# Synthesis of novel 1,3,4-oxadiazole-derived α-aminophosphonates/α-aminophosphonic acids and evaluation of their in vitro antiviral activity against the avian coronavirus infectious bronchitis virus

Shaima Hkiri, Marwa Mekni-Toujani, Elvan Üstün, Karim Hosni, Abdeljelil

Ghram, Soufiane Touil, Ali Samarat, David Sémeril

► To cite this version:

Shaima Hkiri, Marwa Mekni-Toujani, Elvan Üstün, Karim Hosni, Abdeljelil Ghram, et al.. Synthesis of novel 1,3,4-oxadiazole-derived α-aminophosphonates/α-aminophosphonic acids and evaluation of their in vitro antiviral activity against the avian coronavirus infectious bronchitis virus. Pharmaceutics, 2023, 15 (1), pp.114. 10.3390/pharmaceutics15010114. [https://doi.org/10.3390/pharmaceutics15010114](https://doi.org/10.3390/pharmaceutics15010114). [hal-04241251](https://HAL.inistre.fr/hal-04241251v1)

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<figure><img src="image_1.png" /></figure>
1 Article

2 Synthesis of novel 1,3,4-oxadiazole-derived
3 α-aminophosphonates/α-aminophosphonic acids and evalua-
4 tion of their *in vitro* antiviral activity against the avian corona-
5 virus infectious bronchitis virus

6 Shaima Hkiri <fnref n="1" />,<fnref n="2" />, Marwa Mekni-Toujani <fnref n="3" />, Elvan Üstün <fnref n="4" />, Karim Hosni <fnref n="5" />, Abdeljelil Ghram <fnref n="3" />, Soufiane Touil <fnref n="2" />, Ali

7 Samarat <fnref n="2" /> and David Sémeril <fnref n="1" />,*

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Citation: To be added by editorial 28

staff during production. 29

Academic Editor: Firstname Last-
name 30

Received: date 33

Accepted: date 34

Published: date 35

Publisher's Note: MDPI stays neutral with regard to jurisdiction. 36
claims in published maps and 37
institutional affiliations. 38

<figure><img src="image_2.png" /></figure>

Copyright: © 2022 by the authors.
Submitted for possible open access
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**Abstract:** An efficient and simple approach has been developed for the synthesis of eight dialkyl/aryl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(aryl)methyl]phosphonates, through the Pudovik-type reaction of dialkyl/arylphosphite with imines, obtained from 5-phenyl-1,3,4-oxadiazol-2-amine and aromatic aldehydes, under microwave irradiation. Five of them were hydrolyzed to lead to the corresponding phosphonic acids. Selected synthesized compounds were screened for their *in vitro* antiviral activity against the avian bronchitis virus (IBV). In the MTT cytotoxicity assay, the dose-response curve showed that all test compounds were safe in the range concentration of 540-1599 µM. The direct contact of novel synthesized compounds with IBV showed that the diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethoxyphenyl)methyl]phosphonate (5f) (at 33 µM) and the [(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethylphenyl)methyl]phosphonic acid (6a) (at 1.23 µM) strongly inhibited the IBV infectivity indicating their high virucidal activity. However, virus titers from IBV-infected Vero cells remained unchanged in response to treatment with the lowest non-cytotoxic concentrations of synthesized compounds suggesting their incapacity to inhibit the virus replication inside the host cell. The lacks of antiviral activity might presumably be ascribed to their polarity that hampers their diffusion across the lipophilic cytoplasmic membrane. Therefore, the interactions of 5f and 6a were analyzed against coronavirus main protease, papain-like protease and nucleocapsid protein by molecular docking methods. Nevertheless, the novel 1,3,4-oxadiazole-based α-aminophosphonic acids and α-aminophosphonates hold potential for the development of new hygienic virucidal products for domestic, chemical, and medical uses.

**Keywords:** α-aminophosphonate; α-aminophosphonic acid; 1,3,4-oxadiazole; infectious bronchitis virus; virucidal activity; coronavirus.

1. Introduction

Coronaviruses (Family Coronaviridae) are highly infectious RNA viruses that affect respiratory, gastrointestinal, urinary and neurological systems of mammals and birds [1-4]. Depending on their genome, coronaviruses (CoVs) are divided into four groups,

<footnote n="1">¹ University of Strasbourg, Synthèse Organométallique et Catalyse, UMR-CNRS 7177, 4 rue Blaise Pascal, 67008 Strasbourg, France</footnote>

<footnote n="2">² University of Carthage, Faculty of Sciences of Bizerte, LR18ES11, Laboratory of Hetero-Organic Compounds and Nanostructured Materials, 7021, Bizerte, Tunisia</footnote>

<footnote n="3">³ University of Tunis El Manar, Institute Pasteur of Tunis, LR19IP03, Laboratory of Epidemiology and Veterinary Microbiology, BP 74, Tunis-Belvedere 1002, Tunisia</footnote>

<footnote n="4">⁴ University of Ordu, Faculty of Science and Arts, Department of Chemistry, 52200, Ordu, Turkey</footnote>

<footnote n="5">5 Institut National de Recherche et d'Analyse Physico-chimique (INRAP), Laboratoire des Substances Naturelles, Biotechpôle de Sidi Thabet, 2020, Tunisia</footnote>

<footnote>* Correspondence: dsemeril@unistra.fr; Tel.: +33 (0)3 68 85 15 50</footnote>
alpha-, beta-, gamma- and delta-coronaviruses [5]. The former groups' alpha and be-
ta-coronaviruses are particularly worrisome because they can cross the species barriers
and infect humans causing thus diseases ranging from upper respiratory tract infections
to severe acute respiratory syndrome (SARS) [6].

Avian infectious bronchitis virus (IBV), a widespread gamma-coronavirus that in-
fect both wild birds like in Mont-Saint-Michel bay last summer and breeding birds like
chickens, which is one of the foremost causes of economic losses to the poultry industry
as a result of disturbance in feed conversion and weight gain with a concomitant reduc-
tion in egg production and quality, and in some case increased chickens mortality [7]. As
for SARS-CoV-2, IBV exhibited high genomic diversity and elevated frequency of muta-
tion leading to different serotypes of the virus that complicate the control of IBV and
widen its host range [8]. The latter biological property is determined by the spike glyco-
protein (key determinant of virus pathogenicity) which allow virus to bind to different
target cells including respiratory (nose, trachea, lung and pulmonary alveoli), kidneys,
oviduct, testes, and alimentary (oesophagus, proventriculus, duodenum, jejunum, bursa
of Fabricius, rectum and cloaca) [8-10].

For the control of IBV, vaccination using live vaccine, inactivated virus and subunit
vaccine, and vector vaccine represents the main way although its inefficiency in manag-
ing some variant serotypes [7,8]. Therefore, searching for alternative and efficient treat-
ment for IBV controlling is a challenging task. Recent efforts in this direction have suc-
cessfully identified some putative anti-IBV including natural like plant-derived oleores-
ins, essential oils and extracts (i.e. extracts from *Mentha piperita*, *Melissa officinalis*, *Thy-
mus vulgaris*, *Hyssopus officinalis*, *Salvia officinalis*) [7,11,12]. Alternatively, synthetic
chemicals (i.e. lithium chloride) have also been applied as potent anti-IBV [13].

As bioisosters [14] of amino acids, α-aminophosphonates and their
α-aminophosphonic acid derivatives have attracted wide attention in medicinal chemis-
try and agrochemical sciences. Indeed, the tetrahedral conformation of the phosphorus
atom allows it to be used in place of a carbon atom, which makes it possible to obtain
analogous transition states [15] with the receptors to which amino acids normally bind.
Taking into account their broad spectrum of biological activities, α-aminophosphonates
and α-aminophosphonic acids have been the subjects of intense research in recent years
[16,17]. Indeed, these compounds are known as enzyme inhibitor [18-21], anti-human
immunodeficiency virus (anti-HIV) [22], anticancer [23-26], antibacterial [27,28] and as
herbicides [29,31] of which the best known are glyphosate (Round up®) and Dufulin
(Figure 1).

Recently, drugs containing an 1,3,4-oxadiazole ring have emerged as promising antiviral agents [32-34] and some of them are commercialized such as anti-HIV (Raltegravir) or antibiotic (Furamizole) (Figure1).

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Examples of antiviral agents based on α-aminophosphonate/α-aminophosphonic acid and 1,3,4-oxadiazole scaffolds.</figcaption></figure>

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In this context and in connection with our research projects on the synthesis of novel phosphorinated compounds with possible biological properties [35,36], we now report an efficient, fast and simple way to obtain 1,3,4-oxadiazole-derived α-aminophosphonates/α-aminophosphonic acids. A selection of the synthesized compounds was tested for their possible antiviral activity against IBV virus. The activity details of the two most active compounds were analyzed by molecular docking methods against coronavirus main proteinase, papain-like protease, and N/C-terminal domain of coronavirus nucleocapsid protein.

## 2. Materials and Methods

### 2.1. Apparatus, Materials, and Analysis

All manipulations involving phosphorus derivatives were carried out under dry argon. Solvents were dried by conventional methods and were distilled immediately before use. Routine ¹H, ¹³C{¹H}, ³¹P{¹H} and ¹⁹F{¹H} spectra were recorded with Bruker FT instruments (AC 300 and 500). ¹H NMR spectra were referenced to residual protonated solvents (δ = 2.50 ppm for DMSO-d₆). ¹³C NMR chemical shifts are reported relative to deuterated solvents (δ = 39.52 ppm for DMSO-d₆). ³¹P and ¹⁹F NMR spectroscopic data are given relative to external H₃PO₄ and CCl₃F, respectively. Chemical shifts and coupling constants are reported in ppm and Hz, respectively. Microwave irradiation was carried out using the CEM Discover microwave synthesis system equipped with a pressure controller (17 bar). Melting points were determined with a Büchi 535 capillary melting point apparatus. Infrared spectra were recorded on a Bruker FT-IR Alpha-P spectrometer. Elemental analyses were carried out by the Service de Microanalyse, Institut de Chimie, Université de Strasbourg. 5-Phenyl-1,3,4-oxadiazol-2-amine (1) [37], diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethylphenyl)methyl]phosphonate (5a) [38], diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-nitrophenyl)methyl]phosphonate (5b) [38] and diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-fluorophenyl)methyl]phosphonate (5c) [39] were prepared by literature procedures.

### 2.2. General procedure for the synthesis of (E)-1-(aryl)-N-(5-phenyl-1,3,4-oxadiazol-2-yl) methanimine

In a round bottomed flask equipped with a Dean Stark apparatus, a mixture of phenyl-1,3,4-oxadiazol-2-amine (1) (2.0 mmol) and aryl aldehyde (3.0 mmol) in toluene (20 mL) was refluxed for 48 h. After cooling to room temperature, the solvent was evaporated and the crude product was washed with cool ethanol (15 mL), filtered and dried under vacuum to afford the desired imine.

**(E)-1-(3,4,5-Trifluophenyl)-N-(5-phenyl-1,3,4-oxadiazol-2-yl)methanimine (2d).**
White solid, yield 89 %. ¹H NMR (300 MHz, DMSO-d₆): δ = 9.34 (s, 1H, N=CH), 8.11-8.04 (m, 4H, arom. CH), 7.69-7.60 (m, 3H, arom. CH) ppm; ¹³C{¹H} NMR (126 MHz, DMSO-d₆): δ = 167.2 (s, N=CH), 165.5 (s, arom. Cquat C(N)=N), 163.1 (s, arom. Cquat C(Ph)=N), 150.7 (dd, arom. Cquat meta CF, ¹J<sub>CF</sub> = 250.6 Hz, ²J<sub>CF</sub> = 10.1 Hz), 142.4 (dt, arom. Cquat para CF, ¹J<sub>CF</sub> = 258.8 Hz, ²J<sub>CF</sub> = 14.1 Hz), 132.2 (s, arom. CH), 130.9 (s, arom. Cquat of C<sub>6</sub>H<sub>2</sub>F<sub>3</sub>), 129.5 (s, arom. CH), 126.4 (s, arom. CH), 123.4 (s, arom. Cquat of C<sub>6</sub>H<sub>5</sub>), 114.7 (d, arom. CH, ²J<sub>CF</sub> = 21.7 Hz) ppm; ¹⁹F{¹H} NMR (282 MHz, DMSO-d₆): δ = -133.1 (d, meta CF of C<sub>6</sub>H<sub>2</sub>F<sub>3</sub>, ³J<sub>FF</sub> = 20.6 Hz), -152.2 (t, para CF of C<sub>6</sub>H<sub>2</sub>F<sub>3</sub>, ³J<sub>FF</sub> = 20.6 Hz) ppm. IR: ν = 1650 (C=NH), 1614 (C=N) cm⁻¹. Elemental analysis calcd. (%) for C<sub>15</sub>H<sub>8</sub>ON<sub>3</sub>F<sub>3</sub> (303.24): C 59.41, H 2.66, N 13.86; found C 59.45, H 2.69 N, 13.81.

**(E)-1-(3,5-Bis-trifluoromethyl-phenyl)-N-(5-phenyl-1,3,4-oxadiazol-2-yl)methane- mine (2e).**
White solid, yield 73 %. ¹H NMR (300 MHz, DMSO-d₆): δ = 9.58 (s, 1H, N=CH), 8.75 (s, 2H, arom. CH of C<sub>6</sub>H<sub>3</sub>(CF<sub>3</sub>)<sub>2</sub>), 8.47 (s, 1H, arom. CH of C<sub>6</sub>H<sub>3</sub>(CF<sub>3</sub>)<sub>2</sub>), 8.08 (d, 2H, arom. CH of C<sub>6</sub>H<sub>5</sub>, ³J<sub>HH</sub> = 6.0 Hz), 7.69-7.63 (m, 3H, arom. CH of C<sub>6</sub>H<sub>5</sub>) ppm; ¹³C{¹H} NMR (126 MHz, DMSO-d₆): δ = 167.6 (s, N=CH), 165.5 (s, arom. Cquat C(N)=N), 163.2 (s, arom. Cquat C(Ph)=N), 136.6 (s, arom. Cquat of C<sub>6</sub>H<sub>3</sub>(CF<sub>3</sub>)<sub>2</sub>), 132.3 (s, arom. CH),
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131.2 (q, arom. Cquat CCF<sub>3</sub>, <sup>2</sup>J<sub>CF</sub> = 33.5 Hz), 130.2 (s, arom. CH), 129.5 (s, arom. CH), 126.8 (s, arom. CH), 126.5 (s, arom. CH), 123.3 (s, arom. Cquat of C<sub>6</sub>H<sub>5</sub>), 123.0 (q, CF<sub>3</sub>, <sup>1</sup>J<sub>CF</sub> = 273.4 Hz) ppm; <sup>19</sup>F{<sup>1</sup>H} NMR (282 MHz, DMSO-d<sub>6</sub>): δ = -61.5 (s, CF<sub>3</sub>) ppm. IR: ν = 1645 (C=NH), 1615 (C=N) cm<sup>-1</sup>. Elemental analysis calcd. (%) for C<sub>17</sub>H<sub>9</sub>ON<sub>3</sub>F<sub>6</sub> (385.26): C 53.00, H 2.35, N 10.91; found C 52.86, H 2.41, N 10.88.

(E)-1-(4-Trifluoromethoxyphenyl)-N-(5-phenyl-1,3,4-oxadiazol-2-yl)methanimine (2f). White solid, yield 83 %. <sup>1</sup>H NMR (300 MHz, DMSO-d<sub>6</sub>): δ = 9.40 (s, 1H, N=CH), 8.25 (d, 2H, arom. CH of C<sub>6</sub>H<sub>4</sub>OCF<sub>3</sub>, <sup>3</sup>J<sub>HH</sub> = 9.0 Hz), 8.09-8.06 (m, 2H, arom. CH), 7.66-7.59 (m, 5H, arom. CH) ppm; <sup>13</sup>C{<sup>1</sup>H} NMR (126 MHz, DMSO-d<sub>6</sub>): δ = 168.5 (s, N=CH), 166.0 (s, arom. Cquat C(N)=N), 162.9 (s, arom. Cquat C(Ph)=N), 152.1 (s, arom. Cquat COCF<sub>3</sub>), 133.28 (s, arom. Cquat of C<sub>6</sub>H<sub>4</sub>OCF<sub>3</sub>), 132.5 (s, arom. CH), 131.1 (s, arom. CH), 129.5 (s, arom. CH), 126.4 (s, arom. CH), 123.5 (s, arom. Cquat of C<sub>6</sub>H<sub>5</sub>), 121.4 (s, arom. CH), 119.9 (q, OCF<sub>3</sub>, <sup>1</sup>J<sub>CF</sub> = 258.4 Hz) ppm; <sup>19</sup>F{<sup>1</sup>H} NMR (282 MHz, DMSO-d<sub>6</sub>): δ = -56.6 (s, OCF<sub>3</sub>) ppm. IR: ν = 1650 (C=NH), 1612 (C=N) cm<sup>-1</sup>. Elemental analysis calcd. (%) for C<sub>16</sub>H<sub>10</sub>O<sub>2</sub>N<sub>3</sub>F<sub>3</sub> (333.27): C 57.66, H 3.02, N 12.61; found C 57.48, H 2.99, N 12.46.

## 2.3. General procedure for the synthesis of dialkyl/aryl[(5-phenyl-1,3,4-oxadiazol-2-ylamino) (aryl)methyl]phosphonate

In a round bottomed flask, a mixture of (E)-1-(aryl)-N-(5-phenyl-1,3,4-oxadiazol-2-yl)methanimine (1.0 mmol) and dialkyl/arylphosphite (2.0 mmol) was added and irradiated under microwave in neat condition at 115°C for 10 min at 300 W. After cooling to room temperature, the crude product was washed with Et<sub>2</sub>O (3 x 10 mL), filtered and dried under vacuum.

**Dimethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethylphenyl)methyl]phosphonate (3).** Yield 54 %. <sup>1</sup>H NMR (300 MHz, DMSO-d<sub>6</sub>): δ = 9.17 (dd, <sup>1</sup>H, NH, <sup>3</sup>J<sub>HH</sub> = 9.6 Hz, <sup>3</sup>J<sub>PH</sub> = 3.6 Hz), 7.84-7.78 (m, 6H, arom. CH), 7.56-7.53 (m, 3H, arom. CH), 5.46 (dd, 1H, CHP, <sup>2</sup>J<sub>PH</sub> = 22.5 Hz, <sup>3</sup>J<sub>HH</sub> = 9.6 Hz), 3.70 (d, 3H, OCH<sub>3</sub>, <sup>3</sup>J<sub>PH</sub> = 10.5 Hz), 3.61 (d, 3H, OCH<sub>3</sub>, <sup>3</sup>J<sub>PH</sub> = 10.8 Hz) ppm; <sup>13</sup>C{<sup>1</sup>H} NMR (126 MHz, DMSO-d<sub>6</sub>): δ = 162.9 (d, arom. Cquat para of CF<sub>3</sub>, <sup>2</sup>J<sub>CP</sub> = 11.5 Hz), 158.5 (s, arom. Cquat C(Ph)=N), 140.3 (s, arom. Cquat C(NH)=N), 130.8 (s, arom. CH), 129.3 (s, arom. CH), 128.9 (d, arom. CH, <sup>3</sup>J<sub>CP</sub> = 4.8 Hz), 128.5 (q, arom. Cquat CCF<sub>3</sub>, <sup>2</sup>J<sub>CF</sub> = 32.4 Hz), 125.3 (s, arom. CH), 125.3 (s, arom. CH), 124.2 (q, CF<sub>3</sub>, <sup>1</sup>J<sub>CF</sub> = 272.8 Hz), 123.9 (s, arom. Cquat of C<sub>6</sub>H<sub>5</sub>), 53.7 (d, OCH<sub>3</sub>, <sup>2</sup>J<sub>CP</sub> = 25.7 Hz), 53.6 (d, OCH<sub>3</sub>, <sup>2</sup>J<sub>CP</sub> = 25.7 Hz), 53.5 (d, CHP, <sup>1</sup>J<sub>CP</sub> = 153.5 Hz) ppm; <sup>31</sup>P{<sup>1</sup>H} NMR (121 MHz, DMSO-d<sub>6</sub>): δ = 21.7 (q, P(O), <sup>7</sup>J<sub>PF</sub> = 2.3 Hz) ppm; <sup>19</sup>F{<sup>1</sup>H} NMR (282 MHz, DMSO-d<sub>6</sub>): δ = -61.0 (d, CF<sub>3</sub>, <sup>7</sup>J<sub>PF</sub> = 2.3 Hz) ppm. IR: ν = 1618 (C=N) cm<sup>-1</sup>. Elemental analysis calcd. (%) for C<sub>18</sub>H<sub>17</sub>O<sub>4</sub>N<sub>3</sub>F<sub>3</sub>P (427.31): C 50.59, H 4.01, N 9.83; found C 50.48, H 3.94, N 9.75.

**Diphenyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethylphenyl)methyl]phosphonate (4).** Yield 90 %. <sup>1</sup>H NMR (300 MHz, DMSO-d<sub>6</sub>): δ = 9.55 (dd, 1H, NH, <sup>3</sup>J<sub>HH</sub> = 10.2 Hz, <sup>3</sup>J<sub>PH</sub> = 3.0 Hz), 7.97 (d, 2H, arom. CH, <sup>3</sup>J<sub>HH</sub> = 7.5 Hz), 7.86-7.81 (m, 4H, arom. CH), 7.56-7.54 (m, 3H, arom. CH), 7.38-7.31 (m, 4H, arom. CH), 7.19 (t, 1H, arom. CH, <sup>3</sup>J<sub>HH</sub> = 7.4 Hz), 7.19 (t, 1H, arom. CH, <sup>3</sup>J<sub>HH</sub> = 7.4 Hz), 7.10 (d, 2H, arom. CH, <sup>3</sup>J<sub>HH</sub> = 8.4 Hz), 7.02 (d, 2H, arom. CH, <sup>3</sup>J<sub>HH</sub> = 8.4 Hz), 5.95 (dd, 1H, CHP, <sup>2</sup>J<sub>PH</sub> = 23.1 Hz, <sup>3</sup>J<sub>HH</sub> = 10.0 Hz) ppm; <sup>13</sup>C{<sup>1</sup>H} NMR (126 MHz, DMSO-d<sub>6</sub>): δ = 162.8 (d, arom. Cquat para of CF<sub>3</sub>, <sup>2</sup>J<sub>CP</sub> = 11.3 Hz), 158.7 (s, arom. Cquat C(Ph)=N), 149.9 (d, arom. Cquat of OC<sub>6</sub>H<sub>5</sub>, <sup>2</sup>J<sub>CP</sub> = 9.7 Hz), 149.7 (d, arom. Cquat of OC<sub>6</sub>H<sub>5</sub>, <sup>2</sup>J<sub>CP</sub> = 9.2 Hz), 139.0 (s, arom. Cquat C(NH)=N), 130.9 (s, arom. CH), 129.9 (s, arom. CH), 129.3 (s, arom. CH), 129.0 (q, arom. Cquat CCF<sub>3</sub>, <sup>2</sup>J<sub>CF</sub> = 33.4 Hz), 125.5 (s, arom. CH), 125.5 (s, arom. CH), 125.5 (s, arom. CH), 125.4 (s, arom. CH), 124.1 (q, CF<sub>3</sub>, <sup>1</sup>J<sub>CF</sub> = 272.5 Hz), 123.0 (s, arom. Cquat of C<sub>6</sub>H<sub>5</sub>), 120.3 (s, arom. CH), 120.3 (s, arom. CH), 54.6 (d, CHP, <sup>1</sup>J<sub>CP</sub> = 157.1 Hz) ppm; <sup>31</sup>P{<sup>1</sup>H} NMR (121 MHz, DMSO-d<sub>6</sub>): δ = 12.6 (q, P(O), <sup>7</sup>J<sub>PF</sub> = 2.4 Hz) ppm; <sup>19</sup>F{<sup>1</sup>H} NMR (282 MHz, DMSO-d<sub>6</sub>): δ = -61.1 (d, CF<sub>3</sub>, <sup>7</sup>J<sub>PF</sub> = 2.4 Hz) ppm. IR: ν = 1615 (C=N) cm<sup>-1</sup>. Elemental analysis calcd. (%) for C<sub>28</sub>H<sub>21</sub>O<sub>4</sub>N<sub>3</sub>F<sub>3</sub>P (551.45): C 60.98, H 3.84, N 7.62; found C 61.03, H 3.88, N 7.55.

**Diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(3,4,5-trifluophenyl)methyl]phosphonate (5d).** White solid, yield 90 %. <sup>1</sup>H NMR (300 MHz, DMSO-d<sub>6</sub>): δ = 9.00 (dd, 1H,
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NH, ³JHH = 6.0 Hz, ³JPH = 1.8 Hz), 7.85-7.83 (m, 2H, arom. CH), 7.59-7.53 (m, 5H, arom. CH), 5.38 (dd, 1H, CHP, ²JPH = 13.2 Hz, ³JHH = 6.0 Hz), 4.10-3.89 (m, 4H, OCH₂CH₃), 1.18 (t, 3H, OCH₂CH₃, ³JHH = 4.2 Hz), 1.12 (t, 3H, OCH₂CH₃, ³JHH = 4.2 Hz) ppm; ¹³C{¹H} NMR (126 MHz, DMSO-d₆): δ = 162.8 (d, arom. Cquat of C₆H₂F₃, ²JCP = 10.7 Hz), 158.5 (s, arom. Cquat C(Ph)=N), 149.9 (dd, arom. Cquat CF, ¹JCF = 247.5 Hz, ²JCF = 9.2 Hz), 138.3 (dt, arom. Cquat CF, ¹JCF = 250.0 Hz, ²JCF = 14.4 Hz), 133.0 (d, arom. Cquat C(NH)=N, ³JCP = 4.0 Hz), 130.8 (s, arom. CH), 129.3 (s, arom. CH), 125.3 (s, arom. CH), 123.9 (s, arom. Cquat of C₆H₅), 112.9 (dd, arom. CH of C₆H₂F₃, ²JCF = 17.5 Hz, ³JCF = 4.5 Hz), 63.0 (d, CH₂CH₃, ²JCP = 19.1 Hz), 63.0 (d, CH₂CH₃, ²JCP = 18.9 Hz), 53.2 (d, CHP, ¹JCP = 154.7 Hz), 16.2 (d, CH₂CH₃, ³JCP = 14.1 Hz), 16.1 (d, CH₂CH₃, ³JCP = 14.4 Hz) ppm; ³¹P{¹H} NMR (121MHz, DMSO-d₆): δ = 18.9 (dt, P(O), ⁶JPF = 4.8 Hz, ⁵JPF = 1.4 Hz) ppm; ¹⁹F{¹H} NMR (282 MHz, DMSO-d₆): δ = -135.1 (dd, meta CF of C₆H₂F₃, ³JFF = 21.7 Hz, ⁵JPF = 1.2 Hz), -162.0 (td, para CF of C₆H₂F₃, ³JFF = 21.7 Hz, ⁶JPF = 5.0 Hz) ppm. IR: ν = 1598 (C=N) cm⁻¹. Elemental analysis calcd. (%) for C₁₉H₁₈O₄N₃F₃P (440.33): C 51.82, H 4.12, N 9.54; found C 51.78, H 4.07, N 9.41.

**Diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(3,5-bis-trifluoromethyl-phenyl) methyl]phosphonate (5e).** White solid, yield 97 %. ¹H NMR (300 MHz, DMSO-d₆): δ = 9.18 (dd, 1H, NH, ³JHH = 10.2 Hz, ³JPH = 3.3 Hz), 8.36 (s, 2H, arom. CH of C₆H₃(CF₃)₂), 8.10 (s, 1H, arom. CH of C₆H₃(CF₃)₂), 7.87-7.84 (m, 2H, arom. CH), 7.55-7.53 (m, 3H, arom. CH), 5.69 (dd, 1H, CHP, ²JPH = 22.2 Hz, ³JHH = 10.2 Hz), 4.12-3.90 (m, 4H, OCH₂CH₃), 1.16 (t, 3H, OCH₂CH₃, ³JHH = 7.0 Hz), 1.08 (t, 3H, OCH₂CH₃, ³JHH = 7.0 Hz) ppm; ¹³C{¹H} NMR (126 MHz, DMSO-d₆): δ = 162.7 (d, arom. Cquat of C₆H₃(CF₃)₂, ²JCP = 9.7 Hz), 158.5 (s, arom. Cquat C(Ph)=N), 139.3 (s, arom. Cquat C(NH)=N), 130.9 (s, arom. CH), 130.1 (q, arom. Cquat CCF₃, ²JCF = 32.8 Hz), 129.3 (s, arom. CH), 129.1 (s, arom. CH), 125.3 (s, arom. CH), 123.3 (q, CF₃, ¹JCF = 273.4 Hz), 123.9 (s, arom. Cquat of C₆H₅), 121.8 (s, arom. CH), 63.1 (d, OCH₂CH₃, ²JCP = 30.4 Hz), 63.0 (d, OCH₂CH₃, ²JCP = 30.4 Hz), 53.4 (d, CHP, ¹JCP = 152.8 Hz), 16.0 (d, OCH₂CH₃, ³JCP = 22.5 Hz), 16.0 (d, OCH₂CH₃, ³JCP = 22.7 Hz) ppm; ³¹P{¹H} NMR (121 MHz, DMSO-d₆): δ = 18.6 (s, P(O)) ppm; ¹⁹F{¹H} NMR (282 MHz, DMSO-d₆): δ = -61.3 (s, CF₃) ppm. IR: ν = 1608 (C=N) cm⁻¹. Elemental analysis calcd. (%) for C₂₁H₂₀O₄N₃F₆P (523.37): C 48.19, H 3.85, N 8.03; found C 48.35, H 4.01, N 7.92.

**Diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethoxyphenyl)methyl] phosphonate (5f).** White solid, yield 92 %. M.p. 106.6-107.5°C; ¹H NMR (300 MHz, DMSO-d₆): δ = 9.06 (dd, ¹H, NH, ³JHH = 9.9 Hz, ³JPH = 3.6 Hz), 7.85-7.81 (m, 2H, arom. CH), 7.69 (dd, 2H, arom. CH, ³JHH = 8.4 Hz, ⁴JHH = 1.8 Hz), 7.56-7.51 (m, 3H, arom. CH), 7.40 (d, 2H, arom. CH, ³JHH = 8.4 Hz), 5.30 (dd, 1H, CHP, ²JPH = 21.9 Hz, ³JHH = 9.9 Hz), 4.11-3.82 (m, 4H, OCH₂CH₃), 1.17 (t, 3H, OCH₂CH₃, ³JHH = 7.5 Hz), 1.08 (t, 3H, OCH₂CH₃, ³JHH = 7.1 Hz) ppm; ¹³C{¹H} NMR (126 MHz, DMSO-d₆): δ = 163.0 (d, arom. Cquat para of OCF₃, ²JCP = 11.0 Hz), 158.4 (s, arom. Cquat C(Ph)=N), 148.0 (s, arom. Cquat C(NH)=N), 135.0 (s, arom. Cquat COCF₃), 130.8 (s, arom. CH), 130.1 (d, arom. CH, ³JCP = 4.8 Hz), 129.3 (s, arom. CH), 125.3 (s, arom. CH), 123.1 (s, arom. Cquat of C₆H₅), 120.9 (s, arom. CH), 120.1 (q, OCF₃, ¹JCF = 256.8 Hz), 62.8 (d, OCH₂CH₃, ²JCP = 18.8 Hz), 62.8 (d, OCH₂CH₃, ²JCP = 18.5 Hz), 53.7 (d, CHP, ¹JCP = 154.7 Hz), 16.2 (d, OCH₂CH₃, ³JCP = 5.0 Hz), 16.1 (d, OCH₂CH₃, ³JCP = 5.1 Hz) ppm; ³¹P{¹H} NMR (121 MHz, DMSO-d₆): δ = 19.7 (s, P(O)) ppm; ¹⁹F{¹H} NMR (282 MHz, DMSO-d₆): δ = -56.8 (s, OCF₃) ppm. IR: ν = 1607 (C=N) cm⁻¹. Elemental analysis calcd. (%) for C₂₀H₂₁O₅N₃F₃P (471.37): C 50.96, H 4.49, N 8.91; found C 50.83, H 4.37, N 8.86.

## 2.4. General procedure for the synthesis of [(5-phenyl-1,3,4-oxadiazol-2-ylamino)(aryl)methyl] phosphonic acids

In a Schlenk tube, a mixture of diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(aryl) methyl]phosphonate (0.5 mmol) and trimethylsilyl bromide (0.53 mL, 4.0 mmol) were dissolved in CH₂Cl₂ (5 mL). The reaction mixture was stirred at room temperature. After 72 h, the reaction mixture was evaporated to dryness and the crude product was dissolved into a solution of NaOH 2 M (10 mL) and filtrated. The white product was pre-
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[(5-Phenyl-1,3,4-oxadiazol-2-ylamino)(4-trifluoromethylphenyl)methyl]phosphonic acid (6a). White solid, yield 92 %. M.p. > 260°C; ¹H NMR (300 MHz, DMSO-d6): δ = 8.98 (br s, 1H, NH), 7.78-7.71 (m, 6H, arom. CH), 7.52-7.51 (m, 3H, arom. CH), 5.02 (d, 1H, CHP, ²JₚH = 21.7 Hz) ppm; ¹³C{¹H} NMR (126 MHz, DMSO-d6): δ = 163.3 (d, arom. Cquat para of CF₃, ²JₚCP = 11.2 Hz), 158.1 (s, arom. Cquat C(Ph)=N), 142.3 (s, arom. Cquat C(NH)=N), 130.7 (s, arom. CH), 129.3 (s, arom. CH), 128.8 (d, arom. CH, ³JₚCP = 4.0 Hz), 127.8 (q, arom. Cquat CCF₃, ²JₚCF = 32.3 Hz), 125.2 (s, arom. CH), 124.8 (s, arom. CH), 124.4 (q, CF₃, ¹JₚCF = 272.7 Hz), 124.1 (s, arom. Cquat of C₆H₅), 56.0 (d, CHP, ¹JₚCF = 146.8 Hz) ppm; ³¹P{¹H} NMR (121 MHz, DMSO-d6): δ = 14.3 (q, P(O), ⁷JₚF = 1.2 Hz) ppm; ¹⁹F{¹H} NMR (282 MHz, DMSO-d6): δ = -60.8 (d, CF₃, ⁷JₚF = 2.2 Hz) ppm. IR: ν = 1619 (C=N) cm⁻¹. Elemental analysis calcd. (%) for C₁₆H₁₃O₄N₃F₃P (399.26): C 48.13, H 3.28, N 16.03; found C 48.05, H 3.23, N 15.96.

[(5-Phenyl-1,3,4-oxadiazol-2-ylamino)(4-nitrophenyl)methyl]phosphonic acid (6b). White solid, yield 87 %. ¹H NMR (300 MHz, DMSO-d6): δ = 8.92 (s br, 1H, NH), 8.55 (s br, 2H, P(OH)₂), 8.22 (d, 2H, arom. CH, ³JₚHH = 8.7 Hz), 7.81-7.77 (m, 4H, arom. CH), 7.53-7.51 (m, 3H, arom. CH), 5.10 (d, 1H, CHP, ²JₚPH = 22.5 Hz) ppm; ¹³C{¹H} NMR (126 MHz, DMSO-d6): δ = 163.7 (d, arom. Cquat para of NO₂, ²JₚCP = 11.2 Hz), 158.6 (s, arom. Cquat C(Ph)=N), 147.2 (d, arom. Cquat C(NH)=N, ³JₚCP = 2.1 Hz), 146.0 (s, arom. Cquat CNO₂), 131.1 (s, arom. CH), 129.7 (s, arom. CH), 129.7 (d, arom. CH, ³JₚCP = 3.9 Hz), 125.7 (s, arom. CH), 124.5 (s, arom. Cquat of C₆H₅), 123.5 (s, arom. CH), 56.6 (d, CHP, ¹JₚCP = 145.0 Hz) ppm; ³¹P{¹H} NMR (121 MHz, DMSO-d6): δ = 13.8 (s, P(O)) ppm. IR: ν = 1623 (C=N) cm⁻¹. Elemental analysis calcd. (%) for C₁₅H₁₃O₆N₄P (376.26): C 47.88, H 3.48, N 14.89; found C 47.76, H 3.61, N 14.75.

[(5-Phenyl-1,3,4-oxadiazol-2-ylamino)(4-fluorophenyl)methyl]phosphonic acid (6c). White solid, yield 95 %. ¹H NMR (300 MHz, DMSO-d6): δ = 8.73 (br s, 1H, NH), 7.82-7.79 (m, 2H, arom. CH), 7.57-7.51 (m, 5H, arom. CH), 7.17 (t, 2H, arom. CH, ³JₚHH = 8.8 Hz), 4.94 (d, 1H, CHP, ²JₚPH = 21.6 Hz) ppm; ¹³C{¹H} NMR (126 MHz, DMSO-d6): δ = 163.3 (d, arom. Cquat of C₆H₄F, ²JₚCP = 10.8 Hz), 161.5 (d, arom. Cquat CF, ¹JₚCF = 243.3 Hz), 158.0 (s, arom. Cquat C(Ph)=N), 133.5 (d, arom. Cquat C(NH)=N, ³JₚCP = 2.3 Hz), 130.6 (s, arom. CH of C₆H₅), 130.5 (dd, arom. CH of C₆H₄F, ³JₚCF = 7.6 Hz, ³JₚCP = 5.3 Hz), 129.3 (s, arom. CH of C₆H₅), 125.2 (s, arom. CH of C₆H₅), 124.1 (s, arom. Cquat of C₆H₅), 114.7 (d, arom. CH of C₆H₄F, ²JₚCF = 21.3 Hz), 55.4 (d, CHP, ¹JₚCP = 149.6 Hz) ppm; ³¹P{¹H} NMR (121 MHz, DMSO-d6): δ = 15.2 (d, P(O), ⁵JₚF = 4.1 Hz) ppm; ¹⁹F{¹H} NMR (282 MHz, DMSO-d6): δ = -115.6 (d, ⁵JₚF = 4.2 Hz) ppm. IR: ν = 1620 (C=N) cm⁻¹. Elemental analysis calcd. (%) for C₁₅H₁₃O₄N₃FP (349.25): C 51.58, H 3.75, N 12.03; found C 51.45, H 3.67, N 11.96.

[(5-Phenyl-1,3,4-oxadiazol-2-ylamino)(3,4,5-trifluophenyl)methyl]phosphonic acid (6d). White solid, yield 89 %. ¹H NMR (300 MHz, DMSO-d6): δ = 9.91 (s br, 2H, P(OH)₂), 8.74 (s br, 1H, NH), 7.81 (d, 2H, arom. CH, ³JₚHH = 2.7 Hz), 7.52-7.45 (m, 5H, arom. CH), 5.00 (d, 1H, CHP, ²JₚPH = 21.3 Hz) ppm; ¹³C{¹H} NMR (126 MHz, DMSO-d6): δ = 163.1 (d, arom. Cquat of C₆H₂F₃, ²JₚCP = 10.7 Hz), 158.2 (s, arom. Cquat C(Ph)=N), 149.8 (dd, arom. Cquat CF, ¹JₚCF = 247.3 Hz, ²JₚCF = 9.1 Hz), 137.9 (dt, arom. Cquat CF, ¹JₚCF = 250.5 Hz, ²JₚCF = 14.9 Hz), 135.0 (d, arom. Cquat C(NH)=N, ³JₚCP = 3.5 Hz), 130.7 (s, arom. CH), 129.3 (s, arom. CH), 125.3 (s, arom. CH), 124.1 (s, arom. Cquat of C₆H₅), 112.7 (d, arom. CH of C₆H₂F₃, ²JₚCF = 17.1 Hz), 55.2 (d, CHP, ¹JₚCP = 147.8 Hz) ppm; ³¹P{¹H} NMR (121 MHz, DMSO-d6): δ = 13.98 (d, P(O), ⁶JₚF = 4.4 Hz) ppm; ¹⁹F{¹H} NMR (282 MHz, DMSO-d6): δ = -135.9 (d, meta CF of C₆H₂F₃, ³JₚFF = 21.7 Hz), -163.5 (td, para CF of C₆H₂F₃, ³JₚFF = 21.7 Hz, ⁶JₚF = 4.4 Hz) ppm. IR: ν = 1612 (C=N) cm⁻¹. Elemental analysis calcd. (%) for C₁₅H₁₁O₄N₃F₃P (385.23): C 46.77, H 2.88, N 10.91; found C 46.69, H 2.95, N 10.87.

[(5-Phenyl-1,3,4-oxadiazol-2-ylamino)(3,5-bis-trifluoromethyl-phenyl)methyl]phosphonic acid (6e). White solid, yield 68 %. ¹H NMR (300 MHz, DMSO-d6): δ = 9.11 (s br, 1H, NH), 8.23 (s, 2H, arom. CH of C₆H₃(CF₃)₂), 8.01 (s, 1H, arom. CH of C₆H₃(CF₃)₂),
7.79 (d, 2H, arom. CH, ³J<sub>HH</sub> = 8.7 Hz), 7.51-7.50 (m, 3H, arom. CH), 5.99 (s br, 2H, P(OH)₂), 5.24 (d, 1H, CHP, ²J<sub>PH</sub> = 21.9 Hz) ppm; ¹³C{¹H} NMR (126 MHz, DMSO-d₆): δ = 163.2 (d, arom. Cquat of C₆H₃(CF₃)₂, ²J<sub>CP</sub> = 10.3 Hz), 158.2 (s, arom. Cquat C(Ph)=N), 141.5 (s, arom. Cquat C(NH)=N), 130.7 (s, arom. CH), 129.9 (q, arom. Cquat CCF₃, ²J<sub>CF</sub> = 32.6 Hz), 129.2 (s, arom. CH), 128.8 (s, arom. CH), 125.2 (s, arom. CH), 124.0 (s, arom. Cquat of C₆H₅), 123.5 (q, CF₃, ¹J<sub>CF</sub> = 273.3 Hz), 121.0 (s, arom. CH), 55.7 (d, CHP, ¹J<sub>CP</sub> = 144.6 Hz) ppm; ³¹P{¹H} NMR (121 MHz, DMSO-d₆): δ = 13.5 (s, P(O)) ppm; ¹⁹F{¹H} NMR (282 MHz, DMSO-d₆): δ = -61.2 (s, CF₃) ppm. IR: ν = 1622 (C=N) cm⁻¹. Elemental analysis calcd. (%) for C₁₇H₁₂O₄N₃F₆P (467.26): C 43.70, H 2.60, N 8.99; found C 43.58, H 2.74, N 8.92.

## 2.5. X-ray Crystal Structure Analysis

Single crystals of 5b suitable for X-ray analysis were obtained by slow diffusion of hexane into a dichloromethane solution of the α-aminophosphonate. Crystal data: C₁₉H₂₁N₄O₆P, M_r = 432.37 g mol⁻¹, monoclinic, space group P2₁/c, a = 18.472(2) Å, b = 5.257(2) Å, c = 21.00(2) Å, β = 91.268(18)°, V = 2039(4) Å³, Z = 4, D = 1.409 g cm⁻³, μ = 0.180 mm⁻¹, F(000) = 904, T = 120(2) K. The sample (0.220 x 0.100 x 0.100) was studied on a Bruker PHOTON-III CPAD using Mo-Kα radiation (λ = 0.71073 Å). The data collection (2θ<sub>max</sub> = 31.837°, omega scan frames by using 0.7° omega rotation and 30 s per frame, range hkl: h -27,27 k -6,6 l -24,24) gave 37,576 reflections. The structure was solved with SHELXT-2014/5 [40], which revealed the non-hydrogen atoms of the molecule. After anisotropic refinement, all of the hydrogen atoms were found with a Fourier difference map. The structure was refined with SHELXL-2018/3 [41] by the full-matrix least-square techniques (use of F square magnitude; x, y, z, β<sub>ij</sub> for C, N, O and P atoms; x, y, z in riding mode for H atoms); 261 variables and 3,554 observations with I> 2.0 σ(I); calcd. w = 1/[σ²(Fo²) + (0.0946P)² + 3.4472P] where P = (Fo² + 2Fc²)/3, with the resulting R = 0.0763, Rw = 0.2188 and Sw = 1.091, Δρ< 0.915 eÅ⁻³.CCDC entry 2098845 contains the supplementary crystallographic data for 5b. These data can be obtained free of charge from The Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/data_request/cif or by e-mailing data_request@ccdc.cam.ac.uk, or by contacting The Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK

## 2.6. Biological Activities

### 2.6.1. Cells and Virus

Vero cells (CCL-81, American Type Culture Collection, MA, USA), used as a cellular model for antiviral bioassay were cultured in Dulbecco's modified Eagle's medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and gentamycin, and incubated at 37°C with 5% CO₂. They were used to adapt the avian Massachusetts serotype CoV-IBV strain.

### 2.6.2. Cell toxicity and viability

Cell viability was evaluated using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) method [42]. The MTT assay is a sensitive and reliable of the cellular metabolic activity. It is based on the ability of nicotinamide adenine dinucleotide phosphate (NADPH)-dependent cellular oxidoreductase enzyme to reduce the tetrazolium dye to the purple formazan crystals. Sub-confluent Vero cells, seeded in 96-well microplates (2 × 10⁶ cells/microplate; Thermo Fisher Scientific), were incubated with serial dilutions of synthesized molecules, in duplicates for 3 days at 37°C. MTT solution (500 µg/mL) was then added, and the cells were incubated for further 4 h. The formed formazan crystals within metabolically viable cells were then dissolved in 100 µL of DMSO and the absorption, proportional to viable cell number, was measured at 490 nm with a Thermo Scientific Multiskan FC. Results were expressed as percentage of viable
cells relative to the negative control (untreated cells cultured in standard medium and considered as 100 % viable). The half-maximum cytotoxic concentration (CC₅₀), which was defined as the concentration which reduced the OD₄₀₀ of the 1,3,4-oxadiazole-derived α-aminophosphonates/α-aminophosphonic acids-treated cells to 50 % of that of untreated cells, were determined from the plotted curve of percentages of viable cells against compound concentrations.

### 2.6.3. Virucidal assay

Avian CoV-IBV (10⁴ TCID₅₀; 50 % tissue-culture infectious dose of Avian CoV-IBV) was incubated with α-aminophosphonates/α-aminophosphonic acids in 200µL of DMEM containing 5 % of FBS for 2 h at 37°C with 5 % CO₂. The suspensions were then added to confluent Vero cells in 24-well microplates (2 × 10⁶ cells/microplate; Thermo Fisher Scientific), and the mixture was incubated 2 h at 37°C with 5 % CO₂. The cell layers were washed with phosphate buffered saline (PBS; 500 µL). DMEM containing 5 % FBS (500µL) was added and the cells were incubated for 3 days at 37°C with 5 % CO₂. Cells and supernatants were harvested, freeze-dried, and then centrifuged before the determination of virus content. The residual viral infectivity was determined by qRT-PCR and the percentage of inhibition was deduced.

### 2.6.4. Avian CoV-IBV replication inhibition assay

The effect of α-aminophosphonates/α-aminophosphonic acids on avian CoV-IBV infection was evaluated using virus RNA quantification. Vero cells at 2 × 10⁶ cells/plate were seeded in 24-well microplates, inoculated with 10⁴ TCID₅₀ (50 % tissue-culture infectious dose of Avian CoV-IBV) and incubated for 2 hours. The cell supernatants were then removed and the layers washed twice with PBS, (pH 7.2), then serial dilutions of the compounds to be tested were added to the infected cells. Wells containing Vero cells culture medium only were considered as negative control. After incubation for 72 h, culture cells and supernatants were harvested, freeze-dried 3 times and cleared by centrifugation at 500×g for 15 min at 4°C. The collected supernatants were then labeled and stored at -80°C until titration.

### 2.6.5. Quantitative Real-Time PCR (qRT-PCR)

Avian CoV-IBV genomic RNA was extracted from supernatant of infected Vero cells after cell lyse, at 3 days post infection, using the IndiSpin® Pathogen Kit (Indical Bioscience GmbH). The extracted nucleic acids were re-suspended in the elution buffer (100 µL), RNase inhibitor (RiboGrip RNase Inhibitor, Solis BioDyne) was added (0.4 µL) and stored at -80°C until use.

The quantitative Real-Time PCR (qRT-PCR) was carried out using a LightCycler 2.0 (Roche Diagnostics). For detection of genomic avian CoV-IBV, a forward primer IBV5'GU391 (5'-GCT TTT GAG CCT AGC GTT-3'), located at nucleotide positions 391-408, a reverse primer IBV5'GL533 (5'-GCC ATG TTG TCA CTG TCT ATT G-3'), located at nucleotide positions 533-512, and a Taqman® dual-labeled IBV5'G probe (5'-FAM-CAC CAC CAG AAC CTG TCA CCT C-BHQ1-3'), located at nucleotide positions 494-473, were used [43]. The qRT-PCR steps comprised 50°C for 15 min, 95°C for 10 min and 40 cycles of 95°C for 15 s and 60°C for 1 min.

The primers and probe contained 5× One-step Probe Mix (4 µL), 40× One-step SOLIScript® Mix (0.5 µL) and probe (0.5µL). The final volume was adjusted with water nuclease-free to obtain 20µL with a concentration of 0.2 µM. Following the manufacturer's instructions, a standard curve was determined for the assay and used to calculate genomic copy numbers for each sample. The standard curve was generated using 10-fold dilutions of positive control.

The amplification efficiency was calculated using the LightCycler 480 software (Roche Lifescience). The qRT-PCR efficiency is deduced from the standard curve. Briefly, a positive standard curve was generated by performing qRT-PCR on a series of diluted
avian CoV-IBV templates, ranging from 10⁷ to 10¹ 50 % tissue-culture infectious dose (TCID₅₀) per microliter. The PCR efficiency was calculated using the equation E = 10[-1/Slope] × 100 [44]. The qRT- PCR software would provide a standard curve and a slope by measuring the fluorogenic quantification cycles, which are represented as cycle threshold (Ct) values versus Log10 virus gene copies (see the supplementary materials). The resulting standard curve allowed deduction of virus concentrations in cultures of Vero cells after each antiviral activity assay.

2.6.6. Statistical analysis

All data were normally distributed and presented as the mean ± standard deviation of triplicate. Comparison between means were performed using one-way analysis of variance (ANOVA) followed by Bonferonni's post-hoc test at the significance level *p*<0.05.

2.7. *Molecular Docking Method*

DFT-based calculation of molecules **5f** and **6a** were carried out using ORCA version 4.1 before performing molecular docking analysis [45]. The x-ray crystal structure of **5b** was evaluated as raw structure for performing the optimization procedure. The calculations were carried out by using the exchange functional according to BP86 as suggested by Becke and Perdew [46,47]. Tightscf and grid4 options and resolution-of-the-identity (RI) approximation were used with TZV basis set and TZV/J auxiliary basis set was used for speeding up the performing calculations [48]. Crystal structure of SARS coronavirus main proteinase (PDB ID: 1uk4) [49], papain-like protease (PDB ID: 4x2z) [50] and N- and C-terminal domain of coronavirus nucleocapsid protein (PDB ID: 2c86) [51] were downloaded from RCSB protein data bank (https://www.rcsb.org/). AutoDockTools 4.2 was used for molecular docking calculations. Only polar hydrogen and Kollman charges were evaluated in target molecules and molecules of water inside proteins were removed. While Lamarkian genetic algorithms were applied, the genetic algorithm population was recorded as 150. Randomized starting positions, Gasteiger charges, torsions have been evaluated for molecules **5f** and **6a** [52,53]. Discovery Studio 4.1.0 were used for illustrations.

**3. Results and discussion**

3.1. *Synthesis of α-aminophosphonates and α-aminophosphonic acids*

It is well known that the Kabachnik-Fields reaction [54,55], which involves the one-pot three-component condensation of amines, aldehydes and dialkylphosphites in the presence of acid catalysts, is undoubtedly the most straightforward approach to form α-aminophosphonates [56]. First, we attempted this classical method to access to the target α-aminophosphonates from 5-phenyl-1,3,4-oxadiazol-2-amine (1) in the presence of benzaldehyde and diethyl phosphite with various acid catalysts such as ZnCl₂, FeCl₃ or AcOH with or without solvent [57,58]. Unfortunately, under these conditions, the desired α-aminophosphonate could only be isolated in very low yields (< 5 %). Furthermore, caring out the reaction under microwave irradiation did not lead to the desired product but to the exclusive formation of a α-hydroxyphosphonate resulting of the condensation between the benzaldehyde and the diethyl phosphite. This latter result suggests that the formation of the expected imine intermediate did not occur under these operating conditions.

To circumvent this problem, the Pudovik reaction [59], which is the stepwise version of the Kabachnik-Fields condensation, requiring the isolation of the imine intermediate was attempted. Thus, imine intermediates **2a-f** were first prepared and isolated from the reaction of 5-phenyl-1,3,4-oxadiazol-2-amine (1) with aromatic aldehydes (1.5 equiv.) in refluxing toluene for 48 h (Scheme 1). Imines **2a-f** were isolated as solids in good to excellent yields (73-90 %), and fully characterized by elemental analysis, infra-
red and NMR spectroscopies (¹H, ¹³C and ¹⁹F); ¹H NMR spectra, in particular, displayed a signal around 9.34-9.58 ppm attributable to the N=CH proton (see supplementary materials).

<figure><img src="image_4.png" /><figcaption>**Scheme 1.** Synthesis of (E)-1-(aryl)-N-(5-phenyl-1,3,4-oxadiazol-2-yl)methanimines **2a-f**.</figcaption></figure>

With imines **2a-f** in hand, we then studied their reactivity in the presence of dialkyl/arylphosphites, in order to access to the target α-aminophosphonates. For the Pudovik reaction, we chose to use an environmentally friendly protocol. The condensation was realized under microwave irradiation in solvent- and catalyst-free conditions, which have become a modern approach for the synthesis of bioactive heterocyclic molecules [60-62]. The optimization was carried out with imine **2a** and dimethylphosphite. After 10 min at 80°C and 60 W, the expected α-aminophosphonate **3** was isolated with a moderate yield of 20 % (Table 1, entry 1). A longer irradiation time did not increase the yield of the desired product (Table 1, entry 2). A significantly improved isolated yield of 54 % was obtained when the reaction was conducted at higher temperature (115°C) with a higher power (300 W) for only 10 min (Table 1, entry 3). It is important to note that under these operating conditions and using either diphenylphosphite or diethylphosphite the reaction provided the corresponding α-aminophosphonates **4** and **5a** with isolated yields of 90 and 92 % respectively (Table 1, entries 4 and 5).

<figure><img src="image_5.png" /><figcaption>**Table 1.** Synthesis of α-aminophosphonates - optimization. <fnref n="1" /></figcaption></figure>

<table><thead><tr><th>Entry</th><th>HP(O)(OR)<sub>2</sub></th><th>Time (min)</th><th>T (°C)</th><th>Power (W)</th><th>Isolated yield (%)</th></tr></thead><tbody><tr><td>1</td><td>HP(O)(OMe)<sub>2</sub></td><td>10</td><td>80</td><td>60</td><td>20 (3)</td></tr><tr><td>2</td><td>HP(O)(OMe)<sub>2</sub></td><td>40</td><td>80</td><td>60</td><td>20 (3)</td></tr><tr><td>3</td><td>HP(O)(OMe)<sub>2</sub></td><td>10</td><td>115</td><td>300</td><td>54 (3)</td></tr><tr><td>4</td><td>HP(O)(OPh)<sub>2</sub></td><td>10</td><td>115</td><td>300</td><td>90 (4)</td></tr><tr><td>5</td><td>HP(O)(OEt)<sub>2</sub></td><td>10</td><td>115</td><td>300</td><td>92 (5a)</td></tr></tbody></table>

Under optimized conditions (Table 1, entry 5), the six imines **2a-f** bearing either electron-donating or electron-withdrawing substituents on the phenyl ring, were put in the presence of diethylphosphite to generate the corresponding 1,3,4-oxadiazole-derived α-aminophosphonates **5a-f** (Scheme 2). It is interesting to note that imines with electron-withdrawing substituents (**2a**, **2b** and **2e**) led to corresponding α-aminophosphonates (**5a**, **5b** and **5e**) in lower isolated yields (76-92 %) than substrates bearing electron-donating substituents, mesomeric effect (**2c**, **2d**, and **2f**; yields 90-97 %). The difference of reactivity could be attributed to the substituent's effects, such as strong electron-donating groups on the phenyl ring would increase the electron density on the imine nitrogen atom and therefore enhance the electrophile of the carbon atom, which

<footnote n="1">¹ Reaction conditions: Imine **2a** (1 mmol), dialkyl/arylphosphite (2 mmol).</footnote>
favors the hydrophosphonylation reaction as suggested by the mechanism earlier reported by the group of Cherkasov [63].

<figure><img src="image_6.png" /><figcaption>**Schema 2.** Synthesis of diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(aryl)methyl] phosphonates **5a-f**.</figcaption></figure>

The synthesized 1,3,4-oxadiazole-derived α-aminophosphonates **3**, **4** and **5a-f** were fully characterized by elemental analysis, infrared and multinuclear NMR spectroscopies (see the experimental part). Their ¹H NMR spectra in DMSO-d₆ revealed for each NH signal a doublet of doublet in the range 9.00-9.55 ppm (³JHH = 6.0-10.2 Hz and ³JPH = 1.5-3.9 Hz and) and for each CHP signal a doublet of doublet in the range 5.24-5.95 ppm (²JPH = 13.2-23.1 Hz and ³JHH = 6.0-10.2 Hz). Their ³¹P{¹H} NMR spectra show a signal centered in the attempt range 12.6-21.7 ppm (see supplementary materials).

Furthermore, the structure of α-aminophosphonates was confirmed by an X-ray diffraction study carried out on compound **5b** (Figure 2). The α-aminophosphonate crystallizes in the monoclinic asymmetric space group P2₁/c with four distinct enantiomeric molecules (A and B molecules in which C1 has an R and an S configuration). The oxadiazole and phenyl aromatic rings are slightly inclined with a dihedral angle of 12.86°. The aromatic ring bearing the nitro substituent is perpendicular to the oxadiazole ring; the dihedral angle between these two aromatic rings being 88.73° (Figure 2). Bond lengths and angles of the 1,3,4-oxadiazole ring are consistent with those of **5a**, previously reported by our group [38] (see supplementary materials).

<figure><img src="image_7.png" /><figcaption>**Figure 2.** ORTEP drawing of **5b**, 50 % probability thermal ellipsoids. Important bond lengths (Å) and angles (°): N2-N3 1.438(4), N3-C3 1.214(4), C3-O1 1.422(4), O1-C2 1.267(4), C2-N2 1.286(5), C2-N1 1.385(5), C2-N2-N3 110.4(3), N2-N3-C3 101.0(3), N3-C3-O1 114.9(3), C3-O1-C2 103.8(3), O1-C2-N2 109.9(3).</figcaption></figure>

α-Aminophosphonates **5a-e** were then hydrolyzed into their corresponding α-aminophosphonic acids **6a-e**. The dealkylation was carried out in mild conditions using 8 equivalents of trimethylsilyl bromide (TMSBr) at room temperature for 3 days. Af-
ter an acid hydrolysis, the α-aminophosphonic acids **6a-e** were isolated in good to excel-
lent yields and purities (68-95 %; Scheme 3). Compounds **6a-e** were fully characterized
by elemental analysis, infrared and multinuclear NMR spectroscopies (see experimental
section and supplementary materials). Their ¹H NMR spectra showed in particular the
disappearance of the ethoxyl groups while their ³¹P{¹H} NMR spectra displayed a down-
field of about 5 ppm.

<figure><img src="image_8.png" /><figcaption>**Schema 3.** Synthesis of diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(aryl)methyl]phosphonic acids **6a-e**.</figcaption></figure>

3.2. *Biological activity*

A selection of synthesized α-aminophosphonates **5b,c,f** and α-aminophosphonic acids **6a-e** were screened for their antiviral activity against avian coronavirus IBV.

3.2.1. Determination of cell growth and viability

The MTT-based (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) cytotoxicity assay [42] was used to determine the safety profiles of the test compounds on Vero cells. As shown in Figure 3, the α-aminophosphonate **5c** and the α-aminophosphonic acids **6a**, **6b**, **6d** and **6e** were the highest cytotoxic compounds with $CC_{50}$ values of around 550 µM. The diethyl[(5-phenyl-1,3,4-oxadiazol-2-ylamino) (4-nitrophenyl)methyl]phosphonate (**5b**) was the lowest cytotoxic compound having a $CC_{50}$ value of 1598.8 ± 4.73 µM. The other 1,3,4-oxadiazole-derivatives showed intermediates $CC_{50}$ values (> 660 µM). The cytotoxic profile of the test compounds was established as follows: **6d** ≥ **6b** > **5c** > **6a** > **5f** > **6e** > **6c** > **5b**. The important $CC_{50}$ values (≥ 540 µM) encourage their use as safe and non-cytotoxic bioactive compounds.

The safety of α-aminophosphonates has already been reported for a series of ribo-
nucleosides of 1,2,3-triazolylbenzyl-aminophosphonates, which have minimum cytotox-
ic concentrations higher than 100 µM in Vero cell cultures [64]. The group of Rezaei ob-
served, a moderate cytotoxicity profile of some α-aminophosphonates (> 100 µM mini-
mum cytotoxic concentration) against the three cancer cell lines JURKAT (T-cell lym-
phoma), RAJI (Burkit's lymphoma) and MCF-7 (breast cancer) [65]. In contrast, a strong
inhibitory effect of a family of thiazolyl α-aminophosphonates against five human can-
cer cell lines, including breast (MCF-7 and MDA-MB-231), prostate (DU-145), liver
(HepG2) and HeLa cancer cell lines has been reported by the group of Cirandur [66].

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<figure><img src="image_9.png" /><figcaption>**Figure 3.** Cytotoxicity (CC₅₀) of α-aminophosphonates **5b,c,f** and α-aminophosphonic acids **6a-e** against uninfected Vero cells. Results are expressed as mean ± standard deviation of triplicate (n = 3). Means followed by different superscript letters (a-e) are significantly different at p<0.05 according to ANOVA followed by Bonferroni's post-hoc test.</figcaption></figure>

### 3.2.2. Determination of cell growth and viability

The inhibitory effect of compounds **5b,c,f** and **6a-e** on avian CoV-IBV was first tested using the direct assay which consists of mixing a known amount of virus with aliquots containing the organic molecule. In this aim, the inhibition of the replication of the virus in Vero cells was measured. Data from Table 2 showed that the direct contact of test compounds with virus particles resulted in 22.73-86.11 % inhibition of viral replication. The α-aminophosphonate **5f** (at 33 µM) and the α-aminophosphonic acid **6a** (at 1.23 µM) proved to be by far the most effective compounds exhibiting 86.11 ± 1.58 and 75.00 ± 0.75 % inhibition of IBV, respectively. This indicates that both compounds have virucidal activities as they are able to inactivate the IBV infectivity outside cells.

<table><caption>**Table 2.** Virucidal and antiviral activities of α-aminophosphonates **5b,c,f** and α-aminophosphonic acids **6a-e** against avian CoV-IBV.</caption><thead><tr><th>Compounds</th><th>Concentration (µM)</th><th>Virucidal activity<sup>1</sup> Inhibition (%)</th><th>Antiviral activity<sup>2</sup></th></tr></thead><tbody><tr><td>5b</td><td>33</td><td>35.01 ± 0.53</td><td>Not active</td></tr><tr><td>5c</td><td>3.66</td><td>46.02 ± 1.49</td><td>Not active</td></tr><tr><td>5f</td><td>33</td><td>86.11 ± 1.58</td><td>Not active</td></tr><tr><td>6a</td><td>1.23</td><td>75.00 ± 0.75</td><td>Not active</td></tr><tr><td>6b</td><td>3.66</td><td>31.38 ± 2.40</td><td>Not active</td></tr><tr><td>6c</td><td>33</td><td>59.59 ± 1.34</td><td>Not active</td></tr><tr><td>6d</td><td>3.66</td><td>25.03 ± 1.15</td><td>Not active</td></tr><tr><td>6e</td><td>33</td><td>22.73 ± 3.25</td><td>Not active</td></tr><tr><td>Negative Control<sup>3</sup></td><td>0</td><td>0</td><td>Not active</td></tr></tbody></table>

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<footnote n="1">¹ The virucidal assay was presented as mean ± standard error of the mean of triplicate and expressed as % inhibition of avian CoV-IBV after 2 h incubation at 37°C. One-way analysis of variance (ANOVA) followed by the Bonferroni's test was used to compare means at the significance level of p < 0.05.</footnote>

<footnote n="2">² The antiviral activity determined through IBV titration in infected Vero cells using quantitative reverse transcription PCR (qRT-PCR).</footnote>

<footnote n="3">³ Negative control consists on Vero cells medium culture (DMEM supplemented with 10 % FBS) only.</footnote>
To further evaluate whether the compounds **5b,c,f** and **6a-e** have antiviral activity, the IBV genomic RNA was extracted from infected Vero cells after three days post infection and the virus titer was quantified using a quantitative Real-Time PCR (qRT-PCR). As reported in Table 2, all tested compounds failed to inhibit the replication of the IBV virus into the host cells, which demonstrates their ineffectiveness as antiviral agents. At this stage, the inability of the test compounds to penetrate the host cell due to their polar character may explain these negative results. These results were consistent with those observed by the group of Lazrek, which tested a ten nucleosides of 1,2,3-triazolylbenzyl-aminophosphonates against a broad range of DNA and RNA viruses in Vero cell cultures and measured the absence or a very weak anti-viral activity of the test products [64]. Similarly the group of Song obtained low antiviral activity against the Tobacco Mosaic Virus using α-aminophosphonates. Moreover the activities drastically depend on the nature of the substituents of the nitrogen and phosphorus atoms [67].

Compared to lithium chloride (LiCl), a potent inhibitor of the DNA of the virus, the tested α-aminophosphonates **5b,c,f** and α-aminophosphonic acids **6a-e** were found to be far effective virucidal agents against IBV at the concentration range used. Thus, it has been shown that incubation of LiCl at concentrations ranging from 5 to 50 mM with the IBV Beaudette strain did not modify the title of the virus indicating its inability to generate a virucidal activity against IBV. Further more, LiCl had the ability to suppress the replication of IBV in Vero cell in a dose dependent manner without virucidal effect. The authors attributed the observed activity to the inhibitory effect of LiCl on host cell protein, such as glycogen synthase kinase 3, thus suppressing the IBV nucleocapsid (N) protein [13]. In other comparative studies using ethanolic extracts from 15 plant species. High virucidal activities against the IBV Beaudette strain was observed for extracts mainly derived from the *Lamiacaea* family, including *Satureja Montana*, *Origanum vulgare*, *Mentha piperita*, *Melissa officinalis*, *Thymus vulgaris*, *Hyssopus officinalis*, *Salvia officinalis* and *Desmodium canadens* with 50 % effective concentrations (EC₅₀) in the microgram range (0.003-0.076 µg) with strong inhibitory effects on IBV replication in Vero cells at concentrations of 0.27-0.63 µg [11]. More recently, a formulation made from plant essential oils of cinnamaldehyde and glycerol monolaurate (5:95) was found to have, *in vivo*, a great potential as an anti-IBV agent. In their animal model (Yellow feather broiler chicks), the group of Mo demonstrated that the mixture of essential oils inhibits virus replication, promotes immune function and reduces the release of the pro-inflammatory cytokine interleukin 6 [12].

### 3.2.3. Molecular Docking

The recent developments in computational chemistry have strengthened the agreement between experimental and computed results [68]. Theoretical methods, which initially focused on structural analysis of the molecules, are also currently used on the evaluation of intermolecular interactions. The modern methods as molecular docking that examine the interactions between biomacromolecules and drug candidates are an important part of structure-based drug design [69,70]. In the aim to obtain information on the mode of action, we studied the interactions between α-aminophosphonate **5f** and α-aminophosphonic acid **6a**, which have the highest virucidal activities against avian CoV-IBV, with SARS coronavirus main proteinase, papain-like protease and N- and C-terminal domain of coronavirus nucleocapsid protein were analyzed.

First, the most stable structures of **5f** and **6a** were optimized with the ORCA package [45]. Both compounds were recorded in pdbqt format. For the molecular docking study, the macromolecules were downloaded from the CBSN protein database, then blinded docking was performed to determine initial interactions, and the detected regions were examined in detail. Exposures with the lowest standard deviation and binding constant were recorded.

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The coronavirus polyprotein encodes two proteases, the main protease and the papain-like protease [71]. These two enzymes have been frequently chosen as target molecules in drug development studies related to COVID-19 pandemic [72]. Although both experimental and theoretical analyzes have been performed for many types of molecule during the COVID-19 pandemic, to the best of our knowledge, α-aminophosphonates and α-aminophosphonic acids have not been studied by molecular docking for interactions with coronavirus main protease and papain-like protease. Nevertheless, molecular docking interactions of phosphonimidates, containing chitosan moieties, against protease of SARS coronavirus were analyzed and displayed remarkable binding affinity (-7.9 to -7.3 kcal/mol) [73]. On the other hand, in 2006, the grouped Wu investigated the action of 58855 compounds on inhibition of the main protease of the coronavirus and detected, through a virtual structure-based analysis, two types of effective compounds, one of which has an oxadiazole moiety [74]. Therefore, interactions of α-aminophosphonate 5f and α-aminophosphonic acid 6a with the main protease enzyme of the coronavirus were first investigated. The two drugs interacted with the same region of the crystalline structure, the interaction of the phosphonic acid 6a is more important than with the phosphonate 5f. A inhibition concentration of 16.4 µM corresponding to a binding affinity of -6.53 kcal/mol was calculated for 6a, while a binding affinity of -5.98 kcal/mol and an inhibition constant of 41.28 µM were evaluated for 5f. In addition to the van der Waals interactions, H-bonds between Gln110, Thr111, Asn151 and Thr292 and the phosphonic acid region, and effective π-interactions between Leu202 and Phe294 and the conjugated cyclic regions of the molecule were detected for 6a. H-bonds with Ile106, Thr111 and Asn151 were observed for 5f. Other notable interactions for the latter drug are the halogenic interactions between Arg105, Gln110 and the CF₃ moiety. These interactions are represented in Figure 4.

<figure><img src="image_10.png" /><figcaption>**Figure 4.** Interaction type and interaction residues of 5f and 6a with SARS coronavirus main proteinase (PDB ID: 1uk4) crystal structure.</figcaption></figure>

Slightly higher interactions for **6a** (binding affinity of -5.85 kcal/mol and inhibition constant of 43.36 µM) than **5f** (binding affinity of -5.08 kcal/mol and inhibition constant of 187.73 µM) were calculated with papain-like protease. In the case of **6a**, H-bonds were observed with Asn155 and Ala237, while with **5f**, only H-bonds with Ser152 were recorded. The observed halogenic interactions for **5f** and **6a** played a positive role in the formation of inclusion complexes (see supplementary materials).

Enveloped viruses such as coronavirus have a filamentous nucleocapsid structure formed by interaction between nucleocapsid (N) protein and single-stranded viral RNA [75]. The nucleocapsid protein plays an important role in viral genome replication and cell signaling pathways [76]. Therefore, in this study, we also examined the interactions
of 5f and 6a with the N- and C-terminal domains of coronavirus nucleocapsid protein. In the case of α-aminophosphonate 5f, a binding affinity of ~5.01 kcal/mol and an inhibition constant of 214.16 µM were calculated, the highest contribution to these values comes from H-bonds with Ser29, Asn32, Ser34, Gln74, Tyr94 and Arg132. For the α-aminophosphonic acid 6a, the measured interactions with the protein are more important (binding affinity of ~6.56 kcal/mol and inhibition constant of 15.6 µM) and mainly halogenic interaction with Met28 and H-bonds with Ser29, Asn32, Ser34, Arg72, and Tyr94 were observed (see supplementary materials).

**4. Conclusions**

In summary, we have described the synthesis of a family of dialkyl/aryl[(5-phenyl-1,3,4-oxadiazol-2-ylamino)(aryl)methyl]phosphonates, through the Pudovik-type reaction of dialkyl/arylphosphite with imines under microwave irradiation, and their hydrolyzed phosphonic acids derivatives. A selection of non-cytotoxic compounds was screened for their *in vitro* antiviral activity against the avian bronchitis virus and displayed moderate virucidal inhibition. The more efficient 1,3,4-oxadiazole-based drugs, α-aminophosphonate 5f (at 33 µM) and α-aminophosphonic acid 6a (at 1.23 µM) could be useful for the prevention of IBV infection with virucidal inhibition of 86.11 ± 1.58 and 75.00 ± 0.75 %, respectively, allowing, additional future studies, their incorporation into cleaning products, disinfectants, wipes and nasal sprays. In order to better understand the mechanism of action of 5f and 6a, their interactions with the main protease, the pain-like protease and the nucleocapsid protein were investigated by molecular docking. Remarkable binding and inhibition constants were recorded for these two compounds. However, the tested compounds were unable to inhibit virus replication in IBV-infected Vero cells. The lack of antiviral activity could probably be attributed to their polarity, which hinders their diffusion through the lipophilic cytoplasmic membrane. To circumvent this problem and promote their diffusion into the host cell and thus their antiviral efficacy, chemical modification for instance monoesterification of α-aminophosphonic acids [77] or encapsulation of the most promising compounds in liposomes/lipid-based carriers/nanocarriers [78-81] will be the subject of future investigations.

**Supplementary Materials:** The following supporting information can be downloaded at: www.mdpi.com/xxx/s1, ¹H, ¹³C, ¹⁹F and ³¹P NMR spectra of compounds **1**, **2a-f**, **3**, **4**, **5a-f** and **6a-e**, and bond lengths and angles of X-ray structure of **5b** are given. Interaction type and interaction residues of **5f** and **6a** with Papain-Like Protease (PDB ID: 4x2z) and with N- and C-terminal domain of coronavirus nucleocapsid protein (PDB ID: 2c86) crystal structures.

**Author Contributions:** Conceptualization, A.L., K.H. and D.S.; methodology, S.H., K.H. and D.S.; software, K.H. and E.Ü; validation, K.H. and D.S.; formal analysis, S.H., K.H. and D.S.; investigation, S.H. and M.M.-T.; resources, A.G.; writing-original draft preparation, A.L., E.Ü, K.H. and S.T.; writing-review and editing, D.S.; supervision, K.H. and D.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** We gratefully acknowledge the University of Carthage and the Tunisian Ministry of Higher Education and Scientific Research for the financial support (grant for S.H. and PRFCOV19-DDP1 project for K.H.). We also thank the Institute Pasteur of Tunis (LEMV) for facilities.

**Conflicts of Interest:** The authors declare no conflict of interest.
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