New nitric oxide-releasing indomethacin derivatives with 1,3-thiazolidine-4-one scaffold: Design, synthesis, in silico and in vitro studies

Biomedicine & Pharmacotherapy 139 (2021) 111678

Contents lists available at ScienceDirect

Biomedicine & Pharmacotherapy

journal homepage: www.elsevier.com/locate/biopha

New nitric oxide-releasing indomethacin derivatives with 1,3-thiazolidi-

ne-4-one scaffold: Design, synthesis, in silico and in vitro studies

,

,1,

Alexandru Sava^{a b}, Frederic Buron^b, Sylvain Routier^{b *}, Alina Panainte^a, Nela Bibire^a,

,1,*

Lenuta Proﬁre^c

¸

^aDepartment of Analytical Chemistry, Faculty of Pharmacy, “Grigore T. Popa” University of Medicine and Pharmacy of Iași, 16 University Street, Iasi, Romania

b

´

Institut de Chimie Organique et Analytique ICOA, CNRS UMR 7311, Universite d’Orleans, Orleans, France

^cDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, “Grigore T. Popa” University of Medicine and Pharmacy of Iași, 16 University Street, Iasi, Romania

A R T I C L E I N F O

A B S T R A C T

Keywords:

In this study we present design and synthesis of nineteen new nitric oxide-releasing indomethacin derivatives

with 1,3-thiazolidine-4-one scaffold (NO-IND-TZDs) (6a–s), as a new safer and efﬁcient multi-targets strategy for

inﬂammatory diseases. The chemical structure of all synthesized derivatives (intermediaries and ﬁnals) was

proved by NMR and mass spectroscopic analysis. In order to study the selectivity of NO-IND-TZDs for COX

isoenzymes (COX-1 and COX-2) a molecular docking study was performed using AutoDock 4.2.6 software. Based

on docking results, COX-2 inhibitors were designed and 6o appears as the most selective derivative which

showed an improved selective index compared with indomethacin (IND) and diclofenac (DCF), used as reference

drugs. The biological evaluation of 6a–s, using in vitro assays has included the anti-inﬂammatory and antioxidant

effects as well as the nitric oxide (NO) release. Referring to the anti-inﬂammatory effects, the most active

compound was 6i, which was more active than IND and aspirin (ASP) in term of denaturation effect, on bovine

serum albumin (BSA), as indirect assay to predict the anti-inﬂammatory effect. An appreciable anti-inﬂammatory

effect, in reference with IND and ASP, was also showed by 6k, 6c, 6q, 6o, 6j, 6d. The antioxidant assay revealed

the compound 6n as the most active, being 100 times more active than IND. The compound 6n showed also the

most increase capacity to release NO, which means is safer in terms of gastro-intestinal side effects. The ADME-

Tox study revealed also that the NO-IND-TZDs are generally proper for oral administration, having optimal

physico-chemical and ADME properties. We can conclude that the compounds 6i and 6n are promising agents

and could be included in further investigations to study in more detail their pharmaco-toxicological proﬁle.

Indomethacin

1

3-thiazolidine-4-one

Nitric oxide

Docking study

Cyclooxygenase

Inﬂammation

1. Introduction

which are used to treat different conditions where inﬂammation is

involved [19]. They inhibit cyclooxygenase (COX) enzyme in a wide

The inﬂammation is a crucial physiological response of the human

body to any kind of aggression in order for restoring the body homeo-

stasis [1–3]. Moreover, uncontrolled and persistent inﬂammation may

develop itself into a chronic malady [4,5], which becomes the funda-

mental basis for the pathogenesis of many chronic diseases such as

rheumatoid arthritis [6], inﬂammatory bowel disease [7], metabolic

disorders (diabetes mellitus and obesity) [8–10], cardiovascular disor-

ders (ischemic heart disease, atherosclerosis) [11,12], neurodegenera-

tive diseases (Parkinson’s and Alzheimer’s disease) [13–15] and cancer

[16], many of which are life-threatening [17,18].

variety of systems, ranging from microsomal enzyme synthesis to

different cells and tissues [20]. Therefore, COX inhibition has become

deﬁnitively the main mechanism that is responsible for both therapeutic

and side effects of these drugs [21,22]. Brieﬂy, despite the clinical

beneﬁts of NSAIDs through nonselective COX inhibition or to the rela-

tive COX-1 and COX-2 inhibition, long term use of them increase the

incidence of side effects (gastrointestinal, renal, allergic skin reactions,

increased risk of acute coronary syndromes, bleeding) [21,23–25].

Indomethacin (IND) is one of the most relevant NSAID used world-

wide for the treatment of acute and chronic rheumatoid arthritis and

other inﬂammatory rheumatic diseases, episodes of acute gout and acute

Non-steroidal anti-inﬂammatory drugs (NSAIDs) are principal drugs

* Corresponding authors.

E-mail addresses: sylvain.routier@univ-orleans.fr (S. Routier), lenuta.proﬁre@umﬁasi.ro (L. Proﬁre).

1

Both authors contributed equally as senior authors.

https://doi.org/10.1016/j.biopha.2021.111678

Received 4 March 2021; Received in revised form 22 April 2021; Accepted 27 April 2021

Available online 6 May 2021

Published by Elsevier Masson SAS. This is an open access article under the CC BY license

(http://creativecommons.org/licenses/by/4.0/).

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

musculoskeletal pain [26,27]. However, it was documented that

long-term clinical usage of IND is responsible for a wide range of side

effects including gastrointestinal (GI) irritation, bleeding and ulceration,

digestive disorders (nausea, vomiting, abdominal pain, diarrhea or

constipation), increased anxiety, headache, dizziness, peripheral edema,

arterial hypertension, tachycardia, kidney and liver dysfunction, allergic

and anaphylactic reactions [26,28].

presents in several drugs, such as rimonabant (the ﬁrst selective

cannabinoid receptor blocker, with anorectic anti-obesity effect)

[56–58], nitrofurantoin (antibacterial drug used to treat bladder in-

fections), carbazochrome (haemostatic agent), nifuroxazide (a nitro-

furan antibiotic) [59,60] and dantrolene (a postsynaptic muscle

relaxant) (Fig. 2) [53,54,61].

Based on arguments presented above we have developed new nitric

oxide-releasing indomethacin derivatives with 1,3-thiazolidine-4-one

scaffold (NO-IND-TZDs), as safer and efﬁcient multi-targets therapeu-

tic strategy. In this study we present the design and synthesis of NO-IND-

TZDs as well as biological evaluation in terms of COX isoenzyme inhi-

bition selectivity, based on molecular docking study, anti-inﬂammatory

and antioxidant effects and also their capacity to release NO.

To improve clinical effectiveness of NSAIDs, two platform strategies

are currently emerging [29,30]. The ﬁrst one is drugs combination, in

order to reduce the side effects of NSAIDs, and the second one is refer-

ring to a single compound can hit multiple targets (single drug-multiple

targets).

Nitric oxide releasing non-steroidal anti-inﬂammatory drugs (NO-

NSAIDs) are a new class of anti-inﬂammatory drugs consisting of a

traditional NSAID to which a NO releasing moiety has been covalently

attached by a spacer [31,32]. NO is an endogenous short-lived free

radical, produced in mammalians cells through nitric oxide synthase

mediated conversion of L-arginine to L-citruline [33–36]. It is known

that NO has a key role in a wide variety of physiological and patho-

physiological processes, such as inﬂammation, vasodilatation, platelet

adhesion, thrombosis neurotransmission, neuronal communication and

wound healing [37–39]. Referring to NO-NSAIDs is recognized that NO

is a critical mediator of GI mucosal defense and suppresses the NSAIDs

side effects mediated by COX-1 inhibition such as suppression of

reduction in mucosal blood ﬂow, synthesis of mucosal cytoprotective

prostaglandins (especially PGE₂) and over-expression of inﬂammatory

2. Material and methods

2.1. Chemistry

2.1.1. General information

All solvents used for chemical synthesis were of analytical grade or

HPLC p.a. quality unless otherwise stated. The anhydrous solvents

needed for certain reactions have been distilled or dried according to

standard procedures prior to use: dichloromethane has been distilled

under P₂O₅and acetonitrile has been dried on cartridge by a GT S100

station. The progress of the reaction and purity of synthesized com-

pounds were monitored by thin layer chromatography (TLC), using

2 × 5 cm pre-coated silicagel 60 F254 aluminium plate (Merck) and UV

lamp exposure at 254 nm. Column chromatography puriﬁcations are

performed on Merck 40–70 mM silicagel (230–240 mesh) or on C18 AIT

40–60 m silicagel. The ¹H NMR (250 MHz or 400 MHz), ¹³C NMR

(63 MHz or 101 MHz) and ¹⁹F NMR (376 MHz) spectra were recorded

on Bruker Avance DPX250 (250, 131 MHz) or Bruker Avance II

(400 MHz) spectrometer. Chemical shifts (δ H, δ C) and coupling con-

stant values (J) are given in ppm and Hz, respectively. An internal

standard of tetramethylsilane (TMS, δ H = 0, δ C = 0) in CDCl₃and

DMSO-d₆was used. High resolution mass spectrometry (HRMS) has

been made on a Bruker maXis mass spectrometer within the "Research

Federation" platform between ICOA and CBM (FR2708).

mediators (plasma tumor necrosis factor alpha (TNF-α) and the leuko-

cyte–endothelial cell adherence) [37,40,41]. Moreover, based the

beneﬁcial cardiovascular effect of NO, were developed nitric-oxide se-

lective COX-2 inhibitors (NO-COXIBs) which proved to be safer than

classical COX-2 inhibitors [21,42].

On other hand, the 1,3-thiazolidine-4-one scaffold serve as a core for

many synthetic compounds of great interest in medicinal chemistry

[43]. This scaffold is a structural component of various natural products,

like thiamine (vitamin B₁), acidomycin (isolated from Strepomyces

strains) [44,45] and many metabolic products (cytotoxic cyclopeptides)

of fungi and primitive marine animals [46]. Several thiazolidine-4-one

based drugs such as ralitoline (anticonvulsant), etozoline (loop

diuretic) and pioglitazone (oral anti-diabetic drug) have already been

approved for therapeutic use (Fig. 1). The literature reports for

thiazolidine-4-one scaffold other important biological effects such as

anti-inﬂammatory, antioxidant, platelet-activating factor (PAF) antag-

onist, cyclooxygenase (COX) inhibition, tumor necrosis factor antago-

nist as well as anticancer, anticonvulsant, antimicrobial, antiviral and

anti HIV effects [47–52].

2.1.2. General procedure for the synthesis of the halide-ethoxy-

benzaldehyde derivatives (2a-s)

To a solution of hydroxybenzaldehydes (1a-s) (1.0 Eq.) in acetoni-

trile (150 mL), 1,2-dibromoethane or bis(2-chloroethyl)ether (10.0 Eq.)

and potassium carbonate (2.0 Eq.) were added, according to the method

reported in the literature [62–64], which was adapted to our synthesis in

terms of the ratio of reagents, solvent, time of reaction, puriﬁcation

method. The mixture was stirred overnight under reﬂux and progress of

reaction was monitored by TLC. After cooling to room temperature,

distilled water (4 ×50 mL) was added and the mixture was extracted

with diethyl ether (50 mL). The combined organic layers were washed

with brine (50 mL), dried over anhydrous magnesium sulfate and

concentrated under reduced pressure. The crude products were puriﬁed

To attach a thiazolidine-4-one scaffold to indomethacin structure a

carbohydrazide linker was used. This group is often used in medicinal

chemistry due to its: (i) synthetic feasibility and functionalization by

reacting with a variety of chemical substances, (ii) plasma stability and

(iii) stereo chemical nature which allows the conjugated hydrophobic

subunit to link at binding pocket in a favorable and stable conﬁguration

[53–55]. Carbohydrazide group is an important pharmacophore,

Fig. 1. Representative drugs containing the thiazolidinone core.

2

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

Fig. 2. Representative drugs containing the carbohydrazide group.

by ﬂash chromatography (silicagel, petroleum ether/ethyl acetate) to

give the pure products (2a-s).

J = 8.2 Hz, 2.0 Hz, 1H), 9.85 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆)

δ = 30.9 (CH₂), 55.6 (CH₃), 68.6 (CH₂), 110.0 (CH_Ar), 112.6 (CH_Ar),

125.9 (CH_Ar), 130.1 (C_q), 149.2 (C_q), 152.7 (3C_q), 191.5 (C = O). HRMS

(EI-MS) m/z calcd for C₁₀H₁₁BrO₃: 257.9892 [M-H]⁺, found: 257.9962.

Rf (petroleum ether/ethyl acetate = 6/4) 0.55.

4-(2-Bromoethoxy)benzaldehyde (2a) was obtained from 4-hydroxy-

benzaldehyde (1a) as a yellow oil in 96% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ = 3.77–3.90 (m, 2H), 4.39–4.49 (m, 2H), 7.16 (d,

J = 8.7 Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 9.88 (s, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ = 31.5 (CH₂), 68.5 (CH₂), 115.5 (2CH_Ar), 130.4

(C_q), 132.2 (2CH_Ar), 163.2 (C_q), 191.7 (C = O). HRMS (EI-MS) m/z calcd

for C₉H₉BrO₂: 227.9786 [M-H]⁺, found: 227.9855. Rf (petroleum ether/

ethyl acetate = 6/4) 0.68.

4-(2-Bromoethoxy)-3-ethoxybenzaldehyde (2f) was obtained from 3-

ethoxy-4-hydroxybenzaldehyde (1e) as yellowish white needle-shaped

crystals in 65% yield, m.p. 65–67 ^◦C. ¹H NMR (400 MHz, DMSO-d₆)

δ = 1.35 (t, J = 6.9 Hz, 3H), 3.83 (dd, J = 6.1 Hz, 5.0 Hz, 2H), 4.12 (q,

J = 6.9 Hz, 2H), 4.43 (dd, J = 6.1 Hz, 5.0 Hz, 2H), 7.20 (d, J = 8.3 Hz,

1H), 7.41 (d, J = 1.9 Hz, 1H), 7.53 (dd, J = 8.2 Hz, 1.9 Hz, 1H), 9.84 (s,

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 14.6 (CH₃), 30.9 (CH₂), 64.1

(CH₂), 68.6 (CH₂), 111.7 (CH_Ar), 113.2 (CH_Ar), 125.6 (CH_Ar), 130.2 (C_q),

148.5 (C_q), 153.0 (C_q), 191.4 (C = O). HRMS (EI-MS) m/z calcd for

4-(2-Bromoethoxy)-3-ﬂuorobenzaldehyde (2b) was obtained from 3-

ﬂuoro-4-hydroxybenzaldehyde (1b) as a white solid in 91% yield, m.

p. 61–63 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 3.87 (dd, J = 6.0 Hz,

4.7 Hz, 2H), 4.53 (dd, J = 6.1 Hz, 4.7 Hz, 2H), 7.41 (t, J = 8.3 Hz, 1H),

7.71 (dd, J = 11.3 Hz, 2.0 Hz, 1H), 7.77 (ddd, J = 8.3 Hz, 2.0 Hz,

1.0 Hz, 1H), 9.87 (d, J = 2.0 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆)

δ = 30.7 (CH₂), 69.0 (CH₂), 114.9 (d, J = 1.7 Hz, CH_Ar), 115.4 (d,

J = 18.4 Hz, CH_Ar), 128.2 (d, J = 3.1 Hz, CH_Ar), 130.1 (d, J = 5.0 Hz,

C_q), 151.0 (d, J = 10.7 Hz, C_q), 151.5 (d, J = 247.3 Hz, C_qF), 190.8 (d,

J = 2.2 Hz, C = O).

C

₁₁H₁₃BrO₃: 272.0048 [M-H]⁺, found: 272.0119. Rf (petroleum ether/

ethyl acetate = 6/4) 0.55.

4-(2-Bromoethoxy)-3-nitrobenzaldehyde (2g) was obtained from 4-hy-

droxy-3-nitrobenzaldehyde (1g) as greenish yellow solid in 85% yield,

1

m.p. 68–70 ^◦C. H NMR (400 MHz, DMSO-d₆) δ = 3.80–3.88 (m, 2H),

4.60–4.68 (m, 2H), 7.59 (d, J = 8.7 Hz, 1H), 8.18 (dd, J = 8.7 Hz,

2.0 Hz, 1H), 8.43 (d, J = 2.0 Hz, 1H), 9.96 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 30.3 (CH₂), 69.8 (CH₂), 115.8 (CH_Ar), 126.3 (CH_Ar),

129.1 (C_q), 134.8 (CH_Ar), 139.6 (C_q), 154.6 (C_q), 190.4 (C = O). HRMS

(EI-MS) m/z calcd for C₉H₈BrNO₄: 272.9637 [M-H]⁺, found: 272.9708.

Rf (petroleum ether/ethyl acetate = 7/3) 0.70.

¹⁹F NMR (376 MHz, DMSO-d₆) δ = ꢀ 133.32 (ddd, J = 10.9 Hz,

8.3 Hz, 2.1 Hz). HRMS (EI-MS) m/z calcd for C₉H₈FBrO₂: 245.9692

[M–H]⁺, found: 245.9762. Rf (petroleum ether/ethyl acetate = 7/3)

0.65.

4-(2-Bromoethoxy)-3-chlorobenzaldehyde (2c) was obtained from 3-

chloro-4-hydroxybenzaldehyde (1c) as a white solid in 95% yield, m.

p. 62–64 ^◦C.¹H NMR (400 MHz, DMSO-d₆) δ = 3.82–3.92 (m, 2H),

4.50–4.59 (m, 2H), 7.38 (d, J = 8.5 Hz, 1H), 7.89 (dd, J = 8.5 Hz,

2.0 Hz, 1H), 7.97 (d, J = 2.0 Hz, 1H), 9.88 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 30.6 (CH₂), 69.1 (CH₂), 114.0 (CH_Ar), 122.3 (C_q), 130.4

(C_q), 130.6 (CH_Ar), 130.8 (CH_Ar), 157.9 (C_q), 190.7 (C = O). HRMS (EI-

MS) m/z calcd for C₉H₈BrClO₂: 261.9396 [M–H]⁺, found: 261.9468. Rf

(petroleum ether/ethyl acetate = 5/5) 0.74.

4-(2-Bromoethoxy)-3,5-dimethoxybenzaldehyde (2h) was obtained

from 4-hydroxy-3,5-dimethoxybenzaldehyde (1h) as white solid in 95%

yield, m.p. 64–66 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 3.69 (t,

J = 5.9 Hz, 2H), 3.87 (s, 6H), 4.27 (t, J = 5.9 Hz, 2H), 7.27 (s, 2H), 9.89

(s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 31.6 (CH₂), 56.2 (2CH₃),

72.4 (CH₂), 106.7 (2CH_Ar), 131.9 (C_q), 141.2 (C_q), 153.2 (2C_q), 191.9 (C

= O). HRMS (EI-MS) m/z calcd for C₁₁H₁₃BrO₄: 287.9997 [M-H]⁺,

found: 288.0068. Rf (petroleum ether/ ethyl acetate =7/3) 0.50.

2.1.1.9. 4-(2-Bromoethoxy)-3,5-dichlorobenzaldehyde (2i) was ob-

tained from 3,5-dichloro-4-hydroxybenzaldehyde (1h) as white solid in

95% yield, m.p. 67–69 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 3.76–3.92

(m, 2H), 4.35–4.49 (m, 2H), 8.0 (s, 2H), 9.91 (s, 1 H).¹³C NMR

(101 MHz, DMSO-d₆) δ = 31.2 (CH₂), 73.4 (CH₂), 129.4 (2C_q), 130.0

(2CH_Ar), 133.6 (C_q), 154.7 (C_q), 190.2 (C = O). HRMS (EI-MS) m/z calcd

for C₉H₇BrCl₂O₂: 295.9006 [M-H]⁺, found: 295.9072. Rf (petroleum

ether/ethyl acetate = 8/2) 0.66.

3-Bromo-4-(2-bromoethoxy)benzaldehyde (2d) was obtained from 3-

bromo-4-hydroxybenzaldehyde (1d) as a white solid in 86% yield, m.

p. 64–66 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 3.82–3.90 (m, 2H),

4.50–4.58 (m, 2H), 7.33 (d, J = 8.5 Hz, 1H), 7.91 (dd, J = 8.5 Hz,

2.0 Hz, 1H), 8.11 (d, J = 2.0 Hz, 1H), 9.86 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 30.6 (CH₂), 69.2 (CH₂), 111.7 (C_q), 113.8 (CH_Ar), 130.9

(C_q), 131.1 (CH_Ar), 134.1 (CH_Ar), 158.8 (C_q), 190.5 (C = O). HRMS (EI-

MS) m/z calcd for C₉H₈Br₂O₂: 305.8891 [M-H]⁺, found: 305.8962. Rf

(petroleum ether/ethyl acetate = 6/4) 0.57.

4-(2-Bromoethoxy)-3-chloro-5-methoxybenzaldehyde (2j) was ob-

tained from 3-chloro-4-hydroxy-5-methoxybenzaldehyde (1j) as white

needle-shaped crystals in 86% yield, m.p. 65–67 ^◦C. ¹H NMR (400 MHz,

DMSO-d₆) δ = 3.67–3.83 (m, 2H), 3.92 (s, 3H), 4.30–4.49 (m, 2H), 7.52

(d, J = 1.8 Hz, 1H), 7.65 (d, J = 1.8 Hz, 1H), 9.89 (s, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ = 31.6 (CH₂), 56.4 (CH₃), 72.9 (CH₂), 111.1

4-(2-Bromoethoxy)ꢀ 3-methoxybenzaldehyde (2e) was obtained from

4-hydroxy-3-methoxybenzaldehyde (1e) as white needle-shaped crys-

tals in 86% yield, m.p. 66–68 ^◦C. ¹H NMR (400 MHz, DMSO-d₆)

δ = 3.84 (dd, J = 6.0 Hz, 4.9 Hz, 2H), 3.85 (s, 3H), 4.43 (dd, J = 6.1 Hz,

4.8 Hz, 2H), 7.20 (d, J = 8.2 Hz, 1H), 7.42 (d, J = 2.0 Hz, 1H), 7.54 (dd,

3

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

(CH_Ar), 124.1 (CH_Ar), 127.6 (C_q), 132.6 (C_q), 148.2 (C_q), 153.6 (C_q),

191.1 (C = O). HRMS (EI-MS) m/z calcd for C₁₀H₁₀BrClO₃: 291.9502

[M–H]⁺, found: 291.9573. Rf (petroleum ether/ethyl acetate = 6/4)

0.64.

(petroleum ether/ethyl acetate = 6/4) 0.50.

4-(2-(2-Chloroethoxy)ethoxy)-3-methoxybenzaldehyde (2r) was ob-

tained from 4-hydroxy-3-methoxybenzaldehyde (1e) and bis(2-

chloroethyl)ether as a ﬁne white needle crystals in 88% yield, m.p.

64–66 ^◦C. ¹H NMR (250 MHz, DMSO-d₆) δ = 3.64–3.80 (m, 4H),

3.78–3.89 (m, 5H), 4.15–4.28 (m, 2H), 7.18 (d, J = 8.7 Hz, 1H), 7.40 (d,

J = 1.9 Hz, 1H), 7.53 (dd, J = 8.3 Hz, 1.9 Hz, 1H), 9.84 (s, 1H).¹³C NMR

(101 MHz, DMSO-d₆) δ = 43.5 (CH₂), 55.6 (CH₃), 68.1 (CH₂), 68.6

(CH₂), 70.7 (CH₂), 109.8 (CH_Ar), 112.3 (CH_Ar), 125.9 (CH_Ar), 129.8 (C_q),

149.2 (C_q), 153.4 (C_q), 191.3 (C = O). HRMS (EI-MS) m/z calcd for

C₁₂H₁₅ClO₄: 258.0659 [M-H]⁺, found: 258.0731. Rf (petroleum ether/

ethyl acetate = 5/5) 0.46.

3-(2-Bromoethoxy)benzaldehyde (2k) was obtained from 3-hydroxy-

benzaldehyde (1k) as a colorless oil in 91% yield. ¹H NMR (400 MHz,

DMSO-d₆) δ = 3.76 – 3.90 (m, 2H), 4.32 – 4.47 (m, 2H), 7.26–7.33 (m,

1H), 7.44 (d, J = 2.7 Hz, 1H), 7.49–7.56 (m, 2H), 9.97 (s, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ = 31.1 (CH₂), 68.0 (CH₂), 114.0 (CH_Ar), 121.4

(CH_Ar), 122.7 (CH_Ar), 130.4 (CH_Ar), 137.6 (C_q), 158.4 (C_q), 192.7 (C =

O). HRMS (EI-MS) m/z calcd for C₉H₉BrO₂: 227.9786 [M-H]⁺, found:

227.9856. Rf (petroleum ether/ethyl acetate = 7/3) 0.57.

3-(2-Bromoethoxy)-4-methoxybenzaldehyde (2l) was obtained from 3-

hydroxy-4-methoxybenzaldehyde (1 l) as white solid in 88% yield, m.p.

64–66 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 3.74–3.85 (m, 2H), 3.89 (s,

3H), 4.31–4.44 (m, 2H), 7.19 (d, J = 8.3 Hz, 1H), 7.41 (d, J = 1.9 Hz,

1H), 7.58 (dd, J = 8.3 Hz, 1.9 Hz, 1H), 9.83 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 31.1 (CH₂), 56.0 (CH₃), 68.6 (CH₂), 111.6 (CH_Ar), 111.8

(CH_Ar), 126.4 (CH_Ar), 129.6 (C_q), 147.7 (C_q), 154.4 (C_q), 191.3 (C = O).

HRMS (EI-MS) m/z calcd for C₁₀H₁₁BrO₃: 257.9892 [M-H]⁺, found:

257.9965. Rf (petroleum ether/ethyl acetate = 5/5) 0.64.

4-(2-(2-Chloroethoxy)ethoxy)-3-ethoxybenzaldehyde (2s) was ob-

tained from 3-ethoxy 4-hydroxybenzaldehyde (1f) and bis(2-

chloroethyl)ether as a ﬁne white needle crystals in 83% yield, m.p.

64–66 ^◦C.¹H NMR (250 MHz, DMSO-d₆) δ = 1.35 (t, J = 6.9 Hz, 3H),

3.68–3.81 (m, 4H), 3.81–3.88 (m, 2H), 4.10 (q, J = 6.9 Hz, 2H),

4.17–4.27 (m, 2H), 7.19 (d, J = 8.3 Hz, 1H), 7.39 (d, J = 1.9 Hz, 1H),

7.52 (dd, J = 8.3 Hz, 1.9 Hz, 1H), 9.83 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 14.6 (CH₃), 43.5 (CH₂), 63.9 (CH₂), 68.3 (CH₂), 68.6

(CH₂), 70.8 (CH₂), 111.2 (CH_Ar), 112.6 (CH_Ar), 125.7 (CH_Ar), 129.8 (C_q),

148.5 (C_q), 153.6 (C_q), 191.4 (C = O). HRMS (EI-MS) m/z calcd for

C₁₃H₁₇ClO₄: 272.0815 [M-H]⁺, found: 272.0889. Rf (petroleum ether/

ethyl acetate = 4/6) 0.66.

3-(2-Bromoethoxy)-4-nitrobenzaldehyde (2m) was obtained from 3-

hydroxy-4-nitrobenzaldehyde (1m) as brown solid in 80% yield, m.p.

69–71 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 3.76–3.88 (m, 2H),

4.54–4.70 (m, 2H), 7.68 (dd, J = 8.2 Hz, 1.5 Hz, 1H), 7.86 (d, J =

1.5 Hz, 1H), 8.07 (d, J = 8.2 Hz, 1H), 10.06 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 30.5 (CH₂), 69.5 (CH₂), 115.6 (CH_Ar), 122.2 (CH_Ar),

125.4 (CH_Ar), 139.5 (C_q), 143.1 (C_q), 150.4 (C_q), 191.9 (C = O). HRMS

(EI-MS) m/z calcd for C₉H₈BrNO₄: 272.9637 [M-H]⁺, found: 272.9714.

Rf (petroleum ether/ethyl acetate = 4/4) 0.65.

2.1.3. General procedure for the synthesis of the nitrate ester benzaldehyde

derivatives (3a-s)

To a solution of the appropriate halide derivative, 2a–s (1.0 Eq.), in

acetonitrile (50 mL), silver nitrate (1.5 Eq.) was added and the mixture

was stirred under reﬂux and darkness for approximately 12 h, according

to the method reported in the literature [65–67], which was adapted to

our synthesis in terms of the ratio of reagents, solvent, time of reaction,

puriﬁcation method. Then brine was added to precipitate the excess of

silver nitrate. After ﬁltration, the mixture was extracted twice with

diethyl ether (50 mL). The combined organic layers were washed with

water (50 mL) and brine (50 mL), dried over anhydrous magnesium

sulfate, ﬁltered and concentrated under reduced pressure. The crude

products were puriﬁed by ﬂash chromatography (silicagel, petroleum

ether/ethyl acetate) to give the pure products (3a-s).

5-(2-Bromoethoxy)-2-nitrobenzaldehyde (2n) was obtained from 5-hy-

droxy-2-nitrobenzaldehyde (1n) as green solid in 86% yield, m.p.

67–69 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 3.81–3.89 (m, 2H),

4.51–4.62 (m, 2H), 7.29 (d, J = 2.8 Hz, 1H), 7.40 (dd, J = 9.0 Hz,

2.9 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 10.29 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 30.8 (CH₂), 68.9 (CH₂), 114.3 (CH_Ar), 118.7 (CH_Ar),

127.3 (CH_Ar), 134.2 (C_q), 142.1 (C_q), 162.1 (C_q), 189.8 (C = O). HRMS

(EI-MS) m/z calcd for C₉H₈BrNO₄: 272.9637 [M-H]⁺, found: 272.9709.

Rf (petroleum ether/ethyl acetate = 6/4) 0.65.

2-Bromo-3-(2-bromoethoxy)-4-methoxybenzaldehyde (2o) was ob-

tained from 2-bromo-3-hydroxy-4-methoxybenzaldehyde (1o) as a

white solid in 90% yield, m.p. 70–72 ^◦C. ¹H NMR (400 MHz, DMSO-d₆)

δ = 3.79 (t, J = 5.8 Hz, 2H), 3.94 (s, 3H), 4.29 (t, J = 5.8 Hz, 2H), 7.27

(dd, J = 8.7 Hz, 0.8 Hz, 1H), 7.68 (d, J = 8.7 Hz, 1H), 10.10 (d, J =

0.8 Hz, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 31.4 (CH₂), 56.6 (CH₃),

72.5 (CH₂), 112.2 (CH_Ar), 121.4 (C_q), 126.7 (C_q), 126.9 (CH_Ar), 144.3

(C_q), 158.1 (C_q), 190.4 (C = O). HRMS (EI-MS) m/z calcd for

2-(4-Formylphenoxy)ethyl nitrate (3a) was obtained from 4-(2-bro-

moethoxy) benzaldehyde (2a) as yellow oil in 95% yield. ¹H NMR

(400 MHz, DMSO-d₆) δ = 4.39–4.50 (m, 2H), 4.88–4.95 (m, 2H),

7.11–7.20 (m, 2H), 7.84–7.93 (m, 2H), 9.88 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 64.5 (CH₂), 71.7 (CH₂), 115.0 (2CH_Ar), 130.1 (C_q), 131.8

(2CH_Ar), 162.7 (C_q), 191.3(C = O). HRMS (EI-MS) m/z calcd for

C₉H₉NO₅: 211.0481 [M-H]⁺, found: 211.0554. Rf (petroleum ether/

ethyl acetate = 6/4) 0.42.

C

₁₀H₁₀Br₂O₃: 335.8997 [M–H]⁺, found: 335.9070. Rf (petroleum ether/

2-(2-Fluoro-4-formylphenoxy)ethyl nitrate (3b) was obtained from 4-

(2-bromoethoxy)-3-ﬂuorobenzaldehyde (2b) as borrow oil in 99% yield.

¹H NMR (400 MHz, DMSO-d₆) δ = 4.46–4.61 (m, 2H), 4.86–5.03 (m,

2H), 7.43 (t, J = 8.3 Hz, 1H), 7.71 (dd, J = 11.3 Hz, 1.9 Hz, 1H), 7.78

(ddd, J = 8.3 Hz, 1.9 Hz, 1.0 Hz, 1H), 9.87 (d, J = 2.1 Hz, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ = 65.6 (CH₂), 71.5 (CH₂), 114.8 (d, J = 1.6 Hz,

CH_Ar), 115.4 (d, J = 18.2 Hz, CH_Ar), 128.2 (d, J = 3.2 Hz, CH_Ar), 130.2

(d, J = 5.0 Hz, C_q), 151.0 (d, J = 10.7 Hz, C_q), 151.6 (d, J = 247.4 Hz,

C_qF), 190.8 (d, J = 2.1 Hz, C = O). ¹⁹F NMR (376 MHz, DMSO-d₆)

δ = ꢀ 133.32 (ddd, J = 10.9 Hz, 8.3 Hz, 2.1 Hz). HRMS (EI-MS) m/z

calcd for C₉H₈FNO₅: 229.0387 [M–H]⁺, found: 229.0459. Rf (petroleum

ether/ethyl acetate = 5/5) 0.45.

ethyl acetate = 6/4) 0.66.

2-(2-Bromoethoxy)benzaldehyde (2p) was obtained from 2-hydroxy-

benzaldehyde (1p) as a greenish yellow oil in 92% yield. ¹H NMR

(400 MHz, DMSO-d₆) δ = 3.82–3.93 (m, 2 H), 4.42–4.54 (m, 2H), 7.11

(ddt, J = 7.3 Hz, 0.9 Hz, 1H), 7.26 (ddd, J = 8.4 Hz, 0.9 Hz, 0.5 Hz, 1H),

7.61–7.75 (m, 2H), 10.45 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆)

δ = 31.3 (CH₂), 68.5 (CH₂), 114.1 (CH_Ar), 121.3 (CH_Ar), 124.5 (C_q),

127.4 (CH_Ar), 136.4 (CH_Ar), 160.4 (C_q), 189.1 (C = O). HRMS (EI-MS)

m/z calcd for C₉H₉BrO₂: 227.9786 [M–H]⁺, found: 227.9856. Rf (pe-

troleum ether/ethyl acetate = 7.5/2.5) 0.56.

4-(2-(2-Chloroethoxy)ethoxy)benzaldehyde (2q) was obtained from 4-

hydroxy-benzaldehyde (1a) and bis(2-chloroethyl)ether as a ﬁne white

needle crystals in 79% yield, m.p. 65–67 ^◦C. ¹H NMR (400 MHz, DMSO-

d₆) δ = 3.74 (s, 4H), 3.77–3.88 (m, 2H), 4.16–4.29 (m, 2H), 7.08–7.17

(m, 2H), 7.79–7.91 (m, 2H), 9.87 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆)

δ = 43.5 (CH₂), 67.6 (CH₂), 68.6 (CH₂), 70.6 (CH₂), 114.9 (2CH_Ar),

129.7 (C_q), 131.8 (2CH_Ar), 163.4 (C_q), 191.3 (C = O). HRMS (EI-MS) m/z

2-(2-Chloro-4-formylphenoxy)ethyl nitrate (3c) was obtained from 4-

(2-bromoethoxy)-3-chlorobenzaldehyde (2c) as pale yellow solid in

85% yield, m.p. 58–60 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 4.51–4.58

(m, 2H), 4.91–5.00 (m, 2H), 7.40 (d, J = 8.5 Hz, 1H), 7.89 (dd, J =

8.5 Hz, 2.0 Hz, 1H), 7.96 (d, J = 2.0 Hz, 1H), 9.87 (s, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ = 65.8 (CH₂), 71.3 (CH₂), 113.9 (CH_Ar), 122.3

(C_q), 130.5 (C_q), 130.6 (CH_Ar), 130.7 (CH_Ar), 157.8 (C_q), 190.7 (C = O).

calcd for

C

₁₁H₁₃ClO₃: 228.0553 [M-H]⁺, found: 228.0626. Rf

4

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

1

HRMS (EI-MS) m/z calcd for C₉H₈ClNO₅: 245.0091 [M-H]⁺, found:

245.0162. Rf (petroleum ether/ethyl acetate = 4/6) 0.68.

bromoethoxy) benzaldehyde (2k) as yellow oil in 86% yield. H NMR

(400 MHz, DMSO-d₆) δ = 4.33–4.44 (m, 2H), 4.86–4.95 (m, 2H),

7.25–7.35 (m, 1H), 7.39–7.49 (m, 1H), 7.48–7.61 (m, 2H), 9.97 (s, 1H).

¹³C NMR (101 MHz, DMSO-d₆) δ = 64.3 (CH₂), 71.8 (CH₂), 113.8

(CH_Ar), 121.4 (CH_Ar), 122.9 (CH_Ar), 130.4 (CH_Ar), 137.7 (C_q), 158.4

(C_q), 192.8 (C = O). HRMS (EI-MS) m/z calcd for C₉H₉NO₅: 211.0481

[M-H]⁺, found: 211.0554. Rf (petroleum ether/ethyl acetate = 8/2)

0.38.

2-(2-Bromo-4-formylphenoxy)ethyl nitrate (3d) was obtained from 3-

bromo-4-(2-bromo-ethoxy)benzaldehyde (2d) as yellow oil in 85%

yield ¹H NMR (400 MHz, DMSO-d₆) δ = 4.50–4.57 (m, 2H), 4.90–5.00

(m, 2H), 7.35 (d, J = 8.6 Hz, 1H), 7.92 (dd, J = 8.5 Hz, 2.0 Hz, 1H), 8.10

(d, J = 2.0 Hz, 1H), 9.86 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆)

δ = 65.9 (CH₂), 71.3 (CH₂), 111.6 (C_q), 113.8 (CH_Ar), 130.9 (C_q), 131.0

(CH_Ar), 134.0 (CH_Ar), 158.7 (C_q), 190.5 (C = O). HRMS (EI-MS) m/z

calcd for C₉H₈BrNO₅: 288.9586 [M-H]⁺, found: 288.9657. Rf (petro-

leum ether/ethyl acetate = 7/3) 0.23.

2-(5-Formyl-2-methoxyphenoxy)ethyl nitrate (3l) was obtained from 3-

(2-bromo-ethoxy)-4-methoxybenzaldehyde (2l) as pale yellow solid in

1

87% yield, m.p. 50–52 ^◦C. H NMR (400 MHz, DMSO-d₆) δ = 3.88 (s,

2-(4-Formyl-2-methoxyphenoxy)ethyl nitrate (3e) was obtained from

4-(2-bromoethoxy)-3-methoxybenzaldehyde (2e) as yellow oil in 99%

yield. ¹H NMR (400 MHz, DMSO-d₆) δ = 3.84 (s, 3H), 4.39–4.46 (m,

2H), 4.88–4.95 (m, 2H), 7.22 (d, J = 8.3 Hz, 1H), 7.42 (d, J = 1.9 Hz,

1H), 7.55 (dd, J = 8.3 Hz, 1.9 Hz, 1H), 9.85 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 55.6 (CH₃), 65.0 (CH₂), 71.7 (CH₂), 109.9 (CH_Ar), 112.5

(CH_Ar), 125.8 (CH_Ar), 130.2 (C_q), 149.2 (C_q), 152.6 (C_q), 191.5 (C = O).

HRMS (EI-MS) m/z calcd for C₁₀H₁₁NO₆: 241.0586 [M-H]⁺, found:

241.0659. Rf (petroleum ether/ethyl acetate = 6/4) 0.43.

3H), 4.34–4.41 (m, 2H), 4.87–4.94 (m, 2H), 7.20 (d, J = 8.3 Hz, 1H),

7.44 (d, J = 1.9 Hz, 1H), 7.60 (dd, J = 8.3 Hz, 1.9 Hz, 1H), 9.84 (s, 1H).

¹³C NMR (101 MHz, DMSO-d₆) δ = 56.0 (CH₃), 65.0 (CH₂), 71.8 (CH₂),

111.5 (CH_Ar), 111.7 (CH_Ar), 126.6 (CH_Ar), 129.6 (C_q), 147.6 (C_q), 154.4

(C_q), 191.3 (C = O). HRMS (EI-MS) m/z calcd for C₁₀H₁₁NO₆: 241.0586

[M-H]⁺, found: 241.0660. Rf (petroleum ether/ethyl acetate = 7/3)

0.35.

2-(5-Formyl-2-nitrophenoxy)ethyl nitrate (3m) was obtained from 3-

(2-bromoethoxy)-4-nitrobenzaldehyde (2m) as orange solid in 99%

yield, m.p. 56–58 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 4.58–4.65 (m,

2H), 4.89–4.96 (m, 2H), 7.70 (dd, J = 8.2 Hz, 1.2 Hz, 1H), 7.87 (d, J =

1.3 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 10.07 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 66.3 (CH₂), 71.2 (CH₂), 115.3 (CH_Ar), 122.4 (CH_Ar),

125.5 (CH_Ar), 139.5 (C_q), 143.0 (C_q), 150.5 (C_q), 192.0 (C = O). HRMS

(EI-MS) m/z calcd for C₉H₈N₂O₇: 256.0332 [M-H]⁺, found: 256.0406. Rf

(petroleum ether/ethyl acetate = 5/5) 0.58.

2-(2-Ethoxy-4-formylphenoxy)ethyl nitrate (3f) was obtained from 4-

(2-bromoethoxy)-3-ethoxybenzaldehyde (2f) as yellow oil in 92% yield

¹H NMR (400 MHz, DMSO-d₆) δ = 1.34 (t, J = 6.9 Hz, 3H), 4.09 (q, J =

6.9 Hz, 2H), 4.39–4.46 (m, 2H), 4.89–4.96 (m, 2H), 7.21 (d, J = 8.3 Hz,

1H), 7.40 (d, J = 1.9 Hz, 1H), 7.53 (dd, J = 8.2 Hz, 1.9 Hz, 1H), 9.84 (s,

1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 14.5 (CH₃), 64.0 (CH₂), 65.2

(CH₂), 71.6 (CH₂), 111.4 (CH_Ar), 113.1 (CH_Ar), 125.5 (CH_Ar), 130.3 (C_q),

148.5 (C_q), 152.8 (C_q), 191.4 (C = O). HRMS (EI-MS) m/z calcd for

2-(3-formyl-4-nitrophenoxy)ethyl nitrate (3n) was obtained from 5-(2-

bromoethoxy)-2-nitrobenzaldehyde (2n) as brown solid in 99% yield,

C

₁₁H₁₃NO₆: 255.0743 [M-H]⁺, found: 255.0816. Rf (petroleum ether/

1

ethyl acetate = 6/4) 0.52.

m.p. 52–54 ^◦C. H NMR (400 MHz, DMSO-d₆) δ = 4.46–4.62 (m, 2H),

2-(4-Formyl-2-nitrophenoxy)ethyl nitrate (3g) was obtained from 4-(2-

bromoethoxy)-3-nitrobenzaldehyde (2g) as pale yellow solid in 60%

yield, m.p. 55–57 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 4.59–4.70 (m,

2H), 4.89–5.00 (m, 2H), 7.61 (d, J = 8.7 Hz, 1H), 8.19 (dd, J = 8.7 Hz,

2.0 Hz, 1H), 8.43 (d, J = 2.0 Hz, 1H), 9.96 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 66.6 (CH₂), 71.1 (CH₂), 115.8 (CH_Ar), 126.4 (CH_Ar),

129.2 (C_q), 134.9 (CH_Ar), 139.6 (C_q), 154.7 (C_q), 190.4 (C = O). HRMS

(EI-MS) m/z calcd for C₉H₈N₂O₇: 256.0332 [M–H]⁺, found: 256.0404.

Rf (petroleum ether/ethyl acetate = 3/7) 0.57.

4.85–5.01 (m, 2H), 7.30 (d, J = 2.9 Hz, 1H), 7.40 (dd, J = 9.0 Hz,

2.9 Hz, 1H), 8.19 (d, J = 9.0 Hz, 1H), 10.29 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 65.4 (CH₂), 71.5 (CH₂), 114.2 (CH_Ar), 118.8 (CH_Ar),

127.2 (CH_Ar), 134.2 (C_q), 142.2 (C_q), 162.0 (C_q), 189.8 (C = O). HRMS

(EI-MS) m/z calcd for C₉H₈N₂O₇: 256.0332 [M-H]⁺, found: 256.0404. Rf

(petroleum ether/ethyl acetate = 7/3) 0.40.

2-(2-Bromo-3-formyl-6-methoxyphenoxy)ethyl nitrate (3o) was ob-

tained from 2-bromo-3-(2-bromoethoxy)-4-methoxybenzaldehyde (2o)

as brown solid in 99% yield, m.p. 66–68 ^◦C. ¹H NMR (400 MHz, DMSO-

d₆) δ = 3.93 (s, 3H), 4.25–4.34 (m, 2H), 4.80–4.92 (m, 2H), 7.29 (dd, J =

8.7 Hz, 0.7 Hz, 1H), 7.69 (d, J = 8.7 Hz, 1H), 10.10 (d, J = 0.7 Hz, 1H).

¹³C NMR (101 MHz, DMSO-d₆) δ = 56.7 (CH₃), 68.8 (CH₂), 72.7 (CH₂),

112.2 (CH_Ar), 121.3 (C_q), 126.7 (C_q), 127.1 (CH_Ar), 144.3 (C_q), 158.1

(C_q), 190.3 (C = O). HRMS (EI-MS) m/z calcd for C₁₀H₁₀BrNO₆:

318.9691 [M-H]⁺, found: 318.9765. Rf (petroleum ether/ethyl acetate

= 7/3) 0.38.

2-(4-Formyl-2,6-dimethoxyphenoxy)ethyl nitrate (3h) was obtained

from 4-(2-bromo-ethoxy)-3,5-dimethoxybenzaldehyde (2h) as pale

yellow solid in 92% yield, m.p. 52–54 ^◦C. ¹H NMR (400 MHz, DMSO-d₆)

δ = 3.85 (s, 6H), 4.18–4.35 (m, 2H), 4.70–4.87 (m, 2H), 7.26 (s, 2H),

9.89 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 56.1 (2CH₃), 68.7

(CH₂), 72.8 (CH₂), 106.6 (2CH_Ar), 132.1(C_q), 141.0 (C_q), 153.3 (2C_q),

191.9 (C = O). HRMS (EI-MS) m/z calcd for C₁₁H₁₃NO₇: 271.0692

[M–H]⁺, found: 271.0765. Rf (petroleum ether/ethyl acetate = 2/8)

0.20.

2-(2-Formylphenoxy)ethyl nitrate (3p) was obtained from 2-(2-bro-

moethoxy) benzaldehyde (2p) as yellow oil in 83% yield. ¹H NMR

(250 MHz, DMSO-d₆) δ = 4.40–4.54 (m, 2H), 4.92–5.05 (m, 2H), 7.09

(tt, J = 7.5 Hz, 0.9 Hz, 1H), 7.18–7.30 (m, 1H), 7.58–7.66 (m, 1H),

7.66–7.76 (m, 1H), 10.36 (d, J = 0.8 Hz, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 66.5 (CH₂), 71.3 (CH₂), 114.1 (CH_Ar), 121.3 (CH_Ar),

124.5 (C_q), 127.4 (CH_Ar), 136.4 (CH_AR), 160.4 (C_q), 189.1 (C = O).

HRMS (EI-MS) m/z calcd for C₉H₉NO₅: 211.0481 [M–H]⁺, found:

211.0551. Rf (petroleum ether/ethyl acetate = 8/2) 0.21.

2-(2,6-Dichloro-4-formylphenoxy)ethyl nitrate (3i) was obtained from

4-(2-bromo-ethoxy)-3,5-dichlorobenzaldehyde (2i) as white solid in

54% yield, m.p. 49–51 ^◦C. ¹H NMR (250 MHz, DMSO-d₆) δ = 4.33–4.55

(m, 2H), 4.79–5.04 (m, 2H), 8.02 (s, 2H), 9.91 (s, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ = 68.5 (CH₂), 72.4 (CH₂), 129.4 (2C_q), 130.0

(2CH_Ar), 133.6 (C_q), 154.7 (C_q), 190.2 (C = O). HRMS (EI-MS) m/z calcd

for C₉H₇Cl₂NO₅: 278.9701 [M–H]⁺, found: 278.9775. Rf (petroleum

ether/ethyl acetate = 8/2) 0.52.

2-(2-(4-Formylphenoxy)ethoxy)ethyl nitrate (3q) was obtained from

4-(2-(2-chloro-ethoxy)ethoxy)benzaldehyde (2q) as yellow needle-

shaped crystals in 86% yield, m.p. 51–53 ^◦C. ¹H NMR (400 MHz,

DMSO-d₆) δ = 3.69–3.89 (m, 4H), 4.13–4.28 (m, 2H), 4.60–4.76 (m,

2H), 7.08–7.18 (m, 2H), 7.79–7.93 (m, 2H), 9.87 (s, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ = 66.6 (CH₂), 67.6 (CH₂), 68.7 (CH₂), 72.9

(CH₂), 115.0 (2CH_Ar), 129.7 (C_q), 131.8 (2CH_Ar), 163.4(C_q), 191.3 (C =

O). HRMS (EI-MS) m/z calcd for C₁₁H₁₃NO₆: 255.0743 [M–H]⁺, found:

255.0817. Rf (petroleum ether/ethyl acetate = 4/6) 0.58.

2-(2-Chloro-4-formyl-6-methoxyphenoxy)ethyl nitrate (3j) was ob-

tained from 4-(2-bromoethoxy)-3-chloro-5-methoxybenzaldehyde (2j)

as borrow oil in 98% yield. ¹H NMR (400 MHz, DMSO-d₆) δ = 3.91 (s,

3H), 4.33–4.45 (m, 2H), 4.79–4.90 (m, 2H), 7.52 (d, J = 1.8 Hz, 1H),

7.64 (d, J = 1.8 Hz, 1H), 9.89 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆)

δ = 56.4 (CH₃), 69.2 (CH₂), 72.7 (CH₂), 111.1 (CH_Ar), 124.0 (CH_Ar),

127.6 (C_q), 132.8 (C_q), 148.0 (C_q), 153.6 (C_q), 191.0 (C = O). HRMS (EI-

MS) m/z calcd for C₁₀H₁₀ClNO₆: 275.0197 [M–H]⁺, found: 275.0270. Rf

(petroleum ether/ethyl acetate = 6/4) 0.55.

2-(2-(4-Formyl-2-methoxyphenoxy)ethoxy)ethyl nitrate (3r) was ob-

tained from 4-(2-(2-chloroethoxy)ethoxy)-3-methoxybenzaldehyde (2r)

2-(3-Formylphenoxy)ethyl nitrate (3k) was obtained from 3-(2-

5

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

as brown oil in 86% yield. ¹H NMR (400 MHz, DMSO-d₆) δ = 3.71–3.87

(m, 7H), 4.15–4.27 (m, 2H), 4.63–4.74 (m, 2H), 7.18 (d, J = 8.3 Hz, 1H),

7.40 (d, J = 1.9 Hz, 1H), 7.53 (dd, J = 8.3 Hz, 1.9 Hz, 1H), 9.84 (s, 1H).

¹³C NMR (101 MHz, DMSO-d₆) δ = 55.5 (CH₃), 66.6 (CH₂), 68.0 (CH₂),

68.7 (CH₂), 72.9 (CH₂), 109.7 (CH_Ar), 112.2 (CH_Ar), 125.9 (CH_Ar), 129.8

(C_q), 149.2 (C_q), 153.3 (C_q), 191.3 (C = O). HRMS (EI-MS) m/z calcd for

was successively washed with a diluted aqueous solution of sodium bi-

carbonate (20 mL), water and ﬁnally with brine. The combined organic

layers were dried over anhydrous magnesium sulfate and solvent was

removed under reduced pressure to get a crude product that was puriﬁed

by column chromatography (silicagel, petroleum ether/ethyl acetate or

acetonitrile/toluene) to give the pure products (6a-s).

C

₁₂H₁₅NO₇: 285.0849 [M–H]⁺, found: 285.0918. Rf (petroleum ether/

2-(4-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)phenoxy)ethyl nitrate (6a) was ob-

tained from 2-(4-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-

indol-3-yl)acetyl)hydrazineylidene)methyl)phenoxy)ethyl nitrate (5a)

as pale yellow solid in 56% yield, m.p. 169–171 ^◦C. ¹H NMR (250 MHz,

DMSO-d₆) δ = 2.10 (s, 3H), 3.49 (s, 2H), 3.70 (s, 3H), 3.70 (d, J =

15.9 Hz, 1H), 3.86 (dd, J = 15.9 Hz, 1.7, 1H), 4.22–4.34 (m, 2H),

4.83–4.94 (m, 2H), 5.71 (s, 1H), 6.68 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.88

(d, J = 8.7 Hz, 2H), 6.94 (d, J = 9.0 Hz, 1H), 7.00 (d, J = 2.5 Hz, 1H),

7.28 (d, J = 8.7 Hz, 2H), 7.65 (s, 4H), 10.35 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 13.2 (CH₃), 28.7 (CH₂), 29.2 (CH₂), 55.3 (CH₃), 61.1

(CH), 64.1 (CH₂), 71.9 (CH₂), 101.6 (CH_AR), 111.6 (CH_Ar), 113.2 (C_q),

114.4 (3CH_Ar), 129.0 (2CH_Ar), 129.2 (2CH_Ar), 130.1 (C_q), 130.4 (C_q),

130.5 (C_q), 131.1 (2CH_Ar), 134.2 (C_q), 135.2 (C_q), 137.6 (C_q), 155.5

(C_q), 158.3 (C_q), 167.8 (C_q), 168.4 (C_q), 168.7 (C_q). HRMS (EI-MS) m/z

calcd for C₃₀H₂₇ClN₄O₈S: 638.1238, [M–H]⁺found: 638.1299. Rf (pe-

troleum ether/ethyl acetate = 2/8) 0.46.

ethyl acetate = 4/6) 0.59.

2-(2-(2-Ethoxy-4-formylphenoxy)ethoxy)ethyl nitrate (3s) was ob-

tained from 4-(2-(2-chloroethoxy)ethoxy)-3-ethoxybenzaldehyde (2s)

as brown solid in 60% yield, m.p. 55–57 ^◦C. ¹H NMR (400 MHz, DMSO-

d₆) δ = 1.35 (t, J = 6.9 Hz, 3H), 3.77–3.88 (m, 4H), 4.10 (q, J = 6.9 Hz,

2H), 4.16–4.28 (m, 2H), 4.63–4.74 (m, 2H), 7.18 (d, J = 8.2 Hz, 1H),

7.39 (d, J = 1.9 Hz, 1H), 7.52 (dd, J = 8.2 Hz, 1.9 Hz, 1H), 9.83 (s, 1H).

¹³C NMR (101 MHz, DMSO-d₆) δ = 14.5 (CH₃), 63.9 (CH₂), 66.7 (CH₂),

68.2 (CH₂), 68.7 (CH₂), 72.9 (CH₂), 111.2 (CH_Ar), 112.6 (CH_Ar), 125.7

(CH_Ar), 129.8 (C_q), 148.4 (C_q), 153.5 (C_q), 191.4 (C = O). HRMS (EI-MS)

m/z calcd for C₁₃H₁₇NO₇: 299.1005 [M-H]⁺, found: 299.1083. Rf (pe-

troleum ether/ethyl acetate = 4/6) 0.62.

2.1.4. Synthesis of 2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-

3-yl)acetohydrazide (4)

To a suspension of indomethacin (1.0 Eq., 45.5 mmol) in acetonitrile

(300 mL), hydroxybenzotriazole (HOBt, 1.1 Eq., 54.6 mmol) and 1-

ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 1.0 Eq.,

54.6 mmol) were added and the mixture was stirred at room tempera-

ture for 3 h, according to the method reported in the literature [68,69],

which was adapted to our synthesis in terms of the ratio of reagents,

solvent, time of reaction, puriﬁcation method. The resulting mixture was

then slowly added drop wise, at 0–5 ^◦C, to a solution of hydrazine (2.0

Eq., 91 mmol) and cyclohexene (1 mL) in acetonitrile (200 mL). The

mixture was kept at this temperature for 1 h and then was diluted with

distilled water (500 mL), when a precipitated is formed, which was

collected by ﬁltration. The indomethacin hydrazide was obtained in

95% yield, m.p. 202–204 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 2.26 (s,

3H), 3.46 (s, 2H), 3.78 (s, 3H), 4.25 (s, 2H), 6.71 (dd, J = 9.0 Hz, 2.5 Hz,

1H), 6.92 (d, J = 9.0 Hz, 1H), 7.16 (d, J = 2.5 Hz, 1H), 7.61–7.75 (m,

4H), 9.22 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 13.3 (CH₃), 29.3

(CH₂), 55.4(CH₃), 102.1 (CH_Ar), 111.1 (CH_Ar), 114.0 (C_q), 114.5 (CH_Ar),

129.0 (2CH_Ar), 130.2 (C_q), 130.8 (C_q), 131.1 (2CH_Ar), 134.2 (C_q), 135.2

(C_q), 137.5(C_q), 155.5 (C_q), 167.8 (C_q), 168.8 (C_q). HRMS (EI-MS) m/z

calcd for C₁₉H₁₈ClN₃O₃: 371.1037 [M-H]⁺, found: 371.1106. Rf

(dichloromethane/methanol = 9.6/0.4) 0.57.

2-(4-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)-2-ﬂuorophenoxy)ethyl nitrate (6b) was

obtained from 2-(4-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-

1H-indol-3-yl)acetyl)hydrazineylidene)methyl)-2-ﬂuoro-phenoxy)ethyl

nitrate (5b) as pale yellow solid in 45% yield, m.p. 154–156 ^◦C. ¹H NMR

(400 MHz, DMSO-d₆) δ = 2.12 (s, 3H), 3.50 (s, 2H), 3.70 (s, 3H), 3.70 (d,

J = 15.8 Hz, 1H), 3.90 (dd, J = 15.8 Hz, 1.8 Hz, 1H), 4.28–4.43 (m, 2H),

4.80–5.00 (m, 2H), 5.72 (s, 1H), 6.67 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.92

(d, J = 9.0 Hz, 1H), 6.98 (d, J = 2.5 Hz, 1H), 7.00–7.10 (m, 2H), 7.29

(dd, J = 12.2 Hz, 1.8 Hz, 1H), 7.65 (s, 4H), 10.38 (s, 1H). ¹⁹F NMR

(376 MHz, DMSO-d₆) δ = 133.67 (dd, J = 12.1 Hz, 7.4 Hz). ¹³C NMR

(101 MHz, DMSO-d₆) δ = 13.2 (CH₃), 28.7 (CH₂), 29.1 (CH₂), 55.3

(CH₃), 60.6 (CH), 65.3 (CH₂), 71.7 (CH₂), 101.5 (CH_Ar), 111.5 (CH_Ar),

113.1 (C_q), 114.4 (C_q), 114.5 (CH_Ar), 115.2 (d, J =19.1 Hz, CH_Ar), 124.3

(d, J =3.3 Hz, CH_Ar), 129.0 (2CH_Ar), 130.1 (C_q), 130.5 (C_q), 131.1

(2CH_Ar), 131.9 (d, J =5.9 Hz, C_q), 134.1 (C_q), 135.3 (C_q), 137.6 (C_q),

146.1 (d, J =10.7 Hz, C_q), 151.4 (d, J =244.9 Hz, C_qF), 155.5 (C_q), 167.8

(C_q), 168.4 (C_q), 168.7 (C_q). ¹⁹F NMR (376 MHz, DMSO-d₆)

δ = ꢀ 133.67 (dd, J =12.1 Hz, 7.4 Hz). HRMS (EI-MS) m/z calcd for

C

₃₀H₂₆ClFN₄O₈S: 656.1144, [M-H]⁺found: 656.1206. Rf (petroleum

ether/ethyl acetate = 2/8) 0.54.

2.1.5. General procedure for the synthesis of the indomethacin hydrazone

derivatives (5a–s)

2-(2-Chloro-4-(3-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-

indol-3-yl)acetamido)-4-oxothiazolidin-2-yl)phenoxy)ethyl nitrate (6c)

was obtained from 2-(2-chloro-4-((2-(2-(1-(4-chlorobenzoyl)-5-

methoxy-2-methyl-1H-indol-3-yl)acetyl)hydrazineylidene)methyl)phe-

noxy) ethyl nitrate (5c) as pale yellow solid in 40% yield, m.p.

137–139 ^◦C. ¹H NMR (250 MHz, DMSO-d₆) δ = 2.12 (s, 3H), 3.51 (s,

2H), 3.69 (s, 3H), 3.70 (dd, J = 15.9 Hz, 0.8 Hz, 1H), 3.91 (dd, J =

15.9 Hz, 1.8 Hz, 1H), 4.28–4.45 (m, 2H), 4.79–5.02 (m, 2H), 5.72 (s,

1H), 6.67 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.94 (d, J = 9.0 Hz, 0.4 Hz, 1H),

6.99 (d, J = 2.5 Hz, 1H), 7.03 (d, J = 8.6 Hz, 1H), 7.23 (dd, J = 8.6 Hz,

To a suspension of indomethacin hydrazide (4, 1.0 Eq.) in absolute

ethanol, the corresponding nitrate ester benzaldehyde (3a–s) (1.1 Eq.)

was added in presence of catalytic amount of hydrochloric acid. The

mixture was stirred for 12 h under reﬂux when an extensive precipitated

was formed. After cooling at room temperature, the precipitate was

collected by ﬁltration, washed with cold water (80 mL), ethyl ether

(30 mL), dried over anhydrous magnesium sulfate, ﬁltered and

concentrated under reduced pressure. The corresponding indomethacin

hydrazone derivatives (5a-e) were obtained in near quantitative yields

(90–98%) and were used without isolation for the ﬁnal step of synthesis.

2.2 Hz, 1H), 7.49 (d, J = 2.2 Hz, 1H), 7.65 (s, 4H), 10.40 (s, 1H). ¹³

C

NMR (101 MHz, DMSO-d₆) δ = 13.7 (CH₃), 29.2 (CH₂), 29.6 (CH₂), 55.8

(CH₃), 61.0 (CH), 65.9 (CH₂), 72.0 (CH₂), 102.0 (CH_Ar), 112.0 (CH_Ar),

113.6 (C_q), 114.0 (CH_Ar), 114.9 (CH_Ar), 122.0 (C_q), 128.4 (CH_Ar), 129.5

(2CH_Ar), 129.6 (CH_Ar), 130.6 (C_q), 130.9 (C_q), 131.6 (2CH_Ar), 132.6

(C_q), 134.6 (C_q), 135.7 (C_q), 138.1 (C_q), 154.0 (C_q), 156.0 (C_q), 168.3

(C_q), 169.0 (C_q), 169.1 (C_q). HRMS (EI-MS) m/z calcd for

2.1.6. General procedure for the synthesis of the nitric oxide-releasing

indomethacin derivatives with 1,3-thiazolidine-4-one scaffold (6a-s)

To a suspension of indomethacin hydrazone derivatives (5a–s) (1.0

Eq.) in dry toluene (80 mL), mercaptoacetic acid (1.5 Eq.) was added in

presence of few amount of anhydrous magnesium sulfate (2–3 g), using

a Dean Stark equipment and applying a modiﬁed method [49,70,71].

The mixture was stirred for 14 h at 110 ^◦C, after that was cooled at room

temperature and the solvent was removed under reduced pressure. The

residue was taken up with ethyl acetate (30 mL), ﬁltered and the ﬁltrate

C

₃₀H₂₆Cl₂N₄O₈S: 672.0848, [M-H]⁺found: 672.0914. Rf (toluene/

acetonitrile = 6/4) 0.60.

2-(2-Bromo-4-(3-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-

indol-3-yl)acetamido)-4-oxothiazolidin-2-yl)phenoxy)ethyl nitrate (6d)

was obtained from 2-(2-bromo-4-((2-(2-(1-(4-chlorobenzoyl)-5-

6

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

methoxy-2-methyl-1H-indol-3-yl)acetyl)hydrazineylidene)methyl)phe-

noxy) ethyl nitrate (5d) as pale yellow solid in 38% yield, m.p.

167–169 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 2.11 (s, 3H), 3.50 (s,

2H), 3.69 (s, 3H), 3.70 (d, J = 15.9 Hz, 1H), 3.91 (dd, J = 15.9 Hz,

1.8 Hz, 1H), 4.27–4.42 (m, 2H), 4.81–5.00 (m, 2H), 5.72 (s, 1H), 6.67

(dd, J = 9.0 Hz, 2.6 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.98 (s, 1H), 7.00

(d, J = 6.2 Hz, 1H), 7.27 (dd, J = 8.6 Hz, 2.2 Hz, 1H), 7.63 (d, J =

2.2 Hz, 1H), 7.59–7.71 (m, 4H), 10.40 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 13.2 (CH₃), 28.7 (CH₂), 29.2 (CH₂), 55.3 (CH₃), 60.4

(CH), 65.5 (CH₂), 71.5 (CH₂), 101.5 (CH_Ar), 111.1 (CH_Ar), 111.61

(CH_Ar), 113.12 (C_q), 113.3 (CH_Ar), 114.4 (C_q), 128.6 (CH_Ar), 129.0

(2CH_Ar), 130.08 (CH_Ar), 130.5 (C_q), 131.1 (2CH_Ar), 132.1 (C_q), 132.5

(C_q), 134.2 (C_q), 135.2 (C_q), 137.6 (C_q), 154.4 (C_q), 155.5 (C_q), 167.8

(C_q), 168.44 (C_q), 168.7 (C_q). HRMS (EI-MS) m/z calcd for

(C_q), 133.7 (C_q), 134.1 (C_q), 135.3 (C_q), 137.6 (C_q), 139.1 (C_q), 150.9

(C_q), 155.5 (C_q), 167.8 (C_q), 168.5 (C_q), 168.6 (C_q). HRMS (EI-MS) m/z

calcd for C₃₀H₂₆ClN₅O₁₀S: 683.1089, [M-H]⁺found: 683.1154. Rf

(toluene/acetonitrile = 6/4) 0.53.

2-(4-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)-2,6-dimethoxyphenoxy)ethyl

nitrate

(6h) was obtained from 2-(4-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-

methyl-1H-indol-3-yl)acetyl)hydrazineylidene)methyl)-2,6-dimethox-

yphenoxy)ethyl nitrate (5h) as pale yellow solid in 42% yield, m.p.

174–176 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 2.08 (s, 3H), 3.55 (s,

2H), 3.70 (s, 10H), 3.88 (dd, J = 15.9 Hz, 1.7 Hz, 1H), 4.01–4.17 (m,

2H), 4.62–4.84 (m, 2H), 5.75 (s, 1H), 6.67 (dd, J = 8.9 Hz, 2.5 Hz, 1H),

6.69 (s, 2H), 6.91 (d, J = 8.9 Hz, 1H), 7.02 (d, J = 2.5 Hz, 1H), 7.65 (s,

4H), 10.39 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 13.1 (CH₃), 28.8

(CH₂), 29.2 (CH₂), 55.3 (CH₃), 55.9 (2CH₃), 61.7 (CH), 68.5 (CH₂), 72.8

(CH₂), 101.6 (CH_Ar), 104.3 (2CH_Ar), 111.5 (CH_Ar), 113.1 (C_q), 114.5

(CH_Ar), 129.0 (2CH_Ar), 130.1 (C_q), 130.6 (C_q), 131.1 (2CH_Ar), 134.1

(C_q), 134.4 (C_q), 135.3 (C_q), 135.9 (C_q), 137.6 (C_q), 152.8 (2 C_q), 155.5

(C_q), 167.8 (C_q), 168.5 (C_q), 169.1 (C_q). HRMS (EI-MS) m/z calcd for

C

₃₀H₂₆BrClN₄O₈S: 716.0343, [M-H]⁺found: 716.0404. Rf (petroleum

ether/ethyl acetate = 2/8) 0.50.

2-(4-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)-2-methoxyphenoxy)ethyl nitrate (6e)

was obtained from 2-(4-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-

methyl-1H-indol-3-yl)acetyl)hydrazineylidene)methyl)-2-methoxy-phe-

noxy)ethyl nitrate (5e) as pale yellow solid in 35% yield, m.p.

174–176 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 2.09 (s, 3H), 3.51 (s,

2H), 3.69 (s, 3H), 3.70 (s, 3H), 3.71 (d, J = 15.3 Hz, 1H), 3.85 (dd, J =

15.9 Hz, 1.7 Hz, 1H), 4.19–4.29 (m, 2H), 4.83–4.90 (m, 2H), 5.73 (s,

1H), 6.67 (dd, J = 9.0 Hz, 2.6 Hz, 1H), 6.80–6.89 (m, 2H), 6.92 (d, J =

C

₃₂H₃₁ClN₄O₁₀S: 698.1449, [M-H]⁺found: 698.1512. Rf (petroleum

ether/ethyl acetate = 2/8) 0.57.

2-(2,6-Dichloro-4-(3-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-

indol-3-yl)aceta-mido)-4-oxothiazolidin-2-yl)phenoxy)ethyl nitrate (6i)

was obtained from 2-(2,6-dichloro-4-((2-(2-(1-(4-chlorobenzoyl)-5-

methoxy-2-methyl-1H-indol-3-yl)acetyl)hydrazineylidene)methyl) phe-

noxy)ethyl nitrate (5i) as pale yellow solid in 53% yield, m.p.

138–140 ^◦C. ¹H NMR (250 MHz, DMSO-d₆) δ = 2.12 (s, 3H), 3.54 (s,

2H), 3.70 (d, J = 15.8 Hz, 1H), 3.72 (s, 3H), 3.98 (dd, J = 15.8 Hz,

1.8 Hz, 1H), 4.20–4.38 (m, 2H), 4.79–4.98 (m, 2H), 5.77 (d, J = 1.6 Hz,

1H), 6.67 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.92 (d, J = 9.0 Hz, 0.4 Hz, 1 H),

7.02 (d, J = 2.5 Hz, 1H), 7.55 (s, 2H), 7.59–7.70 (m, 4H), 10.45 (s, 1H).

¹³C NMR (101 MHz, DMSO-d₆) δ = 13.1 (CH₃), 28.8 (CH₂), 29.0 (CH₂),

55.3 (CH₃), 59.8 (CH), 69.4 (CH₂), 72.4 (CH₂), 101.5 (CH_Ar), 111.5

(CH_Ar), 113.0 (C_q), 114.5 (CH_Ar), 128.2 (2C_q), 128.3 (2CH_Ar), 129.0

(2CH_Ar), 130.1 (C_q), 130.5 (C_q), 131.1 (2CH_Ar), 134.1 (C_q), 135.2 (C_q),

137.5 (C_q), 137.6 (C_q), 150.1 (C_q), 155.5 (C_q), 167.8 (C_q), 168.5 (C_q),

168.7 (C_q). HRMS (EI-MS) m/z calcd for C₃₀H₂₅Cl₃N₄O₈S: 706.0459, [M-

H]⁺found: 707.0519. Rf (toluene/acetonitrile = 6/4) 0.64.

9.0 Hz, 1H), 6.99 (s, 2H), 7.65 (d, J = 1.4 Hz, 4H), 10.34 (s, 1H). ¹³

C

NMR (101 MHz, DMSO-d₆) δ = 13.2 (CH₃), 28.7 (CH₂), 29.3 (CH₂),

39.99 (CH₂), 55.3 (CH₃), 55.5 (CH₃), 61.5 (CH), 64.9 (CH₂), 72.0 (CH₂),

101.6 (CH_Ar), 111.0 (CH_Ar), 111.5 (CH_Ar), 113.1 (CH_Ar), 113.2 (C_q),

114.4 (CH_Ar), 120.1 (CH_Ar), 129.0 (2CH_Ar), 130.1 (C_q), 130.5 (C_q), 131.1

(2CH_Ar), 134.2 (C_q), 135.2 (C_q), 137.6 (C_q), 147.8 (C_q), 149.1 (C_q),

155.5 (C_q), 167.8 (C_q), 168.4 (C_q), 168.9 (C_q). HRMS (EI-MS) m/z calcd

for C₃₁H₂₉ClN₄O₉S: 668.1344, [M–H]⁺found: 668.1407. Rf (petroleum

ether/ethyl acetate = 2/8) 0.43.

2-(4-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1 H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)-2-ethoxyphenoxy)ethyl nitrate (6f) was

obtained from 2-(4-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-

1H-indol-3-yl)acetyl)hydrazineylidene)methyl)-2-ethoxy-phenoxy)

ethyl nitrate (5 f) as pale yellow solid in 32% yield, m.p. 170–172 ^◦C. ¹H

NMR (400 MHz, DMSO-d₆) δ = 1.27 (t, J = 6.9 Hz, 3H), 2.09 (s, 3H),

3.50 (s, 2H), 3.69 (s, 3H), 3.70 (d, J = 15.9 Hz, 1H), 3.80–4.02 (m, 3H),

4.22–4.29 (m, 2H), 4.83–4.90 (m, 2H), 5.71 (s, 1H), 6.67 (dd, J =

9.0 Hz, 2.5 Hz, 1H), 6.81–6.90 (m, 2H), 6.91 (d, J = 9.0 Hz, 1H), 6.97

(d, J = 2.0 Hz, 1H), 7.00 (d, J = 2.5 Hz, 1H), 7.65 (s, 4H), 10.33 (s, 1H).

¹³C NMR (101 MHz, DMSO-d₆) δ = 13.1 (CH₃), 14.6 (CH₃), 28.7 (CH₂),

29.3 (CH₂), 55.3 (CH₃), 61.4 (CH), 63.9 (CH₂), 65.3 (CH₂), 721.0 (CH₂),

101.6 (CH_Ar), 111.5 (CH_Ar), 112.5 (CH_Ar), 113.2 (C_q), 113.9 (CH_Ar),

114.4 (CH_Ar), 120.2 (CH_Ar), 129.0 (2CH_Ar), 130.1 (C_q), 130.5 (C_q), 131.1

(2CH_Ar), 131.3 (C_q), 134.1 (C_q), 135.2 (C_q), 137.6 (C_q), 148.0(C_q), 148.5

(C_q), 155.5 (C_q), 167.8 (C_q), 168.4 (C_q), 168.8 (C_q). HRMS (EI-MS) m/z

calcd for C₃₂H₃₁ClN₄O₉S: 682.1500, [M-H]⁺found: 682.1561. Rf

(toluene/acetonitrile = 6/4) 0.58.

2-(2-Chloro-4-(3-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-

indol-3-yl)aceta-mido)-4-oxothiazolidin-2-yl)-6-methoxyphenoxy)ethyl ni-

trate (6j) was obtained from 2-(2-chloro-4-((2-(2-(1-(4-chlorobenzoyl)-

5-methoxy-2-methyl-1H-indol-3-yl)acetyl)hydrazineyli-dene)methyl)-

6-methoxyphenoxy)ethyl nitrate (5j) as pale yellow solid in 35% yield,

m.p. 144–146 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 2.10 (s, 3H), 3.54

(d, J = 2.3 Hz, 2H), 3.70 (s, 3H), 3.71 (d, J = 15.9 Hz, 1H), 3.73 (s, 3H),

3.92 (dd, J = 15.9 Hz, 1.8 Hz, 1H), 4.18–4.25 (m, 2H), 4.78–4.85 (m,

2H), 5.75 (s, 1H), 6.67 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.91 (d, J = 9.0 Hz,

1H), 7.01 (d, J = 2.5 Hz, 1H), 7.04 (d, J = 2.0 Hz, 1H), 7.10 (d, J =

2.0 Hz, 1H), 7.60–7.70 (m, 4H), 10.43 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 13.1 (CH₃), 28.7 (CH₂), 29.1 (CH₂), 55.3 (CH₃), 56.2

(CH₃), 60.7 (CH), 68.8 (CH₂), 72.6 (CH₂), 101.5 (CH_Ar), 110.9 (CH_Ar),

111.5 (CH_Ar), 113.1 (C_q), 114.4 (CH_Ar), 120.1 (CH_Ar), 126.9 (C_q), 129.0

(2CH_Ar), 130.1 (C_q), 130.5 (C_q), 131.1 (2CH_Ar), 134.1 (C_q), 135.3 (C_q),

135.9 (C_q), 137.6 (C_q), 143.2 (C_q), 153.2 (C_q), 155.5 (C_q), 167.9 (C_q),

168.5 (C_q), 168.8 (C_q). HRMS (EI-MS) m/z calcd for C₃₁H₂₈Cl₂N₄O₉S:

702.0954, [M-H]⁺found: 702.1018. Rf (petroleum ether/ethyl acetate

= 2/8) 0.47.

2-(4-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)-2-nitrophenoxy)ethyl nitrate (6g) was

obtained from 2-(4-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-

1H-indol-3-yl)acetyl)hydrazineylidene)methyl)-2-nitro-phenoxy)ethyl

nitrate (5g) as pale yellow solid in 25% yield, m.p. 165–167 ^◦C. ¹H NMR

(250 MHz, DMSO-d₆) δ = 2.13 (s, 3H), 3.50 (s, 2H), 3.68 (s, 3H), 3.73 (d,

J = 16.0 Hz, 1H), 3.94 (dd, J = 16.0 Hz, 1.8 Hz, 1H), 4.37–4.54 (m, 2H),

4.80–4.99 (m, 2H), 5.82 (s, 1H), 6.66 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.90

(d, J = 9.0 Hz, 1H), 6.95 (d, J = 2.5 Hz, 1H), 7.26 (d, J = 8.8 Hz, 1H),

7.60 (dd, J = 8.8 Hz, 2.3 Hz, 1H), 7.65 (s, 4H), 7.93 (d, 2.3 Hz, 1H),

10.41 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 13.2 (CH₃), 28.8

(CH₂), 29.1 (CH₂), 55.3 (CH₃), 60.1 (CH₂), 66.1 (CH₂), 71.2 (CH₂),

101.4 (CH_Ar), 111.(CH_Ar), 113.0 (C_q), 114.4 (CH_Ar), 115.2 (CH_Ar), 124.3

(CH_Ar), 129.0 (2CH_Ar), 130.0 (C_q), 130.4 (C_q), 131.1 (2CH_Ar), 131.5

2-(3-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)phenoxy)ethyl nitrate (6k) was ob-

tained from 2-(3-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-

indol-3-yl)acetyl)hydrazineylidene)methyl)phenoxy)ethyl nitrate (5k)

as pale yellow solid in 63% yield, m.p. 170–172 ^◦C. ¹H NMR (400 MHz,

DMSO-d₆) δ = 2.09 (s, 3H), 3.52 (s, 2H), 3.70 (s, 3H), 3.71 (d, J =

15.8 Hz, 1H), 3.88 (dd, J = 15.9 Hz, 1.8 Hz, 1H), 4.25 (q, J = 3.8 Hz,

2H), 4.86 (t, J = 4.3 Hz, 2H), 5.75 (d, J = 1.5 Hz, 1H), 6.67 (dd, J =

9.0 Hz, 2.5 Hz, 1H), 6.92 (d, J = 9.0 Hz, 1H), 6.85–7.01 (m, 3H), 7.01

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A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

(d, J = 2.5 Hz, 1H), 7.25 (t, J = 7.9 Hz, 1H), 7.65 (s, 4H), 10.43 (s, 1H).

¹³C NMR (101 MHz, DMSO-d₆) δ = 13.1 (CH₃), 28.7 (CH₂), 29.1 (CH₂),

55.3 (CH₃), 61.2 (CH), 64.0 (CH₂), 72.0 (CH₂), 101.6 (CH_Ar), 111.6

(CH_Ar), 113.0 (CH_Ar), 113.1 (CH_Ar), 114.4 (C_q), 115.1 (CH_Ar), 120.2

(CH_Ar), 129.0 (2CH_Ar), 129.8 (C_q), 130.1 (CH_Ar), 130.5 (C_q), 131.1

(2CH_Ar), 134.1 (C_q), 135.2 (C_q), 137.6 (C_q), 140.2 (C_q), 155.5 (C_q),

158.0 (C_q), 167.8 (C_q), 168.5 (C_q), 169.0 (C_q). HRMS (EI-MS) m/z calcd

for C₃₀H₂₇ClN₄O₈S: 638.1238, [M-H]⁺found: 638.1304. Rf (petroleum

ether/ethyl acetate = 2/8) 0.56.

5-methoxy-2-methyl-1H-indol-3-yl)acetyl)hydrazineyli-dene)methyl)-

6-methoxyphenoxy)ethyl nitrate (5o) as pale yellow in yield 52%, m.p.

184–186 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 2.10 (s, 3H), 3.55 (d, J =

4.5 Hz, 2H), 3.70 (s, 3H), 3.83 (s, 3H), 3.65–3.96 (m, 2H), 4.04–4.33 (m,

2H), 4.82 (t, J = 4.3 Hz, 2H), 6.02 (s, 1H), 6.66 (dd, J = 9.0 Hz, 2.5 Hz,

1H), 6.91 (d, J = 9.0 Hz, 1H), 7.02 (d, J = 2.5 Hz, 1H), 7.21 (d, J =

51.3 Hz, 2H), 7.65 (d, J = 1.4 Hz, 4H), 10.54 (s, 1H). ¹³C NMR

(101 MHz, DMSO-d₆) δ = 13.1(CH₃), 28.8 (2CH₂), 55.3 (CH₃), 56.2

(CH₃), 60.7 (CH), 68.7 (CH₂), 72.7 (CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar),

112.5 (CH_Ar), 113.1 (CH_Ar), 114.4 (CH_Ar), 117.7 (C_q), 129.0 (2CH_Ar),

130.1 (C_q), 130.5 (C_q), 131.1 (2CH_Ar), 134.1 (C_q), 135.3 (C_q), 137.6

(C_q), 144.1 (C_q), 153.0 (C_q), 155.5 (C_q), 167.8 (C_q), 168.7 (C_q), 169.3

(C_q). HRMS (EI-MS) m/z calcd for C₃₁H₂₈BrClN₄O₉S: 746.0449, [M-H]⁺

found: 746.0506. Rf (petroleum ether/ethyl acetate = 2/8) 0.62.

2-(2-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)phenoxy)ethyl nitrate (6p) was ob-

tained from 2-(2-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-

indol-3-yl)acetyl)hydrazineylidene)methyl)phenoxy)ethyl nitrate (5p)

as pale yellow in yield 34%, m.p. 175–177 ^◦C. ¹H NMR (250 MHz,

DMSO-d₆) δ = 2.11 (s, 3H), 3.54 (s, 2H), 3.65 (d, J = 15.8 Hz, 1H), 3.68

(s, 3H), 3.78 (dd, J = 15.8 Hz, 1.8 Hz, 1H), 3.99–4.35 (m, 2H),

4.50–4.88 (m, 2H), 5.99 (d, J = 1.5 Hz, 1H), 6.68 (dd, J = 9.0 Hz,

2.5 Hz, 1H), 6.93 (dd, J = 9.0 Hz, 0.4 Hz, 1H), 6.96–7.08 (m, 3H),

7.23–7.43 (m, 2H), 7.65 (s, 4H), 10.44 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 13.1 (CH₃), 28.7 (CH₂), 28.8 (CH₂), 55.3 (CH, CH₃) 64.4

(CH₂), 71.8 (CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar), 112.3 (CH_Ar), 113.2 (C_q),

114.4 (CH_Ar), 121.1 (CH_Ar), 126.8 (CH_Ar), 127.5 (C_q), 129.0 (2CH_Ar),

129.9 (CH_Ar), 130.1 (C_q), 130.5 (C_q), 131.1 (2CH_Ar), 134.2 (C_q), 135.3

(C_q), 137.6 (C_q), 155.6 (C_q), 155.6 (C_q), 167.8 (C_q), 168.6 (C_q), 169.2

(C_q). HRMS (EI-MS) m/z calcd for C₃₀H₂₇ClN₄O₈S: 639.1238, [M-H]⁺

found: 638.1303. Rf (petroleum ether/ethyl acetate = 2/8) 0.64.

2-(2-(4-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-

yl)aceta-mido)-4-oxothiazolidin-2-yl)phenoxy)ethoxy)ethyl nitrate (6q)

was obtained from 2-(2-(4-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-

methyl-1H-indol-3-yl)acetyl)hydrazineylidene)methyl)phenoxy)

ethoxy)ethyl nitrate (5q) as pale yellow in yield 27%, m.p. 132–134 ^◦C.

¹H NMR (400 MHz, DMSO-d₆) δ = 2.09 (s, 3H), 3.49 (s, 2H), 3.70 (d, J =

15.9 Hz, 1H), 3.71 (s, 3H), 3.73–3.82 (m, 4H), 3.84 (dd, J = 15.9 Hz,

1.7 Hz, 1H), 3.97–4.28 (m, 2H), 4.56–4.78 (m, 2H), 5.71 (s, 1H), 6.69

(dd, J = 8.9 Hz, 2.5 Hz, 1H), 6.86 (d, J = 8.5 Hz, 2H), 6.94 (d, J =

8.9 Hz, 1H), 7.01 (d, J = 2.5 Hz, 1H), 7.27 (d, J = 8.5 Hz, 2H), 7.65 (s,

4H), 10.34 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 13.2 (CH₃), 28.7

(CH₂), 29.3 (CH₂), 55.3 (CH₃), 61.2 (CH), 66.5 (CH₂), 67.1 (CH₂), 68.9

(CH₂), 72.9 (CH₂), 101.7 (CH_Ar), 111.5 (CH_Ar), 113.2 (C_q), 114.4

(2CH_Ar), 114.4 (CH_Ar), 129.0 (2CH_Ar), 129.1 (2CH_Ar), 129.9 (C_q), 130.1

(C_q), 130.5 (C_q), 131.1 (2CH_Ar), 134.2 (C_q), 135.2 (C_q), 137.6 (C_q),

155.5 (C_q), 158.9 (C_q), 167.8 (C_q), 168.4 (C_q), 168.8 (C_q). HRMS (EI-MS)

m/z calcd for C₃₂H₃₁ClN₄O₉S: 682.1500, [M-H]⁺found: 682.1561. Rf

(toluene/acetonitrile = 6/4) 0.67.

2-(5-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1 H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)-2-methoxyphenoxy)ethyl nitrate (6l)

was obtained from 2-(5-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-

methyl-1H-indol-3-yl)acetyl)hydrazineylidene)methyl)-2-methoxy-phe-

noxy)ethyl nitrate (5l) as pale yellow solid in 25% yield, m.p.

176–178 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 2.07 (s, 3H), 3.50 (s,

2H), 3.70 (s, 3H), 3.71 (d, J = 15.8 Hz, 1H), 3.74 (s, 3H), 3.84 (dd, J =

15.9 Hz, 1.7 Hz, 1H), 4.04–4.33 (m, 2H), 4.84 (t, J = 4.4 Hz, 2H), 5.71

(s, 1H), 6.68 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.86 (s, 1H), 6.81–6.93 (m,

1H), 6.93 (d, J = 9.0 Hz, 1H), 7.00 (d, J = 4.9 Hz, 1H), 7.00 (d, J =

4.3 Hz, 1H), 7.65 (s, 4H), 10.31 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.1 (CH₃), 28.7 (CH₂), 29.3 (CH₂), 55.3 (CH₃), 55.5 (CH₃), 61.4

(CH), 64.9 (CH₂), 72.0 (CH₂), 101.6 (CH_Ar), 111.5 (CH_Ar), 111.6 (CH_Ar),

112.5 (C_q), 113.1 (CH_Ar), 114.4 (CH_Ar), 121.3 (CH_Ar), 129.0 (2CH_Ar),

130.1 (C_q), 130.1 (C_q), 130.5 (C_q), 131.1 (2CH_Ar), 134.1 (C_q), 135.3

(C_q), 137.6 (C_q), 147.3 (C_q), 149.6 (C_q), 155.5 (C_q), 167.8 (C_q), 168.3

(C_q), 168.8 (C_q). HRMS (EI-MS) m/z calcd for C₃₁H₂₉ClN₄O₉S: 668.1344,

[M-H]⁺found: 668.1407. Rf (toluene/acetonitrile = 6/4) 0.58.

2-(5-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)-2-nitrophenoxy)ethyl nitrate (6m) was

obtained from 2-(5-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-

1H-indol-3-yl)acetyl)hydrazineylidene)methyl)-2-nitro-phenoxy)ethyl

nitrate (5m) as pale yellow in 20% yield 17%, m.p. 152–154 ^◦C. ¹H NMR

(400 MHz, DMSO-d₆) δ = 2.14 (s, 3H), 3.54 (s, 2H), 3.66 (s, 3H), 3.75 (d,

J = 15.9 Hz, 1H), 3.95 (dd, J = 15.9 Hz, 1.8 Hz, 1H), 4.24–4.44 (m, 2H),

4.87 (t, J = 4.4 Hz, 2H), 5.85 (d, J = 1.6 Hz, 1H), 6.65 (dd, J = 9.0 Hz,

2.5 Hz, 1H), 6.88 (d, J = 9.0 Hz, 1H), 6.93 (d, J = 2.5 Hz, 1H), 7.17 (dd,

J = 8.4 Hz, 1.7 Hz, 1H), 7.35 (d, J = 1.7 Hz, 1H), 7.65 (d, J = 1.4 Hz,

4H), 7.85 (d, J = 8.3 Hz, 1H), 10.49 (s, 1H). ¹³C NMR (101 MHz, DMSO-

d₆) δ = 13.1 (CH₃), 28.8 (CH₂), 29.1 (CH₂), 55.3 (CH₃), 60.4 (CH), 66.0

(CH₂), 71.2 (CH₂), 101.6 (CH_Ar), 111.3 (CH_Ar), 113.0 (CH_Ar), 114.0

(CH_Ar), 114.4 (C_q), 119.9 (CH_Ar), 125.5 (CH_Ar), 129.0 (2CH_Ar), 130.1

(C_q), 130.4 (C_q), 131.1 (2CH_Ar), 134.1 (C_q), 135.4 (C_q), 137.6 (C_q),

139.1 (C_q), 145.6 (C_q), 150.7 (C_q), 155.5 (C_q), 167.8 (C_q), 168.6 (C_q),

168.8 (C_q). HRMS (EI-MS) m/z calcd for C₃₀H₂₆ClN₅O₁₀S: 683.1089, [M-

H]⁺found: 683.1155. Rf (toluene/acetonitrile = 6/4) 0.6.

2-(3-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)

acetamido)-4-oxo-thiazolidin-2-yl)-4-nitrophenoxy)ethyl nitrate (6n) was

obtained from 2-(3-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-

1H-indol-3-yl)acetyl)hydrazineylidene)methyl)-4-nitrophe-noxy)ethyl

nitrate (5n) as pale yellow in yield 35%, m.p. 180–182 ^◦C. ¹H NMR

(400 MHz, DMSO-d₆) δ = 2.12 (s, 3H), 3.56 (d, J = 4.7 Hz, 2H), 3.64 (d,

J = 15.8 Hz, 1H), 3.69 (s, 3H), 3.93 (dd, J = 15.8 Hz, 1.9 Hz, 1 H),

4.44–4.57 (m, 2H), 4.93 (t, J = 4.4 Hz, 2H), 6.18 (d, J = 1.8 Hz, 1H),

6.67 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.90 (d, J = 9.0 Hz, 1H), 6.98 (d, J =

2.5 Hz, 1H), 7.09–7.20 (m, 2H), 7.58–7.70 (m, 4 H), 8.12 (d, J = 9.0 Hz,

1.1 Hz, 1H), 10.63 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 13.1

(CH₃), 28.1 (CH₂), 28.9 (CH₂), 55.3 (CH₃), 57.4 (CH), 65.0 (CH₂), 71.6

(CH₂), 101.5 (CH_Ar), 111.5 (CH_Ar), 112.5 (C_q), 113.0 (CH_Ar), 114.5

(CH_Ar), 114.8 (CH_Ar), 128.3 (CH_Ar), 129.0 (2CH_Ar), 130.1 (C_q), 130.4

(C_q), 131.1 (2CH_Ar), 134.1 (C_q), 135.3 (C_q), 137.6 (C_q), 138.4 (C_q),

140.2 (C_q), 155.5 (C_q), 162.5 (C_q), 167.8 (C_q), 168.7 (C_q), 169.3 (C_q).

HRMS (EI-MS) m/z calcd for C₃₀H₂₆ClN₅O₁₀S: 683.1089, [M-H]⁺found:

683.1151. Rf (petroleum ether/ethyl acetate = 2/8) 0.73.

2-(2-(4-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-

yl)acetamido)-4-oxothiazolidin-2-yl)-2-methoxyphenoxy)ethoxy)ethyl ni-

trate (6r) was obtained from 2-(2-(4-((2-(2-(1-(4-chlorobenzoyl)-5-

methoxy-2-methyl-1H-indol-3-yl)acetyl)hydrazineylidene)methyl)-2-

methoxyphenoxy)ethoxy)ethyl nitrate (5r) as pale yellow in yield 25%,

m.p. 135–136 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 2.08 (s, 3H), 3.51

(s, 2H), 3.70 (s, 6H), 3.63–3.89 (m, 6H), 3.98–4.10 (m, 2H), 4.53–4.82

(m, 2H), 5.72 (s, 1H), 6.68 (dd, J = 8.9 Hz, 2.4 Hz, 1H), 6.83 (s, 2H),

6.93 (d, J = 8.9 Hz, 1H), 6.97 (s, 1H), 7.01 (d, J = 2.4 Hz, 1H), 7.65 (s,

4H), 10.33 (s, 1H). ¹³C NMR (101 MHz, DMSO-d₆) δ = 13.2 (CH₃), 28.7

(CH₂), 29.3 (CH₂), 55.3 (CH₃), 55.4 (CH₃), 61.5 (CH), 66.6 (CH₂), 67.8

(CH₂), 68.9 (CH₂), 72.9 (CH₂), 101.6 (CH_Ar), 110.9 (CH_Ar), 111.5

(CH_Ar), 112.6 (C_q), 113.2 (CH_Ar), 114.4 (CH_Ar), 120.2 (CH_Ar), 129.0

(2CH_Ar), 130.1 (C_q), 130.4 (C_q), 130.6 (C_q), 131.1 (2CH_Ar), 134.2 (C_q),

135.2 (C_q), 137.6 (C_q), 148.4 (C_q), 149.0 (C_q), 155.5 (C_q), 167.8 (C_q),

168.4 (C_q), 168.9 (C_q). HRMS (EI-MS) m/z calcd for C₃₃H₃₃ClN₄O₁₀S:

712.1606, [M–H]⁺found: 712.1667. Rf (toluene/acetonitrile = 6/4)

2-(2-Bromo-3-(3-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1 H-

indol-3-yl)aceta-mido)-4-oxothiazolidin-2-yl)-6-methoxyphenoxy)ethyl ni-

trate (6o) was obtained from 2-(2-bromo-3-((2-(2-(1-(4-chlorobenzoyl)-

8

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

0.49.

molecule to dock, what is its center and number of torsions, which

docking algorithm to use and how many runs to do. For doing the

conformation search, was applied the Lamarckian Genetic Algorithm

(LGA) with the following parameters: number of individuals in the

population (300), the maximum number of 27,000 generations simu-

lated during each LGA run, the maximum number of evaluation at

25,000,000, a mutation rate of 0.02 and a cross over rate of 0.80, while

remaining docking parameters were set to default. The ligands were

allowed to move within the target proteins to achieve the lowest energy

conformations and the number of runs for each docking procedure was

set to 200. The selected ligands were docked against COX-1 and COX-2

using AutoDock 4.2.6 to identify their selectivity. After performing

molecular docking simulation of the selected ligand molecules against

the COX isoenzymes, the best ligand molecules were evaluated on the

basis of their binding energy against the COX receptor. All the results

obtained by molecular docking simulation were evaluated on the basis

of hydrophilic and lipophilic interactions obtained between the binding

residues present in the active ligand binding site of the macromolecule

and ligand. The dockings experiments were clustered with a root mean

square deviation (RMSD) of 0.5 Å and evaluated by PyMOL software.

Most energetically favored orientations were selected for next research.

2-(2-(4-(3-(2-(1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-

yl)acetamido)-4-oxothiazolidin-2-yl)-2-ethoxyphenoxy)ethoxy)ethyl nitrate

(6s) was obtained from 2-(2-(4-((2-(2-(1-(4-chlorobenzoyl)-5-methoxy-

2-methyl-1H-indol-3-yl)acetyl)hydrazineylidene)methyl)-2-ethox-

yphenoxy)ethoxy)ethyl nitrate (5s) as pale yellow in yield 27%, m.p.

136–138 ^◦C. ¹H NMR (400 MHz, DMSO-d₆) δ = 1.27 (t, J = 6.9 Hz, 3H),

2.08 (s, 3H), 3.50 (s, 2H), 3.70 (d, J = 15.9 Hz, 1H), 3.70 (s, 3H), 3.77 (t,

J = 4.7 Hz, 2H), 3.79–3.86 (m, 3H), 3.86–4.02 (m, 2H), 4.05 (t, J =

4.8 Hz, 2H), 4.65–4.72 (m, 2H), 5.70 (s, 1H), 6.67 (dd, J = 8.9 Hz,

2.4 Hz, 1H), 6.76–6.88 (m, 2 H), 6.92 (d, J = 8.9 Hz, 1H), 6.95 (s, 1H),

7.01 (d, J = 2.4 Hz, 1H), 7.65 (s, 4H), 10.32 (s, 1H). ¹³C NMR (101 MHz,

DMSO-d₆) δ = 13.1 (CH₃), 14.6 (CH₃), 28.7 (CH₂), 29.3 (CH₂), 55.3

(CH₃), 61.5 (CH), 63.9 (CH₂), 66.6 (CH₂), 68.1 (CH₂), 68.9 (CH₂), 72.9

(CH₂), 101.6 (CH_Ar), 111.5 (CH_Ar), 112.5 (CH_Ar), 113.1 (C_q), 113.2

(CH_Ar), 114.4 (CH_Ar), 120.3 (CH_Ar), 129.0 (2CH_Ar), 130.1 (C_q), 130.5

(C_q), 130.6 (C_q), 131.1 (2CH_AR), 134.2 (C_q), 135.2 (C_q), 137.6 (C_q),

148.3 (C_q), 148.7 (C_q), 155.5 (C_q), 167.8 (C_q), 168.3 (C_q), 168.8 (C_q).

HRMS (EI-MS) m/z calcd for C₃₄H₃₅ClN₄O₁₀S: 726.1762, [M-H]⁺found:

726.1823. Rf (toluene/acetonitrile = 6/4) 0.55.

2.2. In silico docking study

2.3. In silico ADME-Tox study

The selectivity of the new NO-IND-TZDs (6a–s) for COX isoenzymes

(COX-1 and COX-2) was studied using AutoDock 4.2.6 software. The

results expressed as docking score were compared to indomethacin

(IND), diclofenac (DCF) and celecoxib (CCB), used as reference drugs.

ADME-Tox (Absorption, Distribution, Metabolism, Excretion and

Toxicity) study offers important data to predict the pharmacokinetic

properties and toxicity degree of new candidates, during the drug dis-

covery process [72–74]. The ADME-Tox proﬁle of the new NO-IND-TZDs

(6a–s) was predicted using SwissADME (http://www.swissadme.ch) and

pkCSM-pharmacokinetics (http://structure.bioc.cam.ac.uk/pkcsm ) tools.

SwissADME provide an overall assessment of the pharmacokinetics

proﬁle of small molecules by most relevant computational methods [75,

76] and pkCSM-pharmacokinetics is a new method based on graph sig-

natures and experimental data [77]. The most important ADME-Tox

properties provided from the ADME-Tox tools were selected to predict

toxicity proﬁle of the NO-IND-TZDs.

2.2.1. Generate the receptor coordinate ﬁle (RCF)

X-ray crystallographic structure of the ligand-enzyme complex were

downloaded from RCSB Protein Data Bank, for COX-1 (pdb code: 4o1z)

and COX-2 (pdb code: 3nt1) and processed prior to docking. The cor-

responding ligand-enzyme complexes were used to remove the ligands,

the water molecules, cofactors and ions that should not be included in

the receptor by a text editor. After that, each receptor was converted to

PDBQT format ﬁle using AutoDock 4.2.6 by reading the coordinates,

adding charges, merging non-polar hydrogens and assigning appropriate

atom types.

2.4. In vitro radical scavenging assay

2.2.2. Generate the ligand coordinate ﬁle (LCF)

The free radical scavenging activity of the new NO-IND-TZDs (6a–s)

was evaluated using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay

with slight modiﬁcation [78–80]. The IND and aspirin (ASP), as refer-

ence drugs, and vitamin C, as standard antioxidant, were used.

Dimensional structures of the compounds (6a–s) were sketched in

ChemDraw and after that were converted to PDB coordinate ﬁles using

Chimera 1.14. Each LCF contain special keywords recognized by Auto-

Dock 4.2.6 like ROOT, ENDROOT, BRANCH, and ENDBRANCH that

establish a rigid set of atoms and rotatable groups of atoms that are

connected to the rigid root. As well, TORSDOF is used in estimating the

change in free energy caused by the loss of torsional degrees of freedom

upon binding. Each structure was energy minimized and converted to

PDBQT format ﬁle using AutoDock4.2.6. A docking method for study the

interaction between a single ligand with a single receptor, with explicit

calculation of afﬁnity maps, was applied. The receptors were kept rigid

and the ligands were allowed to be ﬂexible.

2.4.1. Preparation of DPPH and test solutions

A weighed amount of DPPH (29.71 mg, 75.34 μmol) was dissolved

by sonication in 50 mL methanol of analytical grade and kept in dark-

ness. After 30 min, a sample of 10 mL was taken and made up to 100 mL

with methanol. The resulted DPPH solution (150.68 μM) was stored in

the darkness at room temperature and used up on the day of preparation.

The stock solutions (2600 M) of tested derivatives (6a–s) were pre-

pared in DMSO, then serially diluted with methanol to obtain different

concentrations (2600 M, 1500 M, 700 M, 620 M, 530 M, 440 M,

M and 120

μ

2.2.3. Preparing the grid parameter ﬁle (GPF)

350

μ

M, 260

μ

μM). The serially diluted solutions of IND and

The GPF speciﬁes the PDBQT ﬁles for the receptor and the parame-

ters for generating the atomic afﬁnity maps. For COX-1 was used a grid

box of 73x78x82 points with a spacing of 0.375 Å between grid points

and the grid box center was put on x = 251.00, y = 104.00 and

z = 1.364. COX-2 was enclosed in a 74x72x86 grid box having 0.375 Å

spacing and ꢀ 37.882, ꢀ 50.853 and ꢀ 21.24 as x, y and z center. The

ligand binding site of COX-1 and COX-2 respectively is identiﬁed by

using protein visualization software such as DSvisualizer, PyMol and

Chimera 1.14.

ASP were prepared in the same manner with tested derivatives (6a–s).

The serially diluted solutions of vitamin C were prepared by dilution of

freshly prepared solution (2619 μM) with methanol to make different

concentrations (152.26

μ

M, 132.7

μM, 112.27 μM, 95.26 μM, 77.62 μM,

54.63 M, 40.3 M, 20.54 μ

μ

M).

2.4.2. DPPH assay procedure

500 L from each sample of the tested compounds (6a-s), reference

drug (IND, ASP) and vitamin C was added to 1000 L of DPPH solution.

Two blanks (blank 1:500 L of methanol and 1000 L of DPPH and

blank 2: 1500 L methanol) were also used. The mixture was kept for 3 h

in the darkness at room temperature. Thereafter, a 270 L aliquot of

μ

2.2.4. Preparing the docking parameter ﬁle (DPF)

μ

The DPF consist of which grid map ﬁles to use, which ligand

μ

9

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

each sample tube was added in a 96 well plate. The absorbance was then

measured at 517 nm using Tecan Sunrise Remote Microplate Reader

TW/mL-Abbott F039306. All tests were performed in quadruplicate.

The DPPH radical-inhibiting capacity (inhibition/scavenging activ-

ity) (%) was calculated using the following formula [63]:

2.6.1. Preparation of sodium nitrite and test solutions

A fresh sodium nitrite stock solution (0.1 M sodium nitrite in distilled

water) was prepared and was standardized using the procedure reported

in the European Pharmacopoeia (real molarity = 0.0999, molarity factor

= 0.999)[85]. Next, a solution of 100 μM sodium nitrite was prepared by

diluting of 1 mL of the stock solution to 1000 mL with MeOH/H₂O

Inhibition (Scavenging activity) % = [(A_CS-A_s)/A_CS] x 100

(1)

= 1/1 (v/v) mixture. Then, serially diluted solutions, containing

different concentrations (100 μM, 50 μM, 25 μM, 12.50 μM, 6.25 μM,

where: A_csis the difference between absorbance of blank 1 and blank 2

and A_sis the difference between the absorbance of tested sample and

blank 2.

3.125 μM, 1.56 μM and 0.78 μM), were prepared by dilution with

MeOH/H₂O = 1/1 (v/v) mixture. The tested compounds (6a–s) and

reference NO donors (SNAP, SNP, NTG) were dissolved in DMSO and

To calculate IC₅₀(f_(x)= 50) of each tested compounds, the inhibition

ratios (f_(x)) were plotted against the sample concentration (x). The re-

sults for each experiment were represented by a dose-response curve and

were used two types of regression lines (f(x)): a sigmoid curve and a

quadratic line.

water, respectively, to afford a stock solution of 2600 μM.

2.6.2. The preparation of nitrite standard curve

An aliquot of 170 μL sodium nitrite solution (in the range of

0.78–100

After 10 min, 50

the formed pink-red azo dye was measured at 540 nm, after 20 min. A

blank sample (contain 170 L PBS, 50 L SULF and 50 L NED) was

μ

M) was added to 50

μL solution of SULF in a 96 well plate.

The DPPH radical scavenging activity of each tested compounds was

expressed also as the vitamin C equivalent antioxidant capacity (CEAC)

and was calculated using the following formula [64]:

μ

L of NED solution was added and the absorbance of

μ

CEAC = IC_50(vit⋅C)/IC_{50(sam ple)}

(2)

performed under the similar conditions. All tests were performed ﬁve

times for each concentration and the average absorbance was calcu-

lated. The calibration curve was constructed by plotting the average

absorbance value in relation with the corresponding sodium nitrite

concentration.

here: IC_{50(vit. C)}is the concentration of vitamin C required for 50%

inhibition and IC_50(sample)is concentration of tested compounds (6a-s)

and reference drugs, respectively, required for 50% inhibition. The

higher CEAC value means the higher DPPH radical scavenging activity.

2.6.3. NO release assay

2.5. In vitro anti-inﬂammatory assay

A solution of 100

μ

M NO-IND-TZDs (6a–s) and reference NO donors

L of each stock solution

M) with 2 mL of PBS, PBS-GSH, HCl and HCl-GSH. These solu-

tions were kept at 37–38 ^◦C for 120 min, after that an aliquot of 170

of the each solution was measured and added to 50 L of SULF and

SULF-HgCl₂respectively in a 96 well plate. After 10 min, 50 L of NED

solution was added and the absorbance of the formed pink-red azo dye

was measured at 540 nm, after 20 min. A blank sample (contain 170

(SNAP, SNP, NTG) was prepared by dilute 80

(2600

μ

To predict the anti-inﬂammatory effects of the new NO-IND-TDZs

(6a–s), a modiﬁed Mizushima’s test was used [81,82]. The test which

assures a signiﬁcant correlation between the in vitro and in vivo effects is

based on denaturing effect on speciﬁc proteins. A solution of bovine

serum albumin (BSA) 0.2% in 0.9% NaCl/DMSO = 6/4 was used. Two

controls (positive and negative), as well as reference drugs (IND, ASP),

were also used. The positive control consisted of the action of 0.1 M

hydrochloric acid, as denaturing agent, in 0.9% NaCl, on the 0.2% BSA

solution. The negative control consisted of untreated 0.2% BSA solution.

Each sample (6a–s, IND, ASP) was tested at different concentration

μ

L

μ

μL

PBS/PBS-GSH/HCl/HCl-GSH, 50 μL SULF/SULF-HgCl₂and 50 μL NED)

was performed under the similar conditions. All tests were performed in

quadruplicate.

The percentage (%) of NO release was calculated using the following

formula [83]:

(50 μM, 100 μM and 150 μM). The test samples and controls were

incubated at 38 ^◦C for 5 h. The degree of denaturation of BSA was

evaluated by measuring the increase in optical density at 450 nm,

measured using Tecan Sunrise Remote Microplate Reader

TW/mL-Abbott F039306. All tests were performed in quadruplicate. The

maximum value of the absorbance at 450 nm of the positive control was

considered as the 100% effect. Results, expressed as averages of the

percentage values (% effect), were plotted versus the concentration of

tested sample.

% NO =(C_{f NO}x100)/C_{t NO}

(3)

where: C_{f NO}is the found concentration of NO (

μ

M) and C_{t NO}is the

theoretical concentration of NO (μM).

2.7. Statistical analysis

The results were expressed as mean value ± standard deviation (SD)

and the analysis was performed using IBM SPSS Statistics 23 for Win-

dows. The statistical signiﬁcance of the results was assessed by the one-

way and two-way analysis of variance (ANOVA’s test) followed by

Tukey’s HSD test used to compare the differences among samples. A p

value ˂ 0.05 was considered statistically signiﬁcant.

2.6. In vitro nitric oxide release measurement

To evaluate the nitric oxide release, the new NO-IND-TZDs (6a–s)

were subjected to Griess colorimetric method [83,84]. The Griess re-

agents consists of 0.34% (wt/v) N-(1-naphthyl)ethylenediamine (NED)

solution in DMSO, 3.4% (wt/v) sulfanilamide (SULF) in 10% (wt/v)

phosphoric acid and a mixture between of 3.4% (wt/v) SULF and 1.07%

(wt/v) mercuric chloride (SULF-HgCl₂) in 10% (wt/v) phosphoric acid.

The NO released from the sample is spontaneous oxidized to NO^-₂, which

subsequently reacts with the Griess reagents to form an azo dye.

S-nitroso-N-acetyl-penicillamine (SNAP), sodium nitropruside (SNP)

and nitroglycerine (NTG), were used as reference NO donors.

3. Results and discussion

3.1. Chemistry

The synthesis of new NO-IND-TZDs (6a–s) was based on chemical

modulation of IND at carboxylic group and involved several steps: (i)

synthesis of the halide-ethoxy-benzaldehyde derivatives (2a–s); (ii)

synthesis of the nitrate ester benzaldehyde derivatives (3a-s); (iii) syn-

thesis of the IND hydrazone derivatives (5a–s) and (iv) condensation of

IND hydrazone derivatives (5a–s) with mercaptoacetic acid to obtain

the ﬁnally derivatives (6a–s) (Fig. 3).

The experiments were carried out in neutral (phosphate buffer so-

lution - PBS) and acidic (hydrochloric acid solution - HCl) experimental

conditions, in presence or absence of L-glutathione (GSH): PBS (pH 7.5),

PBS-GSH (pH 7.51), HCl (pH 1.55) and HCl-GSH (pH 1.56).

For the synthesis of the nitrate ester benzaldehydes (3a-s) different

10

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

Fig. 3. The synthesis of the new NO-IND-TZDs (6a–s).

substituted hydroxy-benzaldehydes (1a–s) were reacted with 1,2-dibro-

moethane or bis(2-chloroethyl)ether respectively, when corresponding

halide-ethoxy-benzaldehydes (2a–s) were obtained, based on an SN₂

Williamson ether synthesis. Then the intermediaries (2a–s) were reacted

with silver nitrate (AgNO₃) to form the compounds 3a–s. In the next

step, the IND hydrazide (4), obtained by a peptide coupling reaction

between IND and hydrazine hydrate, was reacted with nitrate ester

benzaldehydes (3a-s), when new IND hydrazone derivatives (5a–s)

were obtained. These intermediaries were subjected to an annelation

with mercaptoacetic acid to obtain the ﬁnal derivatives (6a–s).

The chemical structure of all synthesized compound (intermediaries

and ﬁnally derivatives) was proved on the basis of NMR and HR mass

spectral methods.

In the ¹H NMR spectra of nitrate ester benzaldehydes (3a–s) it was

found the characteristic multiplet signals corresponding to methylene

protons close to the electron withdrawing nitrate ester group

(-CH₂–ONO₂) that appear more deshielded (δ 4.80–5.00, m, 2H)

compared with the protons close to halide (-CH₂-X) (δ 3.75–3.90, m, 2H)

from halide-ethoxy-benzaldehyde derivatives (2a-s). At the same time,

¹³C NMR signal for -CH₂–ONO₂was observed in range of

δ

11

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

71.06–72.66 ppm compared to the signal of the same carbon from de-

rivatives (2a-s) (in range of δ 30.40–31.60 ppm).

Table 1

The Gibbs free energies values (ΔG) and inhibition effect rate (Ki) of NO-IND-

TZDs (6a-s) and of reference drugs (IND, DCF, CCB) for COX-1 and COX-2.

The ﬁnal NO-IND-TZDs (6a-s) showed the multiplet signals of pro-

tons and secondary carbon of methylene (-CH₂–ONO₂) in the range of δ

4.80–4.90 ppm and δ 64.00–69.00 ppm, respectively. The presence of

1,3-thiazolidine-4-one scaffold in the structure of NO-IND-TZDs (6a-s)

was proved in the NMR spectra by the characteristic signals of protons

and carbons corresponding to methine (=CH-) and methylene (-CH₂-)

group. In ¹H NMR spectra, the signal of =CH- was observed in the range

of δ 5.70–6.00 ppm. For -CH₂- there were diastereotopic protons, that

have different chemical shifts δ 3.70 (d, J = 15.9 Hz, 1 H) respectively δ

3.90 (dd, J = 15.9 Hz, 1.7 Hz, 1H) ppm. In ¹³C NMR spectra, signal of

the =CH- group was observed in the range of δ 60.0–61.4 ppm and of the

-CH₂- group at δ 28.9–29.6 ppm. The protons and carbon signals of other

aliphatic and aromatic fragments of 6a–s were observed at expected

values of chemical shift. The presence of the signals above mentioned

conﬁrmed correct cyclization reaction of IND hydrazone derivatives

(5a–s) to form 1,3-thiazolidine-4-one ring and preserved the nitrate

ester moiety in the 6a–s. Moreover the NMR spectral data coupled with

mass spectra strong support the proposed structures of the all synthe-

sized compounds.

Ligand

COX-1

COX-2

Selectivity index

log₁₀(Ki_COX-1/Ki_COX-

ΔG (kcal/

Ki (nM)

ΔG (kcal/

Ki (nM)

)

2

mol)

6a

6b

6c

ꢀ 12.23

ꢀ 10.96

ꢀ 13.08

ꢀ 11.90

ꢀ 10.96

ꢀ 11.06

ꢀ 10.62

ꢀ 9.56

1.09

9.18

ꢀ 8.27

872.40

41.59

1.94

ꢀ 2.903

ꢀ 0.656

ꢀ 0.877

ꢀ 0.609

ꢀ 1.635

0.005

ꢀ 10.07

ꢀ 11.89

ꢀ 11.07

ꢀ 8.73

0.26

6d

6e

6f

1.90

7.74

9.20

397.62

7.74

7.83

ꢀ 11.07

ꢀ 8.06

6g

6h

6i

16.32

98.66

0.77

1240

ꢀ 1.880

ꢀ 1.376

ꢀ 1.957

ꢀ 0.841

ꢀ 3.387

ꢀ 1.286

ꢀ 0.419

ꢀ 1.632

0.779

ꢀ 7.68

2350

ꢀ 12.43

ꢀ 11.60

ꢀ 13.72

ꢀ 11.87

ꢀ 10.91

ꢀ 11.29

ꢀ 10.34

ꢀ 12.42

ꢀ 11.73

ꢀ 12.01

ꢀ 11.29

ꢀ 9.98

ꢀ 9.76

70.25

21.85

213.93

38.28

26.56

229.29

4.41

6j

3.15

ꢀ 10.45

ꢀ 9.10

6k

6l

0.09

1.98

ꢀ 10.12

ꢀ 10.34

ꢀ 9.06

6m

6n

6o

6p

6q

6r

10.12

5.34

26.52

0.79

ꢀ 11.40

ꢀ 8.52

568.34

22.22

41.36

266.20

25.72

468.54

27.52

ꢀ 2.855

ꢀ 0.947

ꢀ 1.420

ꢀ 1.701

0.273

2.51

ꢀ 10.44

ꢀ 10.07

ꢀ 8.97

1.57

6s

5.29

IND

DCF

CCB

48.23

1120.00

737.59

ꢀ 10.35

ꢀ 8.63

3.2. In silico docking study

ꢀ 8.12

0.378

ꢀ 8.37

ꢀ 10.31

1.428

The X-ray crystallographic structures used in this study were selected

in term of the quality of the atomic model obtained from the crystallo-

graphic data [86]. A murine COX-2 (PDB ID 3nt1) and an ovine COX-1

(PDB ID 4o1z) X-ray crystallographic structures were selected to study

the predicted binding mode of the ligands with COX isoenzymes. The

PDB 3nt1 and PDB 4o1z were described as COX-2 and COX-1 structures

respectively with highest resolution to date, 1.7 Å and 2.4 Å, respec-

tively [87], which means that there is a more conﬁdence in the location

of atoms in the electron density map. Also the R-value was less 0.16,

indicating a strong agreement between the crystallographic model and

the experimental X-ray diffraction data [88,89].

In order to calculate the Gibbs free energies (ΔG) of NO-IND-TZDs

(6a–s) each ligand-receptor complex was subjected to careful analysis

for ideal docked poses on the basis of least binding energy scores and

maximum number of cluster conformations and the results are presented

in Table 1.

In order to establish the statistical signiﬁcance of the difference be-

tween the receptors while removing ligand variances from the overall

error variance, a two-way ANOVA analysis was applied. In the same

manner, the difference between the ligands while removing the receptor

variances from the overall error variance term was also analyzed. The

results established a statistically signiﬁcant difference between re-

ceptors (F(0.5, 1, 21) = 17.4616, p = 0.0004, Fcrit = 4.3247) and no

difference between ligands (F(0.5, 21, 21) = 1.3707, p = 0.2380, Fcrit

= 2.0841).

To validate the computational method, it was used the RSMD vari-

ation (less than 2 Å) for IND, DCF and CCB individually, as reference

drugs, into the active site of both COX-1 and COX-2 isoforms and we

found the same binding cleft residues reported by other authors [90–92]

The IND and DCF are non selective COX inhibitors, that inhibit both

types of the COX enzymes, whereas CCB is a preferentially COX-2 se-

lective inhibitor. It was found also that all reference drugs have polar

interactions with the catalytic site of both COX-1 and COX-2.

As expected, IND was approximately equipotent against COX-1 and

COX-2, but more active against COX-2 than DCF (Table 1), although the

both drugs belong to the same chemical class, being acetic acid de-

rivatives. More speciﬁc, it was showed that IND and DCF bound deeply

into the COX-2 active site in very similar conformation, when their

corresponding carboxylate moiety forms two strong hydrogen bonds to

the side chain of Arg120 (1.8 Å) and Tyr355 (2.7 Å). In addition, IND

formed an extra hydrogen bond with Ser530 (3.2 Å) through benzoyl

moiety, which explains why IND showed an enhancing afﬁnity to COX

isoenzymes compared to DCF.

Therefore, the ligand’s (6a–s) effect over the COX-1 and COX-2

isoenzymes don’t differ signiﬁcantly (the compounds have afﬁnity to

the both receptors), but the accessibility of COX-1 and COX-2 for binding

the ligands differ signiﬁcantly. The estimated free binding energy of the

reference drugs (IND, DCF and CCB) has a higher negative value for the

COX-2 than COX-1, so less energy is needed to stabilize the drug at the

ligand binding center (Table 1).

To estimate the selectivity of docked NO-IND-TZDs (6a–s) for COX

isoenzymes a selectivity index was calculated based on logarithm of

ratio between calculated inhibitory constant (Ki) for COX-1 and COX-2

(log₁₀Ki_COX-1/Ki_COX-2) (Table 1, Fig. 4).

It was noted that studied compounds are COX-1 selective except 6o,

which is COX-2 selective, with improved selective index in reference

with IND and DCF (Fig. 4).

It was noted that the free carboxylate moiety, common for IND and

DCF, is responsible for non selective inhibition of the COX isoenzymes,

being in accordance with the literature data [93,94]. That’s why it forms

a salt bridge with the basic nitrogen of Arg120, being responsible for the

conformational change of COX isoenzymes.

The COX binding pocket is a hydrophobic channel that is extended

from the membrane binding domain of the COX isoenzymes that com-

prises four α-helices that create a hydrophobic surface to the core of the

catalytic domain. At the apex of the channel, both isoenzymes have two

important amino acids Ser530 and Tyr385. Ser530 is the amino acid

targeted by different NSAIDs and inﬂuences the COX stereochemistry in

prostaglandins synthesis. The catalytic residue Tyr385 is located at the

top of the cannel and is involved in the hydroperoxidase activity. Arg120

and Tyr355 are two charged amino acids present in the COX active site

of both isoenzymes, which form together a narrow constriction in the

channel towards the bottom of the COX active site. The main difference

Referring to the CCB, which is a substituted pyrazole derivative, it

was showed that the most important for binding to COX are its phenyl

sulfonamide substituent and pyrazole ring. Sulfonamide group forms

four strong hydrogen bonds with the side chains of Gly519 (3.3 Å),

His90 (2.2 Å), Gly354 (3.3 Å) and Leu352 (2.1 Å) of the enlarged polar

pocket from COX-2. Moreover, pyrazole ring interacts with Tyr355

(3.3 Å) via an extra hydrogen bond, which explain high COX-2 selec-

tivity of CCB.

12

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

Fig. 4. The COX selectivity of NO-IND-TZDs (6a–s) in respect to references drugs.

between the active sites of both COX isoenzymes is the replacement of

Ile (Ile434 and Ile523) in COX-1 by less bulky amino acid Val (Val434

and Val523) in COX-2. The loss of a single methylene group (Ile vs Val) is

sufﬁcient to open a secondary internal hydrophobic side pocket in COX-

2 that enlarging the volume of the active site by approximately 25% and

giving access to Arg513 replaced in COX-1 by a His.

Referring to the binding of NO-IND-TZDs (6a-s) to COX-2 it was

noted differences between compounds. However it was observed that all

compounds interact with amino acids both from the narrow constriction

and the inside of COX-2 active site, especially with the site pocket

(delimited by Val523, Phe518, Arg513, Ala516, Gln192, His90, Tyr355)

and extra space (delimited by Leu384, Leu503, Tyr385, Trp387)

(Fig. 5b).

The binding mode of NO-IND-TZDs (6a-s) to COX-1 side pocket is

quite similar (Fig. 5a) for all compounds, they having afﬁnity for hy-

drophobic channel. It was noted that NO releasing chain and 1,3-thiazo-

lidine-4-one moiety interact with amino acids from entrance of the

active site (Arg120, Tyr355, Glu524, Val116) while the indole structure

(IND) interacts with the amino acids from inside of pocket (Ala527,

Ser530, Tyr385, Ile523).

Based on docking results we can appreciate that NO-IND-TZDs (6a-s)

are bulky and can block the access of arachidonic acid (physiological

substrate) to the active site of COX isoenzymes.

Fig. 5. Binding mode of 6a–s to the active site of COX-1 (a) and COX-2 (b).

13

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

3.3. In silico ADME-Tox study

permeability. More speciﬁc, a LogBB value higher 0.3 means that drug

readily cross the blood brain barrier while a value less than ꢀ 1, in-

dicates a reduced permeability for this barrier [77]. BBB permeability

values of NO-IND-TZDs were predicted to range from – 2.41 to - 1.49

which means that are poorly distributed to the brain.

Using the ADME-Tox evaluation, the pharmacokinetic properties and

toxicity degree of the synthesized NO-IND-TZDs (6a–s) were predicted

and the results are summarized in Table 2.

It is known that oral absorption of the drug is inﬂuenced by solubi-

lity, permeability (active and passive) across the gastrointestinal tract

wall and ﬁrst pass metabolism [73]. The absorption of our compounds

was predicted from lipophilicity, water solubility, percentage of intes-

tinal human absorption (HIA) properties and P-glycopretein (P-gp) in-

hibitor properties.

The metabolism of NO-IND-TZDs, based on inhibition of the main

cytochromes (CYP) of the P450 family (CYP1A2, CYP2C19, CYP2C9,

CYP2D6 and CYP3A4), descriptors (SwissADME) was also estimated.

CYP2C19 is involved in detoxifying of potential carcinogens or bio-

activating environmental procarcinogens [98], CYP2C9 is the major

enzyme that metabolizes drugs with a low therapeutic index [99] and

CYP2D6 is very polymorphic and it metabolizes ~20% of drugs [100].

Referring to the CYP2D6, it is known that reduced or lacked activity of

this enzyme reduce efﬁcacy of the drugs or increase the occurrence of

adverse effects [100]. Our compounds were predicted to inhibit all

tested CYP enzymes except CYP1A2 and CYP2D6 (Table 2).

The water solubility was predicted using the Silicos IT LogSw

descriptor (SwissADME) depending on which was gave a qualitative

estimation of the solubility class [75]. LogSw values for NO-IND-TZDs

were predicted to range from – 9.36 to – 7.54, being considered

poorly soluble.

The lipophilicity was predicted using the Consensus LogPo/w

descriptor (SwissADME), which is the arithmetic mean of ﬁve compu-

tational methods values [75]. This parameter has a signiﬁcant inﬂuence

on various pharmacokinetic properties such as the absorption, distri-

bution, permeability, as well as the routes of drugs clearance [95].

Compounds with a LogPo/w higher than 1 or less than 4 are generally

considered to have optimal physico-chemical and ADME properties for

oral route [96] whereas logPo/w > 5 (high lipophilicity) often con-

tributes to high metabolic turnover, low solubility and poor oral ab-

sorption [96]. For our compounds, the predicted values of LogPo/w

ranged from 2.88 to 4.74 (Table 2).

The excretion of the drugs is measured by total clearance (CLtot) and

can be achieved by either the kidney and/or the liver where drugs are

eliminated in the form of urine or bile, respectively [95]. Using the CLtot

descriptor of pkCSM pharmacokinetics it was forecast that excretion of

NO-IND-TZDs varies between – 0.174 and 0.401 log(mL/min/kg).

The assessment of drugs toxicity, in terms of hepatotoxicity, oral acute

toxicity (LD₅₀) values, maximum tolerated dose (MRTD) and mutagenic

potential using bacteria (AMES), was predicted by using pkCSM-

pharmacokinetics. The hepatotoxicity descriptor predicted that all

molecules could present hepatotoxicity. Predicted LD₅₀values ranged

from 2.492 to 2.982 mol/kg and for MRTD between 0.47 and 0.58 log

(mg/kg/day), which means the tested compounds could be toxic, but

many studies, including in vivo, must be performed in order to conﬁrm it.

Also, most of the tested compounds showed negative results for AMES

toxicity (excepting 6g, 6m and 6n) indicating that they do not have

mutagenic potential.

The pkCSM pharmacokinetics prediction showed a good human in-

testinal absorption for NO-IND-TZDs, which ranged from 91.23% to

100%. These results support their good absorption in the small intestine

and predict also their capacity to inhibit P-glycoprotein I/II transport.

Using pkCSM-pharmacokinetics, the drug distribution in terms of the

blood brain barrier (BBB) permeability, the volume of distribution

(VDss) and the fraction unbound, could be also predicted. For tested NO-

IND-TZDs, the predicted values of VDss ranged from – 1.28 to – 0.66 and

of fraction unbound ranged from 0.127 to 0.273. The lower VDss (<

ꢀ 0.15) are in agreement with lower value for the fraction of drug

molecules in the plasma which are unbound (free) to proteins. It is

known that only the unbound fraction of the drug is active whereas the

high binding to proteins can affect both the pharmacokinetics and

pharmacodynamics of drugs [95,97].

Brieﬂy, the ADME-Tox proﬁle predicted for NO-IND-TZDs (6a–s)

consisted of optimal physico-chemical and ADME properties for oral

administration, good absorption in the small intestine, low fraction

unbound value and poorly distributed to the brain. As for metabolism

the tested compounds do not inhibit the CYP2D6 which is determinant

in biotransformation processes and could have some degree of

hepatotoxicity.

3.4. In vitro radical scavenging assay

The BBB permeability is assessed using two parameters, one quali-

tative (BBB) and another one quantitative (LogBB). Depending of the

BBB parameter value the drugs are classiﬁed as drugs with high and low

The most used method to evaluate the radical scavenging effect is

based on the reduction of DPPH, which is violet in ethanol solution, to

Table 2

The ADME-Tox proﬁle of NO-IND-TZDs (6a-s) predicted using SwissADME and pkCSM-pharmacokinetics tools.

Comp.

Cons. Log

Silicos-IT

HIA (%)

VDss (human)

(Log L/kg)

Fraction unbound

(human)

BBB permeability

(Log BB)

CLtot (Log mL/

min/kg)

MRTD (human) (Log

mg/kg/day)

LD₅₀

Po/w

LogSw

(mol/kg)

6a

6b

6c

6d

6e

6f

3.79

4.01

4.21

4.36

3.77

4.06

2.99

3.75

4.76

4.22

3.8

ꢀ 8.23

ꢀ 8.48

ꢀ 8.79

ꢀ 8.96

ꢀ 8.31

ꢀ 8.7

100

ꢀ 0.867

ꢀ 0.972

ꢀ 0.83

0.129

0.136

0.135

0.136

0.25

ꢀ 1.496

ꢀ 1.68

ꢀ 0.053

ꢀ 0.096

0.007

ꢀ 0.174

0.086

0.04

0.509

0.527

0.525

0.527

0.514

0.524

0.472

0.514

0.493

0.488

0.515

0.53

2.674

2.714

2.639

2.971

2.953

2.528

2.969

2.656

2.725

2.67

100

ꢀ 1.648

ꢀ 1.656

ꢀ 2.111

ꢀ 2.135

ꢀ 1.924

ꢀ 2.34

100

ꢀ 0.817

ꢀ 0.869

ꢀ 0.804

ꢀ 1.236

ꢀ 0.876

ꢀ 0.767

ꢀ 0.845

ꢀ 0.864

ꢀ 0.865

ꢀ 1.18

100

0.256

0.211

0.262

0.147

0.155

0.128

0.152

0.208

0.238

0.156

0.127

0.142

0.266

0.273

6g

6h

6i

ꢀ 7.54

ꢀ 8.39

ꢀ 9.36

ꢀ 8.88

ꢀ 8.23

ꢀ 8.31

ꢀ 7.54

ꢀ 9.04

ꢀ 8.23

ꢀ 8.71

ꢀ 8.8

96.219

100

0.012

0.224

0.044

0.142

ꢀ 0.047

0.084

ꢀ 0.048

0.112

0.069

0.074

0.261

0.401

0.355

100

ꢀ 2.267

ꢀ 2.327

ꢀ 1.493

ꢀ 1.708

ꢀ 1.976

ꢀ 1.915

ꢀ 1.868

ꢀ 1.49

6j

100

6k

6l

95.014

91.227

92.26

97.895

100

3.7

2.733

2.523

2.492

2.719

2.659

2.634

2.982

2.962

6m

6n

6o

6p

6q

6r

6s

3.06

2.88

4.29

3.75

3.84

3.85

4.04

0.473

0.527

0.545

0.52

ꢀ 1.276

ꢀ 0.811

ꢀ 0.885

ꢀ 0.774

ꢀ 0.722

ꢀ 0.66

95.132

95.338

100

ꢀ 1.713

ꢀ 2.386

ꢀ 2.41

0.58

0.55

ꢀ 9.18

100

0.557

14

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

yellow, in the presence of a proton or electron donating agent [101]. In

order to measure the anti radicalic effect of NO-IND-TZDs, the concen-

tration needed to decrease by 50% the initial DPPH concentration (IC₅₀),

was calculated. A lower IC₅₀means a higher antioxidant effect. The

values found for tested compounds (6a-s), reference drugs (IND and

ASP) and vitamin C are showed in Table 3.

protein (BSA), an increase in optical density will occur as a result of

protein precipitation [81]. Literature data report that the native states of

the proteins are stable in hydrophobic solvents, whereas the proteins

tend to lose their native structure in polar organic solvents like DMSO,

DMFA or triﬂuoroethanol [114]. In addition, it was suggested that the

effect of DMSO on the proteins stability depends on the concentration

[115].

Moreover, to compare the antioxidant effect of the tested compounds

(6a-s) to vitamin C, the CEAC value, that represents how many times

tested compounds are more active than vitamin C, was also calculated

(Fig. 6). Using one-way ANOVA test there was determined that there is a

statistically signiﬁcant difference (F(20, 63) = 11891.16, p < 0.05) be-

tween tested compounds. Analysis of the results revealed that all tested

compounds have improved antioxidant effect compared to reference

drugs (IND, ASP), the CEAC values ranging between 0.266 and 0.137. It

can appreciate the majority of compounds are around 23 times more

active than IND (CEAC = 0.0014 ± 0.0002) and 2 times more active

than ASP (CEAC = 0.016 ± 0.004). The most active compound was 6 n

(CEAC = 0.137 ± 0.01), which has on the second position of 1,3-thiazo-

lidne-4-one scaffold the 2-(4-nitrophenoxy)-ethyl nitrate as substituent,

being around 100 times more active than IND. Compared to vitamin C,

used as a positive control, all tested compounds were less active, the

CEAC values being less than 1.

Because the tested compounds (6a-s) have poor solubility in water,

DMSO (40%) was used as solvent. The our study showed that BSA, 0.2%

in 0.9% NaCl/DMSO = 6/4 remained stable after incubation at 38 ^◦C for

5 h and no turbidity was observed. In similar conditions, the tested

compounds (6a-s) cause an intense precipitation of BSA, resulting a

cloudy solution.

The results, expressed as denaturation effects (%) at different con-

centration (50 μM, 100 μM and 150 μM) in reference with positive

control (100%) are presented in Fig. 7. Using two-way ANOVA test there

was determined that there is a statistically signiﬁcant interaction (F(40,

189) = 49644.649, p < 0.05) between concentration and type of tested

compounds on BSA denaturation process.

It was noted that the tested compounds (6a–s) were able to increase

the albumin denaturation in a concentration dependent manner, the

higher concentrations increase the denaturation effect (Fig. 8). For

example, the compounds 6a, 6k and 6p showed a statistically signiﬁcant

increasing of denaturation effect (p < 0.05) at 150 µM but there were no

difference (p = 0.701) at 50 µM and 100 µM respectively. At 150 µM,

compound 6k was more active (15.04 ± 0.02%) compared with 6a

(8.44 ± 0.02%) and 6p (3.33 ± 0.02%). Therefore, in relation with

chemical structure, we can conclude that the presence of 3-phenoxy

moiety on second position of thiazolidine-4-one scaffold enhance the

anti-inﬂammatory activity.

3.5. In vitro anti-inﬂammatory assay

The discovery of new drugs is focused primary to identiﬁcation of a

ligand that binds to the target protein with high afﬁnity [102–104]. It is

known that NSAIDs are strong ligands to enzymatic and non-enzymatic

proteins, being proved that their anionic radicals interact with the polar

amino acids of proteins whereas their lipophilic moieties are ﬁxed into

hydrophobic site of proteins [105]. Referring to the COX, different

mechanisms of inhibition are known. For example, ASP has a unique

therapeutic action and irreversibly inhibits COX-1 and COX-2, due to

covalent acetylation of the enzymes [106–109] while IND and other aryl

acetic acid derivatives inhibit COX by a conformational change of pro-

tein [106]. In order to study the mechanism of inhibition of newly

synthesized NO-IND-TZDs, IND and ASP were used as reference NSAIDs.

Literature notes that ASP also induce acetylation of multiple proteins

(such as human serum albumin, ﬁbrinogen, p53, cellular protein) by in

vitro and in vivo assays and extensive ASP therapy give rise of

anti-acetylated serum albumin antibodies [110–112].

As expected, ASP (4.78 ± 0.18%) bonded BSA signiﬁcantly stronger

(p < 0.05) than IND (2.65 ± 0.23%). We also noted that, comparing

with the reference drugs (IND and ASP), that have the denaturation

effect less 5%, the derivatives 6k, 6c, 6q, 6o, 6j, 6d and 6i showed to be

more active, with value between 15% and 37%.

The analysis of the results revealed that the substitution of the aro-

matic ring on 1,3-thiazolidne-4-one scaffold with electron withdrawing

groups (like Cl, Br) as well as increase of -O–CH₂-CH₂- spacer length

(from 1 unit to 2 units) increase the denaturation effect and thus pro-

mote interaction with non enzymatic proteins. The most active com-

pound, in term of anti-inﬂammatory effects was 6i which contain a (2,6-

dichloro-phenoxy)ethyl nitrate moiety.

These interactions make changes on the native structure of the pro-

teins as the modiﬁcation of the secondary, tertiary or quaternary

structure without the breaking of covalent bonds. As result, a variety of

easily quantiﬁable physico-chemical effects are produced, which are

used to predict the anti-inﬂammatory effects of different compounds

[82].

3.6. In vitro nitric oxide release measurement

For detection and quantiﬁcation of in vitro NO release by NO-IND-

TZDs (6a-s), a modiﬁed colorimetric Griess assay, based on decompo-

sition of nitrate ester moiety in presence of Hg²⁺and thiol based com-

pounds, was used. The experiment included also reference NO donors,

such as SNAP, SNP, NTG, which belong at different classes. So, NTG is a

The Mizushima’s test used to evaluate the anti-inﬂammatory effects

of the new NO-IND-TZDs (6a-s) is a nephelometric assay based on

thermal denaturation of BSA [113]. When a chemical is incubated with a

Table 3

The IC₅₀and CEAC values of NO-IND-TZDs (6a–s) referring to the DPPH radical scavenging effect.

Comp.

IC₅₀(mM)^a

CEAC^a

Comp.

IC₅₀(mM)^a

CEAC^a

6a

6b

6c

6d

6e

6f

2.63 ± 0.02

2.18 ± 0.01

2.28 ± 0.01

2.21 ± 0.02

2.46 ± 0.06

2.58 ± 0.04

2.20 ± 0.01

15.64 ± 1.00

2.32 ± 0.01

2.25 ± 0.01

2.78 ± 0.04

0.0280 ± 0.003

0.0339 ± 0.002

0.0324 ± 0.002

0.0334 ± 0.002

0.0300 ± 0.008

0.0286 ± 0.004

0.0335 ± 0.002

0.0047 ± 0.003

0.0318 ± 0.002

0.0329 ± 0.003

0.0266 ± 0.003

6l

2.22 ± 0.01

2.32 ± 0.01

0.54 ± 0.01

2.17 ± 0.02

1.82 ± 0.05

2.78 ± 0.12

2.14 ± 0.01

2.20 ± 0.01

4.58 ± 0.09

54.36 ± 7.93

0.07 ± 0.01

0.0333 ± 0.002

0.0318 ± 0.001

0.1370 ± 0.010

0.0341 ± 0.004

0.0405 ± 0.003

0.0266 ± 0.012

0.0345 ± 0.004

0.0336 ± 0.003

0.0162 ± 0.004

0.0014 ± 0.0002

1 ± 0

6m

6n

6o

6p

6q

6g

6h

6i

6r

6 s

ASP

IND

Vit C

6j

6k

a

The data are expressed as the mean ± SD, n = 4.

15

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

Fig. 6. The DPPH radical scavenging activity of NO-IND-TZDs (6a–s) and of reference drugs (IND, ASP) expressed as CEAC values (* mean ± SD, n = 4).

Fig. 7. The effects of NO-IND-TZDs (6a–s) and of reference drugs (IND, ASP) on BSA denaturation process.

representative organic nitrate which requires speciﬁc thiol and/or

enzymatic activation to generate NO, SNP is a metal nitrosyl compound

that spontaneously releases NO at physiological pH and SNAP is an S-

nitrosothiol which is rapidly decompose in presence of metal ions, such

as Cu⁺, Fe²⁺, Hg²⁺and Ag⁺.

of Hg²⁺in Griess reagents increased signiﬁcantly the quantiﬁed amount

of NO (p = 0.002). These results suggest that the Hg²⁺decompose the S-

nitrosothiol from SNAP. In addition, the presence of GSH in the exper-

imental medium can ﬁx the free NO to form a stable S-nitrosothiol (GS-

NO). It was noted also that the experimental pH did not inﬂuence

signiﬁcantly the amount of NO released from reference NO donors.

In order to study the interaction effects of used Griess reagents and

experimental conditions on NO released from nitrate ester moiety of

tested compounds (6a–s), two-way ANOVA test was applied. It was

found that there is no statistically signiﬁcant interaction between them

(F(3, 600) = 1.454, p = 0.226). Moreover, it was noted that the adding

of HgCl₂to SULF solution do not increase signiﬁcantly the total amount

of NO released from nitrate ester moiety (F(1, 600) = 0.629, p = 0.428).

A statistically signiﬁcant interaction between structure of compounds

(6a–s) and experimental conditions (F(54, 532) = 29.908, p < 0.05)

was noted. All these results suggest that the presence of Hg²⁺in Griess

reagents do not inﬂuence the amount of NO released by NO-IND-TZDs

but it is strongly inﬂuenced by the structure of tested compounds and

experimental conditions (pH and GSH presence).

The NO releasing from the NO-IND-TZDs (6a–s) was studied in

different experimental conditions in order to mimics the intestinal (PBS,

PBS-GSH), and gastric (HCl, HCl-GSH) environmental conditions and

the results are presented in Figs. 9 and 10.

As expected, there was a statistically signiﬁcant difference between

reference NO donors, as determined by one-way ANOVA (F(2,93)

= 60.729, p < 0.05). Post hoc comparisons using the Tukey HSD test

indicated that the mean score for SNAP (52.70 ± 16.82) was signiﬁ-

cantly different than the SNP (3.97 ± 1.21) and NTG (0.49 ± 0.18)

(Fig. 10). Also, by two way ANOVA it was found there was not a sta-

tistically signiﬁcant interaction between used Griess reagents (NED –

SULF/SULF-HgCl₂) and experimental conditions (PBS, PBS-GSH, HCl,

HCl-GSH) on NO released ((F(3,88) = 1.029, p = 0.384). Moreover,

there was no statistically signiﬁcant difference in mean of NO released

by changing the experimental conditions (p = 0.669), but the presence

By estimated marginal mean of NO released it was showed that the

16

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

Fig. 8. The marginal mean of denaturation effect of NO-IND-TZDs (6a–s) and of reference drugs (IND, ASP).

Fig. 9. The percentage (%) of NO released by NO-IND-TZDs (6a–s) in different experimental conditions (PBS, PBS-GSH, HCl HCl-GSH), relative to theoretical

maximum release of 1.0 mol NO/mol tested compound) (n = 4).

most of the tested compounds (6a–s) released more NO compared with

NTG (Fig. 10). It was noted that the amount of NO released is inﬂuence

by the position of nitrate ester moiety on aromatic ring and also by the

number and type of the substituents on aromatic ring. The most proper

position of nitrate ester moiety is ortho, 6p (2-oxy-ethylnitrate) being

more active than 6k (3-oxy-ethylnitrate) and 6a (4-oxy-ethylnitrate).

Referring to the substitution of phenoxy-ethylnitrate moiety the pres-

ence of electron withdrawing groups (Cl, Br, NO₂) on aromatic ring

increase the NO, the most active compounds being 6o, 6g, 6q, 6i, 6c and

6n (Figs. 9 and 10). Of these, the best compound was 6n that consist of

an oxy-ethylnitrate (meta) and nitro (ortho) radicals on aromatic ring.

In this research it was noted a strong correlation between the

17

A. Sava et al.

Biomedicine & Pharmacotherapy 139 (2021) 111678

Fig. 10. The estimated marginal means of NO (%) release by NO-IND-TZDs (6a–s) compared to reference NO donors (NTG, SNP and SNAP).

theoretical studies (in silico) with the biological tests (in vitro). Based on

in vitro assay results the compounds 6k, 6c, 6q, 6o, 6j, 6d and 6i showed

to be the most active in term of the anti-inﬂammatory effects, interacting

strongly with non-enzymatic proteins (BSA). These results are supported

also by in silico docking study when the same compounds showed the

higher afﬁnity for COX isoenzyme, having thus an improved anti-

inﬂammatory effect compared to IND. In addition, the ADME-Tox

study revealed that the NO-IND-TZDs are generally proper for oral

administration, having optimal physico-chemical and ADME properties.

These results are in agreement with the in vitro assays results when an

improved antioxidant effect and increase NO release were observed for

tested NO-IND-TZDs, when compared to IND and NTG, respectively.

in vivo inﬂammation model assays.

Declaration of Competing Interest

The authors declare that they have no known competing ﬁnancial

interests or personal relationships that could have appeared to inﬂuence

the work reported in this paper.

Acknowledgments

The authors would like to thank the Agence Universitaire de la

Francophonie and Institute of Atomic Physics (AUF-IFA 2019–2020

grant, contract no. 28/2019), University of Medicine and Pharmacy

“Grigore T. Popa” of Iasi grant (contract no. 6985/2020), the Region

Centre Val de Loire (RTR Motivhealth), the SFR neuroimagerie (SFR FED

4224), the Labex programs SYNORG (ANR-11-LABX-0029) and IRON

(ANR-11-LABX-0018-01), the FEDER TECHSAB, the Ligue contre le

4. Conclusion

In order to improve the clinical efﬁciency of IND, especially for long-

term use, new nitric oxide-releasing indomethacin derivatives with 1,3-

thiazolidine-4-one scaffold (NO-IND-TZDs) have been synthesized,

based on the beneﬁcial effects of 1,3-thiazolidine-4-one scaffold and NO,

the last one being known as an important endogenous molecule,

including its critical role in GI mucosal defense. The synthesized NO-

IND-TZDs were biologically evaluated using in silico and in vitro as-

says. The molecular docking results revealed that most of the synthe-

sized NO-IND-TZDs are selective COX-1 inhibitors, except 6o (with (2-

bromo-6-methoxyphenoxy)ethyl nitrate moiety) which is COX-2 selec-

tive. Based on ADME-Tox proﬁle, it can be concluded that NO-IND-TZDs

are suitable for oral administration, showing a good absorption in the

small intestine, a low fraction unbound value and a reduced distribution

to the brain. The tested compounds also showed improved radical

scavenging effects, the highest radical scavenging effect being noted for

6n, which contains a 2-(4-nitro-phenoxy)ethyl nitrate moiety. In addi-

tion, for this compound it was also noted the highest NO release ca-

pacity, which means it could have reduced side effects to GI level such as

irritation, bleeding or ulceration. Moreover, the best predicted anti-

inﬂammatory effect, measured as BSA denaturation, was showed by 6i

which contains a (2,6-dichloro-phenoxy)ethylnitrate moiety, which

supports the good inﬂuence of chloro substituent for anti-inﬂammatory

effects.

´

`

Cancer du Grand Ouest (comites des Deux Sevres, du Finistere, de l’Ile et

Villaine, du Loir et Cher, de Loire Atlantique, du Loiret, de la Vienne),

and the Canceropole Grand Ouest for their ﬁnancial support.

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New nitric oxide-releasing indomethacin derivatives with 1,3-thiazolidine-4-one scaffold: Design, synthesis, in silico and in vitro studies

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