Antiproliferative Activity of Polyether Antibiotic - Cinchona Alkaloid Conjugates Obtained via Click Chemistry

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/cbdd.12523

This article is protected by copyright. All rights reserved.

Received Date : 09-Oct-2014

Revised Date : 12-Dec-2014

Accepted Date : 13-Jan-2015

Article type : Research Letter

Antiproliferative activity of polyether antibiotic – Cinchona alkaloid

conjugates obtained via click chemistry

Iwona Skiera1, Michał Antoszczak1, Justyna Trynda2, Joanna Wietrzyk2,

Przemysław Boratyński3, Karol Kacprzak1,* and Adam Huczyński1

1Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland

2Ludwik Hierszfeld Institute of Immunology and Experimental Therapy, Polish Academy of Sciences,

Rudolfa Weigla 12, 53-114 Wroclaw, Poland

3Faculty of Chemistry, Wrocław University of Technology, Wyspiańskiego 27, 50-370 Wroclaw Poland

Abstract

A series of eight new conjugates of salinomycin or monensin and Cinchona alkaloids were obtained

by the Cu(I)-catalyzed 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,

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HepG2) showed that four of the compounds exhibited high antiproliferative activity (IC50 below 3.00

μM) and appeared to be less toxic and more selective against normal cells than two standard

anticancer drugs.

Key words: anticancer activity; click chemistry, salinomycin, monensin, Cinchona alkaloids

*Corresponding author: [email protected], tel. +48 618291367, fax. +48 618291555 (K.

Kacprzak)

Introduction

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 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).

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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 leukemic, colon carcinoma, prostate cancer, including

those that display multi-drug 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 toward 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 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 μM, respectively (22). Nevertheless, quinine and especially cinchonine

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have been successfully used in reversing of multidrug resistance (MDR) in cancer patients 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, 1M)

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

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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 the Supplementary

material.

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 click-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. 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

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prepared by diastereoselective Corey−Chaykovsky 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)-9-azido-(9-

deoxy)epiquinidine (Q4) were completed using standard Sharpless protocol with in situ generation of

Cu(I) from copper(II) sulfate and sodium ascorbate in methanol-water system (Scheme 1). We found

that despite multifunctional nature of ionofores 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 isolated 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 two-dimensional (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 the Supplementary

material.

The major evidence of formation of 1,2,3-triazole linked conjugates is the absence of three

characteristic bands at about 2100 cm−1, 3316 cm−1 and 2125 cm−1 in the FT-IR spectra of all

products. The first one located near 2100 cm−1 is assigned to the ν(N3) stretching vibrations

and is observed in the FT-IR spectra of all four azides (Q1-Q4). Two further bands at 3316

and 2125 cm−1 attributed to the alkyne ν(≡C–H) and ν(C≡C) 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

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linkage in all conjugates was also directly supported by the 1H NMR spectra which

showeding a typical singlet for triazole proton in the range 7.60-8.44 ppm.

Wavenumber [cm-1]

80012001600200024002800320036004000

Tra

nsm

ittan

ce [%

]

0

50

100

SAL-prop SAL-Q3

Q3

2253

20963319

Fig. 1. FT-IR spectra of SAL-Q3 and its precursors Q3 and SAL-prop recorded in KBr.

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

cytotoxic effect was also studied on the normal murine embryonic fibroblast cell line (BALB/3T3) in

order 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

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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 compounds against the cells with MDR (multi-

drug 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 to 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 μM and 0.07 μM, 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 μM and IC50 = 12.44 μM 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 greater than 3 is considered as

highly selective. The calculated SI values for MON and SAL for human colon adenocarcinoma cell

lines (LoVo and LoVo/DX) indicate that these compounds can be recognised as the potential

anticancer drugs.

Contrary, all Cinchona alkaloid azides (Q1-Q4) display rather low cytotoxicity against all tested cancer

cells (IC50 in range from 63.25 μM to 114.73 μM). Therefore, it was interesting to check the

anticancer activity exhibited by conjugates of highly active polyether ionophores (activities of N-

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proprgylated 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 µM) against the same

three human cancer cell lines and, simultaneously, relatively low toxicity against normal murine

embryonic fibroblast cell line. Derivatives SAL-Q3, SAL-Q4, MON-Q3 and MON-Q4 were active in low

micromolar concentration range (IC50 below 3.00 μM).

These active conjugates contained the 9-epiquinine (SAL-Q3 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 µM) 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

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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.

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.

Acknowledgements

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.

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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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Table 1: Antiproliferative activity of polyether antibiotic – Cinchona alkaloid conjugates and their

precursors. Data are given as IC50 [µM].

Compound Cancer cells Normal cells

LoVo LoVo/DX HepG2 BALB/3T3

Q1 107.26 ± 34.28 97.76 ± 9.10 94.64 ± 3.98 109.66 ± 5.47

Q2 67.31 ± 7.58 68.23 ± 15.05 70.51 ± 37.98 83.54 ± 12.13

Q3 63.25 ± 21.78 84.14 ± 6.30 81.19 ± 10.59 94.61 ± 4.81

Q4 66.88 ± 0.74 100.28 ± 26.73 114.73 ± 18.95 130.01 ± 41.07

SAL 0.61 ± 0.36 0.52 ± 0.17 12.44 ± 6.34 35.18 ± 6.86

SAL-Q1 11.61 ± 3.92 16.72 ± 3.00 19.57 ± 5.64 34.04 ± 1.48

SAL-Q2 11.96 ± 2.37 2.79 ± 1.12 6.89 ± 0.75 18.69 ± 2.60

SAL-Q3 2.18 ± 0.18 2.28 ± 0.44 2.66 ± 0.48 3.09 ± 0.37

SAL-Q4 2.51 ± 0.16 2.05 ± 0.57 2.59 ± 0.63 2.81 ± 0.68

MON 0.06 ± 0.03 0.07 ± 0.03 0.76 ± 0.04 6.54 ± 1.09

MON-Q1 6.75 ± 2.19 4.43 ± 1.98 5.25 ± 1.17 33.60 ± 1.28

MON-Q2 8.81 ± 2.04 4.76 ± 2.47 4.08 ± 0.90 32.76 ± 1.19

MON-Q3 2.66 ± 0.32 2.03 ± 0.29 2.34 ± 1.03 4.58 ± 1.04

MON-Q4 2.56 ± 0.74 2.86 ± 0.36 2.61 ± 1.21 7.15 ± 0.76

doxorubicin 0.28 ± 0.11 6.73 ± 0.81 0.77 ± 0.22 0.53 ± 0.20

cisplatin 4.40 ± 0.87 5.67 ± 0.50 8.93 ± 1.37 12.43 ± 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.

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Table 2: The calculated values of the indexes of resistance (IR) and selectivity (SI) of polyether

antibiotic – Cinchona alkaloid conjugates and their precursors.

Compound LoVo LoVo/DX HepG2

SI SI IR SI

Q1 1.02 1.12 0.91 1.16

Q2 1.24 1.22 1.01 1.18

Q3 1.50 1.12 1.33 1.17

Q4 1.94 1.27 1.50 1.13

SAL 57.67 67.65 0.85 2.83

SAL-Q1 2.93 2.04 1.44 1.74

SAL-Q2 1.56 6.70 0.23 2.71

SAL-Q3 1.42 1.36 1.05 1.16

SAL-Q4 1.12 1.37 0.82 1.08

MON 109.00 93.43 1.17 8.61

MON-Q1 4.98 7.58 0.66 6.40

MON-Q2 3.72 6.88 0.54 8.03

MON-Q3 1.72 2.26 0.76 1.96

MON-Q4 2.79 2.50 1.11 2.74

doxorubicin 1.89 0.08 24.04 0.69

cisplatin 2.83 2.19 1.29 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.

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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.

Scheme 1. Reagents and conditions: (a) aq. CuSO4, sodium ascorbate, MeOH/H2O, rt, 24 h

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Antiproliferative Activity of Polyether Antibiotic - Cinchona Alkaloid Conjugates Obtained via Click Chemistry