Potential anti-herpes and cytotoxic action of novel semisynthetic digitoxigenin-derivatives

Article abstract of DOI:10.1016/j.ejmech.2019.01.076

In recent years, new therapeutic possibilities were proposed for cardiac glycosides traditionally used to treat heart diseases, such as anticancer and antiviral activities. In this sense, this work aimed to synthesize the readily accessible 3β-azido-3-deoxydigitoxigenin (5) from digitoxigenin (1). Two new series of compounds were obtained from derivative (5): (i) O-glycosyl trizols through click chemistry with propargyl glycosides; and (ii) compounds substituted in the alpha carbonyl position with different residues linked via an amino-group. All obtained derivatives have their chemical structures confirmed, and their anti-herpes (against HSV-types 1 and 2 replication) and cytotoxic (against PC3, A549, HCT-8 and LNCaP cell lines) activities evaluated. Compounds 10 and 11 exhibited the most promising results against HSV-1 (KOS and 29-R strains) and HSV-2 (333 strain) replication with SI values > 1000. Both compounds were also the most cytotoxic for the human cancer cell lines tested with IC₅₀ values similar to those of paclitaxel. They also presented reduced toxicity toward non-cancerous cell lines (MRC-5 and HGF cells). Promising compounds were tested in regard to their ability to inhibit Na⁺/K⁺-ATPase. The inhibition rate correlates suitably with the bioactivity demonstrated by those both compounds against the different human cancer cells tested as well as against HSV replication. Moreover, the results showed that specific chemical features of compound 10 and 11 influenced the bioactivities tested. In summary, it was possible to obtain novel digitoxigenin-derivatives with remarkable cytotoxic and anti-herpes activities as well as low toxicity and high selectivity. In this way, they could be considered potential molecules for the development of new drugs.

Full text of DOI:10.1016/j.ejmech.2019.01.076

Accepted Manuscript
Potential anti-herpes and cytotoxic action of novel semisynthetic digitoxigenin-
derivatives
Laurita Boff, Jennifer Munkert, Flaviano Melo Ottoni, Naira Fernanda Zanchett
Schneider, Gabriela Silva Ramos, Wolfgang Kreis, Saulo Fernandes de Andrade,
José Dias de Souza Filho, Fernão Castro Braga, Ricardo José Alves, Rodrigo Maia
de Pádua, Cláudia Maria Oliveira Simões
PII:
S0223-5234(19)30096-0
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.076
EJMECH 11083
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 26 April 2018
Revised Date: 5 September 2018
Accepted Date: 29 January 2019
Please cite this article as: L. Boff, J. Munkert, F.M. Ottoni, N.F. Zanchett Schneider, G.S. Ramos, W.
Kreis, S. Fernandes de Andrade, José. Dias de Souza Filho, Fernã.Castro. Braga, Ricardo.José. Alves,
R. Maia de Pádua, Clá.Maria. Oliveira Simões, Potential anti-herpes and cytotoxic action of novel
semisynthetic digitoxigenin-derivatives, European Journal of Medicinal Chemistry (2019), doi: https://
doi.org/10.1016/j.ejmech.2019.01.076.
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ACCEPTED MANUSCRIPT
Graphical abstract
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Anti-herpes
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Digitoxigenin
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Digitalis lanata
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Potential anti-herpes and cytotoxic action of novel semisynthetic digitoxigenin-derivatives
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c*
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Laurita Boff , Jennifer Munkert , Flaviano Melo Ottoni , Naira Fernanda Zanchett Schneider ,
c
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Gabriela Silva Ramos , Wolfgang Kreis , Saulo Fernandes de Andrade , José Dias de Souza Filho ,
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Fernão Castro Braga , Ricardo José Alves , Rodrigo Maia de Pádua , Cláudia Maria Oliveira Simões
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a
Laboratório de Virologia Aplicada, Programa de Pós-Graduação em Farmácia, Universidade Federal de
Santa Catarina (UFSC), Florianópolis, SC, 88040-970, Brazil.
b
Friedrich-Alexander-Universität, Lehrstuhl für Pharmazeutische Biologie, Staudtstr. 5, D-91058
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Erlangen, Germany.
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Departamento de Produtos Farmacêuticos, Faculdade de Farmácia, Universidade Federal de Minas
Gerais (UFMG), Belo Horizonte, MG, 31270-901, Brazil.
d
Departmento de Produção de Matéria-Prima, Faculdade de Farmácia, Universidade Federal de Rio
Grande do Sul (UFRGS), Porto Alegre, RS, 90610-000, Brazil.
e
Departmento de Química, Instituto de Ciências Exatas, Universidade Federal de Minas Gerais (UFMG),
Belo Horizonte, MG, 31270-901, Brazil.
*
These authors contributed equally to this work.
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*
Corresponding author:
E-mail address: claudia.simoes@ufsc.br (C. M. O. Simões)
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Abstract
In recent years, new therapeutic possibilities were proposed for cardiac glycosides traditionally used to
treat heart diseases, such as anticancer and antiviral activities. In this sense, this work aimed to synthesize
the readily accessible 3β-azido-3-deoxydigitoxigenin (5) from digitoxigenin (1). Two new series of
compounds were obtained from derivative (5): (i) glycosylated triazoles through click chemistry with
propargyl glycosides; and (ii) compounds substituted in the alpha carbonyl position with different amines.
All obtained derivatives have their chemical structures confirmed, and their anti-herpes (against HSV-
types 1 and 2 replication) and cytotoxic (against PC3, A549, HCT-8 and LNCaP cell lines) activities
evaluated. Compounds 10 and 11 exhibited the most promising results against HSV-1 (KOS and 29-R
strains) and HSV-2 (333 strain) replication with SI values >1,000. Both compounds were also the most
cytotoxic for the human cancer cell lines tested with IC50 values similar to those of paclitaxel. They also
presented reduced toxicity toward non-cancerous cell lines (MRC-5 and HGF cells). All compounds were
+
+
tested in regard to their ability to inhibit Na /K ATPase. The inhibition rate correlates suitably with the
bioactivity demonstrated by those both compounds against the different human cancer cells tested as well
as against HSV replication. Moreover, the results showed that specific chemical features influenced the

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bioactivities tested. In summary, it was possible to obtain novel digitoxigenin-derivatives with remarkable
cytotoxic and anti-herpes activities as well as low toxicity and selectivity. In this way, they could be
considered potential molecules for the development of new drugs.
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Keywords
Cardenolides, digitoxigenin-derivatives, anti-herpes, cytotoxic.
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List of Abbreviations
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A549: non-small cell lung cancer
ACV: acyclovir
Ac O: acetic anhydride
2
AcOH: acetic acid
ACN: acetonitrile
AgCO : silver carbonate
3
AgOTf: silver triflate
BF .Et O: boron trifluoride diethyl etherate
3
2
CC : 50% cytotoxic concentration
50
CHCl : chloroform
3
CH Cl : dichloromethane
2
2
CMC: carboxymethylcellulose
CrO : chromium trioxide
3
CuSO .5H O: cupric sulfate
4
2
DIPEA: N,N-diisopropylethylamine
DMEM: Dulbecco's modified eagle's medium
DMF: N,N-dimethylformamide
DMSO: Dimethyl sulfoxide
EDTA: Ethylenediamine tetraacetic acid
ETA: ethanolamine
EtOAc: Ethyl acetate
H460: non-small cell lung cancer
H O: distilled water
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H SO : sulfuric acid
2 4
HBr: hydrobromic acid
HCl: hydrochloric acid
HCT-8: human ileocecal adenocarcinoma
HGF: human gingival fibroblasts
HSV-1: Herpes Simplex Virus type 1
HSV-2: Herpes Simplex Virus type 2
IC : concentration that inhibited 50% of viral replication or cell viability
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IR: infrared spectroscopy
K CO : potassium carbonate
2
3
KI: potassium iodide
LiOH.H O: lithium hydroxide monohydrate
2
LNCaP: androgen-sensitive human prostate adenocarcinoma cells
MEM: Eagle’s minimum essential medium
MeOH: methanol
MRC-5: human fetal lung fibroblast cell line
Na CO : sodium carbonate
2
3
Na SO : sodium sulfate anhydrous
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4
NaBH : sodium borohydride
4
NaHCO : sodium bicarbonate
3
NaN : sodium azide
3
NI: no inhibitory activity
NSCLC: non-small lung cancer cells
NT: not tested
NMR: nuclear magnetic resonance
PBS: phosphate-buffered saline
PC3: no hormone-sensitive human prostate adenocarcinoma cells
PFU: plaque-forming units
PI: propidium iodide
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Ph P: triphenylphosphine
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RPMI: Roswell Park Memorial Institute
THF: Tetrahydrofuran
TsCl: p-toluenesulfonyl chloride
TPP: triphenylphosphine
SD: Standard Deviation
SI: Selectivity Index
SRB: sulforhodamine B
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1. Introduction
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Bioactive compounds from natural sources have great relevance in the development of new drugs used to
treat different diseases, including those from microbial and parasitic origins, different types of cancers,
and for the control of blood lipid levels [1]. In addition, natural compounds are frequently used as
templates for the total synthesis or semisynthesis of derivatives, an useful tool widely explored for drug
development [2].
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Cardiac glycosides (CGs), classified as cardenolides and bufadienolides, are natural compounds found in
species of Apocynaceae (e.g. Nerium oleander L.) and Plantaginaceae (e.g. Digitalis lanata Ehrh. and D.
purpurea L.) families [3] among others. They have been used to treat heart diseases for more than 200
years [4] and are characterized by their high specific and potent cardiotonic action. The mechanism of the
+
+
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cardiotonic effects occurs through the inhibition of Na /K ATPase responsible for regulate the ions Na
+
and K that promote cardiac muscle contraction [5].
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Despite the widespread use of cardenolides as positive inotropic agents, the investigation of their effects
on other pathological conditions has been intensified in recent years offering new therapeutic possibilities
[6]. One of them is their anticancer action since several authors have already reported cytotoxic and
antitumor effects [7-16] (recently reviewed by De et al. [17] and Schneider et al. [18], as well as their
potential antiviral activity [19-28].
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Cancer is a global disease that accounts for almost 13% of deaths worldwide. It is estimated that by 2020
there will be 15 to 17 million new cases every year [29]. The treatment of cancer depends on several
factors, which is generally adapted to the stage of the disease and the characteristics of the tumor. Several
chemotherapy drugs are currently available and, as previously mentioned, many are derived from natural
products (e.g. paclitaxel from Taxus brevifolia; vinblastine and vincristine from Catharanthus roseus;

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and camptothecin from Camptotheca acuminata) [2]. In this sense, several CGs showed potent effects in
vitro in non-adherent and adherent cancer cell lines e.g. AMANTADIG [9, 10, 13], convallatoxin [12],
digitoxigenin [30], ouabain [31], glucoevatromonoside [32, 33] and digoxin [34-36]. Also, some of them
have been investigated for cancer treatment in phases I and II clinical trials (eg. extracts rich in different
TM
CGs: Anvirzel , PBI-05204, HuaChanSu; and the cardenolides UNBS1450 and digoxin) [17, 18, 37].
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Besides cancer, some important human diseases are from viral origin, such as those caused by Herpes
Simplex Virus (HSV-1 and HSV-2), which cause oral, esophageal, genital and rectal lesions [38]. It is
estimated that the majority of the population is infected by at least one of HSV. Acyclovir is the gold
standard therapy for HSV infections [39-41]. Although this drug as well as other available ones are
effective and selective, the emergence of resistant strains has hampered herpes infections treatment since
most drugs share the same mechanism of action implying cross-resistance [42, 43]. In this context, natural
products can provide an important source of bioactive compounds playing a key role in the research and
development of novel anti-herpes products. Several CGs have been tested in vitro against HSV replication
(eg. Digoxin [25], digitoxin [27], ouabain [44], evatromonoside [45], glucoevatromonoside [20]), and
showed to be potent inhibitors of viral replication. Their powerful effects against DNA viruses were well
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correlated with the inhibition of sodium transport by Na /K ATPase [46].
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In view of the promising results obtained by the aforementioned researchers, this work aimed to
synthesize the readily accessible 3β-azido-3-deoxydigitoxigenin (5) from digitoxigenin (1). Two new
series of cardenolides were obtained from derivative (5): (i) glycosylated triazoles through click
chemistry with propargyl glycosides; and (ii) compounds substituted in the alpha carbonyl position with
different amines, which can be prepared from compound 5 by reduction to 3β-amino-3-
deoxydigitoxigenin (8), coupling with chloroacetyl chloride and subsequent substitution at alpha-position
with different amines.
1
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54
55
56
57
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In the first series (i), the designed glycosides can be considered as analogues of glucoevatromonoside in
which the D-digitoxose moiety was replaced by a triazole in order to facilitate synthesis and to evaluate
the influence of the chosen carbohydrates and triazole ring in the activity of digitoxigenin. Besides
improving aqueous solubility, the carbohydrate moiety can contribute to the interaction with the
biological target (receptor), and can direct the bioactive molecule to cells in view of the presence of
carbohydrate receptors in cells surface. For example,
D-glucosides can be taken up by cancer cells over
expressing -glucose transporters in their cell surfaces. This lead to the accumulation of glucosylated
D

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compounds inside the cells and therefore can contribute to enhance their activity [47].
D
-galactose is a C-
4 epimer of
evaluation of the influence of sugar configuration on bioactivity.
two 1-4-β-linked -glucose units presenting a higher number of hydroxyl groups affecting water
D
-glucose, while D-mannose is a C-2 epimer, so the corresponding glycosides allow for the
D-cellobiose is a disaccharide formed by
D
solubility and offers the possibility to investigate the influence of an additional
biological response.
D-glucose residue on the
1
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The second series (ii) was designed based on the structure of AMANTADIG, a potent cytotoxic
cardenolide derivative [9, 10, 13]. The coupling with different hydrophobic, hydrophilic, or small mimic
of 1-adamantyl-amine residues might help understanding the mechanism of action of bioactive
compounds.
1
1
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All obtained derivatives have their chemical structures confirmed unequivocally, and their anti-HSV-1,
anti-HSV-2, and cytotoxic activities against different human cancer cell lines were evaluated.
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2. Results and discussion
2.1 Chemistry
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The propargyl glycosides of
D
-glucose,
D
-galactose,
D
-mannose and -cellobiose used to synthesize the
D
1
triazol glycosides derivatives of digitoxigenin (1) were prepared as shown in Scheme 1.
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The peracetylated propargyl glycosides IIa, IIb and IIc were obtained by glycosylation of propargyl
alcohol with the β anomer of peracetylated
D
-glucose,
D
-galactose and α anomer of peracetylated -
D
mannose, respectively, in dry dichloromethane using boron trifluoride etherate as catalyst, at room
temperature [48]. The propargyl glycoside III was prepared by the Koenigs-Knorr method, which
consisted in the glycosylation of propargyl alcohol with 2,3,6,2',3',4',6'-hepta-O-acetyl-α- -cellobiosyl
D
bromide (IId) in dry dichloromethane, using a mixture of silver carbonate and silver triflate as
glycosylation promoters [49].
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Compound 5 was prepared in four steps from digitoxigenin (1) following mainly a literature procedure
(Scheme 2) [50]. Digitoxigenin was oxidized to the corresponding digitoxigenone (2) by the Jones
reagent with 84% yield; reduction of 2 with sodium borohydride in aqueous dioxane at 5 °C furnished 3α-
digitoxigenin (3) in a stereoselective manner with 94% yield on molar basis. Tosylation of 3 with tosyl
chloride in dry pyridine gave the corresponding 3-α-tosylate 4 in 50% yield which, upon reaction with

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sodium azide in N,N-dimethylformamide (DMF) at 75 °C, furnished 3β-azido-3-deoxydigitoxigenin (5)
with a yield of 85% on molar basis [50].
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The click reaction of the propargyl glycosides IIa-c and III with 3β-azido-3-deoxydigitoxigenin (5) in a
mixture of tetrahydrofuran/water, in the presence of copper (II) sulfate and sodium ascorbate, gave the
glycosylated triazol derivatives of digitoxigenin 6a-d [51]. Deacetylation with lithium hydroxide in
water/methanol afforded the corresponding deprotected derivatives 7a-d [52, 53].
1
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Reduction of compound 5 with triphenylphosphine, in a mixture of tetrahydrofuran/water (70 °C),
furnished 3β-amino-3-deoxydigitoxigenin (8) with 60% yield [50, 54]. The catalytic reduction of
derivative 5 was performed based on the patent WO2013000286 [54] and the Staudinger reaction;
however, the extraction of 3β-amino-3-deoxydigitoxigenin (8) followed the procedure described by
Sawlewicz et al. [50]. The reaction of compound 8 with chloroacetyl chloride, in tetrahydrofuran and
potassium carbonate, gave 3-β-(chloroacetylamino)-3-deoxydigitoxigenin (9) [54, 55] (90% yield).
2
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Compound 9 was used for synthesis of derivatives 10-14, as shown in Scheme 2. Reaction of 9 with
ethanolamine in tetrahydrofuran gave derivative 10 (85% yield). Finally, one-pot reactions or those
occurring in two steps of derivative 9 with potassium iodide (KI) in a mixture of acetonitrile/water, gave
the iodine derivative, which was used to obtain the amino- and aromatic-amino-digitoxigenin and
hydroxyl derivatives 11-14, through the reaction with the appropriate compound in acetonitrile using
DIPEA as base [56, 57]. After chromatographic purification, all derivatives were obtained with yields
ranging from 23% (12) and 25% (11, 13) to 46% (14).
2
2
2
2
2
2
2
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2
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09
10
11
12
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The obtained compounds were characterized by IR, NMR and ESI-MS spectroscopy. The infrared spectra
-1
of the propargyl glycosides IIa-c and III showed bands in the region of 3255-3282 cm (C-H, sp carbon),
-1
-1
1
2117-2119 cm (C≡C), and 1732-1754 cm (C=O). The H NMR spectra of these compounds showed
signals at δH 1.91-2.10 ppm ascribed to the CH CO groups, as well as signals at δH 2.41-2.44 ppm
3
attributed to terminal acetylenic hydrogens. The duplets centered at δH 4.73, 4.67 and 4.67 ppm (d, J=7.9-
8.0 Hz; 1H, H-1) were assigned to the anomeric hydrogens of the propargyl glycosides IIa, IIb and III,
respectively. The J-coupling values are compatible with β-type glycosides (trans-diaxial coupling) [58].
The anomeric hydrogen of the propargyl glycoside IIc ressonates at δH 4.96 ppm (d, J=1.6 Hz; 1H, H-1),
13
and its J-coupling indicates α-configuration (diequatorial coupling). The C NMR spectra showed signals

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at δC 54.9-55.9 ppm ascribed to the methylene group of the propargyl moiety, and δC 96.2-100.6 ppm
attributed to the anomeric carbon.
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2
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The spectral data of digititoxigenin derivatives 2-5 are in accordance with their structures. The infrared
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spectrum of the key intermediate compound 5 showed a strong absorption band at 2097 cm due to
13
N=N=N stretching of the azido group. The C resonance signal at δC 58.6 ppm (CH, C-3) was ascribed to
the corresponding C-N of this compound, thus confirming the structure.
3
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The infrared spectra of the peracetylated glycosylated triazol derivatives of digitoxigenin 6a-d showed
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bands at 3412-3468 cm (OH, C-14), 1739-1746 cm (C=O, ester) and 1218-1228 cm (C-O, ester). The
1
H NMR spectra of these compounds showed signals at δH 0.82-0.94 ppm (methyl groups at C-18 and C-
19 of the aglycone), δH 1.91-2.16 ppm (CH C=O), δH 3.50-5.50 ppm (pyranosidic protons), δH 5.81-5.89
3
ppm (s,1H, CH, H-22) and 7.60-7.66 ppm (hydrogen of the triazole ring). These structural features were
1
3
confirmed by their C NMR spectra, which showed signals at δC 15.6-15.9 ppm (CH , C-18), δC 20.4-
3
20.8 ppm (CH , CH =O), δC 22.4-23.5 ppm (CH , C-19), δC 96.8-100.6 ppm (CH, C-1'), δC 117.4-117.5
3
3
3
ppm (CH, C-22), and C
δ
169.0-170.6 ppm (C=O, ester). The β-configuration at the anomeric carbon of
glycosides 6a, 6b e 6d was confirmed by the resonance signals at δC 99.8-100.6 (CH, C-1'). On its turn,
the α-configuration at the anomeric carbon of glycoside 6c was indicated by the signal at δC 96.8 ppm
(CH, C-1').
2
2
2
2
2
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The infrared spectra of the deprotected glycosylated triazol derivatives of digitoxigenin 7a-d showed, as
-1
expected, absorption bands in the region of 3253-3397 cm . This is due to OH stretching of the
-1
carbohydrate moiety, as well as absence of carbonyl absorption bands at circa 1750 cm , found in the
spectra of the peracetylated precursors. Similarly, the signals of hydrogen and carbon of the acetyl groups
1
13
are absent in the H and C NMR spectra of compounds 7a-d.
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1
2
2
2
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The infrared spectrum of compound 8 showed a band in 3356 cm that is characteristic of NH stretching,
2
-1
and a band in 3280 cm due to OH stretching. The resonance signals at δH 4.05 ppm (tt, J =4.4 Hz, J
1
13
=11.9 Hz, 1H, H-3) and at δC 52.6 ppm (C-3) in the H and C NMR spectra of derivative 8 allowed us to
confirm the structure of this key intermediate.

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The infrared spectrum of compound 9 showed bands at 3335-3455 cm due to NH and C=O stretching of
the amide group. The formation of the 2’-chloroacetamide derivative 9 was further confirmed by the
NMR signals at δH 7.33 ppm (d, J =5.5 Hz, 1H, N-H) and δC 166.0 ppm (C-1’).
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1
2
2
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In the same way, infrared data obtained for compound 10 showed bands at 3304 and 1643 cm related,
respectively, to NH and C=O stretching of the amide group. The resonance signals at δH 2.91 ppm (t, J
=6.5 Hz, 1H, H3’), 3.73 (t, J =6.5 Hz, 1H, H4’) and δC 52.2 (C-3’) and 64.0 (C-4’) further evidenced the
structure of the 2’-hydroxyethyl derivative 10.
2
2
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2
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The NMR data obtained for compound 11 were very similar to those of derivative 9. The difference is
owing to the substitution of the chloride atom at compound 9 for the more electronegative oxygen at
compound 11, which resulted in deshielding of the resonance signal of C-2’ at derivative 11 in
comparison to compound 9 (62.8 vs. 43.8 ppm, respectively).
2
2
2
2
2
53
54
55
56
57
The spectral data of derivatives 12 and 14 were in agreement with their structures and the infrared
-1
spectrum of compound 12 showed the characteristic C≡N stretching band at 2214 cm , while the
-1
1
13
spectrum of derivative 14 exhibited the C-Cl bending at 820 cm . The H and C NMR spectra of
compounds 12 and 14 demonstrated the aromatic nature of the amine group bound at C-2’ as well as the
presence of the 4-phenylpiperidine group in compound 13.
2
2
58
59
The purity of all synthesized compounds were ≥ 95%, and their mass spectra showed molecular weight
values compatible with the proposed structures (Supplementary Data available).
2
2
2
2
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2
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63
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65
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67
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70
This synthesis approach included in total 16 new cardenolide derivatives that were all derived from 3β-
azido-3-deoxydigitoxigenin (5), and could be potentially cytotoxic similar to other structures based on the
3β-amino-3-deoxydigitoxigenin (8), which were already tested and showed promising cytotoxic effects
[eg. AMANTADIG (3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin) against leukemia (K562 and
SEM), prostate cancer (PC-3, DU145, LNCaP) and renal tumor cells (A498, 786-O and Caki-1) [9, 10,
13]. Further, bufogenin derivatives containing similar residues also showed cell growth inhibition of
different cancer cell lines, such as non-small lung (A549), breast (MCF-7), colon (LoVo) and prostate
(PC3) [54]. Compound (8) was coupled to residues individually, which were chosen following [55] or
presenting small mimics of the basic AMANTADIG structure. Moreover, we describe herein, for the first
time, the synthesis of peracetylated and non-acetylated glycosylated triazol derivatives of digitoxigenin
(1) as well their bioactive effects. Since the triazol group shifts the sugar moieties, its presence in the

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72
steroid scaffold can also influences bioactivities. In summary, the newly synthesized compounds will help
to identify functional groups that are important for their cytotoxicity and anti-herpes effects.
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2.2 Biological activities
2.2.1 Anti-herpes in vitro activity
2.2.1.1 Plaque number reduction assay
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According to the CC50 values (Table 1), only compounds 9 (83.98 ± 9.03 µM) and 15 (36.07 ± 6.58 µM)
presented moderate toxic effects, while the other derivatives showed low toxicity on Vero cells ranging
from 111.9 ± 7.01 to >300 µM. Regarding the plaque number reduction assay, compounds 10 and 11
exhibited the most promising results against HSV-1 (KOS and 29-R strains) and HSV-2 (333 strain) with
SI values >1,000. Comparing to acyclovir (ACV), used as positive control, these results are similar for
HSV-1(KOS strain) and much better for HSV-2. Concerning to the anti-HSV-1 activity (KOS strain), the
obtained IC50 values of compounds 10 and 11 were 0.23 ± 0.01 and 0.24 ± 0.03 µM, respectively, almost
six-folder more potent than ACV (1.38 ± 0.46 µM). On its turn, the obtained IC50 values of compounds
10 and 11 against HSV-2 replication were 0.27 ± 0.01 and 0.30 ± 0.04 µM, respectively, twelve and
eleven-folder higher than ACV (3.23 ± 0.89 µM), respectively. Regarding the anti-HSV-1 activity (29-R
strain, which is resistant to ACV), both compounds were still more active showing IC50 values of 0.18 ±
0.01 and 0.19 ± 0.02 µM, respectively. It is worth mentioning that compounds 7a, 7b, 7c, 12, 13 and 14
also showed encouraging results with SI values >100 for all viruses tested.
2
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Anti-herpes activity of other cardenolides has been reported against HSV-1 and HSV-2 [20, 27, 46]
replication. Results found for compounds 10 and 11 were similar to those of glucoevatromonoside (GEV)
[20] and digitoxin [27]. On the other hand, regarding to cardenolides toxicity that show narrow
therapeutic index [18], our compounds were less cytotoxic on Vero cells when compared to GEV [20]
and digitoxin [27] which can be considered an advantage.
2
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Herein, digitoxigenin (1) was used as scaffold for the semisynthesis of novel cardenolide derivatives, and
then assayed for all viruses tested. Digitoxigenin (1) alone was one of the most cytotoxic compounds on
Vero cells (27.54 ± 4.29 µM) (Table 1), while presented potent IC50 values against the three tested HSV
strains. These results disclosed digitoxigenin (1) as the tested compound with the lowest SI values,

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whereas its derivatives here described showed higher SI values. This reinforces the importance of the
synthesis/semisynthesis of new derivatives more active and less cytotoxic.
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2.2.2 Cytotoxic activity
2.2.2.1 Cell viability
3
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06
07
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09
For the cytotoxic screening, the 16 new cardenolide derivatives were tested on human A549, HCT-8,
LNCaP and PC3 cell lines for 48 h, and stained with sulforhodamine B (SRB) that measure total protein
mass, which is related to cell viability. Fig. 1 shows the color grid effects, where compounds labeled with
shades of green were considered the most active and those with red shades were less active. Compounds
6c, 7c, 10, 11, 12, 13, 14 and 16 inhibited cell proliferation, wherein once again compounds 10 and 11
appeared to be the most cytotoxic, with IC values similar to those of the anti-cancer drug paclitaxel here
50
used as a positive control. The remaining compounds were less cytotoxic showing IC values up to 29.46
50
µM after 48 h [Table S1 in Supplementary Data contains IC values and Standard Deviation (SD)].
50
3
3
3
3
3
3
3
3
3
10
11
12
13
14
15
16
17
18
During the initial cytotoxic screening, A549 cell line was the most sensitive for the tested cardenolide
derivatives, with IC50 values ranging from 0.11 to 2.86 µM. For this reason, the most cytotoxic
compounds on A549 cells were tested, at the same concentrations, on other NSCLC, the H460 cell line.
Taken together (Table 2), these results showed that the most promising cardenolide derivatives were
compounds 7c, 10, 11, 12 and 16. To verify the selectivity on non-tumoral cells, the selected compounds
were tested on MRC-5 and HGF cells. They were all less cytotoxic for HGF cells than for A549 or H460
cells (Table 2). Digitoxigenin (1), the aglycone of digitoxin, was used as scaffold for the semisynthesis of
the novel derivatives herein tested, and it showed a moderate activity for all human cancer cells assayed,
when compared to their derivatives (shades of yellow, Fig. 1).
3
3
3
3
19
20
21
22
Our results are in line with previous investigations showing the ability for same CGs to induce cytotoxic
effects mainly in lung cancer cells, as it has been showed for digoxin, ouabain [59], convallatoxin [60],
and glucoevatromonoside [33]. Moreover, these potent cardenolides showed IC50 values at nM
concentration range, as it was also identified herein for compounds 7c, 10, 11, 12 and 16.
3
3
3
23
24
25
Additionally, the IC value of compound 10 on the non-tumor MRC-5 cells was similar to that of digoxin
5
0
(~150 nM) [59] and even better for compound 11 (IC 250 nM). This could be an important finding since
50
16 clinical trials with digoxin are in progress to treat several cancer types, including lung cancer [18].

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26
2.2.3 Effects on Na+/K+ ATPase
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The most promising anti-herpes compounds were the same most cytotoxic (compounds 10 and 11)
+
+
suggesting that they share the same primary target (i.e. Na /K ATPase). As recognized and explored in
+
+
the literature, Na /K ATPase plays essential roles in ionic homeostasis and is the main target of
cardenolides through their binding to the alpha-subunit [61, 62]. All compounds evaluated for their
+
+
cytotoxic activity were also tested in regard to their ability to inhibit Na /K ATPase. The IC values
50
found for the inhibition of this enzyme were ranging from 0.84 µM (11), 1.99 µM (10), 2.04 µM (16),
4.16 µM (12), 4.46 µM (6c) 5.47 µM (7c), 6.60 µM (13) to 10.82 µM (14) (Fig. S1 in Supplementary
Data).
3
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48
In most cases, it is possible to establish a correlation between the cytotoxicity of the individual CGs and
+
+
their ability to bind to the alpha subunits and inhibit Na /K ATPase even at in low concentrations [10].
Within our study, we only determined the inhibition rate of the new derivatives using a mixture of α1,2,3
+
+
subunits of Na /K ATPase. Therefore, we cannot distinguish between the selective affinities of the
derivatives on the individual α subunits. It has been previously demonstrated that different CGs and some
derivatives have distinct affinities to the four individual α subunits. Several studies addressed the
selective affinities of CGs, such ouabain towards α1 and α3 subunits or digoxin towards α2 subunit [63-
65]. In addition, Clifford and Kaplan [66] demonstrated that malignant or oncogene-transfected cells are
less sensitive than non-tumor breast cells to ouabain-mediated inhibition of proliferation and induction of
apoptosis. A further study clearly demonstrated that proliferation and survival of malignant and non-
tumor cell lines in the presence of ouabain are depending on the cell surface abundance and the
+
+
expression rates of the different α subunits of the Na /K ATPase, also influencing its selectivity index
[67]. To evaluate the affinity of compounds 10 and 11 towards the individual α subunits will be an
interesting and challenging further project.
3
3
3
3
3
49
50
51
52
53
The inhibition rate correlates suitably with the bioactivity demonstrated by compounds 10 and 11 that
were the most actives against the different tested human cancer cell lines as well as against HSV
replication. This correlation was also showed for other cardenolide derivates such as AMANTADIG [10,
13] whose bioactivity was not discussed only based on the cardenolide scaffold but also on the antiviral
action of 1-adamantyl-amine residue [9].

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54
2.3 Influence of specific chemical features on bioactivity of new digitoxigenin-derivatives
3
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70
In this work, two series of digitoxigenin derivatives were obtained and evaluated against different cancer
cell lines. Series (i) is formed by glycosylated triazole derivatives (compounds 6a-d and 7a-d) in which
compounds 6c and 7c presented promising results for cytotoxicity and compounds 7b and 7c for anti-
herpes activity. Compound 7c was the best one showing a lower IC50 value than digitoxigenin (1)
suggesting that groups bound to the position H-3β of digitoxigenin (1) may influence positively both
activities. Furthermore, compound 7c has a mannose bound to the tetrazolic nucleus. Thus, the
configuration of the hydroxyl group at positions C-1 (alpha-configuration) and C-2 (beta-configuration)
of mannose may influence positively the activities, when compared to compounds 7a, 7b and 7d, which
do not present hydroxyl groups with these configurations. Moreover, the deacetylated glycosylated
triazole derivatives (7a-d) were more active than the corresponding acetylated compounds (6a-d)
indicating that the free hydroxyl group of the sugar may improve both activities. This finding is aligned
with previous reports that demonstrated the influence of sugar residues attached to digitoxigenin scaffold
on the antitumor activity [68, 69]. It is also noteworthy to mention that triazole derivatives possess an
additional structural feature that may favor bioactivity: the presence of mannose receptor in cell
membranes (Endo 180 - endocytic recycling glycoprotein) [70] that can facilitate absorption of those
derivatives by the cells [71].
3
3
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3
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3
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83
Derivatives of series (ii) (compounds 9-15) are based on the bioactive semisynthetic cardenolide
AMANTADIG (3β-[2-(1-amantadine)-1-on-ethylamine]-digitoxigenin. Compounds 10 and 11, presenting
a hydroxyl group and an ethanolamine group, both bound to the alpha-carbonyl position, were the most
active ones relating the anti-herpes and cytotoxic activities. On the other hand, hindering groups bounding
at an electronegative atom, as those found in compounds 12, 13 and 14, interfere negatively in both
activities. Consequently, the presence of an electronegative atom at the alpha-carbonyl position of the
amide group at the side chain at C-3 seems to be important to reduce the IC50 values in comparison to
digitoxigenin (1). In addition, lipophilicity of the compounds may interfere with the biological response,
+
+
since the target of cardenolides is the membrane bound Na /K ATPase enzyme. In order to investigate
the relationship between lipophilicity and bioactivity, we calculated the LogP values [72] of some of the
bioactive compounds of series (ii), and compared the obtained values with the ability of the derivatives to
+
+
inhibit Na /K ATPase. Lower LogP values (1.59 and 1.96) were obtained for the highly active cytotoxic
compounds 10 and 11 in comparison to the values calculated for the less cytotoxic compounds 12 (3.78),

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85
13 (5.16), 14 (4.53) and 15 (3.50). These results suggest that lipophilicity may influence the binding of
+
+
the tested derivatives to the Na /K ATPase and therefore their cytotoxic effects.
3
3
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00
Furthermore, it was recently demonstrated that the IC values of the derivative AMANTADIG (3β-[2-(1-
50
amantadine)-1-on-ethylamine]-digitoxigenin were 0.68 and 0.186 µM for PC-3 and LNCaP cell lines,
respectively [9, 13]. Compounds 10 and 11 proved to be nearly as cytotoxic or even more cytotoxic for
PC-3 or LNCaP prostate cancer cell lines. In this work, compound 11, which contains the smallest and
simplest hydroxyl group as residue, showed similar IC50 value for PC-3 cell line (0.42 µM). Compound
10 containing a N-(2-hydroxyetil)aminoacetyl residue at C-3 was even more cytotoxic for both cell lines
(PC-3: 0.18 µM and LNCaP: 0.13 µM) than AMANTADIG, leading to the conclusion that the coupling
of the cardenolide scaffold to the 1-adamantyl-amine residue is not essential for the reduction of cancer
cells viability. This conclusion was strengthened by evaluating the 1-adamantyl-amine residue inhibition
+
+
potency of the Na /K ATPase and its bioactivity against two carcinoma cell lines [DU145 and 786-O
(data not shown)]. These results indicated that the 1-adamantyl-amine residue neither has the ability to
+
+
inhibit Na /K ATPase nor to reduce the viability of both tested cell lines. However, AMANTADIG as
well as the novel cardenolide derivatives reported here, whose advantage is to have a selective influence
on cell viability of different cancer cell lines, are all very promising compounds. This was shown by the
different IC values of the same compound obtained on different cell lines (Table 2 and Fig. S1).
5
0
4
01
3. Conclusions
4
4
4
4
4
02
03
04
05
06
Two new series of compounds were obtained from 3β-azido-3-deoxydigitoxigenin (5) from digitoxigenin
(1): (i) glycosylated triazoles through click chemistry with propargyl glycosides; and (ii) compounds
substituted in the alpha carbonyl position with different amines. These synthesis approaches generated 16
new cardenolide derivatives, and regarding their anti-herpes and cytotoxic activities, the most potent
compounds were 10 and 11.
4
4
4
4
4
4
07
08
09
10
11
12
In relation to anti-herpes action, both compounds were very active against all tested HSV strains, but they
were even more active against a resistant ACV strain (HSV-1, 29-R). It is well known that the emergence
of resistant strains to ACV has hampered the treatment of herpes infections, since most available antiviral
drugs share the same mechanism of action implying cross-resistance. In this way, the search for new
alternatives is an important goal to follow, and our findings seem to be in this direction. Complementary
approaches to better understand how they inhibit HSV replication cycle have to be performed.

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15
16
Regarding the cytotoxic activity against different cancer cell lines, our findings suggest that lung cancer
cells were more sensitive to compounds 10 and 11, and the results obtained allow us to conclude that they
could be seen as promising molecules as well, and deserve further studies to elucidate their mechanisms
of action.
4
4
4
17
18
19
4. Experimental Section
4.1 General methods
4.1.1 TLC analysis
4
4
4
4
4
4
20
21
22
23
24
25
TLC plates (Merck, Silica gel 0.063-0.2 mm) were used and spots were detected under natural light, after
straining with either anisaldehyde or Kedde reagent. As mobile phase 100% ethyl acetate (items 4.2.1 and
4.2.2), hexane: ethyl acetate (3:2, v/v; items 4.2.3 to 4.2.5) or methanol/ethyl acetate/triethylamine
(4.99/4.99/0.02, v/v; items 4.2.10 and 4.2.11) and methanol/ethyl acetate (0.1/9.9, v/v, items 4.2.12 to
4.2.14) were used. Fifteen µL of samples and standards (digitoxigenin and digitoxin at 2 mg/ mL) were
assayed.
4
26
4.2 Synthesis
4
4
4
4
4
4
4
4
4
27
28
29
30
31
32
33
34
35
4.2.1 Hydrolysis of methanolic CGs extract to obtain digitoxigenin (1)
Digitoxigenin was obtained as described by Pádua et al. [73] with few modifications. Instead of digitoxin,
10g of Digitalis lanata methanolic extract enriched in A-series cardenolides were used for hydrolysis
reaction. The dried residue (7 g) was dissolved in dichloromethane (100 mL) and filtrated under reduced
pressure over silica gel (0.04-0.063 mm, Büchner funnel size 4). Then, silica gel was washed with the
following solvents of different polarities (100% dichloromethane 3 × 100 mL ‰ dichloromethane/ethyl
acetate – 9:1, 15 × 50 mL ‰ dichloromethane/ethyl acetate – 7:3, 15 × 50 mL‰ dichloromethane/ethyl
acetate – 1:1, 7 × 50 mL). Fractions 21 to 26 contained digitoxigenin, and the combined fractions resulted
in 1.3 g of digitoxigenin.
4
4
4
4
36
37
38
39
4.2.1.1 Digitoxigenin (1)
2
7
25
mp 233.5 – 240.3 ºC (Lit: 248-253 °C [74]). [α]D +14.5 ° (c 0.55; MeOH). Lit:[α]D +14.1 ° (c 0.55;
-1
-1
3
-1
-1
MeOH [74]). IR: ῡ 3522 cm (OH), 2864–2932 cm (C-H sp ), 1733 cm (C=O), 1632 cm (C=C), 1260
-1
-1
1
cm (O-C=O), 1035 cm (C-O). H NMR (400 MHz, DMSO-d ): δ 0.77 (s, 3H, CH , H-18), 0.87 (s, 3H,
6
3

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48
CH , H-19), 1.05−1.79 (m, 19H, H-1, H-2, H-4, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-16), 1.98−2.08
3
(m, 2H, H-15), 2.72 (dd, J = 5.4 Hz, J = 9.0 Hz, 1H, H-17), 3.89 (s, 1H, H-3), 4.04 (s, 1H, OH), 4.16 (d, J
= 2.6 Hz, 1H, OH), 4.86 (dd, J = 1.5 Hz, J = 18.4 Hz, 1H, H-21a), 4.96 (dd, J =1.4 Hz, J =18.4 Hz, 1H,
1
3
H-21b), 5.89 (s, 1H, H-22). C NMR (100 MHz, DMSO-d ): 15.7 (CH , C-18), 20.8 (CH , C-7), 21.1
6
3
2
(CH , C-11), 23.7 (CH , C-19), 26.4 (CH , C-6), 26.5 (CH , C-16), 27.5 (CH , C-2), 29.5 (CH , C-15),
2
3
2
2
2
2
32.2 (CH , C-4), 33.1 (CH , C-1), 34.8 (CH, C-9), 35.0 (C , C-10), 35.7 (CH, C-5), 39.0 (CH , C-12),
2
2
0
2
40.9 (CH, C-8), 49.4 (C , C-13), 50.2 (CH, C-17), 64.6 (CH, C-3), 73.1 (CH , C-21), 83.8 (C , C-14),
0
2
0
+
116.2 (CH, C-22), 173.8 (C=O, C-23), 176.3 (C , C-20); HRMS-ESI: calcd for C H O [M+H]
0
23 34
4
375.5211, found: 375.45
4
49
4.2.2 Oxidation of digitoxigenin (1) to digitoxigenone (2)
4
4
4
4
4
4
4
4
50
51
52
53
54
55
56
57
1 g of digitoxigenin (1) was dissolved in 112 mL acetone, and the solution was cooled down to 0 °C by
stirring on ice. Then, 1.4 mL of Kiliani reagent were added drop by drop until a yellow/orange color was
maintained. The reaction was stirred for 20 min on an ice bath. After adding 20 to 25 mL of methanol to
remove surplus CrO , the reaction mixture was further stirred for 20 min at 0 °C. Next, adding 40 mL of
3
water, the acetone was removed under vacuo. The reaction mixture was extracted with 4 × 30 mL
dichloromethane, neutralized with 20 mL of 3% w/v Na CO aqueous solution, and washed with 3 × 30
2
3
mL water. The organic layer was dried over anhydrous sodium sulfate, evaporated to dryness, and the
reaction product was analyzed by TLC.
4
4
4
4
4
4
4
4
4
4
58
59
60
61
62
63
64
65
66
67
4.2.2.1 Digitoxigenone (2)
3
0
20
Yield: 90%; mp 187.2 – 191.8 ºC (Lit:191-194 °C [75]). [α]D +22 ° (c 1.00; MeOH). Lit: [α]D +27.6 °
-1
-1
3
-1
-1
(c 1.0; MeOH [50]). IR: ῡ 3486 cm (OH), 2867–2941 cm (C-H sp ), 1704–1738 cm (C=O), 1618 cm
-1
1
(C=C), 1026–1283 cm (C-O). H NMR (400 MHz, CDCl ): δ 0.87 (s, 3H, CH , H-18), 0.98 (s, 3H, CH ,
3
3
3
H-19), 1.21−2.17 (m, 19H, H-1, H-2α, H-4α, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-15, H-16), 2.30 (td,
J = 5.4 Hz, J = 14.6 Hz, 1H, H-2β), 2.59 (t, J = 14.3 Hz, 1H, H-4β), 2.74−2.78 (m, 1H, H-17), 4.78 (dd, J
13
= 1.3 Hz, J = 18.1 Hz, 1H, H-21a), 4.96 (d, J =18.0 Hz, 1H, H-21b), 5.84 (s, 1H, H-22). C NMR (100
MHz, CDCl ): 15.9 (CH , C-18), 21.0 (CH , C-7), 21.3 (CH , C-11), 22.6 (CH , C-19), 26.6 (CH , C-6),
3
3
2
2
3
2
27.0 (CH , C-16), 33.1 (CH , C-15), 35.3 (C , C-10), 36.7 (CH, C-9), 36.8 (CH , C-12), 37.2 (CH , C-2),
2
2
0
2
2
39.8 (CH , C-1), 41.6 (CH, C-8), 42.2 (CH , C-4), 43.8 (CH, C-5), 49.8 (C , C-13), 50.9 (CH, C-17), 73.7
2
2
0

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69
(CH , C-21), 85.2 (C , C-14), 117.7 (CH, C-22), 174.7 (C=O, C-23), 174.9 (C , C-20), 212.8 (C , C-3);
2 0 0 0
+
HRMS-ESI: calcd for C H O [M+H] 373.5052, found: 373.45
2
3
32
4
4
70
4.2.3 Reduction of digitoxigenone (2) to 3α-digitoxigenin (3)
4
4
4
4
4
4
4
4
71
72
73
74
75
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77
78
1 g of digitoxigenone (2) was dissolved in 40 mL dioxane and 10 mL water. Then, 365 mg NaBH in 37.5
4
mL of 80% dioxane (30 mL dioxane + 7.5 mL H O) were added to the solution and stirred for 30 min at
2
20 °C. The reaction was neutralized with 30 mL of 5% acetic acid and the pH was adjusted to 5. Hence,
the reaction color turned from yellow to white after adding the acid. For stopping the reaction and
avoiding the formation of additional reaction products, the fast reducing of dioxane under vacuo is
essential. The aqueous residue was extracted with 5 × 30 mL chloroform/ethanol (3:1) and the organic
layer was washed 3 × 30 mL of water, and the organic layer was dried over anhydrous sodium sulfate and
evaporated to dryness.
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4.2.3.1 3α-Digitoxigenin (3)
3
0
22
Yield: 60%; mp 266.4 – 269.5 °C (Lit: 268-270 °C [76]). [α]D +5.7 ° (c 0.35; MeOH). Lit:[α]D +26.8 °
-
1
-1
3
-1
(c 0.33; MeOH [77]). IR: ῡ 3419–3507 cm (OH), 2859–2929 cm (C-H sp ), 1733 cm (C=O), 1632
-
1
-1
1
cm (C=C), 1036 cm (C-O). H NMR (400 MHz, DMSO-d ): δ 0.77 (s, 3H, CH , H-18), 0.85 (s, 3H,
6
3
CH , H-19), 0.89−1.83 (m, 19H, H-1, H-2, H-4, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-16), 2.00−2.08
3
(m, 2H, H-15), 2.72−2.74 (m, 1H, H-17), 3.36−3.41 (m, 1H, H-3), 4.06 (s, 1H, OH), 4.43 (d, J = 4.4 Hz,
13
1H, OH), 4.87 (d, J = 18.6 Hz, 1H, H-21a), 4.97 (d, J =18.2 Hz, 1H, H-21b), 5.90 (s, 1H, H-22).
C
NMR (100 MHz, DMSO-d ): 15.7 (CH , C-18), 20.6 (CH , C-7), 21.2 (CH , C-11), 23.1 (CH , C-19),
6
3
2
2
3
26.4 (CH , C-6), 26.9 (CH , C-16), 30.4 (CH , C-2), 32.2 (CH , C-1), 34.5 (C , C-10), 34.9 (CH , C-15),
2
2
2
2
0
2
35.5 (CH, C-9), 36.2 (CH , C-4), 39.0 (CH , C-12), 41.0 (CH, C-5), 41.3 (CH, C-8), 49.4 (C , C-13), 50.2
2
2
0
(CH, C-17), 69.8 (CH, C-3), 73.1 (CH , C-21), 83.7 (C , C-14), 116.2 (CH, C-22), 173.8 (C=O, C-23),
2
0
+
176.3 (C , C-20); HRMS-ESI: calcd for C H O [M+H] 375.5211, found: 375.45
0
23 34
4
4
91
4.2.4 Tosylation of 3α-digitoxigenin (3)
4
4
4
4
92
93
94
95
500 mg of 3α-digitoxigenin (3) were dissolved in 12.5 mL of dried pyridine. Then, 465 mg of
tosylchloride in 3 mL pyridin were added, and the reaction mixture was stirred for 15 h at 20 °C. The pH
was adjusted to 4 by adding 140 to 180 mL of 1 M HCl aqueous solution. The reaction mixture was
sequentially extracted with chloroform (6 × 30 mL), neutralized with 3% w/v Na CO aqueous solution (2
2
3

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96
97
× 10 mL), and washed with water (3 × 30 mL). In the sequence, the organic layer was dried over
anhydrous sodium sulfate and evaporated to dryness.
4
4
5
5
5
5
5
5
5
5
5
5
5
5
98
99
00
01
02
03
04
05
06
07
08
09
10
11
4.2.4.1 3α-O-Tosyldigitoxigenin (4)
2
9
25
Yield: 90%; mp 107.0 – 110.2 °C (Lit: 155 – 156 °C [50]). [α]D +28 ° (c 1,0; CHCl ). Lit:[α] +35.0 °
3
D
-
1
-1
3
-1
-1
(CHCl [50]). IR: ῡ 3534 cm (OH), 2939 cm (C-H sp ), 1753 cm (C=O), 1628 cm (C=C), 1167 e
3
-1
-1
1
1339 cm (S=O), 911 cm (S-O). H NMR (400 MHz, CDCl ): δ 0.85 (s, 3H, CH , H-18), 0.88 (s, 3H,
3
3
CH , H-19), 0.99−1.80 (m, 19H, H-1, H-2, H-4, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-16), 2.14−2.17
3
(m, 2H, H-15), 2.45 (s, 3H, CH , H-7’), 2.75−2.77 (m, 1H, H-17), 4.45 (ddd, J = 4.8 Hz, J = 11.0 Hz, J =
3
16.0 Hz, 1H, H-3), 4.80 (d, J = 18.1 Hz, 1H, H-21a), 4.98 (d, J =18.9 Hz, 1H, H-21b), 5.86 (s, 1H, H-22),
13
7.34 (d, J =7.8 Hz, 2H, H-3’), 7.79 (d, J =8.0 Hz, 2H, H-2’). C NMR (100 MHz, CDCl ): 15.9 (CH , C-
3
3
18), 21.1 (CH , C-7), 21.5 (CH , C-11), 21.8 (CH , C-7’), 23.1 (CH , C-19), 26.8 (CH , C-6), 27.0 (CH ,
2
2
3
3
2
2
C-2), 27.8 (CH , C-16), 33.2 (CH , C-15), 33.3 (CH , C-4), 34.8 (C , C-10), 34.9 (CH , C-1), 36.3 (CH,
2
2
2
0
2
C-9), 40.0 (CH , C-12), 41.8 (CH, C-5), 42.0 (CH, C-8), 49.7 (C , C-13), 51.0 (CH, C-17), 73.6 (CH , C-
2
0
2
21), 82.9 (CH, C-3), 85.5 (C , C-14), 117.8 (CH, C-22), 127.8 (2CH, C-2’), 130.0 (2CH, C-3’), 134.8 (C ,
0
0
+
C-4’), 144.7 (C , C-1’), 174.7 (C=O, C-23), 174.8 (C , C-20); HRMS-ESI: calcd for C H O S [M+H]
0
0
30 40
6
529.7074, found: 529.56
5
12
4.2.5 Azidation of 3α-Tosyl-digitoxigenin (4) to 3β-azido-3-deoxydigitoxigenin (5)
5
5
5
5
5
13
14
15
16
17
500 mg of 3α-tosyl-digitoxigenin (4) were dissolved in 43 mL dimethylformamide (DMF) and 700 mg of
NaN were added. The reaction mixture was heated to 75 °C and stirred for 3 h. After 16 h at 20 °C, the
3
remaining NaN was removed by filtering twice through cotton, and three volumes of EtOAc were added
3
to the clear filtration residue. The organic layer was extracted 8 × 20 mL of water, and the DMF remained
in the water and the organic layer was dried over anhydrous sodium sulfate and evaporated to dryness.
5
5
5
5
5
5
18
19
20
21
22
23
4.2.5.1 3β-azido-3-deoxydigitoxigenin (5)
29
25
Yield: 85%; mp 191.5 – 194.5 ºC (Lit: 213-217 °C [50]). [α]D +18.0 ° (c 1.00; CHCl ). Lit:[α] +22.0
3
D
-
1
-1
-1
-1
-
(CHCl 0 [50]). IR: ῡ 3473 cm (OH), 2930 cm (C-H), 2097 cm (N=N=N), 1737 cm (C=O), 1620 cm
3
1
-1
1
(C=C), 1025 cm (C-O). H NMR (400 MHz, CDCl ): δ 0.81 (s, 3H, CH , H-18), 0.88 (s, 3H, CH , H-
3
3
3
19), 1.17−1.85 (m, 19H, H-1, H-2, H-4, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-16), 2.01−2.11 (m, 2H,
H-15), 2.69−2.71 (m, 1H, H-17), 3.89 (s, 1H, H-3), 4.74 (d, J = 18.1 Hz, 1H, H-21a), 4.93 (d, J =18.1 Hz,

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3
5
5
5
5
5
5
24
25
26
27
28
29
1H, H-21b), 5.80 (s, 1H, H-22). C NMR (400 MHz, CDCl ): 15.9 (CH , C-18), 21.2 (CH , C-11), 21.5
3 3 2
(CH , C-7), 23.8 (CH , C-19), 24.9 (CH , C-6), 26.5 (CH , C-2), 27.0 (CH , C-16), 30.3 (CH , C-4), 30.5
2
3
2
2
2
2
(CH , C-1), 33.2 (CH , C-15), 35.4 (C , C-10), 36.0 (CH, C-8), 36.9 (CH, C-5), 40.1 (CH , C-12), 41.9
2
2
0
2
(CH, C-9), 49.8 (C , C-13), 51.1 (CH, C-17), 58.6 (CH, C-3), 73.7 (CH , C-21), 85.5 (C , C-14), 117.7
0
2
0
+
(CH, C-22), 174.8 (C=O, C-23), 175.0 (C , C-20); HRMS-ESI: calcd for C H N O [M+H] 400.5338,
0
23 33
3
3
found: 400.49
5
30
4.2.6 General procedure for the synthesis of peracetylated propargyl glycosides (IIa-c)
To a solution of peracetylated -glucose, -galactose or -mannose (1.0 g, 2.56 mmol) in 20 mL of dry
dichloromethane at 0 °C, 0.5 mL of BF , Et O 46% v/v and 0.6 mL (10.4 mmol) of propargyl alcohol
5
5
5
5
5
5
5
5
5
31
32
33
34
35
36
37
38
39
D
D
D
3
2
were added in a stepwise manner. The reaction mixture was stirred at room temperature for 24 h, water
(50 mL) was added and the mixture transferred to a separatory funnel. Next, the organic layer was
separated and the aqueous phase was extracted with 3 × 50 mL of dichloromethane The combined
.
organic layers were washed with water (3 × 25 mL), dried over anhydrous sodium sulfate and evaporated
to dryness. The crude propargyl glycosides obtained from D-glucose and D-mannose were purified by
recrystallization from ethanol. The propargyl glycoside obtained from
column chromatography using hexane-ethyl acetate 7:3 as eluent.
D
-galactose was purified by
5
5
5
5
5
5
5
5
5
5
40
41
42
43
44
45
46
47
48
49
4.2.6.1 Propargyl 2,3,4-6-tetra-O-acetyl-β-D-glucopyranoside (IIa)
28
20
Yield: 60%; mp 105.7 – 107.6 °C (Lit. 102-104 ºC [78]). [α]D -32.7 ° (c 1.10; CHCl ). Lit. [α] -43.4 °
3
D
-1
-1
-1
(c 0.9; CHCl [79]). IR: ῡ 3273 cm (C-H sp), 1732–1754 cm (C=O), 1366–1379 cm (α-CH ), 1207–
3
3
-1
-1
1
1233 cm (O-C=O), 1037 cm (C-O). H NMR (400 MHz, CDCl ): 1.96−2.04 (s, 12H, CH C=O), 2.44
3
3
(t, J = 2.3 Hz, 1H, H-9), 3.69 (ddd, J = 2.3 Hz, J = 4.5 Hz, J = 9.9 Hz, 1H, H-5), 4.10 (dd, J = 2.2 Hz, J =
12.3 Hz, 1H, H-6a), 4.22 (dd, J = 4.6 Hz, J = 12.3 Hz, 1H, H-6b), 4.32 (d, J = 2.3 Hz, 2H, H-7), 4.73 (d, J
= 8.0 Hz, 1H, H-1), 4.96 (dd, J =7.9 Hz, J =9.6 Hz, 1H, H-2), 5.05 (t, J = 9.7 Hz, 1H, H-3), 5.19 (t, J =
1
3
9.4 Hz, 1H, H-4). C NMR (100 MHz, CDCl ): 20.4−20.8 (4CH , CH C=O), 55.9 (CH , C-7), 61.8
3
3
3
2
(CH , C-6), 68.4 (CH, C-3), 69.8 (CH, C-9), 71.0 (CH, C-2), 72.0 (CH, C-4), 72.8 (CH, C-5), 78.1 (C , C-
2
0
8), 98.2 (CH, C-1).
5
50
4.2.6.2 Propargyl 2,3,4-6-tetra-O-acetyl-β-D-galactopyranoside (IIb)

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4
20
-1
5
5
5
5
5
5
5
5
51
52
53
54
55
56
57
58
Yield: 70%. [α]D -32.0 ° (c 1.10; CHCl ). Lit. [α] -23.0 ° (c 1.00; CHCl [80]). IR: ῡ 3275 cm (C-H,
3
D
3
-1
3
-1
-1
-1
-1
sp), 2981 cm (C-H, sp ), 2119 cm (C≡C), 1740 cm (C=O), 1368 cm (α-CH ), 1211 cm (O-C=O),
3
-1
1
1016–1043 cm (C-O). H NMR (400 MHz, CDCl ): 1.92−2.08 (s, 12H, CH C=O), 2.42 (t, J = 2.2 Hz,
3
3
1H, H-9), 3.88 (t, J = 6.4 Hz, 1H, H-5), 4.06 (dd, J =6.8 Hz, J =11.2 Hz, 1H, H-6a), 4.12 (dd, J =6.8 Hz, J
=11.3 Hz, 1H, H-6b), 4.31 (d, J = 2.2 Hz, 2H, H-7), 4.67 (d, J = 8.0 Hz, 1H, H-1), 4.99 (dd, J = 3.4 Hz, J
13
= 10.4 Hz, 1H, H-3), 5.14 (dd, J = 8.0 Hz, J = 10.3 Hz, 1H, H-2), 5.33 (d, J = 2.3 Hz, 1H, H-4).
C
NMR (100 MHz, CDCl ): 20.5−20.7 (4CH , CH C=O), 55.9 (CH , C-7), 61.2 (CH , C-6), 67.0 (CH, C-
3
3
3
2
2
4), 68.5 (CH, C-2), 70.8 (2CH, C-3, C-5), 75.4 (CH, C-9), 78.2 (C , C-8), 98.6 (CH, C-1).
0
5
5
5
5
5
5
5
5
5
5
59
60
61
62
63
64
65
66
67
68
4.2.6.3 Propargyl 2,3,4-6-tetra-O-acetyl-α-D-mannopyranoside (IIc)
29
20
Yield: 50%; mp 101.5 – 103.1 °C (Lit. 103-104 ºC [81]). [α]D +48.7 ° (c 1.15; CHCl ). Lit. [α] +68.0
3
D
-1
-1
-1
-1
(c 1.00; CHCl [81]). IR: ῡ 3255 cm (C-H, sp), 2117 cm (C≡C), 1739 cm (C=O), 1367 cm (α-CH ),
3
3
-1
-1
1
1217–1231 cm (O-C=O), 1056–1078 cm (C-O). H NMR (400 MHz, CDCl ): 1.92−2.10 (s, 12H,
3
CH C=O), 2.43 (t, J = 2.4 Hz, 1H, H-9), 3.96 (ddd, J = 2.4 Hz, J = 5.2 Hz, J = 9.2 Hz, 1H, H-5), 4.05 (dd,
3
J =2.4 Hz, J =12.3 Hz, 1H, H-6a), 4.21 (d, J = 2.4 Hz, 2H, H-7), 4.22 (dd, J =5.1 Hz, J =12.4 Hz, 1H, H-
6b), 4.96 (d, J = 1.6 Hz, 1H, H-1), 5.20 (dd, J = 1.9 Hz, J = 3.1 Hz, 1H, H-2), 5.23−5.29 (m, 2H, H-3, H-
1
3
4). C NMR (100 MHz, CDCl ): 20.6−20.8 (4CH , CH C=O), 54.9 (CH , C-7), 62.3 (CH , C-6), 66.0
3
3
3
2
2
(CH, C-4), 68.9 (CH, C-2), 69.0 (CH, C-3), 69.4 (CH, C-5), 75.6 (CH, C-9), 77.9 (C , C-8), 96.2 (CH, C-
0
1).
5
69
4.2.7 General procedure for the synthesis of peracetylated propargyl cellobioside (III)
5
5
5
5
5
5
5
5
70
71
72
73
74
75
76
77
To a 100 mL round bottom flask containing activated 4A molecular sieves, Ag CO (0.64 g, 2.30 mmol),
2
3
AgOTf (0.06 g, 0.22 mmol) and dry dichloromethane (10 mL) were added. The flask was kept at -10 °C
for 20 min. Then, propargyl alcohol (0.6 g, 5.0 mmol) was added; the mixture was allowed to reach room
temperature and stirred for 30 min. Finally, hepta-O-acetyl-α- -cellobiosyl bromide (0.5 g, 0.72 mmol)
D
was added and the reaction mixture was stirred at room temperature for 24 h. The mixture was filtered
through Celite, transferred to a separatory funnel, washed with saturated aqueous NaHCO solution (3 ×
3
30 mL) and brine (3 × 50 mL), dried over anhydrous sodium sulfate and evaporated to dryness. The crude
propargyl cellobioside was recristallized from ethanol.
5
78
4.2.7.1 Propargyl 2,3,6,2´,3´,4´,6´-hepta-O-acetyl-β-D-cellobioside (III)

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5
5
5
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5
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79
80
81
82
83
84
85
86
87
88
89
Yield: 50%; mp 155.0 – 158.1 °C. [α]D -44.0 ° (c 0.50; CHCl ). Lit. [α] -52.2 ° (c 0.50; CHCl [82]).
3 D 3
-1
-1
3
-1
-1
-1
IR: ῡ 3282 cm (C-H, sp), 2938 cm (C-H, sp ), 1742 cm (C=O), 1366 cm (α-CH ), 1218 cm (O-
3
-1
1
C=O), 1039 cm (C-O). H NMR (400 MHz, CDCl ): 1.91−2.10 (s, 21H, CH C=O), 2.41 (t, J = 2.2 Hz,
3
3
1H, H-15), 3.55−3.62 (m, 2H, H-5, H-11), 3.73 (t, J = 9.6 Hz, 1H, H-8), 3.98 (dd, J = 1.9 Hz, J = 12.4 Hz,
1H, H-6a), 4.04 (dd, J = 4.9 Hz, J = 12.0 Hz, 1H, H-6b), 4.26 (d, J = 2.3 Hz, 2H, H-13), 4.29 (dd, J = 4.3
Hz, J =12.5 Hz, 1H, H-12a), 4.46 (d, J = 8.2 Hz, 2H, H-7, H-12b), 4.67 (d, J =7.9 Hz, 1H, H-1), 4.85 (t, J
=8.7 Hz, 2H, H-3, H-10), 4.99 (t, J =9.9 Hz, 1H, H-2), 5.08 (t, J = 9.3 Hz, 1H, H-4), 5.14 (t, J = 9.3 Hz,
13
1H, H-9). C NMR (100 MHz, CDCl ): 20.6−20.9 (7CH , CH C=O), 56.0 (CH , C-13), 61.7 (CH , C-6),
3
3
3
2
2
61.9 (CH , C-12), 68.1 (CH, C-2), 71.4 (CH, C-3), 71.8 (CH, C-10), 72.1 (CH, C-4), 72.6 (CH, C-5), 73.0
2
(CH, C-9), 73.1 (CH, C-11), 75.6 (CH, C-14), 76.4 (CH, C-8), 78.2 (CH, C-15), 98.1 (CH, C-1), 100.8
(CH, C-7), 169.1−170.8 (7C=O, CH C=O).
3
5
5
5
5
5
5
5
5
5
5
90
91
92
93
94
95
96
97
98
99
4.2.8 General procedure for the synthesis of peracetylated digitoxigenin triazolyl glycosides (6a-d)
To a 50 mL round bottom flask, 3β-azido-3-deoxydigitoxigenin (5, 0.16 mmol) dissolved in 1 mL of
tetrahydrofuran was added, followed by the appropriate propargyl glycoside (0.2 mmol) dissolved in 0.5
mL of tetrahydrofuran. Then, CuSO .5H O, 50% mol, dissolved in 0.5 mL of water and sodium ascorbate
4
2
(60 % mol) was dissolved in 1 mL of water and added in a stepwise manner. The reaction mixture was
stirred at room temperature for 4 h. After, the tetrahydrofuran was removed by distillation at reduced
pressure. The reaction residues were solubilized in 50 mL CH Cl , washed with 2 × 50 mL H O and
2
2
2
subsequently washed with 3 × 50 mL alkaline EDTA 20% w/v. The organic phase was dried over
Na SO , filtered and removed by distillation at reduced pressure. The derivatives 3a-d were added in
2
4
Florisil and purified by silica column chromatography using CH Cl : ethyl acetate /4:6 as eluent.
2
2
6
6
6
6
6
6
6
6
00
01
02
03
04
05
06
07
4.2.8.1 3β-[4-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxymethyl]-1,2,3-triazol-1-yl]-3-
deoxydigitoxigenin (6a)
2
6
-1
-1
Yield: 65%; mp 106.2 – 108.5 °C. [α]D -9.00 (c 2.00; CH Cl ). IR: ῡ 3412 cm (OH), 2935 cm (C-H
2
2
3
-1
-1
-1
-1
-1
1
sp ), 1746 cm (C=O), 1619 cm (C=C), 1374 cm (α-CH ), 1228 cm (O-C=O), 1038 cm (C-O). H
3
NMR (400 MHz, CDCl ): δ 0.89 (s, 3H, CH , H-18), 0.92 (s, 3H, CH , H-19), 1.17−2.24 (m, 20H, H-1,
3
3
3
H-2, H-4α, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-15, H-16), 1.98−2.09 (s, 12H, CH C=O), 2.32 (td, J
3
= 4.2 Hz, J = 14.2 Hz, 1H, H-4β), 2.79−2.82 (m, 1H, H-17), 3.76 (ddd, J = 2.1 Hz, J = 4.2 Hz, J = 9.8 Hz,
1H, H-5’), 4.15 (dd, J =1.9 Hz, J =12.4 Hz, 1H, H-6’a), 4.28 (dd, J =4.7 Hz, J =12.3 Hz, 1H, H-6’b), 4.71

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6
6
6
6
6
6
6
6
6
08
09
10
11
12
13
14
15
16
(d, J = 7.9 Hz, 2H, H-1’, H-3), 4.82 (d, J =16.2 Hz, 2H, H-7’a, H-21a), 4.93−5.03 (m, 3H, H-4’, H-7’b,
H-21b), 5.10 (t, J = 9.6 Hz, 1H, H-2’), 5.21 (t, J = 9.4 Hz, 1H, H-3’), 5.88 (s, 1H, H-22), 7.63 (s, 1H, H-
1
3
9’). C NMR (100 MHz, CDCl ): 15.9 (CH , C-18), 20.7−20.8 (4CH , CH C=O), 21.2 (CH , C-7), 21.4
3
3
3
3
2
(CH , C-11), 23.8 (CH , C-19), 25.1 (CH , C-6), 26.5 (CH , C-16), 27.0 (CH , C-2), 30.0 (CH , C-4),
2
3
2
2
2
2
30.6 (CH , C-1), 33.2 (CH , C-15), 35.2 (C , C-10), 36.4 (CH, C-9), 36.9 (CH, C-5), 40.0 (CH , C-12),
2
2
0
2
41.9 (CH, C-8), 49.8 (C , C-13), 51.0 (CH, C-17), 56.7 (CH, C-3), 62.0 (CH , C-6’), 63.2 (CH , C-7’),
0
2
2
68.5 (CH, C-2’), 71.5 (CH, C-4’), 72.0 (CH, C-5’), 72.9 (CH, C-3’), 73.6 (CH , C-21), 85.4 (C , C-14),
2
0
100.1 (CH, C-1’), 117.8 (CH, C-22), 169.5−170.8 (4C=O, CH C=O), 174.7 (C=O, C-23), 174.8 (C , C-
3
0
+
20); HRMS-ESI: calcd for C H N O [M+H] 786.8844, found: 787.45.
40
55
3
13
6
6
6
6
6
17
18
19
20
21
4.2.8.2 3β-[4-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxymethyl]-1,2,3-triazol-1-yl]-3-
deoxydigitoxigenin (6b)
2
6
-1
-1
Yield: 75%; mp 121.1 – 123.1 °C. [α]D -6.00 (c 2.00; CH Cl ). IR: ῡ 3455 cm (OH), 2938 cm (C-H
2
2
3
-1
-1
-1
-1
-1
1
sp ), 1739 cm (C=O), 1622 cm (C=C), 1368 cm (α-CH ), 1218 cm (O-C=O), 1045 cm (C-O).
H
3
NMR (400 MHz, CDCl ): δ 0.82 (s, 3H, CH , H-18), 0.86 (s, 3H, CH , H-19), 1.12−2.17 (m, 20H, H-1,
3
3
3
6
6
6
6
6
6
6
6
6
6
6
22
23
24
25
26
27
28
29
30
31
32
H-2, H-4α, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-15, H-16), 1.91−2.10 (s, 12H, CH C=O), 2.27 (t, J =
3
12.7 Hz, 1H, H-4β), 2.72−2.75 (m, 1H, H-17), 3.91 (t, J = 5.8 Hz, 1H, H-5’), 4.09 (d, J = 5.0 Hz, 2H, H-
6’), 4.62−4.98 (m, 3H, H-1’, H-3, H-3’, H-7’, H-21), 5.15 (dd, J = 8.0 Hz, J = 9.9 Hz, 1H, H-2’), 5.33 (d,
13
J = 2.7 Hz, 1H, H-4’), 5.81 (s, 1H, H-22), 7.60 (s, 1H, H-9’). C NMR (100 MHz, CDCl ): 15.9 (CH , C-
3
3
18), 20.6−20.9 (4CH , CH C=O), 21.2 (CH , C-7), 21.4 (CH , C-11), 23.8 (CH , C-19), 25.0 (CH , C-6),
3
3
2
2
3
2
26.4 (CH , C-16), 27.0 (CH , C-2), 30.0 (CH , C-4), 30.6 (CH , C-1), 33.2 (CH , C-15), 35.2 (C , C-10),
2
2
2
2
2
0
36.4 (CH, C-9), 36.9 (CH, C-5), 40.0 (CH , C-12), 41.8 (CH, C-8), 49.8 (C , C-13), 51.0 (CH, C-17), 56.9
2
0
(CH, C-3), 61.4 (CH , C-6’), 63.2 (CH , C-7’), 67.2 (CH, C-4’), 69.0 (CH, C-2’), 70.9 (CH, C-5’), 71.0
2
2
(CH, C-3’), 73.6 (CH , C-21), 85.4 (C , C-14), 100.7 (CH, C-1’), 117.8 (CH, C-22), 169.6−170.5 (4C=O,
2
0
+
CH C=O), 174.7 (C=O, C-23), 174.8 (C , C-20); HRMS-ESI: calcd for C H N O [M+H] 786.8844,
3
0
40 55
3
13
found: 787.58.
6
6
6
6
33
34
35
36
4.2.8.3 3β-[4-[(2,3,4,6-tetra-O-acetyl-α-D-mannopyranosyl)oxymethyl]-1,2,3-triazol-1-yl]-3-
deoxydigitoxigenin (6c)
2
6
-1
-1
Yield: 68%; mp 114.9 – 117.5 °C. [α]D +29.3 (c 2.12; CH Cl ). IR: ῡ 3458 cm (OH), 2940 cm (C-H
2
2
3
-1
-1
-1
-1
-1
1
sp ), 1740 cm (C=O), 1612 cm (C=C), 1364 cm (α-CH ), 1220 cm (O-C=O), 1044 cm (C-O). H
3

23
ACCEPTED MANUSCRIPT
6
37
NMR (400 MHz, CDCl ): δ 0.89 (s, 3H, CH , H-18), 0.94 (s, 3H, CH , H-19), 1.20−2.36 (m, 21H, H-1,
3 3 3
6
6
6
6
6
6
6
6
6
6
6
38
39
40
41
42
43
44
45
46
47
48
H-2, H-4, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-15, H-16), 1.99−2.16 (s, 12H, CH C=O), 2.81 (dd, J =
3
5.4 Hz, J = 8.8 Hz, 1H, H-17), 4.08−4.10 (m, 1H, H-5’), 4.12 (dd, J =2.3 Hz, J =12.3 Hz, 1H, H-6’a),
4.31 (dd, J =5.3 Hz, J =12.4 Hz, 1H, H-6’b), 4.67−5.03 (m, 6H, H-1’, H-3, H-7’, H-21), 5.24−5.34 (m,
13
3H, H-2’, H-3’, H-4’), 5.89 (s, 1H, H-22), 7.66 (s, 1H, H-9’). C NMR (100 MHz, CDCl ): 15.9 (CH , C-
3
3
19), 20.8−20.9 (4CH , CH C=O), 21.2 (CH , C-7), 21.4 (CH , C-11), 23.7 (CH , C-19), 25.0 (CH , C-6),
3
3
2
2
3
2
26.4 (CH , C-16), 27.0 (CH , C-2), 29.9 (CH , C-4), 30.6 (CH , C-1), 33.2 (CH , C-15), 35.2 (C , C-10),
2
2
2
2
2
0
36.4 (CH, C-9), 36.8 (CH, C-5), 40.0 (CH , C-12), 41.8 (CH, C-8), 49.8 (C , C-13), 51.0 (CH, C-17), 57.0
2
0
(CH, C-3), 61.1 (CH , C-6’), 62.5 (CH , C-7’), 66.2 (CH, C-4’), 68.8 (CH, C-2’), 69.2 (CH, C-5’), 69.6
2
2
(CH, C-3’), 73.6 (CH , C-21), 85.4 (C , C-14), 97.0 (CH, C-1’), 117.7 (CH, C-22), 169.8−170.8 (4C=O,
2
0
+
CH C=O), 174.7 (C=O, C-23), 174.8 (C , C-20); HRMS-ESI: calcd for C H N O [M+H] 786.8844,
3
0
40 55
3
13
found: 787.58.
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
4.2.8.4 3β-[4-[[4-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)-2,3,6-tri-O-acetyl-β-D-
glucopyranosyl]oxymethyl]-1,2,3-triazol-1-yl]-3-deoxydigitoxigenin (6d)
2
6
-1
-1
Yield: 70%; mp 104.2 – 107.1 °C. [α]D -4.05 (c 2.00; CH Cl ). IR: ῡ 3468 cm (OH), 2933 cm (C-H
2
2
3
-1
-1
-1
-1
-1
1
sp ), 1739 cm (C=O), 1619 cm (C=C), 1367 cm (α-CH ), 1219 cm (O-C=O), 1034 cm (C-O). H
3
NMR (400 MHz, CDCl ): δ 0.89 (s, 3H, CH , H-18), 0.92 (s, 3H, CH , H-19), 1.15−2.23 (m, 20H, H-1,
3
3
3
H-2, H-4α, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-15, H-16), 1.98−2.14 (s, 21H, CH C=O), 2.32 (td, J
3
= 4.2 Hz, J = 14.3 Hz, 1H, H-4β), 2.79−2.83 (m, 1H, H-17), 3.63−3.70 (m, 2H, H-5’, H-11’), 3.80 (t, J =
9.5 Hz, 1H, H-8’), 4.04 (dd, J =1.8 Hz, J =12.4 Hz, 1H, H-6’a), 4.12 (dd, J =4.8 Hz, J =12.0 Hz, 1H, H-
6’b), 4.37 (dd, J =4.3 Hz, J =12.5 Hz, 1H, H-12’a), 4.52−5.19 (m, 13H, H-1’, H-2’, H-3’, H-4’, H-7’, H-
13
9’, H-10’, H-3; H-12’b, H-13’, H-21’), 5.89 (s, 1H, H-22), 7.61 (s, 1H, H-15’). C NMR (100 MHz,
CDCl ): 15.9 (CH , C-18), 20.6−21.0 (7CH , CH C=O), 21.2 (CH , C-7), 21.4 (CH , C-11), 23.8 (CH ,
3
3
3
3
2
2
3
C-19), 25.0 (CH , C-6), 26.4 (CH , C-16), 27.0 (CH , C-2), 29.9 (CH , C-4), 30.5 (CH , C-1), 33.2 (CH ,
2
2
2
2
2
2
C-15), 35.2 (C , C-10), 36.3 (CH, C-9), 36.8 (CH, C-5), 40.0 (CH , C-12), 41.8 (CH, C-8), 49.8 (C , C-
0
2
0
13), 51.0 (CH, C-17), 56.7 (CH, C-3), 61.7 (CH , C-12’), 61.9 (CH , C-6’), 63.1 (CH , C-13’), 68.0 (CH,
2
2
2
C-2’), 71.6 (CH, C-9’), 71.7 (CH, C-4’), 72.0 (CH, C-11’), 72.6 (CH, C-5’), 72.9 (CH, C-10’), 73.0
(CH, C-3’), 73.6 (CH , C-21), 76.4 (CH, C-8’), 85.4 (C , C-14), 99.8 (CH, C-7’), 100.8 (CH, C-1’), 117.7
2
0

24
ACCEPTED MANUSCRIPT
6
6
65
66
(CH, C-22), 122.1 (CH, C-15’), 143.7 (C , C-14’), 169.2−170.6 (7C=O, CH C=O), 174.7 (C=O, C-23),
0 3
+
174.8 (C , C-20); HRMS-ESI: calcd for C H N O [M+H] 1075.1350, found: 1075.54.
0
52 71
3
21
6
67
4.2.9 General procedure for the synthesis of deacetylated digitoxigenin triazoly glycosides (7a-d)
To a solution of 0.1 g of the appropriate digitoxigenin triazolyl glycoside (0.13 mmol) of compounds 6a-c
6
6
6
6
6
68
69
70
71
72
and 0.093 mmol of 6d in 5 mL of methanol, LiOH.H O (0.60 mmol) dissolved in 2 mL of water were
2
+
added. The reaction mixture was stirred at 0 °C for 2 h, neutralized with resin Amberlite IRA 120 H ,
filtered, and the resin washed with methanol. Finally, the filtrate was concentrated in vacuo to furnish the
desired glycosides.
6
6
6
73
74
75
4.2.9.1 3β-[4-[(β-D-glucopyranosyl)oxymethyl]-1,2,3-triazol-1-yl]-3-deoxydigitoxigenin (7a)
2
5
-1
-1
Yield: 80%; mp >104 °C (decomp.). [α]D -0.96 (c 2.08; MeOH). IR: ῡ 3363 cm (OH), 2930 cm (C-H
3
-1
-1
-1
1
sp ), 1732 cm (C=O), 1621 cm (C=C), 1022 cm (C-O). H NMR (400 MHz, MeOD-d ): δ 0.89 (s,
4
6
6
6
6
6
6
6
6
6
6
6
76
77
78
79
80
81
82
83
84
85
86
3H, CH , H-18), 0.92 (s, 3H, CH , H-19), 1.05−2.28 (m, 20H, H-1, H-2, H-4α, H-5, H-6, H-7, H-8, H-9,
3
3
H-11, H-12, H-15, H-16), 2.44 (t, J = 13.8 Hz, 1H, H-4β), 2.85 (s, 1H, H-17), 3.21−3.38 (m, 4H, H-4’, H-
5’, H-6’), 3.69 (d, J = 11.0 Hz, 1H, H-3’), 3.75−3.84 (m, 1H, OH), 3.90 (d, J = 11.6 Hz, 1H, H-2’), 4.40
(d, J = 7.1 Hz, 1H, H-1’), 4.48−5.12 (m, 7H, H-3, H-7’, H-21, OH), 5.48 (s, 1H, OH), 5.91 (s, 1H, H-22),
1
3
8,12 (s, 1H, H-9’). C NMR (100 MHz, MeOD-d ): 16.5 (CH , C-18), 22.4 (CH , C-7), 22.6 (CH , C-
4
3
2
2
11), 24.4 (CH , C-19), 25.8 (CH , C-6), 27.7 (CH , C-16), 28.2 (CH , C-2), 30.9 (CH , C-4), 31.8 (CH ,
3
2
2
2
2
2
C-1), 33.6 (CH , C-15), 36.4 (C , C-10), 37.4 (CH, C-9), 38.5 (CH, C-5), 41.0 (CH , C-12), 42.8 (CH, C-
2
0
2
8), 51.2 (C , C-13), 52.2 (CH, C-17), 58.5 (CH, C-3), 62.9 (CH , C-6’), 63.3 (CH , C-7’), 71.8 (CH, C-
0
2
2
2’), 75.1 (CH, C-4’), 75.5 (CH , C-21), 78.1 (CH, C-5’), 78.2 (CH, C-3’), 86.4 (C , C-14), 103.7 (CH, C-
2
0
1’), 117.9 (CH, C-22), 124.8 (CH, C-9’), 177.4 (C=O, C-23), 178.6 (C , C-20); HRMS-ESI: calcd for
0
+
C H N O [M+H] 618.7377, found: 618.43.
3
2
47
3
9
6
6
6
6
6
6
87
88
89
90
91
92
4.2.9.2 3β-[4-[(β-D-galactopyranosyl)oxymethyl]-1,2,3-triazol-1-yl]-3-deoxydigitoxigenin (7b)
2
5
-1
-1
Yield: 88%; mp >138 °C (decomp.). [α]D -0.94 (c 2.12; MeOH). IR: ῡ 3354 cm (OH), 2932 cm (C-H
3
-1
-1
-1
1
sp ), 1732 cm (C=O), 1621 cm (C=C), 1024 cm (C-O). H NMR (400 MHz, MeOD-d ): δ 0.89 (s,
4
3H, CH , H-18), 0.92 (s, 3H, CH , H-19), 1.05−2.24 (m, 20H, H-1, H-2, H-4α, H-5, H-6, H-7, H-8, H-9,
3
3
H-11, H-12, H-15, H-16), 2.46 (dt, J = 3.2 Hz, J = 14.2 Hz, 1H, H-4β), 2.84−2.87 (m, 1H, H-17), 3.48
(dd, J = 2.9 Hz, J = 9.5 Hz, 1H, H-3’) 3.55−3.59 (m, 2H, H-6’), 3.72−3.82 (m, 3H, H-2’, H-5’, OH), 3.85

25
ACCEPTED MANUSCRIPT
6
6
6
6
6
6
6
7
93
94
95
96
97
98
99
00
(d, J = 2.3 Hz, 1H, H-4’), 4.36 (d, J = 8.9 Hz, 1H, H-1’), 4.50−5.07 (m, 8H, H-3, H-7’; H-21, OH), 5.91
1
3
(s, 1H, H-22), 8,12 (s, 1H, H-9’). C NMR (100 MHz, MeOD-d ): 16.5 (CH , C-18), 22.4 (CH , C-7),
4
3
2
22.6 (CH , C-11), 24.4 (CH , C-19), 25.8 (CH , C-6), 27.7 (CH , C-16), 28.2 (CH , C-2), 30.9 (CH , C-
2
3
2
2
2
2
4), 31.8 (CH , C-1), 33.6 (CH , C-15), 36.4 (C , C-10), 37.4 (CH, C-9), 38.5 (CH, C-5), 41.0 (CH , C-12),
2
2
0
2
42.8 (CH, C-8), 51.2 (C , C-13), 52.2 (CH, C-17), 58.5 (CH, C-3), 62.7 (CH , C-6’), 63.3 (CH , C-7’),
0
2
2
70.4 (CH, C-2’), 72.6 (CH, C-4’), 75.0 (CH, C-5’), 75.5 (CH , C-21), 76.9 (CH, C-3’), 86.4 (C , C-14),
2
0
104.4 (CH, C-1’), 117.9 (CH, C-22), 177.3 (C=O, C-23), 178.5 (C , C-20); HRMS-ESI: calcd for
0
+
C H N O [M+H] 618.7377, found: 618.62.
3
2
47
3
9
7
7
7
01
02
03
4.2.9.3 3β-[4-[(α-D-mannopyranosyl)oxymethyl]-1,2,3-triazol-1-yl]-3-deoxydigitoxigenin (7c)
2
5
-1
-1
Yield: 82%; mp >169 °C (decomp.). [α]D +21.82 (c 1.92; MeOH). IR: ῡ 3397 cm (OH), 2932 cm (C-
3
-1
-1
-1
1
H sp ), 1729 cm (C=O), 1626 cm (C=C), 1026 cm (C-O). H NMR (400 MHz, MeOD-d ): δ 0.89 (s,
4
7
7
7
7
7
7
7
7
7
7
04
05
06
07
08
09
10
11
12
13
3H, CH , H-18), 0.92 (s, 3H, CH , H-19), 1.13−2.30 (m, 20H, H-1, H-2, H-4α, H-5, H-6, H-7, H-8, H-9,
3
3
H-11, H-12, H-15, H-16), 2.45 (t, J = 12.6 Hz, 1H, H-4β), 2.85−2.87 (m, 1H, H-17), 3.60−3.97 (m, 6H,
H-2’, H-3’, H-4’, H-5’, H-6’), 4.52−4.87 (m, 4H, H-3, H-7’, H-1’), 4.92 (d, J = 18.4 Hz, 1H, H-21a), 5.04
13
(d, J = 18.3 Hz, 1H, H-21b), 5.91 (s, 1H, H-22), 8.16 (s, 1H, H-9’). C NMR (100 MHz, MeOD-d ): 16.5
4
(CH , C-18), 22.4 (CH , C-7), 22.6 (CH , C-11), 24.4 (CH , C-19), 25.8 (CH , C-6), 27.7 (CH , C-16),
3
2
2
3
2
2
28.2 (CH , C-2), 30.9 (CH , C-4), 31.8 (CH , C-1), 33.6 (CH , C-15), 36.4 (C , C-10), 37.4 (CH, C-9),
2
2
2
2
0
38.5 (CH, C-5), 41.0 (CH , C-12), 42.8 (CH, C-8), 51.2 (C , C-13), 52.3 (CH, C-17), 58.6 (CH, C-3), 61.1
2
0
(CH , C-7’), 63.2 (CH , C-6’), 68.8 (CH, C-2’), 72.2 (CH, C-4’), 72.7 (CH, C-3’), 75.1 (CH, C-5’), 75.5
2
2
(CH , C-21), 86.5 (C , C-14), 101.0 (CH, C-1’), 118.0 (CH, C-22), 177.3 (C=O, C-23), 178.5 (C , C-20);
2
0
0
+
HRMS-ESI: calcd for C H N O [M+H] 618.7377, found: 618.56.
3
2
47
3
9
7
7
7
7
7
7
7
7
14
15
16
17
18
19
20
21
4.2.9.4 3β-[4-[[4-O-(β-D-glucopyranosyl)-β-D-glucopyranosyl]oxymethyl]-1,2,3-triazol-1-yl]-3-
deoxydigitoxigenin (7d)
2
5
-1
-
Yield: 82%; mp >163 °C (decomp.). [α]D +2.10 (c 1.90; MeOH). IR: ῡ 3253 cm (OH), 2853–2923 cm
1
3
-1
-1
-1
-1
1
(C-H sp ), 1735 cm (C=O), 1670 cm (C=C), 1259 cm (O-C-O), 1023 cm (C-O). H NMR (400
MHz, MeOD-d ): δ 0.89 (s, 3H, CH , H-18), 0.92 (s, 3H, CH , H-19), 1.05−2.31 (m, 20H, H-1, H-2, H-
4
3
3
4α, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-15, H-16), 2.44 (td, J = 3.8 Hz, J = 14.2 Hz, 1H, H-4β),
2.85−2.87 (m, 1H, H-17), 3.23 (t, J = 8.4 Hz, 1H, H-11’), 3.33−3.60 (m, 8H, H-2’, H-3’, H-4’, H-5’, H-
8’, H-9’, H-10’, H-12’a), 3.66 (dd, J = 5.2 Hz, J = 11.8 Hz, 1H, H-6’a), 3.86−3.95 (m, 3H, H-3, H-6’b, H-

26
ACCEPTED MANUSCRIPT
7
7
7
7
7
7
7
7
7
22
23
24
25
26
27
28
29
30
12’b), 4.43 (t, J = 7.9 Hz, 2H, H-1’, H-7’), 4.90−5.07 (m, 4H, H-13’, H-21), 5.91 (s, 1H, H-22), 8.13 (s,
1
3
1H, H-15’). C NMR (100 MHz, MeOD-d ): 16.5 (CH , C-18), 22.4 (CH , C-7), 22.6 (CH , C-11), 24.4
4
3
2
2
(CH , C-19), 25.8 (CH , C-6), 27.7 (CH , C-16), 28.2 (CH , C-2), 30.9 (CH , C-4), 31.8 (CH , C-1), 33.6
3
2
2
2
2
2
(CH , C-15), 36.4 (C , C-10), 37.4 (CH, C-9), 38.5 (CH, C-5), 41.0 (CH , C-12), 42.8 (CH, C-8), 51.2
2
0
2
(C , C-13), 52.3 (CH, C-17), 58.6 (CH, C-3), 62.0 (CH , C-12’), 62.6 (CH , C-7’), 63.4 (CH , C-6’), 71.5
0
2
2
2
(CH, C-2’), 74.9 (CH, C-9’), 75.1 (CH, C-4’), 75.5 (CH , C-21), 76.5 (CH, C-11’), 76.7 (CH, C-5’), 78.0
2
(CH, C-10’), 78.2 (CH, C-3’), 80.9 (CH, C-8’), 86.5 (C , C-14), 103.6 (CH, C-7’), 104.8 (CH, C-1’),
0
+
118.0 (CH, C-22), 177.4 (C=O, C-23), 178.6 (C , C-20); HRMS-ESI: calcd for C H N O [M+H]
0
38 57
3
14
780.8783, found: 780.41.
7
7
7
7
7
7
7
7
7
7
7
31
32
33
34
35
36
37
38
39
40
41
4.2.10 Synthesis of 3β-amino-3-deoxydigitoxigenin (8)
300 mg of 3β-azido-3-deoxydigitoxigenin (5) (0.75 mmol, EQ 1) and 236 mg TPP (triphenylphosphine,
0.9 mmol, EQ 1.2) were dissolved in 5 mL of tetrahydrofuran. 1 mL of water was added and the reaction
was kept under reflux at 70 °C overnight. After, the reaction was dissolved in 80 mL dichloromethane
and the organic layer (CH Cl I, containing mainly the intermediate ring product) was extracted with 4 ×
2
2
100 mL with a mixture of 390 mL of H O and 10 mL 2 M HCl. The acid aqueous phases were extracted
2
with 8 × 30 mL dichloromethane and the organic phase (CH Cl II) was dried over anhydrous sodium
2
2
sulfate and evaporated. Next, the aqueous phases were combined, pH adjusted to 8-9 (13 to 15 mL of 3%
NH water solution) and submitted to extraction with dichloromethane (8 × 30 mL). The organic layer
3
was washed with 2 × 50 mL water (pH adjusted to 8 – 9 by 3% NH water solution), dried over
3
anhydrous sodium sulfate and evaporated to dryness furnish a yellow oil.
7
7
7
7
7
7
7
7
7
42
43
44
45
46
47
48
49
50
4.2.10.1 3β-amino-3-deoxydigitoxigenin (8)
26
20
Yield: 60%; mp 207.0 – 209.5 ºC (Lit: 216-217 °C [50]). [α]D +26.0 ° (c 1.00; acetone). Lit:[α]D +17.0
-1
-1
-1
-1
° (CHCl [50]). IR: ῡ 3356 cm (NH), 2862−2933 cm (C-H), 1740−1783 cm (C=O lactone), 1635 cm
3
-1
-1
1
(C=C), 1450 cm (N-H), 1036 cm (C-O). H NMR (400 MHz, CDCl ): δ 0.87 (s, 3H, CH , H-19), 0.92
3
3
(s, 3H, CH , H-18), 1.09 (td, J =3.6 Hz, J =14.2 Hz, 1H, H-1a), 1.16−1.58 (m, 8H, H-5, H-6α, H-7β, H-8,
3
H-11, H-12), 1.68−1.75 (m, 3H, H-2β, H-7α, H-9), 1.80−1.92 (m, 5H, H-1α, H-4α, H-6β, H-15α, H-16β),
2.03−2.06 (m, H, H-2α), 2.14−2.25 (m, 3H, H-4β, H-15β, H-16α), 2.79 (dd, J =6.0 Hz , J =8.6 Hz, 1H, H-
17), 4,06 (tt, J =4.4 Hz, J =12.0 Hz, 1H, H-3), 4.81 (dd, J =1.6 Hz, J =18.0 Hz, 1H, H-21a), 4.99 (dd, J
13
=1.2 Hz, J =18.0 Hz, 1H, H-21b), 5.88 (s, 1H, H-22). C NMR (400 MHz, CDCl ): 16.0 (CH , C-18),
3
3

27
ACCEPTED MANUSCRIPT
7
7
7
7
7
51
52
53
54
55
21.1 (CH , C-11), 21.7 (CH , C-7), 23.5 (CH , C-19), 27.1 (2CH , C-6, C-16), 33.5 (CH , C-15), 33.6
2 2 3 2 2
(CH , C-2), 34.9 (C , C-10), 36.5 (CH, C-9), 37.9 (CH , C-1), 39.1 (CH , C-4), 40.1 (CH , C-12), 42.1
2
0
2
2
2
(CH, C-8), 44.8 (CH, C-5), 49.8 (C , C-13), 51.1 (CH, C-17), 52.8 (CH, C-3), 73.6 (CH , C-21), 85.6 (C ,
0
2
0
C-14), 117.9 (CH, C-22), 174.6 (C , C-20), 174.7 (C=O, C-23); HRMS-ESI: calcd for C H NO
3
0
23 35
+
[M+H] 374.5363, found: 374.35
7
7
7
7
7
7
7
56
57
58
59
60
61
62
4.2.11 Synthesis of 3β-(chloroacetyl)amino-3-deoxydigitoxigenin (9)
A suspension of 100 mg of 3β-amino-3-deoxydigitoxigenin (8) (0.27 mmol) in 2 mL tetrahydrofuran was
added dropwise over 30 min to a stirred mixture of [48 µL chloroacetyl chloride (0.6 nmol) and 149 mg
K CO (1.08 mmol) in tetrahydrofuran (200 µL)] at room temperature. Next, the reaction mixture was
2
3
stirred for 18 h at room temperature, filtrated through cotton to remove the K CO and diluted with 80 mL
2
3
CH Cl . Finally, the organic layer was washed 3 × 30 mL of water, dried over anhydrous sodium sulfate
2
2
and evaporated.
7
7
7
7
7
63
64
65
66
67
4.2.11.1 3β-(Chloroacetyl)amino-3-deoxydigitoxigenin (9)
2
6
-1
Yield: 95%; mp 221.0-223.3 ºC. [α]D +8.0 ° (c 0.50; acetone). IR: ῡ 3335–3455 cm (N-H amide),
-1
-1
-1
-1
-1
2865–2932 cm (C-H), 1732 cm (C=O), 1677 cm (C=O amide), 1615 cm (C=C), 1532 cm (N-H),
-1
-1
1
1019 cm (C-O), 779 cm (C-Cl). H NMR (400 MHz, acetone-d ): δ 0.91 (s, 3H, CH , H-18), 0.96 (s,
6
3
3H, CH , H-19), 1.23−1.96 (m, 18H, H-1, H-2, H-4β, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-15β, H-
3
7
7
7
7
7
7
7
7
7
68
69
70
71
72
73
74
75
76
16β), 2.09−2.28 (m, 3H, H-4α, H-H-15α, H-16α), 2.85−2.87 (m, 1H, H-17), 3.26 (s, 1H, OH), 4.04 (s,
2H, H-2’), 4.13 (t, J =3.2 Hz, 1H, H-3), 4.85 (dd, J =1.7 Hz, J =18.1 Hz, 1H, H-21a), 5.01 (dd, J =1.3 Hz,
13
J =18.1 Hz, 1H, H-21b), 5.86 (dd, J =1.2 Hz, J =1,6 Hz, 1H, H-22), 7.33 (d, J = 5.5 Hz, 1H, N-H).
C
NMR (400 MHz, acetone-d ): 16.3 (CH , C-18), 22.0 (CH , C-11), 22.2 (CH , C-7), 24.2 (CH , C-19),
6
3
2
2
3
25.4 (CH , C-2), 27.7 (2CH , C-6, C-16), 31.1 (CH , C-4), 31.6 (CH , C-1), 33.6 (CH , C-15), 36.2 (C ,
2
2
2
2
2
0
C-10), 36.3 (CH, C-9), 38.2 (CH, C-5), 40.5 (CH , C-12), 42.6 (CH, C-8), 43.8 (CH , C-2’), 46.7 (CH,
2
2
C-3), 50.6 (C , C-13), 51.9 (CH, C-17), 74.0 (CH , C-21), 85.5 (C , C-14), 117.9 (CH, C-22), 166.0
0
2
0
+
(C=O, C-1’), 174.5 (C=O, C-23), 176.4 (C , C-20); HRMS-ESI: calcd for C H ClNO [M+H]
0
25 36
4
451.0180, found: 450.50 and 452.44

28
ACCEPTED MANUSCRIPT
7
77
4.2.12 Synthesis of 3β-[(N-(2-hydroxyetil)aminoacetyl]amino-3-deoxydigitoxigenin (10)
7
7
7
7
7
7
7
7
78
79
80
81
82
83
84
85
To a solution of 50 mg (0.11 mmol) of 3β-(Chloroacetyl)amino-3-deoxydigitoxigenin (9) in 5 mL of
THF, 24 mg (0.39 mmol) ethanolamine were added and stirred for 72 h at room temperature. Then, the
reaction mixture was diluted with 80 mL of CH Cl , washed with H O (3 ×10 mL), dried over anhydrous
2
2
2
sodium sulfate and evaporated to residue under reduced pressure. The silica gel (0.04 – 0.63 mm; 15 g to
reaction mixture) was washed with the following solvents of different polarities: ethyl acetate:hexane 7:3,
(20 × 10 mL fractions), ethyl acetate (20 × 10 mL fractions) ‰ ethyl acetate: acetone 8:2 (20 × 10 mL
fractions) ‰ ethyl acetate: acetone 1:1 (20 × 10 mL fractions) ‰ ethyl acetate: acetone 2:8 (20 × 10 mL
fractions) ‰ acetone (20 × 10 mL fractions). Compound 10 eluted in fractions 80-105.
7
7
7
7
7
86
87
88
89
90
4.2.12.1 3β-[(N-(2-hydroxyetil)aminoacetyl]amino-3-deoxydigitoxigenin (10)
2
5
-1
-1
Yield: 85%; mp 88.0-89.4 ºC. [α]D +8.3 ° (c 0.24; acetone). IR: ῡ 3304 cm (O-H), 2863–2940 cm (C-
-1
-1
-1
-1
1
H), 1719-1755 cm (C=O), 1643 cm (C=O amide), 1544 cm (N-H), 1024-1067 cm (C-O). H NMR
(400 MHz, acetone-d ): δ 0.78 (s, 3H, CH , H-18), 0.85 (s, 3H, CH , H-19), 1.00−1.81 (m, 19H, H-1, H-
6
3
3
2, H-4β, H-5, H-6, H-7, H-8, H-9, H-11, H-12, H-15α, H-16β), 2.00−2.15 (m, 3H, H-4α, H-15β, H-16α),
2.70-2.74 (br, 1H, H-17), 2.91 (t, J =6.5 Hz, 1H, H-3’), 2.96 (s, 1H, H-2’), 3.12−3.15 (m, 2H, N-H),
3.41−3.46 (m, 2H, OH), 3.73 (t, J =6.5 Hz, 1H, H-4’), 3.98 (s, 1H, H-3), 4.72 (d, J =18.1 Hz, 1H, H-21a),
7
7
7
7
7
7
7
7
7
91
92
93
94
95
96
97
98
99
13
4.88 (d, J =18.1 Hz, 1H, H-21b), 5.73 (s, 1H, H-22). C NMR (400 MHz, acetone-d ): 16.3 (CH , C-18),
6
3
22.1 (CH , C-11), 22.3 (CH , C-7), 24.6 (CH , C-19), 25.8 (CH , C-6), 27.7 (CH , C-16), 27.8 (CH , C-
2
2
3
2
2
2
2), 31.5 (CH , C-4), 32.1 (CH , C-1), 33.7 (CH , C-15), 36.3 (C , C-10), 36.4 (CH, C-9), 38.9 (CH, C-5),
2
2
2
0
40.5 (CH , C-12), 42.6 (CH, C-8), 45.4 (CH, C-3), 50.6 (C , C-13), 51.9 (CH, C-17), 52.2 (CH , C-3’),
2
0
2
54.1 (CH , C-2’), 64.0 (CH , C-4’), 74.0 (CH , C-21), 85.5 (C , C-14), 117.9 (CH, C-22), 169.6-169.7
2
2
2
0
+
(C=O, C-1’), 174.5 (C=O, C-23), 176.3 (C , C-20); HRMS-ESI: calcd for C H N O [M+H] 475.6402,
0
27 42
2
5
found: 475.48
8
00
4.2.13 Synthesis of 3β-(hydroxyacetyl)amino-3-deoxydigitoxigenin (11)
8
8
8
8
8
01
02
03
04
05
To a solution of 5 mg (0.011 mmol) of 3β-(Chloroacetyl)amino-3-deoxydigitoxigenin (9) in 500 µL
acetonitrile, 50 mg KI (0.3 mmol) in 100 µL H O and 50 µL of DIPEA were added. The reaction was
2
stirred for 120 h at 70 °C. Next, the reaction mixture was diluted with 80 mL of CH Cl , washed 3 × 10
2
2
mL 2M HCl and 3 × 10 mL H O, dried over anhydrous sodium sulfate and evaporated. Compound 11
2
was purified by flash column chromatography. The silica gel (0.04 – 0.63 mm; 10 g to 30 g reaction