Synthesis and in vitro investigation of potential antiproliferative monosaccharide–D-secoestrone bioconjugates

Article abstract of DOI:10.1016/j.bmcl.2017.03.029

The syntheses of monosaccharide–D-secoestrone conjugates are reported. They were prepared from 3-(prop-2-inyloxy)-D-secoestrone alcohol or oxime and monosaccharide azides via Cu(I)-catalyzed azide–alkyne cycloaddition reactions (CuAAC). The antiproliferat

Full text of DOI:10.1016/j.bmcl.2017.03.029

Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and in vitro investigation of potential antiproliferative
monosaccharide–D-secoestrone bioconjugates
Brigitta Bodnár a, Erzsébet Mernyák b, Johanna Szabó b, János Wölfling b, Gyula Schneider b, István Zupkó c,
Zoltán Kupihár a, , Lajos Kovács a,
⇑
⇑
a Department of Medicinal Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary
b Department of Organic Chemistry, University of Szeged, Dóm tér 8, H-6720 Szeged, Hungary
c Department of Pharmacodynamics and Biopharmacy, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
The syntheses of monosaccharide–D-secoestrone conjugates are reported. They were prepared from
Received 17 January 2017
Revised 11 March 2017
Accepted 14 March 2017
Available online xxxx
3-(prop-2-inyloxy)-D-secoestrone alcohol or oxime and monosaccharide azides via Cu(I)-catalyzed
azide–alkyne cycloaddition reactions (CuAAC). The antiproliferative activities of the conjugates were
investigated in vitro against a panel of human adherent cancer cell lines (HeLa, A2780 and MCF-7) by
means of MTT assays. The protected
D-glucose-containing D-secoestrone oxime bioconjugate (24b)
proved to be the most effective with an IC50 value in the low micromolar range against A2780 cell line.
Keywords:
Monosaccharide
Ó 2017 Elsevier Ltd. All rights reserved.
D-Secoestrone
Oxime
Bioconjugate
CuAAC
Literature precedents reveal that different synthetic modifica-
metabolic stability. On the other hand, making bioconjugates is a
potential strategy to enhance the antiproliferative effect or to
increase the selectivity of a compound. Preparation of natural pro-
duct conjugates is a very promising approach, since the biological
potency of the new hybrids may exceed that of the parent com-
pounds.14 In general, in the compounds constructed from diverse
molecular entities, the components may result in synergic action,
with better tolerability or the conjugate may possess more selec-
tive cellular uptake or may influence the pharmacokinetic proper-
ties.14 Based on our previous observations and the potential
tions of estrone (1, Scheme 1) lead to anticancer compounds.1 In
order to suppress their hormonal action, substitutions at C-2,
opening of ring D or inversion of configuration at C-13 are usually
carried out.2–7 We recently reported that 3-O-benzyl ethers of
D-
secoestrone alcohol or oxime (3a and 3b, Scheme 1) display sub-
stantial in vitro antiproliferative action against certain cancer cell
lines in the low micromolar range.8,9
The starting compounds bearing phenolic hydroxy groups (2a
and 2b) did not influence the proliferation of the investigated cell
lines. The cytostatic potential of benzyl ethers (3a and 3b) was suc-
cessfully improved by the introduction of a 1,2,3-triazole moiety
between the benzyl and the hydroxy groups. The heterocyclic ring
was introduced to C-3 via a short oxymethylene group applying
copper(I)-catalyzed alkyne–azide click reaction (CuAAC). The
resulting 3-O-[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl] derivatives
(5a and 5b) displayed submicromolar IC50 values against certain
human reproductive adherent cancer cell lines.10 There are already
a number of literature examples of the synthesis of antiprolifera-
tive steroidal triazoles9,11–13 and the triazole moiety is also used
as a linker arm in bioconjugates owing to its high proteolytic and
advantages of modifying our most active
bioconjugates, here we aimed at synthesizing novel
bioconjugates, stemming from -secoestrones, retaining the 3-O-
D
-secoestrones by making
D
-secoestrone
D
[(1,2,3-triazol-4-yl)methyl] moiety, and introducing a monosac-
charide unit instead of the previously used benzyl group on the
triazole ring. The rationale behind this aim was the fact that
although sugars per se have no therapeutic action in glycosylated
steroid conjugates, they have a dramatic effect on the physical,
chemical and biological properties of bioconjugates and the sugar
moieties act as molecular elements that control the pharmacoki-
netics of a drug, such as absorption, distribution, metabolism and
excretion.15 As it was reported, the number, location and type of
sugars in steroidal glycoalkaloids, even with identical aglycon, play
an important role in the antiproliferative activity.16 Similarly,
subtle sugar modifications can dramatically, and independently,
⇑
Corresponding authors.
E-mail addresses: kupihar.zoltan@med.u-szeged.hu (Z. Kupihár), kovacs.lajos@
med.u-szeged.hu (L. Kovács).
http://dx.doi.org/10.1016/j.bmcl.2017.03.029
0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Bodnár B., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.03.029

2
B. Bodnár et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Some of the most abundant monosaccharides in the nature,
D-
glucose, -mannose, -galactose and -ribose were chosen to pre-
D
D
D
pare these building blocks. We aimed at synthesizing their azide
derivatives in which the azide group is built into their glycosidic
position or in place of their primary hydroxy groups (position 6
in hexopyranoses and position 5 in ribofuranose). The most com-
mon ways for the preparation of glycosyl azides are either an SN2
substitution of a protected glycosyl halide by sodium azide at high
temperature in DMF18 or an SN1 substitution of a peracetylated
carbohydrate under mild conditions using a Lewis acid catalyst
and trimethylsilyl azide.19–21 We have chosen the latter method
as all of our sugars contained a neighbouring participation group
at position 2 (O-acetyl or O-benzoyl) that can ensure the desired
stereoselectivity. The primary hydroxy groups of the monosaccha-
rides can be replaced to azides in a two-step procedure22 involving
the introduction of a good leaving group (e.g. a tosyl) to the pri-
mary hydroxy and a subsequent azide substitution of the tosylate
by sodium azide in DMF.
First, the hexoses studied were peracetylated according to a lit-
erature method23 to yield compounds 6–8 (Scheme 2). Next, the
glycosidic O-acetyl groups were replaced with the azide group
using tin tetrachloride as a Lewis acid catalyst and trimethylsilyl
azide as the source of the nucleophilic azide ion to afford com-
pounds 9–11.
The SN1 type substitution resulted in only 1,2-trans products
due to the neighbouring group participation of the O-acyl group
at position 2. For
D-glucose and D-galactose the b-anomer, for D-
mannose the -anomer formed in this way in 72–79% yield. The
a
purity of the azide products and their quantities were sufficient
for the subsequent conjugation reactions.
In order to introduce the azide group to positions 6 (hexopyra-
noses) and 5 (pentofuranose), first the methyl glycosides of
D-glu-
cose, -mannose and -ribose (12–14, Scheme 3) were selectively
D
D
tosylated in pyridine on their primary hydroxy groups without
the protection of the secondary hydroxy groups. This distinction
was allowed by the higher reactivity of the primary hydroxy
groups over the secondary ones.
Unfortunately, the direct replacement of the tosyl group with
azide in compounds 15–17 was not successful probably due to sol-
ubility reasons, therefore the secondary hydroxy groups were ben-
zoylated first, then the tosyl–azide exchange has successfully
occurred in all the fully protected monosaccharides 18–20 and
resulted in the fully protected 6-azido-6-deoxy- and 5-azido-5-
deoxymonosaccharides (21–23).
The azide group-containing monosaccharides (9–11 and 21–23)
were coupled to the alkyne-containing D-secoalcohol (4a) and D-
secooxime (4b) using similar CuAAC conditions that we used pre-
viously (Scheme 4) applying copper(I) iodide, triphenylphosphine
and DIPEA as a base with a slight access of propargyl-D-secosteroid
in toluene at boiling temperature until TLC showed quantitative
conversions.24
Scheme 1. The synthesis of estrone derivatives 2–5 obtained earlier.
In case of two glucose-containing bioconjugates (24a and 24b),
which showed the best biological activities, the acetyl protecting
groups were removed by the Zemplén’s method25 using sodium
methylate in methanol to obtain their unprotected derivatives
30a and 30b.
modulate both the cytotoxic properties and the Na+/K+-ATPase
inhibitory properties of cardiac glycosides.17 In this vein, we
planned to perform CuAAC reactions of steroidal alkynes (4a and
4b) with protected monosaccharide azides and to investigate the
in vitro antiproliferative activities of these bioconjugates by means
of MTT assays against a panel of human adherent cancer cell lines
(HeLa, MCF-7 and A2780).
The antiproliferative properties of the D-secoestrone–carbohy-
drate conjugates (24–30) were characterized in vitro on a panel
of human adherent cancer cell lines (HeLa, A2780 and MCF-7) by
means of MTT assays (Table 1).
The antiproliferative properties of some of the presented com-
pounds proved to comparable to that of reference agent cisplatin
that is utilized clinically in the treatment of certain gynaecological
malignancies.26,27 The most potent compounds (24b, 25b and 26b)
exhibited remarkable activities with IC50 values in the range 5.3–
As our aim was to synthesize carbohydrate–
conjugates from our previously reported 3-O-propargyl
D
-secoestrone bio-
-secoe-
D
strones using CuAAC, this conjugation reaction required the
synthesis of azide-containing carbohydrate building blocks and
their CuAAC reaction with propargylated D-secoestrones (4a and
4b, Scheme 1).
20.5 lM, exerting their best effects against A2780 cells. Among
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B. Bodnár et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
3
Scheme 2. Synthesis of protected glycosyl azides 9–11. Reagents: (i) Ac2O, NaOAc; (ii) SnCl4, Me3SiN3, CH2Cl2.
Scheme 3. Synthesis of 6-azido-6-deoxy-D-hexopyranose (21, 22) and 5-azido-5-deoxy-D-ribofuranose (23) derivatives. Reagents: (i) TsCl, pyridine; (ii) BzCl, pyridine; (iii)
NaN3, LiBr, DMF.
the potent compounds the glucoside conjugate (24b) displayed the
highest, the mannoside derivative (25b) the lowest cell-line selec-
tivity. Considering the results of Table 1, important structure–
activity relationships appear. The antiproliferative activities of
the compounds greatly depend on the attachment site of the
monosaccharide unit, and on the nature of the functional group
at C-13, but do not really depend on the type of monosaccharide
attached. It can be stated that the glycoside derivatives (24–26)
are more potent than their methyl glycoside analogues (27–29)
which were attached to the triazole ring at the 60- or 50-positions
of the carbohydrate units. The removal of the protecting groups
from the most potent 24b compound results in a compound
(30b) with decreased antiproliferative properties showing the
importance of the nonpolar property of the moiety attached to
the triazole ring for the bioactivity. Comparison of the results for
achieved by incorporation of a triazole ring between the 3-hydroxy
and the benzyl group. An additional determining factor is the nat-
ure (type, attachment position and polarity) of the substituent on
the triazole ring. The benzyl to monosaccharide exchange
decreases the cell growth-inhibition. Even the most potent hexose
conjugates (24b, 25b and 26b) displayed one order of magnitude
higher IC50 values than certain recently described 3-O-[(1-ben-
zyl-1H-1,2,3-triazol-4-yl)methyl] derivatives (5a and 5b). Cancer
selectivity is a critical parameter determining the fate of a poten-
tial drug candidate. A viability assay on mouse fibroblasts cannot
substitute the toxicological evaluation. However, it seems advanta-
geous that our most potent compounds (24b, 25b and 26b) exert
substantially less growth inhibiting action on fibroblasts than on
cancerous cell lines.9
acetylated conjugates formed from the
D-secooxime and the D-
secoalcohol reveals that the presence of the oxime function gener-
ally improves the growth-inhibitory properties of the conjugates.
Extending the discussion on our recent results,9 it seems that both
the polarity and the size of the fragment at C-3 position greatly
influence the antiproliferative properties. The presence of the less
polar, but bulky benzyl function is advantageous over the phenolic
hydroxy group, and further enhancement in the activity is
Conclusions
Fourteen monosaccharide–D-secoestrone bioconjugates were
prepared by CuAAC reaction and their antiproliferative properties
were investigated. Although the antiproliferative activities of the
bioconjugates were not as high as the activities of the parent com-
pounds, the structure–activity relationships provided by the
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4
B. Bodnár et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
Scheme 4. The synthesis of monosaccharide–D-secoestrone bioconjugates 24–30. Reagents: (i) Cu(I), (Ph)3P, DIPEA, toluene, reflux; (ii) NaOMe, MeOH.
Table 1
Anticancer activity of monosaccharide–D-secoestrone conjugates 24–30 against different cell lines.
Compd. No. or
namea
Monosaccharide
configuration
Attachment site in
monosaccharide
Concn. (mM)
Inhibition (%) SEM
[calculated IC50 (mM)]b
HeLa
A2780
MCF-7
NIH/3T3 (mouse fibroblast)
24a
24b
b-
b-
D
-Glcp
-Glcp
10
10
10
30
10
30
[IC50
10
30
10
30
[IC50
10
30
10
30
[IC50
10
30
10
30
10
34.5 0.7
23.9 0.9
20.4 1.8
40.0 2.2
[ > 30]
51.9 1.0
41.5 1.7
52.4 1.7
89.6 0.5
[8.9]
23.9 1.1
31.8 1.2
31.5 2.0
61.9 2.0
[20.5]
<20
<20
<20
<20
<20
<20
<20
<20
21.0 1.6
19.5 1.4
69.5 0.9
76.7 0.8
[5.3]
34.9 2.3
36.5 1.4
69.5 0.6
86.4 0.8
[6.6]
27.4 2.8
45.5 1.5
46.7 1.3
67.7 1.6
[10.7]
26.3 2.2
54.3 0.6
53.9 0.9
65.9 1.2
[9.4]
D
<20
28.4 0.9
]
]
]
25a
25b
a-
D
-Manp
-Manp
10
10
a-
D
27.3 0.5
34.8 0.3
26a
26b
b-
b-
D
-Galp
-Galp
10
10
<20
<20
32.7 1.0
59.3 0.9
85.2 0.4
[8.8]
<20
<20
28.4 0.8
45.7 2.1
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
61.9 0.9
59.1 2.2
71.5 1.5
[8.0]
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
<20
D
<20
25.5. 1.4
27a
27b
28a
28b
29a
29b
30a
30b
a
a
a
a
-
D
D
D
D
-Glcp
60
60
60
60
50
50
10
10
-
-
-
-Glcp
-Manp
-Manp
30
10
30
10
30
10
30
10
b-
b-
b-
b-
D-Ribf
D-Ribf
D-Glcp
D-Glcp
<20
<20
24.3 2.6
<20
<20
30
10
<20
<20
<20
30
[IC50
10
30
[IC50
31.5 0.4
[>30]
42.6 2.3
99.9 0.3
[12.4]
57.9 0.6
[27.4]
83.6 1.2
95.0 0.3
[1.3]
<20
[>30]
66.9 1.8
96.8 0.4
[5.8]
]
]
Cisplatin
–
–
94.2 0.4
96.4 0.2
[3.2]
a
For structures see Scheme 4. The compound series a contain hydroxymethyl, series b oxime moieties at C-13 position of the steroid skeleton.
Mean value from two independent determinations with five parallel wells; standard deviation <15%.
b
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B. Bodnár et al. / Bioorganic & Medicinal Chemistry Letters xxx (2017) xxx–xxx
5
9. Mernyák E, Fiser G, Szabó J, et al. Synthesis and in vitro antiproliferative
evaluation of D-secooxime derivatives of 13b- and 13a-estrone. Steroids.
2014;89:47.
results deliver very important information for the design of
antiproliferative estrone derivatives in the future.
10. Szabó J, Jerkovics N, Schneider G, et al. Synthesis and in vitro antiproliferative
evaluation of C-13 epimers of triazolyl-D-secoestrone alcohols: the first potent
Acknowledgments
13a-D-secoestrone derivative. Molecules. 2016;21:611.
11. Mernyák E, Kovács I, Minorics R, et al. Synthesis of trans-16-triazolyl-13alpha-
methyl-17-estradiol diastereomers and the effects of structural modifications
The authors thank the Hungarian Scientific Research Fund –
Hungary (OTKA K113150 and K109293), the New Hungary Devel-
opment Plan (TÁMOP-4.2.2.A-11/1/KONV-2012-0047) for financial
support. This research was also supported by the European Union
and the State of Hungary, co-financed by the European Social Fund
on their in vitro antiproliferative activities.
2015;150:123.
J Steroid Biochem Mol Biol.
12. Szabó J, Pataki Z, Wölfling J, et al. Synthesis and biological evaluation of
13alpha-estrone derivatives as potential antiproliferative agents. Steroids.
2016;113:14.
13. Szabó J, Bacsa I, Wölfling J, et al. Synthesis and in vitro pharmacological
evaluation of N-[(1-benzyl-1,2,3-triazol-4-yl)methyl]-carboxamides on D-
secoestrone scaffolds. J Enzyme Inhib Med Chem. 2016;31:574.
14. Tietze LF, Bell HP, Chandrasekhar S. Natural product hybrids as new leads for
drug discovery. Angew Chem Int Ed. 2003;42:3996.
–
Belgium and Hungary (TÁMOP-4.2.4.A/2-11/1-2012-0001
‘National Excellence Program’).
15. Pellissier H. The glycosylation of steroids. Tetrahedron. 2004;60:5123.
16. Ding X, Zhu F, Yang Y, Li M. Purification, antitumor activity in vitro of steroidal
glycoalkaloids from black nightshade (Solanum nigrum L.). Food Chem.
2013;141:1181.
17. Langenhan JM, Peters NR, Guzei IA, Hoffmann FM, Thorson JS. Enhancing the
anticancer properties of cardiac glycosides by neoglycorandomization. Proc
Natl Acad Sci USA. 2005;102:12305.
A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.
03.029.
18. Györgydeák Z, Szilágyi L, Paulsen H. Synthesis, structure and reactions of
glycosyl azides. J Carbohydr Chem. 1993;12:139.
19. Hayashi M, Kawabata H. Preparation of 1-azide sugars. In: Anonymous, ed.
Recent Developments in Carbohydrate Research, Vol. 1. Research Signpost;
2003:195–208.
20. Györgydeák Z, Thiem J. Synthesis and transformation of glycosyl azides. In:
Anonymous, ed. Advances in Carbohydrate Chemistry and Biochemistry, Vol.
60. Elsevier; 2006:103–182.
21. Beckmann HSG, Wittmann V. Azides in carbohydrate chemistry. In: Brase S,
Banert K, eds. Organic Azides: Syntheses and Applications. Chichester: John Wiley
and Sons; 2010:469–490.
22. Sicherl F, Wittmann V. Orthogonally protected sugar diamino acids as building
blocks for linear and branched oligosaccharide mimetics. Angew Chem Int Ed.
2005;44:2096.
23. Dutta D, Pulsipher A, Yousaf MN. Selective tethering of ligands and proteins to a
microfluidically patterned electroactive fluid lipid bilayer array. Langmuir.
2010;26:9835.
References
1. Gupta A, Kumar BS, Negi AS. Current status on development of steroids as
anticancer agents. J Steroid Biochem Mol Biol. 2013;137:242.
2. Cushman M, He H-M, Katzenellenbogen JA, et al. Synthesis of analogs of 2-
methoxyestradiol with enhanced inhibitory effects on tubulin polymerization
and cancer cell growth. J Med Chem. 1997;40:2323.
3. Möller G, Deluca D, Gege C, et al. Structure-based design, synthesis and in vitro
characterization of potent 17beta-hydroxysteroid dehydrogenase type
1
inhibitors based on 2-substitutions of estrone and D-homo-estrone. Bioorg
Med Chem Lett. 2009;19:6740.
4. Hillisch A, Peters O, Gege C, Siemeister G, Unger E, Menzenbach B. Antitumoral
D-homoestra-1,3,5(10)-trien-3-yl 2-substituted sulfamates; 2007. US Patent
7,244,762.
5. Ayan D, Roy J, Maltais R, Poirier D. Impact of estradiol structural modifications
(18-methyl and/or 17-hydroxy inversion of configuration) on the in vitro and
in vivo estrogenic activity. J Steroid Biochem Mol Biol. 2011;127:324.
6. Schönecker B, Lange C, Kötteritzsch M, et al. Conformational design for
13alpha-steroids. J Org Chem. 2000;65:5487.
7. Jovanovic´-Santa S, Petrovic´ J, Andric´ S, et al. Synthesis, structure, and screening
of estrogenic and antiestrogenic activity of new 3, 17-substituted-16, 17-seco-
estratriene derivatives. Bioorg Chem. 2003;31:475.
24. Bodnár B, Mernyák E, Wölfling J, et al. Synthesis and biological evaluation of
triazolyl 13a-estrone–nucleoside bioconjugates. Molecules. 2016;21:1212.
25. Anon. Zemplén Deacetylation. In: Wang Z, ed. Comprehensive Organic Name
Reactions and Reagents. Wiley Online Library; 2010:3123–3128.
26. Lippert B, ed. Cisplatin. Chemistry and biochemistry of
a leading anticancer
drug. Zürich, Weinheim: Verlag Helvetica Chimica Acta, Wiley-VCH; 1999.
27. Ferenc G, Pádár P, Szolomájer J, Howarth NM, Kovács L. Transition metal ion
complexes of N-alkylguanines. Curr Org Chem. 2011;15:2871.
8. Mernyák E, Szabó J, Bacsa I, et al. Syntheses and antiproliferative effects of D-
homo- and D-secoestrones. Steroids. 2014;87:128.
Please cite this article in press as: Bodnár B., et al. Bioorg. Med. Chem. Lett. (2017), http://dx.doi.org/10.1016/j.bmcl.2017.03.029