Three-component synthesis of chromeno β-lactam hybrids for inflammation and cancer screening

Article abstract of DOI:10.1016/j.ejmech.2019.06.036

Highly diastereoselective synthesis of chromeno β-lactam hybrids was achieved by an efficient one-pot three-component reaction. With this procedure, the desired β-lactam products were obtained in good yields and with exclusive cis stereoselection, by combining a variety of benzaldehydes, malononitrile, and either 5,5-dimethylcyclohexane-1,3-dione or 4-hydroxycoumarin in the presence of 1,4-diazabicyclo [2.2.2]octane under reflux conditions. These adducts were structurally characterized on the basis of IR, 1D and 2D NMR spectra, X-ray analysis, H–H COSY and H–C HSQC two-dimensional NMR experiments, and elemental analysis. Each of the synthesized compounds was screened for anti-inflammatory and anticancer activities. β-Lactams 5b and 8b showed a 53.4 and 19.8 anti-inflammatory ratio, respectively, and 5b appeared more active than the well-known dexamethasone corticosteroid used for the treatment of rheumatoid and skin inflammation. β-Lactams 5a, 5b, 5e, 5f, 5g, 8c, 8j and 8p also showed good antitumor activity against the SW1116 (colon cancer) cell line without notable cytotoxicity towards the HepG2 control cell line.

Full text of DOI:10.1016/j.ejmech.2019.06.036

Accepted Manuscript
Three-component synthesis of chromeno β-lactam hybrids for inflammation and
cancer screening
Nassim Borazjani, Saghi Sepehri, Maryam Behzadi, Aliasghar Jarrahpour, Javad
Ameri Rad, Maryam Sasanipour, Milad Mohkam, Younes Ghasemi, Amin Reza
Akbarizadeh, Carole Digiorgio, Jean Michel Brunel, Mohammad Mehdi Ghanbari,
Gyula Batta, Edward Turos
PII:
S0223-5234(19)30563-X
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.036
EJMECH 11439
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 April 2019
Revised Date: 12 June 2019
Accepted Date: 13 June 2019
Please cite this article as: N. Borazjani, S. Sepehri, M. Behzadi, A. Jarrahpour, J.A. Rad, M.
Sasanipour, M. Mohkam, Y. Ghasemi, A.R. Akbarizadeh, C. Digiorgio, J.M. Brunel, M.M. Ghanbari,
G. Batta, E. Turos, Three-component synthesis of chromeno β-lactam hybrids for inflammation and
cancer screening, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.036.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Three-Component Synthesis of Chromeno β-Lactam Hybrids for
Inflammation and Cancer Screening
Nassim Borazjania, Saghi Sepehrib, Maryam Behzadic, Aliasghar Jarrahpoura*, Javad Ameri
Rada, Maryam Sasanipoura, Milad Mohkamd, Younes Ghasemic,e, Amin Reza Akbarizadehf,
Carole Digiorgiog, Jean Michel Brunelg, Mohammad Mehdi Ghanbarih, Gyula Battai, and
Edward Turosj
aDepartment of Chemistry, College of Sciences, Shiraz University, Shiraz, 71946-84795, Iran.
bDepartment of Medicinal Chemistry, School of Pharmacy, Ardabil University of Medical Sciences, Ardabil, Iran
cPharmaceutical Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
dBiotechnology Research Center, Shiraz University of Medical Sciences, Shiraz, Iran
eDepartment of Biotechnology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz, Iran
fDepartment of Pharmacology and Toxicology, School of Pharmacy, Shiraz University of Medical Sciences, Shiraz,
Iran
gCentre de Recherche en Cancérologie de Marseille (CRCM), CNRS, UMR7258 ; Institut Paoli Calmettes; Ai x-
Marseille Université, UM 105; Inserm, U1068, Faculté de Pharmacie, Bd Jean Moulin, F-13385, Marseille, France.
hDepartment of Chemistry,Sarvestan Branch, Islamic Azad University, Sarvestan, Iran.
iDepartment of Chemistry, University of Debrecen, Debrecen, Egyetem tér 1, 4032, Debrecen, Hungary.
jCenter for Molecular Diversity in Drug Design, Discovery, and Delivery, Department of Chemistry, CHE 205, 4202
East Fowler Avenue, University of South Florida, Tampa, FL 33620, USA.
Abstract: Highly diastereoselective synthesis of chromeno β-lactam hybrids was achieved by
an efficient one-pot three-component reaction. With this procedure, the desired β-lactam
products were obtained in good yields and with exclusive cis stereoselection, by combining a
variety of benzaldehydes, malononitrile, and either 5,5-dimethylcyclohexane-1,3-dione or 4-
hydroxycoumarin in the presence of 1,4-diazabicyclo[2.2.2]octane under reflux conditions.
These adducts were structurally characterized on the basis of IR, 1D and 2D NMR spectra, X-ray
analysis, H-H COSY and H-C HSQC two-dimensional NMR experiments, and elemental
analysis. Each of the synthesized compounds was screened for anti-inflammatory and anticancer
activities. β-Lactams 5b and 8b showed a 53.4 and 19.8 anti-inflammatory ratio, respectively,
and 5b appeared more active than the well-known dexamethasone corticosteroid used for the
treatment of rheumatoid and skin inflammation. β-Lactams 5a, 5b, 5e, 5f, 5g, 8c, 8j and 8p also
showed good antitumor activity against the SW1116 (colon cancer) cell line without notable
cytotoxicity towards the HepG2 control cell line.
Keywords: Anticancer, Anti-inflammatory, β-Lactams, Colon cancer, Cytotoxicity, DABCO.
1

ACCEPTED MANUSCRIPT
1. Introduction
In the recent past, our laboratories have pursued research exploring the synthetic combination
of several prominent classes of bioactive compounds to create new scaffolds with
pharmacological potential. Pharmacophore hybridization has emerged as a paradigm for
pharmaceutical chemists. The main motivations for using this strategy are the significant
improvement in the therapeutic potential, strength, mode of action and pharmacokinetics [1-4].
Multicomponent reactions (MCRs) have recently been exploited as a means to synthesize a
variety of chemically-hybridized compounds, including those of interest for biological
evaluation. Among the synthetic methods utilized for this purpose, single-pot multi-component
reactions provide the most efficient routes to the desired products, avoiding the need to isolate
and purify intermediate products while improving atom economy and product yields [5].
Medicinal chemists have further exploited multi-component synthesis in order to construct
compounds of pharmaceutical interest, by combining two or more structural subunits that have
their own biological activities. In some cases, these biological activities are complementary to
one another or aid in the enrichment of some desired properties, such as antimicrobial activity
(bifunctional or even trifunctional prodrugs), or completely orthogonal (such as anticancer with
antimicrobial). Recently our laboratories have explored applications of the MCR approach to
uncover new chemical structures with intriguing biological properties. In this study, we explore
the unique combination of two different classes of bioactive structures, namely fused chromenes
and β-lactams (Figure 1).
Fig. 1. Generalized structure of a fused chromene-β-lactam hybrid.
Tetrahydro-4H-chromenes and dihydropyrano[c]-chromenes have diverse pharmacological
potential as anti-allergenic, anti-proliferative, antibacterial, antiviral, antifungal, antioxidant,
antitumor, and anticancer agents [6-9]. Examples include benzopyrans A and B, which possess
anticancer activity [10], and compound C, which has antibacterial activity [11] (Figure 2).
2

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Fig. 2. Structures of biologically-active fused chromenes.
The discovery and clinical development of β-lactam antibiotics remains one of the major
advances in modern medicine [12]. The commonly used bicyclic β-lactam antibiotics such as the
penicillins and cephalosporins prevent bacterial transpeptidases from crosslinking the
polysaccharide cell wall [13]. More recently, functionalized monocyclic β-lactams have been
developed having a host of other pharmaceutical capabilities, such as anti-HIV [14], antifungal
[15], anticancer [16,17] and antimalarial [18] (Figure 3), anti-inflammatory, or analgesic
activities [19]. Thus, the β-lactam framework presents a rich source of diverse biological
properties.
Fig. 3. Structures of some biologically active monocyclic β-lactam derivatives.
In this current study, we set our focus on structurally unique β-lactams 8a-p bearing
tetrahydro-4H-chromene and dihydropyrano[c]-chromene pharmacophores, through the
development of a single-pot, multi-component coupling strategy illustrated in Scheme 1.
3

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Scheme 1. Multi-component synthesis of aldehydic β-lactams 5a-j and chromeno-β-lactam hybrids 8a-p.
2. Results and discussion
2.1. Chemistry
In this study, β-lactams 5a-j were synthesized by a diastereoselective ketene-imine
cycloaddition reaction (Scheme 1) [18,20]. The reaction of 4-hydroxybenzaldehyde (1) with
bromoacetic acid (2) in DMF at 70oC in the presence of solid K2CO3 afforded 2-(4-
formylphenoxy)acetic acid (3). Treatment of 3 with Schiff bases 4a-j in the presence of
triethylamine and p-toluenesulfonyl chloride produced the cis-β-lactams 5a-j in isolated yields
varying from 68-80% after column chromatography (Table 1).
Table 1. Isolated yields of aldehydic
β-lactam hybrids 5a-j.
Cpd
5a
R
OMe
Ar
4-ClPh
Isolated yield (%)
80
Me
Cl
4-ClPh
75
77
70
80
5b
5c
5d
5e
3-NO2Ph
3-NO2Ph
4-ClPh
OMe
OEt
OEt
NMe2
OEt
4-NO2Ph
4-NO2Ph
80
88
74
80
80
5f
5g
5h
5i
4-NMe2Ph
anthracene
anthracene
OEt
OMe
5j
4

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1
13
Structures of the cycloadducts 5a-j were established on the basis of their IR, H NMR, C
NMR spectral data and elemental analysis (see supporting information). The IR spectra of 5b
(R=Me, Ar=4-ClPh) showed absorption band at υ 1759 cm-1 for the β-lactam carbonyl group and
absorption band at υ 1689 cm-1 for the aldehyde carbonyl group. Compound 5b showed doublets
at δ 5.79 ppm and δ 6.03 ppm for the H-2 and H-3 β-lactam ring protons. The aldehydic
13
hydrogen resonated as a singlet at δ 9.81 ppm. The C NMR spectral data for compound 5b
exhibited signals at δ 60.9 and δ 80.4 ppm for the C-2 and C-3 β-lactam ring protons,
respectively, the aromatic carbons at appropriate shifts, the β-lactam carbonyl carbon at δ 161.4
ppm and the aldehyde carbonyl carbon at δ 190.5 ppm. The cis stereochemistry was assigned
from the vicinal coupling constants of the two β-lactam ring protons, H-2 and H-3 (J2,3 ≥4.0 Hz)
[18]. Single crystal X-ray analysis of β-lactam 5b confirmed the cis stereochemistry (Figure 4).
Crystallographic data, details of the data collection and structure refinement can be found in the
supplementary material [21].
Fig. 4. ORTEP diagram of β-lactam 5b.
From aldehydic β-lactams 5, we further developed an expedient multi-component approach
for constructing chromeno β-lactam hybrids 8 (Scheme 2).
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Scheme 2. Multi-component synthesis of chromeno β-lactam hybrids 8a-p.
To optimize the reaction conditions, β-lactam 5a was stirred in ethanol with equimolar
amounts of malononitrile (6) and 5,5-dimethylcyclohexane-1,3-dione (7a) in the presence of
different base catalysts (Table 2). A higher yield and shorter reaction time were observed when
the reaction was carried out in the presence of a molar equivalent of 1,4-
diazabicyclo[2.2.2]octane (DABCO) under reflux conditions (entry 6) [22,23].
Table 2. E
ff
ects of reagent and solvent on the reaction of
β-lactam 5a, malononitrile (6) and 5,5-
dimethylcyclohexane-1,3-dione (7a) under di
ff
erent reaction conditions.
Entry
Reagent
Reagent (mmol)
Solvent
Temp (οC)
Time (h)
Yield
1
2
——
——
1.0
1.0
0.5
1.5
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
EtOH
EtOH
EtOH
EtOH
EtOH
EtOH
MeCN
H2O
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
Reflux
r.t.
48
5
——
70
Et3N
3
K2CO3
8
62
4
DABCO
DABCO
DABCO
DABCO
DABCO
DABCO
DABCO
DABCO
DABCO
DABCO
DABCO
1.5
1.5
1.5
10
10
10
10
10
8
75
5
95
6
95
7
20
8
50
9
MeOH
CHCl3
EtOAc
EtOH
——
55
10
11
12
13
14
Trace
24
60
80
10
10
33
——
100
46
Subsequently, these optimized reaction conditions were used for the condensations of β-
lactams 5a-j (Table 3).
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Table 3. Isolated yields of chromeno β-lactam hybrids 8a-p under the optimized reaction conditions.
Cpd
R
Ar
Time (h)
Yield
(%)
Cpd
R
Ar
Time (h)
Yield
(%)
OMe
Me
4-ClPh
4-ClPh
1.5
2.5
2.2
3.5
2.5
1.6
3.2
3.0
95
90
92
80
94
95
85
90
OEt
OMe
OEt
Anthracene
Anthracene
4-ClPh
1.5
1.5
3.0
2.6
4.0
3.8
1.8
2.0
92
90
85
92
82
90
95
90
8a
8b
8c
8d
8e
8f
8i
8j
Cl
3-NO2Ph
3-NO2Ph
4-ClPh
8k
8l
OMe
OEt
OEt
NMe2
OEt
OMe
NMe2
OEt
4-ClPh
8m
8n
8o
8p
4-NO2Ph
4-NMe2Ph
Anthracene
Anthracene
4-NO2Ph
4-NO2Ph
4-NMe2Ph
OEt
8g
8h
OMe
1
Structures of the chromeno β-lactam hybrids 8a-p were established from their IR, H NMR,
13C NMR spectral data, H-H COSY and H-C HSQC two-dimensional NMR studies, and
elemental analysis (see supporting information). The IR spectra showed the anticipated NH2
stretching vibration, the CN stretching, and the β-lactam carbonyl signals in regions similar to
those for the precursor β-lactams 5a-j. We also noted the absence of the aldehyde carbonyl group
at 1689 cm-1, and the appearance of a sharp band at 1666 cm-1 for the vinylogous carbonyl
(ketone or lactone). The 1H NMR spectra showed doublets with J = 5.0 Hz for the two β-lactam
ring protons, H-2 and H-3, and NH2 protons in the aromatic region. For compound 8b as a
representative example, these NH2 protons deuterium-exchanged with D2O and the integration of
4Hs in the region of δ 6.88-6.95 (m, ArH, NH2) decreased to 2H, producing a sharp doublet at δ
13
6.94 (J = 10.0 Hz). The C NMR spectral data for each of the compounds exhibited carbonyl,
aliphatic and aromatic carbons at appropriate chemical shifts. H-H COSY and H-C HSQC NMR
experiments also corroborated the stereochemical assignments, in that the correlations observed
in the two-dimensional spectra for lactams 8a, 8b, 8c, 8f, 8g, 8i, 8j, 8l, 8n, 8o, and 8p all were in
alignment with the structure having cis-disubstitution on the β-lactam rings.
2.2. Biological activities
2.2.1. Anti-inflammatory activity assay
Inflammation contributes to the degenerative processes of host of human ailments and
diseases, such as cancer, arthritis, atherosclerosis, autoimmune diseases and aging of skin and
other tissues and organs. Treatment with anti-inflammatory compounds may reduce the severity
of inflammation. Non-steroidal anti-inflammatory drugs (NSAIDs) are the most popular
pharmaceutical agents for the treatment of chronic inflammatory diseases, pain and fever that
exert their therapeutic effect via blockade of cyclooxygenase isoenzymes (arachidonic acid
pathway) to prevent the production of prostaglandins and other inflammatory mediators. Nitric
oxide (NO) plays significant roles in the pathophysiology of inflammatory illnesses. It gives an
anti-inflammatory effect under normal physiological conditions. NO is synthesized and released
7

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into the endothelial cells by the help of nitric oxide synthases that convert arginine into citrulline
producing NO in the process. Also, NO is considered as a pro-inflammatory mediator that
induces inflammation because of over production in abnormal situations. Therefore, NO
inhibitors represent important therapeutic advance in the management of inflammatory diseases
[24]. In order to identify new anti-inflammatory candidates, the RAW 264.7 murine macrophage
assay was used to examine anti-inflammatory behavior of the new β-lactam hybrids we
synthesized. This assay monitors the inflammatory cascade leading to an overproduction of NO
in the endothelial lining of blood vessels. In the present study, it was performed to evaluate the
capacity of the compounds to inhibit the pro-inflammatory cascade leading to NO production in
mouse macrophages. Results are reported in Table 4. Compound 8b was the most active of all
the chromeno β-lactam hybrids tested, with a 19.8 anti-inflammatory ratio.
Table 4. Anti-inflammatory activity of chromeno
β
-lactam hybrids 8a-p.
IC50-NO release
Sample (µM)
IC50-cell viability
Sample (µM)
Anti-inflammatory
ratio
Sample
ND
ND
ND
19.8
2.3
1.1
2.7
1.4
4.1
3.8
1.6
2.4
ND
1.4
1.0
ND
1.3
1.2
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
6.24 + 1.28
53.41 + 5.13
100.02 + 6.21
73.36 + 7.94
89.42 + 6.81
60.08 + 5.24
38.10 + 4.31
101.65 + 23.24
56.41 + 5.64
ND
89.56 + 3.81
161.36 + 21.54
ND
168.23 + 11.41
164.57 + 8.84
5.02 + 1.34 µM
123.47 + 13.24
124.21 + 8.23
114.31 + 10.31
201.45 + 11.56
124.31 + 11.39
251.48 + 8.37
146.25 + 7.23
165.23 + 10.87
134.63 + 8.24
ND
124.31 + 8.33
163.24 + 11.42
ND
224.34 + 11.84
198.56 + 12.09
159.2 + 26.35 µM
Dexamethasone
31.9
ND: Not determined due to poor solubility
For comparison, we also checked anti-inflammatory activity for the aldehydic β-lactams 5a-j
(Table 5). As observed for lactam 8b, the p-tolyl group on N-1 of the β-lactam ring of 5b
enhances anti-inflammatory activity in this assay, more active than the corticosteroid drug,
dexamethasone.
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Table 5. Anti-inflammatory activity of derivatives 5a-j.
IC50-NO release
Sample (µM)
IC50-cell viability
Sample (µM )
Anti-inflammatory
ratio
Sample
92.42 + 6.81
2.31 +0.56
63.65 + 4.07
2.91 + 0.84
11.32 + 0.89
97.73 + 9.95
32.86 + 3.35
125.20 + 8.15
42.58 + 4.27
69.11 + 19.45
5.02 + 1.34 µM
131.31 + 9.39
123.78 + 7.49
132.48 + 6.37
4.15 + 1.66
1.4
53.4
2.1
1.4
4.9
1.9
3.6
1.9
2.6
1.0
31.9
5a
5b
5c
5d
5e
5f
5g
5h
56.28 + 6.31
189.65 + 8.18
116.84 + 14.58
241.78 + 11.42
112.56 + 10.36
68.32 + 6.87
5i
5j
159.2 + 26.35 µM
Dexamethasone
2.2.2. Anticancer activity and cytotoxicity assays
In vitro cytotoxicity and anti-cancer activity were also evaluated for each of the compounds
synthesized. Three of the chromeno β-lactam hybrid compounds (8c, 8j and 8p) showed elevated
anticancer activity against the SW1116 (colon cancer) cell lines, with IC50 values of 7.29, 8.83,
and 7.43 µM, respectively, in comparison to the anticancer agent methotrexate (IC50 of 2.49
µM). The remainder of the lactams 8 afford no anticancer activity at or below 200 µM. Since all
of these lactams carry a tetrahydro-4H-chromene or dihydropyrano[c]-chromene group on C-3 of
the β-lactam ring, the aryl substituents at N-1 and C-2 of the β-lactam ring are responsible for
these differences in activity. A tentative structure-activity relationship exists, in that the presence
of a p-nitrophenyl or p-chlorophenyl group on C-2 of the β-lactam ring and a p-methoxyphenyl,
p-tolyl, p-ethoxyphenyl or p-N,N-dimethylaminophenyl ring on N-1 of the β-lactam provide the
best anticancer bioactivity. Additionally, five of the aldehydic β-lactams (5a, 5b, 5e, 5f and 5g)
possess comparable IC50 values. IC50 values are 10.26, 10.00, 6.71, 8.19, and 6.93 µM,
respectively. Conversely, none of the lactams (5a-j or 8a-p) have cytotoxicity against HepG2
cells at or below 200 µM (Table 6).
9

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Table 6. Anticancer and cytotoxic activity assays of selected
β
-lactams 5 and 8, assessed by the MTT
SW1116 and HepG2
reduction assay against
cells.
Compound
SW1116
HepG2
IC50 (µM)
IC50 (µM)
5a
10.26
˃
˃
˃
˃
˃
˃
˃
˃
200
200
200
200
200
200
200
200
5b
10.00
6.71
8.19
6.93
7.29
8.83
7.43
2.49
5e
5f
5g
8c
8j
8p
Methotrexate
0.30
2.2.3. Molecular docking studies
2.2.3.1. Validation of molecular docking
The performance of a common computational docking technique can be tested by checking its
power to foresee the most significant binding mode of a cognate (co-crystallographic) ligand
[25]. This method was carried out by eliminating the structure of a cognate ligand and re-docking
it into its protein (self-docking). Root-mean-square deviation (RMSD) of the Cartesian
coordinates of the atoms of the ligand in the docked and crystallographic conformations is the
principle of the docking validation (RMSD < 2 Å). Validation of molecular docking showed
RMSD values were for PDB ID: 4NOS = 0.4 Å and for PDB ID: 3LN1 = 0.01 Å (Supplementary
Information).
2.2.3.2. Study of the binding mode
Molecular docking experiments suggest that chromeno β-lactams 8a-p may bind to human
inducible nitric oxide synthase protein via hydrogen bond and hydrophobic interactions with
specific active site amino acid residues. The integration of computational and experimental
techniques is an acutely attractive strategy for the design and optimization of drug candidates.
Molecular docking studies showed that synthesized compounds 5a-j and 8a-p could inhibit
human inducible nitric oxide synthase and cyclooxygenase-2 via hydrogen bond and
hydrophobic interactions (Tables 7 and 8). According to molecular docking results, all
compounds exhibited higher free binding energy values with human inducible nitric oxide
synthase enzyme compared with cyclooxygenase-2. Therefore, these derivatives showed anti-
inflammatory activity through inhibition of release of nitric oxide. Thus, we focused on
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investigation of molecular docking results and binding modes of synthesized compounds at the
human inducible nitric oxide synthase active site.
Table 7. Docking results of 5a-j and 8a-p derivatives docked into the human inducible nitric oxide
synthase target (PDB ID: 4NOS).
Code
∆Gbinding
Hydrogen
Hydrophobic
interaction
π-π
Cation-π
(Kcal/mol) bond
-
Gly371, Ala351,
Pro350, Asn370,
Val352, Glu377,
Asp382, Arg266,
Trp346, Tyr347,
Gln263, Ala262,
Asn354
5a
5b
-7.54
-
-
Gln263
Ala351, Asp382,
Arg381, Glu377,
Tyr373, Pro350,
Ala262, Val352,
Met355, Asn354
-10.30
-
-
Cys110
Cys110
Gly117, Leu116,
Ser118, Cys115,
Ile462, Gln478,
Trp461, Glu479
-9.30
-9.23
5c
Trp461
-
-
Gln478, Trp461,
Glu479, Ile462,
Gly117, Cys115,
Ser118, Leu116
5d
-
Arg388,
Tyr373
Arg266, Arg388,
Ala262, Val352,
Gln263, Pro350,
Gly371, Asn370,
Phe369, Glu377
-9.91
-8.35
5e
5f
-
Arg266
Tyr373,
Arg388
Asp385, Arg381,
Ala282, Arg266,
Asn354, Ala262,
Gln263, Met355,
Val352, Pro350,
Glu377, Trp372,
Gly371
Tyr491
-
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Cys110
His477, Gln478,
Glu479, Met480,
Phe476, Trp461,
Cys115, Ser118,
Ile462, Gly117
-9.65
-8.19
5g
5h
-
-
-
Tyr373,
Arg388
Asp382, Glu377,
Gln263, Val352,
Ala351, Phe369,
Pro350, Gly371,
Asn370
Hem
-
-
Trp461, Pro466,
Ile462, Val465,
Pro467, Asp385,
Arg381, Trp463
-8.64
-8.53
5i
5j
Hem
Hem
Arg381
Arg381
Trp461, Ile462,
Pro466, Val465,
Trp463, Pro467,
Arg381, Asp385
Arg381,
Asp382
Gln263, Arg266,
Arg388, Glu377,
Val352, Met120,
Trp463, Pro466,
Pro467, Ile462
-9.23
-10.01
-8.80
8a
8b
8c
Hem
-
-
-
Arg381,
Asp382
Gln263, Val352,
Glu377, Arg388,
Trp463, Met120,
Pro467, Ile462,
Pro466, Val465
-
Gly470,
Trp463
Met120, Arg381,
Ile462, Pro467,
Val465, Pro466,
Trp461, Phe476,
Ser469, Met468
-
Arg381
Val352, Glu377,
Gln263, Arg388,
Met120, Val465,
Trp463, Pro466,
Pro467, Ile462
-8.82
-9.18
8d
8e
-
-
-
-
-
Asp412, Cys384,
Leu392, Lys411,
12

ACCEPTED MANUSCRIPT
Val386, Val415,
Pro467, Val380,
Arg381, Asp385,
Val465, Pro466,
Ile462, Trp461
Arg388,
Tyr373
Asp385, Arg381,
Ala282, Arg266,
Asn354, Ala262,
Met355, Val352,
Gln263, Gly371,
Pro350, Trp372,
Glu377
-8.99
-9.71
8f
Arg266
-
-
Ala351, Glu377,
Val352, Gln263,
Arg266, Tyr491,
Met355, Asn354,
Ala262, Gln492,
Glu494, Lys497,
Thr121
8g
Hem
-
Gly371,
Tyr373,
Gln263
Arg388, Met120,
Thr121, Asn354,
Ala262, Met355,
Trp463, Val352,
Glu377, Ala351,
Phe369, Pro350,
Asn370
-9.13
-8.52
8h
8i
-
-
Lys411
Val465, Trp463,
Met120, Arg381,
Ile462, Pro467,
Met468, Val415,
Val380, Asp385,
Cys384
-
Arg381
-
Met120, Arg381,
Ile462, Pro466,
Trp463, Val465,
Pro467, Asp385,
Cys384, Val380,
Val415, Lys411,
Met468
-9.16
-9.61
8j
-
-
Arg381
Arg381,
Asp382
Ala351, Pro350,
Val352, Pro467,
Gln263, Arg388,
8k
-
13

ACCEPTED MANUSCRIPT
Trp463, Met120,
Ile462
Tyr373,
Arg388,
Arg381
Arg266, Glu377,
Met120, Ala282,
Asn354, Val352,
Gln263, Ala262,
Pro350, Gly371,
Asn370
-8.79
-8.53
8l
Hem
Hem
Arg266
Arg381
Arg381
Pro350, Ala351,
Arg388, Val352,
Met374, Gln263,
Arg266, Pro466,
Pro467, Val465,
Met120, Trp463,
Ile462
8m
-
Met355, Asn354,
Ala262, Tyr491,
Arg266, Val352,
Gln263, Met120,
Glu377, Met374,
Ile462, Trp461,
Val465, Trp463,
Pro467
Tyr491
, Hem
-8.49
-9.44
8n
8o
-
-
Gln263
Arg381
Glu285, Ala282,
Gln387, Ala262,
Asn354, Phe369,
Tyr491, Val352,
Met120, Trp463
-
Val352, Tyr491,
Met355, Asn354,
Trp463, Met120,
Pro467, Ile462
-9.39
-8.01
8p
Hem
-
-
Tyr373,
Arg388,
Gln263,
Glu377
Asp382, Val352,
Phe369, Asn370,
Pro350, Gly371
Dexamethasone
-
14

ACCEPTED MANUSCRIPT
Table 8. Docking results of 5a-j and 8a-p derivatives docked into the cyclooxygenase-2 target (PDB ID:
3LN1).
Code
∆Gbinding
Hydrogen
bond
Hydrophobic
interaction
π-π
Cation-π
(Kcal/mol)
Arg499
Arg106
-
Tyr371, Phe367,
Leu370, Gly512,
Ser516, Trp373,
Ala513, Ser339,
Val335, Phe504,
Val509, Leu338,
Ala502, His75,
Tyr341
Phe504
,
-8.43
5a
Trp373
,
Tyr371
-
Leu370, Gly512,
Met508, Trp373,
Val509, Val335,
Phe504, Ala502,
Leu338, Ser339,
Tyr341, Leu345,
Leu517, His75,
Ala513, Val102
-9.53
5b
-
-
Trp373, Phe367,
Gly512, Leu370,
Tyr371, Ser516,
Val509, Ala513,
Leu338, Phe504,
Leu345, His75,
Ser339, Val335,
Tyr341, Val102,
Arg106, Ala502,
Arg499
Phe504
,
Trp373
-9.54
5c
-
Tyr371,
Tyr341,
Gln178, Ser339,
Val509, Ala502,
Arg106, His75 Leu338, Ala513,
Phe504, Gly512,
5d
5e
-9.26
-9.18
Phe504
Tyr371
-
Ser516, Val335,
Tyr334, Val330
Arg499
Ser339, Val509,
Tyr341, His75,
Ala502, Phe504,
-
15

ACCEPTED MANUSCRIPT
Ala513, Leu338,
Leu517, Leu520,
Val335, Tyr334,
Tyr371, Ser516,
Phe191, Phe367
-
His75, Ala502,
Tyr341, Ser339,
Gln178, Val509,
Leu338, Phe504,
Tyr371, Leu517,
Val102, Ser516,
Leu345, Ala513,
Val335, Tyr334,
Gly512, Phe367,
Leu520, Phe191
-8.61
5f
Tyr371
-
-
Leu370, Val509,
Met508, Trp373,
Phe504, Ser516,
Leu338, Gly512,
Val335, Ser339,
Ala513, Leu517,
Tyr341, Arg106,
Leu345, Val102
5g
-8.35
-
-
Arg499
His75, Ser339,
Tyr341, Val509,
Phe504, Ala502,
Ala513, Leu338,
Tyr371, Val102,
Leu345, Val335,
Leu517, Ser516,
Phe367, Tyr334,
Phe191
-8.82
5h
Tyr371
-
His75
Pro500, Thr79,
Asp501, Gln178,
Gln336, Gly340,
Tyr341, His337,
Pro177, His342,
Val568, Asn567
-7.25
-7.11
5i
5j
His342
His342
-
-
-
Pro500, Ala502,
Thr79, His75,
Gly340, Pro177,
Gln178, Asp501,
His337, Tyr341,
16

ACCEPTED MANUSCRIPT
His342, Gln336,
Val568, Asn567,
Gly179
-
Pro500, Arg499,
Ala502, Thr79,
Asp501, Gly340,
His75, His342,
Gln336, Ser339,
Gln178, Asp333,
Asn567, His337
-6.14
8a
His342
-
Gln336, Thr79 Arg499, Ala502,
His75, Pro500,
Gln178, Ser339,
Tyr341, Gly340,
Asp501, His337,
Asp333, Glu332,
Asn567
-6.07
-6.59
-6.31
8b
8c
8d
-
-
-
-
-
-
Gln336, Thr79 Pro500, Asp501,
Arg499, His75,
Ser339, Ala502,
His342, Gly340,
Gln178, Ser565,
His337, Asn567
Gln336
His75, Pro500,
Gln178, Thr79,
Asp501, Gly340,
Ser339, Ala502,
Tyr341, His337,
Asn567, Asp333,
Glu332
His75, Gln178 Gly179, Asn567,
His342, Pro177,
-5.85
-6.79
8e
8f
Pro500, Gly340,
Asp501, Ala502,
Thr79, Tyr76, His80
His80
-
-
Gln178
Asn567, Gly340,
Gly179, Pro177,
His342, Pro500,
Asp501, Ala502,
Thr79, His80, His75,
Tyr76
-
17

ACCEPTED MANUSCRIPT
Gln336, Thr79 His75, Pro500,
His342, Asp501,
Ala502, Arg499,
Gly340, His337,
Gly179, Gln178,
Asn567, Asp333
-5.64
-5.65
8g
8h
-
-
-
-
Pro500
Thr79, Ala502,
Asp501, His342,
Gly340, Ser339,
His337, Gln178,
Val568, Phe566,
Asp333, Ile550,
Ser565, Asn567,
Gln336, Gly179
Gln178
Thr79
His337, Asn567,
Gln336, Gly340,
Ser339, Thr79,
Tyr76, Asn72,
Ala502, His75,
Arg499, His80,
Pro500
-1.59
8i
-
-
His75, Ala502,
Thr79, Gln178,
Pro500, Gly340,
Asp501, Ser339,
His80, Gln336,
Phe566, Asp333,
His337, Asn567
-5.45
-7.18
8j
-
-
Gly340
Gln336,
Thr79, His80,
Pro500, His75,
His342, Asp501,
His337, Gln178,
Gln336, Asn567
8k
-
-
His75, His80,
Gly340, Thr79 Pro500, Asp501,
His342, Gln178,
-7.26
-5.37
8l
-
-
-
-
Asn567, His337,
Asp333
Gln178
Asn567, Leu338,
Gln336, His337,
Arg499, Ser339,
Gly179, Gly340,
8m
18

ACCEPTED MANUSCRIPT
His75, Ala502,
Pro500, Thr79,
Asn72, Tyr56, His80
Thr79
His80, Pro500,
His75, His342,
Asp501, Gly340,
His337, Gln336,
Gln178, Val568,
Asn567, Gly179,
Asp333, Ile550
-5.53
-7.19
8n
8o
His75
-
Pro500, His75,
Thr79, Gln178,
Ala502, Asp501,
Gly340, Asn567,
Phe566, His337,
Asp333, Ile550,
Gln336, His342,
Tyr341
-
-
-
Asp501, Thr79,
His75, His80,
Pro500, Ala502,
Lys82, Gly340,
Asn567, Gln569,
Val568, His337,
Gln336, Gln178
8p
-5.86
-7.23
-
Lys82
-
Tyr373,
Glu377
-
Asp382, Val352,
Phe369, Asn370,
Pro350, Gly371
Dexamethasone
-
Members of this group exhibit weak to good in vitro anti-inflammatory activity, such that
higher lipophilicity enhances these particular hydrophobic interactions. Indeed, 8b exhibited the
best docking score from the computational studies, coinciding with the most potent in vitro anti-
inflammatory activity. Interactions of 8b with the enzyme active site are depicted in Figure 5.
The carbonyl and 4-oxyphenyl oxygen atoms interact through hydrogen bonds with Arg381
(O…NH, 1.96 Å and O…NH, 1.98 Å). Also the amine nitrogen atom of the C-2 chromeno
moiety is involved in a hydrogen bond with Asp382 amino acid (NH…O, 1.96 Å). Compound
8b was found to also interact with a hydrophobic surface formed by Gln263, Val352, Glu377,
Arg388, Trp463, Met120, Pro467, Ile462, Pro466 and Val465 residues.
19

ACCEPTED MANUSCRIPT
Fig. 5: Compound 8b in the human inducible nitric oxide synthase active site. The 3D (left) and 2D
(right) ligand interaction diagrams.
Replacement of the p-chlorophenyl group at C-4 position of the lactam ring in 8e with a p-
nitrophenyl in 8f substantially decreased the in vitro activity. The calculated CLogP value for 8e
(6.75) indicates that it is somewhat more lipophilic than 8f (CLogP = 5.78) and can thus
experience stronger interactions with hydrophobic amino acids, accounting for its better anti-
inflammatory activity and free binding energy (Table 4 & 7).
Replacing the p-(N,N-dimethylamino)phenyl ring of 8h with an anthracene moiety at the C-4
position (8i) decreased the in vitro activity. The lower anti-inflammatory activity of 8i can be
attributed to steric hindrance and a change in the orientation of compound 8i in the active site,
which weakens the intermolecular interactions.
Dexamethasone was likewise computationally docked into the protein and showed a similar
binding mode to the β-lactams, forming hydrogen bonds and hydrophobic interactions with key
residues in the enzyme active site. (Figure 6).
20

ACCEPTED MANUSCRIPT
Fig. 6: Hydrogen bonds and hydrophobic interactions of dexamethasone within the human inducible
nitric oxide synthase active site.
For comparison, we also examined the docking of aldehydic β-lactams 5 into the human
inducible nitric oxide synthase active site. Compounds 5b and 5e gave the best docking scores
that correspond with their strong anti-inflammatory activity (Table 5). The carbonyl oxygen
atom of lactam ring of 5b formed a hydrogen bond interaction with NH of Gln263 (O…HN, 1.84
Å). Moreover, 5e formed two hydrogen bonds with amino acid active sites. The carbonyl oxygen
atom of lactam ring displayed a hydrogen bond with OH group of Tyr373 (O…HO, 2.59 Å) and
the carbonyl oxygen atom of aldehyde showed a hydrogen bond with NH group of Arg388
(O…HN, 2.18 Å). The binding mode of compounds 5b and 5e to the human inducible nitric
oxide synthase active site is revealed in Figure 7.
Fig. 7: Binding modes of compound 5b (A) and 5e (B) with human inducible nitric oxide synthase active
site (PDB ID: 4NOS).
The three aryl rings of 5b were located within the hydrophobic pocket enclosed by Ala351,
Asp382, Arg381, Glu377, Tyr373, Pro350, Ala262, Val352, Met355 and Asn354. Exposed
21

ACCEPTED MANUSCRIPT
residues Arg266, Arg388, Ala262, Val352, Gln263, Pro350, Gly371, Asn370, Glu377 and
Phe369 also formed hydrophobic interactions with 5e. Docking calculations revealed that 5e
forms a cation-π interaction with the Arg266 residue. All these interactions help anchor 5b and
5e in the binding site of the human inducible nitric oxide synthase protein. In addition, anti-
inflammatory activity of 5b was higher than dexamethasone as positive control (IC50-NO release
5.02 µM).
=
Replacing the 4-chlorophenyl ring (5e) with the 4-nitrophenyl ring (5f) and 4-(N,N-
dimethylamino)phenyl ring (5h) decreased the anti-inflammatory activity. The increased
polarization and strong electron-donating characters reduced the potency of compounds but
addition of hydrophobic groups had a positive effect. Also, docking results exposed that
compounds 5e and 5f formed a cation-π and π-π stacking interactions with Arg266 and Tyr491
amino acids, respectively. While compound 5h did not participate in these contacts (Figure 8).
Fig. 8: Close up of the calculated binding of 5e (A) and 5f (B) within the human inducible nitric oxide
synthase enzyme active site.
A methoxy or methyl group at the para position of the aryl ring induced different
conformations, leading to a change in interactions with the hydrophobic pocket of the protein.
This suggests that this is the reason for compound 5b showing higher anti-inflammatory activity
and ∆Gbind value than compound 5a. In addition, the carbonyl oxygen atom of lactam ring of 5b
formed a hydrogen bond interaction with NH of Gln263 (O…HN, 1.84 Å), whereas 5a did not
have this interaction. The superimposition of 5a and 5b in the enzyme binding site is illustrated
in Figure 9.
22

ACCEPTED MANUSCRIPT
Fig. 9: Superimposition of 5a (violet) and 5b (green) within the enzyme binding site.
Compound 5a, having a methoxy group at the para position of the aryl ring, showed lower
inhibitory activity than compound 5e that has an ethoxy group at the same position (Table 5).
The lower anti-inflammatory activity of 5a can be attributed to the improper geometric
orientation in the active site of enzyme. Also, compound 5e shows two hydrogen bonds with
Arg388 and Tyr373 residues and a cation-π stacking interaction with Arg266, while compound
5a did not have these interactions (Figure 10). Docking calculations indicate that 5e has stronger
interactions than 5a with active site amino acids, accounting for its elevated anti-inflammatory
activity.
Fig. 10: Calculated binding modes of compound 5a (A) and 5e (B) within the human inducible nitric
oxide synthase active site (PDB ID: 4NOS).
It is also interesting to compare β-lactam 5e with 5i. While 5e has a p-chlorophenyl ring at the
C-2 position of lactam ring, 5i has a bulkier anthracene moiety (Figure 11). The computational
modeling suggests that steric differences between these two aryl groups, along with the
differences in the calculated ∆Gbind values for lactams 5e and 5i, may account for the different
anti-inflammatory potencies.
Fig. 11: Calculated binding mode of compound 5e (A) and 5i (B) within the human inducible nitric oxide
synthase active site (PDB ID: 4NOS).
Replacing the N-aryl ethoxy substituent (5f) with an N,N-dimethylamino (5g) increased anti-
23

ACCEPTED MANUSCRIPT
inflammatory activity, suggesting that increased electron-donation into the N-aryl ring improves
potency. This is corroborated by computational docking experiments showing that while
compound 5f forms looser hydrogen bonds with Tyr373 and Arg388, compound 5g forms a
tighter hydrogen bond to the Cys110 residue in the enzyme binding pocket.
3. Conclusion
This describes the first diastereoselective synthesis of chromeno-substituted β-lactams. The
compounds were accessed by an expedient single-pot three-component synthesis approach, to
give exclusively the cis-disubstituted adducts. The series of chromeno β-lactam hybrids 8a-p, as
well as their aldehydic precursors 5a-j, were evaluated for anti-inflammatory activity. The best
anti-inflammatory activity was observed for compounds 5b and 8b (53.4 and 19.8 anti-
inflammatory ratio, respectively). Moreover, based on the results of anti-inflammatory activity
and molecular docking calculations of the synthesized compounds, the anti-inflammatory
activity is closely dependent on the substituents at the N1 and C-2 centers of the lactam ring.
Chromeno lactams 8c, 8j and 8p, as well as aldehydic lactams 5a, 5b, 5e, 5f, 5g, display in vitro
anticancer activity against the SW1116 colon cancer cell line, without noticeable cytotoxicity
towards healthy cells. Studies are now underway to improve the biological activity and
determine the mechanism of action.
4. Experimental Section
4.1 General experimental methods
All needed chemicals were purchased from Fluka, Aldrich, Merck and Across chemical
companies and used without further purification. Solvents and reagents that used such as CH2Cl2
and Et3N were dried before to use by distillation over CaH2. All products were characterized by
comparison of FT-IR 8300 spectrophotometer using potassium bromide pellets (ʋ in cm-1). The
13
1H NMR (250 MHz) and C NMR (100 MHz) spectra, as well as the two-dimensional H-H
COSY and H-C HSQC experiments, were run on a BrukerAvance400 in CDCl3 using a Bruker
Avance DPX instrument. Chemical shifts were reported in parts per million (δ) downfield from
tetramethylsilane. Coupling constants (J) are reported in hertz (Hz). Splitting patterns are
indicated as s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, dd: doublet of doublet.
Melting points were recorded on a Buchi 510 melting point apparatus in open capillary tubes.
Elemental analyses were run on a Thermo Finnigan Flash EA-1112 series. The mass spectra
were recorded on a Shimadzu GC-MS QP 1000 EX instrument. Thin-layer chromatography
(TLC) was carried out on silica gel 254. X-ray data were collected on a Bruker D8 VENTURE
diffractometer.
4.2. General procedure for the synthesis of some new
5a-j:
β-lactams containing benzaldehydes
To a solution of 4-hydroxybenzaldehyde (1) (1.00 mmol) in 5 mL DMF in a 250 mL beaker,
was added bromoacetic acid (1.50 mmol) (2) and potassium carbonate (5.00 mmol). The mixture
was stirred at room temperature for 24 h. Then the reaction mixture was poured into 20 mL water
with stirring. The solution was adjusted to pH 2.0 with 2.0M hydrochloric acid, where upon
precipitation occurred. The product was collected and washed with water (20 mL). The crude
24

ACCEPTED MANUSCRIPT
product was purified by recrystallization from ethanol to give 2-(4-formylphenoxy)acetic acid
(3). A solution of corresponding imine 4a-j (1.00 mmol) was stirred with the corresponding
substituted 2-(4-formylphenoxy)acetic acid (3) (1.50 mmol), p-toluenesulfonyl chloride (0.69
mL, 1.5 mmol) and triethylamine (5.00 mmol) in dry CH2Cl2 at room temperature overnight.
Then it was washed with HCl 1.0 N (20 mL), saturated NaHCO3 (20 mL), brine (20 mL), dried
over Na2SO4 and the solvent was evaporated under reduced pressure to give the crude product. It
was then purified by recrystallization from ethanol to afford pure β-lactams containing
benzaldehydes 5a-j.
4.2.1. 4-((2-(4-Chlorophenyl)-1-(4-methoxyphenyl)-4-oxoazetidin-3-yl)oxy)benzaldehyde (5a):
1
White solid; Mp. 175-177 °C; IR (KBr, cm-1): 1766 (CO β-lactam), 1689 (CO aldehyde); H-
NMR (250 MHz, DMSO): 3.68 (3H, s, OCH3), 5.77 (1H, d, J = 4.0 Hz, H-2 β-lactam), 6.02 (1H,
d, J = 4.0 Hz, H-3 β-lactam), 6.91 (2H, d, J = 8.2 Hz, ArH), 7.02 (2H, d, J = 8.0 Hz, ArH), 7.22
(2H, d, J = 8.2 Hz, ArH), 7.30 (2H, d, J = 7.7 Hz , ArH), 7.37 (2H, d, J = 8.0 Hz , ArH), 7.75
(2H, d, J = 8.0 Hz , ArH), 9.81 (1H, s, CHO); 13C-NMR (100 MHz, DMSO) δ 191.3 (CO
aldehyde), 161.1 (CO β-lactam), 160.7, 156.1, 132.3, 131.4, 131.1, 130.6, 130.2, 129.7, 121.7,
118.5, 115.4, 114.6 (aromatic carbons), 80.1 (C-3 β-lactam), 59.9 (C-2 β-lactam) , 55.2 (O-CH3);
GC-MS m/z = 407 [M+]; Analysis calculated for C23H18ClNO4: C, 67.73; H, 4.45; N, 3.43%.
Found: C, 66.18; H, 4.35; N, 3.62%.
4.2.2. 4-((2-(4-Chlorophenyl)-4-oxo-1-(p-tolyl)azetidin-3-yl)oxy)benzaldehyde (5b):
1
White solid; Mp. 178-180 °C; IR (KBr, cm-1): 1759 (CO β-lactam), 1689 (CO aldehyde); H-
NMR (250 MHz, DMSO): 2.21 (3H, s, CH3), 5.79 (1H, d, J = 4.7 Hz, H-2 β-lactam), 6.03 (1H,
d, J = 4.7 Hz, H-3 β-lactam), 7.02 (2H, d, J = 8.7 Hz, ArH), 7.13 (2H, d, J = 8.7 Hz, ArH), 7.18
(2H, d, J = 8.2 Hz, ArH), 7.29 (2H, d, J = 8.7 Hz, ArH), 7.36 (2H, d, J = 8.5 Hz, ArH), 7.75
(2H, d, J = 8.7 Hz , ArH), 9.81 (1H, s, CHO); 13C-NMR (100 MHz, DMSO) δ 190.5 (CO
aldehyde), 161.4 (CO β-lactam), 159.0, 131.6, 131.1, 130.7, 129.7, 129.3, 128.8, 122.7, 120.9,
119.9, 117.4, 115.6 (aromatic carbons), 80.4 (C-3 β-lactam), 60.9 (C-2 β-lactam), 20.9 (CH3);
GC-MS m/z = 391 [M+]; Analysis calculated for C23H18ClNO3: C, 70.50; H, 4.63; N, 3.57%.
Found: C, 70.33; H, 4.31; N, 3.61%.
4.2.3. 4-((1-(4-Chlorophenyl)-2-(3-nitrophenyl)-4-oxoazetidin-3-yl)oxy)benzaldehyde (5c):
1
White solid; Mp. 232-234 °C; IR (KBr, cm-1): 1751 (CO β-lactam), 1697 (CO aldehyde); H-
NMR (250 MHz, DMSO): 6.02 (1H, d, J = 5.0 Hz, H-2 β-lactam), 6.15 (1H, d, J = 5.0 Hz, H-3
β-lactam), 7.01 (2H, d, J = 8.5 Hz, ArH), 7.34 (2H, d, J = 8.7 Hz, ArH), 7.42 (2H, d, J = 9.0 Hz,
ArH) 7.55 (1H, t, J = 8.0 Hz, ArH), 7.73 (2H, d, J = 8.5 Hz, ArH), 7.80 (1H, d, J = 7.7 Hz,
13
ArH), 8.08 (1H, d, J = 8.2 Hz, ArH), 8.23 (1H, s, ArH), 9.79 (1H, s, CHO); C-NMR (100
MHz, DMSO) δ 190.2 (CO aldehyde), 161.4 (CO β-lactam), 160.6, 147.9, 134.6, 134.3, 133.7,
131.4, 130.9, 129.9, 129.5, 129.2, 123.7, 122.9, 118.4, 115.3 (aromatic carbons) , 80.4 (C-3 β-
lactam), 60.2 (C-2 β-lactam); GC-MS m/z = 422 [M+]; Analysis calculated for C22H15ClN2O5: C,
62.50; H, 3.58; N, 6.63%. Found: C, 61.93; H, 3.45; N, 6.80%.
25

ACCEPTED MANUSCRIPT
4.2.4. 4-((1-(4-Methoxyphenyl)-2-(3-nitrophenyl)-4-oxoazetidin-3-yl)oxy)benzaldehyde (5d):
1
Cream solid; Mp. 205-207 °C; IR (KBr, cm-1): 1751 (CO β-lactam), 1689 (CO aldehyde); H-
NMR (250 MHz, DMSO): 3.68 (3H, s, OCH3), 5.97 (1H, d, J = 4.7 Hz, H-2 β-lactam), 6.11 (1H,
d, J = 4.7 Hz, H-3 β-lactam), 6.91 (2H, d, J = 8.7 Hz, ArH), 7.01 (2H, d, J = 8.5 Hz, ArH), 7.27
(2H, d, J = 8.7 Hz, ArH), 7.54 (1H, t, J = 7.7 Hz, ArH), 7.72 (2H, d, J = 8.5 Hz, ArH), 7.79 (1H,
13
d, J = 7.7 Hz, ArH), 8.07 (1H, d, J = 8.2 Hz, ArH), 8.22 (1H, s, ArH), 9.79 (1H, s, CHO); C-
NMR (100 MHz, DMSO) δ 191.3 (CO aldehyde), 161.0 (CO β-lactam), 160.5, 156.2, 147.4,
135.3, 134.4, 131.4, 130.6, 129.8, 129.5, 123.4, 122.9, 118.5, 115.4, 114.6 (aromatic carbons),
80.2 (C-3 β-lactam), 56.5 (C-2 β-lactam), 55.2 (O-CH3); GC-MS m/z = 418 [M+]; Analysis
calculated for C23H18N2O6: C, 66.03; H, 4.34; N, 6.70%. Found: C, 66.72; H, 4.43; N, 6.87%.
4.2.5. 4-((2-(4-Chlorophenyl)-1-(4-ethoxyphenyl)-4-oxoazetidin-3-yl)oxy)benzaldehyde (5e):
1
White solid; Mp. 193-195 °C; IR (KBr, cm-1): 1735 (CO β-lactam), 1689 (CO aldehyde); H-
NMR (250 MHz, DMSO): 1.24 (3H, t, J = 6.7 Hz, CH3), 3.93 (2H, q, J = 6.7 Hz, OCH2), 5.77
(1H, d, J = 4.2 Hz, H-2 β-lactam), 6.01 (1H, d, J = 4.2 Hz, H-3 β-lactam), 6.89 (2H, d, J = 7.5
Hz, ArH), 7.02 (2H, d, J = 7.7, ArH), 7.20 (2H, d, J = 7.5 Hz, ArH), 7.30 (2H, d, J = 8.2 Hz,
13
ArH), 7.37 (2H, d, J = 7.5 Hz, ArH), 7.74 (2H, d, J = 7.5 Hz, ArH), 9.81 (1H, s, CHO); C-
NMR (100 MHz, DMSO) δ 191.3 (CO aldehyde), 161.1 (CO β-lactam), 160.7, 155.3, 133.0,
131.9, 131.4, 130.5, 129.9, 129.6, 128.2, 118.5, 115.4, 115.0 (aromatic carbons) , 80.1 (C-3 β-
lactam), 63.2 (O-CH2), 59.8 (C-2 β-lactam), 14.5 (CH3); GC-MS m/z = 421 [M+]; Analysis
calculated for C24H20ClNO4: C, 68.33; H, 4.78; N, 3.32%. Found: C, 68.27; H, 4.60; N, 3.59%.
4.2.6. 4-((1-(4-Ethoxyphenyl)-2-(4-nitrophenyl)-4-oxoazetidin-3-yl)oxy)benzaldehyde (5f):
1
White solid; Mp. 198-200 °C; IR (KBr, cm-1): 1751 (CO β-lactam), 1697 (CO aldehyde); H-
NMR (250 MHz, DMSO): 1.25 (3H, t, J = 7.0 Hz, CH3), 3.93 (2H, q, J = 7.0 Hz, OCH2) 5.93
(1H, d, J = 4.7 Hz, H-2 β-lactam), 6.10 (1H, d, J = 4.7 Hz, H-3 β-lactam), 6.90 (2H, d, J = 9.0
Hz, ArH ), 7.02 (2H, d, J = 8.5, ArH), 7.22 (2H, d, J = 9.0 Hz, ArH), 7.64 (2H, d, J = 8.5 Hz,
13
ArH), 7.74 (2H, d, J = 8.5 Hz, ArH), 8.10 (2H, d, J = 8.7 Hz, ArH), 9.80 (1H, s, CHO); C-
NMR (100 MHz, DMSO) δ 190.5 (CO aldehyde), 161.0 (CO β-lactam), 160.8, 156.4, 148.2,
139.9, 131.7, 131.3, 129.4, 128.9, 123.7, 118.7, 115.6, 115.2 (aromatic carbons), 80.6 (C-3 β-
lactam), 63.7 (O-CH2), 60.7 (C-2 β-lactam), 14.7 (CH3); GC-MS m/z = 432 [M+]; Analysis
calculated for C24H20N2O6: C, 66.66; H, 4.66; N, 6.48%. Found: C, 65.61; H, 4.45; N, 6.35%.
4.2.7. 4-((1-(4-(Dimethylamino)phenyl)-2-(4-nitrophenyl)-4-oxoazetidin-3-yl)oxy)benzaldehyde
(5g):
1
Cream solid; Mp. 160-162 °C; IR (KBr, cm-1): 1743 (CO β-lactam), 1689 (CO aldehyde); H-
NMR (250 MHz, DMSO): 2.81 (6H, s, NCH3), 5.89 (1H, d, J = 4.7 Hz, H-2 β-lactam), 6.08 (1H,
d, J = 4.7 Hz, H-3 β-lactam), 6.67 (2H, d, J = 9.0 Hz, ArH), 7.02 (2H, d, J = 8.5, ArH), 7.14
(2H, d, J = 9.0 Hz, ArH), 7.62 (2H, d, J = 8.5 Hz, ArH), 7.74 (2H, d, J = 8.5 Hz, ArH), 8.10
(2H, d, J = 8.5 Hz, ArH), 9.80 (1H, s, CHO); 13C-NMR (100 MHz, DMSO) δ 191.3 (CO
26

ACCEPTED MANUSCRIPT
aldehyde), 160.7 (CO β-lactam), 160.4, 147.8, 147.4, 141.1, 131.5, 130.6, 129.4, 125.9, 123.2,
118.3, 115.4, 112.7 (aromatic carbons), 80.4 (C-3 β-lactam), 59.6 (C-2 β-lactam), 40.1 (N-CH3);
GC-MS m/z = 431 [M+]; Analysis calculated for C24H21 N3O5: C, 66.81; H, 4.91; N, 9.74%.
Found: C, 66.51; H, 4.64; N, 9.83%.
4.2.8.
4-((2-(4-(Dimethylamino)phenyl)-1-(4-ethoxyphenyl)-4-oxoazetidin-3-
yl)oxy)benzaldehyde (5h):
1
White solid; Mp. 230-232 °C; IR (KBr, cm-1): 1735 (CO β-lactam), 1689 (CO aldehyde); H-
NMR (250 MHz, DMSO): 1.20 (3H, t, J = 6.7 Hz, CH3), 2.72 (6H, s, NCH3), 3.80 (2H, q, J =
6.7 Hz, OCH2) , 5.53 (1H, d, J = 4.2 Hz, H-2 β-lactam), 5.84 (1H, d, J = 4.2 Hz, H-3 β-lactam),
6.48 (2H, d, J = 8.2 Hz, ArH ), 6.81 (2H, d, J = 8.5, ArH), 6.96 (2H, d, J = 8.2 Hz, ArH), 7.09
(2H, d, J = 8.5 Hz, ArH), 7.14 (2H, d, J = 8.7 Hz, ArH), 7.68 (2H, d, J = 8.5 Hz, ArH), 9.74
(1H, s, CHO); 13C-NMR (100 MHz, DMSO) δ 191.7 (CO aldehyde), 161.6 (CO β-lactam),
159.8, 155.6, 143.1, 139.7, 131.8, 130.4, 129.4, 119.5, 118.9, 115.9, 115.4, 112.0 (aromatic
carbons), 80.7 (C-3 β-lactam), 63.6 (O-CH2), 61.1 (C-2 β-lactam), 40.5 (N-CH3), 15.0 (CH3);
GC-MS m/z = 430 [M+]; Analysis calculated for C26H26 N2O4: C, 72.54; H, 6.09; N, 6.51%.
Found: C, 72.28; H, 5.96; N, 6.61%.
4.2.9. 4-((2-(Anthracen-9-yl)-1-(4-ethoxyphenyl)-4-oxoazetidin-3-yl)oxy)benzaldehyde (5i):
1
Cream solid; Mp. 200-202 °C; IR (KBr, cm-1): 1751 (CO β-lactam), 1697 (CO aldehyde); H-
NMR (250 MHz, DMSO): 1.17 (3H, t, J = 7.0 Hz, CH3), 3.81 (2H, q, J = 7.0 Hz, OCH2), 6.46
(1H, d, J = 5.0 Hz, H-3 β-lactam ), 6.74 (4H, dd, J 1= 10.2 Hz, J2 = 2.0 Hz, ArH), 7.04 (2H, d, J
= 8.7 Hz, ArH), 7.20 (1H,d, J = 5.0 Hz, H-2 β-lactam ), 7.36-7.45 (4H, m, ArH), 7.54 (1H, t, J =
7.7 Hz, ArH), 7.70 (1H, t, J = 7.7 Hz, ArH), 7.97-8.07 (2H, m, ArH), 8.49 (1H, d, J = 9.7 Hz,
ArH), 8.56 (1H, s, ArH), 8.79 (1H, d, J = 9.0 Hz ArH), 9.60 (1H, s, CHO); 13C-NMR (100 MHz,
DMSO) δ 191.0 (CO aldehyde), 161.6 (CO β-lactam), 160.7, 155.3, 131.3, 131.0, 130.9, 130.8,
130.6, 130.3, 130.2, 130.0, 129.5, 129.3, 127.3, 126.2, 125.0, 124.7, 124.4, 122.8, 121.7, 117.8,
115.0, 114.9 (aromatic carbons), 82.1 (C-3 β-lactam), 63.0 (O-CH2) , 58.2 (C-2 β-lactam), 14.4
(CH3); GC-MS m/z = 487 [M+]; Analysis calculated for C32H25NO4: C, 78.83; H, 5.17; N,
2.87%. Found: C, 76.93; H, 5.01; N, 3.02%.
4.2.10. 4-((2-(Anthracen-9-yl)-1-(4-methoxyphenyl)-4-oxoazetidin-3-yl)oxy)benzaldehyde (5j):
1
Cream solid; Mp. 170-172 °C; IR (KBr, cm-1): 1759 (CO β-lactam), 1689 (CO aldehyde); H-
NMR (250 MHz, DMSO) δ 3.55 (3H, s, OCH3), 6.47 (1H, d, J = 4.7 Hz, H-3 β-lactam), 6.75
(4H, d, J = 8.5 Hz, ArH), 7.06 (2H, d, J = 8.7 Hz, ArH), 7.21 (1H, d, J = 4.7 Hz, H-2 β-lactam),
7.35-7.44 (4H, m, ArH), 7.53 (1H, t, J = 7.0 Hz, ArH), 7.70 (1H, t, J = 8.2 Hz, ArH), 7.98 (1H,
d, J = 8.2 Hz, ArH), 8.05 (1H, d, J = 8.5 Hz, ArH), 8.51 (1H, d, J = 8.5 Hz, ArH), 8.56 (1H, s,
13
ArH), 8.79 (1H, d, J = 9.0 Hz, ArH), 9.60 (1H, s, ArH); C-NMR (100 MHz, DMSO): 191.0
(CO aldehyde), 161.6 (CO β-lactam), 160.7, 156.0, 131.3, 130.9, 130.8, 130.6, 130.4, 130.2,
130.0, 129.5, 129.3, 128.3, 127.3, 126.2, 125.0, 124.7, 124.4, 122.8, 121.7, 117.8, 114.9, 114.6
(aromatic carbons), 82.2 (C-2 β-lactam), 58.2 (C-3 β-lactam), 55.0 (O-CH3); GC-MS m/z = 473
[M+]; Analysis calculated for C31H23NO4: C, 78.63; H, 4.90; N, 2.96; O, 13.51%. Found: C,
79.03; H, 4.71; N, 3.15%.
27

ACCEPTED MANUSCRIPT
4.3. A typical procedure for the synthesis of novel
chromene and dihydropyrano[ ]-chromene 8a-p:
β-lactam hybrids with tetrahydro-4H-
c
To a mixture of cis-β-lactam benzaldehyde 5a-j (1.00 mmol), malonitrile 6 (1.00 mmol) and
cyclic ketone 7a,b (1.00 mmol) in 3 mL of ethanol, DABCO (1.00 mmol) was added and the
mixture was refluxed for appropriate time. After completion of the reaction, as indicated by
TLC, ethanol (10 mL) was added and the reaction mixture was filtered. The crude products 8a-p
was purified by recrystallization from ethanol (95%).
4.3.1. 2-Amino-4-(4-((2-(4-chlorophenyl)-1-(4-methoxyphenyl)-4-oxoazetidin-3-yl)oxy)phenyl)-
7,7-dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (8a):
White solid; Mp. 250-252 °C; IR (KBr, cm-1): 3386 (NH2), 2191 (CN), 1743 (CO β-lactam),
1666 (CO ketone); 1H-NMR (250 MHz, DMSO): 0.90 (3H, s, CH3), 1.00 (3H, s, CH3), 2.03 (1H,
d, J = 16.0 Hz, CH2), 2.20 (1H, d, J = 16.0 Hz, CH2), 2.32-2.48 (2H, m, CH2), 3.65 (3H, s, CH3),
4.06 (1H, s, CH), 5.64 (1H, d, J = 4.7 Hz, H-2 β-lactam), 5.77 (1H, d, J = 4.7 Hz, H-3 β-lactam),
6.72 (2H, d, J = 8.2 Hz, ArH), 6.81 (2H, d, J = 8.0, ArH), 6.94 (4H, br, NH2, ArH), 7.21-7.28
(4H, m, ArH), 7.38 (2H, d, J = 8.5 Hz, ArH); 13C-NMR (100 MHz, DMSO) δ 195.9 (CO
ketone), 162.5, 158.9, 156.4, 155.5, 138.7, 133.2, 131.5, 130.5, 130.2, 128.5, 121.9, 120.1, 118.8,
115.5, 115.0, 113.3, 113.2 (CO β-lactam, vinylic carbon, aromatic carbons and CN), 81.2 (C-3 β-
lactam), 60.5 (C-2 β-lactam), 58.5 (C-CN), 55.6 (O-CH3), 50.3 (CH2), 35.0 (CH2), 32.1 (CH),
28.8 (CH3), 27.0 (CH3); GC-MS m/z = 595 [M+]; Analysis calculated for C34H30ClN3O5: C,
68.51; H, 5.07; N, 7.05%. Found: C, 63.64; H, 4.69; N, 6.90%.
4.3.2.
2-Amino-4-(4-((2-(4-chlorophenyl)-4-oxo-1-(p-tolyl)azetidin-3-yl)oxy)phenyl)-7,7-
dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (8b):
White solid; Mp. 208-210 °C; IR (KBr, cm-1): 3386, 3325 (NH2), 2198 (CN), 1743 (CO β-
1
lactam), 1666 (CO ketone); H-NMR (250 MHz, DMSO): 0.88 (3H, s, CH3), 1.00 (3H, s, CH3),
2.04 (1H, d, J = 16.2 Hz, CH2), 2.18-2.25 (4H, m, CH3 and CH2), 2.38-2.47 (2H, m, CH2), 4.07
(1H, s, CH), 5.69 (1H, d, J = 5.0 Hz, H-2 β-lactam), 5.82 (1H, d, J = 5.0 Hz, H-3 β-lactam), 6.72
(2H, d, J = 7.0, ArH), 6.88-6.96 (4H, m, ArH, NH2), 7.10-7.18 (4H, m, ArH), 7.27-7.34 (4H, m,
13
ArH); C-NMR (100 MHz, DMSO) δ 195.9 (CO ketone), 162.9, 162.5, 158.9, 155.5, 139.7,
138.7,134.4, 134.0, 133.3, 132.7, 130.2, 128.5, 120.1, 118.8, 117.4, 115.5, 113.3 (CO β-lactam,
vinylic carbon, aromatic carbons and CN), 81.2 (C-3 β-lactam), 60.5 (C-2 β-lactam), 58.5 (C-
CN), 50.3 (CH2), 35.1 (CH2), 32.1 (CH), 28.8 (CH3), 27.0 (CH3), 20.8 (CH3); GC-MS m/z = 579
[M+]; Analysis calculated for C34H30ClN3O4: C, 70.40; H, 5.21; N, 7.24%. Found: C, 69.93; H,
5.11; N, 7.61 %.
4.3.3. 2-Amino-4-(4-((1-(4-chlorophenyl)-2-(3-nitrophenyl)-4-oxoazetidin-3-yl)oxy)phenyl)-7,7-
dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (8c):
White solid; Mp. 219-221 °C; IR (KBr, cm-1): 3417, 3332 (NH2), 2191 (CN), 1759 (CO β-
1
lactam), 1674 (CO ketone); H-NMR (250 MHz, DMSO): 0.87 (3H, s, CH3), 1.00 (3H, s, CH3),
2.03 (1H, d, J = 16.0 Hz, CH2), 2.20 (1H, d, J = 16.0 Hz, CH2), 2.36-2.48 (2H, m, CH2), 4.04
(1H, s, CH ), 5.93 (2H, br, H-2 and H-3 β-lactam), 6.71 (2H, d, J = 8.2 Hz, ArH), 6.91-6.94 (4H,
m, ArH, NH2), 7.32 (2H, d, J = 8.2 Hz, ArH), 7.40 (2H, d, J = 8.2 Hz, ArH), 7.54 (1H, t, J = 7.7
28