- Email: [email protected]
Synthesis and cytotoxicity evaluation of glycosidic derivatives of lawsone against breast cancer cell lines
Journal Pre-proofs Synthesis and cytotoxicity evaluation of glycosidic derivatives of lawsone against breast cancer cell lines Flaviano M. Ottoni, Eliza R Gomes, Rodrigo M. Pádua, Mônica C. Oliveira, Izabella T. Silva, Ricardo J. Alves PII: DOI: Reference:
S0960-894X(19)30786-3 https://doi.org/10.1016/j.bmcl.2019.126817 BMCL 126817
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
9 August 2019 6 November 2019 8 November 2019
Please cite this article as: Ottoni, F.M., Gomes, E.R., Pádua, R.M., Oliveira, M.C., Silva, I.T., Alves, R.J., Synthesis and cytotoxicity evaluation of glycosidic derivatives of lawsone against breast cancer cell lines, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126817
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Ltd.
Synthesis and cytotoxicity evaluation of glycosidic derivatives of lawsone against breast cancer cell lines Flaviano M Ottoni a*, Eliza R Gomesa*, Rodrigo M Páduaa, Mônica C Oliveiraa, Izabella T Silvaa,b**, Ricardo J Alvesa** a
Department of Pharmaceutical Products, Faculty of Pharmacy, Federal University of Minas Gerais, 6627 Antônio Carlos,
Belo Horizonte, MG 31.270-901, Brazil. b
Department of Pharmaceutical Sciences, Federal University of Santa Catarina, Florianópolis, SC, 88.040-970, Brazil.
* These authors contributed equally to this manuscript. ** Corresponding authors. Tel.: +55 031 34096955; Fax: +55 031 3409 6934. E-mail addresses: [email protected]; [email protected]
Abstract Breast cancer is the most incident and mortal cancer type in women, with an estimated 2 million new cases expected by 2020 worldwide, with 600,000 deaths. As not all breast cancer types respond to the anti-hormonal therapy, the development of new antineoplastic drugs is necessary. Lawsone (2hydroxy-1,4-naphtoquinone) is a natural bioactive naphtoquinone displaying a range of activities, with dozens of derivatives described in the literature, including some glycosides possessing antitumor activity. Here, a series of glycosides of lawsone are reported for the first time and all compounds displayed good activity against the SKBR-3 cell line, with IC50 below 10 µM. The most promising derivative was the glycosyl triazole derived from peracetylated D-glucose (11), which showed better cytotoxicity against SKBR-3 (IC50=0.78 µM), being the most selective toward this tumoral cell (SI>20). All compounds described in this work were more active than lawsone, indicating the importance of the carbohydrate and glycosyl triazole moiety for activity.
Keywords Lawsone glycosides, breast cancer, cytotoxicity, copper catalyzed azido-alkyne cycloaddition Cancer is currently one of the major public health problems worldwide and the number of cases will increase in the next decades, mainly in developing countries. According to WHO, more than 17 million cases are predicted in 2020 with a mortality rate higher than 10 million, with 60% of cases occurring in developing countries.1 Cancer is the main cause of death in underdeveloped countries (>300,000 deaths) and the second in developed countries (almost 200,000 deaths).2 In Brazil 600,000 new cases of cancer were estimated for the years 2018-2019, the most prevalent being prostate cancer in men (31.7%) and breast cancer in women (29.5%).3 Breast cancer is the most incident and mortal cancer type in women, with an estimated 2 million new cases expected by 2020 worldwide. This represents almost 25% of all cancer types diagnosed in women with 600,000 deaths (15% of all cancer deaths).1,3 Breast cancer is one of the most usual cancer types in the female population. Breast cancer is considered a heterogeneous disease with regard to the morphology and clinic, and the WHO recognizes
more than 20 breast cancer subtypes. The majority of breast tumors are formed in the ductal epithelium and are known to be invasive ductal carcinoma. Besides, the number of cases is higher in women up to 50 years old, with a reduction after this age, so it is believed that there is participation of hormones in the etiology of the disease. The overexpression of hormone receptors has been identified in a high number of breast cancer patients.4 The activation of these receptors by female hormones induces the proliferation of tumor cells, so blocking it with receptor-specific therapy is an effective therapeutic strategy.5 However, not all breast cancer subtypes overexpress hormone receptors or become resistant, and thus do not respond to receptor-specific therapy6,7, necessitating the use of anticancer drugs. The high cancer mortality rate is due, mostly, to the inefficient current treatment available (radiotherapy, chemotherapy, and surgical procedures).8 Although preventive measures are taken and several drugs have been developed, it is still necessary to develop new antineoplastic drugs more effective and selective toward tumoral cells. Many modern chemotherapeutic agents currently used originate from plants and the examples include taxol from Taxus baccata (Yew), vincristine and vinblastine from Catharanthus roseus (Sadabahar), and podophyllotoxin from Podophyllum peltatum (Mayapple).9 The importance of the 1,4-naphtoquinonic scaffold in medicinal chemistry has been widely described in the last decades. Many of these compounds have been screened as anticancer agents and have shown cytotoxic activity against several tumoral cell lines. An example is lapachol, which has already been extensively studied as an antitumor agent and submitted to Phase I clinical trials, but due to several adverse effects, the studies were discontinued.10,11 Another interesting naphthoquinonic derivative is atovaquone, an approved antimicrobial drug, whose potent antitumor activity has been indicated in current studies.12 A commercially available naphtoquinone is lawsone 1 (2-hydroxy-1,4naphtoquinone), a natural bioactive compound isolated from plants of the genus Lawsonia displaying a range of activities, e.g., antimicrobial and antitumor13, with lawsone 1 being active against HCT-15 (human colon cancer cells).9 Naphthoquinones cytotoxicity has been mainly associated to the inhibition of the human DNA topoisomerase II and production of reactive oxygen species (ROS). It is likely that the naphthoquinones kill or induce cell death by more than one mechanism.14 The enzyme topoisomerases II is mostly expressed during the G2 and M phases from mythosis.15 Naphthoquinones can be reduced to form semiquinones and hydroxyquinones, which can be reoxidized leading to ROS formation and causing damage to macromolecules.16 The NQO1 (NADPH: quinone oxidoreductase 1) is a enzyme responsible for reduction of naphthoquinones in anaerobic conditions. The overexpression of this enzyme in cancer cells besides be crucial for naphthoquinones cytotoxicity can explain their selectivity.17 Herein, we hypothesize that carbohydrate moiety could facilitate the transport of the compounds to the target cell via hexose transportes.18 Lawsone 1 can be easily obtained by synthesis (60% of global yield) and thus is an interesting starting material towards the preparation of bioactive derivatives.19 Dozens of derivatives of 1 are described in the literature, including some glycosides possessing antitumor activity (Figure 1).20,21
O
OAc
HO
N N
O
AcO AcO
O
H N
N OAc
O
Lawsone (1)
OAc
2
OAc
OAc O
AcO AcO
OAc O
O
AcO
O
O
O
OAc
O
3
4
O
O OAc O
AcO AcO
O
OAc
O
5
O
Figure 1. Lawsone (1) and some glycosidic derivatives that show antitumoral activity. The synthesis of glycosidic derivatives of lawsone 1 can furnish bioactive compounds and the variation of carbohydrate moiety allows a structure-activity relationship (SAR) study. Therefore, this work aimed to synthesize peracetylated and deacetylated glycosides from lawsone 1. The designed compounds were classical glycosides (carbohydrates linked directly to lawsone) and glycosyl triazoles (a triazolic ring connecting the saccharidic moiety to lawsone), investigated for their activity against three breast cancer cell lines (SKBR-3, MDA-MB-231, and MCF-7). The chemical structures of the classical glycosides and glycosyl triazoles of lawsone designed in the present work are shown in Figure 2. AcO AcO
OAc O
AcO AcO
O
OAc
O
OAc O
AcO AcO
O
OAc
O
OAc O AcHN
O O O O
3 () 4 ()
OR RO RO
5 () 6 ()
O
O
OR N
O N
OR R= Ac; 11 R= H; 15
7 () 8 ()
O O
N N
RO OR
R= Ac; 12 R= H; 16 O
O
N
O O
RO RO
N NHAc
OAc
N
O
R= Ac; 13 R= H; 17 O
O
AcO
OR
OR O
N
H3C
OAc
O 9 () 10 ()
N N
O
O
H3C
O RO O
N
N OR
O O
OR R= Ac;14 R= H; 18
Figure 2. Designed classical glycosides and glycosyl triazoles of lawsone. The synthetic route to peracetylated glycosides 5-10 and glycosyl triazoles 11-18 of lawsone is shown in Scheme 1. The key intermediates, peracetylated glycosyl halides a-d, were prepared from the respective carbohydrate (D-glucose, D-galactose, D-N-acetylglucosamine, and L-fucose) according to literature procedures.22-25 Then, glycosyl halides a-d were used as glycosyl donors in the glycosylation step using phase transfer catalysis in alkaline medium to prepare peracetylated glycosides of lawsone as mixtures of α and β anomers, which were separated by column chromatography.26,27 The pure anomers 5-10 were isolated in 21-50% yield. Stereochemistry at the anomeric carbon was unequivocally established using NMR spectroscopy. In the 1H NMR spectra, the H-1/H-2 coupling constant (3J) for α anomers were 2-4 Hz and for β anomers 8-10 Hz.28 For all β glycosides, HSQC experiments were used to assign H-1 in the 1H NMR spectra from the corresponding C-1 signal in the 13C NMR spectra, which
O
is observed at circa 98 ppm for β anomers and 95 ppm for α anomers.29 The peracetylated glycosyl azides e-h were also obtained from glycosyl halides a-d by reaction with NaN3 in acetone/H2O at room temperature.30,31 The deacetylated glycosyl azides i-l were obtained from deacetylation of corresponding peracetylated glycosyl azides, using a transesterification reaction with KOH/MeOH.32,33 The 2-O-propargyllawsone (11) was obtained in 70% yield from reaction of lawsone 1 with propargyl bromide in alkaline medium, using potassium carbonate as base and N,N-dimethylformamide (DMF) as solvent under reflux.8,34 Finally, the propargylic derivative 11 reacted with the glycosyl azides e-l under [2+3] alkyne-azide cycloaddition reaction conditions (CuSO4.5H2O, sodium ascorbate) in THF/water as solvent at room temperature, affording the glycosyl triazole derivatives of lawsone 11-18 in 60-70% yield.35-37 O
O
HO
AcO
i
O
O AcO
N3
HO
iii
N3
i-l
e-h
a-d
OH
D-glicose D-galactose
ii
X X= Br ou Cl
80 - 90 %
50 - 80 %
D-N-acetilglicosamina L-fucose O
AcO
O
O O
iv
OAc OAc O
3 (35%) OAc O OAc
4 (40%)
AcO
5 (34%) OAc O
6 (40%)
AcNH
AcO
O
O
11-18
11 (72%)
R= AcO
OAc
12 (72%) HO
OH HO R= HO
OAc OAc O
OH
OAc O
R=
NHAc
H3C
R=
O
AcO
13(64%)
14 (51%)
O OH
16 (44%)
HO R= HO
O NHAc
17 (67%)
H3C
R=
OH
O OH
18(65%)
NHAc
H3C OAc
OAc
9 (31%)
AcO
O
OAc
OAc
OH
OH
O
R= HO
AcO AcO
8 (50%)
7 (21%) H3C
OAc
15 (49%)
OAc O
AcO AcO
OAc O
R=
OAc O
AcO AcO
R
O
19 (70%)
AcO AcO
OAc
vi
O
N
N
O
1
AcO
AcO
AcO AcO
v
OAc OAc O
AcO
AcO AcO
O
O
O
3-10
N
O
HO
OAc
OAc
10 (40%)
Scheme 1. Reagents and conditions: i) a) Ac2O, AcONa, 100°C; b) HBr/AcOH, CH2Cl2, r.t; or c) CH3COCl, r.t; ii) NaN3, H2O/acetone, r.t, 3 h; iii) KOH, MeOH, 0oC, 1 h; iv) Glycosyl halide (a-d), CH2Cl2, Na2CO3 10% p/v (1:1), n-Bu4NBr, r.t., 24h; v) Propargyl bromide, K2CO3, DMF, 80-100o C, 20 h; vi) Glycosyl azide e-l, CuSO4.5H2O 50% mol, sodium ascorbate 60% mol, THF:H2O, r.t, 2-5h.
To evaluate the cytotoxic activity, lawsone 1 and all synthesized glycosides were screened against three human breast adenocarcinoma cell lines (SKBR-3, MDA-MB-231, and MCF-7) and one nontumor human fibroblast from primary gingival tissue culture (HGF) kindly supplied by Dra. Cláudia Maria Oliveira Simões (Laboratory of Virology, UFSC, Florianópolis, Brazil) according to the Research Ethics Committee of the UFSC, 021/2009. The difference among the tumoral cell lines are related to the presence or absence of estrogen (ER), progesterone (PR), and HER2 receptors.38-40 The breast cancer cell lines SKBR-3 (HER2+), MCF-7 (ER+), and MDA-MB-231 (triple-negative) were
OH
purchased from American Type Culture Collection (ATCC) and grown in McCoy’s 5A Modified Medium (McCoy), Eagle’s Minimum Essential Medium (MEM) supplemented with 0.01 mg/mL human recombinant insulin and Dulbecco’s Modified Eagle’s Medium (DMEM), respectively. All cell lines were supplemented with 10% fetal bovine serum (FBS, GibcoTM) and maintained at 37°C and 5% CO2 in a humidified atmosphere. The compounds were evaluated against 1x104 cells in 96-well plates by Sulforhodamine B assay.41 The concentrations that cause 50% inhibitory concentration (IC50) were determined using GraphPad Prism software (version 6.0, San Diego, USA) and are shown in Table 1.
Table 1. Effect of glycosidic derivatives from lawsone on breast cancer and non-cancer cell growth (µM).
Compound
SKBR-3 IC50a (CI 95%)
MCF-7 IC50a (CI 95%)
MDA-MB-231 IC50a (CI 95%)
HGF IC50a (CI 95%)
3
2.51 (2.17-2.91)
4.58 (3.29-6.37)
9.18 (8.35-10.10)
4
8.86 (7.17-10.95)
13.32 (11.87-14.94)
5
2.96 (2.63-3.32)
13.63 (11.4916.17) 4.76 (3.26-6.96)
6
2.11 (1.86-2.39)
3.52 (2.61-4.75)
8.29 (7.11-9.87)
7
3.41 (2.94-3.938)
6.59 (5.60-7.78)
19.78 (15.52-25.19)
8
8.30 (7.42-9.29)
27.64 (25.37-30.11)
9 10
9.52 (6.90-13.14) 2.41(2.18-2.67)
16.32 (11.5923.00) 11.01 (9.80-12.37) 5.02 (3.82-6.60)
35.68 (26.6947.71) 34.64 (22.8252.58) 19.14 (13.3827.37) 20.22 (12.3533.10) 40.19 (31.8350.67) >50
11
0.78 (0.67-0.90)
3.33 (2.61-4.24)
13.99 (9.95-19.65)
12
1.27 (1.05-1.53)
4.22 (3.77-4.72)
16.72 (11.73-23.83)
13
16.99 (14.17-20.38)
>50
14
3.32 (2.76-3.99)
28.89 (26.0232.09) 9.74 (8.73-10.88)
15
34.74 (29.38-41.08)
16 17 18 lawsone (1) Doxorubicinb
>50 >50 >50 >50 0.28 (0.25-0.31)
38.85 (34.3543.95) >50 >50 >50 >50 0.38 (0.32-0.46)
12.64 (11.61-13.76)
18.17 (14.51-22.76) 8.65 (7.63-9.81)
23.39 (17.24-31.73)
>50 19.77 (14.1927.53) 17.65 (12.9823.75) 27.61 (23.6032.29) >50
>50
38.15 (33.6743.21) >50
>50 >50 >50 >50 1.23 (0.99-1.52)
>50 >50 >50 >50 >50
aIC
50 (μM): inhibitory concentration of 50% cell growth was calculated through a nonlinear fit-curve (log of compound concentration versus normalized response—variable slope). bPositive control.
Among the classical glycosides of lawsone 5-10, the α-glycosides of
D
series were more cytotoxic
than the corresponding β-glycosides for the three cancer cell lines, with the exception of D-glucose
derivatives 5 and 6 (anomers showing comparable cytotoxicity). Fedorov and coworkers (2011) reported that α anomer 3 was more cytotoxic than anomer 4 against the human leukemic HL-60 cell line.16 On the other hand, L-fucoside 10 (β anomer) was more cytotoxic than L-fucoside 9 (α anomer). Regarding the toxicity against non-tumor cell line HGF, anomers of the same compound of D series showed comparable toxicity while fucoside 10 (β anomer, IC50>50 µM) was less cytotoxic than fucoside 9 (α anomer, IC50=19.77 µM), indicating that the D or L configuration influenced cytotoxicity against this cell line. The saccharidic moiety also seems to have influenced cytotoxicity since compound 5 (β-glycoside) was more active than the corresponding anomer from the other monosaccharides. Glycosides 7 and 8, derived from N-acetylglucosamine, were considerably less active than the same anomer of the glycoside derived from
D-glucose
(compounds 6 and 5,
respectively), which belongs to the same stereochemical series (D-gluco). These results indicate that substitution of acetoxy (OAc) by acetamido (NHAc) at C-2 decreases activity. Compound 3 showed the highest selectivity index (SI) for MCF-7 (SI~8) and MDA-MB-231 (SI~4) cell lines, the latter being more resistant because it is triple negative to hormone receptors (ER, PR, and HER2) and does not respond to blocking by anti-hormones.38,39 Glycosyl triazoles 11 and 12 showed greater cytotoxicity among all tested compounds against SKBR-3 (CI50=0.78 µM and 1.27 µM, respectively), showing a comparable selectivity index (SI~22). Finally, the only deacetylated glycosyl triazole that showed cytotoxicity against tumor cells was compound 15 (derived from D-glucose), being cytotoxic against SKBR-3 (CI50=34.74 µM) and MCF-7 (CI50=38.85 µM), and it was non-cytotoxic to non-tumor cells HGF (CI50>50 µM). The greater activity of peracetylated glycosides and glycosyl triazoles as compared to lawsone is probably due to the more favorable lipophilic-hydrophilic balance that has been achieved with the peracetylated glycosyl derivatives which could be absorbed by tumor cells more easily.42
D-glucose-based
6 and glycosyl triazole 11 were the most active compounds suggesting that
classical glycoside
D-glucose
transporters
(GLUTs) may be target by these compounds.43 In conclusion, we have synthesized two series of glycosidic derivatives of lawsone, corresponding to classical glycosides and glycosyl triazoles. All compounds were more active than lawsone, which did not show cytotoxicity against the tumor nor the non-tumor cell lines, although there are reports on the cytotoxicity of lawsone against other cell lines.9 Thus, the modification of lawsone by direct glycosylation or glycosyl triazole formation improved the cytotoxic activity. The anomer mixture obtained in the synthesis of classical glycosides 5-10 provided the compounds for evaluation of the influence of C-1 configuration, allowing for a structure-activity relationship (SAR) study. For glycosyl triazoles 11-18, the presence of the triazole ring permitted the evaluation of its influence on activity. Glycosyl triazoles 11 and 12 were more active against SKBR-3 than the corresponding classical glycosides 3-6. The deacetylated glycosyl triazoles were not active against the three tumor cell
lines and none was cytotoxic against the non-tumor cells (HGF), except for compound 15, which was cytotoxic against SKBR-3 and MCF-7. It was also observed that the stereochemical series of the carbohydrate moiety influenced both activity and the selectivity index. The most promising derivatives were the classical glycosides 3 and 6, which showed low IC50 values against SKBR-3 (<5 µM), MCF-7 (<5 µM), and MDA-MB-231 (<10 µM). Compound 6 was the most cytotoxic of all against MDA-MB-231, a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer (TNBC). Compound 3 was the most selective towards MCF-7 (SI~8) and MDA-MB-231 (SI~4). The glycosyl triazoles derived from peracetylated D-glucose (11) and D-galactose (12) were the most selective toward SKBR-3 (SI>20), with compound 11 being the most potent against this cell line (IC50=0.78 µM), which is considered a poor prognostic tumor.44 Furthermore, its deacetylated derivative (compound 15) was the only one among the deacetylated glycosyl triazoles that did show any cytotoxic activity for tumor cells. Altogether, the present work allowed for the obtention of two series of glycosidic derivatives of lawsone with relevant activity against three breast cancer cell lines with good selectivity in some cases, indicating that this approach deserves further attention. Work in this direction is underway and results will be reported in due time.
Acknowledgments This work was supported by grants and fellowships from CAPES, CNPq, and FAPEMIG.
Reference 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
GLOBOCAN 2012. http://globocan.iarc.fr/Default.aspx, accessed in May 2018. WHO. http://www.who.int/news-room/fact-sheets/detail/cancer, accessed in May 2018. INCA 2018. http://www.inca.gov.br/estimativa/2018/, accessed in May 2018. Sommer, S.; Fuqua, S. A. Semin. Cancer Biol. 2001, 11, 339. Jordan, V. C.; Brodie, A. M. Steroids. 2007, 72, 7. Smith, L.; Watson, M. B.; O'Kane, S. L.; Drew, P. J.; Lind, M. J.; Cawkwell, L. Mol. Cancer Ther. 2006, 5, 2115. Neve, R. M.; Chin, K..; Fridlyand, J.; Yeh, J.; Baehner, F. L.; Fevr, T.; Speed, T. Cancer cell 2006, 10, 515. Valença, W. O.; Baiju, T. V., Brito; F. G., Araujo, M. H.; Pessoa, C.; Cavalcanti, B. C.; Da Silva Júnior, E. N. ChemistrySelect 2017, 2, 4301. Singh, D. K.; Luqman, S. Biomed. Res. Ther. 2014, 1, 112. Block, J.B.; Serpick, A. A.; Miller. W.; Wiernik, P.H. Cancer Chemother. Rep. 1974, 4, 27. Hussain, H.; Krohn, K.; Ahmad, V. U.; Miana, G. A.; Green, I. R. Arkivoc 2007, 2, 145. Xiang, M.; Kim, H.; Ho, V. T.; Walker, S. R.; Bar-Natan, M.; Liu, S.; Heppler, L. N. Blood 2016, 128, 1845. Jordão, A. K.; Vargas, M. D.; Pinto, A. C.; Da Silva, F. D. C.; Ferreira, V. F. RSC Adv. 2015, 5, 67909. Pereyra, C. E., Dantas, R. F., Ferreira, S. B., Gomes, L. P., Silva-Jr, F. P. Cancer Cell Int. 2019 19, 1. Hung D. T., Jamison T. F., Schreiber S. L. Chem Biol. 1996, 3, 623–39. Veskoukis A. S., Tsatsakis A. M., Kouretas D. Cell Stress Chaperones. 2012, 17, 11. Lee H. Oh E-T.; Choi B-H.; Park M-T.; Lee J-K. Lee J-S. Park H. J. Sci Rep. 2015, 5, 7769. Cloherty, E. K., Sultzman, L. A., Zottola, R. J., Carruthers, A. Biochemistry 1995, 34, 15395. Li, H.; Liu, R. Adv. Mat. Res. 2012, 550, 85. Fedorov, S. N.; Shubina, L. K.; Kuzmich, A. S.; Polonik, S. G. Open Glycosci. 2011, 4, 1. Da Cruz, E. H.; Hussene, C. M.; Dias, G. G.; Diogo, E. B.; De Melo, I. M.; Rodrigues, B. L.; De Paiva, Y. G. Bioorg. Med. Chem. 2014, 22, 1608. Conchie, J.; Levvy, G. A.; Marsh, C. A. Adv. Carbohydr. Chem. 1957 12, 157. Horton, D. Org. Synth. Coll. 1973, 5, 1. Šardzík, R.; Noble, G. T.; Weissenborn, M. J.; Martin, A.; Webb, S. J.; Flitsch, S. L. Beilstein J. Org. 2010, 6, 699. Adiyala, P. R.; Tekumalla, V.; Sayeed, I. B.; Nayak, V. L.; Nagarajan, A., Shareef, M. A.; Kamal, A. Bioorg. Chem. 2017, 76, 288. Starks, C. M. J. Am. Chem. Soc. 1971, 93, 199. Ngameni, B.; Patnam, R.; Sonna, P.; Ngadjui, B. T.; Roy, R.; Abegazd, B. M. Arkivoc 2008, 6, 152. Karplus, M. J. Am. Chem. Soc. 1963, 85, 2870 Bubb, W. A. Concept. Magnetic Res. Part A 2003, 19, 1. Ibatullin, F. M.; Shabalin, K. A. Synth. Comm. 2000. 30, 2819. Tang, Y.; Zhang, S.; Chang, Y.; Fan, D.; Agostini, A. D.; Zhang, L.; Jiang, T. J. Med. Chem. 2018, 61, 2937.
32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.
Zemplén, G.; Pacsu, E. Ber. Dtsch. Chem. Ges. 1929, 62, 1613. Xu, W.; Yang, H.; Liu, Y.; Hua, Y.; He, B.; Ning, X.; Liu, F. W. Synthesis 2017, 49, 3686. Raja, R.; Kandhasamy, S.; Perumal, P. T.; SubbiahPandi, A. Acta Crystallogr. Sect. E: Crystallogr. Comm. 2015, 71, 231. Huisgen, R. Proc. Chem. Soc. 1961, 357. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. 2002, 114, 2708. Bodnár, B.; Mernyák, E.; Szabó, J.; Wölfling, J.; Schneider, G.; Zupkó, I.; Kovács, L. Bioorg. Med. Chem. Lett. 2017, 27, 1938. Lacroix, M.; Leclercq, G. Breast Cancer Res. Treat. 2004, 83, 249. Mitri, Z.; Constantine, T.; O'Regan, R. Chemother. Res. Pract. 2012, 2012, 1. Sweeney, E. E.; McDaniel, R. E.; Maximov, P. Y.; Fan, P.; Jordan, V. C. Horm. Mol. Biol. Clin. Investig. 2012, 9, 143. Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112. Linardi, M. C. F.; De Oliveira, M. M.; Sampaio, M. R. P. J. Med. Chem. 1975, 18, 1159. Hossain, F.; Andreana, P.R. Pharmaceuticals 2019, 12, 84. Tseng, P. H.; Wang, Y. C.; Weng, S. C.; Weng, J. R.; Chen, C. S.; Brueggemeier, R. W.; Chen, C. S. Mol. Pharmacol. 2006, 70, 1534.
Table 1. Effect of glycosidic derivatives from lawsone on breast cancer and non-cancer cell growth (µM).
Compound
SKBR-3 IC50a (CI 95%)
MCF-7 IC50a (CI 95%)
MDA-MB-231 IC50a (CI 95%)
HGF IC50a (CI 95%)
3
2.51 (2.17-2.91)
4.58 (3.29-6.37)
9.18 (8.35-10.10)
4
8.86 (7.17-10.95)
13.32 (11.87-14.94)
5
2.96 (2.63-3.32)
13.63 (11.4916.17) 4.76 (3.26-6.96)
6
2.11 (1.86-2.39)
3.52 (2.61-4.75)
8.29 (7.11-9.87)
7
3.41 (2.94-3.938)
6.59 (5.60-7.78)
19.78 (15.52-25.19)
8
8.30 (7.42-9.29)
27.64 (25.37-30.11)
9 10
9.52 (6.90-13.14) 2.41(2.18-2.67)
16.32 (11.5923.00) 11.01 (9.80-12.37) 5.02 (3.82-6.60)
35.68 (26.6947.71) 34.64 (22.8252.58) 19.14 (13.3827.37) 20.22 (12.3533.10) 40.19 (31.8350.67) >50
11
0.78 (0.67-0.90)
3.33 (2.61-4.24)
13.99 (9.95-19.65)
12
1.27 (1.05-1.53)
4.22 (3.77-4.72)
16.72 (11.73-23.83)
13
16.99 (14.17-20.38)
>50
14
3.32 (2.76-3.99)
28.89 (26.0232.09) 9.74 (8.73-10.88)
15
34.74 (29.38-41.08)
16 17 18 lawsone (1) Doxorubicinb
>50 >50 >50 >50 0.28 (0.25-0.31)
aIC
38.85 (34.3543.95) >50 >50 >50 >50 0.38 (0.32-0.46)
12.64 (11.61-13.76)
18.17 (14.51-22.76) 8.65 (7.63-9.81)
23.39 (17.24-31.73)
>50 19.77 (14.1927.53) 17.65 (12.9823.75) 27.61 (23.6032.29) >50
>50
38.15 (33.6743.21) >50
>50 >50 >50 >50 1.23 (0.99-1.52)
>50 >50 >50 >50 >50
50 (μM): inhibitory concentration of 50% cell growth was calculated through a nonlinear fit-curve (log of compound concentration versus normalized response—variable slope). bPositive control.
Glycosylation of lawsone enhances its antitumor activity Glucosyl triazole of lawsone displays submicromolar IC50 against SKBR-3 breast cancer cell line Glycosylation of lawsone under phase transfer catalysis yields separable mixture of alpha and beta glycosides
S0960-894X(19)30786-3 https://doi.org/10.1016/j.bmcl.2019.126817 BMCL 126817
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
9 August 2019 6 November 2019 8 November 2019
Please cite this article as: Ottoni, F.M., Gomes, E.R., Pádua, R.M., Oliveira, M.C., Silva, I.T., Alves, R.J., Synthesis and cytotoxicity evaluation of glycosidic derivatives of lawsone against breast cancer cell lines, Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.126817
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
© 2019 Published by Elsevier Ltd.
Synthesis and cytotoxicity evaluation of glycosidic derivatives of lawsone against breast cancer cell lines Flaviano M Ottoni a*, Eliza R Gomesa*, Rodrigo M Páduaa, Mônica C Oliveiraa, Izabella T Silvaa,b**, Ricardo J Alvesa** a
Department of Pharmaceutical Products, Faculty of Pharmacy, Federal University of Minas Gerais, 6627 Antônio Carlos,
Belo Horizonte, MG 31.270-901, Brazil. b
Department of Pharmaceutical Sciences, Federal University of Santa Catarina, Florianópolis, SC, 88.040-970, Brazil.
* These authors contributed equally to this manuscript. ** Corresponding authors. Tel.: +55 031 34096955; Fax: +55 031 3409 6934. E-mail addresses: [email protected]; [email protected]
Abstract Breast cancer is the most incident and mortal cancer type in women, with an estimated 2 million new cases expected by 2020 worldwide, with 600,000 deaths. As not all breast cancer types respond to the anti-hormonal therapy, the development of new antineoplastic drugs is necessary. Lawsone (2hydroxy-1,4-naphtoquinone) is a natural bioactive naphtoquinone displaying a range of activities, with dozens of derivatives described in the literature, including some glycosides possessing antitumor activity. Here, a series of glycosides of lawsone are reported for the first time and all compounds displayed good activity against the SKBR-3 cell line, with IC50 below 10 µM. The most promising derivative was the glycosyl triazole derived from peracetylated D-glucose (11), which showed better cytotoxicity against SKBR-3 (IC50=0.78 µM), being the most selective toward this tumoral cell (SI>20). All compounds described in this work were more active than lawsone, indicating the importance of the carbohydrate and glycosyl triazole moiety for activity.
Keywords Lawsone glycosides, breast cancer, cytotoxicity, copper catalyzed azido-alkyne cycloaddition Cancer is currently one of the major public health problems worldwide and the number of cases will increase in the next decades, mainly in developing countries. According to WHO, more than 17 million cases are predicted in 2020 with a mortality rate higher than 10 million, with 60% of cases occurring in developing countries.1 Cancer is the main cause of death in underdeveloped countries (>300,000 deaths) and the second in developed countries (almost 200,000 deaths).2 In Brazil 600,000 new cases of cancer were estimated for the years 2018-2019, the most prevalent being prostate cancer in men (31.7%) and breast cancer in women (29.5%).3 Breast cancer is the most incident and mortal cancer type in women, with an estimated 2 million new cases expected by 2020 worldwide. This represents almost 25% of all cancer types diagnosed in women with 600,000 deaths (15% of all cancer deaths).1,3 Breast cancer is one of the most usual cancer types in the female population. Breast cancer is considered a heterogeneous disease with regard to the morphology and clinic, and the WHO recognizes
more than 20 breast cancer subtypes. The majority of breast tumors are formed in the ductal epithelium and are known to be invasive ductal carcinoma. Besides, the number of cases is higher in women up to 50 years old, with a reduction after this age, so it is believed that there is participation of hormones in the etiology of the disease. The overexpression of hormone receptors has been identified in a high number of breast cancer patients.4 The activation of these receptors by female hormones induces the proliferation of tumor cells, so blocking it with receptor-specific therapy is an effective therapeutic strategy.5 However, not all breast cancer subtypes overexpress hormone receptors or become resistant, and thus do not respond to receptor-specific therapy6,7, necessitating the use of anticancer drugs. The high cancer mortality rate is due, mostly, to the inefficient current treatment available (radiotherapy, chemotherapy, and surgical procedures).8 Although preventive measures are taken and several drugs have been developed, it is still necessary to develop new antineoplastic drugs more effective and selective toward tumoral cells. Many modern chemotherapeutic agents currently used originate from plants and the examples include taxol from Taxus baccata (Yew), vincristine and vinblastine from Catharanthus roseus (Sadabahar), and podophyllotoxin from Podophyllum peltatum (Mayapple).9 The importance of the 1,4-naphtoquinonic scaffold in medicinal chemistry has been widely described in the last decades. Many of these compounds have been screened as anticancer agents and have shown cytotoxic activity against several tumoral cell lines. An example is lapachol, which has already been extensively studied as an antitumor agent and submitted to Phase I clinical trials, but due to several adverse effects, the studies were discontinued.10,11 Another interesting naphthoquinonic derivative is atovaquone, an approved antimicrobial drug, whose potent antitumor activity has been indicated in current studies.12 A commercially available naphtoquinone is lawsone 1 (2-hydroxy-1,4naphtoquinone), a natural bioactive compound isolated from plants of the genus Lawsonia displaying a range of activities, e.g., antimicrobial and antitumor13, with lawsone 1 being active against HCT-15 (human colon cancer cells).9 Naphthoquinones cytotoxicity has been mainly associated to the inhibition of the human DNA topoisomerase II and production of reactive oxygen species (ROS). It is likely that the naphthoquinones kill or induce cell death by more than one mechanism.14 The enzyme topoisomerases II is mostly expressed during the G2 and M phases from mythosis.15 Naphthoquinones can be reduced to form semiquinones and hydroxyquinones, which can be reoxidized leading to ROS formation and causing damage to macromolecules.16 The NQO1 (NADPH: quinone oxidoreductase 1) is a enzyme responsible for reduction of naphthoquinones in anaerobic conditions. The overexpression of this enzyme in cancer cells besides be crucial for naphthoquinones cytotoxicity can explain their selectivity.17 Herein, we hypothesize that carbohydrate moiety could facilitate the transport of the compounds to the target cell via hexose transportes.18 Lawsone 1 can be easily obtained by synthesis (60% of global yield) and thus is an interesting starting material towards the preparation of bioactive derivatives.19 Dozens of derivatives of 1 are described in the literature, including some glycosides possessing antitumor activity (Figure 1).20,21
O
OAc
HO
N N
O
AcO AcO
O
H N
N OAc
O
Lawsone (1)
OAc
2
OAc
OAc O
AcO AcO
OAc O
O
AcO
O
O
O
OAc
O
3
4
O
O OAc O
AcO AcO
O
OAc
O
5
O
Figure 1. Lawsone (1) and some glycosidic derivatives that show antitumoral activity. The synthesis of glycosidic derivatives of lawsone 1 can furnish bioactive compounds and the variation of carbohydrate moiety allows a structure-activity relationship (SAR) study. Therefore, this work aimed to synthesize peracetylated and deacetylated glycosides from lawsone 1. The designed compounds were classical glycosides (carbohydrates linked directly to lawsone) and glycosyl triazoles (a triazolic ring connecting the saccharidic moiety to lawsone), investigated for their activity against three breast cancer cell lines (SKBR-3, MDA-MB-231, and MCF-7). The chemical structures of the classical glycosides and glycosyl triazoles of lawsone designed in the present work are shown in Figure 2. AcO AcO
OAc O
AcO AcO
O
OAc
O
OAc O
AcO AcO
O
OAc
O
OAc O AcHN
O O O O
3 () 4 ()
OR RO RO
5 () 6 ()
O
O
OR N
O N
OR R= Ac; 11 R= H; 15
7 () 8 ()
O O
N N
RO OR
R= Ac; 12 R= H; 16 O
O
N
O O
RO RO
N NHAc
OAc
N
O
R= Ac; 13 R= H; 17 O
O
AcO
OR
OR O
N
H3C
OAc
O 9 () 10 ()
N N
O
O
H3C
O RO O
N
N OR
O O
OR R= Ac;14 R= H; 18
Figure 2. Designed classical glycosides and glycosyl triazoles of lawsone. The synthetic route to peracetylated glycosides 5-10 and glycosyl triazoles 11-18 of lawsone is shown in Scheme 1. The key intermediates, peracetylated glycosyl halides a-d, were prepared from the respective carbohydrate (D-glucose, D-galactose, D-N-acetylglucosamine, and L-fucose) according to literature procedures.22-25 Then, glycosyl halides a-d were used as glycosyl donors in the glycosylation step using phase transfer catalysis in alkaline medium to prepare peracetylated glycosides of lawsone as mixtures of α and β anomers, which were separated by column chromatography.26,27 The pure anomers 5-10 were isolated in 21-50% yield. Stereochemistry at the anomeric carbon was unequivocally established using NMR spectroscopy. In the 1H NMR spectra, the H-1/H-2 coupling constant (3J) for α anomers were 2-4 Hz and for β anomers 8-10 Hz.28 For all β glycosides, HSQC experiments were used to assign H-1 in the 1H NMR spectra from the corresponding C-1 signal in the 13C NMR spectra, which
O
is observed at circa 98 ppm for β anomers and 95 ppm for α anomers.29 The peracetylated glycosyl azides e-h were also obtained from glycosyl halides a-d by reaction with NaN3 in acetone/H2O at room temperature.30,31 The deacetylated glycosyl azides i-l were obtained from deacetylation of corresponding peracetylated glycosyl azides, using a transesterification reaction with KOH/MeOH.32,33 The 2-O-propargyllawsone (11) was obtained in 70% yield from reaction of lawsone 1 with propargyl bromide in alkaline medium, using potassium carbonate as base and N,N-dimethylformamide (DMF) as solvent under reflux.8,34 Finally, the propargylic derivative 11 reacted with the glycosyl azides e-l under [2+3] alkyne-azide cycloaddition reaction conditions (CuSO4.5H2O, sodium ascorbate) in THF/water as solvent at room temperature, affording the glycosyl triazole derivatives of lawsone 11-18 in 60-70% yield.35-37 O
O
HO
AcO
i
O
O AcO
N3
HO
iii
N3
i-l
e-h
a-d
OH
D-glicose D-galactose
ii
X X= Br ou Cl
80 - 90 %
50 - 80 %
D-N-acetilglicosamina L-fucose O
AcO
O
O O
iv
OAc OAc O
3 (35%) OAc O OAc
4 (40%)
AcO
5 (34%) OAc O
6 (40%)
AcNH
AcO
O
O
11-18
11 (72%)
R= AcO
OAc
12 (72%) HO
OH HO R= HO
OAc OAc O
OH
OAc O
R=
NHAc
H3C
R=
O
AcO
13(64%)
14 (51%)
O OH
16 (44%)
HO R= HO
O NHAc
17 (67%)
H3C
R=
OH
O OH
18(65%)
NHAc
H3C OAc
OAc
9 (31%)
AcO
O
OAc
OAc
OH
OH
O
R= HO
AcO AcO
8 (50%)
7 (21%) H3C
OAc
15 (49%)
OAc O
AcO AcO
OAc O
R=
OAc O
AcO AcO
R
O
19 (70%)
AcO AcO
OAc
vi
O
N
N
O
1
AcO
AcO
AcO AcO
v
OAc OAc O
AcO
AcO AcO
O
O
O
3-10
N
O
HO
OAc
OAc
10 (40%)
Scheme 1. Reagents and conditions: i) a) Ac2O, AcONa, 100°C; b) HBr/AcOH, CH2Cl2, r.t; or c) CH3COCl, r.t; ii) NaN3, H2O/acetone, r.t, 3 h; iii) KOH, MeOH, 0oC, 1 h; iv) Glycosyl halide (a-d), CH2Cl2, Na2CO3 10% p/v (1:1), n-Bu4NBr, r.t., 24h; v) Propargyl bromide, K2CO3, DMF, 80-100o C, 20 h; vi) Glycosyl azide e-l, CuSO4.5H2O 50% mol, sodium ascorbate 60% mol, THF:H2O, r.t, 2-5h.
To evaluate the cytotoxic activity, lawsone 1 and all synthesized glycosides were screened against three human breast adenocarcinoma cell lines (SKBR-3, MDA-MB-231, and MCF-7) and one nontumor human fibroblast from primary gingival tissue culture (HGF) kindly supplied by Dra. Cláudia Maria Oliveira Simões (Laboratory of Virology, UFSC, Florianópolis, Brazil) according to the Research Ethics Committee of the UFSC, 021/2009. The difference among the tumoral cell lines are related to the presence or absence of estrogen (ER), progesterone (PR), and HER2 receptors.38-40 The breast cancer cell lines SKBR-3 (HER2+), MCF-7 (ER+), and MDA-MB-231 (triple-negative) were
OH
purchased from American Type Culture Collection (ATCC) and grown in McCoy’s 5A Modified Medium (McCoy), Eagle’s Minimum Essential Medium (MEM) supplemented with 0.01 mg/mL human recombinant insulin and Dulbecco’s Modified Eagle’s Medium (DMEM), respectively. All cell lines were supplemented with 10% fetal bovine serum (FBS, GibcoTM) and maintained at 37°C and 5% CO2 in a humidified atmosphere. The compounds were evaluated against 1x104 cells in 96-well plates by Sulforhodamine B assay.41 The concentrations that cause 50% inhibitory concentration (IC50) were determined using GraphPad Prism software (version 6.0, San Diego, USA) and are shown in Table 1.
Table 1. Effect of glycosidic derivatives from lawsone on breast cancer and non-cancer cell growth (µM).
Compound
SKBR-3 IC50a (CI 95%)
MCF-7 IC50a (CI 95%)
MDA-MB-231 IC50a (CI 95%)
HGF IC50a (CI 95%)
3
2.51 (2.17-2.91)
4.58 (3.29-6.37)
9.18 (8.35-10.10)
4
8.86 (7.17-10.95)
13.32 (11.87-14.94)
5
2.96 (2.63-3.32)
13.63 (11.4916.17) 4.76 (3.26-6.96)
6
2.11 (1.86-2.39)
3.52 (2.61-4.75)
8.29 (7.11-9.87)
7
3.41 (2.94-3.938)
6.59 (5.60-7.78)
19.78 (15.52-25.19)
8
8.30 (7.42-9.29)
27.64 (25.37-30.11)
9 10
9.52 (6.90-13.14) 2.41(2.18-2.67)
16.32 (11.5923.00) 11.01 (9.80-12.37) 5.02 (3.82-6.60)
35.68 (26.6947.71) 34.64 (22.8252.58) 19.14 (13.3827.37) 20.22 (12.3533.10) 40.19 (31.8350.67) >50
11
0.78 (0.67-0.90)
3.33 (2.61-4.24)
13.99 (9.95-19.65)
12
1.27 (1.05-1.53)
4.22 (3.77-4.72)
16.72 (11.73-23.83)
13
16.99 (14.17-20.38)
>50
14
3.32 (2.76-3.99)
28.89 (26.0232.09) 9.74 (8.73-10.88)
15
34.74 (29.38-41.08)
16 17 18 lawsone (1) Doxorubicinb
>50 >50 >50 >50 0.28 (0.25-0.31)
38.85 (34.3543.95) >50 >50 >50 >50 0.38 (0.32-0.46)
12.64 (11.61-13.76)
18.17 (14.51-22.76) 8.65 (7.63-9.81)
23.39 (17.24-31.73)
>50 19.77 (14.1927.53) 17.65 (12.9823.75) 27.61 (23.6032.29) >50
>50
38.15 (33.6743.21) >50
>50 >50 >50 >50 1.23 (0.99-1.52)
>50 >50 >50 >50 >50
aIC
50 (μM): inhibitory concentration of 50% cell growth was calculated through a nonlinear fit-curve (log of compound concentration versus normalized response—variable slope). bPositive control.
Among the classical glycosides of lawsone 5-10, the α-glycosides of
D
series were more cytotoxic
than the corresponding β-glycosides for the three cancer cell lines, with the exception of D-glucose
derivatives 5 and 6 (anomers showing comparable cytotoxicity). Fedorov and coworkers (2011) reported that α anomer 3 was more cytotoxic than anomer 4 against the human leukemic HL-60 cell line.16 On the other hand, L-fucoside 10 (β anomer) was more cytotoxic than L-fucoside 9 (α anomer). Regarding the toxicity against non-tumor cell line HGF, anomers of the same compound of D series showed comparable toxicity while fucoside 10 (β anomer, IC50>50 µM) was less cytotoxic than fucoside 9 (α anomer, IC50=19.77 µM), indicating that the D or L configuration influenced cytotoxicity against this cell line. The saccharidic moiety also seems to have influenced cytotoxicity since compound 5 (β-glycoside) was more active than the corresponding anomer from the other monosaccharides. Glycosides 7 and 8, derived from N-acetylglucosamine, were considerably less active than the same anomer of the glycoside derived from
D-glucose
(compounds 6 and 5,
respectively), which belongs to the same stereochemical series (D-gluco). These results indicate that substitution of acetoxy (OAc) by acetamido (NHAc) at C-2 decreases activity. Compound 3 showed the highest selectivity index (SI) for MCF-7 (SI~8) and MDA-MB-231 (SI~4) cell lines, the latter being more resistant because it is triple negative to hormone receptors (ER, PR, and HER2) and does not respond to blocking by anti-hormones.38,39 Glycosyl triazoles 11 and 12 showed greater cytotoxicity among all tested compounds against SKBR-3 (CI50=0.78 µM and 1.27 µM, respectively), showing a comparable selectivity index (SI~22). Finally, the only deacetylated glycosyl triazole that showed cytotoxicity against tumor cells was compound 15 (derived from D-glucose), being cytotoxic against SKBR-3 (CI50=34.74 µM) and MCF-7 (CI50=38.85 µM), and it was non-cytotoxic to non-tumor cells HGF (CI50>50 µM). The greater activity of peracetylated glycosides and glycosyl triazoles as compared to lawsone is probably due to the more favorable lipophilic-hydrophilic balance that has been achieved with the peracetylated glycosyl derivatives which could be absorbed by tumor cells more easily.42
D-glucose-based
6 and glycosyl triazole 11 were the most active compounds suggesting that
classical glycoside
D-glucose
transporters
(GLUTs) may be target by these compounds.43 In conclusion, we have synthesized two series of glycosidic derivatives of lawsone, corresponding to classical glycosides and glycosyl triazoles. All compounds were more active than lawsone, which did not show cytotoxicity against the tumor nor the non-tumor cell lines, although there are reports on the cytotoxicity of lawsone against other cell lines.9 Thus, the modification of lawsone by direct glycosylation or glycosyl triazole formation improved the cytotoxic activity. The anomer mixture obtained in the synthesis of classical glycosides 5-10 provided the compounds for evaluation of the influence of C-1 configuration, allowing for a structure-activity relationship (SAR) study. For glycosyl triazoles 11-18, the presence of the triazole ring permitted the evaluation of its influence on activity. Glycosyl triazoles 11 and 12 were more active against SKBR-3 than the corresponding classical glycosides 3-6. The deacetylated glycosyl triazoles were not active against the three tumor cell
lines and none was cytotoxic against the non-tumor cells (HGF), except for compound 15, which was cytotoxic against SKBR-3 and MCF-7. It was also observed that the stereochemical series of the carbohydrate moiety influenced both activity and the selectivity index. The most promising derivatives were the classical glycosides 3 and 6, which showed low IC50 values against SKBR-3 (<5 µM), MCF-7 (<5 µM), and MDA-MB-231 (<10 µM). Compound 6 was the most cytotoxic of all against MDA-MB-231, a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer (TNBC). Compound 3 was the most selective towards MCF-7 (SI~8) and MDA-MB-231 (SI~4). The glycosyl triazoles derived from peracetylated D-glucose (11) and D-galactose (12) were the most selective toward SKBR-3 (SI>20), with compound 11 being the most potent against this cell line (IC50=0.78 µM), which is considered a poor prognostic tumor.44 Furthermore, its deacetylated derivative (compound 15) was the only one among the deacetylated glycosyl triazoles that did show any cytotoxic activity for tumor cells. Altogether, the present work allowed for the obtention of two series of glycosidic derivatives of lawsone with relevant activity against three breast cancer cell lines with good selectivity in some cases, indicating that this approach deserves further attention. Work in this direction is underway and results will be reported in due time.
Acknowledgments This work was supported by grants and fellowships from CAPES, CNPq, and FAPEMIG.
Reference 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31.
GLOBOCAN 2012. http://globocan.iarc.fr/Default.aspx, accessed in May 2018. WHO. http://www.who.int/news-room/fact-sheets/detail/cancer, accessed in May 2018. INCA 2018. http://www.inca.gov.br/estimativa/2018/, accessed in May 2018. Sommer, S.; Fuqua, S. A. Semin. Cancer Biol. 2001, 11, 339. Jordan, V. C.; Brodie, A. M. Steroids. 2007, 72, 7. Smith, L.; Watson, M. B.; O'Kane, S. L.; Drew, P. J.; Lind, M. J.; Cawkwell, L. Mol. Cancer Ther. 2006, 5, 2115. Neve, R. M.; Chin, K..; Fridlyand, J.; Yeh, J.; Baehner, F. L.; Fevr, T.; Speed, T. Cancer cell 2006, 10, 515. Valença, W. O.; Baiju, T. V., Brito; F. G., Araujo, M. H.; Pessoa, C.; Cavalcanti, B. C.; Da Silva Júnior, E. N. ChemistrySelect 2017, 2, 4301. Singh, D. K.; Luqman, S. Biomed. Res. Ther. 2014, 1, 112. Block, J.B.; Serpick, A. A.; Miller. W.; Wiernik, P.H. Cancer Chemother. Rep. 1974, 4, 27. Hussain, H.; Krohn, K.; Ahmad, V. U.; Miana, G. A.; Green, I. R. Arkivoc 2007, 2, 145. Xiang, M.; Kim, H.; Ho, V. T.; Walker, S. R.; Bar-Natan, M.; Liu, S.; Heppler, L. N. Blood 2016, 128, 1845. Jordão, A. K.; Vargas, M. D.; Pinto, A. C.; Da Silva, F. D. C.; Ferreira, V. F. RSC Adv. 2015, 5, 67909. Pereyra, C. E., Dantas, R. F., Ferreira, S. B., Gomes, L. P., Silva-Jr, F. P. Cancer Cell Int. 2019 19, 1. Hung D. T., Jamison T. F., Schreiber S. L. Chem Biol. 1996, 3, 623–39. Veskoukis A. S., Tsatsakis A. M., Kouretas D. Cell Stress Chaperones. 2012, 17, 11. Lee H. Oh E-T.; Choi B-H.; Park M-T.; Lee J-K. Lee J-S. Park H. J. Sci Rep. 2015, 5, 7769. Cloherty, E. K., Sultzman, L. A., Zottola, R. J., Carruthers, A. Biochemistry 1995, 34, 15395. Li, H.; Liu, R. Adv. Mat. Res. 2012, 550, 85. Fedorov, S. N.; Shubina, L. K.; Kuzmich, A. S.; Polonik, S. G. Open Glycosci. 2011, 4, 1. Da Cruz, E. H.; Hussene, C. M.; Dias, G. G.; Diogo, E. B.; De Melo, I. M.; Rodrigues, B. L.; De Paiva, Y. G. Bioorg. Med. Chem. 2014, 22, 1608. Conchie, J.; Levvy, G. A.; Marsh, C. A. Adv. Carbohydr. Chem. 1957 12, 157. Horton, D. Org. Synth. Coll. 1973, 5, 1. Šardzík, R.; Noble, G. T.; Weissenborn, M. J.; Martin, A.; Webb, S. J.; Flitsch, S. L. Beilstein J. Org. 2010, 6, 699. Adiyala, P. R.; Tekumalla, V.; Sayeed, I. B.; Nayak, V. L.; Nagarajan, A., Shareef, M. A.; Kamal, A. Bioorg. Chem. 2017, 76, 288. Starks, C. M. J. Am. Chem. Soc. 1971, 93, 199. Ngameni, B.; Patnam, R.; Sonna, P.; Ngadjui, B. T.; Roy, R.; Abegazd, B. M. Arkivoc 2008, 6, 152. Karplus, M. J. Am. Chem. Soc. 1963, 85, 2870 Bubb, W. A. Concept. Magnetic Res. Part A 2003, 19, 1. Ibatullin, F. M.; Shabalin, K. A. Synth. Comm. 2000. 30, 2819. Tang, Y.; Zhang, S.; Chang, Y.; Fan, D.; Agostini, A. D.; Zhang, L.; Jiang, T. J. Med. Chem. 2018, 61, 2937.
32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44.
Zemplén, G.; Pacsu, E. Ber. Dtsch. Chem. Ges. 1929, 62, 1613. Xu, W.; Yang, H.; Liu, Y.; Hua, Y.; He, B.; Ning, X.; Liu, F. W. Synthesis 2017, 49, 3686. Raja, R.; Kandhasamy, S.; Perumal, P. T.; SubbiahPandi, A. Acta Crystallogr. Sect. E: Crystallogr. Comm. 2015, 71, 231. Huisgen, R. Proc. Chem. Soc. 1961, 357. Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. 2002, 114, 2708. Bodnár, B.; Mernyák, E.; Szabó, J.; Wölfling, J.; Schneider, G.; Zupkó, I.; Kovács, L. Bioorg. Med. Chem. Lett. 2017, 27, 1938. Lacroix, M.; Leclercq, G. Breast Cancer Res. Treat. 2004, 83, 249. Mitri, Z.; Constantine, T.; O'Regan, R. Chemother. Res. Pract. 2012, 2012, 1. Sweeney, E. E.; McDaniel, R. E.; Maximov, P. Y.; Fan, P.; Jordan, V. C. Horm. Mol. Biol. Clin. Investig. 2012, 9, 143. Vichai, V.; Kirtikara, K. Nat. Protoc. 2006, 1, 1112. Linardi, M. C. F.; De Oliveira, M. M.; Sampaio, M. R. P. J. Med. Chem. 1975, 18, 1159. Hossain, F.; Andreana, P.R. Pharmaceuticals 2019, 12, 84. Tseng, P. H.; Wang, Y. C.; Weng, S. C.; Weng, J. R.; Chen, C. S.; Brueggemeier, R. W.; Chen, C. S. Mol. Pharmacol. 2006, 70, 1534.
Table 1. Effect of glycosidic derivatives from lawsone on breast cancer and non-cancer cell growth (µM).
Compound
SKBR-3 IC50a (CI 95%)
MCF-7 IC50a (CI 95%)
MDA-MB-231 IC50a (CI 95%)
HGF IC50a (CI 95%)
3
2.51 (2.17-2.91)
4.58 (3.29-6.37)
9.18 (8.35-10.10)
4
8.86 (7.17-10.95)
13.32 (11.87-14.94)
5
2.96 (2.63-3.32)
13.63 (11.4916.17) 4.76 (3.26-6.96)
6
2.11 (1.86-2.39)
3.52 (2.61-4.75)
8.29 (7.11-9.87)
7
3.41 (2.94-3.938)
6.59 (5.60-7.78)
19.78 (15.52-25.19)
8
8.30 (7.42-9.29)
27.64 (25.37-30.11)
9 10
9.52 (6.90-13.14) 2.41(2.18-2.67)
16.32 (11.5923.00) 11.01 (9.80-12.37) 5.02 (3.82-6.60)
35.68 (26.6947.71) 34.64 (22.8252.58) 19.14 (13.3827.37) 20.22 (12.3533.10) 40.19 (31.8350.67) >50
11
0.78 (0.67-0.90)
3.33 (2.61-4.24)
13.99 (9.95-19.65)
12
1.27 (1.05-1.53)
4.22 (3.77-4.72)
16.72 (11.73-23.83)
13
16.99 (14.17-20.38)
>50
14
3.32 (2.76-3.99)
28.89 (26.0232.09) 9.74 (8.73-10.88)
15
34.74 (29.38-41.08)
16 17 18 lawsone (1) Doxorubicinb
>50 >50 >50 >50 0.28 (0.25-0.31)
aIC
38.85 (34.3543.95) >50 >50 >50 >50 0.38 (0.32-0.46)
12.64 (11.61-13.76)
18.17 (14.51-22.76) 8.65 (7.63-9.81)
23.39 (17.24-31.73)
>50 19.77 (14.1927.53) 17.65 (12.9823.75) 27.61 (23.6032.29) >50
>50
38.15 (33.6743.21) >50
>50 >50 >50 >50 1.23 (0.99-1.52)
>50 >50 >50 >50 >50
50 (μM): inhibitory concentration of 50% cell growth was calculated through a nonlinear fit-curve (log of compound concentration versus normalized response—variable slope). bPositive control.
Glycosylation of lawsone enhances its antitumor activity Glucosyl triazole of lawsone displays submicromolar IC50 against SKBR-3 breast cancer cell line Glycosylation of lawsone under phase transfer catalysis yields separable mixture of alpha and beta glycosides