Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
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Dehydroabietylamine Ureas and Thioureas as Tyrosyl-DNA Phosphodiesterase 1 Inhibitors That Enhance the Antitumor Effect of Temozolomide on Glioblastoma Cells Kseniya Kovaleva,†,‡,# Olga Oleshko,‡,# Evgeniya Mamontova,§ Olga Yarovaya,*,†,‡ Olga Zakharova,§ Alexandra Zakharenko,§ Alena Kononova,‡ Nadezhda Dyrkheeva,§ Sergey Cheresiz,‡,⊥ Andrey Pokrovsky,‡ Olga Lavrik,‡,§ and Nariman Salakhutdinov†,‡ Downloaded via STOCKHOLM UNIV on August 21, 2019 at 00:02:16 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
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N. N. Vorozhtsov Novosibirsk Institute of Organic Chemistry, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russian Federation ‡ Novosibirsk State University, Novosibirsk, 630090, Russian Federation § Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, 630090, Russian Federation ⊥ State Scientific Research Institute of Physiology and Basic Medicine, P.O. Box 237, Novosibirsk, 630117, Russian Federation S Supporting Information *
ABSTRACT: A new class of tyrosyl-DNA phosphodiesterase 1 (TDP1) inhibitors was found among resin acid derivatives. Several novel ureas and thioureas derived from dehydroabietylamine were synthesized and tested for TDP1 inhibition. The synthesized compounds showed IC50 values in the range of 0.1 to 3.7 μM and demonstrated low cytotoxicity against the human tumor cell lines U-937, U-87MG, MDA-MB, SK-Mel8, A-549, MCF7, T98G, and SNB19. Several compounds showed enhancement of the cytotoxic activity of the alkylating agent temozolomide, which is used as a first line therapy against glioblastoma (GBM), in the GBM cell lines U-87MG and SNB19.
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researched. The resistance to chemotherapy takes place due to a number of factors, such as the ability of tumor cells to actively pump out the chemotherapeutic drugs, the expression of antiapoptotic factors, or the altered activity of the DNA repair system. The activity of TMZ, which acts as an alkylating agent mediating methyl group transfer to purine residues (to N-7 or O-6 of guanine or N-3 of adenine), is known to depend on DNA repair systems. For instance, 6-O-methylguanine (6O-MeG) lesions are repaired by direct reversal by methylguanine-DNA-methyltransferase (MGMT) in MGMT-expressing tumors. In addition, 6-O-MeG may be lacking its damaging effect in the tumors deficient of the mismatch repair (MMR-) system.2 Thus, the key enzymes of the DNA repair system represent important targets for the development of chemotherapeutic drugs. The tyrosyl-DNA-phosphodiesterase 1 (TDP1) enzyme is an attractive target in antitumor therapies based on
he search for inhibitors of key DNA repair enzymes is a promising field of medicinal chemistry, which opens up avenues to the development of efficient therapies for cardiovascular, neurodegenerative, and oncological diseases.1 Malignant glioma, the most prevalent adult primary CNS tumor, represents an example of an oncological disease for which the search for new therapies is indispensable. The current standard of care for patients with recently diagnosed glioblastoma multiforme (GBM, grade IV glioma) includes maximal surgical resection of the tumor, followed by radiotherapy (RT) and adjuvant temozolomide (TMZ) chemotherapy. However, despite this multidisciplinary approach to GBM treatment, the median survival time is 12−15 months. Of note, the median survival of patients who received the complete treatment regimen is not significantly longer than of those for whom the chemotherapy was excluded (14.6 vs 12.2 months). Taking into account the low efficiency of GBM therapy, strategies aimed at overcoming resistance and enhancing the response to TMZ treatment are currently being actively © XXXX American Chemical Society and American Society of Pharmacognosy
Received: January 14, 2019
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DOI: 10.1021/acs.jnatprod.8b01095 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
topoisomerase 1 inhibitors (Top1).1,3−5 TDP1 plays a key role in removing Top1-DNA adducts stabilized by Top1 inhibitors, such as camptothecin and its clinically used derivatives.6,7 The mutation of the TDP1 gene, which leads to a significant loss of activity, also makes the cancer cells hypersensitive to camptothecin, and conversely the overexpression of TDP1 results in DNA protection against damage caused by camptothecin.3,8 The suppression of TDP1 activity may thus significantly enhance the therapeutic effect of the Top1 inhibitors.9 In addition to its ability to remove the covalent Top1-DNA adducts, TDP1 can hydrolyze apurinic (AP) sites, thus inducing their repair.10 This is the key enzymatic activity for the repair of DNA lesions induced by the monofunctional alkylating agents, such as methylmethanesulfonate or TMZ, as well as by ionizing radiation. Additionally, GBM cancer cells become resistant to TMZ by overexpressing methyl guanine methyl transferase. Depletion of TDP1 in these cells sensitizes them to TMZ.11 Therefore, TDP1 inhibition may enhance the sensitivity of tumor cells to the drugs used in standard therapies for malignant tumors, including GBM.12 The importance of the search for new inhibitors among natural compounds and their derivatives is due to both the significant chemical diversity of these molecules and their complementarity to biological targets, which is inherent in many natural compounds. The propriety of this approach has been shown in the development of TDP1 inhibitors based on natural compounds such as usnic acid,13,14 coumarin,15 varacin,16 monoterpenes,17 and disaccharide nucleosides.18 The effectiveness of the accompanying treatment of Lewis carcinoma with a Tdp1 inhibitor based on an usnic acid scaffold in combination with topotecan has also been demonstrated.19 Dehydroabietylamine (DHAm, or leelamine) is a diterpenetype primary amine obtained from dehydroabietic acid (DHA). The latter is a component of the resins of coniferous plants; for example, a high DHA content (71%) is found in the resin of Picea obovata.20 It can be directly obtained from the resin by the reduction of dehydroabietyl nitrile. A wide range of biological activities (bactericidal, antiviral, antimalarial, etc.) have been demonstrated for different DHAm derivatives.21−24 Recently, the study of anticancer activity of DHAm derivatives attracted considerable research interest. DHAm hydrochloride was found to have high cytotoxicity against certain cancer cell lines and to effectively destroy melanoma cells by reducing the cell proliferation rate and inducing apoptosis.25,26 The drug directly affects several cell-signaling pathways, which makes it a promising candidate for the treatment of resistant forms of cancer. The study of DHAm derivatives is thus important due to both its unique biological activities and also the availability and renewability of its feedstock.
Scheme 1. Synthesis of Ureas and Thioureas
cytostatic properties of some DHAm thioureas have also been investigated.30 The ureas and thioureas synthesized in this work have not been previously described. Bisureas 10−12 were synthesized according to Scheme 2. DHAm and diisocyanates were used in a 2:1 ratio, the reactions being carried out under the same conditions. Bisureas 10 and 12 were obtained earlier and used for supramolecular organogel formation.31 Nevertheless, the biological properties of this structural class and the effect of DHAm derivatives on DNA repair enzymes have never been studied. All the compounds obtained were purified by column chromatography. The structures of the compounds were determined by 1 H and 13C NMR spectroscopy and high-resolution mass spectrometry. Biology. The recently designed simple fluorophorequencher-coupled DNA-biosensor for real-time measurement of TDP1 cleavage activity was used to assess the TDP1 inhibition by the synthesized DHAm ureas and thioureas.32 The biosensor is a 16-mer single-stranded oligonucleotide containing both the 5′-FAM fluorophore donor and the quenching 3′-BHQ1 (black hole quencher 1) moiety. The results for the DHAm ureas, thioureas, and bisureas are collated in Table 1. IC50 values ranged from 0.1 to 3.7 μM. As shown in Table 1, compounds 10−12 showed the best inhibitory activity against both the wild-type TDP1 and its mutant form with the H493R substitution. These DHAm derivatives that belong to the bisurea class had IC50 values ranging from 0.1 to 0.2 μM for TDP1 and from 5.4 to 9.3 μM for its mutant. The mutation represented by a His493 to Arg493 substitution in the TDP1 active site results in a severe neurodegenerative disease known as SCAN1 (spinocerebellar ataxia syndrome with axonal neuropathy).33 This mutation leads to the accumulation of covalent enzyme−DNA adducts due to the inability to carry out the final resolution step of catalysis.3,34 The exact molecular mechanisms underlying SCAN1 disease are still unclear. However, despite TDP1 being a DNA repair enzyme, the frequency of oncological or other diseases associated with defects in the DNA repair system is not increased in patients with SCAN1. It is likely that the pathology is caused by the accumulation of mutant TDP1DNA covalent cleavage complexes formed during the reactions.35 Therefore, the suppression of TDP1(H493R) activity can improve the condition of SCAN1 patients and prevent the disease progression. The study of the effect of leelamines on TDP1 (H493R) is a simple functional test, which allows the determination of the stage of catalysis at which the inhibitors act. Those compounds that inhibit both wild-type TDP1 and H493R TDP1 affect the binding of an enzyme to DNA or the formation of a transition
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RESULTS AND DISCUSSION Chemistry. The ureas and thioureas 1−9 were prepared by interaction of DHAm with the appropriate isocyanate or isothiocyanate (Scheme 1). The reactions were carried out by refluxing in CHCl3 for 3 h, which resulted in the target ureas and thioureas 1−9. The compounds were obtained with yields of 73−90%. The structures of the R substituents are shown in Table 1. Previously, various DHAm thioureas were used as stereoselective asymmetric catalysts and were able to effectively catalyze such reactions as Michael addition27,28 and asymmetric inverse-electron-demand Diels−Alder reactions.29 The B
DOI: 10.1021/acs.jnatprod.8b01095 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 1. Influence of DHAm Ureas and Thioureas on the Activity of Purified DNA Repair Enzymes
a
The positions of the substituents X, R, and R1 are indicated in Schemes 1 and 2. bConcentration of a compound required to reduce the enzyme activity by 50%. cResidual activity in the presence of 100 μM inhibitor, %. The average of a minimum of two repeats. The error did not exceed 20%.
type enzyme mimic the effect of this mutation or, in other words, stabilize this complex. The IC50 values for TDP1 do not depend significantly on the choice between the urea or thiourea moiety, but rather correlate with the structure of R. Compounds 1, 6, and 7, containing methyl or ethyl groups, showed higher IC50 concentrations (1.5−3.7 μM) than other compounds with the group containing bulkier aromatic or aliphatic substituents (0.3−1.0 μM). The same relation was observed for H493R TDP1, with IC50 values for compounds 2−5 and 9, containing bulky aromatic or aliphatic substituents, ranging from 19 to 39 μM, while the IC50 values for compounds 1 and 6−8, with methyl, ethyl, or allyl groups, exceeded 50 μM and were indeterminable. Compound 3, with the bulky adamantane moiety, inhibited wild-type TDP1 as effectively as bisureas (0.1 μM), while the mutant form of TDP1 (H493R) was inhibited similarly to the compounds with monocyclic substituents (19.3 μM). Thus, bisureas 10−12 have significant inhibitory properties against the mutant form and are inhibitors of the first stage of catalysis. Compounds 1 and 6−8 did not affect the activity of the mutant form and were poisons that stabilized the covalent complex. The remaining compounds inhibited the mutant in
Scheme 2. Synthesis of Bisureas
covalent complex where the participation of H493 is not required. Since the mutant form of the enzyme is devoid of H493, which is necessary for the release of the enzyme from the covalent complex, compounds that affect only the wildC
DOI: 10.1021/acs.jnatprod.8b01095 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Article
Table 2. Effects of DHAm Ureas and Thioureas on Cell Viability IC50,a μM compound 1 2 3 4 5 6 7 8 9 10 11 12
U-937 34.8 ± >100 NT >100 15.0 ± 16.8 ± 18.7 ± 22.2 ± >100 >100 >100 >100
0.5
0.7 0.5 2.2 2.3
U-87MG
MDA-MB
SK-Mel8
A-549
WI-38
MCF7
T98G
SNB19
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 19.7 ± 0.8 >100 100 ± 11 >100 >100 >100 >100
NT 52.0 ± 5.0 NT >100 >100 NT NT 50 ± 5 70 ± 9 >100 >100 >100
NT >100 NT >100 52 ± 7 NT NT 10 ± 1 26.0 ± 3.0 >100 >100 >100
>100 >100 >100 >100 21.2 ± 0.5 73.3 ± 1.1 >100 >100 NT >100 >100 >100
