
position on the phenyl ring showed 63, 62 and 65% lysis, respectively, for NINOA strain
which is higher than compounds C-6 to C-8. In a comparison of compound D-6, compound
B-6 showed a similar lysis but compound D-7 and D-8 showed a higher lysis than B-7 and B-
8
. These result of series D showed that the –CH substituent at 4-position on the phthaloyl
3
moiety increases the lysis of halogenated substituents. The –CH (electron donating) on the
3
phthaloyl moiety produced a good effect on the lytic activity of compound D-11 which has a
naphthyl group, showing 63% lysis in NINOA compared to C-11 compound which has a –
COOH group at 4-position on the phthaloyl ring and showed lysis similar to the B-11
compound of series B.
For INC-5 strain, compounds D-1 to D-3 with the lipophilic substituents OCH , –CH
3
3
and –C H at para position on the phenyl ring of the propionic acid part, showed 57, 67 and
2
5
5
9% lysis activity which is better than compounds B-1 to B-3, and approximately similar to
compounds C-1 to C-3 and the reference drugs Bzn (56%) and Nfx (50%) (Table 2). This
finding indicates that the substituent on the phthaloyl moiety can enhance the lytic effect for
INC-5 strain, compared to series B but no significant difference in lytic activity was observed
for the –COOH and –CH substituent on the phthaloyl moiety. Compound D-11 with a
3
naphthyl group showed 65% lysis which is greater than a C-11 compound in series C and
similar to compound B-11. This provides evidence that the addition of a more lipophilic
substituent on the phthaloyl moiety can increase the lytic effect in INC-5 strain. A decrease in
lysis of compound D-12, with a bi-phenyl substituent, was observed compared to C-12. The
remaining compounds showed consistent behaviour as they have shown in series B and C for
INC-5.
The different lytic effects of compounds in both NINOA and INC-5 strains showed
that there are different protein expression and biochemical pathways [42] with different
mechanisms of action for each compound in each series except in compounds with a naphthyl

ACCEPTED MANUSCRIPT
group in series A, B and D, which have shown the same consistency in results for both
strains.
2
.4.5. LC determination
50
The LC was determined only in the compounds with a percentage lysis >50 in both
5
0
strains and whose value was lower than those of the reference drugs Bzn and Nfx, which
showed LC values (LC = 129.56 ± 5.1 µM for NINOA and 102 ± 3.54 for INC-5 µM) and
5
0
50
(
LC = 89.52 ± 4.6 µM for NINOA and 77.23 ± 2.11 µM for INC-5), respectively.
50
From series A, seven compounds A-1, A-2, A-3, A-4, A-5, A-11 and A-14 were
selected for LC determination (See supporting material section). From these compounds, A-
5
0
1
1 showed the best biological activity with LC values (98.66 ± 2.78 µM and 102.88 ±
50
3
.542) similar in NINOA and INC-5 strains, respectively and better than reference drug Bzn.
From series B, compound B-4, B-5, B-6 and B-11 were selected to determine LC50.
All these compounds showed better LC values on both strains compared to reference drug
5
0
Bzn, but less than the Nfx (Table 2). Most prominent LC was showed by compound B-5
5
0
(
LC = 53.49 ± 2.98 µM) for NINOA strain.
50
From series C, compound C-2 showed the best trypanocidal activity on INC-5 strain
LC = 39.88 ± 3.25 µM), compared to the reference drugs, but on NINOA this compound
(
5
0
showed a low trypanocidal activity (LC = 109.50 ± 1.62 µM). Compound C-14 showed
5
0
LC = 77.57 ± 2.85 µM for NINOA strain, which is better than the reference drugs, but on
5
0
the INC-5 strain, the value was found greater than the reference drugs (LC = 101.61 ± 2.01
50
µM) (Table 2).
In series D, six compounds D-1, D-2, D-6, D-7, D-8 and D-11 were selected to
determine the LC values (Table. 2). Compound D-8 showed the most potent LC for both
5
0
50
strains, NINOA and INC-5 (LC = 24.59 ± 4.3 and 23.02 ± 3.9 µM, respectively), while
5
0

ACCEPTED MANUSCRIPT
compound D-11 which also have the highest TcTS inhibition, showed LC = 52.70 ± 2.1 µM
5
0
for NINOA and 46.19 ± 2.3 µM for INC-5, which is better than the references.
Table 2. %age lysis of trypomastigotes of NINOA and INC-5 strains, LC and C log P
5
0
values of representative compounds of series A, B, C and D.
Compound
C log P
T. cruzi NINOA
T. cruzi INC-5
%
Lysis
% Lysis
at 10 µg/mL
54 ± 2
59 ± 3
50 ± 7
76 ± 2
60 ± 3
68 ± 1
80 ± 1
52 ± 3
62 ±4
68 ± 5
32 ± 3
55 ± 2
24 ± 4
55± 3
19 ± 2
57 ± 4
67 ± 6
59 ± 5
56 ± 6
51 ± 7
50 ± 6
65 ± 2
56 ± 7
50 ± 6
LC50 (µM)
LC (µM)
50
at 10 µg/mL
83 ± 6
78 ± 4
65 ± 3
50 ± 4
60 ± 6
77 ± 4
62 ± 5
63 ± 2
57 ± 2
56 ± 6
72 ± 4
85 ± 5
42 ± 4
77 ± 6
67 ± 5
78 ± 7
73 ±8
65 ± 4
63 ± 6
52 ± 6
65 ± 6
63 ± 4
46.30 ± 13.12
40.74 ± 6.4
A-1
A-2
A-3
A-4
A-11
B-4
B-5
B-6
B-11
B-13
C-1
0.59
0.92
1.20
0.15
1.38
1.61
1.32
2.34
2.96
1.26
1.59
1.91
1.91
0.91
1.57
2.36
2.69
3.01
2.99
2.77
3.08
3.34
101.53 ± 1.15
183.68 ± 2.81
113.09 ± 1.58
151.52 ± 2.35
98.66 ± 2.78
71.38 ± 2.88
53.49 ± 2.98
90.81 ± 2.99
97.27 ± 1.62
Nd
256.12 ± 2.22
290.14 ± 3.10
263.90 ± 1.68
127.38 ± 2.53
102.88 ± 3.542
90.96 ± 2.10
118.63 ± 3.86
81.41 ± 2.56
117.26 ± 5.32
Nd
Nd
Nd
C-2
109.50 ± 1.62
Nd
77.57 ± 2.85
Nd
65.57 ± 3.7
85.57 ± 2.8
Nd
45.18 ± 4.0
37.70 ± 3.9
24.59 ± 4.3
52.70 ± 2.71
129.65 ± 5.1
89.52 ± 4.6
39.88 ± 3.25
Nd
101.61 ± 2.01
Nd
59.46 ± 3.9
43.57 ± 2.9
Nd
74.63 ± 6.5
52.01 ± 2.6
23.02 ± 3.9
46.19 ± 2.36
123.68 ± 4.51
77.23 ± 2.11
C-11
C-14
C-15
D-1
D-2
D-3
D-6
D-7
D-8
D-11
Bzn
Nfx
Nd= not determined
3
. Conclusions
In summary, fifteen 3-amino-3-aryl propionic acid derivatives (series A) and forty-
five novel phthaloyl derivatives of 3-amino-3-aryl propionic acid (series B, C and D) were
designed and synthesised with an excellent yield of 80-90%. The molecular docking analysis
of these compounds on the TcTS enzyme revealed that compound C-11 (series C) and D-11
(series D) showed the highest predicted binding affinity -11.1 Kcal/mol by hydrogen
bonding, π-π stacking and Van der Waals forces with key amino acid residues Tyr342,

ACCEPTED MANUSCRIPT
Trp312, Arg53 and Glu230. HPAEC-PAD was used to determine the inhibition of selected
compounds for the TcTS enzyme and found the following trend of inhibition D >B >C >A.
Compounds D-11 and D-4 showed the highest predicted binding affinities 86.9% and 82.6%,
respectively. This trend suggests that the high C log P and lipophilic substituents (-CH ) on
3
the phthaloyl moiety are important to increase TcTS inhibition. An in vitro test showed no
correlation in trypanocidal activity between NINOA and INC-5 strain, due to the possibility
of different protein expression, but series D compounds showed better lysis activity and LC50
for both strains. Series A (3-amino-3-arylpropionic acid) also showed good LC values but
5
0
poor enzyme inhibition, which suggests that these compounds have another mechanism of
action.
A strong correlation between molecular docking and enzyme inhibition was found for
compound D-11 (86.9% inhibition and -11.1 Kcal/mol binding affinity) with good
trypanocidal activity for both strains. ADME prediction showed that these compounds have
high gastrointestinal absorption (GI) values; some even have positive BBB (blood brain
barrier) values and follow Lipinski properties for drug development. Therefore, these
phthaloyl derivatives could be used as TcTS inhibitors to develop new anti-Chagas drugs.
4
. Experimental
4
.1. Chemistry
All reactive, solvents were purchased from Sigma-Aldrich and were used without
further purification. Melting points were determined on Mel-Temp capillary apparatus and
are uncorrected. The solvent evaporation under vacuum was carried out by using BUCHI
Rotavapor® R-100. The purity and reactions were monitored by thin-layer chromatography
(TLC) performed on silica gel plates prepared with silica gel 60 (PF-245 with gypsum,
Merck Japan), of the thickness of 0.25 nm. The developed chromatograms were visualized

ACCEPTED MANUSCRIPT
under ultraviolet light at 254-265 nm. Infrared spectra were recorded using OPUS_7.5.18
software with PLATINUM-ATR Bruker Alpha FT-IR spectrometer. NMR data were
1
collected with Bruker Avance 500 spectrometer operating at 500 MHz ( H), and 126 MHz
1
3
1
(
C). H proton NMR spectra were obtained in DMSO-d , with TMS as an internal standard.
6
Chemical shifts are given on the δ scale (ppm). Multiplicities are indicated as follows: s
singlet), d (doublet), t (triplet), q (quartet), m (multiplet) or br (broadened).
(
4
.1.1. General procedure for preparation series A compounds A-1 to A-15
A mixture of appropriate aldehyde 2.40 g (1-15), 2.44 g of malonic acid and 3.54 g of
ammonium acetate (1:1.1:2.3), in 200 mL of the 1-butanol was refluxed for 1.5-2 hours until
the evolution of CO ceased. The precipitate formed was filtered and washed with boiling 1-
2
butanol (2 x 50 mL), boiling ethanol (2 x 50 mL) and 100 mL of water. Precipitates were
o
dried at 80-100 C for 8-10 hours. Purity of product was checked by TLC, and yield obtained
about 65-80% in each reaction.
4
.1.1.1.
3-amino-3-(4-methoxyphenyl)propanoic acid (A-1)
o
-1
+
2
Yield: 70%, mp 236-8 C, FT IR (υ cm ): 3250-2900 (NH ), 3050 (CH sp ),
3
1
1
613asym (C=O), 1255 (C-O), 1156 (C-N). H NMR (500 MHz, DMSO-d6): δ 7.11 (d, 2H,
C H ), 6.77 (d, 2H, C H ), 4.12 (q, 1H, CHN), 3.61 (s, 3H, OCH ), 2.32-2.41 (d, 2H,
6
4
6
4
3
13
CHCH ). C NMR (126 MHz, DMSO-d6): δ 179.3, 161.8, 137.5, 129.7, 118.4, 59.8, 53.3,
2
4
4
9.1.
.1.1.2.
3-amino-3-(p-tolyl)propanoic acid (A-2)
o
-1
+
2
Yield: 67%, mp 223-5 C, FT IR (υ cm ): 3200-2970 (NH ), 3070 (CH sp ),
3
1
1
619asym (C=O), 1268 (C-O), 1145 (C-N). H NMR (500 MHz, DMSO-d6): δ 7.21-7.30 (m,
H, C H ), 6.90-6.77 (m, 2H, C H ), 4.31 (q, 1H, CHN), 2.27-2.39 (d, 2H, CHCH ), 2.11 (s,
2
6
4
6
4
2

ACCEPTED MANUSCRIPT
13
3
3
4
H, CH ). C NMR (126 MHz, DMSO-d6): δ 170.9, 139.4 134.8, 128.1, 126.0, 119.9, 51.0,
3
9.2, 18.9.
.1.1.3.
3-amino-3-(4-ethylphenyl)propanoic acid (A-3)
o
-1
+
2
Yield: 71%, mp 240-2 C, FT IR (υ cm ): 3240-2885 (NH ), 3055 (CH sp ),
3
1
1
2
2
1
4
647asym (C=O), 1265 (C-O), 1162 (C-N). H NMR (500 MHz, DMSO-d6): δ 7.20-7.31 (m,
H, C H ), 6.90-6.79 (m, 2H, C H ), 4.38 (q, 1H, CHN), 2.32-2.30 (d, 2H, CHCH ), 2.11 (q,
6
4
6
4
2
13
H,CH CH ), 1.39 (t, 3H, CH CH ); C NMR (126 MHz, DMSO-d6): δ 178.3, 143.4,
2
3
2
3
41.3, 131.5, 127.8, 127.7, 126.3,126.0, 50.1,33.4, 19.1
.1.1.4. 3-amino-3-(4-hydroxyphenyl)propanoic acid (A-4)
o
-1
+
2
Yield: 70%, mp 194-6 C, FT IR (υ cm ): 3190-2910 (NH ), 2999 (CH sp ),
3
1
1
2
2
3
4
620asym (C=O), 1258 (C-O), 1147 (C-N). H NMR (500 MHz, DMSO-d6): δ 7.16-7.20 (m,
H, C H ), 6.74-6.80 (m, 2H, C H ), 4.60 (s, 1H, C H OH), 4.39 (q, 1H, CHN), 2.49-2.38 (d,
6
4
6
4
6
4
1
3
H, CHCH ). C NMR (126 MHz, DMSO-d6): δ 171.2, 142.3, 134.5, 130.8, 123.6, 51.1,
2
8.2.
.1.1.5.
3-amino-3-(4-nitrophenyl)propanoic acid (A-5)
o
-1
+
2
Yield: 69%, mp 211-3 C, FT IR (υ cm ): 3210-2990 (NH ), 3020 (CH sp ),
3
1
1
637asym (C=O), 1557 (NO ), 1275 (C-O), 1173 (C-N). H NMR (500 MHz, DMSO-d6): δ
2
7
.19-7.23 (m, 2H, C H ), 6.78-6.86 (m, 2H, C H ), 4.41 (q, 1H, CHN), 3.32-2.21 (d, 2H,
6
4
6
4
1
3
CHCH ). C NMR (126 MHz, DMSO-d6): δ 174.2, 151, 145.3, 132.5, 129.8, 121.6, 50.1,
2
3
4
7.1.
.1.1.6.
3-amino-3-(4-fluorophenyl)propanoic acid (A-6)
o
-1
+
2
Yield: 72%, mp 231-3 C, FT IR (υ cm ): 3290-2916 (NH ), 3038 (CH sp ),
3
1
1
620asym (C=O), 1268 (C-O), 1163 (C-N), 832 (C-F). H NMR (500 MHz, DMSO-d6): δ
7
.15-7.25 (m, 2H, C H ), 7.01-7.11 (m, 2H, C H ), 4.21 (q, 1H, CHN), 2.31-2.62 (d, 2H,
6
4
6
4
13
CHCH ); C NMR (126 MHz, DMSO-d6): δ 172.9, 164.8, 131.5, 130.4, 117.7, 51.4, 38.8.
2