# Synthesis of isomeric β-cycloalkoxyphosphonated hydrazones containing a dioxaphosphorinane ring: Configurational and conformational investigation and molecular docking analysis

Dorra Kanzari-Mnallah, Sirine Salhi, Michael Knorr, Jan-Lukas Kirchhoff, Carsten Strohmann, Med Lotfi Efrit, Azaiez Ben Akacha

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Dorra Kanzari-Mnallah, Sirine Salhi, Michael Knorr, Jan-Lukas Kirchhoff, Carsten Strohmann, et al.. Synthesis of isomeric β-cycloalkoxyphosphonated hydrazones containing a dioxaphosphorinane ring: Configurational and conformational investigation and molecular docking analysis. Journal of Molecular Structure, 2024, 1317, pp.139035. 10.1016/j.molstruc.2024.139035. hal-04651499

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# Synthesis of isomeric β-cycloalkoxyphosphonated hydrazones containing a dioxaphosphorinane ring: Configurational and conformational investigation and molecular docking analysis

Dorra Kanzari-Mnallah<sup>a, *</sup>, Sirine Salhi<sup>a</sup>, Michael Knorr<sup>b, *</sup>, Jan-Lukas Kirchhoff<sup>c</sup>, Carsten Strohmann<sup>c</sup>, Med Lotfi Efrit<sup>a</sup>, Azaiez Ben Akacha<sup>a</sup>

<figure><img src="image_3.png" /></figure>

## ARTICLE INFO

### Keywords:

Allenylphosphonate

Hydrazone

Dioxaphosphorinane

Azine

Conformers, <sup>31</sup>P NMR

DFT calculation

SCXRD

Molecular Docking

ADMET

## ABSTRACT

A series of Z/E isomeric β-cycloalkoxyphosphonated hydrazones [R¹R²C(CH₂O)₂P(=O)CH2-C(=N-N(H)R⁵}C (H)R³R⁴] 3 bearing a six-membered dioxaphosphorinane ring was prepared, structurally analysed, and subjected to *in silico* studies to assess their Acetylcholinesterase (AChE) and Butyrylcholinesterase (BuChE) inhibition activity. Furthermore, their ADMET (Absorption, Distribution, Metabolism, Excretion, Toxicity) properties were determined. Hydrazones 3 were prepared from their corresponding allenylphosphonates [R¹R²C(CH₂O)₂P(=O)C (H)=C=CR³R⁴] 2 by addition of various hydrazines NH₂-NHR⁵. Since NMR studies indicated the occurrence of several isomeric species in solution, conformational and configurational studies, based on mechanistic elucidation and energy stability using Density functional theory (DFT) calculation at the B3LYP/6-311 G++ (d,p) level of theory, were performed to determine their relative predominance. The hydrazone 1-(2-oxo-1,3,2-dioxaphosphoranyl)-3-phenylpropanone 3c was crystallographically analysed by single-crystal X-ray diffraction (SCXRD), revealing the occurrence of strong intra- and intermolecular N-H···O hydrogen bonding. These secondary interactions were further examined by a Hirshfeld surface analysis. The experimental SCXRD parameters are compared with the theoretical calculated ones and the preferred configuration of product 3c was determined. Furthermore, the crystal structure of the symmetric azine 2,2'-(((2E,2'E)-hydrazine-1,2-diylidenebis(3-phenylpropan-1-yl-2-ylidene))bis(5-methyl-5-propyl-1,3,2-dioxaphosphinane 2-oxide) 4, resulting from the reaction between hydrazone 3a and allene 2a, has been determined. To predict whether hydrazones 3 are appropriate as anticholinesterase agents exhibiting a potential activity for treatment of the Alzheimer disease, *in silico* molecular docking simulations were employed to investigate the interaction modes between hydrazones 3 and the active sites of the BuChE and AChE enzymes. Additionally, an ADMET-properties prediction was carried out for selected hydrazones to determine their pharmacokinetics and drug-likeness properties.

## 1. Introduction

Hydrazones derivatives featuring the motif R₁R₂C=N-N(H)R₃ (R = H, alkyl, aryl) represent attractive targets that continue to receive attention due to their significant biological and therapeutic properties [1]. The presence of an imine-type C≡N function makes them also interesting as N-donor ligand in coordination chemistry [2-4]. Some representatives of this class display also important biological activities such as analgesic [5,6], anti-microbial [7], anti-cancer [8-11],

anti-convulsant [12], anti-inflammatory [13] properties. Previous studies [14-16] have also revealed that some hydrazone derivatives may have a significant inhibitory activity against Butyrylcholinesterase (BuChE) and/or Acetylcholinesterase (AChE) (Fig. 1).

BuChE and AChE are enzymes involved in Acetylcholine (ACh = 2-acetoxyethyl-trimethylammonium) hydrolysis yielding the inactive metabolites choline and acetate. ACh is an important neurotransmitter for the regulation of cognition in humans and the activation of muscles. Cholinesterase inhibitors (https://en.wikipedia.org/wiki/Acetylcholi

<footnote><sup>a</sup> Laboratory of Selective Organic and Heterocyclic Synthesis-Biological Activity Evaluation, SOHES, (LR17ES01), Department of Chemistry, Faculty of Science of Tunis, University Tunis El Manar 2092 Tunis Tunisia</footnote>

<footnote><sup>b</sup> Institut UTINAM-UMR CNRS 6213, Université de Franche-Comté, 16, route de Gray 25030 Besançon, France</footnote>

<footnote><sup>c</sup> Anorganische Chemie, Technische Universität Dortmund, Otto-Hahn-Str. 6 44227 Dortmund, Germany</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: michael.knorr@univ-fcomte.fr (M. Knorr).</footnote>
<figure><img src="image_4.png" /><figcaption>Fig. 1. Examples of previously reported hydrazones [14-16] featuring significant inhibitory activity against BuChE and/or AChE.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>Scheme 1. Synthetic pathway leading to allenes 2 and hydrazones 3.</figcaption></figure>

nesterase inhibitor) are employed for the treatment of the Alzheimer disease [17-19]. Despite all the progress and discoveries made for treatment of this neurodegenerative disease, further research is needed to enlarge the library of more effective agents. Consequently, synthesizing new molecules whose structural motifs are close to that of chemical entities having proven activity and assessing whether they present potential activity, remains a challenge.

In this context, a series of hydrazones [R¹R²C(CH₂O)₂P(=O)CH₂-C{=N-N(H)R⁵}C(H)R³R⁴] 3 bearing a six-membered 1,3,2-dioxaphosphorinane heterocycle was synthesised from their corresponding phosphonated allenes 2 and obtained as diastereomeric product mixtures. With the aim to determine the predominant configurations of the diastereomeric allene [Me(Pr)C(CH₂O)₂P(=O)C(H)=C=C(H)Ph] 2a and its derived diastereomeric hydrazone 3a, a computational method was used. In addition, the structural characterisation of a single crystal of hydrazone 3c by X-Ray diffraction is reported in this paper. A DFT calculation allowed to investigate the geometrical properties and to compare them with the experimental data of the analyzed compound. With the intention to screen if the prepared hydrazones could be employed as potential candidates acting as AChE or BuChE inhibitors for use in the treatment of Alzheimer disease, an in silico simulation was performed. To analyse any secondary interactions and bonding modes occurring between the β-cycloalkoxyphosphonated hydrazones 3 and the active sites of the AChE and BuChE enzymes, a molecular docking study was conducted. To predict their pharmacokinetics and drug-

<table><caption>**Table 1**
Substitution pattern, yield and ³¹P{¹H} NMR data of allenes 2a-e.</caption><thead><tr><th></th><th>a</th><th>b</th><th>c</th><th>d</th><th>e</th><th>f</th></tr></thead><tbody><tr><td>R<sup>1</sup></td><td>Me</td><td>H</td><td>H</td><td>Me</td><td>Et</td><td>Et</td></tr><tr><td>R<sup>2</sup></td><td>Pr</td><td>H</td><td>H</td><td>Me</td><td>Et</td><td>Et</td></tr><tr><td>R<sup>3</sup></td><td>H</td><td>Me</td><td>Me</td><td>H</td><td>H</td><td>Me</td></tr><tr><td>R<sup>4</sup></td><td>Ph</td><td>Me</td><td>Ph</td><td>Ph</td><td>Ph</td><td>Me</td></tr><tr><td>Yield%</td><td>84</td><td>75</td><td>78</td><td>70</td><td>90</td><td>72</td></tr><tr><td><sup>31</sup>P NMR (ppm)</td><td>6.7; 7.4</td><td>9.6</td><td>7.6</td><td>6.5</td><td>7.4</td><td>11.2</td></tr><tr><td colspan="7">cis/trans ratio 27: 73</td></tr></tbody></table>

likeness properties, an ADMET profile was established for some selected hydrazones.

## 2. Results and discussion

### 2.1. Chemistry

#### 2.1.1. Preparation of allenylphosphonates 2a-e and β-cycloalkoxyphosphonated hydrazones 3a-f

In continuation of previous work published by our laboratory [20-23], a series of allenylphosphonates 2 was synthesised based on a method developed by Boiselle et al. (Scheme 1) [24-26]. The structural modifications concern substitutions of R¹ and R² on the dioxaphosphorinane ring. Also, R³ and R⁴ were modified owing the use of different
<table><caption>**Table 2**

Substitution pattern, yield, ³¹P{¹H} NMR data and diastereomeric ratios of hydrazones 3a-f.</caption><thead><tr><th>3</th><th>a</th><th>b</th><th>c</th><th>d</th><th>e</th><th>f</th></tr></thead><tbody><tr><td>R<sup>1</sup></td><td>Me</td><td>H</td><td>H</td><td>Me</td><td>Et</td><td>Et</td></tr><tr><td>R<sup>2</sup></td><td>Pr</td><td>H</td><td>H</td><td>Me</td><td>Et</td><td>Et</td></tr><tr><td>R<sup>3</sup></td><td>H</td><td>Me</td><td>Me</td><td>H</td><td>H</td><td>H</td></tr><tr><td>R<sup>4</sup></td><td>Ph</td><td>Me</td><td>Ph</td><td>Ph</td><td>Ph</td><td>Ph</td></tr><tr><td>R<sup>5</sup></td><td>H</td><td>H</td><td>H</td><td>H</td><td>H</td><td>Me</td></tr><tr><td>Yield%</td><td>74</td><td>80</td><td>75</td><td>70</td><td>70</td><td>80</td></tr><tr><td>³¹P NMR (ppm)</td><td>3a<sub>1</sub><br>21.12; 21.37;</td><td>3a<sub>2</sub><br>21.69; 21.55</td><td>21.5; 21.4</td><td>20.8</td><td>20.9; 20.7</td><td>21.7; 21.9</td><td>21.8; 21.3</td></tr><tr><td>Z/E ratio</td><td>61: 39</td><td>57:43</td><td>96: 4</td><td>100:0</td><td>86: 14</td><td>62: 38</td><td>85: 15</td></tr></tbody></table>

<figure><img src="image_6.png" /><figcaption>**Fig. 2.** Comparison of characteristic allenic carbon's signals of 2d and 2a in the ¹³C NMR spectra. (left) Part of the ¹³C{¹H} NMR spectrum 2d. (right) Part of ¹³C{¹H} NMR spectrum of allene 2a.</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Scheme 2.** Proposed mechanism for the formation of 2a.</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>Fig. 3. Computed preferred conformations for conformers 2a₁, 2a₁'; 2a₂, 2a₂'.</figcaption></figure>

propargylic alcohols. The first step in the synthetic pathway involves the addition of propargylic alcohol to 2-chloro-5,5-dialkyldioxaphosphorinane 1 in an equimolar quantity at 0 °C using an anhydrous solvent, in the presence of triethylamine as an additional base. The allenyl-phosphonates 2 were thus prepared in high yield varying from 72 % to 90 % and were isolated as white solids (Table 1). To convert them into the corresponding hydrazones 3, the respective hydrazines were added at room temperature. After heating the mixture for 40 min, the targeted hydrazones 3 were isolated in good yields ranging from 70 % to 80 % (Scheme 1 and Table 2). All prepared hydrazones were collected as white solids, except for 3b, which has a pasty consistency.

### 2.1.2. Spectroscopic characterisation and mechanistic elucidation of 2a.

In the proton-decoupled ³¹P NMR spectrum of allene 2a, two signals are observed at 6.7 and 7.4 ppm, whereas for the other products 2b-f, only a single resonance appears in the range between 6.5–10.75 ppm (Table 1). This indicates that allene 2a (in which the dioxaphosphorinane ring substituents R¹ ≠ R²), exists as a mixture of two diastereomers. This is confirmed by the duplication of the doublet resonance (due to ²Jₚ-C coupling) characteristic of the allenic carbon =C= in the ¹³C{¹H} NMR spectrum of 2a, which is absent in that of 2d (where R¹ = R²) (Fig. 2).

A mechanistical proposal for the formation of 2a is depicted in Scheme 2. The reaction begins with a nucleophilic attack of the hydroxyl group of the propargylic alcohol on the axial chlorine atom of chlorophosphite precursor **1a**: **1a**₁ (**trans**-configuration) and **1a**₂ (**cis**-configuration), followed by elimination of HCl. Four possibilities of intermediates were formed in pairwise equilibrium. After heating, the

resulting allene 2a was obtained with hypothetically four possibilities of conformations 2a₁, 2a₁', 2a₂, 2a₂'. However, only two diastereomers were detected by ³¹P{¹H} NMR. Therefore, we turned our attention to theoretical calculation to determine the predominant conformations. In this sense, the optimized molecular structures and energies of conformations were determined using Density Functional Theory DFT/ (B3LYP 6–311++ G (d,p) basis set) calculations, which revealed that the 2a₁ conformers with the *trans* configuration and 2a₂ adopting a *cis* configuration are more stable by –3.05 Kcal/mol and –5.66 Kcal/mol, respectively (Fig. 3). The common point between 2a₁ *trans* and 2a₂ *cis* is that the P=O group is in the axial position. This is in agreement with our previous study [27] concerning 2-benzylamino-5-methyl-5-propyl-2-oxo-1,3,2-dioxaphosphorinane, where theoretical calculations demonstrated that conformers with the P=O group in the axial position are more stable than those with the P=O in the equatorial position. This result can be explained by a conformational stabilization due to the anomeric effect [28–29]. Moreover, the energetic stability of diastereomers 2a₁ and 2a₂ was evaluated, revealing that 2a₁ *Trans* is more stable by –0.14 Kcal/mol (Fig. 4).

### 2.1.3. Spectroscopic characterisation and mechanistic elucidation of 3a

Hydrazone 3a, synthesized from the reaction between allene 2a and hydrazine, was also obtained as a mixture of diastereomers, but four distinct signals were detected in the ³¹P NMR spectrum at 21.12; 21.37; 21.55 and 21.69, respectively (Fig. 5).

To determine the optimized structures of the four detected diastereomers, mechanistic and theoretical studies were employed. Mechanistically, the hydrazine derivative reacts by nucleophilic

<figure><img src="image_9.png" /><figcaption>Fig. 4. Computed preferred configuration between configurations 2a₁ and 2a₂.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>Fig. 5. ³¹P{$^{1}$H} NMR spectrum of hydrazone 3a (CDCl₃) revealing the presence of 4 diastereomers.</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>Scheme 3. Proposed mechanism for the formation of 3a.</figcaption></figure>

addition on the central allenic carbon of diastereomeric 2a (2a₁+2a₂) to afford two enamine intermediates. Each intermediate formed undergoes rearrangement to form two isomers with different configurations: Z and E due to presence of the C=N group. In solution Z/E isomers coexist due to inversion processes at C=N bond [21,30,31]. E/Z isomerization

mechanism in intramolecularly hydrogen-bonded arylhydrazones has been also assessed by DFT computing [32]. Accordingly, we obtained a mixture of four hydrazones isomers: 3a₁Z, 3a₁E, 3a₂Z, 3a₂E (Scheme 3).

Assignment of the signals to the corresponding structure was
<figure><img src="image_12.png" /><figcaption>Fig. 6. Computed preferred configuration for hydrazone 3a (3a₁ Z; 3a₁ E; 3a₂ Z; 2a₂ E).</figcaption></figure>

therefore a difficult task. For this reason, our next goal was the determination of the relative stability of the four hydrazone isomers by a DFT investigation in order to assign the signals in ³¹P NMR spectrum of 3a and to deduce the preferred configuration. First, we studied the relative energetic stability between the couple of isomers (3a₁ Z and 3a₁ E) formed from 2a₁. It was found that 3a₁ Z is more stable by –3.56 Kcal/mol. Similarly, we studied the relative energetic stability of the second pair of isomers (3a₂ Z and 3a₂ E) formed from 2a₂. It was found that 3a₂ Z is more stable by –1.28 Kcal/mol. As 2a₁ has the preferred configuration over 2a₂, the hydrazones (3a₁ Z and 3a₁ E) obtained from 2a₁

predominate over the hydrazones (3a₂ Z and 3a₂ E) obtained from 2a₂ (Fig. 6). Hence, the following classification of the 4 diastereomers in ascending order of energy was established as follows: 3a₁ Z > 3a₁ E > 3a₂ Z > 3a₂ E. Based on this classification, signals at 21.12 ppm, 21.37 ppm, 21.55 ppm and 21.69 ppm were attributed to 3a₁ Z (50%), 3a₁ E (34%), 3a₂ E (7%) and 3a₂ Z (9%), respectively (Fig. 7).

When analysing the reaction mixture resulting from reaction of allene 2a with hydrazine, we observed in the ³¹P{¹H} NMR an additional resonance at δ = 19.09 ppm due to another compound formed in minor amounts (Fig. S26 in Supplemental Materials). During our
<figure><img src="image_13.png" /><figcaption>Fig. 7. Attribution of the ³¹P{¹H} resonances of hydrazone **3a** to their corresponding configurations **3a₁Z; 3a₁E; 3a₂Z; 3a₂E**.</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>**Scheme 4.** Proposed mechanism accounting for the formation of azine 4 as secondary product.</figcaption></figure>

attempt to obtain X-ray suitable crystals of **3a**, we failed, unfortunately, to grow crystal of **3a**. However, we noticed the formation of some colorless crystals, whose elemental analysis and crystallographic characterization (see below Fig. 9) revealed the formation of azine 4. We suppose that formation of this symmetric azine 4 occurring in CH₃Cl solution is the result of a nucleophilic addition reaction followed by a H-shift (see Scheme 3) between the starting material, allenylphosphonate **2a** and *in situ* formed hydrazone **3a**, as outlined in (Scheme 4). Azines, with RR'C=N-N=CRR' connectivity are commonly the products of hydrazine condensation with ketone or stem from reaction of a hydrazone with a chalcone [33].

They are also used as N,N donor ligands in coordination chemistry [34]. So, the formation of 4 found by serendipity constitutes an alternative approach to this class of compound. Since this finding is not the topic of the present manuscript, we haven't it investigated in more detail. But this finding obtained by serendipity will be developed in future work.

## 2.2. Molecular structures of 1-(2-oxo-1,3,2-dioxaphosphoranyl)-3-phenylpropanone **3c** and azine 4

### 2.2.1. Crystallographic characterisation of hydrazone **3c**

In the ³¹P{¹H} NMR spectrum of compound **3c**, just a single resonance was observed, unlike for the other hydrazone products where at least two signals related to *E* and *Z* isomers are detected (Table 2). With the aim to explain this observation, a structural study of compound **3c** was carried out by single-crystal X-ray diffraction at 100 K to determine the *Z/E* configuration of **3c** and the relative orientation of the dioxaphosphorinane ring substituents. After slow evaporation of a solution of **3c** in diethyl ether, white single-crystals crystallizing in the P2₁/c monoclinic space group were formed (see Table S1). Fig. 8 (top) reveals that the molecule adopts a Z-configuration with respect to the C=N bond, stabilized by a strong intramolecular hydrogen bond giving rise to a seven-membered ring. The connection occurs between the N-H···O=P groups (2.630 (17) Å), forming a N1-H1A···O angle of 143.6(13)°. On the other hand, the dioxaphosphorinane ring adopts a chair conformation with the P=O group in the axial position and C1 oriented in the equatorial position. A similar conformation of the dioxaphosphorinane cycle was crystallographically found for other allenylphosphonate-
<figure><img src="image_15.png" /><figcaption>**Fig. 8.** (top) Molecular structure of 3c in the crystal. Selected bond lengths (Å) and angles (°): P–O3 1.5809(6), P–O2 1.5860 (6), P–O1 1.4781(6), P–C1 1.7915 (8), O3–C13 1.4665 (10), O2–C11 1.4645 (10), N2–C2 1.2835 (10), N2–N1 1.3879(10), C2–C1 1.5165 (11), C2–C3 1.5240 (11); O3–P–O2 105.17(3), O3–P–C1 104.22 (3), O2–P–C1 103.59 (4), O1–P–O3 114.29 (4), O1–P–O2 113.84 (4), O1–P–C1 114.54 (4), P–O3–C13 117.68 (5), P–O2–C11 117.45(5), N1–N2–C2 118.35 (7). (bottom) View of a segment of the supramolecular 1D chain of 3c generated by intermolecular N1–H1A···O3¹ hydrogen bonding. Symmetry operation to generate equivalent atoms: ¹1-x, 1-y, 1-z; ²2-x, 1-y, 1-z; ³_1 + x, -1 + y, +z.</figcaption></figure>

derived organic molecules and for phosphates bearing this ring motif [35-40].

For example, the group of Sartillo-Piscil [35] demonstrated that the 2-phenoxy-2-oxo-1,3,2-dioxaphosphorinane of sugar 1,2-O-iso-propylidene-3,5-O-phenoxyphosphoryl-α-d-glucofuranose adopts a chair conformation with the P=O group in the axial and the phenoxy group in equatorial position. This conformation is stabilized by the seven-membered intramolecular hydrogen-bonded ring structure due to the formation of O-H···O=P hydrogen bond (2.17 Å). Thus, the strong tendency of the phenoxy group to be in the axial position is diminished

due to the intramolecular H-bonding. Ethyl-3-[1-(5,5-dimethyl-2-oxo-1,3,2dioxaphosphorin-2-yl)propan-2-ylidene]carbazate is another example of a hydrazone featuring an intramolecular seven-membered N-H···O=P cycle with N-H···O 2.05 Å forcing the molecule into a cis-chair conformation [36].

In addition to this intramolecular hydrogen bonding, an even stronger intermolecular N-H···O=P bonding of 2.118(15) Å occurs in the crystal between N1-H1···O1² of a neighboring molecule generating a dimeric macrocyclic motif. This hydrogen bond is further extended by a second intermolecular N1-H1A···O3¹ interaction of 2.508(16) Å
<figure><img src="image_16.png" /><figcaption>**Fig. 9.** Molecular structure of **4** in the crystal. Selected bond lengths (Å) and angles (°): P–O1 1.467(4), P–O3 1.585(5), P–O2 1.580(5), P–C1 1.804(4), O3–C16 1.466 (5), O2–C10 1.475(4), N–C2 1.285(4), N–N' 1.412(5); O1–P–O3 112.32(15), O1–P–O2 111.72(15), O1–P–C1 113.61(18), O3–P–C1 107.1(2), O2–P–O3 105.52(17), O2–P1–C1 106.0(2), C16–O3–P1 118.5(2), C10–O2–P 120.4(2), C2–C1–P1 112.2(2), N–C2–C1 125.3(3), N1–C2–C3 116.7(3). Symmetry operation to generate equivalent atoms: ¹₁-x, 1-y, 1-z.</figcaption></figure>

between the N-H function and a O3 atom of the dioxaphosphorinane cycle of an adjacent molecule, generating a supramolecular 1D ribbon (Fig. 8 bottom). A further 2D extension is secured by a short C12-H12B...N2³ contact of 2.584(1) Å and a C1-H1D...O1¹ contact of 2.467 (14) Å.

Note that the occurrence of intra- and intermolecular N-H···O bonding is quite common for hydrazones having O-atoms as acceptor sites. For example, 3-ethanehydrazonoyl-4-hydroxy-6-methyl-2H-pyran-2-one exhibits intermolecular N-H···O contacts of 2.221 Å along with a intramolecular hydrogen bonding between a O-H function and the sp² N atom of the hydrazone (1.821 Å, CSD refcode RUZWI) [41] Another example is ethyl 7-chloro-3-(2-ethoxy-1-hydrazinylidene-2-oxoethyl)-1,1-dioxo-2, 3-dihydro-1H-1,2-benzothiazole-3-carboxylate displaying intramolecular — N-H···O bonding of 1.924 Å giving rise to a six-membered cycle (CSD refcode FIDDII) [42]. A third example is N-(2-(1-hydrazonoethyl)-3-benzofuranyl)-p-toluenesulfonamide forming a supramolecular 1D chain through intermolecular N-H···O=S bonding of 2.450 Å (CSD refcode TELZUF) [43].

### 2.2.2. Crystallographic characterisation of azine 4

The centrosymmetric molecular structure of the condensation product **4** crystallizing like **3c** in the monoclinic space group P2₁/c is shown in (Fig. 9). The mid-point of the azine-type N–N bond forms an inversion center. The bond length is elongated with respect to that of **3c** (1.412(5) vs. 1.3879(10) Å) and matches that of other symmetric azines such as 2'-hydroxyacetophenone azine (1.415(3) Å) or (E,E)2,5-bis(-5-chloro-2-methoxyphenyl)-3,4-diazahexa-2,4-diene (1.1415(5) Å). For

(E,E)-4,4'-dimethyl-2,2'-(1,1'-dibenzylazino)diphenol bearing like **4** a benzyl substituent at the C=N carbon, a somewhat shorter N–N bond length of 1.399(3) Å is reported [44-46].

As in the latter three examples, there is a *transoid* (or *E,E*) conformation around the C=N-N=C motif forming a torsion angle of 180°. There is no intramolecular contact worth mentioning, some weaker secondary intermolecular hydrogen bonding occurs between C10-H10A...O1¹ (2.54(4) Å), C10-H10B...O2² (2.54(4) Å), and C15-H15B...O1¹ (2.56(4) Å).

### 2.2.3. Hirshfeld surface analyses of 3c

To better clarify the presence of strong and weak intermolecular contacts occurring in the crystalline state, that may be complementary and relevant to the docking simulation shown in Section II.3, a Hirshfeld surface analysis was carried out utilizing the *CrystalExplorer21* software package by Spackman *et al.* [47,48]. Both intramolecular and intermolecular H-bonding was analyzed, with varying intensity and strength of these bonds. The strongest and most significant H-bond was identified as an intermolecular N1-H1B...O1 bond, which exhibits a length of 2.118 (15) Å. This bond is characterized by high linearity, indicating a significant ionic character. Apart from the very short bond length, this explains why the intermolecular interaction in the crystal is comparatively strong. In contrast, an intramolecular N1-H1A...O1 contact having a more elongated bond length of 2.630(17) Å was observed, which is weaker due to the molecular structure. Furthermore, C-H···O interactions were identified to be less pronounced on the Hirshfeld surface than the N-H···O contacts (Fig. 10). These findings indicate that in the
<figure><img src="image_17.png" /><figcaption>**Fig. 10.** Hirshfeld-surface of **3c**, indicating a strong intermolecular bonding between two **3c** molecules in the elementary cell with *d*<sub>norm</sub> -0.4503 to 1.4815 Å (left); some weaker intermolecular interactions occurring in **3c** on the same Hirshfeld surface with *d*<sub>norm</sub> -0.4503 to 1.4815 Å (right).</figcaption></figure>

<figure><img src="image_18.png" /><figcaption>**Fig. 11.** 2D-fingerprint plots of **3c** showing all contributions (a) and resolved into O···H/H···O- percentages (b).</figcaption></figure>

present molecule, especially the N-H···O contacts are crucial for supra-molecular interactions with other molecules.

**Fig. 11** depicts the 2D-fingerprint plots showing all contribution plot and O···H/H···O- contacts. The plots depicting the percentages participations to the total Hirshfeld surface area due to N···H/H···N-, C···H/H···N- and H···H-contacts are presented in the Supporting Material as Fig. S45.

### 2.2.4. Theoretical study of the Z/E **3c** isomer stability and comparison of theoretical and experimental geometric properties of **3c** Z

To study the relative stability of the Z/E configuration of **3c** in the gas and solvent (chloroform) phases, a DFT calculation was performed using the B3LYP/6-311++G (d,p) basis set. The optimized molecular geometries of **3c** both with Z and E configuration are shown in Fig. 12. These calculations in both gas and solvent phases confirm that the Z-isomer is more stable than the E-isomer. In fact, **3c** Z is more stable by -11.82 Kcal/mol and by -9.11 Kcal/mol in gas-phase and solvent phase, respectively. This result agrees with the crystal structure revealing a Z-configuration with a strong intramolecular H-bond (Fig. 8). Accordingly, the singlet observed in the ³¹P NMR spectrum at

20.8 ppm is attributed to **3c** with Z configuration (Table 2). A comparison of the theoretical optimized structure of **3c** Z investigated by DFT in the gas-phase, in CHCl₃ and the experimental structure obtained by SCXRD was conducted. The selected geometrical parameters are as follows: the bond length and angles values of **3c** Z isomer found by DFT (in the gas-phase and in CHCl₃) and by SCXRD are outlined in Table 3. It can be deduced that they match well with correlation coefficients of R² = 0.9892, R² = 0.9930 for bond length (DFT (gas)/ SCXRD), (DFT (CHCl₃)/ SCXRD), respectively and R² = 0.9514 and R² = 0.9147 for angles (DFT (gas)/ SCXRD) and (DFT(CHCl₃) / SCXRD), as displayed in Fig. 13.

Table 4 lists the O···H distance (SCXRD/DFT) and angle θ (O···H-N) (SCXRD/DFT) in hydrazone **3c** Z. The distances between O and H calculated by DFT/B3LYP are 2.094 Å and 2.115 Å for the gas-phase and chloroform, respectively. These values match with those reported in the literature [49,50]. The angles (O···H-N) calculated by DFT/B3LYP in both phases are around 154°. These values are among 90–180° that is determined for hydrogen-bond. These results reflect that there is an intramolecular hydrogen bond between oxygen and hydrogen in the optimized structure of **3c** Z (Fig. 12). The geometrical properties of **3c** Z
<figure><img src="image_19.png" /><figcaption>Fig. 12. Optimized geometries for the Z/E isomers of hydrazone 3c.</figcaption></figure>

**Table 3**

<table><caption>Selected bond lengths (Å) and angles (°) from X-ray diffraction and DFT optimization for 3c Z isomer.</caption><thead><tr><th>Bond lengths (Å)</th><th>Exp SCXRD</th><th>Calc. in gas</th><th>Calc. in CHCl<sub>3</sub></th><th>Angles (°)</th><th>Exp SCXRD</th><th>Calc. in gas</th><th>Calc. in CHCl<sub>3</sub></th></tr></thead><tbody><tr><td>P7=O8</td><td>1.478(6)</td><td>1.488</td><td>1.493</td><td>C14=N28-N29</td><td>118.35(7)</td><td>119.01</td><td>118.98</td></tr><tr><td>P7-C11</td><td>1.7915(8)</td><td>1.805</td><td>1.808</td><td>P7-C11-C14</td><td>111.12(5)</td><td>113.82</td><td>114.05</td></tr><tr><td>P7=O10</td><td>1.5809(6)</td><td>1.626</td><td>1.619</td><td>C11-C14=N28</td><td>124.77(7)</td><td>123.93</td><td>124.03</td></tr><tr><td>P7=O9</td><td>1.5860(6)</td><td>1.626</td><td>1.619</td><td>O8=P7-C11</td><td>114.54(4)</td><td>115.16</td><td>114.30</td></tr><tr><td>C14=N28</td><td>1.283(10)</td><td>1.278</td><td>1.278</td><td>O8=P7=O9</td><td>113.84(4)</td><td>114.76</td><td>114.78</td></tr><tr><td>N28-N29</td><td>1.387(10)</td><td>1.384</td><td>1.388</td><td>O8=P7=O10</td><td>114.29(4)</td><td>114.59</td><td>114.60</td></tr><tr><td>C11-C14</td><td>1.5165(11)</td><td>1.522</td><td>1.520</td><td>C11-C14-C15</td><td>117.14(7)</td><td>116.61</td><td>116.43</td></tr><tr><td>C14-C15</td><td>1.5240(11)</td><td>1.522</td><td>1.522</td><td>N28=C14-C15</td><td>118.04(7)</td><td>119.35</td><td>119.44</td></tr><tr><td>C15-C35</td><td>1.5267(13)</td><td>1.536</td><td>1.536</td><td>C14-C15-C35</td><td>111.61(7)</td><td>113.20</td><td>113.49</td></tr></tbody></table>

alter slightly in the gas-phase or in the presence of chloroform as solvent. On the other hand, a comparison of the calculated and experimental SCXRD geometrical properties shows that they are quite similar.

### 2.2.5. Theoretical/ experimental infrared characterisation of 3c

The experimental IR spectral data of 3c (black in Fig. 14, see also Fig. S49) show characteristic absorption bands in the range of 2900–3500, 1600–1700, 1250–1300 and 1000–1200 cm⁻¹ which may be assigned to ν(NH), ν(C=N), ν(P=O) and ν(P-O-C), respectively. This is consistent with the literature data [51-55] and also with the computed wave-numbers used to plot the theoretical spectrum (red in Fig. 14, see also Fig. S51). The theoretical spectrum was determined after optimization of the 3c structure using Gaussian 09 with B3LYP/ 6-311++ G (d.p)

basis set. In the experimental spectrum, the N-H bond gives rise to broad band between 2900 and 3500 cm⁻¹ indicating that the N-H groups are engaged in intramolecular and intermolecular hydrogen bonding, in line with the crystal structure (Fig. 8). This broadness is in accordance with the IR features described in related work dealing on compounds exhibiting a H-N···O hydrogen bonding [56-58].

### 2.3. Molecular docking of the β-cycloalkoxyphosphonated hydrazones

The current work investigated by molecular docking the interactions of β-cycloalkoxyphosphonated hydrazones 3a-f as hosts with the active binding site of both enzymes Butyrilcholinesterase (BuChE) (PDB ID: 4BDS) and Acetylcholinesterase (AChE) (PDB ID: 4EY7). Tacrine

<footnote>*The atom numbering is shown in the optimized Z configuration of 3c.</footnote>
<figure><img src="image_20.png" /><figcaption>**Fig. 13.** DFT/SCXRD graphical correlation for bond lengths of **3c Z** (a: calculated bond lengths in the gas-phase b: calculated bond lengths in CHCl<sub>3</sub>) and bond angles (c: calculated angles gas-phase d: calculated angles in CHCl<sub>3</sub>).</figcaption></figure>

**Table 4**

Comparison of **3c Z** isomer H-bond distance (Å) and angle (°) from X-ray diffraction and DFT optimization.

<table><thead><tr><th></th><th></th><th>d (O···H-N) (Å)</th><th>θ (O···H-N) (°)</th></tr></thead><tbody><tr><td rowspan="2">DFT (3c Z)</td><td>gas-phase</td><td>2.094</td><td>154.44</td></tr><tr><td>in CHCl<sub>3</sub></td><td>2.115</td><td>154.14</td></tr><tr><td>SCXRD (3c Z)</td><td></td><td>2.630 (17)</td><td>143.60(13)</td></tr></tbody></table>

(https://en.wikipedia.org/wiki/Tacrine) and Donepezil (https://en.wikipedia.org/wiki/Donepezil), two acetylcholinesterase inhibitors employed for the treatment of Alzheimer disease [59-62], were used as reference standards in our study. One of the common features occurring in these reference molecules and hydrazones **3** is that they contain nitrogen atoms in their scaffolds. They also incorporate heterocyclic rings and six-membered aromatic cycles capable of interacting with the active site (Fig. 15). In the literature, there are compounds with some or all of these features that have shown confirmed inhibitory activity for one or both proteins [14-16,63-65]. For example, tert-butyl *N*-(3-(2, 3-dimethoxyphenyl)-2-phenylpropyl)sulfamoylcarbamate with a phenyl group and a N-atom in its backbone shows a high inhibitory effect against AChE [64]. Further examples [14-16] are given in Fig. 1.

Hence, we decided to employ *in silico* molecular docking simulations to test our synthesized hydrazones. To assess whether hydrazones **3**

could represent potential candidates for inhibition of both proteins, an investigation based on free energies released as the ligands bind to the proteins as well as their binding mechanism to key residues, has been conducted.

### 2.3.1. Active sites of AChE and BuChE

According to previous studies [66-68], it is confirmed that the structure of AChE possesses a catalytic active site (CAS), a 20 Å-deep gorge and a peripheral anionic site (PAS). The active site residues of the catalytic triad are: SER203, HIS447, GLU 334. This active site has a subside involving TRP86. The PAS of AChE contains TYR72, ASP74, TYR124, TRP286, SER293 and TYR341. The overall structure of BuChE is similar to that of AChE with 65 % of the amino acids in common. The difference lies in the acyl-binding pocket which is larger in BuChE than in AChE. The catalytic triads of BuChE consist of the following amino acids: HIS438, SER198, GLU325. There are key residues: TYR332, PHE329, TRP82 belonging to the anionic site, which is essential for the reaction. In addition, the oxyanion hole, incorporating GLY116, GLY117 and ALA119, stabilizes the transition-state through hydrogen-bond interactions [69,70]. These amino acids are important for the active site of both AChE and BuChE.

### 2.3.2. Interactions of references and compounds with BuChE and AChE receptors

The interactions of Tacrine with BuChE as well as that of Donepezil
<figure><img src="image_21.png" /><figcaption>Fig. 14. Theoretical (red) and experimental (black) ATR-IR spectra of 3c.</figcaption></figure>

<figure><img src="image_22.png" /><figcaption>Fig. 15. Structures of reference molecules (Donepezil and Tacrine), and previously reported benzylhydrazones [14] and our phosphonated hydrazones 3.</figcaption></figure>

with AChE were analysed to predict their binding mode using the AutoDock vina Software [71]. Results are reported in Table 5 and 6. All compounds were docked into the same groove of the native co-crystallized binding site of both proteins.

Firstly, we studied the interaction between the hydrazone derivatives and BuChE. It is important to note that all **3a-f** compounds exhibit different types of interaction (H-bond, Van der Waals...) with key residues of the BuChE active site. All six **3a-f** hydrazones subjected
<table><caption>**Table 5**
Interactions occurring between the ligand and BuChE: binding affinity, number of conventional hydrogen bonds and interacting amino acid residues.</caption><thead><tr><th rowspan="2">Ligand</th><th rowspan="2">Estimated free energy of<br>binding (kcal/mol)</th><th colspan="3">Intermolecular interactions</th></tr><tr><th>Interacting amino acid residues</th><th colspan="2">Conventional hydrogen bonds</th></tr><tr><th></th><th></th><th></th><th>Number of H-bonds</th><th>Involved amino<br>acid Residue</th><th>Distance<br>(Å)</th></tr></thead><tbody><tr><td rowspan="2">3a</td><td rowspan="2">-7.8</td><td rowspan="2">TRP82, GLY116*, GLY117, GLN119, THR120**, SER198, TRP231, PRO285,<br>LEU286, VAL288, ALA328, PHE329, PHE398, HIS438, GLY439,</td><td rowspan="2">3</td><td>GLY116</td><td>1.71</td></tr><tr><td>THR120</td><td>2.85</td></tr><tr><td rowspan="2">3b</td><td rowspan="2">-5.5</td><td rowspan="2">TRP82, GLY115, GLY116, THR120, TYR128, GLU197*, SER198, ALA199,<br>ALA328, PHE32<br>9, TRP430, MET437, HIS438*, GLY439, TYR440</td><td rowspan="2">2</td><td>GLU197</td><td>2.89</td></tr><tr><td>HIS438</td><td>2.09</td></tr><tr><td rowspan="2">3c</td><td rowspan="2">-7.1</td><td rowspan="2">TRP82, GLY116*, GLY117, THR120*, SER198, TRP231, LEU286, VAL288,<br>PHE329, ALA328, TYR332, PHE398, HIS438</td><td rowspan="2">2</td><td>GLY116</td><td>2.93</td></tr><tr><td>THR120</td><td>2.36</td></tr><tr><td rowspan="2">3d</td><td rowspan="2">-7.4</td><td rowspan="2">ILE69, ASP70, SER79, TRP82, ASN83, GLY116*, GLY117, GLN119, THR120**,<br>SER198, TRP231, PRO285, LEU286, VAL288, PHE329, PHE 398</td><td rowspan="2">3</td><td>GLY116</td><td>1.85</td></tr><tr><td>THR120</td><td>3.03</td></tr><tr><td rowspan="2">3e</td><td rowspan="2">-7.7</td><td rowspan="2">TRP82, GLY116*, GLY117, GLN119, THR120*, SER198, TRP231, PRO285,<br>LEU286, VAL288, ALA328, PHE329, TYR332, PHE 398, HIS438,</td><td rowspan="2">2</td><td>GLY116</td><td>2.88</td></tr><tr><td>THR120</td><td>2.49</td></tr><tr><td rowspan="2">3f</td><td rowspan="2">-7.5</td><td rowspan="2">ASP70, SER79, TRP82, GLY116, GLY117, SER198, TRP231, PRO285*, LEU286,<br>VAL288, PHE329, PHE398, HIS438</td><td rowspan="2">1</td><td>PRO285</td><td>2.58</td></tr><tr><td></td><td></td><td></td></tr><tr><td>Tacrine<sup>R1</sup></td><td>-8.1</td><td>TRP82, GLY115, GLY116, GLU197, SER198, ALA328, TYR332, TRP430,<br>MET437, HIS438, GLY439, TYR440</td><td>0</td><td>-</td><td>-</td></tr></tbody></table>

R1: Reference 1; *: Amino acid residue involved in H-bond formation.

<table><caption>**Table 6**
Interactions occurring between the ligand and AChE: binding affinity, number of conventional hydrogen bonds and interacting amino acid residues.</caption><thead><tr><th rowspan="2">Ligand</th><th rowspan="2">Estimated free energy of<br>binding (kcal/mol)</th><th colspan="3">Intermolecular interactions</th></tr><tr><th>Interacting amino acid residues</th><th colspan="2">Conventional hydrogen bonds</th></tr><tr><th></th><th></th><th></th><th>Number of H-bonds</th><th>Involved amino<br>acid Residue</th><th>Distance<br>(Å)</th></tr></thead><tbody><tr><td rowspan="2">3a</td><td rowspan="2">-7.2</td><td rowspan="2">TYR72, TYR124, TRP286, LEU289, GLN291, GLU292, SER293*, VAL294,<br>PHE295, ARG296*, TYR341</td><td rowspan="2">2</td><td>SER293</td><td>2.56</td></tr><tr><td>ARG296</td><td>2.80</td></tr><tr><td rowspan="2">3b</td><td rowspan="2">-6.4</td><td rowspan="2">ASP74*, THR83, TRP86, GLY121, GLY122, TYR124**, SER125, SER203,<br>PHE297, TYR337, PHE338, TYR341*, HIS447</td><td rowspan="2">4</td><td>ASP74</td><td>2.10</td></tr><tr><td>TYR124</td><td>3.12</td></tr><tr><td rowspan="2">3c</td><td rowspan="2">-8.9</td><td rowspan="2">TRP86, GLY121, GLY122, TYR124*, SER125, TRP286, VAL294, PHE295,<br>PHE297, TYR337, PHE338, TYR341, HIS447</td><td rowspan="2">1</td><td>TYR341</td><td>2.36</td></tr><tr><td>TYR124</td><td>3.25</td></tr><tr><td rowspan="2">3d</td><td rowspan="2">-9.2</td><td rowspan="2">ASP74*, THR83, TRP86, GLY121, TYR124*, TRP286, VAL294, PHE295,<br>PHE297, TYR337, PHE338, TYR341*, HIS447</td><td rowspan="2">3</td><td>ASP74</td><td>1.99</td></tr><tr><td>TYR124</td><td>3.03</td></tr><tr><td rowspan="2">3e</td><td rowspan="2">-7.0</td><td rowspan="2">TYR124, TRP286, LEU289, GLN291, GLU292, SER293*, VAL294, PHE295,<br>ARG296*, PHE297, PHE338, TYR341</td><td rowspan="2">2</td><td>SER293</td><td>2.51</td></tr><tr><td>ARG296</td><td>2.59</td></tr><tr><td rowspan="2">3f</td><td rowspan="2">-9.4</td><td rowspan="2">ASP74*, THR83, TRP86, GLY120, GLY121, TYR124*, GLU202, TRP286,<br>VAL294, PHE295, PHE297, TYR337, PHE338, TYR341*, HIS447, GLY448</td><td rowspan="2">3</td><td>ASP74</td><td>2.28</td></tr><tr><td>TYR124</td><td>3.07</td></tr><tr><td rowspan="2">Donepezil<sup>R2</sup></td><td rowspan="2">-11.6</td><td rowspan="2">TYR72, ASP74, TRP86, GLY121, TYR124, GLU202, TRP286, LEU289, VAL294,<br>PHE295*, ARG296, PHE297, TYR337, PHE338, TYR341, HIS447, GLY448</td><td rowspan="2">1</td><td>TYR341</td><td>2.10</td></tr><tr><td>PHE295</td><td>3.05</td></tr></tbody></table>

R2: Reference; *: Amino acid residue involved in H-bond formation.

to molecular docking display H-bonding whereas the reference drug does not present any H-bonding. According to the literature [72], a hydrogen bond with a bond distance < 3.1 Å is considered as a strong one. Thus, compounds **3a** and **3d** showed the highest number of conventional H-bonds (3 strong H-bonds). In fact, compound **3a** is bonded to the oxyanion hole amino acid GLY116 by one strong hydrogen bond (bond distance = 1.71 Å) and to another THR120 residue with two strong hydrogen bonds (bond distances = 2.85 Å; 2.89 Å). Similarly, compound **3d** establishes a strong hydrogen bond with GLY116 (bond distance = 1.85 Å) and two strong H-bond with THR120 (bond distances = 2.88 Å; 3.03 Å). In addition to hydrogen bonding, compounds **3a-f** exhibit other types of interaction with important residues of the active site. In fact, compound **3a** interacts with residues of the catalytic triad as follows: Van der Waals interaction with amino acids SER198 and HIS438. Also, we note interactions with the anionic site: a π-sigma

interaction with amino acid TRP82 and a T-shaped π-π interaction with PHE329. Compound **3d** exhibits a Van der Waals contact with the SER198 residue of the catalytic triad, a π-alkyl interaction and a T-shaped π-π interaction with the anionic site residues PHE329 and TRP82, respectively. These described interactions indicate that compounds **3a** and **3d** can be stable within the BuChE active site. With regard to the binding energy score, Tacrine showed a lower binding energy score –8.1 kcal/mol compared to the six hydrazone derivatives (–7.1 kcal/mol to –7.8 kcal/mol). **3a** owns the best binding energy score (–7.8 kcal/mol), followed by compound **3e** (–7.7 kcal/mol) and **3d** (–7.4 kcal/mol), reflecting high affinity with the active site.

Based on these findings, it appeared promising for forthcoming studies to assess the *in vitro* inhibitory activity of **3a** and **3d** against BuChE. **Table 5** summarizes the docking results for BuChE: binding energy score, conventional H-bonds, distances and interacting amino
<figure><img src="image_23.png" /><figcaption>Fig. 16. 2D binding patterns of hydrazone derivatives 3a-f with BuChE (PDB: 4BDS).</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>Fig. 17. 2D binding patterns of hydrazone derivatives 3a-f with AChE (PDB: 4EY7).</figcaption></figure>
<figure><img src="image_25.png" /><figcaption>**Fig. 18.** Presentation of the best docking conformations of compounds **3** and references into BuChE and AChE active sites. **I:** Compound **3a** (blue), **3d** (yellow) and **Tacrine** (purple) are inserted into BuChE (PDB ID: 4BDS); **II:** Compound **3f** (blue), **3d** (yellow) and **Donepezil** (purple) are inserted into AChE (PDB ID: 4EY7).</figcaption></figure>

acid residues.

Fig. 16

Then, we proceeded to study the interaction between hydrazones **3a-f** and AChE (Fig. 17). We found that most of tested compounds form more than one H-bonds with certain key amino acid residues in the active site compared to Donepezil, which present only one H-bond with the amino acid PHE295 (bond distance = 3.05 Å). However, Donepezil has a better binding energy score of -11.6 kcal/mol compared to compounds **3a-f** (-6.4 kcal/mol to -9.4 kcal/mol) (Table 6). The **3d** derivative has a binding energy score of -9.2 kcal/mol and generates 3

strong H-bonds with amino acids of the PAS pocket as follows: ASP74 (bond distance = 1.99 Å), TYR124 (bond distance = 3.03 Å), TYR341 (bond distance = 2.31 Å). On the other hand, compound **3f** exhibits -9.4 kcal/mol as binding energy score and establishes 3 strong H-bonds with PAS pocket residues: ASP 74 (bond distance = 2.28 Å), TYR124 (bond distance = 3.07 Å), TYR341 (bond distance = 2.10 Å). Additionally, both **3d** and **3f** interact via a Carbon-Hydrogen contact with HIS447 residue of the catalytic triad and make a π-sigma contact with a key amino acid: TRP86.

Because of their high binding energy values and several interactions
<table><caption>**Table 7**
ADMET and drug-likeness properties of compounds **3a**, **3d** and **3f**.</caption><thead><tr><th>Entry</th><th>MW<sup>a</sup><br>(g/mol)</th><th>TPSA<sup>b</sup><br>(Å<sup>2</sup>)</th><th>n-<br>ROTB<sup>c</sup></th><th>MLog P<br>d<br>WLog<br>p<sup>d</sup></th><th>n- H bond<br>acceptors<sup>e</sup></th><th>n- H bond<br>donors<sup>f</sup></th><th>Lipinski's<br>violation</th><th>Verber<br>violation</th><th>Egan<br>violations</th><th>PAINS<sup>g</sup><br>alert</th></tr></thead><tbody><tr><td></td><td>&lt;500</td><td>&lt;140</td><td>&lt;11</td><td>≤ 5</td><td>&lt;10</td><td>&lt;5</td><td>≤1</td><td>≤1</td><td>≤1</td><td></td></tr><tr><td>3a</td><td>324.36</td><td>83.72</td><td>6</td><td>2.08<br>3.59</td><td>5</td><td>1</td><td>Accepted</td><td>Accepted</td><td>Accepted</td><td>0 alert</td></tr><tr><td>3d</td><td>296.30</td><td>83.72</td><td>4</td><td>1.57<br>2.81</td><td>5</td><td>1</td><td>Accepted</td><td>Accepted</td><td>Accepted</td><td>0 alert</td></tr><tr><td>3f</td><td>338.38</td><td>69.73</td><td>7</td><td>2.32<br>3.85</td><td>5</td><td>1</td><td>Accepted</td><td>Accepted</td><td>Accepted</td><td>0 alert</td></tr><tr><td>ADMET</td><td>Absorption<br>Caco2<sup>h</sup> (10<sup>-6</sup><br>cm/s)</td><td>HIA<sup>i%</sup></td><td>Distribution<br>BBB<sup>j</sup> (log BB)</td><td>Metabolism<br>CYP2D6<sup>k</sup><br>substrate</td><td>CYP1A2<sup>l</sup><br>inhibitor</td><td>Excretion<br>Total Clearance (mL/min/kg)</td><td>Toxicity<br>Hepato-toxicity<sup>m</sup></td><td></td><td></td><td></td></tr><tr><td>3a</td><td>0.894</td><td>92.97</td><td>-0.53</td><td>No</td><td>Yes</td><td>0.53</td><td></td><td></td><td>No</td><td></td></tr><tr><td>3d</td><td>0.901</td><td>92.43</td><td>-0.50</td><td>No</td><td>Yes</td><td>0.51</td><td></td><td></td><td>No</td><td></td></tr><tr><td>3f</td><td>1.351</td><td>91.51</td><td>-0.27</td><td>No</td><td>Yes</td><td>0.62</td><td></td><td></td><td>No</td><td></td></tr></tbody></table>

a: Molecular weight, b: Topological Polar Surface Area, c: number of Rotable Bonds, d, d': logarithm of partition coefficient of compound between n-octanol and water, e: number of hydrogen bond acceptors, f: number of hydrogen bond donors, g: Pan Assay Interference Structures, h: colon adenocarcinoma, i: Human Intestinal Absorption, j: Blood-Brain Barrier permeability, k: Cytochrome P450 2D6 inhibitor, l: Cytochrome P450 1A2 inhibitor, m: Hepatic toxicity.

with key amino acids in the AChE active site, compounds **3d** and **3f** appears as potential candidates for AChE inhibition activity. The best docking conformations of compounds **3** and references (Tacrine and Donepezil) are shown into BuChE and AChE active sites (Fig. 18).

## 2.4. ADMET properties

A computational study was performed to determine the ADMET properties of the three candidates **3a**, **3d** and **3f** using the SwissADME and pkCSM-pharmacokinetics servers [73,74]. According to the results achieved reported in Table 7, the three tested compounds have some physicochemical properties in common. In fact, their molecular weights are inferior than 500 g/mol, they contain less than 5 H-bond donors and 10 H-bond acceptors. Also, they exhibit TPSA (Topological Polar Surface Area see https://en.wikipedia.org/wiki/Polar surface area) values less than 140 Å². In addition, the logarithm of the partition coefficient of compound between n-octanol and water MLogP and WLogP values are under 5 and the nROTB (number of Rotable Bonds) values are below 11. Based on these results, the three tested hydrazones meet Lipinski's rule of five [75], Veber's [76] and Egan's [77] rules with zero violations and no pan-assay interference structures (PAINS) alerts were detected.

Furthermore, the ADMET-absorption was evaluated and is presented in Table 7. The Caco-2 cell permeability is an indication of intestinal absorption [78]. Values obtained for all compounds ranged between 0.894 0.10⁻⁶ and 1.351 0.10⁻⁶ cm/s, indicating good permeability. Human intestinal absorption (HIA) values varied from 91.51 to 92.97 % (> 80 %) showing that compounds can have a high gastro-intestinal permeation. As seen in the ADMET- distribution section, The Blood Brain Barrier (BBB) permeability values of our compounds were predicted to range from -0.53 to -0.27 with log BB > -1 indicating that they are BBB permeable [79]. Metabolism-prediction was estimated according to the inhibition of the cytochrome P450 (CYP) superfamily of enzymes. In fact, CYP inhibition may cause toxicity or lack of efficacy for a drug [80]. The results reported in Table 7 indicate that molecules **3a**, **3d** and **3f** were predicted to likely inhibit CYP1A2. In contrast, they were predicted to not inhibit CYP2D6. Excretion values were evaluated using the total clearance descriptor. Values ranged from 0.51 to 0.62 mL/min/kg (< 5 mL/min/kg). The toxicity level was predicted to assess hepatotoxicity. None of the candidates was found to cause liver damage.

# 3. Experimental

## 3.1. Apparatus and general information

NMR spectra were recorded on 300 and 400 MHz Brucker

spectrometers. Chemical shifts were reported in ppm relative to the residual solvent peak (7.27 ppm for CHCl₃) for ¹H spectra, (77.00 ppm for CDCl₃) for ¹³C spectra and referenced to H₃PO₄ for the ³¹P spectra. The multiplicity of signals is indicated by the following abbreviations: s (singlet), d (doublet), m (multiplet). Column chromatography separations were performed using silica gel (0.040–0.060 mm). Infrared spectra were obtained with a Shimadzu IRS Spirit spectrometer using ATR technique in the 4000–400 cm⁻¹ range.

## 3.2. General procedure of the synthesis of 1-(5,5-dialkyl-2-oxo-1,3,2-dioxaphosphoranyl)-propa-1,2-diene 2a-f

In an ice bath, A mixture of propargylalcohol (1.5. 10⁻² mol) and triethylamine (1.6. 10⁻² mol, 2.16 mL) in 100 mL of anhydrous THF was added to a three-neck oven dried round bottom flask and maintained at 0 °C. Next, a solution of 2-chloro-5,5-dialkyl-1,3,2-dioxaphosphorinane 1 (1.5. 10⁻² mol) was added dropwise using an addition funnel. When all the reagent 1 was added, a white precipitate of triethylamine hydrochloride appears, and the reaction mixture was allowed to stir for 1 h at room temperature. After that, the resulting mixture was heated in THF under reflux for 1 h. The triethylamine hydrochloride was removed by filtration and THF was evaporated in vacuo. A resulting white solid was obtained and washed by hexane to afford the final compound.

Allenylphosphonates **2d**, **2e** were synthesized according to literature procedures [22,23].

### 1-(5-methyl-5-propyl-2-oxo-1,3,2-dioxaphosphoranyl-3-phenyl-prop-1,2-diene) (2a)

M = 292.00 g/mol, Yield = (3.67 g, 12.6 mmol, 84 %), White solid, mp. (°C ± 2) = 116 °C. *Cis/Trans* ratio 27: 73, Diastereomer *Cis*: ³¹P NMR: δ ³¹P = 6.67, ¹H NMR: δ ¹H = 0.69 (s, 3H, CH3-C), 0.88 (t, 3H, CH3-CH2-CH2), 1.18 (m, 2H, C-CH2), 1.26 (m, 2H, C-CH2-CH2), 3.82–4.08 (m, 4H, [AB]₂), 5.76 (d, 1H, ⁴JₚH =6.7 Hz, =CH-C6H5), 6.44 (dd, ²JₚH = 13.3 Hz, ⁴JₚH = 6.7 Hz, P-CH=), 7.16 (m, 5H, arom-H). ¹³C NMR: δ ¹³C= 14.3 (s, CH3-CH2-CH2), 16.5 (s, CH2-CH2-C), 17.7 (s, CH3-C), 35.1 (d, ³JₚC = 6.4 Hz, -C-), 35.8 (s, CH2-C), 75.5 (d, ²JₚC = 6.6 Hz, CH2-O-P), 75.9 (d, ²JₚC = 6.8 Hz, CH2-O-P), 81.7 (s, P-CH=), 95.9 (d, ³JₚC = 16.6 Hz, =CH-C₆H₅), 127.2 (d, ⁶JₚC = 2.7 Hz, CH=arom), 128.1 (d, ⁷JₚC = 1.8 Hz, CH=arom), 129.1 (d, ⁵JₚC = 1.6 Hz, CH=arom), 131.0 (d, ⁴JₚC = 8.5 Hz, CH=arom), 213.2 (d, ²JₚC = 1.8 Hz, =C=).

Diastereomer *trans*: ³¹P NMR: δ ³¹P = 7.4, ¹H NMR: δ ¹H = 0.74 (t,3H, CH3-CH2-CH2), 0.89 (t, 2H, CH3-CH2-CH2), 1.07 (s, 3H, CH3-C), 1.10 (m, 2H, C-CH2-CH2), 3.90–4.01 (m, 4H, [AB]₂), 5.76 (d, 1H, ⁴JₚH = 6.7 Hz, =CH-C₆H₅), 6.44 (dd, ²JₚH = 13.3 Hz, ⁴JₚH = 6.7 Hz, P-CH=), 7.18 (m, 5H, arom-H). ¹³C NMR: δ ¹³C= 14.6 (s, CH3-CH2-CH2), 16.2 (s, CH2-CH2-C), 19.0 (s, CH3-C), 35.1 (d, ³JₚC = 6.3 Hz, -C-), 36.7 (s, -CH2-C-
)), 75.8 (d, $^2J_{PC}$ = 6.6 Hz, CH$_2$-O-P), 76.3 (d, $^2J_{PC}$ = 6.8 Hz, CH$_2$-O-P), 81.8 (s, P-CH=), 95.9 (d, $^3J_{PC}$ = 16.6 Hz, =CH-C$_6$H$_5$), 127.2 (d, $^6J_{PC}$ = 2.7 Hz, CH=arom), 128.1 (d, $^7J_{PC}$ = 1.8 Hz, CH=arom), 128.9 (d, $^5J_{PC}$ = 1.6 Hz, CH=arom), 131.0 (d, $^4J_{PC}$ = 8.5 Hz, CH=arom), 213.4 (d, $^2J_{PC}$ = 1.9 Hz, =C=). GC-MS: *m/z* = 292 [*M*+], 196 [(O)P(OH)$_2$-HC=C=CH-Ph], 178 [O-P(O)-CH=C=CH-Ph], 115 [Ph-CH=C=CH], 89 [P(=O)-O-CH$_2$-C], 69 [CH$_2$-CH-CH$_2$-CH$_2$-CH$_3$], 55 [CH$_3$-C-CH$_2$-CH$_2$], 41 [CH$_3$-C-CH$_2$]. IR-ATR (cm$^{-1}$): 2973, 1948, 1468, 1269, 1064, 1008, 975, 815, 538, 460 cm$^{-1}$.

### 1-(2-oxo-1,3,2-dioxapohosphoranyl-3,3-dimethylprop-1,2-diene) (2b)

M = 188.16 g/mol, Yield = (2.11 g, 11.21 mmol, 75 %), White solid, mp. (°C ± 2) = 49 °C, ³¹P NMR: δ ³¹P = 9.6, ¹H NMR: δ ¹H = 1.71 (d, 3H, Me), δ = 1.74 (d, 3H, Me), (2.12–2.24) (m, 2H, CH2-CH2-O), (4.21-4.44) (m, 4H, (CH$_2$-O) cycl), (5.13–5.17) (m, P-CH=). ¹³C NMR: δ ¹³C = 19.2 (d, $^4J_{PC}$ = 6.7 Hz, Me), 26.4 (d, $^3J_{PC}$ = 8.2 Hz, -CH$_2$-), 67.6 (d, $^2J_{CP}$ = 6.7 Hz, CH2-O-P), 76.6 (d, $^1J_{PC}$ = 192.1 Hz, P-CH=), 97.3 (d, $^3J_{PC}$ = 17.2 Hz, =C), 210.1 (s, =C=). IR-ATR (cm$^{-1}$): 2973, 1950, 1475, 1269, 1064, 1008, 815, 538, 460 cm$^{-1}$.

### 1-(2-oxo-1,3,2-dioxapohosphoranyl-3-phenylprop-1,2-diene) (2c)

M = 236.21 g/mol, Yield = (2.76 g, 11.70 mmol, 78 %), White solid, mp. (°C ± 2) = 60 °C, ³¹P NMR: δ ³¹P = 7.6, ¹H NMR: δ ¹H = (2.13–2.24) (m, 2H, CH2-CH2-O), 2.44 (s, 3H, CH$_3$), (4.27-4.39) (m, 4H, (CH$_2$-O) cycl), (5.70–5.75) (m, 1H, P-CH=), 7.16 (m, 5H, arom-H). ¹³C NMR: δ ¹³C = 16.0 (s, CH$_3$), 26.5 (d, $^3J_{PC}$ = 8.3 Hz, -CH$_2$-), 67.7 (d, $^2J_{CP}$ = 7.0 Hz, =CH-P), 79.9 (s, CH$_2$-O-P), 82.5 (s, CH$_2$-O-P), 102.5 (d, $^3J_{PC}$ = 17.3, =C-Ph), (125.9–133.9) (m, Ph), 213.3 (s, =C=).

### 1-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphoranyl-3-phenylprop-1,2-diene) (2d)

M = 264.26 g/mol, Yield = (2.85 g, 10.80 mmol, 72 %), White solid, mp. (°C ± 2) = 104 °C. ³¹P NMR: δ ³¹P = 6.5, ¹H NMR: δ ¹H = 0.86 (s, 3H, Me), 1.21 (s, 3H, Me), 3.94–4.07 (m, 4H, [AB]$_2$), 5.80 (d, $^4J_{PH}$ = 6.7 Hz, 1H, =CH-C$_6$H$_5$), (6.49–6.55) (dd, 1H, $^2J_{PH}$ = 12.0 Hz, $^4J_{HH}$ = 6.0 Hz =CH-P), (7.28–7.38) (m, 5H, Ph). ¹³C NMR: δ ¹³C = 20.8 (s, CH$_3$ax), 21.7 (s, CH$_3$eq), 23.3 (s, CH$_2$ax), 32.4 (d, $^3J_{PC}$ = 6.7 Hz, -C-), 76.8 (d, $^1J_{PC}$ = 6.7, =CH-P), 81.51 (s, CH$_2$-O-P), 84.04 (s, CH$_2$-O-P), 95.9 (d, $^3J_{PC}$ = 16.5, =CH-Ph), (127.23–131.05) (m, Ph), 213.3 (s, =C=). IR-ATR (cm$^{-1}$): 2973, 1941, 1470, 1269, 1064, 1008, 975, 815, 538, 460 cm$^{-1}$.

### 1-(5,5-diethyl-2-oxo-1,3,2-dioxaphosphoranyl-3-phenylprop-1,2-diene) (2e)

M = 292.00 g/mol, Yield = (2.76 g, 13.50 mmol, 90%), White solid, mp. (°C ± 2) = 114 °C. ³¹P NMR: δ ³¹P = 7.4, ¹H NMR: δ ¹H = 0.75 (t, 3H, CH$_3$CH$_2$), 0.87 (t, 3H, CH$_3$-CH$_2$), 1.24 (m, 2H, CH$_2$-C), 1.60 (m, 2H, CH$_2$-C), 3.93–4.19 (m, 4H, [AB]$_2$), 5.80 (d, 1H, $^4J_{PH}$ = 6.8 Hz, =CH-C$_6$H$_5$), 6.47 (dd, $^2J_{PH}$ = 13.3 Hz, $^4J_{HH}$ = 6.5 Hz, P-CH=), 7.30 (m, 5H, arom-H). ¹³C NMR: δ ¹³C = 7.1 (s, CH$_3$ax), 7.2 (s, CH$_3$eq), 23.2 (s, CH$_2$ax), 23.5 (s, CH$_2$eq), 37.7 (d, $^3J_{PC}$ = 5.9 Hz, -C-), 74.2 (d, $^2J_{PC}$ = 6.8 Hz, C CH$_2$-O-P), 83.2 (d, $^1J_{PC}$ = 192.2 Hz, P-CH=), 96.0 (d, $^3J_{PC}$ = 16.7 Hz, =CH-C$_6$H$_5$), 127.4 (d, $^5J_{PC}$ = 2.8 Hz, C-O), 129.1 (d, $^7J_{PC}$ = 1.6 Hz, C-p), 214.2 (s, =C=).

### 1-(5,5-diethyl-2-oxo-1,3,2-dioxaphosphoranyl-3,3-dimethyl-prop-1,2-diene) (2f)

M = 244 g/mol, Yield = (2.63 g, 10.80 mmol, 72 %), White solid, mp. (°C ± 2) = 110 °C, ³¹P NMR: δ ³¹P = 11.2, ¹H NMR: δ ¹H = 0.75 (m, 3H, CH$_3$CH$_2$), 0.81 (m, 3H, CH$_3$CH$_2$), 1.25 (m, 2H, CH$_2$-C), 1.56 (m, 2H, CH$_2$-C), 1.71 (d, $^5J_{PH}$ = 4.0 Hz, 3H, C-CH$_3$), 1.73 (d, $^5J_{PH}$ = 4.0 Hz, 3H, C-CH$_3$), 3.47–4.10 (m, 4H, [AB]$_2$), 5.14 (m, P-CH=), ¹³C NMR: δ ¹³C = 7.1 (s, CH$_3$ax), 7.2 (s, CH$_3$eq), 19.1 (s, CH$_2$ax), 22.2 (s, CH$_2$eq), 22.5 (s, Me), 23.0 (s, Me), 37.3 (d, $^3J_{PC}$ = 6.0 Hz, -C-), 73.8 (d, $^2J_{PC}$ = 7.0 Hz, CH$_2$-O-P), 75.2 (d, $^1J_{PC}$ = 202.0 Hz, P-CH=), 97.3 (d, $^3J_{PC}$ = 16.1 Hz, =C-CH$_3$), 210.9 (s, =C=).

### 3.3. General procedure of the synthesis of β-cycloalkoxyphosphonated hydrazones 3a-f

To a solution of allenylphosphonate (10⁻³ mol) in 20 mL of CHCl$_3$, 10⁻³ mol of the respective hydrazine derivative was added dropwise at room temperature. The mixture was stirred for 20 min. Then, the solution was heated at 62 °C for 40 min-6 h. After concentration under reduced pressure, the crude material was washed by a 50:50 hexane/ether mixture. Hydrazones 3d, 3e were synthesized according to literature procedures [22,23].

### 1-(5-methyl-5-propyl-2-oxo-1,3,2-dioxaphosphoranyl)-3-phenylpropanone (3a)

M = 324.36 g/mol. Yield = (0.24 g, 0.74 mmol, 74 %), White solid, mp. (°C ± 2) = 160 °C, 3a$_1$ (Z/E) ratio 61: 39, 3a$_2$ (Z/E) ratio 57: 43, ³¹P NMR: δ ³¹P$_1$ = 21.12, δ ³¹P$_2$ = 21.37, δ ³¹P$_3$ = 21.55, δ ³¹P$_4$ = 21.69, ¹H NMR: δ ¹H = [0.82–0.86] (s, 3H, CH$_3$-C), [0.90–0.96] (m, 3H, CH$_3$-CH$_2$-CH$_2$), [1.16–1.24] (m, 2H, C-CH$_2$), [1.25–1.34] (m, 2H, C-CH$_2$-CH$_2$), [2.72–2.83] (d, 2H, P-CH$_2$), 3.50 (s, 2H, CH$_2$-Ph), [3.65–4.18] (m, 4H, [AB]$_2$), [5.19–5.60] (s, 2H, NH$_2$), [7.11–7.25] (m, 5H, Ph), ¹³C NMR: δ ¹³C = [14.6–14.9] (CH$_3$-CH$_2$-CH$_2$), [16.1–16.6] (CH$_2$CH$_2$C), [18.2–18.8] (CH$_3$-C), [24.2–26.1] (CH$_2$-P), 35.1 (-C-), 36.5 (-CH$_2$-C), 44.3 (CH$_2$-Ph), 73.7 (CH$_2$-O-P), 74.5 (CH$_2$-O-P), 126.9–137.4 (Ph) [143.0–146.2] (CN). GC-MS: *m/z* = 324 [*M*⁺], 130 [(CH$_3$-CH$_2$) (CH$_3$) C (CH$_2$O)$_2$], 103 [C$_6$H$_5$-CH$_2$-C], 91 [C$_6$H$_5$-CH$_2$], 55 [CH$_3$-C(CH$_2$)-CH$_2$]. IR-ATR (cm$^{-1}$): 3478, 2960, 1650, 1500, 1265, 696, 1006, 527 cm$^{-1}$.

### 1-(2-oxo-1,3,2-dioxaphosphoranyl)-3,3-dimethylpropanone (3b)

M = 220.21 g/mol, Yield = (0.17 g, 0.80 mmol, 80 %), Pasty solid, Z/E ratio 96: 4, Z isomer: ³¹P NMR: δ ³¹P = 21.5, ¹H NMR: δ ¹H = 1.11 (d, 6H, CH$_3$), 1.63 (m, 1H, -CH-CH$_3$), 1.68 (m, 2H, CH$_2$-CH$_2$-O), 3.17 (d, 2H, $^2J_{PH}$ = 22.3 Hz, CH$_2$-P), 4.07–4.38 (m, 4H, (CH$_2$-O) cycl), 7.31 (s, 2H, NH$_2$), ¹³C NMR: δ ¹³C = 20.1 (s, Me), 25.1 (CH$_2$-CH$_2$-O), 26.4 (d, $^1J_{PC}$ = 7.5 Hz, P-CH$_2$), 36.2 (CH-Me), 66.2 (d, $^2J_{PC}$ = 6.7 Hz, CH$_2$-O-P), 149.1 (d, $^2J_{PC}$ = 11.3 Hz, CN). E isomer: ³¹P NMR: δ ³¹P = 21.4, ¹H NMR: δ ¹H = 1.18 (d, 6H, CH$_3$), 1.85 (m, 1H, -CH-CH$_3$), 1.85 (m, 2H, CH$_2$-CH$_2$-O), 2.18 (d, 2H, $^2J_{PH}$ = 8.3 Hz, CH$_2$-P), 3.80–3.95 (m, 4H, (CH$_2$-O) cycl), 7.24 (s, 2H, NH$_2$). ¹³C NMR: δ ¹³C = 20.1 (s, Me), 25.1 (CH$_2$-CH$_2$-O), 26.4 (d, $^1J_{PC}$ = 7.5 Hz, P-CH$_2$), 36.2 (CH-Me), 66.2 (d, $^2J_{PC}$ = 6.7 Hz, CH$_2$-O-P), 149.1 (d, $^2J_{PC}$ = 11.3 Hz, CN).

### 1-(2-oxo-1,3,2-dioxaphosphoranyl)-3-phenylpropanone (3c)

M = 282.11 g/mol. Yield = (0.21 g, 0.75 mmol, 75 %), White crystal, mp. (°C ± 2) = 170 °C, Z/E ratio 100: 0, Z isomer: ³¹P NMR: δ ³¹P = 20.8, ¹H NMR: δ ¹H = 1.34 (d, 3H, CH$_3$), 1.94 (m, 2H, CH$_2$-CH$_2$-O), 2.51 (dd, $^2J_{PH}$ = 24.0 Hz, $^2J_{HH}$ = 15.0 Hz, 1H, CH-P), 2.86 (dd, $^2J_{PH}$ = 24.0 Hz, $^2J_{HH}$ = 15.0 Hz, 1H, CH-P), 3.57 (m, 1H, CH-CH$_3$), (4.01–4.54) (m, 4H, (CH$_2$-O) cycl), 5.62 (s, 2H, NH$_2$), (7.08–7.27) (m, 5H, Ph), ¹³C NMR: δ ¹³C = 19.6 (s, CH$_3$), 25.5 (s, CH$_2$-CH$_2$-O), 26.3 (d, $^1J_{PC}$ = 7.7 Hz, P-CH$_2$), 47.7 (s, HC=CH$_3$), 66.5 (dd, $^2J_{PC}$ = 1.8 Hz, CH$_2$-O-P), (126.6–143.2) (m, Ph), 146.1 (d, $^2J_{PC}$ = 10.9 Hz, CN). GC-MS: *m/z* = 282 [*M*⁺], 144 [Ph-CH-C(C=N-NH$_2$)] 105 [CH$_2$-CH$_2$-O-P-O-CH$_2$], 91 [CH$_2$-O-P-O-CH$_2$], 77 [Ph], 44 [CH$_2$-CH$_2$-O]. IR-ATR (cm$^{-1}$): 3300, 2950, 1633, 1500, 1266, 1054, 515 cm$^{-1}$.

### 1-(5,5-dimethyl-2-oxo-1,3,2-dioxaphosphoranyl)-3-phenylpropanone (3d)

M = 296.31 g/mol, Yield = (0.21 g, 0.70 mmol, 70 %), White solid, mp. (°C ± 2) = 150 °C, Z/E ratio 86:14, Z isomer: ³¹P NMR: δ ³¹P = 21.0, ¹H NMR: δ ¹H = 0.92 (s, 3H, CH$_3$), 1.04 (s, 3H, CH$_3$), 2.75 (d, $^2J_{PH}$ = 24.0 Hz, 2H, P-CH$_2$), 3.54 (s, 2H, CH$_2$-Ph), 3.70–4.20 (m, 4H, [AB]$_2$), 5.62 (s, 2H, NH$_2$), 7.14–7.6 (m, 5H, Ph). ¹³C NMR: δ ¹³C = 21.1 (s, CH$_3$e), 21.8 (s, CH$_3$a), 24.3 (d, $^1J_{PC}$ = 126.03 Hz, P-CH$_2$), 32.5 (d, $^3J_{PC}$ = 5.8, C-Me$_2$), 44.3 (s, CH$_2$-Ph), 75.0 (d, $^2J_{PC}$ = 6.3 Hz, CH$_2$-O-P), 126.0–136.2 (m, Ph), 161.7 (d, $^2J_{PC}$ = 3.0 Hz, CN). E isomer: ³¹P NMR: δ ³¹P = 21.9, ¹H NMR: δ ¹H = 1.11 (s, 3H, CH$_3$), 1.14 (s, 3H, CH$_3$), 2.82 (d, $^2J_{PH}$ = 24.0 Hz, 2H, P-CH$_2$), 3.48 (s, 2H, CH$_2$-Ph), 3.70–4.21 (m, 4H, [AB]$_2$), 5.24 (s, 2H, NH$_2$), 7.13–8.25 (m, 5H, Ph). ¹³C NMR: δ ¹³C = 21.4 (s, CH$_3$e), 21.4 (s, CH$_3$a), 26.3 (d, $^1J_{PC}$ = 127.04 Hz, P-CH$_2$), 32.2 (d, $^3J_{PC}$ = 127.04 Hz, P-CH$_2$).
= 7.0, C-Me₂), 43.6 (s, CH₂-Ph), 75.7 (d, ²JₚC = 6.9 Hz, CH₂-O-P), 126.0–136.1 (m, Ph), 169.91(d, ²JₚC = 3.0 Hz, CN). GC–MS: *m/z* = 234 [C(CH₂-O)₂-P-CH₂-C(N)-CH₂-Ph], 219 [(Me)₂C(CH₂-O)₂-P(O)-CH₂-C (CH₂)=N-NH₂], 173 [(Me)₂C(CH₂-O)₂-P-CH₂-C=N], 91 [C₆H₅-CH₂], 77 [Ph], 65 [P(OH)₂], 41 [CH₃-C-CH₂]. IR-ATR (cm⁻¹): 3300, 2973, 1633, 1490, 1266, 1054, 515 cm⁻¹.

### 1-(5,5-diethyl-2-oxo-1,3,2-dioxaphosphoranyl)-3-phenylpropanone (3e)

M = 324.00 g/mol, Yield = (0.23 g, 0.70 mmol, 70 %), White solid, mp. (°C ± 2) = 138°C, Z/E ratio 62: 38, *Cis* (Z) isomer: ³¹P NMR: δ ³¹P = 21.6, ¹H NMR: δ¹H = 0.99 (m, 3H, CH₃C), 1.10 (m, 3H, CH₃-C), 1.30 (m, 2H, C-CH₂), 1.31 (m, 2H, CH₂-C), 2.82 (d, ³JₚH = 12.6 Hz, CH₂-CN), 3.61 (d, ²JₚH = 2.50 Hz, P-CH₂), 3.82–4.00 (m, 4H, [AB]₂), 5.58 (s, 2H, NH₂), 7.20 (m, 5H, arom-H). ¹³C NMR: δ ¹³C = 6.9 (CH₃-C), 7.1 (CH₃-C), 22.5 (C-CH₂), 23.1 (C-CH₂), 25.2 (CH₂-Ph), 29.0 (P-CH₂), 37.5 (-C-), 73.1 (d, ²JₚC = 6.4 Hz, CH₂-O-P), 113.0–128.5 (CH-arom), 128.8 (d, ⁵JₚC = 4.6 Hz, C-i), 145.7 (CN).

*Trans* (E) isomer: ³¹P NMR: δ ³¹P = 21.8. ¹H NMR: δ¹H = 0.82 (m, 3H, CH₃C), 0.88 (m, 3H, CH₃-C), 1.50 (m, 2H, CH₂), 1.52 (m, 2H, CH₂), 2.90 (d, ³JₚH = 11.3, CH₂-CN), 3.73 (d, ²JₚH = 1.8 Hz, 2H, P-CH₂), 4.17–4.26 (m, 4H, [AB]₂), 5.58 (s, 2H, NH₂), 7.32 (m, 5H, arom-H). ¹³C NMR: δ ¹³C = 7.0 (CH₃-C), 7.1 (CH₃-C), 22.8 (C-CH₂), 23.1 (C-CH₂), 25.2 (CH₂-Ph), 29.0 (P-CH₂), 37.5(-C-), 73.1(d, ²JₚC = 6.4 Hz, CH₂-O-P), 113.0–128.5 (CH-arom), 129.6 (d, ⁵JₚC = 11.2 Hz, C-i), 145.7 (CN). GC–MS: *m/z*: 324 (M⁺), 130 (Et₂C(CH₂O)₂), 103 (C₆H₅-CH₂-C), 91 (C₆H₅-CH₂), 55 ((CH₃)₂C=CH). IR-ATR (cm⁻¹): 3200, 2980, 1633, 1490, 1054, 500 cm⁻¹.

### 1-(5,5-diethyl-2-oxo-1,3,2-dioxaphosphoranyl)-N-methyl-3-phenylpropanone (3f)

M = 338.00 g/mol, Yield = (0.27 g, 0.80 mmol, 80%), White solid, mp. (°C ± 2) = 108°C, Z/E ratio 85: 15, *Cis* (Z) isomer: ³¹P NMR: δ ³¹P = 21.3, ¹H NMR: δ¹H = 0.80 (m, 3H, CH₃-C), 0.84 (m, 3H, CH₃-C), 1.32 (m, 2H, C-CH₂), 1.41 (m, 2H, C-CH₂), 2.72 (s, Me), 3.08 (s, CH₂-CN), 3.61 (d, ²JₚH = 2.3 Hz, P-CH₂), 3.74–4.32 (m, 4H, [AB]₂), 5.56 (s, 1H, NH), 7.20 (m, 5H, arom-H). ¹³C NMR: δ ¹³C = 4.8 (CH₃-C), 5.0 (CH₃-C), 22.4 (C-CH₂), 23.1 (C-CH₂), 25.2 (CH₂-CN), 30.0 (P-CH₂), 30.7 (Me), 37.6 (-C-), 43.8 (CH₂-Ph) 73.0 (CH₂-O-P), 126.3–137.4 (Cₐarom), 139.8 (CN).

*Trans* (E) isomer: ³¹P NMR: δ ³¹P = 21.7 ¹H NMR: δ ¹H = 0.83 (m, 3H, CH₃-C), 0.88 (m, 3H, CH₃-C), 1.34 (m, 2H, C-CH₂), 1.47 (m, 2H, C-CH₂), 2.78 (s, Me), 3.10 (s, CH₂-CN), 3.61 (d, ²JₚH = 2.3 Hz, P-CH₂), 3.82–4.27 (m, 4H, [AB]₂), 5.59 (s, 1H, NH), 7.78 (m, 5H, arom-H). ¹³C NMR: δ ¹³C = 4.9 (CH₃-C), 5.1 (CH₃-C), 22.5 (C-CH₂), 23.2 (C-CH₂), 25.3 (CH₂-CN), 30.1 (P-CH₂), 30.8 (Me), 37.7 (-C-), 43.9 (CH₂-Ph), 73.1 (CH₂-O-P), 126.3–137.4 (Cₐarom), 139.9 (CN).

### 3.4. Theoretical calculations

All computations were performed with the Gaussian 09 program [81, 82]. The Conformations were optimized using DFT geometry optimizations using hybrid B3LYP [83] functional and the 6311++ G (d, p) basis set. To be sure that all optimized structures lay at a local point on the potential energy surface, harmonic vibrational frequencies of all structures were performed. None of the predicted spectra has any imaginary frequencies.

### 3.5. General X-ray structure analysis and refinement

Data collection was performed on a Bruker D8 Venture four-circle diffractometer from Bruker AXS GmbH (Karlsruhe, Germany). CPAD detectors used were Photon II from Bruker AXS GmbH; X-ray sources:

Microfocus source IµS; and microfocus source IµS Mo and Cu, respectively, from Incoatec GmbH with mirror optics HELIOS and a single hole collimator from Bruker AXS GmbH. Programs used for data collection were APEX4 Suite (v2021.10–0) and integrated programs SAINT (V8.40B; integration) and SADABS (2016/2; Bruker, 2016/2 absorption correction) from Bruker AXS GmbH [84]. The SHELX programs were used for further processing [85]. The solution of the crystal structures was done with the help of the program SHELXT [86], the structure refinement with SHELXL [87]. The processing and finalization of the crystal structure data was done with program OLEX2 v1.5 [88]. All non-hydrogen atoms were refined anisotropically. For the hydrogen atoms, the standard values of the SHELXL program were used with *U*<sub>iso</sub>(H) = -1.2 *U*<sub>eq</sub>(C) for CH₂ and CH and with *U*<sub>iso</sub>(H) = -1.5 *U*<sub>eq</sub>(C) for CH₃. All H atoms were refined freely using independent values for each *U*<sub>iso</sub>(H).

Crystal data for C₁₃H₁₉FN₂O₃P (3c), *M* = 282.27 g. mol⁻¹, colorless blocks, crystal size 0.415 × 0.326 × 0.117 mm³, monoclinic, space group P2₁/c, *a* = 5.9823(4) Å, *b* = 7.5563(5) Å, *c* = 30.729(2), *α* = 90°, *β* = 94.802 (2)°, *γ* = 90°; *V* = 1384.19(16) Å³, *Z* = 4, *D*<sub>calc</sub> = 1.355 g/cm³, *T* = 100 K, *R*₁ = 0.0388, *wR*₂ = 0.0972 for 195,798 reflections with *I* ≥ 2σ (*I*) and 7495 independent reflections. GOF = 1.110. Largest diff. peak/hole/e Å⁻³ 0.53/–0.39.

Crystal data for C₁₆H₂₃FNO₃P (4), *M* = 308.32 g. mol⁻¹, colorless needles, crystal size 0.382 × 0.092 × 0.063 mm³, monoclinic, space group P2₁/c, *a* = 10.89(3) Å, *b* = 6.113(11) Å, *c* = 25.02(7), *α* = 90°, *β* = 97.36 (12)°, *γ* = 90°; *V* = 1651(7) Å³, *Z* = 4, *D*<sub>calc</sub> = 1.240 g/cm³, *T* = 100 K, *R*₁ = 0.0666, *wR*₂ = 0.1902 for 12,302 reflections with *I* ≥ 2σ (*I*) and 3246 independent reflections. GOF = 1.047. Largest diff. peak/ hole/e Å⁻³ 0.42/–0.48. Other crystallographic data and structure refinement details for hydrazone 3c and azine 4 are presented in Table S1.

Data were collected using graphite monochromated MoK<sub>α</sub> radiation λ = 0.71073 Å and have been deposited at the Cambridge Crystallographic Data Centre as CCDC 2,323,102 (3c) and 2,333,377 (4). (Supplementary Materials). The data can be obtained free of charge from the Cambridge Crystallographic Data Centre via http://www.ccdc.cam.ac.uk/getstructures, accessed on 21 January 2024.

### 3.6. Molecular docking

The 3D crystal structures of PDB were obtained from the RSCB protein data bank: Butyrylcholinesterase (BuChE) in complex with Tacrine (PDB: 4BDS) [89], Acetylcholinesterase (AChE) in complex with Donepezil (PDB: 4EY7) [90] All water molecules were removed, hydrogen atoms were added before docking using Discovery Studio Visualizer D.S. (2017) (Biovia San Diego, CA, USA) Gasteiger charges were also added to the system during the preparation of the receptor input file. Docking studies were carried out using AutodockTools (ADT version 1.5.6) [71] to prepare enzyme and ligands files (PDBQT). The optimization of all the geometries was performed with ACD (3D viewer) software [91] Cocrystalized ligands into proteins were used as reference ligands and redocked into the active site for energy comparison. 2D pictures were captured using Discovery Studio Visualizer (2017) (Biovia San Diego, CA, USA).

In order to test the validation of the docking performance and the accuracy of co-crystallized ligands for both targets, Donepezil and Tacrine were re-docked into the binding site pocket of AChE and BuChE, respectively. Root-Mean-Square Deviation (RMSD) values between the docking and initial poses, docking scores, and properties of AChE and BuChE were given in Table 8.
<table><caption>**Table 8**
Information details related to proteins AChE and BuChE.</caption><thead><tr><th>Proteins</th><th>Methods</th><th>Resolution (Å)</th><th>Amino acids number</th><th>Co-crystallized ligand</th><th>Docking score (Kcal/mol)</th><th>RMSD</th></tr></thead><tbody><tr><td>4EY7</td><td>X-ray diffraction</td><td>2.35</td><td>542</td><td>Donepezil</td><td>-11.6</td><td>0.748</td></tr><tr><td>4BDS</td><td>X-ray diffraction</td><td>2.10</td><td>529</td><td>Tacrine</td><td>-8.1</td><td>0.469</td></tr></tbody></table>

The co-crystallized ligands of 4EY7 and 4BDS are: Donepezil (C<sub>24</sub>H<sub>29</sub>N O<sub>3</sub>) and Tacrine (C<sub>13</sub>H<sub>14</sub>N<sub>2</sub>) respectively.

### 3.7. ADME-Toxicity and drug-likeness prediction

ADMET properties (TPSA, n-ROTB, Log P, n-H bond donors and acceptors) were predicted using the SwissADME server (http://www.swissadme.ch/, accessed on 02/05/2024) [73] in order to evaluate Lipinski, Vaber, Egan and PAINS filters. Moreover, the pkCSM server (https://biosig.unimelb.edu.au/pkcsm/prediction, accessed on 02/05/2024) [74] was employed to predict Absorption-, Distribution-, Metabolism-, Excretion- and Toxicity properties.

### 4. Conclusion

In the present work, we have prepared allenylphosphonates 2a-f and β-cycloalkoxyphosphonated hydrazones 3a-f bearing a dioxaphosphorinane ring. Most of them coexist in solution as diastereomeric mixtures. An attribution of the signals in the ³¹P NMR spectra of allenylphosphonate 2a and hydrazone 3a to their corresponding diastereomers was attempted based on mechanistic elucidation and theoretical calculations. Unlike the other hydrazones, the ³¹P NMR spectrum of the hydrazone 3c displays only a singlet. X-ray diffraction was therefore conducted to understand this difference by investigating the structure of hydrazone 3c. The analyzed single crystal shows a Z configuration stabilized by a strong intramolecular hydrogen bond. DFT calculation was conducted and confirmed that the Z-configuration is the more stable one, rationalizing the observation of a just singlet in the ³¹P NMR spectrum. We have also found by serendipity that hydrazones 3 may serve as precursors of azines like 4. The potential of this condensation will be further exploited in future studies. Also, the potential of compounds 3 and 4 acting as N-donor ligands in coordination chemistry deserves forthcoming investigations. The experimental IR spectrum of 3c was compared with the computed one and found to fit well. Docking simulations performed on AChE and BuChE enzymes revealed that all hydrazones 3a-f exhibit high affinity for the binding sites of AChE and BuChE. Compounds 3a and 3d were identified as very potent for BuChE enzyme inhibition. On the other hand, compounds 3d and 3f showed the highest affinity with the active site of AChE enzyme. The ADMET prediction revealed that hydrazones 3a, 3d and 3f exhibit drug-likeness according to Lipinski's, Veber's and Egan's rules. These in silico simulations allowed us to select the most promising candidates that will be the subject of in vivo experiments concerning AChE and BuChE inhibitory activity in a subsequent work.

### CRediT authorship contribution statement

**Dorra Kanzari-Mnallah:** Writing – original draft, Software, Investigation, Formal analysis. **Sirine Salhi:** Investigation. **Michael Knorr:** Writing – review & editing. **Writing – original draft, Project administration, Conceptualization.** **Jan-Lukas Kirchhoff:** Software, Investigation. **Carsten Strohmann:** Resources, Data curation. **Med Lotfi Efrit:** Resources. **Azaiez Ben Akacha:** Conceptualization, Writing – review & editing.

### Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

### Data availability

Data will be made available on request.

### Acknowledgements

This work was supported financially by the general directorate of scientific research, Tunisian ministry of scientific research.

### Supplementary materials

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.molstruc.2024.139035.

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