Synthesis of 2,5-Dialkyl-1,3,4-oxadiazoles Bearing Carboxymethylamino Groups

A series of new symmetrical 2,5-dialkyl-1,3,4-oxadiazoles containing substituted alkyl groups at the terminal positions with substituents, such as bromine, isopropyloxycarbonylmethylamino, and carboxymethylamino, were successfully synthesized. The developed multistep method employed commercially available acid chlorides differing in alkyl chain length and terminal substituent, hydrazine hydrate, and phosphorus oxychloride. The intermediate bromine-containing 2,5-dialkyl-1,3,4-oxadiazoles were easily substituted with diisopropyl iminodiacetate, followed by hydrolysis in aqueous methanol solution giving the corresponding 1,3,4-oxadiazoles bearing carboxymethylamino substituents. The structure of all products was confirmed by conventional spectroscopic methods including 1H NMR, 13C NMR, and HRMS.

According to the above data, we assumed that the combination of the 1,3,4-oxadiazole core containing oxygen and two nitrogen atoms in the ring with aminopolycarboxylic groups allows for the development of a new family of organic ligands with potential applications as complexing agents in medicine and agriculture. Herein, we developed an effective method for the preparation of new symmetrically substituted 1,3,4-oxadiazole derivatives containing carboxymethylaminoalkyl groups at the 2 and 5 positions. Initially, a three-step transformation was conducted using commercially available reagents (Scheme 1). In the first step, the acid chlorides were reacted with hydrazine hydrate to form symmetrical N,N -diacylhydrazine derivatives with different alkyl chain lengths. In the second step, they were cyclized using POCl 3 , a known cyclodehydration reagent. Finally, the substitution reaction between bromine-containing 1,3,4-oxadiazole derivatives and iminodiacetic acid was studied.
Molecules 2022, 27, x FOR PEER REVIEW 2 of 12 (PbO2) [32], chloramine T [33], 2,3-dichloro-5,6-dicyano-1,4-benzowuinone (DDQ) [34], and hypervalent iodine reagents [35]. 1,3,4-Oxadiazoles containing alkyl chains at the 2 and 5 positions and substituted with carboxymethylamino groups are of particular interest. Generally, the presence of aminopolycarboxylic functionalities promote complexing properties in such derivatives, allowing for binding to metal cations. The literature shows a range of organic ligands of this type, some of which have been approved for use in medicine and agriculture. These include ethylenediaminetetraacetic acid (EDTA) [36], N-(hydroxyethyl)ethylenediaminetriacetic acid (HEEDTA) [37], ethylenediamine-N,N′-bis(o-hydroxy-pmethylphenyl)acetic acid (EDDHMA) [38], diethylenetriaminepentaacetic acid (DTPA) [39], nitrilotriacetic acid (NTA) [40], glucoheptanoic acid [41], and citric acid [42,43]. Unfortunately, despite the excellent chelating properties, not all fertilizing chelates show the adequate biodegradation. Some of the most common coordination compounds based on EDTA and DTPA are characterized by very high stability, but they are resistant to biodegradation. Numerous studies revealed that they are present in waters of lakes or ponds, as well as in the soil for a long time, which may result in eutrophication of waters and introduction of metals into the food chain [44]. On the other hand, there has recently been an emphasis on chelating agents based on sugar molecules in order to increase the effectiveness of the micronutrients. There is a possibility that sugar acid derivatives, condensed tannis, and glucohetonates could effectively replace the traditionally used EDTA for the production of chelated micronutrients [43]. Heterocyclic compounds composed of carbon, oxygen, and nitrogen atoms could also constitute another alternative scaffold for the construction of new chelating agents.
According to the above data, we assumed that the combination of the 1,3,4-oxadiazole core containing oxygen and two nitrogen atoms in the ring with aminopolycarboxylic groups allows for the development of a new family of organic ligands with potential applications as complexing agents in medicine and agriculture. Herein, we developed an effective method for the preparation of new symmetrically substituted 1,3,4-oxadiazole derivatives containing carboxymethylaminoalkyl groups at the 2 and 5 positions. Initially, a three-step transformation was conducted using commercially available reagents (Scheme 1). In the first step, the acid chlorides were reacted with hydrazine hydrate to form symmetrical N,N′-diacylhydrazine derivatives with different alkyl chain lengths. In the second step, they were cyclized using POCl3, a known cyclodehydration reagent. Finally, the substitution reaction between bromine-containing 1,3,4-oxadiazole derivatives and iminodiacetic acid was studied. Scheme 1. Initial concept of the synthetic pathway.

Results
The target derivatives containing carboxymethylaminoalkyl groups were obtained in a multistep transformation reaction. The first step consisted of the synthesis of symmetrical N,N′-diacylhydrazine derivatives (2a-d) (Scheme 2) using commercially available acid chloride derivatives (1a-d) (Scheme 2) differing in alkyl chain length and bearing a Scheme 1. Initial concept of the synthetic pathway.

Results
The target derivatives containing carboxymethylaminoalkyl groups were obtained in a multistep transformation reaction. The first step consisted of the synthesis of symmetrical N,N -diacylhydrazine derivatives (2a-d) (Scheme 2) using commercially available acid chloride derivatives (1a-d) (Scheme 2) differing in alkyl chain length and bearing a bromine atom at the terminal position. The model reaction employed bromoacetyl chloride (1a) as the starting material. The optimization study consisted of examining the base (triethylamine (TEA) and sodium carbonate), solvent (chloroform, and diethyl ether), and the influence of temperature. The best results were obtained at low temperature (0 • C) in diethyl ether using bromine atom at the terminal position. The model reaction employed bromoacetyl chloride (1a) as the starting material. The optimization study consisted of examining the base (triethylamine (TEA) and sodium carbonate), solvent (chloroform, and diethyl ether), and the influence of temperature. The best results were obtained at low temperature (0 °C ) in diethyl ether using aqueous sodium carbonate as the base. The products were purified via recrystallization from either methanol or ethanol. The yields of the obtained hydrazine derivatives (2a-d) were 65-79% (Table 1,  The synthesis of 1,3,4-oxadiazole derivatives (3a-d) (Scheme 2) involved reacting with POCl3, a cyclodehydration reagent. The reaction was conducted in anhydrous toluene or solvent-free conditions and monitored by TLC. In toluene, product was formed in lower yield compared to the solvent-free reaction ( Table 2, entries 2, 4, 6, and 8). The final products (3a-d) were obtained in 40-76% yield and were used for the subsequent reactions without purification (Table 2).  The synthesis of 1,3,4-oxadiazole derivatives (3a-d) (Scheme 2) involved reacting with POCl 3 , a cyclodehydration reagent. The reaction was conducted in anhydrous toluene or solvent-free conditions and monitored by TLC. In toluene, product was formed in lower yield compared to the solvent-free reaction ( Table 2, entries 2, 4, 6, and 8). The final products (3a-d) were obtained in 40-76% yield and were used for the subsequent reactions without purification ( Table 2). As part of the synthesis, we planned to use iminodiacetic acid (4), which could directly react with the formed 2,5-dialkyl-1,3,4-oxadiazole derivatives (3a-d) containing bromine substituents (Scheme 1). After testing a series of bases, including TEA, sodium hydroxide, and sodium carbonate, as well as several organic solvents (chloroform, methanol, acetonitrile) and their mixtures [45], the desired products 7 were not generated. Therefore, the iminodiacetic acid was converted into a more reactive ester, which could be subjected to a substitution reaction with 2,5-bis(bromoalkyl)-1,3,4-oxadiazoles (3a-d), followed by hydrolysis to restore the carboxyl groups. Hence, the esterification reactions were performed using ethanol (a) and isopropanol (b) as substrates, and in the presence of sulfuric acid as the catalyst (Scheme 3). The final esters (5a,b) were obtained in good yields (37-52%).

6
-44 7 3d 4 Toluene 59 8 -76 As part of the synthesis, we planned to use iminodiacetic acid (4), which could directly react with the formed 2,5-dialkyl-1,3,4-oxadiazole derivatives (3a-d) containing bromine substituents (Scheme 1). After testing a series of bases, including TEA, sodium hydroxide, and sodium carbonate, as well as several organic solvents (chloroform, methanol, acetonitrile) and their mixtures [45], the desired products 7 were not generated. Therefore, the iminodiacetic acid was converted into a more reactive ester, which could be subjected to a substitution reaction with 2,5-bis(bromoalkyl)-1,3,4-oxadiazoles (3a-d), followed by hydrolysis to restore the carboxyl groups. Hence, the esterification reactions were performed using ethanol (a) and isopropanol (b) as substrates, and in the presence of sulfuric acid as the catalyst (Scheme 3). The final esters (5a,b) were obtained in good yields (37-52%). Scheme 3. Synthesis of diethyl iminodiacetate (5a) and diisopropyl iminodiacetate (5b). Reaction conditions: iminodiacetic acid (4, 15.0 g, 0.11 mol), alcohol (120 mL), H2SO4 (7.5 mL), reflux, 12 h. The next step was to perform the substitution reaction involving the appropriate ester (5a,b), oxadiazole derivative (3a-d), and base in an aprotic solvent (Scheme 4). In order to determine the optimal conditions, 2,5-bis(bromomethyl)-1,3,4-oxadiazole (3a) and diethyl iminodiacetate (5a) were first examined. When the reaction was conducted at room temperature, main product 6a was produced but in a low yield, at 23% (Table 3, entry 11). The further study revealed that the optimal temperature was in the range of 50-60 °C , giving the product 6a in a 91% yield (Table 3, entry 12). Scheme 3. Synthesis of diethyl iminodiacetate (5a) and diisopropyl iminodiacetate (5b). Reaction conditions: iminodiacetic acid (4, 15.0 g, 0.11 mol), alcohol (120 mL), H 2 SO 4 (7.5 mL), reflux, 12 h. The next step was to perform the substitution reaction involving the appropriate ester (5a,b), oxadiazole derivative (3a-d), and base in an aprotic solvent (Scheme 4). In order to determine the optimal conditions, 2,5-bis(bromomethyl)-1,3,4-oxadiazole (3a) and diethyl iminodiacetate (5a) were first examined. When the reaction was conducted at room temperature, main product 6a was produced but in a low yield, at 23% (Table 3, entry 11). The further study revealed that the optimal temperature was in the range of 50-60 • C, giving the product 6a in a 91% yield (  60 91 Among the solvents tested, acetonitrile gave the best result (Table 3, entry 12) owing to its characteristic aprotic polarity and its relatively low boiling point in relation to dimethylformamide (DMF) or DMSO. The inorganic weak base sodium carbonate enhanced the removal of inorganic compounds during the extraction process. Additionally, the optimal reaction time was 8-12 h, which was determined by TLC. Having the optimized conditions in hand, a series of substitution reactions were conducted using oxadiazoles (3b-d), and producing products (6a-e) in a 71-91% yield ( Table 4, entries 1-5). All products (6a-e) were purified by column chromatography on silica gel using ethyl acetate as the eluent. The last step in the synthetic pathways was the restoration of the carboxyl groups from esters groups (6). Various literature methods were examined, including anhydrous lithium chloride [46] and the classical method of hydrolysis in an acidic and alkaline environment [47]. It was found that the alkaline hydrolysis reaction gave desired 1,3,4-oxadiazole derivatives bearing carboxymethylaminoalkyl groups (7b, 7d) at the side alkyl chains (Scheme 5). The lithium chloride method was ineffective, and hydrolysis under acidic conditions gave the desired product, but excessive heating promoted decomposition of the oxadiazole. Optimization of the hydrolysis reaction involved different amounts of NaOH and solvents. The obtained results showed that a significant excess of NaOH and conducting the reaction in a relatively high temperature caused the decomposition. However, we observed the formation of the intended final product (7b) when the molar ratio between ester 6 and NaOH was 1:10. Methanol was found to be the best solvent owing to its ability to dissolve the substrate. The final products (7b, 7d) were purified by recrystallization from methanol, providing the pure products in a 54-68% yield (Table 4, entries 6 and 7). Among the solvents tested, acetonitrile gave the best result (Table 3, entry 12) owing to its characteristic aprotic polarity and its relatively low boiling point in relation to dimethylformamide (DMF) or DMSO. The inorganic weak base sodium carbonate enhanced the removal of inorganic compounds during the extraction process. Additionally, the optimal reaction time was 8-12 h, which was determined by TLC. Having the optimized conditions in hand, a series of substitution reactions were conducted using oxadiazoles (3b-d), and producing products (6a-e) in a 71-91% yield ( Table 4, entries 1-5). All products (6a-e) were purified by column chromatography on silica gel using ethyl acetate as the eluent.  The last step in the synthetic pathways was the restoration of the carboxyl groups from esters groups (6). Various literature methods were examined, including anhydrous lithium chloride [46] and the classical method of hydrolysis in an acidic and alkaline environment [47]. It was found that the alkaline hydrolysis reaction gave desired 1,3,4oxadiazole derivatives bearing carboxymethylaminoalkyl groups (7b, 7d) at the side alkyl chains (Scheme 5). The lithium chloride method was ineffective, and hydrolysis under acidic conditions gave the desired product, but excessive heating promoted decomposition of the oxadiazole. Optimization of the hydrolysis reaction involved different amounts of NaOH and solvents. The obtained results showed that a significant excess of NaOH and conducting the reaction in a relatively high temperature caused the decomposition. However, we observed the formation of the intended final product (7b) when the molar ratio between ester 6 and NaOH was 1:10. Methanol was found to be the best solvent owing to its ability to dissolve the substrate. The final products (7b, 7d) were purified by recrystallization from methanol, providing the pure products in a 54-68% yield (Table 4, entries 6 and 7).  The structure of all obtained intermediates and final products was confirmed by 1 H and 13 C NMR spectroscopy (see Supplementary Materials. Both ester and acid derivatives were symmetrical molecules; hence, the number of signals was reduced. Among 2,5-dialkyl-1,3,4-oxadiazole derivatives (6a-e), and not described so far in the literature, characteristic 1 H NMR signals included the doublet at 1.25 ppm and septet at 5.00 ppm corresponding to the isopropyl group. The singlet at 3.50 ppm was related to the iminodiacetate moiety. The remaining signals at 1.00-3.00 ppm corresponded to the alkyl side chain between the oxadiazole and diisopropyl ester. In the case of 13 C NMR, the characteristic C2 and C5 signals of the heterocyclic 1,3,4-oxadiazole ring were found at 165.0 ppm. The remaining peaks from carbonyl groups were located at 170.0 ppm, while two signals from isopropyloxy group were found at 22.0 ppm (CH 3 ) and 68.0 ppm (-CH-), respectively. Signals at 50.0-55.0 ppm corresponded to the iminodiacetate part of the molecule (-N(CH 2 ) 2 <). Finally, carbons of the alkyl chain were in the range of 22.0-60.0 ppm. 1 H and 13 C spectra of the final products, containing carboxyl groups (7b, 7d), showed no visible signals of the ester residue. High-resolution mass spectra further confirmed the structure of the obtained intermediates and final products.

General Information
All reagents were purchased from commercial sources and used without further purification. Melting points were measured using a Stuart SMP3 melting point apparatus (Staffordshire, UK). NMR spectra were recorded at 25 • C using an Agilent 400-NMR spectrometer (Agilent Technologies, Waldbronn, Germany) at 400 MHz for 1 H and 100 MHz for 13 C, with CDCl 3 or DMSO as solvent, and TMS as the internal standard. Highresolution mass spectra were acquired using a Waters ACQUITY UPLC/Xevo G2QT instrument (Waters Corporation, Milford, MA, USA). Thin-layer chromatography (TLC) was performed using silica gel 60 F254 (Merck, Merck KGaA, Darmstadt, Germany) thinlayer chromatography plates, with ethyl acetate, chloroform/ethyl acetate (5:1 v/v), or methanol/chloroform (4:1 v/v) as the mobile phases.

Synthesis of N,N -Diacylhydrazine Derivatives (2a-d)
Hydrazine hydrate (4.6 mL, 0.06 mol) was dissolved in diethyl ether (50 mL), and the mixture was cooled to 0 • C. The appropriate amount of acid chloride (1a-d, 0.06 mol) was dissolved in diethyl ether (20 mL) and added dropwise to the mixture. The temperature was carefully monitored, keeping it below 35 • C. Then, after 30 min, sodium carbonate (6.36 g, 0.06 mol) dissolved in water (40 mL) was added. After the evolution of carbon dioxide had ceased, acid chloride (1a-d, 0.06 mol) dissolved in diethyl ether (10 mL) was added dropwise. The reaction mixture was stirred at room temperature for 2 h. The resulting precipitate was filtered, and dried products was recrystallized from methanol to obtain the desired pure products.

2-Bromo-N -(2-bromoacetyl)acetohydrazide (2a)
The product was obtained as white powder (10. .007 mol). The mixture was heated to reflux for 6-24 h. The progress of the reaction was monitored by TLC using methanol/chloroform (4:1 v/v) as the mobile phase. Excess phosphorus oxychloride was evaporated, and the residue in the flask was dissolved in diethyl ether (40 mL) and poured into water (100 mL). The mixture was neutralized using sodium carbonate, extracted with diethyl ether (40 mL), dried over anhydrous magnesium sulfate, and evaporated to dryness.

Synthesis of Iminodiacetic Acid Ester Derivatives (5a,b)
Ethanol (a) or isopropanol (b) (120 mL) and concentrated H 2 SO 4 (7.5 mL) were added to iminodiacetic acid (4) (15.0 g, 0.11 mol). The reaction mixture was heated to reflux for 12 h. Excess alcohol was then evaporated using a rotary evaporator, and the mixture was neutralized with sodium bicarbonate solution. Then, the resulted solution was extracted with ethyl acetate (30 mL), dried over anhydrous magnesium sulfate, and evaporated to dryness.