RU2454420C1 - Cytotoxic semi-synthetic macrolide antibiotic oligomycin a derivatives and synthesis method thereof - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to novel antibiotic oligomycin A derivatives, having anti-tumour activity and low toxicity, of formula:

, where R is a methanesulphonic acid (OSO₃CH₃) residue or an azide group (N₃) and synthesis method and use thereof.

EFFECT: obtaining novel antibiotic oligomycin A derivatives.

3 cl, 1 dwg, 1 tbl, 4 ex

Description

FIELD OF THE INVENTION

The invention relates to the pharmaceutical industry and relates to new derivatives of the inhibitor F ₀ F ₁ ATP synthases - macrolide antibiotic oligomycin A - and a method for their preparation.

State of the art

Macrolide antibiotics of the oligomycin family, the mechanism of action of which is associated with the suppression of the activity of F ₀ F ₁ ATP synthases, are natural substances that exhibit a cytotoxic effect against tumor cells, fungi and bacteria (Salomon AR, Voehringer DW, Herzenberg LA, Khosla C. Understanding and exploiting the mechanistic basis for selectivity of polyketide inhibitors of F ₀ F ₁ -ATPase. Proc Natl Acad Sci USA. 2000, 97 (26): 14766-14771; Senior AE, Nadanaciva S, Weber J. The molecular mechanism of ATP synthesis by F1F0-synthase. Biochim Biophys Acta. 2002 Feb 15; 1553 (3): 188-211; Devenish RJ, Prescott M., Boyle GM, Nagley P. The Oligomycin Axis of Mitochondrial ATP Synthase: OSCP and the Proton Channel. J Bioenerg Biomembr. 2000, 32 (5): 507-515; Salomon AR, Voehrin ger DW, Herzenberg LA, Khosla C. Apoptolidin, a selective cytotoxic agent, is an inhibitor of F ₀ F ₁ -ATPase. Chemistry & Biology, 2001, 8, 71-80; Comelli M., Di PF, Mavelli I. Apoptosis is induced by decline of mitochondrial ATP synthesis in erythroleukemia cells. Free Radicals Biol. Med. 2003; 34 (9): 1190-1199; Li YC, Fung KP, Kwok TT, Lee CY, Suen YK, Kong SK Mitochondria-targeting drug oligomycin blocked P-glycoprotein activity and triggered apoptosis in doxorubicin-resistant HepG2 cells. Chemotherapy. 2004, 50 (2): 55-62; MGAlekseeva, SMElizarov, O.V. Bekker, IKLubimova and VNDanilenko. F0F1ATP Synthase of Streptomycetes: Modulation of Activity and Oligomycin Resistance by Protein Ser / Thr Kinases. Biologicheskie Membrany, 2009, Vol. 26, No.1, pp. 41-49). Oligomycins affect important cellular processes, such as proton translocation associated with ATPase.

The most important representatives of this chemical class are oligomycin A (formula 1, R ₁ = CH ₂ , R ₂ = CH ₃ , R ₃ = H, R ₄ = OH), oligomycin B (formula 1 R ₁ = C = O, R ₂ = H, R ₃ = CH ₃ , R ₄ = OH), oligomycin C (formula 1, R ₁ = CH ₂ , R ₂ = H, R ₃ = CH ₃ , R ₄ = H), rutamycin A (formula 2, R = OH), rutamycin B (formula 2, R = H), cytovaricin (formula 3) and several others.

Oligomycin A is a cytotoxic macrolide antibiotic. In structure, it is a 26-membered α, β-unsaturated lactone articulated with a bicyclic spiroketal structure. The structure and absolute configuration of oligomycin A were established by comparing them with the products of rutamycin degradation (G.T. Carter. Structure Determination of Oligomycins A and C.J. Org. Chem. 1986, 51, 4264-4271). The well-studied mechanism of action of macrolide antibiotics of the oligomycin group is based on interaction with the F ₀ complex F ₀ F ₁ ATP synthase of eukaryotic mitochondria and the cytoplasmic complex F ₀ F ₁ ATP synthase of certain actinobacteria (http://www.rpi.edu/dept/bcbpp /molbiochem/MBWeb/mb1/part2/f1fo.htm). At the same time, it is suggested that the family of this antibiotic group may have other biological targets [Wender PA, Jankowski OD, Longcore, K, Tabet EA, Seto H, Tomikawa T. Correlation of F ₀ F ₁ -ATPase inhibition and antiproliferative activity of apoptolidin analog . Org Lett. 2006, 8 (4): 589-592]. Oligomycin is specific for the C-subunit F _{0 of} the ATP synthase complex and, in micromolar concentrations, effectively blocks proton transport through the F ₀ subunit and ATP synthesis. The enzyme complex F0F1 ATP synthase is currently regarded as a promising molecular target in anti-cancer and infectious therapy. [LA Shchepina, O. Y. Pletjushkina, A.V. Avetisyan, LE Bakeeva, E.K Fetisova, DS Izyumov, V. B Saprunova, M.Yu Vyssokikh, B.V Chernyak, and VP Skulachev, Oligomycin, inhibitor of the F0 part of H + -ATP-synthase, suppresses the TNF-induced apoptosis, Oncogene (2002), 21, 8149-8157; Cole ST, Alzari PM Microbiology. TV - a new target, a new drug. Science. 2005 Jan 14; 307 (5707): 214-215].

Chemical modification of natural antibiotics is the most important way to obtain new drugs that have advantages over the original antibiotics. Chemical modification of antibiotics of the oligomycin group was practically not carried out. Partial acetylation of oligomycin A is described, which led to the formation of 5,9,33-tri-O-acetyl and 5,9,13,33-tetra-O-acetyl derivatives, whose complete structures were established on the basis of ¹ H and ¹³ C NMR spectra . These derivatives did not show inhibitory activity when tested on Aspergillus niger spores [László Szilágyi; János Samu; Ilona Harsányi, Structure Elucidation of Two Acetylated Derivatives of Oligomycin A, Spectroscopy Letters, 28, Issue 5 July 1995, 699-707]. Oligomycin A contains several keto groups and their reactivity was studied [Fausto Rfmires, James F. Marecek, Shu-I Tu, Thomas V. Kantor, and Hiroshi Okazaki, Effects of Borohydride-Treated Oligomycins on Processes of Energy Transduction in Mitochondria , Eur. J. Biochem. (1982), 121, 275-2279]. The authors showed that the gradual addition of sodium borohydride to the ethanol solution of rutamycin and oligomycin restores the keto groups at the C-7 and C-11 positions of the macrolide aglycon to the corresponding hydroxyl groups without further changing the lactone cycle. The reduced compounds, like the original antibiotics, inhibit ADP-dependent respiration and proton emission associated with ATP hydrolysis, but do not affect other respiration-related activities in intact rat liver mitochondria.

Methods for the chemical modification of oligomycin A, which selectively change the 7-keto group and the C-2, C-3 double bond, have been previously described. The interaction of oligomycin A with hydroxylamine led to the formation of a 6-membered nitron annelated with the original antibiotic at positions 3-7. Reactions with 1-aminopyridinium iodide gave pyrazolo [1,5-a] pyridines conjugated to oligomycin A at positions C-2 and C-3, followed by spontaneous oxidation of the coupling product at the C2-C3 double bond. The resulting derivatives are less toxic than the original. [L.N. Lysenkova, K.F. Turchin, V.N. Danilenko, A.M. Korolev and M.N. Preobrazhenskaya, The first examples of chemical modification of oligomycin A, The Journal of Antibiotics (2010), 63, 17-22].

Disclosure of invention

Compared to the semi-synthetic derivatives described above, the 33-hydroxyl group is replaced in the present invention. The invention includes a method for producing new semi-synthetic analogues of the antibiotic oligomycin A, which consists in the selective esterification of a hydroxyl group in the side chain of oligomycin A at position C-33 with methanesulfonic acid chloride to form 33-O-methanesulfonyl-oligomycin A (formula 4), followed by nucleophilic substitution of the anion methanesulfonic acid per azido group (Scheme 1). The azidation reaction of 33-O-mesyl-oligomycin A (formula 5) with sodium azide to obtain 33-deoxy-33-azido-oligomycin A was carried out in aprotic solvents - N, N-dimethylformamide and dimethyl sulfoxide.

Scheme 1

In addition, the invention relates to pharmaceutical compositions comprising a therapeutically effective amount of at least one compound corresponding to Formula 4 or 5 in a mixture with at least one acceptable carrier.

The term "Pharmaceutically acceptable" means a material used in the preparation of a pharmaceutical composition, usually safe, non-toxic and acceptable for use in pharmaceuticals and veterinary medicine.

Examples of the preparation of oligomycin A derivatives of the present invention

Example 1

33-O-Mesyl-oligomycin A (Formula 4).

Oligomycin in an amount of 1.0 g was placed in a round bottom flask with a magnetic stirrer and dissolved in 30 ml of dry pyridine. With vigorous stirring, 0.5 g of dimethylaminopyridine in powder form and 0.3 ml methanesulfonyl chloride were added to the flask. The reaction mixture was stirred for 1.5 hours at room temperature and 0.2 ml of methanesulfonyl chloride was added, after an additional 0.1 ml of methanesulfonyl chloride was added. The reaction was monitored by thin-layer chromatography in a hexane-acetone system (1: 1), at the end of the reaction, the reaction mixture was poured onto ice, acidified with 1 N hydrochloric acid to pH 3 and extracted with ethyl acetate (2 × 50 ml). The extract was washed with water until a neutral pH, dried and concentrated in vacuo. The substance was purified by column chromatography on silica gel in a hexane-acetone system (4: 1). Received 0.6 mg (58%) of the oligomycin derivative in the form of a colorless amorphous powder, R _f 0.47 in the hexane-acetone system, R _t 12.4 min (mobile phase - water - acetonitrile. Elution in gradient mode, in which the percentage the acetonitrile content varied from 80 to 95% in 10 minutes and remained 20 minutes at 95% at a flow rate of 1 ml / min). Found: m / z 891.4939 [M + Na] +. C ₄₆ H ₇₆ NaO ₁₃ S. Calculated: M = 891.4904. Found: m / z 907.4638 [M + K] +. C ₄₆ H ₇₆ KO ₁₃ S. Calculated: M = 907.4643. Using ¹ H and ¹³ C NMR spectroscopy in versions 1D and 2D (correlation spectra ¹ H- ¹ H COSY and ¹ H- ¹³ C HETCOR)] the structure of oligomycin A mesylate was studied. Significant weak-field shift of the C ₃₃ H signal was detected (by ~ 0.9 m .d., assignment based on ¹ H- ¹ H COZY) and signal C ₃₃ (by ~ 14 ppm, assignment based on ¹ H- ¹³ C HETCOR) relative to the corresponding signals in the starting oligomycin A. The indicated effect, as well as the presence in the ¹ H NMR spectrum of a singlet mesylate at δ 3.00 with an intensity of 3 bp consistent with the electron-acceptor properties of the substituent OS (O ₂ ) CH ₃ and confirms its presence at C ₃₃ in oligomycin A mesylate.

Example 2

33-Deoxy-33-azido-oligomycin A (Formula 5)

0.5 g of 33-O-Mesyloligomycin A was placed in a round bottom flask equipped with a thermometer and a magnetic stirrer, and dissolved in 10 ml of dry dimethylformamide. Sodium azide 0.25 g was sprinkled in dry form, the reaction mixture was heated to 60-65 ° C with stirring for 2 hours. The reaction was monitored by thin layer chromatography in a hexane-acetone system, 1: 1, and chloroform-methanol, 10: 0.5. Then 0.125 g of sodium azide was added. At the end of the reaction, the reaction mixture was extracted with a 1: 1 mixture of toluene and ethyl acetate, washed with water, dried with sodium sulfate. Oligomycin azide was isolated on silica gel using a chloroform-methanol chromatographic system (30: 0.5). Oligomycin azide was obtained as an amorphous white powder with R _f 0.68 in the chloroform-methanol system (10-0.5) with a yield of 60%. R _t 23.0 min (mobile phase - water - acetonitrile. Elution in gradient mode, in which the percentage of acetonitrile changed from 80 to 95% in 10 minutes and remained 20 minutes at 95% at a flow rate of 1 ml / min). IR spectrum (powder), v / cm ^-1 : 2103 (azido group). Found: m / z 838.5178 [M + Na] +. C ₄₅ H ₇₃ N ₃ O ₁₀ Na. Calculated: M = 838.5194.

Example 3

The biological activity of 33-O-mesylate and 33-deoxy-33-azido oligomycin A

Table 1 presents the results of a comparative measurement of the activity of oligomycin A and its derivatives 4 and 5 on the tumor cell lines HCT116 and K562, in the cell test system Streptomyces sp. (Hypersensitive to oligomycin) and proteoliposomes of this strain. 33-Deoxy-33-azido-oligomycin showed an IC ⁵⁰ comparable to oligomycin A, showing 1000 times less activity in the bacterial test system Streptomyces sp., And almost proportionally reduced activity on proteoliposomes containing F0F1ATP synthase. 33-o-mesyloligomycin is less active compared to oligomycin A in relation to cancer cell lines HCT116 and K562 6 and 45 times, respectively, and also 100 times in the cell test system Streptomyces sp., Like the previous derivative, it was active against F0F1ATP synthase in proteoliposomes.

Table 1 Toxicity of oligomycin A and its new derivatives. Compound Streptomyces sp *. Tumor cell lines **, IC ₅₀ , µM ATPase activity in proteoliposomes *** Concentration, nmol / disk Zone mm HCT116 K562 Concentration, nm Activity, pmol ^32P _i / min Oligomycin A 0.001 10 1.0 + 0.2 0.2 + 0.01 5.0 412 ± 50 50.0 249 ± 25 250,0 124 ± 12 1000,0 69 ± 7 33-o- 0.1 9.5 6.4 ± 0.9 5.8 ± 2.1 5,0 467 ± 68 Mesyloligomycin 50,0 454 ± 59 A4 250,0 463 ± 45 1000,0 408 ± 23 33-deoxy-33- one 7.5 1.0 + 0.3 0.1 + 0.03 5,0 451 ± 46 azido 50,0 452 ± 43 oligomycin A5 250,0 483 ± 72 1000,0 450 ± 65 * The sensitivity level was determined by applying discs with different concentrations of antibiotic on Petri dishes with agar medium, as described previously [M. G. Alekseeva, SMElizarov, O. Bekker, IKLubimova, and VNDanilenko. F0F1-ATP Synthase of Streptomycetes: Modulation of Activity and Oligomycin Resistance by Protein Ser / Thr Kinases. Biochemistry (Moscow) SUPPLEMENT SERIES A: MEMBRANE AND CELL BIOLOGY. Vol.3, No.1, P.16-22, 2009].
** Cells of lines HCT116 (colon adenocarcinoma) and K562 (promyelocytic leukemia) were incubated for 72 hours with the addition of various concentrations of the studied substances (MTT test was used to analyze cell survival). *** ATPase activity in the preparation of ^{proteoliposomes} , pmol ^32P _i , released in 1 min in the presence of 1 μg of protein. In the control (DMSO only), the ATPase activity in the proteoliposome preparation is 462 ± 34 pmol ^32P _i / min.

Thus, new derivatives of the antibiotic oligomycin A were obtained, which have high cytotoxic activity against tumor cell lines. Since the new derivatives do not inhibit ATPase in proteoliposomes, it should be considered that the biological target for exposure to 4 and 5 is not ATPase. Such a statement is valid, because ATPase-independent target has previously been shown for derivatives of apoptolidine, a compound close in structure to oligomycin [Wender PA, Jankowski OD, Longcore K, Tabet EA, Seto H, Tomikawa T. Correlation of F ₀ F ₁ -ATPase inhibition and antiproliferative activity of apoptolidin analogues. Org Lett. 2006, 8 (4): 589-592]. The absence of the effects of synthesized substances 4 and 5 on F0F1 ATP synthase while maintaining high cytotoxicity and the ability to induce apoptosis suggests their lower toxicity to humans. The compounds 4 and 5 obtained by us can be used as hit compounds to create new antitumor drugs based on them.

Claims (3)

1. Derivatives of the antibiotic oligomycin A (compounds 4 and 5, Scheme 1), corresponding to the formula:

where: R represents a methanesulfonic acid residue (4, R = OSO ₃ CH ₃ ) or an azido group (5, R = N ₃ ). 2. The method of producing derivatives of the antibiotic oligomycin A according to claim 1, which consists in the selective esterification of the hydroxy group with methanesulfonic acid chloride located in the C-33 position of the antibiotic side chain, followed by nucleophilic substitution of the methanesulfonic acid residue in the resulting methanesulfonate with an azido group (Scheme 1 ) 3. The use of compounds of formulas 4 and 5 of claim 1 as chemotherapeutic biologically active substances for the manufacture of drugs for the treatment of cancer.