Synthesis and antimalarial activity of new nanocopolymer β-lactams and molecular docking study of their monomers

Article abstract of DOI:10.1007/s00044-015-1477-7

This report describes the preparation of some new β-lactam nanocopolymers. These nanoparticles are synthesized in water by emulsion polymerization of an acrylate β-lactam pre-dissolved in a mixture of co-monomers in the presence of sodium dodecyl sulfate as a surfactant and potassium persulfate as a radical initiator. Dynamic light scattering analysis and electron microscopy images of these emulsions show that the nanoparticles are approximately 30-70 nm in diameter. These compounds have been evaluated for their antimalarial activities against chloroquine-resistant Plasmodium faliparum K1 strain demonstrating IC₅₀ varying from 14 to 50 μM. The interactions between these β-lactam nanocopolymers and the P. falciparum single-stranded DNA-binding proteins have been studied by molecular docking calculations.

Full text of DOI:10.1007/s00044-015-1477-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2016) 25:247–262
DOI 10.1007/s00044-015-1477-7
ORIGINAL RESEARCH
Synthesis and antimalarial activity of new nanocopolymer
b-lactams and molecular docking study of their monomers
2
Edris Ebrahimi1 Aliasghar Jarrahpour1 Nahid Heidari1 Veronique Sinou Christine Latour2
•
•
•
•
•
´
Jean M. Brunel3 Amin R. Zolghadr1 Edward Turos4
•
•
Received: 17 June 2015 / Accepted: 30 October 2015 / Published online: 12 November 2015
Ó Springer Science+Business Media New York 2015
Abstract This report describes the preparation of some
new b-lactam nanocopolymers. These nanoparticles are
synthesized in water by emulsion polymerization of an
acrylate b-lactam pre-dissolved in a mixture of co-mono-
mers in the presence of sodium dodecyl sulfate as a sur-
factant and potassium persulfate as a radical initiator.
Dynamic light scattering analysis and electron microscopy
images of these emulsions show that the nanoparticles are
approximately 30–70 nm in diameter. These compounds
have been evaluated for their antimalarial activities against
chloroquine-resistant Plasmodium faliparum K1 strain
demonstrating IC50 varying from 14 to 50 lM. The
interactions between these b-lactam nanocopolymers and
the P. falciparum single-stranded DNA-binding proteins
have been studied by molecular docking calculations.
Keywords b-Lactams Á Nanoparticles Á Polyacrylates Á
Emulsion polymerization Á Molecular docking
Introduction
b-Lactams form a class of antibiotics characterized by the
presence of a b-lactam (2-azetidinone) ring (George,
1992). This four-membered ring is responsible for bio-
logical activity in penicillins, cephalosporins, carbapen-
ems, nocardicins and monobactams, which have been
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-015-1477-7) contains supplementary
material, which is available to authorized users.
1
& Aliasghar Jarrahpour
jarrah@susc.ac.ir;
aliasghar6683@yahoo.com
Department of Chemistry, College of Sciences, Shiraz
University, Shiraz 71946-84795, Iran
2
ˆ
UMR-MD3 Relation hote-parasites, Physiopathologie and
Edris Ebrahimi
edris.ebrahimi@yahoo.com
´
Pharmacologie, Faculte de pharmacie, Aix-Marseille
Universite, Bd Jean Moulin, 13385 Marseille, France
´
3
4
Nahid Heidari
nahidheidari88@yahoo.com
´
Centre de Recherche en Cancerologie de Marseille (CRCM),
UM 105, CNRS, UMR7258, Institut Paoli Calmettes, Inserm,
´
´
U1068, Faculte de pharmacie, Aix-Marseille Universite, Bd
´
Veronique Sinou
veronique.sinou@gmail.com
Jean Moulin, 13385 Marseille, France
Department of Chemistry, University of South Florida, 4202
E. Fowler Avenue, CHE 205, Tampa, FL, USA
Christine Latour
crislatour@gmail.com
Jean M. Brunel
bruneljm@yahoo.fr
Amin R. Zolghadr
a.r.zolghadr@gmail.com
Edward Turos
eturos@usf.edu
123

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Med Chem Res (2016) 25:247–262
widely used as chemotherapeutic agents for treating
microbial diseases (Marchand-Brynaert and Brule, 2008;
Alcide et al., 2008; Halve and Bhadauria, 2007). Many
other interesting biological properties, such as cholesterol
absorption inhibitors (Rosenblum et al., 1998), human
cytomegalovirus protease inhibitors (Borthwick et al.,
1998), thrombin inhibitors (Han et al., 1995), anti-hy-
perglycemic (Goel et al., 2004), anti-tumor (Veinberg
et al., 2004), anti-HIV (Sperka et al., 2005), anti-inflam-
matory, analgesic activities (Saturnino et al, 2000), anti-
fungal (Zarei and Mohamadzadeh, 2011), antimalarial
activities (Jarrahpour et al., 2014) and serine-dependent
enzyme inhibitors, have been highlighted (Konaklieva,
2002). Moreover, b-lactams are very useful as key inter-
mediates in organic synthesis (Ojima and Delaloge, 1997)
and provide building blocks (Alcaide et al., 2007) for the
construction of many nitrogen-containing organic mole-
cules with fundamental biologically active novel com-
pounds (Gerona-Navarro et al., 2003). Unfortunately, the
microorganisms have developed resistance against b-lac-
tam antibiotic drugs so organic chemists should design
new b-lactam drugs displaying broader antibacterial
activity (Fisher et al., 2005). So, many synthetic methods
for synthesizing b-lactams are now available (Staudinger,
1907). The most convenient method for the construction
of the b-lactam ring is the (2 ? 2) cycloaddition of
ketenes to imines known as the Staudinger reaction
(Ebrahimi and Jarrahpour, 2014). The use of nanotech-
nology in medicinal chemistry has undergone rapid
development in the last several decades (Peer et al.,
2007). Nanomedicine is designed and synthesized drug
delivery vehicles that can carry drug and efficiently cross
physiological membranes to reach target sites (El-Ansary
and Al-Daihan, 2009). The most quintessential nanoscale
carriers include liposomes (Cavalcanti et al., 2005),
polymeric nanoparticles (Langer, 1998), quantum dots
(Smith et al., 2004), gold nanoparticles (Han et al., 2007),
magnetic nanoparticles (McCarthy et al., 2007), carbon
nanotubes (Jarrahpour et al., 2012), dendrimers, nucleic
acid-based nanoconstructs (Jain and Asthana, 2007;
Yevdokimov et al., 2005) and engineered viral nanopar-
ticles (Singh et al., 2006). The aqueous solubility of
poorly water-soluble drugs can be increased by using
nanoemulsions. Enhanced drug solubility, perfect ther-
modynamic stability, ease of manufacturing and perme-
and carbohydrate (Abeylath et al., 2008)- and antibiotic
(Garay-Jimenez and Turos, 2011)-conjugated polyacrylate
nanoparticles formed by emulsion polymerization in water
has been reported. The preparation of polyacrylate-based
nanoparticle emulsions that contain penicillin or N-thio-
lated b-lactam has been reported by Turos (Abeylath and
Turos, 2007). These nanoparticles were prepared by free
radical emulsion polymerization in water. Enhanced anti-
MRSA activity of the nanoparticle polymers together with
easy preparation and facile control of nanoparticle size
are some advantages of these nanoparticles (Turos et al.,
2007). Furthermore, the first in vivo study of the peni-
cillin-conjugated nanoparticle emulsion has been reported
(Greenhalgh and Turos, 2009). This report describes the
synthesis of nanoparticle b-lactams based on well-prece-
dent emulsion polymerization procedures. Docking stud-
ies are performed to propose a molecular target for these
potential antimalarial compounds.
Experimental
General
All required chemicals were purchased from the Fluka,
Merck and Acros chemical companies. CH2Cl2 and Et3N
were dried by distillation over CaH2 and then stored over
4-A molecular sieves. 1H-NMR and 13C-NMR spectra
˚
were recorded in CDCl3 or DMSO-d6 using a Bruker
Avance DPX instrument (operating at 250 MHz for 1H
and 62.9 MHz for 13C). Chemical shifts were reported in
ppm (d) downfield from TMS. All the coupling constants
(J) are in Hertz. IR spectra were run on a Shimadzu FT-IR
8300 spectrophotometer. The mass spectra were recorded
on a Shimadzu GC–MS QP 1000 EX instrument. Ele-
mental analyses were run on a Thermo Finnigan Flash
EA-1112 series. Melting points were determined in open
capillaries with a Buchi 510 melting point apparatus and
are not corrected. The distribution morphology of the
product was analyzed by CM10 transmission electron
microscope (TEM; Philips 120 kV). Sample size and
distribution of the products were checked by dynamic
light scattering using a HORIBA DLS instrument. Column
chromatography was performed on Merck Kieselgel
(230–270 mesh). Thin-layer chromatography (TLC) was
carried out on silica gel 254.
ation
over
conventional
formulations
convert
nanoemulsions to important drug delivery systems (Tha-
kur et al., 2013). The unique properties of polymeric
micelles as carriers of hydrophobic drugs have been
reported in recent years (Lee et al., 2012). The prepara-
tion of different ampicillin (Fontana et al., 1998)-,
amoxicillin (Fontana et al., 2001)- or penicillin (Henry-
Michelland et al., 1987)-entrapped polycyanoacrylates
General procedure for the preparation of Schiff
bases 2a–h
A mixture of amine 1a–d (1.00 mmol) and aromatic
aldehydes (1.00 mmol) was heated at reflux in ethanol
(20 mL) for 5–6 h. Evaporation of the solvent afforded
123

Med Chem Res (2016) 25:247–262
249
(OH), 1735 (CO, b-lactam). 1H NMR (CDCl3) d 1.44– 1.69
(CH2, m, 2H), 2.87 (CH2, dt, 1H, J = 13.9, 6.9 Hz), 3.27–
3. 47 (CH2, m, 3H), 3.63 (OMe, s, 3H), 4.46 (OH, t, 1H,
J = 4.8 Hz), 5.06 (H-4, d, 1H, J = 4.3 Hz), 5.52 (H-3, d,
1H, J = 4.3 Hz), 6.78 (ArH, ddd, 5H, J = 17.7, 8.1,
0.8 Hz), 7.11 (ArH, dd, 2H, J = 11.3, 4.7 Hz), 7.16–7.31
(ArH, m, 2H). 13C-NMR (CDCl3) d 30.1 (CH2), 37.1
(CH2), 54.8 (OMe), 58.1 (CH2), 60.6 (C-4), 81.0 (C-3),
113.4, 114.9, 121.4, 125.5, 129.1, 129.6, 156.5, 159.0
(aromatic carbons), 165.0 (CO b-lactam). MS m/z = 327
[M?]. Anal. Calcd. for C19H21NO4: C, 69.71; H, 6.47; N,
4.28 %. Found: C, 69.79; H, 6.56; N, 4.35 %.
imines 2a–h as either oil or solid, which were used for the
next step without further purification.
General procedure for the synthesis of N-substituted
hydroxyl b-lactams 3a–h
A mixture of Schiff base 3a–h (1.00 mmol) and triethy-
lamine (7.00 mmol) in dry CH2Cl2 (15 mL) was stirred at
0 °C. Chlorotrimethylsilane (1.20 mmol) was added drop-
wise to the mixture and stirred for 1 h. Then, phenoxy-
acetic acid (1.50 mmol) and tosyl chloride (1.50 mmol)
were added and the mixture was stirred at room tempera-
ture for 12 h. Then, it was washed with HCl 1 M (20 mL),
saturated NaHCO3 (20 mL), and brine (20 mL). The
organic layer was dried (Na2SO4) and filtered, and the
solvent was evaporated to give the crude b-lactams 3a–
h. Then, desired products were purified by silica gel col-
umn chromatography (eluent 8:2 petroleum ether:EtOAc)
or recrystallization from EtOAc.
1-(3-Hydroxylpropyl)-4-(4-nitrophenyl)-3-phenoxyaze-
tidin-2-one (3d) Recrystallized from EtOAc to give white
solid (yield 83 %). Mp: 88–90 °C. IR (KBr) cm-1: 3394
(OH), 1751 (CO, b-lactam). 1H NMR (CDCl3) d 1.65–1.86
(CH2, m, 2H), 2.58 (OH, s, 1H), 3.07–3.27 (CH2, m, 1H),
3.82–3.57 (CH2, m, 3H), 5.12 (H-4, d, 1H, J = 4.1 Hz),
5.52 (H-3, d, 1H, J = 4.1 Hz), 6.71 (ArH, d, 2H,
J = 8.4 Hz), 6.90 (ArH, t, 1H, J = 7.0 Hz), 7.13 (ArH, t,
2H, J = 7.7 Hz), 7.53 (ArH, d, 2H, J = 8.3 Hz), 8.15
(ArH, d, 2H, J = 8.1 Hz, 2H). 13C-NMR (CDCl3) d 8.1
(CH2), 37.4 (CH2), 60.2 (CH2), 60. 7 (C-4), 85.1 (C-3),
113.7, 115.3 121.9, 124.6, 128.0, 129.2, 129.9, 131.1,
156.8, 159.8 (aromatic carbons), 166.0 (CO b-lactam). MS
m/z = 342 [M?]. Anal. Calcd. for C18H18N2O5: C, 63.15;
H, 5.30; N, 8.18 %. Found: C, 63.24; H, 5.38; N, 8.15 %.
1-(2-Hydroxylethyl)-4-(4-methoxyphenyl)-3-phenoxyaze-
tidin-2-one (3a) Purified by column chromatography to
give yellow oil (yield 87 %). IR (neat) cm-1: 3415 (OH),
1758 (CO, b-lactam). 1H NMR (DMSO) d 2.74–2.99 (CH2,
m, 1H), 3.25–3.51 (CH2, m, 3H), 3.62 (OMe, s, 3H), 4.81
(OH, t, 1H, J = 4.2 Hz), 5.12 (H-4, d, 1H, J = 4.1 Hz),
5.52 (H-3, d, 1H, J = 4.1 Hz), 6.66–6.92 (ArH, m, 5H),
7.11 (ArH, t, 2H, J = 7.5 Hz), 7.22 (ArH, d, 1H,
J = 8.2 Hz). 13C-NMR (DMSO) d 42.5 (CH2), 54.8
(OMe), 57.9 (CH2), 60.9 (C-4), 81.2 (C-3), 113.3, 114.9,
121.4, 125.2, 129.2, 129.5, 156.5, 159.0 (aromatic car-
bons), 165.2 (CO b-lactam). MS m/z = 313 [M?]. Anal.
Calcd. for C18H19NO4: C, 69.00; H, 6.11; N, 4.47 %.
Found: C, 69.12; H, 6.25; N, 4.52 %.
Cis and trans 1-(4-hydroxylphenyl)-4-(4-methoxyphenyl)-
3-phenoxyazetidin-2-one (3e) Recrystallized from EtOAc
to give white solid (yield 91 %). Mp: 148–150 °C. IR
1
(KBr) cm-1: 3386 (OH), 1743 (CO, b-lactam). H NMR
(DMSO) d 3.66 (OMe, s, 3H), 5.60 (H-4, d, 1H,
J = 4.6 Hz), 5.70 (H-4, d, 1H, J = 4.7 Hz), 5.76 (H-3, d,
1H, J = 4.6 Hz), 5.85 (H-3, d, 1H, J = 4.7 Hz), 6.73
(ArH, d, 1H, J = 8.8 Hz), 6.83 (ArH, dd, 4H, J = 8.5,
3.3 Hz), 6.87–6.96 (ArH, m, 1H), 7.01 (ArH, d, 1H,
J = 8.7 Hz), 7.18 (ArH, dd, 3H, J = 16.3, 8.0 Hz), 7.33
(ArH, dd, 3H, J = 17.3, 8.7 Hz), 9.44 (OH, s, 1H). 13C-
NMR (DMSO) d 55.5 (OMe), 64.9 (C-4), 80.9 (C-3),
114.0, 114.9, 115.5, 118.7, 119.0, 121.8, 122.2, 123.1,
124.9, 125.3, 129.8, 135.1, 146.4, 154.6, 156.9, 159.6 8
(aromatic carbons), 163.2 (CO b-lactam). MS m/z = 361
[M?]. Anal. Calcd. for C22H19NO4: C, 73.12; H, 5.30; N,
3.88 %. Found: C, 73.27; H, 5.41; N, 3.79 %.
1-(2-Hydroxylethyl)-4-(4-nitrophenyl)-3-phenoxyazetidin-
2-one (3b) Recrystallized from EtOAc to give light yel-
low solid (yield 83 %). Mp: 80–82 °C. IR (KBr) cm-1
:
3382 (OH), 1766 (CO, b-lactam). 1H NMR (CDCl3) d
3.05–3. 20 (CH2, m, 1H), 3.31 (OH, s, 1H), 3.39–3.54
(CH2, m, 1H), 3.78 (CH2, s, 2H), 5.11 (H-4, d, 1H,
J = 4.4 Hz), 5.51 (H-3, d, 1H, J = 4.4 Hz), 6.65 (ArH, d,
2H, J = 7.9 Hz), 6.84 (ArH, t, 1H, J = 7.3 Hz), 7.07
(ArH, t, 2H, J = 6.9 Hz), 7.47 (ArH, d, 2H, J = 8.7 Hz),
8.09 (ArH, d, 2H, J = 8.7 Hz, 2H). 13C-NMR (CDCl3) d
45.2 (CH2), 59.6 (CH2), 62.3 (C-4), 81.5 (C-3), 115.2,
122.5, 123.4, 129.3, 129.4, 140.6, 148.1, 156.2 (aromatic
carbons), 166.4 (CO b-lactam). MS m/z = 328 [M?]. Anal.
Calcd. for C17H16N2O5: C, 62.19; H, 4.91; N, 8.53 %.
Found: C, 62.28; H, 4.99; N, 8.61 %.
1-(4-Hydroxylphenyl)-4-(4-nitrophenyl)-3-phenoxyaze-
tidin-2-one (3f) Recrystallized from EtOAc to give white
solid (yield 87 %). Mp: 204–206 °C. IR (KBr) cm-1: 3393
1
(OH), 1751 (CO, b-lactam). H NMR (DMSO) d 5.63 (H-
4, d, 1H, J = 4.5 Hz), 5.73 (H-3, d, 1H, J = 4.5 Hz), 6.77
(ArH, t, 4H, J = 9.0 Hz), 6.02–7.85 (ArH, m, 1H),
7.23–7.05 (ArH, m, 4H), 7.58 (ArH, d, 2H, J = 8.0 Hz),
1-(3-Hydroxylpropyl)-4-(4-methoxyphenyl)-3-phenoxyaze-
tidin-2-one (3c) Recrystallized from EtOAc to give white
solid (yield 80 %). Mp: 89–91 °C. IR (neat) cm-1: 3417
123

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Med Chem Res (2016) 25:247–262
8.10 (ArH, d, 2H, J = 7.9 Hz), 9.17 (OH, s, 1H). 13C-
NMR (DMSO) d 60.2 (C-4), 80.5 (C-3), 114.8, 115.5,
118.2, 121.8, 121.9, 122.8, 128.1, 128.7, 128.9, 129.1,
140.6, 147.3, 154.3, 155.9 (aromatic carbons), 161.2 (CO
b-lactam). MS m/z = 376 [M?]. Anal. Calcd. for
C21H16N2O5: C, 67.02; H, 4.29; N, 7.44 %. Found: C,
67.15; H, 4.37; N, 7.38 %.
2-((4-Methoxybenzylidene)amino)ethyl acrylate (4a) Yellow
oil (yield 84 %). IR (neat) cm-1: 1728 (CO, acrylate), 1650
1
(C=N). H NMR (CDCl3) d 3.60–3.80 (CH2, m, 4H), 3.92
(OMe, s, 3H), 5.60–5.70 (vinylic, m, 1H), 5.87 (vinylic, dt,
1H, J = 3.10, 1.10 Hz), 6.11 (vinylic, ddd, 1H, J = 7.1,
4.6, 1.9 Hz), 7.03 (ArH, d, 2H, J = 15.0 Hz), 7.84 (ArH, d,
1H, J = 13.2 Hz), 8.45 (CH=N, s, 1H). 13C-NMR (CDCl3)
d 38.9 (CH2), 55.6 (OMe), 62.1 (CH2), 111.8, 114.1, 119.8,
128.2, 131.8, 134.7 (aromatic and vinylic carbons), 160.91
(C=N), 164.17 (CO arylate). MS m/z = 233 [M?]. Anal.
Calcd. for C13H15NO3: C, 66.94; H, 6.48; N, 6.00 %.
Found: C, 67.11; H, 6.59; N, 5.88 %.
1-(4-(Hydroxylmethyl)phenyl)-4-(4-methoxyphenyl)-3-phe-
noxy azetidin-2-one (3g) Purified by column chromatog-
raphy to give yellow oil (yield 88 %). IR (neat) cm-1: 3394
(OH), 1751 (CO, b-lactam). 1H NMR (CDCl3) d 3.69
(OMe, s, 3H), 4.55 (CH2, s, 2H), 4.62 (OH, s, 1H), 5.30 (H-
4, d, 1H, J = 4.8 Hz), 5.46 (H-3, d, 1H, J = 4.8 Hz), 6.76
(ArH, d, 4H, J = 8.6 Hz), 6.82–7.00 (ArH, m, 2H), 7.05
(ArH, d, 1H, J = 7.1 Hz), 7.10–7.19 (ArH, m, 4H),
7.22–7.32 (ArH, m, 3H). 13C-NMR (CDCl3) d 55.1 (OMe),
60.9 (CH2), 65.3, 81.4, 115.6, 118.4, 121.8, 122.1, 123.4,
124.0, 128.0, 128.4, 128.0, 129.0, 133.0, 134.0, 139.9,
146.7, 148.2, 156.6 (aromatic carbons), 161.9 (CO b-lac-
tam). MS m/z = 375 [M?]. Anal. Calcd. for C23H21NO4:
C, 73.58; H, 5.64; N, 3.73 %. Found: C, 73.65; H, 5.70; N,
3.67 %.
2-((4-Nitrobenzylidene)amino)ethyl acrylate (4b) Light yel-
low solid (yield 86 %). Mp: 61–63 °C. IR (KBr) cm-1
:
1751 (CO, acrylate), 1627 (C=N). 1H NMR (CDCl3) d
3.70–4.07 (CH2, m, 4H), 5.44–5. 68 (vinylic, m, 1H),
5.87–6.16 (vinylic, m, 1H), 6.27–6.56 (vinylic, m, 1H),
7.80–8.04 (ArH, m, 2H), 8.25 (ArH, dt, 2H, J = 8.90,
1.80 Hz), 8.42 (CH=N, s, 1H). 13C-NMR (CDCl3)
d = 38.9 (CH2), 63.4 (CH2), 123.1, 123.7, 127.4, 129.0,
141.1, 149.1(aromatic and vinylic carbons), 160.7 (CH=N),
164.1 (CO acrylate). MS m/z = 248 [M?]. Anal. Calcd. for
C12H12N2O4: C, 58.06; H, 4.87; N, 11.29 %. Found: C,
58.21; H, 5.03; N, 11.12 %.
1-(4-(Hydroxylmethyl)phenyl)-4-(4-nitrophenyl)-3-phe-
noxyazeti din-2-one (3h) Recrystallized from EtOAc to
give white solid (yield 85 %). Mp: 126–128 °C. IR (KBr)
cm-1: 3417 (OH), 1751 (CO, b-lactam). 1H NMR (CDCl3) d
4.53 (CH2, s, 2H), 4.79 (OH, s, 1H), 5.67 (H-4, d, 1H,
J = 4.8 Hz), 5.74 (H-3, d, 1H, J = 4.8 Hz), 6.78 (ArH, d,
2H, J = 8.5 Hz), 6.91 (ArH, t, 1H, J = 7.4 Hz), 7.05–7. 20
(ArH, m, 2H), 7.26 (ArH, d, 4H, J = 2.6 Hz), 7.56 (ArH, dd,
2H, J = 8.8, 2.4 Hz), 8.10 (ArH, dd, 2H, J = 8.8, 2.5 Hz).
13C-NMR (CDCl3) d 45.7 (CH2), 61.1 (C-4), 81.3 (C-3),
114.6, 115.3, 118.2, 118.3, 122.3, 122.6, 123.6, 127.5, 129.0,
129.5, 129.7, 133.0, 134.1, 140.1, 147.3, 148.1, 156.3 (aro-
matic carbons), 162.3 (CO b-lactam). MS m/z = 390 [M?].
Anal. Calcd. for C22H18N2O5: C, 67.69; H, 4.65; N, 7.18 %.
Found: C, 68.76; H, 4.75; N, 7.06 %.
3-((4-Methoxybenzylidene)amino)propyl acrylate (4c) Yel-
low oil (yield 82 %). IR (neat) cm-1: 1720 (CO, acrylate),
1650 (C=N). 1H NMR (CDCl3) d 1.44–1.69 (CH2, m, 2H),
2.74–2. 99 (CH2, m, 1H), 3.25–3.51 (CH2, m, 3H), 3.86
(OMe, s, 3H), 5.83–6.10 (vinylic, m, 1H), 5.16–6.39
(vinylic, m, 1H), 5.52–6.70 (vinylic, m, 1H), 7.01 (ArH, dt,
2H, J = 4.4, 2.6 Hz), 7.76–7.92 (ArH, m, 2H), 8.37
(CH=N, s, 1H). 13C-NMR (CDCl3)
d 27.1(CH2),
36.1(CH2), 55.7 (OMe), 60.9(CH2), 123.7, 125.0, 127.5,
128.0, 130.5, 139.6, 144.0 (aromatic and vinylic carbons),
165.4 (C=N), 160.7 (CO arylate). MS m/z = 247 [M?].
Anal. Calcd. for C14H17NO3: C, 68.00; H, 6.93; N, 5.66 %.
Found: C, 68.15; H, 7.05; N, 5.49 %.
3-((4-Nitrobenzylidene)amino)propyl acrylate (4d) Orange
oil (yield 82 %). IR (neat) cm-1: 1712 (CO, acrylate), 1650
(C=N). 1H NMR (CDCl3) d 2.73–1. 13 (CH2, m, 2H), 3.37
(CH2, q, J = 6.3 Hz, 2H), 4.08–4.30 (CH2, m, 2H), 5.57
(vinylic, ddd, 1H, J = 9.8, 2.0, 1.0 Hz), 5.68–5.90 (vinylic,
m, 1H), 6.11 (vinylic, ddd, 1H, J = 10.4, 5.4, 1.0 Hz), 8.04
(ArH, d, 2H, J = 9.8 Hz), 8.34 (ArH, d, 2H, J = 8.2 Hz),
8.71 (CH=N, s, 1H), 13C-NMR (CDCl3) d 25.5(CH2),
36.1(CH2), 61.2(CH2), 124.2, 128.0, 129.6, 130.5, 140.2,
144.2 (aromatic and vinylic carbons), 160.1, (CH=N),
166.3 (CO acrylate). MS m/z = 262 [M?]. Anal. Calcd. for
C13H14N2O4: C, 59.54; H, 5.38; N, 10.68 %. Found: C,
59.63; H, 5.47; N, 10.55 %.
General procedure for the synthesis of acryloyloxy-
Schiff bases (4a–h)
To the solution of imines 2a–h (1.00 mmol) in 10 mL of
CH2Cl2 was added triethylamine (2.20 mmol). Then,
acryloyl chloride (2.20 mmol) was added dropwise to the
solution at 0 °C. The resulting solution was stirred at room
temperature for 30 min and then washed with H2O
(2 9 10 mL). The organic layer was dried with anhydrous
MgSO4, filtered and evaporated. The crude imines 4a–h as
either oil or solid were used for the next step without
further purification.
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251
4-((4-Methoxybenzylidene)amino)phenyl acrylate (4e)
Light brown oil (yield 93 %). IR (neat) cm-1: 1729 (CO,
General procedure for the synthesis of acrylate b-
lactams 5a–h
1
acrylate), 1606 (C=N). H NMR (CDCl3) d 3.86 (OMe, s,
3H), 5.83–6.10 (vinylic, m, 1H), 5.16–6.39 (vinylic, m,
1H), 5.52–6.70 (vinylic, m, 1H), 6.98 (ArH, dt, 3H,
J = 7.4, 3.8 Hz), 7.11–7.24 (ArH, m, 3H), 7.76–7.95
(ArH, m, 2H), 8.37 (CH=N, s, 1H). 13C-NMR (CDCl3) d
55.4 (OMe), 114.2, 114.3, 121.7, 122.1, 127.8, 128.1,
129.0, 130.5, 132.0, 132.6, 148.3, 149.9 (aromatic and
vinylic carbons), 162.3 (CH=N), 164.3 (CO acrylate). MS
m/z = 281 [M?]. Anal. Calcd. for C17H15NO3: C, 72.58;
H, 5.37; N, 4.98 %. Found: C, 72.69; H, 5.51; N, 4.84 %.
A mixture of Schiff base 4a–h (1.00 mmol), triethylamine
(5.00 mmol), substituted acetic acids (1.50 mmol) and
tosyl chloride (1.50 mmol) in dry CH2Cl2 (15 mL) was
stirred at room temperature for overnight. Then, it was
washed with HCl 1 M (20 mL), saturated NaHCO3
(20 mL) and brine (20 mL). The organic layer was dried
(Na2SO4) and filtered, and the solvent was evaporated to
give the crude b-lactams 5a–h. Then, desired products
were purified by silica gel column chromatography (eluent
8:2 n-hexane/EtOAc) or recrystallization from EtOAc.
4-((4-Nitrobenzylidene)amino)phenyl
acrylate
(4f)
Orange solid (yield 94 %). Mp: 118–120 °C. IR (KBr)
cm-1: 1728 (CO, acrylate), 1627 (C=N). 1H NMR (CDCl3)
d 5.96–6.07 (vinylic, m, 1H), 6.26–6.42 (vinylic, m, 1H),
6.61 (vinylic, ddd, 1H, J = 14.5, 6.2, 2.0 Hz), 6.98 (ArH,
dd, 3H, J = 8.9, 2.5 Hz), 7.12–7.24 (ArH, m, 3H), 7.79 –
7.88 (ArH, m, 2H), 8.38 (CH=N, s, 1H). 13C-NMR
(CDCl3) d 113.8, 115.6, 120.9, 121.2, 122.1, 127.7, 128.9,
130.9, 131.8, 133.0, 148.2, 150.1 (aromatic and vinylic
carbons), 160.3, (CH=N), 164.4 (CO acrylate). MS m/
z = 296 [M?]. Anal. Calcd. for C16H12N2O4: C, 64.86; H,
4.08; N, 9.46 %. Found: C, 64.97; H, 4.15; N, 9.35 %.
2-(4-Methoxyphenyl)-4-oxo-3-phenoxyazetidin-1-yl)ethyl
acrylate (5a) Purified by column chromatography to give
yellow oil (yield 81 %). IR (neat) cm-1: 1758 (CO, b-
1
lactam), 1720 (CO, acrylate). H NMR (CDCl3) d 3.07
(CH2, dt, 1H, J = 14.0, 6.8 Hz), 3.58 (CH2, dt, 1H,
J = 17.6, 7.3 Hz), 4.21 (CH2, dd, 2H, J = 12.7, 6.4 Hz),
3.76 (OMe, s, 3H), 4.91 (H-4, d, 1H, J = 4.3 Hz), 5.40 (H-
3, d, 1H, J = 4.3 Hz), 5.76–5.92 (vinylic, m, 1H), 6.10
(vinylic, td, 1H, J = 17.2, 11.5 Hz), 6.28–6.51 (vinylic, m,
1H), 6.73 (ArH, d, 2H, J = 8.6 Hz), 6.82 (ArH, d, 2H,
J = 11.4 Hz), 7.12 (ArH, t, 2H, J = 9.2 Hz), 7.21–7.32
(ArH, m, 3H). 13C-NMR (CDCl3) d 37.7(CH2), 55.1
(OMe), 62.5 (CH2), 67.5 (C-4), 82.1 (C-3), 113.7, 114.6,
115.5, 121.9, 124.6, 125.8, 128.1, 129.2, 129.8, 129.9,
131.1, 156.9, 159.9 (aromatic and vinylic carbons), 161.9
(CO, b-lactam), 166.1 (CO acrylate). MS m/z = 367 [M?].
Anal. Calcd. for C21H21NO5: C, 68.65; H, 5.76; N, 3.81 %.
Found: C, 68.71; H, 5.82; N, 3.76 %.
4-((4-Methoxybenzylidene)amino)benzyl
acrylate
(4g) Yellow oil (yield 87 %). IR (neat) cm-1: 1728 (CO,
1
acrylate), 1604 (C=N). H NMR (CDCl3) d 3.89 (CH2, s,
2H), 5.68–5.44 (vinylic, m, 1H), 6.16–5.87 (vinylic, m,
1H), 6.56–6.27 (vinylic, m, 1H), 6.73–7.10 (ArH, m, 4H),
7.13–7.48 (ArH, m, 4H), 7.84 (ArH, d, 1H, J = 8.7), 8.35
(CH=N, s, 1H). 13C-NMR (CDCl3) d 55.4 (OMe), 65.6
(CH2), 114.1, 127.8, 129.0, 130.6, 132.0, 137.4, 140.8,
159.8 (aromatic and vinylic carbons), 160.0 (CH=N), 165.7
(CO acrylate). MS m/z = 295 [M?]. Anal. Calcd. for
C18H17NO3: C, 73.20; H, 5.80; N, 4.74 %. Found: C,
73.34; H, 5.96; N, 4.67 %.
2-(4-Nitrophenyl)-4-oxo-3-phenoxyazetidin-1-yl)ethyl
acrylate (5b) Recrystallization from EtOAc to give white
solid (yield 85 %). Mp: 120–122 °C. IR (KBr) cm-1: 1766
1
(CO, b-lactam), 1728 (CO, acrylate). H NMR (CDCl3) d
3.31 (CH2, ddd, 1H, J = 15.0, 6.4, 3.3 Hz), 3.89 (CH2,
ddd, 1H, J = 11.8, 10.8, 8.0 Hz), 4.17 (CH2, ddd, 1H,
J = 11.8, 6.4, 3.5 Hz), 4.31–4. 48 (CH2, m, 1H), 5.17 (H-
4, d, 1H, J = 4.6 Hz), 5.52 (H-3, d, 1H, J = 4.5 Hz),
5.81–5.92 (vinylic, m, 1H), 6.03 (vinylic, ddd, 1H,
J = 17.1, 10.4, 0.4 Hz), 6.29–6.48 (vinylic, m, 1H), 6.71
(ArH, d, 2H, J = 7.9 Hz), 6.90 (ArH, t, 1H, J = 7.4 Hz),
7.14 (ArH, dd, 1H, J = 8.4, 7.5 Hz), 7.52 (ArH, d, 2H,
J = 8.7 Hz), 8.15 (ArH, d, 2H, J = 8.6 Hz). 13C-NMR
(CDCl3) d 39.9 (CH2), 61.2 (CH2), 62.0 (C-4), 82.3 (C-3),
115.2, 122.4, 123.4, 127.4, 129.3, 129.4, 132.0, 140.7,
148.1, 156.3 (aromatic and vinylic carbons), 165.6 (CO, b-
lactam), 165.7 (CO acrylate). MS m/z = 382 [M?]. Anal.
Calcd. for C20H18N2O6: C, 62.82; H, 4.75; N, 7.33 %.
Found: C, 62.91; H, 4.83; N, 7.25 %.
4-((4-Nitrobenzylidene)amino)benzyl
acrylate
(4h)
Orange solid (yield 94 %). Mp: 118–120 °C. IR (KBr)
cm-1: 1728 (CO, acrylate), 1627 (C=N). 1H NMR (CDCl3)
d 5.26 (CH2, s, 2H), 5.84–5. 93 (vinylic, m, 1H), 6.07– 6.
27 (vinylic, m, 1H), 6.44 (vinylic, ddd, 1H, J = 12.3, 8.4,
3.6 Hz), 7.28 (ArH, dd, 2H, J = 9.1, 7.1 Hz), 7.45 (ArH, d,
1H, J = 8.2 Hz), 8.08 (ArH, d, 1H, J = 7.9 Hz),
8.27–8.38 (ArH, m, 2H), 8.56 (s, 1H). 13C-NMR (CDCl3) d
65.9 (CH2), 119.9, 121.2, 124.3 128.1, 129.6 130.3, 130.9
134.7, 141.4, 150.8 157.6 (aromatic and vinylic carbons),
161.2 (CH=N), 166.3 (CO acrylate). MS m/z = 310 [M?].
Anal. Calcd. for C17H14N2O4: C, 65.80; H, 4.55; N,
9.03 %. Found: C, 65.97; H, 4.66; N, 8.92 %.
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2-(4-Methoxyphenyl)-4-oxo-3-phenoxyazetidin-1-yl)propyl
acrylate (5c) Purified by column chromatography to give
yellow oil (yield 80 %). IR (neat) cm-1: 1766 (CO, b-
2-(4-Nitrophenyl)-4-oxo-3-phenoxyazetidin-1-yl)phenyl
acrylate (5f) Purified by column chromatography to give
light yellow oil (yield 83 %). IR (neat) cm-1: 1751 (CO, b-
lactam), 1720 (CO, acrylate). 1H NMR (CDCl3) d 5.54 (H-
4, d, 1H, J = 4.9 Hz), 5.67 (H-3, d, 1H, J = 4.9 Hz), 6.01
(vinylic, dd, 1H, J = 10.4, 0.8 Hz), 6.28 (vinylic, ddd, 1H,
J = 17.2, 10.3, 0.7 Hz), 6.58 (vinylic, dd, 1H, J = 17.7,
1.7 Hz), 6.78 (ArH, d, 2H, J = 8.6 Hz), 6.95 (ArH, d, 1H,
J = 6.9 Hz), 7.00–7.11 (ArH, m, 2H), 7.17 (ArH, t, 2H,
J = 7.8 Hz), 7.34 (ArH, q, 2H, J = 2.8 Hz), 7.55 (ArH, d,
2H, J = 8.6 Hz), 8.13 (ArH, d, 2H, J = 8.2 Hz, 2H). 13C-
NMR (CDCl3) d 61.1 (C-4), 81.3 (C-3), 114.6, 115.3,
118.2, 118.3, 122.0, 122.3, 122.6, 123.6, 127.5 129.0,
129.5 129.7, 133.0, 134.1, 140.1, 147.3, 148.1, 156.3
(aromatic and vinylic carbons), 162.3 (CO, b-lactam),
164.4 (CO acrylate). MS m/z = 430 [M?]. Anal. Calcd. for
C24H18N2O6: C, 66.97; H, 4.22; N, 6.51 %. Found: C,
67.05; H, 4.15; N, 6.68 %.
1
lactam), 1728 (CO, acrylate). H NMR (CDCl3) d 1.88
(CH2, dd, 2H, J = 13.3, 6.7 Hz), 2.96– 3.17 (CH2, m, 2H),
3.73 (OMe, s., 3H), 4.16 (CH2, t, 2H, J = 6.2 Hz), 4.94 (H-
4, d, 1H, J = 4.3 Hz), 5.42 (H-3, d, 1H, J = 4.3 Hz), 5.81
(vinylic, dd, 1H, J = 10.4, 1.5 Hz), 5.96–6.19 (vinylic, m,
1H), 6.36 (vinylic, dt, 1H, J = 4.5, 2.3 Hz), 6.66–6.94
(ArH, m, 5H), 7.12 (ArH, dd, 2H, J = 9.9, 5.9 Hz), 7.26
(ArH, dd, 2H, J = 8.5, 3.6 Hz). 13C-NMR (CDCl3) d 7.9
(CH2), 37.4 (CH2), 55.1 (OMe), 61.7 (CH2), 62.0 (C-4),
81.7 (C-3), 113.7, 115.3, 121.9, 124.6, 128.0, 129.1, 129.8,
129.9, 131.1, 157.0, 159.8 (aromatic and vinylic carbons),
166.4 (CO, b-lactam), 169.7 (CO acrylate). MS m/z = 381
[M?]. Anal. Calcd. for C22H23NO5: C, 69.28; H, 6.08; N,
3.67 %. Found: C, 69.37; H, 6.15; N, 3.51 %.
2-(4-Nitrophenyl)-4-oxo-3-phenoxyazetidin-1-yl)propyl
acrylate (5d) Recrystallization from EtOAc to give white
solid (yield 78 %). Mp: 98–100 °C. IR (KBr) cm-1: 1751
2-(4-Methoxyphenyl)-4-oxo-3-phenoxyazetidin-1-yl)benzyl
acrylate (5g) Purified by column chromatography to give
light yellow oil (yield 83 %). IR (neat) cm-1: 1751 (CO, b-
1
(CO, b-lactam), 1725 (CO, acrylate). H NMR (CDCl3) d
1
1.77–2.13 (CH2, m, 2H), 3.01–3.25 (CH2, m, 2H), 4.21
(CH2, dd, 2H, J = 9.8, 5.6 Hz), 5.13 (H-4, d, 1H,
J = 4.3 Hz), 5.54 (H-3, d, 1H, J = 4.4 Hz), 5.74–5.93
(vinylic, m, 1H), 6.08 (vinylic, ddd, 1H, J = 17.3, 10.4,
1.1 Hz), 6.26–6.46 (vinylic, m, 1H), 6.72 (ArH, dd, 2H,
J = 7.7, 0.9 Hz, 2H), 6.81–6.99 (ArH, m, 1H), 6.96–7.36
(ArH, m, 2H), 7.40–7.66 (ArH, m, 2H), 8.01–8.36 (ArH,
m, 2H). 13C-NMR (CDCl3) d 8.4 (CH2), 38.0 (CH2), 61.5
(CH2), 61.6 (C-4), 82.1 (C-3), 115.2, 122.4, 123.4, 127.9,
129.4, 131.3, 140.8, 148.1, 156.3 (aromatic and vinylic
carbons), 165.5 (CO, b-lactam), 165.9 (CO acrylate). MS
m/z = 396 [M?]. Anal. Calcd. for C21H20N2O6: C, 63.63;
H, 5.09; N, 7.07 %. Found: C, 63.75; H, 5.21; N, 7.01 %.
lactam), 1720 (CO, acrylate). H NMR (CDCl3) d 3.73
(OMe, s, 3H), 4.59 (CH2, s, 2H), 5.33 (H-4, d, 1H,
J = 4.8 Hz), 5.52 (H-3, d, 1H, J = 4.8 Hz), 5.90–6.06
(vinylic, m, 1H), 6.29 (vinylic, dt, 1H, J = 17.3, 9.3 Hz),
6.65–6.50 (vinylic, m, 1H), 6.80 (ArH, dd, 4H, J = 11.7,
5.5 Hz), 6.85–6.97 (ArH, m, 3H), 7.03 (ArH, dt, 3H,
J = 6.8, 3.8 Hz), 7.07–7.22 (ArH, m, 3H). 13C-NMR
(CDCl3) d 55.1 (OMe), 61.8 (CH2), 67.7 (C-4), 81.3 (C-3),
113.9, 114.6, 114.8, 114.9, 115.3, 115.6, 118.4, 121.0
122.0 122.1, 122.2, 124.0, 127.6, 127.7, 127.8, 129.2,
129.4, 129.5, 129.6, 129.9, 132.8, 134.7 146.8, 156.9,
159.9 (aromatic and vinylic carbons), 163.03 (CO, b-lac-
tam), 164.0 (CO acrylate). MS m/z = 429 [M?]. Anal.
Calcd. for C26H23NO5: C, 72.71; H, 5.40; N, 3.26 %.
Found: C, 72.85; H, 5.55; N, 3.30 %.
2-(4-Methoxyphenyl)-4-oxo-3-phenoxyazetidin-1-yl)phenyl
acrylate (5e) Purified by column chromatography to give
color less oil (yield 87 %). IR (neat) cm-1: 1751 (CO, b-
2-(4-Nitrophenyl)-4-oxo-3-phenoxyazetidin-1-yl)benzyl
acrylate (5h) Recrystallized from EtOAc to give white
solid (yield 79 %). Mp: 130–132 °C. IR (KBr) cm-1: 1743
1
lactam), 1704 (CO, acrylate). H NMR (CDCl3) d 3.73
(OMe, s, 3H), 5.33 (H-4, d, 1H, J = 4.8 Hz), 5.52 (H-3, d,
1H, J = 4.8 Hz), 5.90–6.06 (vinylic, m, 1H), 6.26 (vinylic,
ddd, 1H, J = 17.3, 10.1, 1.6 Hz), 6.48–6.65 (vinylic, m,
1H), 6.73–6.85 (ArH, m, 3H), 6.95–7.06 (ArH, m, 3H),
7.09–7.22 (ArH, m, 2H), 7.23–7.43 (ArH, m, 5H). 13C-
NMR (CDCl3) d = 55.1 (OMe), 61.8 (C-4), 81.3 (C-3),
113.9, 114.8, 114.9, 115.3, 115.6, 118.4, 121.0, 122.0,
122.1, 122.2, 127.6, 127.8, 129.2, 129.4, 129.6, 129.9,
132.8, 146.8, 156.9, 159.9 (aromatic and vinylic carbons),
163.0 (CO, b-lactam), 164.5 (CO acrylate). MS m/z = 415
[M?]. Anal. Calcd. for C25H21NO5: C, 72.28; H, 5.10; N,
3.37 %. Found: C, 72.36; H, 5.05; N, 3.31 %.
1
(CO, b-lactam), 1704 (CO, acrylate). H NMR (CDCl3) d
4.59 (CH2, s, 2H), 5.33 (H-4, d, 1H, J = 4.8 Hz), 5.52 (H-
3, d, 1H, J = 4.8 Hz), 5.90–6.06 (vinylic, m, 1H), 6.26
(vinylic, ddd, 1H, J = 17.3, 10.1, 1.6 Hz), 6.48–6.65
(vinylic, m, 1H), 6.73–6.85 (ArH, m, 2H), 6.95–7.06 (ArH,
m, 2H), 7.09 –7.22 (ArH, m, 3H), 7.23–7.43 (ArH, m, 6H).
13C-NMR (CDCl3) d 61.8 (CH2), 67.4 (C-4), 87.3 (C-3),
114.0, 115.3, 115.6, 118.7, 120.9 122.4, 124.0, 127.0,
127.7, 129.2, 129.9, 132.4, 134.6, 147.3, 157.2 (aromatic
and vinylic carbons), 163.1 (CO, b-lactam), 164.7 (CO
acrylate). MS m/z = 444 [M?]. Anal. Calcd. for
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253
C25H20N2O6: C, 67.56; H, 4.54; N, 6.30 %. Found: C,
67.68; H, 4.63; N, 6.21 %.
Characterization of the emulsions of prepared
nanoparticles
Sample size and distribution of the emulsions were
checked by dynamic light scattering (DLS) using a HOR-
IBA DLS instrument equipped with a laser beam at
550 nm. The sample was pre-diluted with distilled water
(0.3 mL of emulsion in 24.7 mL of water). Transmission
electron microscopy (TEM) of the samples was captured
by placing a drop of diluted emulsion (1 mL of emulsion in
10 mL of the distilled water and sonicated) onto a Form-
var-coated copper grid and evaporating the solvent by air
blowing, and then, the grid was viewed on the microscope.
Preparation of the polyacrylate nanoparticle
emulsions (6a–h)
Experimental procedure for the emulsion
copolymerization
Poly (butyl acrylate–styrene) nanoparticles were prepared
by emulsion polymerization. b-Lactams 5a–h (10 mg, 1 %
w/w) were dissolved in a 7:3 (w/w) mixture of butyl
acrylate (700 mg) and styrene (300 mg) at 70 °C and
heated for 10 min under Ar atmosphere. Deionized water
(4.0 mL) was then added to the solution, followed by
appropriate amount of sodium dodecyl sulfate (10 mg) and
potassium persulfate (5 mg) as surfactant and radical ini-
tiator, respectively. The mixture was then stirred for 7 h at
70 °C and cooled to room temperature. The samples were
purified and characterized to determine their shape and
average size. Purification of the above emulsion was per-
formed by continuous extraction using ethyl acetate fol-
lowed by centrifugation on a bench-top centrifuge, and
then, samples were filtered with cellulose acetate mem-
brane (200 nm).
General procedure for antimalarial activity
measurements
The chloroquine-resistant P. falciparum strain K1 (South-
east Asia) was in vitro cultured in complete medium con-
sisting of RPMI 1640 (In Vitrogen) supplemented with
27.5 mM NaHCO3, 20 mg/L gentamycin and 10 % human
serum. Parasites were grown at 37 °C in human O? red
blood cells at a 6 % hematocrit under a 5 % CO2, 10 % O2
and 85 % N2 atmosphere. Cultures were synchronized by
sorbitol treatments (Noedl et al., 2005). Stock solutions of
lactam derivatives were prepared in sterile DMSO
(10 mM), and later dilutions were done with complete
culture medium. Increasing concentrations of lactam
derivatives (100 lL/well, top concentration = 50 lM)
were distributed in a 96-well plate; DMSO (0.5 % vol/vol,
top concentration) was distributed for control. Then,
100 lL from a culture containing[95 % ring (0–20 h post
invasion) at a 0.8 % parasitemia and 3 % hematocrit in
complete medium was added per well. The plates were
incubated at 37 °C in the presence of 5 % CO2, 85 % N2
and 10 % O2 for 72 h. After culture the plates were frozen
down at -20 °C. Parasite growth inhibition was quantified
using a homemade HRP2 ELISA assay based on Pf HRP2
detection (Lemkul and Bevan, 2012). Drug concentrations
inhibiting parasite growth by 50 % (IC50) were calculated
by nonlinear regression analysis from the dose–response
relationship as fitted by software—ICEstimator 1.2 (http://
www.antimalarial-icestimator.net) (Nagard et al., 2011;
Kaddouri et al., 2006). Each concentration was estimated
from independent experiments in triplicate.
Results and discussion
In this study, some free hydroxyl imines 2a–h (Chackala-
mannil et al., 2010; Hosseini-Sarvari, 2011; Peng et al.,
2007; Bae et al., 2012; Mu¨ller et al., 2010; Murphy-Jolly
et al., 2010) were synthesized from aliphatic and aromatic
Fig. 1 ORTEP diagram of imine 2f
123

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Med Chem Res (2016) 25:247–262
Scheme 1 Synthesis of N-substituted hydroxy b-lactams 3a–h,
acrylate b-lactam monomers 5a–h and copolymer 6a–h by emulsion
polymerization. Reagents and conditions: a EtOH, reflux; b 1.
TMSiCl, Et3N, CH2Cl2, 1 h, 2. PhOCH2CO2H, TsCl, 12 h; c acryloyl
chloride, Et3N, CH2Cl2, 0.5 h; d PhOCH2CO2H, Et3N, CH2Cl2, TsCl,
15 h; e SDS, K2S2O8, n-butyl acrylate, styrene, H2O, 70 °C, 7 h
amines containing the hydroxyl group 1a–d and aromatic
aldehydes in refluxing ethanol (Scheme 1). The X-ray
crystallography of 2f confirmed the E stereochemistry
(Fig. 1) (Atioglu et al., 2015).
J = 4.2 Hz, 1H) ppm. The b-lactam H-3 and H-4 protons
appeared as two doublets at 5.52 ppm and 5.12, respec-
tively. The observed coupling constants of these protons
confirmed the cis stereochemistry for this compound.
Aromatic protons showed a multiplet, a triplet and a dou-
blet at 6.66–6.92, 7.11 and 7.22, respectively. The 13C-
NMR spectrum of compound 3a displayed the b-lactam
carbonyl peak at 165.2 ppm. In addition, the carbon of
aliphatic part, methoxy group, C-4 and C-3 of b-lactam
ring appeared at 42.5, 57.9, 54.8, 60.9 and 81.2 ppm,
respectively.
To prepare b-lactams 3a–h, hydroxy imines 2a–h were
protected by trimethylsilyl chloride (TMSCl) in the pres-
ence of Et3N in dry CH2Cl2 (Cossfo et al., 1986). Then,
phenoxyacetic acid and p-toluenesulfonyl chloride were
added to the above crude mixture. The new mixture was
stirred for 12 h to afford N-substituted hydroxy b-lactams
3a–h in good to excellent yields. The silyl group was
deprotected during aqueous workup (Scheme 1).
Acrylated b-lactam monomers 5a–h were synthesized
from acrylated-imines 4a–h in the presence of phenoxy-
acetic acid, tosyl chloride and triethylamine in dry CH2Cl2.
The IR spectrum of 5a showed a sharp band at 1758 cm-1
for the b-lactam carbonyl and an absorption at 1720 cm-1
for the carbonyl of the acrylate (Fig. 2b). The disappear-
ance of the hydroxyl peak also confirmed the success of the
The cycloadducts of N-substituted hydroxy b-lactams
3a–h were characterized by spectral analyses (Table 1). As
a model, in the IR spectrum of 3a, the absorption for the
hydroxy group was exhibited at 3415 cm-1. A sharp band
at 1758 cm-1 was assigned to the stretching frequency of
b-lactam carbonyl, which is a proof of the b-lactam ring
1
1
(Fig. 2a). The H-NMR spectrum of 3a displayed a mul-
protection by the acroyl group. The H-NMR spectrum of
tiplet for the two methylene groups at 2.74–2.99 (m, 1H),
3.25–3.51 (m, 3H), a singlet at 3.62 for the methoxy pro-
tons and the hydroxyl proton as a triplet at 4.81 (t,
5a exhibited a singlet peak at 3.76 which is due to the
methoxy protons. The ethyl part of the molecule showed
two doublets of triplets at 3.07 and 3.58. The b-lactam H-3
123

Med Chem Res (2016) 25:247–262
255
Table 1 Structures of b-lactams 3a–h and 5a–h
b-Lactam
Structure
Yield (%)
87
b-Lactam
Structure
Yield (%)
81
3a
5a
OMe
OMe
O
O
O
O
N
N
OH
O
O
NO2
NO2
O
O
O
3b
3c
3d
83
80
81
5b
5c
5d
85
80
78
N
N
O
O
OH
O
OMe
OMe
OH
O
O
O
O
N
N
O
O
NO2
OH
NO2
O
O
O
O
N
N
O
O
123

256
Med Chem Res (2016) 25:247–262
Table 1 continued
b-Lactam
Structure
Yield (%)
91
b-Lactam
Structure
Yield (%)
3e
5e
87
OMe
OMe
O
O
O
O
N
N
OH
O
O
3f
87
5f
83
NO2
NO2
O
O
O
O
N
N
O
O
OH
3g
88
5g
81
OMe
OMe
O
O
O
O
N
N
OH
O
O
3h
85
5h
79
NO2
NO2
O
O
O
O
N
N
OH
O
O
123

Med Chem Res (2016) 25:247–262
257
and H-4 protons exhibited two doublets at 5.40 and 4.91,
by centrifuging on a bench-top centrifuge to remove minor
impurities such as excess surfactant or residual monomers,
without changing the physical characteristics of the
nanoparticles in the emulsion. Finally, to remove any
particles bigger than 200 nm the samples were filtered with
cellulose acetate membrane (0.2 lm).
The IR spectrum of nanocopolymer 6a showed the
characteristic absorption of C=O acrylate at 1724 and C=O
b-lactam at 1743 cm-1 (Fig. 2c). The morphology and the
particle size analyses of the emulsions were performed by
TEM and DLS. DLS analyses of the final emulsion showed
that the average diameter of the 6a–h were regularly about
50, 39, 58, 58, 29, 26, 58, 44 nm, respectively, with size
distributions spanning from about 10–230 nm. The average
diameter of nanocopolymeric emulsions 6e–h bearing an
aromatic ring at the N-1 position of the b-lactam ring is less
than the aliphatic ones 6a–d (Fig. 3).
respectively.
The
observed
coupling
constants
(J = 4.3 Hz) for H-4 and (J = 4.3 Hz) for H-3 confirmed
the cis stereochemistry for this compound. The vinylic
protons were observed as a multiplet at 5.92–5.76 (vinylic,
m, 1H), 6.10 (td, J = 17.2, 11.5 Hz, 1H), 6.28– 6.51 (m,
1H). Aromatic protons showed two doublets, a triplet and a
multiplet at 6.73 (d, J = 8.6 Hz, 2H), 6.82 (d,
J = 11.4 Hz, 2H), 7.12 (t, J = 9.2 Hz, 2H) and 7.21–7.32
(m, 3H) ppm, respectively. The 13C-NMR spectrum of 5a
displayed the b-lactam and acryloyl carbonyl signals at
161.9 and 166.1 ppm, respectively. In addition, the carbon
of methoxy group, C-4 and C-3 of b-lactam ring appeared
at 55.1, 67.5 and 82.1 ppm, respectively.
In the next step, the acrylated b-lactam monomers 5a–
h were used for the preparation of b-lactam nanocopoly-
mers 6a–h. These nanocopolymeric emulsions were pre-
pared easily in water by radical initiator emulsion
polymerization using warmed mixture of butyl acrylate,
styrene and an acrylated b-lactam monomer (Jarrahpour
and Heiran, 2014). For this, the acrylated b-lactam
monomers 5a–h were dissolved in a mixture of butyl
acrylate and styrene 7:3 (w:w) at 70 °C under Ar atmo-
sphere. Then, the mixture was pre-emulsified in deionized
water and sodium dodecyl sulfate (SDS) (1 % w) was
added as the surfactant. After about 30 min, the homoge-
nous solution was treated with potassium persulfate (0.5 %
w/w) to start free radical polymerization for 7 h
(Scheme 1). After that, for the purification the nanopoly-
meric emulsions were extracted with ethyl acetate followed
TEM image of 6a is shown in Fig. 4, and it is seen that
the nanoparticles are essentially spherical and their sizes
are about 30–70 nm.
All of these newly synthesized b-lactams derivatives
were subsequently evaluated for their biological activities.
Firstly, it has been demonstrated that these compounds do
not possess significant antimicrobial activities against
Gram-positive S. aureus and Gram-negative bacteria
E. coli or P. aeruginosa with MICs superior to 50 lM.
Nevertheless, moderate antimalarial activities have been
obtained against chloroquine-resistant P. falciparum K1
strain as outlined in Table 2 with IC50 varying from 14 to
50 lM. On the other hand, a strong influence of the groups
Fig. 2 The IR spectrum of a N-
substituted hydroxyl b-lactam
3a, b acrylated b-lactam
monomer 5a, c b-lactam
nanocopolymer 6a
123

258
Med Chem Res (2016) 25:247–262
bearing on the nitrogen atom of the considered derivative is
noted. Moreover, nanopolymeric emulsions present activ-
ities in a similar range with their monomers.
more important and is extremely cost-effective in drug
discovery because of its ability to quickly screen libraries
of compounds that may potentially act as drugs. Docking
methods usually use an energy-based scoring function to
identify the energetically most favorable ligand–protein
binding. The calculated energy of interaction is reflective
of the affinity of the ligand for binding to the macro-
molecule active site. The lower energy scores represent
better protein–ligand bindings compared to higher energy
values (Lemkul and Bevan, 2012). Proteins–ligand com-
plexes can be considered as an ensemble of representative
conformational structures, and the binding events may
undergo a wide range of motions in the ensemble structures
ranging from small changes in binding site residues to
large-scale motions of entire protein (Verkhivker et al.,
2002).
Structure activity relationship (SAR) study of b-lactams
reveals that the methoxyphenyl on C-4 of b-lactam ring (3e
and 3g) enhances the antimalarial activity. Also the phe-
nolic 2-azetidinones (3e and 3f) and those bearing the
benzyl alcohol moiety (3g and 3h) showed more activity
than the aliphatic alcohols.
Molecular docking
Molecular docking technique is used to predict the bound
conformations of organic ligands into a protein target of
known structure. This technique is becoming increasingly
Fig. 3 DLS analyses of 6a–h
123

Med Chem Res (2016) 25:247–262
259
(poses) to determine their favorability. It requires high-
quality structures of a receptor molecule, typically an
enzyme or receptor protein to which the ligand will bind.
The candidate drug molecule may be treated as flexible,
while the receptor is predominantly rigid, though most
modern algorithms allow for a subset of residues to be
treated as flexible, as well.
Atomic coordinates for the three-dimensional protein
˚
models (a crystal structure at 2.1 A resolution of the Pf-
SSB tetramer bound to two (dT)35 molecules; PDB code
3ULP) (Antony et al., 2012) are obtained from the Protein
Data Bank ‘‘(http://www.rcsb.org/pdb/) DNA residues,’’
and all crystallographic waters are removed from the
atomic coordinate data files.
The structures of the compounds used in this study (see
Table 1) were optimized at the B3LYP/6-31G* level of
theory by using Gaussian 03 program (Frisch et al., 2009).
The Autodock Vina program (Trott and Olson, 2010) was
used to study the binding of the 3a–h and 5a–h to the Pf-
SSB tetramer. The docking energies are calculated from a
set of energy grids centered in the active site of the
enzyme. The protein coordinates are fixed during calcula-
tions, while the inhibitor is flexible and moves on the grid.
Grid searching was performed by genetic algorithm to
locate the ligand in the best binding orientation and con-
formation based on the binding energy.
Fig. 4 TEM image of 6a
Efficient development of new therapeutics for targeting
Plasmodium falciparum K1 requires detailed information
about the mechanism of action of these compounds.
Information obtained from molecular docking simulations
will likely accelerate the process of novel antimalarial drug
development.
The single-stranded DNA-binding (SSB) protein from
P. falciparum [P. falciparum SSB (Pf-SSB)] is encoded in
the nucleus and transported to the apicoplast where it likely
functions in the replication and maintenance of the api-
coplast genome. SSB proteins are present in nearly all
organisms and bind to single-stranded DNA (ssDNA)
intermediates produced transiently during replication,
repair and recombination.
The results revealed that the best inhibitor is 3g with a
docking energy of -130 kcal/mol, while 3e was slightly
less potent (with a docking energy of -128 kcal/mol).
Docking energies of the 3a–h compounds ranged from
-27 to -130 kcal/mol. These results are in accordance
with the IC50 variations. The same trends are also observed
for 5a–h compounds. For instance, the docking energy of
5g compound is about -38 kcal/mol less than that of 3g
due to lack of fit in the cavity of the target protein. The
lowest energy models of 3g and 5g bound to Pf-SSB tet-
ramer are shown in Fig. 5a and 5b. A hydrogen bond
between 3g and Asn28 of Pf-SSB tetramer is shown in
Fig. 5b.
Therefore, we have performed docking to study the
probable interaction between our synthetic compounds and
Pf-SSB. Docking is usually carried out in two steps: the
generation of configurations of the ligand in the binding
site and the energetic scoring of those configurations
Table 2 Antimalarial activities of derivatives 3a–h, 5a–h and 6a–6h against chloroquine-resistant Plasmodium falciparum K1 strain
Product
IC50 (lM)
Product
IC50 (lM)
Product
IC50 (lM)
Chloroquine
0.804
[50
[50
33
3a
3b
3c
3d
3e
3f
5a
5b
5c
5d
5e
5f
[50
[50
39
6a
6b
6c
6d
6e
6f
[50
[50
30
36
32
29
14
27
27
21
30
36
3g
3h
14
5g
5h
25
6g
6h
22
19
20
39
123

260
Med Chem Res (2016) 25:247–262
Fig. 5 One mode of binding of a 3g and b 5g to the Pf-SSB tetramer. c A hydrogen bond between 3g and Asn83 of Pf-SSB tetramer is shown as
black dashed line
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