Synthesis and biological evaluation of new 2-azetidinones with sulfonamide structures

Molecules 2013, 18, 4140-4157; doi:10.3390/molecules18044140

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molecules

ISSN 1420-3049

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Article

Synthesis and Biological Evaluation of New 2-Azetidinones with

Sulfonamide Structures

Oana Maria Dragostin ¹, Florentina Lupascu ¹, Cornelia Vasile ², Mihai Mares ³,

Valentin Nastasa ³, Ramona Florina Moraru ³, Dragos Pieptu ^4,* and Lenuta Profire ^1,*

1

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Grigore T. Popa University of

Medicine and Pharmacy, 16 University Street, Iasi 700115, Romania;

E-Mails: farmacist_oanamaria@yahoo.com (O.M.D.); geani0407@yahoo.com (F.L.)

2

Department of Physical Chemistry of Polymers, Petru Poni Institute of Macromolecular Chemistry,

Romanian Academy, 41A Grigore Ghica Voda Alley, Iasi 700487, Romania;

E-Mail: cvasile@icmpp.ro

3

Laboratory of Antimicrobial Chemotherapy, Faculty of Veterinary Medicine, Ion Ionescu de la

Brad University of Agricultural Sciences and Veterinary Medicine, 8 Mihail Sadoveanu Alley,

Iasi 700489, Romania; E-Mails: mycomedica@gmail.com (M.M.); alvali2003@yahoo.com (V.N.);

moraru82@gmail.com (R.F.M.)

4

Department of Plastic Surgery, Faculty of Medicine, Grigore T. Popa University of Medicine and

Pharmacy, 16 University Street, Iasi 700115, Romania

* Authors to whom correspondence should be addressed; E-Mails: dpieptu@chpla.umfiasi.ro (D.P.);

nprofire@yahoo.com (L.P.); Tel.: +40-232-412375 (L.P.); Fax: +40-232-211818 (L.P.).

Received: 26 February 2013; in revised form: 26 March 2013 / Accepted: 3 April 2013 /

Published: 8 April 2013

Abstract: New series of N-(arylidene)hydrazinoacetyl sulfonamides 4a_1–6, 4b_1–6and

N-(4-aryl-3-chloro-2-oxoazetidin-1-yl)aminoacetyl sulfonamides 5a_1–6, 5b_1–6were synthesized.

The structures of the new derivatives was confirmed using spectral methods (FT-IR,

¹H-NMR, ¹³C-NMR). The antibacterial activities of these compounds against Gram

positive (Staphyloccoccus aureus ATCC 6583, Staphyloccoccus epidermidis ATCC 12228,

Enterococcus faecalis ATCC 25912) and Gram negative (Klebsiella pneumoniae CIP

53153, Proteus vulgaris CIP 104989, Citrobacter freundii CIP 5732, Enterobacter cloacae

CIP 103475, Escherichia coli ATCC 25922, Pseudomonas aeruginosa CIP 82118)

bacterial strains were evaluated using the broth micro-dilution method. Compound 4a₂

displayed the highest antibacterial activity, especially against Staphyloccoccus epidermidis,

Enterococcus faecalis and Pseudomonas aeruginosa. The antioxidant potential of the

Molecules 2013, 18

synthesized compounds was also investigated according to ferric reducing power, total

4141

antioxidant activity and DPPH radical scavenging assays. All tested compounds showed

excellent antioxidant activity in comparison with sulfadiazine and sulfisoxazole which

were used as parent sulfonamides. Moreover, some of them showed an antioxidant activity

comparable with that of ascorbic acid. In general, the compounds designed based on a

sulfadiazine skeleton (compounds 4a_1–6, 5a_1–6) are more active than those obtained from

sulfisoxazole (compounds 4b_1–6, 5b_1–6), and the N-(arylidene)hydrazinoacetyl sulfonamide

derivatives 4a_1–6, 4b_1–6are more active than their azetidionone analogues 5a_1–6, 5b_1–6.

Keywords: sulfonamide; azetidinone; synthesis; antimicrobial activity; antioxidant effect

1. Introduction

The 2-azetidinone skeleton, otherwise known as the β-lactam ring, has been recognized as a useful

building block in the synthesis of biologically important compounds. Azetindin-2-one derivatives

display interesting biological activities such as antifungal, antimicrobial [1–4], antitubercular [5,6],

analgesic, anti-inflammatory [7,8], chymase inhibitory [9], antitumoral [10–12], antiviral, antidiabetic

and cholesterol absorption inhibitory properties [13]. The activity of famous antibiotic classes such as

the penicillins, cephalosporins, carumonam, aztreonam, thienamicine, nocardicins and carbapenems is

attributed to the presence of an 2-azetidinone ring [2]. Unfortunately, the most widely used of them

exert selective pressure on bacteria and permit the proliferation of resistant organisms. Several

synthetic and semi-synthetic β-lactam antibiotics were developed due to the growing resistance of

bacteria towards the classical β-lactam antibiotics and the need for drugs with a more specific

antibacterial activity [1]. The biological activity of sulfonamides is also well documented. They have

be found to be useful in a variety of applications, including antibacterial, antifungal, antitumor agents,

diuretics, carbonic anhydrase inhibitors, hypoglycemic agents, thyroid inhibitors, anticonvulsants and

protease inhibitors [14,15]. Among antibacterial sulfonamides, sulfadiazine and its silver and cerium

salts have an important place. They are widely used as topical agents for the management of burns

where they prevent infections and promote rapid healing with minimal scarring [15].

Wounds are physical injuries that result in an opening of the skin. Proper healing of wounds is

essential for the restoration of disrupted anatomical continuity and disturbed functional status of the

skin [16]. Normal healing of wounds is a dynamic process following three phases: inflammation,

granulation (tissue formation) and re-epithelization (tissue remodeling), which overlap in time [17]. It was

proven that reactive oxygen species (ROS) and bacterial infections are deleterious to the wound

healing process due to their harmful effects on cells and tissues [18]. ROS are produced in high

amounts at wound sites as a defense mechanism against invading bacteria. At the same time, the

process of wound healing may be hampered by the presence of free radicals, which can damage the

cells surrounding the wound, or by microbial infection [19] and recent data has proved the beneficial

effects of antioxidants in the wound healing process [20–22]. In the present study, we are reporting the

design, synthesis and biological evaluation of some new 2-azetidinone derivatives of sulfadiazine and

sulfisoxazole with potential use in wound healing processes.

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2. Results and Discussion

2.1. Chemistry

Azetidinone derivatives 5a_1–6, 5b_1–6were prepared using the method summarized in Scheme 1.

First, sulfadiazine (4-amino-N-pyrimidin-2-yl-benzensulfonamide, 1a) and sulfisoxazole [4-amino-N-

(3,4-dimethyl-1,2-oxazol-5-yl)benzensulfonamide, 1b] were reacted with chloroacetyl chloride whereby

the corresponding chloracetyl derivatives 2a–b were obtained. Compounds 2a–b on amination with

hydrazine hydrate afforded hydrazinoacetyl sulfonamide derivatives 3a–b [23]. The condensation

reaction of compounds 3a–b with various aromatic aldehydes yielded N-(arylidene)hydrazinoacetyl

sulfonamide derivatives 4a_1–6, 4b_1–6. Finally, the compounds 4a_1–6, 4b_1–6upon reaction with

chloracetyl chloride in the presence of triethylamine afforded N-(4-aryl-3-chloro-2-oxoazetidin-1-

yl)aminoacetyl sulfonamides 5a_1–6, 5b_1–6.

Scheme 1. Synthesis of azetidinone derivatives (5a_1–6, 5b_1–6).

O

C

O

NH₂

Cl

NH

S

CH₂

NH C CH₂NH NH₂

NH C CH₂NH N CH

a

b

c

O

S

NH R₁

S

NH R₁

S

NH R₁

R₂

O

2a-b

4a_1-6

4b_1-6

O

3a-b

1a-b

H

C

R₂

NH

O

NH C CH₂

N

Cl

d

H₃C

;

CH₃

N

O

N

R₁=

S

NH R₁

O

5a_1-6

5b_1-6

R₂= H, F, Cl, Br, OH, -NO₂

Reagents and Conditions: (a) chloracetyl chloride, dry acetone, anhydrous K₂CO_3,heating 12 h;

(b) hydrazine hydrate 99%, ethanol, heating 10 h; (c) aromatic aldehydes, acetic acid, ethanol 50%,

heating 8 h; (d) chloracetyl chloride, anhydrous 1,4-dioxane, triethylamine, room temperature,

stirrer 3 h.

The structure of the compounds was assigned on the basis of spectral (IR, ¹H-NMR, ¹³C-NMR) data.

The IR spectra of compounds 4a_1–6(sulfadiazine series) showed absorption bands for the -CH₂-NH-

group in the range of 2830–2853 cm⁻¹, for the NH-CO group in the range of 1622–1623 cm⁻¹and for

the characteristic azomethine group (CH=N) in the 1534–1539 cm⁻¹range. In the spectra of the

sulfisoxazole derivatives 4b_1–6the characteristic absorption bands were observed in the region of

2842–2870 cm⁻¹(-CH₂-NH-), 1622–1629 cm⁻¹(NH-CO) and 1507–1540 cm⁻¹(CH=N). In the ¹H-NMR

spectra of the N-(arylidene)hydrazinoacetyl sulfonamides 4a_1–6, 4b_1–6the -CH₂-NH methylene protons

resonated as a doublet in the 3.56–3.79 ppm region, while the proton of the azomethine group (N=CH)

Molecules 2013, 18

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appeared as a singlet in the 8.06–8.22 ppm region. In the IR spectra of the azetidinone derivatives 5a_1–6,

5b_1–6the carbonyl group of the β-lactam ring appeared as a characteristic absorption band in the range

1

of 1739–1745 cm⁻¹and 1739–1752 cm⁻¹, respectively. The IR absorption bands and H-NMR signals

characteristic of the azomethine group disappeared from the spectra of the azetidinone derivatives,

which confirms that the cyclization reaction with chloracetyl chloride took place. The ¹H-NMR spectra of

5a_1–6(sulfadiazine series) and 5b_1–6(sulfisoxazole series) showed two doublets, which are

characteristic for N-CH and CH-Cl that appear in the range of 5.32–5.45 and 5.02–5.23 ppm,

respectively. In ¹³C-NMR spectra of the azetidinone derivatives, the characteristic signals for a β-lactam

ring (CH-NH, CH-Cl, CO cyclic) appeared in the range of 67.8–76.1, 61.04–64.3 and 160.3–162.8 ppm

(5a_1–6series) and 75.9–79.1, 64.2–67.0 and 160.3–168.2 ppm (5b_1–6series) respectively. The spectral

data lend strong support to the proposed structures of all the synthesized compounds.

2.2. Biological Evaluation

2.2.1. Antibacterial Activity

The antibacterial activities of the title compounds were evaluated using the broth micro-dilution

method [24] and the results are listed Tables 1 and 2.

Table 1. MIC values (µg/mL) of the sulfadiazine derivatives 4a_1–6, 5a_1–6.

MICs * values (µg/mL)

Sample

SA

SE

EF

KP

PV

CF

EC ^a

EC ^b

PA

256

128

4a₁

4a₂

4a₃

4a₄

4a₅

4a₆

5a₁

5a₂

5a₃

5a₄

5a₅

5a₆

S

>512 >512 >512 >512 >512 >512 >512 >512

>512 128 256 >512 >512 >512 >512 >512

>512 >512 >512 >512 >512 >512 >512 >512 >512

>512 >512 >512 >512 >512 >512 >512 >512 128

>512 >512 >512 >512 >512 >512 >512 >512 >512

>1000 >1000 >1000 >1000 >1000 >1000 >1000 >1000 800

A

35.8

3

16

-

8

35.8

128

* Mean values (n = 3); SA: Staphyloccoccus aureus ATCC 6583; SE: Staphyloccoccus epidermidis ATCC

12228; EF: Enterococcus faecalis ATCC 25912; KP: Klebsiella pneumoniae CIP 53153; PV: Proteus

a

b

vulgaris CIP 104989; CF: Citrobacter freundii CIP 5732; EC : Enterobacter cloacae CIP 103475; EC :

Escherichia coli ATCC 25922; PA: Pseudomonas aeruginosa CIP 82118; S: Sulfanilamide; A: Ampicillin.

Molecules 2013, 18

Table 2. MIC values (µg/mL) of the sulfisoxazole derivatives 4b_1–6, 5b_1–6.

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MICs * values (µg/mL)

Sample

SA

>512

>1000

35.8

SE

>512

>1000

3

EF

>512

>1000

3

KP

>512

>1000

16

PV

>512

>1000

-

CF

>512

>1000

-

EC ^a

>512

>1000

8

EC ^b

>512

>1000

35.8

PA

4b₁

4b₂

4b₃

4b₄

4b₅

4b₆

5b₁

5b₂

5b₃

5b₄

5b₅

5b₆

S

>512

256

>512

800

A

128

* Mean values (n = 3); SA: Staphyloccoccus aureus ATCC 6583; SE: Staphyloccoccus epidermidis ATCC

12228; EF: Enterococcus faecalis ATCC 25912; KP: Klebsiella pneumoniae CIP 53153; PV: Proteus

a

b

vulgaris CIP 104989; CF: Citrobacter freundii CIP 5732; EC : Enterobacter cloacae CIP 103475; EC :

Escherichia coli ATCC 25922; PA: Pseudomonas aeruginosa CIP 82118; S: Sulfanilamide; A: Ampicillin.

The minimum inhibitory concentrations (MICs) of almost all compounds were more than 512 μg/mL.

The N-(arylidene)hydrazinoacetyl derivative of sulfadiazine (compound 4a₂) was the most active

compound, as it was active on Staphyloccoccus epidermidis ATCC 12228 (128 μg/mL), Enterococcus

faecalis ATCC 25912 (256 μg/mL) and Pseudomonas aeruginosa CIP 82118 (128 μg/mL). The

compounds 4a₁(256 μg/mL), 4a₄(128 μg/mL) and 4b₅(256 μg/mL) were active against Pseudomonas

aeruginosa. All tested compounds are more active than sulfanilamide, but less active than ampicillin

used as positive controls.

2.2.2. Antioxidant Activity

2.2.2.1. Ferric Reducing Power

The measurement of reducing power defines an important aspect of the antioxidant activity of the

compounds. In this assay, the presence of a reducing agent in the sample results in reducing of the

ferric/ferricyanide complex to its ferrous (Fe²⁺) form. The amount of Fe²⁺is then quantitatively

monitored by measuring the intensity of Perl’s Prussian blue colour complex at 695 nm [25]. The

results expressed as EC₅₀values (mg/mL) are presented in Tables 3 and 4. The small value of the EC₅₀

indicates a higher ferric reducing power.

Molecules 2013, 18

Table 3. Ferric reducing power (EC₅₀mg/mL) of the sulfadiazine derivatives 4a_1–6, 5a_1–6.

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Sample

4a₁

EC₅₀mg/mL

0.0663 ± 0.0056

0.0756 ± 0.0040

0.0856 ± 0.0051

0.0790 ± 0.0026

0.0510 ± 0.0036

0.0503 ± 0.0025

2.6140 ± 0.0301

Sample

5a₁

EC₅₀mg/mL

0.1553 ± 0.0152

0.1376 ± 0.0002

0.1745 ± 0.0125

0.1798 ± 0.0018

0.0945 ± 0.0085

0.2277 ± 0.0037

0.0075 ± 0.0002

4a₂

5a₂

4a₃

5a₃

4a₄

5a₄

4a₅

5a₅

4a₆

5a₆

1a

AA

Data are mean ± SD (n = 3, p  0.05).

As it can be seen both N-(arylidene)hydrazinoacetyl and azetidinone derivatives are more active

than their sulfonamide parents, sulfadiazine (1a) and sulfizoxazole (1b). In the N-(arylidene)

hydrazinoacetyl series of sulfadiazine (compounds 4a_1–6) it was observed that the most active

compounds were those which resulted from reaction of condensation with 4-hydroxybenzaldehyde

(compound 4a₅) and 4-nitrobenzaldehyde (compound 4a₆). The values of EC₅₀for these compounds

were 0.0510 ± 0.0036 (compound 4a₅) and 0.0503 ± 0.0025 (compound 4a₆), which means that they

are about 50 times more active than sulfadiazine (EC₅₀= 2.6140 ± 0.0301). Concerning the azetidinone

series the most active compound was 5a₅, which is the analogue of 4a₅in the azetidinone series. This

compound is approximately 28 time more active (EC₅₀= 0.0945 ± 0.0085) then sulfadiazine

(EC₅₀= 2.6140 ± 0.0301) (Table 3). The ferric reducing power of the compounds resulting through

modulation of sulfisoxazole is lower than that of the the analogues of the sulfadiazine series. In

reference to sulfisoxazole (1b), all tested compounds 4b_1–6, 5b_1–6are more active. The most active

compounds were 4b₅[N-(arylidene)hydrazinoacetyl series] and 5b₅(azetidinone series), which have in

their structure the 4-hydroxyphenyl radical. These compounds are 46 times (4b₅, EC₅₀= 0.0210 ± 0.0065)

and 10 times (5b₅, EC₅₀= 0.0935 ± 0.0098) more active, respectively, than sulfisoxazole

(1b, EC₅₀= 0.9640 ± 0.0443) (Table 4).

Table 4. Ferric reducing power (EC₅₀mg/mL) of the sulfisoxazole derivatives 4b_1–6, 5b_1–6.

Sample

4b₁

EC₅₀mg/mL

0.0756 ± 0.0055

0.2043 ± 0.0055

0.0610 ± 0.0036

0.0612 ± 0.0040

0.0210 ± 0.0065

0.1173 ± 0.0066

0.9640 ± 0.0443

Sample

5b₁

EC₅₀mg/mL

0.1450 ± 0.0003

0.1164 ± 0.0025

0.1915 ± 0.0216

0.3182 ± 0.0411

0.0935 ± 0.0098

0.1106 ± 0.0149

0.0075 ± 0.0002

4b₂

5b₂

4b₃

5b₃

4b₄

5b₄

4b₅

5b₅

4b₆

5b₆

1b

AA

Data are mean ± SD (n = 3, p  0.05).

The chemical modulation of the parent sulfonamides improve their ferric reducing power and all

tested compounds are more active than sulfadiazine and sulfisoxazole, respectively, but they are less

active than ascorbic acid (AA) at the same concentration.

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2.2.2.2. Total Antioxidant Activity

The total antioxidant activity was determined using phophomolybdenum blue complex with a

maximum absorption at 695 nm [26]. The data presented in Tables 5 and 6 show that the tested

compounds are more active than sulfadiazine and sulfisoxazole, respectively, and moreover, the

sulfadiazine derivatives are more active than sulfisoxazole compounds.

Table 5. Total antioxidant activity (EC₅₀mg/mL) of the sulfadiazine derivatives 4a_1–6, 5a_1–6.

Sample

4a₁

EC₅₀mg/mL

0.0180 ± 0.0044

0.0280 ± 0.0067

0.0110 ± 0.0007

0.0360 ± 0.0089

0.0440 ± 0.0050

0.0220 ± 0.0072

6.6483 ± 0.0180

Sample

5a₁

EC₅₀mg/mL

0.0398 ± 0.0022

0.0498 ± 0.0015

0.0330 ± 0.0098

0.0507 ± 0.0037

0.0563 ± 0.0009

0.0341 ± 0.0055

0.0067 ± 0.0003

4a₂

5a₂

4a₃

5a₃

4a₄

5a₄

4a₅

5a₅

4a₆

5a₆

1a

AA

Data are mean ± SD (n = 3, p  0.05).

Table 6. Total antioxidant activity (EC₅₀mg/mL) of the sulfisoxazole derivatives 4b_1–6, 5b_1–6.

Sample

4b₁

EC50 mg/mL

0.0481 ± 0.0042

0.0612 ± 0.0078

0.0330 ± 0.0009

0.0332 ± 0.0047

0.0551 ± 0.0086

0.0794 ± 0.0091

21.658 ± 0.0224

Sample

5b₁

EC50 mg/mL

0.0433 ± 0.0009

0.0756 ± 0.0033

0.0574 ± 0.0025

0.0385 ± 0.0078

0.0718 ± 0.0008

0.0825 ± 0.0045

0.0067 ± 0.0003

4b₂

5b₂

4b₃

5b₃

4b₄

5b₄

4b₅

5b₅

4b₆

5b₆

1b

AA

Data are mean ± SD (n = 3, p  0.05).

The most favorable influence seems to be the presence of halogen on the phenyl ring, especially the

presence of chlorine in the sulfadiazine series (compounds 4a₃, 5a₃) and the presence of chlorine and

bromine in the sulfisoxazole series (compounds 4b_3–4, 5b_3–4). The compound 4a₃(EC₅₀= 0.0110 ± 0.0007)

is about 600 times more active than sulfadiazine (1a) (EC₅₀= 6.6483 ± 0.0180) and its antioxidant

activity is comparable with the activity of ascorbic acid (AA) (EC₅₀= 0.0067 ± 0.0003). Although its

azetidinone analogue 5a₃has a lower activity, it remains significant in reference with sulfadiazine

(Table 5). In the sulfisoxazole series the compounds 4b₃(EC₅₀= 0.0330 ± 0.0009) and 4b₄

(EC₅₀= 0.0332 ± 0.0047) are approximately 650 time more active than sulfisoxazole (1b)

(EC₅₀= 21.658 ± 0.0224). Their azetidinone analogues are 380 times (5b₃, EC₅₀= 0.0574 ± 0.0025) and

560 times (5b₄, EC₅₀= 0.0385 ± 0.0078) more active than sulfisoxazole.

2.2.2.3. DPPH Radical Scavenging Assay

DPPH is a well-know radical which demonstrates a strong absorption band centered at about

517 nm, and it becomes colorless or pale yellow when it is neutralized. DPPH radical is scavenged by

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antioxidants through the donation of proton forming the reduced DPPH, and it is commonly used to

evaluate the radical scavenging capacity of antioxidants [27]. The scavenging activities of the

N-(arylidene)hydrazinoacetyl sulfonamides 4a_1–6, 4b_1–6and N-(4-aryl-3-chloro-2-oxoazetidin-1-

yl)aminoacetyl sulfonamides 5a_1–6, 5b_1–6, are presented in Tables 7 and 8. All tested compounds are

more active than their parent (sulfadiazine and sulfisoxazole) sulfonamides and some of them have a

scavenging ability comparable with the scavenging ability of ascorbic acid.

Table 7. DPPH radical scavenging ability of sulfadiazine derivatives 4a_1–6, 5a_1–6.

Sample Scavenging ability (%) Sample Scavenging ability (%)

4a₁

4a₂

4a₃

4a₄

4a₅

4a₆

1a

87.73 ± 0.69

92.22 ± 0.89

73.09 ± 0.50

85.66 ± 0.89

93.17 ± 0.64

77.28 ± 0.83

11.15 ± 0.24

5a₁

5a₂

5a₃

5a₄

5a₅

5a₆

AA

67.18 ± 0.14

78.25 ± 0.49

80.94 ± 0.74

71.52 ± 0.48

61.28 ± 0.13

71.61 ± 0.33

97.08 ± 0.52

Table 8. DPPH radical scavenging ability of sulfisoxazole derivatives 4b_1–6, 5b_1–6.

Sample Scavenging ability (%) Sample Scavenging ability (%)

4b₁

4b₂

4b₃

4b₄

4b₅

4b₆

1b

48.17 ± 0.63

6.43 ± 0.41

67.39 ± 0.52

42.95 ± 0.23

61.20 ± 0.68

82.65 ± 0.18

36.59 ± 0.08

5b₁

5b₂

5b₃

5b₄

5b₅

5b₆

AA

64.28 ± 0.49

44.03 ± 0.13

44.01 ± 0.86

35.52 ± 0.48

61.28 ± 0.13

33.81 ± 0.09

97.08 ± 0.52

The compounds obtained starting from sulfadiazine (compounds 4a_1–6, 5a_1–6, Table 7) are more

active than sulfisoxazole derivatives 4b_1–6, 5b_1–6(Table 8). In reference with sulfadiazine (1a), its

N-(arylidene)hydrazinoacetyl derivatives 4a_1–6are 6.5–8.4 times more active. Under similar conditions

the azetidinone derivatives 5a_1–6are slightly less active, being 5.5–7.3 more active than sulfadiazine.

The most active compound is 4a₅[N⁴-(4-hydroxybenzylidene)hydrazinoacetylamino-N¹-(pyrimidin-2-

yl)benzensulfonamide]; its scavenging ability (93.17 ± 0.64) being 8.4 time higher than sulfadiazine

(11.15 ± 0.24) and comparable with ascorbic acid (97.08 ± 0.52).

3. Experimental

3.1. General Procedures

Melting points were measured using a Buchi Melting Point B-540 apparatus and are uncorrected.

The FT-IR spectra were recorded on an ABB Bomen MB3000 spectrometer, over a 500–4000 cm⁻¹

range, after 32 scans at a resolution of 4 cm⁻¹. The spectra processing was carried out with Horizon

1

13

MB^TMFTIR Software. The H-NMR (300 MHz) and C-NMR (75 MHz) spectra were obtained on a

Bruker Avance ARX-300 spectrometer using tetramethylsilane as internal standard and DMSO-d₆as

Molecules 2013, 18

4148

solvent. The chemical shifts are shown in  values (ppm). The progress of the reaction was monitored

on TLC, using pre-coated Kieselgel 60 F254 plates (Merck) and the compounds were visualized by

UV light.

3.2. Synthetic Procedures

3.2.1. Preparation of N-(arylidene)hydrazinoacetyl Sulfonamides 4a_1–6; 4b_1–6

To a solution of hydrazinoacetyl sulfonamide derivatives (10 mmol) in ethanol 50% (200 mL),

glacial acetic acid (0.5 mL) and the appropriate aldehyde (10 mmol) were added. The mixture was

heated under reflux for 8 h, and then it was cooled at room temperature. The solid was filtered off,

dried and recrystallized from isopropyl alcohol.

N⁴-(Benzylidene)hydrazinoacetylamino-N¹-(pyrimidin-2-yl)benzensulfonamide (4a₁). Yield 87%, m.p.

200–202 C; IR (KBr, cm⁻¹): 3450 (-NH), 2853 (CH₂-NH), 2924 (=CH- pyrimidine ring), 1693

(C=O), 1623 (HN-CO), 1591 (-C=C- aromatic ring), 1534 (N=CH), 1448 (-C=C- pyrimidine ring),

1

1313 (C-O), 1128 (-NH-SO₂), 1097 (CH aromatic ring), 947 (S-N), 840 (S-C); H-NMR δ: 8.72–8.91

(dm, 3H, pyrimidine ring), 8.27 (s, 1H, CO-NH), 8.08 (s, 1H, N=CH), 7.15–7.90 (dm, 9H, Ar-H), 4.19

(s, 1H, -NH-SO₂), 3.60 (d, 2H, CH₂), 3.43 (m, 1H, HN-N).

N⁴-(4-Fluorobenzylidene)hydrazinoacetylamino)-N¹-(pyrimidin-2-yl)benzensulfonamide (4a₂). Yield

90%, m.p. 20–210 C; IR (KBr, cm⁻¹): 3350 (-NH), 2833 (CH₂-NH), 2933 (=CH- pyrimidine ring),

1693 (C=O), 1622 (HN-CO), 1592 (-C=C- aromatic ring), 1535 (N=CH), 1455 (-C=C- pyrimidine

ring), 1314 (C-O), 1229 (C-F), 1130 (-NH-SO₂), 1099 (CH aromatic ring), 928 (S-N), 836 (S-C); ¹H-NMR

δ: 8.63-8.90 (dm, 3H, pyrimidine ring), 8.29 (s, 1H, CO-NH), 8.10 (s, 1H, N=CH), 7.1–7.95 (d, 8H, Ar-H),

4.20 (s, 1H, -NH-SO₂), 3.79 (d, 2H, CH₂), 3.45 (m, 1H, HN-N).

N⁴-(4-Chlorobenzylidene)hydrazinoacetylamino-N¹-(pyrimidin-2-yl)benzensulfonamide (4a₃). Yield

83% m.p. 20–207 C; IR (KBr, cm⁻¹): 3448 (-NH), 2830 (CH₂-NH), 2930 (=CH- pyrimidine ring),

1695 (C=O), 1623 (HN-CO), 1591 (-C=C- aromatic ring), 1536 (N=CH), 1496 (-C=C- pyrimidine

ring), 1312 (C-O), 1173 (-NH-SO₂), 1084 (CH aromatic ring), 940 (S-N), 839 (S-C), 751 (C-Cl);

¹H-NMR δ: 8.7–8.89 (dm, 3H, pyrimidine ring), 8.22 (s, 1H, CO-NH), 8.15 (s, 1H, N=CH), 7.0–7.76

(d, 8H, Ar-H), 4.17 (s, 1H, -NH-SO₂), 3.61 (d, 2H, CH₂), 3.40 (m, 1H, HN-N).

N⁴-(4-Bromobenzylidene)hydrazinoacetylamino-N¹-(pyrimidin-2-yl)benzensulfonamide (4a₄). Yield

81%, m.p. 212–213 C; IR (KBr, cm⁻¹): 3446 (-NH), 2850 (CH₂-NH), 2940 (=CH- pyrimidine ring),

1690 (C=O), 1623 (HN-CO), 1591 (-C=C- aromatic ring), 1535 (N=CH), 1454 (-C=C- pyrimidine

ring), 1314 (C-O), 1130 (-NH-SO₂), 1079 (CH aromatic ring), 959 (S-N), 840 (S-C), 550 (C-Br); ¹H-NMR

δ: 8.71–8.89 (dm, 3H, pyrimidine ring), 8.21 (s, 1H, CO-NH), 8.18 (s, 1H, N=CH), 7.19–7.84 (d, 8H,

Ar-H), 4.16 (s, 1H, -NH-SO₂), 3.56 (d, 2H, CH₂), 3.40 (m, 1H, HN-N).

N⁴-(4-Hydroxybenzylidene)hydrazinoacetylamino-N¹-(pyrimidin-2-yl)benzensulfonamide (4a₅). Yield

86%, m.p. 208–209 C; IR (KBr, cm⁻¹): 3505 (OH), 3335 (-NH), 2850 (CH₂-NH), 2932 (=CH-

pyrimidine ring), 1677 (C=O), 1622 (HN-CO), 1590 (-C=C- aromatic ring), 1539 (N=CH), 1514

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(-C=C- pyrimidine ring), 1304 (C-O), 1168 (-NH-SO₂), 1078 (CH aromatic ring), 965 (S-N), 839 (S-C);

¹H-NMR δ: 8.56–8.89 (dm, 3H, pyrimidine ring), 8.24 (s, 1H, CO-NH), 8.07 (s, 1H, N=CH), 7.35–7.89

(d, 8H, Ar-H), 5.05 (s, 1H, Ar-OH), 4.17 (s, 1H, -NH-SO₂), 3.63 (d, 2H, CH₂), 3.44 (m, 1H, HN-N).

N⁴-(4-Nitrobenzylidene)hydrazinoacetylamino-N¹-(pyrimidin-2-yl)benzensulfonamide (4a₆). Yield 75%,

m.p. 227–230 C; IR (KBr, cm⁻¹): 3445 (-NH), 2851 (CH₂-NH), 2932 (=CH- pyrimidine ring), 1689

(C=O), 1623 (HN-CO), 1592 (-C=C- aromatic ring), 1536 (N=CH), 1514 (-C=C- pyrimidine ring), 1343

1

(C-NO₂), 1316 (C-O), 1174 (-NH-SO₂), 1078 (CH aromatic ring), 951 (S-N), 837 (S-C); H-NMR δ:

8.50–8.87 (dm, 3H, pyrimidine ring), 8.21 (s, 1H, CO-NH), 8.06 (s, 1H, N=CH), 7.33–7.90 (d, 8H, Ar-H),

4.16 (s, 1H, -NH-SO₂), 3.70 (d, 2H, CH₂), 3.41 (m, 1H, HN-N).

N⁴-(Benzylidene)hydrazinoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-5-yl)benzensulfonamide (4b₁). Yield

87%, m.p. 118–120 C; IR (KBr, cm⁻¹): 3465 (-NH), 2864 (CH₂-NH), 1686 (C=O), 1625 (HN-CO),

1592 (-C=C- aromatic ring), 1518 (N=CH), 1496 (-C=C- oxazole ring), 1310 (C-O), 1151 (-NH-SO₂),

1

1093 (CH aromatic ring), 956 (S-N), 830 (S-C); H-NMR δ: 8.41 (s, 1H, CO-NH), 8.12 (s, 1H,

N=CH), 7.21–7.93 (dm, 9H, Ar-H), 4.62 (s, 1H, -NH-SO₂), 3.59 (d, 2H, CH₂), 3.24 (m, 1H, HN-N),

2.01–2.38 (s, 6H, 2CH₃).

N⁴-(4-Fluorobenzylidene)hydrazinoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-5-yl)benzensulfonamide

(4b₂). Yield 68%, m.p. 187–199 C; IR (KBr, cm⁻¹): 3452 (-NH), 2863 (CH₂-NH), 1697 (C=O), 1631

(HN-CO), 1601 (-C=C- aromatic ring), 1507 (N=CH), 1482 (-C=C- oxazole ring), 1320 (C-O), 1225

1

(C-F), 1153 (-NH-SO₂), 1097 (CH aromatic ring), 962 (S-N), 824 (S-C); H-NMR δ: 8.36 (s, 1H,

CO-NH), 8.21 (s, 1H, N=CH), 7.18–7.91 (d, 8H, Ar-H), 4.65 (s, 1H, -NH-SO₂), 3.62 (d, 2H, CH₂),

3.38 (m, 1H, HN-N), 2.08–2.34 (s, 6H, 2CH₃).

N⁴-(4-Chlorobenzylidene)hydrazinoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-5-yl)benzensulfonamide (4b₃).

Yield 62%, m.p. 175–178 C; IR (KBr, cm⁻¹): 3451 (-NH), 2850 (CH₂-NH), 1702 (C=O), 1622

(HN-CO), 1586 (-C=C- aromatic ring), 1540 (N=CH), 1483 (-C=C- oxazole ring), 1312 (C-O), 1169

1

(-NH-SO₂), 1086 (CH aromatic ring), 957 (S-N), 830 (S-C), 814 (C-Cl); H-NMR δ: 8.31 (s, 1H,

CO-NH), 8.16 (s, 1H, N=CH), 7.25–7.98 (d, 8H, Ar-H), 4.74 (s, 1H, -NH-SO₂), 3.64 (d, 2H, CH₂),

3.39 (m, 1H, HN-N), 2.08–2.40 (s, 6H, 2CH₃).

N⁴-(4-Bromobenzylidene)hydrazinoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-5-yl)benzensulfonamide

(4b₄). Yield 57%, m.p. 219 C; IR (KBr, cm⁻¹): 3439 (-NH), 2870 (CH₂-NH), 1669 (C=O), 1625

(HN-CO), 1591 (-C=C- aromatic ring), 1522 (N=CH), 1481 (-C=C- oxazole ring), 1315 (C-O), 1156

1

(-NH-SO₂), 1095 (CH aromatic ring), 940 (S-N), 832 (S-C), 555 (C-Br); H-NMR δ: 8.32 (s, 1H,

CO-NH), 8.18 (s, 1H, N=CH), 7.36-7.94 (d, 8H, Ar-H), 4.61 (s, 1H, -NH-SO₂), 3.65 (d, 2H, CH₂), 3.39

(m, 1H, HN-N), 2.10–2.32 (s, 6H, 2CH₃).

N⁴-(4-Hydroxybenzylidene)hydrazinoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-5-yl)benzensulfonamide (4b₅).

Yield 63%, m.p. 190–192 C; IR (KBr, cm⁻¹): 3495 (C-OH), 3479 (-NH), 2859 (CH₂-NH), 1659

(C=O), 1622 (HN-CO), 1593 (-C=C- aromatic ring), 1530 (N=CH), 1476 (-C=C- oxazole ring), 1323

1

(C-O), 1150 (-NH-SO₂), 1094 (CH aromatic ring), 962 (S-N), 825 (S-C); H-NMR δ: 8.36 (s, 1H,

Molecules 2013, 18

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CO-NH), 8.22 (s, 1H, N=CH), 7.31–7.84 (d, 8H, Ar-H), 4.92 (s, 1H, Ar-OH), 4.62 (s, 1H, -NH-SO₂),

3.58 (d, 2H, CH₂), 3.42 (m, 1H, HN-N), 2.08–2.32 (s, 6H, 2CH₃).

N⁴-(4-Nitrobenzylidene)hydrazinoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-5-yl)benzensulfonamide (4b₆).

Yield 71%, m.p. 280 C; IR (KBr, cm⁻¹): 3433 (-NH), 2842 (CH₂-NH), 1683 (C=O), 1629 (HN-CO),

1593 (-C=C- aromatic ring), 1516 (N=CH), 1480 (-C=C- oxazole ring), 1342 (C-NO₂), 1329 (C-O),

1

1161 (-NH-SO₂), 1106 (CH aromatic ring), 951 (S-N), 838 (S-C); H-NMR δ: 8.28 (s, 1H, CO-NH),

8.21 (s, 1H, N=CH), 7.27–7.88 (d, 8H, Ar-H), 4.64 (s, 1H, -NH-SO₂), 3.56 (d, 2H, CH₂), 3.21 (m, 1H,

HN-N), 2.11–2.34 (s, 6H, 2CH₃).

3.2.2. Preparation of N-(4-aryl-3-chloro-2-oxoazetidin-1-yl)aminoacetyl Sulfonamides 5a_1–6; 5b_1–6

To a solution of N-(arylidene)hydrazinoacetyl sulfonamides 4a_1–6; 4b_1–6(2 mmol) in anhydrous

1,4-dioxane (50 mL), chloracetyl chloride (3 mmol) and triethylamine (2 mmol) were added dropwise

at 0–5 °C. The mixture of reaction was stirred at room temperature for 3 h and the solid (triethylamine

hydrochloride) was removed. The solution was heated under reflux for 5 h and then the solvent was

evaporated under reduced pressure. The solid product was washed with water (20 mL), filtered off,

dried and recrystallized from absolute ethanol. The progress of the reaction was monitored by silica gel

coated TLC plates.

N⁴-(2-Phenyl-3-chloro-4-oxoazetidin-1-yl)aminoacetylamino-N¹-(pyrimidin-2-yl)benzensulfonamide (5a₁).

Yield 75%, m.p. 218–220 C; IR (KBr, cm⁻¹): 3450 (-NH), 2869 (CH₂-NH), 2945 (=CH- pyrimidine

ring), 1745 (CO β-lactam), 1622 (HN-CO), 1591 (-C=C- aromatic ring), 1449 (-C=C- pyrimidine

ring), 1314 (C-O), 1131 (-NH-SO₂), 1078 (CH aromatic ring), 945 (S-N), 841 (S-C), 635 (C-Cl);

¹H-NMR δ: 8.49–8.72 (dm, 3H, pyrimidine ring), 8.25 (s, 1H, CO-NH), 7.21–7.96 (dm, 9H, Ar-H),

5.45 (d, 1H, CH-Ar, azetidinone ring), 5.18 (d, 1H, CH-Cl), 4.27 (s, 1H, -NH-SO₂), 3.75 (d, 2H, CH₂),

13

3.39 (m, 1H, HN-N); C-NMR δ: 170.4 (CONH), 162.8 (CO, β-lactam), 126.7–140.9 (12 aromatic

carbons), 118.9 (1C pyrimidine ring), 154.7 (3C pyrimidine ring), 67.8 (CH), 62.7 (CH-Cl), 57.7

(CH₂); Anal. calcd for C₂₁H₁₉O₄N₆S: C 55.87, H 4.24, N 18.61; found: C 56.02, H 4.38, N 18.48.

N⁴-[2-(4-Fluoro)phenyl-3-chloro-4-oxoazetidin-1-yl]aminoacetylamino-N¹-(pyrimidin-2-yl)benzen-sulfonamide

(5a₂). Yield 90%, m.p. 213–215 C; IR (KBr, cm⁻¹): 3355 (-NH), 2831 (CH₂-NH), 2943 (=CH-

pyrimidine ring), 1744 (CO β-lactam), 1624 (HN-CO), 1593 (-C=C- aromatic ring), 1508 (-C=C-

pyrimidine ring), 1315 (C-O), 1230 (C-F), 1131 (-NH-SO₂), 1100 (CH aromatic ring), 926 (S-N), 836 (S-

1

C), 613 (C-Cl); H-NMR δ: 8.47–8.70 (dm, 3H, pyrimidine ring), 8.22 (s, 1H, CO-NH),

7.23–7.97 (d, 8H, Ar-H), 5.35 (d, 1H, CH-Ar, azetidinone ring), 5.04 (d, 1H, CH-Cl), 4.37 (s, 1H,

-NH-SO₂), 3.79 (d, 2H, CH₂), 3.39 (m, 1H, HN-N); ¹³C-NMR δ: 169.2 (CONH), 162.5 (CO, β-lactam),

124.9–141.06 (12 aromatic carbons), 118.8 (1C pyrimidine ring), 151.8 (3C pyrimidine ring), 69.2

(CH), 61.04 (CH-Cl), 55.6 (CH₂); Anal. calcd for C₂₁H₁₈O₄N₆SF: C 53.73, H 3.86, N 17.90; found: C

53.96, H 4.06, N 18.14.

N⁴-[2-(4-Chloro)phenyl-3-chloro-4-oxoazetidin-1-yl]aminoacetylamino-N¹-(pyrimidin-2-yl)benzen-

sulfonamide (5a₃). Yield 85%, m.p. 201–203 C; IR (KBr, cm⁻¹): 3355 (-NH), 2830 (CH₂-NH), 2944

Molecules 2013, 18

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(=CH- pyrimidine ring), 1745 (CO β-lactam), 1624 (HN-CO), 1592 (-C=C- aromatic ring), 1490

(-C=C- pyrimidine ring), 1314 (C-O), 1174 (-NH-SO₂), 1088 (CH aromatic ring), 958 (S-N), 825 (S-C),

610 (C-Cl); ¹H-NMR) δ: 8.48–8.74 (dm, 3H, pyrimidine ring), 8.24 (s, 1H, CO-NH), 7.19–7.96 (d, 8H,

Ar-H), 5.38 (d, 1H, CH-Ar, azetidinone ring), 5.00 (d, 1H, CH-Cl), 4.4 (s, 1H, -NH-SO₂), 3.75 (d, 2H,

CH₂), 3.39 (m, 1H, HN-N); ¹³C-NMR δ: 167.6 (CONH), 160.5 (CO, β-lactam), 126.7–142.6

(12 aromatic carbons), 120.5 (1C pyrimidine ring), 155.1 (3C pyrimidine ring), 68.7 (CH), 62.8

(CH-Cl), 54.4 (CH₂); Anal. calcd for C₂₁H₁₈O₄N₆SCl: C 51.91, H 3.73, N 17.29; found: C 52.14, H

3.95, N 17.04.

N⁴-[2-(4-Bromo)phenyl-3-chloro-4-oxoazetidin-1-yl]aminoacetylamino-N¹-(pyrimidin-2-yl)benzen-

sulfonamide (5a₄). Yield 88%, m.p. 206–208 C; IR (KBr, cm⁻¹): 3445 (-NH), 2848 (CH₂-NH), 2943

(=CH- pyrimidine ring), 1739 (CO β-lactam), 1624 (HN-CO), 1590 (-C=C- aromatic ring), 1485 (-C=C-

pyrimidine ring), 1315 (C-O), 1130 (-NH-SO₂), 1067 (CH aromatic ring), 961 (S-N), 840 (S-C), 630

(C-Cl), 572 (C-Br); ¹H-NMR δ: 8.47–8.71 (dm, 3H, pyrimidine ring), 8.22 (s, 1H, CO-NH), 7.16–7.85

(d, 8H, Ar-H), 5.36 (d, 1H, CH-Ar, azetidinone ring), 5.05 (d, 1H, CH-Cl), 4.27 (s, 1H, -NH-SO₂), 3.79

13

(d, 2H, CH₂), 3.39 (m, 1H, HN-N); C-NMR δ: 165.3 (CONH), 160.7 (CO, β-lactam), 126.6–136.6

(12 aromatic carbons), 118.7 (1C pyrimidine ring), 156.6 (3C pyrimidine ring), 68.2 (CH), 62.09

(CH-Cl), 57.5 (CH₂); Anal. calcd for C₂₁H₁₈O₄N₆SBr: C 47.56, H 3.42, N 15.85; found: C 47.28, H

3.71, N 15.67.

N⁴-[2-(4-Hydroxy)phenyl-3-chloro-4-oxoazetidin-1-yl]aminoacetylamino-N¹-(pyrimidin-2-yl)benzen-

sulfonamide (5a₅). Yield 80%, m.p. 231–233 C; IR (KBr, cm⁻¹): 3497 (C-OH), 3355 (-NH), 2843

(CH₂-NH), 2943 (=CH- pyrimidine ring), 1740 (CO β-lactam), 1622 (HN-CO), 1590 (-C=C- aromatic

ring), 1495 (-C=C- pyrimidine ring), 1315 (C-O), 1175 (-NH-SO₂), 1067 (CH aromatic ring), 961

(S-N), 840 (S-C), 636 (C-Cl), ¹H-NMR δ: 8.54–8.75 (dm, 3H, pyrimidine ring), 8.26 (s, 1H, CO-NH),

7.30–7.90 (d, 8H, Ar-H), 5.34 (d, 1H, CH-Ar, azetidinone ring), 5.06 (d, 1H, CH-Cl), 4.95 (s, 1H,

Ar-OH), 4.25 (s, 1H, -NH-SO₂), 3.64 (d, 2H, CH₂), 3.40 (m, 1H, HN-N); ¹³C-NMR δ: 167.7 (CONH),

160.3 (CO, β-lactam), 114.7–132.1 (12 aromatic carbons), 119.5 (1C pyrimidine ring), 153.4

(3C pyrimidine ring), 72.9 (CH), 63.3 (CH-Cl), 54.2 (CH₂); Anal. calcd for C₂₁H₁₉O₅N₆S: C 53.95,

H 4.10, N 17.98; found: C 54.21, H 4.32, N 18.19.

N⁴-[2-(4-Nitro)phenyl-3-chloro-4-oxoazetidin-1-yl]aminoacetylamino-N¹-(pyrimidin-2-yl)benzen-

sulfonamide (5a₆). Yield 86%, m.p. 200–203 C; IR (KBr, cm⁻¹): 3350 (-NH), 2846 (CH₂-NH), 2942

(=CH- pyrimidine ring), 1743 (CO β-lactam), 1624 (HN-CO), 1593 (-C=C- aromatic ring), 1496 (-C=C-

pyrimidine ring), 1343 (C-NO₂), 1316 (C-O), 1131 (-NH-SO₂), 1079 (CH aromatic ring), 951 (S-N),

1

839 (S-C), 635 (C-Cl); H-NMR) δ: 8.36–8.87 (dm, 3H, pyrimidine ring), 8.29 (s, 1H, CO-NH),

7.19–7.99 (d, 8H, Ar-H), 5.33 (d, 1H, CH-Ar, azetidinone ring), 5.02 (d, 1H, CH-Cl), 4.24 (s, 1H,

-NH-SO₂), 3.62 (d, 2H, CH₂), 3.37 (m, 1H, HN-N); ¹³C-NMR δ: 168.3 (CONH), 161.9 (CO, β-lactam),

118.5–137.2 (12 aromatic carbons), 118.6 (1C pyrimidine ring), 155.8 (3C pyrimidine ring), 76.1

(CH), 64.3 (CH-Cl), 58.0 (CH₂); Anal. calcd for C₂₁H₁₈O₆N₇S: C 50.80, H 3.65, N 19.75; found: C

50.64, H 3.89, N 19.97.

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N⁴-(2-Phenyl-3-chloro-4-oxoazetidin-1-yl)aminoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-5-yl)benzen-

sulfonamide (5b₁). Yield 51%, m.p. 212–213 C; IR (KBr, cm⁻¹): 3342 (-NH), 2860 (CH₂-NH), 1743

(CO β-lactam), 1624 (HN-CO), 1591 (-C=C- aromatic ring), 1495 (-C=C- oxazole ring), 1318 (C-O),

1153 (-NH-SO₂), 1092 (CH aromatic ring), 957 (S-N), 836 (S-C), 663 (C-Cl); ¹H-NMR δ: 8.34 (s, 1H,

CO-NH), 7.13–7.86 (dm, 9H, Ar-H), 5.37 (d, 1H, CH-Ar, azetidinone ring), 5.19 (d, 1H, CH-Cl), 4.57

13

(s, 1H, -NH-SO₂), 3.51 (d, 2H, CH₂), 3.11 (m, 1H, HN-N), 1.95–2.30 (s, 6H, 2CH₃); C-NMR δ:

168.5 (CONH), 164.0 (CO, β-lactam), 151.4–155.8 (2C oxazole ring), 101.05 (1C oxazole ring),

118.8–135.3 (12 aromatic carbons), 77.2 (CH), 66.3 (CH-Cl), 59.2 (CH₂), 8.48 (2CH₃); Anal. calcd for

C₂₂H₂₂O₅N₅S: C 56.40, H 4.73, N 14.95; found: C 56.63, H 4.94, N 15.18.

N⁴-[2-(4-Fluorophenyl)-3-chloro-4-oxoazetidin-1-yl]aminoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-

5-yl)benzensulfonamide (5b₂). Yield 76%, m.p. 209–211 C; IR (KBr, cm⁻¹): 3450 (-NH), 2861

(CH₂-NH), 1739 (CO β-lactam), 1633 (HN-CO), 1591 (-C=C- aromatic), 1497 (-C=C- oxazole ring),

1322 (C-O), 1232 (C-F), 1153 (-NH-SO₂), 1094 (CH aromatic ring), 961 (S-N), 836 (S-C), 662 (C-Cl);

¹H-NMR δ: 8.24 (s, 1H, CO-NH), 7.10–7.85 (d, 8H, Ar-H), 5.38 (d, 1H, CH-Ar, azetidinone ring),

5.23 (d, 1H, CH-Cl), 4.57 (s, 1H, -NH-SO₂), 3.57 (d, 2H, CH₂), 3.41 (m, 1H, HN-N), 2.02–2.27 (s, 6H,

13

2CH₃); C-NMR δ: 169.6 (CONH), 160.3 (CO, β-lactam), 118.6–139.9 (12 aromatic carbons), 99.56

(1C oxazole ring), 151.8–153.3 (2C oxazole ring), 75.9 (CH), 64.2 (CH-Cl), 56.2 (CH₂), 10.84 (2CH₃);

Anal. calcd for C₂₂H₂₁O₅N₅SF: C 54.31, H 4.35, N 14.40; found: C 54.09, H 4.58, N 14.68.

N⁴-[2-(4-Chlorophenyl)-3-chloro-4-oxoazetidin-1-yl]aminoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-

5-yl)benzensulfonamide (5b₃). Yield 72%, m.p. 211–213 C; IR (KBr, cm⁻¹): 3361 (-NH), 2869

(CH₂-NH), 1750 (CO β-lactam), 1620 (HN-CO), 1591 (-C=C- aromatic), 1495 (-C=C oxazole ring),

1318 (C-O), 1153 (-NH-SO₂), 1092 (CH aromatic ring), 957 (S-N), 836 (S-C), 663 (C-Cl); ¹H-NMR δ:

8.23 (s, 1H, CO-NH), 7.22–7.92 (d, 8H, Ar-H), 5.35 (d, 1H, CH-Ar, azetidinone ring), 5.20 (d, 1H,

CH-Cl), 4.68 (s, 1H, -NH-SO₂), 3.52 (d, 2H, CH₂), 3.33 (m, 1H, HN-N), 2.05–2.36 (s, 6H, 2CH₃);

¹³C-NMR (DMSO) δ in ppm: 170.3 (CONH), 163.6 (CO, β-lactam), 118.8–132.7 (12 aromatic

carbons), 100.97 (1C oxazole ring), 150.4–152.7 (2C oxazole ring), 77.4 (CH), 66.3 (CH-Cl), 53.1

(CH₂), 8.50 (2CH₃); Anal. calcd for C₂₂H₂₁O₅N₅SCl: C 52.54, H 4.21, N 13.92; found: C 52.38, H

4.08, N 13.71.

N⁴-[2-(4-Bromophenyl)-3-chloro-4-oxoazetidin-1-yl]aminoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-

5-yl)benzensulfonamide (5b₄). Yield 80%, m.p. 212–214 C; IR (KBr, cm⁻¹): 3386 (-NH), 2866

(CH₂-NH), 1748 (CO β-lactam), 1626 (HN-CO), 1589 (-C=C- aromatic ring), 1483 (-C=C- oxazole

ring), 1315 (C-O), 1158 (-NH-SO₂), 1095 (CH aromatic ring), 960 (S-N), 859 (S-C), 660 (C-Cl), 556

1

(C-Br); H-NMR δ: 8.21 (s, 1H, CO-NH), 7.34–7.98 (d, 8H, Ar-H), 5.33 (d, 1H, CH-Ar, azetidinone

ring), 5.19 (d, 1H, CH-Cl), 4.56 (s, 1H, -NH-SO₂), 3.60 (d, 2H, CH₂), 3.36 (m, 1H, HN-N), 2.08–2.28

(s, 6H, 2CH₃); ¹³C-NMR δ: 171.3 (CONH), 160.7 (CO, β-lactam), 128.8–142.7 (12 aromatic carbons),

102.37 (1C oxazole ring), 151.7–153.5 (2C oxazole ring), 78.8 (CH), 67.0 (CH-Cl), 55.0 (CH₂), 8.50

(2CH₃); Anal. calcd for C₂₂H₂₁O₅N₅SBr: C 48.27, H 3.87, N 12.79; found: C 48.54, H 3.94, N 12.97.

N⁴-[2-(4-Hydroxyphenyl)-3-chloro-4-oxoazetidin-1-yl]aminoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-

5-yl)benzensulfonamide (5b₅). Yield 55%, m.p. 210–212 C; IR (KBr, cm⁻¹): 3561 (C-OH), 3461

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(-NH), 2855 (CH₂-NH), 1746 (CO β-lactam), 1621 (HN-CO), 1590 (-C=C- aromatic), 1480 (-C=C-

oxazole ring), 1308 (C-O), 1151 (-NH-SO₂), 1093 (CH aromatic ring), 989 (S-N), 835 (S-C), 659 (C-Cl);

¹H-NMR δ in ppm: 8.31 (s, 1H, CO-NH), 7.28–7.81 (d, 8H, Ar-H), 5.32 (d, 1H, CH-Ar, azetidinone

ring), 5.10 (d, 1H CH-Cl), 4.84 (s, 1H, Ar-OH), 4.57 (s, 1H, -NH-SO₂), 3.55 (d, 2H, CH₂), 3.39 (m,

1H, HN-N), 2.04–2.28 (s, 6H, 2CH₃); ¹³C-NMR δ: 175.2 (CONH), 168.2 (CO, β-lactam), 115.8–126.8

(12 aromatic carbons), 101.79 (1C oxazole ring), 148.9–150.5 (2C oxazole ring), 77.3 (CH), 64.8

(CH-Cl), 55.2 (CH₂), 8.51 (2CH₃); Anal. calcd for C₂₂H₂₂O₆N₅S: C 54.54, H 4.58, N 14.45; found: C

54.73, H 4.63, N 14.71.

N⁴-[2-(4-Nitrophenyl)-3-chloro-4-oxoazetidin-1-yl]aminoacetylamino-N¹-(3,4-dimethyl-1,2-oxazol-5-

yl)benzensulfonamide (5b₆). Yield 59%, m.p. 218–220 C; IR (KBr, cm⁻¹): 3415 (-NH), 2863

(CH₂-NH), 1752 (CO β-lactam), 1624 (HN-CO), 1591 (-C=C- aromatic ring), 1490 (-C=C- oxazole

ring), 1337 (C-O), 1329 (C-NO₂) 1154 (-NH-SO₂), 1092 (CH aromatic ring), 987 (S-N), 836 (S-C),

1

662 (C-Cl); H-NMR δ: 8.18 (s, 1H, CO-NH), 7.19–7.86 (d, 8H, Ar-H), 5.36 (d, 1H, CH-Ar,

azetidinone ring), 5.20 (d, 1H, CH-Cl), 4.55 (s, 1H, -NH-SO₂), 3.49 (d, 2H, CH₂), 3.11 (m, 1H,

HN-N); 2.09–2.27 (s, 6H, 2CH₃); ¹³C-NMR δ: 170.7 (CONH), 165.1 (CO, β-lactam), 118.7–130.6 (12

aromatic carbons), 101.20 (1C oxazole ring), 148.8–150.4 (2C oxazole ring), 79.1 (CH), 65.4 (CH-Cl),

60.7 (CH₂), 9.54 (2CH₃); Anal. calcd for C₂₂H₂₁O₇N₆S: C 51.46, H 4.12, N 16.37; found: C 51.29, H

4.36, N 16.09.

3.3. Biological Evaluation

3.3.1. Antibacterial Assay

In order to evaluate the antibacterial activity of the synthesized compounds 4a_1–6, 4b_1–6, 5a_1–6and

5b_1–6, a panel of three Gram positive (Staphyloccoccus aureus ATCC 6583, Staphyloccoccus epidermidis

ATCC 12228, Enterococcus faecalis ATCC 25912) and six Gram negative (Klebsiella pneumoniae

CIP 53153, Proteus vulgaris CIP 104989, Citrobacter freundii CIP 5732, Enterobacter cloacae CIP

103475, Escherichia coli ATCC 25922, Pseudomonas aeruginosa CIP 82118) bacterial strains were

used. Minimum inhibitory concentrations (MICs) were assessed according to the guidelines of

EUCAST Def. 3.1 [24]. Briefly, stock solutions were prepared by solving the substances mentioned

above (200 mg) in dimethyl sulfoxide (DMSO, 19.5 mL). Using these solutions, series of two-fold

dilutions were subsequently obtained. In a 9 cm diameter Petri dish, one milliliter of each dilution was

mixed thoroughly with Mueller-Hinton agar (19 mL), sterilized by autoclaving and cooled to 50 °C.

After this, the concentrations of the substances inside the medium were 512, 256, 128, 64, 32, 16, 8, 4,

2, and 1 μg/mL respectively. A blank plate (control of growth) was also prepared by mixing DMSO

(1 mL) with molten agar (19 mL). From each bacterial strain, a 0.5 McFarland suspension was

prepared in 0.85% saline solution and after that, the inoculum was standardized in order to assure 10⁴

colony-forming units (CFU) per spot (5 μL). All inoculated plates were incubated for 18 h at 36 °C.

The MIC was interpreted as the lowest concentration of the substance that completely inhibits the

growth of bacteria in the spot area. Each determination was performed in triplicate in order to

accurately confirm the MIC values.

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3.3.2. Antioxidant Assays

The antioxidant activity was estimated using in vitro tests: ferric reducing power, total antioxidant

capacity and radical scavenging ability.

3.3.2.1. Ferric Reducing Power

The ferric reducing power of the compounds was quantified by the method described by [25] with

slight modifications. The sample solution (1 mL, 5 mg/mL in DMSO) was mixed with sodium

phosphate buffer (1 mL, 0.2 M, pH 6.6) and potassium ferricyanide (1 mL, 1% w/v) in a test tube. The

reaction mixture was incubated at 50 °C for 20 min in a water bath and then the reaction was stopped

by adding trichloroacetic acid (1 mL, 10% w/v). After centrifugation of the mixture at 4500 rpm for

15 min, the upper layer of the solution (1mL) was collected and diluted further by adding deionised water

(1 mL) and ferric chloride (0.2 mL, 0.1% w/v). After 5 min of incubation, the absorbance was

measured at 700 nm against a blank (the mixture of DMSO with the reagents). A higher absorbance

indicates a higher reducing power. For each sample it was calculated the effective concentration (EC₅₀)

and the reducing power was expressed in reference with ascorbic acid (AA) in the same concentration.

3.3.2.2. Total Antioxidant Activity

The antioxidant activity of tested compounds was evaluated using the phosphomolybdenum method

according to the procedure of [26] with minor modifications. The method is based on the reduction of

Mo(VI) to Mo(V) by the tested compounds followed by the formation of a green phosphate/Mo(V)

complex at acid pH. An aliquot of sample solution (50 µL, 5 mg/mL in DMSO) is mixed with the

reagent solution (2 mL, 0.6 M sulphuric acid, 28 mM sodium phosphate and 4 mM ammonium

molybdate). The samples were incubated at 95 °C for 90 min and then were cooled to room

temperature. The absorbance was measured at 695 nm against a blank (DMSO mixed with reagent

solution). For each sample the effective concentration (EC₅₀) was calculated and the antioxidant

activity was expressed in reference with ascorbic acid (AA) in the same concentration.

3.3.2.3. DPPH Radical Scavenging Assay

The radical scavenging activity of the tested compounds towards the radical 1,1-diphenyl-2-

picrylhydrazyl (DPPH) was measured as described by [27] with slight modifications. The sample

solution (50 µL, 20 mg/mL in DMSO) was mixed thoroughly with a solution of DPPH in methanol

(2.95 mL, 0.1 mM). The sample was left for 30 min at room temperature, in the dark, and after that the

absorbance was measured at 517 nm (A_s). A methanol solution of DPPH was used as control sample

(A_c). The ability to scavenge the DPPH radical was calculated using the following formula:

% Inhibition = 100 × (Ac−As)/Ac

and it was expressed in reference with the radical scavenging activity of ascorbic acid (AA) in the

same concentration.

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3.3.3. Statistical Analysis

All assays (antimicrobial and antioxidant) were carried out in triplicate. Data were analysed by an

analysis of variance (ANOVA) (p < 0.05) and were expressed as means ± SD. The total antioxidant

antivity (EC₅₀values) were calculated by linear interpolation between values above and below 50%

activity.

4. Conclusions

In this study new N-(arylidene)hydrazinoacetyl and new 2-azetidionone derivatives have been

designed and synthesized starting from sulfadiazine and sulfizoxazole. The structures of all new

compounds were proved using spectral methods. The compounds were evaluated for their

antimicrobial and antioxidant activity. Although their antimicrobial potential was reduced, they shown

excellent antioxidant properties; for some of them the potential is comparable with the antioxidant

activity of ascorbic acid. These results support the antioxidant potential of the synthesized compounds

and their applications in several disease mediated by reactive oxygen species (ROS) including the

healing of the wounds.

Acknowledgments

This work was supported in part by the project “Doctoral Scholarships for increasing

competitiveness in the medical and pharmaceutical field” (POSDRU/88/1.5/S/58965) and the grant of

the Romanian National Authority for Scientific Research, CNCS – UEFISCDI, project number PN-II-

ID-PCE-2011-3-0906 (Contract No. 274/31.10.2011). The authors thank Polymer Research Group,

Department of Organic Chemistry, University of Ghent, Belgium for their help to perform NMR analyses.

Conflict of Interest

The authors declared no conflict of interest.

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Sample Availability: Samples of the compounds 4a_1–6, 4b_1–6, 5a_1–6, 5b_1–6are available from the authors.

distributed under the terms and conditions of the Creative Commons Attribution license

(http://creativecommons.org/licenses/by/3.0/).

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Synthesis and biological evaluation of new 2-azetidinones with sulfonamide structures

DOI: 10.3390/molecules18044140

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