Bioorganic & Medicinal Chemistry Letters 25 (2015) 3511–3514
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiproliferative activity of new bioconjugates of Salinomycin with amino acid esters Michał Antoszczak a, Maria Sobusiak a, Ewa Maj b, Joanna Wietrzyk b, Adam Huczyn´ski a,⇑ a b
´ , Poland Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89 b, 61-614 Poznan Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland
a r t i c l e
i n f o
Article history: Received 25 May 2015 Revised 25 June 2015 Accepted 26 June 2015 Available online 2 July 2015
a b s t r a c t New Salinomycin (SAL) bioconjugates with amino acid methyl esters were obtained and their antiproliferative activity against cancer cell lines including drug-resistant ones was studied. New compounds exhibit antiproliferative activity towards leukemia and doxorubicin-resistant colon adenocarcinoma cell line and are more effective and less toxic than the commonly currently used anticancer drugs. Ó 2015 Elsevier Ltd. All rights reserved.
Keywords: Salinomycin bioconjugates Anticancer activity Doxorubicin-resistant cells Amino acid derivatives
Salinomycin (SAL, Scheme 1), a polyether antibiotic isolated from Streptomyces albus, has been successfully used for decades in veterinary medicine as a non-hormonal growth-promoting and coccidiostatic agent.1 However, only a few years ago it was documented that SAL can be potentially used in the fight against neoplastic diseases.2 In 2009, Gupta et al. proved that this compound is nearly 100-fold more effective against breast cancer stem cells (CSCs) than the commonly used cytostatic drug—Paclitaxel. Detailed studies have been performed on about 16,000 biologically active substances, of which only 32 were destroying CSCs studied and the most effective proved to be SAL.2 Since then, a high activity of this compound against a variety of cancer types has been evidenced, including colorectal, ovarian, lung, gastric and prostate cancer.3 Since 2012 SAL has been approved for testing in the screening studies on a small group of patients with invasive carcinoma of the head, neck, breast and ovary. According to the publication,4 patients were treated with 200–250 lg/kg of SAL intravenously every second day for three weeks. The therapy of patients with SAL resulted in inhibition of disease progress over an extended period of time. Moreover, acute side effects were rare and the serious long-term adverse side effects were not observed.4 The antiproliferative activity of SAL has become the subject of interest of other authors. For example, it has been documented that SAL ⇑ Corresponding author. Tel.: +48 618291297; fax: +48 618291555. E-mail address: [email protected] (A. Huczyn´ski). http://dx.doi.org/10.1016/j.bmcl.2015.06.086 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
inhibits hepatocellular carcinoma as well as pancreatic cancer cell invasion and migration, and the tumor growth of glioma CSCs.5 In view of the above, a very interesting direction of research has become chemical modification of SAL, which can lead to obtaining its various biologically active derivatives. Up to now, a series of SAL derivatives, such as amides, esters, carbamates and carbonates, have been successfully synthesized.6 What is important, the compounds obtained show very interesting biological activity, including high anticancer activity and selectivity.6 Furthermore, different SAL bioconjugates with floxuridine,7 Cinchona alkaloids8 and silybin9 have been synthesized. Results of the anticancer activity tests of these bioconjugates, especially floxuridine bioconjugates, have clearly shown that this type connection of SAL with other biologically active substances leads to the formation of interesting compounds, which may be used in the nearest future in the fight against cancer.7–9 However, to the best of our knowledge, no attempts have been made to synthesize SAL bioconjugates with amino acid esters. The synthesis of the compounds of this type is very interesting, because it is well known that the use of amino acid prodrugs and their derivatives improves poor solubility, poor permeability, sustained release, intravenous delivery, drug targeting as well as metabolic stability of the parent substances.10 Taking into account the above information, we have undertaken for the first time the synthesis of SAL bioconjugates with methyl esters of selected naturally occurring amino acids (Scheme 1) and proposed an efficient method for this synthesis. The structures
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M. Antoszczak et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3511–3514 35 35
OH
O
34 15
39
17
O
11
9 40
O
O 20
33
OH 31
24
O
O
OH
38
O
5 1
OH
37
30
34 15 17
39 32
28
O
OH
O
11
9 40
O
(a)
O
31
24
20
O
32 28
O
OH
OH
38
37
O
5
33
1
HN
Salinomycin (SAL)
H
30
R 44
O O Salinomycin bioconjugates with L-amino acid methyl esters (1-5) 42
42
R=
1 (75%)
H
2 (56%)
OH
3 (51%)
N HN
4 (68%)
5 (70%) OH
Scheme 1. Reagents and conditions: (a) SAL (1 equiv), DCC (1.2 equiv), HOBt (0.5 equiv), appropriate methyl ester of amino acid (2.5 equiv), triethylamine (2.5 equiv, if the amino acid methyl ester is in the form of a hydrochloride), DMF, 0 °C—1 h, then rt—24 h. Time for completion of the reaction at rt as indicated by TLC. Yield of isolated and purified products are given in the brackets.
of all obtained compounds were fully characterized using spectroscopic methods. It has been very well documented that SAL sensitizes cancer cells to the effect of doxorubicin treatment by many different ways, that is, by increasing DNA damage, reducing p21 protein, decreasing the efflux of doxorubicin and/or inhibiting the b-catenin/TCF complex association via FOXO3a activation, even at sub-lethal concentrations.11 These observations prompted us also to determine the antiproliferative activity of SAL bioconjugates obtained against different drug-sensitive and drug-resistant cancer cell lines, especially against doxorubicin-resistant cancer cell line (here it is LoVo/DX). Additionally, as equally important we studied here the toxicity of the compounds obtained against normal cells and we determined the Selectivity Index (SI).12 The isolation procedure of Salinomycin (SAL) has been described by us previously.13 Because SAL is very sensitive to acidic conditions and heating, the mild reaction condition for amide bond formation was chosen. Five new bioconjugates of SAL were synthesized in the reaction between SAL and an appropriate blocked amino acid (its methyl esters) in the presence of DCC (N,N0 -dicyclohexylcarbodiimide) as a coupling agent and HOBt (1-hydroxybenzotriazole) as an activator of this reaction. In addition, when the hydrochlorides of amino acid methyl esters were applied, an equimolar amount of triethylamine was also added (Scheme 1). To facilitate the structural activity relationship (SAR) resolution, five L-amino acid methyl esters with different kind of side chains were chosen giving bioconjugates with different substituents, that is, serine methyl ester with aliphatic side chain (2), histidine and phenylalanine methyl esters with aliphatic-aromatic side chains (3 and 4) and tyrosine methyl ester with aliphatic-aromatic side chain with an additional hydroxyl group (5). For comparison, a SAL bioconjugate with glycine methyl ester with only one hydrogen atom in the side chain has been also synthesized (1). All SAL derivatives can be easily purified by Dry Vacuum Column Chromatography14 using CH2Cl2/THF mixture as a mobile phase. This method gave bioconjugates of SAL in moderate to good yields (51–75%, Scheme 1).15 The purity and structures of SAL derivatives obtained were determined on the basis of elemental, FT-IR, NMR and ESI-MS analysis. The 1H and 13C NMR signals were assigned using one- as well as two-dimensional (1H–1H COSY,
1
H–13C HETCOR and 1H–13C HMBC) spectra. The FT-IR, 1H, 13C and 2D NMR, as well as ESI-MS spectra of selected bioconjugates of SAL are included in the Supplementary material (Figs. S1–S8). The main evidence for the formation of SAL bioconjugates with methyl esters of amino acids is the presence of new characteristic bands in their FT-IR spectra (Fig. S1). In the FT-IR spectrum of SAL a characteristic broad band arises with a maximum at 1711 cm1 due to the overlapping of the m(C@O) stretching vibrations of ketone and carboxylic groups. In the spectrum of its exemplary derivative 2 three additional characteristic bands are observed. The first band at 1746 cm1 is assigned to the m(C@O) stretching vibrations of the ester group. The two next bands with maxima at 1659 cm1 and 1533 cm1, are identified as amide I and amide II bands, respectively. In the spectra of other SAL bioconjugates these high-intensity bands are observed in the narrow ranges of 1729–1750 cm1, 1656–1664 cm1 and 1523–1541 cm1 for the m(C@O) of the ester group, amide I and amide II, respectively (Table S1, Supplementary material). Additionally, in the FT-IR spectra of SAL derivatives, the intensity of the band at 1711 cm1 decreased indicating that COOH group had been consumed during the amide synthesis. In the 13C NMR spectra (all performed in CDCl3) of SAL bioconjugates, a characteristic signal of carbon atom of the amide group is observed in the narrow range of 171.0–175.3 ppm. On the other hand, a characteristic signal of the ester group of the amino acid moieties is observed in the range of 167.7–172.6 ppm. The signal of the carbon atom from the carboxyl group of chemically unmodified SAL is observed at 177.7 ppm. The characteristic signal of proton of NH(amide) group in the 1H NMR spectra of these derivatives is in the range of 6.70–7.89 ppm (Table 1). The exemplary 1H, 13C and 2D NMR spectra of selected bioconjugates of SAL are included in the Supplementary material (Figs. S2–S6). The ESI mass spectrometry studies have clearly demonstrated that SAL derivatives are able to form complexes with Na+ cation of 1:1 stoichiometry (Supplementary material, Figs. S7–S8). This is an important property, because the biological activity of SAL and its derivatives is strictly connected with their abilities of transporting monovalent metal cations through lipid bilayers.1 ESI MS experiment has shown that compound 3 also forms a complex with proton, because the signal of a protonated species is observed.
M. Antoszczak et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3511–3514 Table 1 Anticancer activity of SAL and its bioconjugates (1–5). Data are given as IC50 [lM] Compound
SAL 1 2 3 4 5 Doxorubicin Cisplatin
Cancer cells
Normal cells
LoVo
LoVo/DX
MV4-11
BALB/3T3
0.53 ± 0.03 5.29 ± 0.83 17.65 ± 10.63 3.67 ± 0.49 3.49 ± 0.57 14.72 ± 5.59
0.59 ± 0.04 3.93 ± 0.15 7.77 ± 2.57 0.85 ± 0.14 3.49 ± 0.23 7.12 ± 1.24
0.33 ± 0.11 3.65 ± 0.83 5.81 ± 2.20 0.80 ± 0.16 2.89 ± 0.50 3.55 ± 0.63
27.76 ± 8.42 42.49 ± 3.31 40.09 ± 1.80 37.01 ± 1.01 17.01 ± 8.08 43.37 ± 8.49
0.09 ± 0.06 2.00 ± 0.47
7.26 ± 2.21 2.53 ± 0.80
0.05 ± 0.02 2.33 ± 0.60
0.31 ± 0.13 7.37 ± 2.23
The IC50 value is defined as the concentration of a compound that corresponds to a 50% growth inhibition. Human colon adenocarcinoma cell line (LoVo) and doxorubicin-resistant subline (LoVo/DX); Human biphenotypic myelomonocytic leukemia cell line (MV4-11); normal murine embryonic fibroblast cell line (BALB/3T3). Data are expressed as the mean ± SD.
SAL, all its new bioconjugates and two reference anticancer drugs were evaluated for their in vitro anticancer activity against three cancer cell lines (LoVo, LoVo/DX and MV4-11) following the procedure described by us previously.16 The cytotoxic effect was also studied on the normal murine embryonic fibroblasts (BALB/3T3) in order to estimate the toxicity of the studied bioconjugates. The mean IC50 ± SD of the tested compounds are collected in Table 1. Doxorubicin and cisplatin are the conventional and effective chemotherapy drugs used in the therapy against many types of cancer cells. However, during long-term monotherapy these cells may eventually develop acquired-resistance to doxorubicin or cisplatin, which results in recurrence and poor prognosis.11 Human colon adenocarcinoma cell line (LoVo) and its doxorubicin-resistant subline (LoVo/DX), pair of cell lines displaying various levels of drug-resistance, were used for evaluation of the activity of the studied derivatives against the cells with MDR (multi-drug resistance) phenotype.17 The resistance indices (RI) were calculated for these lines and are presented in Table 2. The RI value indicates how many times more resistant is the subline in comparison to its parental cell line.17–19 As shown in Table 1, three of the five obtained derivatives (1, 3 and 4) show high in vitro anticancer activity against LoVo cell line with IC50 = 5.29 lM, 3.67 lM and 3.49 lM, respectively. What is interesting, this activity is comparable to that exhibited by a
Table 2 The calculated values of the Resistance Index (RI) and Selectivity Index (SI) of tested compounds Compound
SAL 1 2 3 4 5 Doxorubicin Cisplatin
LoVo
LoVo/DX
MV4-11
SI
SI
RI
SI
52.38 8.03 2.27 10.08 4.87 2.95
47.05 10.81 5.16 43.54 4.87 6.09
1.11 0.74 0.44 0.23 1.00 0.48
84.12 11.64 6.90 46.26 5.89 12.22
3.44 3.69
0.04 2.91
80.67 1.27
6.20 3.16
The RI (Resistance Index) indicates how many times a resistant subline is chemoresistant relative to its parental cell line. The RI was calculated for each compound using the formula: RI = IC50 for LoVoDX/IC50 for LoVo cell line. When RI is 0–2 the cells are sensitive to the compound tested; RI in the range 2–10 means that the cell shows moderate sensitivity to the drug tested; RI above 10 indicates strong drug-resistance. The SI (Selectivity Index) was calculated for each compound using the formula: SI = IC50 for normal cell line BALB3T3/IC50 for respective cancerous cell line. A beneficial SI >1.0 indicates a drug with efficacy against tumor cells greater than the toxicity against normal cells.
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widely used drug in chemotherapy—cisplatin (IC50 = 2.00 lM). The other two compounds (2 and 5) show a lower activity in this assay (IC50 = 17.65 lM and 14.72 lM, respectively). However, the anticancer in vitro activity of all bioconjugates obtained significantly increases against doxorubicin-resistant subline LoVo/DX with IC50 in the range 0.85–7.77 lM. What is worth noting, in some cases this activity is equal or higher to that exhibited by the reference compounds (IC50 = 2.53 lM and 7.26 lM for cisplatin and doxorubicin, respectively). Particularly noteworthy is the very high activity of derivative 3 against LoVo/DX cancer cell line with IC50 = 0.85 lM, which is several times higher than that of the reference compounds (about threefold and ninefold higher than cisplatin and doxorubicin, respectively). It confirms the high selectivity of the obtained compounds against this type of doxorubicin-resistant cancer cell line. These observations are also confirmed by the data collected in Table 2. The RI values confirmed that the obtained compounds strongly break the drug-resistance of cancer cell line tested, more strongly than the reference compounds (RI = 0.23–1.00, RI = 1.27 and RI = 80.67 for all bioconjugates, cisplatin and doxorubicin, respectively) and parental compound (RI = 1.11). In addition, all obtained SAL derivatives are highly active against human biphenotypic myelomonocytic leukemia cell line (MV4-11, Table 1) with IC50 values in the range from 0.80 lM (compound 3) to 5.81 lM (compound 2). This activity is comparable to that exhibited by cisplatin (IC50 = 2.33 lM). Simultaneously, as shown in Table 2, SAL bioconjugates are much less toxic to normal murine fibroblasts (BALB/3T3) than cisplatin and doxorubicin, which is confirmed by their high SI values (SI >4.00 in most cases). Until now, we have successfully synthesized several tens of SAL derivatives with modified carboxylic group, including secondary amides and esters, which have been characterized for their biological activity.6–9,12,15 Among all obtained SAL amides the most in vitro anticancer active are those with aliphatic-aromatic moieties such as benzylamine (IC50 = 3.31–6.02 lM), 4-fluorophenethylamine (IC50 = 2.3–6.7 lM), dopamine (IC50 = 2.8– 6.9 lM) and tryptamine (IC50 = 2.78–4.31 lM).6 The tests performed on the series of mono-substituted N-benzyl amides of SAL containing fluorine, chlorine, bromine atoms as well as nitro groups clearly showed that the most anticancer active, except one compound, are those substituted at –ortho position and the least active derivatives are those substituted at –para position.6 Among all synthesized ester derivatives of SAL, the most anticancer active are two esters with saturated aliphatic chain, that is, butyl (IC50 = 3.58–4.15 lM) and 2,2,2-trifluoroethyl (IC50 = 0.47–3.05 lM), and two esters with aromatic substituents, that is, b-naphthylethyl (IC50 = 3.73–9.33) and benzotriazole (IC50 = 1.84–6.61).6 It is worth noting that almost all obtained secondary amides and esters of SAL exhibit lower toxicity toward normal cells of the body than the commonly used cytostatic agents.6 In this context, the SAL amides obtained with blocked amino acid substituent (1–5) belong to high anticancer active SAL derivatives with modified carboxylic group. The most anticancer active compounds are these with aliphatic-aromatic substituents in amino acid moieties, that is, amides of SAL with L-histidine methyl ester (3) and L-phenylalanine methyl ester (4) with IC50 = 0.80– 3.67 lM and IC50 = 2.89–3.49 lM, respectively. Moreover, all newly synthesized SAL amides are less toxic to healthy cells of the body, much less than the majority of already obtained SAL derivatives. On the other hand, the bioconjugates of SAL with amino acid methyl esters are characterized by the highest values of SI as well as the lowest values of RI indexes from among all already obtained bioconjugates of this antibiotic. Only the ester of SAL with floxuridine is characterized by improved values of these parameters.7–9 It is worth noting that up to now, amide of SAL with L-
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histidine methyl ester (3) has been one of the most anticancer active derivative of SAL with modified carboxylic group. It is postulated that SAL derivatives with modified carboxylic group transport metal cations via an electrogenic or biomimetic mechanism, while unmodified ionophore transports metal cations only by an electroneutral mechanism as a result of a negatively charged carboxylate group.1 Therefore, the chemical modification of SAL carboxylic group changes the ionophoretic properties of its derivatives and the mechanism of cation transport across the lipid bilayers. Finally, it induces changes in biological activity of obtained derivatives. To summarize, for the first time, a simple and efficient method for the synthesis of Salinomycin (SAL) bioconjugates with amino acid methyl esters has been described. All these derivatives were examined for their anticancer in vitro activity against different drug-sensitive and drug-resistant cancer cell lines. Results of our study have clearly shown that unmodified SAL and its studied derivatives exhibit interesting anticancer in vitro activity against human cancer cells at low micromolar concentration. It is worth noting that all compounds tested, similarly to the unmodified parent compound,11 overcome the resistance of doxorubicin-resistant cancer cell line (LoVo/DX), which confirms the high selectivity of these compounds against the cancer cells of this type. Moreover, for the first time, the potent anticancer activity of SAL derivatives against leukemia cell line (MV4-11) has been proved. Simultaneously, almost all bioconjugates were shown to be less toxic for normal murine fibroblast cells than the currently used anticancer drugs, such as cisplatin and doxorubicin. All these results demonstrate strong potential and advantages of bioconjugates of SAL and are a good starting point for further studies, based on other active compounds, which are currently underway in our group. The obtained compounds seem to be attractive in the fight against cancer, because they are able to overcome a strong drug-resistance of the cancer cell lines. Moreover, SAL derivatives reversed the resistance of doxorubicin, suggesting that chemotherapy in combination with these bioconjugates may benefit the MDR cancer therapy. These findings support a strategy to decrease the doxorubicin concentration in combination with SAL amino acid esters in order to reduce the toxic side effects. Acknowledgments Financial support from the budget funds for science in years 2013–2015—grant ‘Iuventus Plus’ of the Polish Ministry of Science and Higher Education—No. IP2012013272, is gratefully acknowledged. Michał Antoszczak wishes to thank the Polish National Science Centre (NCN) for doctoral scholarship ‘ETIUDA’—No. 2014/12/T/ST5/00710. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.06.
086. These data include MOL files and InChiKeys of the most important compounds described in this article. References and notes 1. Antoszczak, M.; Rutkowski, J.; Huczyn´ski, A. Structure and biological activity of polyether ionophores and their semi-synthetic derivatives. In Bioactive Natural Products. Chemistry and Biology; Brahmachari, G., Ed., 1st ed.; Wiley-VCH Verlag GmbH, 2015; pp 107–170. 2. Gupta, P. B.; Onder, T. T.; Jiang, G.; Tao, K.; Kuperwasser, C.; Weinberg, R. A.; Lander, E. S. Cell 2009, 138, 645. 3. Antoszczak, M.; Huczyn´ski, A. Anticancer Agents Med. Chem. 2015, 15, 575. 4. Naujokat, C.; Steinhart, R. J. Biomed. Biotechnol. 2012, 2012, 950658. 5. (a) Xu, L.; Wang, T.; Meng, W.-Y.; Wei, J.; Ma, J.-L.; Shi, M.; Wang, Y.-G. Oncol. Rep. 2015, 1057, 33; (b) Chen, T.; Yi, L.; Li, F.; Hu, R.; Hu, S.; Yin, Y.; Lan, C.; Li, Z.; Fu, C.; Cao, L.; Chen, Z.; Xian, J.; Feng, H. Mol. Med. Rep. 2015, 11, 2407; (c) Schenk, M.; Aykut, B.; Teske, C.; Giese, N. A.; Weitz, J.; Welsch, T. Cancer Lett. 2015, 358, 161. 6. (a) Huczyn´ski, A.; Janczak, J.; Antoszczak, M.; Stefan´ska, J.; Brzezinski, B. J. Mol. Struct. 2012, 1022, 197; (b) Antoszczak, M.; Maj, E.; Stefan´ska, J.; Wietrzyk, J.; Janczak, J.; Brzezinski, B.; Huczyn´ski, A. Bioorg. Med. Chem. Lett. 2014, 24, 1724; (c) Antoszczak, M.; Popiel, K.; Stefan´ska, J.; Wietrzyk, J.; Maj, E.; Janczak, J.; Michalska, G.; Brzezinski, B.; Huczyn´ski, A. Eur. J. Med. Chem. 2014, 76, 435; (d) Antoszczak, M.; Maj, E.; Napiórkowska, A.; Stefan´ska, J.; Augustynowicz-Kopec´, E.; Wietrzyk, J.; Janczak, J.; Brzezinski, B.; Huczyn´ski, A. Molecules 2014, 19, 19435; (e) Borgström, B.; Huang, X.; Pošta, M.; Hegardt, C.; Oredsson, S.; Strand, D. Chem. Commun. 2013, 9944; (f) Huang, X.; Borgström, B.; Mänsson, L.; Persson, L.; Oredsson, S.; Hegardt, C.; Strand, D. ACS Chem. Biol. 2014, 9, 1587. 7. Huczyn´ski, A.; Antoszczak, M.; Kleczewska, N.; Baraniak, D.; Maj, E.; Stefan´ska, J.; Wietrzyk, J.; Janczak, J.; Celewicz, L. Eur. J. Med. Chem. 2015, 93, 33. 8. Skiera, I.; Antoszczak, M.; Trynda, J.; Wietrzyk, J.; Boratyn´ski, P.; Kacprzak, K.; Huczyn´ski, A. Chem. Biol. Drug. Des. 2015 (in press). DOI:10.1111/cbdd.12523. 9. Antoszczak, M.; Kleiborowska, G.; Kruszyk, M; Maj, E.; Wietrzyk, J.; Huczyn´ski, A.;, Chem. Biol. Drug. Des. 2015 (in press). DOI:10.1111/cbdd.12602. 10. Vig, B. S.; Huttunen, K. M.; Laine, K.; Rautio, J. Adv. Drug Deliv. Rev. 2013, 65, 1370. 11. (a) Kim, K.-Y.; Kim, S.-H.; Yu, S.-N.; Park, S.-K.; Choi, H.-D.; Yu, H.-S.; Ji, J.-H.; Seo, Y.-K.; Ahn, S.-C. Mol. Med. Rep. 1898, 2015, 12; (b) Zhou, Y.; Liang, C.; Xue, F.; Chen, W.; Zhi, X.; Feng, X.; Bai, X.; Liang, T. Oncotarget 2015, 6, 10350; (c) Liffers, S.-T.; Tilkorn, D. J.; Stricker, I.; Junge, C. G.; Al-Benna, S.; Vogt, M.; Verdoodt, B.; Steina, H.-U.; Tannapfel, A.; Tischoff, I.; Mirmohammadsadegh, A. BMC Cancer 2013, 13, 490; (d) Kim, J.-H.; Chae, M.; Kim, W. K.; Kim, Y.-J.; Kang, H. S.; Kim, H. S.; Yoon, S. Br. J. Pharmacol. 2011, 162, 773. 12. Badisa, R. B.; Darling-Reed, S. F.; Joseph, P.; Cooperwood, J. S.; Latinwo, L. M.; Goldman, C. B. Anticancer Res. 2009, 29, 2993. 13. Huczyn´ski, A.; Janczak, J.; Stefan´ska, J.; Antoszczak, M.; Brzezinski, B. Bioorg. Med. Chem. Lett. 2012, 22, 4697. 14. Pedersen, D. S.; Rosenbohm, C. Synthesis 2001, 16, 2431. 15. To a mixture of SAL (500 mg, 0.66 mmol) in DMF (25 ml) the following compounds were added: DCC (206 mg, 1.0 mmol), HOBt (45 mg, 0.33 mmol), Lamino acid methyl ester (2.0 mmol) and, if the L-amino acid methyl ester was in the form of a hydrochloride, triethylamine (2.2 mmol). The mixture was first stirred at a temperature below 0 °C for 1 h and then for further 24 h at room temperature. The solvent was subsequently evaporated under reduced pressure to dryness. The residue was suspended in hexane and filtered off. The filtrate was evaporated under reduced pressure and the residue was purified chromatographically on silica gel (Fluka type 60) to give bioconjugates of SAL (yield from 51% to 75%, see Scheme 1 and Table 1) as a yellow oil state. The 1H, 13C, 2D NMR, FT-IR and ESI-MS spectra of selected bioconjugates of SAL are included in the Supplementary material. 16. Huczyn´ski, A.; Janczak, J.; Antoszczak, M.; Wietrzyk, J.; Maj, E.; Brzezinski, B. Bioorg. Med. Chem. Lett. 2012, 22, 7146. 17. Grandi, M.; Geroni, C.; Giuliani, F. C. Br. J. Cancer 1986, 54, 515. 18. Hang, X.; Yashiro, M.; Qiu, H.; Nishii, T.; Matsuzaki, T.; Hirakawa, K. Anticancer Res. 2010, 30, 915. 19. Lukawska, M.; Wietrzyk, J.; Opolski, A.; Oszczapowicz, J.; Oszczapowic, I. Invest. New Drugs 2010, 28, 600.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antiproliferative activity of new bioconjugates of Salinomycin with amino acid esters Michał Antoszczak a, Maria Sobusiak a, Ewa Maj b, Joanna Wietrzyk b, Adam Huczyn´ski a,⇑ a b
´ , Poland Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89 b, 61-614 Poznan Ludwik Hirszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wrocław, Poland
a r t i c l e
i n f o
Article history: Received 25 May 2015 Revised 25 June 2015 Accepted 26 June 2015 Available online 2 July 2015
a b s t r a c t New Salinomycin (SAL) bioconjugates with amino acid methyl esters were obtained and their antiproliferative activity against cancer cell lines including drug-resistant ones was studied. New compounds exhibit antiproliferative activity towards leukemia and doxorubicin-resistant colon adenocarcinoma cell line and are more effective and less toxic than the commonly currently used anticancer drugs. Ó 2015 Elsevier Ltd. All rights reserved.
Keywords: Salinomycin bioconjugates Anticancer activity Doxorubicin-resistant cells Amino acid derivatives
Salinomycin (SAL, Scheme 1), a polyether antibiotic isolated from Streptomyces albus, has been successfully used for decades in veterinary medicine as a non-hormonal growth-promoting and coccidiostatic agent.1 However, only a few years ago it was documented that SAL can be potentially used in the fight against neoplastic diseases.2 In 2009, Gupta et al. proved that this compound is nearly 100-fold more effective against breast cancer stem cells (CSCs) than the commonly used cytostatic drug—Paclitaxel. Detailed studies have been performed on about 16,000 biologically active substances, of which only 32 were destroying CSCs studied and the most effective proved to be SAL.2 Since then, a high activity of this compound against a variety of cancer types has been evidenced, including colorectal, ovarian, lung, gastric and prostate cancer.3 Since 2012 SAL has been approved for testing in the screening studies on a small group of patients with invasive carcinoma of the head, neck, breast and ovary. According to the publication,4 patients were treated with 200–250 lg/kg of SAL intravenously every second day for three weeks. The therapy of patients with SAL resulted in inhibition of disease progress over an extended period of time. Moreover, acute side effects were rare and the serious long-term adverse side effects were not observed.4 The antiproliferative activity of SAL has become the subject of interest of other authors. For example, it has been documented that SAL ⇑ Corresponding author. Tel.: +48 618291297; fax: +48 618291555. E-mail address: [email protected] (A. Huczyn´ski). http://dx.doi.org/10.1016/j.bmcl.2015.06.086 0960-894X/Ó 2015 Elsevier Ltd. All rights reserved.
inhibits hepatocellular carcinoma as well as pancreatic cancer cell invasion and migration, and the tumor growth of glioma CSCs.5 In view of the above, a very interesting direction of research has become chemical modification of SAL, which can lead to obtaining its various biologically active derivatives. Up to now, a series of SAL derivatives, such as amides, esters, carbamates and carbonates, have been successfully synthesized.6 What is important, the compounds obtained show very interesting biological activity, including high anticancer activity and selectivity.6 Furthermore, different SAL bioconjugates with floxuridine,7 Cinchona alkaloids8 and silybin9 have been synthesized. Results of the anticancer activity tests of these bioconjugates, especially floxuridine bioconjugates, have clearly shown that this type connection of SAL with other biologically active substances leads to the formation of interesting compounds, which may be used in the nearest future in the fight against cancer.7–9 However, to the best of our knowledge, no attempts have been made to synthesize SAL bioconjugates with amino acid esters. The synthesis of the compounds of this type is very interesting, because it is well known that the use of amino acid prodrugs and their derivatives improves poor solubility, poor permeability, sustained release, intravenous delivery, drug targeting as well as metabolic stability of the parent substances.10 Taking into account the above information, we have undertaken for the first time the synthesis of SAL bioconjugates with methyl esters of selected naturally occurring amino acids (Scheme 1) and proposed an efficient method for this synthesis. The structures
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OH
O
34 15
39
17
O
11
9 40
O
O 20
33
OH 31
24
O
O
OH
38
O
5 1
OH
37
30
34 15 17
39 32
28
O
OH
O
11
9 40
O
(a)
O
31
24
20
O
32 28
O
OH
OH
38
37
O
5
33
1
HN
Salinomycin (SAL)
H
30
R 44
O O Salinomycin bioconjugates with L-amino acid methyl esters (1-5) 42
42
R=
1 (75%)
H
2 (56%)
OH
3 (51%)
N HN
4 (68%)
5 (70%) OH
Scheme 1. Reagents and conditions: (a) SAL (1 equiv), DCC (1.2 equiv), HOBt (0.5 equiv), appropriate methyl ester of amino acid (2.5 equiv), triethylamine (2.5 equiv, if the amino acid methyl ester is in the form of a hydrochloride), DMF, 0 °C—1 h, then rt—24 h. Time for completion of the reaction at rt as indicated by TLC. Yield of isolated and purified products are given in the brackets.
of all obtained compounds were fully characterized using spectroscopic methods. It has been very well documented that SAL sensitizes cancer cells to the effect of doxorubicin treatment by many different ways, that is, by increasing DNA damage, reducing p21 protein, decreasing the efflux of doxorubicin and/or inhibiting the b-catenin/TCF complex association via FOXO3a activation, even at sub-lethal concentrations.11 These observations prompted us also to determine the antiproliferative activity of SAL bioconjugates obtained against different drug-sensitive and drug-resistant cancer cell lines, especially against doxorubicin-resistant cancer cell line (here it is LoVo/DX). Additionally, as equally important we studied here the toxicity of the compounds obtained against normal cells and we determined the Selectivity Index (SI).12 The isolation procedure of Salinomycin (SAL) has been described by us previously.13 Because SAL is very sensitive to acidic conditions and heating, the mild reaction condition for amide bond formation was chosen. Five new bioconjugates of SAL were synthesized in the reaction between SAL and an appropriate blocked amino acid (its methyl esters) in the presence of DCC (N,N0 -dicyclohexylcarbodiimide) as a coupling agent and HOBt (1-hydroxybenzotriazole) as an activator of this reaction. In addition, when the hydrochlorides of amino acid methyl esters were applied, an equimolar amount of triethylamine was also added (Scheme 1). To facilitate the structural activity relationship (SAR) resolution, five L-amino acid methyl esters with different kind of side chains were chosen giving bioconjugates with different substituents, that is, serine methyl ester with aliphatic side chain (2), histidine and phenylalanine methyl esters with aliphatic-aromatic side chains (3 and 4) and tyrosine methyl ester with aliphatic-aromatic side chain with an additional hydroxyl group (5). For comparison, a SAL bioconjugate with glycine methyl ester with only one hydrogen atom in the side chain has been also synthesized (1). All SAL derivatives can be easily purified by Dry Vacuum Column Chromatography14 using CH2Cl2/THF mixture as a mobile phase. This method gave bioconjugates of SAL in moderate to good yields (51–75%, Scheme 1).15 The purity and structures of SAL derivatives obtained were determined on the basis of elemental, FT-IR, NMR and ESI-MS analysis. The 1H and 13C NMR signals were assigned using one- as well as two-dimensional (1H–1H COSY,
1
H–13C HETCOR and 1H–13C HMBC) spectra. The FT-IR, 1H, 13C and 2D NMR, as well as ESI-MS spectra of selected bioconjugates of SAL are included in the Supplementary material (Figs. S1–S8). The main evidence for the formation of SAL bioconjugates with methyl esters of amino acids is the presence of new characteristic bands in their FT-IR spectra (Fig. S1). In the FT-IR spectrum of SAL a characteristic broad band arises with a maximum at 1711 cm1 due to the overlapping of the m(C@O) stretching vibrations of ketone and carboxylic groups. In the spectrum of its exemplary derivative 2 three additional characteristic bands are observed. The first band at 1746 cm1 is assigned to the m(C@O) stretching vibrations of the ester group. The two next bands with maxima at 1659 cm1 and 1533 cm1, are identified as amide I and amide II bands, respectively. In the spectra of other SAL bioconjugates these high-intensity bands are observed in the narrow ranges of 1729–1750 cm1, 1656–1664 cm1 and 1523–1541 cm1 for the m(C@O) of the ester group, amide I and amide II, respectively (Table S1, Supplementary material). Additionally, in the FT-IR spectra of SAL derivatives, the intensity of the band at 1711 cm1 decreased indicating that COOH group had been consumed during the amide synthesis. In the 13C NMR spectra (all performed in CDCl3) of SAL bioconjugates, a characteristic signal of carbon atom of the amide group is observed in the narrow range of 171.0–175.3 ppm. On the other hand, a characteristic signal of the ester group of the amino acid moieties is observed in the range of 167.7–172.6 ppm. The signal of the carbon atom from the carboxyl group of chemically unmodified SAL is observed at 177.7 ppm. The characteristic signal of proton of NH(amide) group in the 1H NMR spectra of these derivatives is in the range of 6.70–7.89 ppm (Table 1). The exemplary 1H, 13C and 2D NMR spectra of selected bioconjugates of SAL are included in the Supplementary material (Figs. S2–S6). The ESI mass spectrometry studies have clearly demonstrated that SAL derivatives are able to form complexes with Na+ cation of 1:1 stoichiometry (Supplementary material, Figs. S7–S8). This is an important property, because the biological activity of SAL and its derivatives is strictly connected with their abilities of transporting monovalent metal cations through lipid bilayers.1 ESI MS experiment has shown that compound 3 also forms a complex with proton, because the signal of a protonated species is observed.
M. Antoszczak et al. / Bioorg. Med. Chem. Lett. 25 (2015) 3511–3514 Table 1 Anticancer activity of SAL and its bioconjugates (1–5). Data are given as IC50 [lM] Compound
SAL 1 2 3 4 5 Doxorubicin Cisplatin
Cancer cells
Normal cells
LoVo
LoVo/DX
MV4-11
BALB/3T3
0.53 ± 0.03 5.29 ± 0.83 17.65 ± 10.63 3.67 ± 0.49 3.49 ± 0.57 14.72 ± 5.59
0.59 ± 0.04 3.93 ± 0.15 7.77 ± 2.57 0.85 ± 0.14 3.49 ± 0.23 7.12 ± 1.24
0.33 ± 0.11 3.65 ± 0.83 5.81 ± 2.20 0.80 ± 0.16 2.89 ± 0.50 3.55 ± 0.63
27.76 ± 8.42 42.49 ± 3.31 40.09 ± 1.80 37.01 ± 1.01 17.01 ± 8.08 43.37 ± 8.49
0.09 ± 0.06 2.00 ± 0.47
7.26 ± 2.21 2.53 ± 0.80
0.05 ± 0.02 2.33 ± 0.60
0.31 ± 0.13 7.37 ± 2.23
The IC50 value is defined as the concentration of a compound that corresponds to a 50% growth inhibition. Human colon adenocarcinoma cell line (LoVo) and doxorubicin-resistant subline (LoVo/DX); Human biphenotypic myelomonocytic leukemia cell line (MV4-11); normal murine embryonic fibroblast cell line (BALB/3T3). Data are expressed as the mean ± SD.
SAL, all its new bioconjugates and two reference anticancer drugs were evaluated for their in vitro anticancer activity against three cancer cell lines (LoVo, LoVo/DX and MV4-11) following the procedure described by us previously.16 The cytotoxic effect was also studied on the normal murine embryonic fibroblasts (BALB/3T3) in order to estimate the toxicity of the studied bioconjugates. The mean IC50 ± SD of the tested compounds are collected in Table 1. Doxorubicin and cisplatin are the conventional and effective chemotherapy drugs used in the therapy against many types of cancer cells. However, during long-term monotherapy these cells may eventually develop acquired-resistance to doxorubicin or cisplatin, which results in recurrence and poor prognosis.11 Human colon adenocarcinoma cell line (LoVo) and its doxorubicin-resistant subline (LoVo/DX), pair of cell lines displaying various levels of drug-resistance, were used for evaluation of the activity of the studied derivatives against the cells with MDR (multi-drug resistance) phenotype.17 The resistance indices (RI) were calculated for these lines and are presented in Table 2. The RI value indicates how many times more resistant is the subline in comparison to its parental cell line.17–19 As shown in Table 1, three of the five obtained derivatives (1, 3 and 4) show high in vitro anticancer activity against LoVo cell line with IC50 = 5.29 lM, 3.67 lM and 3.49 lM, respectively. What is interesting, this activity is comparable to that exhibited by a
Table 2 The calculated values of the Resistance Index (RI) and Selectivity Index (SI) of tested compounds Compound
SAL 1 2 3 4 5 Doxorubicin Cisplatin
LoVo
LoVo/DX
MV4-11
SI
SI
RI
SI
52.38 8.03 2.27 10.08 4.87 2.95
47.05 10.81 5.16 43.54 4.87 6.09
1.11 0.74 0.44 0.23 1.00 0.48
84.12 11.64 6.90 46.26 5.89 12.22
3.44 3.69
0.04 2.91
80.67 1.27
6.20 3.16
The RI (Resistance Index) indicates how many times a resistant subline is chemoresistant relative to its parental cell line. The RI was calculated for each compound using the formula: RI = IC50 for LoVoDX/IC50 for LoVo cell line. When RI is 0–2 the cells are sensitive to the compound tested; RI in the range 2–10 means that the cell shows moderate sensitivity to the drug tested; RI above 10 indicates strong drug-resistance. The SI (Selectivity Index) was calculated for each compound using the formula: SI = IC50 for normal cell line BALB3T3/IC50 for respective cancerous cell line. A beneficial SI >1.0 indicates a drug with efficacy against tumor cells greater than the toxicity against normal cells.
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widely used drug in chemotherapy—cisplatin (IC50 = 2.00 lM). The other two compounds (2 and 5) show a lower activity in this assay (IC50 = 17.65 lM and 14.72 lM, respectively). However, the anticancer in vitro activity of all bioconjugates obtained significantly increases against doxorubicin-resistant subline LoVo/DX with IC50 in the range 0.85–7.77 lM. What is worth noting, in some cases this activity is equal or higher to that exhibited by the reference compounds (IC50 = 2.53 lM and 7.26 lM for cisplatin and doxorubicin, respectively). Particularly noteworthy is the very high activity of derivative 3 against LoVo/DX cancer cell line with IC50 = 0.85 lM, which is several times higher than that of the reference compounds (about threefold and ninefold higher than cisplatin and doxorubicin, respectively). It confirms the high selectivity of the obtained compounds against this type of doxorubicin-resistant cancer cell line. These observations are also confirmed by the data collected in Table 2. The RI values confirmed that the obtained compounds strongly break the drug-resistance of cancer cell line tested, more strongly than the reference compounds (RI = 0.23–1.00, RI = 1.27 and RI = 80.67 for all bioconjugates, cisplatin and doxorubicin, respectively) and parental compound (RI = 1.11). In addition, all obtained SAL derivatives are highly active against human biphenotypic myelomonocytic leukemia cell line (MV4-11, Table 1) with IC50 values in the range from 0.80 lM (compound 3) to 5.81 lM (compound 2). This activity is comparable to that exhibited by cisplatin (IC50 = 2.33 lM). Simultaneously, as shown in Table 2, SAL bioconjugates are much less toxic to normal murine fibroblasts (BALB/3T3) than cisplatin and doxorubicin, which is confirmed by their high SI values (SI >4.00 in most cases). Until now, we have successfully synthesized several tens of SAL derivatives with modified carboxylic group, including secondary amides and esters, which have been characterized for their biological activity.6–9,12,15 Among all obtained SAL amides the most in vitro anticancer active are those with aliphatic-aromatic moieties such as benzylamine (IC50 = 3.31–6.02 lM), 4-fluorophenethylamine (IC50 = 2.3–6.7 lM), dopamine (IC50 = 2.8– 6.9 lM) and tryptamine (IC50 = 2.78–4.31 lM).6 The tests performed on the series of mono-substituted N-benzyl amides of SAL containing fluorine, chlorine, bromine atoms as well as nitro groups clearly showed that the most anticancer active, except one compound, are those substituted at –ortho position and the least active derivatives are those substituted at –para position.6 Among all synthesized ester derivatives of SAL, the most anticancer active are two esters with saturated aliphatic chain, that is, butyl (IC50 = 3.58–4.15 lM) and 2,2,2-trifluoroethyl (IC50 = 0.47–3.05 lM), and two esters with aromatic substituents, that is, b-naphthylethyl (IC50 = 3.73–9.33) and benzotriazole (IC50 = 1.84–6.61).6 It is worth noting that almost all obtained secondary amides and esters of SAL exhibit lower toxicity toward normal cells of the body than the commonly used cytostatic agents.6 In this context, the SAL amides obtained with blocked amino acid substituent (1–5) belong to high anticancer active SAL derivatives with modified carboxylic group. The most anticancer active compounds are these with aliphatic-aromatic substituents in amino acid moieties, that is, amides of SAL with L-histidine methyl ester (3) and L-phenylalanine methyl ester (4) with IC50 = 0.80– 3.67 lM and IC50 = 2.89–3.49 lM, respectively. Moreover, all newly synthesized SAL amides are less toxic to healthy cells of the body, much less than the majority of already obtained SAL derivatives. On the other hand, the bioconjugates of SAL with amino acid methyl esters are characterized by the highest values of SI as well as the lowest values of RI indexes from among all already obtained bioconjugates of this antibiotic. Only the ester of SAL with floxuridine is characterized by improved values of these parameters.7–9 It is worth noting that up to now, amide of SAL with L-
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histidine methyl ester (3) has been one of the most anticancer active derivative of SAL with modified carboxylic group. It is postulated that SAL derivatives with modified carboxylic group transport metal cations via an electrogenic or biomimetic mechanism, while unmodified ionophore transports metal cations only by an electroneutral mechanism as a result of a negatively charged carboxylate group.1 Therefore, the chemical modification of SAL carboxylic group changes the ionophoretic properties of its derivatives and the mechanism of cation transport across the lipid bilayers. Finally, it induces changes in biological activity of obtained derivatives. To summarize, for the first time, a simple and efficient method for the synthesis of Salinomycin (SAL) bioconjugates with amino acid methyl esters has been described. All these derivatives were examined for their anticancer in vitro activity against different drug-sensitive and drug-resistant cancer cell lines. Results of our study have clearly shown that unmodified SAL and its studied derivatives exhibit interesting anticancer in vitro activity against human cancer cells at low micromolar concentration. It is worth noting that all compounds tested, similarly to the unmodified parent compound,11 overcome the resistance of doxorubicin-resistant cancer cell line (LoVo/DX), which confirms the high selectivity of these compounds against the cancer cells of this type. Moreover, for the first time, the potent anticancer activity of SAL derivatives against leukemia cell line (MV4-11) has been proved. Simultaneously, almost all bioconjugates were shown to be less toxic for normal murine fibroblast cells than the currently used anticancer drugs, such as cisplatin and doxorubicin. All these results demonstrate strong potential and advantages of bioconjugates of SAL and are a good starting point for further studies, based on other active compounds, which are currently underway in our group. The obtained compounds seem to be attractive in the fight against cancer, because they are able to overcome a strong drug-resistance of the cancer cell lines. Moreover, SAL derivatives reversed the resistance of doxorubicin, suggesting that chemotherapy in combination with these bioconjugates may benefit the MDR cancer therapy. These findings support a strategy to decrease the doxorubicin concentration in combination with SAL amino acid esters in order to reduce the toxic side effects. Acknowledgments Financial support from the budget funds for science in years 2013–2015—grant ‘Iuventus Plus’ of the Polish Ministry of Science and Higher Education—No. IP2012013272, is gratefully acknowledged. Michał Antoszczak wishes to thank the Polish National Science Centre (NCN) for doctoral scholarship ‘ETIUDA’—No. 2014/12/T/ST5/00710. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2015.06.
086. These data include MOL files and InChiKeys of the most important compounds described in this article. References and notes 1. Antoszczak, M.; Rutkowski, J.; Huczyn´ski, A. Structure and biological activity of polyether ionophores and their semi-synthetic derivatives. In Bioactive Natural Products. Chemistry and Biology; Brahmachari, G., Ed., 1st ed.; Wiley-VCH Verlag GmbH, 2015; pp 107–170. 2. Gupta, P. B.; Onder, T. T.; Jiang, G.; Tao, K.; Kuperwasser, C.; Weinberg, R. A.; Lander, E. S. Cell 2009, 138, 645. 3. Antoszczak, M.; Huczyn´ski, A. Anticancer Agents Med. Chem. 2015, 15, 575. 4. Naujokat, C.; Steinhart, R. J. Biomed. Biotechnol. 2012, 2012, 950658. 5. (a) Xu, L.; Wang, T.; Meng, W.-Y.; Wei, J.; Ma, J.-L.; Shi, M.; Wang, Y.-G. Oncol. Rep. 2015, 1057, 33; (b) Chen, T.; Yi, L.; Li, F.; Hu, R.; Hu, S.; Yin, Y.; Lan, C.; Li, Z.; Fu, C.; Cao, L.; Chen, Z.; Xian, J.; Feng, H. Mol. Med. Rep. 2015, 11, 2407; (c) Schenk, M.; Aykut, B.; Teske, C.; Giese, N. A.; Weitz, J.; Welsch, T. Cancer Lett. 2015, 358, 161. 6. (a) Huczyn´ski, A.; Janczak, J.; Antoszczak, M.; Stefan´ska, J.; Brzezinski, B. J. Mol. Struct. 2012, 1022, 197; (b) Antoszczak, M.; Maj, E.; Stefan´ska, J.; Wietrzyk, J.; Janczak, J.; Brzezinski, B.; Huczyn´ski, A. Bioorg. Med. Chem. Lett. 2014, 24, 1724; (c) Antoszczak, M.; Popiel, K.; Stefan´ska, J.; Wietrzyk, J.; Maj, E.; Janczak, J.; Michalska, G.; Brzezinski, B.; Huczyn´ski, A. Eur. J. Med. Chem. 2014, 76, 435; (d) Antoszczak, M.; Maj, E.; Napiórkowska, A.; Stefan´ska, J.; Augustynowicz-Kopec´, E.; Wietrzyk, J.; Janczak, J.; Brzezinski, B.; Huczyn´ski, A. Molecules 2014, 19, 19435; (e) Borgström, B.; Huang, X.; Pošta, M.; Hegardt, C.; Oredsson, S.; Strand, D. Chem. Commun. 2013, 9944; (f) Huang, X.; Borgström, B.; Mänsson, L.; Persson, L.; Oredsson, S.; Hegardt, C.; Strand, D. ACS Chem. Biol. 2014, 9, 1587. 7. Huczyn´ski, A.; Antoszczak, M.; Kleczewska, N.; Baraniak, D.; Maj, E.; Stefan´ska, J.; Wietrzyk, J.; Janczak, J.; Celewicz, L. Eur. J. Med. Chem. 2015, 93, 33. 8. Skiera, I.; Antoszczak, M.; Trynda, J.; Wietrzyk, J.; Boratyn´ski, P.; Kacprzak, K.; Huczyn´ski, A. Chem. Biol. Drug. Des. 2015 (in press). DOI:10.1111/cbdd.12523. 9. Antoszczak, M.; Kleiborowska, G.; Kruszyk, M; Maj, E.; Wietrzyk, J.; Huczyn´ski, A.;, Chem. Biol. Drug. Des. 2015 (in press). DOI:10.1111/cbdd.12602. 10. Vig, B. S.; Huttunen, K. M.; Laine, K.; Rautio, J. Adv. Drug Deliv. Rev. 2013, 65, 1370. 11. (a) Kim, K.-Y.; Kim, S.-H.; Yu, S.-N.; Park, S.-K.; Choi, H.-D.; Yu, H.-S.; Ji, J.-H.; Seo, Y.-K.; Ahn, S.-C. Mol. Med. Rep. 1898, 2015, 12; (b) Zhou, Y.; Liang, C.; Xue, F.; Chen, W.; Zhi, X.; Feng, X.; Bai, X.; Liang, T. Oncotarget 2015, 6, 10350; (c) Liffers, S.-T.; Tilkorn, D. J.; Stricker, I.; Junge, C. G.; Al-Benna, S.; Vogt, M.; Verdoodt, B.; Steina, H.-U.; Tannapfel, A.; Tischoff, I.; Mirmohammadsadegh, A. BMC Cancer 2013, 13, 490; (d) Kim, J.-H.; Chae, M.; Kim, W. K.; Kim, Y.-J.; Kang, H. S.; Kim, H. S.; Yoon, S. Br. J. Pharmacol. 2011, 162, 773. 12. Badisa, R. B.; Darling-Reed, S. F.; Joseph, P.; Cooperwood, J. S.; Latinwo, L. M.; Goldman, C. B. Anticancer Res. 2009, 29, 2993. 13. Huczyn´ski, A.; Janczak, J.; Stefan´ska, J.; Antoszczak, M.; Brzezinski, B. Bioorg. Med. Chem. Lett. 2012, 22, 4697. 14. Pedersen, D. S.; Rosenbohm, C. Synthesis 2001, 16, 2431. 15. To a mixture of SAL (500 mg, 0.66 mmol) in DMF (25 ml) the following compounds were added: DCC (206 mg, 1.0 mmol), HOBt (45 mg, 0.33 mmol), Lamino acid methyl ester (2.0 mmol) and, if the L-amino acid methyl ester was in the form of a hydrochloride, triethylamine (2.2 mmol). The mixture was first stirred at a temperature below 0 °C for 1 h and then for further 24 h at room temperature. The solvent was subsequently evaporated under reduced pressure to dryness. The residue was suspended in hexane and filtered off. The filtrate was evaporated under reduced pressure and the residue was purified chromatographically on silica gel (Fluka type 60) to give bioconjugates of SAL (yield from 51% to 75%, see Scheme 1 and Table 1) as a yellow oil state. The 1H, 13C, 2D NMR, FT-IR and ESI-MS spectra of selected bioconjugates of SAL are included in the Supplementary material. 16. Huczyn´ski, A.; Janczak, J.; Antoszczak, M.; Wietrzyk, J.; Maj, E.; Brzezinski, B. Bioorg. Med. Chem. Lett. 2012, 22, 7146. 17. Grandi, M.; Geroni, C.; Giuliani, F. C. Br. J. Cancer 1986, 54, 515. 18. Hang, X.; Yashiro, M.; Qiu, H.; Nishii, T.; Matsuzaki, T.; Hirakawa, K. Anticancer Res. 2010, 30, 915. 19. Lukawska, M.; Wietrzyk, J.; Opolski, A.; Oszczapowicz, J.; Oszczapowic, I. Invest. New Drugs 2010, 28, 600.
