Design, synthesis, in silico and in vitro studies for new nitric oxide‐releasing indomethacin derivatives with 1,3,4‐oxadiazole‐2‐thiol scaffold

International Journal of

Molecular Sciences

Article

Design, Synthesis, In Silico and In Vitro Studies for New Nitric

Oxide-Releasing Indomethacin Derivatives with

1,3,4-Oxadiazole-2-thiol Scaffold

Alexandru Sava ^1,2, Frederic Buron ², Sylvain Routier ^2,*^,†, Alina Panainte ¹, Nela Bibire ¹,

Sandra Mădălina Constantin ³, Florentina Geanina Lupascu ³, Alin Viorel Focsa ³and Lenu¸ta Proﬁre ^{3, ,†}

*

,

1

Department of Analytical Chemistry, Faculty of Pharmacy, “Grigore T. Popa” University of Medicine and

Pharmacy of Iasi, 16 University Street, 700115 Iasi, Romania; alexandru.i.sava@umﬁasi.ro (A.S.);

,

alina-diana.panainte@umﬁasi.ro (A.P.); nela.bibire@umﬁasi.ro (N.B.)

Institut de Chimie Organique et Analytique ICOA, CNRS UMR 7311, Université d’Orléans,

45067 Orléans, France; frederic.buron@univ-orleans.fr

2

3

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

and Pharmacy of Iasi, 16 University Street, 700115 Iasi, Romania; constantin.sandra@umﬁasi.ro (S.M.C.);

,

ﬂorentina-geanina.lupascu@umﬁasi.ro (F.G.L.); alin-viorel-v-focsa@d.umﬁasi.ro (A.V.F.)

Correspondence: sylvain.routier@univ-orleans.fr (S.R.); lenuta.proﬁre@umﬁasi.ro (L.P.)

Both authors contributed equally as senior authors.

*

†

Abstract: Starting from indomethacin (IND), one of the most prescribed non-steroidal anti-inflammatory

drugs (NSAIDs), new nitric oxide-releasing indomethacin derivatives with 1,3,4-oxadiazole-2-thiol

scaffold (NO-IND-OXDs, 8a–p) have been developed as a safer and more efficient multitarget therapeutic

strategy. The successful synthesis of designed compounds (intermediaries and finals) was proved by

complete spectroscopic analyses. In order to study the in silico interaction of NO-IND-OXDs with

cyclooxygenase isoenzymes, a molecular docking study, using AutoDock 4.2.6 software, was performed.

Moreover, their biological characterization, based on in vitro assays, in terms of thermal denaturation

of serum proteins, antioxidant effects and the NO releasing capacity, was also performed. Based on

docking results, 8k, 8l and 8m proved to be the best interaction for the COX-2 (cyclooxygense-2) target

site, with an improved docking score compared with celecoxib. Referring to the thermal denaturation

of serum proteins and antioxidant effects, all the tested compounds were more active than IND and

ꢀꢁꢂꢀꢃꢄꢅꢆꢇ

ꢀꢁꢂꢃꢄꢅꢆ

Citation: Sava, A.; Buron, F.; Routier,

S.; Panainte, A.; Bibire, N.;

Constantin, S.M.; Lupascu, F.G.;

,

Focsa, A.V.; Proﬁre, L. Design,

,

Synthesis, In Silico and In Vitro Studies

for New Nitric Oxide-Releasing

Indomethacin Derivatives with

1,3,4-Oxadiazole-2-thiol Scaffold. Int. J.

Mol. Sci. 2021, 22, 7079. https://

doi.org/10.3390/ijms22137079

aspirin, used as references. In addition, the compounds 8c, 8h, 8i, 8m, 8n and 8o showed increased

capacity to release NO, which means they are safer in terms of gastrointestinal side effects.

Academic Editor: Antonio

Palumbo Piccionello

Keywords: indomethacin; 1,3,4-oxadiazole-2-thiol; nitric oxide; docking study; cyclooxyge-

Received: 25 May 2021

Accepted: 25 June 2021

Published: 30 June 2021

nase; inflammation

Publisher’s Note: MDPI stays neutral

with regard to jurisdictional claims in

published maps and institutional afﬁl-

iations.

1. Introduction

The non-steroidal anti-inﬂammatory drugs (NSAIDs) are the most prescribed drugs

for management of different pathological conditions where inﬂammation is involved, based

on their analgesic, anti-inﬂammatory and antipyretic effects [1,2].

The inﬂammation is a defense reaction of the human body to various harmful agents

in restoring the body homeostasis [

holding the body in a constant state of alert, it may become chronic, with a negative impact

on tissue and organs [ ]. The clinical consequences of chronic inﬂammation-driven damage

can be severe and include increased risk of many chronic diseases such as: rheumatoid

arthritis [ ], inﬂammatory bowel disease [ ], metabolic disorders (diabetes mellitus and

obesity) [10 12], cardiovascular disorders (ischemic heart disease, atherosclerosis) [13 14],

neurodegenerative diseases (Parkinson’s and Alzheimer’s disease) [15 17] and cancer [18],

many of these conditions being life-threatening [19 20]. The current use of NSAIDs in the

European Union is associated with a 19% increased risk of hospital admissions for heart

3–6]. When inﬂammation persists for a long time,

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8

9

–

,

–

,

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Int. J. Mol. Sci. 2021, 22, 7079

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failure compared to periods before 1999–2012, as is well documented [21

–23]. Despite the

clinical beneﬁts, the chronic use of NSAIDs is also associated with increased risk of side

effects, including gastrointestinal toxicity, renal injury, hepatotoxicity, hypertension, as well

as allergic skin reactions [24–27]. The main mechanism of NSAIDs’ action, which is respon-

sible for both therapeutic and side effects, is inhibition of cyclooxygenase (COX) enzymes

in a wide variety of systems, ranging from microsomal enzyme synthesis to different cells

and tissues [24,28,29]. Moreover, in the last two decades, based on the beneﬁcial effects

of endogenous nitric oxide (NO), NSAIDs with NO-releasing moiety were developed as

a new therapeutic strategy for a variety of clinical conditions which involve acute and

chronic inﬂammation [30

is produced in mammalian cells through nitric oxide synthase-mediated conversion of

L-arginine to L-citroline [35 38]. This important signaling molecule has a key role in a wide

–34]. Nitric oxide (NO), an endogenous short-lived free radical,

–

variety of biological processes such as immune defenses, inﬂammation, neurotransmission,

vasodilatation, platelet adhesion, thrombosis and wound healing [39–41]. In addition, NO

is intimately involved in regulating all aspects of our lives from waking, digestion, sexual

function, perception of pain and pleasure, memory recall and sleeping [40,41]. Moreover,

NO is known to have a protective effect on the gastrointestinal tract (GT), based on its

properties in stimulating gastric mucus secretion to increase the mucosal blood ﬂow and to

inhibit the leukocyte adherence to the vascular endothelium [39,42,43].

The design of hybrid molecules, able to reduce the inﬂammation by both COX in-

hibition and NO release could be of great interest. The aim of our study was to develop

new nitric oxide-releasing indomethacin derivatives with 1,3,4-oxadiazole-2-thiol scaffold

(NO-IND-OXDs), as a safer and more efﬁcient multitarget therapeutic strategy. Among the

library of COX inhibitors, indomethacin (IND) retains our attention as it is one of the most

prescribed NSAIDs in the management of osteoarthritis, rheumatoid arthritis, episodes of

acute gout, ankylosing spondylitis and acute musculoskeletal pain [44,45]. The long-term

use of IND could increase the risk for a wide range of side effects such as gastrointestinal

irritation, bleeding and ulceration, dizziness, peripheral edema, arterial hypertension,

tachycardia, kidney and liver dysfunction, allergic and anaphylactic reactions, increased

anxiety, headache [44,46].

On the other hand, the oxadiazole scaffold serves as core for many synthetic com-

pounds of great interest in medicinal chemistry [47–49]. Its use offers several advantages:

(i) it is an essential part of the pharmacophore, based on its ligand binding role; (ii) it

acts as a ﬂat aromatic linker assuring an appropriate molecule orientation; (iii) it induces

metabolic stability, water solubility and lower lipophilicity; (iv) it can easily chemically

modulate the compounds which contain carbonyl groups such as amides, carbamates,

esters and hydoxamic esters [32

containing the oxadiazole core, generally 1,3,4-oxadiazole motif, have important biological

effects such as anti-inﬂammatory [32 52 53], antioxidant [54 55], antidiabetic [56], anticon-

vulsant [57], anticancer [58], antitubercular [59 60], antiviral [61], antidepressant [62]. There

,50,51]. According to the literature data, the compounds

,

have already been several approved oxadiazole-based drugs such as: furamizole (strong

antibacterial activity, an antibiotic), butalamine (a vasodilator), oxolamine (a cough suppres-

sant), pleconaril (an antiviral), fasiplon (a non-benzodiazepine anxiolitic drug), raltegravir

(an antiretroviral drug for the treatment of HIV infection), nesapidil (an anti-arrhythmic

drug), zibotentan (an anticancer drug) as well as tiodazosin (an antihypertensive drug)

(Figure 1).

In nature, the oxadiazole scaffold has been reported to occur in quisqualic acid (from

Quiqualis fructus, strong agonist of (AMPA)-subtype glutamate receptor) and phidianidines

A and B (from primitive marine organisms, high cytotoxicity against tumor mammalian

cell lines) [63,64].

Moreover, the designed compounds contain, between NO precursor and oxadiazole

unit, an aromatic linker, substituted with different electron-withdrawing and electron-

donating groups. This fragment may serve as a decoy that promotes the binding of the COX

to the designed NO-IND-OXDs, according to the reported substrate trap strategies [65–68]

.

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Figure 1. Representative drugs and natural products containing the oxadiazole core.

Based on presented aspects, the chemical modulation of the free carboxyl group of

conventional NSAIDs, such as indomethacin, with 1,3,4-oxadiazole scaffold, can provide

new drugs with an improved pharmacological proﬁle, in terms of increased efﬁciency,

fewer side effects and reduced ulcerogenic potential. Moreover, the presence of two

pharmacophors (indol and 1,3,4-oxadiazole moieties) could increase the ability to inhibit

COX enzymes, especially COX-2.

Herein, we present the design and synthesis of some new NO-IND-OXDs, in silico

interaction with COX isoenzymes, based on a molecular docking study, as well as the

biological characterization using in vitro assays in terms of thermal denaturation of serum

proteins, antioxidant effects and the NO-releasing capacity.

2. Results and Discussion

2.1. Chemistry

Based on reactivity of the carboxyl group of IND, new NO-IND-OXDs 8a–p were

prepared in a few steps (Scheme 1). Reaction of hydroxy-benzaldehydes 1a–o and 4-(2-

hydroxyethyl)phenol 2p with 1,2-dibromoethane, afforded on an SN₂Williamson ether

synthesis, gave the corresponding (bromoethoxy)benzaldehyde derivatives 2a–o and 2-

(4-(2-bromoethoxy)phenyl)ethan-1-ol 3p, respectively. In the next step, the intermediaries

2a–o were reduced by standard procedure with NaBH₄into their corresponding (bro-

moethoxy)aromatic primary alcohols 3a–o in a good yield (86–99%). This method offered

signiﬁcant advantages such as mild reaction conditions and easy isolation (simple neutral-

ization and extraction) of the product.

By the metathesis reaction of 3a–p with silver nitrate in acetonitrile, the corresponding

(hydroxyalkyl)phenoxy nitrates 4a–p were obtained, which were further reacted with

thionyl chloride and iodine, respectively, to form the corresponding (halidealkyl)phenoxy

nitrates 5a–p. In turn, the IND derivative

tween IND and hydrazine hydrate when the IND hydrazide

was reacted with carbon disulﬁde in presence of triethylamine leading to (4-chlorobenzoyl)-

5-methoxy-2-methyl-1H-indol-3-yl)methyl)-1,3,4-oxadiazol . Finally, the intermediates

5a–p were used to alkylate the IND derivative in presence of triethylamine to form the

7

was obtained by peptide coupling reaction be-

6

was obtained, which further

7

desired NO-IND-OXDs 8a–p. The common methods for conversion of alcohols to the cor-

responding alkyl chloride involve activation of hydroxyl group before treatment with the

chlorination agent [69]. In the present study we used thionyl chloride in combination with

benzotryazole as the activation agent. Benzotryazole is a mild base as well as acid and of-

fers several advantages. It can be removed easily from the reaction mixture by acid or alkali

and its hydrochloride salt is insoluble in organic solvents such as dichloromethane [70

Using this method, the desired compounds 5a–m were obtained at room temperature

in high yields which ranged between 85 and 99%.

,71].

, t, u

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Scheme 1. Synthesis of NO-IND-OXDs 8a–p. Reagents and conditions: (i) C₂H₄Br₂, K₂CO₃/CH₃CN,

◦

95 C, 18–26 h, (ii) NaBH₄/THF, RT, 12–24 h, (iii) AgNO₃/CH₃CN, 95 C, 8–10 h, (iv) SOCl₂, BTA/dry

CH₂Cl₂, RT, 20–30 min, (v) imidazole, PPh₃, I₂/CH₂Cl₂, RT, 6 h, (vi) HOBt, EDC/CH₃CN, RT, 3h,

◦

N₂H₄(0–5 C, 1 h), (vii) Et₃N, CS₂/CH₃CN, reﬂux, 3 h, (viii) 5a–p, Et₃N/CH₃CN, RT, 3–6 h.

The compound 7 was obtained in near quantitative yield (92%) and was somewhat

easier (reaction time of 3 h) while NO-IND-OXDs were obtained at room temperature in

good yields ranged between 70 and 91%.

The chemical structure of all synthesized compounds (intermediate and ﬁnal com-

pounds) was proven on the basis of nuclear magnetic resonance (¹H NMR, ¹³C NMR) and

high-resolution mass spectrometry (HRMS) analysis (Supplementary Materials).

In the ¹H NMR spectra of (halidealkyl)phenoxy nitrates 5a–p it was found that the mul-

tiple signal of protons (-CH₂ONO₂) that are in the vicinity of electron-withdrawing nitrate

ester group appear more unshielded (

in the vicinity of bromide (-CH₂Br) ( = 3.7–3.90 ppm, m, 2H) from (bromoethoxy)aromatic

alcohol derivatives 3a–p. At the same time, ¹³C NMR signal for -CH₂ONO₂was ob-

served at = 72–73 ppm, compared to the signal of the same carbon from derivatives 3a–p

(δ = 31–32 ppm). In ¹H NMR spectra, the ﬁnal NO-IND-OXDs 8a–p showed signals for

CH₂ONO₂and CH₂S in the range of = 4.7–4.9 ppm, and = 4.3–4.5 ppm, respectively.

Whereas in ¹³C NMR spectra the signal for CH₂ONO₂group was observed in the range

of = 71–72 ppm, the signal for CH₂S was observed in the range of = 34–36 ppm and

δ = 4.7–4.9 ppm, m, 2H), compared with the protons

δ

for quaternary carbon from second position of 1,3,4-oxadiazol scaffold (C_qS) in the range

of δ = 162–163 ppm. The proton and carbon signals of other aliphatic and aromatic frag-

Int. J. Mol. Sci. 2021, 22, 7079

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ments of 8a–p were observed at the expected values of chemical shift. The presence of

the abovementioned signals have conﬁrmed the presence of 2-mercapto-1,3,4-oxadiazole

scaffold and the preserved nitrate ester moiety in the ﬁnal compounds. Moreover, the

NMR spectral data coupled with mass spectra support the proposed structures of the

all-synthesized compounds.

2.2. In Silico Docking Study

To predict the binding mode of the synthesized compounds with the COX isoenzymes,

two X-ray crystallographic structures, obtained from crystallographic data, were used [72].

The PDB 4o1z and PDB 3nt1 were described as the COX-1 and COX-2 structures with the

highest resolution to date, 2.4 Å and 1.7 Å, respectively [73], which means that there is

more conﬁdence in the location of atoms in the electron density map. Furthermore, the

reliability factor was less than 0.16, indicating a strong agreement between the simulated

and the experimentally observed diffraction patterns [74,75].

The used computational method was validated using RSMD variation at less than 2 Å

for reference drug (indomethacin—IND, diclofenac—DCF and celecoxib—CCB, individ-

ually) into the active site of both COX-1 and COX-2 isoforms and the same binding cleft

residues reported by other authors were found [76–79]. The CCB is a preferential COX-2

selective inhibitor whereas IND and DCF are non-selective COX inhibitors, inhibiting both

types of the COX enzymes.

Moreover, the docking results showed that all reference drugs have polar interactions

with the catalytic site of both COX-1 and COX-2 isoenzymes. More speciﬁcally, they act as

hydrogen bond acceptors with Arg120 and Tyr355, and have long-range polar interactions

with Tyr385 and Ser530 and non-polar interactions with Val349, Trp387, Phe-518, Ile/Val523

and Ala527 [80,81].

For estimating the docking score of NO-IND-OXDs 8a–p each ligand–receptor complex

was subjected to careful analysis for the ideal docked positions, based on the least binding

energy scores and maximum number of cluster conformations.

In order to establish the statistical signiﬁcance of the difference between the ligands

while removing the receptor variances from the overall error variance term, a two-way

ANOVA test was applied. The results established a statistically signiﬁcant difference

between ligands (F(0.5, 18, 37) = 3.2648, p = 0.0079, Fcrit = 2.2171). The value of the

estimated free binding energy of the compounds 8a–p was less than

−

10 kcal/mol and less

than that of the reference drugs (IND, DCF and CCB) (Table 1 ). Therefore, less energy is

needed to stabilize the synthesized compounds 8a–p at the ligand-binding center of the

COX receptor.

Based on inhibitory constant (Ki) for COX-1 and COX-2 [82], a selectivity index

(log₁₀Ki_COX-1/Ki_COX-2) was also calculated for docked NO-IND-OXDs 8a–p (Table 1 and

Figure 2 ). It was noted that the studied compounds have a better interaction with the

COX-2 target site, with improved selective index in reference to IND and DCF, except 8n,

8f and 8h, which have a better docking score for COX-1 target site. Even more, it was

observed that 8m, 8l and 8k proved to be the best interaction COX-2 target sites in reference

with CCB (Figure 2).

The COX binding pocket, for both COX-1 and COX-2, is a hydrophobic channel which

comprises four α-helices, thus creating a hydrophobic surface to the core of the catalytic

domain. In the upper part of the channel, both isoenzymes have two important amino

acids, Ser530 and Tyr385. Ser530 inﬂuences the COX stereochemistry in prostaglandin

synthesis while Tyr385 is involved in the hydroperoxidase activity. Moreover, towards the

bottom of the COX active site there are two polar amino acids, Arg120 and Tyr355, which

together form a narrow constriction at the entrance of the channel.

The main difference between the active sites of COX isoenzymes consists in the

replacement of Ile (Ile434 and Ile523) in COX-1 by the less bulky amino acid Val (Val434

and Val523) in COX-2. A single methylene group (Ile vs. Val) is enough to open a secondary

Int. J. Mol. Sci. 2021, 22, 7079

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internal hydrophobic side pocket in COX-2 that enlarges the volume of the active site by

approximately 25% and gives access to Arg513 replaced in COX-1 by a His.

Table 1. Docking score (

∆G, kcal/mol) and inhibition effect rate (Ki) during docking simulation of

NO-IND-OXDs 8a–p and reference drugs (IND, DCF, CCB) for COX-1 and COX-2.

COX-1

COX-2

Selectivity Index

log₁₀(Ki_COX-1/Ki_COX-2

Ligand

)

∆G(kcal/mol) Ki (nM)

8a

8b

8c

8d

8e

8f

8g

8h

8i

8j

8k

8l

8m

8n

8o

8p

IND

DCF

CCB

−12.68

−11.52

−11.15

−12.37

−11.50

−11.91

−12.27

−12.60

−12.76

−12.03

−11.03

−10.75

−11.42

−11.85

−12.55

−12.72

−9.98

0.508

3.590

6.710

0.860

3.700

1.860

1.010

0.584

0.442

1.520

8.240

13.140

4.270

2.050

0.631

0.470

48.230

1120

−13.18

−12.32

−13.14

−13.45

−12.67

−10.18

−13.84

−11.66

−13.37

−12.79

−13.51

−13.2

0.219

0.925

0.232

0.137

0.512

34.800

0.072

2.840

0.157

0.419

0.124

0.209

0.110

1680

0.364

0.589

1.461

0.796

0.859

−1.272

1.146

−0.687

0.449

0.559

1.821

1.796

1.587

−2.914

0.476

1.129

0.273

0.378

1.428

−13.58

−7.88

−13.2

0.211

0.034

25.720

468.540

27.520

−14.27

−10.35

−-8.63

−10.31

−8.12

−8.37

737.590

CCB

DCF

IND

8k

8l

8m

8c

8g

8p

8e

8d

8b

8j

8o

8i

8a

8h

8f

8n

-3

-2

-1

0

1

2

3

Increasing COX-1 interaction

Increasing COX-2 interaction

Range for COX-1 and COX-2 interaction (log₁₀Ki_COX-1/Ki_COX-2

)

Figure 2. The COX interaction of NO-IND-OXDs 8a–p in respect to reference drugs.

Referring to the binding of NO-IND-OXDs 8a–p to COX-1, differences were noted

between compounds. However, it was observed that all compounds interact with amino

acids both from the narrow constriction and the inside of COX-1 active site (Figure 3a). The

binding mode of NO-IND-OXDs 8a–p to COX-2 side pocket is quite similar (Figure 3b) for

all compounds, as they have afﬁnity for the hydrophobic channel. It was noted that the

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NO-releasing chain interacts with the site pocket (delimited by Val523, Phe518, Arg513,

Ala516, Gln192, Tyr355) and extra space (delimited by Leu384, Leu503, Tyr385, Trp387).

The oxadiazole scaffold interacts by hydrogen bonds with Arg120 and Tyr355 from the

narrow constriction while the indole structure interacts with amino acids from the entrance

of the active site.

Figure 3. Binding mode of 8a–p to the active site of COX-1 (a) and COX-2 (b).

The analysis of the results revealed that the COX-1/COX-2 selectivity of 8a–p is

inﬂuenced by the position of nitrate ester moiety on aromatic ring and by the number

and type of the substituents on aromatic ring. The most proper positions of nitrate ester

moiety are meta and para, 8i (3-oxy-ethylnitrate) and 8a (4-oxy-ethylnitrate), respectively,

being more active on COX-2 than 8n (2-oxy-ethylnitrate), which is more COX-1-selective.

Referring to the substitution of phenoxy-ethylnitrate moiety, the presence of halogens (F,

Cl, Br) or NO₂on aromatic ring increases the COX-2 selectivity index, in contrast with

only OCH₃(8h, 8f), which increases COX-1 selectivity. Moreover, it was observed that the

elongation of distance between oxadiazole moiety and aromatic ring, from methylene (8a

)

to ethylene (8p), seems to be responsible for better ligand ﬂexibility and selectivity for the

COX-2 active site. Based on the obtained results, we can say that the synthesized NO-IND-

OXDs have the theoretical premises to be promising anti-inﬂammatory agents. This is in

agreement with the literature which reported that the replacement of the free carboxylic

acid group of conventional NSAID with 1,3,4-oxadiazole heterocycle may provide new

drugs with increased anti-inﬂammatory activity, improved efﬁciency and with fewer side

effects, reducing ulerogenic potential [53,83].

2.3. In Vitro Radical Scavenging Assay

The most used radical scavenging assay is based on the reduction of the DPPH (2,2-

diphenyl-1-picrylhydrazyl radical), in the presence of a proton- or electron-donating agent,

when there is a color changing from violet to yellow [79]. In order to evaluate the scav-

enging effect of NO-IND-OXDs, the concentration needed to decrease the initial DPPH

concentration (IC₅₀) by 50% was calculated and the results are presented in Table 2. Fur-

thermore, the vitamin C equivalent antioxidant capacity (CEAC) value was also calculated,

in order to compare the antioxidant effect of 8a–p to vitamin C (Figure 4).

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Table 2. The IC₅₀and CEAC values of NO-IND-OXDs 8a–p referring to the DPPH radical scaveng-

ing effect.

Comp.

IC₅₀(mM) ^a

CEAC ^a

Comp.

IC₅₀(mM) ^a

CEAC ^a

8a

8b

8c

8d

8e

8f

8g

8h

8i

2.38±0.02

4.35 ± 0.34

2.37 ± 0.01

2.46 ± 0.02

2.16 ± 0.03

2.42 ± 0.02

2.98 ± 0.06

2.49 ± 0.02

2.56 ± 0.01

1.99 ± 0.01

0.0309 ± 0.0002

0.0170 ± 0.0012

0.0311 ± 0.0001

0.0299 ± 0.0003

0.0341 ± 0.0004

0.0305 ± 0.0003

0.0248 ± 0.0005

0.0296 ± 0.0003

0.0288 ± 0.0001

0.0369 ± 0.0003

8k

8l

8m

8n

8o

8p

ASP

IND

Vit C

2.19 ± 0.01

5.29 ± 0.21

2.9 ± 0.31

0.0336 ± 0.0001

0.0139 ± 0.0005

0.0256 ± 0.0029

0.0258 ± 0.0005

0.0192 ± 0.0047

0.0265 ± 0.0003

0.0162 ± 0.0004

0.0014 ± 0.0002

1 ± 0

2.85 ± 0.06

4.03 ± 1.11

2.77 ± 0.03

4.58 ± 0.10

54.37 ± 7.94

0.07 ± 0.00

8j

a

The data are expressed as the mean ± SD, n = 4.

Figure 4. The DPPH radical scavenging activity of NO-IND-OXDs (8a–p) and of reference drugs (IND, ASP) expressed as

CEAC values (* signiﬁcant differences than ASP (p< 0.05); ** mean ± SD, n = 4).

Using one-way ANOVA test, a statistically signiﬁcant difference (F(17, 71) = 164.778,

p < 0.05) between tested compounds (8a–p, ASP, IND) was highlighted. Post hoc com-

parisons using the Tukey HSD test revealed that all tested compounds have signiﬁcantly

improved antioxidant effect in reference to IND (p< 0.05). Moreover, it was noted that most

of the tested compounds (except 8b

(ASP) (p< 0.05). It can be appreciated that the majority of compounds are from 10.0 to

26.7 times more active than IND (CEAC = 0.0014 0.0002) and from 1.5 to 2.3 times more

active than ASP (CEAC = 0.0162 0.0004). Compared to vitamin C, used as a positive

, 8l and 8o) are signiﬁcantly more active than aspirin

±

control, all tested compounds were less active, the CEAC values being less than 1.

Moreover, it was reported that 1,3,4-oxadiazole scaffold has been found to be a potent

antioxidant pharmacophore, and chemical modulation of the conventional drugs with this

scaffold resulted in a synergistic effect, enhancing the antioxidant efﬁciency of the parent

drug [47,49,50,83,84]. In our research, the presence of 1,3,4-oxadiazole scaffold signiﬁcantly

enhanced antioxidant activity for the majority of the NO-IND-OXDs.

2.4. Thermal Denaturation of Serum Proteins

The Mizushima’s test, which is based on thermal denaturation of proteins, was used to

predict the anti-inﬂammatory effects of the new NO-IND-OXDs 8a–p [85]. The NSAIDs are

strong ligands to enzymatic and non-enzymatic proteins and it has been proven that their

hydrophilic part, as an anionic radical, interacts with the polar amino acids of proteins while

their lipophilic part is ﬁxed into the hydrophobic site of proteins [86]. As a result of these

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interactions, various changes in the native structure of the proteins, such as modiﬁcation

of the secondary, tertiary or quaternary structure without breaking the covalent bonds,

occur [87,88]. It was reported that the native state of the proteins is stable in hydrophobic

solvents, whereas the polar organic solvents such as triﬂuoroethanol, DMFA or DMSO

affect their structure, the degree of injury being concentration-dependent [89,90].

When a compound is incubated with non-enzymatic proteins, such as bovine serum

albumin (BSA), an increase in optical density will occur because of protein precipitation [88].

Because the tested compounds 8a–p are less soluble in water, DMSO (40%) was used as

solvent. The results 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

compounds 8a

The results, expressed as denaturation effect (%) at different concentration (25

and 100 M) in reference to positive control (100% denaturation of BSA induced by 0.1 M

hydrochloric acid) are presented in Figure 5.

–

p

cause an intense precipitation of BSA and a cloudy solution was obtained.

µM, 50

µM

µ

Figure 5. The effects of NO-IND-OXDs (8a–p) and of reference drugs (IND, ASP) on BSA denaturation process (* 8n

and 8i, p < 0.05).

< 8a

Using two-way ANOVA test, it was determined that there is a statistically signiﬁ-

cant interaction (F(30, 144) = 2060.794, p < 0.05) between concentration and structure of

compounds referring to the denaturation effect. It was noted that the tested compounds

8a

the higher concentration increases the denaturation effect. For example, the compounds

8a 8i and 8n showed a statistically signiﬁcant increase of denaturation effect (p < 0.05)

–p were able to increase the albumin denaturation in a concentration-dependent manner;

,

with concentration increasing, but at the same level of each concentration there was no

difference (p = 0.150) between 8a and 8i compared with 8n, for which the denaturation

effect increased signiﬁcantly less (p < 0.05). At 100

µM, compound 8n was less active

(

20.7 ± 0.4%) compared with 8a (33.7

±

1.0%) and 8i (33.0

±

0.9%). Therefore, in relation to

chemical structure, we can also note that the presence of nitrate ester moiety in para and meta

positions was more favorable than ortho, 8a (4-oxy-ethylnitrate) and 8i (3-oxy-ethylnitrate)

being more active than 8n (2-oxy-ethylnitrate).

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Compared to the reference drugs, IND and ASP, which have a denaturation effect of

less than 5%, all the tested compounds were shown to be more active, with the value of the

denaturation effect ranging between 20% and 40% (at 100 µM).

The results of our study also revealed that the presence of halogens (F, Cl, Br) or NO₂

on phenoxy-ethylnitrate moiety, as well as the elongation of distance between oxadiazole

moiety and aromatic ring from methylene group (8a) to ethylene group (8p) increases the

denaturation effect and thus promotes interaction with non-enzymatic proteins.

In addition, it is known that ASP is able to produce acetylation of proteins (such as

human serum albumin, ﬁbrinogen, p53, cellular protein) and the ASP therapy is associated

with the rise of the anti-acetylated serum albumin antibodies [91–93]. It was also reported

that these interactions could be used to predict the anti-inﬂammatory properties of the

compounds [94]. The results of our study revealed that the chemical modulation of free

carboxylic group of IND with oxadiazole moiety enhances the drug–protein interactions

that are in agreement with previous in silico results, which showed good interaction with

COX enzyme sites.

2.5. In Vitro Nitric Oxide Release Measurement

For detection and quantiﬁcation of NO released by NO-IND-OXDs 8a–p, a modiﬁed

colorimetric Griess assay was used, based on decomposition of nitrate ester moiety in

presence of Hg²⁺and thiol-based compounds. The study was performed using different

NO donors as reference, such as sodium nitropruside (SNP), nitroglycerine (NTG) and

S-nitroso-N-acetyl-penicillamine (SNAP), which belong to different classes. SNP is a

metal nitrosyl compound that spontaneously releases NO at physiological pH, NTG is a

representative organic nitrate which requires speciﬁc thiol and/or enzymatic activation to

generate NO and SNAP is an S-nitrosothiol which is rapidly decomposed in the presence

of metal ions, such as Cu⁺, Fe²⁺, Hg²⁺and Ag⁺.

In order to appropriately simulate the body conditions, the NO releasing from

the NO-IND-OXDs 8a–p was evaluated in intestinal (PBS, PBS-GSH) and gastric (HCl,

HCl-GSH) environmental conditions, respectively, and the results are presented in

Figures 6 and 7. As expected, using one-way ANOVA analysis, a statistically significant

difference (F(2,93) = 60.729, p < 0.05) between NO donors, used as reference, was found.

In addition, Tukey HSD post hoc comparison test indicated that the NO percentage

released for SNAP (52.70 ± 16.82) was significantly higher than the SNP (3.97

±

1.21)

and NTG (0.49 ± 0.18) (Figure 7). There was no statistically significant interaction

between experimental conditions (PBS, PBS-GSH, HCl, HCl-GSH) and used Griess

reagents (NED—SULF/SULF-HgCl₂), as determined by two-way Anova (F(3.88) = 1.029,

p = 0.384). Moreover, the presence of Hg²⁺in Griess reagents increased by a statistically

significant value the mean of NO released (p = 0.002), but changing the experimental

conditions did not significantly influence (p = 0.669) the quantified amount of NO. These

results suggest that the GSH, present in the experimental medium, fixes the free NO,

forming a stable S-nitrosothiol (GS-NO) and so prevents the oxidation of NO into stable

NO₃⁻, which cannot be detected using Griess reagents. It was also noted that the

Hg²⁺produces the cleavage of the S-NO bond from SNAP. Therefore, the GSH and Hg²⁺

are appropriate reagents to measure the total amount of released NO. In addition, the

experimental pH has no significant influence on NO release from NO donors.

Referring to the tested compounds 8a

–p, no statistically significant interaction

(

F(3, 504) = 0.465, p = 0.707) between Griess reagents and experimental conditions, ap-

plied to study the NO release, was noted. Moreover, it was noted that the adding

of HgCl₂to SULF solution did not significantly increase the total amount of NO re-

leased from nitrate ester moiety (F(1, 504) = 0.122, p = 0.727). A statistically signifi-

cant interaction between structure of compounds 8a

F(45, 448) = 69.945, p < 0.05) was noted. All these results suggest that the presence of

Hg²⁺in Griess reagents did not influence the amount of NO released by NO-IND-OXDs

–p and experimental conditions

(

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but it was strongly influenced by experimental conditions (pH and GSH presence) and

the structure of tested compounds.

Figure 6. The percentage (%) of NO released by NO-IND-OXDs 8a–p 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).

Figure 7. The estimated marginal means of NO (%) release by NO-IND-OXDs (8a–p) compared to reference NO donors

(NTG, SNP, SNAP) (* signiﬁcantly more NO released than SNP, p < 0.05).

By estimated marginal mean of NO released it was shown that all of the tested com-

pounds 8a–p released more NO compared with NTG (Figure 7). Using one-way ANOVA

test it was determined that there is a statistically signiﬁcant difference (F(16, 255) = 33.054

p < 0.05) between tested compounds. Post hoc comparisons using the Tukey HSD test

revealed that the tested compounds 8h 8n 8c 8i 8m and 8o, respectively, released sig-

niﬁcantly more NO than SNP (p < 0.05). It was noted that the amount of NO released is

,

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inﬂuenced by the position of nitrate ester moiety on aromatic ring and by the number and

type of the substituents on aromatic ring. The most proper positions of nitrate ester moiety

are meta and ortho, 8i (3-oxy-ethylnitrate) and 8n (2-oxy-ethylnitrate) being more active

than 8a (4-oxy-ethylnitrate). In addition, the elongation of distance between oxadiazole

moiety and aromatic ring from methylene group (8a) to ethylene group (8p) increase the

percentage of NO released.

It was reported that NO, at low level (less than 300 nM) exerts beneﬁcial effects (e.g.,

anti-inﬂammatory, antioxidant, wound healing effects) while at high level (1000–3000 nM)

it has pro-inﬂammatory effects in pathological conditions [40,95]. The NO-IND-OXDs

could act as new drugs because of their balanced inhibition of COX enzymes and their

ability to release NO at a submicromolar level, which was proven to be beneﬁcial for

inﬂammation conditions.

3. Materials and Methods

3.1. Chemistry

3.1.1. General Information

All chemicals used in this research were of analytical grade or HPLC p.a. quality,

certiﬁed by the commercially available sources and were used without further puriﬁcation

unless otherwise speciﬁed. The anhydrous solvents were distilled or dried in accordance

with standard procedures before use: dichloromethane was distilled under P₂O₅and

acetonitrile was dried on cartridge by a GT S100 station. The reaction progress was

monitored by thin layer chromatography (TLC), using 2

×

5 cm precoated UV ﬂuorescent

silicagel aluminum plate (type Merck 60 F254) and UV lamp exposure (254 nm). The

synthesized compounds were puriﬁed by ﬂash column chromatography on silicagel 60

(0.063

0.200 mm, Merck), using appropriate solvents. 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 (

respectively. The chemical shifts were referenced to tetramethylsilane (TMS,

C = 0) in CDCl₃and DMSO-d₆. The standard abbreviation for signals is as follows:

δ

H,

δ

C) and coupling constant values (J) were given in ppm and Hz,

δ

H = 0,

δ

singlet (s), doublet (d), doublet of doublets (dd), doublet of doublets of doublets (ddd),

triplet (t), triplet of doublets (dt), quartet (q), hexuplet (h), multiplet (m). For detailed

peak assignments, 2D NMR analysis: gradient-selected correlation spectroscopy (gCOSY),

gradient-selected heteronuclear multiple bond coherence (gHMBC) and gradient-selected

heteronuclear multiple quantum coherence (gHMQC) were used. High-resolution mass

spectrometry (HRMS) was recorded on a Bruker maXis mass spectrometer within the

“Research Federation” platform between ICOA and CBM (FR2708). The samples were

ionized by electrospray ion source (ESI), using N₂for nebulization (pressure of 0.6 bar) and

drying (ﬂow of 7 L/min, temperature of 200 ^◦C). The capillary potential was set at 4500 V

and collision cell RF at 1800.0 Vpp. The ion polarities were positive and were analyzed

using scanning range between 50–2500 m/z. Melting points (m.p.) were measured on

a Koﬂer heating bench. A microplate reader (Tecan Sunrise Remote Microplate Reader

TW/ML-Abbott F039306) and Hanna Model Pack pH 21 digital pHmeter were also used.

3.1.2. General Procedure for the Synthesis of the (bromoethoxy)benzaldehyde

Derivatives (2a–o)

The intermediates 2a–o were obtained by reaction of hydroxybenzaldehydes 1a–o

(1.0 Eq.) in acetonitrile (150 mL for 15 mmol scale) with 1,2-dibromoethane (10.0 Eq.) in

the presence of potassium carbonate (2.0 Eq.), according to the method reported in the

literature [96–98], which was reported in our previous paper [99].

3.1.3. General Procedure for the Synthesis of the (bromoethoxy)aromatic Alcohol

Derivatives (3a–p)

To a solution of appropriate (bromoethoxy)benzaldehydes 2a–o (1_◦.0 Eq.), in dry THF

(30 mL for 10 mmol scale), sodium borohydride (1.1 Eq.) was added at 0 C and the mixture

Int. J. Mol. Sci. 2021, 22, 7079

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was stirred at room temperature for 12–24 h, according to the method reported in the

literature [100–103]. Then, the excess of sodium borohydride was quenched by addition

of 1M HCl and extracted twice with EtOAc (30 mL). The combined organic layers were

washed with water and brine, dried over anhydrous magnesium sulfate, ﬁltered and

concentrated under reduced pressure. To obtain 2-(4-(2-bromoethoxy)phenyl)ethanol (3p),

4-(2-hydroxyethyl)phenol (2p) (1.0 Eq.) was reacted with 1,2-dibromoethane (10.0 Eq.)

in CH₃CN (50 mL for 15 mmol scale) in the presence of potassium carbonate (2.0 Eq.).

The resulting mixture was heated to reﬂux for 20 h. After cooling to room temperature,

distilled water (100 mL) was added and the crude product was extracted with diethyl ether

(3 × 50 mL).

The combined organic layers were washed with distilled water (50 mL), brine (50 mL)

and dried over anhydrous magnesium sulfate. The crude products were puriﬁed by ﬂash

column chromatography on silicagel with petroleum ether/EtOAc or CH₂Cl₂/CH₃OH to

give the pure compounds 3a–p.

◦

(4-(2-Bromoethoxy)phenyl)methanol (3a): white solid, yield 95%, m.p. 73–75 C; ¹H NMR

(250 MHz, DMSO-d₆)

δ = 3.68–3.80 (m, 2H), 4.18–4.30 (m, 2H), 4.37 (d, J = 5.7 Hz, 2H), 5.01

(t, J = 5.7 Hz, 1H), 6.81–6.93 (m, 2H), 7.13–7.25 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 31.5 (CH₂), 62.5 (CH₂OH), 67.7 (CH₂), 114.3 (2CH_Ar), 127.9 (2CH_Ar), 135.1 (C_q), 156.8

(C_q); HRMS (ESI-MS) m/z calcd for C₉H₁₁BrNaO₂[M+Na]⁺: 252.9840, found: 252.9835; Rf

(CH₂Cl₂/CH₃OH = 9.8/0.2) 0.53, according to [104].

(4-(2-Bromoethoxy)-3-ﬂuorophenyl)methanol (3b): pale yellow oil, yield 93%; ¹H NMR

(250 MHz, DMSO-d₆)

δ = 3.76–3.84 (m, 2H), 4.32–4.40 (m, 2H), 4.42 (dd, J = 5.7 Hz, 0.7 Hz,

2H), 5.19 (t, J = 5.7 Hz, 1H), 7.00–7.22 (m, 3H); ¹³C NMR (101 MHz, DMSO-d₆) δ = 31.2

(CH₂), 61.9 (CH₂OH), 69.0 (CH₂), 114.2 (d, J = 18.2 Hz, CH_Ar), 115.3 (d, J = 1.9 Hz, CH_Ar),

122.4 (d, J = 3.4 Hz, CH_Ar), 136.6 (d, J = 5.6 Hz, C_q), 144.3 (d, J = 10.9 Hz, C_q), 151.6 (d,

J = 243.9 Hz, C_qF); ¹⁹F NMR (376 MHz, DMSO-d₆)

δ = -134.7 (dd, J = 12.5 Hz, 8.6 Hz);

HRMS (ESI-MS) m/z calcd for C₉H₁₀BrFNaO₂[M+Na]⁺: 270.9746, found: 270.9740; Rf

(CH₂Cl₂/CH₃OH = 9.8/0.2) 0.61.

(4-(2-Bromoethoxy)-3-chlorophenyl)methanol (3c): pale yellow oil, yield 97%; ¹H NMR

(400 MHz, DMSO-d₆)

5.19 (t, J = 5.8 Hz, 1H), 7.11 (d, J = 8.4 Hz, 1H), 7.22 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.37 (d,

J = 2.1 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 31.0 (CH₂), 61.8 (CH₂OH), 68.8 (CH₂),

δ = 3.75–3.83 (m, 2H), 4.35–4.40 (m, 2H), 4.42 (d, J = 5.8 Hz, 2H),

δ

114.1 (CH_Ar), 121.2 (C_q), 126.3 (CH_Ar), 128.1 (CH_Ar), 136.6 (C_q), 152.0 (C_q). HRMS (ESI-

MS) m/z calcd for C₉H₁₀BrClNaO₂[M+Na]⁺: 286.9450, found: 286.9835; Rf (petroleum

ether/EtOAc = 5/5) 0.58.

(4-(2-Bromoethoxy)-3,5-dichlorophenyl)methanol (3d): white solid, yield 96%, m.p.

65–67 ^◦C; ¹H NMR (250 MHz, DMSO-d₆)

δ = 3.72–3.86 (m, 2H), 4.18–4.34 (m, 2H), 4.46

(d, J = 5.8 Hz, 2H), 5.40 (t, J = 5.8 Hz, 1H), 7.40 (s, 2H); ¹³C NMR (101 MHz, DMSO-

d₆) δ = 31.2 (CH₂), 61.3 (CH₂OH), 72.9 (CH₂), 126.8 (2CH_Ar), 128.0 (C_q), 141.3 (C_q),

148.5 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₉BrCl₂NaO₂[M+Na]⁺: 320.9061, found:

320.9055; Rf (CH₂Cl₂/CH₃OH = 9.8/0.2) 0.64.

(4-(2-Bromoethoxy)-3-nitrophenyl)methanol (3e): brown oil, yield 86%; ¹H NMR (400 MHz,

DMSO-d₆)

δ = 3.72–3.84 (m, 2H), 4.41–4.56 (m, 4H), 5.35 (t, J = 5.7 Hz, 1H), 7.34 (d, J = 8.7 Hz,

1H), 7.57 (dd, J = 8.6 Hz, 2.1 Hz, 1H), 7.79 (d, J =2.1 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 30.7 (CH₂), 61.4 (CH₂OH), 69.4 (CH₂), 115.4 (CH_Ar), 122.6 (CH_Ar), 132.2 (CH_Ar), 135.9

(C_q), 139.4 (C_q), 149.3 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₁₁BrNO₄[M+H]⁺: 275.9871,

found: 275.9865; Rf (petroleum ether/EtOAc = 4/6) 0.56.

1

(4-(2-Bromoethoxy)-3-methoxyphenyl)methanol (3f): white solid, yield 91%; H NMR

(250 MHz, DMSO-d₆)

2H), 5.08 (t, J = 5.7 Hz, 1H), 6.81 (ddt, J = 8.1 Hz, 1.9 Hz, 0.72, 1H), 6.92 (d, J = 8.1 Hz, 1H),

6.95 (d, J = 1.9 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 31.5 (CH₂), 55.5 (CH₃), 62.7

δ = 3.70–3.82 (m, 2H), 3.77 (s, 3H), 4.26 (m, 2H), 4.42 (d, J = 5.7 Hz,

δ

(CH₂OH), 69.0 (CH₂), 111.1 (CH_Ar), 114.2 (CH_Ar), 118.6 (CH_Ar), 136.3 (C_q), 146.0 (C_q), 149.1

(C_q);. HRMS (ESI-MS) m/z calcd for C₁₀H₁₃BrNaO₃[M+Na]⁺: 282.9946, found: 282.9940;

Rf (petroleum ether/EtOAc = 3/7) 0.53, according to [105,106].

Int. J. Mol. Sci. 2021, 22, 7079

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◦

(4-(2-Bromoethoxy)-3-ethoxyphenyl)methanol (3g): white solid, yield 93%, m.p. 53–55 C;

¹H NMR (250 MHz, DMSO-d₆)

δ

= 1.32 (t, J = 6.9 Hz, 3H), 3.75 (dd, J = 6.0 Hz, 5.2 Hz, 2H),

4.03 (q, J = 6.9 Hz, 2H), 4.27 (t, J = 6.0 Hz, 5.22 Hz, 2H), 4.41 (d, J = 5.7 Hz, 0.6 Hz, 2H),

5.07 (t, J = 5.7 Hz, 1H), 6.81 (ddt, J = 8.0 Hz, 1.8 Hz, 0.6 Hz, 1H), 6.89–6.96 (m, 2H); ¹³

NMR (101 MHz, DMSO-d₆) = 14.8 (CH₃), 31.5 (CH₂), 62.6 (CH₂OH), 64.0 (CH₂), 69.2

C

δ

(CH₂), 112.8 (CH_Ar), 115.1 (CH_Ar), 118.8 (CH_Ar), 136.5 (C_q), 146.4 (C_q), 148.5 (C_q); HRMS

(ESI-MS) m/z calcd for C₁₁H₁₅BrNaO₃[M+Na]⁺: 297.0102, found: 297.0096; Rf (petroleum

ether/EtOAc = 3/7) 0.55, according to [107].

(4-(2-Bromoethoxy)-3,5-dimethoxyphenyl)methanol (3h): white solid, yield 96%, m.p.

1

71–73 ^◦C; H NMR (400 MHz, DMSO-d₆)

δ = 3.64 (td, J = 6.2 Hz, 1.3 Hz, 2H), 3.77 (d,

J = 1.3 Hz, 6H), 4.11 (td, J = 6.2 Hz, 1.3 Hz, 2H), 4.44 (d, J = 5.6 Hz, 2H), 5.16 (td, J = 5.7 Hz,

1.3 Hz, 1H), 6.64 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 31.6 (CH₂), 55.9 (2CH₃),

62.9 (CH₂OH), 72.3 (CH₂), 103.4 (2CH_Ar), 134.4 (C_q), 138.7 (C_q), 152.6 (2C_q); HRMS (ESI-

MS) m/z calcd for C₁₁H₁₅BrNaO₄[M+H]⁺: 291.0232, found: 291.0228; Rf (petroleum

ether/EtOAc = 2/8) 0.50, according to [108].

(3-(2-Bromoethoxy)phenyl)methanol (3i): colorless oil, yield 90%; ¹H NMR (250 MHz,

DMSO-d₆)

1H), 6.76–6.86 (m, 1H), 6.86–6.96 (m, 2H), 7.24 (t, J = 8.0 Hz, 1H); ¹³C NMR (101 MHz,

DMSO-d₆) = 31.5 (CH₂), 62.7 (CH₂OH), 67.6 (CH₂), 112.5 (CH_Ar), 112.8 (CH_Ar), 119.0

δ = 3.70–3.87 (m, 2H), 4.16–4.34 (m, 2H), 4.48 (d, J = 5.8 Hz, 2H), 5.17 (t, J = 5.8 Hz,

δ

(CH_Ar), 129.2 (CH_Ar), 144.4 (C_q), 157.9 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₁₁BrNaO₂

[M+Na]⁺: 252.9840, found: 252.9834; Rf (petroleum ether/EtOAc = 5/5) 0.58, according

to [109].

1

(3-(2-Bromoethoxy)-4-methoxyphenyl)methanol (3j): colorless oil, yield 91%; H NMR

(250 MHz, DMSO-d₆)

4.40 (d, J = 5.7 Hz, 2H), 5.05 (t, J = 5.7 Hz, 1H), 6.84–6.90 (m, 1H), 6.91 (s, 1H), 6.92–6.96

(m, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 31.4 (CH₂), 55.7 (CH₃), 62.6 (CH₂OH), 68.7

δ = 3.75 (s, 3H), 3.63–3.90 (m, 2H), 4.27 (dd, J = 6.1 Hz, 5.1 Hz, 2H),

δ

(CH₂), 112.2 (CH_Ar), 112.9 (CH_Ar), 119.6 (CH_Ar), 135.2 (C_q), 147.1 (C_q), 148.0 (C_q); HRMS

(ESI-MS) m/z calcd for C₁₀H₁₃BrNaO₃[M+Na]⁺: 282.9946, found: 282.9937; Rf (petroleum

ether/EtOAc = 3/7) 0.64.

(3-(2-Bromoethoxy)-4-nitrophenyl)methanol (3k): greenish yellow oil, yield 97%; ¹H NMR

(250 MHz, DMSO-d₆)

5.48 (t, J = 5.7 Hz, 1H), 7.09 (ddt, J = 8.3 Hz, 1.5 Hz, 0.8 Hz, 1H), 7.23–7.34 (m, 1H), 7.86

(d, J = 8.3 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 30.7 (CH₂), 62.0 (CH₂OH), 69.2

δ = 3.75–3.86 (m, 2H), 4.44–4.53 (m, 2H), 4.57 (d, J = 5.7 Hz, 2H),

δ

(CH₂), 112.6 (CH_Ar), 118.4 (CH_Ar), 125.1 (CH_Ar), 138.0 (C_q), 150.3 (C_q), 150.7 (C_q); HRMS

(ESI-MS) m/z calcd for C₉H₁₁BrNO₄[M+H]⁺: 275.9871, found: 275.9866; Rf (petroleum

ether/EtOAc = 3/7) 0.54.

(3-(2-Bromoethoxy)-2-chloro-4-methoxyphenyl)methanol (3l): white solid, yield 99%, m.p.

76–78 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ

= 3.73 (t, J = 5.8 Hz, 2H), 3.82 (s, 3H), 4.24 (t,

J = 5.8 Hz, 2H), 4.48 (d, J = 5.0 Hz, 2H), 5.24 (t, J = 5.6 Hz, 1H), 7.05 (d, J = 8.6 Hz, 1H), 7.23

(t, J = 8.5 Hz, 0.9 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 31.5 (CH₂), 56.1 (CH₃), 60.2

δ

(CH₂OH), 72.4 (CH₂), 111.2 (CH_Ar), 123.1 (CH_Ar), 125.2 (C_q), 132.3 (C_q), 142.9 (C_q), 151.9

(C_q); HRMS (ESI-MS) m/z calcd for C₁₀H₁₂BrClNaO₃[M+Na]⁺: 316.9556, found: 316.9552;

Rf (petroleum ether/EtOAc = 3/7) 0.66.

(2-Bromo-3-(2-bromoethoxy)-4-methoxyphenyl)methanol (3m): white solid, yield 99%, m.p.

68–70^◦C; ¹H NMR (250 MHz, DMSO-d₆)

δ = 3.75 (dd, J = 6.2 Hz, 5.6 Hz, 2H), 3.82 (s, 3H),

4.23 (dd, J = 6.2 Hz, 5.6 Hz, 2H), 4.45 (dd, J = 5.6 Hz, 0.9 Hz, 2H), 5.29 (t, J = 5.6 Hz, 1H),

7.10 (d, J = 8.6 Hz, 1H), 7.24 (dt, J = 8.5 Hz, 0.8 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 31.5 (CH₂), 56.2 (CH₃), 62.5 (CH₂OH), 72.3 (CH₂), 111.8 (CH_Ar), 116.4 (C_q), 123.3 (CH_Ar),

133.7 (C_q), 143.9 (C_q), 151.7 (C_q); HRMS (ESI-MS) m/z calcd for C₁₀H₁₂Br₂NaO₃[M+Na]⁺:

360.9051, found: 360.9039; Rf (petroleum ether/EtOAc = 3/7) 0.63.

(2-(2-Bromoethoxy)phenyl)methanol (3n): pale yellow oil, yield 88%; ¹H NMR (250 MHz,

DMSO-d₆)

(t, J = 5.6 Hz, 1H), 6.88–7.02 (m, 2H), 7.20 (dddt, J = 8.1 Hz, 7.5 Hz, 1.8 Hz, 0.6 Hz, 1H),

7.40 (dddd, J = 7.4 Hz, 1.9 Hz, 1.0 Hz, 0.6 Hz, 1H; ¹³C NMR (101 MHz, DMSO-d₆)

= 31.8

δ = 3.72–3.87 (m, 2H), 4.24–4.38 (m, 2H), 4.56 (dd, J = 5.5 Hz, 0.8 Hz, 2H), 4.97

δ

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(CH₂), 57.7 (CH₂OH), 67.9 (CH₂), 111.5 (CH_Ar), 120.7 (CH_Ar), 126.9 (CH_Ar), 127.5 (CH_Ar),

130.9 (C_q), 154.5 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₁₁BrNaO₂[M+Na]⁺: 252.9840,

found: 252.9834; Rf (petroleum ether/EtOAc = 5/5) 0.49.

(5-Bromo-2-(2-bromoethoxy)phenyl)methanol (3o): pale yellow oil, yield 89%; ¹H NMR

(250 MHz, DMSO-d₆)

2H), 5.19 (t, J = 5.6 Hz, 1H), 6.93 (d, J = 8.6 Hz, 1H), 7.36 (ddt, J = 8.6 Hz, 2.6 Hz, 0.7 Hz,

1H), 7.50 (dt, J = 2.6 Hz, 0.9 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 31.5 (CH₂), 57.3

δ = 3.74–3.84 (m, 2H), 4.26–4.36 (m, 2H), 4.53 (dt, J = 5.67 Hz, 0.85 Hz,

δ

(CH₂OH), 68.1 (CH₂), 112.5 (C_q), 113.8 (CH_Ar), 129.1 (CH_Ar), 129.8 (CH_Ar), 133.7 (C_q), 153.6

(C_q); HRMS (ESI-MS) m/z calcd for C₉H₁₀Br₂NaO₂[M+Na]⁺: 330.8945, found: 330.8953;

Rf (petroleum ether/EtOAc = 5/5) 0.47

2-(4-(2-Bromoethoxy)phenyl)ethanol (3p): white needle crystals, yield82%, m.p. 65–68 ^◦C

;

¹H NMR (250 MHz, DMSO-d₆)

3.72–3.83 (m, 2H), 4.21–4.33 (m, 2H), 4.58 (t, J = 5.2 Hz, 1H), 6.79–6.93 (m, 2H), 7.06–7.23

(m, 2H); ¹³C NMR (101 MHz, DMSO-d₆)

= 31.5 (CH₂), 38.1 (CH₂), 62.4 (CH₂OH), 67.7

δ = 2.65 (t, J =7.1 Hz, 2H), 3.55 (td, J = 7.1 Hz, 5.2 Hz, 2H),

δ

(CH₂), 114.4 (2CH_Ar), 129.8 (2CH_Ar), 132.0 (C_q), 156.2 (C_q); HRMS (ESI-MS) m/z calcd for

C₁₀H₁₄BrO₂[M+H]⁺: 245.0177, found: 245.0171; Rf (petroleum ether/EtOAc = 3/7) 0.60.

3.1.4. General Procedure for the Synthesis of the (hydroxyalkyl)phenoxy Nitrates (4a–p)

To a solution of the appropriate (bromoethoxy)aromatic alcohol derivatives 3a–p

(1.0 Eq.), in acetonitrile (50 mL for 10 mmol scale), silver nitrate (1.5 Eq.) was added. The

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

methods reported in the literature [110–117], which were adapted to our synthesis in terms

of the ratio of reagents, solvent, time of reaction, puriﬁcation method. At the end of the

reaction, brine was added to precipitate the excess of silver nitrate. After ﬁltration through

celite, the mixture was extracted with diethyl ether (2

×

50 mL). The combined organic

layers were washed with distilled 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/EtOAc) to obtain the

pure products 4a–p.

2-(4-(Hydroxymethyl)phenoxy)ethyl nitrate (4a): white solid, yield 97%, m.p. 55–57 ^◦C;

¹H NMR (250 MHz, DMSO-d₆)

δ

= 4.23–4.34 (m, 2H), 4.42 (d, J = 5.6 Hz, 2H), 4.81–4.91

(m, 2H), 5.05 (t, J = 5.6 Hz, 1H), 6.79–6.98 (m, 2H), 7.15–7.31 (m, 2H); ¹³C NMR (101 MHz,

DMSO-d₆) δ = 62.5 (CH₂OH), 64.0 (CH₂), 72.1 (CH₂), 114.1 (2CH_Ar), 127.9 (2CH_Ar), 135.2

(C_q), 156.7 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₁₁NNaO₅[M+Na]⁺: 236.0535, found:

236.0526; Rf (petroleum ether/EtOAc = 6/4) 0.62.

2-(2-Fluoro-4-(hydroxymethyl)phenoxy)ethyl nitrate (4b): greenish yellow oil, yield 95%;

¹H NMR (400 MHz, DMSO-d₆)

δ

= 4.33–4.39 (m, 2H), 4.42 (d, J = 5.6 Hz, 2H), 4.84–4.94

(m, 2H), 5.19 (t, J = 5.7Hz, 1H), 7.03–7.09 (m, 1H), 7.11–7.18 (m, 2H); ¹³C NMR (101 MHz,

DMSO-d₆) = 61.9 (CH₂OH), 65.4 (CH₂), 71.9 (CH₂), 114.2 (d, J = 18.0 Hz, CH_Ar), 115.1

(d, J = 1.8 Hz, CH_Ar), 122.4 (d, J = 3.5 Hz, CH_Ar), 136.7 (d, J = 5.5 Hz, C_q), 144.2 (d,

δ

J = 10.7 Hz, C_q), 151.5 (d, J = 243.9 Hz, C_qF); ¹⁹F NMR (376 MHz, DMSO-d₆)

δ

=

−

134.94

(dd, J = 12.1 Hz, 8.3 Hz); HR.MS (ESI-MS) m/z calcd for C₉H₁₀FNNaO₅[M+Na]⁺: 254.0441,

found: 254.0436; Rf (CH₂Cl₂/CH₃OH = 9.8/0.2) 0.66.

2-(2-Chloro-4-(hydroxymethyl)phenoxy)ethyl nitrate (4c): orange oil, yield 97%; ¹H NMR

(400 MHz, DMSO-d₆)

(t, J = 5.8 Hz, 1H), 7.14 (d, J = 8.4 Hz, 1H), 7.23 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.36 (d, J = 2.0 Hz,

1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 61.8 (CH₂OH), 65.4 (CH₂), 71.7 (CH₂), 114.0 (CH_Ar),

δ = 4.33–4.39 (m, 2H), 4.42 (d, J = 5.8 Hz, 2H), 4.85–4.95 (m, 2H), 5.20

δ

121.2 (C_q), 126.3 (CH_Ar), 128.1 (CH_Ar), 136.7 (C_q), 151.9 (C_q);HRMS (ESI-MS) m/z calcd for

C₉H₁₀ClNNaO₅[M+Na]⁺: 270.0145, found: 270.0140; Rf (CH₂Cl₂/CH₃OH = 9.8/0.2) 0.63.

2-(2,6-Dichloro-4-(hydroxymethyl)phenoxy)ethyl nitrate (4d): greenish yellow oil, yield

1

99%; H NMR (250 MHz, DMSO-d₆)

δ = 4.20–4.34 (m, 2H), 4.46 (dt, J = 5.8 Hz, 0.8 Hz,

2H), 4.78–4.94 (m, 2H), 5.40 (t, J = 5.8 Hz, 1H), 7.41 (t, J = 0.8 Hz 2H); ¹³C NMR (101 MHz,

DMSO-d₆) = 61.2 (CH₂OH), 69.4 (CH₂), 72.5 (CH₂), 126.8 (2CH_Ar), 127.9 (2C_q), 141.4

δ

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(C_q), 148.4 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₉Cl₂NNaO₅[M+Na]⁺: 303.9755, found:

303.9747; Rf (CH₂Cl₂/CH₃OH = 9.8/0.2) 0.53.

2-(4-(Hydroxymethyl)-2-nitrophenoxy)ethyl nitrate (4e): brown greenish yellow solid,

yield 95%, m.p. 56–58 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ

= 4.44–4.53 (m, 4H), 4.86–4.93

(m, 2H), 5.36 (t, J = 5.7 Hz, 1H), 7.36 (d, J = 8.6 Hz, 1H), 7.59 (dd, J = 8.6 Hz, 2.1 Hz, 1H),

7.80 (d, J = 2.1 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 61.4 (CH₂OH), 66.1 (CH₂),

δ

71.4 (CH₂), 115.4 (CH_Ar), 122.7 (CH_Ar), 132.3 (CH_Ar), 136.0 (C_q), 139.3 (C_q), 149.3 (C_q);

HRMS (ESI-MS) m/z calcd for C₉H₁₀N₂NaO₇[M+Na]⁺: 281.0386, found: 281.0381; Rf

(CH₂Cl₂/CH₃OH = 9.8/0.2) 0.52.

2-_◦(4-(hydroxymethyl)-2-methoxyphenoxy)ethyl nitrate (4f): brown solid, yield 99%, m.p.

1

51–52 C; H NMR (250 MHz, DMSO-d₆)

δ = 3.75 (s, 3H), 4.17–4.31 (m, 2H), 4.42 (d,

J = 5.5 Hz, 2H), 4.78–4.91 (m, 2H), 5.09 (t, J = 5.6 Hz, 1H), 6.81 (ddt, J = 8.0 Hz, 1.9 Hz, 0.7 Hz,

1H), 6.87–7.00 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 55.4 (CH₃), 62.7 (CH₂OH), 65.2

(CH₂), 72.2 (CH₂), 110.9 (CH_Ar), 114.0 (CH_Ar), 118.5 (CH_Ar), 136.4 (C_q), 146.0 (C_q), 149.0

(C_q); HRMS (ESI-MS) m/z calcd for C₁₀H₁₃NNaO₆[M+Na]⁺: 266.0641, found: 266.0632;

Rf (petroleum ether/EtOAc = 6/4) 0.55.

2-(2-ethoxy-4-_◦(hydroxymethyl)phenoxy)ethyl nitrate (4g): greenish yellow solid, yield

1

99%, m.p. 57–59 C; H NMR (250 MHz, DMSO-d₆)

δ

= 1.31 (t, J = 6.98 Hz, 3H), 4.01

(q, J = 6.96 Hz, 2H), 4.17–4.29 (m, 2H), 4.41 (d, J = 5.1 Hz, 2H), 4.76–4.91 (m, 2H), 5.08 (t,

J = 5.6 Hz, 1H), 6.81 (ddt, J = 8.1 Hz, 1.9 Hz, 0.7 Hz, 1H), 6.94 (d, J = 8.1 Hz, 1H), 6.94

(d, J = 1.9 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 14.7 (CH₃), 62.7 (CH₂OH), 63.8

(CH₂), 65.7 (CH₂), 72.2 (CH₂), 112.4 (CH_Ar), 114.9 (CH_Ar), 118.7 (CH_Ar), 136.6 (C_q), 146.3

(C_q), 148.5 (C_q); HRMS (ESI-MS) m/z calcd for C₁₁H₁₅NNaO₆[M+Na]⁺: 280.0797, found:

280.0794; Rf (petroleum ether/EtOAc = 6/4) 0.48.

2-(4-(Hydroxymethyl)-2,6-dimethoxyphenoxy)ethyl nitrate (4h): greenish yellow solid,

yield 99%, m.p. 56–58 ^◦C; ¹H NMR (250 MHz, DMSO-d₆)

δ

= 3.74 (s, 6H), 4.04–4.15 (m,

2H), 4.43 (dt, J = 5.8 Hz, 0.6 Hz, 2H), 4.67–4.78 (m, 2H), 5.17 (t, J = 5.8 Hz, 1H), 6.63 (d,

J = 0.6 Hz, 2H);¹³C NMR (101 MHz, DMSO-d₆)

= 55.8 (2CH₃), 62.9 (CH₂OH), 68.4 (CH₂),

δ

72.9 (CH₂), 103.3 (2CH_Ar), 134.2 (C_q), 138.9 (C_q), 152.6 (2C_q); HRMS (ESI-MS) m/z calcd for

C₁₁H₁₆NO₇[M+H]⁺: 274.0927, found: 274.0922; Rf (petroleum ether/EtOAc = 7/3) 0.43.

1

2-(3-(Hydroxymethyl)phenoxy)ethyl nitrate (4i): colorless oil, yield 89%; H NMR

(250 MHz, DMSO-d₆)

5.17 (t, J = 5.8 Hz, 1H), 6.76–6.86 (m, 1H), 6.86–6.96 (m, 2H), 7.24 (t, J =8.0 Hz, 1H); ¹³

NMR (101 MHz, DMSO-d₆) = 62.8 (CH₂OH), 67.6 (CH₂), 72.1 (CH₂),112.5 (CH_Ar), 112.8

δ = 4.16–4.34 (m, 2H), 4.48 (d, J = 5.8 Hz, 2H), 4.81–4.90 (m, 2H),

C

δ

(CH_Ar), 119.0 (CH_Ar), 129.2 (CH_Ar), 144.4 (C_q), 157.9 (C_q); HRMS (ESI-MS) m/z calcd for

C₉H₁₁NNaO₅[M+Na]⁺: 236.0535, found: 236.0529; Rf (CH₂Cl₂/CH₃OH = 9.6/0.4) 0.39.

2-_◦(5-(Hydroxymethyl)-2-methoxyphenoxy)ethyl nitrate (4j): yellow solid, yield 80%, m.p.

1

51–53 C; H NMR (250 MHz, DMSO-d₆)

δ = 3.74 (s, 3H), 4.21–4.30 (m, 2H), 4.41 (d,

J = 5.7 Hz, 2H), 4.81–4.90 (m, 2H), 5.06 (t, J = 5.7 Hz, 1H), 6.82–6.98 (m, 3H); ¹³C NMR

(101 MHz, DMSO-d₆)

δ

= 55.6 (CH₃), 62.6 (CH₂OH), 65.0 (CH₂), 72.1 (CH₂), 112.0 (CH_Ar),

112.8 (CH_Ar), 119.7 (CH_Ar), 135.1 (C_q), 147.0 (C_q), 148.0 (C_q); HRMS (ESI-MS) m/z calcd for

C₁₀H₁₃NNaO₆[M+Na]⁺: 266.0641, found: 266.0635; Rf (petroleum ether/EtOAc = 7/3) 0.63.

2-(5-(Hydroxymethyl)-2-nitrophenoxy)ethyl nitrate (4k): brown oil, yield 94%; ¹H NMR

(250 MHz, DMSO-d₆)

5.49 (t, J = 5.7 Hz, 1H), 7.10 (ddt, J = 8.3 Hz, 1.5 Hz, 0.7 Hz, 1H), 7.28–7.33 (m, 1H),

7.87 (d, J = 8.3 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 62.0 (CH₂OH), 65.9 (CH₂),

δ = 4.43–4.53 (m, 2H), 4.58 (d, J = 5.7 Hz, 2H), 4.85–4.96 (m, 2H),

δ

71.3 (CH₂), 112.5 (CH_Ar), 118.5 (CH_Ar), 125.1 (CH_Ar), 137.9 (C_q), 150.4 (C_q), 150.7 (C_q);

HRMS (ESI-MS) m/z calcd for C₉H₁₀N₂NaO₇[M+Na]⁺: 281.0386, found: 281.0380; Rf

(CH₂Cl₂/CH₃OH = 9.6/0.4) 0.53.

2-(2-Chloro-3-(hydroxymethyl)-6-methoxyphenoxy)ethyl nitrate (4l): white solid, yield 91%,

m.p. 57–59 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ = 3.80 (s, 3H), 4.15–4.29 (m, 2H), 4.48 (dd,

J = 5.6 Hz, 0.8 Hz, 2H), 4.75–4.90 (m, 2H), 5.25 (t, J = 5.6 Hz, 1H), 7.06 (d, J = 8.6 Hz, 1H), 7.24

(dt, J = 8.7 Hz, 0.9 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆) δ = 56.0 (CH₃), 60.2 (CH₂OH),

68.7 (CH₂), 72.7 (CH₂), 111.1 (CH_Ar), 123.2 (CH_Ar), 125.2 (C_q), 132.3 (C_q), 142.8 (C_q), 151.9

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(C_q); HRMS (ESI-MS) m/z calcd for C₁₀H₁₂ClNNaO₆[M+Na]⁺: 300.0251, found: 300.0246;

Rf (CH₂Cl₂/CH₃OH = 9.6/0.4) 0.55.

2-(2-Bromo-3-(hydroxymethyl)-6-methoxyphenoxy)ethyl nitrate (4m): white solid, yield

◦

92%, m.p. 58–60 C; ¹H NMR (400 MHz, DMSO-d₆)

δ

= 3.81 (s, 3H), 4.16–4.25 (m, 2H), 4.44

(dd, J = 5.6 Hz, 0.8 Hz, 2H), 4.76–4.87 (m, 2H), 5.29 (t, J = 5.6 Hz, 1H), 7.10 (d, J = 8.5 Hz,

1H), 7.25 (dt, J = 8.5 Hz, 0.8 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 56.0 (CH₃), 62.5

δ

(CH₂OH), 68.6 (CH₂), 72.7 (CH₂), 111.8 (CH_Ar), 116.4 (C_q), 123.4 (CH_Ar), 133.7 (C_q), 143.8

(C_q), 151.7 (C_q); HRMS (ESI-MS) m/z calcd for C₁₀H₁₂BrNNaO₆[M+Na]⁺: 343.9746, found:

343.9737; Rf (CH₂Cl₂/CH₃OH = 9.6/0.4) 0.56.

2-(2-(Hydroxymethyl)phenoxy)ethyl nitrate (4n): orange oil, yield 93%; ¹H NMR (400 MHz,

DMSO-d₆)

1H), 6.97 (t, J = 7.4 Hz, 2H), 7.21 (td, J = 7.8 Hz, 1.8 Hz, 1H), 7.39 (dd, J = 7.1 Hz, 1.2 Hz, 1H);

¹³C NMR (101 MHz, DMSO-d₆)

= 57.6 (CH₂OH), 64.4 (CH₂), 72.0 (CH₂), 111.4 (CH_Ar), 120.8

δ = 4.25–4.31 (m, 2H), 4.47 (d, J = 5.6 Hz, 2H), 4.86–4.92 (m, 2H), 4.98 (t, J = 5.6 Hz,

δ

(CH_Ar), 127.0 (CH_Ar), 127.5 (CH_Ar), 130.7 (C_q), 154.5 (C_q); HRMS (ESI-MS) m/z calcd for

C₉H₁₁NNaO₅[M+Na]⁺: 236.0535, found: 236.0530; Rf (CH₂Cl₂/CH₃OH = 9.6/0.4) 0.62.

2-(4-Bromo-2-(hydroxymethyl)phenoxy)ethyl nitrate (4o): greenish yellow oil, yield 95%;

¹H NMR (250 MHz, DMSO-d₆)

δ

= 4.26–4.34 (m, 2H), 4.45 (dt, J = 5.6 Hz, 0.8 Hz, 2H),

4.85–4.92 (m, 2H), 5.19 (t, J = 5.6 Hz, 1H), 6.95 (d, J = 8.7 Hz, 1H), 7.38 (ddt, J = 8.7 Hz,

2.6 Hz, 0.7 Hz, 1H), 7.49 (dt, J = 2.6 Hz, 0.9 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 57.2

δ

(CH₂OH), 64.8 (CH₂), 71.8 (CH₂), 112.6 (C_q), 113.7 (CH_Ar), 129.2 (CH_Ar), 129.9 (CH_Ar),

133.6 (C_q), 153.7 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₁₀BrNNaO₅[M+Na]⁺: 313.9640,

found: 313.9634; Rf (CH₂Cl₂/CH₃OH = 9.6/0.4) 0.55.

2-(4-(2-Hydroxyethyl)phenoxy)ethyl nitrate (4p): white solid, yield 98%, m.p. 53–55 ^◦C;

¹H NMR (250 MHz, DMSO-d₆)

δ

= 2.65 (t, J = 7.1 Hz, 2H), 3.55 (td, J = 7.1 Hz, 5.2 Hz,

2H), 4.21–4.31 (m, 2H), 4.58 (t, J = 5.2 Hz, 1H), 4.82–4.89 (m, 2H), 6.82–6.90 (m, 2H),

7.08–7.18 (m, 2H); ¹³C NMR (101 MHz, DMSO-d₆)

= 38.1 (CH₂), 62.4 (CH₂OH), 64.0

δ

(CH₂), 72.1 (CH₂), 114.2 (2CH_Ar), 129.9 (2CH_Ar), 132.1 (C_q), 156.1 (C_q); HRMS (ESI-

MS) m/z calcd for C₁₀H₁₃NNaO₅[M+Na]⁺: 250.0691, found: 250.0697; Rf (petroleum

ether/EtOAc = 6/4) 0.63.

3.1.5. General Procedure for the Synthesis of the (halidealkyl)phenoxy Nitrates (5a–p)

To a stirred solution of the appropriate (hydroxyalkyl)phenoxy nitrates 4a–o (1.0 Eq.)

in dry CH₂Cl₂(35 mL for 7 mmol scale), 10 mL solution of thionyl chloride (1.2 Eq.)

and benzotriazole (BTA, 1.2 Eq.) in dry CH₂Cl₂was slowly added, into small portions,

according to the methods reported in the literature [70,71], which were adapted to our

synthesis in terms of the ratio of reagents, solvent, time of reaction, puriﬁcation method.

Before the reaction was complete, benzotriazole hydrochloride started separating out as

a solid. Reaction mixture was stirred further for 20–30 min and after that the solid was

ﬁltered off. The ﬁltrate was successively washed with distilled water (2

×

50 mL) and

brine (50 mL). To obtain 2-(4-(2-iodooethyl)phenoxy)ethyl nitrate (5p), to a solution of

(hydroxyethyl)phenoxy)ethyl nitrate (4p) (1.0 Eq.) in CH₂Cl₂(30 mL), imidazole (1.3 Eq.),

triphenylphosphine (PPh₃, 1.3 Eq.) and iodine (1.3 Eq.) were sequentially added at 0 ^◦C,

according to the method descried in the literature [118–120]. The resulting mixture was

stirred at room temperature for 6 h and reaction was quenched by addition of 10 mL

of saturated aqueous Na₂S₂O₃solution. The combined organic layers were dried over

magnesium sulfate, ﬁltered and concentrated under reduced pressure to obtain a crude

product that was puriﬁed by ﬂash chromatography (silicagel, petroleum ether/EtOAc) to

produce the pure products 5a–p.

2-(4-(Chloromethyl)phenoxy)ethyl nitrate (5a): yellow oil, yield 99%; ¹H NMR (400 MHz,

DMSO-d₆) δ = 4.28–4.36 (m, 2H), 4.73 (s, 2H), 4.85–4.93 (m, 2H), 6.97 (d, J = 8.2 Hz, 2H), 7.38 (d,

J = 8.2 Hz, 2H); ¹³C NMR (101 MHz, DMSO-d₆) δ = 46.1 (CH₂), 64.0 (CH₂), 72.0 (CH₂), 114.6

(2CH_Ar), 130.3 (C_q), 130.4 (2CH_Ar), 157.8 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₁₀ClNNaO₄

[M+Na]⁺: 254.0196, found: 254.0185; Rf (petroleum ether/EtOAc = 7/3) 0.69.

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1

2-(4-(Chloromethyl)-2-ﬂuorophenoxy)ethyl nitrate (5b): pale yellow oil, yield 97%; H

NMR (400 MHz, DMSO-d₆)

δ

= 4.36–4.43 (m, 2H), 4.72 (s, 2H), 4.86–4.93 (m, 2H), 7.16–7.26

(m, 2H), 7.33 (dd, J = 12.3 Hz, 1.7 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 45.3 (CH₂),

65.3 (CH₂), 71.7 (CH₂), 115.2 (d, J = 1.9 Hz, CH_Ar), 116.7 (d, J = 18.7 Hz, CH_Ar), 125.5 (d,

J = 3.5 Hz, CH_Ar), 131.3 (d, J = 6.6 Hz, C_q), 145.7 (d, J = 10.6 Hz, C_q), 151.19 (d, J = 244.7 Hz,

C_qF); ¹⁹F NMR (376 MHz, DMSO-d₆)

δ

=

−

134.2 (dd, J = 12.3 Hz, 7.5 Hz); HRMS (ESI-

MS) m/z calcd for C₉H₉ClFNNaO₄[M+Na]⁺: 272.0102, found: 272.0096; Rf (petroleum

ether/EtOAc = 7/3) 0.69.

2-(2-Chloro-4-(chloromethyl)phenoxy)ethyl nitrate (5c): white solid, yield 95%, m.p. 52–54 ^◦C;

¹H NMR (400 MHz, DMSO-d₆)

δ

= 4.38–4.46 (m, 2H), 4.73 (s, 2H), 4.89–4.95 (m, 2H), 7.19 (d,

J = 8.5 Hz, 1H), 7.39 (dd, J = 8.5 Hz, 2.2 Hz, 1H), 7.54 (d, J = 2.1 Hz, 1H); ¹³C NMR (101 MHz,

DMSO-d₆)

δ = 45.1 (CH₂), 65.4 (CH₂), 71.6 (CH₂), 114.1 (CH_Ar), 121.3 (C_q), 129.0 (CH_Ar),

130.5 (CH_Ar), 131.7 (C_q), 153.1 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₉Cl₂NNaO₄[M+Na]⁺:

287.9806, found: 287.9798; Rf (petroleum ether/EtOAc = 7/3) 0.65.

2-(2,6-Dichloro-4-(chloromethyl)phenoxy)ethyl nitrate (5d): white solid, yield 85%,

m.p. 49–51 ^◦C; H NMR (400 MHz, DMSO-d₆)

δ

= 4.27–4.38 (m, 2H), 4.74 (s, 2H),

1

4.86–4.93 (m, 2H), 7.62 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 43.9 (CH₂), 69.5

(CH₂), 72.4 (CH₂), 128.2 (2C_q), 129.7 (2CH_Ar), 136.3 (C_q), 149.9 (C_q); HRMS (ESI-MS)

m/z calcd for C₉H₈Cl₃NNaO₄[M+Na]⁺: 321.9417, found: 321.9406; Rf (petroleum

ether/EtOAc = 8/2) 0.70.

2-(4-(Chloromethyl)-2-nitrophenoxy)ethyl nitrate (5e): greenish yellow solid, yield 90%,

◦

m.p. 56–58 C; ¹H NMR (400 MHz, DMSO-d₆)

δ

= 4.49–4.55 (m, 2H), 4.81 (s, 2H), 4.86–4.94

(m, 2H), 7.42 (d, J = 8.7 Hz, 1H), 7.74 (dd, J = 8.7 Hz, 2.3 Hz, 1H), 8.00 (d, J = 2.3 Hz, 1H);

¹³C NMR (101 MHz, DMSO-d₆)

= 44.4 (CH₂), 66.1 (CH₂), 71.3 (CH₂), 115.8 (CH_Ar), 125.3

δ

(CH_Ar), 131.0 (C_q), 133.0 (CH_Ar), 139.2 (C_q), 150.4 (C_q); HRMS (ESI-MS) m/z calcd for

C₉H₉ClN₂NaO₆[M+Na]⁺: 299.0047, found: 299.0044; Rf (CH₂Cl₂) 0.70.

2-(4-(Chloromethyl)-2-methoxyphenoxy)ethyl nitrate (5f): white solid, yield 85%, m.p.

54–56 ^◦C; ¹H NMR (250 MHz, DMSO-d₆)

δ

= 3.77 (s, 3H), 4.20–4.35 (m, 2H), 4.71 (s, 2H),

4.80–4.91 (m, 2H), 6.96–6.99 (m, 2H), 7.07 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 46.5

(CH₂), 55.6 (CH₃), 65.0 (CH₂), 72.0 (CH₂), 112.9 (CH_Ar), 113.7 (CH_Ar), 121.4 (CH_Ar), 131.0

(C_q), 147.3 (C_q), 149.0 (C_q). HRMS (ESI-MS) m/z calcd for C₁₀H₁₂ClNNaO₅[M+Na]⁺:

284.0302, found: 284.0299; Rf (petroleum ether/EtOAc = 5/5) 0.76.

2-(4-(Chloromethyl)-2-ethoxyphenoxy)ethyl nitrate (5g): white solid, yield 84%, m.p.

57–60 ^◦C; ¹H NMR (250 MHz, DMSO-d₆)

δ

= 1.32 (t, J = 6.9 Hz, 3H), 4.02 (q, J = 6.9 Hz,

2H), 4.24–4.33 (m, 2H), 4.70 (s, 2H), 4.81–4.92 (m, 2H), 6.95–6.99 (m, 2H), 7.06 (d, J = 1.7 Hz,

0.6 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 14.6 (CH₃), 46.4 (CH₂), 63.9 (CH₂), 65.4

δ

(CH₂), 72.0 (CH₂), 114.4 (CH_Ar), 114.5 (CH_Ar), 121.5 (CH_Ar), 131.2 (C_q), 147.6 (C_q), 148.4

(C_q); HRMS (ESI-MS) m/z calcd for C₁₁H₁₄ClNNaO₅[M+Na]⁺: 298.0458, found: 298.0456;

Rf (petroleum ether/EtOAc = 7/3) 0.72.

2-_◦(4-(Chloromethyl)-2,6-dimethoxyphenoxy)ethyl nitrate (5h): white solid, yield 86%, m.p.

1

59–61 C; H NMR (400 MHz, DMSO-d₆)

δ = 3.76 (s, 6H), 4.09–4.17 (m, 2H), 4.70 (s,

2H), 4.72–4.78 (m, 2H), 6.78 (s, 2H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 46.6 (CH₂), 55.9

(2CH₃), 68.5 (CH₂), 72.9 (CH₂), 106.1 (2CH_Ar), 133.6 (C_q), 135.6 (C_q), 152.7 (2C_q); HRMS

(ESI-MS) m/z calcd for C₁₁H₁₅ClNO₆[M+H]⁺: 292.0588, found: 292.0580; Rf (petroleum

ether/EtOAc = 7/3) 0.61.

2-(3-(Chloromethyl)phenoxy)ethyl nitrate (5i): pale yellow oil, yield 88%; ¹H NMR

(250 MHz, DMSO-d₆)

δ = 4.26–4.37 (m, 2H), 4.72 (s, 2H), 4.83–4.95 (m, 2H), 6.94 (ddd,

J = 8.2 Hz, 2.6 Hz, 1.0 Hz, 1H), 7.00–7.09 (m, 2H), 7.24–7.38 (m, 1H); ¹³C NMR (101 MHz,

DMSO-d₆)

δ = 45.9 (CH₂), 64.0 (CH₂), 71.9 (CH₂), 114.4 (CH_Ar), 114.9 (CH_Ar), 121.6 (CH_Ar),

129.8 (CH_Ar), 139.2 (C_q), 157.9 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₁₁ClNO₄[M+H]⁺:

232.0377, found: 232.0371; Rf (petroleum ether/EtOAc = 8/2) 0.60.

2-(5-(Chloromethyl)-2-methoxyphenoxy)ethyl nitrate (5j): yellow oil, yield 92%, ¹H NMR

(250 MHz, DMSO-d₆)

δ = 3.77 (s, 3H), 4.22–4.33 (m, 2H), 4.70 (s, 2H), 4.79–4.92 (m,

2H), 6.97 (d, J = 8.2 Hz, 1H), 7.02 (d, J = 1.9 Hz, 1H), 7.09 (d, J = 1.9 Hz, 1H); ¹³C

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NMR (101 MHz, DMSO-d₆)

δ

= 46.5 (CH₂), 55.6 (CH₃), 65.1 (CH₂), 72.0 (CH₂), 112.1

(CH_Ar), 114.7 (CH_Ar), 122.5 (CH_Ar), 129.9 (C_q), 147.1 (C_q), 149.2 (C_q); HRMS (ESI-MS)

m/z calcd for C₁₀H₁₂ClNNaO₅[M+Na]⁺: 284.0302, found: 284.0297; Rf (petroleum

ether/EtOAc = 8/2) 0.45.

2-(5-(Chloromethyl)-2-nitrophenoxy)ethyl nitrate (5k): brown solid, yield 91%, m.p.

51–53 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ

= 4.47–4.56 (m, 2H), 4.81 (s, 2H), 4.88–4.97

(m, 2H), 7.23 (dd, J = 8.3 Hz, 1.6 Hz, 1H), 7.50 (d, J = 1.6 Hz, 1H), 7.91 (d, J = 8.3 Hz,

1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 44.7 (CH₂), 66.1 (CH₂), 71.3 (CH₂), 115.6

δ

(CH_Ar), 121.5 (CH_Ar), 125.5 (CH_Ar), 139.0 (C_q), 144.3 (C_q), 150.5 (C_q); HRMS (ESI-MS)

m/z calcd for C₉H₉ClN₂NaO₆[M+Na]⁺: 299.0047, found: 299.0043; Rf (petroleum

ether/CH₂Cl₂= 3/7) 0.67. 2-(2-Chloro-3-(chloromethyl)-6-methoxyphenoxy)ethyl nitrate (5l):

1

pale yellow oil, yield 96%; H NMR (400 MHz, DMSO-d₆)

δ = 3.83 (s, 3H), 4.21–4.30

(m, 2H), 4.79 (s, 2H), 4.80–4.87 (m, 2H), 7.08 (d, J = 8.6 Hz, 1H), 7.36 (d, J = 8.6 Hz,

1H);. ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 44.2 (CH₂), 56.2 (CH₃), 68.8 (CH₂), 72.7 (CH₂),

111.4 (CH_Ar), 126.9 (CH_Ar), 127.6 (C_q), 127.7 (C_q), 143.4 (C_q), 153.5 (C_q); HRMS (ESI-

MS) m/z calcd for C₁₀H₁₁Cl₂NNaO₅[M+Na]⁺: 317.9912, found: 317.9906; Rf (petroleum

ether/EtOAc = 7/3) 0.67.

2-(2-Bromo-3-(chloromethyl)-6-methoxyphenoxy)ethyl nitrate (5m): greenish yellow oil,

yield 95%; ¹H NMR (400 MHz, DMSO-d₆)

δ

= 3.83 (s, 3H), 4.19–4.30 (m, 2H), 4.80 (s, 2H),

4.81–4.88 (m, 2H), 7.12 (d, J = 8.5 Hz, 1H), 7.38 (d, J = 8.5 Hz, 1H); ¹³C NMR (101 MHz,

DMSO-d₆) = 46.8 (CH₂), 56.2 (CH₃), 68.7 (CH₂), 72.7 (CH₂), 112.1 (CH_Ar), 119.3 (C_q), 127.3

δ

(CH_Ar), 129.3 (C_q), 144.5 (C_q), 153.3 (C_q); HRMS (ESI-MS) m/z calcd for C₁₀H₁₁BrClNNaO₅

[M+Na]⁺: 361.9407, found: 361.9401; Rf (petroleum ether/EtOAc = 6/4) 0.71.

2-(2-(Chloromethyl)phenoxy)ethyl nitrate (5n): yellow oil, yield 83%; ¹H NMR (250 MHz,

DMSO-d₆)

δ

= 4.34–4.42 (m, 2H), 4.69 (s, 2H), 4.89–4.97 (m, 2H), 6.92–7.11 (m, 2H), 7.29–7.46

(m, 2H); ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 40.5 (CH₂), 64.8 (CH₂), 72.0 (CH₂), 111.4

(CH_Ar), 114.8 (CH_Ar), 127.5 (CH_Ar), 129.5 (CH_Ar), 132.7 (C_q), 154.5 (C_q); HRMS (ESI-

MS) m/z calcd for C₉H₁₀ClNNaO₄[M+Na]⁺: 254.0196, found: 254.0193; Rf (petroleum

ether/EtOAc = 8/2) 0.64.

1

2-(4-Bromo-2-(bromomethyl)phenoxy)ethyl nitrate (5o): pale yellow oil, yield 89%; H

NMR (400 MHz, DMSO-d₆)

J = 8.7 Hz, 1H), 7.52 (dd, J = 8.8 Hz, 2.5 Hz, 1H), 7.62 (d, J = 2.6 Hz, 1H); ¹³C NMR (101 MHz,

DMSO-d₆) = 40.4 (CH₂), 65.2 (CH₂), 71.7 (CH₂), 112.3 (C_q), 114.9 (CH_Ar), 128.3 (C_q),

δ = 4.33–4.41 (m, 2H), 4.67 (s, 2H), 4.87–4.96 (m, 2H), 7.07 (d,

δ

132.7 (CH_Ar), 133.1 (CH_Ar), 155.2 (C_q); HRMS (ESI-MS) m/z calcd for C₉H₉BrClNNaO₄

[M+Na]⁺: 331.9301, found: 331.9310; Rf (petroleum ether/EtOAc = 8/2) 0.55.

2-(4-(2-Iodooethyl)phenoxy)ethyl nitrate (5p): white solid, yield 79%, m.p. 57–59 ^◦C; ¹H

NMR (400 MHz, DMSO-d₆)

δ = 3.05 (t, J = 7.4 Hz, 2H), 3.42 (t, J = 7.4 Hz, 2H), 4.23–4.31 (m,

2H), 4.81–4.91 (m, 2H), 6.89 (d, J = 7.4Hz, 2H), 7.18 (d, J = 7.6 Hz, 2H); ¹³C NMR (101 MHz,

DMSO-d₆)

δ = 8.7 (CH₂), 38.4 (CH₂), 63.9 (CH₂), 72.0 (CH₂), 114.4 (2CH_Ar), 129.6 (2CH_Ar),

133.3 (C_q), 156.5 (C_q); HRMS (ESI-MS) m/z calcd for C₁₀H₁₃INO₄[M+H]⁺: 337.9889, found:

337.9884; Rf (petroleum ether/EtOAc = 8/2) 0.60.

3.1.6. Synthesis of (4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)methyl)-1,3,

4-oxadiazol (7)

To a suspension of indomethacin hydrazide (

12 mmol scale), Et₃N (2.0 Eq.) and CS₂(2.0 Eq.) were slowly added, into small portions,

according to the method reported in the literature [121 124], which was adapted to our

6) [99] (1.0 Eq.) in CH₃CN (150 mL for

–

synthesis in terms of the ratio of reagents, solvent, time of reaction and puriﬁcation method.

The mixture was stirred for 3 h under reﬂux till hydrogen sulﬁde formation was stopped.

After cooling at room temperature, the solvent was removed under reduced pressure and

the residue was dissolved in EtOAc (40 mL) and acidiﬁed with aqueous diluted HCl 0.5 M

solution (10 mL). The organic layer was separated and was successively washed with

distilled water (3

×

50 mL) and ﬁnally with brine (50 mL). The combined organic layers

were dried over anhydrous magnesium sulfate, ﬁltered and concentrated under reduced

pressure. The crude product was puriﬁed by ﬂash column chromatography to give the

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◦

pure product: white solid, yield 92%, m.p. 199–201 C; ¹H NMR (400 MHz, DMSO-d₆)

= 2.29 (s, 3H), 3.76 (s, 3H), 4.25 (s, 2H), 6.74 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.90 (d, J = 9.0 Hz,

1H), 7.12 (d, J = 2.5 Hz, 1H), 7.56–7.76 (m, 4H), 14.36 (s, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

= 13.1 (CH₃), 20.4 (CH₂), 55.4 (CH₃), 101.6 (CH_Ar), 111.1 (C_q), 111.6 (CH_Ar), 114.7 (CH_Ar),

δ

129.1 (2CH_Ar), 129.9 (C_q), 130.2 (C_q), 131.2 (2CH_Ar), 133.9 (C_q), 136.1 (C_q), 137.7 (C_q), 155.6

(C_q), 162.3 (C_qS), 167.8 (C_q), 177.8 (C_q); HRMS (ESI-MS) m/z calcd for C₂₀H₁₇ClN₃O₃S

[M+H]⁺: 414.0679, found: 414.0680;Rf (CH₂Cl₂/CH₃OH = 9.8/0.2) 0.52.

3.1.7. General Procedure for the Synthesis of the Nitric Oxide-Releasing Indomethacin

Derivatives with 2-mercapto-1,3,4-oxadiazol Scaffold (8a–p)

To a suspension of (4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)methyl)-

1,3,4-oxadiazol (7) (1.0 Eq.) in acetonitrile (70 mL for 4 mmol scale), the correspond-

ing (halidealkyl)phenoxy nitrates 5a–p (1.1 Eq.) were added in one portion followed by

Et₂N being added drop wise(1.5 Eq.), according to the method reported in the literature,

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

reaction and puriﬁcation method [125

for 3–6 h and then the solvent was removed under reduced pressure. The residue was

taken up with EtOAc (50 mL) and successively washed with distilled water (2 50 mL)

,126]. The mixture was stirred at room temperature

×

and ﬁnally with brine (50 mL). The combined organic layers were dried over magnesium

sulfate and solvent was removed under reduced pressure to obtain a crude product that

was puriﬁed by ﬂash column chromatography to produce the pure products 8a–p.

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

oxadiazol-2-yl)thio)methyl)phenoxy)ethyl nitrate (8a): pale yellow solid, yield 88%,

m.p. 108–110 ^◦C; ¹H NMR (250 MHz, DMSO-d₆)

δ = 2.29 (s, 3H), 3.74 (s, 3H), 4.19–4.28

(m, 2H), 4.36 (s, 2H), 4.37 (s, 2H), 4.79–4.91 (m, 2H), 6.74 (dd, J = 9.0 Hz, 2.5 Hz, 1H),

6.73–6.86 (m, 2H), 6.93 (dd, J = 9.0 Hz, 0.5 Hz, 1H), 7.09 (dd, J = 2.6 Hz, 0.5 Hz, 1H),

7.14–7.30 (m, 2H), 7.54–7.76 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.1 (CH₃), 20.0

(CH₂), 35.4 (CH₂), 55.4 (CH₃), 64.0 (CH₂), 71.9 (CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar), 111.9

(C_q), 114.4 (2CH_Ar), 114.7 (CH_Ar), 128.9 (C_q), 129.1 (2CH_Ar), 129.9 (C_q), 130.3 (2CH_Ar),

131.2 (2CH_Ar), 133.9 (C_q), 135.7 (C_q), 137.8 (C_q), 155.6 (C_q), 157.3 (C_q), 163.0 (C_q), 166.1

(2C_q), 167.9 (C_q); HRMS (ESI-MS) m/z calcd for C₂₉H₂₆ClN₄O₇S [M+H]⁺: 609.1211,

found: 609.1205; Rf (petroleum ether/EtOAc = 4/6) 0.66.

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

oxadiazol-2-yl)thio)methyl)-2-fluorophenoxy)ethyl nitrate (8b): pale yellow solid, yield

86%, m.p. 105–107 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ = 2.28 (s, 3H), 3.74 (s, 3H), 4.29–

4.34 (m, 2H), 4.36 (s, 2H), 4.38 (s, 2H), 4.81–4.93 (m, 2H), 6.74 (dd, J = 9.0 Hz, 2.5 Hz, 1H),

6.92 (d, J = 9.0 Hz, 1H), 7.01 (t, J = 8.5 Hz, 1H), 7.07 (dd, J = 9.0 Hz, 2.0 Hz, 2H), 7.25 (dd,

J = 12.2 Hz, 2.0 Hz, 1H), 7.59–7.73 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.0 (CH₃),

20.0 (CH₂), 34.9 (CH₂), 55.4 (CH₃), 65.2 (CH₂), 71.7 (CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar),

111.8 (C_q), 114.7 (CH_Ar), 114.9 (d, J =1.8 Hz, CH_Ar), 116.6 (CH_Ar), 116.8 (C_q), 125.3 (d,

J = 3.4 Hz, CH_Ar), 129.0 (2CH_Ar), 129.9 (C_q), 130.2 (d, J = 3.3 Hz, C_q), 131.2 (2CH_Ar), 133.9

(C_q), 135.7 (C_q), 137.7 (C_q), 145.2 (d, J = 10.6 Hz, C_q), 151.1 (d, J = 244.6 Hz, C_qF), 155.6

(C_q), 162.9 (C_q), 166.2 (C_q), 167.9 (C_q); ¹⁹F NMR (376 MHz, DMSO-d₆)

δ

=

−

134.2 (dd,

J = 12.2 Hz, 8.6 Hz); HRMS (ESI-MS) m/z calcd for C₂₉H₂₅ClFN₄O₇S [M+H]⁺: 627.1117,

found: 627.1102; Rf (petroleum ether/EtOAc = 3/7) 0.66.

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

3,4-oxadiazol-2-yl)thio)methyl)phenoxy)ethyl nitrate (8c): pale yellow solid, yield 78%, m.p.

◦

95–97 C; ¹H NMR (400 MHz, DMSO-d₆)

δ = 2.28 (s, 3H), 3.73 (s, 3H), 4.29–4.37 (m, 2H), 4.35

(s, 2H), 4.38 (s, 2H), 4.84–4.92 (m, 2H), 6.74 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.92 (d, J = 9.0 Hz,

1H), 6.99 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 2.5 Hz, 1H), 7.23 (dd, J = 8.5 Hz, 2.2 Hz, 1H), 7.47

(d, J = 2.2 Hz, 1H), 7.59–7.75 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.1 (CH₃), 20.0

(CH₂), 34.7 (CH₂), 55.4 (CH₃), 65.4 (CH₂), 71.5 (CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar), 111.8 (C_q),

113.8 (CH_Ar), 114.7 (CH_Ar), 121.2 (C_q), 128.9 (CH_Ar), 129.0 (2CH_Ar), 129.9 (C_q), 130.3 (C_q),

130.5 (CH_Ar), 130.6 (C_q), 131.2 (2CH_Ar), 133.9 (C_q), 135.7 (C_q), 137.8 (C_q), 152.6 (C_q), 155.6

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(C_q), 162.9 (C_q), 166.2 (C_q), 167.9 (C_q); HRMS (ESI-MS) m/z calcd for C₂₉H₂₅Cl₂N₄O₇S

[M+H]⁺: 643.0821, found: 643.0816; Rf (petroleum ether/EtOAc = 4/6) 0.56.

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

1,3,4-oxadiazol-2-yl)thio)methyl)phenoxy)ethyl nitrate (8d): pale yellow solid, yield 89%, m.p.

125–127 ^◦C; ¹H NMR (250 MHz, DMSO-d₆)

δ = 2.28 (s, 3H), 3.73 (s, 3H), 4.22–4.32 (m, 2H),

4.35 (s, 2H), 4.42 (s, 2H), 4.80–4.91 (m, 2H), 6.72 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.91 (dd, J = 9.0 Hz,

0.5 Hz, 1H), 7.07 (dd, J = 2.5 Hz, 0.5 Hz, 1H), 7.54 (s, 2H), 7.59–7.74 (m, 4H); ¹³C NMR (101 MHz,

DMSO-d₆) δ = 13.1 (CH₃), 20.0 (CH₂), 34.0 (CH₂), 55.4 (CH₃), 69.4 (CH₂), 72.4 (CH₂), 101.5

(CH_Ar), 111.6 (CH_Ar), 111.8 (C_q), 114.7 (CH_Ar), 128.0 (2C_q), 129.0 (2CH_Ar), 129.7 (2CH_Ar),

129.9 (C_q), 130.2 (C_q), 131.2 (2CH_Ar), 133.9 (C_q), 135.7 (C_q), 135.7 (C_q), 137.7 (C_q), 149.4 (C_q),

155.6 (C_q), 162.8 (C_q), 166.3 (C_q), 167.8 (C_q); HRMS (ESI-MS) m/z calcd for C₂₉H₂₄Cl₃N₄O₇S

[M+H]⁺: 677.0431, found: 677.0723; Rf (petroleum ether/EtOAc = 5/5) 0.49.

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

oxadiazol-2-yl)thio)methyl)-2-nitrophenoxy)ethyl nitrate (8e): pale yellow solid, yield

1

93%, m.p. 92–94 ^◦C;. H NMR (400 MHz, DMSO-d₆)

δ = 2.27 (s, 3H), 3.73 (s, 3H), 4.35 (s,

2H), 4.39–4.51 (m, 2H), 4.47 (s, 2H), 4.78–4.95 (m, 2H), 6.73 (dd, J = 9.0 Hz, 2.5 Hz, 1H),

6.91 (d, J = 9.0 Hz, 1H), 7.06 (d, J = 2.5 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.55–7.74 (m,

5H), 7.96 (d, J = 2.3 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.0 (CH₃), 20.0 (CH₂),

34.2 (CH₂), 55.4 (CH₃), 66.0 (CH₂), 71.3 (CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar), 111.8 (C_q),

114.7 (CH_Ar), 115.4 (CH_Ar), 125.5 (CH_Ar), 129.0 (2CH_Ar), 129.9 (C_q), 130.1 (C_q), 130.2 (C_q),

131.2 (2CH_Ar), 133.9 (C_q), 134.9 (CH_Ar), 135.7 (C_q), 137.7 (C_q), 139.0 (C_q), 150.0 (C_q), 155.6

(C_q), 162.8 (C_q), 166.2 (C_q), 167.9 (C_q); HRMS (ESI-MS) m/z calcd for C₂₉H₂₅ClN₅O₉S

[M+H]⁺: 654.1062, found: 654.1053; Rf (CH₂Cl₂/CH₃OH = 9.8/0.2) 0.73.

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

oxadiazol-2-yl)thio)methyl)-2-methoxyphenoxy)ethyl nitrate (8f): pale yellow solid,

yield 83%, m.p. 95–97 ^◦C; ¹H NMR (250 MHz, DMSO-d₆)

δ = 2.29 (s, 3H), 3.69 (s, 3H),

3.74 (s, 3H), 4.14–4.27 (m, 2H), 4.36 (s, 2H), 4.38 (s, 2H), 4.76–4.92 (m, 2H), 6.74 (dd,

J = 9.0 Hz, 2.5 Hz, 1H), 6.78 (d, J = 1.1 Hz, 2H), 6.93 (dd, J = 9.0 Hz, 0.5 Hz, 1H), 7.03

(d, J = 1.2 Hz, 1H), 7.08 (d, J = 2.5 Hz, 1H), 7.58–7.73 (m, 4H); ¹³C NMR (101 MHz,

DMSO-d₆)

δ = 13.1 (CH₃), 20.0 (CH₂), 35.9 (CH₂), 55.4 (CH₃), 55.4 (CH₃), 64.9 (CH₂), 72.0

(CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar), 111.9 (C_q), 113.0 (CH_Ar), 113.5 (CH_Ar), 114.7 (CH_Ar),

121.2 (CH_Ar), 129.0 (2CH_Ar), 129.6 (C_q), 129.9 (C_q), 130.3 (C_q), 131.2 (2CH_Ar), 133.9 (C_q),

135.7 (C_q), 137.7 (C_q), 146.8 (C_q), 148.9 (C_q), 155.6 (C_q), 163.1 (C_q), 166.1 (C_q), 167.9 (C_q);

HRMS (ESI-MS) m/z calcd for C₃₀H₂₈ClN₄O₈S [M+H]⁺: 639.1316, found: 639.1311; Rf

(petroleum ether/EtOAc = 4/6) 0.66.

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

oxadiazol-2-yl)thio)methyl)-2-ethoxyphenoxy)ethyl nitrate (8g): pale yellow solid, yield

91%, m.p. 122–124 ^◦C; ¹H NMR (250 MHz, DMSO-d₆)

δ = 1.27 (t, J = 6.9 Hz, 3H), 2.29 (s,

3H), 3.74 (s, 3H), 3.94 (q, J = 6.9 Hz, 2H), 4.14–4.27 (m, 2H), 4.36 (s, 2H), 4.37 (s, 2H), 4.75–

4.91 (m, 2H), 6.74 (dd, J = 9.0 Hz, 2.5 Hz, 1H), 6.79 (d, J = 1.1 Hz, 2H), 6.93 (dd, J = 9.0 Hz,

0.5 Hz, 1H), 7.02 (d, J = 1.1Hz, 1H), 7.08 (dd, J = 2.5 Hz, 0.5 Hz, 1H), 7.57–7.73 (m, 4H);

¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.1 (CH₃), 14.6 (CH₃), 20.0 (CH₂), 35.8 (CH₂), 55.4

(CH₃), 63.9 (CH₂), 65.3 (CH₂), 71.9 (CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar), 111.9 (C_q), 114.3

(CH_Ar), 114.5 (CH_Ar), 114.7 (CH_Ar), 121.3 (CH_Ar), 129.0 (2CH_Ar), 129.8 (C_q), 129.9 (C_q),

130.3 (C_q), 131.2 (2CH_Ar), 133.9 (C_q), 135.7 (C_q), 137.8 (C_q), 147.1 (C_q), 148.3 (C_q), 155.6

(C_q), 163.1 (C_q), 166.0 (C_q), 167.9 (C_q); HRMS (ESI-MS) m/z calcd for C₃₁H₃₀ClN₄O₈S

[M+H]⁺: 653.1473, found: 653.1459; Rf (petroleum ether/EtOAc = 4/6) 0.58.

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

oxadiazol-2-yl)thio)methyl)-2,6-dimethoxyphenoxy)ethyl nitrate (8h): pale yellow solid,

◦

1

yield 91%, m.p. 110–112 C; H NMR (250 MHz, DMSO-d₆)

δ = 2.28 (s, 3H), 3.68 (s,

6H), 3.74 (s, 3H), 4.03–4.18 (m, 2H), 4.36 (s, 2H), 4.42 (s, 2H), 4.64–4.80 (m, 2H), 6.73 (dd,

J = 9.0 Hz, 2.5 Hz, 1H), 6.73 (s, 2H), 6.92 (dd, J = 9.0 Hz, 0.5 Hz, 1H), 7.09 (d, J = 2.3 Hz,

1H), 7.57–7.74 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 13.1 (CH₃), 20.0 (CH₂), 36.3

(CH₂), 55.4 (CH₃), 55.8 (2CH₃), 68.5 (CH₂), 72.8 (CH₂), 101.5 (CH_Ar), 106.2 (2CH_Ar), 111.5

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(CH_Ar), 111.8 (C_q), 114.7 (CH_Ar), 129.0 (2CH_Ar), 129.9 (C_q), 130.2 (C_q), 131.2 (2CH_Ar),

132.3 (C_q), 133.9 (C_q), 135.3 (C_q), 135.7 (C_q), 137.7 (C_q), 152.7 (2C_q), 155.6 (C_q), 163.1 (C_q),

166.1 (C_q), 167.9 (C_q); HRMS (ESI-MS) m/z calcd for C₃₁H₃₀ClN₄O₉S [M+H]⁺: 669.1422,

found: 669.1411; Rf (petroleum ether/EtOAc = 4/6) 0.57.

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

oxadiazol-2-y_◦l)thio)methyl)phenoxy)ethyl nitrate (8i): pale yellow solid, yield 80%,

1

m.p. 98–100 C; H NMR (250 MHz, DMSO-d₆)

δ

= 2.28 (s, 3H), 3.74 (s, 3H), 4.15–

4.31 (m, 2H), 4.35 (s, 2H), 4.41 (s, 2H), 4.76–4.93 (m, 2H), 6.73 (dd, J = 9.0 Hz, 2.5 Hz,

1H), 6.79 – 6.98 (m, 2H), 6.92 (dd, J = 9.0 Hz, 0.5 Hz, 1H), 6.98–7.03 (m, 1H), 7.08 (dd,

J = 2.6 Hz, 0.5 Hz, 1H), 7.10–7.20 (m, 1H), 7.58–7.73 (m, 4H); ¹³C NMR (101 MHz, DMSO-

d₆)

δ = 13.0 (CH₃), 20.0 (CH₂), 35.7 (CH₂), 55.4 (CH₃), 63.9 (CH₂), 71.9 (CH₂), 101.5

(CH_Ar), 111.6 (CH_Ar), 111.9 (C_q), 113.6 (CH_Ar), 114.7 (CH_Ar), 115.3 (CH_Ar), 121.6 (CH_Ar),

129.1 (2CH_Ar), 129.6 (CH_Ar), 129.9 (C_q), 130.2 (C_q), 131.2 (2CH_Ar), 133.9 (C_q), 135.7 (C_q),

137.7 (C_q), 138.1 (C_q), 155.6 (C_q), 157.8 (C_q), 163.0 (C_q), 166.1 (C_q), 167.9 (C_q). HRMS (ESI-

MS) m/z calcd for C₂₉H₂₆ClN₄O₇S [M+H]⁺: 609.1211, found: 609.1207; Rf (petroleum

ether/EtOAc = 4/6) 0.63.

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

oxadiazol-2-yl)thio)methyl)-2-methoxyphenoxy)ethyl nitrate (8j): pale yellow solid,

◦

1

yield 75%, m.p. 104–106 C; H NMR (250 MHz, DMSO-d₆)

δ = 2.28 (s, 3H), 3.70 (s,

3H), 3.74 (s, 3H), 4.10 – 4.26 (m, 2H), 4.36 (s, 4H), 4.75–4.92 (m, 2H), 6.70–6.76 (m, 1H),

6.77 (s, 1H), 6.83 (dd, J = 8.3 Hz, 1.9 Hz, 1H), 6.93 (dd, J = 9.0 Hz, 0.5 Hz, 1H), 7.06 (d,

J = 1.9 Hz, 1H), 7.08 (dd, J = 2.5 Hz, 0.5 Hz, 1H), 7.59–7.74 (m, 4H); ¹³C NMR (101 MHz,

DMSO-d₆)

δ = 13.1 (CH₃), 20.0 (CH₂), 35.8 (CH₂), 55.4 (CH₃), 55.5 (CH₃), 65.0 (CH₂),

71.9 (CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar), 111.9 (C_q,CH_Ar), 114.7 (CH_Ar), 114.8 (CH_Ar),

122.3 (CH_Ar), 128.5 (C_q), 129.1 (2CH_Ar), 129.9 (C_q), 130.3 (C_q), 131.2 (2CH_Ar), 133.9 (C_q),

135.7 (C_q), 137.7 (C_q), 147.0 (C_q), 148.8 (C_q), 155.6 (C_q), 163.0 (C_q), 166.0 (C_q), 167.9 (C_q);

HRMS (ESI-MS) m/z calcd for C₃₀H₂₈ClN₄O₈S [M+H]⁺: 639.1316, found: 639.1313; Rf

(petroleum ether/EtOAc = 4/6) 0.53.

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

oxadiazol-2-yl)thio)methyl)-2-nitrophenoxy)ethyl nitrate (8k): pale yellow solid, yield

88%, m.p. 90–92 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ = 2.27 (s, 3H), 3.72 (s, 3H), 4.35 (s,

2H), 4.40–4.46 (m, 2H), 4.50 (s, 2H), 4.81–4.95 (m, 2H), 6.73 (dd, J = 9.0 Hz, 2.5 Hz, 1H),

6.90 (d, J = 9.0 Hz, 1H), 7.05 (d, J = 2.5 Hz, 1H), 7.09 (dd, J = 8.3 Hz, 1.6 Hz, 1H), 7.47 (d,

J = 1.6 Hz, 1H), 7.60–7.71 (m, 4H), 7.73 (d, J = 8.3 Hz, 1H); ¹³C NMR (101 MHz, DMSO-d₆)

δ

= 13.0 (CH₃), 20.0 (CH₂), 35.1 (CH₂), 55.4 (CH₃), 66.0 (CH₂), 71.2 (CH₂), 101.5 (CH_Ar),

111.5 (CH_Ar), 111.8 (C_q), 114.7 (CH_Ar), 115.9 (CH_Ar), 121.5 (CH_Ar), 125.2 (CH_Ar), 129.0

(2CH_Ar), 129.9 (C_q), 130.2 (C_q), 131.2 (2CH_Ar), 133.9 (C_q), 135.8 (C_q), 137.8 (C_q), 138.6

(C_q), 144.0 (C_q), 150.5 (C_q), 155.6 (C_q), 162.6 (C_q), 166.3 (C_q), 167.9 (C_q); HRMS (ESI-

MS) m/z calcd for C₂₉H₂₅ClN₅O₉S [M+H]⁺: 654.1062, found: 654.1049; Rf (petroleum

ether/EtOAc = 3/7) 0.48.

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

3,4-oxadiazol-2-yl)thio)_◦methyl)-6-methoxyphenoxy)ethyl nitrate (8l): pale yellow solid,

yield 87%, m.p. 117–119 C; ¹H NMR (400 MHz, DMSO-d₆)

δ = 2.29 (s, 3H), 3.74 (s, 3H), 3.76

(s, 3H), 4.17–4.27 (m, 2H), 4.37 (s, 2H), 4.44 (s, 2H), 4.71–4.86 (m, 2H), 6.74 (dd, J = 9.0 Hz,

2.5 Hz, 1H), 6.82 (d, J = 8.6 Hz, 1H), 6.93 (d, J = 8.9 Hz, 1H), 7.10 (dd, J = 5.5 Hz, 3.0 Hz, 2H),

7.60–7.73 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.1 (CH₃), 20.0 (CH₂), 34.3 (CH₂),

55.4 (CH₃), 56.1 (CH₃), 68.8 (CH₂), 72.7 (CH₂), 101.5 (CH_Ar), 111.0 (CH_Ar), 111.6 (CH_Ar),

111.8 (C_q), 114.7 (CH_Ar), 126.2 (C_q), 126.4 (CH_Ar), 127.4 (C_q), 129.0 (2CH_Ar), 129.9 (C_q),

130.2 (C_q), 131.2 (2CH_Ar), 133.9 (C_q), 135.8 (C_q), 137.8 (C_q), 143.4 (C_q), 152.9 (C_q), 155.6 (C_q),

162.6 (C_q), 166.3 (C_q), 167.9 (C_q); HRMS (ESI-MS) m/z calcd for C₃₀H₂₇Cl₂N₄O₈S [M+H]⁺:

673.0927, found: 673.0918; Rf (petroleum ether/EtOAc = 4/6) 0.53.

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

3,4-oxadiazol-2-yl)thio)methyl)-6-methoxyphenoxy)ethyl nitrate (8m): pale yellow solid,

◦

yield 88%, m.p. 94–96 C; ¹H NMR (400 MHz, DMSO-d₆)

δ = 2.29 (s, 3H), 3.74 (s, 3H), 3.77

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(s, 3H), 4.16–4.24 (m, 2H), 4.37 (s, 2H), 4.46 (s, 2H), 4.75–4.87 (m, 2H), 6.74 (dd, J = 9.0 Hz,

2.5 Hz, 1H), 6.86 (d, J = 8.7 Hz, 1H), 6.93 (d, J = 8.9 Hz, 1H), 7.10 (d, J = 2.5 Hz, 1H), 7.12 (d,

J = 8.6 Hz, 1H), 7.60–7.72 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.1 (CH₃), 20.0 (CH₂),

36.9 (CH₂), 55.4 (CH₃), 56.1 (CH₃), 68.6 (CH₂), 72.7 (CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar), 111.7

(CH_Ar), 111.9 (C_q), 114.7 (CH_Ar), 119.2 (C_q), 126.6 (CH_Ar), 127.7 (C_q), 129.0 (2CH_Ar), 129.9

(C_q), 130.3 (C_q), 131.2 (2CH_Ar), 133.9 (C_q), 135.8 (C_q), 137.8 (C_q), 144.5 (C_q), 152.7 (C_q), 155.6

(C_q), 162.6 (C_q), 166.3 (C_q), 167.9 (C_q); HRMS (ESI-MS) m/z calcd for C₃₀H₂₇BrClN₄O₈S

[M+H]⁺: 717.0421, found: 717.0409; Rf (petroleum ether/EtOAc = 4/6) 0.55.

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

oxadiazol-2-yl)thio)methyl)phenoxy)ethyl nitrate (8n): pale yellow solid, yield 70%,

◦

m.p. 89–91 C; ¹H NMR (400 MHz, DMSO-d₆)

δ = 2.28 (s, 3H), 3.74 (s, 3H), 4.28–4.40 (m,

2H), 4.34 (s, 2H), 4.35 (s, 2H), 4.79–4.98 (m, 2H), 6.74 (dd, J = 8.9 Hz, 2.5 Hz, 1H), 6.78

(td, J = 7.5 Hz, 1.0 Hz, 1H), 6.92 (d, J = 8.9 Hz, 1H), 7.01 (dd, J = 8.4 Hz, 1.0 Hz, 1H), 7.08

(d, J = 2.5 Hz, 1H), 7.19 (dd, J = 7.5 Hz, 1.7 Hz, 1H), 7.25 (ddd, J = 8.2 Hz, 7.4 Hz, 1.7 Hz,

1H), 7.58–7.72 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.1 (CH₃), 20.0 (CH₂), 31.4

(CH₂), 55.4 (CH₃), 64.7 (CH₂), 71.8 (CH₂), 101.5 (CH_Ar), 111.6 (CH_Ar), 111.9 (C_q), 112.2

(CH_Ar), 114.7 (CH_Ar), 120.8 (CH_Ar), 124.3 (C_q), 129.0 (2CH_Ar), 129.6 (CH_Ar), 129.9 (C_q),

130.3 (C_q), 130.3 (CH_Ar), 131.2 (2CH_Ar), 133.9 (C_q), 135.7 (C_q), 137.7 (C_q), 155.6 (C_q), 155.7

(C_q), 163.3 (C_q), 166.1 (C_q), 167.9 (C_q); HRMS (ESI-MS) m/z calcd for C₂₉H₂₆ClN₄O₇S

[M+H]⁺: 609.1211, found: 609.1195; Rf (petroleum ether/EtOAc = 4/6) 0.68.

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

3,4-oxadiazol-2-yl)thio)methyl)phenoxy)ethyl nitrate (8o): pale yellow solid, yield 87%,

m.p. 91–93 ^◦C; ¹H NMR (400 MHz, DMSO-d₆)

δ = 2.28 (s, 3H), 3.74 (s, 3H), 4.26–4.41 (m,

2H), 4.34 (s, 4H), 4.79–4.92 (m, 2H), 6.73 (dd, J = 8.9 Hz, 2.5 Hz, 1H), 6.92 (d, J = 8.9 Hz,

1H), 7.00 (d, J = 8.8 Hz, 1H), 7.07 (d, J = 2.5 Hz, 1H), 7.44 (dd, J = 8.7 Hz, 2.5 Hz, 1H), 7.50

(d, J = 2.5 Hz, 1H), 7.59–7.72 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.1 (CH₃), 20.0

(CH₂), 30.7 (CH₂), 55.4 (CH₃), 65.1 (CH₂), 71.7 (CH₂), 101.4 (CH_Ar), 111.6 (CH_Ar), 111.8

(C_q), 112.0 (C_q), 114.4 (CH_Ar), 114.7 (CH_Ar), 127.1 (C_q), 129.0 (2CH_Ar), 129.9 (C_q), 130.2 (C_q),

131.2 (2CH_Ar), 131.9 (CH_Ar), 132.7 (CH_Ar), 133.9 (C_q), 135.7 (C_q), 137.7 (C_q), 155.0 (C_q), 155.6

(C_q), 163.1 (C_q), 166.2 (C_q), 167.8 (C_q); HRMS (ESI-MS) m/z calcd for C₂₉H₂₅BrClN₄O₇S

[M+H]⁺: 687.0316, found: 687.0302; Rf (petroleum ether/EtOAc = 4/6) 0.70.

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

oxadiazol-2-yl)thio)ethyl)phenoxy)ethyl nitrate (8p): yellow solid, yield 82%, m.p. 92–

94 ^◦C; ¹H NMR (250 MHz, DMSO-d₆)

δ = 2.29 (s, 3H), 2.91 (t, J = 7.5 Hz, 2H), 3.25–3.45

(m, 2H), 3.74 (s, 3H), 4.18–4.31 (m, 2H), 4.36 (s, 2H), 4.79–4.93 (m, 2H), 6.74 (dd, J = 9.0 Hz,

2.5 Hz, 1H), 6.81–6.89 (m, 2H), 6.92 (dd, J = 9.0 Hz, 0.5 Hz, 1H), 7.04–7.16 (m, 3H),

7.56–7.73 (m, 4H); ¹³C NMR (101 MHz, DMSO-d₆)

δ = 13.1 (CH₃), 20.0 (CH₂), 33.4 (CH₂),

34.0 (CH₂), 55.4 (CH₃), 63.9 (CH₂), 72.0 (CH₂), 101.5 (CH_Ar), 111.5 (CH_Ar), 111.9 (C_q),

114.4 (2CH_Ar), 114.7 (CH_Ar), 129.0 (2CH_Ar), 129.7 (2CH_Ar), 129.9 (C_q), 130.2 (C_q), 131.2

(2CH_Ar), 131.7 (C_q), 133.9 (C_q), 135.7 (C_q), 137.7 (C_q), 155.6 (C_q), 156.5 (C_q), 163.4 (C_q),

165.9 (C_q), 167.9 (C_q); HRMS (ESI-MS) m/z calcd for C₃₀H₂₈ClN₄O₇S [M+H]⁺: 623.1367,

found: 623.1364; Rf (petroleum ether/EtOAc = 5/5) 0.63.

3.2. In Silico Docking Study

In order to study the selectivity of the new NO-IND-OXDs 8a–p for COX isoenzymes

(COX-1 and COX-2), AutoDock 4.2.6 software was used [82,127–129]. The results, expressed

as docking score, were compared to IND, DCF and CCB, used as reference drugs.

3.2.1. Generating the Receptor Coordinate File (RCF)

X-ray crystallographic structures for COX-1 (pdb code: 4o1z) and COX-2 (pdb code:

3nt1)) used in this research were selected in terms of the quality of the model obtained

from RCSB Protein Data Bank and were processed prior to docking. From downloaded

experimental structures, the following were removed: the ligands, the water molecules,

cofactors and ions that should not be included in the receptor with a text editor. After

Int. J. Mol. Sci. 2021, 22, 7079

24 of 32

that, by reading the coordinates, adding charges, merging nonpolar hydrogens and as-

signing appropriate atom types, each receptor was converted to PDBQT format ﬁle using

AutoDock 4.2.6.

3.2.2. Generating the Ligand Coordinate File (LCF)

The PDB coordinate ﬁles of compounds 8a–p were generated using Chimera 1.14

software. The LCF describes the ligand structure through several types of records (such

as ROOT, ENDROOT, BRANCH, ENDBRANCH and TORSDOF) that are recognized by

AutoDock. Each structure was energy minimized and converted to PDBQT format ﬁle. To

study the interaction between a single ligand with a single receptor, with explicit calculation

of afﬁnity maps, a docking method was used. The receptors were kept rigid and the ligands

were allowed to be ﬂexible.

3.2.3. Preparing the Grid Parameter File (GPF)

The GPF speciﬁes the parameters for generating the atomic afﬁnity maps and the

PDBQT ﬁles for the receptor. For COX-2 we used a grid box of 74

a spacing of 0.375 Å between grid points and the grid box center was put on x = –37.882,

y = –50.853 and z = –21.24. COX-1 was enclosed in a 73 78 82 grid box with 0.375 Å

×

72

×

86 points with

×

spacing and 251.00, 104.00 and 1.364 as x, y and z center. The binding site of COX-1 and

COX-2 ligands is respectively identiﬁed by using protein visualization software such as

PyMOL, DS visualizer and Chimera 1.14.

3.2.4. Preparing the Docking Parameter File (DPF)

To prepare the DPF ﬁle, different parameters were selected: the ligand molecule to

dock and its center and number of torsions and how many runs as well as the grid map

ﬁles and the docking algorithm to use. For conformation search, the Lamarckian genetic

algorithm (LGA) was applied, with the following parameters: number of runs for each

docking procedure (200), number of individuals in the population (300), the maximum

number of 27,000 generations simulated during each LGA run, the maximum number of

evaluations at 25,000,000, a mutation rate of 0.02 and a cross over rate of 0.80, while the

remaining docking parameters were set to default.

To identify the COX selectivity of ligands 8a–p, AutoDock 4.2.6 software was applied.

After performing molecular docking simulation, the best ligand molecules were evaluated

on the basis of their docking score against the COX receptors. The dockings experiments

were clustered with a root mean square deviation (RMSD) of 0.5 Å and were evaluated by

PyMOL software. Finally, the most energetically favored orientations were selected for

subsequent study.

3.3. In Vitro Radical Scavenging Assay

The antioxidant activity, as radical scavenging effect, of the new NO-IND-OXDs

8a–p was evaluated using 2,2-diphenyl-1-picrylhydrazyl radical (DPPH) assay with slight

modiﬁcation [130–132]. The results were analyzed using IND and ASP as reference drugs

and vitamin C as standard antioxidant.

Preparation of DPPH and test solutions. A weighed amount of DPPH (29.8 mg,

0.076 mmol) was dissolved by sonication in 50 mL methanol of analytical grade. Af-

ter 30 min at darkness, a sample of 10 mL was taken and made up to 100 mL with

methanol. The resulting DPPH solution (151.14

temperature and used up on the day of preparation. The stock solutions (2600

tested derivatives 8a–p were prepared in DMSO, then serially diluted with methanol to

obtain different concentrations (2600 M, 1500 M, 700 M, 620 M, 530 M, 440 M,

350 M, 260 M and 120 M). In the same manner with tested derivatives 8a–p, the

serially diluted solutions of IND and ASP were prepared. By dilution of vitamin C,

freshly prepared solution (2612 M) with methanol, serially diluted solutions (152.3 M,

133.1 µM, 112.4 µM, 96.1 µM, 75.3 µM, 55.1 µM, 40.1 µM, 20.5 µM) were also prepared.

µ

M) was stored in the darkness at room

µ

M) of

µ

Int. J. Mol. Sci. 2021, 22, 7079

25 of 32

DPPH assay procedure. First, 500

reference drugs (IND, ASP) and vitamin C was added to 1000

blanks (blank 1: 500 L of methanol and 1000 L of DPPH and blank 2: 1500

were also used. The mixture was kept for 3 h in the darkness at room temperature and then

a 270 L aliquot of each sample tube was added in a 96-well plate. The absorbance was

µ

L from each sample of the tested compounds 8a–p

L of DPPH solution. Two

L of methanol)

,

µ

then measured at 517 nm using Tecan Sunrise Remote Microplate Reader TW/ML-Abbott

F039306. All tests were performed in quadruplicate.

To calculate the DPPH radical-inhibiting capacity (scavenging activity, %), the follow-

ing formula was used [133,134]:

Inhibition (Scavenging activity) % = [(A_CS− A_s)/A_CS] × 100

(1)

where: A_sis the difference between the absorbance of tested sample and blank 2 and A_csis

the difference between absorbance of blank 1 and blank 2.

The IC₅₀(f_(x)= 50) of each tested compound was calculated by plotting the inhibition

ratios (f_(x)) against the sample concentration (x). The results for each experiment were

represented by a dose–response curve and two types of regression lines (f(x)) were used: a

sigmoid curve and a quadratic line.

The DPPH radical scavenging activity of each tested compound was also expressed

as the vitamin C equivalent antioxidant capacity (CEAC), which was calculated using the

following formula [135,136]:

CEAC = IC_50(vit

.

_C)/IC_50(sample)

(2)

where: IC_50(sample)is concentration of tested compounds 8a–p and reference drugs, respec-

tively, necessary for 50% inhibition and IC_{50(vit C)}is the concentration of vitamin C needed

.

for 50% inhibition. The higher CEAC value means higher DPPH radical scavenging activity.

3.4. Thermal Denaturation of Serum Proteins

The anti-inﬂammatory effects of the new NO-IND-OXZs 8a–p were predicted by a

modiﬁed Mizushima’s test [87,88]. The test consists of the denaturing effect on speciﬁc

proteins and assures a signiﬁcant correlation between the in vitro and in vivo effects. 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 0.1M hydrochloric acid, as denaturing agent,

in 0.9% NaCl, on the 0.2% BSA solution while the negative control consisted of untreated

0.2% BSA solution. Each sample (8a–p, IND, ASP) was tested at different concentrations

◦

(20

µM, 50

µM and 100

µ

M). The test samples and controls were incubated at 38 C for 5 h.

The degree of denaturation of BSA was quantiﬁed based on absorbance value, measured

at 450 nm, 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. The results were expressed as

averages of the percentage values (% effect) and were plotted versus the concentration of

tested sample.

3.5. In Vitro Nitric Oxide Release Measurement

To assess the capacity of new NO-IND-OXDs 8a–p to to release the nitric oxide

(NO), the Griess colorimetric method was applied [137,138]. The Griess reagents consist

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 3.4% (wt/v)

SULF and 1% (wt/v) mercuric chloride (SULF-HgCl₂) in 10% (wt/v) phosphoric acid. The

NO released from the sample was spontaneously oxidized to NO₂⁻, which subsequently

reacted with the Griess reagents to form an azo dye. As reference NO donors, S-nitroso-

N-acetyl-penicillamine (SNAP), sodium nitropruside (SNP) and nitroglycerine (NTG)

were used.

Int. J. Mol. Sci. 2021, 22, 7079

26 of 32

The experiments were performed in neutral (phosphate buffer solution—PBS) and

acidic (hydrochloric acid solution—HCl) experimental conditions, in the presence or ab-

sence of L-glutathione (GSH): PBS (pH 7.5), PBS-GSH (pH 7.51), HCl (pH 1.55), HCl-GSH

(pH 1.56).

Preparation of sodium nitrite and test solutions

A fresh sodium nitrite stock aqueous solution (0.1 M) was prepared and was stan-

dardized according to the procedure reported in the European Pharmacopoeia (real molar-

ity = 0.0998, molarity factor = 0.998) [139]. Then, the stock solution was serially diluted with

MeOH/H₂O = 1/1 (v/v) mixture obtaining different concentrations (100

µM, 50 µM, 25 µM,

12.50 M, 6.25 M, 3.125 M, 1.56 M and 0.78 M). The tested compounds (8a–p) and

µ

reference NO donors (SNAP, SNP, NTG) were dissolved in DMSO and water, respectively,

to afford a stock solution of 2600 µM.

The preparation of nitrite standard curve

An aliquot of 170

to 50 L solution of SULF in a 96-well plate. After 10 min, 50

added and then after another 20 min, the absorbance at 540 nm of the formed pink-red azo

dye was measured. A blank sample (containing 170 L PBS, 50 L SULF and 50 L NED)

µL sodium nitrite solution (in the range of 0.78–100

µM) was added

µ

µL of NED solution was

µ

was prepared under similar conditions. All tests were performed in quintuplicate and the

average absorbance was calculated. The calibration curve was constructed by graphical

representation of the mean absorbance value in relation to the corresponding concentration

of sodium nitrite.

NO release assay

A solution of 100

NTG) was prepared by diluting 80

PBS-GSH, HCl and HCl-GSH. These above solutions were kept at 37–38 ^◦C for 120 min;

after that, an aliquot of 170 L of each solution was measured and added to 50 L of SULF

and SULF-HgCl₂, respectively, in a 96-well plate. After 10 min, 50 L of NED solution was

added and after another 20 min, the absorbance at 540 nm of the formed pink-red azo dye

was measured. A blank sample (containing 170 L PBS/PBS-GSH/HCl/HCl-GSH, 50

SULF/SULF-HgCl₂and 50 L NED) was prepared under similar conditions. All tests were

µM NO-IND-OXDs 8a–p and reference NO donors (SNAP, SNP,

µ

L of each stock solution (2600 M) with 2 mL of PBS,

µ

µL

µ

performed in quadruplicate. The percentage (%) of NO release was calculated using the

following formula [137]:

% NO = (C_{f NO}× 100)/C_{t NO}

(3)

where: C_fNOis the found concentration and C_tNOis the theoretical concentration of

NO (µM).

3.6. Statistical Analysis

The results were expressed as mean value

±

standard deviation (SD) and the analysis

was performed using IBM SPSS Statistics 23 for Windows. The statistical signiﬁcance of the

results was assessed using the one-way and two-way analysis of variance (ANOVA’s test).

To compare the differences among samples, the Tukey’s HSD test was used. A p value less

than 0.05 was considered statistically signiﬁcant.

4. Conclusions

Based on the beneﬁcial effects of 1,3,4-oxadiazole-2-thiol scaffold and NO, new nitric

oxide-releasing indomethacin derivatives with 1,3,4-oxadiazole-2-thiol scaffold (NO-IND-

OXDs) have been synthesized. It is known that NO is an important endogenous molecule,

having a critical role in protecting the GI mucosa while 1,3,4-oxadiazole-2-thiol scaffold

is associated with important pharmacological effects, including anti-inﬂammatory and

antioxidant ones. The molecular docking study revealed that 8k, 8l and 8m are COX-2

inhibitors, more selective than celecoxib. It was noted that COX-2 selectivity is inﬂuenced

by the position of nitrate ester moiety, meta and para being more favorable than ortho,

as well as by the nature of the substituents from phenoxy-ethylnitrate moiety, the better

inﬂuence being associated with halogens (F, Cl, Br) and NO₂. The tested compounds also

Int. J. Mol. Sci. 2021, 22, 7079

27 of 32

showed improved radical scavenging as well as anti-inﬂammatory properties, the last one

measured as albumin denaturation effect. While for IND and ASP, a denaturation effect of

less than 5% was recorded, all the tested compounds showed a denaturation effect ranging

between 20% and 40% (at 100 µM). The NO release capacity of tested compounds is strongly

inﬂuenced by the position of nitrate ester moiety and by the nature of the substituents

from phenoxy-ethylnitrate moiety, being in good agreement with the COX-selectivity. The

results of our study, related to COX-2 in silico interaction, DPPH^•radical-scavenging

capacity, PBS denaturation-promoting effect and NO-releasing property, encourage us to

continue our research with in vivo inﬂammation model assay to prove the potential effect

of NO-IND-OXDs as multitarget strategy.

Supplementary Materials: The following are available online at https://ww w .mdpi.com/article/

10.3390/ijms22137079/s1.

Author Contributions: Conceptualization, L.P., S.R., F.B. and A.S.; methodology, A.S., A.P. and A.V.F.;

formal analysis, N.B. and A.P.; investigation, A.S., S.M.C. and F.G.L.; resources, A.S., S.R. and L.P.;

data curation, A.S., F.G.L. and A.V.F.; writing, A.S. and S.M.C.; writing—review and editing, A.S. and

F.B.; supervision, S.R. and L.P.; funding acquisition, S.R. and L.P. All authors have read and agreed to

the published version of the manuscript.

Funding: This research was funded by 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 FEDER under

projects Valbiocosm FEDER-FSE 2017-EX003202, CHembio FEDER-FSE 2014–2020-EX003677, Techsab

FEDER-FSE 2014–2020-EX011313; the SFR neuroimagerie (SFR FED 4224), the Labex programs

SYNORG (ANR-11-LABX-0029) and IRON (ANR-11-LABX-0018-01), the Ligue contre le Cancer du

Grand Ouest (comités des Deux Sèvres, du Finistère, de l’Ile et Villaine, du Loir et Cher, de Loire

Atlantique, du Loiret, de la Vienne).We thank the above for their ﬁnancial support.

Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.

Data Availability Statement: Samples of the compounds are not available from the authors.

Conﬂicts of Interest: The authors declare no conﬂict of interest.

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Design, synthesis, in silico and in vitro studies for new nitric oxide‐releasing indomethacin derivatives with 1,3,4‐oxadiazole‐2‐thiol scaffold

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