Spirooxindol‐1,3‐oxazine Alkaloids: Highly Potent and Selective Antitumor Agents Evolved from Iterative Structure Optimization

Abstract

Spirooxindole‐1,3‐oxazines are a small and structurally unique class of spirooxindole alkaloids. To date, only four of these compounds have been isolated from natural sources, and their biological properties remained unknown thus far. Dioxyreserpine is a synthetic spirooxindole‐1,3‐oxazine, that can readily be prepared from the Rauvolfia alkaloid (–)‐reserpine by catalytic photooxygenation. While dioxyreserpine itself was now identified as a moderately effective antitumoral agent, structurally modified analogs of it emerged as a new class of highly potent and selective growth inhibitors of various human cancers, including pancreatic cancers. Systematic structural optimization ultimately led to an inhibitor displaying low‐micromolar IC₅₀‐values against six cancer cell lines as well as selective apoptosis induction in vitro.

Six birds with one stone: A highly effective and selective inhibitor of six human cancer cell lines, including pancreatic cancer, evolved after the systematic structural optimization of alkaloids that possess the unusual spirooxindol‐1,3‐oxazine core structure.

Introduction

Small molecules, together with biologicals, form the foundation of targeted therapies in oncology and offer the possibility of establishing therapy protocols tailored to the individual patient. ^[1] Despite the successful introduction of various small molecules into different oncological therapy concepts, a significant proportion of patients show a non‐response or a recurrence of the disease. ^[2] A possible reason for this problem is with high probability the different genetic background of oncological diseases of the same entity (tumor heterogeneity) at the beginning of therapy, as well as the accumulation of genetic changes of the initial neoplasia during therapy itself. ^[3] Many nitrogen‐containing heterocycles are considered privileged structural motifs, and some drugs derived from them are also used therapeutically in oncology. ^[4]

Naturally occurring indole alkaloids offer a plethora of useful biological properties, and since ancient times traditional medicine has made use of them. ^[5] Spirooxindoles constitute a structurally intriguing and medicinally highly valuable class of indole alkaloids. Roughly 75 % of all known spirooxindoles possess the most common core structure, the spiro[pyrrolidin‐3,3’‐oxindole] motif A (Scheme 1a). ^[6] A large number of these compounds display analgesic, antibiotic and antitumoral activities. ^[7] Regarding the latter, the natural product mitraphylline (1), active against leukemia, sarcoma, and breast cancer, ^[8] as well as the synthetic inhibitor of the P53‐MDM2 interaction, MI‐888 (2) ^[9] are prominent examples.

Scheme 1.

a) Structures of spiro[pyrrolidin‐3,3’‐oxindole] alkaloids. b) Natural spiro[(1,3)oxazinan‐3,6’‐oxindole] alkaloids. c) Catalytic photooxygenation of reserpine (7) to dioxyreserpine (8). 1,5‐AAQ=1,5‐Diaminoanthraquinone.

A small and quite peculiar class of spirooxindoles is made up of compounds that possess a spiro[(1,3)‐oxazinan‐3,6’‐oxindole] core structure B (Scheme 1b). By 2022, the isolation of only four natural products of this genus had been reported, all of which originate from South East Asian shrubs and flowering plants: Mitradiversifoline 3, ^[10] uncarialine D 4, ^[11] nauclealomide A 5 ^[12] and melodinoxanine 6. ^[13] The biosynthetic pathways leading to these alkaloids remain to be elucidated as well as their potential medicinal uses. However we can anticipate the isolation of more representatives of this interesting compound family.

A purely synthetic and thus readily available spirooxindole‐1,3‐oxazine is dioxyreserpine (8), that can be prepared in larger quantities by the catalytic photooxygenation[ ¹⁴ , ¹⁵ ] of the Rauvolfia alkaloid reserpine (7), which is sold commercially at relatively low cost (Scheme 1c). Dioxyreserpine (8) equals the polycyclic natural products 3–6 in their level of molecular complexity, and also its absolute configuration at the C‐3 and C‐7 stereocenters of the 1,3‐oxazine ring is identical. Its cis‐fused D and E rings, a structural specialty that gives the whole molecule a strong curvature, are also present in melodinoxanine 6. These features make dioxyreserpine (8) an ideal starting point for a biological study that, if it would establish any desirable medicinal properties, would suggest that natural products of the spirooxindole‐1,3‐oxazine structure B are of general medicinal interest.

Results and Discussion

Our investigations began with an initial screening of dioxyreserpine (8) for its growth inhibitory activity against six human cancer cell lines, i. e. the B‐ALL cell lines RS4;11 and NALM‐6, the lymphoma cell lines SU‐DHL‐4 and SUP−T1, and the pancreatic carcinoma cell lines MIA‐PaCa‐2 and Capan‐1. After 72 h of incubation, a cell count was carried out for the haematological cell lines, while for the solid cell lines, the cell mass was determined using crystal violet staining. The metabolic activity was also quantified with the WST‐1 assay (see further below). Of all cell lines, NALM‐6 appeared to be most responsive to incubation with dioxyreserpine (8), showing a moderate growth inhibition and an IC₅₀ of 24.39±19.26 μM after 72 h (Table 1, 1st row). Hence we continued our investigation with NALM‐6 and employed structurally modified derivatives of dioxyreserpine (8) as shown in Scheme 2. The reductive ring‐opening of 8 with NaBH₄ leads to compound 9, ^[14] that showed no significant effects, clearly confirming that the spirooxindol‐1,3‐oxazine structure is essential for activity. On the other hand, the C‐14‐oxidized derivative trioxyreserpine (10), which is accessible from 8 by another catalytic photooxygenation, ^[14] showed a markedly increased growth inhibition, with a superior IC₅₀ of 3.68±1.46 μM (Scheme 2a).

Table 1.

Collected IC₅₀ values [μM] of compounds 8–18 for growth inhibition of six human carcinoma cell lines after 72 h incubation time.^[a]

Compound	SU‐DHL‐4	SUP−T1	NALM‐6	RS4;11	MIA‐PaCa‐2	Capan‐1
8	61.40±16.26	74.47±50.78	24.39±19.26	n.c.	n.c.	n.c.
9	n.c.	n.c.	n.c.	63.73±35.77	n.c.	n.c.
10	2.93±0.66	2.85±1.03	3.68±1.46	2.97±1.66	3.99±0.30	23.01±12.40
11	12.62±7.15	19.24±17.97	4.02±1.61	10.25±5.85	21.21±2.42	88.14±38.46
12	31.92±14.60	n.c.	4.03±1.29	10.23±7.24	21.81±12.85	61.92±21.20
13	1.39±0.07	4.72±1.52	2.38±0.10	3.71±0.87	4.18±1.32	22.15±11.01
14	15.93±6.00	n.c.	7.33±3.31	21.26±39.35	n.c.	n.c.
15	2.26±0.33	7.01±0.05	2.37±1.16	3.52±4.26	7.04±1.99	28.91±59.53
16	1.73±0.17	3.64±3.48	1.79±0.62	1.95±0.53	6.11±2.07	n.c.
17	n.c.	n.c.	4.45±0.47	n.c.	n.c.	n.c.
18	1.14±0.20	1.62±0.13	0.61±0.38	0.32±0.06	1.90±0.88	4.21±0.95

[a] Cell count was carried out for the haematological cell lines. For the solid cell lines, the cell mass was determined using crystal violet staining. n.c.=not calculable.

Scheme 2.

Initial screening, structural variation and iterative optimization of the growth inhibition of NALM‐6 leukemia cells. IC₅₀ values determined after 72 h incubation.

The subsequent course of the investigation was decisively altered by the discovery that functionalization of dioxyreserpine (8) in the C‐10 position had a pronounced positive effect (Scheme 2b). Electrophilic bromination of 8 leads to C‐10 bromide 11, ^[14] that showed strong growth inhibition with an IC₅₀ of 4.02±1.61 μM. Hence we hypothesized that the introduction of other hydrophobic groups in this position would be beneficial. Bromide 11 undergoes clean Suzuki arylations with arylboronic acids when using (PhCN)₂PdCl₂ as the Pd(0) precursor, in combination with the CataCXium® A ligand. ^[16] The C‐10 biaryl derivatives 12–16 are obtained in 64–88 % yield under these conditions, and pleasingly, they show strongly improved inhibitory activity compared to the parent dioxyreserpine (8), with IC₅₀ ranging from 4.03±1.29 to 1.79±0.62 μM. Up to this point, all compounds tested contained the hydrophobic 3,4,5‐trimethoxyphenyl ester moiety attached to the C‐18 hydroxyl group on the E‐ring. As shown exemplary for compound 14, the saponification of the ester only has little influence on the growth inhibition of NALM‐6, as IC₅₀ decreased only slightly from 7.33±3.31 μM for compound 14 to 4.45±0.47 μM for compound 17. Other cell lines however responded more strongly to this structural change (Table 1, compare rows 7 and 10).

Based on our observations that introduction of a hydrophobic aromatic group at C‐10 and the carbonyl group at C‐14 both strongly improved activity, we reasoned that combining these two modifications potentially would lead to an even more potent inhibitor. Hence, 4‐trifluoromethylphenyl‐substituted compound 16, most active among the biaryls 12–16, was subjected to C‐14 methylene oxidation. Gratifyingly, when the C−H oxidation is performed under the photooxygenation conditions developed previously for the oxidation of dioxyreserpine (8) to trioxyreserpine (10), compound 18 is isolated in a reproducibly high yield of 75 %.

In the subsequent screening, the C‐10, C‐14 difunctionalized derivative, 10‐(4‐trifluoromethylphenyl)trioxyreserpine (18), emerged as a highly potent growth inhibitor of all six cell lines investigated (Table 1, last row). Its IC₅₀ values significantly fall below all of those of trioxyreserpine (10) and 4‐trifluoromethylphenyl‐substituted biaryl 16 for SU‐DHL‐4 (1.14±0.20 μM), SUP−T1 (1.62±0.13 μM), NALM‐6 (0.61±0.38 μM) as well as RS4;11 which suddenly is the most sensitive cell line (0.32±0.06 μM) and MIA‐PaCa‐2 (1.90±0.88 μM). Of particular importance, of all compounds tested, compound 18 is the only one to effectively inhibit the pancreatic cancer cell line Capan‐1, with an IC₅₀ of 4.21±0.95 μM; this represents a 90‐fold increase in activity compared to dioxyreserpine (8), the starting point of the structure optimization.

Metabolic activity

The new spirooxindole derivatives were tested for their in vitro metabolism‐modulating effects on lymphoma cell lines SU‐DHL‐4 and SUP−T1, the B‐ALL cell lines NALM‐6 and RS4;11 and moreover pancreatic cancer cell lines MIA‐PaCa‐2 and Capan‐1, by using the WST‐1 assay after 72 h of incubation (Figure 1a). Regarding the results of the WST‐1 assay we could observe an extraordinary increase of the metabolism‐modulating effect on all cell lines, due to the structural modification of the parent compound dioxyreserpine (8). For B‐ALL cell line RS4;11 we identified compounds 10, 13, and 15–18 to have a significant reducing effect on metabolic activity (Figure 1b). Compound 18 showed the strongest effect for all tested concentrations. 1 μM of compound 18 resulted in a reduction of metabolic activity to 19,04 %±5,44 %, 5 μM to 1,09 %±1,06 %, respectively. Compounds 10 and 17 showed similar results for the highest concentration of 10 μM.

Figure 1.

a) Results of WST‐1 assay after 72 h of concentration‐dependent spirooxindole exposure on B‐ALL cell line RS4;11. Data are presented as mean±SD (n≥3). Statistical significance was calculated by one‐way ANOVA. b) Haemolytic activity of spirooxindole derivatives after an incubation period of 2 h. Shown are means with±SD (n≥3).

Haemolysis

To preclude a potential haemolytic activity of the new spirooxindole derivatives, we measured the haemolysis of whole blood from three donors, 2 h after substance application (Figure 1b). None of the tested spirooxindoles showed a significant increase of haemolysis compared to negative control (only whole blood) or zero control (whole blood+PBS), which indicates the absence of erythrocyte toxicity.

Apoptosis induction and morphological characterization

Flow‐cytometric analysis after Annexin‐V FITC/PI double staining was used to observe a potential pro‐apoptotic effect after 24, 48 and 72 h of the spirooxindole‐1,3‐oxazine derivatives (Figure 2a). The results indicate a significant increase of late apoptosis after all tested time points for the highest concentration of compound 10. Here the portion of late apoptotic cells increased from 9.82 %±2.97 % in the control up to 82.71 %±2.81 % after 72 h exposure. For compound 15 at concentrations of 5 μM and 10 μM we could also observe a significant increase of late apoptotic cells up to 48.70 %±35.16 % and 53.68±30.18 % and early apoptosis from 5.07 %±2.31 % in the control to 21.56 %±13.11 % and 23.51 %±9.33 % after 72 h, respectively. Incubation of RS4;11 cells with Compound 18 led to a significant increase of late apoptosis for all tested concentrations to 45.89 %±2.78 %, 80.45 %±10.00 % and 82.60 %±8.80 % for the highest concentration of 10 μM.

Figure 2.

a) Apoptosis induction in RS4;11 cells after concentration‐dependent spirooxindole exposure. Apoptosis induction was determined by using Annexin‐V FITC/PI double staining after an incubation period of 72 h. Shown are means with±SD (n≥3). b) Morphological characterization of RS4;11 cells after exposure to 10 μM of each compound for 72 h. DMSO was used as a control. The red arrows point to the membrane blebs and the black arrows to the cell detritus.

Microscopic analysis after May‐Gruenwald‐Giemsa staining revealed the presence of membrane blebs in case of compounds 11, 13 and 15 (Figure 2b, red arrows). Morphological characterization of RS4;11 cells after exposure to 10 μM of compounds 10, 16 and 18 showed the presence of detritus and cell debris (Figure 2b, black arrows), but no more intact and vital cells compared to DMSO control cells.

Cell‐cycle analysis

In addition to the flow cytometric apoptosis measurements, the cell cycle was also analyzed after 48 h and 72 h. The results of the cell cycle analyzes for the B‐ALL cell line RS4;11 after 72 h are shown below (Figure 3). The data were evaluated using the FlowJo™ software. For 10 μM of compound 10, 5 μM and 10 μM of compound 13, 10 μM of compound 15, as well as 5 μM and 10 μM of compound 16, no evaluation with the software could be carried out, since the cells are totally fragmented, so that cell cycle classification was not possible (Figure 3a).

Figure 3.

a) Cell cycle analysis after 72‐hour derivative exposure of RS4;11 cells. The cell cycle phases are divided into sub‐G1, G1, S and G2 phases. Shown is the percentage of cells in the corresponding phase in mean values with standard deviations (n=3). Results that cannot be evaluated (because of complete cell fragmentation) are marked with n.a.. Significance compared to the DMSO control *=G2 phase, #=S phase, +=G1 phase and $=sub‐G1 phase. Significance level: p <0.05. b) Exemplary representation of the cell cycle analysis of RS4;11 cells after 72 hours exposure to 5 μM (left) and 10 μM (right) of compound 15. In the illustration on the left, the FlowJo™ software enables a clear classification into the cell cycle phases based on the PI content of the cells. On the right, at a concentration of 10 μM of compound 15, the cells are too fragmented, so that it is no longer possible to divide them into cell cycle phases.

Compared to the DMSO control, the application of compound 11 at a concentration of 5 μM resulted in a significant reduction in the number of cells in the S phase. The proportion was reduced here from 40.73 %±7.46 % in the DMSO control to 27.67 %±8.62 %. At a concentration of 10 μM, the proportion of the S phase decreased significantly to 17.83 %±5.28 %. The proportion of the G1 phase also increased significantly from 45.13 %±4.48 % in the DMSO control to 66.43 %±6.92 %. At a concentration of 5 μM, compound 10 showed a significant increase in the G1‐phase population from 45.13 %±4.48 % to 63.37 %±2.02 % and an associated reduction in the S‐phase population of 40.73 %±7.46 % to 21.83 %±2.52 %. Through the application of derivatives 10 and 11, a substance‐induced G1 phase arrest could be observed in RS4;11 cells. Derivative 15 showed a significant increase in sub‐G1 phase, confirming the induction of apoptosis and the results shown previously.

Conclusions

In summary, a series of newly synthesized spirooxindole‐1,3‐oxazines based on the parent compound dioxyreserpine (8) was examined for its inhibitory effect on a set of haematological and solid human cancer cell lines. For dioxyreserpine (8) itself, we found only moderate biological effects on the investigated cell lines. Through the iterative structural modification of dioxyreserpine (8), we achieved however an enormous increase in biological activity, and the C‐10‐arylated and C‐14‐oxidized spirooxindole‐1,3‐oxazine 18 emerged as the most effective new inhibitor. 10‐(4‐Trifluoromethylphenyl)trioxyreserpine (18) showed strong antiproliferative effects for all tested cell lines, including pancreatic cancer cell lines MIA‐PaCa‐2 (IC₅₀=1.90±0.88 μM) and Capan‐1 (IC₅₀₌4.21±0.95 μM). The strongest effects of inhibitor 18 were detected for the B‐ALL cell line RS4;11 (IC₅₀=0.32±0.06 μM). For the spirooxindoles 10, 15, 16 and 18 we detected the induction of late apoptosis/necrosis and for compound 16 also early apoptosis of RS4;11 cells. To exclude a toxic effect on human erythrocytes, a haemolysis assay on human whole blood was performed, which confirmed the absence of any toxic effect for all the new spirooxindol‐1,3‐oxazine derivatives evaluated.

Experimental Section

Chemistry

Full experimental detail is provided in the Supporting Information.

Representative synthetic procedures : Suzuki arylation of bromide 11 to 4‐(trifluoromethylphenyl)‐substituted derivative 16 and photooxygenation of 16 to compound 18.

(−)‐10‐(4‐Trifluoromethylphenyl)dioxyreserpine (16). 10‐Bromodioxyreserpine ^[14] (11, 100.0 mg, 139.0 μmol), K₃PO₄ (44.5 mg, 209.6 μmol) and (4‐trifluoromethylphenyl)boronic acid (53.0 mg, 279.1 μmol) were dissolved in a PhCH₃/EtOH/H₂O mixture (4 : 1 : 1, 10.0 mL) and the mixture was degassed using the freeze‐pump‐thaw method. (PhCN)₂PdCl₂ (5.0 mg, 13.0 μmol, 10 mol‐%) and CataCXium A (Di‐(1‐adamantyl)‐n‐butylphosphine, 10.0 mg, 27.9 μmol, 20 mol‐%) were added and the mixture was stirred at 90 °C for 17 h. After cooling to r.t., the mixture was diluted with EtOAc and washed with NaCl aq. solution. The organic phase was dried over MgSO₄, filtered and concentrated under reduced pressure. The crude product was purified by column chromatography (silica gel, PhCH₃/EtOAc 3 : 2) to give compound 16 (70.6 mg, 65 %) as a colorless solid. R _f 0.45 (PhCH₃/EtOAc 3 : 2). [α]_D ²² =−2.5° (c 0.7 CHCl₃). m.p. 160–162 °C. ¹ H‐NMR (500 MHz, CDCl₃): δ=1.44 (dt, J=3.5, 13.2 Hz, 1 H, 14^a‐H), 1.71 (m_c, 1 H, 14^b‐H), 1.85 (dt, J=2.6, 13.7 Hz, 1 H, 6^a‐H), 1.94–2.04 (m, 2 H, 19^a‐H, 20‐H), 2.26–2.34 (m, 2 H, 15‐H, 19^b‐H), 2.40 (td, J=4.8, 13.7 Hz, 1 H, 6^b‐H), 2.54 (dd, J=3.2, 12.1 Hz, 1 H, 21^a‐H), 2.63–2.79 (m, 3 H, 5^a‐H, 16‐H, 21^b‐H), 3.04 (m_c, 1 H, 5^b‐H), 3.49 (s, 3 H, 33‐H), 3.67 (s, 3 H, 34‐H), 3.80 (s, 3 H, 35‐H), 3.85 (m_c, 1 H, 17‐H), 3.91 (s, 9 H, 30‐H, 31‐H, 32‐H), 4.60 (dd, J=2.6, 9.6 Hz, 1 H, 3‐H), 5.02 (ddd, J=4.7, 9.6, 12.0 Hz, 1 H, 18‐H), 6.51 (s, 1 H, 12‐H), 7.32 (s, 3 H, 9‐H, 25‐H, 29‐H), 7.47 (s, 1 H, 1‐H), 7.59 (d, J=8.2 Hz, 2 H, 37‐H, 41‐H), 7.63 (d, J=8.2 Hz, 2 H, 38‐H, 40‐H) ppm. ¹³ C‐NMR (125 MHz, CDCl₃): δ=29.4 (C‐14), 29.8 (C‐19), 31.9 (C‐6), 34.2 (C‐20), 36.3 (C‐15), 47.9 (C‐5), 51.88 (C‐16), 51.93 (C‐34), 56.2 (C‐35), 56.4 (C‐30, C‐32), 57.4 (C‐21), 61.0 (C‐33), 61.1 (C‐31), 74.7 (C‐7), 77.9 (C‐17), 78.2 (C‐18), 87.3 (C‐3), 94.6 (C‐12), 107.0 (C‐25, C‐29), 122.7 (C‐8), 124.5 (q, ¹ J _CF=272.1 Hz, C‐42), 125.1 (q, ³ J _CF=3.7 Hz, C‐38, C‐40), 125.48 (C‐10), 125.54 (C‐24), 126.8 (C‐9), 128.9 (q, ² J _CF=30.7 Hz, C‐39), 129.8 (C‐37, C‐41), 141.4 (C‐13), 141.9 (C‐36), 142.5 (C‐27), 153.1 (C‐26, C‐28), 158.3 (C‐11), 165.6 (C‐23), 172.0 (C‐22), 178.3 (C‐2) ppm. ¹⁹ F‐NMR (470 MHz, CDCl₃): δ=−62.39 ppm. HRMS (ESI‐TOF) m/z: calc. for C₄₀H₄₄F₃N₂O₁₁, [M+H]⁺: 785.2897, found 785.2899. IR: ṽ=2950, 2840, 1715, 1615, 1585, 1455, 1415, 1330, 1105, 1065, 845, 760 cm⁻¹.

(–)‐10‐(4‐Trifluoromethylphenyl)trioxyreserpine (18). In a 50 mL Pyrex tube (ø 3 cm), compound 16 (61.0 mg, 77.7 μmol) and 1,5‐diaminoanthraquinone (1.1 mg, 4.6 μmol, 6 mol‐%) were dissolved in MeCN (4.20 mL). The vial was sealed with a septum and O₂ was bubbled through the solution via cannula for 3 min. An O₂ balloon was fitted (septum pierced by needle). The mixture was stirred vigorously with irradiation by blue LEDs (10.1 W, 460±15 nm) at r.t. for 3 h. The mixture was concentrated to dryness and the crude product was purified by column chromatography (silica gel, PhCH₃/EtOAc 3 : 4) to give compound 18 (46.5 mg, 75 %) as a colorless solid. R _f 0.22 (PhCH₃/EtOAc 3 : 4). [α]_D ²² =−0.4° (c 1.0 CHCl₃). m.p. 174–175 °C. ¹ H‐NMR (300 MHz, CDCl₃): δ=1.84 (dt, J=2.4, 14.0 Hz, 1 H, 6^a‐H), 1.89 (q, J=12.8 Hz, 1 H, 19^a‐H), 2.01–2.13 (m, 1 H, 19^b‐H), 2.36–2.50 (m, 2 H, 6^b‐H, 20‐H), 2.53 (dd, J=5.3, 10.5 Hz, 1 H, 16‐H), 2.83–2.95 (m, 2 H, 5^a‐H, 21^a‐H), 3.12 (dd, J=2.5, 12.2 Hz, 1 H, 21^b‐H), 3.40 (m_c, 1 H, 5^b‐H), 3.49–3.57 (m, 1 H, 15‐H), 3.63 (s, 3 H, 33‐H), 3.66 (s, 3 H, 34‐H), 3.75 (s, 3 H, 35‐H), 3.92 (s, 9 H, 30‐H, 31‐H, 32‐H), 4.04 (dd, J=9.3, 10.5 Hz, 1 H, 17‐H), 4.97 (ddd, J=4.8, 9.3, 12.0 Hz, 1 H, 18‐H), 5.35 (s, 1 H, 3‐H), 6.42 (s, 1 H, 12‐H), 7.31 (s, 3 H, 9‐H, 25‐H, 29‐H), 7.50 (s, 1 H, 1‐H), 7.56 (d, J=8.5 Hz, 2 H, 37‐H, 41‐H), 7.61 (d, J=8.5 Hz, 2 H, 38‐H, 40‐H) ppm. ¹³ C‐NMR (125 MHz, CDCl₃): δ=31.3 (C‐6), 31.8 (C‐19), 36.0 (C‐20), 46.7 (C‐16), 48.3 (C‐5), 51.1 (C‐15), 52.1 (C‐34), 55.9 (C‐21), 56.1 (C‐35), 56.4 (C‐30, C‐32), 60.99 (C‐33), 61.07 (C‐31), 75.0 (C‐7), 77.3 (C‐17), 78.2 (C‐18), 87.9 (C‐3), 94.6 (C‐12), 106.9 (C‐25, C‐29), 121.6 (C‐8), 124.5 (q, ¹ J _CF=272.1 Hz, C‐42), 125.0 (q, ³ J _CF=3.7 Hz, C‐38, C‐40), 125.2 (C‐24), 125.4 (C‐10), 126.9 (C‐9), 128.9 (q, ² J _CF=32.7 Hz, C‐39), 129.8 (C‐37, C‐41), 141.7 (C‐13), 141.8 (C‐36), 142.5 (C‐27), 153.1 (C‐26, C‐28), 158.5 (C‐11), 165.6 (C‐23), 171.4 (C‐22), 177.5 (C‐2), 201.1 (C‐14) ppm. ¹⁹ F‐NMR (470 MHz, CDCl₃): δ=−62.39 ppm. HRMS (ESI‐TOF) m/z: calc. for C₄₀H₄₂F₃N₂O₁₂, [M+H]⁺: 799.2690, found 799.2690. IR: ṽ=3300, 2945, 2835, 1715, 1325, 1220, 1120, 1105, 725 cm⁻¹.

Molecular and cell biology

Cell lines and cell culture methods . The human ALL cell lines NALM‐6, RS4;11 (both B‐ALL) and the human lymphoma cell lines SU‐DHL‐4 (B‐cell) and SUP−T1 (T‐cell) as well as the pancreatic cancer cell lines MIA‐PaCa‐2 and Capan‐1 were purchased from DSMZ (Braunschweig, Germany). The cells were cultivated as recommended by the manufactural protocol. All the cell lines were cultivated at 37 °C and 5 % CO₂ in the corresponding media with 10–15 % heat‐inactivated FKS (Biochrom, Berlin, Germany) and 100 μg/ml penicillin and streptomycin (Biochrom, Berlin, Germany).

Drug exposure experiments . The suspension cell lines (3.3×10⁵ cells) and adherent cell lines were treated with each substance in three different concentrations (1 μM, 5 μM and 10 μM). Therefore the cells were cultured in the appropriate medium containing 0.1 % (v/v) DMSO as a control or dose ranges of the different derivatives as single substance for 24 h, 48 h and 72 h, depending on the experimental assay. After the incubation period, the effect on cell proliferation (trypan blue staining) or cell mass (crystal violet staining) in case of solid cells, metabolism (WST‐1 assay), apoptosis/necrosis (annexin V/PI staining), cell cycle analysis, live cell imaging and morphology were determined. All experiments were performed at least in three biological replicates.

WST‐1 assay . Haematological cell lines NALM‐6, RS4;11, SU‐DHL‐4 and SUP−T1 were seeded in a 96‐well plate at a density of 5×10⁴ cells per well in 150 μl media containing the substances or DMSO (0.1 % v/v) as a control. Pancreatic cancer cell lines MIA‐PaCa‐2 and Capan‐1 were seeded at a density of 5×10³ cells and were allowed to attach overnight in the incubator (37 °C, 5 % CO₂). After attachment of cells, the media was removed and 150 μl of substance/DMSO containing media was added. All experiments were carried out in biological and technical replicates. After incubation period of 72 hours, 15 μl of WST‐1 reagent was added to each well and the plates were again incubated for the next 3 h. The absorbance at 450 nm and a reference wavelength at 750 nm were determined using the GloMax‐Multi Microplate Multimode Reader (Promega, Madison, WI, USA). For background control, 150 μl of the appropriate media containing 15 μl WST‐1 was used.

Proliferation assay . To evaluate the impact of newly synthesized spirooxindoles, cell count was performed for the haematological cell lines NALM‐6, RS4;11, SU‐DHL‐4 and SUP−T1. Therefore 5×10⁵ cells were seeded in 24‐well plates and exposed to all substances (1 μM, 5 μM, 10 μM). DMSO (0.1 % v/v) exposed cells served as control. After 72 h of incubation, the cells were harvested and washed with PBS. The number of viable cells were determined by cell count after trypan blue staining.

Crystal violet staining . In case of solid cancer cell lines MIA‐PaCa‐2 and Capan‐1, crystal violet staining was carried out, to determine the cell mass after substance application. For both cell lines 5×10⁴ cells were seeded in 96‐well‐plates and let them adhere overnight. After adding the substances and 72 h of incubation, the supernatant was discarded and the cells were washed with 150 μl PBS. Thereafter 50 μl of crystal violet staining solution was added for 10 minutes. After two washing steps with 150 μl PBS, the cells were dried for 2 h. To detach the cells, 100 μl of 1 % SDS was used. The absorbance at 560 nm were determined using the GloMax‐Multi Microplate Multimode Reader (Promega, Madison, WI, USA).

Haemolysis assay . Haemolytic activity of selected spirooxindole compounds was determined by haemoglobin release from whole blood cells. Shortly, whole blood of healthy donors (n≥3) was seeded in 96‐well plates (round bottom) and incubated with 1 μM, 5 μM and 10 μM of each substance for 120 min. Positive control cells (=maximum lysis) were treated with 1 % Sodium Dodecyl Sulfate (SDS, Merck KGaA). Following the incubation period of 2 h, cell‐free supernatants were transferred into a new 96‐well plate (flat bottom) and absorption was measured on GloMax‐Multi Microplate Multimode Reader (Promega, Madison, WI, USA ) at 540 nm. Haemolytic activity was quantified according to the following formula: % haemolysis=[(OD_{540 nm sample} − OD_{540 nm buffer}) / (OD_{540 nm max} − OD_{540 nm buffer})] ×100.

Morphological characterization . Morphological changes were determined by May‐Gruenwald‐Giemsa staining. Therefore the cells (0,5×10⁶) were seeded in a 24‐well plate (flat bottom) and incubated with the compounds in concentration range of 1 μM to 10 μM for 24 h, 48 h and 72 h. After incubation period, 0,5×10⁴ cells were immobilized to cover slides with the cytospin 3 technology (Shandon, Frankfurt/Main, Germany). After staining, the morphological changes were analysed by microscopic analysis with EVOS® XL Core Imaging System (AMG, Washington, DC, USA).

Analysis of apoptosis . Determination of apoptosis induction was carried out by flow cytometric analysis using a FACS Calibur (BD Biosciences, Heidelberg, Germany). After incubation with each substance for 24 h, 48 h and 72 h in different concentrations (1 μM, 5 μM and 10 μM), cells were double stained with fluorescent dyes Annexin V‐FITCS and propidium iodide. During apoptosis the membrane phospholipids were translocated to the cell surface and Annexin V binds to it. Propidium iodide is a DNA‐intercalator, which binds to cellular nucleic acids of late apoptotic and necrotic cells with no intact cell membrane. The cells were harvested and washed twice with cold PBS (10 min, 180 g, 4 °C) and the pellet was resuspended in 100 μl Annexin Binding Buffer. 5 μl Annexin V FITCS were added to cell suspension and incubated for 15 min in the dark (RT). After incubation period, 400 μl Annexin Binding Buffer were added.

Cell‐cycle analysis . Changes within the rates of cell cycle phases (G0/G1, S and G2/M) after 48 h and 72 h of incubation with each substance were examined by flow cytometric analysis using FACS Calibur (BD Bioscience, Heidelberg, Germany). After an RNase digest of ethanol fixed cells, cells were stained with the DNA intercalator propidium iodide. Emission signals are proportional to DNA mass. Signal peaks were identified for each phase due to the amount of DNA. Thereby G0/G1 phase cells has one set of paired chromosomes per cell, G2/M phase cells two sets of paired chromosomes per cell, prior to cell division and S‐phase cells with variable DNA amount during DNA synthesis. Data analysis was carried out by using the FloJo™ software (BD Bioscience, Heidelberg, Germany).

Statistical analysis . All values are constituted as mean±standard deviation. After testing for normal contribution, the differences between treated cells and control cells were evaluated using the one‐way ANOVA. A p‐value <0,05 was set as statistically significant. IC₅₀ values were calculated according to the mathematical model (Prism 8.0.2, dose‐response‐inhibition vs. normalized response‐ variable slope).

Conflict of interest

The authors declare no conflict of interest.

Supporting information

As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

Supporting Information

A. Eichhorst, M. Gallhof, A. Voss, A. Sekora, L. Eggers, L. T. Huyen, C. Junghanss, H. Murua Escobar, M. Brasholz, ChemMedChem 2022, 17, e202200162.

Data Availability Statement

The data that support the findings of this study are available in the Supporting Information of this article.