Cytotoxic effect of levoglucosenone and related derivatives against human hepatocarcinoma cell lines

Article abstract of DOI:10.1016/j.bmcl.2016.07.007

Levoglucosenone has been used as template for the synthesis of a wide variety of compounds with an impressive structural variability. However, scarce work has been done regarding the generation of new bioactive entities. Here we report the cytotoxic effec

Full text of DOI:10.1016/j.bmcl.2016.07.007

Accepted Manuscript
Cytotoxic effect of levoglucosenone and related derivatives against human hep-
atocarcinoma cell lines
Germán F. Giri, Mauro Danielli, Raúl A. Marinelli, Rolando A. Spanevello
PII:
S0960-894X(16)30709-0
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.07.007
BMCL 24051
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
3 May 2016
30 June 2016
2 July 2016
Please cite this article as: Giri, G.F., Danielli, M., Marinelli, R.A., Spanevello, R.A., Cytotoxic effect of
levoglucosenone and related derivatives against human hepatocarcinoma cell lines, Bioorganic & Medicinal
Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.07.007
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Cytotoxic effect of levoglucosenone and
related derivatives against human
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Germán F. Giri, Mauro Danielli, Raúl A. Marinelli, Rolando A. Spanevello

Bioorganic & Medicinal Chemistry Letters
Cytotoxic effect of levoglucosenone and related derivatives against human
hepatocarcinoma cell lines
Germán F. Giri,a Mauro Danielli,b Raúl A. Marinelli,b Rolando A. Spanevelloa*
a Instituto de Quimica de Rosario, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario-CONICET, Suipacha 531, S2002LRK
Rosario, Argentina
b Instituto de Fisiología Experimental, Facultad de Ciencias Bioquimicas y Farmaceuticas, Universidad Nacional de Rosario-CONICET, Suipacha 531,
S2002LRK Rosario, Argentina
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Levoglucosenone has been used as template for the synthesis of a wide variety of compounds
with an impressive structural variability. However, scarce work has been done regarding the
generation of new bioactive entities. Here we report the cytotoxic effect of levoglucosenone and
some related derivatives against hepatocarcinoma cell lines. Compounds were obtained in only
one synthetic step and one of them showed an activity within the range of IC50 values of
cisplatin, a frequently administered chemotherapy drug.
Keywords:
2009 Elsevier Ltd. All rights reserved.
Levoglucosenone
Cytotoxic activity
Michael addition
Hepatocarcinoma
Cancer cell lines
in order to develop a wide variety of applications of this
It is generally recognized that the resources of the world are
limited and sustainability has become a crucial point for the
development of chemical products. These circumstances
impose a great urgency to find renewable sources of raw
material for their transformation into useful products
including chemicals, fuels, and materials for replacing the
enormous demand for fossil resources.
chiral template for the synthesis of enantiomerically pure
compounds. The plethora of derivatives synthesized from
levoglucosenone includes chiral catalysts and auxiliaries and
natural products, many of them with known biological
activities such as tetrodotoxin1. However, there was little
investigation reported regarding the use of levoglucosenone
as scaffold for the generation of novel bioactive molecular
structures. Peri´s group reported the synthesis of Ras protein
inhibitors as potential antitumoral drugs3 and employed a 1,3
dipolar addition to levoglucosenone`s double bond as
synthetic strategy. In this work, the authors showed that the
aromatic moieties were highly relevant for the observed
activity.
Biomass has received particular attention because it
represents the only abundant source of renewable organic
carbon. More importantly, its oxygenated nature, chemical
diversity, and chirality render biomass a highly suitable raw
material to manufacture a wide array of high-added-value
compounds1. By far, carbohydrates are the leading annually
renewable feedstocks from which to develop viable organic
chemicals that can compete or eventually replace those
derived from petrochemical industries.
Although a result obtained more than 25 years ago showed
that
1
could arrest cell cycle of hamsters’ cells4, surprisingly
there has been no additional reports about their activity until
now. Working with bio-oils from soybean hulls we recently
observed that
1 was active against the pathogen Salmonella
The pyrolytic treatment of microcrystalline cellulose or
cellulose-containing materials, such as waste paper,
degrades the cellulose polymeric chain into a useful building
block named levoglucosenone (1,6-anhydro-3,4-dideoxy-β-
enterica but this compound resulted to be cytotoxic against
CHO cells. In this context, we realized that the carbonyl
group was important for the observed cytotoxic effect and
the substitution of
1 with a bromine atom at C-3 highly
improved the activity5.
D-glycero-hex-3-enopyranos-2-ulose)
(1). The highly
functionalized structure of makes it an attractive chiral
1
synthon for the synthesis of a broad diversity of natural and
unnatural compounds1.
Based on these data, we decided to reinvestigate the
bioactivity of levoglucosenone and its brominated
derivative against neoplastic cells. We also synthesized a
set of thio-derivatives through Michael type additions to the
-unsaturated carbonyl system present in and tested
1
2
Since the first report on the preparation of levoglucosenone
about 40 years ago2, different studies have been conducted
1

their activities. Levoglucosenone was obtained by pyrolysis
of microcrystalline cellulose according with previous studies
with an overall yield between 7-10%.6 The reaction of
1
with bromine in CH2Cl2 at 0 °C followed by the addition of
Et3N afforded 3-bromolevoglucosenone (2)7 in 92% yield
(Scheme 1).
Scheme 1 Synthesis 3-bromolevoglucosenone 2
Scheme 3 Ketone reduction
With respect to the thioconjugates derivatives, we carried
out reactions between
1 with some representative thiols in
the presence of a base (Scheme 2)8. Table 1 showed the
thiols and reaction conditions used to obtain the Michael
adducts9 which were then employed to assess the influence
of the different types of substituents on the biological
activity (Figure 1, compounds 3-7).
All derivatives were synthesized in very good to excellent
yields with the exception of compound 5N which was
obtained in 57% in a serendipitous manner. The conjugate
addition of 2-mercaptobenzoxazole was expected to afford
adduct 5S with an S-C linkage; however, its tautomer was
the reacting species instead (Scheme 4). A careful analysis
of the 1H and 13C NMR spectra revealed that the
nucleophilic addition proceeded through
a
different
pathway. The first spectral evidence for this outcome was
the H-4 signal, which appeared at lower fields than the
expected one. Also, its 13C NMR spectrum showed a signal
at 180 ppm attributed to C-2´, although a comparison with
similar compounds indicated that the signal for this type of
carbon usually appeared at 170 ppm.11 These mismatching
elements allowed us to suspect the possibility that the
substitution had occurred through the N atom attack to yield
compound 5N. The HMBC correlation experiment showed a
crosspeak a proton signal at 5.58 ppm with an aromatic C at
129.7 ppm which were assigned to H-4 and C-3´a. This
correlation was only possible for the derivative with a C-N
bond and not for that with a C-S bond (Scheme 4). These
spectroscopic evidences allowed us to establish that the
structure obtained was the N-substituted compound 5N and
not the expected adduct 5S. At this point, we considered
Scheme 2 Michael type additions
Table 1
Reagents and reactions conditions
Yield
(%)
78
Entry
Reagent
Base/Eq.
Et3N/0.035
Et3N/0.035
Prod.
1
2
thiophenol
p-methyl
thiophenol
2-mercapto-
benzoxazole
1-butanethiol
1-hexanethiol
3
4
75
57
3
DIPEA/0.05
5N
4
5
DIPEA/0.05
DIPEA/0.04
85
83
6
7
reasonable to test the biological behavior of compound 5N
.
Scheme 4 Addition of 2-mercaptobenzoxazole tautomers
The hepatocarcinoma or hepatocellular carcinoma (HCC) is
one of the most common causes of cancer deaths worldwide.
The HCC is highly resistant to systemic chemotherapy.
Thus, the available and most commonly used
chemotherapeutic agents, i.e., doxorubicin, cisplatin, and
sorafenib show low effectiveness in clinical settings12. This
situation highlights the need for searching new cytotoxic
drugs against HCC.
Figure 1
Additionally, we reduced compounds
evaluate the role of the carbonyl group in the cytotoxic
4
and
6
in order to
effect (Scheme 3)10.

Therefore, we decided to test the above mentioned
compounds against two commonly used human HCC cell
lines: Huh-7 and HepG2.13 Cytotoxicity was assessed by
methyl-thiazolyl tetrazolium bromide (MTT) quantitative
derivative firstly obtained nearly 4 decades ago. With this
starting material, we synthesized a set of structurally related
compounds in only one simple step with very good yields
and some of them showed a very interesting activity against
two HCC cell lines. We also noted the key role exerted by
the carbonyl group in the biological activity. Finally, this
report shows a new horizon in the field of levoglucosenone´s
chemistry, allowing the synthesis of a novel class of
bioactive compounds with original structural features.
colorimetric assay, as described.14 In
a
preliminary
were active but
bearing an aromatic ring at C-4 showed
cytotoxicity. In contrast, compounds showed no
screening, we found that compounds
1 and 2
also derivatives
3-5
6-9
apparent cytotoxicity. Based on this outcome, the IC50
values for compounds 1-5 were determined after 48 h of
incubation with cells. The results of these assays are
summarized in Table 2.
Acknowledgments
Table 2
This research was supported by Agencia Nacional de Promoción
Científica y Tecnológica, CONICET and UNR from Argentina.
G. F. G. thanks ANPCyT and CONICET for the award of the
fellowships.
Cytotoxic assays against hepatocarcinoma cell lines
*
IC ( M)

50
Entry
Compound
Huh-7
12.94
4.68
11.02
12.79
8.79
HepG2
39.39
13.96
17.00
27.53
22.20
12.0
1
2
3
4
5
6
7
1
2
3
Supplementary Data
4
5N
Supplementary data (cytotoxic activity graphics, experimental
procedures and spectroscopic characterizations data of the
compounds) associated with this article can be found, in the
online version.
Sorafenib15a
11.3
Cisplatin15b
Doxorubicin15b
5-FU15b
3.09
7.28
0.68
8
9
0.24
390.00
205.19
References and notes
*See graphics in supplementary data
1. a) Corne, V.; Botta, M. C.; Giordano, E. D. V.; Giri, G. F.; Llompart,
D. F.; Biava, H. D.; Sarotti, A. M.; Mangione, M. I.; Mata, E. G.;
Suárez, A. G.; Spanevello, R. A. Pure Appl. Chem. 2013, 85, 1683; b)
Sarotti, A. M.; Zanardi, M. M.; Spanevello, R. A.; Suárez, A. G. Curr.
Org. Synth. 2012, 9, 439 and references therein cited.
2. Halpern, J.; Riffer, R.; Broido, A. J. Org. Chem. 1973, 38, 204.
3. Müller, C.; Gómez-Zurita Frau, M. A.; Ballinari, D.; Colombo, S.;
Bitto, A.; Martegani, E.; Airoldi, C.; van Neuren, A. S.; Stein, M.;
Weiser, J.; Battistini, C.; Peri, F. Chem. Med. Chem. 2009, 4, 524.
4. Nishi, Y.; Miyakawa, Y.; Kato, K.; Mutat. Res. 1989, 227, 117.
5. Giri G. F. Ph.D. Thesis, Universidad Nacional de Rosario, August
2015.
6. a) Sarotti, A. M.; Spanevello, R. A.; Suárez, A. G. Green Chem. 2007,
9, 1137. b) Morin, C. In Levoglucosenone and Levoglucosans:
Chemistry and Applications; Witczak, Z. J., Ed.; ATL Press, Mount
Prospect, 1994. Chapter 2.
7. Ward, D.; Shafizadeh, F. Carbohydr. Res. 1981, 93, 284.
8. Becker, B.; Thimm, J.; Thiem, J. J. Carbohydr. Chem. 1996, 15, 1179.
9. General Procedure for the conjugated addition: Levoglucosenone was
dissolved in anhydrous chloroform (5.5 mL) under Ar atmosphere,
followed by the sequential addition of the corresponding nucleophile
and base. The reaction was stirred at room temperature until the TLC
showed complete consumption of the Michael acceptor. Solvent was
evaporated under reduced pressure. The crude product obtained was
purified by column chromatography.
10. General Procedure for reduction: Ketones 4 and 6 were dissolved in
MeOH and NaBH4 98% was then added. The reaction was stirred at
room temperature until the TLC showed complete consumption of the
substrate. Acetone (0.5 mL) was then added and the mixture was
filtered through a celite pad. Solvent was evaporated under reduced
pressure. The crude product was purified by column chromatography
to separate the epimeric alcohols.
11. a) Xie, J-G.; Quan, J.; Li, S-B.; Zheng, Y.; Zhu, L-M. Synth. Commun.,
2011, 41, 871. b) Melzig, L.; Metzger, A.; Knochel, P. Chem. Eur. J.,
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Table 2 showed that the activity of the new synthetic
derivatives was better than the one observed with
Compounds and 5N were even more active than Sorafenib
1.
2
and the first one was also comparable in activity with
Cisplatin (entry 2 with Huh-7 cell line),15 two of the drugs
clinically used nowadays to treat HCC. Another observation
derived from these experiments was that the Huh-7 cell line
resulted to be more susceptible to the in vitro therapy than
HepG2 cell line. Although the reason for this effect is still
unknown, it might be related to the fact that Huh-7 cells
possess a mutation in the p53 tumor suppressor gene while
HepG2 cells, conversely, has a normal p53. Interestingly,
mutations of the p53 tumor suppressor are present in the
majority of tumors, including HCC.16
Meanwhile, during the development of this research, a new
report in literature showed that some thio-sugars derivates
obtained from Michael type additions to
1 were active
against some tumoral cell lines, although the activities were
relatively low.17 The authors mentioned the relevance of C-
S-C bond angle in activity determination. In our examples
this assumption could be true for compounds
3 and 4 but in
the case of compound 5-N which has a C-N bond it could
not be applied. On the other hand, aliphatic analogs
which possess a C-S-C bond similar to and
6
4
and
were
7
3
inactive. A similar lost of activity was observed when the
carbonyl group was reduced to the corresponding alcohols
and . Based on the data collected with this small library of
8
9
12. Stotz, M.; Gerger, A.; Haybaeck, J.; Kiesslich, T.; Bullock, M. D.;
Pichler, M.; Anticancer Res. 2015, 35, 5737.
13. Krelle, A. C., Okoli, A. S. Mendz, G. L., J. Cancer Therapy, 2013, 4,
606.
compounds we could infer that in our case, the two main
features that were essential for the observed cytotoxic effect
were the aromatic moiety and the carbonyl group.
14. Marchissio, M. J.; Francés, D. E. A.; Carnovale, C. E.; Marinelli, R. A.
Toxicol. Appl. Pharmacol. 2012, 264, 246.
15. a) Cervello, M.; Bachvarov, D.; Lampiasi, N.; Cusimano, A.; Azzolina,
A.; McCubrey, J. A.; Montalto, G. Cell Cycle, 2012, 11, 2843. b)
Brito, A. F.; Abrantes, A. M.; Pinto-Costa, C.; Gomes, A. R.; Mamede,
In summary, in this work we demonstrated the potential
antitumor activity for levoglucosenone, a glucose-related

A. C.; Casalta-Lopes, J.; Gonçalves, A. C.; Sarmento-Ribeiro, A. B.;
Tralhão, J. G.; Botelho, M. F. Chemotherapy 2012, 58, 381.
16. Chen, G. G.; Merchant, J. L.; Lai, P. B.; Ho, R. L.; Hu, X.; Okada, M.;
Huang, S. F.; Chui, A. K.; Law, D. J.; Li, Y. G.; Lau, W. Y.; Li, A. K.
Am. J. Pathol., 2003, 162, 1823.
17. Witczak, Z. J.; Sarnik, J.; Czubatka, A.; Forma, E.; Poplawski, T.
Bioorg. Med. Chem. Lett. 2014, 24, 5606.

Highlights
Sulfur containing carbohydrates possess distinctive
biological and physicochemical properties.
Thiosugars are useful tools to probe carbohydrate-
protein interactions within enzymes systems and as
prospective therapeutic agents
Levoglucosenone is a high added- value chiral
building-block in organic synthesis.