RU2724882C1 - 1,1'-(hexane-1,6-diyl)bis(3-(((1r,4as,10ar)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenantaren-1-yl)methyl)urea, which exhibits inhibiting action on human tyrosyl-dna-phosphodiesterase 1 enzyme and increases temozolomide activity on glioblastoma cells - Google Patents
1,1'-(hexane-1,6-diyl)bis(3-(((1r,4as,10ar)-7-isopropyl-1,4a-dimethyl-1,2,3,4,4a,9,10,10a-octahydrophenantaren-1-yl)methyl)urea, which exhibits inhibiting action on human tyrosyl-dna-phosphodiesterase 1 enzyme and increases temozolomide activity on glioblastoma cells Download PDFInfo
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- RU2724882C1 RU2724882C1 RU2019138671A RU2019138671A RU2724882C1 RU 2724882 C1 RU2724882 C1 RU 2724882C1 RU 2019138671 A RU2019138671 A RU 2019138671A RU 2019138671 A RU2019138671 A RU 2019138671A RU 2724882 C1 RU2724882 C1 RU 2724882C1
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- Russia
- Prior art keywords
- dna
- phosphodiesterase
- enzyme
- tdp1
- temozolomide
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Abstract
Description
Изобретение относится к молекулярной биологии, биохимии, медицине и фармакологии, конкретно к соединению, являющемуся производным дегидроабиетиламина формулы I, у которого выявлена способность ингибировать фермент тирозил-ДНК-фосфодиэстеразу 1 человека (Tdp1) и способность усиливать действие алкилирующего химиопрепарата темозоломида на перевиваемых клеточных линиях глиобластомы U-87MG и SNB-19.The invention relates to molecular biology, biochemistry, medicine and pharmacology, specifically to a compound derived from dehydroabietylamine of the formula I, which has the ability to inhibit the human tyrosyl DNA phosphodiesterase 1 enzyme (Tdp1) and the ability to enhance the effect of the alkylating chemotherapy drug temozolomide on transplantable gliast cell lines U-87MG and SNB-19.
Традиционная химиотерапия онкологических заболеваний направлена на повреждение ДНК злокачественных клеток, и ее результат часто зависит от эффективности систем репарации ДНК. Во многих раковых клетках нарушен уровень экспрессии отдельных ферментов и факторов репарации ДНК, что в сочетании с ускоренной клеточной пролиферацией может приводить к резистентности таких опухолей к лекарственным препаратам. Развитие молекулярной биологии позволило принципиально изменить подходы к созданию новых противоопухолевых препаратов. Важным стратегическим направлением лечения онкологических заболеваний является избирательное подавление активности ряда ферментов, вовлеченных в регуляцию жизнедеятельности клеток. В последнее время проводятся интенсивные исследования ингибиторов репарации ДНК как потенциальных лекарств [Hosoya N., Miyagawa K., Targeting DNA damage response in cancer therapy. // Cancer Sci. 2014, 105, 370-388]. При ограничении способности раковой клетки к восстановлению поврежденной ДНК, существенно возрастает эффективность традиционных методов лечения. Поиск ингибиторов ключевых ферментов репарации ДНК относится к перспективным направлениям медицинской химии и является одним из путей создания эффективной терапии сердечно-сосудистых, нейродегенеративных и онкологических заболеваний [Laev S.S., Salakhutdinov N.F., Lavrik О.I., Tyrosyl-DNA phosphodiesterase inhibitors: Progress and potential // Bioorg. Med. Chem. 2016, 24, 5017-5027].Traditional cancer chemotherapy is aimed at damaging the DNA of malignant cells, and its outcome often depends on the effectiveness of DNA repair systems. In many cancer cells, the expression level of individual enzymes and DNA repair factors is impaired, which, in combination with accelerated cell proliferation, can lead to drug resistance of such tumors. The development of molecular biology has made it possible to fundamentally change approaches to the creation of new antitumor drugs. An important strategic direction in the treatment of cancer is the selective suppression of the activity of a number of enzymes involved in the regulation of cell activity. Recently, intensive studies of DNA repair inhibitors as potential drugs have been conducted [Hosoya N., Miyagawa K., Targeting DNA damage response in cancer therapy. // Cancer Sci. 2014, 105, 370-388]. By limiting the ability of a cancer cell to repair damaged DNA, the effectiveness of traditional methods of treatment significantly increases. The search for inhibitors of key DNA repair enzymes is one of the promising areas of medical chemistry and is one of the ways to create effective therapy for cardiovascular, neurodegenerative and oncological diseases [Laev SS, Salakhutdinov NF, Lavrik O.I., Tyrosyl-DNA phosphodiesterase inhibitors: Progress and potential // Bioorg. Med. Chem. 2016, 24, 5017-5027].
Имеющим особую значимость примером заболеваний, для которых поиск новых терапевтических подходов является необходимым, может служить злокачественная глиома - наиболее распространенная среди взрослого населения первичная опухоль центральной нервной системы (ЦНС). Современный стандарт терапии для пациентов с недавно диагностированной мультиформной глиобластомой (ГБМ, глиома IV степени) включает хирургическую резекцию опухоли, лучевую терапию и адъювантную химиотерапию темозоломидом (ТМЗ). Однако, несмотря на мультимодальный подход к терапии данного заболевания, медиана выживаемости с момента постановки диагноза составляет около 12-15 месяцев. Важно отметить, что медиана выживаемости при использовании полного комплекса терапевтических мер составляет 14,6 месяцев по сравнению с 12,2 месяцами для пациентов, при лечении которых химиотерапия не применялась.A particularly significant example of diseases for which the search for new therapeutic approaches is necessary is malignant glioma, the most common primary tumor of the central nervous system (CNS) among adults. The current standard of therapy for patients with newly diagnosed glioblastoma multiforme (GBM, grade IV glioma) includes surgical resection of the tumor, radiation therapy, and adjuvant chemotherapy with temozolomide (TMZ). However, despite the multimodal approach to the treatment of this disease, the median survival from the moment of diagnosis is about 12-15 months. It is important to note that the median survival when using the full range of therapeutic measures is 14.6 months compared with 12.2 months for patients in whose treatment chemotherapy was not used.
С учетом низкой эффективности терапии, в настоящее время активно изучаются стратегии, направленные на преодоление резистентности и усиления ответа на ТМЗ. Резистентность к химиотерапии может быть обусловлена различными факторами, например, способностью опухолевых клеток к активному выведению химиотерапевтических препаратов, экспрессией антиапоптотических белков, а также изменением активности систем репарации ДНК. Известно, что эффективность ТМЗ (алкилирующего агента, который осуществляет перенос метальной группы на пуриновые основания ДНК (O6-гуанин, N7-гуанин и N3-аденин)), зависит от работы системы репарации ДНК. Так, 06-метилгуанин (О6-MeG), может удаляться метилгуанин метилтрансферазой (MGMT, прямая репарация). Кроме того, в опухолях с дефектом в системе репарации неправильно спаренных оснований (MMR-) О6-MeG может не оказать повреждающего действия [Zhang, J., Stevens, M.F.G., Bradshaw, T.D. Temozolomide: mechanisms of action, repair and resistance. // Curr. Molecular Pharm. 2012, 5, 102-114]Given the low effectiveness of therapy, strategies are currently being actively studied to overcome resistance and enhance the response to TMZ. Resistance to chemotherapy can be due to various factors, for example, the ability of tumor cells to actively excrete chemotherapeutic drugs, the expression of anti-apoptotic proteins, as well as changes in the activity of DNA repair systems. It is known that the effectiveness of TMZ (an alkylating agent that transfers the methyl group to purine DNA bases (O6-guanine, N7-guanine and N3-adenine)) depends on the functioning of the DNA repair system. So, 06-methylguanine (O6-MeG) can be removed with methylguanine by methyltransferase (MGMT, direct repair). In addition, in tumors with a defect in the misrepresented base repair system (MMR-), O6-MeG may not have a damaging effect [Zhang, J., Stevens, M.F.G., Bradshaw, T.D. Temozolomide: mechanisms of action, repair and resistance. // Curr. Molecular Pharm. 2012, 5, 102-114]
В связи с этим, ключевые ферменты систем репарации являются важнейшими мишенями для создания химиотерапевтических препаратов. К числу перспективных мишеней относится фермент тирозил-ДНК-фосфодиэстераза1 (Tdp1). Этот фермент является важной мишенью в случае противоопухолевой терапии, основанной на применении ингибиторов топоизомеразы 1 (Top1) [Negrini S., Gorgoulis V.G., Halazonetis T.D., Genomic instability-an evolving hallmark of cancer. // Nat. Rev. Mol. Cell. Biol. 2010, 11, 220-228]. Tdp1 играет ключевую роль в удалении аддуктов Top1-ДНК, стабилизированных ингибиторами Top1, такими как камптотецин и его клинические производные. Мутация в гене Tdp1 делает клетки гиперчувствительными к камптотецину - каноническому ингибитору Top1 [El-Khamisy S.F., Masutani М., Suzuki Н., Caldecott K.W., A requirement for PARP-1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage. // Nucleic Acids Res., 2003, 31, 5526-5533]. Мыши, нокаутные по Tdp1, гиперчувствительны к камптотецину и его производным. При подавлении экспрессии Tdp1 с помощью миноциклина усиливается антиметастатический эффект иринотекана - ингибитора Tdp1 и увеличивается продолжительность жизни экспериментальных животных [Huang Н. -С., Liu J., Baglo Y., Rizvi I., Anbil S., Pigula M., Hasan Т., Mechanism-informed Repurposing of Minocycline Overcomes Resistance to Topoisomerase Inhibition for Peritoneal Carcinomatosis. // Mol. Cancer Ther., 2018, 17, 508-520]. И, наоборот, гиперэкспрессия Tdp1 приводит к защите клеток от повреждений ДНК, вызванных камптотецином [Nivens М.С., Pouliot J.J., Felder Т., Pena М.М.О., Spencer Н.Т., Galloway, А.Н., Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase. // Cancer Chemother. Pharmacol., 2004, 53, 107-115; Alagoz M., Gilbert D.C., El-Khamisy S., Chalmers A.J., DNA Repair and Resistance to Topoisomerase I Inhibitors: Mechanisms, Biomarkers and Therapeutic Targets. // Curr. Med. Chem., 2012, 19, 3874-3885]. Более того, в опухолях кишечника с гиперэкспрессией Tdp1 снижен ответ на терапию иринотеканом [Meisenberg С, Gilbert D.C., Chalmers A., Haley V., Gollins S., Ward S.E., El-Khamisy, S.F., Clinical and Cellular Roles for TDP1 and TOP1 in Modulating Colorectal Cancer Response to Irinotecan. // Mol. Cancer Ther. 2015, 14, 575-585]. Следовательно, подавление активности Tdp1 может значительно усилить терапевтический эффект ингибиторов Top1.In this regard, the key enzymes of the repair systems are the most important targets for the creation of chemotherapeutic drugs. Among the promising targets is the enzyme tyrosyl-DNA-phosphodiesterase 1 (Tdp1). This enzyme is an important target in the case of antitumor therapy based on the use of topoisomerase 1 (Top1) inhibitors [Negrini S., Gorgoulis V.G., Halazonetis T. D., Genomic instability-an evolving hallmark of cancer. // Nat. Rev. Mol. Cell. Biol. 2010, 11, 220-228]. Tdp1 plays a key role in the removal of Top1 DNA adducts stabilized by Top1 inhibitors such as camptothecin and its clinical derivatives. A mutation in the Tdp1 gene makes cells hypersensitive to camptothecin, a canonical Top1 inhibitor [El-Khamisy SF, Masutani M., Suzuki N., Caldecott KW, A requirement for PARP-1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage. // Nucleic Acids Res., 2003, 31, 5526-5533]. Tdp1 knockout mice are hypersensitive to camptothecin and its derivatives. When suppressing the expression of Tdp1 using minocycline, the antimetastatic effect of irinotecan, a Tdp1 inhibitor, is enhanced and the lifespan of experimental animals is increased [Huang N.-C., Liu J., Baglo Y., Rizvi I., Anbil S., Pigula M., Hasan T ., Mechanism-informed Repurposing of Minocycline Overcomes Resistance to Topoisomerase Inhibition for Peritoneal Carcinomatosis. // Mol. Cancer Ther., 2018, 17, 508-520]. Conversely, overexpression of Tdp1 leads to the protection of cells from DNA damage caused by camptothecin [Nivens M.S., Pouliot JJ, T. Felder, M. M. Pena, Spencer N.T., Galloway, A.N. , Engineered resistance to camptothecin and antifolates by retroviral coexpression of tyrosyl DNA phosphodiesterase-I and thymidylate synthase. // Cancer Chemother. Pharmacol., 2004, 53, 107-115; Alagoz M., Gilbert D.C., El-Khamisy S., Chalmers A.J., DNA Repair and Resistance to Topoisomerase I Inhibitors: Mechanisms, Biomarkers and Therapeutic Targets. // Curr. Med. Chem., 2012, 19, 3874-3885]. Moreover, in intestinal tumors with overexpression of Tdp1, the response to irinotecan therapy is reduced [Meisenberg C, Gilbert DC, Chalmers A., Haley V., Gollins S., Ward SE, El-Khamisy, SF, Clinical and Cellular Roles for TDP1 and TOP1 in Modulating Colorectal Cancer Response to Irinotecan. // Mol. Cancer Ther. 2015, 14, 575-585]. Therefore, suppression of Tdp1 activity can significantly enhance the therapeutic effect of Top1 inhibitors.
Помимо способности удалять ковалентные аддукты Top1 и ДНК, Tdp1 может гидролизовать в ДНК АР-сайты и индуцировать их репарацию [Речкунова Н.И., Лебедева Н.А., Лаврик О.И.. Тирозил-ДНК-фосфодиэстераза 1 - новый участник репарации апуриновых/апиримидиновых сайтов в ДНК. // Биоорг. химия 2015, 41 (5), 531-538]. Эта активность особенно актуальна для репарации повреждений ДНК, инициированных монофункциональными алкилирующими агентами, включая метилметансульфонат и темозоломид, а также ионизирующим излучением. Таким образом, подавление активности Tdp1, может повышать чувствительность опухолевых клеток к препаратам, входящим в стандарт терапии онкологических заболеваний, в том числе глиобластомы [Pommier Y., Huang S.N., Gao R., Das B.B., Murai J., Marchand, C., Tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2). // DNA Repair 2014, 19, 114-29].In addition to the ability to remove covalent adducts of Top1 and DNA, Tdp1 can hydrolyze AP sites in DNA and induce their repair [Rechkunova NI, Lebedeva NA, Lavrik OI. Tyrosyl DNA phosphodiesterase 1 - a new participant in repair apurinic / apyrimidine sites in DNA. // Bioorg. Chemistry 2015, 41 (5), 531-538]. This activity is especially relevant for the repair of DNA damage initiated by monofunctional alkylating agents, including methyl methanesulfonate and temozolomide, as well as ionizing radiation. Thus, the suppression of the activity of Tdp1, can increase the sensitivity of tumor cells to drugs that are part of the standard treatment of cancer, including glioblastoma [Pommier Y., Huang SN, Gao R., Das BB, Murai J., Marchand, C., Tyrosyl-DNA-phosphodiesterases (TDP1 and TDP2). // DNA Repair 2014, 19, 114-29].
В литературе имеются данные о некоторых ингибиторах Tdp1, эффективных при различных концентрациях в диапазоне значений IC50 0.15-100 мкМ. Известны ингибиторы производные гликозидов: неомицина, нетилмицина, спектиномицина [Liao Z., Thibaut L., Jobson A., Pommier Y., Inhibition of Human Tyrosyl-DNA Phosphodiesterase by Aminoglycoside Antibiotics and Ribosome Inhibitors // Mol. Pharmacol. 2006, 70, 366-372]. Однако для ингибирования необходимы достаточно высокие концентрации, кроме того, селективность ингибирования данного фермента очень низкая. В том же диапазоне концентраций ингибируют тирозил-ДНК-фосфодиэстеразу 1 диамидины [Huang S.N., Pommier Y., Marchand С., Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) inhibitors // Expert Opin. Ther. Pat. 2011, 9, 1285-1292]. Активно изучается способность ингибировать Tdp1 производных инденоизохинолина [Elsayed M.S.A., Su Y., Wang P., Sethi Т., Agama K., Ravji A., Redon C.E., Kiselev E., Horzmann K.A., Freeman J.L., Yves Pommier Y., Cushman M., Design and Synthesis of Chlorinated and Fluorinated 7 Azaindenoisoquinolines as Potent Cytotoxic Anticancer Agents That Inhibit Topoisomerase I // J. Med. Chem. 2017, 60, 5364-5376]. Исследована ингибирующая активность в отношении Tdp1 производных 2-циано-3-фенилпроп-2-ентиоамида. Соединения ингибируют фермент со значениями IC50=0.9-100 мкМ [Sirivolu V.R., Vernekar S.K., Marchand С., Naumova A., Chergui A., Renaud А., Stephen A.G., Chen F., Sham Y.Y., Pommier Y., Wang Z., 5-Arylidenethioxothiazolidinones as inhibitors of tyrosyl-DNA phosphodiesterase I // J. Med. Chem. 2012, 55, 8671-8684]. Были обнаружены такие новые классы ингибиторов как аналоги бензопентатиепина [Zakharenko A., Khomenko Т., Zhukova S., Koval О., Zakharova О., Anarbaev R., Lebedeva N., Korchagina D., Komarova N., Vasiliev V., Reynisson J., Volcho K., Salakhutdinov N., Lavrik O. // Bioorg. Med. Chem. 2015, 23, 2044-2052], производные кумаринов [Khomenko Т., Zakharenko A., Odarchenko Т., Arabshahi H.J., Sannikova V., Zakharova O., Korchagina D., Reynisson J., Volcho K., Salakhutdinov N., Lavrik O. // Bioorg. Med. Chem. 2016, 24, 5573-5581] и производные усниновой кислоты [Zakharenko A., Luzina О., Koval О., Nilov D., Gushchina I., Dyrkheeva N., Svedas V., Salakhutdinov N., Lavrik O. // J. Nat. Prod. 2016, 79, 2961-2967].In the literature, there are data on some Tdp1 inhibitors effective at various concentrations in the range of IC 50 values of 0.15-100 μM. Inhibitors of glycoside derivatives are known: neomycin, netilmycin, spectinomycin [Liao Z., Thibaut L., Jobson A., Pommier Y., Inhibition of Human Tyrosyl-DNA Phosphodiesterase by Aminoglycoside Antibiotics and Ribosome Inhibitors // Mol. Pharmacol 2006, 70, 366-372]. However, sufficiently high concentrations are necessary for inhibition, in addition, the selectivity of inhibition of this enzyme is very low. In the same concentration range, tyrosyl DNA phosphodiesterase 1 diamidines are inhibited [Huang SN, Pommier Y., Marchand C., Tyrosyl-DNA Phosphodiesterase 1 (Tdp1) inhibitors // Expert Opin. Ther. Pat. 2011, 9, 1285-1292]. The ability to inhibit Tdp1 of indenoisoquinoline derivatives [Elsayed MSA, Su Y., Wang P., Sethi T., Agama K., Ravji A., Redon CE, Kiselev E., Horzmann KA, Freeman JL, Yves Pommier Y., Cushman is being actively studied M., Design and Synthesis of Chlorinated and Fluorinated 7 Azaindenoisoquinolines as Potent Cytotoxic Anticancer Agents That Inhibit Topoisomerase I // J. Med. Chem. 2017, 60, 5364-5376]. The inhibitory activity against Tdp1 of 2-cyano-3-phenylprop-2-entioamide derivatives was studied. The compounds inhibit the enzyme with IC 50 values of 0.9-100 μM [Sirivolu VR, Vernekar SK, Marchand C., Naumova A., Chergui A., Renaud A., Stephen AG, Chen F., Sham YY, Pommier Y., Wang Z., 5-Arylidenethioxothiazolidinones as inhibitors of tyrosyl-DNA phosphodiesterase I // J. Med. Chem. 2012, 55, 8671-8684]. Such new classes of inhibitors were found as analogues of benzopentathiepine [Zakharenko A., Khomenko T., Zhukova S., Koval O., Zakharova O., Anarbaev R., Lebedeva N., Korchagina D., Komarova N., Vasiliev V., Reynisson J., Volcho K., Salakhutdinov N., Lavrik O. // Bioorg. Med. Chem. 2015, 23, 2044-2052], coumarin derivatives [Khomenko T., Zakharenko A., Odarchenko T., Arabshahi HJ, Sannikova V., Zakharova O., Korchagina D., Reynisson J., Volcho K., Salakhutdinov N. , Lavrik O. // Bioorg. Med. Chem. 2016, 24, 5573-5581] and derivatives of usnic acid [Zakharenko A., Luzina O., Koval O., Nilov D., Gushchina I., Dyrkheeva N., Svedas V., Salakhutdinov N., Lavrik O. // J. Nat. Prod. 2016, 79, 2961-2967].
Наиболее близким к заявляемым соединениям - прототипом, является инденоизохинолины, представляющие собой полициклическую структуру формулы II [Nguyen TX, Morrell A, Conda-Sheridan M, Marchand C, Agama K, Bermingham A, Stephen AG, Chergui A, Naumova A, Fisher R, O'Keefe BR, Pommier Y, Cushman M. // J Med Chem. 2012, 23, 55, 7297].Closest to the claimed compounds, the prototype is indenoisoquinolines, which are a polycyclic structure of the formula II [Nguyen TX, Morrell A, Conda-Sheridan M, Marchand C, Agama K, Bermingham A, Stephen AG, Chergui A, Naumova A, Fisher R, O'Keefe BR, Pommier Y, Cushman M. // J Med Chem. 2012, 23, 55, 7297].
Недостатками известного соединения являются невысокая активность в отношении очищенного фермента Tdp1 (IC50 29.5 мкмоль) и значительная цитотоксичность.The disadvantages of the known compounds are low activity against purified Tdp1 enzyme (IC 50 29.5 μmol) and significant cytotoxicity.
Задачей изобретения является выявление нового эффективного ингибитора фермента Tdp1, обладающего высокой активностью и низкой токсичностью и проявляющего синергетический эффект с темозоломидом в отношении клеток глиобластомы.The objective of the invention is to identify a new effective inhibitor of the enzyme Tdp1, which has high activity and low toxicity and exhibits a synergistic effect with temozolomide against glioblastoma cells.
Технический результат: повышение эффективности ингибирования фермента Tdp1, расширение ассортимента ингибиторов фермента Tdp1 и усиление цитотоксического действия алкилирующего химиопрепарата темозоломида на культуры клеток глиобластомы человека.Effect: increasing the efficiency of inhibition of the Tdp1 enzyme, expanding the range of inhibitors of the Tdp1 enzyme and enhancing the cytotoxic effect of the alkylating chemotherapy drug temozolomide on human glioblastoma cell cultures.
Поставленная задача решается применением производного дегидроабиетиламина формулы I:The problem is solved by the use of a dehydroabietylamine derivative of the formula I:
у которого выявлена высокая ингибирующая активность в отношении фермента репарации Tdp1 (IC50 0.19 мкмоль), низкая токсичность в отношении ряда опухолевых клеток и усиление эффективности действия темозоломида в отношении перевиваемых клеточных линий глиобластомы.which revealed a high inhibitory activity against the Tdp1 repair enzyme (IC 50 0.19 μmol), low toxicity to a number of tumor cells, and increased efficacy of temozolomide against transplantable glioblastoma cell lines.
Нами был синтезирован ряд производных природного сексвитерпеноида дегидроабиетиламина, содержащих в остове структурные блоки мочевины и тиомочевины, и различные заместители. В том числе были получены соединения с двумя фрагментами дегидроабиетиламина и двумя уреидными группировками, соединенные различными линкерами. [Kovaleva K., Oleshko О., Mamontova Е., Yarovaya О., Zakharova О., Zakharenko A., Kononova A., Dyrkheeva N., Cheresiz S., Pokrovsky A., Lavrik O., Salakhutdinov N. Dehydroabietylamine Ureas and Thioureas as Tyrosyl-DNA Phosphodiesterase 1 Inhibitors That Enhance the Antitumor Effect of Temozolomide on Glioblastoma Cells // J Nat Prod. 2019, 82, 9, 2443-2450]We have synthesized a number of derivatives of the natural sexwiterpenoid dehydroabietylamine containing in the core structural blocks of urea and thiourea, and various substituents. In particular, compounds with two fragments of dehydroabietylamine and two ureide moieties connected by different linkers were obtained. [Kovaleva K., Oleshko O., Mamontova E., Yarovaya O., Zakharova O., Zakharenko A., Kononova A., Dyrkheeva N., Cheresiz S., Pokrovsky A., Lavrik O., Salakhutdinov N. Dehydroabietylamine Ureas and Thioureas as Tyrosyl-DNA Phosphodiesterase 1 Inhibitors That Enhance the Antitumor Effect of Temozolomide on Glioblastoma Cells // J Nat Prod. 2019, 82, 9, 2443-2450]
Преимущество поиска новых ингибиторов среди природных соединений и их производных заключается как в значительной вариативности структур исходных молекул, так и в присущей многим природным соединениям комплементарности к биологическим мишеням [Bermingham A., Price Е., Marchand С., Chergui A., Naumova A., Whitson Е. L., Krumpe L. R. Н., Goncharova Е. I., Evans J. R., McKee Т.С., Henrich C.J., Pommier Y., Identification of Natural Products That Inhibit the Catalytic Function of Human Tyrosyl-DNA Phosphodiesterase (TDP1) // SLAS disc. 2017, 22, 9, 1093-1105]. Кроме того, важным фактором является коммерческая доступность смоляных кислот. Исходным соединением является дегидроабиетиламин - дитерпеновый первичный амин, получаемый в промышленности из дегидроабиетиновой кислоты, содержащейся в живице хвойных деревьев. Важно отметить, что дегидроабиетиламин можно получать непосредственно из доступной живичной канифоли восстановлением нитрила дегидроабиетиновой кислоты.The advantage of the search for new inhibitors among natural compounds and their derivatives lies both in the significant variability of the structures of the starting molecules and in the complementarity to biological targets inherent in many natural compounds [Bermingham A., Price E., Marchand C., Chergui A., Naumova A. , Whitson E. L., Krumpe LR N., Goncharova E. I., Evans JR, McKee T.S., Henrich CJ, Pommier Y., Identification of Natural Products That Inhibit the Catalytic Function of Human Tyrosyl-DNA Phosphodiesterase ( TDP1) // SLAS disc. 2017, 22, 9, 1093-1105]. In addition, the commercial availability of resin acids is an important factor. The starting compound is dehydroabietylamine, a diterpene primary amine obtained in industry from dehydroabietic acid found in the resin of conifers. It is important to note that dehydroabietylamine can be obtained directly from available gum rosin by reduction of dehydroabietic acid nitrile.
Поскольку ингибиторы ферментов репарации ДНК предполагается использовать в "коктейлях" с известными противоопухолевыми препаратами, обладающими высокой токсичностью, важно, чтобы применение новых соединений в лекарственных формах не приводило бы к дополнительной токсической нагрузке на организм. В связи с этим был выполнен анализ цитотоксичности предлагаемого соединения на широком спектре перевиваемых клеточных линий. Использовали культуры клеток человека, имеющие как опухолевое, так и не опухолевое происхождение. К числу первых относятся такие перевиваемые клеточные линии, как культуры клеток аденокарциномы молочной железы человека (MCF-7, MDA-MB-231), меланомы (Mel-8), линия карциномы легких (А-549) и глиобластома человека (U-87MG, SNB-19, T98G). Неопухолевые клеточные культуры были представлены линией клеток фибробластов легких человека WI-38.Since inhibitors of DNA repair enzymes are supposed to be used in “cocktails” with well-known antitumor drugs that are highly toxic, it is important that the use of new compounds in dosage forms does not lead to additional toxic stress on the body. In this regard, an analysis of the cytotoxicity of the proposed compound was performed on a wide range of transplantable cell lines. Used human cell cultures having both tumor and non-tumor origin. The former include transplantable cell lines such as human mammary adenocarcinoma cell cultures (MCF-7, MDA-MB-231), melanoma (Mel-8), lung carcinoma line (A-549), and human glioblastoma (U-87MG , SNB-19, T98G). Non-tumor cell cultures were represented by the human lung fibroblast cell line WI-38.
На втором этапе, также с помощью стандартного МТТ-теста, цитотоксическая активность темозоломида (ТМЗ) и его комбинации с предлагаемым соединением оценивалась на клеточных линиях глиобластомы человека U-87MG и SNB-19. На линии U-87MG цитотоксичность ТМЗ оценивали при концентрации 2500 мкмоль, а на SNB-19 при концентрациях 2000 мкмоль. Ингибитор Tdp1 использовали в концентрации 100 мкмоль для клеточной линии U-87MG, для экспериментов с SNB-19 концентрации ингибитора Tdp1 75 мкмоль. В результате было показано, что комбинация ТМЗ с соединением I в указанных концентрациях приводила к снижению жизнеспособности клеточной линии U-87MG до 40% по сравнению с монотерапией ТМЗ, а линии SNB-19 - до 30%. Таким образом, можно говорить о потенцировании ТМЗ предлагаемым соединением I (рисунок).At the second stage, also using the standard MTT test, the cytotoxic activity of temozolomide (TMZ) and its combination with the proposed compound was evaluated on human glioblastoma cell lines U-87MG and SNB-19. On the U-87MG line, TMZ cytotoxicity was evaluated at a concentration of 2500 μmol, and on SNB-19 at a concentration of 2000 μmol. The Tdp1 inhibitor was used at a concentration of 100 μmol for the U-87MG cell line; for experiments with SNB-19, the concentration of the Tdp1 inhibitor was 75 μmol. As a result, it was shown that the combination of TMZ with compound I at the indicated concentrations reduced the viability of the U-87MG cell line by 40% compared with TMZ monotherapy, and the SNB-19 line by 30%. Thus, we can talk about the potentiation of TMZ by the proposed compound I (figure).
Изобретение иллюстрируется следующими примерами:The invention is illustrated by the following examples:
Пример 1. Методика синтеза соединения I.Example 1. The method of synthesis of compound I.
В круглодонной колбе 50 мл в хлороформе смешали 0,5 г (0.00175 моль) дегидроабиетиламина с 0,147 г (0.000875 моль) гексаметилендиизоцианата. Кипятили с обратным холодильником в течение 3 часов. Контроль реакции проводили методом тонкослойной хроматографии, по окончании реакции охладили до комнатной температуры. Реакционную смесь промыли дважды водой (2×15 мл). Органический слой сушили над прокаленным Na2SO4, после чего упарили на ротационном испарителе. Очищали методом колоночной хроматографии на 10 г силикагеля, элюент хлороформ/метанол. Вещество выделено в виде желтовато-белого порошка с выходом 90% (0,58 г). Спектральные исследования выполнены в Химическом Сервисном Центре коллективного пользования СО РАН. Спектры ЯМР 1Н и 13С регистрировали на спектрометрах Bruker DRX-500 (1Н: 500.13 МГц, 13С: 125.76 МГц). В качестве внутреннего стандарта использовали остаточные сигналы растворителя - хлороформа (1H 7.24, 13С 76.90 м.д.). Отнесение сигналов в спектрах ЯМР проводилось с привлечение стандартных одномерных и двумерных экспериментов (COSY, HETCOR, COLOC, НМВС, HSQC). Нумерация атомов в соединениях дана для отнесения сигналов в спектрах ЯМР и не совпадает с нумерацией атомов в номенклатурном названии. Масс-спектры высокого разрешения записывали на спектрометре DFS ThermoScientific в режиме полного сканирования в диапазоне m/z 0-500, ионизация электронным ударом 70 эВ при прямом вводе образца. Разделение продуктов реакций проводили с помощью колоночной хроматографии на силикагеле (60-200 μ, Masherey-Nagel).In a 50 ml round-bottom flask in chloroform, 0.5 g (0.00175 mol) of dehydro-diethylamine and 0.147 g (0.000875 mol) of hexamethylene diisocyanate were mixed. Boiled under reflux for 3 hours. The reaction was monitored by thin layer chromatography; after the completion of the reaction, it was cooled to room temperature. The reaction mixture was washed twice with water (2 × 15 ml). The organic layer was dried over calcined Na 2 SO 4 , and then evaporated on a rotary evaporator. Purified by column chromatography on 10 g of silica gel, eluent chloroform / methanol. The substance was isolated as a yellowish-white powder with a yield of 90% (0.58 g). Spectral studies were performed at the Chemical Service Center for Collective Use of the SB RAS. 1 H and 13 C NMR spectra were recorded on Bruker DRX-500 spectrometers ( 1 H: 500.13 MHz, 13 C: 125.76 MHz). The residual signals of the solvent, chloroform ( 1 H 7.24, 13 C 76.90 ppm), were used as the internal standard. The assignment of signals in the NMR spectra was carried out using standard one-dimensional and two-dimensional experiments (COSY, HETCOR, COLOC, NMVS, HSQC). The numbering of atoms in the compounds is given for assigning signals in the NMR spectra and does not coincide with the numbering of atoms in the nomenclature name. High-resolution mass spectra were recorded on a DFS ThermoScientific spectrometer in the full scan mode in the range m / z 0-500, electron impact ionization of 70 eV with direct injection of the sample. The reaction products were separated by silica gel column chromatography (60-200 μm, Masherey-Nagel).
1,1'-(Гексан-1,6-диил)бис(3-(((1R,4aS,10aR)-7-изопропил-1,4а-диметил-1,2,3,4,4а,9,10,10а-октагидрофенантрен-1-ил)метил)мочевина1,1 '- (Hexane-1,6-diyl) bis (3 - (((1R, 4aS, 10aR) -7-isopropyl-1,4a-dimethyl-1,2,3,4,4a, 9, 10,10a-octahydrophenanthren-1-yl) methyl) urea
ЯМР 1Н (500 МГц, CDCl3, δ, м.д., J/Гц): 6.84 (2Н, с, Н-14, Н-14'), 6.95 (2Н, д, J11, 12=8.2, Н-12, Н-12'), 7.13 (2Н, д, J11, 12=8.2 Н-11, Н-11'), 0.85 (6Н, с, Ме-19, Ме-19'),
1.19 (12Н, д, J16, 15=6.9, Ме-16, Ме-17, Ме-16', Ме-17'), 1.17 (6Н, с, Ме-20, Ме-20'), 2.79 (2Н, септ, J15, 16=6.9, Н-15, Н-15'), 2.23 (2Н, д, 2J=12.4, H-1e, 1e'), 1.45 (2Н, д, 2J=12.4, Н-1а, Н-1а'), 2.98-3.12 (4Н, м, Н-22, Н-22'), 2.79-2.84 (4Н, м, Н-7, Н-7'), 2.89-2.99 (4Н, м, Н-18, Н-18'), 1.78-1.87 (4Н, м, Н-23, Н-23'), 1.26-1.39 (4Н, м, Н-24, Н-24'), 1.45-1.53 (6Н, м, Н-3, Н-5a, Н-3', Н-5а'), 1.65-1.74 (8Н, м, Н-2, Н-6, Н-2', Н-6'). ЯМР 13С (125 МГц, CDCl3, δ, м.д 159.26 (С-21, С-21'), 147.25 (С-9, С-9'), 145.31 (С-13, С-13'), 134.85 (С-8, С-8'), 126.71 (С-14, С-14'), 124.07 (С-11, С-11'), 123.58 (С-12, С-12'), 50.55 (С-18, С-18'), 18.49 (Ме-19, Ме-19'), 23.91 (Ме-17, Ме-17', Ме-16, Ме-16'), 25.23 (Ме-20, Ме-20'), 33.31 (С-15, С-15'), 44.82 (С-5, С-5'), 18.62 (С-2, С-2'), 18.81 (С-6, С-6'), 30.11 (С-7, С-7'), 25.66 (С-24, С-24') 29.73 (С-23, С-23'), 39.94 (С-4, С-4'), 38.28 (С-1, С-1'), 37.37 (С-10, С-10'), 35.94 (С-3, С-3'), 37.27 (С-22, С-22'). MS m/z 738.5789 [М]+ (calcd for C48H74O2N4, 738.5806).1.19 (12H, d, J 16 , 15 = 6.9, Me-16, Me-17, Me-16 ', Me-17'), 1.17 (6H, s, Me-20, Me-20 '), 2.79 ( 2H, sept., J 15, 16 = 6.9, H-15, H-15 '), 2.23 (2H, d, 2 J = 12.4, H-1e, 1e'), 1.45 (2H, d, 2 J = 12.4 , Н-1а, Н-1а '), 2.98-3.12 (4Н, m, Н-22, Н-22'), 2.79-2.84 (4Н, m, Н-7, Н-7 '), 2.89-2.99 (4H, m, H-18, H-18 '), 1.78-1.87 (4H, m, H-23, H-23'), 1.26-1.39 (4H, m, H-24, H-24 ') , 1.45-1.53 (6Н, m, Н-3, Н-5 a , Н-3 ', Н-5 а '), 1.65-1.74 (8Н, m, Н-2, Н-6, Н-2 ' , H-6 '). 13 C NMR (125 MHz, CDCl 3 , δ, ppm 159.26 (C-21, C-21 '), 147.25 (C-9, C-9'), 145.31 (C-13, C-13 ') , 134.85 (С-8, С-8 '), 126.71 (С-14, С-14'), 124.07 (С-11, С-11 '), 123.58 (С-12, С-12'), 50.55 (С-18, С-18 '), 18.49 (Ме-19, Ме-19'), 23.91 (Ме-17, Ме-17 ', Ме-16, Ме-16'), 25.23 (Ме-20, Me-20 '), 33.31 (С-15, С-15'), 44.82 (С-5, С-5 '), 18.62 (С-2, С-2'), 18.81 (С-6, С- 6 '), 30.11 (С-7, С-7'), 25.66 (С-24, С-24 ') 29.73 (С-23, С-23'), 39.94 (С-4, С-4 ') 38.28 (С-1, С-1 '), 37.37 (С-10, С-10'), 35.94 (С-3, С-3 '), 37.27 (С-22, С-22'). MS m / z 738.5789 [M] + (calcd for C 48 H 74 O 2 N 4 , 738.5806).
Пример 2. Исследование влияния соединения I на активность Tdp1.Example 2. The study of the effect of compound I on the activity of Tdp1.
Рекомбинантная тирозил-ДНК-фосфодиэстераза 1 человека (КФ 3.1.4.) экспрессированная в системе Escherichia coli (плазмида рЕТ 16B-Tdp1 предоставлена доктором Кальдекотт К.У., Университет Сассекса, Великобритания) и выделена как описано [Interthal Н., Pouliot J.J., Champoux J.J., The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily. // Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12009-12014; Lebedeva N.A., Rechkunova N.I., Lavrik O.I., AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1. // FEBS Lett., 2011, 585, 683-686.].Recombinant human tyrosyl DNA phosphodiesterase 1 (EC 3.1.4.) Expressed in the Escherichia coli system (plasmid pET 16B-Tdp1 provided by Dr. Caldecott K.U., University of Sussex, UK) and isolated as described [Interthal N., Pouliot JJ , Champoux JJ, The tyrosyl-DNA phosphodiesterase Tdp1 is a member of the phospholipase D superfamily. // Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12009-12014; Lebedeva N.A., Rechkunova N.I., Lavrik O.I., AP-site cleavage activity of tyrosyl-DNA phosphodiesterase 1. // FEBS Lett., 2011, 585, 683-686.].
В качестве тест-системы для определения ингибирующих свойств исследуемых соединений использована реакция удаления тушителя флуоресценции Black Hole Quencher 1 (BHQ1) с 3'-конца олигонуклеотида, катализируемая Tdp1. На 5'-конце олигонуклеотида находится (5,6)-FAM - флуорофор, интенсивность флуоресценции которого возрастает при удалении тушителя. Для измерения флуоресценции использовался флуориметр POLARstar OPTIMA производства BMG LABTECH.As a test system for determining the inhibitory properties of the studied compounds, the reaction of removal of the Black Hole Quencher 1 fluorescence quencher (BHQ1) from the 3'-end of the oligonucleotide catalyzed by Tdp1 was used. At the 5'-end of the oligonucleotide is (5,6) -FAM - a fluorophore, the fluorescence intensity of which increases with the removal of the quencher. To measure fluorescence, a POLARstar OPTIMA fluorometer manufactured by BMG LABTECH was used.
Реакционные смеси объемом 200 мкл содержали буфер (50 мМ Tris-HCl, рН 8,0; 50 мМ NaCl; 7 мМ меркаптоэтанол), 50 нМ олигонуклеотид и различные концентрации ингибиторов. Реакция запускалась добавлением Tdp1 до конечной концентрации 1,3 нМ. Измерения проводились в линейном диапазоне зависимости скорости реакции от времени (до 8 минут) через каждые 55 секунд. Влияние предлагаемых соединений оценивали по величине IC50 (концентрация ингибитора, при которой активность фермента снижена наполовину). Обсчет значений IC50 проводили с помощью программы MARS Data Analisys 2.0 (BMG LABTECH).200 μl reaction mixtures contained a buffer (50 mM Tris-HCl, pH 8.0; 50 mM NaCl; 7 mM mercaptoethanol), 50 nM oligonucleotide, and various inhibitor concentrations. The reaction was initiated by the addition of Tdp1 to a final concentration of 1.3 nM. The measurements were carried out in a linear range of the reaction rate versus time (up to 8 minutes) every 55 seconds. The effect of the proposed compounds was evaluated by the value of IC 50 (inhibitor concentration at which the enzyme activity is reduced by half). IC 50 values were calculated using the MARS Data Analisys 2.0 software (BMG LABTECH).
Величина IC50 для соединения I составляет 0.1 мкМ, что в 300 раз ниже, чем у соединения-прототипа II (29.5 мкМ).The IC 50 value for compound I is 0.1 μM, which is 300 times lower than that of the prototype compound II (29.5 μM).
Пример 3. Цитотоксичность изучаемого соединения. Индуцированную соединением клеточную гибель оценивали с помощью стандартного МТТ-теста [Mosmann Т. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. // J. Immunol. Meth., 1983, 65, 55-63.] путем колориметрического измерения количества формазана, конвертированного из 3-(4,5-диметилтиазол-2-ил)-2,5-дифенил-2Н-тетразолия бромида (МТТ) клетками, подвергшимися воздействию соединения. Клетки культивировали в среде IMDM, с 40 мкг/мл гентамицина, 100 ед/мл пенициллина, 0,1 мг/мл стрептомицина и 0,25 мкг/мл амфотерицина и в присутствии 10% эмбриональной бычьей сыворотки производства фирмы "Биолот" в атмосфере с 5% СО2. После формирования 50% монослоя в культуральную среду добавляли исследуемый препарат и контролировали пролиферацию клеточной культуры в течение 72 часов. В качестве контроля использовали клетки, культивируемые при соответствующей концентрации DMSO.Example 3. Cytotoxicity of the studied compounds. Compound-induced cell death was assessed using a standard MTT assay [Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. // J. Immunol. Meth., 1983, 65, 55-63.] By colorimetric measurement of the amount of formazan converted from 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-2H-tetrazolium bromide (MTT) by cells exposed exposure to the compound. Cells were cultured in IMDM medium, with 40 μg / ml gentamicin, 100 u / ml penicillin, 0.1 mg / ml streptomycin and 0.25 μg / ml amphotericin and in the presence of 10% Biolot bovine serum in an atmosphere with 5% CO 2 . After the formation of a 50% monolayer, the studied preparation was added to the culture medium and the proliferation of the cell culture was monitored for 72 hours. As a control, cells cultured at an appropriate concentration of DMSO were used.
Токсичность соединения отсутствовала во всем диапазоне изученных концентраций (до 100 мкмоль).The toxicity of the compound was absent in the entire range of studied concentrations (up to 100 μmol).
Важно отметить, что ТМЗ обладал субоптимальной цитотоксичностью, в то время как ингибитор Tdp1 не проявил цитотоксической активности. Как показано в Таблице 2 и на Рисунке 1, комбинация ТМЗ с соединением в указанных концентрациях приводила к снижению жизнеспособности обеих клеточных линий: до 40% для линии U-87MG и до 30% на линии SNB-19 по сравнению с монотерапией ТМЗ. Таким образом, можно говорить о потенцировании ТМЗ предлагаемым соединением.It is important to note that TMZ had suboptimal cytotoxicity, while the Tdp1 inhibitor did not show cytotoxic activity. As shown in Table 2 and Figure 1, the combination of TMZ with the compound at the indicated concentrations reduced the viability of both cell lines: up to 40% for the U-87MG line and up to 30% on the SNB-19 line compared to TMZ monotherapy. Thus, we can talk about the potentiation of TMZ by the proposed compound.
Таким образом, предложено соединение, представляющее собой производное дегидроабиетиламина формулы I, у которого выявлена биологическая активность, заключающаяся в способности ингибировать действие фермента тирозил-ДНК-фосфодиэстеразы 1 человека (Tdp1). Предлагаемое соединение, являющееся низкотоксичным эффективным ингибитором тирозил-ДНК-фосфодиэстеразы 1, возможно рассматривать как перспективный агент для комбинированной химиотерапии онкологических заболеваний.Thus, a compound is proposed, which is a dehydroabietylamine derivative of the formula I, in which a biological activity has been identified consisting in the ability to inhibit the action of the human tyrosyl DNA phosphodiesterase 1 enzyme (Tdp1). The proposed compound, which is a low toxic effective inhibitor of tyrosyl DNA phosphodiesterase 1, can be considered as a promising agent for combined chemotherapy of cancer.
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