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Synthesis and Biological Evaluation of Furanoallocolchicinoids Yuliya V. Voitovich, Ekaterina S. Schegravina, Nikolay S. Sitnikov, Vladimir I. Faerman, Valery V. Fokin, Hans-Guenther Schmalz, Sebastien Combes, Diane Allegro, Pascale Barbier, Irina P. Beletskaya, Elena V. Svirshchevskaya, and Aleksey Yurievich Fedorov J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Dec 2014 Downloaded from http://pubs.acs.org on December 12, 2014
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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The Synthesis and Biological Evaluation of Furanoallocolchicinoids Yuliya V. Voitovicha, Ekaterina S. Shegravinaa, Nikolay S. Sitnikova, Vladimir I. Faermana, Valery V. Fokina, Hans-Gunther Schmalzb, Sebastien Combesc, Diane Allegrod, Pascal Barbierd, Irina P. Beletskayae, Elena V. Svirshchevskayaf*, Alexey Yu. Fedorova*
a
Department of Organic Chemistry, Nizhny Novgorod State University, Gagarina av. 23, Nizhny Novgorod 603950, Russian Federation, Fax: +7 831-462-30-85, E-mail: [email protected] b
Department of Chemistry, University of Cologne, Greinstrasse 4, 50939 Koln (Germany) Fax: +49 221-470-3064, Tel: +49 221 470 3063, E-mail: [email protected] c
CRCM, CNRS UMR7258, Laboratory of Integrative Structural and Chemical Biology
(ISCB), INSERM, U1068, Institut Paoli-Calmettes, Aix-Marseille Universit´e, UM105, F-13009, Marseille, France, Fax: +33 (0)491 164 540; Tel: +33 (0)86 97 73 31, E-mail: [email protected], d
Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, CRO2 INSERM UMR 911, Universite d’Aix-Marseille, Faculte de Pharmacie, 27 Boulevard Jean Moulin, Marseille 13005, France Tel : +33 (0)491 835 616, E-mail : [email protected]
e
Department of Chemistry, M.V. Lomonosov Moscow State University, Vorobyevy Gory, 119992 Moscow, Russia, Tel: +7 (495) 938 36 18, E-mail: [email protected]
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Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Laboratory of Cell
Interactions, GSP-7, Miklukho-Maklaya Street, 16/10, 117997 Moscow, Russian Federation, Tel/Fax +7 (495) 330 40-11, E-mail: [email protected]
Corresponding authors Prof. Fedorov Alexey Yu. a
Department of Organic Chemistry, Nizhny Novgorod State University, Gagarina av. 23, Nizhny Novgorod 603950, Russian Federation, Fax: +7 831-462-30-85, E-mail: [email protected] Dr. Svirshchevskaya Elena V. e
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Laboratory of Cell
Interactions, GSP-7, Miklukho-Maklaya Street, 16/10, 117997 Moscow, Russian Federation, Tel/Fax (495) 330 40-11, E-mail: [email protected]
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Keywords: colchicine, allocolchicinoids, cross-coupling, antitumor activity, tubulin binding agents. Abstract A series of conformationally flexible furan-derived allocolchicinoids was prepared from commercially available colchicine in good to excellent yields using a three-step reaction sequence. Cytotoxicity studies indicated potent activity of two compounds against human epithelial and lymphoid cell lines (AsPC-1, HEK293 and Jurkat) as well as against Wnt-1 related murine epithelial cell line W1308. The results of in vitro experiments demonstrated that the major effect of these compounds was the induction of cell cycle arrest in the G2/M phase as a direct consequence of effective tubulin binding. In vivo testing of the most potent furanoallocolchicinoid 10c using C57BL/6 mice inoculated with Wnt-1 tumor cells indicated significant inhibition of the tumor growth.
1. Introduction Due to the essential role of microtubules in cell growth and division, inhibition of microtubule formation is considered promising in the treatment of cancer.1 Microtubules are formed via a polymerization process involving α- and β-tubulin protein subunits.2 Interference with the dynamic equilibrium between the assembly of tubulin into microtubules or, inversely, the depolymerization of microtubules leads to the arrest of cell division and eventually to apoptosis. Compounds inhibiting either of these important processes are referred to as spindle poisons or antimitotics.1,2
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The search for selective inhibitors of tubulin assembly or disassembly has led to the development of some of the most useful antitumor drugs currently in clinical use.1,2 These are the naturally occurring Vinca alkaloids3 as well as the taxoids,4 which are widely used for the treatment of breast, ovarian, and non-small cell lung carcinomas. However, these structurally rather complex molecules share several drawbacks: they are difficult to synthesize (thus expensive) and prone to the development of resistance.1,5 Therefore, the development of novel small-molecule antimitotics with low-toxicity remains a clinically relevant challenge. Colchicine (1) (Figure 1), isolated from Colchicum autumnale, was the first tubulin-destabilizing agent ever discovered.6 Although it has been employed since ancient times in the treatment of acute gout and familial Mediterranean fever, high cardio- and neurotoxic adverse effects prevent its use in cancer chemotherapy.7 However, structural analogs of colchicine, such as allocolchicine (2),8 combretastatin A-4 (3),9 4-arylcoumarins (4),10 (Figure 1) and their derivatives, represent promising leads in the search for new anticancer agents addressing the colchicine binding site of tubulin.11 The ability of colchicine and its analogs to block mitosis by affecting the microtubule dynamics is well studied,12,13 and several colchicine analogs such as AbT-751 and indibulin are currently in clinical trials for patients suffering from advanced solid tumors.14-16 Clinical trials of other colchicine analogues have been discontinued due to poorly controlled drug absorption, neurotoxicity17 or low efficacy.18 Nevertheless, the search for novel colchicinoids remains an interesting challenge because, besides acting as mitosis inhibitors, recent studies have shown that microtubule-targeting agents may also exhibit effects in the areas of (i) mitosis-independent cell death and metastasis, (ii) tumor angiogenesis, and (iii) vasculardisrupting activity.12 Moreover, the development of drugs overcoming Pgp/β-III-tubulinmediated drug resistance is a key task in cancer research.11d, 19
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A molecular modeling study suggested that a key structural feature of colchicine binding site inhibitors is the presence of two closely neighbored non-coplanar hydrophobic aromatic fragments A and B, bearing hydrogen bond donor or acceptor functionalities (Figure 1).11a Indeed, the structural similarity of compounds 1 to 4 arises from the presence of two adjacent polyoxygenated aromatic rings A and B, which are non-coplanar.11 Figure 1 For a long time, the unique substitution pattern of ring B in molecules such as 1-4 was considered essential for the high affinity towards the colchicine binding site of tubulin. However, recent investigations by the groups of Wang,20 Simonini,21 and in our laboratories22 demonstrated that replacement of the aromatic B-ring by a hydrophobic benzoheterocyclic moiety23 leads to highly active compounds (Figure 2). For instance, the indole derivatives 520 and 722a were found to inhibit in vitro microtubule assembly in a substoichiometric mode of action. The phosphate prodrugs 6 demonstrated high antitumor activity both in vitro and in vivo, notwithstanding a minimal effect on microtubule organization with respect to 3 (CA-4).21 The indole-derived allocolchicine analogs 8 and 9 caused proliferation inhibition and apoptosis induction at nanomolar or even in sub-nanomolar concentrations.22b These derivatives actually represent the first examples of heteroarene-based allocolchicinoids.24 Figure 2 As structural analogs of pyrrole-fused colchicinoids 8 and 922b we considered the corresponding furan-derived allocolchicinoids of type 10 to be promising compounds for biological testing. We herein report a short and convenient semi-synthetic access to such compounds (of type 10) and
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demonstrate their promising antitumor activity (including in vivo mice experiments) in comparison with colchicine (1) and combretastatin A-4 (3).
2. Results and Discussion Chemistry Compounds of type 10 were prepared in only four steps starting from commercially available colchicine (1) by making use of the previously described colchiceine - N-acetyliodocolchinol rearrangement24,25 and transition metal-catalyzed methods for the construction of the furan ring (Scheme 1). First, the tropolone ether function of colchicine (1) was hydrolyzed by heating with HCl in AcOH to afford colchiceine (11) in 97% yield.26 The oxidative degradation of 11 to iodocolchinol (12) was also achieved in high yield using the Windaus procedure (NaOH, I2, KI, H2O).27,28 The Sonogashira coupling of iodocolchinol (12) with terminal alkynes (13)29 then afforded the alkyne intermediates of type 14, which under the reaction conditions (Pd/Cu catalysis, base) directly underwent cyclisation30 to afford the desired benzofurans of type 10. Scheme 1 Table 1 The Sonogashira coupling of iodocolchinol (12) with phenylacetylene (13a) or 2pyridylacetylene (13b) was best performed at 65 °C using Pd(OAc)2/CuI/PPh3 (5:10:15 mol%) as the catalytic system in MeCN in the presence of K3PO4 as a base to afford the cyclization products 10a and 10b, respectively, in 86 and 83% isolated yield (Table 1). In contrast, when iodocolchinol 12 was reacted with non-(hetero)aryl-substituted terminal alkynes, such as
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propargyl alcohol (13c), under the same conditions, the cyclization products were obtained in much lower yield (e.g. 30% of 10c). However, as the yield of the furanoallocolchicinoids 10 proved to be strongly base-dependent, different bases (NEt3, DIPEA, K2CO3, Cs2CO3, t-BuOK, or AcOK) were tested in the reaction between 12 and 13c using the same catalytic system as before. Under the optimized conditions (Pd(OAc)2/CuI/PPh3 (5:10:15 mol%), AcOK (3 equiv.), MeCN, 70 oC) the reaction of iodocolchinol (12) with different functionalized terminal alkynes (13c-k) afforded the products 10a-i in good to high yields (Table 1). Thus, the desired benzofurans of type 10 were efficiently obtained in only three preparative steps starting from commercially available colchicine (1). An attempt to prepare benzofuran 10j with an aminomethyl group in the side chain revealed that this compound was rather unstable. Noteworthy, we did not succeed at all in synthesizing the TMS-substituted furanoallocolchicine 10k, because the reaction of 12 with trimethylsilylacetylene (13k) stopped at the stage of intermediate 14k, which was isolated in 62% yield. This correlates well with the literature reports that the Pd/Cu-catalyzed cyclization of ortho-trimethylsilylethynyl- and ortho-ethynyl-substituted phenols, anilines or thiols require rather harsh conditions (thermolysis or photolysis).31 The pyrrolidinomethyl-substituted benzofuran 10l was obtained in 80% yield by reacting 12 with propargylbromide 13l in the presence of catalytic amounts of Pd(PPh3)2Cl2 and CuI in pyrrolidine as a solvent (Scheme 2). This type of reaction might also be useful for the preparation of related aminomethyl-substituted furanoallocolchicinoids employing different primary and secondary amines. Scheme 2 One should note that furan-derived allocolchicinoids 10 were prepared using semi-synthetic pathway in three steps from commercially available colchicine with the retention of the absolute
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configuration at C7. Proposed methodology seems to be advantageous in comparison with the eight-to-twelve-step synthesis used for the preparation of allocolchicinoids 8 and 9, affording the target compounds as a racemic mixture.22b
Biology In vitro cytotoxicity of new compounds against cancer cell lines The in vitro cytotoxicity of all synthesized compounds against human cancer cell lines was investigated by MTT assay after the incubation of the cells with the compounds for 72 h. The representative inhibitory curves for HEK293, Jurkat, and AsPC-1 cells are shown in Figure 3. For comparison, effects of all 10 compounds synthesized are shown only for HEK293 cells in two sets (Figure 3 A and B), where A demonstrates the effects of the compounds with high activity, and B – with low activity. The results for Jurkat and AsPC-1 cells are presented only for the compounds with high activity (Figures 3C and 3D). Figure 3 All compounds, including 10c and 10d as the most toxic ones, induced ca. 60% inhibition even at very high concentrations (Figure 3, black dotted lines) showing the absence of direct necrotic or pro-apoptotic toxicity. The same is true for many previously published colchicine analogues.32 Activity of compounds inducing 50% of the maximal cell growth inhibition (IC50, Figure 3, grey dotted lines) are summarized in Table 2. The sensitivity of different cell lines to these compounds slightly varied due to variations in cell concentrations, different rates of division, and experimental errors. For the comparison we have also included IC50 for
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indoloallocolchicinoids 8a and 9a synthesized by us earlier, the most active compounds from 8 and 9 series.22b Table 2 Compounds 10a and 10b bearing an aromatic (or heteroaromatic) moiety in the side chain of the benzofuran fragment were not active. In contrast, compounds 10c and 10d, with a hydroxymethyl or a 1-hydroxyethyl substituent at the furan ring, exhibited potent antiproliferative activity with IC50 values in the nanomolar range. While allocolchicinoid 10e also contains a hydroxyl group in the pseudo-benzylic position (like 10c and 10d) it exhibited lower cytotoxicity, possibly because the hydrophobic cyclopentane ring shields the OH group. Interestingly, compound 10f with a 2-hydroxyethyl side chain showed a two-order of magnitude lower activity in comparison with the hydroxylated compounds 10c and 10d. Compound 10g, which can be regarded as an ester prodrug of 10c, exhibits a significantly lower activity in comparison with the “parent” compound 10c.33 A low cytotoxicity was also observed for the hydrophobic ester 10h. The most cytotoxic among the 8-series compounds22b was 8a, an indolyl analog of furanoallocolchicinoid 10.
It was less active than 10c. On the other hand, the isomeric
colchicinoid 9a manifested the same order of activity as 10c (Table 2). These results demonstrate that a hydroxymethyl fragment in the α-position of the furan ring of type 10 compounds significantly improves the pharmacophore. Therefore it was interesting to also investigate the cytotoxic activity of nitrogen-containing analogs of 10c and 10d. While the aminomethyl-substituted
allocolchicinoid
10j
turned
out
to
be
unstable,
the
N,N-
dialkylaminomethyl derivatives 10i and 10l were successfully synthesized and tested. However, both compounds were found to be inactive up to a concentration of 1000 µM.
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Among all compounds tested, 10c and 10d were identified as the most active ones and were therefore used for further in vitro and in vivo studies.
Effects of compounds 10c and 10d on tubulin polymerization As structural analogues of colchicine, compounds of type 10 were expected to interact with the colchicine binding site of tubulin and, consequently, to inhibit the formation of microtubules. To prove this, compounds 10c and 10d were examined in a tubulin polymerization assay. Figures 4A and 4B show the effects on the time-resolved turbidity change reflecting the microtubule assembly from pure tubulin. For both compounds a clear inhibition of polymerization was noted, and the rate of assembly as well as the final amount of microtubules was lower in the presence of the compounds than in the control experiment. Figures 4C and 4D show that the extent of inhibition by 10c and 10d, respectively, increases monotonically with the increase in the molar ratio (R) of the total ligand to total tubulin in the solution. From this it becomes evident that 50% inhibition occurs at a molar ratio of ca. 0.08 and 0.3 for 10c and 10d, respectively. A similar experiment performed with combretastatin A-4 (3) revealed an R value of 0.09 mol (data not shown). It should be mentioned that the “substoichiometric” poisoning of microtubule assembly by colchicine (R = 0.38) is well established.34 Figure 4 Although less potent in tubulin assembly inhibition, 10d also exhibited a “substoichiometric” mode of action, like 10c which in turn showed a slightly higher activity than combretastatin A-4 (3) and was almost one order of magnitude more effective than colchicine (1). Interestingly, compound 10c, which was identified as the most potent cytotoxic agent in this series, also showed the strongest tubulin depolymerization activity. This suggests that the cytotoxic effect of
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compounds of type 10 indeed is a consequence of their tubulin-binding properties.
Effects of compound 10c on apoptosis induction As the cell proliferation inhibition assay has revealed (Figure 3), compound 10c does not show an acute (non-specific) toxicity against both epithelial and lymphoid cells. To support this conclusion, we also studied the induction of apoptosis in W1308 cells incubated with compounds 10c, 10d, and 9a at 200 nM for 48 hrs. For this purpose, cells were stained with DiOC6 and PI dyes.35 DiOC6 is a mitochondria membrane potential-sensitive dye, while PI is able to penetrate the membranes of dead and apoptotic cells. Double staining permits the identification of a population of cells with a decreased DiOC6 and increased PI fluorescence (Figure 5) specific to apoptotic cells. After 48 h of incubation the rate of apoptosis in 10c culture was comparable with the control one (Figure 5 A and B), while both 10d and 9a induced significant apoptosis. These results demonstrate that compounds of type 10, in particular 10c and 10d, partially differ in the mechanisms of action. Similar to 10c effects were described for colchicine and its analogues, which were able to induce early apoptosis only in the presence of tumor necrosis factor alpha (TNF-alpha), a direct apoptosis inducer.36 However, the inhibition of microtubule assembly inevitably results in late stage apoptosis.
Figure 5 Late apoptosis was studied simultaneously with cell cycle arrest after incubating cells with compound 10c for 72 hours. Representative histograms for AsPC-1 and W1308 are shown in Figure 6 A-D and the data for the different cell lines tested are summarized in Table 3. Treatment of HEK293, Jurkat, AsPC-1, and W1308 cells with 10c for 72 hours led to profound changes in
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the cell cycle state (Figure 6). The flow cytometric analysis using PI-stained cells indicates that 10c induces a massive accumulation of cells in the G2/M phase (Table 3). At the same time, even after 72 h of incubation with 10c, the apoptotic rate was not very high (up to 6% depending on cell line) (Table 3). The absence of apoptotic cells in 72 cultures could be a result of dead cell elimination during sample preparation. To visualize the full population of cells after 10c treatment monolayer cultures were analyzed by confocal microscopy. Figure 6 Different stages of apoptosis were observed in cell cultures after 72 h of incubation with 10c: double nucleated cells (Figure 6, C-D), cell fractionation and apoptotic body formation (Figure 6, E-F), mitochondria enlargement and depolarization (Figure 6, C-E). These results clearly indicate that the cell cycle arrest caused by 10c, as evidenced by the inability of the incubated cells to pass through the G2/M phase (see G1/G2 ratio, Table 3), leads to a consequent apoptosis (Figure 6). Table 3
In vivo antitumor activity of the compound 10c The results of the in vitro experiments revealed tubulin as a target of furanoallocolchicine 10c, which induces late apoptosis as a result of cell cycle arrest. As compound 10c exhibited higher antitubulin polymerization activity than the natural products colchicine (1) and combretastatin A4 (3) (Figures 4, 5C) it may have pharmacological advantages.1 In addition, compound 10c is configurationally stable, in contrast to 3, which tends to undergo Z/E isomerization.37 Therefore, 10c was chosen for the preliminary in vivo acute toxicity and antitumor activity investigation
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to define the therapeutic dose of 10c, we first determined the LD100 and LD50 acute toxicity doses by injecting single doses of 10c either intraperitoneally (i.p.) or intravenously (i.v.) into C57BL/6 mice. On average injection of 10c i.p. was more toxic than i.v. and resulted in 0.8 and 2 mg/kg LD100, respectively. Corresponding LD50 for i.p. and i.v. injections were 0.3 and 0.8 mg/kg. Lethality was observed 24-48 hrs after injections. To analyze the therapeutic activity of 10c, C57BL/6 mice inoculated with breast tumors Wnt-135 were injected once a week for 4 weeks with 0.4 mg/kg (i.v.) of 10c dissolved in saline. At the initiation of 10c treatment both control and experimental groups had palpable tumors with a comparable average volume (42±12 and 32±11 mm3 accordingly). The selected dose significantly (p 95%. Separation by column chromatography was performed using Merck Kieselgel 60 (70 – 230 mesh). All reactions were performed with commercially available reagents («Aldrich», «Alfa Aesar», «Acros», «Serva»). Phenylacetylene was distilled under low pressure (b.p. 75 °C/80 mm Hg). Solvents were purified according to standard procedures. The petroleum spirit refers to the fraction with distillation range 40 – 70 °C.
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4.2. Preparation of compounds 10-12. Synthesis of colchiceine (11).28A mixture of colchicine (1) (300.0 mg, 0.75 mmol), glacial acetic acid (3 mL) and hydrochloric acid (0.1 N 18 mL) was stirred for 3 h at 100 °C, then solid Na2CO3 was added until pH become neutral. Resulting solution was extracted with CHCl3 (3×250 mL), the combine organic layer was washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure. Colchiceine (11) was obtained as yellow-green crystals (275.0 mg, 95%) and directly used at the next step. mp 150 ºС. mp lit28 150 ºС. 1Н NMR: (400 MHz, CDCl3) δ 7.49 (s, 1H), 6.87 (s, 1H), 6.54 (s, 1H), 5.91 (s, 1H), 5.75 (d, J = 8.1 Hz, 1H), 4.81 – 4.72 (m, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.53 (s, 3H), 2.54 – 2.26 (m, 4H), 2.04 (s, 3H). 13C NMR (101 MHz, CDCl3): δ = 29.64, 37.26, 52.61, 55.94, 61.22, 61.27, 107.22, 119.95, 122.53, 125.71, 134.31, 136.37, 141.32, 150.69, 151.57, 153.47, 170.36, 175.59. Synthesis of iodocolchinol (12)28 Colchiceine (11) (275.0 mg, 0.71 mmol) was dissolved in water (37 mL), solid NaOH (300.0 mg, 7.50 mmol) was added and the solution was cooled to 0 °C. The solution of NaI×2H2O (2,439.0 mg, 16.00 mmol) and iodine (543.0 mg, 2.14 mmol) in water (190 mL) was added dropwise to the reaction mixture and the resulting solution was stirred for 1 h at 0 °C. The temperature was allowed to rise until 20 °C. Corresponding quantity of Na2SO3 was added to the solution to remove the excess of iodine. Then the mixture was acidified with conc. HCl to pH=2. The obtained yellow-green precipitate was isolated by filtration and dried under reduced pressure at 50 – 60 °C. The residual solution was extracted with ethyl acetate (3×250 ml), the combined organic layer was washed with brine, dried over Na2SO4. The crude product obtained after the solvent removing together with the precipitate were purified by column chromatography, eluent petroleum spirit - ethyl acetate - ethanol (4:1:1), to afford 338.0 mg, 93% of iodocolchinol (12). mp 238 ºС. mp lit28 238 ºС. 1H NMR (400 MHz, DMSO-d6) δ
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9.95 (s, 1H), 8.37 (d, J = 8.1 Hz, 1H), 7.17 (s, 1H), 6.86 (s, 1H), 6.76 (s, 1H), 4.42 (dt, J = 12.3, 6.2 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.42 (s, 3H), 2.08 (s, 4H), 1.87 (s, 3H). 13С NMR (101 MHz, DMSO-d6): 22.51, 29.87, 37.78, 48.13, 55.72, 60.41, 81.29, 107.98, 109.96, 123.14, 126.49, 134.65, 139.06, 140.41, 142.30, 150.06, 152.10, 155.52, 168.20. General procedure for the synthesis of compounds 10a-i. Iodocolchinol (12) (100.0 mg, 0.21 mmol), Pd(OAc)2 (2.3 mg, 0.01 mmol), CuI (3.9 mg, 0.02 mmol), PPh3 (8.1 mg, 0.03 mmol) and AcOK (60.6 mg, 0.62 mmol) were placed into the two-neck flask under an inert atmosphere. In the case of 10a and 10b compounds synthesis 131.1 mg (0.62 mmol) of K3PO4 was used as a base instead of AcOK. Then anhydrous acetonitrile (4 mL) and the corresponding alkyne (0.25 mmol) were added. The mixture was stirred at 60 °C for 1 – 1.5 h, and then the temperature was raised up to 80 °C until the completeness of the reaction (TLC control). The solvent was removed under reduce pressure, a solid residue was purified by column chromatography to afford the target products. N-((1S)-2"-phenyl-1 ,2 ,3 -trimethoxy-6,7-dihydro-1H-benzo[5',6':5,4]cyclohepta[3,2f]benzofuran-1-yl)acetamide (10a). Purified by column chromatography, eluent petroleum spiritEtOAc-EtOH (5:1:1), as brownish-yellow powder, 86%, mp 140 ºС. 1H NMR (400 MHz, DMSO-
d6) δ 8.50 (d, J = 8.5 Hz, 1H), 7.93 (s, 1H), 7.91 (s, 1H), 7.57 (s, 2H), 7.52 (t, J = 7.7 Hz, 2H), 7.44 (s, 1H), 7.41 (t, J = 7.3 Hz, 1H), 6.81 (s, 1H), 4.66 – 4.59 (m, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.46 (s, 3H), 2.29 – 1.97 (m, 4H), 1.93 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.53, 168.50, 155.13, 153.73, 152.36, 150.41, 138.26, 134.80, 132.42, 129.91, 129.30, 129.09, 128.96, 126.98, 124.57, 121.98, 108.08, 105.75, 101.99, 60.62, 60.56, 55.85, 48.54, 30.05, 22.75, 22.72. MS: m/z (%) = 354 (19), 367 (40), 383 (45), 398 (100), 399 (30), 458 (81). Found С, 73.45; Н, 5.97. С28Н27NO5 requires: C, 73.51; H, 5.95%.
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N-((1S)-2"-(pyridin-2-yl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
(10b).
Purified
by
column
chromatography, eluent petroleum spirit-ethyl acetate-ethanol (4:1:1), as brown polycrystalline powder, 83%, mp 140 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.69 (d, J = 4.7 Hz, 1H), 8.51 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 3.6 Hz, 2H), 7.65 (s, 1H), 7.60 (d, J = 4.9 Hz, 2H), 7.41 (dd, J = 8.9, 4.5 Hz, 1H), 6.81 (s, 1H), 4.66 – 4.60 (m, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.46 (s, 3H), 2.26 – 1.95 (m, 4H), 1.93 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.47, 154.76, 154.10, 152.38, 150.40, 150.00, 148.32, 140.61, 139.20, 137.35, 134.76, 129.53, 126.56, 124.43, 123.43, 122.64, 119.40, 108.06, 105.97, 104.87, 60.60, 60.56, 55.83, 48.57, 30.95, 30.01, 22.70. MS: m/z (%) = 368 (35), 384 (39), 399 (100), 400 (27), 415 (12), 458 (62). Found С, 70.81; Н, 5.67. С28Н26N2O5 requieres C, 70.73; H, 5.72%. N-((1S)-2"-(hydroxymethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
(10c).
Purified
by
column
chromatography, eluent petroleum spirit-EtOAc-EtOH (3:1:1), as yellow polycrystalline powder, 84%, mp 110 ºС. 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 8.5 Hz, 1H), 7.51 (s, 1H), 7.46 (s, 1H), 6.79 (s, 1H), 6.75 (s, 1H), 5.43 (t, J = 5.8 Hz, 1H), 4.64 – 4.54 (m, 3H), 3.84 (s, 3H), 3.79 (s, 3H), 3.41 (s, 3H), 2.22 – 1.70 (m, 7H).
13
C NMR (101 MHz, DMSO-d6) δ 168.39,
158.42, 153.73, 152.23, 150.37, 140.59, 137.42, 134.75, 128.69, 126.18, 124.73, 121.81, 108.01, 105.52, 103.21, 60.59, 60.45, 56.22, 55.82, 48.40, 30.95, 30.05, 22.67. MS: m/z (%) = 294 (17), 321 (57), 337 (43), 352 (100), 353 (23), 411 (55). Found С, 67.07; Н, 6.24. С23H25NO6 requires C, 67.14; H, 6.12%. N-((1S)-2"-(1-hydroxyethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
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(10d). Purified by column chromatography, eluent petroleum spirit-ethyl acetate-ethanol (4:1:1), as yellow polycrystalline powder, 93%, mp 80 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 8.4 Hz, 1H), 7.50 (s, 1H), 7.45 (s, 1H), 6.79 (s, 1H), 6.70 (s, 1H), 5.51 (s, 1H), 4.84 (q, J = 6.5 Hz, 1H), 4.63 – 4.55 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.41 (s, 3H), 2.29 – 1.93 (m, 4H), 1.90 (s, 3H), 1.47 (d, J = 6.5 Hz, 3H).
13
C NMR (101 MHz, DMSO-d6) δ 168.43, 162.01, 161.98,
153.48, 152.24, 150.39, 140.61, 137.24, 134.77, 128.65, 126.17, 124.79, 121.79, 108.03, 105.55, 101.22, 62.30, 60.60, 60.46, 56.03, 55.83, 48.43, 30.06, 26.45, 22.68, 18.56. MS: m/z (%) = 321 (24), 335 (42), 351 (42), 366 (100), 367 (26), 425 (62). Found С, 67.76; Н, 6.42. С24H27NO6 requires C, 67.75; H, 6.40%. N-((1S)-2''-(1-hydroxyсусlopentyl)-1',2',3'-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
(10e).
Purified
by
column
chromatography, eluent petroleum spirit-ethyl acetate-ethanol (6:2:1), as dark-yellow oil, 86%. 1
H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 8.5 Hz, 1H), 7.48 (s, 1H), 7.44 (s, 1H), 6.79 (s,
1H), 6.70 (s, 1H), 5.25 (s, 1H), 4.62 – 4.55 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.41 (s, 3H), 2.16 (td, J = 12.1, 5.9 Hz, 2H), 2.05 (dd, J = 12.4, 6.5 Hz, 2H), 1.99 (s, 1H), 1.90 (s, 3H), 1.74 (dd, J = 12.2, 8.0 Hz, 3H), 1.68 – 1.55 (m, 1H), 1.54 – 1.43 (m, 1H), 1.20 – 0.99 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 168.40, 163.40, 153.53, 152.21, 150.37, 140.61, 136.98, 134.76, 128.56, 126.32, 124.83, 121.61, 108.02, 105.49, 100.87, 78.09, 60.60, 60.45, 55.82, 48.42, 34.19, 30.05, 26.34, 24.78, 23.48, 22.68, 13.96. Found C, 69.85; H, 6.64. C27H31NO6 requieres C, 69.66; H, 6.71%. N-((1S)-2"-(2-hydroxyethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
(10f).
Purified
by
column
chromatography, eluent petroleum spirit-EtOAc-EtOH (4:1:1), as yellow polycrystalline powder,
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74%, mp 125 ºС. 1H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 8.4 Hz, 1H), 7.45 (s, 1H), 7.42 (s, 1H), 6.78 (s, 1H), 6.62 (s, 1H), 4.81 (t, J = 5.4 Hz, 1H), 4.63 – 4.56 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.78 – 3.73 (m, 2H), 3.41 (s, 3H), 2.91 (t, J = 6.6 Hz, 2H), 2.31 – 1.91 (m, 4H), 1.90 (s, 3H).
13
C NMR (101 MHz, DMSO-d6) δ 168.37, 156.91, 153.46, 152.18, 150.36, 140.59,
136.65, 134.75, 128.49, 126.69, 124.85, 121.21, 107.99, 105.25, 102.91, 62.78, 60.60, 60.48, 59.03, 55.80, 48.38, 31.93, 30.06, 22.67. MS: m/z (%) = 321 (15), 335 (57), 351 (36), 366 (100), 367 (26), 425 (57). Found С, 67.82; Н, 6.36. С24H27NO6 requires C, 67.75; H, 6.40%. N-((1S)-2"-(acetoxymethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
(10g).
Purified
by
column
chromatography, eluent petroleum spirit-EtOAc-EtOH (6:1:1), as light-beige polycrystalline powder, 41%. mp 82 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 8.3 Hz, 1H), 7.56 (s, 1H), 7.49 (s, 1H), 6.99 (s, 1H), 6.79 (s, 1H), 5.22 (s, 2H), 4.58 (dd, J = 11.8, 7.5 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.42 (s, 3H), 2.23 – 2.11 (m, 2H), 2.08 (s, 3H), 2.03 (dd, J = 12.7, 5.6 Hz, 2H), 1.91 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 169.95, 168.45, 153.95, 152.33, 152.12, 150.37, 140.60, 138.54, 134.74, 129.11, 125.70, 124.51, 122.29, 108.05, 107.03, 105.75, 60.59, 60.49, 57.95, 55.83, 48.50, 30.61, 29.98, 22.66, 20.56. MS: m/z (%) = 334 (30), 351 (45), 363 (33), 394 (100), 395 (26), 453 (77). Found С, 66.11; Н, 6.12. С25H27NO7 requires C, 66.21; H, 6.00%. N-((1S)-2"-(8-(metoxycarbonyl)octyl)-1′,2′,3′-trimetoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl) acetamide (10h). Purified by column chromatography, eluent petroleum spirit-ethyl acetate-ethanol (5:1:1), as brownish oil, 49%. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 8.5 Hz, 1H), 7.44 (s, 1H), 7.42 (s, 1H), 6.78 (s, 1H), 6.57 (s, 1H), 4.63 – 4.54 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.57 (s, 3H), 3.42 (s, 3H), 2.75 (t, J = 7.4 Hz, 2H), 2.22 – 1.76 (m, 7H), 1.68 (dd, J = 14.3, 7.1 Hz, 2H), 1.54 – 1.47 (m, 2H), 1.41 –
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1.15 (m, 10H).
13
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C NMR (101 MHz, DMSO-d6) δ 168.41, 156.02, 153.73, 152.22, 150.35,
140.57, 137.19, 134.76, 128.63, 126.17, 124.75, 121.55, 120.59, 108.02, 105.52, 104.90, 62.78, 60.57, 60.51, 55.83, 48.90, 48.38, 46.47, 30.05, 22.70, 12.07. MS: m/z (%) = 447 (10), 461 (19), 478 (19), 492 (100), 493 (30), 551 (38). Found С, 69.73; Н, 7.47. С32H41NO7 requires C, 69.67; H, 7.49%. N-((1S)-2"-((diethylamino)methyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)
acetamide (10i). Purified by column
chromatography, eluent petroleum spirit-ethyl acetate-ethanol (2:1:1), as light-brown polycrystalline powder, 72%, mp 148 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 8.6 Hz, 1H), 7.47 (d, J = 6.8 Hz, 2H), 6.79 (s, 1H), 6.75 (s, 1H), 4.59 (dd, J = 6.9, 3.9 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.75 (s, 2H), 3.43 (s, 3H), 2.56 – 2.51 (m, 4H), 2.21 – 2.10 (m, 2H), 2.04 (dd, J = 12.5, 7.1 Hz, 2H), 1.90 (s, 3H), 1.03 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 168.41, 156.02, 153.73, 152.22, 150.35, 140.57, 137.19, 134.76, 128.63, 126.17, 124.75, 121.55, 108.02, 105.52, 104.90, 62.78, 60.57, 60.51, 55.83, 48.90, 48.38, 46.47, 30.05, 22.70, 12.07. MS: m/z (%) = 335 (44), 336 (18), 368 (16), 394 (100), 395 (34), 466 (16). Found С, 69.46; Н, 7.38. С27H34N2O5 requires C, 69.50; H, 7.35%. Synthesis
of
N-((1S)-2"-(pyrollidino)-1′,2′,3′-trimethoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl) acetamide (10l). Iodocolchinol (12) (100.0 mg, 0.21 mmol), Pd(PPh3)2Cl2 (7.2 mg, 0.01 mmol), CuI (3.9 mg, 0.02 mmol) were added into the two-neck flask, and the flask was filled with argon. Pyrrolidine (4 ml) was appended to the mixture and the solution was cooled to 0°С, then the solution of propargylbromide (29.4 mg, 0.25 mmol) in toluene was added dropwise. The mixture was stirred at 50 °С. After the completeness of the reaction the resulting solution was poured into 15 mL of water and the
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resulting mixture was extracted with ethyl acetate (3×10 ml). The combined organic layer was washed with brine, dried over Na2SO4. The solvent was removed under reduced pressure. A solid residue was purified by column chromatography, eluent petroleum spirit-ethyl acetate-ethanol (3:1:1) to afford product 10l as brownish polycrystalline powder, 80%, mp 123 °С. 1H NMR (400 MHz, DMSO-d6) δ 8.60 (d, J = 8.5 Hz, 1H), 7.48 (s, 2H), 6.78 (s, 1H), 6.74 (s, 1H), 4.61 – 4.56 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.76 (d, J = 3.7 Hz, 2H), 3.42 (s, 3H), 2.56 (s, 4H), 2.18 – 2.09 (m, 2H), 2.04 (dd, J = 12.4, 6.6 Hz, 2H), 1.90 (s, 3H), 1.71 (s, 4H). 13C NMR (101 MHz, DMSO-d6) δ 168.43, 156.10, 153.66, 152.23, 150.36, 140.58, 137.33, 134.77, 128.67, 126.19, 124.75, 121.63, 108.02, 105.55, 104.28, 60.58, 60.50, 55.82, 53.29, 51.73, 51.59, 48.43, 42.53, 30.06, 23.33, 23.21, 22.68. Found C, 69.88; H, 6.89. C27H32N2O5 requires C, 69.81; H, 6.94%.
Preparation of lamb brain tubulin. Tubulin was purified from lamb brain by ammonium sulfate fractionation and ion-exchange chromatography. The pure protein was stored in liquid nitrogen
and
prepared
as
described.44
Protein
concentrations
were
determined
spectrophotometrically with a Perkin Elmer spectrophotometer Lambda 800 and an extinction coefficient at 275 nm of 1.07 L.g-1.cm-1 in 0.5% SDS in neutral aqueous buffer or 1.09 L.g-1.cm-1 in 6 M guanidine hydrochloride. Tubulin polymerization. Microtubule assembly was performed in 20 mM sodium phosphate buffer, 1 mM EGTA, 10 mM MgCl2, and 3.4 M glycerol, 1 mM GTP pH 6.5. The reaction was started by warming the samples at 37 °C and the mass of polymer formed was monitored by turbidimetry at 350 nm with a POLARstar BMG Labtech spectrophotometer using 96-well plate. Samples containing the compound and controls had less than 1% residual Me2SO.
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Cell cultures. HEK-293, human embryonic kidney cell line (ATCC, CRL-1573), Jurkat human T-lymphoma (ATCC, TIB-152), AsPC-1 pancreatic human adenocarcinoma cell line (ATCC, CRL-1682), and Wnt-1 related murine epithelial cell line W1308, generated by us earlier,35 were used in the study. HEK-293 and AsPC-2 were grown in DMEM supplemented with 10% fetal calf serum (FCS), pen-strep-glut, and 2-ME 5 x 10–5 M (all from PanEco, Moscow, Russian Federation). Jurkat and W1308 cells were grown in RPMI-1640 with the same supplements. Cells were passed by trypsinization using Trypsin /EDTA solution (PanEco, Moscow, Russian Federation) twice a week. Twenty four hours before assays, cells were seeded in the appropriate plates (96- or 24-well plates) adjusted to 3x105 cells/mL and incubated overnight to achieve standardized growth conditions. Confocal analysis. For confocal analysis AsPC-1 cells were grown overnight on sterile cover slips in 200 µl of complete culture medium in 6-well plates (Costar). Compound 10c in different concentrations was dissolved in 4 ml of complete medium and added to the wells. Cells were cultivated for 72 hrs. Nuclei dye Hoecst 33342 (Sigma) and mitochondrial tracker MitoTreckRed (Invitrogen) were added for 1 hr. Finally cells were fixed with 1% paraformaldehyde, washed, and polymerized with Mowiol 4.88 medium (Calbiochem, Germany). Slides were analyzed using Eclipse TE2000 confocal microscope (Nikon, Japan). MTT-assay. Cytotoxic effect of the prodrugs was estimated by a standard 3-(4, 5-dimethyl-2thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) test as was described earlier.45 In short, different dilutions of the new compounds using series of 10 times dilutions from 10 µM to 1 nM were prepared on a separate plate and then transferred in 100 ul to the plates with the cells. Non-treated cells served as controls. Plates were incubated for 72 hrs. For the last 6 hours 250 µg/ml of MTT was added in 10 ul to each well. After the incubation culture medium was
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removed and 100 µl dimethylsulfoxide was added to each well. Plates were incubated at shaking for 15 min to dissolve formazan. Optical density was read on spectrophotometer Titertek (UK) at 540 nm. Results were analyzed by Excel package (Microsoft). Cytotoxic concentration giving 50% of dead cells (IC50) was calculated from the titration curves. The inhibition of proliferation (inhibition index, II) was calculated as [1 – (ODexperiment / ODcontrol)], where OD was MTT optical density. Analysis of apoptosis by flow cytometry. Percentage of apoptotic cell was analyzed by 3,3'dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide (PI) (Sigma Chemical Co) double staining. The cationic lypophilic fluorochrome DiOC6 (Invitrogen) was used to evaluate transmembrane potential in mitochondria.46 Cells were harvested, washed in pre-warmed PBS supplemented with 2% of FCS, resuspended at 106 cells/ml in PBS/2%FCS, 40 µM DiO C6 and 5 ug/ml PI and incubated for 30 minutes at 37oC. Cells were washed with PBS/2% FCS and analyzed by flow cytometry using FACScan device (BD, USA). Total 2000 events were collected. The results were analyzed using CellQuest software (BD Biosciences). Cell cycle analysis. Cell cycle was analyzed using PI-stained DNA. Two million cells were harvested at indicated time, washed in ice-cold PBS, fixed by the addition of 70% ethanol and left for 2 hours at -20oC. Thereafter, the cells were washed twice in PBS, stained with 50 μg/ml of PI (Sigma Chemical Co) in PBS and analyzed by flow cytometry. Animals. C57BL/6 mice were purchased from Pushchino Affiliation of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow. All mice were 6–8 wk old and maintained in minimal pathogen animal facility at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow. All studies were conducted in an AAALAC accredited facility in compliance with the PHS Guidelines for the Care and Use of Animals in Research.
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Wnt-1 tumor growth and treatment in vivo. Wnt-1 tumor cells (106/mouse) were obtained as described,47 and inoculated subcutaneously into the left inguinal mouse fat pad (MFP #4). The injection of cells in 50 µl of PBS was performed through the skin of anesthetized mice. Tumor size was measured with vernier calipers twice a week and calculated using the formula (W2xL)/2, where W and L corresponded to width and length of tumors. This protocol was efficient in the induction of tumors in 100% of mice approximately on day 30 after the cell inoculation. Experiment was started only in mice (n=7 per group) with palpable tumors at day 27. The volume of tumors was comparable (100 mm3) in both groups before 10c treatment. Compound 10c was injected in 200µl of saline intravenously into the orbital sinus of mice once a week at days 27, 34, 41, and 48. Control group was injected with saline only. The experiment was terminated at day 51 when two control mice died. Short term effect of colchicine analogues on endothelial cells (interphase effect). Colchicine, combretastatin A4, compound 10c were dissolved in dimethyl sulfoxide at 20 mM concentration. Four µl of each were transferred to 200 µl of saline (1 mg/kg of weight), mixed with 50 µl of rhodamine B (100 µl/ml of water) and injected i.v. into C57BL/6 intact mice (3 per group). Control animals were injected with dimethyl sulfoxide and rhodamine B only. Four hours later mice were sacrificed by cervical dislocation. Major organs were collected, cut in pieces, and extracted with 0.3N acidic acid in ethanol for 2 hours at 37oC. Supernatants were collected, centrifuged 10 min at 10000 rpm, and cleared fraction was transferred into 96-well black plates (SPL). Fluorescence was measured using GlomaxMulti reader (Promega) with 540 nm filter. For confocal analysis small pieces (1x1 mm) of nonfixed tissue were transferred on the microscopic glass, layed with Mowiol 4.88 medium (Calbiochem, Germany), covered with the cover glass and squeezed. Slides were analyzed using Eclipse TE2000 confocal microscope (Nikon, Japan).
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Statistical analysis Statistical analysis was performed using Excel Student's t-test. Comparison values of p
Article
Synthesis and Biological Evaluation of Furanoallocolchicinoids Yuliya V. Voitovich, Ekaterina S. Schegravina, Nikolay S. Sitnikov, Vladimir I. Faerman, Valery V. Fokin, Hans-Guenther Schmalz, Sebastien Combes, Diane Allegro, Pascale Barbier, Irina P. Beletskaya, Elena V. Svirshchevskaya, and Aleksey Yurievich Fedorov J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 11 Dec 2014 Downloaded from http://pubs.acs.org on December 12, 2014
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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The Synthesis and Biological Evaluation of Furanoallocolchicinoids Yuliya V. Voitovicha, Ekaterina S. Shegravinaa, Nikolay S. Sitnikova, Vladimir I. Faermana, Valery V. Fokina, Hans-Gunther Schmalzb, Sebastien Combesc, Diane Allegrod, Pascal Barbierd, Irina P. Beletskayae, Elena V. Svirshchevskayaf*, Alexey Yu. Fedorova*
a
Department of Organic Chemistry, Nizhny Novgorod State University, Gagarina av. 23, Nizhny Novgorod 603950, Russian Federation, Fax: +7 831-462-30-85, E-mail: [email protected] b
Department of Chemistry, University of Cologne, Greinstrasse 4, 50939 Koln (Germany) Fax: +49 221-470-3064, Tel: +49 221 470 3063, E-mail: [email protected] c
CRCM, CNRS UMR7258, Laboratory of Integrative Structural and Chemical Biology
(ISCB), INSERM, U1068, Institut Paoli-Calmettes, Aix-Marseille Universit´e, UM105, F-13009, Marseille, France, Fax: +33 (0)491 164 540; Tel: +33 (0)86 97 73 31, E-mail: [email protected], d
Centre de Recherche en Oncologie Biologique et en Oncopharmacologie, CRO2 INSERM UMR 911, Universite d’Aix-Marseille, Faculte de Pharmacie, 27 Boulevard Jean Moulin, Marseille 13005, France Tel : +33 (0)491 835 616, E-mail : [email protected]
e
Department of Chemistry, M.V. Lomonosov Moscow State University, Vorobyevy Gory, 119992 Moscow, Russia, Tel: +7 (495) 938 36 18, E-mail: [email protected]
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Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Laboratory of Cell
Interactions, GSP-7, Miklukho-Maklaya Street, 16/10, 117997 Moscow, Russian Federation, Tel/Fax +7 (495) 330 40-11, E-mail: [email protected]
Corresponding authors Prof. Fedorov Alexey Yu. a
Department of Organic Chemistry, Nizhny Novgorod State University, Gagarina av. 23, Nizhny Novgorod 603950, Russian Federation, Fax: +7 831-462-30-85, E-mail: [email protected] Dr. Svirshchevskaya Elena V. e
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Laboratory of Cell
Interactions, GSP-7, Miklukho-Maklaya Street, 16/10, 117997 Moscow, Russian Federation, Tel/Fax (495) 330 40-11, E-mail: [email protected]
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Keywords: colchicine, allocolchicinoids, cross-coupling, antitumor activity, tubulin binding agents. Abstract A series of conformationally flexible furan-derived allocolchicinoids was prepared from commercially available colchicine in good to excellent yields using a three-step reaction sequence. Cytotoxicity studies indicated potent activity of two compounds against human epithelial and lymphoid cell lines (AsPC-1, HEK293 and Jurkat) as well as against Wnt-1 related murine epithelial cell line W1308. The results of in vitro experiments demonstrated that the major effect of these compounds was the induction of cell cycle arrest in the G2/M phase as a direct consequence of effective tubulin binding. In vivo testing of the most potent furanoallocolchicinoid 10c using C57BL/6 mice inoculated with Wnt-1 tumor cells indicated significant inhibition of the tumor growth.
1. Introduction Due to the essential role of microtubules in cell growth and division, inhibition of microtubule formation is considered promising in the treatment of cancer.1 Microtubules are formed via a polymerization process involving α- and β-tubulin protein subunits.2 Interference with the dynamic equilibrium between the assembly of tubulin into microtubules or, inversely, the depolymerization of microtubules leads to the arrest of cell division and eventually to apoptosis. Compounds inhibiting either of these important processes are referred to as spindle poisons or antimitotics.1,2
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The search for selective inhibitors of tubulin assembly or disassembly has led to the development of some of the most useful antitumor drugs currently in clinical use.1,2 These are the naturally occurring Vinca alkaloids3 as well as the taxoids,4 which are widely used for the treatment of breast, ovarian, and non-small cell lung carcinomas. However, these structurally rather complex molecules share several drawbacks: they are difficult to synthesize (thus expensive) and prone to the development of resistance.1,5 Therefore, the development of novel small-molecule antimitotics with low-toxicity remains a clinically relevant challenge. Colchicine (1) (Figure 1), isolated from Colchicum autumnale, was the first tubulin-destabilizing agent ever discovered.6 Although it has been employed since ancient times in the treatment of acute gout and familial Mediterranean fever, high cardio- and neurotoxic adverse effects prevent its use in cancer chemotherapy.7 However, structural analogs of colchicine, such as allocolchicine (2),8 combretastatin A-4 (3),9 4-arylcoumarins (4),10 (Figure 1) and their derivatives, represent promising leads in the search for new anticancer agents addressing the colchicine binding site of tubulin.11 The ability of colchicine and its analogs to block mitosis by affecting the microtubule dynamics is well studied,12,13 and several colchicine analogs such as AbT-751 and indibulin are currently in clinical trials for patients suffering from advanced solid tumors.14-16 Clinical trials of other colchicine analogues have been discontinued due to poorly controlled drug absorption, neurotoxicity17 or low efficacy.18 Nevertheless, the search for novel colchicinoids remains an interesting challenge because, besides acting as mitosis inhibitors, recent studies have shown that microtubule-targeting agents may also exhibit effects in the areas of (i) mitosis-independent cell death and metastasis, (ii) tumor angiogenesis, and (iii) vasculardisrupting activity.12 Moreover, the development of drugs overcoming Pgp/β-III-tubulinmediated drug resistance is a key task in cancer research.11d, 19
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A molecular modeling study suggested that a key structural feature of colchicine binding site inhibitors is the presence of two closely neighbored non-coplanar hydrophobic aromatic fragments A and B, bearing hydrogen bond donor or acceptor functionalities (Figure 1).11a Indeed, the structural similarity of compounds 1 to 4 arises from the presence of two adjacent polyoxygenated aromatic rings A and B, which are non-coplanar.11 Figure 1 For a long time, the unique substitution pattern of ring B in molecules such as 1-4 was considered essential for the high affinity towards the colchicine binding site of tubulin. However, recent investigations by the groups of Wang,20 Simonini,21 and in our laboratories22 demonstrated that replacement of the aromatic B-ring by a hydrophobic benzoheterocyclic moiety23 leads to highly active compounds (Figure 2). For instance, the indole derivatives 520 and 722a were found to inhibit in vitro microtubule assembly in a substoichiometric mode of action. The phosphate prodrugs 6 demonstrated high antitumor activity both in vitro and in vivo, notwithstanding a minimal effect on microtubule organization with respect to 3 (CA-4).21 The indole-derived allocolchicine analogs 8 and 9 caused proliferation inhibition and apoptosis induction at nanomolar or even in sub-nanomolar concentrations.22b These derivatives actually represent the first examples of heteroarene-based allocolchicinoids.24 Figure 2 As structural analogs of pyrrole-fused colchicinoids 8 and 922b we considered the corresponding furan-derived allocolchicinoids of type 10 to be promising compounds for biological testing. We herein report a short and convenient semi-synthetic access to such compounds (of type 10) and
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demonstrate their promising antitumor activity (including in vivo mice experiments) in comparison with colchicine (1) and combretastatin A-4 (3).
2. Results and Discussion Chemistry Compounds of type 10 were prepared in only four steps starting from commercially available colchicine (1) by making use of the previously described colchiceine - N-acetyliodocolchinol rearrangement24,25 and transition metal-catalyzed methods for the construction of the furan ring (Scheme 1). First, the tropolone ether function of colchicine (1) was hydrolyzed by heating with HCl in AcOH to afford colchiceine (11) in 97% yield.26 The oxidative degradation of 11 to iodocolchinol (12) was also achieved in high yield using the Windaus procedure (NaOH, I2, KI, H2O).27,28 The Sonogashira coupling of iodocolchinol (12) with terminal alkynes (13)29 then afforded the alkyne intermediates of type 14, which under the reaction conditions (Pd/Cu catalysis, base) directly underwent cyclisation30 to afford the desired benzofurans of type 10. Scheme 1 Table 1 The Sonogashira coupling of iodocolchinol (12) with phenylacetylene (13a) or 2pyridylacetylene (13b) was best performed at 65 °C using Pd(OAc)2/CuI/PPh3 (5:10:15 mol%) as the catalytic system in MeCN in the presence of K3PO4 as a base to afford the cyclization products 10a and 10b, respectively, in 86 and 83% isolated yield (Table 1). In contrast, when iodocolchinol 12 was reacted with non-(hetero)aryl-substituted terminal alkynes, such as
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propargyl alcohol (13c), under the same conditions, the cyclization products were obtained in much lower yield (e.g. 30% of 10c). However, as the yield of the furanoallocolchicinoids 10 proved to be strongly base-dependent, different bases (NEt3, DIPEA, K2CO3, Cs2CO3, t-BuOK, or AcOK) were tested in the reaction between 12 and 13c using the same catalytic system as before. Under the optimized conditions (Pd(OAc)2/CuI/PPh3 (5:10:15 mol%), AcOK (3 equiv.), MeCN, 70 oC) the reaction of iodocolchinol (12) with different functionalized terminal alkynes (13c-k) afforded the products 10a-i in good to high yields (Table 1). Thus, the desired benzofurans of type 10 were efficiently obtained in only three preparative steps starting from commercially available colchicine (1). An attempt to prepare benzofuran 10j with an aminomethyl group in the side chain revealed that this compound was rather unstable. Noteworthy, we did not succeed at all in synthesizing the TMS-substituted furanoallocolchicine 10k, because the reaction of 12 with trimethylsilylacetylene (13k) stopped at the stage of intermediate 14k, which was isolated in 62% yield. This correlates well with the literature reports that the Pd/Cu-catalyzed cyclization of ortho-trimethylsilylethynyl- and ortho-ethynyl-substituted phenols, anilines or thiols require rather harsh conditions (thermolysis or photolysis).31 The pyrrolidinomethyl-substituted benzofuran 10l was obtained in 80% yield by reacting 12 with propargylbromide 13l in the presence of catalytic amounts of Pd(PPh3)2Cl2 and CuI in pyrrolidine as a solvent (Scheme 2). This type of reaction might also be useful for the preparation of related aminomethyl-substituted furanoallocolchicinoids employing different primary and secondary amines. Scheme 2 One should note that furan-derived allocolchicinoids 10 were prepared using semi-synthetic pathway in three steps from commercially available colchicine with the retention of the absolute
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configuration at C7. Proposed methodology seems to be advantageous in comparison with the eight-to-twelve-step synthesis used for the preparation of allocolchicinoids 8 and 9, affording the target compounds as a racemic mixture.22b
Biology In vitro cytotoxicity of new compounds against cancer cell lines The in vitro cytotoxicity of all synthesized compounds against human cancer cell lines was investigated by MTT assay after the incubation of the cells with the compounds for 72 h. The representative inhibitory curves for HEK293, Jurkat, and AsPC-1 cells are shown in Figure 3. For comparison, effects of all 10 compounds synthesized are shown only for HEK293 cells in two sets (Figure 3 A and B), where A demonstrates the effects of the compounds with high activity, and B – with low activity. The results for Jurkat and AsPC-1 cells are presented only for the compounds with high activity (Figures 3C and 3D). Figure 3 All compounds, including 10c and 10d as the most toxic ones, induced ca. 60% inhibition even at very high concentrations (Figure 3, black dotted lines) showing the absence of direct necrotic or pro-apoptotic toxicity. The same is true for many previously published colchicine analogues.32 Activity of compounds inducing 50% of the maximal cell growth inhibition (IC50, Figure 3, grey dotted lines) are summarized in Table 2. The sensitivity of different cell lines to these compounds slightly varied due to variations in cell concentrations, different rates of division, and experimental errors. For the comparison we have also included IC50 for
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indoloallocolchicinoids 8a and 9a synthesized by us earlier, the most active compounds from 8 and 9 series.22b Table 2 Compounds 10a and 10b bearing an aromatic (or heteroaromatic) moiety in the side chain of the benzofuran fragment were not active. In contrast, compounds 10c and 10d, with a hydroxymethyl or a 1-hydroxyethyl substituent at the furan ring, exhibited potent antiproliferative activity with IC50 values in the nanomolar range. While allocolchicinoid 10e also contains a hydroxyl group in the pseudo-benzylic position (like 10c and 10d) it exhibited lower cytotoxicity, possibly because the hydrophobic cyclopentane ring shields the OH group. Interestingly, compound 10f with a 2-hydroxyethyl side chain showed a two-order of magnitude lower activity in comparison with the hydroxylated compounds 10c and 10d. Compound 10g, which can be regarded as an ester prodrug of 10c, exhibits a significantly lower activity in comparison with the “parent” compound 10c.33 A low cytotoxicity was also observed for the hydrophobic ester 10h. The most cytotoxic among the 8-series compounds22b was 8a, an indolyl analog of furanoallocolchicinoid 10.
It was less active than 10c. On the other hand, the isomeric
colchicinoid 9a manifested the same order of activity as 10c (Table 2). These results demonstrate that a hydroxymethyl fragment in the α-position of the furan ring of type 10 compounds significantly improves the pharmacophore. Therefore it was interesting to also investigate the cytotoxic activity of nitrogen-containing analogs of 10c and 10d. While the aminomethyl-substituted
allocolchicinoid
10j
turned
out
to
be
unstable,
the
N,N-
dialkylaminomethyl derivatives 10i and 10l were successfully synthesized and tested. However, both compounds were found to be inactive up to a concentration of 1000 µM.
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Among all compounds tested, 10c and 10d were identified as the most active ones and were therefore used for further in vitro and in vivo studies.
Effects of compounds 10c and 10d on tubulin polymerization As structural analogues of colchicine, compounds of type 10 were expected to interact with the colchicine binding site of tubulin and, consequently, to inhibit the formation of microtubules. To prove this, compounds 10c and 10d were examined in a tubulin polymerization assay. Figures 4A and 4B show the effects on the time-resolved turbidity change reflecting the microtubule assembly from pure tubulin. For both compounds a clear inhibition of polymerization was noted, and the rate of assembly as well as the final amount of microtubules was lower in the presence of the compounds than in the control experiment. Figures 4C and 4D show that the extent of inhibition by 10c and 10d, respectively, increases monotonically with the increase in the molar ratio (R) of the total ligand to total tubulin in the solution. From this it becomes evident that 50% inhibition occurs at a molar ratio of ca. 0.08 and 0.3 for 10c and 10d, respectively. A similar experiment performed with combretastatin A-4 (3) revealed an R value of 0.09 mol (data not shown). It should be mentioned that the “substoichiometric” poisoning of microtubule assembly by colchicine (R = 0.38) is well established.34 Figure 4 Although less potent in tubulin assembly inhibition, 10d also exhibited a “substoichiometric” mode of action, like 10c which in turn showed a slightly higher activity than combretastatin A-4 (3) and was almost one order of magnitude more effective than colchicine (1). Interestingly, compound 10c, which was identified as the most potent cytotoxic agent in this series, also showed the strongest tubulin depolymerization activity. This suggests that the cytotoxic effect of
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compounds of type 10 indeed is a consequence of their tubulin-binding properties.
Effects of compound 10c on apoptosis induction As the cell proliferation inhibition assay has revealed (Figure 3), compound 10c does not show an acute (non-specific) toxicity against both epithelial and lymphoid cells. To support this conclusion, we also studied the induction of apoptosis in W1308 cells incubated with compounds 10c, 10d, and 9a at 200 nM for 48 hrs. For this purpose, cells were stained with DiOC6 and PI dyes.35 DiOC6 is a mitochondria membrane potential-sensitive dye, while PI is able to penetrate the membranes of dead and apoptotic cells. Double staining permits the identification of a population of cells with a decreased DiOC6 and increased PI fluorescence (Figure 5) specific to apoptotic cells. After 48 h of incubation the rate of apoptosis in 10c culture was comparable with the control one (Figure 5 A and B), while both 10d and 9a induced significant apoptosis. These results demonstrate that compounds of type 10, in particular 10c and 10d, partially differ in the mechanisms of action. Similar to 10c effects were described for colchicine and its analogues, which were able to induce early apoptosis only in the presence of tumor necrosis factor alpha (TNF-alpha), a direct apoptosis inducer.36 However, the inhibition of microtubule assembly inevitably results in late stage apoptosis.
Figure 5 Late apoptosis was studied simultaneously with cell cycle arrest after incubating cells with compound 10c for 72 hours. Representative histograms for AsPC-1 and W1308 are shown in Figure 6 A-D and the data for the different cell lines tested are summarized in Table 3. Treatment of HEK293, Jurkat, AsPC-1, and W1308 cells with 10c for 72 hours led to profound changes in
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the cell cycle state (Figure 6). The flow cytometric analysis using PI-stained cells indicates that 10c induces a massive accumulation of cells in the G2/M phase (Table 3). At the same time, even after 72 h of incubation with 10c, the apoptotic rate was not very high (up to 6% depending on cell line) (Table 3). The absence of apoptotic cells in 72 cultures could be a result of dead cell elimination during sample preparation. To visualize the full population of cells after 10c treatment monolayer cultures were analyzed by confocal microscopy. Figure 6 Different stages of apoptosis were observed in cell cultures after 72 h of incubation with 10c: double nucleated cells (Figure 6, C-D), cell fractionation and apoptotic body formation (Figure 6, E-F), mitochondria enlargement and depolarization (Figure 6, C-E). These results clearly indicate that the cell cycle arrest caused by 10c, as evidenced by the inability of the incubated cells to pass through the G2/M phase (see G1/G2 ratio, Table 3), leads to a consequent apoptosis (Figure 6). Table 3
In vivo antitumor activity of the compound 10c The results of the in vitro experiments revealed tubulin as a target of furanoallocolchicine 10c, which induces late apoptosis as a result of cell cycle arrest. As compound 10c exhibited higher antitubulin polymerization activity than the natural products colchicine (1) and combretastatin A4 (3) (Figures 4, 5C) it may have pharmacological advantages.1 In addition, compound 10c is configurationally stable, in contrast to 3, which tends to undergo Z/E isomerization.37 Therefore, 10c was chosen for the preliminary in vivo acute toxicity and antitumor activity investigation
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to define the therapeutic dose of 10c, we first determined the LD100 and LD50 acute toxicity doses by injecting single doses of 10c either intraperitoneally (i.p.) or intravenously (i.v.) into C57BL/6 mice. On average injection of 10c i.p. was more toxic than i.v. and resulted in 0.8 and 2 mg/kg LD100, respectively. Corresponding LD50 for i.p. and i.v. injections were 0.3 and 0.8 mg/kg. Lethality was observed 24-48 hrs after injections. To analyze the therapeutic activity of 10c, C57BL/6 mice inoculated with breast tumors Wnt-135 were injected once a week for 4 weeks with 0.4 mg/kg (i.v.) of 10c dissolved in saline. At the initiation of 10c treatment both control and experimental groups had palpable tumors with a comparable average volume (42±12 and 32±11 mm3 accordingly). The selected dose significantly (p 95%. Separation by column chromatography was performed using Merck Kieselgel 60 (70 – 230 mesh). All reactions were performed with commercially available reagents («Aldrich», «Alfa Aesar», «Acros», «Serva»). Phenylacetylene was distilled under low pressure (b.p. 75 °C/80 mm Hg). Solvents were purified according to standard procedures. The petroleum spirit refers to the fraction with distillation range 40 – 70 °C.
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4.2. Preparation of compounds 10-12. Synthesis of colchiceine (11).28A mixture of colchicine (1) (300.0 mg, 0.75 mmol), glacial acetic acid (3 mL) and hydrochloric acid (0.1 N 18 mL) was stirred for 3 h at 100 °C, then solid Na2CO3 was added until pH become neutral. Resulting solution was extracted with CHCl3 (3×250 mL), the combine organic layer was washed with brine and dried over Na2SO4. The solvent was removed under reduced pressure. Colchiceine (11) was obtained as yellow-green crystals (275.0 mg, 95%) and directly used at the next step. mp 150 ºС. mp lit28 150 ºС. 1Н NMR: (400 MHz, CDCl3) δ 7.49 (s, 1H), 6.87 (s, 1H), 6.54 (s, 1H), 5.91 (s, 1H), 5.75 (d, J = 8.1 Hz, 1H), 4.81 – 4.72 (m, 1H), 3.92 (s, 3H), 3.88 (s, 3H), 3.53 (s, 3H), 2.54 – 2.26 (m, 4H), 2.04 (s, 3H). 13C NMR (101 MHz, CDCl3): δ = 29.64, 37.26, 52.61, 55.94, 61.22, 61.27, 107.22, 119.95, 122.53, 125.71, 134.31, 136.37, 141.32, 150.69, 151.57, 153.47, 170.36, 175.59. Synthesis of iodocolchinol (12)28 Colchiceine (11) (275.0 mg, 0.71 mmol) was dissolved in water (37 mL), solid NaOH (300.0 mg, 7.50 mmol) was added and the solution was cooled to 0 °C. The solution of NaI×2H2O (2,439.0 mg, 16.00 mmol) and iodine (543.0 mg, 2.14 mmol) in water (190 mL) was added dropwise to the reaction mixture and the resulting solution was stirred for 1 h at 0 °C. The temperature was allowed to rise until 20 °C. Corresponding quantity of Na2SO3 was added to the solution to remove the excess of iodine. Then the mixture was acidified with conc. HCl to pH=2. The obtained yellow-green precipitate was isolated by filtration and dried under reduced pressure at 50 – 60 °C. The residual solution was extracted with ethyl acetate (3×250 ml), the combined organic layer was washed with brine, dried over Na2SO4. The crude product obtained after the solvent removing together with the precipitate were purified by column chromatography, eluent petroleum spirit - ethyl acetate - ethanol (4:1:1), to afford 338.0 mg, 93% of iodocolchinol (12). mp 238 ºС. mp lit28 238 ºС. 1H NMR (400 MHz, DMSO-d6) δ
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9.95 (s, 1H), 8.37 (d, J = 8.1 Hz, 1H), 7.17 (s, 1H), 6.86 (s, 1H), 6.76 (s, 1H), 4.42 (dt, J = 12.3, 6.2 Hz, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.42 (s, 3H), 2.08 (s, 4H), 1.87 (s, 3H). 13С NMR (101 MHz, DMSO-d6): 22.51, 29.87, 37.78, 48.13, 55.72, 60.41, 81.29, 107.98, 109.96, 123.14, 126.49, 134.65, 139.06, 140.41, 142.30, 150.06, 152.10, 155.52, 168.20. General procedure for the synthesis of compounds 10a-i. Iodocolchinol (12) (100.0 mg, 0.21 mmol), Pd(OAc)2 (2.3 mg, 0.01 mmol), CuI (3.9 mg, 0.02 mmol), PPh3 (8.1 mg, 0.03 mmol) and AcOK (60.6 mg, 0.62 mmol) were placed into the two-neck flask under an inert atmosphere. In the case of 10a and 10b compounds synthesis 131.1 mg (0.62 mmol) of K3PO4 was used as a base instead of AcOK. Then anhydrous acetonitrile (4 mL) and the corresponding alkyne (0.25 mmol) were added. The mixture was stirred at 60 °C for 1 – 1.5 h, and then the temperature was raised up to 80 °C until the completeness of the reaction (TLC control). The solvent was removed under reduce pressure, a solid residue was purified by column chromatography to afford the target products. N-((1S)-2"-phenyl-1 ,2 ,3 -trimethoxy-6,7-dihydro-1H-benzo[5',6':5,4]cyclohepta[3,2f]benzofuran-1-yl)acetamide (10a). Purified by column chromatography, eluent petroleum spiritEtOAc-EtOH (5:1:1), as brownish-yellow powder, 86%, mp 140 ºС. 1H NMR (400 MHz, DMSO-
d6) δ 8.50 (d, J = 8.5 Hz, 1H), 7.93 (s, 1H), 7.91 (s, 1H), 7.57 (s, 2H), 7.52 (t, J = 7.7 Hz, 2H), 7.44 (s, 1H), 7.41 (t, J = 7.3 Hz, 1H), 6.81 (s, 1H), 4.66 – 4.59 (m, 1H), 3.85 (s, 3H), 3.80 (s, 3H), 3.46 (s, 3H), 2.29 – 1.97 (m, 4H), 1.93 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.53, 168.50, 155.13, 153.73, 152.36, 150.41, 138.26, 134.80, 132.42, 129.91, 129.30, 129.09, 128.96, 126.98, 124.57, 121.98, 108.08, 105.75, 101.99, 60.62, 60.56, 55.85, 48.54, 30.05, 22.75, 22.72. MS: m/z (%) = 354 (19), 367 (40), 383 (45), 398 (100), 399 (30), 458 (81). Found С, 73.45; Н, 5.97. С28Н27NO5 requires: C, 73.51; H, 5.95%.
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N-((1S)-2"-(pyridin-2-yl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
(10b).
Purified
by
column
chromatography, eluent petroleum spirit-ethyl acetate-ethanol (4:1:1), as brown polycrystalline powder, 83%, mp 140 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.69 (d, J = 4.7 Hz, 1H), 8.51 (d, J = 8.4 Hz, 1H), 7.96 (d, J = 3.6 Hz, 2H), 7.65 (s, 1H), 7.60 (d, J = 4.9 Hz, 2H), 7.41 (dd, J = 8.9, 4.5 Hz, 1H), 6.81 (s, 1H), 4.66 – 4.60 (m, 1H), 3.85 (s, 3H), 3.81 (s, 3H), 3.46 (s, 3H), 2.26 – 1.95 (m, 4H), 1.93 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 168.47, 154.76, 154.10, 152.38, 150.40, 150.00, 148.32, 140.61, 139.20, 137.35, 134.76, 129.53, 126.56, 124.43, 123.43, 122.64, 119.40, 108.06, 105.97, 104.87, 60.60, 60.56, 55.83, 48.57, 30.95, 30.01, 22.70. MS: m/z (%) = 368 (35), 384 (39), 399 (100), 400 (27), 415 (12), 458 (62). Found С, 70.81; Н, 5.67. С28Н26N2O5 requieres C, 70.73; H, 5.72%. N-((1S)-2"-(hydroxymethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
(10c).
Purified
by
column
chromatography, eluent petroleum spirit-EtOAc-EtOH (3:1:1), as yellow polycrystalline powder, 84%, mp 110 ºС. 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 8.5 Hz, 1H), 7.51 (s, 1H), 7.46 (s, 1H), 6.79 (s, 1H), 6.75 (s, 1H), 5.43 (t, J = 5.8 Hz, 1H), 4.64 – 4.54 (m, 3H), 3.84 (s, 3H), 3.79 (s, 3H), 3.41 (s, 3H), 2.22 – 1.70 (m, 7H).
13
C NMR (101 MHz, DMSO-d6) δ 168.39,
158.42, 153.73, 152.23, 150.37, 140.59, 137.42, 134.75, 128.69, 126.18, 124.73, 121.81, 108.01, 105.52, 103.21, 60.59, 60.45, 56.22, 55.82, 48.40, 30.95, 30.05, 22.67. MS: m/z (%) = 294 (17), 321 (57), 337 (43), 352 (100), 353 (23), 411 (55). Found С, 67.07; Н, 6.24. С23H25NO6 requires C, 67.14; H, 6.12%. N-((1S)-2"-(1-hydroxyethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
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(10d). Purified by column chromatography, eluent petroleum spirit-ethyl acetate-ethanol (4:1:1), as yellow polycrystalline powder, 93%, mp 80 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.46 (d, J = 8.4 Hz, 1H), 7.50 (s, 1H), 7.45 (s, 1H), 6.79 (s, 1H), 6.70 (s, 1H), 5.51 (s, 1H), 4.84 (q, J = 6.5 Hz, 1H), 4.63 – 4.55 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.41 (s, 3H), 2.29 – 1.93 (m, 4H), 1.90 (s, 3H), 1.47 (d, J = 6.5 Hz, 3H).
13
C NMR (101 MHz, DMSO-d6) δ 168.43, 162.01, 161.98,
153.48, 152.24, 150.39, 140.61, 137.24, 134.77, 128.65, 126.17, 124.79, 121.79, 108.03, 105.55, 101.22, 62.30, 60.60, 60.46, 56.03, 55.83, 48.43, 30.06, 26.45, 22.68, 18.56. MS: m/z (%) = 321 (24), 335 (42), 351 (42), 366 (100), 367 (26), 425 (62). Found С, 67.76; Н, 6.42. С24H27NO6 requires C, 67.75; H, 6.40%. N-((1S)-2''-(1-hydroxyсусlopentyl)-1',2',3'-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
(10e).
Purified
by
column
chromatography, eluent petroleum spirit-ethyl acetate-ethanol (6:2:1), as dark-yellow oil, 86%. 1
H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 8.5 Hz, 1H), 7.48 (s, 1H), 7.44 (s, 1H), 6.79 (s,
1H), 6.70 (s, 1H), 5.25 (s, 1H), 4.62 – 4.55 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.41 (s, 3H), 2.16 (td, J = 12.1, 5.9 Hz, 2H), 2.05 (dd, J = 12.4, 6.5 Hz, 2H), 1.99 (s, 1H), 1.90 (s, 3H), 1.74 (dd, J = 12.2, 8.0 Hz, 3H), 1.68 – 1.55 (m, 1H), 1.54 – 1.43 (m, 1H), 1.20 – 0.99 (m, 2H). 13C NMR (101 MHz, DMSO-d6) δ 168.40, 163.40, 153.53, 152.21, 150.37, 140.61, 136.98, 134.76, 128.56, 126.32, 124.83, 121.61, 108.02, 105.49, 100.87, 78.09, 60.60, 60.45, 55.82, 48.42, 34.19, 30.05, 26.34, 24.78, 23.48, 22.68, 13.96. Found C, 69.85; H, 6.64. C27H31NO6 requieres C, 69.66; H, 6.71%. N-((1S)-2"-(2-hydroxyethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
(10f).
Purified
by
column
chromatography, eluent petroleum spirit-EtOAc-EtOH (4:1:1), as yellow polycrystalline powder,
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74%, mp 125 ºС. 1H NMR (400 MHz, DMSO-d6) δ 8.43 (d, J = 8.4 Hz, 1H), 7.45 (s, 1H), 7.42 (s, 1H), 6.78 (s, 1H), 6.62 (s, 1H), 4.81 (t, J = 5.4 Hz, 1H), 4.63 – 4.56 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.78 – 3.73 (m, 2H), 3.41 (s, 3H), 2.91 (t, J = 6.6 Hz, 2H), 2.31 – 1.91 (m, 4H), 1.90 (s, 3H).
13
C NMR (101 MHz, DMSO-d6) δ 168.37, 156.91, 153.46, 152.18, 150.36, 140.59,
136.65, 134.75, 128.49, 126.69, 124.85, 121.21, 107.99, 105.25, 102.91, 62.78, 60.60, 60.48, 59.03, 55.80, 48.38, 31.93, 30.06, 22.67. MS: m/z (%) = 321 (15), 335 (57), 351 (36), 366 (100), 367 (26), 425 (57). Found С, 67.82; Н, 6.36. С24H27NO6 requires C, 67.75; H, 6.40%. N-((1S)-2"-(acetoxymethyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)acetamide
(10g).
Purified
by
column
chromatography, eluent petroleum spirit-EtOAc-EtOH (6:1:1), as light-beige polycrystalline powder, 41%. mp 82 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 8.3 Hz, 1H), 7.56 (s, 1H), 7.49 (s, 1H), 6.99 (s, 1H), 6.79 (s, 1H), 5.22 (s, 2H), 4.58 (dd, J = 11.8, 7.5 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.42 (s, 3H), 2.23 – 2.11 (m, 2H), 2.08 (s, 3H), 2.03 (dd, J = 12.7, 5.6 Hz, 2H), 1.91 (s, 3H). 13C NMR (101 MHz, DMSO-d6) δ 169.95, 168.45, 153.95, 152.33, 152.12, 150.37, 140.60, 138.54, 134.74, 129.11, 125.70, 124.51, 122.29, 108.05, 107.03, 105.75, 60.59, 60.49, 57.95, 55.83, 48.50, 30.61, 29.98, 22.66, 20.56. MS: m/z (%) = 334 (30), 351 (45), 363 (33), 394 (100), 395 (26), 453 (77). Found С, 66.11; Н, 6.12. С25H27NO7 requires C, 66.21; H, 6.00%. N-((1S)-2"-(8-(metoxycarbonyl)octyl)-1′,2′,3′-trimetoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl) acetamide (10h). Purified by column chromatography, eluent petroleum spirit-ethyl acetate-ethanol (5:1:1), as brownish oil, 49%. 1H NMR (400 MHz, DMSO-d6) δ 8.42 (d, J = 8.5 Hz, 1H), 7.44 (s, 1H), 7.42 (s, 1H), 6.78 (s, 1H), 6.57 (s, 1H), 4.63 – 4.54 (m, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.57 (s, 3H), 3.42 (s, 3H), 2.75 (t, J = 7.4 Hz, 2H), 2.22 – 1.76 (m, 7H), 1.68 (dd, J = 14.3, 7.1 Hz, 2H), 1.54 – 1.47 (m, 2H), 1.41 –
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1.15 (m, 10H).
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C NMR (101 MHz, DMSO-d6) δ 168.41, 156.02, 153.73, 152.22, 150.35,
140.57, 137.19, 134.76, 128.63, 126.17, 124.75, 121.55, 120.59, 108.02, 105.52, 104.90, 62.78, 60.57, 60.51, 55.83, 48.90, 48.38, 46.47, 30.05, 22.70, 12.07. MS: m/z (%) = 447 (10), 461 (19), 478 (19), 492 (100), 493 (30), 551 (38). Found С, 69.73; Н, 7.47. С32H41NO7 requires C, 69.67; H, 7.49%. N-((1S)-2"-((diethylamino)methyl)-1′,2′,3′-trimethoxy-6,7-dihydro-1Hbenzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl)
acetamide (10i). Purified by column
chromatography, eluent petroleum spirit-ethyl acetate-ethanol (2:1:1), as light-brown polycrystalline powder, 72%, mp 148 ºC. 1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 8.6 Hz, 1H), 7.47 (d, J = 6.8 Hz, 2H), 6.79 (s, 1H), 6.75 (s, 1H), 4.59 (dd, J = 6.9, 3.9 Hz, 1H), 3.84 (s, 3H), 3.79 (s, 3H), 3.75 (s, 2H), 3.43 (s, 3H), 2.56 – 2.51 (m, 4H), 2.21 – 2.10 (m, 2H), 2.04 (dd, J = 12.5, 7.1 Hz, 2H), 1.90 (s, 3H), 1.03 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ 168.41, 156.02, 153.73, 152.22, 150.35, 140.57, 137.19, 134.76, 128.63, 126.17, 124.75, 121.55, 108.02, 105.52, 104.90, 62.78, 60.57, 60.51, 55.83, 48.90, 48.38, 46.47, 30.05, 22.70, 12.07. MS: m/z (%) = 335 (44), 336 (18), 368 (16), 394 (100), 395 (34), 466 (16). Found С, 69.46; Н, 7.38. С27H34N2O5 requires C, 69.50; H, 7.35%. Synthesis
of
N-((1S)-2"-(pyrollidino)-1′,2′,3′-trimethoxy-6,7-dihydro-1H-
benzo[5',6':5,4]cyclohepta[3,2-f]benzofuran-1-yl) acetamide (10l). Iodocolchinol (12) (100.0 mg, 0.21 mmol), Pd(PPh3)2Cl2 (7.2 mg, 0.01 mmol), CuI (3.9 mg, 0.02 mmol) were added into the two-neck flask, and the flask was filled with argon. Pyrrolidine (4 ml) was appended to the mixture and the solution was cooled to 0°С, then the solution of propargylbromide (29.4 mg, 0.25 mmol) in toluene was added dropwise. The mixture was stirred at 50 °С. After the completeness of the reaction the resulting solution was poured into 15 mL of water and the
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resulting mixture was extracted with ethyl acetate (3×10 ml). The combined organic layer was washed with brine, dried over Na2SO4. The solvent was removed under reduced pressure. A solid residue was purified by column chromatography, eluent petroleum spirit-ethyl acetate-ethanol (3:1:1) to afford product 10l as brownish polycrystalline powder, 80%, mp 123 °С. 1H NMR (400 MHz, DMSO-d6) δ 8.60 (d, J = 8.5 Hz, 1H), 7.48 (s, 2H), 6.78 (s, 1H), 6.74 (s, 1H), 4.61 – 4.56 (m, 1H), 3.83 (s, 3H), 3.79 (s, 3H), 3.76 (d, J = 3.7 Hz, 2H), 3.42 (s, 3H), 2.56 (s, 4H), 2.18 – 2.09 (m, 2H), 2.04 (dd, J = 12.4, 6.6 Hz, 2H), 1.90 (s, 3H), 1.71 (s, 4H). 13C NMR (101 MHz, DMSO-d6) δ 168.43, 156.10, 153.66, 152.23, 150.36, 140.58, 137.33, 134.77, 128.67, 126.19, 124.75, 121.63, 108.02, 105.55, 104.28, 60.58, 60.50, 55.82, 53.29, 51.73, 51.59, 48.43, 42.53, 30.06, 23.33, 23.21, 22.68. Found C, 69.88; H, 6.89. C27H32N2O5 requires C, 69.81; H, 6.94%.
Preparation of lamb brain tubulin. Tubulin was purified from lamb brain by ammonium sulfate fractionation and ion-exchange chromatography. The pure protein was stored in liquid nitrogen
and
prepared
as
described.44
Protein
concentrations
were
determined
spectrophotometrically with a Perkin Elmer spectrophotometer Lambda 800 and an extinction coefficient at 275 nm of 1.07 L.g-1.cm-1 in 0.5% SDS in neutral aqueous buffer or 1.09 L.g-1.cm-1 in 6 M guanidine hydrochloride. Tubulin polymerization. Microtubule assembly was performed in 20 mM sodium phosphate buffer, 1 mM EGTA, 10 mM MgCl2, and 3.4 M glycerol, 1 mM GTP pH 6.5. The reaction was started by warming the samples at 37 °C and the mass of polymer formed was monitored by turbidimetry at 350 nm with a POLARstar BMG Labtech spectrophotometer using 96-well plate. Samples containing the compound and controls had less than 1% residual Me2SO.
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Cell cultures. HEK-293, human embryonic kidney cell line (ATCC, CRL-1573), Jurkat human T-lymphoma (ATCC, TIB-152), AsPC-1 pancreatic human adenocarcinoma cell line (ATCC, CRL-1682), and Wnt-1 related murine epithelial cell line W1308, generated by us earlier,35 were used in the study. HEK-293 and AsPC-2 were grown in DMEM supplemented with 10% fetal calf serum (FCS), pen-strep-glut, and 2-ME 5 x 10–5 M (all from PanEco, Moscow, Russian Federation). Jurkat and W1308 cells were grown in RPMI-1640 with the same supplements. Cells were passed by trypsinization using Trypsin /EDTA solution (PanEco, Moscow, Russian Federation) twice a week. Twenty four hours before assays, cells were seeded in the appropriate plates (96- or 24-well plates) adjusted to 3x105 cells/mL and incubated overnight to achieve standardized growth conditions. Confocal analysis. For confocal analysis AsPC-1 cells were grown overnight on sterile cover slips in 200 µl of complete culture medium in 6-well plates (Costar). Compound 10c in different concentrations was dissolved in 4 ml of complete medium and added to the wells. Cells were cultivated for 72 hrs. Nuclei dye Hoecst 33342 (Sigma) and mitochondrial tracker MitoTreckRed (Invitrogen) were added for 1 hr. Finally cells were fixed with 1% paraformaldehyde, washed, and polymerized with Mowiol 4.88 medium (Calbiochem, Germany). Slides were analyzed using Eclipse TE2000 confocal microscope (Nikon, Japan). MTT-assay. Cytotoxic effect of the prodrugs was estimated by a standard 3-(4, 5-dimethyl-2thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT, Sigma) test as was described earlier.45 In short, different dilutions of the new compounds using series of 10 times dilutions from 10 µM to 1 nM were prepared on a separate plate and then transferred in 100 ul to the plates with the cells. Non-treated cells served as controls. Plates were incubated for 72 hrs. For the last 6 hours 250 µg/ml of MTT was added in 10 ul to each well. After the incubation culture medium was
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removed and 100 µl dimethylsulfoxide was added to each well. Plates were incubated at shaking for 15 min to dissolve formazan. Optical density was read on spectrophotometer Titertek (UK) at 540 nm. Results were analyzed by Excel package (Microsoft). Cytotoxic concentration giving 50% of dead cells (IC50) was calculated from the titration curves. The inhibition of proliferation (inhibition index, II) was calculated as [1 – (ODexperiment / ODcontrol)], where OD was MTT optical density. Analysis of apoptosis by flow cytometry. Percentage of apoptotic cell was analyzed by 3,3'dihexyloxacarbocyanine iodide (DiOC6) and propidium iodide (PI) (Sigma Chemical Co) double staining. The cationic lypophilic fluorochrome DiOC6 (Invitrogen) was used to evaluate transmembrane potential in mitochondria.46 Cells were harvested, washed in pre-warmed PBS supplemented with 2% of FCS, resuspended at 106 cells/ml in PBS/2%FCS, 40 µM DiO C6 and 5 ug/ml PI and incubated for 30 minutes at 37oC. Cells were washed with PBS/2% FCS and analyzed by flow cytometry using FACScan device (BD, USA). Total 2000 events were collected. The results were analyzed using CellQuest software (BD Biosciences). Cell cycle analysis. Cell cycle was analyzed using PI-stained DNA. Two million cells were harvested at indicated time, washed in ice-cold PBS, fixed by the addition of 70% ethanol and left for 2 hours at -20oC. Thereafter, the cells were washed twice in PBS, stained with 50 μg/ml of PI (Sigma Chemical Co) in PBS and analyzed by flow cytometry. Animals. C57BL/6 mice were purchased from Pushchino Affiliation of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow. All mice were 6–8 wk old and maintained in minimal pathogen animal facility at the Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, Moscow. All studies were conducted in an AAALAC accredited facility in compliance with the PHS Guidelines for the Care and Use of Animals in Research.
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Wnt-1 tumor growth and treatment in vivo. Wnt-1 tumor cells (106/mouse) were obtained as described,47 and inoculated subcutaneously into the left inguinal mouse fat pad (MFP #4). The injection of cells in 50 µl of PBS was performed through the skin of anesthetized mice. Tumor size was measured with vernier calipers twice a week and calculated using the formula (W2xL)/2, where W and L corresponded to width and length of tumors. This protocol was efficient in the induction of tumors in 100% of mice approximately on day 30 after the cell inoculation. Experiment was started only in mice (n=7 per group) with palpable tumors at day 27. The volume of tumors was comparable (100 mm3) in both groups before 10c treatment. Compound 10c was injected in 200µl of saline intravenously into the orbital sinus of mice once a week at days 27, 34, 41, and 48. Control group was injected with saline only. The experiment was terminated at day 51 when two control mice died. Short term effect of colchicine analogues on endothelial cells (interphase effect). Colchicine, combretastatin A4, compound 10c were dissolved in dimethyl sulfoxide at 20 mM concentration. Four µl of each were transferred to 200 µl of saline (1 mg/kg of weight), mixed with 50 µl of rhodamine B (100 µl/ml of water) and injected i.v. into C57BL/6 intact mice (3 per group). Control animals were injected with dimethyl sulfoxide and rhodamine B only. Four hours later mice were sacrificed by cervical dislocation. Major organs were collected, cut in pieces, and extracted with 0.3N acidic acid in ethanol for 2 hours at 37oC. Supernatants were collected, centrifuged 10 min at 10000 rpm, and cleared fraction was transferred into 96-well black plates (SPL). Fluorescence was measured using GlomaxMulti reader (Promega) with 540 nm filter. For confocal analysis small pieces (1x1 mm) of nonfixed tissue were transferred on the microscopic glass, layed with Mowiol 4.88 medium (Calbiochem, Germany), covered with the cover glass and squeezed. Slides were analyzed using Eclipse TE2000 confocal microscope (Nikon, Japan).
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Statistical analysis Statistical analysis was performed using Excel Student's t-test. Comparison values of p
