Exploration of 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives effect on triple-negative breast, prostate cancer and melanoma cell 2D and 3D cultures

Guoda Pranaitytė; Birutė Grybaitė; Ugne Endriulaityte; Vytautas Mickevičius; Vilma Petrikaitė

doi:10.1038/s41598-025-02106-8

. 2025 May 21;15:17590. doi: 10.1038/s41598-025-02106-8

Exploration of 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives effect on triple-negative breast, prostate cancer and melanoma cell 2D and 3D cultures

Guoda Pranaitytė ¹, Birutė Grybaitė ¹, Ugne Endriulaityte ², Vytautas Mickevičius ^1,^✉, Vilma Petrikaitė ^2,^3,^✉

PMCID: PMC12095558 PMID: 40399317

Abstract

1-Substituted 5-oxopyrrolidine-3-carboxylic acid and its derivatives play an important role as components of many biologically active molecules. This study describes the synthesis of 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives and their anticancer properties. The target compounds were prepared using 2,4-difluoroniline as a starting material; in this way, derivatives of benzimidazoles, hydrazones and azoles were formed. Investigation of the anticancer activity of all synthesized compounds showed that the hydrazones had the strongest effect on cancer cell lines. Compounds were tested for their cytotoxic effect by the MTT assay in human triple-negative breast cancer MDA-MB-231, prostate adenocarcinoma PPC1, melanoma A375 and human foreskin fibroblasts CRL-4001 after 72 hours of incubation. The impact of the compounds on cancer cell migration was assessed using a ‘wound healing assay’. Activity in 3D cultures was determined by evaluating changes in spheroid size and assessing cell viability. Overall, the selected compounds 7b, 9c, 9e, 9f and 10 exhibited greater activity in the A375 cell line and were less active against the MDA-MB-231 cell line. Compounds 9c, 9e and 10 showed relatively higher selectivity for cancer cells over fibroblasts. Hydrazone 9f, bearing N’-(4-methylbenzylidene) moiety, was identifiedasthe most cytotoxic compound in both prostate adenocarcinoma PPC-1 and melanoma A375 cells in monolayer and 3D culture models. Compound 9e, with N’-(4-bromobenzylidene) moiety, exhibited the most pronounced inhibitory effect on cell migration as determined by the ‘wound healing’ assay.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-02106-8.

Keywords: Azoles, Hydrazones, Triple-negative breast cancer, Prostate carcinoma, Melanoma, Cytotoxicity, Cell migration, Tumor spheroids

Subject terms: Pharmacodynamics, Drug discovery and development

Introduction

Cancer remains one of the major public health challenges today. According to data from the World Health Organization, nearly 10 million people died from this disease in 2020¹. Chemotherapy is the most common form of cancer treatment; however, it also affects healthy cells and is associated with serious side effects. Treatment with low molecular weight compounds that target specific enzymes or receptors is becoming increasingly popular, as these agents block oncogenic signalling pathways and interfere with cancer cell proliferation². Consequently, the search for novel synthetic molecules with high activity and low toxicity has become an important key focus in the development of new therapies for malignant tumors.

β-Amino acid derivatives are of considerable interest in the field of organic synthesis due to their broad spectrum of biological activity. These compounds play a role in metabolic and immune system regulatory processes, and are therefore used in the treatment of diabetes, cardiovascular diseases and other ailments^2,3. For example, five-membered 2-pyrrolidinones represent a class of heterocyclic compounds with a wide variety of biologically active derivatives. This structural motif is common in many natural products that exhibit diverse biological effects. 2-Pyrrolidinone-based compound pyrrocidine A is a known antimicrobial agent produced by the endophytic fungus Sarocladium zeae⁴. Its significant role is highlighted not only by its antimicrobial properties^5–7 but also by its potent cytotoxic activity against human acute promyelocytic leukemia HL60 cells. This activity is approximately 70-fold higher than that of its analog, pyrrocidine B, which lacksthe α,β-unsaturated carbonyl group⁴.

Hydrazones, due to their significant biological potential, are increasingly attracting the attention of researchers involved in the discovery and development of effective pharmaceuticals. This fragment is utilized in the synthesis of compounds with anticonvulsant, analgesic, anticancer, and antiviral properties^8–10. Compounds containing a hydrazone moiety have demonstrated activity against gastric, lung and breast cancer cell lines without exerting toxic effects on normal liver cell lines. Therefore, these compounds represent promising candidates for the development of novel anticancer agents. Additionally, the hydrazone group enables the formation of hydrogen bonds, which enhances interactions between organic molecules and amino acids – the primary components of biological targets^9,11.

Our inclusion of the 2,4-difluorophenyl fragment in the molecule was guided by the understanding that fluorine atoms play a distinct and significant role in influencing molecular conformation. From a steric perspective, the impact of fluorine is expected to be minimal, as it is a small atom with a van der Waals radius of 1.47 Å—comparable to that of hydrogen (1.20 Å). Additionally, fluorine’s high electronegativity results in strongly polarized C–F bonds, which can create opportunities for hydrogen bonding interactions with biological targets. Furthermore, several FDA-approved fluorinated drugs—such as nirogacestat (approved for the treatment of progressing desmoid tumors), alpelisib (for breast cancer), and sotorasib (for non-small cell lung cancer)—demonstrate the clinical relevance and therapeutic potential of fluorine-containing compounds in modern drug development. For these reasons, we have incorporated the 2,4-difluorophenyl fragment into our molecules^12,13.

To evaluate the anticancer activity of the synthesized 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives, several cancer types were selected. Triple-negative breast cancer (TNBC) is a highly aggressive and metastatic subtype, characterized by a high recurrence rate and mortality¹⁴. The MDA-MB-231 is commonly employed as a model system for screening compounds targeting this cancer type. The limited therapeutic options for TNBC highlight the urgent need for more effective and less toxic novel agents.

Although prostate carcinoma is generally considered less lethal, it remains the second most frequently diagnosed cancer in men, and certain metastatic subtypes exhibit resistance to current treatment strategies¹⁵. The PPC-1 cell line was selected for this study due to its well-established metastatic properties and widespread use as a model for evaluating compound efficacy against prostate carcinoma¹⁶.

Melanoma is the most lethal form of skin cancer, and its associated mortality continues to rise each year¹⁷. Although therapeutic advances have accelerated since 2011, the high aggressiveness of melanoma and the frequent development of treatment resistance underscore the urgent need for novel therapeutic options¹⁸. Depending on the melanoma subtype, disease stage, patient health status, and other factors, a range of treatment strategies may be employed, including surgery, radiotherapy, chemotherapy, and drug combinations – such as immune checkpoint inhibitors and targeted therapies¹⁹. However, the primary chemotherapeutic agent used to treat metastatic melanoma remains the non-specific alkylating agent dacarbazine²⁰. The A375 cell line, known for its aggressive and rapidly proliferating phenotype²¹, was therefore included in this study to identify potential candidate compounds for melanoma traetment.

For screening the synthesized compounds, we chose to assess their cytotoxicity using the MTT assay, a widely employed method in the initial stages of in vitro activity evaluation due to its simplicity²². Additionally, considering the metastatic potential of the selected cancer types, the effects of the compounds on cell migration were evaluated using the ‘wound healing’ assay. Finally, given that three-dimensional (3D) cell cultures more accurately represent the tumor microenvironment compared to traditional monolayer cultures, this model was employed chosen for a more detailed analysis of the most active candidate compounds.

Results and discussion

Chemistry

In our previous studies, we reported the synthesis and in vitro anticancer activity of 5-oxo-1-arylpyrrolidine-3-carbohydrazide derivatives²³. The results were promising for further exploration of this scaffold to enhance the anticancer activity and establish the structure–activity relationship (SAR) properties of 5-oxo-1-arylpyrrolidine-3-carbohydrazide derivatives. In the present work, 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carboxylic acid (2) was prepared according to the established method²², by refluxing 2,4-difluoroaniline (1) with itaconic acid in water. The intermediate product – 4-arylamino-3-carboxybutanoic acid – was not isolated from the reaction mixture because it lost a water molecule and underwent cyclization to form 3-carboxy-5-oxopyrrolidine (2) during the reaction (Scheme 1).

Scheme 1 — Synthesis of compounds **2–4**. 3a R¹ = H; 3b R¹ = F; 3c R¹ = Cl. Reagents and conditions: (i) itaconic acid, water, Δ, 22 h, 73%; (ii) corresponding o-phenylenediamine, 4 M HCl, Δ, 72 h, 5% Na₂CO₃, H₂O, 36−49%; (iii) CH₃CH₂I, KOH, K₂CO₃, DMF, RT, 1 h, 68%.

The target compounds 3a-c were synthesized by the Philips method (heating of both reagents in 4 M hydrochloric acid), and a sufficient yield of benzimidazoles was obtained. N-Substituted benzimidazole 4 was synthesized by alkylation of 4-(1H-benzo[d]imidazol-2-yl)-1-(2,4-difluorophenyl)pyrrolidine-2-one 3a with iodoethane in DMF in the presence of potassium carbonate and potassium hydroxide. In the ¹H NMR spectrum of the alkylated compound 4, compared to the spectrum of the non-alkylated compound 3a, no proton signal characteristic of the NH fragment of the imidazole ring is observed in weak magnetic fields, and proton signals of the ethyl group appear at 1.33 and 2.88–3.06 ppm. In the ¹³C NMR spectrum of the alkylated compound 4, the signals of the carbon atoms of the ethyl group are observed at 15.20 and 29.76 ppm.

Methyl 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carboxylate (5) was synthesized by esterification of acid 2 with an excess of methanol under reflux in the presence of a catalytic amount of sulphuric acid. The reaction of ester 5 with hydrazine hydrate in 2-propanol under reflux yielded 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (6), which crystallized from the reaction mixture upon cooling (Scheme 2). Condensation of hydrazide 6 with heterocyclic, aromatic aldehydes and ketones produced hydrazones 7–9. The hydrazones gave rise to two sets of spectral lines (intensity ratio ~ 35:65) in the ¹H and ¹³C NMR spectra (DMSO-d₆). Thus, the hydrazones form stable E/Z isomers due to the restricted rotation of the amide group around the CO-NH bond. This has been discussed in more detail in previous works^23,24.

Scheme 2 — Synthesis of compounds **5–10**. 7a R² = H; 7b R² = NO₂; 8a R³ = CH₃; 8b R³ = C₂H₅; 8c R³ = 4-NH₂-C₆H₄; 9a R⁴, R⁵, R⁶, R⁷, R⁸ = H; 9b R⁴, R⁵, R⁷, R⁸ = H, R⁶ = F; 9c R⁴, R⁵, R⁷, R⁸ = H, R⁶ = Cl; 9d R⁴, R⁵, R⁷ = H, R⁶, R⁸ = F; 9e R⁴, R⁵, R⁷, R⁸ = H, R⁶ = Br; 9f R⁴, R⁵, R⁷, R⁸ = H, R⁶ = CH₃; **9 g** R⁴, R⁵, R⁷, R⁸ = H, R⁶ = N(CH₃)₂; **9 h** R⁴, R⁵, R⁷, R⁸ = H, R⁶ = NO₂. Reagents and conditions: (i) MeOH, H₂SO₄, Δ, 2 h, 76%; (ii) N₂H₄.H₂O, Δ, 8 h, i-PrOH, 62%; (iii) coresponding thiophene-2-carbaldehyde, i-PrOH, Δ, 2 h, 72% (7a), 96% (7b); (iv) acetone or ethyl methyl ketone, CH₃COOH, Δ, 1 h, 85% (8a), 65% (8b) or 4’-aminoacetophenone, CH₃COOH, i-PrOH, Δ, 5 h, 56% (8c); (v) coresponding aromatic aldehyde, i-PrOH, Δ, 1 h, 70−92%; (vi) CH₃CH₂I, KOH, K₂CO₃, DMF, RT, 1 h, 54%.

Alkylation of N’-(4-bromobenzylidene)-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (9e) with iodoethane was investigated. The reaction was carried out in a large excess of iodoethane in the presence of potassium hydroxide and potassium carbonate, and compound 10 was synthesized. The absorption band characteristic of the NH group is absent in the IR spectra compared to the initial compounds.

The reaction of acid hydrazides with β- and γ-diketones usually provides cyclic compounds (Scheme 3). The condensation of hydrazide 6 with 2,5-hexanedione in 2-propanol in the presence of a catalytic amount of acetic acid resulted in the formation of 1-(2,4-difluorophenyl)-N-(2,5-dimethyl-1H-pyrrol-1-yl)-5-oxopyrrolidine-3-carboxamide (11). The intense singlets at 1.91–2.11 and 5.67 ppm, attributed to the CH₃ and CH groups of the pyrrole ring, were present in the ¹H NMR spectra. The double intensity resonances at 10.88, 103.10, and 126.73 ppm in the ¹³C NMR spectra pointed to the existence of a pyrrole ring.

The condensation of hydrazide 6 with 2,4-pentanedione in 2-propanol in the presence of a catalytic amount of hydrochloric acid resulted in the formation of pyrazole 12. The ¹³C NMR spectra of this compound exhibited three resonances at 111.15-111.48, 143.45, and 151.73 ppm, assigned to the pyrazole ring.

The interaction of carbohydrazide 6 with phenyl isocyanate and phenyl isothiocyanate in methanol under reflux gave (thio)semicarbazides 13, 15. A precipitate formed during the reaction. In the ¹H NMR spectrum of compound 15, three singlets at 9.58, 9.85 and 10.18 ppm, show the presence of three NH groups. The formation of the C(O)-NH-NH-C(S)-NHPh fragment is further confirmed by the resonances at 172.04 and 181.16 ppm assigned to C = O and C = S groups and additional spectral lines in the aromatic region in the ¹³C NMR spectrum.

The final step of the study involved ring closure reactions, in which (thio)semicarbazides 13, 15 were heated under reflux in aqueous sodium hydroxide solution. This reaction afforded 3-substituted 1,2,4-triazole derivatives; however, simultaneous opening of the pyrrolidinone cycle occured, forming the corresponding gamma amino acids 14, 16. Ring opening was confirmed by ¹H NMR spectra, where proton signals at 5.58 and 5.60 ppm were assigned to the NH fragment of the amino acids.

Biological activity

Cytotoxicity

The effect of compounds 2–16 on the viability of selected cancer cell lines varied considerably between both cell lines and compounds (Fig. 1). In general, the compounds were less active against the melanoma cell line, and only seven of them reduced the viability of A375 cells by more than 50%. The cytotoxicity against prostate and TNBC was comparable across different compounds. However, the same six compounds that showed the most substantial viability-reducing effect against melanoma cells – 7b, 9a, 9c, 9e, 9f, and 10 - were also more active against the other two cell lines (The most active compound 7b reduced A375 cell viability to as low as 4.41%.

The most active compounds were hydrazone derivatives bearing the N’-((5-nitrothiophen-2-yl)methylene) (7b), N’-benzylidene (9a), N’-(4-chlorobenzylidene) (9c), N’-(4-bromobenzylidene) (9e), N’-(4-methylbenzylidene) (9f) fragments, and alkylated hydrazone with N’-(4-bromobenzylidene) (10) moiety. Moreover, functionalization of the carboxyl group to heterocyclic compounds 2 − 6 and 11 − 16 resulted in a reduced anticancer activity.

The six most active compounds were included in further research, and their EC₅₀ values against cancer cell lines and human fibroblasts were determined (Fig. 2). Compound 9a was identified as non-toxic to fibroblasts and showed relatively low activity against all tested cancer cell lines, as its EC₅₀ was higher than 200 µM.

Fig. 2 — EC₅₀ values of the most active compounds 7b, 9a, 9c, 9e, 9f and 10 after 72 h of incubation in human triple-negative breast cancer MDA-MB-231, human prostate carcinoma PPC, human melanoma A375 cell lines and human fibroblasts HF, established by the MTT assay, n = 3 (A). The dose-effect correlation has been calculated using the Hill equation (B). Data points are experimental values (averages of three repeats), while the lines are fit of the standard inhibition model with the Hill coefficient of 2.0 (MDA-MB-231, A375 and PPC-1 cells) and 2.6 (for HF).

Hydrazones 7b and 9f were identified as the most active compounds, particularly against the melanoma A375 and prostate carcinoma PPC-1 cell lines (EC₅₀ values ranged from 1.0 to 13.1 µM). The activity of 7b was similar across all tested cell lines, including cytotoxicity against fibroblasts. In contrast, compound 9f exhibited significantly lower activity against the MDA-MB-231 cell line (it was from 11 to 22-fold less active compared to its activity against the other cancer cell lines). Notably, it was 3.9 times more selective towards A375 and 1.6 times more selective toward PPC-1 than fibroblasts. Compounds 9c, 9e and 10 were considerably less active against the tested cancer cells (EC₅₀ values ranged rom 14.5 to 36.6 µM), but they demonstrated 1.6- to 3.0-fold selectivity for cancer cells over fibroblasts. Compound 9f, bearing N’-(4-methylbenzylidene) moiety, was the most active against the melanoma cell line (EC₅₀ = 1.0 ± 0.2 µM) and the prostate adenocarcinoma cell line (EC₅₀ = 2.5 ± 0.6 µM). The most active compound against the TNBC cell line was hydrazone 7b, bearing an N’-(5-nitrotiophen-2-yl) moiety (EC₅₀ = 6.4 ± 1.1 µM).

Additionally, we compared the cytotoxicity of the compounds with the kinase inhibitor sunitinib, the platinum derivative cisplatin and anthracycline-class drug doxorubicin (Table 1). These drugs have different mechanisms of action and are characterized by varying levels of cytotoxicity across different cancer cell lines. Sunitinib has been extensively studied in clinical trials for its potential in treating melanoma, metastatic breast cancer, prostate cancer and other cancer types²⁴. Despite its high toxicity, cisplatin is frequently used as a first—line chemotherapy, either alone or in combination with other anticancer drugs, to treat testicular cancer, head and neck cancer, lung cancer, breast cancer, melanoma, prostate cancer, and other malignancies²⁵. Doxorubicin has been used to treat a wide range of cancers for more than five decades and remains a component of many chemotherapy regimens²⁶. All the tested compounds were several hundred times less active than doxorubicin and several-fold less active than sunitinib, except compound 9f, which exhibited approximately three-fold higher cytotoxicity against the A375 cell line than sunitinib (Table 1). Compound 9f was also about ten-fold more active against the PPC-1 cell line compared to cisplatin. Additionally, the activity of compound 7b was comparable to that of cisplatin against melanoma cells and was approximately two-fold more active against the prostate cancer cell line.

Table 1.

Established EC₅₀ values for the most active compounds and anticancer drugs.

Compound	EC₅₀ (µM)
Compound	A375	MDA-MB-231	PPC-1	HF
7b	6.4 ± 1.1	10.9 ± 1.0	13.1 ± 3.4	10.6 ± 2.1
9a	275.9 ± 45.1	255.5 ± 49.3	455.2 ± 30.0	> 500.0
9c	36.6 ± 11.0	29.9 ± 6.4	36.6 ± 6.1	66.0 ± 7.2
9e	14.5 ± 1.5	22.6 ± 4.7	27.3 ± 2.3	43.3 ± 5.8
9f	1.0 ± 0.2	22.8 ± 3.9	2.5 ± 0.6	3.9 ± 0.2
10	36.6 ± 4.0	32.9 ± 2.1	28.9 ± 3.1	91.7 ± 14.3
Sunitinib	2.9 ± 0.99	3.4 ± 0.1	1.45 ± 0.07	8.17 ± 1.5
Doxorubicin	0.007 ± 0.0026	0.022 ± 0.0023	0.088 ± 0.01	0.33 ± 0.04
Cisplatin	8.13 ± 3.18	ND	27.8 ± 11.09	26.0 ± 10.8

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ND – not determined.

Overall, compounds 7b, 9e and 9f demonstrated greater activity against the melanoma cell line than dacarbazine, a chemotherapeutic agent used in clinical practice to treat melanoma, whose EC₅₀ values range from 25 to 100 µM in vitro²⁷. However, newer compounds with significantly higher potency have been reported, such as the investigational proteasome inhibitor salinosporamide A (EC₅₀ = 16.79 nM after 48 h)²⁸ and FDA-approved BRAF inhibitor dabrafenib (EC₅₀ = 9.5 nM in colony formation assay)²⁹, approved in 2013 for the treatment of metastatic melanoma.

Our compounds were not more active than docetaxel, a drug approved for metastatic castration-resistant and hormone-sensitive prostate cancer, which exhibits nanomolar potency in PC-3 prostate cancer cells (EC₅₀ = 15 ± 4 nM after 72 h of incubation).

Moreover, none of the tested compounds exhibited high activity against MDA-MB-231, which could be attributed to the intrinsic resistance and heterogeneity of this TNBC cell line³⁰. The anthracycline drug doxorubicin, clinically used for the treatment of TNBC, has shown a wide range of activity in vitro, from nanomolar to micromolar concentrations^31,32, depending on variable experimental conditions. Considering the urgent need for more selective therapeutic options for TNBC, it may be hypothesized that the most active compound, hydrazone 7b, holds potential for further investigation.

It should be emphasized that the cytotoxic activity determined in monolayer cultures represent only an initial step in compound development, as this in vitro model does not adequately reflect the complexity of the in vivo tumor microenvironment. Therefore, based on the observed selectivity and activity in the two-dimensional system, we proceeded to investigate the effects of these compounds on cancer cell migration, as well as their activity in a more physiologically relevant three-dimensional cell model (cancer cell spheroids).

Effect on cell migration

The most active and relatively more cancer cell-selective hydrazones – 7b, 9c, 9e and 10 – were tested in a ‘wound healing’ assay at a concentration of 10 µM. To avoid confounding effects due to cytotoxicity, cell migration was evaluated up to 36 h for MDA-MB-231 and 48 h for PPC-1 and A375 cells (Fig. 3).

In general, none of the tested compounds inhibited TNBC cell (MDA-MB-231) migration after 12 and 24 h (Fig. 3, A and D). Only compound 9e, after 36 h, exhibited a statistically significant anti-migratory effect compared to the control (p < 0.05). In contrast, compounds 7b, 9c and 9e significantly inhibited prostate adenocarcinoma PPC-1 cell migration after 12 h of incubation (Fig. 3, B and E). These compounds also demonstrated inhibitory effect on melanoma A375 cell migration after 48 h; however, only compounds 9c and 9e showed a statistically significant effect (p < 0.05) (Fig. 3, C and F).

It should be noted that compound 10 did not exhibit any effect on the migration of the tested cell lines. Although compound 7b demonstrated the highest cytotoxicity against all three cancer cell lines, it did not show superior activity in a ‘wound-healing’ assay compared to the other tested compounds. Interestingly, it was more active in PPC-1 cells even after a shorter incubation period, suggesting that its impact on cell migration is not directly linked to its cytotoxicity. Overall, compound 9e emerged as the most active inhibitor of cell migration, exhibiting a variable but consistent anti-migratory effect across all cell lines at both earlier and later time points.

Overall, the effects of compounds on TNBC, prostate carcinoma and melanoma cell migration were inconsistent. It is important to acknowledge the limitations of the method used to assess cell migration. Although the ‘wound healing’ assay is widely recognised as a simple technique,, it is also associated with low reproducibility and technical challenges related to the creation of the scratch in the cell monolayer³³. In this study, we employed the ‘wound healing’ assay as a preliminary screening tool to evaluate the potential of the compounds to affect cell migration and to identify the most promising candidates for further, more detailed investigation.

Effect in 3D cultures

Experiments in 3D cultures were conducted with hydrazones 7b, 9e, 9f and 10, which were the most effective at reducing cell viability in monolayer cultures. Spheroids were formed from TNBC, prostate adenocarcinoma, and melanoma cell lines, in combination with human fibroblasts at a 1:1 ratio. These conditions previously established by our group based on the observation that fibroblasts support spheroid formation and enhance their development^34,35. Additionally, it is well-known that the incorporation of stromal cells into spheroids imparts in vivo-like properties to the model, thereby making it more translatable to real world conditions³⁶.

At the beginning of the experiment, the spheroids had a diameter of 200–300 μm. The melanoma spheroids grew slightly faster than the other two types, and by the end of the experiment (on the eighth day), their diameter ranged from 350 to 460 μm (Fig. 4, A and B). Overall, compounds at a 10 µM concentration exhibited varying effects on spheroid growth. Compound 9e did not inhibit spheroid growth. In contrast, compound 10 caused the spheroids to become looser, especially the A375 spheroids (Fig. 4A). The most active compound was 9f, which statistically significantly reduced spheroid size, with some spheroids disintegrating after 6–8 days of incubation. However, this effect was not as pronounced as that observed with the cytotoxic drug doxorubicin. Compound 7b showed an effect only on the growth of PPC-1 spheroids (p < 0.05) (Fig. 4B).

Fig. 4 — Effect of the most active compounds 7b, 9e, 9f, 10, and doxorubicin on 3D cell cultures. (A) Photos of human triple-negative breast cancer MDA-MB-231, prostate carcinoma PPC-1 and melanoma A375 tumour spheroids at the end (Day 8) of the experiment (after incubation with 10 µM of compounds). (B) MDA-MB-231, PPC-1 and A375 spheroid size at the end of the experiment. (C) Viability of cells in MDA-MB-231, PPC-1 and A375 spheroids at the end of the experiment by MTT assay. Asterisks (*) indicate p < 0.05 compared to the control (untreated spheroids), (**) indicate p < 0.01 compared to the control (untreated spheroids), crosses (×) indicate means; inner dashes indicate medians; whiskers indicate maximum and minimum values. The scale bar indicates 200 μm.

Considering that spheroid size does not necessarily correlate with cell viability, we performed an MTT assay on the final day of the experiment (Fig. 4C). Doxorubicin remained the most active agent in this assay, while compound 9f was identified as the most cytotoxic compound in 3D cultures. It reduced PPC-1 and A375 spheroid cell viability up to 31.9% and MDA-MB-231 spheroid cell viability up to 53.2% (p < 0.01) (Fig. 4C). Notably, compound 7b also reduced cell viability in all three types of spheroids, despite not having a significant effect on MDA-MB-231 and A375 spheroid growth. This phenomenon further supports previous observations, indicating that assessing compound effects based solely on spheroid size is insufficient³⁷. In addition, compound 10 also significantly reduced PPC-1 spheroid cell viability compared to the control (p < 0.05).

Overall, compounds exhibited variable anticancer properties in different functional assays. Hydrazone 9f was identified as the most cytotoxic compound in prostate adenocarcinoma PPC-1 and melanoma A375 cell monolayer and 3D cultures. Although it was less active than doxorubicin, it reduced PPC-1 and A375 cell viability up to 30% after 8 days of incubation. Additionally, 9f showed the most significant effect among the tested compounds on TNBC cell spheroid growth and viability. In contrast, compound 9e was not active in 3D cultures and was only moderately cytotoxic in cell monolayer against all cell lines. However, it demonstrated an inhibitory effect on cell migration after different incubation durations. Overall, the selected compounds exhibited greater activity in the melanoma A375 cell line and lower activity against the TNBC MDA-MB-231 cell line.

Conclusions

In conclusion, a series of novel 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives containing azole and hydrazone fragments was synthesized and investigated for their anticancer activity.

Hydrazone-containing compounds were identified as the most cytotoxic agents against human TNBC MDA-MB-231, prostate carcinoma PPC1 and melanoma A375 cell lines. Generally, compounds 7b, 9c, 9e, 9f and 10 demonstrated greater activity in the A375 cell line and loweractivity against MDA-MB-231 cells. Among them, compound 9f, bearing N’-(4-methylbenzylidene) moiety, was the most cytotoxic in both prostate adenocarcinoma PPC-1 and melanoma A375 cell monolayer and 3D cultures. It also showed the strongest effect on TNBC cell spheroid growth and viability among the tested compounds. In contrast, compound 9e, with the N’-(4-bromobenzylidene) moiety, was not active in 3D cultures and displayed moderate cytotoxicity in cell monolayer, but it significantly inhibited cell migration after various incubation periods.

Materials and methods

Chemistry

Reagents and solvents were purchased from Sigma–Aldrich (St. Louis, MO, USA) and used without further purification. The progress of reactions and the purity of synthesized compounds were monitored by TLC on aluminium plates precoated with Silica gel with F254 nm (Merck KGaA, Darmstadt, Germany). Melting points were determined with a B-540 melting point analyzer (Büchi Corporation, New Castle, DE, USA) and were uncorrected. NMR spectra were recorded on a Brucker Avance III (400, 101 MHz) spectrometer (Bruker BioSpin AG, Fällanden, Switzerland). Chemical shifts were reported in (δ) ppm relative to tetramethylsilane (TMS) with the residual solvent as internal reference (DMSO-d₆, δ = 2.50 ppm for ¹H and δ = 39.52 ppm for ¹³C). Data were reported as follows: chemical shift, multiplicity, coupling constant (Hz), integration, and assignment. IR spectra (ν, cm⁻¹) were recorded on a Perkin–Elmer Spectrum BX FT–IR spectrometer (Perkin– Elmer Inc., Waltham, MA, USA) using KBr pellets. Elemental analyses (C, H, N) were conducted using the Elemental Analyzer CE-440 (Exeter Analytical, Inc., Chelmsford, MA, USA); their results were found to be in good agreement (± 0.3%) with the calculated values.

1-(2,4-Difluorophenyl)-5-oxopyrrolidine-3-carboxylic acid (2)

A mixture of 2,4-difluoroaniline (1) (15 g, 120 mmol), itaconic acid (22.44 g, 170 mmol) and water (30 mL) was refluxed for 22 h, and then cooled. The formed precipitate was filtered off and dissolved in an aqueous 5% sodium hydroxide solution, sodium dithionite (0.8 g) was added, the solution was filtered off, and the filtrate was acidified with hydrochloric acid to pH 1 to give the title compound 2.

White solid, yield 20.19 g, 73%, m. p. 140–141 °C. IR (KBr): ν 3065 (OH); 1740; 1670 (2x C = O) cm^–1.

¹H NMR (400 MHz, DMSO–d₆) δ 2.57‒2.78 (m, 2 H, COCH₂), 3.35‒3.55 (m, 1H, CHCO), 3.82‒3.99 (m, 2 H, NCH₂), 7.14 (t, J = 8.1 Hz, 1H, H_Ar), 7.26–7.41 (m,, H_Ar), 7.49 (q, J = 8.7 Hz, 1H, H_Ar), 12.80 (br. s, 1H, COOH) ppm.

¹³C NMR (101 MHz, DMSO–d₆) δ 33.38 (CH₂CO), 36.27 (CHCO), 51.13 (NCH₂), 104.67, 104.91, 105.18, 111.67, 111.70, 111.89, 111.92, 122.52, 122.56, 122.64, 122.68, 129.10, 129.20, 129.23, 155.60, 155.73, 158.10, 158.23, 159.34, 159.46, 161.79, 161.90 (C_Ar), 171.96, 174.08 (2x C = O) ppm. Calcd. for C₁₁H₉F₂NO₃, %: C 54.78; H 3.76; N 5.81; Found, %: C 54.63; H 3.73; N 5.80.

General procedure for the Preparation of benzimidazoles 3a−c

A mixture compound 2 (0.7 g, 2.9 mmol) and corresponding o-phenylenediamine (5.8 mmol) was refluxed 4 M HCl (8 mL) for 72 h, then cooled and neutralized with aqueous 5% sodium carbonate solution. Formed crystallines products 3a−c was filtered off, washed with water. Product 3a was recrystallized from 1,4-dioxane. In the case of products 3b, 3c, the obtained compounds was purified by recrystallization from a mixture of 2-propanol and water (1:1, 15 mL). After cooling, the formed crystals were filtered off, washed with diethyl ether to give the title compounds 3b and 3c.

4-(1 H-benzo[d]imidazol-2-yl)-1-(2,4-difluorophenyl)pyrrolidin-2-one (3a)

Yellow solid, yield 0.41 g, 45%, m. p. 162–163 °C. IR (KBr): ν 3018 (NH); 1707 (C = O); 1516 (C = N) cm^–1.

¹H NMR (400 MHz, DMSO–d₆) δ 3.00‒3.23 (m, 2 H, COCH₂), 4.19‒4.36 (m, 2 H, NCH₂), 4.46 (qu, J = 8.1 Hz, 1H, CHCN), 7.19 (t, J = 8.5 Hz, 1H, H_Ar), 7.33–7.45 (m, 1H, H_Ar), 7.46‒7.55 (m, 2 H, H_Ar), 7.62 (dd, J = 15.4, 8.2 Hz, 1H, H_Ar), 7.73‒7.82 (m, 2 H, H_Ar), 15.38 (br. s. 1H, NH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 30.46 (CH₂CO), 35.16 (CHCN), 52.35 (NCH₂), 104.71, 104.98, 105.22, 111.75, 111.97, 112.01, 113.98, 122.24, 122.36, 122.40, 125.19, 129.31, 129.34, 129.41, 129.44, 131.89, 153.46, 155.63, 155.76, 158.14, 158.27, 159.46, 159.58, 161.91, 162.02 (C_Ar), 171.00 (C = O) ppm. Calcd. for C₁₇H₁₃F₂N₃O, %: C 65.17; H 4.18; N 13.41; Found, %: C 64,98; H 4.13; N 13.29.

1-(2,4-Difluorophenyl)-4-(6-fluoro-1 H-benzo[d]imidazol-2-yl)pyrrolidin-2-one (3b)

Dark purple solid, yield 0.35 g, 36%, m. p. 108–109 °C. IR (KBr): ν 3108 (NH); 1699 (C = O); 1515 (C = N) cm^–1.

¹H NMR (400 MHz, DMSO–d₆) δ 2.95 (d, J = 7.8 Hz, 2 H, CH₂CO), 4.00–4.21 (m, 3 H, NCH₂ overlaps with CHCN), 6.95−7.08 (m, 1H, H_Ar), 7.09–7.23 (m, 1H, H_Ar), 7.27–7.62 (m, 4 H, H_Ar), 12.59 (s, 1H, NH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 31.89 (CH₂CO), 35.60 (CHCN), 53.24 (NCH₂), 104.04, 104.66, 104.93, 105.17, 109.31, 110.07, 111.65, 111.69, 111.76, 111.91, 119.28, 122.77, 129.09, 129.19, 131.19, 139.43, 156.66 (C_Ar), 172.03 (C = O) ppm. Calcd. for C₁₇H₁₂F₃N₃O, %: C 61.63; H 3.65; N 12.68; Found, %: C 61.53; H 3.60; N 12.59.

4-(6-Chloro-1 H-benzo[d]imidazol-2-yl)-1-(2,4-difluorophenyl)pyrrolidin-2-one (3c)

Light brown solid, yield 0.49 g, 49%, m. p. 122–123 °C. IR (KBr): ν 3092 (NH); 1699 (C = O); 1514 (C = N) cm^–1.

¹H NMR (400 MHz, DMSO–d₆) δ 2.95 (d, J = 7.2 Hz, 2 H, CH₂CO), 4.02–4.23 (m, 3 H, NCH₂ overlaps with CHCN), 7.10–7.25 (m, 2 H, H_Ar), 7.30–7.45 (m, 1H, H_Ar), 7.47−7.67 (m, 3 H, H_Ar), 12.67 (s, 1H, NH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 31.89 (CH₂CO), 35.57 (CHCN), 104.66, 104.91, 105.17, 111.65, 111.88, 121.89, 122.59, 122.72, 129.06, 129.09, 129.17, 155.53, 155.66, 156.34, 158.04, 158.16, 159.27, 159.39, 161.72 (C_Ar), 172.00 (C = O) ppm. Calcd. for C₁₇H₁₂ClF₃N₃O, %: C 58.72; H 3.48; N 12.08; Found, %: C 58.56; H 3.46; N 12.01.

1-(2,4-Difluorophenyl)-4-(1-ethyl-1 H-benzo[d]imidazol-2-yl)pyrrolidin-2-one (4)

Compound 3a (0.5 g, 1.6 mmol) was dissolved in dimethylformamide (4 mL). Potassium hydroxide (0.27 g, 4.8 mmol) and potassium carbonate (0.66 g, 4.8 mmol) were added and then the mixture was stirred at 30 °C. After 15 min, ethyl iodide (0.89 g, 5.7 mmol) was added dropwise, and the reaction was carried out at room temperature for 1 h. The volatile fractions were separated under reduced pressure, and then the obtained residue was diluted with distilled water (15 mL). After cooling, the precipitated crystals were filtered. The desired product 4 was purified by recrystallization from 2-propanol.

Yellow solid, yield 0.37 g, 68%, m. p. 93–94 °C. IR (KBr): ν 1699 (C = O); 1516 (C = N) cm^–1.

¹H NMR (400 MHz, DMSO–d₆) δ 1.33 (t, J = 7.1 Hz, 3 H, CH₃), 2.88–3.06 (m, 2 H, CH₂CO), 4.11–4.37 (m, 5 H, CH₂CH₃ overlaps with NCH₂ and CHCN), 7.11–7.28 (m, 3 H, H_Ar), 7.31–7.46 (m, 1H, H_Ar), 7.51‒7,66 (m, 3 H, H_Ar) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 15.20 (CH₂CH₃), 29.76 (CH₂CH₃), 35.69 (CH₂CO), 37.82 (CHCN), 53.25 (NCH₂), 104.65, 104.92, 105.16, 110.15, 111.65, 111.91, 118.85, 121.53, 122.02, 122.77, 129.11, 134.97, 142.08, 154.25, 155.99, 155.72, 158.09, 158.22, 159.29, 161.73, 161.85 (C_Ar); 171.94 (C = O) ppm. Calcd. for C₁₉H₁₇F₂N₃O, %: C 66.85; H 5.02; N 12.31; Found, %: C 66.77; H 5.01; N 12.26.

Methyl 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carboxylate (5)

Ester 5 is obtained by dissolving compound 2 (6.03 g, 25 mmol) in methanol (125 mL) for 2 h at the boiling temperature of the mixture and using 0.5 mL of concentrated sulfuric acid as a catalyst. At the end of the reaction, the solvent is distilled under pressure. The residue is neutralized using 5% sodium carbonate solution (150 mL). The formed crystals are filtered and washed with water. Purified by recrystallization from a mixture of methanol and water (1:1).

Yellowish, yield 4.85 g, 76%, m. p. 94–95 °C. IR (KBr): ν 1741; 1699 (C = O) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ 2.60–2.81 (m, 2 H, CH₂CO), 3.46–3.58 (m, 1H, CHCO), 3.68 (s, 3 H, CCH₃), 3.82–3.99 (m, 2 H, NCH₂), 7.04–7.19 (m, 1H, H_Ar), 7.29–7.43 (m, 1H, H_Ar), 7.49 (dd, J = 15.1, 8.7 Hz, 1H, H_Ar) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 33,86 (CH₂CO), 36.66 (CHCO), 51.50 (NCH₂), 52.82 (CH₃), 105.29, 105.53, 105.80, 112.29, 112.32, 112.51, 112.55, 123.06, 123.19, 129.73, 129.86, 156.21, 156.34, 158.71, 158.84, 159.98, 160.10, 162.43, 162.54 (C_Ar), 172.27, 173.63 (2x C = O) ppm. Calcd. for C₁₂H₁₁F₂NO₃, %: C 56.47; H 4.34; N 5.49; Found, %: C 56.40; H 4.31; N 5.45.

1-(2,4-Difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (6)

Compound 5 (5.10 g, 20 mmol) was dissolved in 2-propanol (25 mL), hydrazine monohydrate (2.00 g, 40 mmol) was added dropwise. Mixture of reaction was refluxed for 8 h, then cooled down. The obtained crystalline solid was filtered off, washed with 2-propanol to give the title compound 6.

Yellowish solid, yield 3.16 g, 62%, m. p. 126–127 °C (from 2-propanol). IR (KBr): ν 3283, 3192 (NH + NH₂); 1694; 1637 (2x C = O) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ 2.56–2.64 (m, 2 H, CH₂CO), 3.19‒3.27 (m, 1H, CHCO), 3.75 (t, J = 7.9 Hz, 1H, NCH₂), 3.84 (t, J = 8.4 Hz, 1H, NCH₂), 4.29 (s, 2 H, NHNH₂), 7.03–7.18 (m, 1H, H_Ar), 7.29–7.42 (m, 1H, H_Ar), 7.49 (dd, J = 15.1, 8.7 Hz, 1H, H_Ar), 9.28 (s, 1H, NH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 33.88 (CH₂CO), 35.37 (CHCO), 51.79 (NCH₂), 104.65, 104.89, 105.16, 111.69, 111.91, 122.56, 122.60, 122.68, 122.72, 129.18, 129.21, 129.28, 129.31, 155.59, 155.72, 158.09, 158.22, 159.30, 159.41, 161.75, 161.86 (C_Ar), 171.21, 171.17 (2x C = O) ppm. Calcd. for C₁₁H₁₁F₂N₃O₂, %: C 51.77; H 4.34; N 16.46; Found, %: C 51.64; H 4.31; N 16.39.

General procedure for the Preparation of hydrazones 7a, B and 9a–h

To a solution of hydrazide 6 (0.3 g 1 mmol) in 2-propanol (7 mL), the corresponding aromatic or heterocyclic aldehyde (1.2 mmol) was added and the mixture was heated at reflux for 2 h. After completion of the reaction, the mixture was cooled, and the formed precipitate was filtered off and washed with 2-propanol to give the corresponding compounds 7a, b and 9a‒h.

1-(2,4-Difluorophenyl)-5-oxo-N’-(thiophen-2-ylmethylene)pyrrolidine-3-carbohydrazide (7a)

Orange solid, yield 0.250 g, 72%, m. p. 119–120 °C (from 2-propanol). IR (KBr): ν 3240 (NH); 1703; 1662 (2x C = O); 1509 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.63–2,83 (m, 2 H, CH₂CO), 3.35‒3.44, 3.81‒4.07 (2 m, 3 H, CHCO, NCH₂), 7.07‒7.20 (m, 2 H, H_Ar), 7.33‒7.58 (m, 3 H, H_Ar), 7.64 (dd, J = 14.7, 4.9 Hz, 1H, H_Ar), 8.20, 8.43 (2s, 1H, NHNCH), 11.56 (s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 33.49, 34.42 (CH₂CO), 34.83, 36.67 (CHCO), 51.83, 52.24 (NCH₂), 105.28, 105.55, 105.79, 112.31, 112.50, 112.54, 123.17, 123.29, 123.32, 128.48, 128.59, 129.10, 129.64, 129.85, 129.95, 131.04, 131.71, 139.28, 139.50, 139.50, 139.55, 142.85, 156.24, 156.36, 158.74, 158.87, 159.93, 160.04, 162.38, 162.50 (C_Ar), 168.89 (C = N), 172.73, 173.49 (2x C = O) ppm. Calcd. for C₁₆H₁₃F₂N₃O₂S, %: C 55.01; H 3.75; N 12.03; Found, %: C 54.94; H 3.71; N 11.97.

1-(2,4-Difluorophenyl)-N’-((5-nitrothiophen-2-yl)methylene)-5-oxopyrrolidine-3-carbohydrazide (7b)

Orange solid, yield 0.39 g, 96%, m. p. 197–198 °C (from 2-propanol). IR (KBr): ν 3200 (NH); 1697; 1677 (2x C = O); 1520 (C = N); 1175 cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.64–2.80 (m, 2 H, CH₂CO), 3.38‒3.48, 3.82‒4.13 (2 m, 3 H, CHCO, NCH₂), 7.03–7.21 (m, 1H, H_Ar), 7.26–7.43 (m, 1H, H_Ar), 7.46–7.60 (m, 2 H, H_Ar), 8.06‒8.14 (m, 1H, H_Ar), 8.19, 8.47 (2s, 1H, NHNCH), 11.98 (s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 32.91, 33.68 (CH₂CO), 34.00, 36.11 (CHCO), 51.05, 51.41 (NCH₂), 104.91, 105.16, 111.67, 111.86, 111.90, 111.93, 122.46, 122.50, 122.54, 122.58, 122.62, 122.67, 129.23, 129.31, 129.34, 129.37, 129.79, 130.46, 130.61, 136.92, 140.61, 146.49, 146.51, 150.55, 150.88, 155.60, 155.74, 158.11, 159.31, 159.43, 161.76, 161.88 (C_Ar), 168.97 (C = N), 171.99, 173.60 (2x C = O) ppm. Calcd. for C₁₆H₁₂F₂N₄O₂S, %: C 48.73; H 3.07; N 14.21; Found, %: C 48.64; H 3.03; N 14.20.

N’-Benzylidene-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (9a)

Brownish solid, yield 0.27 g, 78%, m. p. 159–160 °C (from 2-propanol). IR (KBr) ν 3039 (NH); 1703; 1676 (2x C = O); 1515 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.64–2.84 (m, 2 H, CH₂CO), 3.36‒3.46, 3.83‒4.21 (2 m, 3 H, CHCO, NCH₂), 7.03–7.21 (m, 1H, H_Ar), 7.32‒7.48 (m, 4 H, H_Ar), 7.49–7.58 (m, 1H, H_Ar), 7.63‒7.75 (m, 2 H, H_Ar), 8.03, 8.22 (2s, 1H, NHNCH), 11.58, 11.62 (2s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 32.96, 33.80 (CH₂CO), 34.04, 36.06 (CHCO), 51.21, 51.63 (NCH₂), 104.64, 104.88, 104.90, 105.15, 111.62, 111.65, 111.84, 111.88, 111.91, 122.51, 122.58, 122.62, 122.64, 122.70, 122.74, 126.87, 127.09, 128.84, 129.20, 129.28, 129.91, 130.12, 134.10, 134.13, 143.68, 147.04, 155.61, 155.73, 158.11, 158.24, 159.29, 159.40, 159.42, 161.73, 161.75, 161.85 (C_Ar), 168.40 (C = N), 172.13, 173.31 (2x C = O) ppm. Calcd. for C₁₈H₁₅F₂N₃O₂, %: C 62.97; H 4.40; N 12.24; Found, %: C 62.91; H 4.35; N 12.19.

1-(2,4-Difluorophenyl)-N’-(4-fluorobenzylidene)-5-oxopyrrolidine-3-carbohydrazide (9b)

Yellowish solid, yield 0.29 g, 78%, m. p. 167–168 °C (from 2-propanol). IR (KBr) ν 3070 (NH); 1707; 1668 (2x C = O); 1514 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.64–2.83 (m, 2 H, CH₂CO), 3.36‒3.46, 3.82‒4.20 (2 m, 3 H, CHCO, NCH₂), 7.05–7.20 (m, 1H, H_Ar), 7.21–7.32 (m, 2 H, H_Ar), 7.31–7.43 (m, 1H, H_Ar), 7.53 (dd, J = 14.5, 7.9 Hz, 1H, H_Ar), 7.69‒7.81 (m, 2 H, H_Ar), 8.02, 8.21 (2s, 1H, NHNCH), 11.58, 11.63 (2s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 32.96, 33.79 (CH₂CO), 34.00, 36.04 (CHCO), 51.20, 51.62 (NCH₂), 104.64, 104.90, 105.15, 111.62, 111.65, 111.84, 111.87, 111.91, 115.77, 115.99, 122.53, 122.58, 122.63, 122.67, 122.70, 122.74, 129.02, 129.10, 129.15, 129.23, 129.29, 129.31, 130.71, 130.74, 142.55, 145.92, 155.60, 155.73, 158.10, 158.23, 159.26, 159.29, 159.40, 161.85, 161.91, 164.22 (C_Ar), 168.41 (C = N), 172.11, 173.31 (2x C = O) ppm. Calcd. for C₁₈H₁₄F₃N₃O₂, %: C 59.83; H 3.91; N 11.63; Found, %: C 59.78; H 3.82; N 11.59.

N’-(4-Chlorobenzylidene)-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (9c)

Brownish solid, yield 0.32 g, 84%, m. p. 177–178 °C (from 2-propanol). IR (KBr) ν 3216 (NH); 1682 (2x C = O); 1514 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.64–2.79 (m, 2 H, CH₂CO), 3.36‒3.46, 3.83‒4.21 (2 m, 3 H, CHCO, NCH₂), 7.10‒7.19 (m, 1H, H_Ar), 7.33‒7.43 (m, 1H, H_Ar), 7.48‒7.58 (m, 3 H, H_Ar), 7.72 (d, J = 8.2 Hz, 2 H, H_Ar), 8.02, 8.20 (2s, 1H, NHNCH), 11.64, 11.69 (2s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 32.97, 33.76 (CH₂CO), 33.98, 36.07 (CHCO), 51.15, 51.59 (NCH₂), 104.64, 104.91, 105.15, 111.66, 111.88, 122.57, 128.54, 128.72, 128.91, 129.28, 133.05, 133.09, 134.33, 134.56, 142.42, 145.72, 155.50, 155.59, 155.72, 158.10, 158.22, 159.28, 159.42, 161.73, 161.85 (C_Ar), 168.49 (C = N), 172.07, 173.39 (2x C = O) ppm. Calcd. for C₁₈H₁₄ClF₂N₃O₂, %: C 57.23; H 3.74; N 11.12; Found, %: C 57.20; H 3.71; N 11.03.

N’-(2,4-Difluorobenzylidene)-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (9d)

White solid, yield 0.29 g, 76%, m. p. 175–176 °C (from 2-propanol). IR (KBr) ν 3185 (NH); 1708; 1675 (2x C = O); 1512 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.64–2.81 (m, 2 H, CH₂CO), 3.34‒3.45, 3.83‒4.20 (2 m, 3 H, CHCO, NCH₂), 7.09‒7.23 (m, 2 H, H_Ar), 7.30‒7.43 (m, 2 H, H_Ar), 7.47‒7.58 (m, 1H, H_Ar), 7.88‒8.02 (m, 1H, H_Ar), 8.17, 8.38 (2s, 1H, NHNCH), 11.68, 11.75 (2s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 32.93, 33.73 (CH₂CO), 33.99, 36.10 (CHCO), 51.12, 51.52 (NCH₂), 104.24, 104.49, 104.64, 104.91, 105.15, 111.66, 111.88, 112.51, 118.46, 118.50, 118.60, 122.56, 128.03, 129.18, 129.28, 135.89, 139.06, 155.61, 158.11, 158.24, 159.48, 159.60, 162.12, 164.42 (C_Ar), 168.49 (C = N), 172.07, 173.40 (2x C = O) ppm. Calcd. for C₁₈H₁₃F₄N₃O₂, %: C 57.00; H 3.45; N 11.08; Found, %: C 56.93; H 3.41; N 10.94.

N’-(4-Bromobenzylidene)-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (9e)

White solid, yield 0.39 g, 92%, m. p. 179–180 °C (from 2-propanol). IR (KBr) ν 3218 (NH); 1708; 1681 (2x C = O); 1513 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.64–2.79 (m, 2 H, CH₂CO), 3.36‒3.46, 3.82‒4.20 (2 m, 3 H, CHCO, NCH₂), 7.10‒7.19 (m, 1H, H_Ar), 7.33‒7.42 (m, 1H, H_Ar), 7.48‒7.58 (m, 1H, H_Ar), 7.58‒7.69 (m, 4 H, H_Ar), 8.00, 8.19 (2s, 1H, NHNCH), 11.64, 11.69 (2s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 32.97, 33.76 (CH₂CO), 33.97, 36.04 (CHCO), 51.15, 51.56 (NCH₂), 104.65, 104.91, 105.15, 111.66, 111.88, 122.69, 123.10, 123.36, 128.77, 125.95, 129.28, 131.82, 133.39, 142.52, 145.80, 155.60, 155.72, 158.10, 158.22, 159.29, 159.40, 161.74, 161.87 (C_Ar), 168.49 (C = N), 172.07, 173.40 (2x C = O) ppm. Calcd. for C₁₈H₁₄BrF₂N₃O₂, %: C 51.20; H 3.34; N 9.95; Found, %: C 51.15; H 3.28; N 9.88.

1-(2,4-Difluorophenyl)-N’-(4-methylbenzylidene)-5-oxopyrrolidine-3-carbohydrazide (9f)

Yellowish solid, yield 0.25 g, 70%, m. p. 138–139 °C (from 2-propanol). IR (KBr) ν 3224 (NH); 1685 (2x C = O); 1513 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.32 (s, 3 H, CCH₃), 2.64–2.83 (m, 2 H, CH₂CO), 3.35‒3.45, 3.82‒4.19 (2 m, 3 H, CHCO, NCH₂), 7.09‒7.19 (m, 1H, H_Ar), 7.21–7.29 (m, 2 H, H_Ar), 7.33‒7.43 (m, 1H, H_Ar), 7.48‒7.63 (m, 3 H, H_Ar), 7.99, 8.17 (2s, 1H, NHNCH), 11.51, 11.55 (2s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 21.01 (CCH₃), 32.97, 33.80 (CH₂CO), 34.04, 36.05 (CHCO), 51.23, 51.65 (NCH₂), 104.90, 105.15, 111.62, 111.65, 111.67, 111.84, 111.87, 111.91, 126.84, 127.07, 129.18, 129.21, 129.28, 129.30, 129.43, 131.40, 139.70, 139.96, 143.76, 147.07, 155.60, 155.73, 158.10, 158.23, 159.28, 159.40, 161.72, 161.84 (C_Ar), 168.27 (C = N), 172.14, 173.19 (2x C = O) ppm. Calcd. for C₁₉H₁₇F₂N₃O₂, %: C 63.86; H 4.80; N 11.76; Found, %: C 63.80; H 4.74; N 11.72.

1-(2,4-Difluorophenyl)-N’-(4-(dimethylamino)benzylidene)-5-oxopyrrolidine-3-carbohydrazide (9 g)

Light yellow solid, yield 0.30 g, 77%, m. p. 163–164 °C (from 2-propanol). IR (KBr) ν 3067 (NH); 1703; 1671 (2x C = O); 1513 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.64‒3.02 (m, 8 H, CH₂CO overlaps with N(CH₃)₂), 3.30‒3.43, 3.80‒4.16 (2 m, 3 H, CHCO, NCH₂), 6.56–6.80 (m, 2 H, H_Ar), 7.09‒7.56 (m, 5 H, H_Ar), 7.89, 8.06 (2s, 1H, NHNCH), 11.29, 11.31 (2s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 25.49 (N(CH₃)₂), 32.93, 33.88 (CH₂CO), 34.07, 36.03 (CHCO), 51.34, 51.74 (NCH₂), 104.63, 104.90, 105.14, 111.62, 111.65, 111.77, 111.82, 111.87, 111.90, 121.34, 121.47, 122.65, 128.11, 128.42, 129.19, 129.30, 144.46, 147.83, 151.37, 151.53, 155.60, 155.73, 158.10, 158.23, 159.27, 159.39, 161.86 (C_Ar), 167.73 (C = N), 172.23, 172.64 (2x C = O) ppm. Calcd. for C₂₀H₂₀F₂N₄O₂, %: C 62.17; H 5.22; N 14.50; Found, %: C 62.08; H 5.19; N 14.44.

1-(2,4-Difluorophenyl)-N’-(4-nitrobenzylidene)-5-oxopyrrolidine-3-carbohydrazide (9 h)

Bright yellow solid, yield 0.35 g, 89%, m. p. 204–205 °C (from 2-propanol). IR (KBr) ν 3087 (NH); 1701; 1683 (2x C = O); 1514 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.64–2.83 (m, 2 H, CH₂CO), 3.40‒3.51, 3.84‒4.25 (2 m, 3 H, CHCO, NCH₂), 7.10‒7.20 (m, 1H, H_Ar), 7.31‒7.43 (m, 1H, H_Ar), 7.47‒7.59 (m, 1H, H_Ar), 7.97 (d, J = 8.5 Hz, 2 H, H_Ar), 8.09‒8.34 (m, 3 H, H_Ar overlaps with NHNCH), 11.88 (s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 32.98, 33.72 (CH₂CO), 34.00, 36.10 (CHCO), 51.07, 51.52 (NCH₂), 104.66, 104.92, 105.16, 111.66, 111.89, 122.58, 122.67, 124.02, 124.12, 127.85, 128.02, 129.13, 129.17, 129.23, 129.26, 140.39, 140.48, 141.38, 144.58, 147.72, 147.89, 154.43, 155.73, 156.52, 158.10, 158.23, 159.30, 159.42, 159.48, 161.77, 161.82, 161.89 (C_Ar), 168.88 (C = N), 172.00, 173.75 (2x C = O) ppm. Calcd. for C₁₈H₁₄F₂N₄O₄, %: C 55.67; H 3.63; N 14.43; Found, %: C 55.60; H 3.59; N 14.39.

General method of the Preparation of hydrazones 8a, B

A mixture of hydrazide 6 (0.30 g 1 mmol), acetone (3 mL in order to synthesize product 8a) or methylethylketone (3 mL to obtain product 8b) and 1 drop of acetic acid was heated at reflux for 1 h, then cooled and the formed crystalline solid was filtered off, washed with hot hexane to give the title compound 8a and 8b.

1-(2,4-Difluorophenyl)-5-oxo-N’-(propan-2-ylidene)pyrrolidine-3-carbohydrazide (8a)

Brownish solid, yield 0.25 g, 85%, m. p. 124–125 °C (from 2-propanol: water 1:1). IR (KBr) ν 3217 (NH); 1701; 1664 (2x C = O); 1514 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 1.81‒1.97 (m, 6 H, C(CH₃)₂), 2.57–2.76 (m, 2 H, CH₂CO), 3.44‒3.54, 3.71‒4.02 (2 m, 3 H, CHCO, NCH₂), 7.02–7.21 (m, 1H, H_Ar), 7.26–7.42 (m, 1H, H_Ar), 7.51 (dd, J = 14.6, 6.9 Hz, 1H, H_Ar), 10.19, 10.31 (2s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 17.16, 17.31 (CCH₃), 24.96, 25.23 (CCH₃), 32.94, 33.93. 34.00 (CH₂CO), 34.24, 35.67 (CHCO), 51.40, 51.99 (NCH₂), 104.88, 111.65, 111.87, 122.72, 129.25, 151.29, 156.24, 158.09, 159.41, 161.72 (C_Ar), 168.23 (C = N), 172.20, 173.36 (2x C = O) ppm. Calcd. for C₁₄H₁₅F₂N₃O₂, %: C 56.95; H 5.12; N 14.23; Found, %: C 56.87; H 5.10; N 14.18.

N’-(Butan-2-ylidene)-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (8b)

Orange solid, yield 0.20 g, 65%, m. p. 92–93 °C (from 2-propanol: water 1:1). IR (KBr) ν 3209 (NH); 1713; 1699 (2x C = O); 1513 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 0.93‒1.07 (m, 3 H, CH₂CH₃), 1.78‒1.95 (m, 3 H, CCH₃), 2.16‒2.34 (m, 2 H, CH₂CH₃), 2.57–2.79 (m, 2 H, CH₂CO), 3.45‒3.55, 3.72‒4.05 (2 m, 3 H, CHCO, NCH₂), 7.02–7.20 (m, 1H, H_Ar), 7.27–7.43 (m, 1H, H_Ar), 7.51 (dd, J = 15.2, 7.9 Hz, 1H, H_Ar), 10.17, 10.34 (2s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 9.76, 10.33, 10.76 (CH₂CH₃), 16.04, 16.08 (CCH₃), 22.16, 22.54, 22.99, 23.44 (CH₂CH₃), 31.43, 31.54, 32.87, 32.94, 33.97 (CH₂CO), 34.24, 35.65, 35.72 (CHCO), 51.32, 51.34, 51.98 (NCH₂), 104.63, 104.88, 105.14, 111.65, 111.87, 122.60, 122.64, 122.72, 129.18, 129.21, 129.24, 129.31, 154.57, 155.39, 155.59, 155.72, 158.09, 158.22, 159.72, 160.03, 161.72, 161.83 (C_Ar), 168.31 (C = N), 172.29, 173.46 (2x C = O) ppm. Calcd. for C₁₅H₁₇F₂N₃O₂, %: C 58.25; H 5.54; N 13.59; Found, %: C 58.12; H 5.49; N 13.55.

N’-[1-(4-Aminophenyl)ethylidene]-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (8c)

A mixture of hydrazide 6 (0.2 g, 0.78 mmol), 4’-aminoacetophenone (0.11 g, 0.82 mmol) and 3 drops of acetic acid in 2-propanol (5 mL) was heated at reflux for 5 h, later the reaction mixture was cooled and formed solid was filtered off, washed with hot hexane and dried to give the title compound 8c.

Yellowish solid, yield 0.16 g, 56%, m. p. 191–192 °C (from 1,4-dioxane). IR (KBr) ν 3498; 3388 (NH₂, NH); 1691; 1670 (2x C = O); 1513 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ (Z/E 65/35): 2.16 (d, J = 8.4 Hz, 3 H, CCH₃), 2.64–2.84 (m, 2 H, CH₂CO), 3.53‒3.64, 3.79‒4.21 (2 m, 3 H, CHCO, NCH₂), 5.38‒5.51 (m, 2 H, CNH₂), 6.55 (d, J = 8.3 Hz, 2 H, H_Ar), 7.05–7.22 (m, 1H, H_Ar), 7.38 (dd, J = 17.1, 8.2 Hz, 1H, H_Ar), 7.43–7.63 (m, 3 H, H_Ar), 10.34, 10.49 (2s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 13.26, 13.79 (CCH₃), 33.02, 34.05 (CH₂CO), 34.33, 35.88 (CHCO), 51.50, 52.07 (NCH₂), 104.64, 104.90, 105.15, 111.62, 111.65, 111.84, 111.88, 113.12, 113.26, 122.62, 122.66, 122.73, 122.77, 125.04, 125.20, 127.23, 127.61, 129.22, 129.25, 129.32, 129.34, 149.08, 150.05, 150.28, 153.68, 155.62, 155.73, 158.10, 158.25, 159.27, 159.38, 161.71, 161.83 (C_Ar), 168.35 (C = N), 172.35, 173.63 (2x C = O) ppm. Calcd. for C₁₉H₁₈F₂N₄O₂, %: C 61.28; H 4.87; N 15.05; Found, %: C 61.04; H 4.86; N 14.98.

N’-(4-Bromobenzylidene)-1-(2,4-difluorophenyl)-N-ethyl-5-oxopyrrolidine-3-carbohydrazide (10)

Compound 9e (0.25 g, 0.6 mmol) was dissolved in dimethylformamide (2 mL). Pottasium hydroxide (0.10 g, 1.8 mmol) and pottasium carbonate (0.25 g, 1.8 mmol) were added and then the mixture was stirred at 30 °C. After 15 min, ethyl iodide (0.34 g, 2.2 mmol) was added dropwise, and the reaction was carried out at the room temperature for 1 h. The volatile fractions were separated under reduced pressure and then obtained residue was diluted with distilled water (10 mL). After cooling, the precipitated crystals were filtered. The desired product 10 was purified by recrystallization from 1,4-dioxane: water (2:1).

White solid, yield 0.14 g, 54%, m. p. 182–183 °C. IR (KBr) ν 1698; 1673 (2x C = O); 1515 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ 1.09 (t, J = 6.9 Hz, 3 H, CH₂CH₃), 2.75 (d, J = 8.4 Hz, 2 H, CH₂CO), 3.80‒4.39 (3 m, 5 H, CH₂CH₃, CHCO, NCH₂), 7.05–7.21 (m, 1H, H_Ar), 7.27–7.42 (m, 1H, H_Ar), 7.52 (dd, J = 15.2, 8.5 Hz, 1H, H_Ar), 7.65 (d, J = 8.0 Hz, 2 H, H_Ar), 7.75 (d, J = 8.1 Hz, 2 H, H_Ar), 8.08 (s, 1H, NNCH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 10.84 (CH₂CH₃), 33.45 (CH₂CO), 34.52 (CH₂CH₃), 35.30 (CHCO), 51.63 (NCH₂), 104.65, 104.92, 105.16, 111.63, 111.88, 122.92, 128.95, 129.23, 131.81, 134.10, 139.33 (C_Ar), 172.11, 172.60 (2x C = O) ppm. Calcd. for C₂₀H₁₈F₂N₃O₂, %: C 53.25; H 4.05; N 9.33; Found, %: C 53.26; H 4.03; N 9.28.

1-(2,4-Difluorophenyl)-N-(2,5-dimethyl-1 H-pyrrol-1-yl)-5-oxopyrrolidine-3-carboxamide (11)

A mixture of hydrazide 6 (0.50 g, 2 mmol), hexane-2,5-dione (0.42 mL, 3.6 mmol), 2-propanol (9 mL) and glacial acetic acid (3 drops) was refluxed for 2 h, then cooled. The formed precipitate was filtered off, recrystalized from 2-propanol to give the title compound 11.

Yellowish solid, yield 0.45 g, 68%, m. p. 180–181 °C. IR (KBr) ν 3313 (NH); 1711; 1682 (2x C = O) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ 1.91–2.11 (m, 6 H, 2x CH₃), 2.65–2.86 (m, 2 H, CH₂CO), 3.47‒3.60 (m, 1H, CHCO), 3.84–3.92, 3.98–4.09 (2 m, 2 H, NCH₂), 5.65 (s, 2 H, 2x NCCH), 7.16 (t, J = 8.3 Hz, 1H, H_Ar), 7.39 (t, J = 10.0 Hz, 1H, H_Ar), 7.52 (dd, J = 15.4, 8.0 Hz, 1H, H_Ar), 10.89 (s, 1H, CONH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 10.88, 10.94 (2x CCH₃), 33.57 (CH₂CO), 35.26 (CHCO), 51.60, 51.62 (NCH₂), 103.10, 104.67, 104.92, 104.94, 105.18, 111.69, 111.73, 111.92, 111.95, 122.42, 122.46, 122.55, 122.58, 126.73, 126.76, 129.27, 129.30, 129.37, 129.40, 155.65, 155.78, 158.15, 158.28, 159.37, 159.48, 161.82, 161.93 (C_Ar), 171.58, 171.86 (2x C = O) ppm. Calcd. for C₁₇H₁₇F₂N₃O₂, %: C 61.26; H 5.14; N 12.61; Found, %: C 61.17; H 5.12; N 12.55.

1-(2,4-Difluorophenyl)-4-(3,5-dimethyl-1 H-pyrazole-1-carbonyl)pyrrolidin-2-one (12)

A mixture of hydrazide 6 (0.40 g, 1.6 mmol), 2,4-pentanedione (0.25 mL, 2.4 mmol), 2-propanol (7 mL) and hydrochloric acid (3 drops) was heated at reflux for 2 h, then cooled. The formed precipitate was filtered off, recrystalized from 2-propanol, dried to give the title compound 12.

Orange solid, yield 0.20 g, 39%, m. p. 110–111 °C. IR (KBr) ν 1724; 1699 (2x C = O); 1517 (C = N) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ 2.19 (s, 3 H, CCH₃), 2.50 (s, 3 H, CCH₃ overlaps with DMSO–d₆), 2.81 (d, J = 8.2 Hz, 2 H, CH₂CO), 3.88‒3.96, 4.05‒4.17 (2 m, 2 H, NCH₂), 4.47‒4.58 (m, 1H, CHCO), 6.23 (s, 1H, NCCH), 7.07–7.19 (m, 1H, H_Ar), 7.29–7.43 (m, 1H, H_Ar), 7.53 (dd, J = 15.5, 8.1 Hz, 1H, H_Ar) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 13.11, 13.62 (2x CCH₃), 32.81 (CH₂CO), 36.27 (CHCO), 50.89 (NCH₂), 104.22, 104.46, 104.73, 111.15, 111.22, 111.44, 111.48, 121.96, 122.00, 122.09, 122.12, 128.81, 128.84, 128.91, 128.94, 143.45, 151.73, 155.20, 155.33, 157.70, 157.83, 158.93, 159.05, 161.38, 161.50 (C_Ar), 171.21, 171.93 (2x C = O) ppm. Calcd. for C₁₆H₁₅F₂N₃O₂, %: C 60.18; H 4.74; N 13.16; Found, %: C 60.11; H 4.72; N 13.13.

General method of the Preparation of carbo(thio)amides 13, 15

Hydrazide 6 (0.50 g, 2 mmol) is dissolved in methanol (7 mL), then corresponding reagent is added dropwise – phenyl isocyanate (0.67 g, 5.6 mmol) to obtain compound 13, phenyl isothiocyanate (0.27 g, 2 mmol) in order to synthesize compound 15. Reactions were heated at reflux for 3 h, respectively. Reaction mixture was dissolved with water: methanol (1:1), the formed precipitate was filtered off, recrystalized from 1,4-dioxane and dried to give the title compounds 13 and 15.

2-[1-(2,4-Difluorophenyl)-5-oxopyrrolidine-3-carbonyl]-N-phenylhydrazine-1-carboxamide (13)

White solid, yield 0.61 g, 81%, m. p. 174–175 °C. IR (KBr) ν 3319; 3269; 3146 (3x NH); 1724; 1682; 1663 (3x C = O) cm^–1. ¹H NMR (400 MHz, DMSO–d₆) δ 2.58–2.78 (m, 2 H, CH₂CO), 3.34‒3.44 (m, 1H, CHCO), 3.77–3.99 (m, 2 H, NCH₂), 6.87-7.00 (m, 1H, H_Ar), 7.09–7.19 (m, 1H, H_Ar), 7.20–7.29 (m, 2 H, H_Ar), 7.33–7.55 (m, 4 H, H_Ar), 8.11, 8.78, 9.92 (3s, 3 H, 3x NH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 33.66 (CH₂CO), 35.02 (CHCO), 51.58 (NCH₂), 104.67, 104.94, 105.18, 111.67, 111.90, 118.51, 121.97, 122.68, 128.68, 129.18, 129.28, 139.56, 155.25, 155.59, 155.71, 158.09, 158.22, 159.30, 159.42, 161.75, 161.87 (C_Ar), 172.01, 172.22 (3x C = O) ppm. Calcd. for C₁₈H₁₆F₂N₄O₃, %: C 57.75; H 4.31; N 14.97; Found, %: C 57.60; H 4.25; N 14.92.

2-[1-(2,4-Difluorophenyl)-5-oxopyrrolidine-3-carbonyl]-N-phenylhydrazine-1-carbothioamide (15)

White solid, yield 0.48 g, 61%, m. p. 110–111 °C. IR (KBr) ν 3568; 3271; 3169 (3x NH); 1696; 1668 (2x C = O); 1209 (C = S) cm^− 1. ¹H NMR (400 MHz, DMSO–d₆) δ 2.62–2.79 (m, 2 H, CH₂CO), 3.34‒3.46 (m, 1H, CHCO), 3.80–4.00 (m, 2 H, NCH₂), 7.10–7.22 (m, 2 H, H_Ar), 7.27–7.58 (m, 6 H, H_Ar), 9.58, 9.85, 10.18 (3s, 3 H, C = ONH, NHC = S, NHC) ppm.

¹³C NMR (101 MHz, DMSO–d₆) δ 33.63 (CH₂CO), 35.23 (CHCO), 51.50 (NCH₂), 104.95, 105.19, 111.73, 111.95, 116.79, 122.57, 122.70, 125.26, 125.31, 125.37, 125.42, 125.69, 125.64, 126.31, 126.38, 126.63, 128.20, 129.17, 129.28, 139.05, 155.58, 155.70, 158.08, 158.20, 159.30, 159.42, 161.75, 161.86, 161.89 (C_Ar), 172.04 (2x C = O), 181.16 (C = S) ppm. Calcd. for C₁₈H₁₆F₂N₄O₂S, %: C 55.38; H 4.13; N 14.35; Found, %: C 55.32; H 4.10; N 14.29.

4-[(2,4-Difluorophenyl)amino]-3-(5-oxo-4-phenyl-4,5-dihydro-1 H-1,2,4-triazol-3-yl)butanoic acid (14)

Compound 13 (0.35 g, 0.9 mmol) was dissolved in 4% NaOH solution (7 mL). Reaction was heated at reflux for 19 h, later sodium hydroxide was neutralized using diluted hydrochloric acid (water: HCl, 1:1) until pH 6 was reached. Formed crystals were filtered, dried and recrystalized using 1,4-dioxane.

Light brown solid, yield 0.16 g, 43%, m. p. 168–169 °C. IR (KBr) ν 3444; 3312 (2x NH); 3074 (OH); 1716; 1692 (2x C = O); 1519 (C = N) cm^− 1. ¹H NMR (400 MHz, DMSO–d₆) δ 2.59–2.78 (m, 2 H, CH₂COOH), 2.79–3.24 (m, 3 H, CNCH overlaps with NHCH₂), 5.60 (s, 1H, CH₂NH), 5.85 (dd, J = 14.8, 9.3 Hz, 1H, H_Ar), 6.39–6.58 (m, 1H, H_Ar), 6.92–7.02 (m, 1H, H_Ar), 7.31–7.53 (m, 5 H, H_Ar), 11.78 (s, 1H, NHC = O), 12.35 (br. s, 1H, CH₂COOH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 33.61 (CH₂COOH), 34.40 (CH₂CH), 45.75 (NHCH₂), 103.15, 103.41, 103.64, 110.20, 110.34, 110.41, 110.44, 127.92, 128.63, 129.38, 132.78, 148.35, 150.91, 151.03, 151.35, 151.45, 151.41 (C_Ar), 173.18 (NHC = O), 176.66 (CH₂COOH) ppm. Calcd. for C₁₈H₁₆F₂N₄O₂, %: C 36.45; H 3.74; N 9.45; Found, %: C 36.34; H 3.70; N 9.39.

4-[(2,4-Difluorophenyl)amino]-3-(4-phenyl-5-thioxo-4,5-dihydro-1 H-1,2,4-triazol-3-yl)butanoic acid (16)

Compound 15 (0.4 g, 1 mmol) was dissolved in 4% NaOH solution (7 mL). Reaction was heated at reflux for 2 h, later sodium hydroxide was neutralized using glacier acetic acid until pH 6 was reached. Formed crystals were filtered, dried and recrystalized using 1,4-dioxane.

Brownish solid, yield 0.28 g, 71%, m. p. 142–143 °C. IR (KBr) ν 3387; 3138 (2x NH); 3094 (OH); 1696 (C = O); 1523 (C = N); 1269 (C = S) cm^− 1. ¹H NMR (400 MHz, DMSO–d₆) δ 2.29–2.40, 2.67–2.77 (2 m, 2 H, CH₂COOH), 3.02–3.25, 3.54–3.94 (2 m, 3 H, CNCH overlaps with NHCH₂), 5.58 (s, 1H, CH₂NH), 6.02 (dd, J = 14.9, 9.3 Hz, 1H, H_Ar), 6.38–6.55 (m, 1H, H_Ar), 6.91–7.01 (m, 1H, H_Ar), 7.30–7.59 (m, 5 H, H_Ar), 11.65 (s, 1H, NHC = S), 12.27 (br. s, 1H, CH₂COOH) ppm. ¹³C NMR (101 MHz, DMSO–d₆) δ 32.67 (CH₂COOH), 34.52 (CH₂CH), 46.35 (NHCH₂), 51.97, 103.19, 103.24, 110.15, 110.19, 110.36, 110.39, 110.48, 110.53, 110.56, 110.61, 128.56, 128.75, 128.77, 128.88, 129.08, 129.35, 132.64, 132.76, 133.98, 148.46, 148.58, 150.88, 150.98, 151.21, 151.32, 151.92, 155.17, 166.90, 168.32 (C_Ar); 174.78 (CH₂COOH); 182.22 (C = S) ppm. Calcd. for C₁₈H₁₆F₂N₄O₂S, %: C 55.38; H 4.13; N 14.35; Found, %: C 55.29; H 4.09; N 14.29.

Biological activity

Cell culturing

The human triple-negative breast cancer MDA-MB-231 and human melanoma cell line A375 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Human foreskin fibroblasts (HF) CRL-4001 were originally obtained from ATCC and kindly provided by Prof. Helder Santos (University of Helsinki, Finland). The primary prostate carcinoma PPC-1 cell line was kindly provided by Prof. Tambet Teesalu (University of Tartu, Estonia). All cell lines were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) with GlutaMAX (Gibco (Carlsbad, CA, USA)), supplemented with 10,000 U/mL penicillin, 10 mg/mL streptomycin (Gibco), and 10% fetal bovine serum (Gibco). Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂. They were used until the passage of 20.

Cytotoxicity

The compound effect on cancer cell viability was established using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich Co., St Louis, MO, USA) assay, as described elsewhere³⁵. Briefly, the cells were seeded in triplicates in 96-well TC-treated flat bottom plates (MDA-MB-231, A375 and PPC-1: 4 × 10³ cells/well; HF: 5 × 10³ cells/well). The cells were treated with 100 µM of tested compounds or anticancer drugs sunitinib (≥95%, Santa Cruz Biotechnology, TX, USA), doxorubicin (> 97%, Merck, Austria) or cisplatin (Pt 64.5% min, Alfa Aesar, MA, USA) after 24 h of incubation. After 72 h, 20 µl of the MTT reagent was added to each well, and plates were incubated for an additional 3 hours in the incubator. Then, the formed formazan crystals were dissolved in DMSO (Sigma-Aldrich Co., St. Louis, MO, USA). The absorbance was determined with a multidetection microplate reader at 570 and 630 nm.

Using the same MTT procedure, the EC₅₀ values of the most active hydrazones 7b, 9a, 9c, 9e, 9f and 10 were established. The compound 2× serial dilutions, ranging from 50 µM to 1.56 µM, were added to the cells in triplicate (technical repetitions). The cells not treated with compounds were used as a negative control (their viability corresponded to 100%), and the positive control was the empty well without cells, representing the situation when all cells were killed (cell viability corresponded to 0%). After measuring the absorbance of the obtained MTT solutions, the half-maximal effective concentration (EC₅₀) values were calculated using the Hill equation. EC₅₀ value was considered as the compound concentration, which resulted in a 50% reduction in the metabolic activity of cells. All experiments were repeated three times independently (biological repetitions).

Wound healing assay

‘Wound healing’ assay was used to evaluate the compound effect on selected cancer cell migration. In this assay, only the most active compounds 7b, 9a, 9c, 9e, and 10 were tested, and the procedure was performed as published elsewhere³⁸. MDA-MB-231 cells were seeded at a density of 1 × 10⁴ cells/well, and PPC-1 and A375 cells were seeded at a density of 2 × 10⁴ cells/well in 24-well plates. After 2–3 days of incubation, once the cells reached 80–90% confluency, the scratch was made with a 100 µL pipette tip in the middle of each well. After washing the cells with PBS, the fresh medium containing 10 µM of compounds was added to the cells. As a negative control, a medium containing 0.1% DMSO was used. Photos of the ‘wound’ were made at the beginning of the experiment (0 h) and after the selected duration of incubation until the cells in control fully migrated into the ‘wound area’: 12, 24 and 36 h for MDA-MB-231 cells, 24 and 48 h for PPC-1 and A375 cells.

Effect in 3D cell cultures

The magnetic 3D Bioprinting method was used to form cancer cell spheroids as described elsewhere³⁹. Briefly, the cells in a 6-well plate at 70% confluency were incubated with Nanoshuttle (n3D Biosciences, Inc., Houston, TX, USA) for 8–10 h. Then, the cells were trypsinized, centrifuged and seeded into the ultra-low attachment 96-well plate at a ratio of 1.5 × 10³ cancer cells and 1.5 × 10³ human fibroblasts/well. The plate was incubated for two days at 37 °C in a humidified atmosphere on a magnetic drive until the spheroids formed. Next, the medium with 10 µM of selected compounds 7b, 9e, 9f, 10, and doxorubicin hydrochloride (Abcam, Cambridge, UK) was added. Photos of spheroids were taken every two days up to 8 days using an Olympus IX73 inverted microscope (OLYMPUS CORPORATION, Tokyo, Japan), and analysis of spheroid size was performed using ImageJ, version 1.53o (National Institutes of Health, USA) and Microsoft Office Excel 2016 software (Microsoft Corporation, Redmond, WA, USA).

On the last day of the experiment, 20 µL of MTT reagent was added to each well. Following a 10-hour incubation, the medium was aspirated, and the formazan crystals formed were dissolved in 100 µL of DMSO overnight. The absorbance was determined with a multidetector microplate reader at 570 and 630 nm.

Statistical analysis

All biological experiments were repeated at least three times, calculating the mean and standard deviation. The data were processed using Microsoft Office Excel 2016 software (Microsoft Corporation, Redmond, WA, USA). Statistical analysis was performed by using Student’s t-test. The level of significance was set as p < 0.05.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary Material 1^{(3.6MB, docx)}

Author contributions

Author Contributions: Conceptualization, V.M. and V.P.; methodology, V.M., and V.P.; formal analysis, G.P., B.G., U.E., and V.P.; investigation, G.P., B.G., U.E., and V.P.; resources, V.M. and V.P.; writing—original draft preparation, G.P., B.G., and V.P.; writing—review and editing, B.G., and V.P.; visualization, B.G. and V.P.; supervision, V.M. and V.P.; funding acquisition, V.M. and V.P. All authors have read and agreed to the published version of the manuscript.

Data availability

Data is provided within the manuscript or supplementary information files.

Declarations

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Contributor Information

Vytautas Mickevičius, Email: vytautas.mickevicius@ktu.lt.

Vilma Petrikaitė, Email: vilma.petrikaite@lsmuni.lt.

References

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Material 1^{(3.6MB, docx)}

Data Availability Statement

Data is provided within the manuscript or supplementary information files.

[CR1] 1.Cancer Available online: https://www.who.int/news-room/fact-sheets/detail/cancer (accessed on 23 July 2024).

[CR2] 2.Ruf, S. et al. Novel β-Amino acid derivatives as inhibitors of cathepsin A. J. Med. Chem.55, 7636–7649. 10.1021/jm300663n (2012). [DOI] [PubMed] [Google Scholar]

[CR3] 3.Rehman, M., Mahboob Ahmad, F. U. & Naveed Riaz, M. ??-Amino acids: role in human biology and medicinal Chemistry - A review. Med. Chem.710.4172/2161-0444.1000472 (2017).

[CR4] 4.Uesugi, S. et al. Pyrrocidine A, a metabolite of endophytic fungi, has a potent Apoptosis-Inducing activity against HL60 cells through caspase activation via the Michael addition. J. Antibiot.69, 133–140. 10.1038/ja.2015.103 (2016). [DOI] [PubMed] [Google Scholar]

[CR5] 5.Jeličić, M. L., Kovačić, J., Cvetnić, M. & Mornar, A. Amidžić klarić, D. Antioxidant activity of pharmaceuticals: predictive QSAR modeling for potential therapeutic strategy. Pharmaceuticals15, 791. 10.3390/ph15070791 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR6] 6.Afzal, O. et al. Analgesic and anticancer activity of Benzoxazole clubbed 2-Pyrrolidinones as novel inhibitors of monoacylglycerol lipase. Molecules26, 2389. 10.3390/molecules26082389 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR7] 7.Muneer, S., Memon, S., Pahnwar, Q. K., Bhatti, A. A. & Khokhar, T. S. Synthesis and investigation of antimicrobial properties of pyrrolidine appended Calix[4]Arene. J. Anal. Sci. Technol.8 (3). 10.1186/s40543-017-0111-3 (2017).

[CR8] 8.Khalifa, M. E. et al. Synthesis, antibacterial, and anti HepG2 cell line human hepatocyte carcinoma activity of some new potentially Benzimidazole-5-(Aryldiazenyl)Thiazole derivatives. Molecules23 (3285). 10.3390/molecules23123285 (2018). [DOI] [PMC free article] [PubMed]

[CR9] 9.Rollas, S. & Küçükgüzel, S. G. Biological activities of hydrazone derivatives. Molecules12, 1910–1939. 10.3390/12081910 (2007). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR10] 10.Işik, A. et al. Benzimidazole-Hydrazone derivatives: synthesis, in vitro anticancer, antimicrobial, antioxidant activities, in Silico DFT and ADMET studies. J. Mol. Struct.127010.1016/j.molstruc.2022.133946 (2022).

[CR11] 11.Xing, A. et al. Anticancer and antioxidant activities of novel heterocyclic phenolic hydrazone based derivatives: investigation of DFT calculation, molecular Docking and Drug-Likeness studies. J. Mol. Struct.1319, 139523. 10.1016/j.molstruc.2024.139523 (2025). [Google Scholar]

[CR12] 12.Chandra, G., Singh, D. V., Mahato, G. K. & Patel, S. Fluorine-a small magic bullet atom in the drug development: perspective to FDA approved and COVID-19 recommended drugs. Chem. Pap. 77, 4085–4106. 10.1007/s11696-023-02804-5 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR13] 13.Liu, H. N. et al. Synthetic approaches and application of representative clinically approved Fluorine-Enriched Anti-Cancer medications. Eur. J. Med. Chem.276, 116722. 10.1016/j.ejmech.2024.116722 (2024). [DOI] [PubMed] [Google Scholar]

[CR14] 14.Afifi, N. & Barrero, C. A. Understanding breast Cancer aggressiveness and its implications in diagnosis and treatment. JCM12 (1375). 10.3390/jcm12041375 (2023). [DOI] [PMC free article] [PubMed]

[CR15] 15.Bray, F. et al. Global Cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J. Clin.68, 394–424. 10.3322/caac.21492 (2018). [DOI] [PubMed] [Google Scholar]

[CR16] 16.Brothman, A. R., Wilkins, P. C., Sales, E. W. & Somers, K. D. Metastatic properties of the human prostatic cell line, PPC-1, in athymic nude mice. J. Urol.145, 1088–1091. 10.1016/S0022-5347(17)38540-3 (1991). [DOI] [PubMed] [Google Scholar]

[CR17] 17.International Agency for Research on Cancer. Cancer Tomorrow Estimated Number of New Cases from 2020 to 2040 of Melanoma of Skin and Non-Melanoma Skin Cancer. Available online: https://gco.iarc.who.int/today/ (accessed on 25 July 2024).

[CR18] 18.Lopes, J., Rodrigues, C. M. P., Gaspar, M. M. & Reis, C. P. Melanoma management: from epidemiology to treatment and latest advances. Cancers14, 4652. 10.3390/cancers14194652 (2022). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR19] 19.Treating Melanoma Skin Cancer | How Is Melanoma Treated?. Available online: July (2024). https://www.cancer.org/cancer/types/melanoma-skin-cancer/treating.html (accessed on 26.

[CR20] 20.Bouchereau, S. et al. Impact of prior treatment with immune checkpoint inhibitors on Dacarbazine efficacy in metastatic melanoma. Br. J. Cancer. 125, 948–954. 10.1038/s41416-021-01486-8 (2021). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR21] 21.Rossi, S. et al. TNF-Alpha and metalloproteases as key players in melanoma cells aggressiveness. J. Exp. Clin. Cancer Res.37, 326. 10.1186/s13046-018-0982-1 (2018). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR22] 22.Ghasemi, M., Turnbull, T., Sebastian, S. & Kempson, I. The MTT assay: utility, limitations, pitfalls, and interpretation in bulk and Single-Cell analysis. IJMS22 (12827). 10.3390/ijms222312827 (2021). [DOI] [PMC free article] [PubMed]

[CR23] 23.Balandis, B. et al. Synthesis and Structure–Affinity relationship of chlorinated Pyrrolidinone-Bearing benzenesulfonamides as human carbonic anhydrase inhibitors. Bioorg. Chem.97, 103658. 10.1016/j.bioorg.2020.103658 (2020). [DOI] [PubMed] [Google Scholar]

[CR24] 24.SUNITINIB. A Comprehensive Guide for Patients Available online: April (2025). https://clinicaltrials.eu/inn/sunitinib/ (accessed on 22.

[CR25] 25.Brown, A., Kumar, S. & Tchounwou, P. B. Cisplatin-Based chemotherapy of human cancers. J. Cancer Sci. Ther.11, 97 (2019). [PMC free article] [PubMed] [Google Scholar]

[CR26] 26.Kciuk, M. et al. Doxorubicin—An agent with multiple mechanisms of anticancer activity. Cells12, 659. 10.3390/cells12040659 (2023). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR27] 27.Bedia, C., Casas, J., Andrieu-Abadie, N., Fabriàs, G. & Levade, T. Acid ceramidase expression modulates the sensitivity of A375 melanoma cells to Dacarbazine. J. Biol. Chem.286, 28200–28209. 10.1074/jbc.M110.216382 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR28] 28.Piskorz, W. M., Krętowski, R., Cechowska-Pasko, M. & Marizomib Salinosporamide A) promotes apoptosis in A375 and G361 melanoma Cancer cells. Mar. Drugs. 22, 315. 10.3390/md22070315 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR29] 29.Cordaro, F. G. et al. Phenotype characterization of human melanoma cells resistant to Dabrafenib. Oncol. Rep.38, 2741–2751. 10.3892/or.2017.5963 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR30] 30.Januškevičienė, I. & Petrikaitė, V. Heterogeneity of breast cancer: the importance of interaction between different tumor cell populations. Life Sci.239, 117009. 10.1016/j.lfs.2019.117009 (2019). [DOI] [PubMed] [Google Scholar]

[CR31] 31.Januškevičienė, I. & Petrikaitė, V. Interaction of phenotypic sublines isolated from Triple-Negative breast Cancer cell line MDA-MB-231 modulates their sensitivity to Paclitaxel and doxorubicin in 2D and 3D assays. Am. J. Cancer Res.13, 3368–3383 (2023). [PMC free article] [PubMed] [Google Scholar]

[CR32] 32.Bashi, M., Madanchi, H. & Yousefi, B. Investigation of cytotoxic effect and action mechanism of a synthetic peptide derivative of rabbit Cathelicidin against MDA-MB-231 breast Cancer cell line. Sci. Rep.14, 13497. 10.1038/s41598-024-64400-1 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR33] 33.Riahi, R., Yang, Y., Zhang, D. D. & Wong, P. K. Advances in Wound-Healing assays for probing collective cell migration. SLAS Technol.17, 59–65. 10.1177/2211068211426550 (2012). [DOI] [PubMed] [Google Scholar]

[CR34] 34.Daunys, S., Matulis, D. & Petrikaitė, V. Synergistic activity of Hsp90 inhibitors and anticancer agents in pancreatic Cancer cell cultures. Sci. Rep.9, 16177. 10.1038/s41598-019-52652-1 (2019). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR35] 35.Bytautaite, M. & Petrikaite, V. Comparative study of lipophilic Statin activity in 2D and 3D in vitro models of human breast Cancer cell lines MDA-MB-231 and MCF-7. Onco Targets Ther.13, 13201–13209. 10.2147/OTT.S283033 (2020). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR36] 36.Li, L. & Lu, Y. Optimizing a 3D culture system to study the interaction between epithelial breast Cancer and its surrounding fibroblasts. J. Cancer. 2, 458–466. 10.7150/jca.2.458 (2011). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR37] 37.Mittler, F. et al. High-Content monitoring of drug effects in a 3D spheroid model. Front. Oncol.7, 293. 10.3389/fonc.2017.00293 (2017). [DOI] [PMC free article] [PubMed] [Google Scholar]

[CR38] 38.Skaraitė, I., Maccioni, E. & Petrikaitė, V. Anticancer activity of Sunitinib analogues in human pancreatic Cancer cell cultures under normoxia and hypoxia. IJMS24 (5422). 10.3390/ijms24065422 (2023). [DOI] [PMC free article] [PubMed]

[CR39] 39.Šedbarė, R., Janulis, V., Pavilonis, A. & Petrikaite, V. Comparative in vitro study: assessing phytochemical, antioxidant, antimicrobial, and anticancer properties of Vaccinium Macrocarpon Aiton and Vaccinium Oxycoccos L. Fruit extracts. Pharmaceutics16, 735. 10.3390/pharmaceutics16060735 (2024). [DOI] [PMC free article] [PubMed] [Google Scholar]

PERMALINK

Exploration of 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carboxylic acid derivatives effect on triple-negative breast, prostate cancer and melanoma cell 2D and 3D cultures

Guoda Pranaitytė

Birutė Grybaitė

Ugne Endriulaityte

Vytautas Mickevičius

Vilma Petrikaitė

Abstract

Supplementary Information

Introduction

Results and discussion

Chemistry

Scheme 1.

Scheme 2.

Scheme 3.

Biological activity

Cytotoxicity

Fig. 1.

Fig. 2.

Table 1.

Effect on cell migration

Fig. 3.

Effect in 3D cultures

Fig. 4.

Conclusions

Materials and methods

Chemistry

1-(2,4-Difluorophenyl)-5-oxopyrrolidine-3-carboxylic acid (2)

General procedure for the Preparation of benzimidazoles 3a−c

4-(1 H-benzo[d]imidazol-2-yl)-1-(2,4-difluorophenyl)pyrrolidin-2-one (3a)

1-(2,4-Difluorophenyl)-4-(6-fluoro-1 H-benzo[d]imidazol-2-yl)pyrrolidin-2-one (3b)

4-(6-Chloro-1 H-benzo[d]imidazol-2-yl)-1-(2,4-difluorophenyl)pyrrolidin-2-one (3c)

1-(2,4-Difluorophenyl)-4-(1-ethyl-1 H-benzo[d]imidazol-2-yl)pyrrolidin-2-one (4)

Methyl 1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carboxylate (5)

1-(2,4-Difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (6)

General procedure for the Preparation of hydrazones 7a, B and 9a–h

1-(2,4-Difluorophenyl)-5-oxo-N’-(thiophen-2-ylmethylene)pyrrolidine-3-carbohydrazide (7a)

1-(2,4-Difluorophenyl)-N’-((5-nitrothiophen-2-yl)methylene)-5-oxopyrrolidine-3-carbohydrazide (7b)

N’-Benzylidene-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (9a)

1-(2,4-Difluorophenyl)-N’-(4-fluorobenzylidene)-5-oxopyrrolidine-3-carbohydrazide (9b)

N’-(4-Chlorobenzylidene)-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (9c)

N’-(2,4-Difluorobenzylidene)-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (9d)

N’-(4-Bromobenzylidene)-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (9e)

1-(2,4-Difluorophenyl)-N’-(4-methylbenzylidene)-5-oxopyrrolidine-3-carbohydrazide (9f)

1-(2,4-Difluorophenyl)-N’-(4-(dimethylamino)benzylidene)-5-oxopyrrolidine-3-carbohydrazide (9 g)

1-(2,4-Difluorophenyl)-N’-(4-nitrobenzylidene)-5-oxopyrrolidine-3-carbohydrazide (9 h)

General method of the Preparation of hydrazones 8a, B

1-(2,4-Difluorophenyl)-5-oxo-N’-(propan-2-ylidene)pyrrolidine-3-carbohydrazide (8a)

N’-(Butan-2-ylidene)-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (8b)

N’-[1-(4-Aminophenyl)ethylidene]-1-(2,4-difluorophenyl)-5-oxopyrrolidine-3-carbohydrazide (8c)

N’-(4-Bromobenzylidene)-1-(2,4-difluorophenyl)-N-ethyl-5-oxopyrrolidine-3-carbohydrazide (10)

1-(2,4-Difluorophenyl)-N-(2,5-dimethyl-1 H-pyrrol-1-yl)-5-oxopyrrolidine-3-carboxamide (11)

1-(2,4-Difluorophenyl)-4-(3,5-dimethyl-1 H-pyrazole-1-carbonyl)pyrrolidin-2-one (12)

General method of the Preparation of carbo(thio)amides 13, 15

2-[1-(2,4-Difluorophenyl)-5-oxopyrrolidine-3-carbonyl]-N-phenylhydrazine-1-carboxamide (13)

2-[1-(2,4-Difluorophenyl)-5-oxopyrrolidine-3-carbonyl]-N-phenylhydrazine-1-carbothioamide (15)

4-[(2,4-Difluorophenyl)amino]-3-(5-oxo-4-phenyl-4,5-dihydro-1 H-1,2,4-triazol-3-yl)butanoic acid (14)

4-[(2,4-Difluorophenyl)amino]-3-(4-phenyl-5-thioxo-4,5-dihydro-1 H-1,2,4-triazol-3-yl)butanoic acid (16)

Biological activity

Cell culturing

Cytotoxicity

Wound healing assay

Effect in 3D cell cultures

Statistical analysis

Electronic supplementary material

Author contributions

Data availability

Declarations

Competing interests

Footnotes

Contributor Information

References

Associated Data

Supplementary Materials

Data Availability Statement

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