Design, Synthesis and In Vitro Biological Activity of Novel C-7 Methylene Congeners of Furanoallocolchicinoids

A series of novel heterocyclic colchicine derivatives bearing a C-7 methylene fragment were synthesized via Wittig, Horner–Wadsworth–Emmons and Nenajdenko–Shastin olefination approaches. The in vitro biological activities of the most promising compounds were investigated using MTT assays and cell cycle analyses. Compounds with an electron withdrawing group on the methylene fragment exhibited substantial antiproliferative activity towards COLO-357, BxPC-3, HaCaT, PANC-1 and A549 cell lines. The spatial orientation of the substituent at the double bond significantly influenced its biological activity.


Introduction
Colchicine (1) is a naturally occurring alkaloid derived from Colchicum, Merendera or Gloriosa plants. Colchicine is currently used to treat acute gout, Behçet's disease, Mediterranean fever, chondrocalcinosis, systemic scleroderma and amyloidosis [1,2]. Due to its strong anti-inflammatory activity, colchicine [3,4] has been considered for treating numerous inflammatory diseases of different origins, including COVID-19 [5][6][7]. However, the high systemic toxicity of colchicine significantly restricts its clinical application [8]. During recent decades, numerous attempts to change colchicine's structure have been undertaken, aiming to reduce its general toxicity while maintaining its antiproliferative activity [9]. To date, a plethora of new derivatives of colchicine have been synthesized and tested in vitro and in vivo [9]. A number of selected promising compounds are presented in Figure 1. Most of them contain a double carbon-carbon bond that plays an important role as a pharmacophore.
The approach based on the contraction of ring C and formation of heterocycle fragment D led to several promising compounds exhibiting antiproliferative activity in the low nanomolar range. Thus, allocolchicine 2 can be considered as the parent structure for the compounds 4 and 5 with Michael acceptor fragments in their side chains, as well as for highly active molecules 6 and 7 that exhibit a six-fold decreased acute toxicity in comparison with colchicine [10,11]. Another impressive example of colchicine structure modification was demonstrated by Prof. H.-G. Schmalz's research group. They synthesized colchicinoid PT-100 (3), which demonstrated a strong synergetic pro-apoptotic effect in combination with vincristine on resistant cell lines [12]. Based on previous research, we suggest novel colchicinoids 8 and 9, bearing activated double bonds in the pseudo-benzylic position in ring B. This C=C bond can possibly interact with thiol groups in cysteine residues, increasing the efficiency and overcoming multidrug resistance. benzylic position in ring B. This C=C bond can possibly interact with thiol groups in cysteine residues, increasing the efficiency and overcoming multidrug resistance.

General Information
1 H NMR and 13 C NMR spectra were recorded in DMSO-d6 at 25 °С on an Agilent DDR2 400 spectrometer with operating frequencies of 400 MHz for 1 H and 101 MHz for 13 C. Chemical shifts (δ) are reported in parts per million (ppm) from TMS using the residual solvent resonance (DMSO-d6: 2.50 ppm for 1 H NMR, 39.52 ppm for 13 C NMR). Signal assignments in the proton spectra were based on comparison [13,14]. Mass spectra were recorded on a DSQ mass spectrometer with a quadrupole mass analyzer. The temperature of the ion source was 230 °C and ionization was carried out by electrons with an energy of 70 eV. Elemental analyses were performed on an Elementar (Vario Micro Cube) instrument. Column chromatography was performed using Merck Kieselgel 60 (70-230 mesh). Commercially available reagents (Aldrich, AlfaAesar and Acros) were used without additional purification. Solvents were purified according to the standard procedures. The petroleum ether (PE) used corresponds to the 40-70 °C fraction.
BALB/c mice were euthanized by cervical dislocation. Spleens were collected and homogenized in saline. Red blood cells were lysed by 0.83% NH4Cl solution, washed twice with saline, transferred to the complete culture medium and stimulated at 5 × 10 6 /mL of cells with concanavalin A (5 µg/mL) for 72 h. Activated splenocytes were washed in saline, transferred to culture medium and used for the analyses.

General Information
1 H NMR and 13 C NMR spectra were recorded in DMSO-d 6 at 25 • C on an Agilent DDR2 400 spectrometer with operating frequencies of 400 MHz for 1 H and 101 MHz for 13 C. Chemical shifts (δ) are reported in parts per million (ppm) from TMS using the residual solvent resonance (DMSO-d 6 : 2.50 ppm for 1 H NMR, 39.52 ppm for 13 C NMR). Signal assignments in the proton spectra were based on comparison [13,14]. Mass spectra were recorded on a DSQ mass spectrometer with a quadrupole mass analyzer. The temperature of the ion source was 230 • C and ionization was carried out by electrons with an energy of 70 eV. Elemental analyses were performed on an Elementar (Vario Micro Cube) instrument. Column chromatography was performed using Merck Kieselgel 60 (70-230 mesh). Commercially available reagents (Aldrich, AlfaAesar and Acros) were used without additional purification. Solvents were purified according to the standard procedures. The petroleum ether (PE) used corresponds to the 40-70 • C fraction.
BALB/c mice were euthanized by cervical dislocation. Spleens were collected and homogenized in saline. Red blood cells were lysed by 0.83% NH 4 Cl solution, washed twice with saline, transferred to the complete culture medium and stimulated at 5 × 10 6 /mL of cells with concanavalin A (5 µg/mL) for 72 h. Activated splenocytes were washed in saline, transferred to culture medium and used for the analyses.

MTT Assay
The antiproliferative effect of the compounds was estimated by a standard 3-(4,5dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, Sigma, St. Louis, MO, USA) test. To do this, the cells were transferred into a flat-bottomed 96-well plate, 5 × 10 3 per well, in which the preparations were titrated in advance. The plates were incubated in a CO 2 incubator for 72 h, and 250 µg·mL −1 MTT was added for the last 4 h. After incubation, the culture medium was removed and 100 µL of DMSO was added to each well to dissolve the formazan. The plates were analyzed on a plate spectrophotometer at a wavelength of 540 nm. The inhibition index (II) was calculated by the formula II = 1 − OD experiment /OD control , where OD is the optical density of the solution. The IC 50 values were determined as 50% inhibition index.

Confocal Microscopy
K562 cells, 10 5 , were seeded onto sterile cover glasses (10 5 /per well) and incubated in a CO 2 incubator at 37 • C until the cells adhered. Preparation 9Z was added at 500 nM concentration and cells were incubated for 18 h at 37 • C. At the end of the incubation, the cells were fixed with 1% PFA and washed three times with PBS 0.01% Triton X100. The fixed and permeabilized cells were stained with anti-murine β-tubulin antibody (Santa Cruze, CA, USA), phalloidin AlexaFluor 488 (Applied Biosystems, Foster City, CA, USA) and nuclear dye Hoechst 33342 (Merck KGaA, Darmstadt, Germany) for 1 h. Secondary anti-murine IgG-AlexaFluor555 (Merck KGaA, Darmstadt, Germany) was used to vizualize β-tubulin. Cells were polymerized by Mowiol (Calbiochem, Nottingham, UK) and analyzed using a TE 2000 Eclipse confocal microscope (Nikon, Tokyo, Japan).

Cell Cycle Analysis
The cell cycle was analyzed using PI-stained DNA. Cells from colchicinoid-treated cultures were collected, washed in ice-cold PBS, fixed by the addition of 70% ethanol and left for 2 h at −20 • C. Thereafter, the cells were washed twice in PBS, stained with 50 µg/mL of propidium iodide (Merck KGaA, Darmstadt, Germany) in PBS, treated with 10 µg/mL of RNAse and analyzed by flow cytometry using an FACScan device (BD, Franklin Lakes, NJ, USA). A total of 2000 events were collected. The results were analyzed using FlowJo 10 software (BD, Franklin Lakes, NJ, USA).

Chemistry
The synthesis of target furano-allocolchicinoids 8 and 9 bearing a methylene moiety at the C-7 position began with the preparation of ketone 13 in a seven-step reaction sequence, according to a published procedure [14]. Briefly, colchicine 1 was converted into iodo-colchinol 10 under Windaus conditions [15]; then, two acid-labile protecting groups (MOM and Boc) were introduced to facilitate the deacetylation process of colchicinoid 11. After acid-catalyzed cleavage of both protecting groups, N-deacetyliodocolchinol 12 was obtained. The above-mentioned amine was converted into ketone 13 by the Rapoport reaction [16] (total yield: 25%). Olefination of compound 13 under Wittig and Nenajdenko-Shastin [17] reaction conditions gave methylidene 15a and dibromo-(15b) and dichlorosubstituted (15c) derivatives, respectively (Scheme 1). In the latter cases of compounds 15b and 15c, ketone 13 was first converted into hydrazone 14 by reaction with hydrazine.
The transformation of hydrazine 14 into corresponding alkenes 15b and 15c was performed using CX 4 (X = Cl or Br) and a catalytic amount of copper(I) salt. The reaction began with the oxidation of Cu(I) to Cu(II), which, in turn, oxidized the hydrazone 14 to diazoalkane. After the decomposition of the latter, a Cu-carbeniod was formed as the key intermediate. In the last step, this was treated with CX 4 , resulting in the formation of alkene 15b or 15c. Finally, a furan ring D was formed via a domino Sonogashira/cyclization crosscoupling reaction of colchicinoids 15a-c with the corresponding alkynes, which has been described earlier [13]. Target furano-allocolchicinoids 8a-c were obtained with moderate (33-61%) yields (Scheme 1).
For the synthesis of derivative 9, ketone 13 was converted into furane derivative 16. The latter was treated with methyl 2-(dimethoxyphosphoryl)acetate and sodium hydride, resulting in the formation of alkene 9 (Z/E ratio 1:1). For details concerning the assignment of E-and Z-configurations for compounds 9E and 9Z, respectively, see the Supporting Information (2D NMR spectra). The transformation of hydrazine 14 into corresponding alkenes 15b and 15c was performed using CX4 (X = Cl or Br) and a catalytic amount of copper(I) salt. The reaction began with the oxidation of Cu(I) to Cu(II), which, in turn, oxidized the hydrazone 14 to diazoalkane. After the decomposition of the latter, a Cu-carbeniod was formed as the key intermediate. In the last step, this was treated with CX4, resulting in the formation of alkene 15b or 15c. Finally, a furan ring D was formed via a domino Sonogashira/cyclization cross-coupling reaction of colchicinoids 15a-c with the corresponding alkynes, which has been described earlier [13]. Target furano-allocolchicinoids 8a-c were obtained with moderate (33-61%) yields (Scheme 1).
For the synthesis of derivative 9, ketone 13 was converted into furane derivative 16. The latter was treated with methyl 2-(dimethoxyphosphoryl)acetate and sodium hydride, resulting in the formation of alkene 9 (Z/E ratio 1:1). For details concerning the assignment of E-and Z-configurations for compounds 9E and 9Z, respectively, see the Supporting Information (2D NMR spectra).

In Vitro Bioassays
All final compounds were tested on a number of cancer cell lines (COLO-357, BxPC-3, HaCaT, PANC-1 and A549). The antiproliferative activity was measured using the standard MTT assay. The results are summarised in Table 1.

In Vitro Bioassays
All final compounds were tested on a number of cancer cell lines (COLO-357, BxPC-3, HaCaT, PANC-1 and A549). The antiproliferative activity was measured using the standard MTT assay. The results are summarised in Table 1. The results indicated that the antiproliferative activity of the target compounds depends on the substituent on the C=C bond. Unsubstituted alkene 8a demonstrated no activity, whereas the presence of electron withdrawing groups, in particular, an ester group, increases the potency of the compound. However, the geometry of the conjugated ester in Pharmaceutics 2023, 15, 1034 5 of 10 compound 9 also influences the in vitro activity. Due to the significant difference in IC 50 of compounds 9Z and 9E, we investigated the in vitro effects of 9E in more detail.
The antiproliferative activity of 9E was investigated using various cell lines (Figure 2a). Cell lines originating from normal tissue, such as keratinocyte HaCaT or embryo kidney HEK293 cells, are often used as normal controls. However, telomerase activities in all immortalized cells either from cancer or normal tissues make them behave similar to cancer cells. To compare the compound's toxicity towards immortalized and normal cells, we used mitogen-activated murine splenocytes. Activation of lymphocytes with mitogen concanavalin A stimulates their proliferation. It appeared that this compound exhibited 5 times less toxicity against the activated splenocytes in comparison with immortalized cell lines (COLO357, EL-4, HaCaT). Additionally, 9E induces cell cycle arrest, a significant accumulation of cells in G2/M and a restriction of cell population in G0/G1 (Figure 2d,e). In general, the biological in vitro effects of 9E and colchicine 1 share much in common, but colchicinoid 9E can potentially covalently bind with tubulin due the presence of the conjugated ester fragment that can act as a Michael acceptor.
The results indicated that the antiproliferative activity of the target compounds depends on the substituent on the C=C bond. Unsubstituted alkene 8a demonstrated no activity, whereas the presence of electron withdrawing groups, in particular, an ester group, increases the potency of the compound. However, the geometry of the conjugated ester in compound 9 also influences the in vitro activity. Due to the significant difference in IC50 of compounds 9Z and 9E, we investigated the in vitro effects of 9E in more detail.
The antiproliferative activity of 9E was investigated using various cell lines ( Figure  2a). Cell lines originating from normal tissue, such as keratinocyte HaCaT or embryo kidney HEK293 cells, are often used as normal controls. However, telomerase activities in all immortalized cells either from cancer or normal tissues make them behave similar to cancer cells. To compare the compound's toxicity towards immortalized and normal cells, we used mitogen-activated murine splenocytes. Activation of lymphocytes with mitogen concanavalin A stimulates their proliferation. It appeared that this compound exhibited 5 times less toxicity against the activated splenocytes in comparison with immortalized cell lines (COLO357, EL-4, HaCaT). Additionally, 9E induces cell cycle arrest, a significant accumulation of cells in G2/M and a restriction of cell population in G0/G1 (Figure 2d,e). In general, the biological in vitro effects of 9E and colchicine 1 share much in common, but colchicinoid 9E can potentially covalently bind with tubulin due the presence of the conjugated ester fragment that can act as a Michael acceptor.

Conclusions
The design, synthesis and in vitro biological evaluation of novel colchicinoids bearing an activated olefin fragment on ring B are presented. The synthetic protocol described in this paper allows the introduction of olefin fragments with various substituents, with the aim to control the compound's activity. Compound 9E, bearing the conjugated ester moiety, exhibits low nanomolar antiproliferative activity and moderate selectivity towards selected cancer cell lines. It induces cell cycle arrest and cell accumulation in the G2/M stage. In contrast, compound 9Z demonstrates a reduced antiproliferative activity in comparison with its isomer 9E, which clearly indicates the importance of the spatial orientation of the ester group on the olefin fragment. The ability to covalently bond with the target protein and other details of the ligand-protein interaction require further investigation.

Experimental Section Synthetic Procedures
Synthesis of intermediates 10-13 and 16 were performed according to a literature protocol described in [14].