Design and stereoselective synthesis of novel β-lactone and β-lactams as potent anticancer agents on breast cancer cells

Article abstract of DOI:10.1016/j.tet.2017.12.044

To produce a novel class of anticancer compounds, an efficient method for synthesizing novel β-lactone and β-lactam frameworks was developed based on the reaction of a new ketene with C=O and C=N bonds. Functionalized 2-azetidinones were efficiently synthesized by employing 2,4-dichlorophenoxylketene, which was generated in situ. The reaction of the ketene with aldehydes was not successful and in all cases except for 4-nitrobenzaldehyde, a rearranged dimer of the ketene was obtained as a lactone. Anticancer cellular activity of all new β-lactams and lactones on breast cancer cells was studied. All new synthesized compounds exhibited potential anticancer activity which may guarantee their future application in moderate chemotherapy.

Full text of DOI:10.1016/j.tet.2017.12.044

Accepted Manuscript
Design and stereoselective synthesis of novel β-lactone and β-lactams as potent
anticancer agents on breast cancer cells
Mahnoosh Rashidi, Mohammad Reza Islami, Saeed Esmaeili-Mahani
PII:
S0040-4020(17)31325-X
10.1016/j.tet.2017.12.044
TET 29200
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 22 October 2017
Revised Date: 18 December 2017
Accepted Date: 21 December 2017
Please cite this article as: Rashidi M, Islami MR, Esmaeili-Mahani S, Design and stereoselective
synthesis of novel β-lactone and β-lactams as potent anticancer agents on breast cancer cells,
Tetrahedron (2018), doi: 10.1016/j.tet.2017.12.044.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design and Stereoselective Synthesis of Novel
β
-Lactone and
β
-Lactams as
potent Anticancer Agents on Breast cancer cells
Mahnoosh Rashidia, Mohammad Reza Islamia* and Saeed Esmaeili-Mahanib
aChemistry Department, Shahid Bahonar University of Kerman, 76169 Kerman, Iran
bBiology Department, Shahid Bahonar University of Kerman, 76169 Kerman, Iran
*mrislami@uk.ac.ir
KEYWORDS: β-lactam, β-Lactone, 2,4-dichlorophenoxyacetic acid, ketene, Anticancer
ABSTRACT: To produce a novel class of anticancer compounds, an efficient method for synthesizing novel β-
lactone and β-lactam frameworks was developed based on the reaction of a new ketene with C=O and C=N bonds.
Functionalized 2-azetidinones were efficiently synthesized by employing 2,4-dichlorophenoxylketene, which was
generated in situ. The reaction of the ketene with aldehydes was not successful and in all cases except for 4-
nitrobenzaldehyde, a rearranged dimer of the ketene was obtained as a lactone. Anticancer cellular activity of all
new β-lactams and lactones on breast cancer cells was studied. All new synthesized compounds exhibited potential
anticancer activity which may guarantee their future application in moderate chemotherapy.
1. Introduction
Human life is threatened by many diseases, such as allergies, asthma, infections, heart diseases, diabetes, HIV, and
cancer. Cancer one of the most dangerous diseases, appearing in different parts of the human body such as brain,
skin, blood, lung, prostate, and breast. Compounds such as luotonin A, Camptothecin, and Psoralidin, both natural
products1,2 and synthetic compounds,3,4 are used to treat cancers. Although anticancer drugs are available, in many
cases chemotherapy fails to have the required effect and the patients lose their lives. As a consequence, the discovery
and development of novel anticancer compounds which are more effective, have fewer side effects, and are useful to
patients are highly desirable.
O
O
N
O
O
N
N
OH
N
O
HO
O
O
N
OH
Psoralidin
Camptothecin
Luotonin A
In continuation of our research5-9 on the synthesis of novel four-membered heterocyclic rings, we are looking for
new β-lactams and β-lactones with benefits for health. β-Lactams are antimicrobial agents10-13 and are found in
antibacterial drugs such as penicillin and cephalosphorin.14-16 They are also useful building blocks for various amino
acids and peptides.17-18 Additionally, literature review demonstrates that some β-lactams such as compound 1 show
anticancer properties.19
In light of these research results, we were encouraged to prepare novel 2-azetidinones and β-lactones and evaluate
their anticancer activities. Many methods have been reported for their synthesis.20-24 One of the most common
1

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methods for the synthesis of β-Lactams is the formal cycloaddition of ketenes with imines. Ketenes, because of their
unusual physical properties and unique chemical reactivity, are highly regarded in the synthesis of four-membered
rings.25-27 In this study, 2,4-dichlorophenoxyacetic acid 2 was used as a precursor for the preparation of new β-
Lactams and β-lactones with anticancer properties. Here, we wish to report our results.
2. Results and discussion
The reaction of 2,4-dichlorophenoxyacetic acid 2 and Mukaiyama reagent 3 with Et3N 4 in CH2Cl2 in the presence
of imines 5 led to the formation of azetidinones 6 in good to excellent yields (Scheme 1). The reaction was found to
be highly stereoselective, and the cis-isomers were formed as the only products except in cases 6k, 6l and 6m which
were obtained as trans-isomers (Table 2).
Scheme 1. Reaction of 2,4-dichlorophenoxyacetic acid with Mukaiyama’s reagent, imine and Et3N
Table1. Synthesis of 3-(2,4-dichlorophenoxy)-1,4-diphenylazetidin-2-ones
Product
Ar1
Ar2
Yield a
(%)
Ph
Ph
Ph
4-ClC6H4
Ph
74
77
89
74
72
75
6a
6b
6c
6d
6e
6f
4-NO2C6H4
Ph
4-MeC6H4
Ph
4-MeOC6H4
2,4-
Ph
DiClC6H3
6g
6h
6i
4-MeC6H4
4-ClC6H4
4-MeC6H4
Ph
90
75
90
75
85
77
90
76
78
4-MeC6H4
4-MeOC6H4
4-MeC6H4
4-ClC6H4
Ph
4-MeC6H4
Naphthyl
Naphthyl
Naphthyl
4-MeC6H4
4-MeC6H4
6j
6k
6l
4-MeOC6H4
2-ClC6H4
6m
6n
6p
4-ClC6H4
aIsolated yield of pure products
2

ACCEPTED MANUSCRIPT
1
The structures of the products were fully characterized by IR, H NMR, and 13C NMR spectroscopy along with
elemental analysis data (see supporting information). The IR spectra of these compounds showed absorption bands
1
due to the carbonyl group at 1741–1771 cm–1. H NMR spectroscopy is generally used for distinguishing between
cis and trans-β-lactam isomers by using H–H coupling constants. The J value is larger (4.0–6.0 Hz) in a cis-β-lactam
1
than in a trans-β-lactam (1.5–2.5 Hz). The H NMR spectrum of cis-6a exhibited signals corresponding to the
vicinal methine protons at δ = 5.54 and 5.45, ppm which appear as two doublets (3JHH = 4.8 Hz). The aromatic
1
protons appeared as a multiplet at δ = 7.45–7.04 ppm. The H-decoupled 13C NMR spectrum of cis-6a showed 17
distinct resonances in agreement with the suggested structure. Partial assignment of these resonances is given in the
supporting information. Characteristic 13C NMR signals appear due to one carbonyl group at δ = 162.33 and signals
1
at δ = 61.44 and 81.6 ppm for CH groups. The H NMR and 13C NMR spectroscopic data of compounds 6a–p are
presented in the supporting information.
Scheme 2. Thermal in situ formation of Ketene
A proposed explanation for the formation of 2-azetidinones 6 is shown in Schemes 2 and 3. In the first step, salt I
is formed as an intermediate from the reaction of 2,4 dichlorophenoxyacetic acid 2 with Mukaiyama’s reagent 3. In
the presence of triethylamine, it is subsequently converted into the ketene 7 (Scheme 2). The stereochemistry28-30 of
the ketene–imine cycloaddition involves an initial attack of the nitrogen atom of the imine on C-1 of the ketene to
form the zwitterionic intermediate 8a, which has an equilibrium with 8b. The cis-β-lactam is formed as a product
from 8a as a crowded intermediate by a direct electrocyclic ring closure reaction via conrotatory mode. The trans-
isomer is formed from intermediate 8b as a less crowded intermediate by the same electrocyclic ring closure
(Scheme 3). We expected the formation of the trans-isomer as the major product but in all cases except
6k, 6l and
6m the cis-isomer was formed as only product. It seems that the substituent effects can be very important on the
direct ring closure. Thus, the ketene with electron donating substituents such as oxygen accelerates the direct ring
closure, leading to a preference for cis-β-lactam formation.31
Scheme 3: A plausible mechanism for the stereochemistry of the cis-β-lactams
The addition of aldehydes to ketenes is a well-known process in organic chemistry.32-34 These reactions usually
give β-lactones, but can also provide a number of other structural types, including the products of skeletal
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rearrangement of ketene and dimers.35-36 However, reported examples of this kind of cooperativity in a synthetic
protocol are rare37 and largely serendipitous because, among other factors, it is hard to envision systems which
exhibit this cooperation. The reaction of 2,4-dichlorophenoxyacetic acid 2 with aromatic aldehyde 9 in the presence
of Mukaiyama reagent 3 and Et3N 4 in CH2Cl2 led to the formation of the rearranged dimer (Scheme 4). It is
important to note that contrary to expectation compound 11, was not obtained.
Scheme 4. Reaction of 2,4-dichlorophenoxyacetic acid with an aromatic aldehyde in the presence of Mukaiyama’s
reagent and Et3N
We tried to run the same reaction without any aldehyde to see the effect on the formation of ketene dimer. The
reaction was performed without any aldehyde in the same conditions in which the dimer was formed. Compound 10
was formed in a trace yield. Therefore, for the evaluation of the catalytic effect of aldehyde on the formation of
dimer of ketene, we had to use several aromatic aldehydes in the synthesis of the ketene dimer. The results are
shown in Table 2. In all cases except for 4-nitrobenzaldehyde, the procedure was useful and compound 10 was
formed in 31–46% yield. We did not investigate the formation mechanism of the mentioned dimer in practical
methods, but a reasonable mechanism for the formation of ketene dimer is proposed in Scheme 5.
4

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Table 2. Synthesis of 3-(2,4-dichlorophenoxy)-4-[(2,4-dichlorophenoxy)- methyl]-2H-oxet-2-one
Product
Ar1
Yield a (%)
Without aldehyde
trace
10
Ph
4-ClC6H4
37
31
46
34
33
33
41
35
35
36
--
10
10
10
10
10
10
10
10
10
10
10
4-MeC6H4
3-MeC6H4
2-MeC6H4
2-ClC6H4
4-MeOC6H4
2,4-Di Cl C6H4
4-BrC6H4
4-(Dimethylamino)C6H4
4-NO2C6H4
aIsolated yield of pure products
According to this mechanism, the reaction cycle is initiated by the formation of ketene 7 from the reaction of acid
2 with Mukaiyama reagent 3 in the presence of Et3N as a base. The resulting ketene 7 is activated by the addition of
an aldehyde to give the nucleophilic ketene enolate I. Subsequently, enolate I is added to another ketene, leading to
the formation of intermediate II. The catalytic cycle is completed by an intramolecular attack of the enolate oxygen,
which forms β-lactone III and releases aldehyde. Finally, tautomerization of compound III produces compound 10
as a new β-lactone.
Scheme 5. Suggested mechanism for ketene dimerization38
5

ACCEPTED MANUSCRIPT
1
The structure of compound 10 is fully characterized by IR, H NMR, and 13C NMR spectroscopy along with
elemental analysis data (see supporting information). The IR spectra of this compound showed an absorption band
1
due to the carbonyl group at 1769 cm–1. The H NMR spectrum of 10 exhibited a signal corresponding to the CH2
protons at δ = 5.02 ppm, which appear as a singlet. The aromatic protons appeared as a multiplet at δ = 7.49–6.94
1
ppm. The H-decoupled 13C NMR spectrum of 10 showed 13 distinct resonances in agreement with the suggested
structure; partial assignment of these resonances is given in the supporting information. Characteristic 13C NMR
signals were shown due to one carbonyl group at δ = 165.76, and a signal was found at δ = 66.19 for the CH2 group.
DEPT technique was used to determine the peak multiplicity of compound 10. This spectrum exhibited one CH2
group, along with six types of CH groups, which confirms the structure of β-lactone 10.
In order to identify the 2,4-dichlorophenoxylketene as an intermediate in the synthesis of the β-lactams, we have
undertaken further reactions of the mentioned ketene with cyclopentadiene 12. Therefore, the reaction of acid 2 with
cyclopentadiene was performed in the presence of benzenesulfonyl chloride 16 and Et3N in CH2Cl2. The reaction led
to the formation of endo-7-(2,4- dichlorophenoxy)bicyclo[3.2.0]hept-2-en-6-one 17 in 69% yield as the only
observed product (Scheme 6).
Scheme 6. Reaction of acid 2 with cyclopentadiene
Assessment of anticancer activity of lactams and dimer of ketene
All newly synthesized compounds were monitored for their anticancer activity. Cytotoxic study was performed
using the MTT (3,4-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay method (Figure 1).39-41 Viability
was determined by MTT assay. Data are expressed as mean ± SEM; n = 5–6 wells for each group; *P<0.05,
**P<0.01 and ***P<0.001 versus control non-treated cells. +P<0.05, ++P<0.01 and +++P<0.001 versus 2 µg/ml
and 4 µg/mL of same compound. All compounds exhibited moderate to high cytotoxic activity in MCF-7 cell lines,
at concentration of 2 and 4 µg/mL. The obtained data are shown in Table 3.
Table 3. precent of cell viability in β-Lactams synthesised compound treated MCF-7.
Synthesized
compound
Cell viability (%)
4 µg/ml
2µg/ml
control
2,4 D
6i
100 ± 5
80.40± 3.30
53.60± 2.20
53.30± 2.19
54.80 ± 2.25
41.30 ± 1.70
42.15 ±2.20
55.10 ± 2.80
64.40 ± 3.30
21.19 ± 1.00
53.10± 2.18
15.21 ± 0.62
59.88 ± 2.45
19.45 ± 0.8
18.00 ± 0.74
20.80 ± 1.00
33.57 ± 1.70
23.40 ± 1.20
27.35 ± 1.40
6n
6g
6h
6d
6b
6e
6p
6

ACCEPTED MANUSCRIPT
63.53 ± 3.25
47.50 ± 2.4
74.50 ± 4.30
73.00 ± 4.20
83.20 ± 4.90
77.95 ± 4.50
60.60 ± 3.50
33.86 ± 0.40
40.00 ± 2.00
25.50 ± 1.30
67.10 ± 3.90
67.20 ± 3.90
60.80 ± 3.50
71.70 ±4.10
46.90 ± 2.70
10.30 ± 0.12
6m
6l
6a
6k
6j
6c
6f
dimer
The anticancer effects of the synthesized compounds were potent in doses of 4 µg. In addition, the initial acid and
compounds numbers 6a, 6k, 6j, and 6c had similar effects, while other β-Lactams showed stronger toxic effects
compared to the initial acid.
150
150
100
+
**
100
50
0
**
+++
***
***
***
*
+
***
***
2 ug
4 ug
**
+
+
+
***
* *
***
***
**
++
***
***
2 ug
4 ug
***
***
50
0
***
150
150
+
**
*
** **
100
50
0
*** ***
100
50
0
***
2 ug
4 ug
2 ug
4 ug
+
***
***
Cont
dimer
Figure 1. Effects of 2 and 4 µg/ml of synthesized β-lactams on MCF-7 cancer cells
Furthermore, the dimer significantly induced dramatic toxic effect on MCF-7 cells. The most potent effect was
observed for compound 10 as a β-lactone against MCF-7 cell lines at a concentration of 4 µg/mL. This reactivity
could be related to the following aspects: 1. high potency of the β-lactone moiety; 2. presence of the C=C double
bond, and 3. two phenyl rings including four chlorine atoms, when compared to β-lactams and primary acid. The
data showed 2,4-dichlorophenoxyacetic acid (2,4-D), a genotoxic organochlorinated herbicide, and its derivatives
have anticancer effects. The MTT assays cannot give an indication on the mechanism of cytotoxicity. Toxic effects
of organic compounds can occur via apoptotic cell death or necrosis. It is now evident that many environmental
chemicals exert their toxicity via apoptotic cell signaling. Previous study has also demonstrated that 2,4-D induces
apoptosis via a direct effect on the mitochondria and initiates caspase-dependent reactions.42 It seems that a same
mechanism is involved in the observed effects in this study. In addition, it has been demonstrated that 2,4-D can
produce intracellular free radicals which lead to numerous unbeneficial changes in animal tissues. The increase in
free radicals levels cause DNA damage and thus cell death (in apoptotic process). For many years the discussion has
been continuing concerning the mutagenicity of 2,4-D, but now many documented investigations have been
performed from 2000 unequivocally proved its mutagenic action.43
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3. Conclusions
In conclusion, a successful short route towards β-lactams, and a new rearranged ketene dimer from
2,4-diphenoxyacetic acid has been developed. In the key step, a new ketene was generated and reacted with imines
to form β-Lactams or form a new dimer in the presence of an aromatic aldehyde. All new synthesized compounds
exhibited potential anticancer activity, with the strength of the effect depended on the substituent on the phenyl
groups.
4. Experimental section
4.1. Materials and methods
General information: IR spectra were recorded on a commercial Spectrophotometer (Bruker Tensor 27 FT-IR). 1H
1
NMR and 13C NMR spectra were recorded on a Bruker 400 Ultrashield NMR Magnet (400 MHz for H NMR and
100 MHz for 13C NMR) with TMS as the internal standard, and 13C NMR were reported with complete proton
decoupling. Elemental analysis (CHNS) was recorded on a Costech 4110, and melting point were recorded on an
Electrothermal-9200. Data are reported as follows: chemical shift δH and δC (ppm), coupling constants J (Hz),
integration and multiplicity (s = singlet, d = doublet, t = triplet, m = multiplet). Solvents for reaction were distilled
prior to use: Toluene, dichloromethane, chloroform, hexane and ethyl acetate. Triethylamine was predried over
sodium wire. Column chromatography was carried out using Sigma-Aldrich silica gel 40 Angstrom (Ǻ).
4.2. General procedure for the preparation of imines (5)
Distilled benzaldehyde (50 mmol, 5.1 ml) in toluene (8ml) was added to a solution of distilled aniline (50 mmol,
4.56 ml) in dry toluene (50ml). The mixture was heated under reflux for 7 h in the presence of CaSO4. The mixture
was filtered and then the solvent was evaporated. The crude products were obtained as solid and recrystallized from
hexane.
4.3. Typical Procedure for the Preparation of Compounds (6a–p)
A solution of the N-benzylideneaniline 5 (0.22 g, 1.2 mmol) in anhydrous dichloromethane (10 ml) was added to
a suspension of 2,4-dichlorophenoxyacetic acid 2 (0.22 g, 1.0 mmol), 2-chloro-N-methylpyridinium iodide 3 (0.3 g,
1.2 mmol) and triethylamine 4 (0.50 mL, 3.6 mmol) in anhydrous dichloromethane (25 ml) and refluxed under a
nitrogen atmosphere for 17 h. After cooling, the solution was washed with water, 5% HCl aqueous solution, and
then with water. The organic layer was dried over Na2SO4 and the solvent was removed under reduced pressure. The
crude products were purified by column chromatography (silica gel, n-hexane/ethyl acetate, 40:1.) and cis-3(2,4-
dichlorophenoxy)-1,4-diphenylazetidin-2-one was obtained as the only product.
4.3.1. Cis 3-(2,4-dichlorophenoxy)-1,4-diphenylazetidin-2-one (6a)
White solid; yield: 285 mg (74%). mp: 165-166 °C. IR (KBr): 1744 (C=O) cm–1. Calcd for C21H15Cl2NO2
(384.26): C, 65.64; H, 3.93; N, 3.65. Found: C, 66.1; H, 3.8; N, 3.4. 1H NMR (400 MHz, CDCl3): δ = 7.04-7.44 (m,
13 H, Ar) 5.54(d, 1 H, 3JH–H = 4.8 Hz), 5.45 (d, 1 H, 3JH-H = 4.8 Hz) ppm. 13C NMR (100 MHz, CDCl3): δ =162.3
(C=O), 151.5, 136.8, 132.1, 130.0, 129.2, 129.0, 128.6, 128.1, 127.6, 127.4, 124.3, 124.8, 117.6, 116.7, 81.6, 61.4
ppm.
4.3.2. Cis 1-(4-chlorophenyl)-3-(2,4-dichlorophenoxy)-4-phenylazetidin-2-one (6b)
8

ACCEPTED MANUSCRIPT
White solid; yield: 322 mg (77%). mp: 205-206 °C. IR (KBr): 1748 (C=O) cm–1. Calcd for C21H14Cl3NO2
(418.70): C, 60.24; H, 3.37; N, 3.35. Found: C, 60.1; H, 2.9; N, 2.9. 1H NMR (400 MHz, CDCl3): δ = 7.01-7.39 (m,
12 H, Ar) 5.43(d, 1 H, 3JH–H = 4.4 Hz), 5.55 (d, 1 H, 3JH-H = 4.4 Hz) ppm. 13C NMR (100 MHz, CDCl3): δ =162.2
(C=O), 151.4, 135.2, 131.6, 130.1, 130.0, 129.4, 129.2, 128.7, 128.1, 127.7, 127.5, 124.3, 118.8, 116.7, 81.8, 61.6
ppm.
4.3.3. Cis 3-(2,4-dichlorophenoxy)-4-(4-nitrophenyl)-1-phenylazetidin-2-one (6c)
White solid; yield: 375 mg (89%). mp: 212-214 °C. IR (KBr): 1774 (C=O) cm–1. Calcd for C21H14Cl2N2O4
(429.25): C, 58.7; H, 3.2; N, 6.5. Found: C, 58.3; H, 3.1; N, 6.4. 1H NMR (400 MHz, CDCl3): δ = 7-7.19 (m, 12 H,
3
3
Ar) 5.60 (d, 1 H, JH–H = 5.2 Hz), 5.58 (d, 1 H, JH-H = 5.2 Hz) ppm. 13C NMR (100 MHz, CDCl3): δ =161.9
(C=O), 151.3, 148.3, 139.9, 136.2, 130.2, 129.5, 129.0, 128.2, 127.7, 125.4, 124.2, 123.8, 117.4, 116.9, 81.9, 60.4
ppm.
4.3.4. Cis 3-(2,4-dichlorophenoxy)-4-phenyl-1-(p-tolyl)azetidin-2-one (6d)
White solid; yield: 296 mg (74%). mp: 160-162°C. IR (KBr): 1741 (C=O) cm–1. Calcd for C22H17Cl2NO2
(398.28): C, 66.34; H, 4.30; N, 3.52. Found: C, 66.1; H, 3.9; N, 3.5. 1H NMR (400 MHz, CDCl3): δ = 7.03-7.43 (m,
3
3
12 H, Ar) 5.43 (d, 1 H, JH–H = 5.0 Hz), 5.53 (d, 1 H, JH-H = 5.0 Hz), 2.31 (3 H, s, CH3) ppm. 13C NMR (100
MHz, CDCl3): δ =162.0 (C=O), 151.5, 134.6, 134.3, 132.3, 130.0, 129.7, 128.9, 128.5, 128.1, 127,5, 127.4, 124,3,
117.6, 116.7, 81.7, 61.4, 21.0 ppm.
4.3.5. Cis 3-(2,4-dichlorophenoxy)-4-(4-methoxyphenyl)-1-phenylazetidin-2-one (6e)
White solid; yield: 300mg (72%). mp: 157-160 °C. IR (KBr): 1751 (C=O) cm–1. Calcd for C22H17Cl2NO3(414.28):
C, 63.78; H, 4.14; N, 3.38. Found: C, 63.50; H, 4.00; N, 3.20. 1H NMR (400 MHz, CDCl3): δ = 7.02-7.31 (m, 12 H,
3
Ar) 5.49 (d, 1 H, 3JH–H = 5.0 Hz), 5.39 (d, 1 H, JH-H = 5.0 Hz), 3.80 (3 H, s, OCH3). ppm. 13C NMR (100 MHz,
CDCl3): δ =162.1 (C=O), 151.6, 138.8, 134.5, 134.4, 130.0, 129.7, 129.2, 129.1, 128.0, 127.5, 127.4, 124.4, 117.6,
116.9, 81.7, 61.4, 21.3, 21.0 ppm.
4.3.6. Cis 3-(2,4-dichlorophenoxy)-4-(2,4-dichlorophenyl)-1-phenylazetidin-2-one (6f)
White solid; yield: 340 mg (75%). mp: 125-126 °C. IR (KBr): 1751 (C=O) cm–1. Calcd for C21H13Cl4NO2
(453.15): C, 55.66; H, 2.89; N, 3.09. Found: C, 55.3; H, 3.0; N, 2.7. 1H NMR (400 MHz, CDCl3): δ = 7.16-7.48 (m,
3
3
11 H, Ar) 5.86 (d, 1 H, JH–H = 5.0 Hz), 5.60 (d, 1 H, JH-H = 5.0 Hz) ppm. 13C NMR (100 MHz, CDCl3): δ
=162.1 (C=O), 151.4, 136.4, 135.2, 134.3, 130.1, 129.7, 129.5, 128.9, 127.9, 127.7, 127.4, 125.2, 124.2, 117.4,
116.7, 81.5, 57.9 ppm.
4.3.7. Cis 3-(2,4-dichlorophenoxy)-1,4-di-p-tolylazetidin-2-one (6g)
White solid; yield: 372mg (90%). mp: 193-195 °C. IR (KBr): 1758 (C=O) cm–1. Calcd for C23H19Cl2NO2
(412.31): C, 67.00; H, 4.64; N, 3.40. Found: C, 66.6; H, 4.3; N, 3.2. 1H NMR (400 MHz, CDCl3): δ = 7.02-7.31 (m,
11 H, Ar) 5.49 (d, 1 H, 3JH–H = 5.0 Hz), 5.39 (d, 1 H, 3JH-H = 5.0 Hz), 2.34 (3 H, s, CH3), 2.30 (3 H, s, CH3) ppm.
13C NMR (100 MHz, CDCl3): δ =162.1 (C=O), 151.6, 138.8, 134.5, 134.4, 130.0, 129.7, 129.2, 129.1, 128.0, 127.5,
127.4, 124.4, 117.6, 116.9, 81.7, 61.4, 21.3, 21.0 ppm.
9

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4.3.8. Cis 4-(4-chlorophenyl)-3-(2,4-dichlorophenoxy)-1-phenylazetidin-2-one (6h)
White solid; yield: 314 mg (75%). mp: 195-196 °C. IR (KBr): 1746 (C=O) cm–1. Calcd for C21H14Cl3NO2
(418.70): C, 60.24; H, 3.37; N, 3.35. Found: C, 60.1; H, 3.3; N, 3.0. 1H NMR (400 MHz, CDCl3): δ = 6.99-7.29 (m,
12 H, Ar) 5.43(d, 1 H, 3JH–H = 5.2 Hz), 5.33 (d, 1 H, 3JH-H = 5.2 Hz) ppm. 13C NMR (100 MHz, CDCl3): δ =162.2
(C=O), 151.4, 136.5, 134.9, 130.8, 130.1, 129.4, 129.3, 128.9, 127.9, 127.5, 125.0, 124.3, 117.5, 116.8, 81.7, 60.8
ppm.
4.3.9. Cis 3-(2,4-dichlorophenoxy)-1-phenyl-4-(p-tolyl)azetidin-2-one (6i)
White solid; yield: 360 mg (90%). mp: 178-180 °C. IR (KBr): 1744 (C=O) cm–1. Calcd for C22H17Cl2NO2
(398.28): C, 66.34; H, 4.30; N, 3.52. Found: C, 66.4; H, 4.1; N, 3.6. 1H NMR (400 MHz, CDCl3): δ = 7.02-7.40 (m,
3
3
12 H, Ar) 5.51 (d, 1 H, JH–H = 5.0 Hz), 5.41 (d, 1 H, JH-H = 5.0 Hz), 2.35 (3 H, s, CH3) ppm. 13C NMR (100
MHz, CDCl3): δ =162.4 (C=O), 151.6, 138.8, 136.8, 130.0, 129.3, 129.2, 129.0, 128.0, 127.6, 127.4, 124.8, 124.4,
117.6, 116.9, 81.7, 61.4, 21.3 ppm.
4.3.10. Cis 3-(2,4-dichlorophenoxy)-4-(4-methoxyphenyl)-1-(p-tolyl)azetidin-2-one (6j)
White solid; yield: 321 mg (75%). mp: 166-168 °C. IR (KBr): 1757 (C=O) cm–1. Calcd for C23H19Cl2NO3
(428.31): C, 64.50; H, 4.47; N, 3.27. Found: C, 64.7; H, 4.2; N, 2.8. 1H NMR (400 MHz, CDCl3): δ = 6.87-7.35 (m,
11 H, Ar) 5.49 (d, 1 H, 3JH–H = 5.0 Hz), 5.37 (d, 1 H, 3JH-H = 5.0 Hz) 2.30 (3 H, s, CH3), 3.80 (3 H, s, OCH3) ppm.
13C NMR (100 MHz, CDCl3): δ =162.1 (C=O), 160.0, 151.6, 134.5, 134.4, 130.0, 129.7, 129.4, 127.4, 124.3, 124.0,
117.6, 116.6, 114.0, 81.6, 61.1, 55.3, 21.0 ppm.
4.3.11. Trans 3-(2,4-dichlorophenoxy)-1-(naphthalen-2-yl)-4-(p-tolyl)azetidin-2-one (6k)
White solid; yield: 381 mg (85%). mp: 113-115 °C. IR (KBr): 1763 (C=O) cm–1. Calcd for C26H19Cl2NO2
(448.34): C, 69.65; H, 4.27; N, 3.12. Found: C, 69.9; H, 4.2; N, 2.8. 1H NMR (400 MHz, CDCl3): δ = 7.11-8.26 (m,
3
3
14 H, Ar) 5.49 (d, 1 H, JH–H = 6.0 Hz), 5.26 (d, 1 H, JH-H = 6.0 Hz), 2.30 (3 H, s, CH3). ppm. 13C NMR (100
MHz, CDCl3): δ =163.4 (C=O), 151.9, 139.3, 134.4, 132.2, 131.6, 130.2, 129.9, 128.4, 128.0, 127.9, 127.8, 127.7,
126.7, 126.6, 126.6, 125.2, 124.4, 123.8, 119.7, 117.3, 87.3, 64.8, 21.2 ppm.
4.3.12. Trans 4-(4-chlorophenyl)-3-(2,4-dichlorophenoxy)-1-(naphthalen-2-yl)azetidin-2-one (6l)
White solid; yield: 364 mg (77%). mp: 140-142 °C. IR (KBr): 1765 (C=O) cm–1. Calcd for C25H16Cl3NO2
(468.76): C, 64.06; H, 3.44; N, 2.99. Found: C, 63.8; H, 3.5; N, 2.5. 1H NMR (400 MHz, CDCl3): δ = 7.15-8.24 (m,
3
3
14 H, Ar) 5.52 (d, 1 H, JH–H = 5.0 Hz), 5.23 (d, 1 H, JH-H = 5.0 Hz). ppm. 13C NMR (100 MHz, CDCl3): δ
=163.1 (C=O), 151.7, 135.2, 134.5, 133.8, 131.4, 130.3, 129.5, 128.5, 128.3, 128.0, 127.9, 127.6, 126.9, 126.7,
125.2, 124.5, 123.6, 119.5, 117.6, 87.2, 64.1 ppm.
4.3.13. Trans 3-(2,4-dichlorophenoxy)-4-(4-methoxyphenyl)-1-(naphthalen-2-yl)azetidin-2-one (6m)
White solid; yield: 417 mg (90%). mp: 166-168 °C. IR (KBr): 1750 (C=O) cm–1. Calcd for
C26H19Cl2NO3(464.34): C, 67.25; H, 4.12; N, 3.02. Found: C, 67.2; H, 3.8; N, 2.6. 1H NMR (400 MHz, CDCl3): δ =
6.84-8.26 (m, 14 H, Ar) 5.47 (d, 1 H, 3JH–H = 5.0 Hz), 5.27 (d, 1 H, 3JH-H = 5.0 Hz), 3.74 (3 H, s, OCH3). ppm. 13
C
10

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NMR (100 MHz, CDCl3): δ =163.4 (C=O), 160.3, 151.9, 134.4, 131.5, 130.3, 128.4, 128.0, 127.9, 127.8, 127.0,
126.7, 126.6, 125.2, 124.3, 123.8, 119.8, 117.2, 114.6, 87.3, 64.7, 55.3 ppm.
4.3.14. Cis 4-(2-chlorophenyl)-3-(2,4-dichlorophenoxy)-1-(p-tolyl)azetidin-2-one (6n)
White solid; yield: 330 mg (76%). mp: 160-162 °C. IR (KBr): 1757 (C=O) cm–1. Calcd for C22H16Cl3NO2
(432.73): C, 61.06; H, 3.73; N, 3.24. Found: C, 61.4; H, 3.4; N, 3.1. 1H NMR (400 MHz, CDCl3): δ = 7.14-7.44 (m,
3
3
11 H, Ar) 5.89 (d, 1 H, JH–H = 5.2 Hz), 5.60 (d, 1 H, JH-H = 5.2 Hz), 2.33 (3 H, s, CH3). ppm. 13C NMR (100
MHz, CDCl3): δ =162.0 (C=O), 151.6, 134.8, 134.2, 133.7, 130.2, 129.9, 129.8, 129.6, 128.7, 127.5, 127.0, 124.2,
117.4, 116.7, 81.5, 58.2, 21.0 ppm.
4.3.15. Cis 4-(4-chlorophenyl)-3-(2,4-dichlorophenoxy)-1-(p-tolyl)azetidin-2-one (6p)
White solid; yield: 338 mg (78%). mp: 184-185 °C. IR (KBr): 1759 (C=O) cm–1. Calcd for C22H16Cl3NO2
(432.73): C, 61.06; H, 3.73; N, 3.24. Found: C, 60.8; H, 3.5; N, 2.8. 1H NMR (400 MHz, CDCl3): δ = 7.07-7.38 (m,
11 H, Ar) 5.51 (d, 1 H, 3JH–H = 5.2 Hz), 5.40 (d, 1 H, 3JH-H = 5.2 Hz), 2.31 (3 H, s, CH3). ppm. 13C NMR (100
MHz, CDCl3): δ =161.9 (C=O), 151.4, 134.9, 134.8, 134.1, 131.0, 130.1, 129.8, 129.5, 128.8, 127.7, 127.5, 124.2,
117.5, 116.7, 81.6, 60.7, 21.0 ppm.
4.4. Reaction of 2,4-Dichlorophenoxyacetic acid 2 with cyclopentadiene
2,4-Dichlorophenoxyacetic acid 2 (0.44 g, 2.0 mmol), benzenesulfonyl chloride 13 (0.28 ml, 2.2 mmol) and
triethylamine 4 (0.70 mL, 5.0 mmol) in anhydrous CH2Cl2 (10 ml) were stirred in anhydrous CH2Cl2 (25 mL) at
room temperature under nitrogen atmosphere for 2h. Then a solution of cyclopentadiene 12 (2.5 mmol) in anhydrous
CH2Cl2 (10 ml) was added and stirring was continued for an additional 17h. The products were purified by column
chromatography (silica gel, n-hexane/ethyl acetate, 40:1).
4.4.1. Endo-7-(2,4-dichlorophenoxy)bicyclo[3.2.0]hept-2-en-6-one (14)
White solid; yield:187 mg,( 69%); mp: 88-89 °C. Calcd for C13H10Cl2O2 (269.12): C, 58.02; H, 3.75. Found: C,
58.00; H, 3.7. IR (νmax/ cm-1) 1775 (C=O). 1H NMR δH (400 MHz; CDCl3) 7.02-7.37 (3 H, m, Ar), 5.99-6.01 (m,
1 H, CH), 5.82-5.8 (m, 1 H, CH of alkene), 5.36 (d of d, 1 H, CH of alkene), 4.02-4.07 (m, 1 H, CH), 3.61-3.71 (m,
1 H, CH3), 2.82-2.87 (m, 1 H, CH), 2.57-2.64 (m, 1 H, CH). 13C NMR δC (100 MHz, CDCl3) 207.9, 152.0, 135.9,
130.1, 127.7, 127.3, 127.2, 124.1, 116.5, 88.5, 54.8, 45.9, 35.4 ppm.
4.5. Reaction of 2-(2,4-dichlorophenoxy)ethenone ketene (7) with Aldehyde (9)
A solution of the aldehyde 9 (0.1 ml, 1 mmol) in anhydrous dichloromethane (10 ml) was added to a suspension
of 2,4-dichlorophenoxyacetic acid 2 (0.44 g, 2.0 mmol), 2-chloro-N-methylpyridinium iodide 3 (0.56 g, 2.2 mmol)
and triethylamine 4 (0.50 mL, 3.6 mmol) in anhydrous dichloromethane (25 ml) and refluxed under a nitrogen
atmosphere for 17 h. The products were purified by column chromatography (silica gel, n-hexane/ethyl acetate,
40:1).
4.5.1. 3-(2,4-dichlorophenoxy)-4-[(2,4 dichlorophenoxy)methyl]-2H-oxet-2-one (10)
11

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White solid; yield:151 mg,( 37%); mp: 110-111 °C. IR (νmax/ cm-1) 1769 (C=O). Calcd for C16H8Cl4O4 (406.04):
1
C, 47.33; H, 1.99. Found: C, 46.90; H, 2.2. H NMR δH (400 MHz; CDCl3) 6.94-7.49 (6 H, m, Ar), 5.02 (s, 2 H,
CH2). 13C NMR δC (100 MHz, CDCl3) 165.7, 152.2, 144.9, 132.6, 130.5, 130.3, 128.2, 127.7, 127.6, 124.5, 124.33,
115.2, 66.2 ppm.
4.6. Material and Methods cytotoxcity assay
MCF-7 (human breast adenocarcinoma) cell line was purchased from Na¬tional Cell Bank of Iran NCBI. The
cells were grown in Dulbecco’s Modified Eagle Medium DMEM (Biosera, UK) supplemented with 10% heat-
inactivated fetal calf serum and 100 ꢀg/ml streptomycin and 100 U/ml penicillin in humidified 5% CO2 atmosphere
at 37 °C.
4.6.1. Cytotoxicity assay
The cytotoxic activities of all synthesized compounds were assessed in monolayer cultures by using MTT assay.
In brief, cells were seeded into 96 well plates (SPL Lifesciences Inc., South Korea) at 5000 cells/well in 100 ꢀl of
medium and allowed to attach overnight. For evaluating the toxic properties of each compound, the specific
concentrations (2 and 4 µg/ml) of synthesized compounds were added per well and the plates were incubated for a
further 24h. Five wells containing tumor cells cultured in complete medium were used as controls for cell viability.
After incubation, 2.5 mg/ml solution of MTT (Sigma-Aldrich, Germany) was added to each well and the plates were
incubated for another 1 h. The culture medium was then replaced with 100 ꢀl of DMSO and the absorbance was
measured by using a microplate reader at 490 nm. Results are expressed relative to the control value.
Acknowledgements
The authors express appreciation to the Shahid Bahonar University of Kerman Faculty Research committee funds
for its support of this investigation.
Supporting Information Available
The NMR spectra of the compounds are available free of charge via the Internet at:
References
1. Cagir, A.; Jones, S. H.; Gao, R.; Eisenhauer, B.; Hecht, S. H. J. Am. Chem. Soc. 2003, 125, 13628.
2. Pahari, P.; Rohr, J. J. Org. Chem. 2009, 74, 2750.
3. Borman, S. Chem. Eng. News Archive. 2005, 83, 7.
4. Fang, B. J. Med. Chem. 2014, 57(19), 7859.
5. Islami, M. R.; Allen, A. D.; Vukovic, S.; Tidwell, T. T. Org. Lett. 2011, 13, 494.
6. Mortazavi, Z. F. A.; Islami, M. R.; Khaleghi, M. Org. Lett. 2015, 17, 3034.
7. Zigheimat, F.; Islami. M. R.; Nourmohammadian, F. Synlett 2014, 25, 229.
8. Hosseinkhani, B.; Islami, M. R.; Hosseinkhani, S. Synlett 2015, 26, 2277.
9. Babaei, E.; Islami, M. R.; Kalantari,M. Synlett 2013, 24, 1937.
10. Koteva, K.; King, A. M.; Capretta, A.; Wright, G. D. Angew. Chem. Int. Ed. 2016, 128, 2250.
11. Song, W.; Woo, H. J.; Kim, J. S.; Lee, K. M. Int. J. Antimicrob. AgentS. 2003, 21, 8.
12. Zhu, C.; Yang, Q.; Liu, L.; Wang, S. Angew. Chem. Int. Ed. 2011, 50, 9607.
13. Lodise, T. P.; Lomaestro, B. M.; Drusano, G. L. Pharmacotherapy. 2006, 26, 1320.
14. Tidwell, T. T. Angew. Chem. Int. Ed. 2008, 47, 1016.
15. Kosowska-Shick, K.; McGhee, P. L.; Appelbaum, P. C. Antimicrob. Agents Chemother. 2010, 54, 1670.
16. Karageorgou, E. G.; Samanidou, V. F.; Papadoyannis, I. N. J. Sep. Sci. 2012, 35, 2599.
17. Palomo, C.; Aizpurua, J. M.; Ganboa, I.; Oiarbide, M. Pure Appl. Chem. 2000, 72, 1763.
18. Palomo, C.; Aizpurua, J. M.; Benito, A.; Miranda, J. I.; Fratila, R. M.; Matute, C.; Domercq, M.; Gago, F.; Martin-
Santamaria, S.; Linden, A. J. Am. Chem. Soc. 2003, 125, 16243.
19. Banik, B. K.; Banik, L.; Becker, F. F. Eur. J. Med. Chem. 2010, 45, 846.
12

ACCEPTED MANUSCRIPT
20. Palomo, C.; Aizpurua, J. M.; Balentová, E.; Jimenez, A.; Oyarbide, J.; Fratila, R. M.; Miranda, J. I. Org. Lett. 2007, 9, 101.
21. Palomo, C.; Aizpurua, J. M.; Ganboa, I.; Benito, A.; Cuerdo, L.; Fratila, R. M.; Jimenez, A.; Loinaz, I.; Miranda, J. I.;
Pytlewska, K. R.; Micle, A. Org. Lett. 2004, 6, 4443.
22. Palomo, C.; Aizpurua, J. M.; Ganboa, I.; Oiarbide, M. Curr. Med. Chem. 2004, 11, 1837.
23. Cariou, C. C.; Clarkson, G. J.; Shipman, M. Org. Lett. 2008, 73, 9762.
24. Li, W.; Liu, C.; Zhang, H.; Ye, K.; Zhang, G.; Zhang, W.; Duan, Z.; You, S.; Lei, A. Angew. Chem. Int. Ed. 2014, 126,
2475.
25. Brady, W. T.; Saidi, K. J. Org. Chem. 1980, 45, 727.
26. Montaigne, R.; Ghosez, L. Angew. Chem. Int. Ed. Engl. 1968, 7, 221.
27. Allen, A. D.; Tidwell, T. T. Eur. J. Org. Chem. 2012, 2012, 1081.
28. Jiao, L.; Liang, Y.; Xu, J. J. Am. Chem. Soc. 2006, 128, 6060.
29. Huang, Z.; Wang, C.; Tokunaga, E.; Sumii, Y.; Shibata, N. Org. Lett. 2015, 17, 5610.
30. Banik, B. K.; Banik, I.; Becker, F. F. Bioorg. Med. Chem. 2005, 13, 3611.
31. Jiao, L.; Liang, Y.; Xu, J. J. Am. Chem. Soc. 2006, 128, 6060.
32. Tamai, Y.; Yoshiwara, H.; Someya, M.; Fukumoto, J.; Miyano, S. Chem. Commun. 1994, 19, 2281.
33. Hattori, T.; Suzuki, Y.; Uesugi, O.; Oi, S.; Miyano, S. Chem. Commun. 2000, 1, 73.
34. Matsunaga, H.; Ikeda, K.; Iwamoto, K. I.; Suzuki, Y.; Sato, M. Tetrahedron Lett. 2009, 50, 2334.
35. Brady, W. T.; Saidi, K. J. Org. Chem. 1980, 45, 727.
36. Montaigne, R.; Ghosez, L. Angew. Chem., Int. Ed. Engl. 1968, 7,221.
37. Yong, K.; Salim, M.; Capretta, A. J. Org. Chem. 1998, 63, 9828.
38. Marqués‐López, E.; Christmann, M. Angew. Chem. Int. Ed. 2012, 51, 8696.
39. Ulukaya, E.; Ozdikicioglu, F.; Oral, A. Y.; Demirci, M. Toxicol In Vitro. 2008, 22, 232.
40. Hayon, T.; Dvilansky, A.; Shpilberg, O.; Nathan, I. Leuk. Lymphoma. 2003, 44, 1957.
41. Gerlier, D.; Thomasset, N. J. Immunol. Methods. 1986, 94, 57.
42. Tuschl, H.; Schwab, C. Food. Chem. Toxicol. 2003, 41, 385.
43. Bukowska, B. Pol. J. Environ. Stud. 2006, 15, 365.
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