Chem Biol Drug Des 2015; 86: 911–917 Research Letter
Antiproliferative Activity of Polyether Antibiotic – Cinchona Alkaloid Conjugates Obtained via Click Chemistry Iwona Skiera1, Michał Antoszczak1, Justyna Trynda2, Joanna Wietrzyk2, Przemysław ski3, Karol Kacprzak1,* and Adam Boratyn ski1 Huczyn 1
Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland 2 Ludwik Hierszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wroclaw, Poland 3 Faculty of Chemistry, Wrocław University of Technology, skiego 27, 50-370 Wroclaw, Poland Wyspian *Corresponding author: Karol Kacprzak, [email protected] A series of eight new conjugates of salinomycin or monensin and Cinchona alkaloids were obtained by the Cu(I)-catalysed 1,3-dipolar Huisgen cycloaddition (click chemistry) of respective N-propargyl amides of salinomycin or monensin with four different Cinchona alkaloid derived azides. In vitro antiproliferative activity of these conjugates evaluated against three cancer cell lines (LoVo, LoVo/DX, HepG2) showed that four of the compounds exhibited high antiproliferative activity (IC50 below 3.00 lM) and appeared to be less toxic and more selective against normal cells than two standard anticancer drugs. Key words: anticancer activity, Cinchona alkaloids, click chemistry, monensin, salinomycin Received 9 October 2014, revised 12 December 2014 and accepted for publication 13 January 2015
Bioconjugation is a relatively new concept in the drug design based on the covalent combination of diverse bioactive compounds to produce hybrid molecules with improved affinity or efficacy and often may lead to new biological activity profile of such hybrids (1,2). Bioconjugation of functionalized molecules requires suitable synthetic tools which should be selective, reliable, easy to perform and preferably insensitive to moisture or oxygen. One of such method for the conjugation become recently Cu(I) catalysed Huisgen 1,3-dipolar cycloaddition between alkynes and azides (CuAAC) reported in 2002 ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12523
independently by Sharpless and Meldal (3,4). This general, reliable, regioselective and easy to perform reaction produces 1,2,3-triazoles as a linkage, which are rigid, stable and inert, and do not undergo hydrolysis under physiological conditions (5,6). More importantly, 1,2,3-triazole ring roughly mimicking amide functionality and participating in hydrogen bonding is currently considered as an active pharmacophore in medicinal chemistry (7). Natural polyether ionophores, such as salinomycin (SAL) and monensin (MON), have been objects of vast interest because of their antibacterial, antifungal, antiparasitic as well as antiviral activities (8). Recently, high anticancer activity of these compounds has been demonstrated against the proliferation of various cancer cells such as leukaemic, colon carcinoma, prostate cancer, including those that display multidrug resistance (MDR) and against cancer stem cells (CSCs) (9). In 2009, it was announced that SAL is nearly 100-fold more effective towards the breast CSCs than the commonly used cytostatic drug paclitaxel (Taxol). Screening of ca. 16 000 substances provided only 32 compounds able to destroy programmed CSCs and the most effective proved to be SAL (10). Recent studies indicated that SAL induces cell death of ovarian cancer cell lines (11–13). On the other hand, the synergistic antitumor effect of combined therapy using SAL and 5-fluorouracil against hepatocellular carcinoma has been presented (14). Similarly, SAL increased the antiproliferative effects of a tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) on glioblastoma cell lines (15). Antitumor properties of MON include inhibition of the proliferation of human colon cancer cell line, lymphoma cell line and myeloma cell line (16–18). In 2010, screening test of 4910 well-known drugs and drug-like compounds towards prostate cancer identified only four leads, including MON, which selectively inhibits prostate cancer cell growth at nanomolar concentrations (19). In vitro cytotoxicity of MON towards immunotoxins and its beneficial role in overcoming MDR has also been documented (20). On the other hand, Cinchona alkaloids comprising quinine, quinidine, cinchonidine and cinchonine as the major
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members constitute a unique class of natural products used for centuries in medicine for the treatment of malaria or more recently as antiarrhythmic agents (21). Major Cinchona alkaloids have no valuable anticancer activity, for example IC50 of quinine and quinidine for MCF-7 line in vitro was determined as 40 and 113 lM, respectively (22). Nevertheless, quinine and especially cinchonine have been successfully used in reversing of multidrug resistance (MDR) in patients with cancer treated by anticancer drugs such as doxorubicin, ethylprednisolone or vinblastine among other (21,23). Dimeric diester of quinine was shown to be highly active in MDR, completely reversing the P-glycoprotein (P-gp)-mediated paclitaxel resistance phenotype as well as inhibiting its transport in MCF-7/DX1 cell in vitro studies (24). Interestingly, conjugation of Cinchona alkaloids and AZT by CuAAC reaction resulted in a few compounds of marked cytotoxic activity in vitro (25). On the basis of our research on the modification and biological activity of Cinchona alkaloids (25–28) and polyether ionophores derivatives (29–33), we initiated a research project on synthesis and biological evaluation of structurally diverse conjugates of these natural products. Herein, we reported for the first time the use of CuAAC reaction for the covalent modification of SAL and MON. In particular, we prepared a representative 8-membered set of conjugates by linking four structurally diverse Cinchona alkaloid azides with readily available alkyne-derived SAL and MON. Antiproliferative effect of the resulting products was tested in vitro using human liver cancer cell line (HepG2), human colon adenocarcinoma cell line (LoVo) and doxorubicin-resistant subline (LoVo/DX), as well as normal murine embryonic fibroblast cell line (BALB/3T3).
Materials and Methods General procedure for the synthesis of conjugates To a stirred solution of Cinchona alkaloid azide (0.3 mmol, 1 equiv) and salinomycin or monensin N-propargyl amide (0.3 mmol, 1 equiv) in 10 mL of 1:1 MeOH/H2O mixture, aq. CuSO4 (0.5 equiv, 1 M) and sodium ascorbate (1 equiv) were added. The tightly sealed mixture was typically stirred for 24–48 h at 25 °C. After the consumption of the alkaloid azide (TLC control, Dragendorff reagent for visualization), the excess of methanol was removed on evaporator and the aqueous solution was diluted by 10% aq. EDTA solution (10 mL) and extracted twice with 15 mL portions of chloroform. Organic phases were separated and dried over anhydrous MgSO4. The solvent was then evaporated under reduced pressure to give crude product, which after column chromatography on short path of silica gel with the use of chloroform as mobile phase gave pure conjugates SAL-1-4 and MON-1-4 in 50–82% yield. The exemplary spectra of obtained compounds are included in Supporting information. 912
Antiproliferative activity The new conjugates were evaluated for their in vitro antiproliferative effect on three human cancer and one normal cell lines following the previously published procedures (32,33).
Results and Discussion Chemistry The synthesis of desired conjugates began from the preparation of respective alkynes and azides as partners for CuAAC reaction. The alkynes were obtained from SAL and MON. SAL was prepared conveniently by isolation of its sodium salt from commercially available veterinary premix – SACOX following acidic extraction using the procedure described previously (30) whereas MON was purchased from Sigma-Aldrich (St Louis, MO, USA). The respective N-propargyl amides: SAL-prop and MON-prop were synthesized in the reaction between SAL or MON and propargylamine in the presence of DCC (N,N’-dicyclohexylcarbodiimide) as a coupling agent and HOBt (1-hydroxybenzotriazole) as an activator, following our procedure described previously (30). The azide counterparts for CuAAC reaction were conveniently prepared from Cinchona alkaloids. Azides Q2–Q4 were synthesized by the substitution of the corresponding 9-O-mesylates of quinine, 9-epiquinine and quinidine with NaN3 as described previously (26). Homologated azide Q1 was prepared by diastereoselective CoreyChaykovsky 9-epoxymethylation of cinchoninone followed by epoxide ring opening with NaN3/NH4Cl as described in a recent work (28). CuAAC reactions of SAL N-propargyl amide (SAL-prop) or MON N-propargyl amide (MON-prop) and four Cinchona alkaloid azides namely, (8R,9S)-9-azidomethylcinchonine (Q1), (8S,9R)-9-azido-(9-deoxy)quinine (Q2), (8S,9S)-9-azido-(9-deoxy)epiquinine (Q3) and (8R,9R)-9azido-(9-deoxy)epiquinidine (Q4), were completed using standard Sharpless protocol with in situ generation of Cu(I) from copper(II) sulphate and sodium ascorbate in methanol-water system (Scheme 1). We found that despite multifunctional nature of ionophores the reactions proceeded cleanly and desired four MON (MON-Q1 – MON-Q4) and four SAL (SAL-Q1 – SAL-Q4) desired click-conjugates were obtained in 50–82% of yield after isolation. The purity and identity of the obtained compounds were determined on the basis of FT-IR, NMR and ESI MS analysis. The 1H and 13C NMR signals were assigned using one- and twodimensional (1H-1H COSY, 1H-1H NOSY, 1H-13C HETCOR, 1H-13C HMBC) spectra. A set of the representative spectra of the conjugates are included in Supporting information. The major evidence of formation of 1,2,3-triazole linked conjugates is the absence of three characteristic bands at Chem Biol Drug Des 2015; 86: 911–917
Polyether Antibiotic Cinchona Conjugates
cinchona alkaloid azide
N3
H N
+
(a)
salinomycin or monensin
O
N N N
N H
cinchona alkaloid
SAL-prop or MON-prop
Q1 - Q4
O
SAL-Q1 (78%) SAL-Q2 (50%) SAL-Q3 (63%) SAL-Q4 (63%)
salinomycin or monensin
MON-Q1 (71%) MON-Q2 (82%) MON-Q3 (78%) MON-Q4 (59%)
building blocks:
Q2 =
N N3
N OH O
N3
N
N3
N
N N
OH O OH
O
O NH
N3
Q4 =
Q3 =
O O
O
N
N
OH
Q1 =
O
O
O
O
OMe
HN
OH
O
O
O
O
O
OH
OH
monensin N-propargylamide ( MON-prop)
salinomycin N-propargylamide ( SAL-prop)
Scheme 1: Reagents and conditions: (a) aq. CuSO4, sodium ascorbate, MeOH/H2O, rt, 24 h.
Transmittance [%]
100
2253 50
3319 SAL-Q
Figure 1: FT-IR spectra of SAL-Q3 and its precursors Q3 and SAL-prop recorded in KBr.
0 4000
3
Q3 3600
about 2100 cm1, 3316 cm1 and 2125 cm1 in the FTIR spectra of all products. The first one located near 2100 cm1 is assigned to the m(N3) stretching vibrations and is observed in the FT-IR spectra of all four azides (Q1Q4). Two further bands at 3316 and 2125 cm1 attributed to the alkyne m(C–H) and m(CC) stretching vibrations are only observed in MON-prop and SAL-prop substrates. None of those bands appeared in the FT-IR spectra of the products, giving a clear proof that azide and alkyne substrates had been completely consumed in the CuAAC reaction (see Figure 1). The formation of 1,2,3-triazole linkage in all conjugates was also directly supported by the Chem Biol Drug Des 2015; 86: 911–917
2096
SAL-prop
3200
2800
2400
Wavenumber
2000
1600
1200
800
[cm–1]
1
H NMR spectra which showeding a typical singlet for triazole proton in the range 7.60–8.44 ppm.
Antiproliferative activity Four SAL (SAL-Q1 – SAL-Q4) and four MON (MON-Q1 – MON-Q4) conjugates, their precursors (SAL, MON), four Cinchona alkaloid azides (Q1-Q4) as well as two reference anticancer drugs – doxorubicin and cisplatin were evaluated for their in vitro antiproliferative effect on three cancer (LoVo, LoVo/DX and HepG2) and one normal cell lines, following the previously published procedures (31,32). The 913
Skiera et al. Table 1: Antiproliferative activity of polyether antibiotic – Cinchona alkaloid conjugates and their precursors. Data are given as IC50 [lM] Cancer cells Compound
LoVo
Q1 Q2 Q3 Q4 SAL SAL-Q1 SAL-Q2 SAL-Q3 SAL-Q4 MON MON-Q1 MON-Q2 MON-Q3 MON-Q4 Doxorubicin Cisplatin
107.26 67.31 63.25 66.88 0.61 11.61 11.96 2.18 2.51 0.06 6.75 8.81 2.66 2.56 0.28 4.40
LoVo/DX
34.28 7.58 21.78 0.74 0.36 3.92 2.37 0.18 0.16 0.03 2.19 2.04 0.32 0.74 0.11 0.87
97.76 68.23 84.14 100.28 0.52 16.72 2.79 2.28 2.05 0.07 4.43 4.76 2.03 2.86 6.73 5.67
Normal cells BALB/3T3
HepG2 9.10 15.05 6.30 26.73 0.17 3.00 1.12 0.44 0.57 0.03 1.98 2.47 0.29 0.36 0.81 0.50
94.64 70.51 81.19 114.73 12.44 19.57 6.89 2.66 2.59 0.76 5.25 4.08 2.34 2.61 0.77 8.93
3.98 37.98 10.59 18.95 6.34 5.64 0.75 0.48 0.63 0.04 1.17 0.90 1.03 1.21 0.22 1.37
109.66 83.54 94.61 130.01 35.18 34.04 18.69 3.09 2.81 6.54 33.60 32.76 4.58 7.15 0.53 12.43
5.47 12.13 4.81 41.07 6.86 1.48 2.60 0.37 0.68 1.09 1.28 1.19 1.04 0.76 0.20 5.90
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 liver cancer cell line (HepG2); normal murine embryonic fibroblast cell line (BALB/3T3). Data are expressed as the mean SD.
cytotoxic effect was also studied on the normal murine embryonic fibroblast cell line (BALB/3T3) to estimate the toxicity of the studied compounds. The mean IC50 SD of the tested compounds are collected in Table 1. Human colon adenocarcinoma cell line (LoVo) and its doxorubicinresistant subline (LoVo/DX), pair of cell lines displaying various levels of drug resistance were used for evaluation of the activity of the studied compounds against the cells with MDR (multidrug resistance) phenotype. Index of resistance (IR) for such a line was calculated and is presented in Table 2. The IR value indicates how many times more resistant is the subline in comparison with its parental cell line. As shown in Table 1, unmodified MON was highly active against LoVo and LoVo/DX cell lines with IC50 values at low submicromolar concentrations (0.06 and 0.07 lM, respectively). The antiproliferative activity of SAL against these lines is also high but about ten times less potent as compared to MON. Both ionophores have significantly lower activity against HepG2 cell line (IC50 = 0.76 lM and IC50 = 12.44 lM for MON and SAL, respectively). It is important to note that these ionophores exhibit low toxicity against normal murine embryonic fibroblast cell line (BALB/3T3). Thus, the SI values calculated for unmodified MON and SAL are impressive, especially when compared with the SI values of the currently used anticancer drugs, like cisplatin or doxorubicin (Table 2). The selectivity index (SI), an important pharmaceutical parameter that facilitates the estimation of possible future clinical development, was calculated as the ratio of IC50 value for normal cell line (BALB/3T3) to IC50 value for a respective cancerous cell line. Higher values of SI indicate greater anticancer specificity and a compound displaying SI >3 is considered as highly selective. The calculated SI values for MON and 914
Table 2: The calculated values of the indexes of resistance (IR) and selectivity (SI) of polyether antibiotic – Cinchona alkaloid conjugates and their precursors LoVo/DX Compound
LoVo SI
SI
IR
HepG2 SI
Q1 Q2 Q3 Q4 SAL SAL-Q1 SAL-Q2 SAL-Q3 SAL-Q4 MON MON-Q1 MON-Q2 MON-Q3 MON-Q4 Doxorubicin Cisplatin
1.02 1.24 1.50 1.94 57.67 2.93 1.56 1.42 1.12 109.00 4.98 3.72 1.72 2.79 1.89 2.83
1.12 1.22 1.12 1.27 67.65 2.04 6.70 1.36 1.37 93.43 7.58 6.88 2.26 2.50 0.08 2.19
0.91 1.01 1.33 1.50 0.85 1.44 0.23 1.05 0.82 1.17 0.66 0.54 0.76 1.11 24.04 1.29
1.16 1.18 1.17 1.13 2.83 1.74 2.71 1.16 1.08 8.61 6.40 8.03 1.96 2.74 0.69 1.39
The IR (Index of Resistance) indicates how many times a resistant subline is chemoresistant relative to its parental cell line. When IR is 0–2, the cells are sensitive to tested compound; IR of the range 2–10 means that the cell shows moderate sensitivity to a drug; IR above 10 indicates strong drug resistance. The SI (Selectivity Index) was calculated for each compounds using formula: SI = IC50 for normal cell line (BALB/3T3)/IC50 for respective cancerous cell line. A beneficial SI >1.0 indicates a drug with efficacy against tumour cells greater than toxicity against normal cells.
SAL for human colon adenocarcinoma cell lines (LoVo and LoVo/DX) indicate that these compounds can be recognized as the potential anticancer drugs. Chem Biol Drug Des 2015; 86: 911–917
Polyether Antibiotic Cinchona Conjugates
Contrary, all Cinchona alkaloid azides (Q1–Q4) display rather low cytotoxicity against all tested cancer cells (IC50 in range from 63.25 to 114.73 lM). Therefore, it was interesting to check the anticancer activity exhibited by conjugates of highly active polyether ionophores [activities of N-propargylated SAL are moderate as compared to that of unmodified SAL and were reported in our former work (30)] with relatively inactive Cinchona alkaloid azides. Our studies have shown that the majority of the newly synthesized conjugates exerted antiproliferative activity at micromolar concentrations (IC50 from 2.03 to 19.57 lM) against the same three human cancer cell lines and, simultaneously, relatively low toxicity against normal murine embryonic fibroblast cell line. Derivatives SAL-Q3, SALQ4, MON-Q3 and MON-Q4 were active in low micromolar concentration range (IC50 below 3.00 lM). These active conjugates contained the 9-epiquinine (SALQ3 and MON-Q3) or 9-epiquinidine (SAL-4 and MON-4) alkaloid moiety. Other conjugates SAL-Q1, SAL-Q2, MON-Q1 and MON-Q2 containing alkaloidal scaffold of absolute configuration of quinine (Q2) or cinchonine (Q1) showed lower cytotoxicity in these tests. It is worth noting that the active conjugates SAL-Q3, SAL-Q4, MON-Q3 and MON-Q4 were 2–3 times more active than cisplatin and MON-Q4 showed also slightly better selectivity index (SI) as compared to those of cisplatin and doxorubicin. According to the data given in Table 1, five from the eight obtained conjugates were very active against cell lines expressing drug-resistant phenotype (IR below 1.00), while for doxorubicin IR = 24.04. Almost all conjugates (except SAL-Q1 with IC50 = 16.72 lM) showed moderate to high cytotoxic activity against LoVo/DX cancer cell line, which was higher than that of the anticancer drugs used in tests (Table 1). The mechanism of action of the conjugates remains unclear at present. Recent reports shown that monensin treatment can reduce sensitivity of cells to doxorubicin to the level of dox-resistant cells (34), contrary salinomycin has been reported to be a Pgp inhibitor (35) and monensin sensitized resistant MCF-7 cells to Adriamycin (36). These data indicate that interactions of parent monensin or salinomycin with Pgp are complex. The conjugation of these ionophores with Cinchona alkaloids azides led to the formation of relatively large (MW ca. 1100) and multifunctional molecule which posses an unique identity rather than retains the activity of the parent counterparts. The inspection of toxicity (selectivity) of the conjugates, expressed as their SI values, revealed that MON conjugates are less toxic (higher SI values) as compared to the SAL analogues. Generally, it was found that parent polyether ionophores as well as their conjugates appeared to be more selective against cancer cells than cisplatin and doxorubicin.
Chem Biol Drug Des 2015; 86: 911–917
Conclusions To summarize, we demonstrated for the first time that complex polyether ionophores are excellent partners for the CuAAC reaction and their click conjugation with Cinchona alkaloids azides could be completed using very simple and efficient methodology. Although none of the eight conjugates exceeded the very high anticancer activity of parent SAL and MON, four of them showed good antiproliferative effect in the low micromolar concentration range. Moreover, these active conjugates were shown to be less toxic for normal murine fibroblast cells than the currently used anticancer drugs, such as cisplatin and doxorubicin. These results confirm the usefulness of conjugation concept and are a good starting point for further discovery research based on ionophores which is currently undergoing in our group.
Acknowledgments Financial support from 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 received the financial resources for their doctoral thesis from the Polish National Science Centre (NCN) in the framework of a doctoral scholarship funding – No. DEC-2014/12/T/ST5/00710.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. FT-IR spectra of MON-Q2 and its precursors Q2 and MON-prop recorded in KBr.
Chem Biol Drug Des 2015; 86: 911–917
Figure S2. 1H NMR spectrum of SAL-Q3 in CD2Cl2. Figure S3.
13
Figure S4. CD2Cl2.
C NMR spectrum of SAL-Q3 in CD2Cl2.
1
H-13C HETCOR spectrum of SAL-Q3 in
Figure S5. 1H-1H COSY spectrum of SAL-Q3 in CD2Cl2. Figure S6. CD2Cl2.
1
H-13C HMBC spectrum of SAL-Q3 in
Figure S7. CD2Cl2.
1
H-1H NOESY spectrum of SAL-Q3 in
Figure S8. ESI spectra of the complexes of (a) SAL-Q1, (b) SAL-Q2, (c) SAL-Q3 and (d) SAL-Q4 with the Na+ cation recorded at cv = 30 V. Figure S9. ESI spectra of the complexes of (a) MON-Q1, (b) MON-Q2, (c) MON-Q3 and (d) MON-Q4 with the Na+ cation recorded at cv = 30 V.
917
Antiproliferative Activity of Polyether Antibiotic – Cinchona Alkaloid Conjugates Obtained via Click Chemistry Iwona Skiera1, Michał Antoszczak1, Justyna Trynda2, Joanna Wietrzyk2, Przemysław ski3, Karol Kacprzak1,* and Adam Boratyn ski1 Huczyn 1
Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland 2 Ludwik Hierszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Rudolfa Weigla 12, 53-114 Wroclaw, Poland 3 Faculty of Chemistry, Wrocław University of Technology, skiego 27, 50-370 Wroclaw, Poland Wyspian *Corresponding author: Karol Kacprzak, [email protected] A series of eight new conjugates of salinomycin or monensin and Cinchona alkaloids were obtained by the Cu(I)-catalysed 1,3-dipolar Huisgen cycloaddition (click chemistry) of respective N-propargyl amides of salinomycin or monensin with four different Cinchona alkaloid derived azides. In vitro antiproliferative activity of these conjugates evaluated against three cancer cell lines (LoVo, LoVo/DX, HepG2) showed that four of the compounds exhibited high antiproliferative activity (IC50 below 3.00 lM) and appeared to be less toxic and more selective against normal cells than two standard anticancer drugs. Key words: anticancer activity, Cinchona alkaloids, click chemistry, monensin, salinomycin Received 9 October 2014, revised 12 December 2014 and accepted for publication 13 January 2015
Bioconjugation is a relatively new concept in the drug design based on the covalent combination of diverse bioactive compounds to produce hybrid molecules with improved affinity or efficacy and often may lead to new biological activity profile of such hybrids (1,2). Bioconjugation of functionalized molecules requires suitable synthetic tools which should be selective, reliable, easy to perform and preferably insensitive to moisture or oxygen. One of such method for the conjugation become recently Cu(I) catalysed Huisgen 1,3-dipolar cycloaddition between alkynes and azides (CuAAC) reported in 2002 ª 2015 John Wiley & Sons A/S. doi: 10.1111/cbdd.12523
independently by Sharpless and Meldal (3,4). This general, reliable, regioselective and easy to perform reaction produces 1,2,3-triazoles as a linkage, which are rigid, stable and inert, and do not undergo hydrolysis under physiological conditions (5,6). More importantly, 1,2,3-triazole ring roughly mimicking amide functionality and participating in hydrogen bonding is currently considered as an active pharmacophore in medicinal chemistry (7). Natural polyether ionophores, such as salinomycin (SAL) and monensin (MON), have been objects of vast interest because of their antibacterial, antifungal, antiparasitic as well as antiviral activities (8). Recently, high anticancer activity of these compounds has been demonstrated against the proliferation of various cancer cells such as leukaemic, colon carcinoma, prostate cancer, including those that display multidrug resistance (MDR) and against cancer stem cells (CSCs) (9). In 2009, it was announced that SAL is nearly 100-fold more effective towards the breast CSCs than the commonly used cytostatic drug paclitaxel (Taxol). Screening of ca. 16 000 substances provided only 32 compounds able to destroy programmed CSCs and the most effective proved to be SAL (10). Recent studies indicated that SAL induces cell death of ovarian cancer cell lines (11–13). On the other hand, the synergistic antitumor effect of combined therapy using SAL and 5-fluorouracil against hepatocellular carcinoma has been presented (14). Similarly, SAL increased the antiproliferative effects of a tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) on glioblastoma cell lines (15). Antitumor properties of MON include inhibition of the proliferation of human colon cancer cell line, lymphoma cell line and myeloma cell line (16–18). In 2010, screening test of 4910 well-known drugs and drug-like compounds towards prostate cancer identified only four leads, including MON, which selectively inhibits prostate cancer cell growth at nanomolar concentrations (19). In vitro cytotoxicity of MON towards immunotoxins and its beneficial role in overcoming MDR has also been documented (20). On the other hand, Cinchona alkaloids comprising quinine, quinidine, cinchonidine and cinchonine as the major
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members constitute a unique class of natural products used for centuries in medicine for the treatment of malaria or more recently as antiarrhythmic agents (21). Major Cinchona alkaloids have no valuable anticancer activity, for example IC50 of quinine and quinidine for MCF-7 line in vitro was determined as 40 and 113 lM, respectively (22). Nevertheless, quinine and especially cinchonine have been successfully used in reversing of multidrug resistance (MDR) in patients with cancer treated by anticancer drugs such as doxorubicin, ethylprednisolone or vinblastine among other (21,23). Dimeric diester of quinine was shown to be highly active in MDR, completely reversing the P-glycoprotein (P-gp)-mediated paclitaxel resistance phenotype as well as inhibiting its transport in MCF-7/DX1 cell in vitro studies (24). Interestingly, conjugation of Cinchona alkaloids and AZT by CuAAC reaction resulted in a few compounds of marked cytotoxic activity in vitro (25). On the basis of our research on the modification and biological activity of Cinchona alkaloids (25–28) and polyether ionophores derivatives (29–33), we initiated a research project on synthesis and biological evaluation of structurally diverse conjugates of these natural products. Herein, we reported for the first time the use of CuAAC reaction for the covalent modification of SAL and MON. In particular, we prepared a representative 8-membered set of conjugates by linking four structurally diverse Cinchona alkaloid azides with readily available alkyne-derived SAL and MON. Antiproliferative effect of the resulting products was tested in vitro using human liver cancer cell line (HepG2), human colon adenocarcinoma cell line (LoVo) and doxorubicin-resistant subline (LoVo/DX), as well as normal murine embryonic fibroblast cell line (BALB/3T3).
Materials and Methods General procedure for the synthesis of conjugates To a stirred solution of Cinchona alkaloid azide (0.3 mmol, 1 equiv) and salinomycin or monensin N-propargyl amide (0.3 mmol, 1 equiv) in 10 mL of 1:1 MeOH/H2O mixture, aq. CuSO4 (0.5 equiv, 1 M) and sodium ascorbate (1 equiv) were added. The tightly sealed mixture was typically stirred for 24–48 h at 25 °C. After the consumption of the alkaloid azide (TLC control, Dragendorff reagent for visualization), the excess of methanol was removed on evaporator and the aqueous solution was diluted by 10% aq. EDTA solution (10 mL) and extracted twice with 15 mL portions of chloroform. Organic phases were separated and dried over anhydrous MgSO4. The solvent was then evaporated under reduced pressure to give crude product, which after column chromatography on short path of silica gel with the use of chloroform as mobile phase gave pure conjugates SAL-1-4 and MON-1-4 in 50–82% yield. The exemplary spectra of obtained compounds are included in Supporting information. 912
Antiproliferative activity The new conjugates were evaluated for their in vitro antiproliferative effect on three human cancer and one normal cell lines following the previously published procedures (32,33).
Results and Discussion Chemistry The synthesis of desired conjugates began from the preparation of respective alkynes and azides as partners for CuAAC reaction. The alkynes were obtained from SAL and MON. SAL was prepared conveniently by isolation of its sodium salt from commercially available veterinary premix – SACOX following acidic extraction using the procedure described previously (30) whereas MON was purchased from Sigma-Aldrich (St Louis, MO, USA). The respective N-propargyl amides: SAL-prop and MON-prop were synthesized in the reaction between SAL or MON and propargylamine in the presence of DCC (N,N’-dicyclohexylcarbodiimide) as a coupling agent and HOBt (1-hydroxybenzotriazole) as an activator, following our procedure described previously (30). The azide counterparts for CuAAC reaction were conveniently prepared from Cinchona alkaloids. Azides Q2–Q4 were synthesized by the substitution of the corresponding 9-O-mesylates of quinine, 9-epiquinine and quinidine with NaN3 as described previously (26). Homologated azide Q1 was prepared by diastereoselective CoreyChaykovsky 9-epoxymethylation of cinchoninone followed by epoxide ring opening with NaN3/NH4Cl as described in a recent work (28). CuAAC reactions of SAL N-propargyl amide (SAL-prop) or MON N-propargyl amide (MON-prop) and four Cinchona alkaloid azides namely, (8R,9S)-9-azidomethylcinchonine (Q1), (8S,9R)-9-azido-(9-deoxy)quinine (Q2), (8S,9S)-9-azido-(9-deoxy)epiquinine (Q3) and (8R,9R)-9azido-(9-deoxy)epiquinidine (Q4), were completed using standard Sharpless protocol with in situ generation of Cu(I) from copper(II) sulphate and sodium ascorbate in methanol-water system (Scheme 1). We found that despite multifunctional nature of ionophores the reactions proceeded cleanly and desired four MON (MON-Q1 – MON-Q4) and four SAL (SAL-Q1 – SAL-Q4) desired click-conjugates were obtained in 50–82% of yield after isolation. The purity and identity of the obtained compounds were determined on the basis of FT-IR, NMR and ESI MS analysis. The 1H and 13C NMR signals were assigned using one- and twodimensional (1H-1H COSY, 1H-1H NOSY, 1H-13C HETCOR, 1H-13C HMBC) spectra. A set of the representative spectra of the conjugates are included in Supporting information. The major evidence of formation of 1,2,3-triazole linked conjugates is the absence of three characteristic bands at Chem Biol Drug Des 2015; 86: 911–917
Polyether Antibiotic Cinchona Conjugates
cinchona alkaloid azide
N3
H N
+
(a)
salinomycin or monensin
O
N N N
N H
cinchona alkaloid
SAL-prop or MON-prop
Q1 - Q4
O
SAL-Q1 (78%) SAL-Q2 (50%) SAL-Q3 (63%) SAL-Q4 (63%)
salinomycin or monensin
MON-Q1 (71%) MON-Q2 (82%) MON-Q3 (78%) MON-Q4 (59%)
building blocks:
Q2 =
N N3
N OH O
N3
N
N3
N
N N
OH O OH
O
O NH
N3
Q4 =
Q3 =
O O
O
N
N
OH
Q1 =
O
O
O
O
OMe
HN
OH
O
O
O
O
O
OH
OH
monensin N-propargylamide ( MON-prop)
salinomycin N-propargylamide ( SAL-prop)
Scheme 1: Reagents and conditions: (a) aq. CuSO4, sodium ascorbate, MeOH/H2O, rt, 24 h.
Transmittance [%]
100
2253 50
3319 SAL-Q
Figure 1: FT-IR spectra of SAL-Q3 and its precursors Q3 and SAL-prop recorded in KBr.
0 4000
3
Q3 3600
about 2100 cm1, 3316 cm1 and 2125 cm1 in the FTIR spectra of all products. The first one located near 2100 cm1 is assigned to the m(N3) stretching vibrations and is observed in the FT-IR spectra of all four azides (Q1Q4). Two further bands at 3316 and 2125 cm1 attributed to the alkyne m(C–H) and m(CC) stretching vibrations are only observed in MON-prop and SAL-prop substrates. None of those bands appeared in the FT-IR spectra of the products, giving a clear proof that azide and alkyne substrates had been completely consumed in the CuAAC reaction (see Figure 1). The formation of 1,2,3-triazole linkage in all conjugates was also directly supported by the Chem Biol Drug Des 2015; 86: 911–917
2096
SAL-prop
3200
2800
2400
Wavenumber
2000
1600
1200
800
[cm–1]
1
H NMR spectra which showeding a typical singlet for triazole proton in the range 7.60–8.44 ppm.
Antiproliferative activity Four SAL (SAL-Q1 – SAL-Q4) and four MON (MON-Q1 – MON-Q4) conjugates, their precursors (SAL, MON), four Cinchona alkaloid azides (Q1-Q4) as well as two reference anticancer drugs – doxorubicin and cisplatin were evaluated for their in vitro antiproliferative effect on three cancer (LoVo, LoVo/DX and HepG2) and one normal cell lines, following the previously published procedures (31,32). The 913
Skiera et al. Table 1: Antiproliferative activity of polyether antibiotic – Cinchona alkaloid conjugates and their precursors. Data are given as IC50 [lM] Cancer cells Compound
LoVo
Q1 Q2 Q3 Q4 SAL SAL-Q1 SAL-Q2 SAL-Q3 SAL-Q4 MON MON-Q1 MON-Q2 MON-Q3 MON-Q4 Doxorubicin Cisplatin
107.26 67.31 63.25 66.88 0.61 11.61 11.96 2.18 2.51 0.06 6.75 8.81 2.66 2.56 0.28 4.40
LoVo/DX
34.28 7.58 21.78 0.74 0.36 3.92 2.37 0.18 0.16 0.03 2.19 2.04 0.32 0.74 0.11 0.87
97.76 68.23 84.14 100.28 0.52 16.72 2.79 2.28 2.05 0.07 4.43 4.76 2.03 2.86 6.73 5.67
Normal cells BALB/3T3
HepG2 9.10 15.05 6.30 26.73 0.17 3.00 1.12 0.44 0.57 0.03 1.98 2.47 0.29 0.36 0.81 0.50
94.64 70.51 81.19 114.73 12.44 19.57 6.89 2.66 2.59 0.76 5.25 4.08 2.34 2.61 0.77 8.93
3.98 37.98 10.59 18.95 6.34 5.64 0.75 0.48 0.63 0.04 1.17 0.90 1.03 1.21 0.22 1.37
109.66 83.54 94.61 130.01 35.18 34.04 18.69 3.09 2.81 6.54 33.60 32.76 4.58 7.15 0.53 12.43
5.47 12.13 4.81 41.07 6.86 1.48 2.60 0.37 0.68 1.09 1.28 1.19 1.04 0.76 0.20 5.90
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 liver cancer cell line (HepG2); normal murine embryonic fibroblast cell line (BALB/3T3). Data are expressed as the mean SD.
cytotoxic effect was also studied on the normal murine embryonic fibroblast cell line (BALB/3T3) to estimate the toxicity of the studied compounds. The mean IC50 SD of the tested compounds are collected in Table 1. Human colon adenocarcinoma cell line (LoVo) and its doxorubicinresistant subline (LoVo/DX), pair of cell lines displaying various levels of drug resistance were used for evaluation of the activity of the studied compounds against the cells with MDR (multidrug resistance) phenotype. Index of resistance (IR) for such a line was calculated and is presented in Table 2. The IR value indicates how many times more resistant is the subline in comparison with its parental cell line. As shown in Table 1, unmodified MON was highly active against LoVo and LoVo/DX cell lines with IC50 values at low submicromolar concentrations (0.06 and 0.07 lM, respectively). The antiproliferative activity of SAL against these lines is also high but about ten times less potent as compared to MON. Both ionophores have significantly lower activity against HepG2 cell line (IC50 = 0.76 lM and IC50 = 12.44 lM for MON and SAL, respectively). It is important to note that these ionophores exhibit low toxicity against normal murine embryonic fibroblast cell line (BALB/3T3). Thus, the SI values calculated for unmodified MON and SAL are impressive, especially when compared with the SI values of the currently used anticancer drugs, like cisplatin or doxorubicin (Table 2). The selectivity index (SI), an important pharmaceutical parameter that facilitates the estimation of possible future clinical development, was calculated as the ratio of IC50 value for normal cell line (BALB/3T3) to IC50 value for a respective cancerous cell line. Higher values of SI indicate greater anticancer specificity and a compound displaying SI >3 is considered as highly selective. The calculated SI values for MON and 914
Table 2: The calculated values of the indexes of resistance (IR) and selectivity (SI) of polyether antibiotic – Cinchona alkaloid conjugates and their precursors LoVo/DX Compound
LoVo SI
SI
IR
HepG2 SI
Q1 Q2 Q3 Q4 SAL SAL-Q1 SAL-Q2 SAL-Q3 SAL-Q4 MON MON-Q1 MON-Q2 MON-Q3 MON-Q4 Doxorubicin Cisplatin
1.02 1.24 1.50 1.94 57.67 2.93 1.56 1.42 1.12 109.00 4.98 3.72 1.72 2.79 1.89 2.83
1.12 1.22 1.12 1.27 67.65 2.04 6.70 1.36 1.37 93.43 7.58 6.88 2.26 2.50 0.08 2.19
0.91 1.01 1.33 1.50 0.85 1.44 0.23 1.05 0.82 1.17 0.66 0.54 0.76 1.11 24.04 1.29
1.16 1.18 1.17 1.13 2.83 1.74 2.71 1.16 1.08 8.61 6.40 8.03 1.96 2.74 0.69 1.39
The IR (Index of Resistance) indicates how many times a resistant subline is chemoresistant relative to its parental cell line. When IR is 0–2, the cells are sensitive to tested compound; IR of the range 2–10 means that the cell shows moderate sensitivity to a drug; IR above 10 indicates strong drug resistance. The SI (Selectivity Index) was calculated for each compounds using formula: SI = IC50 for normal cell line (BALB/3T3)/IC50 for respective cancerous cell line. A beneficial SI >1.0 indicates a drug with efficacy against tumour cells greater than toxicity against normal cells.
SAL for human colon adenocarcinoma cell lines (LoVo and LoVo/DX) indicate that these compounds can be recognized as the potential anticancer drugs. Chem Biol Drug Des 2015; 86: 911–917
Polyether Antibiotic Cinchona Conjugates
Contrary, all Cinchona alkaloid azides (Q1–Q4) display rather low cytotoxicity against all tested cancer cells (IC50 in range from 63.25 to 114.73 lM). Therefore, it was interesting to check the anticancer activity exhibited by conjugates of highly active polyether ionophores [activities of N-propargylated SAL are moderate as compared to that of unmodified SAL and were reported in our former work (30)] with relatively inactive Cinchona alkaloid azides. Our studies have shown that the majority of the newly synthesized conjugates exerted antiproliferative activity at micromolar concentrations (IC50 from 2.03 to 19.57 lM) against the same three human cancer cell lines and, simultaneously, relatively low toxicity against normal murine embryonic fibroblast cell line. Derivatives SAL-Q3, SALQ4, MON-Q3 and MON-Q4 were active in low micromolar concentration range (IC50 below 3.00 lM). These active conjugates contained the 9-epiquinine (SALQ3 and MON-Q3) or 9-epiquinidine (SAL-4 and MON-4) alkaloid moiety. Other conjugates SAL-Q1, SAL-Q2, MON-Q1 and MON-Q2 containing alkaloidal scaffold of absolute configuration of quinine (Q2) or cinchonine (Q1) showed lower cytotoxicity in these tests. It is worth noting that the active conjugates SAL-Q3, SAL-Q4, MON-Q3 and MON-Q4 were 2–3 times more active than cisplatin and MON-Q4 showed also slightly better selectivity index (SI) as compared to those of cisplatin and doxorubicin. According to the data given in Table 1, five from the eight obtained conjugates were very active against cell lines expressing drug-resistant phenotype (IR below 1.00), while for doxorubicin IR = 24.04. Almost all conjugates (except SAL-Q1 with IC50 = 16.72 lM) showed moderate to high cytotoxic activity against LoVo/DX cancer cell line, which was higher than that of the anticancer drugs used in tests (Table 1). The mechanism of action of the conjugates remains unclear at present. Recent reports shown that monensin treatment can reduce sensitivity of cells to doxorubicin to the level of dox-resistant cells (34), contrary salinomycin has been reported to be a Pgp inhibitor (35) and monensin sensitized resistant MCF-7 cells to Adriamycin (36). These data indicate that interactions of parent monensin or salinomycin with Pgp are complex. The conjugation of these ionophores with Cinchona alkaloids azides led to the formation of relatively large (MW ca. 1100) and multifunctional molecule which posses an unique identity rather than retains the activity of the parent counterparts. The inspection of toxicity (selectivity) of the conjugates, expressed as their SI values, revealed that MON conjugates are less toxic (higher SI values) as compared to the SAL analogues. Generally, it was found that parent polyether ionophores as well as their conjugates appeared to be more selective against cancer cells than cisplatin and doxorubicin.
Chem Biol Drug Des 2015; 86: 911–917
Conclusions To summarize, we demonstrated for the first time that complex polyether ionophores are excellent partners for the CuAAC reaction and their click conjugation with Cinchona alkaloids azides could be completed using very simple and efficient methodology. Although none of the eight conjugates exceeded the very high anticancer activity of parent SAL and MON, four of them showed good antiproliferative effect in the low micromolar concentration range. Moreover, these active conjugates were shown to be less toxic for normal murine fibroblast cells than the currently used anticancer drugs, such as cisplatin and doxorubicin. These results confirm the usefulness of conjugation concept and are a good starting point for further discovery research based on ionophores which is currently undergoing in our group.
Acknowledgments Financial support from 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 received the financial resources for their doctoral thesis from the Polish National Science Centre (NCN) in the framework of a doctoral scholarship funding – No. DEC-2014/12/T/ST5/00710.
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Polyether Antibiotic Cinchona Conjugates
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. FT-IR spectra of MON-Q2 and its precursors Q2 and MON-prop recorded in KBr.
Chem Biol Drug Des 2015; 86: 911–917
Figure S2. 1H NMR spectrum of SAL-Q3 in CD2Cl2. Figure S3.
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Figure S4. CD2Cl2.
C NMR spectrum of SAL-Q3 in CD2Cl2.
1
H-13C HETCOR spectrum of SAL-Q3 in
Figure S5. 1H-1H COSY spectrum of SAL-Q3 in CD2Cl2. Figure S6. CD2Cl2.
1
H-13C HMBC spectrum of SAL-Q3 in
Figure S7. CD2Cl2.
1
H-1H NOESY spectrum of SAL-Q3 in
Figure S8. ESI spectra of the complexes of (a) SAL-Q1, (b) SAL-Q2, (c) SAL-Q3 and (d) SAL-Q4 with the Na+ cation recorded at cv = 30 V. Figure S9. ESI spectra of the complexes of (a) MON-Q1, (b) MON-Q2, (c) MON-Q3 and (d) MON-Q4 with the Na+ cation recorded at cv = 30 V.
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