Introduction

Organophosphorus compounds belong to one of the most essential classes of bioactive chemicals and are extensively distributed in nature. The majority of approved phosphorus-containing pharmaceuticals include a phosphate, a phosphoramide or a phosphonate group, while phosphine oxides are relatively rare1,2,3,4. Tertiary phosphines containing a phosphorus atom binding directly to three carbon atoms feature rendering a more hydrophilic surface and having better chemical stability compared to phosphates. Despite these advantages, the applications of phosphine oxides have been largely neglected in medicinal chemistry5,6. The situation changed in 2016, when Huang and coworkers reported the discovery of Brigatinib, a potent, orally active inhibitor of anaplastic lymphoma kinase (Fig. 1)6. Since that time, the phosphine oxide moiety has gained recognition as a valuable functional group in drug development. It acts as a strong hydrogen bond acceptor, enhances solubility in water, reduces lipophilicity, decreases protein binding, and enhances metabolic stability7,8.

Fig. 1
figure 1

Pharmacologically active phosphinanes and first FDA-approved phosphorus-based drug.

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Six-membered heterocycles, such as piperidines, oxanes, and thianes, are common in biologically active compounds found in both natural and synthetic substances, including various frontline drugs. Among them, piperidines are of great interest due to their broad spectrum of biological activities, which is reflected in the number of pharmacological studies covering almost one-third of known compounds (Figure S1)9. In contrast, analogical six-membered phosphorus-containing heterocycles (phosphinanes), have been largely neglected in medicinal chemistry. To date, only 1.2% of known phosphinanes have been subjected to pharmacological evaluation.

As research has demonstrated, the largest group of pharmacologically active phosphinanes consists of tertiary phosphines (Fig. 1 and S2)10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37. Available studies suggest that phosphinane derivatives may exhibit a broad spectrum of pharmacological activities, including anticancer, antibacterial, antiviral, and anticonvulsant effects. In the context of anticancer activity, phosphinanes operate through diverse mechanisms, frequently involving the inhibition of overexpressed enzymes that drive cancer progression. Although the inhibition of broad-spectrum enzymes by phosphinanes is documented, these compounds have been the subject of limited biological research, primarily due to the synthetic challenges associated with their preparation and structural modification.

It is of paramount importance to highlight that within this group, two primary structural modification strategies can be identified: alterations of the substituents at the phosphorus atom (Fig. 1a), and modifications at the 4-position of the phosphinane ring (Fig. 1b). Nevertheless, systematic studies investigating the impact of such modifications on biological activity are lacking. To address this gap, the aim of this study was to evaluate how specific structural modifications within the phosphinane scaffold influence biological activity, with a particular focus on pharmacokinetic properties and cytotoxicity profiles.

Accordingly, we designed and synthesized a series of small-molecule phosphinane derivatives (< 300 Da), incorporating targeted modifications at the phosphorus atom, the C4-position, and the chalcogen substituent (Fig. 2).

Fig. 2
figure 2

Functionalization strategies and key structural motifs in phosphinanes 1–15.

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To date, all phosphinane derivatives described in the literature have featured exclusively phosphine oxide moieties. In this context, we considered it important to investigate how replacing the oxygen atom with sulfur, selenium, or borane affects biological activity. Modifications at the C4-position were designed to introduce either a carbonyl or hydroxyl group, functionalities known to act as hydrogen bond acceptors and donors, respectively. In parallel, the substitution pattern at the phosphorus atom was varied by replacing a phenyl group with sterically bulky aliphatic moieties, facilitated by a adapted dearomatization strategy of phenyl ring. Considering the nature of the introduced modifications, the tested compounds can be classified into three series: phosphinanes, phosphinan-4-ones and phosphinan-4-ols (Fig. 2).

A detailed investigation of their structure–activity relationships was carried out, along with an initial assessment of pharmacokinetic and toxicity-related properties. The results presented herein are expected to provide valuable insights into the potential of phosphinanes as scaffolds in medicinal chemistry.

Results and discussion

Chemistry

The dearomatization of the phenyl group attached to the phosphorus atom in 1-phenylphosphinane 1-oxide represents a pivotal step in the phosphinane P-substituent modification. A series of novel phosphinane derivatives with an alkyl substituent at the phosphorus atom (2–6) was synthesized by dearomatyzation of the phenyl ring in 1-phenylphosphinane 1-oxide (1). The selective formation of these derivatives was achieved by varying the reduction conditions (Fig. 3). In particular, 1-cyclohexylphosphinane 1-oxide (2) was obtained selectively through a modified Bouveault–Blanc-type reduction, in which sodium was used as the reducing agent in iso-propanol/THF solvent system38.

Fig. 3
figure 3

Synthetic strategy for 1-alkyl phosphinane derivatives 2–6 and 1-phenylphosphinanes 7 and 8. Reagents and conditions: i) 1) Na, i-PrOH, THF 70 °C, 24 h, 2) Pd/C, H2 (1 atm), EtOAc, r.t., 18 h, (46%); ii) 1) Na, NH3, THF, − 40 °C, 0.5 h, 2) MeI, − 40 °C, 0.5 h, (76%); iii) Pd/C, H2 (1 atm), EtOAc, 50 °C, 18 h, (92%); iv) 1) PhSiH3, toluene, 70 °C, 18 h, 2) S8 or BH3·THF, (73% or 60%); v) 1) PhSiH3, toluene, 70 °C, 18 h, 2) S8 or BH3·THF, (69% or 52%).

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The Birch reduction, a method that converts arenes into 1,4-cyclohexadienes using alkali metals in liquid ammonia, achieves reductive dearomatization via forming a cyclohexadienyl carbanion. This intermediate can be alkylated with primary alkyl halides, resulting in the synthesis of novel organophosphorus compounds featuring bulky cyclohexadienyl P-substituents39,40. Application of this method to 1-phenylphosphinane oxide (1) followed by treatment with methyl iodide, resulted in the formation of α-functionalized 1-(1-methylcyclohexa-2,5-dien-1-yl)phosphinane 1-oxide (3) in excellent yield. Subsequent hydrogenation of the double bonds in compound 3 afforded compound 4, bearing a sterically hindered alkyl substituent at the phosphorus atom.

The phosphine–borane and phosphine–sulfide moieties possess unique structural and electronic features that may influence biological activit41,42. For example, replacing oxygen with either BH3 or S leads to a change in the electron density, hydrogen-bond acceptor and donor properties, and bond length. Therefore, evaluating the impact of P=O, P=S, and P → BH3 groups, which share a common structural core, was considered essential to better understand their potential in biological applications. Accordingly, phosphinane oxides 1 and 2 were converted into the corresponding sulfides (5 and 7) and boranes (6 and 8). The reduction of the P=O bond in phosphine oxides 1 and 2 can be effectively achieved through a reaction with phenylsilane. The free phosphines obtained in this manner were subsequently converted to the corresponding sulfides or boranes by the addition of an excess of either elemental sulfur or the borane-THF complex. This approach yields 1-phenylphosphinane 1-sulfide (7) or borane 8, as well as 1-cyclohexylphosphinane 1-sulfide (5) or borane 6, in a good to quantitative yields, as illustrated in Fig. 3.

In consideration of the documented variations in phosphinane structures (see Figs. 1 and S2), it can be observed that the incorporation of various substituents in the C4-position of the six-membered ring has been shown to significantly affect both their chemical properties and potential biological applications. Previous studies have primarily focused on the introduction of amino or alkyl groups at this position (Fig. 1). Therefore, in this work, we evaluated the impact of incorporating carbonyl and hydroxyl functionalities at this position of the phosphinane ring. Furthermore, since all C4-substituted phosphinane derivatives investigated to date in biological contexts have been phosphine oxides, it was also considered essential to assess the role of the P=X moiety in 1-phenylphosphinan-4-one (Fig. 4).

Fig. 4
figure 4

Synthesis of a series of chalcogenides 10–12 and borane complex of phosphinan-4-one 13 from free phosphine 9. Reagents and conditions: i) H2O2, MeOH, r.t. (89%); ii) S8, MeOH, r.t. (91%); iii) BH3·THF, THF, 0 °C (82%); iv) Se, MeOH, r.t. (76%).

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The synthesis of phosphinan-4-ones (10-12) was undertaken to address the lack of data concerning the relationship between the nature of the phosphorus center and the biological activity of phosphinanes. Compounds 10, 11 and 12 were quantitatively obtained by oxidation of free phosphine 9 using hydrogen peroxide, elemental sulfur, or elemental selenium, respectively. Additionally, complexation of phosphine 9 with BH3·THF was employed to afford the corresponding phosphinan-4-one 1-borane derivative (13).

The reduction of the carbonyl group in phosphinan-4-ones offers a route to access the corresponding secondary alcohols. The presence of a tetrahedral phosphorus atom with a P-phenyl substituent incorporated into a six-membered ring distinguishes phosphinan-4-ones as a type of ketone, in which the two faces of the C=O group are sterically and electronically nonequivalent. As a result, their reduction is expected to yield two stereoisomeric alcohols with different relative configurations. To test this hypothesis, we used phosphinan-4-one 1-oxide 10 as a starting material (Fig. 5).

Fig. 5
figure 5

Stereoselectivity of 1-phenylophosphinan-4-one 1-oxide (10) reduction under different conditions and molecular structure of major isomer trans-14.

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As expected, heterocyclic ketone 10 reacted smoothly with hydride reducing agents to afford the corresponding alcohol. Moreover, phosphinan-4-one exhibited high stereoselectivity under low-temperature conditions, favoring hydride attack from the less hindered face of the molecule and yielding the trans isomer in significant excess over the cis isomer (14.2: 1 isomers ratio), as shown in Fig. 5. The observed isomer ratio can be explained by steric interactions of BH4-, which preferentially approaches the less shielded carbonyl face in the more stable chair conformation of the substrate, in which the phenyl group occupies an axial position. The determined molecular structure of the major isomer of alcohol 14 allowed for assigning the trans relative configuration (Table S1).

An analogical reaction was also performed with borane 13 as a substrate, producing the corresponding alcohols 15 with good yield and selectivity (cis : trans = 1: 9.1) as shown in Fig. 6.

Fig. 6
figure 6

Synthesis of borane complex of phosphinan-4-ol 15 via reduction of corresponding ketone 13.

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Biological studies

Cytotoxic activity

The in vitro cytotoxicity studies of the synthesized compounds 1–15 was evaluated against three human colon cancer cell lines of different malignant potential: SW480 cells of low metastatic ability, fast growing HCT116 line and SW620 cells with high metastatic ability43, as well as against metastatic prostate cancer (PC3) cell lines and human immortal keratinocytes (HaCaT) (Table 1). Chemotherapeutic agents, doxorubicin and cisplatin were used as positive controls.

Table 1 Cytotoxic activity (IC50, µM) of studied compounds estimated by the MTT assaya.
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The SW480 cell cultures, followed by SW620 cells, appeared to be the most sensitive to the presence of applied compounds. Within the new phosphinanes, three derivatives were active at ≤ 10 µM against at least one cancer cell line, being more effective than the standard cisplatin. The most potent phosphinan-4-one derivative 11 inhibited the growth of all tested cell lines at 4.4 – 6.3 µM, with selectivity indexes (SI) above 2.2. The of 1-cyclohexylphosphinane-borane complex (8) exerted high cytotoxicity towards both colon cancer cell lines (SW480, SW620) at nearly the same concentration of 7.2–7.6 µM, with beneficial SI (˃ 13). However, its activity towards HCT116 and PC3 cell lines was weak (at 36.7–49.1 µM). The 1-phenylphosphinane 1-oxide (2) acted selectively against SW480 cell line, with IC50 of 5.1 µM, that was twice as strong as cisplatin. The activity of this derivative was significant also against other colon carcinomas, with IC50 ranged from 13.8 to 16.1 µM. In contrast to susceptible colon cancer cell lines, the PC3 line remained vulnerable only to the presence of derivative 11, at the inhibitory concentration of 6.0 µM. The most favorable drug candidates (2, 8) were not cytotoxic against the HaCaT cell line (IC50 ˃ 100 µM), except from the derivative 11 (IC50 = 14.2 µM), as compared to the highly cytotoxic reference compounds (0.3–6.3 µM). The SI of active phosphinane derivatives ranged from 2.2 to 19.6, being higher than these of the standards (0.4 – 1.5).

Structure–activity relationship

Previous studies have highlighted the phosphinane scaffold as a promising structural motif for biologically active compounds (Fig. 1). In the present study, we focused on small-molecule phosphinane derivatives (< 300 Da) with rationally designed modifications, grouped into three distinct series to enable comparative analysis (Fig. 2).

Series A (compounds 1–8) consists of phosphinanes composed exclusively sp3-hybridized carbon atoms, without any additional ring substituents, and their structure is modified by changing the substituents at the phosphorus atom. In Series B, the carbonyl group was incorporated into the compound structure. The carbon atom of the C=O group adopts sp2 hybridization, forming a planar trigonal system. The electronegative oxygen atom contains lone pairs that could participate as hydrogen acceptors in hydrogen bonding. Additionally, the presence of the carbonyl group also reduces ring conformational flexibility and decreases lipophilicity. In the last examined Series C, the modification at the C4-position was targeted by introducing the hydroxyl group, known to act as a hydrogen bond donor (HBD = 1). This modification results in the formation of two stereoisomers with cis or trans configurations relative to the P-phenyl substituent. In both series B and C, the P-phenyl group was selected as the most promising substituent due to its potential to engage in π–π interactions with aromatic or heteroaromatic residues in protein binding sites. Finally, physicochemical properties such as logP and TPSA were modulated across all series by substituting P=X with l.p., O, S, Se, or BH3. The influence of these modifications on the physicochemical properties of the tested phosphinanes is shown in Table 2. With the exception of compound 6, all tested compounds comply with Lipinski’s Rule of Five.

Table 2 Molecular descriptors (logPa and TPSAb), Lipinski parameters and bioactivity scorec of the studied compounds 1–15.
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The modification of the P-substituent in phosphinane 1-oxide (series A) from aryl to alkyl is associated with an increase in lipophilicity and steric hindrance. Notably, biological activity improved upon replacement of the phenyl group with a cyclohexyl group. However, further increases in steric hindrance, as in compounds 3 and 4 by introducing a (1-methylcyclohexa-2,5-dien-1-yl) or 1-(1-methylcyclohexyl) group did not lead to a further enhancement in activity (Fig. 7).

Fig. 7
figure 7

SAR of cytotoxicity of the studied phosphinanes 1–8 (series A); left: directions of changes of molecular descriptors and properties; right: Cytotoxic activity (IC50, µM) of studied compounds.

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Further attempts to modify the biological activity of the compounds involved changing the nature of the P=X moiety in compounds 1 and 2 by replacing oxygen atom with either sulfur or borane. These modifications led to a marked increase in lipophilicity from 1.8 for (oxide 1) to 3.79 and 4.45, from 2.85 for (oxide 2) to 4.84 and 5.5 for the corresponding sulfides and boranes, respectively. Notably, in the case of P-phenyl phosphinanes, these changes resulted in a significant enhancement of biological activity. It was observed that the increased lipophilicity of the phosphinane–borane complex 8 correlated with effective growth inhibition of SW480 and SW620 cell lines, with an IC50 value of ≤ 10 µM. Moderate activity was also observed against PC3 and HCT116 cells, with IC50 values of 37 µM and 49 µM, respectively.

In the context of phosphine-borane complexes, it is known that the P-B σ-bond reduces the electron density on the phosphorus atom, increasing acidity44. Additionally, unlike oxygen and sulfur, BH3 lacks lone pairs and therefore cannot act as a hydrogen bond acceptor or form strong interactions with metal ions. These properties may influence the way compound 8 interacts with biomolecular targets. In contrast, the same modifications applied to P-cyclohexyl phosphinanes did not lead to improved biological activity. Among this series, 1-cyclohexylphosphinane 1-oxide (2) remained the most active compound across all tested cell lines (Fig. 7).

In series B, the incorporation of a carbonyl group into the compound structure reduced ring conformational flexibility and decreased lipophilicity compared to Series A. Comparing the anticancer activity between these series (A and B), a significant increase in activity of sulfides (7 versus 11) can be observed. These findings indicate that the presence of the carbonyl group plays a key role in enhancing biological activity. This effect may be related to the presence of lone pairs on the carbonyl oxygen, which can participate as hydrogen bond acceptors at enzyme active sites. Additionally, the reduced conformational flexibility and increased planarity of the molecule may improve its ability to bind effectively to biological targets.

Another important issue is the influence of the character of P=X moiety on the activity of phospinan-4-ones in series B. The exchange of chalcogen atom (O, S and Se) at the phosphorus center has only a minor effect on the overall geometry and the van der Waals sphere (Fig. S3). However, despite no changes in geometry, replacing oxygen with sulfur or selenium leads to a change in the electron density distribution and P=X bond length (e.g. P=O 1.489 Å; P = S 1.954 Å; P = Se 2.093 Å)45,46. Changes in the polarity of the P = X bond influence the electronic properties of the molecule compared to its oxygen-containing counterpart. Consequently, this has an impact on the electronic structure descriptors of the molecule, such as molecular orbital energies or electronic charges. Additionally, the small formal electronegativity difference between P and S or Se (0.39 and 0.36, respectively) reduces the acceptor capacity of these groups, making P=S and P=Se considerably weaker acceptors than P=O. Biological evaluation suggests that the modifications to the P=X group are responsible for enabling the molecule to adapt to the target binding site and, as a result, inhibit the enzymatic processes that would otherwise proceed.

To better understand this relationship, key bioactivity-related molecular descriptors for phosphinan-4-ones 9–13 were compared and correlated with their observed cytotoxic activity. Molecular descriptors that have been widely recognized as having a significant impact on biological activity include lipophilicity (logP), total polar surface area (TPSA), the presence of hydrogen-bond donors and acceptors, molecular flexibility, and electronic descriptors such as molecular orbital energies and partial atomic charges. It is important to note that the influence of these descriptors on biological activity may vary depending on the structural characteristics of the compounds and the nature of the biological target. A detailed analysis of electronic descriptors revealed that, among the phosphinan-4-ones studied, the partial negative surface area (VdW surface PNSA1) exhibited the strongest correlation with the indicated activity (Table S2 and Fig. S4). The study of the structure–activity relationship (SAR) of the studied phosphinan-4-ones 9–13 is represented in Fig. 8.

Fig. 8
figure 8

SAR of cytotoxicity of the studied phosphinan-4-ones 9–13 and phosphinan-4-oles 14–15 (series B and C).

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Finally, the results of cytotoxicity studies for compounds in Series C were evaluated. Introduction of a hydroxyl group at the C4-position, known as a hydrogen bond donor, led to a reduction in biological activity. Among the four compounds tested, only those containing a P=O moiety exhibited weak inhibition of cancer cell growth. Moreover, the results indicate that the relative configuration of the substituents within the six-membered ring also plays a role. Specifically, only the isomer with a trans configurations of the hydroxyl group relative to the P-phenyl substituent (trans-14) displayed measurable biological activity (Fig. 8).

Antiproliferative activity

To calculate the cell number and percentage of viable cells in cancer and normal cell populations after incubation with compounds 2, 8 and 11, the trypan blue dye exclusion assay was performed. The amount of live cancerous cells treated for 72 h by studied phosphinane derivatives was considerably lower as compared to controls (Fig. 9 and Fig. S5). The compound 11 has reduced PC3 cells number and their viability nearly complete (both in 98%). These observations have proved excellent cytotoxic properties of the derivative 11 against the above-mentioned cells. Similarly, it also diminished the HTC116 cells population by 94%, together limiting their viability by 18%. In addition, the highest decline in SW480 and SW620 cells number, accounted for 98–100%, was denoted also in the presence of the compound 11. However, these colon cancer cells viability did not change, as compared to controls. Substances 2 and 8 significantly diminished both SW480 and SW620 cells amount—they were reduced by 45–53% and 56–60%, respectively. What is more, the derivative 2 diminished PC3 population by 67%, whereas the number of live cells treated with the compound 8 equaled 50%, comparing to control experiments. The phosphinane oxide 2 led to the reduction of live HCT116 cells number by 29%, while the effect of an action of the compound 8 was moderate (decreased by 16%). The above results showed that compounds 2 and 8 exert cytostatic effect on all examined cancer cells, suppressing their growth and proliferation.

Fig. 9
figure 9

The effect of selected compounds (2, 8 and 11) on the live SW480, SW620, PC3 and HCT116 cells number and viability (%), measured by trypan blue assay. Cells were treated with studied compounds at their IC50 for 72 h. Data are expressed as the mean ± SD. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05, as compared to the control.

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As expected, normal HaCaT cell line was the least susceptible to studied derivatives. Target compounds 2 and 8, used at their IC50, did not influence keratinocytes viability. Their effect on HaCaT cells number was only moderate—the percentage of cells reduction equaled 10–20. Although the most potent phosphinanone 11 diminished both the amount and viability of HaCaT cells by 27–31%, its selectivity in contrast to cancerous cells was threefold higher (94–100% of reduced cells).

Activation of apoptosis

In order to assess the potential to induce the death of a cell, the effect of the most cytotoxic derivatives 2, 8, and 11 on processes of apoptosis and necrosis was evaluated by flow cytometry analysis (Figs. 10, 11 and Table S4). The phosphinanone 11, incubated at IC50 doses (4.4–6.3 µM) with studied cancer cells, showed a very high percentage of cells in late apoptosis as compared to the controls. The strongest pro-apoptotic effect was observed in PC3 (95.5% ± 0.70) and SW480 (87.5% ± 0.71) cell lines, as well as in HCT116 cells (80.15% ± 0.21). The compound 11 influenced considerably also SW620 cell apoptosis, triggering the late apoptosis in 48.45% ± 0.77 of cells. The derivative 11 has not induced cellular mechanisms leading to necrosis; that process was registered only for less than 6% of selected colon cancer cells (SW480, SW620). None of the tested compounds indicated a distinct ability to induce early apoptosis in cancer cells. Only in the presence of derivatives 2 and 8, approximately 8% of PC3 cells entered early apoptosis. In contrast to substance 11, the treatment of pathologically changed cells with low doses of phosphinanes 2 or 8 has not revealed noticeably apoptotic or necrotic cell death, as compared to control probes. Interestingly, incubation of normal HaCaT cells with studied derivatives, including the strongest cytotoxic agent 11, revealed negligible levels of apoptosis and necrosis (in fact 4.20–5.55% of cancer cells), comparing with untreated cancerous controls. The presented results agree with the IC50 values of the lead compound 11, determined for studied cancer cell lines.

Fig. 10
figure 10

The effect of derivatives 2, 8 and 11 on early and late apoptosis or necrosis in SW480, SW620, PC3, HCT-116 and HaCaT cells. ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05 as compared to the control.

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Fig. 11
figure 11

Representative results (%) as dot plots from apoptosis analysis of SW480, SW620, PC3, HCT-116 and HaCaT cells after treatment with compound 2, 8 and 11 tested with flow cytometry. Q1—necrotic cells, Q2—late apoptotic cells, Q3—live cells, Q4—early apoptotic cells.

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Inhibition of IL-6 release

Interleukin-6 (IL-6) is an important proinflammatory cytokine, which has a pathological effect on acute and chronic inflammation47,48. It is also considered as a significant tumor-promoting factor in various types of human cancer, including glioma, lymphoma, melanoma, as well as breast, ovarian, pancreatic, prostate and colorectal cancer49. This interleukin participates in cancer progression, influencing tumor cells proliferation, migration, and angiogenesis50. Considering colon cancer, IL-6 expression can be correlated with tumor size, stage, metastasis and in consequence poor prognosis for patients51. However, under physiological conditions, the interleukin-6 takes part in organ/cellular homeostasis in normal tissues52.

Searching for anti-interleukin-6 agents within the new phosphinanes, selected cancer cells were tested at IC50 doses of derivatives 2, 8 and 11 (Fig. 12, Table S5). Significant biological effects, exceeding 30% reduction of cytokine level in cancer cells, were denoted for all compounds. The substance 11 exerted the highest inhibitory activity in both PC3 and HCT116 cells, decreasing IL-6 secretion nearly complete, i.e. by 96.8% and 93.4%, respectively. Its strong effect was also observed on other colon cancer cells, achieving an 89.8–71.8% reduction in SW620 and SW480 cell lines. The compound 2 exhibited significant activity in PC3 cells, with a 60.5% decrease of the IL-6 level, whereas compound 8 reduced the cytokine concentration in these cells by 47.6%. What’s more, the similar efficiency in all studied colon tumor cells, accounted for 31.4–43% reduction, was denoted for derivatives 2 and 8. Interestingly, after treatment of HaCaT cells with these two phosphinanes, IL-6 cytokine level increased by 17.8–26.5%. The most active phosphinanone 11 reduced the interleukin level in HaCaT cells by 38.9%, however studied prostate and colon cancer cell lines demonstrated 2–2.5 enhanced sensitivity to this compound in comparison with normal keratinocytes.

Fig. 12
figure 12

Effects of compounds 2, 8 and 11 on IL‐6 levels, measured by the ELISA test. Data are expressed as the mean ± SD from primary colon cancer (SW480), metastatic colon cancer (SW620), metastatic prostate cancer (PC3), colon carcinoma (HCT116) and immortal keratinocyte cell line from adult human skin (HaCaT). ****p ≤ 0.0001; ***p ≤ 0.001; **p ≤ 0.01; *p ≤ 0.05 as compared to the control. ELISA—enzyme‐linked immunosorbent assay; IL‐6—interleukin 6.

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Conclusions

In summary, we have rationally designed, synthesized, and evaluated a series of small-molecule compounds featuring a six-membered phosphinane ring with the aim of exploring how systematic structural modifications influence their anticancer activity. The applied modifications included: dearomatization of the phenyl ring to enable the introduction of sterically diverse aliphatic substituents at the phosphorus atom, functionalization at the C4-position with either a carbonyl or hydroxyl group, and replacement of the oxygen atom with chalcogen atoms (S, Se) or borane. The nature of the introduced modifications allows for the classification of the tested compounds into three series: phosphinanes, phosphinan-4-ones, and phosphinan-4-oles. In order to gain a deeper understanding of the influence of structural alterations on the biological activity of phosphinanes, systematic studies were carried out on each series of phosphinane derivatives. The results of the conducted research indicate that some of the newly synthesized compounds exhibited notable antiproliferative activity against human cancer cell lines, including SW480, SW620, HCT116, and PC3. Among them, phosphinan-4-ols exhibited no significant antiproliferative activity. Notably, compound 2, a phosphinane bearing a cyclohexyl ring, showed higher anticancer activity than their phenyl-substituted counterparts, highlighting the impact of steric and electronic properties introduced via dearomatization. The introduction of borane 8 and sulfur 11 in place of oxygen also proved beneficial, with compound 11 emerging as the most active derivative. It is noteworthy that the most promising drug candidates (2, 8 and 11) exhibited low or non-cytotoxic effects against the normal HaCaT cell line. The lead compound 11 may act as an encouraging starting point for the reasonable development of new more potent anticancer agents. Its high cytotoxicity towards cancer cells, strong proapoptotic and IL6-inhibiting properties highlight its ability for further studies and clinical application.

Experimental section

Chemistry

General remarks

All reactions were carried out in vacuum- and flame-dried glass reaction flasks under argon atmosphere. All reagents were purchased from commercial sources and used without further purification. Tetrahydrofuran was dried over sodium/benzophenone ketyl. Prior to condensation, ammonia was passed through a column filled with solid potassium hydroxide. All the experiments involving microwave irradiation were performed on a CEM Discover instrument. The course of the reaction was monitored by TLC or/and GC–MS analysis, which was recorded with a Shimadzu GC-MSQP2010S spectrometer. GC–MS analysis was performed using an Inferno capillary column (30 m × 0.25 mm, 0.25 µm film thickness). Helium was used as the carrier gas at a constant flow rate of 1.0 mL/min. The oven temperature was programmed from 50 °C (2 min hold) to 280 °C at 10 °C/min. Injection volume: 1 µL, splitless mode. Mass spectra were recorded under electron impact ionization (EI, 70 eV). Thin-layer chromatography (TLC) was performed with precoated silica gel plates and visualised by exposing the plates to UV light, a potassium permanganate (KMnO4) stain or iodine adsorbed on silica. The reaction mixtures were purified by column chromatography over silica gel (60–240 mesh). The elemental analyses were conducted using a PerkinElmer CHN 2400 analyzer. Melting points were determined on Büchi Melting Point M-560 in a capillary tube and are uncorrected.

The NMR spectra was recorded with Bruker Ascend (500 MHz) spectrometer in CDCl3 as a solvent at room temperature unless otherwise noted. Chemical shifts (δ) are given in ppm relative to residual CHCl3. The following abbreviations are used in reporting NMR data: s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), br (broad). Coupling constants (J) are in Hz.

1-Phenylphosphinane-1-oxide (1)53, 1-Cyclohexylphosphinane 1-oxide (2)38, 1-Phenylphosphinan-4-ones (9, 10, 11 and 13)54 and 1-Phenylphosphinan-4-one 1-selenide (12)55 were prepared according to literature procedures. All data are in according with previously reported.

Single crystal X-ray diffraction data for trans-14 were collected at room temperature using a SuperNova diffractometer (Oxford Diffraction; Agilent56) with graphite-monochromated CuKα radiation (λ = 1.54184 Å). Data collection, unit cell refinement, and data reduction were performed using the CrysAlisPro program system57. The intensities were corrected for Lorentz and polarization effects, and a multi-scan absorption corrections were applied. The crystal structure was solved by direct methods using SHELXT (version 2018/2), followed by full-matrix least-squares refinement on F2 using SHELXL (version 2018/3)58,59,60. Both programs are available from https://shelx.uni-goettingen.de/download.php. The non-hydrogen atoms were refined with anisotropic displacement parameters, while hydrogen atoms were positioned at calculated positions and refined using the riding model. The experimental details and final atomic parameters for the analyzed crystal have been deposited with the Cambridge Crystallographic Data Centre (CCDC No. 2382302) as Supplementary Material. These data are available free of charge by the CCDC Access Structures service at: https://www.ccdc.cam.ac.uk/structures/.

Synthesis of 1-(1-Methylcyclohexa-2,5-dien-1-yl)phosphinane 1-oxide (3)

The synthesis of compound 3 was achieved through a modified Birch reduction, using sodium metal and liquid ammonia39. A flame-dried 50 mL three-necked flask, equipped with an inert gas inlet, a dry-ice condenser, and a cooling bath (− 78 °C), was filled with gaseous ammonia until 10 mL of liquid ammonia had condensed. Next, sodium (0.058 g, 2.5 mmol) was added and the mixture was stirred for 15 min. Subsequently, 1-phenylphosphorinane-1-oxide (1) (0.194 g, 1 mmol) was added, followed 5 min later by methyl iodide (0.156 mL, 2.5 mmol). The reaction mixture was allowed to stir at –78 °C for 30 min. The reaction was quenched by the addition of solid ammonium chloride (NH4Cl, 0.5 g), and ammonia was evaporated. The resulting residue was then filtered and washed with DCM (3 × 15 mL). The collected organic phases were concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel with DCM/MeOH (30:1, v/v) as an eluent to yield 3 (0,160 g, 76%). Colorless oil. 1H NMR (500 MHz, CDCl3) δ ppm 1.11–1.20 (m, 1 H, CH2), 1.34 (d, J = 14.19 Hz, 3 H, CH3), 1.48–1.57 (m, 2 H, CH2), 1.77–1.86 (m, 3 H, CH2), 1.87–1.99 (m, 4 H, CH2), 2.59–2.67 (m, 1 H, CH2), 2.68–2.74 (m, 1 H, CH2), 5.57 (d, J = 9.77 Hz, 2 H, olefinic CH), 5.79–5.94 (m, 2 H, olefinic CH). 13C NMR (126 MHz, CDCl3) δ ppm 21.12 (d, J = 6.4 Hz, CH2), 21.31 (d, J = 3.7 Hz, CH3), 22.07 (d, J = 60.2 Hz, CH2P), 26.38 (d, J = 5.4 Hz, CH2), 26.74 (d, J = 5.4 Hz, CH2), 40.25 (d, J = 64.8 Hz, C–P), 126.44 (d, J = 8.2 Hz, olefinic CH), 127.13 (d, J = 3.6 Hz, olefinic CH). 31P NMR (202 MHz, CDCl3) δ 49.4 (s). Elemental Anal. (calcd., found for C₁₂H₁₉OP): C (68.55, 68.46), H (9.11, 9.17).

Synthesis of 1-(1-Methylcyclohexyl)phosphinane 1-oxide (4)

A solution of 1-(1-Methylcyclohexa-2,5-dien-1-yl)phosphinane 1-oxide (3) (0.105 g, 0.5 mmol) in methanol (15 mL) was placed in a flame-dried Schlenk tube (100 mL). Pd/C (10%, 0.02 g) was then added, the reaction vessel was evacuated three times and filled with hydrogen, and the reaction mixture was heated to 50 °C under hydrogen (1 atm) for 24 h. The reaction mixture was then filtered through Celite, which was washed with DCM (2 × 20 mL), and the organic phase was evaporated. The residue was purified by column chromatography on silica gel with DCM/MeOH (30:1, v/v) as an eluent to yield 1-(1-methylcyclohexyl)phosphinane 1-oxide (4, 0.98 g, 92%) Pale yellow solid; m.p. = 79.8–82.4 °C. 1H NMR (500 MHz, CDCl3) δ ppm 1.13–1.29 (m, 5 H, CH2/CH3), 1.43–1.56 (m, 6 H, CH2), 1.57–1.73 (m, 5 H, CH2), 1.75–1.90 (m, 3 H, CH2), 1.92–2.05 (m, 4 H, CH2). 13C NMR (126 MHz, CDCl3) δ ppm 16.55 (s, CH3), 20.36 (d, J = 10.0 Hz, CH2), 21.07 (d, J = 5.4 Hz, CH2), 21.28 (d, J = 58.1 Hz, CH2P), 25.81 (s, CH3), 27.16 (d, J = 5.4 Hz, CH2), 29.52 (s, CH2), 34.18 (d, J = 68.4 Hz, C–P). 31P NMR (202 MHz, CDCl3) δ 50.3 (s). Elemental Anal. (calcd., found for C₁₂H₂₃OP): C (67.26, 67.39), H (10.82, 10.70).

General procedure for the reduction of phosphine oxides 1 or 2 and subsequent conversion to the corresponding phosphine sulfides or boranes

In a microwave vial equipped with a magnetic stir bar and argon inlet, the phosphine oxide 1 or 2 (1 equiv.) was dissolved in dry-degassed toluene (5 mL). Subsequently, phenylsilane (PhSiH3) (1,5 equiv.) was added, and the vial was flushed with argon, capped and heated in the microwave reactor at 100 °C for 2.5 h to afford the free phosphine. The vial was cooled to room temperature and BH3·THF (2.0 equiv., 1 M in THF) or S8 (1.2 equiv.) was added and the reaction mixture was stirred for further 6 h under argon at 23 °C. The crude product was purified by column chromatography on silica gel (SiO2) with hexane/THF (20:1, v/v) as eluent. After removal of all volatiles under vacuum the desired products (sulfides: 7 or 5 and boranes: 8 or 6) were obtained.

1-Cyclohexylphosphinane 1-sulfide (5)

The synthesis of the title compound was carried out via the General Procedure using 0.1 g (0.5 mmol) of 2, 0.031 g (0.75 mmol) of PhSiH3, and 19 mg (0.6 mmol) of elemental sulfur. Purification on silica eluting with hexane/THF (10:1, v/v) afforded 75 mg (69%) of the sulfide 5 as a white crystals; m.p. = 145.7–147 °C. 1H NMR (500 MHz, CDCl3) δ ppm 1.24–1.43 (m, 4 H, CH2), 1.49–1.64 (m, 2 H, CH2), 1.66–1.77 (m, 3 H, CH2/CH), 1.77–1.94 (m, 10 H, CH2), 2.03–2.17 (m, 2 H, CH2). 13C NMR (126 MHz, CDCl3) δ ppm 21.28 (d, J = 6.4 Hz, CH2), 24.69 (d, J = 1.8 Hz, CH2), 25.68 (s, CH2), 26.12 (d, J = 13.6 Hz, CH2), 26.70 (d, J = 5.4 Hz, CH2), 28.38 (d, J = 47.3 Hz, CH2P), 38.31 (d, J = 51.2 Hz, C–P). 31P NMR (202 MHz, CDCl3) δ 43.9 (s). Elemental Anal. (calcd., found for C₁₁H₂₁PS): C (61.07, 61.19), H (9.78, 9.71).

1-Cyclohexylphosphinane 1-borane (6)

The synthesis of the title compound was carried out via the General Procedure using 0.1 g (0.5 mmol) of 2, 0.031 g (75 mmol) of PhSiH3, and 1 mL (1 M in THF, 1 mmol) of BH3·THF. Purification on silica eluting with hexane/THF (20:1, v/v) afforded 52 mg (52%) of the borane 8 as a white crystals; m.p. = 99.2–100.7 °C. 1H NMR (500 MHz, CDCl3) δ ppm 0.09–0.77 (m, 3 H, BH3), 1.21–1.33 (m, 4 H, CH2), 1.34–1.46 (m, 3 H, CH2), 1.58–1.69 (m, 3 H, CH2), 1.69–1.82 (m, 6 H, CH2), 1.82–1.96 (m, 5 H, CH2/CH). 13C NMR (126 MHz, CDCl3) δ ppm 19.87 (d, J = 33.1 Hz, CH2P), 21.39 (d, J = 4.5 Hz, CH2), 25.62 (s, CH2), 25.87 (s, CH2), 26.52 (d, J = 11.8 Hz, CH2), 26.87 (d, J = 5.4 Hz, CH2), 32.59 (d, J = 34.4 Hz, C–P). 31P NMR (202 MHz, CDCl3) δ 7.4–8.9 (bm). Elemental Anal. (calcd., found for C₁₁H₂₄BP): C (66.69, 66.44), H (12.21, 12.03).

1-Phenylphosphinane 1-sulfide (7)

The synthesis of the title compound was carried out via the General Procedure using 0.155 g (0.8 mmol) of 1, 0.049 g (1.2 mmol) of PhSiH3, and 31 mg (0.96 mmol) of elemental sulfur. Purification on silica eluting with hexane/THF (10:1, v/v) afforded 123 mg (73%) of the sulfide 7 as a white solid; m.p. = 82.3–83.6 °C. 1H NMR (500 MHz, CDCl3) δ ppm 1.39–1.51 (m, 1 H, CH2), 1.77–1.85 (m, 1 H, CH2), 1.86–1.99 (m, 2 H, CH2), 2.00–2.11 (m, 2 H, CH2), 2.18–2.26 (m, 2 H, CH2), 2.26–2.37 (m, 2 H, CH2), 7.45–7.61 (m, 3 H, aromatic CH), 7.84–7.98 (m, 2 H, aromatic CH). 13C NMR (126 MHz, CDCl3) δ ppm 21.75 (d, J = 5.4 Hz, CH2), 26.60 (d, J = 6.4 Hz, CH2), 31.81 (d, J = 51.1 Hz, CH2P), 128.80 (d, J = 11.8 Hz, metaC–P), 130.28 (d, J = 10.0 Hz, ortoC–P), 131.57 (d, J = 2.7 Hz, paraC–P), 133.00 (d, J = 76.8 Hz, ipsoC–P). 31P NMR (202 MHz, CDCl3) δ 35.0 (s). Elemental Anal. (calcd., found for C₁₁H₁₅PS): C (62.83, 62.98), H (7.19, 7.23).

1-Phenylphosphinane 1-borane (8)

The synthesis of the title compound was carried out via the General Procedure using 0.155 g (0.8 mmol) of 1, 0.049 g (1.2 mmol) of PhSiH3, and 1.6 mL (1 M in THF, 1.6 mmol) of BH3·THF. Purification on silica eluting with hexane/THF (20:1, v/v) afforded 92 mg (60%) of the borane 8 as a colorless oil. 1H NMR (500 MHz, CDCl3) δ ppm 0.39–1.07 (m, 3 H, BH3), 1.40–1.48 (m, 1 H, CH2), 1.67–1.75 (m, 1 H, CH2), 1.81–2.10 (m, 8 H, CH2), 7.43–7.49 (m, 3 H, aromatic CH), 7.66–7.72 (m, 2 H, aromatic CH). 13C NMR (126 MHz, CDCl3) δ ppm 21.87 (d, J = 4.5 Hz, CH2), 23.11 (d, J = 34.8 Hz, CH2P), 26.66 (d, J = 5.4 Hz, CH2), 128.86 (d, J = 9.1 Hz, mataC–P), 130.24 (d, J = 52.7 Hz, ipsoC–P), 130.96 (d, J = 2.7 Hz, paraC–P), 131.04 (d, J = 8.2 Hz, ortoC–P). 31P NMR (202 MHz, CDCl3) δ 2.6–4.5 (bm). Elemental Anal. (calcd., found for C₁₁H₁₈BP): C (68.80, 68.91), H (9.45, 9.22).

General procedure for the reduction of phosphinan-4-ones 10 or 13 to the corresponding cis- and trans-phosphinan-4-ols 14 or 15

Into a 50 mL round-bottom flask equipped with magnetic stirrer was placed 10 or 13 (0.005 mol) in methanol (25 mL). The mixture was cooled to 0 °C and sodium borohydride (1.5 equiv.) was added slowly and in small portions and the reaction was allowed to reach room temperature and stirr until TLC showed complete consumption of starting material. The reaction was quenched by addition of saturated NH4Cl solution (10 mL), the mixture was extracted with DCM (3 × 30 mL), the organic layers were collected, dried over MgSO4, filtered and evaporated. The residue was purified by flash chromatography on silica gel. After removal of all volatiles under vacuum the desired phosphinan-4-oles 14 or 15 were obtained as the mixture of two isomers.

1-Phenylphosphinan-4-ol 1-oxide (14)

The synthesis of the title compound was carried out via the General Procedure using 1.4 g (5 mmol) of 10, and 0.28 g (7.5 mmol) of NaBH4. Purification on silica gel eluting with DCM/MeOH (20:1, v/v) afforded 1.15 g (82%) of the expected secondary alcohol 14 as a isomers mixture (cis : trans = 1 : 14.2 ratio).

cis-1-Phenylphosphinan-4-ol 1-oxide (cis-14), white solid; m.p. = 149.6–150.5 °C. 1H NMR (500 MHz, CDCl3) δ ppm 1.85–1.96 (m, 2 H, CH2) 2.04–2.15 (m, 2 H, CH2), 2.21–2.30 (m, 2 H, CH2), 2.31–2.45 (m, 2 H, CH2), 4.16 (br. s., 1 H, CH-OH) 7.43–7.59 (m, 3 H, aromatic CH) 7.72–7.84 (m, 2 H, aromatic CH). 13C NMR (126 MHz, CDCl3) δ ppm 22.5 (d, J = 65.4 Hz, CH2P), 28.3 (d, J = 5.5 Hz, CH2), 65.1 (d, J = 6.15 Hz, CH-OH), 128.7 (d, J = 11.8 Hz, metaC–P), 130.1 (d, J = 9.1 Hz, ortoC–P), 131.9 (s, paraC–P), 132.9 (d, J = 96.7 Hz, ipsoC–P). 31P NMR (202 MHz, CDCl3) δ 32.0 (s). Elemental Anal. (calcd., found for C₁₁H₁₅O₂P): C (62.85, 63.02), H (7.19, 7.92).

trans-1-Phenylphosphinan-4-ol 1-oxide (trans-14), white crystals; m.p. = 132.1–134.8 °C. 1H NMR (500 MHz, CDCl3) d ppm 1.92–2.06 (m, 2 H, CH2), 2.07–2.16 (m, 2 H, CH2), 2.19–2.31 (m, 4 H, CH2), 3.83 (br. s., 1 H, CH-OH), 7.45–7.60 (m, 3 H, aromatic CH), 7.75 (ddd, J = 11.19, 8.20, 1.10 Hz, 2 H, aromatic CH). 13C NMR (126 MHz, CDCl3) δ ppm 24.75 (d, J = 66.00 Hz, CH2P), 30.04 (d, J = 5.45 Hz, CH2), 67.83 (d, J = 5.45 Hz, CH-OH), 128.84 (d, J = 10.90 Hz, metaC–P), 129.98 (d, J = 9.08 Hz, ortoC–P), 131.82 (d, J = 95.50 Hz, ipsoC–P), 132.00 (d, J = 2.40 Hz, paraC–P). 31P NMR (202 MHz, CDCl3) δ 31.7 (s). Elemental Anal. (calcd., found for C₁₁H₁₅O₂P): C (62.85, 62.90), H (7.19, 7.14).

Crystal data for trans-14: formula C₁₁H₁₅O₂P, Mw = 210.20, crystal system monoclinic, space group P21. Unit cell dimensions: a = 8.2493(2) Å, b = 7.0989(2) Å, c = 9.8021(3) Å, β = 97.542(2)°, V = 569.05(3) Å3, Z = 2, D(calcd) = 1.227 g cm−3, μ(CuKα) = 1.928 mm−1, F(000) = 224. Crystal size 0.25 × 0.1 × 0.05 mm3, λ = 1.54184 Å, θ = 4.550 to 72.449°, index ranges − 9 ≤ h ≤ 9, − 7 ≤ k ≤ 8, − 11 ≤ l ≤ 12. Reflections collected/independent: 3594/1701 [R(int) = 0.0314]. Non-hydrogen atoms were refined anisotropically. Hydrogen atoms were positioned geometrically and allowed to ride on their parent atoms. Parameters refined 132; Goodness-of-fit on F2 1.087, final R indices [I > 2σ(I)] R₁ = 0.0308, wR₂ = 0.0834, R indices (all data) R₁ = 0.0324, wR₂ = 0.0852. Absolute structure parameter x = 0.04(3). Δρ max/min 0.16 and − 0.16 e.Å−3. CCDC No. 2382302.

1-Phenylphosphinan-4-ol 1-borane (15)

The synthesis of the title compound was carried out via the General Procedure using 1.4 g (5 mmol) of 13, and 0.28 g (7.5 mmol) of NaBH4. Purification on silica gel eluting with DCM/MeOH (50:1, v/v) afforded 0.95 g (68%) of the expected secondary alcohol 15 as a isomers mixture (cis : trans = 1 : 9.1 ratio).

cis-1-Phenylphosphinan-4-ol 1-borane (cis-15), white solid; m.p. = 96.2–103.1 °C. 1H NMR (500 MHz, CDCl3) δ ppm 0.40–1.12 (m, 3 H, BH3), 1.79–1.88 (m, 2 H, CH2), 1.97–2.07 (m, 2 H, CH2), 2.17–2.27 (m, 2 H, CH2), 2.45 (d, J = 14.82 Hz, 2 H, CH2), 4.08–4.13 (m, 1 H, CH-OH), 7.50–7.53 (m, 3 H, aromatic CH), 7.78 (ddd, J = 10.48, 7.96, 1.73 Hz, 2 H, aromatic CH). 13C NMR (126 MHz, CDCl3) δ ppm 17.87 (d, J = 34.60 Hz, CH2P), 28.88 (d, J = 3.63 Hz, CH2), 65.98 (d, J = 5.45 Hz, CH-OH), 128.91 (d, J = 9.99 Hz, mataC–P), 129.80 (d, J = 53.40 Hz, ipsoC–P), 131.26 (d, J = 8.17 Hz, ortoC–P), 131.25 (s, paraC–P). 31P NMR (202 MHz, CDCl3) δ 1.33–2.56 (bm). Elemental Anal. (calcd., found for C₁₁H₁₈BOP): C (63.50, 63.69), H (8.72, 8.84).

trans-1-Phenylphosphinan-4-ol 1-borane (trans-15), white solid; m.p. = 70.6–72.6 °C. 1H NMR (500 MHz, CDCl3) δ ppm 0.43–1.15 (m, 3 H, BH3), 2.01–2.22 (m, 8 H, CH2), 3.76–3.86 (m, 1 H, CH-OH), 7.45–7.55 (m, 3 H, aromatic CH), 7.71 (ddd, J = 10.32, 7.96, 1.58 Hz, 2 H, aromatic CH). 13C NMR (126 MHz, CDCl3) δ ppm 20.01 (d, J = 34.50 Hz, CH2P), 29.81 (d, J = 4.54 Hz, CH2), 68.68 (d, J = 4.54 Hz, CH-OH), 129.04 (d, J = 53.00 Hz, ipsoC–P), 129.00 (d, J = 9.08 Hz, metaC–P), 131.12 (d, J = 9.08 Hz, ortoC–P), 131.27 (d, J = 1.82 Hz, paraC–P). 31P NMR (202 MHz, CDCl3) δ 1.94–3.44 (bm). Elemental Anal. (calcd., found for C₁₁H₁₈BOP): C (63.50, 63.61), H (8.72, 8.80).

Biological studies

Cell cultures

Human cell lines, including primary (SW480, HCT116) and metastatic (SW620) colon cancer, metastatic prostate cancer (PC3), and immortalized keratinocytes (HaCaT), were obtained from the American Type Culture Collection (ATCC). The cells were cultured in the recommended media: RPMI 1640 for PC3, MEM for SW480, HCT116, and SW620, and DMEM for HaCaT. The media were supplemented with 10% FBS, penicillin (100 U/mL), and streptomycin (100 μg/mL). The cells were maintained in a humidified incubator at 37 °C with 5% CO₂. Once they reached 80%–90% confluence, they were passaged using 0.25% trypsin (Gibco Life Technologies) and subsequently used for further experiments.

MTT test

Synthesized phosphinane derivatives and reference chemotherapeutics—doxorubicin and cisplatin were evaluated at concentrations ranging from 1 to 100 µM. All compounds were dissolved in DMSO prior to use. These compounds were added to 96-well plates containing seeded normal and cancer cells (1 × 104 cells per well) and incubated for 72 h. Cell viability was assessed using the MTT assay, following a previously established protocol56‚61. Absorbance values obtained from the assay were used to determine the relative MTT level (%), which enabled the calculation of cell viability in response to the tested compounds. Viability was expressed as the percentage of MTT reduction in treated cells relative to the control. The relative MTT level was determined using the formula: [100%] = A/B × 100%, where A represents the absorbance of the treated sample, and B corresponds to the absorbance of the control. IC50 values were calculated using GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA, USA; https://www.graphpad.com), which was the version used at the time of data analysis. The institution currently holds an active license for GraphPad Prism version 9.1.0 (build 221) (see Fig. S5).

Trypan blue assay

Cells (1 × 105 per well) were seeded in 12-well plates and incubated for 72 h with IC5050 concentrations of selected compounds. Following incubation, the cells were washed twice with phosphate-buffered saline (PBS) and then harvested. Cell viability was determined using the trypan blue exclusion assay with an automated cell counter (Countess™; Invitrogen, Waltham, MA, USA). Untreated cells served as the control. Each experiment was repeated 3 times.

Annexin binding assay

Cells were plated in 6-well plates (2 × 105 cells per well) and exposed to IC50 concentrations of 2, 8 and 11 derivatives for 72 h. The impact of these compounds on early and late apoptosis, as well as necrosis, was assessed using dual staining with Annexin V:FITC and propidium iodide, following the manufacturer’s instructions (Becton Dickinson), as previously described57‚62. Cells stained positive for Annexin V:FITC but negative for PI were classified as early apoptotic, while those positive for both Annexin V:FITC and PI were identified as either late apoptotic or necrotic.

Interleukin-6 assay

The IL-6 concentration in all analyzed cancer cell lines and the normal HaCaT cell line was determined using an ELISA kit (Diaclone SAS, Besançon CEDEX, France). Cells were seeded in 12-well plates (1 × 105 cells per well) and exposed to IC50 concentrations of compounds 2, 8, 11 for 72 h. The IL-6 levels in the cell culture supernatant were quantified using an enzyme-linked immunosorbent assay (ELISA) according to the previous instructions63.

Statistical analysis

All statistical analyses were conducted using GraphPad Prism (version 8.0; GraphPad Software, San Diego, CA, USA; https://www.graphpad.com). Data are presented as the mean ± standard deviation (SD) derived from a minimum of three independent experiments. Statistical significance between groups was determined by one-way ANOVA, followed by Dunnett’s post hoc test for multiple comparisons. Differences were considered significant at p < 0.05.