Cytotoxicity, anticancer, and antioxidant properties of mono and bis-naphthalimido β-lactam conjugates

MEDICINAL

CHEMISTRY

RESEARCH

Medicinal Chemistry Research

https://doi.org/10.1007/s00044-020-02552-1

ORIGINAL RESEARCH

Cytotoxicity, anticancer, and antioxidant properties of mono

and bis-naphthalimido β-lactam conjugates

1

Nassim Borazjani¹Maryam Behzadi²Marzieh Dadkhah Aseman³Aliasghar Jarrahpour

Javad Ameri Rad¹

●

Sedigheh Kianpour^2,4Aida Iraji⁵S. Masoud Nabavizadeh¹Mohammad Mehdi Ghanbari⁶Gyula Batta⁷

Edward Turos⁸

●

Received: 24 February 2020 / Accepted: 27 April 2020

Abstract

This article reports the diastereoselective synthesis of some novel naphthalimido and bis-naphthalimido β-lactam derivatives

and a preliminary evaluation of their anticancer properties. The reactions were completely diastereoselective, leading

exclusively to the formation of cis-β-lactams 11a–l and trans-bis-β-lactams 16a–g. All of these compounds were obtained in

good to excellent yields and their structures were established based on IR, ¹H NMR, ¹³C NMR spectral data, and elemental

analysis. Each of the β-lactams was screened for antioxidant and anticancer activities. Our results showed that all the

compounds lacked cytotoxicity against HepG2 cells, whereas 16a and 16b exhibited excellent anticancer activity with IC₅₀

values below 191.57 µM on MCF-7 cell line and also, bis-β-lactams 16a–g showed excellent antitumor activity against the

TC-1 cell line. Antioxidant experiments of 16a–d by the diphenylpicrylhydrazyl (DPPH) assay showed IC₅₀values ranging

from 7 to 32.3 µg/ml. Interaction of 16a, 16b, 16d–g with calf-thymus DNA (CT-DNA) was also supported by absorption

titration studies. The compounds exhibit good binding propensity to CT-DNA and the DNA binding afﬁnity (K_b) of the

compounds varies as 16a; 16b; 16e; 16g > 16d; 16f. Interaction of 16d with CT-DNA was also investigated by ﬂuorescence

spectroscopy. The results support an intercalative interaction of 16d and 16f and non-intercalation mechanism for 16a, 16b,

16e, and 16g.

●

Keywords Anticancer Antioxidant Cytotoxicity Diastereoselective β-Lactam

Abbreviations

ORTEP

Oak Ridge Thermal Ellipsoid Plot

MCF-7

TC-1

HepG2

DPPH

Breast cancer cells

CT-DNA Calf thymus-deoxyribonucleic acid

Mouse lung epithelial cells

Liver hepatocellular carcinoma

Diphenylpicrylhydrazyl

K_b

binding afﬁnity

IC50

NI

Half maximal inhibitory concentration

Naphthalimide

NDI

MTT

OD

1,4,5,8-Naphthalenetetracarboxylicdiimide

Methyl thiazol tetrazolium bromide

Optical density

Supplementary information The online version of this article (https://

doi.org/10.1007/s00044-020-02552-1) contains supplementary

material, which is available to authorized users.

* Aliasghar Jarrahpour

5

Central Research Laboratory, Shiraz University of Medical

Sciences, Shiraz, Iran

jarahpor@shirazu.ac.ir

6

7

8

1

Department of Chemistry,Sarvestan Branch, Islamic Azad

University, Sarvestan, Iran

Department of Chemistry, College of Sciences, Shiraz University,

Shiraz 71946-84795 I.R., Iran

2

Department of Chemistry, University of Debrecen, Debrecen

Egyetem tér 1, Debrecen 4032, Hungary

Pharmaceutical Research Center, Shiraz University of Medical

Sciences, Shiraz, Iran

3

Center for Molecular Diversity in Drug Design, Discovery, and

Delivery, Department of Chemistry, University of South Florida,

CHE 205, 4202 East Fowler Avenue, Tampa, FL 33620, USA

Department of Chemistry, Kharazmi University,

Tehran 1983969411, Iran

4

Biotechnology Research Center, Shiraz University of Medical

Sciences, Shiraz, Iran

Medicinal Chemistry Research

Fig. 1 Structures of biologically-

active cyclic imides and selected

DNA-intercalating agents

Introduction

major or minor grooves of DNA, and (3) intercalation

between stacked base pairs (Tomczyk and Walczak 2018).

Two examples of DNA-intercalating agents, amonaﬁde C

and elinaﬁde D (Fig. 1), were initially identiﬁed as having

promising anticancer properties but did not pass Phase II

trials due to elevated toxicity. Several attempts have been

made to overcome this limitation by synthesizing various

analogues (Ge et al. 2017; Tumiatti et al. 2009). 1,8-

Naphthalimide derivatives such as C and D are also known

for their strong ﬂuorescence properties and are used as

pigments in polymer industries, ﬂuorescent probes for

biological purposes and medical, DNA fragmentation fac-

tors, crystalline liquid additions, potential anti-HIV drugs,

and laser colors (Xiao et al. 2010).

The β-lactam antibiotics, most notably the penicillins,

cephalosporins, monobactams, and the penicillinase inhi-

bitor, clavulanic acid, has led to worldwide applications

toward the control of infectious bacterial diseases. Besides

their recognized antibacterial properties, β-lactams are also

known for an even wider range of biological activities such

as anticancer, anti-inﬂammatory, antimalarial, and anti-

tubercular. β-Lactams have also been used as prodrugs to

deliver cancer chemotherapeutic agents directly to a tumor

site (Geesala et al. 2016) and as synthetic building blocks.

β-Lactam derivatives have been also reported to increase

DNA damage and lead to apoptosis of T cells in human

leukemia cells. Interestingly, one of the β-lactams has

inhibited cell proliferation and has induced apoptosis in

several tumor cell lines (Arya et al. 2014; Parul et al. 2010;

Galletti et al. 2014). An emerging strategy in drug discovery

campaigns is that of the pharmacophore hybridization, in

which two independently bioactive moieties are covalently

joined into a single molecular unit. Some examples include

antibacterial hybrid E (Borazjani et al. 2019a, 2019b),

anticancer hybrid F (Borazjani et al. 2019a, 2019b), anti-

inﬂammatory hybrid G (Borazjani et al. 2019a, 2019b), and

Cancer is a collection of diseases characterized by abnormal

cell growth and the possibility of attacking other parts of the

body. In 2015, the World Health Organization reported the

ﬁve most common cancer deaths worldwide, including liver

cancer, colorectal cancer, lung cancer, breast cancer, and

gastric cancer (Miller et al. 2016). Although a plethora of

anticancer drugs are commercially available, most have

serious side effects. In addition, there is an urgent need to

discover and to synthesize new anticancer drugs (Kumar

et al. 2017). The imide moiety is an integral part of the

structures of some of the most important anticancer drugs,

such as uramustine (Baraldi et al. 2002), granulatimide A

(Berlinck et al. 1998), and rebeccamycin B (Zhang et al.

2005) (Fig. 1). Cyclic imide derivatives have a wide range

of biological activities such as antimicrobial (Anizon et al.

1997), antitumor (Henon et al. 2007; Laronze et al. 2005),

anti-inﬂammatory activity (Amr et al. 2007), antioxidative

and anticonvulsant activities (Abdel-Aziz et al. 2011;

Machado et al. 2011; El-Azab et al. 2013), and serve as

inhibitors of N-aminopeptidase (Li et al. 2010) and Myco-

bacterium tuberculosis protein tyrosine phosphatase B (De

Oliveira et al. 2011). Bis cyclic imides likewise have anti-

inﬂammatory, anticancer, analgesic, and anticoagulant

activities (Arya et al. 2013; Said et al. 2009). Naphthalimide

(NI) and 1,4,5,8-naphthalenetetracarboxylic diimide (NDI)

act as duplex DNA intercalators and have shown anticancer

activity against several human cancer cell lines. DNA is the

carrier of genetic information involved in gene expression,

protein synthesis, and cell growth and division. As a result,

DNA is highly regarded as an important target for the

design of new anticancer drugs. Many compounds exert

anticancer effects through binding to duplex DNA, through

three general modes: (1) interactions with the anionic

phosphates in the DNA backbone, (2) interactions with the

Medicinal Chemistry Research

Fig. 2 Examples of hybrid

compounds with antibacterial,

anticancer, anti-inﬂammatory

and antimalarial activity

antimalarial hybrid H (Alborz et al. 2018) (Fig. 2). Impor-

tant motivations for expanding upon this concept include

the creation of new chemical constructs with enhanced

biological activity that can circumvent drug resistance or the

use of active transport mechanisms that add therapeutic

value in terms of potency, breadth of bioactivity, improved

pharmacokinetics, or delay in the onset of drug resistance

(Baraldi et al. 2007; Wang et al. 2012). Therefore, we

decided to use the molecular hybridization strategy by

exploiting the rich chemistry and known biological effec-

tiveness of β-lactams and 1,8-naphthalimides. In addition to

synthesizing representative structures, we also were inter-

ested in studying their anticancer and antioxidant activities

as well as potential cytotoxicity.

acetonitrile to afford 4-(3-(1,3-dioxo-1H-benzo[de]iso-

quinolin-2(3H)-yl)propoxy)benzaldehyde (7) in 95% yield.

1

The structure of compound 7 was characterized by IR, H

NMR, and ¹³C NMR spectroscopy and elemental analysis

data. For example, the IR spectrum of 7 showed the char-

acteristic stretching absorption for the aldehyde carbonyl at

1674 cm⁻¹and twin absorption bands for the naphthalimide

1

carbonyl groups at 1699 and 1657 cm⁻¹. The H NMR

spectrum of compound 7 exhibited a singlet at δ 9.82 for the

aldehyde proton, as well as all the expected resonances for

the other protons. The ¹³C NMR spectral data for compound

7 gave a signal at δ 191.2 for the aldehyde carbon.

Aldehyde 7, when treated with aniline derivatives 8a–c

in ethanol, readily afforded the corresponding N-aryl imines

9a–c. The IR spectrum of 9a shows the expected absorption

for the imine (CH=N) at 1624 cm⁻¹and absorption bands

for the naphthalimide carbonyl groups at 1696 and 1659 cm⁻¹.

Results and discussion

1

The H NMR spectrum of 9a displayed a signal at δ 8.43

Chemistry

corresponding to the imine proton (CH=N). Imines 9a–c

were then subsequently reacted with various phenoxyacetic

acid derivatives 10a–d in the presence of triethylamine and

tosyl chloride, in molar ratios of 1:1.5:5:1.5, in anhydrous

CH₂Cl₂(Scheme 1)_.These reactions led to the stereo-

selective formation of naphthalimido cis-β-lactams 11a–l in

good to excellent yields (75–95%, Table 1).

These cycloaddition reactions were totally diaster-

eoselective and afforded the cis stereoisomers as the only

products, as an unresolved racemic mixture. The structures

of β-lactams 11a–l were characterized by elemental analysis

and IR, ¹H NMR, and ¹³C NMR spectroscopy. As a

representative example, the IR spectrum of 11a showed the

characteristic absorption of a sharp band of β-lactam car-

bonyl at 1751 cm⁻¹. The C=O absorption band of

In this study, twelve novel naphthalimido cis-β-lactams

11a–l were synthesized by the Staudinger reaction of one of

three N-aryl imines 9a–c with various aryloxyacetic acids

10a–d, shown in Scheme 1 (Palomo et al. 2004). In the ﬁrst

step, 1,8-naphthalimide (3) was prepared in 95% yield by

the reaction of commercially-available 1,8-naphthalic

anhydride (1) and ammonium acetate (2) at 60 °C using

DMF as solvent. Then, a mixture of 1,8-naphthalimide 3,

1,3-dibromopropane (4) and K₂CO₃stirred together in

DMF at room temperature afforded bromo-N-propyl-1,8-

naphthalimide (5) in 90% yield (Kamal et al. 2002).

Recrystallized compound

hydroxybenzaldehyde (6) in the presence of K₂CO₃in

5

was treated with 4-

Medicinal Chemistry Research

Scheme 1 Synthesis of naphthalimido cis-β-lactams 11a–l

naphthalimide carbonyl groups appeared at 1697 and

of triethylamine and p-toluenesulfonyl in anhydrous

dichloromethane to afford bis-naphthalimido β-lactams

16a–g in 40–68% yields (Scheme 2).

1658 cm⁻¹. The cis stereochemistry of 11a was deduced

1

readily from the H NMR spectrum, by the β-lactam ring’s

proton H-4 showing up as a doublet at δ 5.59 with J =

4.7 Hz and H-3 appearing as a doublet at δ 5.76 with J =

4.7 Hz (Fig. 3). (J_3,4< 3.0 Hz for the trans and J_3,4> 4.0 Hz

for the cis stereoisomer) (Ameri Rad et al. 2017). The IR,

¹H NMR, and ¹³C NMR spectroscopic data of compounds

11a–l are presented in the supporting information.

The structures of the bis-cycloaddition products were

1

characterized by IR, H NMR, ¹³C NMR spectral data, and

elemental analysis data (see Supplementary Information).

As an illustration, the IR spectrum of bis-β-lactam 16e

showed a sharp β-lactam C=O absorption band centered at

1759 cm⁻¹, as well as twin C=O absorption bands for the

naphthalimide carbonyl groups at 1712 and 1674 cm⁻¹. The

¹H NMR spectrum of 16e exhibited separate doublets at δ

5.42 and 5.94 for the vicinal protons on each β-lactam ring,

whose coupling constants of ³J_HH= 2.5 Hz indicates a trans

disubstitution of the β-lactam ring (Fig. 5). Analogously, the

substitution patterns on both β-lactam rings of all the bis-

β-lactams 16a–f have been assigned trans.

Although we depict only one structure for 16e in which

both β-lactam rings have trans stereochemistry, we cannot

deﬁnitively determine the relative stereochemistry across

the naphthalimide ring system as being cis or trans (or a

mixture of both). In fact, two diastereomeric products 16e

having trans disubstitution on each of the β-lactam rings are

possible (Fig. 6). From the ¹H NMR and ¹³C NMR spectral

data, however, only one set of resonances for each proton

signal is observed, indicative of a single diastereomer.

Single crystal X-ray analysis of β-lactam 11b conﬁrmed

the cis stereochemistry (Fig. 4). Crystallographic data,

details of the data collection, and structure reﬁnement can

be found in the supporting information (Westrip 2010).

Next we turned to preparation of bis-β-lactams 16a–g

built from naphthalenetetracarboxylic diimide (14), which

we synthesized from commercial 1,4,5,8-naphthalenete-

tracarboxylic dianhydride (12). We note that naphthalene-

tetracarboxylic diimide derivatives have previously been

used as semiconductors due to the easy addition of varied

substituents to the imide moiety, as a means to exert elec-

tronic effects and increase the efﬁciency of electron

absorption (Gudeika et al. 2012). Compound 12 reacted

with glycine (13) in DMF to afford naphthalenete-

tracarboxylic diacetic acid (14). Then, compound 14 was

treated with different aromatic imines 15a–g in the presence

Medicinal Chemistry Research

Table 1 Structures and % yields of naphthalimido cis-β-lactams 11a-l

Yield

(%)

Cpd

11a

β-Lactam

Cpd

11b

β-Lactam

(%)

90

85

11c

11e

11g

11i

85

95

90

11d

78

11f

90

75

82

11h

11j

11k

80

11l

75

A plausible mechanism for the formation of the

β-lactams is outlined in Scheme 3. The ﬁrst step of the

reaction involves a nucleophilic attack of the imino nitrogen

on the ketene carbon to form zwitterionic intermediates 17

as a possible mixture of cis and trans species. These species

may interconvert by way of the initial imine–ketene addi-

tion being reversible. The cis and trans β-lactam adducts

result from subsequent ring closure of the zwitterionic

species. The ratio of cis–trans cycloadducts depends on a

variety of experimental factors, including the kinetics of the

Medicinal Chemistry Research

ﬁnal ring closure step, the rate of interconversion of the two

zwitterions 17 as a function of temperature, electronic and

steric nature of the substituents, and solvent (Cossio et al.

1993; Landa et al. 2018).

Antioxidant activity assay

The antioxidant capabilities of the mono and bis-β-lactam

adducts, 11a–l and 16a–g, respectively, was evaluated

using a diphenylpicrylhydrazyl (DPPH) radical-scavenging

assay (Ayati et al. 2018; Kostova et al. 2011; Benzie and

Strain 1999; Kamboj et al. 2019). Each compound was

dissolved in DMSO and added to a solution of the DPPH in

methanol, and the UV absorbance at 517 nm was measured.

Percent radical-scavenging activity was determined mathe-

matically from the absorbance after 5 min versus prior to the

addition of the lactam. Among the compounds tested, bis-

β-lactams 16a–d showed excellent antioxidant activity with

IC₅₀values of 15, 14.8, 7, 32.3 μg/ml, respectively, com-

pared with the control standard (vitamin C) which had an

IC₅₀value of 195 μg/ml. The other compounds showed

much weaker free radical-scavenging activity with IC₅₀

values between 4800 and 10,000 μg/ml. The preliminary

structure-activity relationship (SAR) study of bis-β-lactam

hybrids 16a–d reveals that the presence of an anthracenyl

moiety at the C-4 position of the β-lactam ring substantially

increased antioxidant activity relative to a ﬂuorenyl or 3,4-

dimethoxyphenyl side group (Table 2).

Typically, it is difﬁcult to predict the stereochemical

outcome a priori for new ketene–imine coupling partners,

particularly those bearing large multicyclic aryl sub-

stituents, which can experience unexpectedly larger or

smaller steric interactions due to skewed alignment of the

rings in space, or conversely, steric bumping, as well as π–π

interactions that can be either attractive (π-stacking) or

electronically repulsive in their nature. Our experiments

indicate that aryloxyacetic acids 10a–d combine with the

aryl imines 9 to afford only cis-β-lactams, while the bulky

bis-arylimidoacetic acid 14 undergoes imine cycloaddition

to yield only the trans-β-lactam products. The chemical

shifts of the β-lactam ring protons of 16a–d are near 7 ppm

due to the deshielding effect of the anthracene ring, as

evidenced in our previous publication (Borazjani et al.

2019a, 2019b).

Anticancer activity and cytotoxicity assays

HepG2, MCF-7, and TC-1 cell lines were exposed to the

various concentrations of the synthesized compounds for

24 h using MTT assay for estimation of cytotoxicity. The

compounds 16a and 16b demonstrated excellent anticancer

activity against the MCF-7 with IC50 values of 136.40,

Fig. 3 Assignment of cis-stereochemistry for β-lactam 11a

Fig. 4 ORTEP diagram of

β-lactam 11b

Medicinal Chemistry Research

Scheme 2 Synthesis of bis-β-lactam adducts 16a–g

anticancer activity of 14, 16a, 16b, 16c, 16d, 16e, 16f, and

16g against the TC-1 with IC₅₀values of 85.51, 69.55,

85.34, 89.37, 64.89, 189.16, 108.54, and 231.01 µM,

respectively, in comparison to the anticancer agent Gem-

citabine (IC₅₀of 153.25 µM) (Alami et al. 2007). The

compounds 7 and 9c showed good anticancer activity with

IC₅₀values of 311.29 and 321.95 µM on TC-1 cell lines.

Also, good anticancer activity against the MCF-7 with IC₅₀

values of 400.01, 213.66, and 261.93 µM for compounds of

16c, 16d, and 16e, respectively. Since all of these lactams

Fig. 5 Assignment of dual trans stereochemistry for bis-β-lactam 16e

131.52 µM, respectively, in comparison to the anticancer

agent Gemcitabin (IC₅₀of 191.57 µM) and raised up

Medicinal Chemistry Research

Fig. 6 Structures of the two possible bis-trans β-lactams 16e

Scheme 3 A mechanism for

discriminating between the cis

versus trans stereochemistry of

the β-lactams

Table 2 Antioxidant activity of bis-lactams 16a–g measured as IC₅₀

scavenging of the DPPH radical

Gemcitabine (IC₅₀of 215.01 µM) (Table 3). The possible

mechanism for their anticancer activity might be related to

production of intracellular free radicals, which ultimately

led to cell apoptosis. Therefore, these compounds provide

an opportunistic remedy for curing cancer diseases.

Compounds

IC₅₀(µg/ml)

16a

15

16b

14.8

7

16c

DNA interaction studies

16d

32.3

320

445

275

195

–

16e

UV/Vis titration assays

16f

16g

UV–visible absorption spectroscopy is an informative

method to assess the binding interactions of compounds

with DNA. Electronic absorption signal of compound

undergoes changes when bound to DNA. Hyperchromicity

or hypochromicity of the absorption signal can demonstrate

the mode of interaction with DNA (Mondal et al. 2018). In

this study, to get further insight into the naphthalimide

derivatives-DNA interaction, we carry out a quantitative

analysis of the binding process toward CT-DNA using 16a,

16b, 16d–g, as model compounds. The absorption spectra

of our compound in the presence of varying concentration

of CT-DNA is shown in Fig. 7. Upon addition of calf-

thymus DNA to 16a and 16b, there is an increase in molar

absorptivity (hyperchromism) of the absorption bands at

362, 380, and 398 nm of both compounds. In its absorption

Vitamin C

DMSO blank

with excellent anticancer activity carry bis-naphthalimide

on C-3 of the β-lactam ring, the aryl substituents at N-1 and

C-4 of the β-lactam ring are responsible for these differ-

ences in activity. A tentative SAR exists, in that the pre-

sence of an anthracene moiety on C-4 of the β-lactam ring

and a p-methoxyphenyl, p-ethoxyphenyl, p-N,N-dimethy-

laminophenyl or p-N,N-diethylaminophenyl ring on N-1 of

the β-lactam provide the best anticancer bioactivity. On the

other hand, there is no any cytotoxic effect on HepG2 cell

line based on tested concentrations (5, 10, 50, 100, and

200 µM) in comparison to high cytotoxicity of HepG2 of

Medicinal Chemistry Research

Table 3 Anticancer and cytotoxic activity assays assessed by the MTT

reduction method against MCF-7, TC-1, and HepG2 cell lines

Eq. (1):

½DNA=ðε_aÀ ε_fÞ ¼ ½DNA=ðε_bÀ ε_fÞ þ 1=K_bðε_bÀ ε_fÞ

Compounds MCF-7 IC₅₀

(µM)

TC-1 IC₅₀

(µM)

HepG2 IC₅₀

(µM)

ð1Þ

7

>1000

760.43

508.96

>1000

972.49

>1000

136.40

131.52

400.01

213.66

261.93

718.55

760.69

311.29

>1000

321.95

>1000

85.51

>1000

215.01

where [DNA] stands for the concentration of DNA in base

pairs, ε_acorresponds to the apparent extinction coefﬁcient,

ε_fis the extinction coefﬁcient of the compound in its free

form, and ε_bis the extinction coefﬁcient of the compound

in the bound form. When data ﬁtted into the above

equation, gave a straight line with the intercept of 1/K_b

(ε_b− ε_f) and slope of 1/(ε_b− ε_f) and the corresponding

K_bvalue, are evaluated from the ratio of slope to intercept

(Fig. 8 and Table 4). The observed K_bvalues indicating

strong binding of these compounds with DNA and these

are in the range for that of other naphthalimide derivatives

compounds (Milelli et al. 2012). The binding afﬁnity of

our compounds thus vary in the order of 16a; 16b; 16e;

16g > 16d; 16f.

9a

9b

9c

11a

11b

11c

11d

11e

11f

11g

11h

11i

11j

11k

11l

14

Fluorescence quenching studies

16a

16b

16c

16d

16e

16f

16g

69.55

To further investigate the potential interactions of the bis-

lactams 16a–g with CT-DNA, a standard ﬂuorescence

quenching technique was carried out (Mandegani et al.

2016; Suh and Chaires 1995). Among these seven com-

pounds, 16d provided optimal ﬂuorescence at room tem-

perature in aqueous solution with an emission maxima at

520 nm after excitation at 420 nm. As shown in Fig. 9a, the

emission intensities of compound 16d decreased with

increasing concentration of CT-DNA, and the wavelength

showed a slight blue shift of 2 nm. The observed quenching

is attributed to the strong binding of 16d with CT-DNA,

while the blue shift is consistent with intercalation (Bane-

rjee et al. 2013; Li et al. 2014). The apparent DNA binding

constant (K_b) for 16d was determined from Eq. (2):

85.34

89.37

64.89

189.16

108.54

231.01

153.25

Gemcitabine 191.57

spectra, 16e exhibited two bands at 362 and 382 nm. Upon

addition of CT-DNA, hyperchromism without any appre-

ciable change in peak position was observed. Similarly,

addition of CT-DNA to a solution of 16g led to hyper-

chromic shift in the position of the transitions at 361 and

383 nm and bands did not show any signiﬁcant shift. This

type of behavior suggests a non-intercalative mode of

interaction (Mondal et al. 2018; Kumar Gupta et al. 2013).

Notably, 16d and 16f showed different behavior in CT-

DNA titration studies. Addition of CT-DNA to a solution of

16d led to a hypochromic shift of the band at 361 and

381 nm. 16f exhibited similar behavior under the same

conditions. Increase in the concentration of CT-DNA

caused decrease in the absorbance intensity for the band

at 362 and 382 nm of 16f without any signiﬁcant change in

peak position. Conversely, the observed hypochromism in

16d and 16f suggested that these two compounds could

insert into the base pairs of DNA and thus bind to CT-DNA

by intercalation (Bhat et al. 2010). The DNA binding afﬁ-

nities of 16a, 16b, 16d–g were compared quantitatively by

calculating the intrinsic binding constant K_busing the

logF₀À F=F ¼ log K_bþ n log½Q

ð2Þ

where F₀and F are the ﬂuorescence intensity in the absence

and the presence of the quencher 16d at various concentra-

tions, respectively, K_bis the binding constant, and n is the

binding number. The K_bvalue was found to be 1.896 × 10⁵

(Fig. 9b). These observations are in good agreement with

the above absorption titration data and indicate that 16d

binds to DNA by intercalation.

Molecular docking studies

In order to conﬁrm the biological results and to get insight

into the interaction and binding mode of the most potent

compound, molecular modeling studies were performed.

The binding site and interactions of 16d with DNA are

illustrated in Fig. 10. DNA intercalation can often be seen

Medicinal Chemistry Research

Fig. 7 Absorption titration

spectra of 16a (a), 16b (b), 16e

(c), 16d (d), 16f (e), and 16g (f)

in 50 mM Tris-HCl buffer at pH

7.4, in the absence and presence

of increasing amounts of DNA

(0–7 μM in 16a, 0–16 μM 16e,

0–20 μM 16d, 16f, and 0–12 μM

16b, 16g)

for compounds having a planar polycyclic core (Zanoza

et al. 2019; Arunadevi et al. 2019; Li et al. 2020; Arif

et al. 2020). Unexpectedly, the anthracene motif of 16d

did not completely participate in intercalation between

DNA base pairs; however, it oriented in a way that the

diethylamine group could place within DC21 and DC22

base pairs and demonstrated hydrogen bond interaction

between the nitrogen of diethylamine with the side chains

of the DNA base pairs (DC22). Moreover, the methyl

group of the diethylamine side chain also demonstrated π–

σ interaction with a guanine of the DNA backbone. The

benzene ring of diethylaniline also exhibited π–anion

interactions with the phosphate backbone of DNA

(DC21). The aromatic rings of the anthracene ring were

observed to be involved in π–π stacking and π–lone pair

interactions with the DT19 base pair and phosphate group,

respectively. However, the other side of the molecule

possesses the criteria for interaction with the minor grove,

which is conﬁrmed with molecular docking experiments.

Bis-lactam 16d perfectly orients in a curved shape along

the length of the minor groove. The anthracene rings on

the side chain were involved in three π–π stacking inter-

actions with the DT8 base pair and π–lone pair interac-

tions with the DNA phosphate groups. Moreover, the

N,N-diethylaniline substituent was stabilized through a π–

σ interaction with the DC9 DNA base pairs. Our results of

molecular docking indicated that 16d can potentially bind

to the DNA groove and behave as a DNA intercalator,

with the minimum binding energy of −10.18 kcal/mol.

These data support the interaction of 16d with DNA by

intercalation.

The binding mode of Gemcitabine as a positive control

with

DNA

(PDB

code:453D,

B-DNA

[(5′-D

(*CP*GP*CP*GP*AP*AP*TP*TP*CP*GP*CP*G)]) was

deﬁned by docking study to evaluate the mode of interac-

tion with the target. As shown in Fig. 11, the interaction of

Medicinal Chemistry Research

5 10^-9

(A)

3 10^-9

2.5 10^-9

2 10^-9

1.5 10^-9

1 10^-9

5 10^-10

0

(B)

4 10^-9

3 10^-9

2 10^-9

1 10^-9

0

-1 10^-9

-5 10^-10

-1 10^-6

0

1 10^-62 10^-63 10^-64 10^-65 10^-66 10^-67 10^-6

[DNA], M

-2 10^-6

0

2 10^-64 10^-66 10^-68 10^-61 10^-51.2 10^-5

[DNA], M

2.5 10^-9

-4.5 10^-10

-5 10^-10

(C)

(D)

2 10^-9

1.5 10^-9

1 10^-9

5 10^-10

0

-5.5 10^-10

-6 10^-10

-6.5 10^-10

-7 10^-10

-7.5 10^-10

-8 10^-10

4 10^-6

8 10^-61.2 10^-51.6 10^-5

[DNA], M

8 10^-6

1 10^-51.2 10^-51.4 10^-51.6 10^-51.8 10^-52 10^-5

0

[DNA], M

3.5 10^-9

3 10^-9

2.5 10^-9

2 10^-9

1.5 10^-9

1 10^-9

5 10^-10

0

-5 10^-9

-5.5 10^-9

-6 10^-9

(F)

(E)

-6.5 10^-9

-7 10^-9

-7.5 10^-9

-8 10^-9

-5 10^-10

-8.5 10^-9

-2 10^-6

0

2 10^-64 10^-66 10^-68 10^-61 10^-51.2 10^-5

8 10^-6

1 10^-51.2 10^-51.4 10^-51.6 10^-51.8 10^-52 10^-5

[DNA], M

Fig. 8 Comparative plots of [DNA]/(ε_a− ε_f) vs. [DNA] for 16a (a), 16b (b) 16e (c), 16d (d), 16f (e), and 16g (f)

this compound with nucleobases involved in hydrogen bond

between the NH₂group of Gemcitabine and thymine of

DT7 base pair. Beside, NH of Gemcitabine also formed a

hydrogen bond with base pair of DT20. It seems that

aminopyrimidin-one fragment of Gemcitabine behaves like

DNA-intercalating agents. Noteworthy, interaction with

minor groove observed between hydroxymethyl group of

Gemcitabine and DT10 of the DNA backbone. Another

HB-interaction was observed between OH moiety of

Gemcitabine and DT20 the backbone of DNA.

Conclusions

Although there are an assortment of methods for synthe-

sizing functionalized β-lactam compounds, the Staudinger

acid chloride-imine cycloaddition reaction is most widely

used, because of both the simplicity in the methodology and

the control of relative stereochemistry. In this study, we

synthesized a selection of naphthalimido hybrids having

one or two β-lactam side chains. Mono β-lactams 11a–l are

exclusively formed as cis stereoisomer, while both β-lactam

Medicinal Chemistry Research

1

rings in the bis-β-lactams 16a–g are trans disubstituted.

Each of the β-lactams were evaluated for antioxidant

activity. The best antioxidant activity was observed for

compounds 16a–d (15, 14.8, 7, 32.3 μg/mL, respectively).

The bis-β-lactams 16a–g display in vitro anticancer activity

against the MCF-7 and TC-1 cancer cell lines, without

noticeable cytotoxicity towards healthy cells. UV–vis and

ﬂuorescence spectroscopic studies have revealed the ability

of our compounds to bind to CT-DNA. The activity of these

compounds, based on their calculated DNA binding con-

stant values, indicated that 16a, 16b, 16e, 16g > 16d, 16f.

The obtained results are consistent with a DNA intercalation

mechanism for both 16d and 16f and non-intercalation

mechanism for 16a; 16b; 16e and 16g. These results of

computational evaluations of 16d is in agreement with the

DNA binding studies as well as the cytotoxicity data.

Molecular docking suggested two DNA binding modes for

bis-naphthalimido β-lactam 16d, that of minor roove and

base pair intercalation.

(250 MHz, 400 MHz for H NMR and 100 MHz for ¹³C

NMR). ¹³C NMR spectral data were reported with complete

proton decoupling. Chemical shifts were reported in parts

per million (δ) downﬁeld from tetramethylsilane. Splitting

patterns are indicated as s: singlet, d: doublet, t: triplet,

q: quartet, m: multiplet, dd: doublet of doublet. Coupling

constants (J) are reported in hertz (Hz). Elemental analyses

were run on a Thermo Finnigan Flash EA-1112 series.

Thin-layer chromatography was carried out on silica gel

254. Melting points were recorded on a Buchi 510 melting

point apparatus in open capillary tubes. The mass spectra

were recorded on a Shimadzu GC-MS QP 1000 EX

instrument. CH₂Cl₂and Et₃N were dried before use by

distillation over CaH_2.

General procedure for the synthesis of 4-(3-(1,3-

dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propoxy)

benzaldehyde (7)

1,8-Naphthalimide (3) was synthesized by the reaction of

1,8-naphthalic anhydride (1) (1.00 mmol) and ammonium

acetate (2) (1.20 mmol) in DMF at 60 °C for an appropriate

time. Then the crude was cooled to room temperature and

recrystallized from ethanol to give compound 3. Compound

3 (1.00 mmol), 1,3-dibromopropane (4) (3.00 mmol) and

solid K₂CO₃(3.00 mmol) in DMF was stirred at room

temperature overnight. Water (10 mL) was added to the

mixture and the precipitate was ﬁltered and washed with

petroleum ether. Compound 5 was recrystallized from

ethanol [31]. A mixture of compound 5 (1.00 mmol),

4-hydroxybenzaldehyde (6) (1.20 mmol), solid K₂CO₃

(3.00 mmol) was stirred in acetonitrile at 70–80 °C for 24 h.

After completion of the reaction, the crude was cooled to

room temperature. The obtained precipitate was ﬁltered and

recrystallized from ethanol to give compound 7.

Experimental section

General

Infrared analyses were done on a FT-IR 8300 spectro-

photometer using potassium bromide pellets (ʋ in cm⁻¹). ¹H

NMR and ¹³C NMR spectra were recorded on a Bruker-

Avance400 using a Bruker Avance DPX instrument

Table 4 Binding constant of the interaction of 16a, 16b, 16d–g with

CT-DNA at 25 °C in 50 mM Tris-HCl buffer at pH 7.4

Compound

λ (nm)

K_b(M⁻¹

)

R^2a

16a

16b

16d

16e

16f

380

382

383

2.575 × 10⁺⁶

2.279 × 10⁺⁶

1.552 × 10⁺⁵

2.567 × 10⁺⁶

1.578 × 10⁺⁵

2.329 × 10⁺⁶

0.9906

0.9936

0.9906

0.9936

0.9934

0.9923

4-(3-(1,3-Dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propoxy)

benzaldehyde (7)

White solid; Mp. 188–200 °C; IR (KBr, cm⁻¹): 1699 (CO

16g

1

R^2ais the linear correlated coefﬁcient

Naph), 1674 (CO Aldehyde), 1657 (CO Naph); H NMR

Fig. 9 a Fluorescence emission

spectra of 16d (20 µM) in the

presence of increasing

concentrations of CT-DNA

(0–12 µM). The excitation

wavelength was 420 nm. Spectra

were recorded in the range of

440–820 nm in 50 mM Tris-HCl

buffer at pH 7.4 in 100 mM

aqueous NaCl. b Plot of log

[(F₀− F)/F] versus log[DNA]

at 25 °C

Medicinal Chemistry Research

Fig. 10 Molecular modeling

studies of bis-lactam 16d with

CT-DNA

2-(3-(4-(((4-Methoxyphenyl)imino)methyl)phenoxy)propyl)-

1H-benzo[de]isoquinoline-1,3(2H)-dione (9a)

White solid; Mp. 172–174 °C; IR (KBr, cm⁻¹): 1696 (CO

Naph), 1659 (CO Naph), 1624 (CH=N); ¹H NMR

(250 MHz, DMSO-d₆): 2.08–2.18)2H, m, –CH₂), 3.75 (3H,

s, OCH₃), 4.14 (2H, t, J = 6.0 Hz, –NCH₂), 4.24 (2H, t, J =

6.7 Hz, OCH₂), 6.88–6.95 (4H, m, ArH), 7.21 (2H, d, J =

7.7 Hz, ArH), 7.76 (2H, d, J = 8.2vHz, ArH), 7.85 (2H, t,

J = 7.7 Hz, ArH), 8.43 (1H, s, CH=N), 8.46–8.48 (4H, m,

ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.5 (CO Naph),

160.8, 157.6, 157.5, 144.4, 134.2, 130.6, 130.0, 129.4,

129.0, 127.3, 127.1, 122.2, 122.1, 114.5, 114.3 (aromatic

carbons and imine carbon), 66.1 (–OCH₂), 55.2 (OCH₃),

37.3 (–NCH₂), 27.3 (–CH₂); Analysis calculated for

C₂₉H₂₄N₂O₄: C, 74.98; H, 5.21; N, 6.03%. Found: C, 74.58;

H, 5.25; N, 6.15%.

Fig. 11 Molecular modeling studies of Gemcitabine with CT-DNA

(250 MHz, DMSO-d₆): 2.09–2.19 (2H, m, –CH₂),

4.16–4.27 (4H, m, –NCH_2,–OCH₂), 6.97 (2H, d, J =

8.7 Hz, ArH), 7.78 (2H, d, J = 8.7 Hz, ArH), 7.85 (2H, t,

J = 8.2 Hz, ArH), 8.43–8.48 (4H, m, ArH), 9.82 (1H, s,

CHO); ¹³C NMR (100 MHz, DMSO-d₆) δ 191.2 (CO

Aldehyde), 163.4 (CO Naph), 134.2, 131.6, 131.2, 130.6,

129.4, 127.3, 127.1, 122.0, 114.7 (aromatic carbons), 66.4

(–OCH₂), 37.1 (–NCH₂), 27.2 (–CH₂); Analysis calculated

for C₂₂H₁₇NO₄: C, 73.53; H, 4.77; N, 3.90%. Found: C,

73.12; H, 4.93; N, 3.43%.

2-(3-(4-(((4-Ethoxyphenyl)imino)methyl)phenoxy)propyl)-

1H-benzo[de]isoquinoline-1,3(2H)-dione (9b)

Cream solid; Mp. 184–186 °C; IR (KBr, cm⁻¹): 1701 (CO

Naph), 1663 (CO Naph), 1627 (CH=N); ¹H NMR

(250 MHz, DMSO-d₆): 1.31 (3H, t, J = 7.0 Hz, CH₃),

2.09–2.18 (2H, m, –CH₂), 4.01 (2H, q, J = 7.0 Hz, OCH₂),

4.14 (2H, t, J = 5.7 Hz, –NCH₂), 4.25 (2H, t, J = 6.5 Hz,

–OCH₂), 6.89 (2H, d, J = 4.2 Hz, ArH), 6.92 (2H, d, J =

4.2 Hz, ArH), 7.20 (2H, d, J = 8.7 Hz, ArH), 7.76 (2H, d,

J = 8.5 Hz, ArH), 7.85 (2H, t, J = 7.5 Hz, ArH), 8.43 (1H,

s, CH=N), 8.46–8.48 (4H, m, ArH); ¹³C NMR (100 MHz,

DMSO-d₆) δ 163.6 (CO Naph), 160.4, 157.6, 156.9, 147.5,

134.2, 133.9, 133.7, 131.7, 130.7, 129.9, 129.3, 127.1,

122.1, 114.9, 114.5 (aromatic carbons and imine carbon),

66.2 (–OCH₂), 63.1 (–OCH₂), 36.8 (–NCH₂), 27.0 (–CH₂),

14.4 (CH₃); Analysis calculated for C₃₀H₂₆N₂O₄: C, 75.30;

H, 5.48; N, 5.85 %. Found: C, 75.11; H, 5.13; N, 5.35%.

General procedure for preparation of Schiff bases

9a–c

A mixture of compound 7 (1.00 mmol) and aniline deriva-

tives 8a–c (1.00 mmol) was reﬂuxed in ethanol and 2–3

drops of AcOH for an appropriate time. Then the mixture

was cooled to room temperature. The mixture of reaction

was ﬁltered and the solvent was evaporated under the

reduced pressure. After that precipitate was recrystallized

from ethanol to give Schiff bases 9a–c.

Medicinal Chemistry Research

2-(3-(4-(((4-(Dimethylamino)phenyl)imino)methyl)phenoxy)

2-(3-(4-(3-(4-Chlorophenoxy)-1-(4-methoxyphenyl)-4-

propyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (9c)

oxoazetidin-2-yl)phenoxy)propyl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (11b)

Yellow solid; Mp. 169–171 °C; IR (KBr, cm⁻¹): 1698 (CO

Naph), 1661 (CO Naph), 1620 (CH=N); ¹H NMR

(250 MHz, DMSO-d₆): 2.07–2.17 (2H, m, –CH₂), 2.88 (6H,

s, NCH₃), 4.13 (2H, t, J = 5.5 Hz, –NCH₂), 4.24 (2H, t, J =

6.2 Hz, –OCH₂), 6.72 (2H, d, J = 9.0 Hz, ArH), 6.87 (2H, d,

J = 8.5 Hz, ArH), 7.18 (2H, d, J = 9.0 Hz, ArH), 7.73 (2H,

d, J = 8.5 Hz, ArH), 7.85 (2H, t, J = 8.0 Hz, ArH), 8.43

(1H, s, CH=N), 8.46–8.49 (4H, d, J = 6.0 Hz, ArH); ¹³C

NMR (100 MHz, DMSO-d₆) δ 163.5 (CO Naph), 160.4,

154.7, 149.0, 140.2, 134.2, 131.2, 130.6, 129.6, 129.5,

127.3, 127.1, 122.1, 122.0, 114.4, 112.6 (aromatic carbons

and imine carbon), 66.1 (–OCH₂), 40.2 (NCH₃), 37.3

(–NCH₂), 27.3 (–CH₂); Analysis calculated for

C₃₀H₂₇N₃O₃: C, 75.45; H, 5.70; N, 8.80%. Found: C, 75.64;

H, 5.41; N, 8.15%.

White solid; Mp. 207–209 °C; IR (KBr, cm⁻¹): 1745 (CO

β-lactam), 1702 (CO Naph), 1656 (CO Naph); H NMR

1

(250 MHz, DMSO-d₆): 1.98–2.06 (2H, m, –CH₂), 3.67 (3H,

s, OCH₃), 3.97 (2H, t, J = 5.7 Hz, –NCH₂), 4.17 (2H, t, J =

7.2 Hz, –OCH₂), 5.59 (1H, d, J = 4.7 Hz, H-4 β-lactam),

5.77 (1H, d, J = 4.7 Hz, H-3 β-lactam), 6.70 (2H, d, J =

8.5 Hz, ArH), 6.81 (2H, d, J = 9.2 Hz, ArH), 6.89 (2H, d,

J = 9.0 Hz, ArH), 7.18–7.22 (6H, m, ArH), 7.82 (2H, t, J =

8.2 Hz, ArH), 8.41–8.45 (4H, m, ArH); ¹³C NMR

(100 MHz, DMSO-d₆) δ 163.4 (CO β-lactam), 161.7 (CO

Naph), 158.4, 155.9, 155.2, 134.2, 131.2, 130.6, 129.9,

129.2, 129.0, 127.3, 127.1, 125.4, 124.5, 122.1, 118.4,

116.8, 114.5, 114.0 (aromatic carbons), 80.5 (C-

3 β-lactam), 65.8 (–OCH₂), 60.2 (C-4 β-lactam), 55.2

(OCH₃), 37.2 (–NCH₂), 27.3 (–CH₂); Analysis calculated

for C₃₇H₂₉ClN₂O₆: C, 70.20; H, 4.62; Cl, 5.60; N, 4.42%.

Found: C, 70.08; H, 4.35; N, 4.04%.

General procedure for synthesis of new naphthalimido

β-lactam conjugates 11a–l

A mixture of Schiff base 9a–c (1.00 mmol), triethylamine

(5.00 mmol), substituted acetic acid 10a–d (1.50 mmol) and

tosyl chloride (1.50 mmol) in dry CH₂Cl₂(15 ml) was stir-

red overnight at room temperature. Then it was washed with

1 N aqueous HCl (20 ml), saturated NaHCO₃(20 ml) and

brine (20 ml). The organic layer was dried (anhydrous

Na₂SO₄), ﬁltered and the solvent was evaporated to give

crude product 11a–l. Conjugates 11a–l were puriﬁed by

recrystallization from ethyl acetate.

2-(3-(4-(3-(2,4-Dichlorophenoxy)-1-(4-methoxyphenyl)-4-

oxoazetidin-2-yl)phenoxy)propyl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (11c)

White solid; Mp. 211–213 °C; IR (KBr, cm⁻¹): 1745 (CO

1

β-lactam), 1701 (CO Naph), 1656 (CO Naph); H NMR

(250 MHz, DMSO-d₆): 1.98–2.08 (2H, m, –CH₂), 3.67

(3H, s, OCH₃), 3.97 (2H, t, J = 6.0 Hz, –NCH₂), 4.16 (2H,

t, J = 6.7 Hz, –OCH₂), 5.61 (1H, d, J = 4.5 Hz, H-4

β-lactam), 5.89 (1H, d, J = 4.5 Hz, H-3 β-lactam), 6.66

(2H, d, J = 8.5 Hz, ArH), 6.89 (1H, d, J = 9.0 Hz, ArH),

7.13 (1H, d, J = 9.0 Hz, ArH), 7.19–7.22 (4H, m, ArH),

7.27 (2H, dd, J₁= 9.0 Hz, J₁= 2.5 Hz, ArH), 7.38 (1H, d,

J = 2.5 Hz, ArH), 7.82 (2H, t, J = 8.0 Hz, ArH), 8.41–8.44

(4H, m ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.4

(CO β-lactam), 161.2 (CO Naph), 158.5, 155.9, 150.7,

134.2, 131.2, 130.6, 129.8, 129.3, 128.3, 127.7, 127.3,

127.1, 125.6, 124.6, 124.1, 122.1, 118.5, 116.0, 114.5,

114.1 (aromatic carbons), 80.6 (C-3 β-lactam), 65.8

(–OCH₂), 60.0 (C-4 β-lactam), 55.2 (OCH₃), 37.2

(–NCH₂), 27.3 (–CH₂); Analysis calculated for

C₃₇H₂₈Cl₂N₂O₆: C, 66.57; H, 4.23; N, 4.20%. Found: C,

66.29; H, 4.08; N, 4.06%.

2-(3-(4-(1-(4-Methoxyphenyl)-4-oxo-3-phenoxyazetidin-2-

yl)phenoxy)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione

(11a)

White solid; Mp 200–202 °C; IR (KBr, cm⁻¹): 1751 (CO

1

β-lactam), 1697 (CO Naph), 1658 (CO Naph); H NMR

(250 MHz, DMSO-d₆): 1.97–2.08)2H, m, –CH₂), 3.67 (3H,

s, OCH₃), 3.96 (2H, t, J = 6.2 Hz, –NCH₂), 4.16 (2H, t, J =

6.7 Hz, –OCH₂), 5.59 (1H, d, J = 4.7 Hz, H-4 β-lactam),

5.76 (1H, d, J = 4.7 Hz, H-3 β-lactam), 6.70 (2H, d, J = 8.5

HZ, ArH), 6.78 (2H, d, J = 7.5 Hz, ArH), 6.84–6.95 (3H, m,

ArH), 7.14 (2H, d, J = 7.5 Hz, ArH), 7.19–7.23 (4H, m,

ArH), 7.82 (2H, t, J = 7.7 Hz, ArH), 8.41–8.44 (4H, m,

ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.4 (CO

β-lactam), 162.1 (CO Naph), 158.5, 156.4, 155.8, 137.7,

134.2, 130.6, 130.0, 129.3, 129.1128.0, 127.1, 125.4,

122.0, 120.0, 118.4, 115.0, 114.5, 114.4 (aromatic carbons),

80.4 (C-3 β-lactam), 66.7 (-OCH₂), 62.6 (C-4 β-lactam),

55.2 (OCH₃), 37.3 (–NCH₂), 27.3 (–CH₂); Analysis calcu-

lated for C₃₇H₃₀N₂O₆: C, 74.23; H, 5.05; N, 4.68%. Found:

C, 74.11; H, 4.93; N, 4.25%.

2-(3-(4-(1-(4-Methoxyphenyl)-3-(naphthalen-2-yloxy)-4-

oxoazetidin-2-yl)phenoxy)propyl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (11d)

White solid; Mp. 199–201 °C; IR (KBr, cm⁻¹): 1743 (CO

1

β-lactam), 1702 (CO Naph), 1657 (CO Naph); H NMR

(250 MHz, DMSO-d₆): 1.93–2.04 (2H, m, –CH₂), 3.68 (3H,

Medicinal Chemistry Research

s, OCH₃), 3.92 (2H, t, J = 6.0 Hz, –NCH₂), 4.13 (2H, t, J =

7.0 Hz, –OCH₂), 5.70 (1H, d, J = 4.7 Hz, H-4 β-lactam),

5.91 (1H, d, J = 4.7 Hz, H-3 β-lactam), 6.66 (2H, d, J =

8.7 Hz, ArH), 6.89–6.98 (3H, m, ArH), 7.21–7.34 (6H, m,

ArH), 7.43 (1H, t, J = 8.0 Hz, ArH), 7.69 (1H, d, J =

9.0 Hz, ArH), 7.75 (2H, d, J = 8.7 Hz, ArH), 7.81 (2H, d,

J = 7.7 Hz, ArH), 8.40–8.43 (4H, m, ArH); ¹³C NMR

(100 MHz, DMSO-d₆) δ 163.4 (CO β-lactam), 162.0 (CO

Naph), 158.3, 155.9, 154.2, 134.1, 133.6, 131.2, 130.6,

130.0, 129.2, 128.8, 127.4, 127.3, 127.1, 126.7, 126.4,

124.7, 124.0, 122.0, 118.4, 117.8, 114.5, 114.0, 108.6

(aromatic carbons), 80.5 (C-3 β-lactam), 65.7 (–OCH₂),

60.4 (C-4 β-lactam), 55.2 (OCH₃), 37.2 (–NCH₂), 27.3

(–CH₂); Analysis calculated for C₄₁H₃₂N₂O₆: C, 75.91; H,

4.97; N, 4.32 %. Found: C, 75.77; H, 4.83; N, 4.28%.

129.2, 129.0, 127.3, 127.1, 125.4, 124.5, 122.1, 118.4,

116.8, 114.9, 114.0 (aromatic carbons), 80.5 (C-

3 β-lactam), 65.7 (–OCH₂), 63.1 (–OCH₂), 60.2 (C-

4 β-lactam), 37.2 (–NCH₂), 27.3 (–CH₂), 14.5 (CH₃);

Analysis calculated for C₃₈H₃₁ClN₂O₆: C, 70.53; H, 4.83;

N, 4.33%. Found: C, 70.48; H, 4.23; N, 4.20%.

2-(3-(4-(3-(2,4-Dichlorophenoxy)-1-(4-ethoxyphenyl)-4-

oxoazetidin-2-yl)phenoxy)propyl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (11g)

White solid; Mp. 213–215 °C; IR (KBr, cm⁻¹): 1755 (CO

1

β-lactam), 1701 (CO Naph), 1666 (CO Naph); H NMR

(250 MHz, DMSO-d₆): 1.25)3H, t, J = 7.0 Hz, CH₃),

2.00–2.06 (2H, m, –CH₂), 3.88–3.99 (4H, m, –NCH₂,

–OCH₂), 4.16 (2H, t, J = 6.7 Hz, –OCH₂), 5.60 (1H, d, J =

4.5 Hz, H-4 β-lactam), 5.87 (1H, d, J = 4.5 Hz, H-3

β-lactam), 6.80 (2H, d, J = 8.5 Hz, ArH), 6.87 (2H, d,

J = 9.0 Hz, H-3), 7.12 (1H, d, J = 9.0, ArH), 7.17–7.22

(4H, m, ArH) 7.27 (1H, dd, J₁= 10.7 Hz, J₂= 2.5 Hz,

ArH), 7.37 (1H, d, J = 2.5 Hz, ArH), 7.81 (2H, t, J = 7.7 Hz

ArH), 8.40–8.44 (4H, m, ArH); ¹³C NMR (100 MHz,

DMSO-d₆) δ 163.4 (CO β-lactam), 161.2 (CO Naph), 158.5,

155.2, 150.7, 134.2, 131.2, 130.6, 129.7, 129.3, 129.2,

127.7, 127.3, 127.1, 125.6, 124.1, 122.1, 122.0, 118.5,

116.0, 114.9, 114.1 (aromatic carbons), 80.6 (C-

3 β-lactam), 65.8 (–OCH₂), 63.1 (–OCH₂), 60.0 (C-

4 β-lactam), 37.2 (–NCH₂), 27.3 (–CH₂), 14.5 (CH₃);

Analysis calculated for C₃₈H₃₀Cl₂N₂O₆: C, 66.97; H, 4.44;

N, 4.11%. Found: C, 66.88; H, 3.93; N, 4.00%.

2-(3-(4-(1-(4-Ethoxyphenyl)-4-oxo-3-phenoxyazetidin-2-yl)

phenoxy)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione

(11e)

White solid; Mp. 198–200 °C; IR (KBr, cm⁻¹): 1750 (CO

1

β-lactam), 1705 (CO Naph), 1656 (CO Naph); H NMR

(250 MHz, DMSO-d₆): 1.25 (3H, t, J = 7.0 Hz, CH₃),

1.96–2.07 (2H, m, –CH₂), 3.88–3.98 (4H, m, –NCH₂,

–OCH₂), 4.16 (2H, t, J = 6.5 Hz, –OCH₂), 5.58 (1H, d, J =

4.7 Hz, H-4 β-lactam), 5.75 (1H, d, J = 4.7 Hz, H-3

β-lactam), 6.70 (2H, d, J = 8.5 Hz, ArH), 6.78 (2H, d,

J = 8.0 Hz, ArH), 6.85–6.89 (3H, m, ArH), 7.13–7.23 (6H,

m, ArH), 7.82 (2H, t, J = 7.2 Hz, ArH), 8.41–8.44 (4H, m,

ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.4 (CO

β-lactam), 162.1 (CO Naph), 158.4, 156.5, 155.1, 134.2,

131.2, 130.6, 129.9, 129.3, 127.3, 127.1, 124.8, 122.0,

121.7, 118.4, 115.0, 114.9, 114.0 (aromatic carbons), 80.5

(C-3 β-lactam), 65.7 (–OCH₂), 63.1 (–OCH₂), 60.4 (C-

4 β-lactam), 37.2 (–NCH₂), 27.3 (–CH₂), 14.5 (CH₃);

Analysis calculated for C₃₈H₃₂N₂O₆: C, 74.50; H, 5.26; N,

4.57%. Found: C, 74.38; H, 5.05; N, 4.18%.

2-(3-(4-(1-(4-Ethoxyphenyl)-3-(naphthalen-2-yloxy)-4-

oxoazetidin-2-yl)phenoxy)propyl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (11h)

Cream solid; Mp. 182–184 °C; IR (KBr, cm⁻¹): 1738 (CO

1

β-lactam), 1702 (CO Naph), 1662 (CO Naph); H NMR

(250 MHz, DMSO-d₆): 1.25)3H, t, J = 6.7 Hz, CH₃),

1.96–2.01 (2H, m, –CH₂), 3.86–3.97 (4H, m, –NCH₂,

–OCH₂), 4.13 (2H, t, J = 7.0 Hz, –OCH₂), 5.69 (1H, d, J =

4.2 Hz, H-4 β-lactam), 5.91 (1H, d, J = 4.2 Hz, H-3

β-lactam), 6.66 (2H, d, J = 5.7 Hz, ArH), 6.88 (2H, d,

J = 8.7 Hz, ArH), 6.95 (1H, d, J = 9.7 Hz, ArH), 7.23–7.30

(5H, m, ArH) 7.32 (1H, t, J = 7.7 Hz, ArH), 7.43 (1H, t,

J = 7.7 Hz, ArH), 7.68 (1H, t, J = 9.2 Hz, ArH), 7.74 (2H,

d, J = 8.5 Hz, ArH), 7.81 (2H, d, J = 7.7 Hz, ArH),

8.39–8.42 (4H, m, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ

163.4 (CO β-lactam), 162.0 (CO Naph), 158.3, 155.1,

154.2, 134.1, 133.6, 131.1, 130.5, 129.9, 129.2, 128.8,

127.4, 127.3, 127.0, 126.7126.5, 124.7, 124.0, 122.0,

118.4, 117.8, 114.9, 114.0, 108.6 (aromatic carbons), 80.4

(C-3 β-lactam), 65.7 (–OCH₂), 63.1 (–OCH₂), 60.5 (C-

4 β-lactam), 37.2 (–NCH₂), 27.3 (–CH₂), 14.5 (CH₃);

2-(3-(4-(3-(4-Chlorophenoxy)-1-(4-ethoxyphenyl)-4-

oxoazetidin-2-yl)phenoxy)propyl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (11f)

White solid; Mp. 203–205 °C; IR (KBr, cm⁻¹): 1758 (CO

1

β-lactam), 1662 (CO Naph); H NMR (250 MHz, DMSO-

d₆): 1.25)3H, t, J = 7.0 Hz, CH₃), 1.99–2.08 (2H, m, –CH₂),

3.88–3.99 (4H, m, –NCH₂, –OCH₂), 4.17 (2H, t, J =

6.7 Hz, –OCH₂), 5.57 (1H, d, J = 4.5 Hz, H-4 β-lactam),

5.76 (1H, d, J = 4.5 Hz, H-3 β-lactam), 6.70 (2H, d, J =

8.0 Hz, ArH), 6.81 (2H, d, J = 7.7 Hz, ArH), 6.87 (2H, d,

J = 8.0 Hz, ArH), 7.18–7.21 (6H, m, ArH), 7.82 (2H, t, J =

8.0 Hz, ArH), 8.41–8.44 (4H, m, ArH); ¹³C NMR

(100 MHz, DMSO-d₆) δ 163.4 (CO β-lactam), 161.7 (CO

Naph), 158.4, 155.2, 154.8, 134.2, 131.2, 130.6, 129.8,

Medicinal Chemistry Research

Analysis calculated for C₄₂H₃₄N₂O₆: C, 76.12; H, 5.17; N,

4.23%. Found: C, 76.08; H, 4.98; N, 4.12%.

(250 MHz, DMSO-d₆): 2.00–2.09 (2H, m, –CH₂), 2.82 (6H,

s, NCH₃), 3.99 (2H, t, J = 5.5 Hz, –NCH₂), 4.18 (2H, t, J =

7.0 Hz, –OCH₂), 5.59 (1H, d, J = 4.5 Hz, H-4 β-lactam),

5.87 (1H, d, J = 4.5 Hz, H-3 β-lactam), 6.67 (2H, d, J =

9.0 Hz, ArH), 6.72 (2H, d, J = 8.7 Hz, ArH), 7.12 (2H, d,

J = 3.7 Hz, ArH), 7.16 (2H, d, J = 3.7 Hz, ArH), 7.22 (2H,

d, J = 8.5 Hz, ArH), 7.29 (1H, dd, J₁= 11.0 Hz, J₂=

2.5 Hz, ArH), 7.40 (1H, d, J = 2.5 Hz, ArH), 7.83 (2H, t,

J = 7.7 Hz, ArH), 8.42–8.46 (3H, m, ArH); ¹³C NMR

(100 MHz, DMSO-d₆) δ 163.4 (CO β-lactam), 160.6 (CO

Naph), 158.4, 150.8, 147.5, 134.1, 131.1, 130.6, 129.2,

127.6, 127.3, 127.0, 126.3, 125.5, 124.5, 124.4, 122.1,

122.0, 118.3, 116.0, 114.0, 112.6 (aromatic carbons), 80.5

(C-3 β-lactam), 65.8 (-OCH₂), 59.9 (C-4 β-lactam), 40.1

(NCH₃), 37.2 (–NCH₂), 27.3 (–CH₂); Analysis calculated

for C₃₈H₃₁Cl₂N₃O₅: C, 67.06; H, 4.59; N, 6.17%. Found: C,

66.92.; H, 4.44; N, 5.98%.

2-(3-(4-(1-(4-(Dimethylamino)phenyl)-4-oxo-3-

phenoxyazetidin-2-yl)phenoxy)propyl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (11i)

Cream solid; Mp. 221–223 °C; IR (KBr, cm⁻¹): 1737 (CO

1

β-lactam), 1703 (CO Naph), 1659 (CO Naph); H NMR

(250 MHz, DMSO-d₆): 1.96–2.07 (2H, m, –CH₂), 2.79 (6H,

s, NCH₃), 3.95 (2H, t, J = 6.0 Hz, –NCH₂), 4.16 (2H, t, J =

7.0 Hz, –OCH₂), 5.54 (1H, d, J = 4.7 Hz, H-4 β-lactam),

5.71 (1H, d, J = 4.7 Hz, H-3 β-lactam), 6.64 (2H, d, J =

9.0 Hz, ArH), 6.69 (2H, d, J = 8.5 Hz, ArH), 6.75 (2H, d,

J = 7.7 Hz, ArH), 6.83 (1H, t, J = 7.2 Hz, ArH), 7.09–7.13

(3H, m, ArH), 7.16–7.21 (3H, m, ArH), 7.81 (2H, t, J =

8.2 Hz, ArH), 8.40–8.43(4H, m, ArH); ¹³C NMR

(100 MHz, DMSO-d₆) δ 163.4 (CO β-lactam), 161.6 (CO

Naph), 158.3, 156.5, 147.4, 134.1, 131.1, 130.5, 129.2,

127.3, 127.0, 126.5, 125.0, 122.0, 121.6, 118.3, 115.0,

113.9, 112.7 (aromatic carbons), 80.4 (C-3 β-lactam), 65.7

(-OCH₂), 60.3 (C-4 β-lactam), 40.1 (NCH₃), 37.2 (–NCH₂),

27.3 (–CH₂); Analysis calculated for C₃₈H₃₃N₃O₅: C, 74.61;

H, 5.44; N, 6.87%. Found: C, 74.48; H, 5.38; N, 6.65%.

2-(3-(4-(1-(4-(Dimethylamino)phenyl)-3-(naphthalen-2-

yloxy)-4-oxoazetidin-2-yl)phenoxy)propyl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (11l)

Brown solid; Mp. 192–194 °C; IR (KBr, cm⁻¹): 1736 (CO

1

β-lactam), 1702 (CO Naph), 1662 (CO Naph); H NMR

(250 MHz, DMSO-d₆): 1.93–2.00 (2H, m, –CH₂), 2.80

(6H, s, NCH₃), 3.90 (2H, t, J = 5.7 Hz, –NCH₂), 4.12 (2H,

t, J = 7.0 Hz, –OCH₂), 5.65 (1H, d, J = 4.7 Hz, H-4

β-lactam), 5.87 (1H, d, J = 4.7 Hz, H-3 β-lactam),

6.63–6.68 (4H, m, ArH), 6.95 (1H, dd, J₁= 8.7 Hz, J₂=

2.5 Hz, ArH), 7.13 (2H, d, J = 9.0 Hz, ArH), 7.22–7.33

(5H, m, ArH), 7.42 (1H, t, J = 8.0 Hz, ArH), 7.67 (1H, d,

J = 9.2 Hz, ArH), 7.72–7.81 (4H, m, ArH), 8.38–8.41 (3H,

m, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ 168.1 (CO

β-lactam), 166.2 (CO Naph), 163.0, 159.0, 152.2, 150.6,

138.9, 138.4, 135.9, 135.3, 134.0, 133.5, 132.2, 132.0,

131.8, 131.4, 131.3, 131.2, 129.7, 128.7, 126.8, 123.0,

122.6, 118.7, 117.5, 113.3 (aromatic carbons), 85.1 (C-

3 β-lactam), 70.4 (–OCH₂), 65.1 (C-4 β-lactam), 44.9

(NCH₃), 42.0 (–NCH₂), 32.0 (–CH₂); Analysis calculated

for C₄₂H₃₅N₃O₅: C, 76.23; H, 5.33; N, 6.35%. Found: C,

76.08; H, 5.05; N, 6.12%.

2-(3-(4-(3-(4-Chlorophenoxy)-1-(4-(dimethylamino)phenyl)-

4-oxoazetidin-2-yl)phenoxy)propyl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (11j)

Cream solid; Mp. 215–217 °C; IR (KBr, cm⁻¹): 1734 (CO

1

β-lactam), 1705 (CO Naph), 1658 (CO Naph); H NMR

(250 MHz, DMSO-d₆): 1.97–2.07 (2H, m, –CH₂), 2.79 (6H,

s, NCH₃), 3.96 (2H, t, J = 5.5 Hz, –NCH₂), 4.17 (2H, t, J =

6.5 Hz, –OCH₂), 5.53 (1H, d, J = 4.5 Hz, H-4 β-lactam),

5.73 (1H, d, J = 4.5 Hz, H-3 β-lactam), 6.64 (2H, d, J =

9.0 Hz, ArH), 6.69 (2H, d, J = 10.0 Hz, ArH), 6.81 (2H, d,

J = 9.0 Hz, ArH), 7.10 (2H, d, J = 9.0 Hz, ArH), 7.16–7.21

(4H, m, ArH), 7.82 (2H, t, J = 8.2 Hz, ArH), 8.41–8.45 (4H,

m, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.9 (CO

β-lactam), 161.7 (CO Naph), 158.9, 155.7, 148.0, 134.7,

131.7, 131.1, 129.7, 129.5, 127.8, 127.6, 126.9, 125.8,

125.3, 122.6, 118.8, 117.3, 114.5, 113.2 (aromatic carbons),

80.9 (C-3 β-lactam), 66.3 (–OCH₂), 60.6 (C-4 β-lactam),

40.6 (NCH₃), 37.7 (–NCH₂), 27.8 (–CH₂); Analysis calcu-

lated for C₃₈H₃₂ClN₃O₅: C, 70.64; H, 4.99; N, 6.50%.

Found: C, 70.56; H, 4.87; N, 6.42%.

General procedure for the synthesis of diacetic acid

(14)

A mixture of 1,4,5,8-naphthalenetetracarboxylic dianhy-

dride (12) (1.00 mmol) and glycine (2.20 mmol) was added

in DMF (5 ml) and the mixture was stirred at 60 °C for

several hours (TLC control in a solvent system n-hexane:

ethyl acetate = 2:1). After cooling to room temperature

20 mL water was added to the mixture and the solid diacetic

acid 14 was separated, the product was puriﬁed by recrys-

tallization from ethanol and used for the next step.

2-(3-(4-(3-(2,4-Dichlorophenoxy)-1-(4-(dimethylamino)

phenyl)-4-oxoazetidin-2-yl)phenoxy)propyl)-1H-benzo[de]

isoquinoline-1,3(2H)-dione (11k)

Brown solid; Mp. 222–224 °C; IR (KBr, cm⁻¹): 1739 (CO

1

β-lactam), 1702 (CO Naph), 1664 (CO Naph); H NMR

Medicinal Chemistry Research

2,2’-(1,3,6,8-Tetraoxo-1,3,6,8-tetrahydrobenzo[lmn][3,8]

phenanthroline-2,7-diyl)diacetic acid (14)

2-(2-(Anthracen-9-yl)-1-(4-methoxyphenyl)-4-oxoazetidin-3-

yl)-7-(2-(anthracen-9-yl)-1-(4-methoxyphenyl)-4-

oxoazetidin-3-yl)benzo[lmn][3,8]phenanthroline-1,3,6,8

(2H,7H)-tetraone (16b)

Pink solid; Mp. 220–222 °C; IR (KBr, cm⁻¹): 2368–3400

(OH, acid), 1720 (CO acid and CO Naph), 1666 (CO

Naph); ¹H NMR (400 MHz, DMSO-d₆) δ 4.77 (4H, s,

2CH₂), 8.66 (4H, s, ArH), 13.34 (2H, brs, 2OH); ¹³C NMR

(100 MHz, DMSO-d₆) δ 169.4 (CO acid), 162.5 (CO

Naph), 131.4, 126.6, 126.2 (aromatic carbons), 41.9 (CH₂);

Analysis calculated for C₁₈H₁₀N₂O₈: C, 56.55; H, 2.64; N,

7.33%. Found: C, 56.40; H, 2.55; N, 7.27%.

Brown solid (Yield 55%); Mp. 159–161 °C; IR (KBr, cm

⁻¹): 1759 (CO β-lactam), 1712 (CO Naph), 1674 (CO

Naph); ¹H NMR (250 MHz, DMSO-d₆) δ 3.57)6H, s,

O–CH₃), 6.71 (2H, d, J = 2.5 Hz, H-4 β-lactam), 6.76 (4H,

d, J = 9.0 Hz, ArH), 6.95 (2H, d, J = 2.5 Hz, H-3 β-lactam),

7.09 (4H, d, J = 9.0 Hz, ArH), 7.50–7.58 (8H, m, ArH),

8.17 (4H, d, J = 8.0 Hz, ArH), 8.48–8.54 (4H, m, ArH),

8.63 (4H, s, ArH), 8.74 (2H, s, ArH); ¹³C NMR (100 MHz,

DMSO-d₆) δ 163.1 (CO β-lactam), 162.3 (CO Naph), 159.4,

131.5, 131.2, 130.7, 129.8, 128.1, 127.2, 126.9, 126.8,

126.3, 125.7, 124.3, 118.9, 118.3, 115.1 (aromatic carbons),

62.8 (C-3 β-lactam), 56.1 (C-4 β-lactam), 55.6 (O–CH₃);

Analysis calculated for C₆₂H₄₀N₄O₈: C, 76.85; H, 4.16; N,

5.78%. Found: C, 76.78; H, 4.10; N, 5.62%.

General procedure for the bis-β-lactams 16a–g

preparation (Staudinger reaction)

The appropriate aromatic imines (Schiff bases)

(2.00 mmol), triethylamine (5.00 mmol), 1: 2,2′-(1,3,6,8-

tetraoxo-1,3,6,8-tetrahydrobenzo[lmn][3,8]phenanthro-

line-2,7-diyl)diacetic acid (14) (1 mmol) and tosyl

chloride (1.50 mmol) were added to anhydrous CH₂Cl₂

(5 mL) and the mixture was stirred at room temperature

for 24 h (TLC control in a solvent system n-hexane: ethyl

acetate = 7:3). Upon return to room temperature, the

mixture was washed twice with 1N aqueous HCl solution

(20 mL), and once with saturated aqueous NaHCO₃

solution (50 mL) and brine (20 mL). The organic layer

was dried over anhydrous Na₂SO₄and the solvent was

evaporated under reduced pressure and the crude product

was puriﬁed by column chromatography to obtain bis-

β-lactams 16a–g (see the Supplementary Information for

details).

2-(2-(Anthracen-9-yl)-1-(4-(dimethylamino)phenyl)-4-

oxoazetidin-3-yl)-7-(2-(anthracen-9-yl)-1-(4-

(dimethylamino)phenyl)-4-oxoazetidin-3-yl)benzo[lmn][3,8]

phenanthroline-1,3,6,8(2H,7H)-tetraone (16c)

Green solid (Yield 45%); Mp. 150–152 °C; IR (KBr, cm⁻¹):

1759 (CO β-lactam), 1712 (CO Naph), 1674 (CO Naph); ¹H

NMR (250 MHz, DMSO-d₆) δ 2.70)12H, s, N–CH₃), 6.52

(4H, d, J = 8.7 Hz, ArH), 6.63 (2H, d, J = 2.7 Hz, H-4

β-lactam), 6.68 (2H, d, J = 2.7 Hz, H-3 β-lactam) 7.01 (4H,

d, J = 8.7 Hz, ArH), 7.49–7.61 (8H, m, ArH), 8.15 (4H, d,

J = 8.2 Hz, ArH), 8.49–8.57 (4H, m, ArH), 8.63 (4H, s,

ArH), 8.73 (2H, s, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ

163.4 (CO β-lactam), 162.2 (CO Naph), 146.9, 135.7,

131.4, 130.4, 129.5, 127.4, 126.3, 122.7, 120.8, 118.2,

117.8, 115.3, 115.1, 114.0, 112.8 (aromatic carbons), 63.9

(C-3 β-lactam), 57.5 (C-4 β-lactam), 40.0 (N–CH₃); Ana-

lysis calculated for C₆₄H₄₆N₆O₆: C, 77.25; H, 4.66; N,

8.45%. Found: C, 77.08; H, 4.60; N, 8.12%.

2-(2-(Anthracen-9-yl)-1-(4-ethoxyphenyl)-4-oxoazetidin-3-

yl)-7-(2-(anthracen-9-yl)-1-(4-ethoxyphenyl)-4-oxoazetidin-

3-yl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-tetraone

(16a)

Brown solid (Yield 60%); Mp. 145–147 °C; IR (KBr, cm

⁻¹): 1751 (CO β-lactam), 1705 (CO Naph), 1674 (CO

1

Naph); H NMR (250 MHz, DMSO-d₆) δ 1.27)6H, t, J =

7.0 Hz, 2CH₃), 3.81 (4H, q, J = 7.0 Hz, 2O-CH₂),

6.71–6.86 (6H, m, H-4 β-lactam, ArH), 6.95 (2H, d, J =

2.2 Hz, H-3 β-lactam), 7.08 (4H, d, J = 8.7 Hz, ArH),

7.45–7.66 (8H, m, ArH), 8.16 (4H, d, J = 7.5 Hz, ArH),

8.46–8.55 (4H, m, ArH), 8.63 (4H, s, ArH), 8.74 (2H, s,

ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ 163.1 (CO

β-lactam), 162.2 (CO Naph), 155.6, 132.0, 131.6, 131.4,

131.1, 130.6, 129.1, 128.1, 126.9, 126.8, 125.7, 121.0,

118.3, 115.5, 114.9 (aromatic carbons), 63.6 (O–CH₂), 62.7

(C-3 β-lactam), 56.1 (C-4 β-lactam), 15.0 (CH₃); Analysis

calculated for C₆₄H₄₄N₄O₈: C, 77.10; H, 4.45; N, 5.62%.

Found: C, 77.02; H, 4.25; N, 5.45%.

2-(2-(Anthracen-9-yl)-1-(4-(diethylamino)phenyl)-4-

oxoazetidin-3-yl)-7-(2-(anthracen-9-yl)-1-(4-(diethylamino)

phenyl)-4-oxoazetidin-3-yl)benzo[lmn][3,8]phenanthroline-

1,3,6,8(2H,7H)-tetraone (16d)

brown solid (Yield 40%); Mp. 148–150 °C; IR (KBr, cm⁻¹):

1

1751 (CO β-lactam), 1712 (CO Naph), 1674 (CO Naph); H

NMR (250 MHz, DMSO-d₆) δ 1.37)12H, t, J = 6.7 Hz, CH₃),

3.96)8H, q, J = 6.7 Hz, N–CH₂), 6.48 (4H, d, J = 5.0 Hz,

ArH), 6.71 (2H, d, J = 2.5 Hz, H-4 β-lactam), 6.94 (2H, d,

J = 2.5 Hz, H-3 β-lactam), 7.01 (4H, d, J = 5.0 Hz, ArH),

7.54–7.68 (8H, m, ArH), 8.19 (4H, d, J = 5.0 Hz, ArH),

Medicinal Chemistry Research

8.53–8.54 (4H, m, ArH), 8.66 (4H, s, ArH), 8.76 (2H, s, ArH);

¹³C NMR (100 MHz, DMSO-d₆) δ 163.1 (CO β-lactam),

161.6 (CO Naph), 145.1, 144.6, 135.9, 135.3, 133.0, 132.5,

131.6, 130.4, 128.0, 126.9, 126.8, 121.3, 118.7, 113.0, 112.3,

(aromatic carbons), 62.6 (C-3 β-lactam), 55.8 (C-4 β-lactam),

44.0 (–NCH₂), 12.8 (CH₃); Analysis calculated for

C₆₈H₅₄N₆O₆: C, 77.70; H, 5.18; N, 7.99%. Found: C, 76.98;

H, 4.95; N, 7.52%.

ﬂuoren]-3-yl)benzo[lmn][3,8]phenanthroline-1,3,6,8(2H,7H)-

tetraone (16g)

Dark orang solid (Yield 68%); Mp. 159–161 °C; IR (KBr,

cm⁻¹): 1766 (CO β-lactam), 1712 (CO Naph), 1681 (CO

Naph); ¹H NMR (250 MHz, DMSO-d₆) δ 3.60)6H, s,

O–CH₃), 6.15 (2H, s, H-3 β-lactam), 6.76 (4H, d, J =

8.8 Hz, ArH), 6.85 (4H, d, J = 8.8 Hz, ArH), 7.02–7.06

(2H, m, ArH), 7.27–7.31 (2H, m, ArH), 7.40–7.53 (6H, m,

ArH), 7.73 (2H, d, J = 7.2 Hz, ArH), 7.87–7.95 (4H, m,

ArH), 8.69 (4H, s, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ

162.4 (CO β-lactam), 160.4 (CO Naph), 156.3, 143.3,

141.7, 139.5, 138.1, 130.5, 130.3, 129.0, 126.4, 123.3,

121.5, 118.4, 114.9 (aromatic carbons), 72.7 (C-

3 β-lactam), 67.7 (spiro carbon), 55.6 (O–CH₃); Analysis

calculated for C₅₈H₃₆N₄O₈: C, 75.97; H, 3.96; N, 6.11%.

Found: C, 75.78; H, 3.65; N, 5.92%.

2-(2-(3,4-Dimethoxyphenyl)-1-(4-ethoxyphenyl)-4-

oxoazetidin-3-yl)-7-2-(3,4-dimethoxyphenyl)-1-(4-

ethoxyphenyl)-4-oxoazetidin-3-yl)benzo[lmn][3,8]

phenanthroline-1,3,6,8(2H,7H)-tetraone (16e)

Light brown solid (Yield 65%); Mp. 146–148 °C; IR (KBr,

cm⁻¹): 1759 (CO β-lactam), 1712 (CO Naph), 1674 (CO

1

Naph); H NMR (250 MHz, DMSO-d₆) δ 1.28)6H, t, J =

7.0 Hz, CH₃), 3.69)6H, s, O–CH₃), 3.73)6H, s, O–CH₃),

3.95)4H, q, J = 7.0 Hz, O–CH₂), 5.42 (2H, d, J = 2.5 Hz, H-

4 β-lactam), 5.94 (2H, d, J = 2.5 Hz, H-3 β-lactam), 6.90

(4H, d, J = 9.0 Hz, ArH), 6.94 (2H, d, J = 8.5 Hz, ArH), 7.05

(2H, d, J = 8.7 Hz, ArH), 7.16–7.22 (6H, m, ArH), 8.68 (4H,

s, ArH); ¹³C NMR (100 MHz, DMSO-d₆) δ 162.9 (CO

β-lactam), 162.8 (CO Naph), 155.4, 149.4, 132.1, 131.4,

131.2, 129.3, 129.1, 126.8, 119.6, 118.9, 115.4, 112.3, 111.0

(aromatic carbons), 64.2 (O–CH₂), 63.7 (C-3 β-lactam), 59.5

(C-4 β-lactam), 56.0 (O–CH₃), 55.9 (O–CH₃), 15.1 (CH₃);

Analysis calculated for C₅₂H₄₄N₄O₁₂: C, 68.11; H, 4.84; N,

6.11%. Found: C, 68.01; H, 4.65; N, 5.92%.

DPPH radical scavenging

For evaluating radical scavenging activity, the diphenylpi-

crylhydrazyl (DPPH) scavenging assay was performed as

described previously (Malterud et al. 1993). Brieﬂy, a sui-

table dilution of test compound (0.05 ml) dissolved in

DMSO was mixed with a solution of DPPH in methanol

(A₅₁₇= 1.0; 2.95 ml) and the UV absorbance at 517 nm was

measured both before addition of the test lactam and again

after 5 min. Percent radical scavenging was calculated as

100 × (A_start− A_end)/(A_start), where A_startis the absorbance

before addition of test compound and A_endis the absorbance

value after 5 min of reaction time. Vitamin C in DMSO

(195 µg/ml) and blank DMSO were used as positive and

negative controls, respectively.

2-(2-(3,4-Dimethoxyphenyl)-1-(4-(dimethylamino)phenyl)-

4-oxoazetidin-3-yl)-7-(-2-(3,4-dimethoxyphenyl)-1-(4-

(dimethylamino)phenyl)-4-oxoazetidin-3-yl)benzo[lmn][3,8]

phenanthroline-1,3,6,8(2H,7H)-tetraone (16f)

Method of anticancer activity and cytotoxicity

assays

Dark green solid (Yield 47%); Mp. 165–167 °C; IR (KBr,

cm⁻¹): 1766 (CO β-lactam), 1712 (CO Naph), 1674 (CO

Naph); ¹H NMR (250 MHz, DMSO-d₆) δ 2.82)12H, s,

N–CH₃), 3.69)6H, s, O–CH₃), 3.72)6H, s, O–CH₃), 5.42

(2H, d, J = 2.2 Hz, H-4 β-lactam), 5.91 (2H, d, J = 2.2 Hz,

H-3 β-lactam), 6.69 (4H, d, J = 8.5 Hz, ArH), 6.94 (2H, d,

J = 11.0 Hz, ArH), 7.03 (2H, d, J = 8.0 Hz, ArH),

7.11–7.15 (6H, m, ArH), 8.69 (4H, s, ArH); ¹³C NMR

(100 MHz, DMSO-d₆) δ 163.1 (CO β-lactam), 162.8 (CO

Naph), 155.4, 149.4, 147.6, 131.4, 131.2, 129.3, 126.8,

126.5, 119.6, 118.9, 115.4, 112.3, 111.0 (aromatic carbons),

62.7 (C-3 β-lactam), 59.5 (C-4 β-lactam), 56.0 (O–CH₃),

55.9 (O–CH₃), 40.3 (N–CH₃); Analysis calculated for

C₅₂H₄₆N₆O₁₀: C, 68.26; H, 5.07; N, 9.19%. Found: C,

68.18; H, 4.95; N, 8.92%.

In vitro cytotoxic effect of some compounds was

evaluated using standard 3-(4,5-dimethylthiazol-2-yl)-2, 5-

diphenyltetrazolium bromide (MTT) assay (Mosmann

1983; Riss et al. 2013; Rowan et al. 2001). The MCF-7

(breast cancer cells), TC-1 (mouse lung epithelial cells), and

HepG2 (liver hepatocellular carcinoma cells) cell lines (all

cell lines were purchased from cell culture collection of

Pasteur Institute, Tehran branch, Iran) were seeded in 96-

well plate at a density of 5 × 10³cells/well. Cells were

incubated at 37 °C in humidiﬁed 5% CO₂incubator. After

incubation for 24 h, the cells were incubated with 200, 50,

10, and 5 µM concentrations of compounds. After 24 h, the

medium was removed and the cells were washed with

phosphate buffered saline then, 25 µl of MTT solution

(4 µM) was added to each well and incubated at 37 °C for

4 h. After incubation time, The MTT solution was removed

2-(1-(4-Methoxyphenyl)-4-oxospiro[azetidine-2,9’-ﬂuoren]-

3-yl)-7-((S)-1-(4-methoxyphenyl)-4-oxospiro[azetidine-2,9’-

Medicinal Chemistry Research

from the wells and replaced with 100 µl of dimethylsulf-

oxide (DMSO). After 10 mines incubation at 37 °C, the

optical absorption was read at a wavelength of 540 nm

using a microplate reader (Power wave X52, BioTek

instrument Inc., US). All experiments were accomplished in

triplicate for each concentration (Kianpour et al.

2017, 2018)

model the interaction/binding between the ligand and

duplex. The other parameters were left at program default

values. The ﬁnal binding mode depicted was selected taking

into account the best-ranked scoring functions (Shaikh et al.

2017). Ligand DNA interactions were visualized on the

basis of docking results using Discovery Studio

Visualizer 17.2.

UV–visible and ﬂuorescence spectroscopy

Acknowledgements The authors would like to thank the Shiraz Uni-

versity Research Council for ﬁnancial support (Grant no. 97-GR-SC-

23) and Dr. Attila Benyei for collecting X-ray data.

Calf thymus DNA (CT-DNA) was purchased from Sigma-

Aldrich and used without further puriﬁcation. UV–vis

spectra were obtained using a Perkin-Elmer Lambda

25 spectrophotometer. Temperature was controlled by a

EYELA NCB-3100 constant temperature bath. Fluores-

Compliance with ethical standards

Conﬂict of interest The authors declare that they have no conﬂict of

interest.

cence

determination

was

recorded

on

an

LS45 spectroﬂuorimeter equipped with thermostat bath

and quartz cells (1.0 cm). Experiments were done in

50 mM Tris-HCl buffer containing 100 mM NaCl at pH

7.4. CT-DNA solution was prepared with doubly distilled

water, and it was stored at 4 °C in the dark. UV–visible

absorption titration studies have been performed using a

ﬁxed concentration of the compounds (16a, 16b, and 16e

(20 µM), 16d (28 µM), 16f (40 µM), and 16g (62 µM) all

in DMSO) and varying the CT-DNA concentration. The

concentration of CT-DNA was determined by UV–vis

absorption spectroscopy, and the molar extinction coefﬁ-

cient (6600 M⁻¹cm⁻¹) at ∼260 nm was obtained (Zsila

et al. 2004). The purity of DNA was determined by fol-

lowing the absorption ratio of the bands at 260 and

280 nm. It was found to be 1.8, showing that DNA is

sufﬁciently protein-free (Marmur 1961).

Publisher’s note Springer Nature remains neutral with regard to

jurisdictional claims in published maps and institutional afﬁliations.

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2:2902–2910

Article Doi

Cytotoxicity, anticancer, and antioxidant properties of mono and bis-naphthalimido β-lactam conjugates

DOI: 10.1007/s00044-020-02552-1

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Article abstract of DOI:10.1007/s00044-020-02552-1

Full text of DOI:10.1007/s00044-020-02552-1

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