Colchicine metallocenyl bioconjugates showing high antiproliferative activities against cancer cell lines

Abstract

A series of ferrocenyl and ruthenocenyl conjugates with colchicine bearing a 1,2,3-triazole moiety were synthesized and their anticancer properties were evaluated. We found that the most potent metallocenyl derivatives Rc4 and Rc5 are 6–7 times more cytotoxic toward HepG2 cells, while Fc4 and Fc5 are two times more cytotoxic toward HCT116 cells as colchicine. We also found that compounds Fc4, Fc5, Rc1 and Rc3–Rc5 are able to induce apoptosis, while compound Fc2 arrests mitosis.

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Introduction

Colchicine 1 is a naturally occurring tubulin-binding poison isolated from autumn crocus (Colchicum autumnale). 1 is an antimitotic compound, which binds to β-tubulin, destabilizes microtubules and promotes their depolymerisation leading to cell cycle arrest and – consequently – apoptosis and cell death. Microtubules are highly dynamic structures, which play a crucial role in many important intracellular processes including intramolecular transport and cell division. Therefore, a search for new compounds which disrupt the dynamic equilibrium of microtubules/tubulin is an important goal in search for new promising anticancer agents. Several tubulin-binding agents such as paclitaxel 2 or vincristine 3 (Fig. 1) are widely used in cancer chemotherapy while usage of 1 is limited to the treatment of gout¹ due to its high systemic toxicity.² Nonetheless, a large number of new analogues and conjugates of 1 exhibiting promising anticancer activities have been synthesized.³ For example, colchicine-based amines,^3h amides,^3f 1,2,3-triazoles,^3d glycosides,³ⁱ and 4-halo derivatives^3b demonstrated high cytotoxicity. Colchicine hybrids with α-tubulin binding drugs, e.g. pironetin, were also studied.^3c

Fig. 1 Structures of tubulin-binding agents: colchicine 1, paclitaxel 2, and vincristine 3.

One of the most efficient methods to prepare new anticancer agents bearing novel and often surprising biological properties is the conjugation of an organic vector with an organometallic moiety.⁴ This method was successfully applied in the synthesis of organometallic analogues of anticancer (e.g. tamoxifen,⁵ paclitaxel,⁶ podophyllotoxin,⁷ plinabulin⁸), antiparasitic (e.g. chloroquine,⁹ praziquantel,¹⁰ monepantel¹¹) and antimicrobial agents.¹²

We demonstrated previously that the introduction of a ferrocenyl moiety into a molecule of a tubulin-binding agent has a positive effect on its biological properties. For example, we found that replacement of the N-benzoyl moiety of paclitaxel with ferrocene not only increased the cytotoxicity of this microtubule-stabilizing agent but also augmented its ability to induce tubulin polymerization.^6a We also found that the conjugation of the ferrocenyl moiety with microtubule destabilizers, such as plinabulin and podophyllotoxin, changes their mode of action. For instance, ferrocenyl analogues of plinabulin turned out to be efficient inhibitors of multidrug resistance proteins ABCB1 and ABCG2 ⁸ while ferrocenyl conjugates of podophyllotoxin became highly cytotoxic toward multidrug resistant cancer cells.^7a These results encouraged us to synthesize metallocenyl conjugates of colchicine and to study their biological properties. Herein we are describing the synthesis and introductory biological assessment of two sets of metallocenyl (ferrocenyl and ruthenocenyl) conjugates of colchicine bearing a 1,2,3-triazolyl linker (Fig. 2). We discuss, in particular, their cytotoxic activity towards cancer cell lines, the impact on the cell cycle and their ability to induce tubulin polymerization.

Fig. 2 Structures of colchicine 1 and its ferrocenyl Fc1–5 and ruthenocenyl Rc1–5 conjugates studied herein.

Results and discussion

Synthesis

Ferrocenyl, Fc1–5, and ruthenocenyl, Rc1–5, conjugates with colchicine were prepared in a 1,3-cycloaddition reaction of 7-azido-7-deacetamidocolchicine 5 with ethynylmetallocenes 12–13, ϖ-alkynoylferrocenes 15–18 or ϖ-alkynoylruthenocenes 20–23 by following a previously described method.¹³ Compound 5 was prepared according to a known procedure starting from colchicine (Scheme 1).

Scheme 1 Synthesis of 7-azido-7-deacetamidocolchicine 5.

Ethynylruthenocene 13 was synthesized by applying the same method as previously described for ethynylferrocene 12 (Scheme 2).¹⁴ First, ruthenocene 7 was acetylated in a reaction with acetic acid and trifluoroacetic anhydride catalysed by trifluoromethanesulfonic acid to give acetylruthenocene 9 in 80% yield. Then 9 reacts with phosphorus oxychloride and DMF providing 3-chloro-3-ruthenocenylacroleine 11 in 70%. Further reaction of 11 with potassium hydroxide in 1,4-dioxane gave ethynylruthenocene 13 in 63%.

Scheme 2 Synthesis of ethynylmetallocenes 12–13.

The corresponding ϖ-alkynoylmetallocenes were prepared via the Friedel–Crafts acylation of ferrocene and ruthenocene with ϖ-alkynoic acids in the presence of trifluoroacetic anhydride and trifluoromethanosulfonic acid (Scheme 3).

Scheme 3 Synthesis of ϖ-alkynoylferrocenes and ruthenocenes.

Finally, the reaction of 5 with ferrocenyl 12, 15–18 and ruthenocenyl 13, 20–23 acetylenes, catalysed by a freshly prepared copper(II) sulphate and sodium ascorbate copper(I) complex with TTTA, gave the desired products in moderate to good yields (35–81%) (Scheme 4). The structures of the products were confirmed by 1D and 2D NMR spectra.

Scheme 4 Synthesis of ferrocenyl Fc1–5 and ruthenocenyl Rc1–5 conjugates with colchicine.

Cytotoxic activity

The cytotoxic potency of the investigated compounds was studied by using the neutral red cell uptake assay in a set of human cell lines derived from different tissues: Colo 205 and HCT116 – colorectal adenocarcinomas; HepG2 – hepatocellular carcinoma; MCF7 – breast adenocarcinoma; and A549 – alveolar basal epithelial cell adenocarcinoma. In general, all of the investigated compounds exhibited biological activities at micromolar or nanomolar concentrations. The cytotoxicity of metallocenyl derivatives was generally comparable to the cytotoxicity of the parental compound 1 and no clear pattern between the linker length, or the kind of the metallocene moiety, and the antiproliferative activity could be demonstrated (see Table 1). The overall toxicity of ferrocenyl compounds (calculated as the sum of ranks for IC₅₀ values for specific compounds across cell lines) could be presented as follows Fc4 > Fc2 > Fc1 ≈ Fc5 > Fc3 while ruthenocene derivatives could be arranged in the following order Rc3 > Rc5 ≥ Rc4 ≥ Rc1 > Rc2. Regarding the cell line sensitivity, MCF7 was the most sensitive line towards ferrocenyl colchicines, followed by HCT116, Colo 205, A549 and HepG2. On the other hand, HCT116 was the most prone towards ruthenocenyl colchicines, followed by MCF7, HepG2, Colo 205 and A549. It should be stressed here that A549 cells were virtually insensitive to Rc4 and Rc5 although the reason for such resistance is unclear and requires further investigation. On the other hand both Rc4 and Rc5 are 6–7 times more cytotoxic toward HepG2, while Fc4 and Fc5 are two times more cytotoxic toward HCT116 than 1, however the explanation of the reason for the high cytotoxicity of these compounds requires further investigation.

Table 1 Cytotoxicity of the ferrocenyl Fc1–5 and ruthenocenyl Rc1–5 colchicine conjugates in comparison with colchicine 1 as determined by using the neutral red uptake assay. 95%-confidence intervals are given below (please note that due to the log-transformation of the data required to perform IC₅₀ calculations, these are asymmetrical). The IC₅₀ values were calculated based on three independent experiments. >100 denotes situations in which the IC₅₀ values were not possible to determine (<50% viability was not achieved in the concentration range used (up to 100 μM))

Compound	IC50
	A549	Colo 205	HCT116	HepG2	MCF7
Fc1	0.42	0.16	0.14	0.89	0.059
	0.29–0.59	0.08–0.29	0.11–0.18	0.55–1.44	0.051–0.070
Fc2	0.10	0.068	0.046	0.11	0.017
	0.07–0.16	0.043–0.106	0.033–0.065	0.06–0.19	0.012–0.024
Fc3	1.49	0.83	0.29	2.30	0.008
	0.29–4.17	0.58–1.17	0.20–0.45	0.55–4.01	0.003–0.016
Fc4	0.37	0.11	0.006	0.045	0.002
	0.02–7.44	0.01–1.07	0.004–0.009	0.017–0.121	0.001–0.006
Fc5	1.23	0.20	0.009	1.27	0.033
	0.15–9.88	0.07–0.59	0.004–0.019	0.48–3.34	0.026–0.042
Rc1	0.36	0.40	0.12	0.45	0.31
	0.18–0.71	0.23–0.68	0.07–0.21	0.17–2.17	0.10–1.01
Rc2	0.43	0.64	0.39	4.65	2.96
	0.29–0.62	0.44–0.92	0.28–0.55	1.72–12.6	1.71–5.113
Rc3	0.24	0.24	0.040	0.22	0.072
	0.15–0.36	0.08–0.7	0.029–0.054	0.12–0.44	0.046–0.114
Rc4	>100	0.31	0.058	0.004	0.13
		0.05–2.03	0.021–0.159	0.003–0.007	0.03–0.53
Rc5	>100	0.090	0.048	0.003	0.71
		0.006–1.47	0.007–0.309	0.002–0.004	0.13–3.78
1	0.061	0.017	0.016	0.023	0.005
	0.039–0.097	0.013–0.023	0.014–0.018	0.018–0.030	0.004–0.006

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Effects on the cell cycle

As the principal mechanism of colchicine action is based on tubulin binding, microtubule destabilization and induction of the cell cycle arrest, we wanted to check whether this is also the case for metallocenyl colchicines. We have chosen the Colo 205 cell line as our model for cell cycle analysis as the IC₅₀ value for all the investigated compounds was below 1 μM for these cells. The expected mitotic arrest was observed for 1 and, to a lower extent, for Fc2 (Table 2). However, the subG1 phase (sign of apoptosis) was observed for Fc4, Fc5, Rc1 and Rc3–Rc5. At the same time, the percentage of cells in the G2/M phase was significantly lower for Fc4, Fc5, and Rc3–Rc5 colchicine derivatives. In the case of Rc5 (the most evident case), the results are suggestive for S phase blockage, which may indicate that some vital resources were depleted in cells exposed to this compound. This mechanism however, cannot be excluded for the other compounds mentioned above.

Table 2 Cell cycle phase distribution in Colo 205 cells exposed to 10 nM of ferrocenyl and ruthenocenyl conjugates. Average data ± SEM from 3 independent experiments. Control – cells incubated in a complete medium alone, DMSO – cells incubated in a complete medium with the addition of 0.1% of DMSO (solvent control)

	subG1	G1	S	G2/M
Control	0.0 ± 0.0	59.6 ± 3.5	30.5 ± 2.9	9.5 ± 1.6
DMSO	0.0 ± 0.0	60.4 ± 4.4	29.2 ± 2.6	8.5 ± 1.6
1	3.6 ± 1.3	52.2 ± 5.3	22.1 ± 3.8	20.6 ± 4.4
Fc1	0.0 ± 0.0	61.6 ± 4.7	30.4 ± 3.8	7.1 ± 0.9
Fc2	0.0 ± 0.0	50.2 ± 3.0	37.1 ± 2.5	11.0 ± 1.3
Fc3	0.0 ± 0.0	55.3 ± 4.8	34.8 ± 3.6	8.6 ± 0.9
Fc4	8.0 ± 2.4	56.1 ± 4.6	30.5 ± 3.4	4.6 ± 0.6
Fc5	2.8 ± 0.5	64.1 ± 5.6	28.9 ± 3.8	3.8 ± 0.5
Rc1	6.0 ± 2.3	49.8 ± 9.0	38.1 ± 5.6	5.7 ± 1.4
Rc2	0.0 ± 0.0	58.9 ± 6.3	32.4 ± 4.9	7.9 ± 2.2
Rc3	7.1 ± 1.4	59.4 ± 4.7	27.3 ± 2.0	2.6 ± 1.5
Rc4	7.7 ± 1.6	59.3 ± 3.7	27.5 ± 2.3	2.5 ± 0.4
Rc5	16.3 ± 2.3	32.6 ± 4.9	44.5 ± 3.4	5.4 ± 0.7

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Influence on tubulin polymerisation

To explain the cell cycle observations in a more detailed way, we analysed the ability of the metallocenyl colchicine conjugates to induce or interfere with tubulin polymerization. Generally, all of the investigated compounds were tubulin polymerization inhibitors although weaker than colchicine. All of the studied compounds were able to completely inhibit tubulin polymerization at a rather high concentration (10 μM, Fig. 3). Fc1 and Fc2 were generally the least potent among the compounds studied although the differences were not overwhelming. Nevertheless, no clear correlation between metallocenyl colchicine cytotoxicity and its ability to disrupt tubulin polymerization could be found. Our previous study has shown that the conjugation of tubulin polymerisation inhibitors such as plinabulin⁸ and podophyllotoxin^7a with ferrocene decreased the ability of the conjugates to inhibit tubulin polymerisation.

Fig. 3 Concentration-dependent ability to induce tubulin polymerisation by Fc1–5 and Rc1–5 in comparison with 1, paclitaxel (PCL) and vinblastine (VINBL). Lack of the polymerisation rate bars indicates that the polymerisation rate reached 0 AU per minute (total inhibition).

Reactive oxygen species production

It is postulated that the biological effects of metallocenes can be attributed to their redox properties.^4f,15 We have previously demonstrated that even though ferrocenyl paclitaxel analogues are able to stimulate intracellular reactive oxygen species (ROS) production, the redox effects are not as important for their biological activity as their ability to bind tubulin.¹⁶ We have been intrigued, however, by the differential effects exerted by the investigated colchicine analogues on the cells of different tissue origins (see Table 1). It seems plausible that cells derived from organs that are metabolically active or continuously exposed to reactive oxygen species such as liver¹⁷ or lungs¹⁸ and thus producing higher amounts of glutathione and other antioxidant barrier elements are less sensitive to redox active compounds. Such effects are readily observed for ferrocenyl colchicines (A549 and HepG2 cells were the most resistant to these compounds) but no such correlation can be inferred for ruthenocene colchicine conjugates. Therefore, we decided to study intracellular ROS production both in less (A549) and in more sensitive (HCT116) cells exposed to metallocenyl colchicines. As a marker of ROS production, we decided to choose dihydrorhodamine 123 (DHR123) – a leuco dye, which is known to be readily oxidized by hydrogen superoxide in a reaction catalysed by peroxidases¹⁹ to a highly fluorescent rhodamine 123 further sequestered in the mitochondria. The initial results were quite surprising: ROS production in A549 cells was suppressed by 20 to 40% by metallocene colchicines, irrespective of their chemical structure. Additionally, such an effect was also observed for colchicine itself (Fig. 4A). On the other hand, ROS production was tremendously increased (by 250–475%) in HCT116 cells and again – this effect was observed for all the compounds studied, including parental 1 (Fig. 4C). However, both rhodamine 123 (formed by the oxidation of DHR123) and colchicine are known substrates for ABCB1 – a plasma membrane xenobiotic transporter involved in the multidrug resistance phenomenon.²⁰ Thus, the potential activity of this protein should be excluded, if such results are to be considered biologically relevant. Employing calcein accumulation assay (data not shown) we were able to detect significant ABCB1 activity in HCT116 cells. Therefore, we decided to repeat the experiments but in the presence of 10 μM verapamil – an efficient inhibitor of ABCB1. Such treatment did not significantly alter the results obtained for A549 cells. Both colchicine and its metallocene derivatives (with a notable exception of Rc1 and Rc3 for which the results were statistically insignificant compared to the control) reduced the level of intracellular DHR123 oxidation. The underlying reason for this phenomenon is unknown and requires further investigation. More interesting results were observed in the case of HCT116. The intracellular fluorescence level was much lower than in verapamil-untreated cells, however DHR123 oxidation was statistically more pronounced for Fc4, Rc1, and Rc3–Rc5. It is thus probable that the high antiproliferative activity of Fc4 and Rc3–Rc5 can be at least partially attributed to their redox potential. Another interesting conclusion resulting from the observation of effects of verapamil on rhodamine 123 accumulation in HCT116 cells is that metallocene colchicines do interact with ABCB1. Nevertheless, the ABCB1 activity is not enough to allow the cells escape the fatal effects of metallocene colchicines as HCT116 cells were the second most sensitive cells in terms of antiproliferative potential of the investigated compounds.

Fig. 4 Dihydrorhodamine 123 oxidation by A549 (panels A and B) and HCT116 (panels C and D) cells after 4-hour-exposure to 1, Fc1–5 and Rc1–5. Results are expressed as mean ± SEM, n = 3. Fluorescence of control cells (incubated with DHR123 and an appropriate amount of DMSO) was considered 100% for a given experimental set. Panels A and C – cells incubated in the absence of ABCB1 inhibitor, panels B and D – cells incubated in the presence of ABCB1 inhibitor (10 μM verapamil).

Partition coefficients (log P)

Cytotoxicity is generally directly related to the intracellular concentration of the drug. Accumulation of drugs is dependent on their lipophilicity, therefore, increasing the drug lipophilicity may result in an elevation of the intracellular drug concentration. Such an approach was successfully applied in a synthesis of highly lipophilic anthracycline analogs, such as annamycin and idarubicin.²¹ The octanol–water partition coefficients (log P) of the investigated compounds were determined using the HPLC method.²² All of the studied metallocene conjugates exhibited significantly higher log P values than colchicine (Table 3). Ruthenocene compounds demonstrated slightly higher log P values than their corresponding ferrocenyl counterparts but compound lipophilicity could be arranged as follows for both metallocenes: M5 ≈ M2 > M1 > M4 > M3 (M = Fc, Rc). However, the differences of log P values between compounds were presumably too low to see the effect of drug lipophilicity on the cells.

Table 3 Lipophilicity values of ferrocenyl Fc1–5 and ruthenocenyl Rc1–5 colchicine conjugates in comparison with colchicine 1

Compound	log P
Fc1	3.203 ± 0.002
Fc2	3.307 ± 0.003
Fc3	2.973 ± 0.001
Fc4	3.037 ± 0.003
Fc5	3.246 ± 0.003
Rc1	3.237 ± 0.004
Rc2	3.393 ± 0.009
Rc3	3.060 ± 0.002
Rc4	3.123 ± 0.002
Rc5	3.317 ± 0.002
1	1.30 (ref. 23)

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Conclusions

We have synthesized two series of metallocenyl, ferrocenyl and ruthenocenyl conjugates with colchicine bearing a 1,2,3-triazolylalkyl linker between the colchicine and metallocenyl moieties. Their cytotoxic properties were studied towards a set of cancer cells of different organ origins. We found that the cytotoxicity of the conjugate was generally similar to that of colchicine. However, the mode of action of metallocene colchicine conjugates differs from the mode of action of the parental compound (interactions of the investigated compounds with tubulin and microtubules as well as their effects on the cell cycle were different from the effects of 1). Additionally, our results clearly demonstrated also that the cellular response to ferrocenyl colchicines differs from the one to ruthenocenyl compounds. Some of the synthesised conjugates exhibit higher cytotoxicity than 1. These effects can be at least partially attributed to metallocene colchicine-induced ROS production. Further studies are required to elucidate these differences as well as to explain the basis of cytotoxicity of some of the investigated metallocenyl colchicines.

Experimental section

General

All reactions were carried out using standard Schlenk techniques. Commercially available solvents (HPLC) and reagents were used as received. Dichloromethane (DCM) was distilled from calcium hydride and stored over activated molecular sieves 4A (8–12 mesh). Column chromatography was performed on FLUKA Silica gel 60 for flash chromatography (0.040–0.063 mm, 230–400 mesh). TLC chromatography was performed on aluminium sheets coated with Merck 5735 Kieselgel 60F254. 1D (¹H, ¹³C{¹H}) and 2D (¹H–¹H COSY, ¹H–¹³C HSQC or HMQC, ¹H–¹³C HMBC) NMR spectra were recorded on a Bruker ARX 600 MHz (spectrometer frequency 600.3 MHz for ¹H and 150.9 MHz for ¹³C). Chemical shifts for the NMR spectra are referenced relative to the residual protons in the deuterated solvent (CDCl₃δ = 7.26 ppm for ¹H and δ = 77.00 ppm for ¹³C{¹H}). The spectra were recorded at room temperature (301 K), chemical shifts are in ppm and coupling constants in Hz. ESI-MS spectra were recorded in positive mode on a Varian 500-MS LC ion trap spectrometer (samples were dissolved in acetonitrile). HPLC analyses were performed on a Shimadzu Prominence system equipped with a PDA detector and using a Phenomenex Kinetex 5μ XB-C18 100 Å column 150 × 4.6 mm. Detection was recorded at wavelengths 220 nm, 254 nm and 280 nm. The purity was tested at a 1 ml min⁻¹ flow rate using a mixture of A : B 40 : 60 (v : v), solvents: phase A was 20 mM MOPS, 0.15% 1-decylamine, pH = 7.4; phase B was 0.25% 1-octanol in methanol. Despite numerous attempts, we were unable to obtain correct results of elemental analyses for all colchicine conjugates and the purity of the investigated compound was confirmed by HPLC analysis (purity >95%).

Acetylruthenocene 9. 4.63 g (3.01 ml, 22 mmol) of trifluoroacetic anhydride was added to a solution of 1.31 g (1.25 ml, 21.8 mmol) of acetic acid in 50 ml of anhydrous dichloromethane. Then 4.63 g (20 mmol) of ruthenocene was added and after its complete dissolving 3.0 g (1.76 ml, 20 mmol) of trifluoromethanesulfonic acid was added dropwise. The resulting solution was stirred at RT for 2 h and the reaction was quenched by adding 100 ml of water. The product was extracted with dichloromethane, and the organic solution was washed with water, sodium bicarbonate and brine, dried with sodium sulphate and evaporated. The crude product was purified by column chromatography on silica using dichloromethane as the solvent. A pure product was obtained in 87% yield (4.75 g). This compound was identical with an authentic sample.²⁴

3-Chloro-3-ruthenocenylacroleine 11. 6.28 g (4.0 ml, 41 mmol) of phosphorus(V) oxychloride was added dropwise for 30 min to cool to 0 °C in DMF (25 ml) placed in a round bottom flask. Then a solution of 2.0 g (7.3 mmol) of 9 in a minimal amount of anhydrous DMF was added dropwise and the resulting mixture was stirred at 0 °C for 2 h and poured into 500 ml of water. The resulting mixture was neutralized by adding of solid sodium acetate and the product was extracted with diethyl ether. The organic solution was washed with water, brine and dried over sodium sulphate and evaporated to dryness. Chromatography on silica using dichloromethane–cyclohexane (8–1) as the eluent gave pure 11 in 70% yield (1.64 g). This compound was identical with an authentic sample.²⁵ Mp 97–100 °C; elemental analysis found: C, 49.0; H, 3.8. C₁₃H₁₁ClORu requires C, 48.8; H, 3.5%; IR (KBr) ν_max/cm⁻¹: 3306w, 3086m, 2851m, 2742w, 1663vs, 1603vs, 1445m, 1410s, 1384s, 1350m, 1330m, 1253s, 1241s, 1208m, 1133s, 1100m, 1035s, 997s, 817vs, 787s, 691m, 649m, 534w, 456vs; ¹H NMR (CDCl₃) δ 10.02 (d, J = 7.1 Hz, 1H, CHO), 6.37 (d, J = 7.1 Hz, 1H, C=CH̲), 5.08 (t, J = 1.8 Hz, 2H, Cp), 4.81 (t, J = 1.8 Hz, 2H, Cp), 4.62 (s, 5H, Cp), ¹³C{¹H} NMR (CDCl₃) δ 191.2 (C=O), 153.7 (C̲=CH), 120.2 (C=C̲H), 84.8 (Cp_ipso), 73.3 (Cp), 72.7 (Cp), 70.4 (Cp).

Ethynylruthenocene 13. 714 mg of powdered potassium hydroxide was added to a solution 1.30 g (4.07 mmol) of 11 in 60 ml of 1,4-dioxane and the resulting mixture was refluxed for 2 h. Then the reaction was quenched by adding 100 ml of water and the resulting solution was neutralized with 5% citric acid. The product was extracted with cyclohexane; the organic solution was washed with brine dried over sodium sulphate and evaporated. A pure product was isolated in 64% yield (660 mg) by column chromatography using cyclohexane as the eluent. This compound was identical with an authentic sample.²⁶ Mp 71–73 °C; elemental analysis found: C, 56.6; H, 4.0. C₁₂H₁₀Ru requires C, 56.5; H, 4.0%; IR (KBr) ν_max/cm⁻¹: 3268vs, 3118w, 3102w, 2924w, 2103s, 1442m, 1407m, 1390m, 1260w, 1222s, 1201m, 1098s, 1025s, 904m, 849w, 811vs, 653s, 619s, 556m, 519s, 378m, 428s; ¹H NMR (CDCl₃) δ 4.86 (br s, 2H, Cp), 4.60 (s, 5H, Cp), 4.55 (br s, 2H, Cp), 2.64 (s, 1H, H_acetylene); ¹³C{¹H} NMR (CDCl₃) δ 81.5 (C̲CH), 73.9 (Cp), 73.5 (CC̲H), 71.9 (Cp), 70.6 (Cp).

General procedure A – synthesis of ϖ-alkynoylruthenocenes 19–23

These compounds were prepared by applying the previously described procedure for the synthesis of ϖ-alkynoylferrocenes.²⁷

(3-Trimethylsilyl)propynoylruthenocene 19. 462 mg (305 μl, 2.2 mmol) of trifluoroacetic anhydride was added to a solution of 312 mg (2.2 mmol) of 3-(trimethylsilyl)propynoic acid in 10 ml of anhydrous dichloromethane. To the resulting solution, 462 mg (2.0 mmol) of ruthenocene was added and after its complete dissolution 300 mg (176 μl, 2.0 mmol) of trifluoromethanesulfonic acid was added. The resulting solution was stirred at RT for 2 h and poured into 50 ml of water. The product was extracted with dichloromethane, and the organic solution was washed with water, sodium bicarbonate, brine, dried over sodium sulphate and evaporated. Chromatography on silica using dichloromethane as the eluent gave a pure product in 52% yield (371 mg). Mp 92–94.5 °C; elemental analysis found: C, 54.2; H, 5.2. C₁₆H₁₈ORuSi requires C, 54.1; H, 5.1%; IR (KBr) ν_max/cm⁻¹: 3237m, 3110m, 3098m, 2959s, 2900m, 2168m, 2159m, 1629vs, 1590m, 1505w, 1448vs, 1411s, 1392s, 1372vs, 1350m, 1268vs, 1249vs, 1213w, 1102s, 1083vs, 1021s, 1004vs, 894vs, 847vs, 819vs, 761s, 752s, 725s, 705m, 632s, 569m, 482s, 423m; ESI-MS calc. for C₁₆H₁₈ORuSi 356.0 (M⁺) found 356.9 (M⁺, 41%); ¹H NMR (CDCl₃) δ 5.22 (t, J = 1.6 Hz, 2H, Cp), 4.84 (d, J = 1.6 Hz, 2H, Cp), 4.62 (s, 5H, Cp), 0.27 (s, 9H, TMS); ¹³C{¹H} NMR (CDCl₃) δ 178.2 (C=O), 101.6 (C), 95.6 (C), 85.1 (C), 74.3 (Cp), 72.8 (Cp), 71.8 (Cp), −0.6 (TMS).

(4-Pentyn-1-oyl)ruthenocene 21. This compound was prepared in 59% yield (369 mg) according to general procedure A starting from 192 mg (2.2 mmol) of 4-pentynoic acid. Mp 79.5–82.0 °C; elemental analysis found: C, 56.6; H, 4.0. C₁₂H₁₀Ru requires C, 56.5; H, 4.0%; IR (KBr) ν_max/cm⁻¹: 3306s, 3285m, 3106m, 3090s, 2945m, 2922w, 2905w, 2115m, 1683vs, 1637m, 1456vs, 1426m, 1406s, 1396s, 1477s, 1261vs, 1225m, 1187w, 1098s, 1084vs, 1041w, 1029s, 995s, 980s, 904w, 893w, 863m, 843m, 833m, 815vs, 645vs, 606vs, 544m, 530s, 510m, 450s, 428s; ESI-MS calc. for C₁₅H₁₄ORu 312.0 (M⁺) found 313.3 (M + H, 100%)⁺; ¹H NMR (CDCl₃) δ 5.12 (t, J = 1.7 Hz, 2H, Cp), 4.79 (t, J = 1.8 Hz, 2H, Cp), 4.61 (s, 5H, Cp), 2.87 (t, J = 7.4 Hz, 2H, CH̲₂CH₂CCH), 2.54 (td, J = 7.3, 2.6 Hz, 2H, CH₂CH̲₂CCH), 1.97 (t, J = 2.6 Hz, 1H, CH₂CH₂CCH̲); ¹³C{¹H} NMR (CDCl₃) δ 199.7 (C=O), 83.8 (CH₂CH₂C̲CH), 83.3 (Cp_ipso), 73.6 (Cp), 72.0 (Cp), 70.6 (Cp), 68.6 (CH₂CH₂CC̲H), 37.6 (C̲H₂CH₂CCH), 13.4 (CH₂C̲H₂CCH); elemental analysis calc. for C₁₅H₁₄ORu C-57.9, H-4.5%, found C-57.8, H-4.7%

(5-Hexyn-1-oyl)ruthenocene 22. This compound was prepared in 75% yield (490 mg) according to general procedure A starting from 246 mg (242 μl, 2.2 mmol) of 5-hexynoic acid. Mp 72–73.4 °C; elemental analysis found: C, 58.9; H, 5.1. C₁₆H₁₆ORu requires C, 59.1; H, 5.0%; IR (KBr) ν_max/cm⁻¹: 3253vs, 3111m, 3104m, 3092w, 2957m, 2943m, 2927s, 2906m, 2847m, 2359w, 2343w, 1656vs, 1457s, 1430s, 1402s, 1380s, 1357s, 1316m, 1254s, 1181m, 1166m, 1199s, 1982m, 1052s, 1039m, 1015m, 997m, 987m, 880m, 871m, 841m, 819vs, 796m, 710s, 687s, 648m, 546m, 513s, 455s, 425m; ESI-MS calc. for C₁₆H₁₆ORu 326.0 (M⁺) found 326.0 (M⁺, 60%), 327.0 ((M + H)⁺, 100%); ¹H NMR (CDCl₃) δ 5.12 (t, J = 1.8 Hz, 2H, Cp), 4.77 (t, J = 1.8 Hz, 2H, Cp), 4.60 (s, 5H, Cp), 2.77 (t, J = 7.1 Hz, CH̲₂CH₂CH₂CCH), 2.74 (td, J = 6.8, 2.6 Hz, 2H, CH₂CH₂CH̲₂CCH), 1.99 (t, J = 2.6 Hz, 1H, CH₂CH₂CH₂CCH̲), 1.90–1.85 (m, 2H, CH₂CH̲₂CH₂CCH); ¹³C{¹H} NMR (CDCl₃) δ 201.6, 84.o (2 × C, CH₂CH₂CH₂C̲CH and Cp_ipso), 73.4 (Cp), 72.0 (Cp), 70.7 (Cp), 68.9 (CH₂CH₂CH₂CC̲H), 37.1 (C̲H₂CH₂CH₂CCH), 23.2 (CH₂C̲H₂CH₂CCH), 17.9 (CH₂CH₂C̲H₂CCH).

(6-Heptyn-1-oyl)ruthenocene 23. This compound was prepared in 72% yield (488 mg) according to general procedure A starting from 278 mg (2.2 mmol) of 6-heptynoic acid. Mp 87–89.5 °C; elemental analysis found: C, 60.1; H, 5.5. C₁₇H₁₈ORu requires C, 60.2; H, 5.4%; IR (KBr) ν_max/cm⁻¹: 3256vs, 3086s, 2948m, 2939m, 2919m, 2904m, 2869m, 2360w, 2343w, 1671vs, 1453s, 1409s, 1393m, 1379s, 1357m, 1303m, 1290w, 1524s, 1213m, 1101s, 1096m, 1050m, 1026m, 1000m, 982m, 892m, 876w, 869w, 821s, 787w, 752w, 684s, 525w, 514m, 460s, 434s, 418m; ESI-MS calc. for C₁₇H₁₈ORu 340.0 (M⁺) found 341.0 ((M + H)⁺, 100%); ¹H NMR (CDCl₃) δ 5.10 (t, J = 1.7 Hz, 2H, Cp), 4.77 (t, J = 1.7 Hz, 2H, Cp), 4.59 (s, 5H, Cp), 2.61 (t, J = 7.4 Hz, 2H, CH̲₂CH₂CH₂CH₂CCH), 2.23 (td, J = 7.1, 2.6 Hz, 2H, CH₂CH₂CH₂CH̲₂CCH), 1.96 (t, J = 2.6 Hz, 1H, CH₂CH₂CH₂CH₂CCH̲), 1.81–1.76 (m, 2H, CH₂CH̲₂CH₂CH₂CCH), 1.61–1.56 (m, 2H, CH₂CH₂CH̲₂CH₂CCH); ¹³C{¹H} NMR (CDCl₃) δ 202.1 (C=O), 84.2 (C̲CH or Cp_ipso), 84.0 (C̲CH or Cp_ipso), 73.4 (Cp), 71.9 (Cp), 70.7 (Cp), 68.5 (CH₂CH₂CH₂CH₂CC̲H), 35.3 (C̲H₂CH₂CH₂CH₂CCH), 28.1 (CH₂CH₂C̲H₂CH₂CCH), 24.1 (CH₂C̲H₂CH₂CH₂CCH), 18.3 (CH₂CH₂CH₂C̲H₂CCH).

Propynoylruthenocene 20. A large excess of powdered potassium fluoride (1.28 g) was added to a solution of 320 mg (0.9 mmol) of 19 and 10 mg of 18-crown-6 in 6 ml of tetrahydrofuran. The resulting solution was stirred at RT for 30 min and poured into 50 ml of water. The product was extracted with dichloromethane, and the organic solution was washed with water, brine, dried over sodium sulfate and evaporated to dryness. Chromatography on silica using dichloromethane as the eluent gave pure 20 in 98% yield (250 mg). This compound was identical with an authentic sample.²⁸ Mp 99–101.2 °C; elemental analysis found: C, 55.1; H, 3.7. C₁₃H₁₀ORu requires C, 55.1; H, 3.6%; IR (KBr) ν_max/cm⁻¹: 3212s, 3092w, 2090s, 1625vs, 1449s, 1411w, 1394w, 1375m, 1350w, 1339w, 1324s, 1101m, 1072s, 1034m, 1023w, 1001m, 992m, 882m, 869w, 849w, 841w, 823m, 765m, 725m, 477m, 430m; ¹H NMR (CDCl₃) δ 5.25 (t, J = 1.8 Hz, 2H, Cp), 4.86 (t, J = 1.8 Hz, 2H, Cp), 4.64 (s, 5H, Cp), 3.11 (s, 1H, H_acetylene); ¹³C{¹H} NMR (CDCl₃) δ 177.7 (C=O), 84.4 (Cp_ipso), 81.0 (C̲CH), 76.3 (CC̲H), 74.5 (Cp), 72.8 (Cp), 71.7 (Cp).

General procedure B – synthesis of colchicine-metallocenyl 1,2,3-triazolyl conjugates Fc1–5 and Rc1–5

These compounds were prepared by applying the previously described procedure for the synthesis of 1-aryl-4-ferrocenyl-1,2,3-triazoles.¹³

7-Deacetamido-7-(4-ferrocenyl-1H-1,2,3-triazol-1-yl)-colchicine Fc1. 75 μl of 0.1 M CuSO₄ and 75 μl of 0.1 M sodium ascorbate and 300 μl of 0.01 M of TTTA were placed in a vial. The resulting colourless solution was added to a solution of 30 mg (0.143 mmol) of ethynylferrocene 12 and 55 mg (0.143 mmol) of 7-deacetamido-7-azidocolchicine 5 in 2 ml of tert-butanol. The reaction mixture was stirred at RT for 3 days and the solvents were evaporated. Chromatography on silica using ethyl acetate (or ethyl acetate–cyclohexane 4–1) gave pure Fe1 in 65% yield (55 mg). Mp 143–147 °C; elemental analysis found: C, 63.5; H, 5.6; N, 6.7. C₃₂H₃₁FeN₃O₅·½H₂O requires C, 63.8; H, 5.4; N, 7.0%; IR (KBr) ν_max/cm⁻¹: 3102w, 2934m, 2845w, 1618s, 1587vs, 1488s, 1461s, 1431m, 1399s, 1350s, 1323s, 1283m, 1254vs, 1195m, 1181m, 1139s, 1096s, 1046m, 1020m, 1002m, 984m, 818m, 509m; ESI-MS calc. for C₃₂H₃₁FeN₃O₅ 593.2 (M⁺) found 593.0 (M⁺, 100%), 594.0 ((M + H)⁺, 66%); R_f (HPLC) τ = 9.6 min; ¹H NMR (CDCl₃) δ 7.50 (s, 1H, H_triazolyl), 7.28 (d, J = 10.7 Hz, 1H, H-12), 6.79 (d, J = 10.8 Hz, 1H, H-11), 6.61 (s, 1H, H-4), 6.57 (s, 1H, H-8), 5.41 (dd, J = 11.9, 5.4 Hz, 1H, H-7), 7.72 (m, 1H, Cp), 4.71–4.70 (m, 1H, Cp), 4.29 (br s, 2H, Cp), 4.08 (s, 5H, Cp), 3.97 (s, 3H, 10-OCH₃), 3.95 (s, 3H, OCH₃), 3.93 (s, 3H, OCH₃), 3.79 (s, 3H, OCH₃), 2.86–2.81 (m, 1H, H-5), 2.77–2.74 (m, 1H, H-6), 2.67–2.60 (m, 2H, H-5 and H-6); ¹³C{¹H} (CDCl₃) δ 178.8 (C-9), 164.3 (C-10), 153.9 (C_Ary), 151.1 (C_Ary), 147.6 (C_Ary), 147.2 (C_Ary), 141.7 (C_Ary), 135.2 (C-12), 134.3 (C_Ary), 133.6 (C_Ary), 131.9 (C-8), 125.0 (C_Ary), 119.0 (CH_triazolyl), 111.6 (C-11), 107.5 (C-4), 69.7 (Cp), 68.8 (Cp), 66.8 (Cp), 62.6 (C-7), 61.3 (OCH₃), 61.2 (OCH₃), 56.3 (OCH₃), 56.1 (OCH₃), 35.3 (C-5), 29.7 (C-6).

7-Deacetamido-7-(4-ferrocenoyl-1H-1,2,3-triazol-1-yl)-colchicine Fc2. This compound was prepared in 77% yield (118 mg) according to general procedure B starting from 95 mg (0.248 mmol) of 5 and 59 mg (0.248 mmol) of 15. Mp 152–156 °C; elemental analysis found: C, 64.0; H, 5.1; N, 6.6. C₃₃H₃₁FeN₃O₆ requires C, 63.8; H, 5.0; N, 6.8%; IR (KBr) ν_max/cm⁻¹: 3223w, 2927s, 2853m, 1623vs, 1587vs, 1527m, 1488s, 1459s, 1399m, 1377m, 1351m, 1322m, 1254vs, 1195m, 1180m, 1140s, 1097s, 1047m, 1036m, 1022m, 1001m, 984m, 940w, 925w, 841m, 826m, 770m, 500m; ESI-MS calc. for C₃₃H₃₁FeN₃O₆ 621.2 (M⁺) found 621.0 (M⁺, 74%), 622.0 ((M + H)⁺, 100%); R_f (HPLC) τ = 12.3 min; ¹H NMR (CDCl₃) δ 8.25 (s, 1H, H_triazolyl), 7.32 (d, J = 10.7 Hz, 1H, H-12), 6.81 (d, J = 10.8 Hz, 1H, H-11), 6.61 (s, 1H, H-4), 6.49 (s, 1H, H-8), 5.49–5.46 (m, 3H, Cp and H-7), 4.64 (s, 2H, Cp), 4.17 (s, 5H, Cp), 3.97 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 3.89 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 2.86–2.81 (m, 1H, H-6), 2.79–2.76 (m, 1H, H-5), 2.68–2.60 (m, 2H, H-5 and H-6); ¹³C{¹H} NMR (CDCl₃) δ 188.8 (C̲OFc), 178.7 (C-9), 164.4 (C-10), 154.0 (C_Ary), 151.1 (C_Ary), 149.1 (C_Ary), 147.1 (C_Ary), 141.9 (C_Ary), 135.5 (C-12), 134.2 (C_Ary), 133.4 (C_Ary), 131.8 (C-8), 127.0 (CH_triazolyl), 111.7 (C_Ary), 107.6 (C-4), 77.7 (Cp_ipso), 73.0 (Cp), 71.7 (Cp), 71.5 (Cp), 70.3 (Cp), 62.9 (C-7), 61.2 (2 × OCH₃), 56.4 (OCH₃), 56.1 (OCH₃), 35.3 (C-6), 29.7 (C-5).

7-Deacetamido-7-(4-(3-ferrocenyl-3-oxoprop-1-yl)-1H-1,2,3-triazol-1-yl)-colchicine Fc3. This compound was prepared in 80% yield (98 mg) according to general procedure B starting from 72 mg (0.188 mmol) of 5 and 50 mg (0.188 mmol) of 16. Mp 108–112 °C; elemental analysis found: C, 64.7; H, 6.3; N, 5.6. C₃₅H₃₅FeN₃O₆ requires C, 64.7; H, 5.4; N, 6.5%; IR (KBr) ν_max/cm⁻¹: 3095w, 2926s, 2852m, 1664s, 1618s, 1587vs, 1488s, 1457s, 1430m, 1399s, 1379m, 1350s, 1322s, 1254vs, 1195m, 1180m, 1140s, 1096s, 1086s, 1048m, 1020m, 1003m, 983m, 924w, 900w, 824m, 487m; ESI-MS calc. for C₃₅H₃₅FeN₃O₆ 649.2 (M⁺) found 649.0 (M⁺, 56%), 650.0 ((M + H)⁺, 100%); R_f (HPLC) τ = 7.0 min; ¹H NMR (CDCl₃) δ 7.43 (s, 1H, H_triazolyl), 7.26 (d, J = 10.7 Hz, 1H, H-12), 6.78 (d, J = 10.8 Hz, 1H, H-11), 6.57 (br s, 2H, H-4 and H-8), 5.34 (dd, J = 12.1, 5.9 Hz, 1H, H-7), 4.78 (brs, 2H, Cp), 4.47 (brs, 2H, Cp), 4.13 (s, 5H, Cp), 3.96 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 3.90 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 3.21–3.19 (m, 2H, CH₂CH̲₂COFc), 3.14–3.11 (m, 2H, CH̲₂CH₂COFc), 2.83–2.78 (m, 1H, H-6), 2.70 (dd, J = 14.5, 6.5 Hz, 1H, H-5), 2.62–2.56 (m, 1H, H-5), 2.53–2.47 (m, 1H, H-6); ¹³C{¹H} NMR (CDCl₃) δ 203.2 (CH₂CH₂C̲OFc), 178.9 (C-9), 164.3 (C-10), 153.9 (C_Ary), 151.0 (C_Ary), 147.7 (C_Ary), 147.3 (C_Ary), 141.7 (C_Ary), 135.2 (C-12), 134.5 (C_Ary), 133.6 (C_Ary), 132.0 (C-8), 125.0 (C_Ary), 122.2 (CH_triazolyl), 111.7 (C-11), 107.4 (C-4), 78.8 (Cp_ipso), 72.2 (Cp), 69.8 (Cp), 69.3 (Cp), 69.2 (Cp), 62.5 (C-7), 61.3 (OCH₃), 61.2 (OCH₃), 56.4 (OCH₃), 56.1 (OCH₃), 38.7 (CH₂C̲H₂COFc), 35.4 (C-6), 29.8 (C-5), 20.0 (C̲H₂CH₂COFc).

7-Deacetamido-7-(4-(4-ferrocenyl-4-oxobut-1-yl)-1H-1,2,3-triazol-1-yl)-colchicine Fc4. This compound was prepared in 75% yield (130 mg) according to general procedure B starting from 100 mg (0.261 mmol) of 5 and 73 mg (0.261 mmol) of 17. Mp 102–105 °C; elemental analysis found: C, 65.4; H, 6.3; N, 5.4. C₃₆H₃₇FeN₃O₆ requires C, 65.2; H, 5.6; N, 6.3%; IR (KBr) ν_max/cm⁻¹: 3096w, 1926s, 2853m, 1663s, 1618s, 1587vs, 1572vs, 1488s, 1457vs, 1379m, 1350s, 1322s, 1283m, 1254vs, 1195m, 1180m, 1140s, 1096s, 1048m, 1020m, 1003m, 984m, 924w, 844m, 824m, 754s, 667w, 533w, 488m; ESI-MS calc. for C₃₆H₃₇FeN₃O₆ 663.2 (M⁺) found 663.0 (M⁺, 55%), 664.0 ((M + H)⁺, 100%), 593.0 (M⁺, 100%), 594.0 ((M + H)⁺, 66%); R_f (HPLC) τ = 7.6 min; ¹H NMR (CDCl₃) δ 7.39 (s, 1H, H_triazolyl), 7.27 (d, J = 11.3 Hz, 1H, H-12), 6.78 (d, J = 10.7 Hz, 1H, H-11), 6.58 (s, 1H, H-4), 6.53 (s, 1H, H-8), 5.37 (dd, J = 12.1, 5.7 Hz, 1H, H-7), 4.79–4.78 (m, 1H, Cp), 4.78–4.77 (m, 1H, Cp), 4.47 (brs, 2H, Cp), 4.17 (s, 5H, Cp), 3.97 (brs, 3H, OCH₃), 3.93 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 2.85 (t, J = 7.5 Hz, 2H, CH̲₂CH₂CH₂COFc), 2.81–2.76 (m, 3H, H-6 and CH₂CH̲₂CH₂COFc), 2.72 (dd, J = 13.1, 6.0 Hz, 1H, H-5), 2.60 (dt, J = 13., 6.4 Hz, 1H, H-5), 2.56–2.52 (m, 1H, H-6), 2.12–2.08 (m, 2H, CH₂CH₂CH̲₂COFc); ¹³C{¹H} NMR (CDCl₃) δ 204.1 (COFc), 178.9 (C-9), 164.3 (C-10), 153.9 (C_Ary), 151.1 (C_Ary), 148.0 (C_Ary), 147.8 (C_Ary), 141.8 (C_Ary), 135.2 (C-12), 134.5 (C_Ary), 133.6 (C_Ary), 131.9 (C-8), 125.1 (C_Ary), 121.4 (CH_triazolyl), 111.7 (C-11), 107.5 (C-4), 79.0 (Cp_ipso), 72.2 (Cp), 69.8 (Cp), 69.4 (Cp), 62.5 (C-7), 61.3 (OCH₃), 61.2 (OCH₃), 56.4 (OCH₃), 56.1 (OCH₃), 38.7 (CH₂C̲H₂CH₂COFc), 35.4 (C-6), 29.8 (C-5), 25.2 (C̲H₂CH₂CH₂COFc), 23.9 (CH₂CH₂C̲H₂COFc).

7-Deacetamido-7-(4-(5-ferrocenyl-5-oxopent-1-yl)-1H-1,2,3-triazol-1-yl)-colchicine Fc5. This compound was prepared in 57% yield (100 mg) according to general procedure B starting from 100 mg (0.261 mmol) of 5 and 77 mg (0.261 mmol) of 18. Mp 91–96 °C; elemental analysis found: C, 65.4; H, 6.1; N, 6.0. C₃₇H₃₉FeN₃O₆ requires C, 65.6; H, 5.8; N, 6.2%; IR (KBr) ν_max/cm⁻¹: 3092w, 2930s, 2855m, 1735m, 1663s, 1618s, 1586vs, 1488s, 1456s, 1398s, 1376m, 1350s, 1322s, 1282s, 1253vs, 1195m, 1179m, 1139s, 1095s, 1047m, 1019m, 1002m, 983m, 924w, 890w, 823m, 486m; ESI-MS calc. for C₃₇H₃₉FeN₃O₆ 677.2 (M⁺) found 677.0 (M⁺, 43%), 678.0 ((M + H)⁺, 100%); R_f (HPLC) τ = 10.2 min; ¹H NMR (CDCl₃) δ 7.38 (s, 1H, H_triazolyl), 7.27 (d, J = 9.7 Hz, 1H, H-12), 6.68 (d, J = 10.7 Hz, 1H, H-11), 6.58 (s, 1H, H-4), 6.53 (s, 1H, H-8), 5.36 (dd, J = 11.9, 5.6 Hz, 1H, H-7), 4.76 (br s, 2H, Cp), 4.47 (t, J = 1.6 Hz, 2H, Cp), 4.17 (s, 5H, Cp), 3.97 (br s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 2.81–2.77 (m, 3H, CH̲₂CH₂CH₂CH₂COFc and H-6), 2.75–2.70 (m, 3H, CH₂CH₂CH₂CH̲₂COFc and H-5), 2.67–2.52 (m, 2H, H-5 and H-6), 1.78–1.77 (m, 4H, CH₂CH̲₂CH̲₂CH₂COFc); ¹³C{¹H} NMR (CDCl₃) δ 204.3 (COFc), 171.1 (C-9), 164.3 (C-10), 153.9 (C_Ary), 151.0 (C_Ary), 148.2 (C_Ary), 147.8 (C_Ary), 141.8 (C_Ary), 135.2 (C-12), 134.5 (C_Ary), 133.7 (C_Ary), 131.9 (C-8), 125.1 (C_Ary), 121.2 (CH_triazolyl), 111.7 (C-11), 107.5 (C-4), 79.1 (Cp_ipso), 72.1 (Cp), 69.7 (Cp), 69.3 (2 × C, Cp), 62.5 (C-7), 61.3 (OCH₃), 61.2 (OCH₃), 56.4 (OCH₃), 56.1 (OCH₃), 39.3 (CH₂CH₂CH₂C̲H₂COFc), 35.4 (C-6), 29.8 (C-5), 28.9 (CH₂CH₂C̲H₂CH₂COFc or CH₂C̲H₂CH₂CH₂COFc), 25.6 (C̲H₂CH₂CH₂CH₂COFc), 23.9 (CH₂CH₂C̲H₂CH₂COFc or CH₂C̲H₂CH₂CH₂COFc).

7-Deacetamido-7-(4-ruthenocenyl-1H-1,2,3-triazol-1-yl)-colchicine Rc1. This compound was prepared in 87% yield (145 mg) according to general procedure B starting from 100 mg (0.261 mmol) of 5 and 67 mg (0.261 mmol) of 13. Mp 153–157 °C; elemental analysis found: C, 60.1; H, 5.0; N, 6.3. C₃₄H₃₅N₃O₅Ru requires C, 60.2; H, 4.9; N, 6.6%; IR (KBr) ν_max/cm⁻¹: 3099m, 2930s, 2839m, 1618s, 1586vs, 1488s, 1460s, 1429m, 1398s, 1349s, 1322s, 1282m, 1254vs, 1195m, 1180m, 1138s, 1097vs, 1045s, 1019s, 1001s, 983m, 843m, 810s, 751m, 491m; ESI-MS calc. for C₃₂H₃₁N₃O₅Ru 638.7 (M⁺) found 639.0 (M⁺, 53%), 640.0 ((M + H)⁺, 100%); R_f (HPLC) τ = 10.1 min; ¹H NMR (CDCl₃) δ 7.43 (s, 1H, H_triazolyl), 7.26 (d, J = 10.7 Hz, 1H, H-12), 6.78 (d, J = 10.9 Hz, 1H, H-11), 6.59 (s, 1H, H-4), 6.49 (s, 1H, H-8), 5.37 (dd, J = 12.0, 5.6 Hz, 1H, H-7), 5.13 (s, 1H, Cp), 5.11 (s, 1H, Cp), 4.64 (s, 2H, Cp), 4.49 (s, 5H, Cp), 3.96 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 2.80–2.75 (m, 1H, H-6), 2.74–2.71 (m, 1H, H-5), 2.63–2.53 (m, 2H, H-5 and H-6); ¹³C{¹H} NMR δ 178.8 (C-9), 164.3 (C-10), 153.9 (C_Ary), 151.0 (C_Ary), 147.6 (C_Ary), 146.4 (C_Ary), 141.8 (C_Ary), 135.2 (C-12), 134.3 (C_Ary), 133.6 (C_Ary), 131.9 (C-8), 125.1 (C_Ary), 119.1 (CH_triazolyl), 111.6 (H-11), 107.4 (H-4), 78.8 (Cp_ipso), 71.5 (Cp), 71.3, 70.6 (Cp), 69.3 (Cp), 62.5 (C-7), 61.3 (OCH₃), 61.2 (OCH₃), 56.3 (OCH₃), 56.1 (OCH₃), 35.3 (C-6), 29.7 (C-5).

7-Deacetamido-7-(4-ruthenocenoyl-1H-1,2,3-triazol-1-yl)-colchicine Rc2. This compound was prepared in 35% yield (61 mg) according to general procedure B starting from 100 mg (0.261 mmol) of 5 and 74 mg (0.261 mmol) of 20. Mp 136–140 °C; elemental analysis found: C, 61.4; H, 5.8; N, 5.7. C₃₃H₃₁N₃O₆Ru·½C₆H₁₄ requires C, 60.9; H, 5.4; N, 5.9%; IR (KBr) ν_max/cm⁻¹: 3112w, 2925s, 2852s, 1622s, 1587vs, 1528m, 1488s, 1459s, 1399m, 1376m, 1351m, 1322m, 1255vs, 1195m, 1180m, 1140s, 1088s, 1046m, 1035m, 1022m, 1000m, 984m, 841m, 819m, 768m, 731w, 492w, 468w; ESI-MS calc. for C₃₃H₃₁N₃O₆Ru 667.1 (M⁺) found 667.0 (M⁺, 49%), 668.0 ((M + H)⁺, 100%); R_f (HPLC) τ = 12.5 min; ¹H NMR (CDCl₃) δ 8.19 (s, 1H, H_triazolyl), 7.31 (d, J = 10.7 Hz, 1H, H-12), 6.80 (d, J = 10.9 Hz, 1H, H-11), 6.61 (s, 1H, H-4), 6.44 (s, 1H, H-8), 5.78 (t, J = 1.7 Hz, 2H, Cp), 5.44 (dd, J = 12.0, 5.4 Hz, 1H, H-7), 4.90–4.88 (m, 2H, Cp), 4.54 (s, 5H, Cp), 3.98 (s, 3H, OCH₃), 3.95 (s, 3H, OCH₃), 3.92 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 2.82–2.75 (m, 2H, H-5 and H-6), 2.67–2.56 (m, 2H, H-5 and H-6); ¹³C{¹H} NMR δ 186.4 (CORc), 178.7 (C-9), 164.4 (C-10), 154.0, 151., 148.7, 147.1, 141.9, 135.5 (C-12), 134.2, 133.4, 131.8 (C-8), 127.0 (CH_triazolyl), 125.0, 111.7 (C-11), 107.6 (C-4), 82.5 (Cp_ipso), 74.0 (2 × Cp), 72.9 (Cp), 72.8 (Cp), 72.4 (Cp), 62.8 (C-7), 61.2 (2 × OCH₃), 56.4 (OCH₃), 56.2 (OCH₃), 35.3 (C-6), 29.7 (C-5).

7-Deacetamido-7-(4-(3-ruthenocenyl-3-oxoprop-1-yl)-1H-1,2,3-triazol-1-yl)-colchicine Rc3. This compound was prepared in 45% yield (82 mg) according to general procedure B starting from 100 mg (0.261 mmol) of 5 and 81 mg (0.261 mmol) of 21. Mp 95–99 °C; elemental analysis found: C, 55.8; H, 5.4; N, 5.2. C₃₅H₃₅N₃O₆Ru·3H₂O requires C, 56.1; H, 5.5; N, 5.6%; IR (KBr) ν_max/cm⁻¹: 3096w, 2927s, 2853m, 1670s, 1618s, 1578vs, 1488s, 1457s, 1430m, 1399s, 1377m, 1350m, 1322m, 1254vs, 1195m, 1179m, 1139s, 1097s, 1086s, 1048m, 1019m, 1002m, 923m, 844w, 818m, 488w; ESI-MS calc. for C₃₅H₃₅N₃O₆Ru 695.2 (M⁺) found 696.0 ((M + H)⁺, 95%), 718.0 ((M + Na)⁺ 100%); R_f (HPLC) τ = 7.9 min; ¹H NMR (CDCl₃) δ 7.38 (brs, 1H, H_triazolyl), 7.26 (d, J = 10.6 Hz, 1H, H-12), 6.78 (d, J = 10.8 Hz, 1H, H-11), 6.57 (s, 1H, H-4), 6.55 (s, 1H, H-8), 5.32 (dd, J = 12.1, 5.9 Hz, 1H, H-7), 5.09 (brs, 2H, Cp), 4.75 (brs, 2H, Cp), 4.51 (s, 5H, Cp), 3.96 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 3.10–3.04 (m, 4H, CH̲₂CH̲₂CORc), 2.81 (dt, J = 11.9, 6.5 Hz, 1H, H-6), 2.70 (dd, J = 13.4, 6.5 Hz, 1H, H-5), 2.59 (dt, J = 13.4, 6.5 Hz, 1H, H-5), 2.53–2.49 (m, 1H, H-6); ¹³C{¹H} NMR (CDCl₃) δ 201.2 (CORc), 178.9 (C-9), 164.3 (C-10), 153.9 (C_Ary), 151.0 (C_Ary), 147.7 (C_Ary), 147.1 (C_Ary), 141.7 (C_Ary), 135.2 (C-12), 134.5 (C_Ary), 133.6 (C_Ary), 132.0 (C-8), 125.0 (C_Ary), 122.1 (CH_triazolyl), 111.7 (C-11), 107.4 (C-4), 83.6 (Cp_ipso), 73.5 (Cp), 72.0 (Cp), 71.9 (Cp), 70.7 (Cp), 70.6 (Cp), 62.5 (C-7), 61.2 (OCH₃), 56.4 (OCH₃), 56.1 (OCH₃), 37.9 (C̲H₂CH₂CORc or CH₂C̲H₂CORc), 35.4 (C-6), 29.7 (C-5), 20.3 (C̲H₂CH₂CORc or CH₂C̲H₂CORc).

7-Deacetamido-7-(4-(4-ruthenocenyl-4-oxobut-1-yl)-1H-1,2,3-triazol-1-yl)-colchicine Rc4. This compound was prepared in 86% yield (95 mg) according to general procedure B starting from 60 mg (0.156 mmol) of 5 and 51 mg (0.156 mmol) of 22. Mp 95–100 °C; elemental analysis found: C, 58.7; H, 5.8; N, 5.2. C₃₆H₃₇N₃O₆Ru·½H₂O requires C, 58.8; H, 5.5; N, 5.7%; IR (KBr) ν_max/cm⁻¹: 2925s, 2853s, 1668s, 1618s, 1587vs, 1488s, 1457vs, 1398m, 1376m, 1350m, 1322m, 1232m, 1254vs, 1195m, 1179m, 1139s, 1097s, 1086s, 1084m, 1020m, 1001m, 984m, 843m, 818m, 489w; ESI-MS calc. for C₃₆H₃₇N₃O₆Ru 709.2 (M⁺) found 709.0 (M⁺, 72%), 710.0 ((M + H)⁺, 100%); R_f (HPLC) τ = 8.6 min; ¹H NMR (CDCl₃) δ = 7.38 (br s, 1H, H_triazolyl), 7.27 (d, J = 11.9 Hz, 1H, H-12), 6.70 (d, J = 10.8 Hz, 1H, H-11), 6.58 (s, 1H, H-4), 6.52 (s, 1H, H-8), 5.36 (dd, J = 12.1, 5.7 Hz, 1H, H-7), 5.10 (brs, 1H, Cp), 5.09 (br s, 1H, Cp), 4.75 (s, 2H, Cp), 4.56 (s, 5H, Cp), 3.96 (OCH₃), 3.93 (OCH₃), 3.91 (OCH₃), 3.75 (OCH₃), 2.80–2.77 (m, 3H, H-6 and CH̲₂CH₂CH₂CORc), 2.72 (dd, J = 13.4, 6.5 Hz, 1H, H-5), 2.70–2.67 (m, 2H, CH₂CH₂CH̲₂CORc), 2.63–2.57 (m, 1H, H-5), 2.56–2.51 (m, 1H, H-6), 2.06–2.06 (m, 2H, CH₂CH̲₂CH₂CORc); ¹³C{¹H} NMR (CDCl₃) δ 202.2 (CORc), 178.9 (C-9), 164.3 (C-10), 153.9 (C_Ary), 151.0 (C_Ary), 147.9 (C_Ary), 147.8 (C_Ary), 141.7 (C_Ary), 135.2 (C-12), 134.4 (C_Ary), 133.6 (C_Ary), 131.9 (C-8), 125.0 (CH_triazolyl), 121.3 (C_Ary), 111.7 (C-11), 107.5 (C-4), 83.9 (Cp_ipso), 73.5 (Cp), 71.9 (Cp), 70.7 (Cp), 62.5 (C-7), 61.3 (OCH₃), 61.2 (OCH₃), 56.4 (OCH₃), 56.1 (OCH₃), 38.1 (CH₂CH₂C̲H₂CORc), 35.4 (C-6), 29.7 (C-5), 25.1 (C̲H₂CH₂CH₂CORc), 24.4 (CH₂C̲H₂CH₂CORc).

7-Deacetamido-7-(4-(5-ruthenocenyl-5-oxopent-1-yl)-1H-1,2,3-triazol-1-yl)-colchicine Rc5. This compound was prepared in 29% yield (38 mg) according to general procedure B starting from 70 mg (0.183 mmol) of 5 and 62 mg (0.183 mmol) of 23. Mp 87–92 °C; elemental analysis found: C, 61.4; H, 5.7; N, 5.6. C₃₇H₃₉N₃O₆Ru requires C, 61.5; H, 5.4; N, 5.8%; IR (KBr) ν_max/cm⁻¹: 3096w, 2927s, 2850m, 1667s, 1618s, 1587vs, 1488s, 1457s, 1399s, 1376m, 1350s, 1322s, 1254vs, 1195m, 1180m, 1139s, 1096s, 1047m, 1020s, 1002m, 983m, 843m, 817m, 637w, 489w; ESI-MS calc. for C₃₇H₃₉N₃O₆Ru 723.2 (M⁺) found 723.0 (M⁺, 70%), 724.0 ((M + H)⁺, 100%), 746 ((M + Na)⁺, 55%); R_f (HPLC) τ = 11.2 min; ¹H NMR (CDCl₃) δ 7.38 (s, 1H, H_triazolyl), 7.27 (d, J = 11.0 Hz, 1H, H-12), 6.78 (d, J = 10.7 Hz, 1H, H-11), 6.58 (s, 1H, H-4), 6.53 (s, 1H, H-8), 5.36 (dd, J = 12.0, 5.7 Hz, 1H, H-7), 5.07 (brs, 2H, Cp), 4.74 (brs, 2H, Cp). 4.56 (brs, 5H, Cp), 3.96 (s, 3H, OCH₃), 3.94 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 2.81–2.76 (m, 3H, H-6 and CH₂CH₂CH̲₂CH₂CORc), 2.72 (dd, J = 13.0, 6.1 Hz, 1H, H-5), 2.64–2.53 (m, 4H, H-5 and H-6 and CH₂CH₂CH₂CH̲₂CORc), 1.72–1.71 (m, 4H, CH̲₂CH₂CH̲₂CH₂CORc); ¹³C{¹H} NMR (CDCl₃) δ 202.4 (CORc), 178.9 (C-9), 164.3 (C-10), 153.9 (C_Ary), 151.0 (C_Ary), 148.2 (C_Ary), 147.8 (C_Ary), 141.7 (C_Ary), 135.2 (C-12), 134.5 (C_Ary), 133.6 (C_Ary), 131.9 (C-8), 125.1 (C_Ary), 121.2 (CH_triazolyl), 111.7 (C-11), 107.5 (C-4), 84.0 (Cp_ipso), 73.4 (Cp), 71.9 (Cp), 70.7 (Cp), 62.5 (C-7), 61.3 (OCH₃), 61.2 (OCH₃), 56.4 (OCH₃), 56.1 (OCH₃), 38.5 (CH₂CH₂CH₂CH̲₂CORc), 35.4 (C-6), 29.8 (C-5), 28.8 (CH₂CH₂C̲H₂CH₂CORc or C̲H₂CH₂CH₂CH₂CORc), 25.5 (CH₂CH₂C̲H₂CH₂CORc), 24.3 (CH₂CH₂C̲H₂CH₂CORc or C̲H₂CH₂CH₂CH₂CORc).

Tubulin polymerization

Tubulin polymerization was determined using the commercially available tubulin polymerization assay kit (Cytoskeleton, Inc., Cat. #BK011P). The investigated compounds were dissolved in DMSO and were tested in a range of concentrations (from 0.1 μM to 30 μM). The final concentration of DMSO in all samples was 0.5%. Paclitaxel and vinblastine were used as a positive- and negative-control of tubulin polymerization, respectively. The fluorescence was measured at 37 °C for 120 min at 355/40 nm excitation and 430/8 nm emission wavelengths using an EnVision Multilabel Plate Reader (PerkinElmer).

Viability assay

The toxicity of compounds was determined using the neutral red uptake assay. The cells were seeded at a density of 10 000 cells per well in 100 μl of a complete medium in wells of a 96-well plate. After 24 hours the cells were allowed to attach to the surface, and the investigated compounds were added. Stock solutions were prepared in DMSO, and the final concentration of the solvent was kept identical in all samples and did not exceed 0.1% v/v. The tested compounds were studied in a concentration range between 10 nM and 100 μM. Following 70-hour-incubation, neutral red solution was added to the cell to a final concentration of 0.33% w/v. After further 2 hours, the medium was aspirated and the wells were washed once with phosphate-buffered saline for 5 minutes in a gyratory shaker. Then PBS was aspirated and 200 μl of the solubilizer solution (1% v/v acetic acid in 50% v/v ethanol) was added. The plates were kept on a gyratory shaker for another 5 minutes and then the absorbance was measured at 540 nm using the EnVision Multilabel Plate Reader (PerkinElmer). The results were calculated as a percentage of the controls and the IC₅₀ values for each cell line and substance were calculated with GraphPad Prism 5.02 software (GraphPad Inc.) using the four-parameter non-linear logistic regression.

Cell cycle

Exponentially growing Colo 205 cells (100 000 cells per well seeded in 6-well plates 24 h before time 0) were treated with 10 nM of the tested compound for 48 h. The cells were then harvested by trypsinisation, washed twice with ice-cold PBS and fixed in 70% ethanol. After storing the cells for at least 8 h at 4 °C, they were stained with a propidium iodide staining solution (75 μM propidium iodide and 50 Kunitz units per ml of RNAse A in PBS) for 30 min at 37 °C. The samples were analysed on an LSRII (Becton Dickinson) instrument (excitation 488 nm, emission 575/26 nm) and the cell cycle phase distribution was determined with FlowJo 7.6.1 software (FlowJo, LLC) using a built-in cell cycle analysis module (Watson pragmatic algorithm).

Reactive oxygen species production

The oxidation rate of dihydrorhodamine 123 (DHR123) was considered a marker of intracellular production of reactive oxygen species. Exponentially growing HCT116 and A549 cells were exposed to 1 μM of the tested compound for 4 h in the presence of 1 μM DHR123 and/or 10 μM verapamil (an inhibitor of ABCB1). The cells were then harvested by trypsinisation and suspended in a complete growth medium. The samples were then collected on an LSRII (Becton Dickinson) instrument and median fluorescence (excitation 488 nm, emission 530/30 nm) was analysed. The median fluorescence of cells incubated with DHR123 and DMSO alone was considered 100% in a given experiment. The statistical significance of the differences was assessed by one-way ANOVA and Bonferroni's post-hoc test, assuming 0.05 as the significance level.

Calcein accumulation assay

The ABCB1 activity in HCT116 cells was measured by performing the calcein accumulation assay as described previously.⁸ Briefly, the cells were trypsinized and suspended in a complete cell culture medium pre-warmed to 37 °C at a final density of approx. 1 × 10⁶ ml⁻¹. Samples were supplemented with calcein AM at a 70 nM final concentration. A specific inhibitor (10 μM verapamil) was added to the parallel samples. Intracellular fluorescence was measured immediately after dye addition (time 0) and every 3–5 minutes up to approx. 20 minutes using LSRII (Becton Dickinson) instrument set at 488 nm excitation and 530/30 emission. Accumulation curves were plotted and the accumulation rate was assessed with the GraphPad Prism 5.02 software (GraphPad Inc.). The difference between the accumulation rate for dye only and the inhibitor samples was evaluated as a measure of ABCB1 activity.

log P determination

log P values of the investigated compounds were determined by a HPLC method²² on using a Phenomenex Kinetex 5μ XB-C18 100 Å column 150 × 4.6 mm using two mobile phases: phase A was 20 mM MOPS, 0.15% 1-decylamine, pH = 7.4; phase B was 0.25% 1-octanol in methanol. Briefly, samples dissolved in methanol with uracil as an internal reference were injected in the column and eluted with mobile phase B between 55, 60, 65, and 70%. The log P values were calculated as previously described^22b using 4-methoxyphenol, naphthalene, acetophenone and paclitaxel as standards. These experiments were repeated three times for each of the compounds.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This study was financially supported by the National Science Centre Poland (NCN) based on decision DEC-2012/05/B/ST5/00300.

References

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7dt03229c

‡ These two authors contributed equally to this work.

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