Cyclic analogs of á-amino acids, in particular proline, are interesting as starting materials for synthesizing new drugs. However, they are little studied because of the limited number of available synthetic pathways to them [1, 2]. A new method for synthesizing derivatives of 2-phenylproline that consisted of the synthesis of phenylglycine derivatives and intramolecular cyclization under phase-transfer catalysis conditions was developed by us earlier [3, 4].

Herein we report the synthesis and biological testing of new derivatives of 2-arylpyrrolidinecarboxylic acid that were prepared in a search for antitumor drugs among proline analogs. The starting materials were benzaldehyde and 3,4-dimethoxybenzaldehyde, the reaction of which with sodium cyanide and various aromatic amines gave the corresponding acetonitriles III – IX, acylation of which by 3-chloropropionyl chloride and subsequent intramolecular cyclization under phase-transfer catalysis conditions afforded the target products X – XVI.

figure a

Benzaldehyde and 1-amino-3-hydroxypropane were reacted in the same manner to give the corresponding aminopropanol derivative XXI, which was converted to chloro derivative XXII by SOCl2 [3]. Acylation of XXII by 2-bromo-, 4-bromo-, and 4-methoxy-3-nitrobenzoic acids also under phase-transfer catalysis conditions with intramolecular cyclization produced proline derivatives XXIII – XXV.

Amides XVII – XX and XXVI – XXVIII were synthesized for biological testing from the corresponding pyrrolidinecarbonitriles X – XVI and XXIII – XXV via reaction with cooling of the corresponding nitriles and conc. H2SO4.

Experimental chemical part

The structures of the synthesized compounds were confirmed by PMR spectra recorded on a Mercury-300 instrument (Varian) and by elemental analyses. The course of reactions and purity of products were monitored using TLC on Silufol UV-254 plates and Me2CO:nonane (1:1, a; 2:1, b).

General method for preparing substituted 2-arylacetonitriles (III – IX). A solution of aldehyde I or II (10 mmol) in EtOH (20 mL) was stirred at room temperature, treated with a solution of NaCN (0.5 g, 10 mmol) in H2O (10 mL), stirred for 10 min, treated with HOAc (0.6 g, 10 mmol), stirred for another 10 min, diluted with a solution of the corresponding amine (10 mmol) in EtOH (10 mL), stirred for 2 h, diluted with cold H2O (10 mL), and left over night. The resulting precipitate was filtered off, washed with H2O, dried, and recrystallized from EtOH (Tables 1 and 2).

Table 1 Properties of Synthesized III – XX and XXIII – XXVIII
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Table 2 PMR Spectra of III – XX and XXIII – XXVIII
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General method for preparing 2-aryl-2-pyrrolidinecarbonitriles (X – XVI, XXIII – XXV). A mixture of the corresponding 2-arylacetonitrile III – IX or XXII (10 mmol) [3] in 1,2-dichloroethane (20 mL) and anhydrous K2CO3 (1.4 g, 10 mmol) at 10 – 15°C was treated dropwise with 3-chloropropionyl chloride or substituted benzoic acid (10 mmol), stirred at room temperature for 30 min and at 40 – 45°C for 2 h, cooled, treated with 1,2-dichloroethane (20 mL), washed several times with H2O, and dried over CaCl2. The solvent was distilled off. The residue was treated with anhydrous K2CO3 (1.4 g, 10 mmol), triethylbenzylam monium chloride (0.1 g, 5 mmol), and CH3CN (20 mL), stirred at 45 – 50°C for 4 h, and filtered. The filtrate was evaporated. The residue was dissolved in CHCl3, washed with H2O, and dried over CaCl2. The solvent was distilled off. The residue was recrystallized from EtOH (Tables 1 and 2).

General method for preparing 2-aryl-2-pyrrolidinecarboxamides (XVII – XX and XXVI – XXVIII). The corresponding 2-aryl-2-pyrrolidinecarbonitrile X – XII, XIV,or XXIII – XXV (10 mmol) was dissolved in conc. H2SO4 (10 mL) at 0 – 5°C, left at room temperature for 3 h, and slowly poured into a beaker with ice. The resulting crystals were filtered off, washed with dilute NaHCO3 solution and H2O, and recrystallized from EtOH (Tables 1 and 2).

Experimental biological part

Antibacterial activity of the synthesized compounds was studied using Gram-positive Staphylococcus aureus 209P, 1 and Gram-negative Shigella dysenteriae Flexneri-6858 and Escherichia coli 0 – 55 and the agar-diffusion method with bacterial load 20×106 microbes per millimeter of medium [5]. Compounds were studied at 1:20 concentrations. Results were calculated from the diameter (d, mm) of the microorganism growth inhibition zone at the application site of the compounds after growth for1dat 37°C (thermostatted).

Toxicity and antitumor activity of XV – XX and XXV – XXVIII were tested by the usual methods [6, 7] on 210 white laboratory mice (20 – 22 g) of both sexes.

Acute toxicity was studied in white mice with a single i.p. injection. The absolute lethality (LD100) and maximum tolerated dose (MTD) were determined for each compound.

Antitumor activity was studied in mice with grafted sarcoma 37 and Ehrlich ascites carcinoma (EAC) tumors. Because of their poor solubility, the compounds were administered to the animals as suspensions in carboxymethylcellulose solution (0.5%) daily by i.p. injection for six days at doses from 1/10 to 1/15 of LD100. The criteria for a therapeutic effect were the percent tumor growth inhibition (TGI, %) for sarcoma 37 and the increase of average lifespan (ALS, %) for EAC.

Results were processed statistically using the Student—Fisher method.

It was found during the acute toxicity study that XV – XX and XXV – XXVIII had comparatively low toxicity (LD100 = 2,200 – 2,500 mg/kg).

The chemotherapy tests showed that XVII, XIX, and XX possessed moderate antitumor activity (Table 3). The other compounds had a weak suppressive effect on the TGI of sarcoma 37 and only a few of them, on EAC.

Table 3 Toxicity and Antitumor Activity of XV – XX and XXV – XXVIII
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Thus, changing the nitrile of 5-oxopyrrolidines XV – XX to carboxamide increased the antitumor activity. The toxicity and antitumor activity of substituted 1-benzoylpyrrolidine derivatives XXV – XXVIII were inferior to those of aforementioned 5-oxopyrrolidine derivatives XV – XX.

The study of the antibacterial properties of X – XX and XXV – XXVIII showed that they did not possess antibacterial activity.