Design, synthesis, and biological evaluation of new 1,4-diarylazetidin-2-one derivatives (β-lactams) as selective cyclooxygenase-2 inhibitors

Article abstract of DOI:10.1002/ardp.201900293

A new series of 1,4-diarylazetidin-2-one derivatives (β-lactams) were designed and synthesized to evaluate their biological activities as selective cyclooxygenase-2 (COX-2) inhibitors. In vitro COX-1 and COX-2 inhibition studies showed that all compounds were selective inhibitors of the COX-2 isozyme with IC₅₀ values in the 0.05–0.11 μM range, and COX-2 selectivity indexes in the range of 170–703.7. Among the synthesized β-lactams, 3-methoxy-4-(4-(methylsulfonyl)phenyl)-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (4j) possessing trimethoxy groups at the N-1 phenyl ring exhibited the highest COX-2 inhibitory selectivity and potency, even more potent than the reference drug celecoxib. The analgesic activity of the synthesized compounds was also determined using the formalin test. Compound 4f displayed the best analgesic activity among the synthesized molecules. Molecular modeling studies indicated that the methylsulfonyl pharmacophore group can be inserted into the secondary pocket of the COX-2 active site for interactions with Arg⁵¹³. The structure–activity data acquired indicate that the β-lactam ring moiety constitutes a suitable scaffold to design new 1,4-diarylazetidin-2-ones with selective COX-2 inhibitory activity.

Full text of DOI:10.1002/ardp.201900293

Received: 6 October 2019
Revised: 14 December 2019
Accepted: 17 December 2019
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DOI: 10.1002/ardp.201900293
F U L L P A P E R
Design, synthesis, and biological evaluation of new
1,4‐diarylazetidin‐2‐one derivatives (β‐lactams) as selective
cyclooxygenase‐2 inhibitors
Hadi Arefi1
Mahsa Azami Movahed1
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Nima Naderi2
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Amir B. Irani Shemirani3
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Mina Kiani Falavarjani3
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Afshin Zarghi1
1Department of Pharmaceutical Chemistry,
Shahid Beheshti University of Medical
Sciences, Tehran, Iran
Abstract
A new series of 1,4‐diarylazetidin‐2‐one derivatives (β‐lactams) were designed and
synthesized to evaluate their biological activities as selective cyclooxygenase‐2 (COX‐2)
inhibitors. In vitro COX‐1 and COX‐2 inhibition studies showed that all compounds were
selective inhibitors of the COX‐2 isozyme with IC50 values in the 0.05–0.11 µM range,
and COX‐2 selectivity indexes in the range of 170–703.7. Among the synthesized
β‐lactams, 3‐methoxy‐4‐(4‐(methylsulfonyl)phenyl)‐1‐(3,4,5‐trimethoxyphenyl)azetidin‐2‐
one (4j) possessing trimethoxy groups at the N‐1 phenyl ring exhibited the highest
COX‐2 inhibitory selectivity and potency, even more potent than the reference drug
celecoxib. The analgesic activity of the synthesized compounds was also determined
using the formalin test. Compound 4f displayed the best analgesic activity among the
synthesized molecules. Molecular modeling studies indicated that the methylsulfonyl
pharmacophore group can be inserted into the secondary pocket of the COX‐2 active
site for interactions with Arg513. The structure–activity data acquired indicate that the
β‐lactam ring moiety constitutes a suitable scaffold to design new 1,4‐diarylazetidin‐2‐
ones with selective COX‐2 inhibitory activity.
2Department of Pharmacology and
Toxicology, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
3Student Research Committee, School of
Pharmacy, Shahid Beheshti University of
Medical Sciences, Tehran, Iran
Correspondence
Afshin Zarghi, Department of Pharmaceutical
Chemistry, School of Pharmacy, Shahid
Beheshti University of Medical Sciences,
Tehran 141556153, Iran.
Email: zarghi@sbmu.ac.ir
Funding information
Deputy of Research, School of Pharmacy,
Shahid Beheshti University of Medical
Sciences
K E Y W O R D S
1,4‐diarylazetidin‐2‐ones, antinociceptive activity, cyclooxygenase‐2 inhibition, docking studies,
formalin test, molecular modeling, β‐lactams
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1
INTRODUCTION
this basis, a number of selective COX‐2 inhibitors were developed as
safer NSAIDs with improved gastric safety profile.[3] In addition,
Nonsteroidal anti‐inflammatory drugs (NSAIDs) are among the most
commonly used medications. Through their anti‐inflammatory, anti‐
pyretic, and analgesic activities, they appear as a preferred treatment
in various inflammatory diseases such as arthritis, rheumatisms, as
well as relieving the pains of daily life. Considering that NSAIDs
inhibit both isoforms of cyclooxygenase (COX; constitutive COX‐1,
responsible for cytoprotective effects; inducible COX‐2, responsible
for inflammatory effects), they can have side effects associated with
GI ulcers and renal function suppression.[1,2] Thus, it was thought that
more selective COX‐2 inhibitors would have reduced side effects. On
COX‐2 inhibitors have been shown to reduce the risk of several
cancer types, such as breast, colon, lung, and prostate cancers[4–7]
and neurodegenerative diseases such as Parkinson[8] and
Alzheimer’s[9] diseases. Selective COX‐2 inhibitors such as celecoxib
and rofecoxib (COXIBs) mainly belong to a class of tricyclics that
possess two vicinal diaryl moieties attached to
a five‐ or
six‐membered cyclic scaffold containing a characteristic methane-
sulfonyl, sulfonamido, or azido group on one of the aryl rings that
plays an important role on COX‐2 selectivity.[10–23] However, the
market removal of some COXIBs such as rofecoxib and valdecoxib
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Arch Pharm Chem Life Sci. 2020;e1900293.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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inhibitors having the 1,4‐diarylazetidin‐2‐one (β‐lactam ring) scaffold
as a 4‐membered heterocyclic central ring to evaluate their COX‐2
inhibitory activities. The β‐lactam ring has been widely used as an
active moiety for designing antibiotics,[26] antitumors,[27] thrombin
inhibitors,[28] antihyperglycemic,[29] anti‐HIV,[30] and analgesic
agents[31] due to its safety and activity. As part of our research
program, aimed at discovering new selective COX‐2 inhibitors, we
focused our attention on the synthesis, COX inhibitory, analgesic
activity and some molecular modeling studies of 1,4‐diaryl‐β‐lactams
possessing a methylsulfonyl COX‐2 pharmacophore at the para
position of the C‐4 phenyl ring and different substituents at the N‐1
phenyl ring. The rationale for the design of these compounds was
based on the application of the β‐lactam moiety as a new scaffold for
developing new selective COX‐2 inhibitors.
Celecoxib
Rofecoxib
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2
RESULTS AND DISCUSSION
Chemistry
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2.1
Due to the importance of β‐lactams, several synthetic methods have
been developed for the preparation of the β‐lactam ring.[32] The
Staudinger reaction is regarded as the most fundamental and
versatile method for the synthesis of the β‐lactam ring and is used
for the synthesis of a large number of β‐lactams.[33] This reaction is
known as the [2 + 2] cycloaddition of ketenes to imines. The ketenes
are generally formed in situ by the reaction of acyl halides with
tertiary amines (such as triethylamine, TEA). Due to low stability and
high toxicity of acyl halides, as the ketene precursors, many reagents
such as p‐toluenesulfonyl chloride, ethyl chloroformate, triphosgene,
and Vilsmeier reagent are used to generate ketenes in situ from
acids.[34] In this study, p‐toluenesulfonyl chloride (tosyl chloride) was
used as the acid activator to synthesize 1,4‐diarylazetidin‐2‐ones
from different imine derivatives and methoxy acetic acid in the
presence of trimethylamine (Scheme 1).[34,35]
Designed molecules
FIGURE 1 Chemical structures of celecoxib, rofecoxib, lead
compounds (A and B), and our designed molecules
due to increased risk of heart attack and cardiovascular toxicity[24]
encourages the medicinal chemists to develop new scaffolds for
COX‐2 inhibitory activities with improved safety profiles. For this
reason, novel scaffolds with high selectivity for COX‐2 inhibition
need to be found and evaluated for their biological activities. In this
regard, ring contraction to smaller carbocycles such as cyclobutenes
leads to potent COX‐2 inhibitors (compounds: A and B; Figure 1).
Accordingly, compounds with a cyclobutene central ring show IC50
values for COX‐1 of 0.12 (A) and >5 μM (B), for COX‐2, 0.002 (A) and
0.11 μM (B).[25] Therefore, it is interesting to synthesize new COX‐2
SCHEME 1 Synthesis route for the
target compounds 4a–f. Reagents and
conditions: (a) Dry DMF, 25°C, 24 hr; (b)
dry DMF, TEA, methoxy acetic acid, TsCl,
25°C, 24 hr. DMF, dimethylformamide;
TEA, triethylamine

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FIGURE 3 Superimposition of SC‐558 (blue) and 4j (green) with
COX‐2
FIGURE 2 Docking of 3‐methoxy‐4‐(4‐(methylsulfonyl)phenyl)‐1‐
(3,4,5‐trimethoxyphenyl)azetidin‐2‐one 4j (in green) in the active site
of murine COX‐2. Hydrogen atoms have been removed to improve
clarity
compounds were also represented in Table 1. Overall, most of the
compounds with (3S,4R) stereochemistry showed higher docking scores
than (4S,3R) stereoisomers.
The stereochemistry of products from the Staudinger reaction
depends on numerous factors, including the reaction conditions, the
order of addition of the reagents, and the substituents present on the
imine intermediate. On this basis, we used a modified Staudinger
reaction that leads to β‐lactam with cis selectivity (J3,4 > 4.0 Hz for
the cis and J3,4 < 3.0 Hz for the trans stereoisomers).[36]
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2.3
Biological activity
A group of 1,4‐diarylazetidin‐2‐ones (4a–j) containing SO2Me at the
para position of the C‐4 phenyl ring and a variety of substituents at N‐1
phenyl ring were synthesized to investigate the structure–activity
relationship (SAR) of these compounds. Accordingly, all compounds
were potent and selective inhibitors of the COX‐2 isozyme with IC50
values in the highly potent 0.050–0.108 µM range, and COX‐2
selectivity indices in the 124.7–319.8 range (Table 2). The IC50 values
determined for the in vitro inhibition of COX‐1 and COX‐2 indicated
that the nature and size of substituents on the N‐1 phenyl ring
influenced both selectivity and potency for COX‐2 inhibitory activity.
Our results showed that compounds having methoxy groups (such as
4e, 4i, and 4j) were more potent and selective COX‐2 inhibitors
compared with other analogs. This may be explained by the hydrogen
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2.2
Molecular modeling
The binding interactions of the most selective COX‐2 inhibitor (4j)
within the COX‐2 binding site were investigated. The most
stable enzyme–ligand complex of 3‐methoxy‐4‐(4‐(methylsulfonyl)‐
phenyl)‐1‐(3,4,5‐trimethoxyphenyl)azetidin‐2‐one (4j) possessing
a
methysulfonyl COX‐2 pharmacophore group within the COX‐2 binding
site (Figure 2) shows that the para‐SO2Me substituent inserts into the
secondary pocket present in COX‐2 (Arg513, Phe518, and Val523). One of
the O‐atoms of the SO2Me moiety forms a hydrogen binding with the
NH of Arg513 (distance = 3.3 Å), whereas the other O‐atom is close to
the NH of Phe518 (distance = 3.8 Å). The oxygen atom of the methoxy
group of C‐3 is also close to the NH of Arg120 (distance = 2.5 Å) and,
therefore, can form a hydrogen bond with this amino acid. In addition,
the C═O of the β‐lactam scaffold is almost close to the NH of Ser353
(distance = 4.7 Å), which may form a hydrogen‐bonding interaction. It
was interesting to note that the methoxy groups of N‐1 phenyl ring
formed hydrogen bonds with hydroxyl groups (OH) of Tyr348
(distance = 3.1 Å), Tyr385 (distance = 2.7 Å) and NH of indole ring of
Trp387 (distance = 4.3 Å). Figures 3 and 4 show that (4j) and SC‐558
(co‐crystallized inhibitor) were superimposed tightly in COX‐2 active
site and both had similar interactions with active site amino acids.
Additional docking studies were performed to show the superimposition
of all synthesized compounds and SC‐558, which showed that SO2Me
moiety of all designed compounds was inserted in the secondary pocket
of the COX‐2 active site. The molecular docking scores of synthesized
FIGURE 4 Three‐dimensional superimposed representations of
SC‐558 and the synthesized compounds with COX‐2

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TABLE 1 Docking scores for compounds 4a–j
binding of methoxy groups with COX‐2 active site amino acids.
Subsequently, compounds with mono‐ or di‐Cl or SMe substituent
(4c, 4f, and 4h) showed less selectivity and potency for COX‐2 isozyme,
which may be explained by steric parameters for interaction with
COX‐2 active binding site. In addition, suitable substituents such as F
(4b) or COMe (4g) at the para position of the N‐1 phenyl ring also
increased both selectivity and potency for COX‐2 inhibitory activity. It
seems that
F and COMe would have facilitated charge‐transfer
interaction due to their electron‐withdrawing properties. According to
our results, the ability of the substituent at the N‐1 phenyl ring for
hydrogen binding formation and also charge‐transfer interaction may be
important for COX‐2 inhibitory activity in these series of compounds.
In accordance with our results, 3‐methoxy‐4‐(4‐(methylsulfonyl)phenyl)‐
1‐(3,4,5‐trimethoxyphenyl)azetidin‐2‐one (4j) was the most potent and
selective (IC50 = 0.058 μM, SI = 240.7) COX‐2 inhibitor among the
synthesized compounds. The ability of synthesized compounds for in
vivo analgesic activity, which was evaluated by the formalin test, is also
presented in Table 3. A significant reduction in the AUC of pain score
was seen in groups treated with 4b, 4c, 4d, and 4f (p < 0.001), 4a and 4g
(p < 0.01), 4e and 4j (p < 0.05) compared with the control group.
However, neither 4h nor 4i could change the AUC of pain score
compared with the control group. Therefore, it can be assumed that
disubstituted compounds or compounds with bulky substituents were
not active enough in analgesic activity evaluation. Among active
analgesic compounds, it seems that electron‐donating groups having
Affinity (kcal/ Affinity (kcal/
Compound R1
R2
R3
H
H
H
H
H
H
H
Cl
mol; 3S,4R)
mol; 3R,4S)
4a
4b
4c
4d
4e
4f
H
H
H
H
H
H
H
H
H
H
−7.1
−6.7
F
−7.3
−7.2
Cl
−7.0
−6.8
Me
OMe
SMe
COMe
Cl
−7.2
−5.9
−5.9
−6.0
−5.1
−4.4
4g
4h
4i
−6.4
−6.9
−4.8
−5.5
OMe
OMe −4.4
OMe −4.0
−3.0
4j
OMe OMe
−3.3
TABLE 2 In vitro COX‐1 and COX‐2 enzyme inhibition assay data
IC50 (μM)a
Compound
R1
H
R2
R3
H
COX‐1
11.80
13.82
12.98
12.28
12.84
13.47
13.26
14.23
13.96
15.99
24.30
COX‐2
0.089
0.065
0.083
0.076
0.063
0.108
0.061
0.097
0.058
0.050
0.06
Selectivity index (SI)b
4a
H
132.5
212.5
156.4
186.2
203.8
124.7
217.4
146.7
240.7
319.8
405
4b
H
F
H
4c
H
Cl
H
4d
H
Me
OMe
SMe
COMe
Cl
H
4e
H
H
4f
H
H
4g
H
H
4h
H
Cl
4i
H
OMe
OMe
OMe
OMe
4j
OMe
Celecoxib
aValues are means of two determinations acquired using an ovine COX‐1/COX‐2 assay kit and the deviation from the mean is <10% of the mean value.
bIn vitro COX‐2 selectivity index (COX‐1 IC50/COX‐2 IC50).

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TABLE 3 Effects of pretreatment with celecoxib or new
compounds on pain‐related behaviors of rats in the formalin test
4
EXPERIMENTAL
Chemistry
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4.1
Compound
AUC of pain score (mean SEM)
84.19 4.52
Control
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4.1.1
General
4a
52.95 7.76**
4b
48.58 5.13***
51.75 4.02***
42.73 2.07***
57.90 2.30*
All chemicals and solvents used in this study were purchased from
Merck AG and Aldrich Chemical. Melting points (mps) were
determined with a Thomas–Hoover capillary apparatus. Infrared
(IR) spectra were acquired using a Perkin Elmer Model 1420
4c
4d
4e
4f
34.73 3.33***
50.77 6.84**
spectrometer.
A
Bruker FT‐500 MHz instrument (Bruker
Biosciences) was used to acquire 1H‐NMR spectra with TMS as an
internal standard. Chloroform‐D and dimethyl sulfoxide (DMSO)‐D6
were used as solvents. Coupling constant (J) values are estimated in
hertz (Hz) and spin multiples are given as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), and br (broad). The mass spectral
measurements were performed on a 6410 Agilent LC‐MS triple
quadrupole mass spectrometer (LC‐MS) with an electrospray ioniza-
tion (ESI) interface. Microanalyses, determined for C and H, were
within 0.4% of theoretical values.
4g
4h
98.29 2.11
4i
100.30 1.31
4j
56.82 2.65*
Celecoxib
48.63 10.83***
Note: Data are shown as mean SEM (N = 6).
Abbreviations: AUC, area‐under‐the‐curve; SEM, standard error of the
mean.
*p < 0.05, **p < 0.01, ***p < 0.001—significant difference compared with
the control group.
The original spectra are provided as Supporting Information, as
are the InChI codes of the investigated compounds together with
some biological activity data.
good lipophilicity, such as Me and SMe, could lead to better analgesic
effects, whereas electron‐withdrawing groups, like F, Cl, and COMe,
decreased analgesic effects. Overall, these findings are in good
agreement with the in vitro results. However, in some cases, there
was not a meaningful correlation between in vitro and in vivo results.
This may be explained by the physiochemical and pharmacokinetic
parameters of these compounds, which can influence their in vivo
activities.
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4.1.2
General method for imine formation
A
mixture of 4‐(methylsulfonyl)benzaldehyde 1 (20 mmol) and
appropriate amine 2 (20 mmol) in dry DMF (dimethylformamide;
5 ml) in the presence of molecular sieve was stirred under an argon
atmosphere at 25°C for 48 hr to obtain imine 3. After this time, water
(50 ml) was added and the resulting solution was left to stand until a
solid product crystallized. The resulting imine was recrystallized from
ethanol (yield: 53–72%).
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3
CONCLUSIONS
A series of β‐lactam analogs of 4a–j were synthesized and evaluated
for their COX inhibitory activity using an enzyme chemiluminescent
assay. Our results indicated that 3‐methoxy‐4‐(4‐(methylsulfonyl)‐
phenyl)‐1‐(3,4,5‐trimethoxyphenyl)azetidin‐2‐one 4j was the most
potent (IC50 = 0.05 μM), and selective (SI = 319.8), COX‐2 inhibitor
among the synthesized compounds, which may be due to better
interaction with the COX‐2 active site. It was more potent than
celecoxib (IC50 = 0.06 μM; SI = 405) in terms of COX‐2 inhibitory
activity but showed less selectivity. The analgesic activity of the
synthesized compounds was also determined using the formalin test.
According to our results, 3‐methoxy‐4‐(4‐(methylsulfonyl)phenyl)‐1‐
(4‐(methylthio)phenyl)azetidin‐2‐one (4f) displayed the best analgesic
activity among the synthesized molecules.
N‐(4‐(Methylsulfonyl)benzylidene)aniline (3a)
Yield, 75%; white crystalline powder; mp: 130.5–131.5°C; IR (KBr): ν
(cm−1) 1,624 (C═N), 1,315, and 1,115 (SO2); LC‐MS (ESI) m/z: 260.1
(M+1, 100); Anal. calcd. for C14H13NO2S: C, 64.84; H, 5.05; N, 5.40.
Found: C, 64.62; H, 4.84; N, 5.69.
4‐Fluoro‐N‐(4‐(methylsulfonyl)benzylidene)aniline (3b)
Yield, 75%; off‐white crystalline powder; mp: 140–141°C; IR (KBr): ν
(cm−1) 1,632 (C═N), 1,312, and 1,150 (SO2); LC‐MS (ESI) m/z: 278.1
(M+1, 100); Anal. calcd. for C14H12FNO2S: C, 60.63; H, 4.36; N, 5.05.
Found: C, 60.34; H, 4.24; N, 5.29.
Our results indicated that (i) β‐lactam ring is a suitable scaffold
(template) to design COX‐1/‐2 inhibitors, (ii) COX‐1/‐2 inhibition is
sensitive to the substituent of N‐1 phenyl ring, (iii) small electron‐
donating groups such as Me and SMe could lead to better analgesic
effects.
4‐Chloro‐N‐(4‐(methylsulfonyl)benzylidene)aniline (3c)
Yield, 80%; white crystalline powder; mp: 147–148°C; IR (KBr): ν
(cm−1) 1,635 (C═N), 1,322, and 1,145 (SO2); LC‐MS (ESI) m/z: 294.1
(M+1, 100); Anal. calcd. for C14H12ClNO2S: C, 57.24; H, 4.12; N, 4.77.
Found: C, 57.34; H, 4.29; N, 4.51.

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Methyl‐N‐(4‐(methylsulfonyl)benzylidene)aniline (3d)
3‐Methoxy‐4‐(4‐(methylsulfonyl)phenyl)‐1‐phenylazetidin‐2‐one (4a)
Yield; 65%; off‐white crystalline powder; mp: 187.5–188.5°C;
IR (KBr): ν (cm−1) 1,750 (C═O), 1,312, and 1,153 (SO2); 1H‐NMR
(CDCl3): δ ppm 3.12 (s, 3H, SO2CH3), 3.32 (s, 3H, OCH3), 4.92 (d, 1H,
CH, J = 5.0 Hz), 5.3 (d, 1H, CH, J = 5.0 Hz), 7.14–7.15 (m, 1H, phenyl
H4), 7.30–7.32 (m, 4H, phenyl H2–H6), 7.64 (d, 2H, 4‐
methylsulfonylphenyl H2 and H6, J = 8.3 Hz), and 8.0 (d, 2H, 4‐
methylsulfonylphenyl H3 and H5, J = 8.3 Hz); 13C‐NMR (CDCl3): δ
ppm 44.39, 58.85, 60.92, 84.88, 117.30, 124.85, 127.70, 128.89,
129.32, 136.57, 140.05, 140.76, and 163.88; LC‐MS (ESI) m/z: 332.1
(M+1, 100); Anal. calcd. for C17H17NO4S: C, 61.61; H, 5.17; N, 4.23.
Found: C, 61.47; H, 4.98; N, 3.97.
Yield, 81%; pale yellow crystalline powder; mp: 184–185°C; IR (KBr):
ν (cm−1) 1,632 (C═N), 1,318, and 1,160 (SO2); LC‐MS (ESI) m/z: 274.1
(M+1, 100); Anal. calcd. for C15H15NO2S: C, 65.91; H, 5.53; N, 5.12.
Found: C, 65.77; H, 5.24; N, 5.01.
4‐Methoxy‐N‐(4‐(methylsulfonyl)benzylidene)aniline (3e)
Yield, 82%; off‐white crystalline powder; mp: 216–217°C; IR (KBr): ν
(cm−1) 1,629 (C═N), 1,310, and 1,157 (SO2); LC‐MS (ESI) m/z: 290.1
(M+1, 100); Anal. calcd. for C15H15NO3S: C, 62.26; H, 5.22; N, 4.84.
Found: C, 62.39; H, 5.01; N, 5.03.
4‐Methylthio‐N‐(4‐(methylsulfonyl)benzylidene)aniline (3f)
Yield, 91%; off‐white crystalline powder; mp: 240°C; IR (KBr): ν
(cm−1) 1,631 (C═N), 1,310, and 1,148 (SO2); LC‐MS (ESI) m/z: 306.1
(M+1, 100); Anal. calcd. for C15H15NO2S2: C, 58.99; H, 4.95; N, 4.59.
Found: C, 59.22; H, 4.77; N, 4.33.
1‐(4‐Fluorophenyl)‐3‐methoxy‐4‐(4‐(methylsulfonyl)phenyl)azetidin‐
2‐one (4b)
Yield: 53%; brown crystalline powder; mp: 136.5–138.5°C; IR (KBr): ν
(cm−1) 1,743 (C═O), 1,309, and 1,150 (SO2); 1H‐NMR (CDCl3): δ ppm
3.14 (s, 3H, SO2CH3), 3.32 (s, 3H, OCH3), 4.93 (d, 1H, CH, J = 4.9 Hz),
5.32 (d, 1H, CH, J = 4.9 Hz), 7.28–7.32 (m, 4H, 4‐fluorophenyl H2–H6),
8.09 (d, 2H, 4‐methylsulfonylphenyl H2 and H6, J = 8.4 Hz), and 8.13
(d, 2H, 4‐methylsulfonylphenyl H3 and H5, J = 8.4 Hz); 13C‐NMR
(CDCl3): δ ppm 44.46, 58.88, 61.15, 85.09, 116.23, 122.53, 127.87,
128.89, 129.42, 140.75, 142.45, 157.45, and 163.59; LC‐MS (ESI)
m/z: 350.1 (M+1, 100); Anal. calcd. for C17H16FNO4S: C, 58.44; H,
4.62; N, 4.01. Found: C, 58.71; H, 4.50; N, 4.25.
4‐Acetyl‐N‐(4‐(methylsulfonyl)benzylidene)aniline (3g)
Yield, 75%; white crystalline powder; mp: 188–189°C; IR (KBr): ν
(cm−1) 1,634 (C═N), 1,312, and 1,150 (SO2); LC‐MS (ESI) m/z: 302.1
(M+1, 100); Anal. calcd. for C16H15NO3S: C, 63.77; H, 5.02; N, 4.65.
Found: C, 63.99; H, 4.88; N, 4.87.
3,4‐Dichloro‐N‐(4‐(methylsulfonyl)benzylidene)aniline (3h)
Yield, 80%; white crystalline powder; mp: 179–180°C; IR (KBr): ν
(cm−1) 1,630 (C═N), 1,310, and 1,140 (SO2); LC‐MS (ESI) m/z: 328.0
(M+1, 100); Anal. calcd. for C14H11Cl2NO2S: C, 65.35; H, 4.31; N,
5.44. Found: C, 65.21; H, 4.51; N, 5.77.
1‐(4‐Chlorophenyl)‐3‐methoxy‐4‐(4‐(methylsulfonyl)phenyl)azetidin‐
2‐one (4c)
Yield, 65%; white crystalline powder; mp: 229–231°C; IR (KBr): ν
(cm−1) 1,755 (C═O), 1,322, and 1,164 (SO2); 1H‐NMR (CDCl3): δ ppm
3.12 (s, 3H, SO2CH3), 3.31 (s, 3H, OCH3), 4.93 (d, 1H, CH, J = 4.9 Hz),
5.32 (d, 1H, CH, J = 4.9 Hz), 7.23–7.30 (m, 4H, 4‐Chlorophenyl
H2–H6), 7.61 (d, 2H, 4‐methylsulfonylphenyl H2 and H6, J = 8.2 Hz),
and 7.90 (d, 2H, 4‐methylsulfonylphenyl H3 and H5, J = 8.2 Hz); 13C‐
NMR (CDCl3): δ ppm 44.39, 58.92, 61.06, 85.10, 118.52, 127.80,
128.86, 130.02, 135.06. 139.56, 140.99, and 163.77; LC‐MS (ESI) m/
z: 366.1 (M+1, 100); Anal. calcd. for C17H16ClNO4S: C, 55.81; H, 4.41;
N, 3.83. Found: C, 60.05; H, 4.74; N, 4.12.
3,4‐Dimethoxy‐N‐(4‐(methylsulfonyl)benzylidene)aniline (3i)
Yield, 74%; yellow crystalline powder; mp: 157–158°C; IR (KBr): ν
(cm−1) 1,632 (C═N), 1,313, and 1,144 (SO2); LC‐MS (ESI) m/z: 320.1
(M+1, 100); Anal. calcd. for C16H17NO4S: C, 60.17; H, 5.36; N, 4.39.
Found: C, 60.35; H, 5.55; N, 4.55.
3,4,5‐Trimethoxy‐N‐(4‐(methylsulfonyl)benzylidene)aniline (3j)
Yield, 83%; yellow crystalline powder; mp: 179–180°C; IR (KBr): ν
(cm−1) 1,628 (C═N), 1,310, and 1,158 (SO2); LC‐MS (ESI) m/z: 320.1
(M+1, 100); Anal. calcd. for C17H19NO5S: C, 61.24; H, 5.74; N, 4.20.
Found: C, 61.39; H, 5.50; N, 4.44.
3‐Methoxy‐4‐(4‐(methylsulfonyl)phenyl)‐1‐(p‐tolyl)azetidin‐2‐one
(4d)
Yield, 69%; white crystalline powder; mp: 197–198°C; IR (KBr): ν
(cm−1) 1,754 (C═O), 1,320, and 1,163 (SO2); 1H‐NMR (CDCl3): δ
ppm 2.32 (s, 3H, CH3), 3.11 (s, 3H, SO2CH3), 3.30 (s, 3H, OCH3),
4.91 (d, 1H, CH, J = 4.9 Hz), 5.32 (d, 1H, CH, J = 4.9 Hz), 7.11 (d, 2H,
4‐methylphenyl H2 and H6, J = 8.4 Hz), 7.20 (d, 2H, 4‐methylphenyl
H3 and H5, J = 8.4 Hz), 7.62 (d, 2H, 4‐methylsulfonylphenyl H2 and
H6, J = 8.3 Hz), and 7.99 (d, 2H, 4‐methylsulfonylphenyl H3 and H5,
J = 8.3 Hz); 13C‐NMR (CDCl3): δ ppm 20.92, 44.40, 58.81, 60.89,
84.90, 117.24, 127.66, 128.89, 129.79, 134.13, 134.60, 140.21,
140.69, and 163.61; LC‐MS (ESI) m/z: 346.1 (M+1, 100); Anal. calcd.
for C18H19NO4S: C, 62.59; H, 5.54; N, 4.05. Found: C, 62.88; H,
5.81; N, 4.19.
|
4.1.3
General procedure for the preparation of
β‐lactams (4a–j)
Imine 3 (30 mmol) was dissolved in dry DMF (5 ml) and TEA (100 mmol),
tosyl chloride (30 mmol) and methoxy acetic acid (30 mmol) were added
and stirred at room temperature for 24 hr. After this time, water was
added and the resulting precipitate was filtered and recrystallized
in ethanol (yield: 53–72%). The cis and trans stereochemistries of
2‐azetidinones were concluded from coupling constants of H‐3 and H‐4
(J 3,4 > 4.0 Hz for the cis and J 3,4 < 3.0 Hz for the trans stereoisomer).

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3‐Methoxy‐1‐(4‐methoxyphenyl)‐4‐(4‐(methylsulfonyl)phenyl)‐
azetidin‐2‐one (4e)
4‐methylsulfonylphenyl H3 and H5, J = 8.3 Hz); 13C‐NMR (CDCl3): δ
ppm 44.39, 58.97, 61.18, 85.23, 116.47, 119.08, 122.72, 127.94,
128.82, 129.68, 131.01, 135.79, 139.11, 140.22, and 163.90; LC‐MS
(ESI) m/z: 400.1 (M+1, 100); Anal. calcd. for C17H15Cl2NO4S: C,
61.99; H, 4.59; N, 4.25. Found: C, 70.19; H, 4.28; N, 4.53.
Yield, 70%; light brown crystalline powder; mp: 170–171°C; IR (KBr): ν
(cm−1) 1,750 (C═O), 1,324, and 1,164 (SO2); 1H‐NMR (CDCl3): δ ppm
3.12 (s, 3H, SO2CH3), 3.30 (s, 3H, OCH3), 3.79 (s, 3H, OCH3), 4.91 (d,
1H, CH, J = 4.9 Hz), 5.30 (d, 1H, CH, J = 4.9 Hz), 6.84 (d, 2H,
4‐methoxyphenyl H3 and H5, J = 6.9 Hz), 7.25 (d, 2H, 4‐methoxyphenyl
H2 and H6, J = 6.9 Hz), 7.62 (d, 2H, 4‐methylsulfonylphenyl H2 and H6,
J = 8.3 Hz), and 7.9 (d, 2H, 4‐methylsulfonylphenyl H3 and H5,
J = 8.3 Hz); 13C‐NMR (CDCl3): δ ppm 44.39, 55.45, 58.80. 61.04,
84.98, 114.50, 118.63, 127.67, 129.17, 130.03, 140.22, 140.72,
156.64, and 163.28; LC‐MS (ESI) m/z: 362.1 (M+1, 100); Anal. calcd.
for C18H19NO5S: C, 59.82; H, 5.30; N, 3.97. Found: C, 60.05; H, 5.49;
N, 4.21.
1‐(3,4‐Dimethoxyphenyl)‐3‐methoxy‐4‐(4‐(methylsulfonyl)phenyl)‐
azetidin‐2‐one (4i)
Yield, 58%; yellow crystalline powder; mp: 154–155°C; IR (KBr): ν
(cm−1) 1,750 (C═O), 1,312, and 1,141 (SO2); 1H‐NMR (CDCl3): δ ppm,
3.12 (s, 3H, SO2CH3), 3.13 (s, 3H, OCH3), 3.96 (s, 3H, OCH3), 3.98 (s,
3H, OCH3), 4.91 (d, 1H, CH, J = 4.8 Hz), 5.30 (d, 1H, CH, J = 4.8 Hz),
6.95 (m, 2H, 3,4‐dimethoxyphenyl H5 and H6), 6.98 (s, 1H, 3,4‐
dimethoxyphenyl H2), 7.63 (d, 2H, 4‐methylsulfonylphenyl H2 and H6,
J = 8.1 Hz), and 8.01 (d, 2H, 4‐methylsulfonylphenyl H3 and H5,
J = 8.09 Hz); 13C‐NMR (CDCl3): δ ppm 44.42, 56.12, 56.36, 58.53,
61.05, 84.88, 109.37, 112.32, 114.96, 127.72, 128.15, 135.12,
139.05, 145.58, 148.77, 151.05, and 163.79; LC‐MS (ESI) m/z:
392.1 (M+1, 100); Anal. calcd. for C19H21NO6S: C, 58.30; H, 5.41;
N, 3.58. Found: C, 58.02; H, 5.11; N, 3.22.
3‐Methoxy‐4‐(4‐(methylsulfonyl)phenyl)‐1‐(4‐(methylthio)phenyl)‐
azetidin‐2‐one (4f)
Yield, 72%; light yellow crystalline powder; mp: 169–171°C; IR (KBr): ν
(cm−1) 1,743 (C═O), 1,317, and 1,157 (SO2); 1H‐NMR (CDCl3): δ ppm
2.47 (s, 3H, SCH3), 3.12 (s, 3H, SO2CH3), 3.31 (s, 3H, OCH3), 4.92 (d, 1H,
CH, J = 4.9 Hz), 5.32 (d, 1H, CH, J = 4.9 Hz), 7.19–7.30 (dd, 4H,
4‐methylthiophenyl H2–H6), 7.62 (d, 2H, 4‐methylsulfonylphenyl H2
and H6, J = 8.2 Hz), and 7.9 (d, 2H, 4‐methylsulfonylphenyl H3 and H5,
J = 8.23 Hz); 13C‐NMR (CDCl3): δ ppm 16.03, 44,39, 58.85, 60.95, 84.98,
117.86, 121.69, 127.84, 128.88, 134.00, 134.66, 139.91, 140.81, and
163.63; LC‐MS (ESI) m/z: 378.1 (M+1, 100); Anal. calcd. for
C18H19NO4S2: C, 57.27; H, 5.07; N, 3.71. Found: C, 57.45; H, 5.41;
N, 3.52.
3‐Methoxy‐4‐(4‐(methylsulfonyl)phenyl)‐1‐(3,4,5‐trimethoxyphenyl)‐
azetidin‐2‐one (4j)
Yield, 66%; yellow crystalline powder; mp: 156–157°C; IR (KBr): ν
(cm−1) 1,750 (C═O), 1,303, and 1,146 (SO2); 1H‐NMR (CDCl3): δ
ppm 3.12 (s, 3H, SO2CH3), 3.14 (s, 3H, OCH3), 3.7 (s, 6H,
3,4,5‐trimethoxyphenyl H3 and H5), 3.81 (s, 3H, 3,4,5‐
trimethoxyphenyl H4), 4.91 (d, 1H, CH, J = 4.9 Hz), 5.31 (d, 1H,
CH, J = 4.9 Hz), 6.55 (s, 2H, 3,4,5‐trimethoxyphenyl H2 and H6),
7.64 (d, 2H, 4‐methylsulfonylphenyl H2 and H6, J = 8.3 Hz), and
8.01 (d, 2H, 4‐methylsulfonylphenyl H3 and H5, J = 8.3 Hz);
13C‐NMR (CDCl3): δ ppm 44.46, 56.18, 56.43, 58.84, 61.04,
84.78, 115.11, 126.13, 127.83, 128.19, 129.35, 130.97, 137.77,
141.19, 142.17, 150.05, and 163.71; LC‐MS (ESI) m/z: 422.1
(M+1, 100); Anal. calcd. for C20H23NO7S: C, 57.00; H, 5.50; N,
3.32. Found: C, 56.77; H, 5.79; N, 3.35.
1‐(4‐Acetylphenyl)‐3‐methoxy‐4‐(4‐(methylsulfonyl)phenyl)azetidin‐
2‐one (4g)
Yield, 67%; light yellow crystalline powder; mp: 170–171°C; IR (KBr): ν
(cm−1) 1,740 (C═O), 1,668 (C═O), 1,379, and 1,147 (SO2); 1H‐NMR
(CDCl3): δ ppm 2.58 (s, 3H, COMe), 3.12 (s, 3H, SO2CH3), 3.37 (s,
3H, OCH3), 4.97 (d, 1H, CH, J = 5.1 Hz), 5.40 (d, 1H, CH, J = 5.1 Hz),
7.37 (d, 2H, 4‐acetylphenyl H2 and H6, J = 8.3 Hz), 7.62 (d, 2H,
4‐methylsulfonylphenyl H2 and H6, J = 8.7 Hz), 7.93 (d, 2H,
4‐methylsulfonylphenyl H3 and H5, J = 8.3 Hz), and 8.01 (d, 2H, 4‐
acetylphenyl H3 and H5, J = 8.7 Hz); 13C‐NMR (CDCl3): δ ppm 26.09,
44.38, 58.98, 61.13, 85.10, 120.89, 127.94, 128.85, 129.93, 133.39,
139.37, 140.22, 141.03, 164.21, and 196.55; LC‐MS (ESI) m/z: 374.1
(M+1, 100); Anal. calcd. for C19H19NO5S: C, 61.11; H, 5.13; N, 3.75.
Found: C, 61.38; H, 5.42; N, 4.00.
|
4.2
Molecular modeling
Docking studies were performed using AutoDock software version
3.0. The coordinates of the X‐ray crystal structure of the selective
COX‐2 inhibitor SC‐558 bound to the murine COX‐2 enzyme were
obtained from the RCSB Protein Data Bank (1cx2) and hydrogens
were added. The ligand molecules were constructed using the Builder
module and were energy‐minimized for 1,000 iterations reaching a
convergence of 0.01 kcal/mol Å. The energy‐minimized ligands were
superimposed on SC‐558 in the PDB file 1cx2 after which SC‐558
was deleted. The purpose of docking is to search for favorable
binding configurations between the small flexible ligands and the
rigid protein. Protein residues with atoms greater than 7.5 Å from the
docking box were removed for efficiency. These docked structures
were very similar to the minimized structures obtained initially. The
1‐(3,4‐Dichlorophenyl)‐3‐methoxy‐4‐(4‐(methylsulfonyl)phenyl)‐
azetidin‐2‐one (4h)
Yield, 70%; white crystalline powder; mp 165–167°C; IR (KBr): ν
(cm−1) 1,748 (C═O), 1,383, and 1,151 (SO2); 1H‐NMR (CDCl3): δ ppm
3.12 (s, 3H, SO2CH3), 3.30 (s, 3H, OCH3), 4.94 (d, 1H, CH, J = 5.0 Hz),
5.33 (d, 1H, CH, J = 5.0 Hz), 7.09–7.11 (dd, 1H, 3,4‐dichlorophenyl H6
J = 8.7 Hz, J = 2.4 Hz), 7.36 (d, 1H, 3,4‐dichlorophenyl H5, J = 8.7 Hz),
7.49 (d, 1H, 3,4‐dichlorophenyl H2, J = 2.4 Hz), 7.60 (d, 2H,
4‐methylsulfonylphenyl H2 and H6, J = 8.3 Hz), and 8.01 (d, 2H,

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AREFI ET AL.
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quality of the docked structures was evaluated by measuring the
ACKNOWLEDGMENT
intermolecular energy of the ligand–enzyme assembly.[37,38]
We would like to thank Deputy of Research, School of Pharmacy,
Shahid Beheshti University of Medical Sciences for financial support
of this study as part of Ph.D. Thesis of Hadi Arefi.
|
4.3
Biological assays
|
4.3.1
In vitro COX inhibition assays
CONFLICT OF INTERESTS
The assay was performed using an enzyme chemiluminescent kit
(Cayman Chemical, MI) according to our previously reported
method.[39] The Cayman chemical chemiluminescent COX
(ovine) inhibitor screening assay utilizes the heme‐catalyzed
hydroperoxidase activity of ovine cyclooxygenases to generate
luminescence in the presence of a cyclic naphthalene hydrazide
and the substrate arachidonic acid. Arachidonate‐induced
luminescence was shown to be an index of real‐time catalytic
activity and demonstrated the turnover inactivation of the
enzyme. Inhibition of COX activity, measured by luminescence,
by a variety of selective and nonselective inhibitors showed
potencies similar to those observed with other in vitro and
whole‐cell methods.
The authors declare that there are no conflicts of interests.
ORCID
Afshin Zarghi
http://orcid.org/0000-0003-2477-9533
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How to cite this article: Arefi H, Naderi N, Shemirani ABI,
Kiani Falavarjani M, Azami Movahed M, Zarghi A.
Design, synthesis, and biological evaluation of new
1,4‐diarylazetidin‐2‐one derivatives (β‐lactams) as selective
cyclooxygenase‐2 inhibitors. Arch Pharm Chem Life Sci.
2020;e1900293. https://doi.org/10.1002/ardp.201900293