Synthesis, Cytotoxicity, and Leishmanicidal Evaluation of Ent-beyerene and Ent-kaurene Derivatives

Article abstract of DOI:10.1002/ejoc.202001424

Cutaneous leishmaniasis (CL) is an infectious disease endemic of tropical and subtropical countries, for which current pharmacologic treatments have serious drawbacks. Therefore, new and improved medical treatments are required. On the other side, natural

Full text of DOI:10.1002/ejoc.202001424

European Journal of Organic Chemistry
Accepted Article
Accepted Article
Title: Synthesis, cytotoxicity and leishmanicidal evaluation of ent-
beyerene and ent-kaurene derivatives
Authors: Jilmar Andres Murillo, Luis Fernando Echeverri, Wiston
Escobar Quinones, Luis Fernando Torres, Laura Isaza
Pineda, Sara Maria Robledo, Tatiana Pineda, Horacio Olivo,
and Gustavo Adolfo Escobar
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.202001424
Link to VoR: https://doi.org/10.1002/ejoc.202001424
01/2020

10.1002/ejoc.202001424
European Journal of Organic Chemistry
FULL PAPER
Synthesis, cytotoxicity and leishmanicidal evaluation of ent-
beyerene and ent-kaurene derivatives
Jilmar. A. Murillo†, Fernando Echeverri, [a] Winston Quinones, [a] Fernando Torres, [a] Laura Isaza, [a] Sara
M. Robledo, [b] Tatiana Pineda, [b] Horacio F. Olivo, [c] Gustavo A. Escobar*[a]
[a]
Prof. Dr. F. Echeverri, Prof. Dr. W. Quiñones, Prof. Dr. F. Torres, L. Isaza, Prof. Dr. G. A. Escobar.
Grupo de Química Orgánica de Productos Naturales, Instituto de Química, Universidad de Antioquia, Calle 70 # 52-21, Medellín, Colombia.
E-mail: gustavo.escobar1@udea.edu.co
Web: https://orcid.org/0000-0002-5387-4356
[b]
[c]
†
Prof. Dr. S. M. Robledo, T. Pineda.
PECET, Facultad de Medicina, Universidad de Antioquia, Calle 70 # 52-21, Medellín, Colombia
Prof. Dr. H. Olivo.
Medicinal and Natural Products Chemistry, The University of Iowa, Iowa City, IA 52242, USA
J. A. Murillo, deceased.
Supporting information for this article is given via a link at the end of the document.
[1]
Abstract: Cutaneous leishmaniasis (CL) is an infectious disease
endemic of tropical and subtropical countries. Current pharmacologic
treatments have serious drawbacks. For these reasons, we need new
and improved medical treatments. On the other side, natural products
have been a valuable source of bioactive molecules, but unlike other
products of natural origin, diterpenoids are molecules with only a few
studies as antiparasitic drugs. Continuing our work on syntheses of
beyerenes, and focusing on obtaining more compounds with
antiparasitic activity, we describe in this work the syntheses and
activity of several ent-beyerene and -kaurene type of compounds
against CL, and also their toxicity. Utilizing classical reactions such as
Grignard, Mitsunobu and Williamson, among others, it was possible
to modify rings A and D of ent-beyerenes, specifically C-19 on ring A,
and C-15 and C-16 on ring D. A total of 40 new compounds were
obtained, 21 of them being active against Leishmania (Viannia)
braziliensis with median effective concentration (EC50) values less
than 25.0 g/mL and of these with EC50 values ranging from 1.8 to 7.1
g/mL and Selectivity Index (SI) varying from 1.1 to 5.1. All
compounds showed to be highly cytotoxic at concentrations less than
100 g/mL, except compound 37 that was cytotoxic at 279.7 g/mL
and moderately active against L. braziliensis (EC50 = 33.5 g/mL and
an SI of 8.4. All active compounds were ent-beyerene type.
development of new and better alternatives for their treatment.
One of these therapeutic alternatives are compounds of natural
origin, since several of them such as alkaloids, quinones,
flavonoids and terpenes, among others, have shown promising
leishmanicidal activity. [6,7]
In this regard, diterpenes have been little explored as
leishmanicidal; for this reason, we recently reported the
semisynthesis and leishmanicidal activity of several diterpenes
as well as other
derivatives. One of these compounds exhibited good in vivo
activity against Leishmania (V) braziliensis applied as a topical
[8]
isolated from the plant Baccharis tola,
[8]
cream, with a 50% healing. These compounds were obtained
from stevioside, a low cost and abundant raw material, thereby
ensuring the quantities necessary to prepare more derivatives
optimize a bioactive structure and carry out more confirmatory
tests in animal models and eventually in clinical trials.
To diversify the number of bioactive substances and have a
broader arsenal of leishmanicidal molecules, in this work we
report obtaining other derivatives of the ent-beyerene and ent-
kaurene type from the same raw material, stevioside.
Syntheses of ent-beyerene and ent-kaurene type derivatives
Introduction
Leishmaniasis is a disease transmitted by phlebotomines infected
Through chemical transformations from stevioside (1), several
derivatives of both ent-beyerene and ent-kaurene types were
synthesized, some of them reported for the first time, which
present similar structures, since they possess a ring system
(ABCD), [9,10] differing only by the configuration of the D ring and
the endo and exocyclic position of the double bond (Scheme 1).
[1]
with protozoa from the genus Leishmania.
Depending on the
organ the infection occurs, there are three clinical manifestations
of the disease, Visceral Leishmaniasis (VL), Mucocutaneous (ML)
and Cutaneous Leishmaniasis (CL), the latter the one with the
highest incidence. [2] This disease is endemic to 98 countries, the
most affected regions being Africa, Asia, and Latinamerican; [3] in
the latter, the cutaneous form is the most predominant, for this
reason, WHO recommends the search for topically applied
medications and considers it a Neglected Tropical Disease
(NTD). Around 1.2 million new cases occur annually in the world,
a prevalence that is increasing because of climate change,
deforestation and massive population movements, which lead to
[4]
the alteration of the ecology and habitat of the vector,
thus
increasing the number of transmission foci. Between 26,000 and
Scheme 1. Chemical structures of stevioside (1), steviol (42), isosteviol (2) and
ent-beyer-15-en-19-ol (3).
65,000 deaths are reported per year from VL. [1]
Chemotherapy for leishmaniasis is very deficient. Most of the
available medicines present serious problems, such as toxicity,
long treatments, usually hospital staying, resistance or low
effectiveness. Currently, a few molecules are available to treat the
different clinical forms of the disease: pentavalent antimony,
Results and Discussion
1. Syntheses of Isosteviol Derivatives.
1.1 Ring A Modification
miltefosine, amphotericin B, pentamidine isethionate and
[1,5]
paramomycin.
Due to the risks associated with these
compounds, the WHO established as a priority the search and
1
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10.1002/ejoc.202001424
European Journal of Organic Chemistry
FULL PAPER
Hydrolysis in acidic media of stevioside (1) generated isosteviol
[8]
(2) in a 75% yield.
Thus, structural modifications in ring A,
particularly position C-19 of isosteviol allowed the preparation of
various derivatives (Figure 1). Utilizing Steglich reaction
(DCC/DCM) secondary amides 4 and 5, primary amide 6 and
thioester 7 were obtained. Acyl fluoride 8 was prepared by
reacting isosteviol in the presence of diethylamino sulfur trifluoride
(DAST). [11]
Figure 3. Derivatives of ent-beyer-15-en-19-oic acid on position C-19.
Butylketone 19 was obtained when the 16-tosylhydrazone of 2a
was treated with an excess of nBuLi in THF (Figure 4).
Figure 1. Isosteviol derivatives, ring A, C-19 position.
Figure 4. Formation of butyl ketone (19)
1.2 Ring D Modification
[8]
Carboxylic acid 2b was treated with LiAlH4
to generate ent-
For the transformation of ring D of isosteviol, derivatives were
beyer-15-en-19-ol (3), one of the diastereomers present in the
plant Baccharis tola and from which 22 compounds were
prepared.
initially prepared from the acid
2 or the ethyl ester 2a
((COCl)2/DMF,EtOH), which allowed the preparation, via Baeyer
Villiger oxidation, lactones 9 and 10 (Figure 2). Likewise, oxidation
of ethyl ester 2a with SeO2 yielded diketone 11, [12] and treatment
with HCHO in the presence of CH3CH2ONa/CH3CH2OH yielded
diol 12 [13]. Ethyl ester 2a was reduced with NaBH4/MeOH
delivering the corresponding C-16 alcohol, which was then
acetylated to ester 13, the same treatment was applied to amide
5, yielding alcohol 14. Keto ethyl ester 2a and its C-15 oxime were
reduced with LiAlH4/THF to generate alcohol 15 and
hydroxylamine 16, respectively.
Sulfones 20 and 21 are the products of the reaction between
beyerenol 3 and mesyl and tosyl chlorides respectively, both
reactions in the presence of triethylamine, and dichloromethane
as solvent. Beyerenol acetate 22 was also generated with acetic
anhydride; and methyl 23 and benzyl 24 ethers by Williamson's
reaction with THF as solvent and sodium hydride as base. Again,
beyerenol 3 allowed the preparation of benzoyl beyerenol 25
when it was treated with benzoyl chloride in basic media. The
products obtained are shown in Figure 5.
Figure 5. Derivatives of ent-beyer-15-en-19-ol on position C-19.
Beyerenol 3 via a Mitsunobu reaction (PPh3, DIAD, DPPA in
[15]
THF),
was converted to azide 26, which in turn enabled
beyerenamine 27 formation by Staudinger reduction (PPh3/
MeOH); later this azide 26 reacted with the acetic and maleic
anhydride to generate the corresponding beyerenamides 28 and
29. Acrylamide 30 was also obtained by Steglich reaction (DCC /
DCM) of azide 26 with acrylic acid (Figure 6).
Figure 2. Ring D isosteviol derivatives, positions C-15 and C-16.
2. Syntheses of ent-Beyer-15-en-19-ol (3) derivatives
2.1 Functionalization of Ring A
Figure 6. Derivatives of ent-beyer-15-en-19-azide on position C-19.
The oxidation of beyerenol 3 via Sarret reaction (PCC/DCM)
yielded the corresponding beyerenal (31) which was utilized as
an intermediate for the preparation of alcohols and amines on
position C-19. Therefore, beyerenal (31) was transformed into the
secondary alcohols (32-36) through the reaction with different
Grignard reagents. Olefin reduction of phenylbeyerenol 36 gave
the saturated phenylalcohol 37 (Figure 7).
Isosteviol (2) was transformed, via its 16-tosylhydrazone
[14]
derivative followed by a Bamford-Stevens reaction
to the
corresponding carboxylic acid 2b from which phenylamide 17 and
N,N-dimethylamide 18 were obtained via the corresponding acid
chloride (Figure 3).
2
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10.1002/ejoc.202001424
European Journal of Organic Chemistry
FULL PAPER
73.8 ± 14.16 (179.70 ±
34.5)
7
73.3 ± 9.2 (178.5 ± 22.5)
1.0
8
9
11.7 ± 0.74 (36.60 ± 2.3)
13.0 ± 3.34 (38.93 ± 9.9)
10.1 ± 0.4 (27.9 ± 1.0)
4.5 ± 0.2 (14.0 ± 0.6)
12.4 ± 2.4 (37.1 ± 7.2)
3.9 ± 0.3 (10.8 ± 0.7)
2.6
1.1
2.6
Figure 7. Derivatives of ent-beyer-15-en-19-al on-position C-19.
10
Through reductive amination reactions [16] utilizing NaBH3CN and
methanol, beyerenal 31 allowed the preparation of amines (38-
40), with yields between 48-75% (Figure 8).
126.6 ± 21.4 (351.3 ±
59.5)
11
49.9 ± 10.9 (138.4 ± 30.4)
0.4
12
13
14
10.2 ± 1.8 (27.0 ± 4.7)
60.2 ± 2.2 (154.0 ± 5.6)
21.9 ± 3.9 (63.0 ± 11.1)
7.1 ± 1.1 (18.8 ± 2.8)
18.4 ± 2.3 (47.1 ± 5.9)
28.3 ± 3.4 (81.4 ± 9.6)
1.4
3.3
0.8
145.8 ± 37.7 (475.6 ±
122.9)
15
33.3 ± 1.1 (108.6 ± 3.7)
4.4
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
5.3 ± 0.0 (16.4 ± 0.1)
46.8 ± 5.2 (123.8 ± 13.7)
64.6 ± 10.0 (195.9 ± 30.4)
96.5 ± 6.9 (281.6 ± 20.4)
14.5 ± 0.5 (38.3 ± 1.3)
13.0 ± 0.1 (29.4 ± 0.3)
14.1 ± 1.2 (42.5 ± 3.6)
49.3 ± 12.6 (163.0 ± 41.8)
10.1 ± 0.2 (26.8 ± 0.5)
153.8 ± 4.1 (406.4 ± 10.7)
62.6 ± 2.8 (199.8 ± 8.9)
22.8 ± 0.2 (79.2 ± 0.6)
29.0 ± 1.1 (88.00 ± 3.2)
10.9 ± 0.9 (28.2 ± 2.2)
11.6 ± 0.6 (34.0 ± 1.7)
3.2 ± 0.4 (9.9 ± 1.3)
90.03 ± 2.5 (238.5 ± 6.5)
108.2 ± 5.3 (328.3 ± 15.9)
>50 ± NA (>146)
1.7
0.5
0.6
<1.9
2.3
0.8
1.1
0.6
0.5
<3.1
1.0
0.4
2.7
2.5
0.7
Figure 8. Derivatives of ent-beyer-15-en-19-al on-position C-19.
3. Derivatives of steviol.
Steviol (41) was obtained utilizing an aqueous solution of
stevioside 2 treated with NaIO4 and then with KOH, [17] which was
reduced with LiAlH4 to yield diol (Figure 9).
6.2 ± 0.3 (16.4 ± 0.9)
16.3 ± 3.8 (36.9 ± 8.7)
12.9 ± 2.1 (38.9 ± 6.4)
86.9 ± 9.0 (287.4 ± 29.9)
19.3 ± 4.2 (51.1 ± 11.2)
>50 (>132)
Figure 9. Preparation of steviol 41 from stevioside 1.
61.1 ± 4.2 (194.9 ± 13.5)
54.0 ± 1.9 (187.9 ± 6.8)
10.8 ± 1.2 (32.7 ± 3.6)
4.4 ± 0.2 (11.3 ± 0.5)
16.8 ± 4.7 (49.2 ± 13.8)
4. Cytotoxicity and antileishmanial activity.
Of the synthesized and evaluated molecules, 22 compounds
showed high activity against L. brazilensis at concentrations
below 25 µg/mL. Of these, 11 in a range between 1 and 10 µg/mL
(3, 4, 8, 10, 12, 16, 20, 29, 38, 39, 40), 9 in the range of 10 to 19
µg/mL (9, 13, 21, 22, 24, 28, 30, 33, 35) and 1 in the range of 20
to 25 µg/mL (42) (Table 1). Five compounds showed moderate
activity in a range of 26 to 49 µg/mL. The rest of the compounds
showed low activity, being active at concentrations between 50
and 126 µg/mL. Of the compounds with high activity, all are ent-
beyerene type and none are ent-kaurene type. All compounds
showed to be highly cytotoxic at concentrations less than 100
µg/mL, except compound 37 is potentially non-toxic (279.70
µg/mL) also with a selectivity index (SI) greater than 8.
Compounds 15, 25, and 32, all ent-beyerene, showed moderate
cytotoxicity at concentrations of 145, 153, and 138 µg/mL
respectively (Table 1).
138.4 ± 11.8 (437.2 ±
37.2)
32
33
34
35
36
28.1 ± 0.5 (88.7 ± 1.6)
12.4 ± 0.8 (37.5 ± 2.3)
>5 (>14.5)
4.9
2.9
36.2 ± 10.2 (109.5 ± 30.9)
9.3 ± 0.5 (26.9 ± 1.6)
<1.8
5
39.82 ± 3.8 (121.2 ± 11.6)
12.7 ± 0.8 (38.5 ± 2.5)
49.9 ± 7.6 (137.1 ± 20.7)
3.2
2.2
108.5 ± 11.8 (297.5 ±
32.3)
Table 1. In vitro cytotoxicity and antileishmanial activity of several ent-beyerene
and ent-kaurene derivatives.
279.7 ± 62.6 (762.9 ±
170.7)
37
33.5 ± 2.2 (91.4 ± 6.0)
8.4
Cytotoxicity [LC50
Comp.
Antileishmanial activity
[EC50 µg/mL (µM)]
SI
µg/mL (µM)]
38
39
40
5.1 ± 0.0 (18.9 ± 0.0)
5.4 ± 0.0 (16.9 ± 0.0)
5.1 ± 0.0 (13.5 ± 0.0)
1.9 ± 0.1 (7.2 ± 0.3)
1.9 ± 0.0 (5.9 ± 0.1)
1.8 ± 0.0 (4.9 ± 0.0)
2.6
2.8
2.8
1
4
5
6
19.3 ± 1.3 (66.9 ± 4.4)
14.9± 0.9 (36.1 ± 2.2)
22.6 ± 4.1 (65.5 ± 11.8)
21.4 ± 6.3 (67.3 ± 19.9)
6.1 ± 0.63 (21.0 ± 2.2)
8.7 ± 1.2 (20.9 ± 2.8)
3.2
1.7
0.6
0.2
38.5 ± 9.7 (111.3 ± 27.9)
99.4 ± 9.3 (313.0 ± 29.2)
138.1 ± 11.4 (433.6 ±
35.9)
41
60.0 ± 3.6 (188.5 ± 11.2)
2.3
3
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10.1002/ejoc.202001424
European Journal of Organic Chemistry
FULL PAPER
compounds showed high in vitro activity against L. braziliensis,
despite being relatively poorly functionalized molecules. Electron
withdrawal groups such as amines and alcohols at C-19, have
higher leishmanicidal activity and toxicity when are compared to
carboxylic acid derivatives and carbonyl groups in this same
position. Likewise, the leishmanicidal effect of bulky groups in ring
A is evident.
42
32.3 ± 7.3 (106.0 ± 23.9)
36.6 ± 8.0 (39.6 ± 8.6)
24.2 ± 6.3 (79.4 ± 20.6)
0.3 ± 0.1 (0.3 ± 0.1)
1.3
AmB
122
The data represents the median Lethal Concentration (LC50) mean value for
each compound SD evaluated in the cell lines U937 and Effective
±
Concentration 50 (EC50) in μM for each compound ± DS in L. brazilensis
(intracellular amastigotes). SI: LC50/EC50. AmB: amphotericin B.
Except for the compounds (6, 11, 14, 17, 18, 21, 23, 24, 26, 27
and 30), the LC50/EC50 ratio that determines the SI was favorable,
that is, compounds were obtained more active than cytotoxic,
enabling its possible use as leishmanicidal, and especially
compound 37, which turned out to be potentially non-toxic. The
presence or absence of the double bond in C-15 revealed
contradictory results, however, more molecules are required to
determine its importance in the biological profile.
5. Discussion.
From a commercial raw material, such as stevioside, it was
possible to generate a series of derivatives that allowed its
evaluation as probable leishmanicidal agents in addition to its
toxicity. In general, these derivatives were formed with acceptable
yields and involved widely used methodologies and reagents.
Twenty-one of the 39 synthesized compounds showed high
leishmanicidal activity, even at concentrations below 10 µM, as is
the case of compounds 16 (9.9 µM), 38 (7.2 µM), 39 (5.9 µM) and
40 (4.9 µM), all ent-beyerene derivatives.
Although some of the compounds obtained are more active
than ent-Beyer-15-en-19-ol (3), this is projected as a good
alternative for treatment against CL, since it can be generated
abundantly at a relatively low cost given the source, reagents and
methodologies used for its production.
Being
a nucleus with few functional groups, these are
determinants in the activity and toxicity. The structure-activity
analysis shows that the presence of carbonyls in C-15 and C-16
decreases the activity when compared with derivatives that have
different groups, which is also affected by groups that increase
the electron density on C-19 such as esters, thioesters, and
amides. This trend seems to be confirmed with the fluorinated
derivative 8, the most active in this group. In the case of the
amides (4, 5 and 6), the chain length significantly affects the
activity, since it goes from 313.0 µM for compound 6 to 20.9 µM
for compound 4 (11.4 times more active).
Compounds with a double bond in the D ring, the presence of
carbonyls at C-19 (ketones or acid derivatives) decrease the
activity, as observed in the previous case. If, instead of carbonyls
in this position, functional groups such as amines and alcohols
are coupled, the activity profile improves significantly; however,
both groups show a different trend. For alcohols, none of the
derivatives improved the activity profile, except for mesylated
alcohol (20), which was slightly more active (22.2 µM vs. 16.4 µM).
With amines and their derivatives the situation is different since
most of the derivatives exhibited a better activity profile compared
to the primary amine (27); when this is transformed to the amides
(28, 29 and 30), the activity is increased, however, the secondary
amines (38, 39 and 40) with alkyl or benzyl type electron donor
groups showed a considerable increase in activity, since they
produced approximately 30 times more active compounds, they
were in fact, the most active in the entire series.
Experimental Section
All commercially available reagents and solvents were obtained from
commercial suppliers and used without further purification. Commercial
stevia (100%) was purchased from Amazon (Wholesale Health
Connection, WHC, 502 Fredericksburg Rd., San Antonio, TX 78201, USA).
The reaction progress was monitored with thin layer chromatography on
silica gel TLC aluminum sheets (60F254, Merck, Darmstadt, Germany)
and visualized with phosphomolybdic acid solution in EtOH on heating at
105 °C. The melting points were determined using a Mel-Temp apparatus
(Electrothermal, Staffordshire, UK) and are uncorrected.
FTIR spectra were obtained on a Bruker Alpha FTIR spectrometer (Bruker
Optic GmbH, Ettlingen, Germany). 1H- and 13C-nuclear magnetic
resonance (NMR) spectra were recorded using Bruker Fourier 300
spectrometer (Bruker Bio-Spin GmbH, Rheinstetten, Germany) operating
at 300 MHz for 1H and 75 MHz for 13C. Samples were dissolved in different
solvent CDCl3, DMSO and MeOD, using TMS as internal standard.
In vitro cytotoxicity in human U937 macrophages.
Human promonocytic cells U937 (ATCC CRL-1593.2) cultured in RPMI-
1640 (Invitrogen) enriched with 10% fetal bovine serum (FBS) (Gibco) and
1% penicillin-streptomycin (Sigma) were dispensed into each well of a 96-
well culture plate at a concentration of 104 cells/100 μL. Then, 100 μL of
each compound at one of six concentrations (200, 50, 12.5, 3.1, 0.8 and
0.2 μg/ml) were added and cells were incubated at 37 °C with 5% CO2.
After 72 hours the cytotoxic effect was determined by adding 20 µL/well of
MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide
solution (0.5 mg/mL) (Sigma) and incubating at 37 °C for 3 hours. The
reaction was stopped with 100 μL/well of isopropanol (50%) and sodium
dodecyl sulfate (10%) and the production of formazan was quantified by
reading at 570 nm in a spectrofluorometer (Varioskan Flash Multimode
Reader, Thermo Scientific, Waltham, MA, USA). The color intensity
(absorbance) was registered as O.D. Cells exposed to amphotericin B
(Sigma) were used as assay control for toxicity (positive control) and non
treated cells were used as viability control (negative control). Non-specific
absorbance was corrected by subtracting absorbance (O.D.) of the blank.
Determinations were done by triplicate in at least two independent
experiments. The results are expressed as the median lethal concentration
(LC50) calculated by Probit method. [18]
Regarding cytotoxicity, most of the derivatives are classified as
cytotoxic, except for compounds 15, 25, 32, 36, 37 and 41 with
moderate toxicity. In general, the presence of carbonyls, alcohols
or alkenes in the D ring does not show a differentiable effect on
toxicity, which seems to be more associated with the C-19
position since groups such as carbonyls are less toxic than
amines and alcohols, a situation that is also evident with ent-
kaurenes, however, the reduction of unsaturation in C-15
presents contradictory results, since for compound 26 a notable
decrease in cytotoxicity is observed concerning to the
compound 37, but for compound 3 did the opposite, since its
[8]
absence increases cytotoxicity.
With secondary alcohols, the
effect of chain length is notorious, since propyl (33), butyl (34) and
allyl (35) are much more cytotoxic than ethyl (32) and phenyl (36).
As for the amines, these showed to be considerably more
cytotoxicity than the rest of the derivatives (Table 1).
In vitro antileishmanial activity.
The U-937 macrophages were adjusted to 3x105 cells/ml in complete
RPMI-1640 medium and 0.1 µg/mL of phorbol 12 myristate 13 acetate
(PMA). In each well of a 24 well plate was dispensed 1 mL of cells and
plates were incubated for 72 hours at 37 °C and 5% CO2. Cells were then
infected with promastigotes of L. braziliensis in a 13:1 parasite per cell
ratio. Cells and parasites were incubated for 3 hours at 34 °C, 5% CO2.
Each well was washed three times with warmed phosphate buffer saline
(PBS) to remove non-internalized parasites, and new medium was added.
After 24 hours of infection, cells were exposed to 6 dilutions of each
Conclusion
Of the 39 compounds described here, 25 are reported for the first
time. The synthesized ent-beyerene but not ent-kaurene
4
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10.1002/ejoc.202001424
European Journal of Organic Chemistry
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(CDCl3, 300 MHz) δ (ppm): 0.8 (3H, s), 1.0 (3H, s), 1.1 (3H, t), 1.2 (3H, s), 1.8
(1H, d, J = 3.8 Hz), 1.9 (1H, d, J = 3.7 Hz), 2.7 (1H, dd, J = 18.8, 3.7 Hz), 3.3
(1H, p, J =7.4 Hz). 13C NMR (MeOD, 75 MHz) δ (ppm): 13.7 (C-20), 14.7 (C-22),
19.2 (C-6), 19.9 (C-17), 20.2 (C-2), 22.3 (C-11), 30.2 (C-18), 34.3 (C-21), 37.3
(C-12), 38.0 (C-10), 38.2 (C-7) 39.5 (C-4), 40.1 (C-3), 41.7 (C-1), 43.5 (C-13),
48.4 (C-15). 48.7 (C-8), 54.2 (C-14), 54.7 (C-9), 57.6 (C-5), 176.4 (C-19), 222.6
(C-16). HRMS (ESI): [M+H]+ calcd for C22H35NO2: 346.2740, found: 346.2736.
compound and incubated again for 72 hours at at 34 °C, 5% CO2. Cells
were removed from the bottom well with trypsin / EDTA, washed and
suspended in PBS and then analyzed in the flow cytometer (Cytomics FC
500MPL, Brea, CA), at 488 nm excitation and 525 nm emission and
counting 10,000 events. [19] Amphotericin B (AmB) was used as an internal
assay control for antileishmanial activity.
Data analysis.
ent-17-Beyeran-19-carboxamide (6): The procedure for preparing 6 was the
same as for preparing 4 from 2 (50.0 mg, 0.16 mmol), and ammonia (1.5 mL) in
THF (2 mL) yielding 6 (40 mg, 80 %). %). Physical State: white solid (mp 182-
184 °C); IR (cm-1) 3369, 2920, 2842, 1739, 1660, 1638; 1H NMR (CDCl3, 300
MHz) δ (ppm): 0.9 (3H, s), 1.0 (3H, s), 1.3 (3H, s), 2.7 (1H, dd, J = 18.6, 3.7 Hz).
13C NMR (CDCl3, 75 MHz) δ (ppm): 13.6 (C-20), 19.2 (C-6), 19.9 (C-17), 20.4
(C-2), 22.4 (C-11), 30.3 (C-18), 37.3 (C-12), 38.2 (C-10), 38.1 (C-7), 40.2 (C-3),
40.1 (C-1), 40.6 (C-13), 48.4 (C-15). 48.8 (C-8), 54.2 (C-14), 54.7 (C-9), 57.3
(C-5), 179.8 (C-19), 222.6 (C-16). HRMS (ESI): [M+H]+ calcd for C20H31NO2:
318.2427, found: 318.2423.
Cytotoxicity was determined according to the percentages of mortality
registered for each compound, including AmB using the Equation 1:
% Mortality = 100- [(O.D exposed cells / O.D non-exposed cells) x 100].
[ Eq.1]
Then, the percentage of mortality were used to calculate the LC50 that
corresponds to the concentration necessary to eliminate 50% of cells by
the Probit analysis [18]
.
S-Bencyl-ent-17-beyeran-19-thioesther (7):
2 (100 mg, 0.3 mmol) was
dissolved in dry dichloromethane (4 mL) and DMF (4 drops) was added to the
solution. An excess of Oxalyl chloride (0.16 mL, 18.2 mmol) was added slowly
the mixture was stirred for 10 minutes under argon atmosphere and room
temperature. Then thiophenol (0.13 mL, 1.3 mmol) and TEA (0.1mL) were
added and the reaction mixture was stirred overnight at 40°C. Water 20 mL was
added to the mixture and extracted with CH2Cl2, (2 × 30 mL). The combined
organic layer was washed with brine solution (2 x 30 mL) and dried over Na2SO4,
filtered and evaporated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (hexane/EtOAc, 8:1) yielding 2
(108.2 mg, 84 %). Physical State: pale yellow solid (mp 163-165 °C); IR (cm-1)
2948, 2850, 1731, 1686, 1439; 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.9 (3H, s),
1.0 (3H, m), 1.4 (3H, s), 2.5 (1H, dt, J = 14.4, 3.5 Hz), 2.7 (1H, dd, J = 18.7, 3.7
Hz), 7.3 (5H, m), 13C NMR (CDCl3, 75 MHz) δ (ppm): 15.1 (C-20), 19.3 (C-6),
19.9 (C-17), 20.4 (C-2), 21.8 (C-11), 30.3 (C-18), 37.3 (C-12), , 37.7 (C-10),
38.3 (C-7) 39.4 (C-4) 39.5 (C-3), 41.7 (C-1), 48.4 (C-15), 48.7 (C-13), 48.7 (C-
8), 54.2 (C-14), 54.7 (C-9), 58.4 (C-5), 128.2 (C-24), 129.2, (C-23,25), 135.1 (C-
22,26), 203.3 (C-19), 222.2 (C-16). HRMS (ESI): [M+H]+ calcd for C26H34O2S:
411.2352, found: 411.2351.
In turn, the antileishmanial activity was determined according to the
reduction in the percentage of infected cells and the amount of viable
intracellular parasites. Briefly, positive events were gated in a dot plot
analysis with the green fluorescence (parasites) in y-axis and the Forward
Scatter (FSC) in x-axis. Afterwards, the number of parasites in infected
cells was determined according to the MFI of those fluorescent parasites.
The parasite inhibition percentage was calculated using the Equation 2:
% inhibition of infection = 100- [(MFI exposed infected cells / MFI non-
exposed infected cells) x 100]. [Eq.2]
The median effective concentration (EC50) was calculated using the
percentage of inhibition of infection by the Probit analysis [19]. In addition,
the Selectivity Index (SI), was calculated using the formula: SI = LC50/EC50
.
Values were expressed as mean ± SD.
To compare the different compounds in terms of their biological activity,
both cytotoxicity and antileishmanial activity were classified as high,
moderate or low, according to the LC50 and EC50 values, respectively.
Thus, the cytotoxicity was considered high when the LC50 was < 100
μg/mL; moderate when the LC50 was between 100 and 200 μg/mL and low
when the LC50 was > 200 μg. On the other hand, the antileishmanial
activity was considered high when the EC50 was < 25 μg/mL, moderate
when the EC50 was between 25 and 50 μg/mL and low when the EC50 was
> 50 μg/mL. [8]
ent-17-Beyeran-19-carbonyl fluoride (8): 2 (50 mg, 0.16 mmol) was dissolved
in dry CH2Cl2 (4 mL). The resulting solution was cooled at -78 °C and treated
dropwise with DAST (0.023 mL, 0.17 mmol) for 45 minutes and 3 h at 0 °C.
Water (15 mL) was added to the mixture and extracted with CH2Cl2 (20 mL). The
combined organic layer was washed sodium bicarbonate 10 % solution (15 mL)
and dried over Na2SO4, filtered, evaporated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
(hexane/EtOAc, 10:1) yielding 8 (31.9 mg, 63.5 %). Physical State: white solid
(mp 143-145 °C); IR (cm-1) 2928, 2843, 1815, 1729, 1456 cm-1. 1H NMR (CDCl3,
300 MHz) δ (ppm): 0.9 (3H, s), 1.0 (3H, s), 1.4 (3H, s), 1.8 (1H, m) 1.9 (1H, d, J
= 5.0 Hz), 2.2 (1H, dd, J = 13.1, 3.8 Hz), 2.7 (1H, dd, J = 18.6, 3.8 Hz). 13C NMR
(CDCl3, 75 MHz) δ (ppm): 13.3 (C-20), 18.4 (C-6), 19.9 (C-17), 20.3 (C-2), 21.4
(C-11), 27.6 (C-18), 37.0 (C-10) 37.2 (C-12), 37.7 (C-7), 39.3 (C-3), 41.1 (C-1),
43.5 (C-4), 44.0 (C-13), 48.4 (C-15), 48.7 (C-8), 54.2 (C-14), 54.6 (C-9), 56.5
(C-5), 167.7 (C-19, d, J = 375 Hz), 222.2 (C-16). HRMS (ESI): [M+H]+ calcd for
C20H29FO2: 321.2224, found: 321.2213.
ent-Beyer-15-en-19-ol (3): Carboxylic acid 2b (500 mg, 1.5 mmol) was
dissolved in dry THF (5 mL) and was added an excess of LiAlH4 (175 mg, 3.8
mmol). The reaction mixture was refluxed for 5 h. Then the mixture was filtered
through celite and evapored under reduced pressure. The residue was purified
by flash column chromatography on silica gel (hexane/EtOAc, 10:1) obtaining
369 mg in 85% yield. Physical State: white solid (mp 105-107 °C); 1H NMR
(CDCl3, 300 MHz) δ (ppm): 0.8 (3H, s), 1.0 (3H, s), 1.0 (3H, s), 1.7 (2H, m), 1.8
(2H, m), 3.5 (1H, d, J = 10.9 Hz), 3.8 (1H, d, J = 10.9 Hz), 5.5 (1H, d, J = 5.6
Hz), 5.7 (1H, d, J = 5.6 Hz). 13C NMR (CDCl3, 75 MHz) δ (ppm): 15.8 (C-20),
19.3 (C-6), 20.2 (C-2), 20.3 (C-11), 25.0 (C-17), 27.0 (C-18), 33.2 (C-12), 35.6
(C-7), 37.2 (C-10), 37.7 (C-3), 38.5 (C-4), 39.2 (C-1), 43.6 (C-13), 49.2 (C-8),
52.9 (C-9), 56.8 (C-5), 61.1 (C-14), 65.5 (C-19), 135.1 (C-16), 136.5 (C-15).
HRMS (ESI): [M+H]+ calcd for C20H32O: 289.2525, found: 289.2522.
4-Carboxy-13-hydroxy-13,16-seco-ent-norbeyeran-19-oic acid (9):
2
(130 mg, 0.38 mmol) was dissolved in CH2Cl2 (6 mL) then was added m-CPBA
(105 mg, 0.8 mmol) in small portions to room temperature. The reaction mixture
was stirred overnight to reflux. Wáter (20 mL) was added to the mixture and
extracted with CH2Cl2, (2 × 15 mL). The combined organic layer was washed
sodium bicarbonate 10 % solution (2 x 15 mL) and dried over Na2SO4, filtered
and evaporated under reduced pressure. The residue was purified by flash
column chromatography on silica gel (hexane/EtOAc, 3:1) yielding 9 (40.4 mg,
30%). Physical State: white solid (mp: 253-255 °C); IR (cm-1) 3264, 2921, 2851,
1716, 1589; 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.9 (3H, s), 1.3 (3H, s), 1.4
(3H, s), 2.05 (1H, s), 2.1 (1H, d, J = 10.0, Hz), 2.2 (1H, d, J = 13.1, Hz), 3.5 (1H,
dd, J = 18, 2,5 Hz). 13C NMR (CDCl3, 75 MHz) δ (ppm): 13.6 (C-20), 18.6 (C-6),
18.9 (C-2), 19.5 (C-11), 28.3 (C-17), 28.9 (C-18), 34.9 (C-4), 37.6 (C-12), 38.5
(C-7), 38.7 (C-3), 40.0 (C-1), 43.5 (C-15), 43.7 (C-8), 47.7 (C-14), 80.4 (C-13),
55.8 (C-9), 57.2 (C-5), 173.0 (C-16), 183.8 (C-19). HRMS (ESI): [M+H]+ calcd
for C20H30O4: 335.2216, found: 335.2211.
N-Hexyl-ent-17-beyeran-19-carboxamide (4): 2 (550 mg, 1.7 mmol) was
dissolved in dry dichloromethane (4 mL) and. DMF three drops were added to
the solution. An excess of Oxalyl chloride (0.5 mL, 5.7 mmol) was added slowly
and the resulting mixture was stirred for 40 minutes under argon
atmosphere.and at room temperature. Then hexylamine (0.7 mL, 5.3 mmol) was
added and the reaction mixture was stirred overnight. Water (30 mL) was added
to the mixture and extracted with EtOAc, (2 × 35 mL). The combined organic
layer was washed with HCl 10 % solution (2 x 30 mL) and dried over Na2SO4,
filtered, and evaporated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (hexane/EtOAc, 4:1) yielding 4 (500
mg, 72.4 %). Physical State: white solid (mp 123-125 °C); IR (cm-1) 3363, 2918,
2843, 1735, 1632, 1519, 1453; 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.8 (3H, s),
0.9 (3H, m), 1.0 (3H, s), 1.2 (3H, s), 1.8 (1H, d, J = 2.4 Hz), 1.9 (1H, d, J = 3.7
Hz), 2.7 (1H, dd, J = 18.7, 3.7 Hz), 3.2 (1H, tdd, J =7.1, 5.6, 3.9 Hz). 13C NMR
(CDCl3, 75 MHz) δ (ppm): 13.6 (C-20), 14.0 (C-26), 19.2 (C-6), 19.7 (C-17), 20.3
(C-2), 22.3 (C-11), 22.6 (C-25), 26.8 (C-24), 29.3 (C-23), 30.3 (C-18), 31.4 (C-
22), 37.3 (C-12), 38.1 (C-10), 35.1 (C-7) 39.4 (C-4) 39.5 (C-21), 40.2 (C-3), 41.7
(C-1), 43.6 (C-13), 48.4 (C-15). 48.7 (C-8), 54.3C-14), 54.7 (C-9), 57.6 (C-5),
176.5 (C-19), 222.6 (C-16). HRMS (ESI): [M+H]+ calcd for C26H43NO2:
402.3366, found: 402.3368.
Ethyl-4-carboxy-13-hydroxy-13,16-seco-ent-norbeyeran-19-oate (10):
The procedure for preparing 10 was the same as for preparing 9 from 2a ethyl
isosteviol ester (300 mg, 0.83 mmol), and m-CPBA (220 mg, 0.63 mmol) to get
a white solid 10 (116 mg, 37 %). Physical State: white solid (mp: 159-161°C);
IR (cm-1) 2964, 2848, 1708, 1236, 1149; 1H NMR (CDCl3, 300 MHz) δ (ppm):
0.8 (3H, s), 1.2 (3H, s), 1.3 (3H, t) 1.4 (3H, s), 2.0 (1H, d, J = 2.0, Hz), 2.1 (1H,
d, J = 2.0, Hz), 2.2 (1H, m) 3.1 (1H, dd, J = 18,6 2,5 Hz), 4.1 (3H, m). 13C NMR
(CDCl3, 75 MHz) δ (ppm): 13.6 (C-20), 14.2 (C-22), 18.6 (C-6), 18.9 (C-2), 19.6
(C-11), 28.3 (C-17), 28.7 (C-18), 34.9 (C-4), 37.9 (C-12), 38.5 (C-7), 38.7 (C-3),
39.9 (C-1), 43.5 (C-15), 43.7 (C-8), 47.6 (C-14), 80.4 (C-13), 55.8 (C-9), 57.2
(C-5), 172.8 (C-16), 177.2 (C-19), 60.1 (C-21). HRMS (ESI): [M+H]+ calcd for
C22H34O4: 363.2529, found: 363.2535.
N-Ethyl-ent-17-beyeran-19-carboxamide (5): The procedure for preparing 5
was the same as for preparing 4 from 2 (300 mg, 0.9 mmol), and ethylamine
(0.26 mL, 4,6 mmol) yielding 5 (297 mg, 91 %). Physical State: white solid (mp
106-108 °C); IR (cm-1) 3363, 2918, 2843, 1735, 1632, 1519, 1453 cm-1. 1H NMR
5
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202001424
European Journal of Organic Chemistry
FULL PAPER
Ethyl-ent-15,16-dioxo-beyeran-19-oate (11): 2a (100 mg, 0.28 mmol) and
SeO2 (144 mg, 4.5 eq) were dissolved in xilene/Ac2O (8:2 mL) and refluxed
overnight. The mixture was filtered through celite and evapored under reduced
pressure. The residue was purified by flash column chromatography on silica
gel (hexane/EtOAc, 9:1) obtaining 11 (58.3 mg, 56 % yield). Physical State:
orange crystals (mp: 183-185 °C); IR (cm-1) 2926, 2848, 1721, 1456. 1149, 1021.
1H NMR (CDCl3, 300 MHz) δ (ppm): 0.6 (3H, s), 1.2 (3H, s), 1.3 (3H, s), 1.4 (3H,
t, J = 7.1 Hz), 2.2 (1H, d, J = 13.2, Hz), 2.4 (1H, m), 4.2 (2H, m). 13C NMR (CDCl3,
75 MHz) δ (ppm): 11.7 (C-20), 14.2 (C-22), 19.0 (C-6), 20.1 (C-17), 20.9 (C-2),
21.5 (C-11), 28.8 (C-18), 34.2 (C-12) 38.1 (C-7), 39.0 (C-10), 40.0 (C-3), 43.7(C-
4), 46,7 (C-13), 47.4 (C-1), 50.5 (C-8), 54.2 (C-14), 56.3 (C-9), 59.6 (C-5), 177.2
(C-19), 209.1 (C-16), 210.3 (C-15). HRMS (ESI): [M+H]+ calcd for C22H32O4:
361.2373, found: 361.2362.
residue was puritied by flash column chromatography on silica gel (EtOAc)
obtaining alcohol in C19, 16 (90.7 mg, 61.3 %). Physical State: white solid (mp:
191-193 °C); IR (cm-1) 3399, 2930, 2837, 1737, 1527, 1451, 1029; 1H NMR
(CDCl3, 300 MHz) δ (ppm): 0.9 (3H, s), 1.0 (3H, s), 1.1 (3H, s), 1.7 (2H, d, J =
3.4 Hz), 1.8 (1H, m), 2.0 (1H, d, J = 18.7 Hz), 3.0 (1H, dd, J = 18.7, 3.3 Hz), 3.5
(1H, d, J = 11.0 Hz), (1H, dd, J = 10.9, 4.8 Hz). 13C NMR (CDCl3, 75 MHz) δ
(ppm): 15.5 (C-20), 18.1 (C-6), 20.2 (C-2), 20.7 (C-11), 22.2 (C-17), 27.1 (C-18),
35,5 (C-12), 37.0 (C-7), 37.6 (C-10), 38.5 (C-4), 39.5 (C-3), 39.6 (C-1), 40.1 (C-
13), 41.1 (C-15), 43.8 (C-8), 55.7 (C-9), ), 56.5 (C-14), 57.0 (C-5), 66.5 (C-19),
170.9 (C-16). HRMS (ESI): [M+H]+ calcd for C20H33NO2: 320.2584, found:
320.2582.
N-phenyl-ent-beyer-15-en-19-carboxamide (17): 2b (60.4 mg, 0.2 mmol) was
dissolved in dichloromethane (8 mL). Then 1-Hidroxybenzotriazole (40.4 mg,
0.3 mmol), aniline (0.036 mL, 0.4 mmol), DCC (61.8 mg, 0.3 mmol) were added
successively. The resulting mixture was stirred at room temperature overnight.
HCl (1N, 15 mL) was added to the reaction mixture and extracted with CH2Cl2
(2 × 15 mL). The combined organic layer was washed with brine (2 x 15 mL)
and dried over Na2SO4, filtered and evaporated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
(hexane/EtOAc, 7:1) to give compound 17 (34.5 mg, 82% yield) Physical State:
yellow oil. IR (cm-1): 2927, 2849, 1796, 1446, 1000. 1H NMR (CDCl3, 300 MHz)
δ (ppm): 0.9 (3H, s), 1.0 (3H, s), 1.3 (3H, s), 2.0 (2H, m), 2.5 (2H, d, J = 13.5,
Hz), 5.6 (1H, d, J = 5.7 Hz), 5.8 (1H, d, J = 5.7 Hz), 7.4 (1H, m), 7.6 (2H, m), 8.1
(2H, dd, J = 8.4, 1.2 Hz) 13C NMR (CDCl3, 75 MHz) δ (ppm): 15.7 (C-20), 18.3
(C-6), 20.4 (C-2), 21.5 (C-11), 24.8 (C-17), 29.1(C-18), 32.9 (C-12), 37.3 (C-7),
38.0 (C-10), 38.1 (C-3), 38.9 (C-4), 39.2 (C-1), 43.7 (C-13), 49.0 (C-8), 52.2 (C-
9), 57.2 (C-5), 60.9 (C-14), 120.6 (C-24), 124.6 (C-23,25), 128.5 (C-23,25),
134.5 (C-16), 136.9(C-15), 143.6(C-21), 173.8(C-19).
Ethyl-ent-15-hydroxymethyl-16-hydroxybeyeran-19-oate (12): To
a
solution of 2a (200 mg, 0.58 mmol) in anhydrous ethanol (15 mL) and C2H5ONa
(0.136 g, 2 mmol) was added an aqueous formaldehyde solution (37%, 2 mL)
and stirring for 24 h at 60 °C. The reaction mixture was extracted with CH2Cl2
(2 x 40 mL). The combined organic solvents were washed with brine solution
(30 mL), dried over Na2SO4, filtered and evaporated under reduced pressure.
The residue was purified by flash column chromatography on silica gel
(Hexane/EtOAc, 4:1) yielding diol 12 (78 mg, 24% yield). Physical State: white
solid (mp: 138-140 °C); IR (cm-1) 3419, 2941, 2843, 1716, 1452, 1376, 1323 cm-
1. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.8 (3H, s), 1.0 (3H, s), 1.2 (3H, s), 1.3
(3H, t), 2.1(1H, dt, J = 10.4, 4.9 Hz), 2.2 (1H, d, J = 13.2, Hz), 2.5 (1H, m), 3.5
(1H, m), 3.7 (1H, d, J = 4.7 Hz), 4.0 (1H, dd, J = 9.9, 4.9 Hz), 3.7 (1H, q, J = 7.1
Hz). 13C NMR (CDCl3, 75 MHz) δ (ppm): 13.3 (C-20); 14.1 (C-22), 18.9 (C-6),
19.5 (C-2), 22.2 (C-11), 25.1 (C-17), 29.0 (C-18), 33.2 (C-12), 34.9 (C-7), 37.9
(C-3), 38.3 (C-10), 39.6 (C-1), 40.9 (C-4), 42.6 (C-13), 43.7 (C-8), 50.2 (C-9).
54.2 (C-14), 57.1 (C-15), 57.7, (C-5), 60.1 (C-21), 64.9, (C-23), 86.7 (C-16),
177.5 (C-19). HRMS (ESI): [M+H]+ calcd for C23H38O4: 379.2842, found:
379.2841.
N,N-Dimethyl ent-beyer-15-en-19-carboxamide (18): 2b (94 mg, 0.2 mmol)
was dissolved in dicloromethane (4 mL) and then DCC (129 mg, 0.6 mmol),
dimethylamine (0.048 mL, 0.7 mmol), DMAP (9.4 mg, 0.06 mmol) were added
successively. The reaction mixture was stirred at room temperature overnight.
HCl (1N, 20 mL) was added to the reaction mixture and extracted with CH2Cl2
(2 × 20 mL). The combined organic layer was washed with brine solution (2x15
mL) and dried over Na2SO4, filtered and evaporated under reduced pressure.
The residue was purified by flash column chromatography on silica gel
(hexane/EtOAc, 7:1) yielding 18 (17.8 mg, 17.4% yield). Physical State: white
solid mp: 152-154 °C; IR (cm-1) 2944, 2844, 2361, 1794, 1450. 1H NMR (CDCl3,
300 MHz) δ (ppm): 0.7 (6H, s), 1.0 (3H, s), 1.2 (3H, s), 1.2 (3H, s), 1.7 (1H, m),
1.8 (2H, dt, J = 9.8, 3.7 Hz), 2.2 (2H, dq, J = 13.4, 2.8 Hz), 5.5 (1H, d, J = 5.6
Hz), 5.7 (1H, d, J = 5.7 Hz. 13C NMR (CDCl3, 75 MHz) δ (ppm): 14.4 (C-20),
18.0 (C-2), 19.0 (C-6), 20.4 (C-2), 21.6 (C-11), 24.8 (C-17), 28.1 (C-18), 29.7
(C-12), 32.9 (C-21,22), 37.5 (C-7), 37.8 (C-10), 38.1 (C-3), 39.0 (C-1), 43.6 (C-
13), 45.3 (C-4), 49.0 (C-8), 52.0 (C-9), 57.2 (C-5), 60.9 (C-14), 134.5 (C-16),
136.6 (C-15), 173.5 (C-19).
Ethyl-ent-16-acetoxy-beyeran-19-oate (13):
A
solution of Ethyl-ent-16-
[8]
hydroxy-beyeran-19-oate
(40 mg, 0.2 mmol), pyridine (0,4 mL) and Acetic
anhhydride (0.2 mL, 1.8 mmol) was stirred to room temperature for 2.5 h. Water
(20 mL) was added to the reaction mixture and extracted with CH2Cl2 (2 × 20
mL). The combined organic layer was washed with HCl 2M (3 x 15 mL) and
dried over Na2SO4, filtered and evaporated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
(hexane/EtOAc, 5:1) to afford 13 (40 mg, 91%, yield). Physical State: colorless
oil; IR (cm-1) 2934, 1720, 1452, 1370, 1238 cm-1. 1H NMR (CDCl3, 300 MHz) δ
(ppm): 0.8 (3H, s), 0.9 (3H, s), 1.2 (3H, s), 1.3 (3H, s), 1.8 (2H, dd, J = 4.9, 1.9
Hz), 2.1 (3H, s) 4.1 (1H, m), 4.7 (1H, dd, J = 10.3, 4.9 Hz). 13C NMR (CDCl3, 75
MHz) δ (ppm): 13.3 (C-20), 14.1 (C-22), 18.9 (C-6), 20.2 (C-2), 21.3 (C-17), 21.7
(C-11), 24.9 (C-1), 28.9 (C-24), 34,6 (C-12), 39.1 (C-7), 39.9 (C-3), 40.6 (C-10),
40.5 (C-1), 41.5 (C-4), 41.5 (C-15), 42.3 (C-13), 43.7 (C-8), 54.8 (C-21), 55.7
(C-9), 57.1 (C-5), 60.0 (C-14), 81.7 (C-16), 171.5 (C-23), 177.6 (C-19). HRMS
(ESI): [M+H]+ calcd for C24H38O4: 391.2842, found: 391.2837.
Butyl-ent-Beyer-15-en-19-one (19): Tosylhydrazone of 2a 480 mg was
dissolved in dry THF (15.0 mL). The mixture was stirred under an argon
atmosphere at room temperature for 10 minutes then the mixture was cooled to
-78 °C, and an excess of n-BuLi (2.5 M in Hexanes, 3.2 mL, 8 mmol) was added
dropwise. The reaction mixture was stirred for 2 h and quenched by the addition
of NH4Cl 10% (20 mL) and extracted with CH2Cl2 (3 × 40 mL) The combined
organic layers were washed with brine solution, over dried Na2SO4, and
evaporated under reduced pressure. The residue was purified by flash column
chromatography on silica gel (hexane/EtOAc, 8:1) yielding 19 (180 mg, 58 %
yield). Physical State: yellow solid (mp: 59-61 °C); IR (cm-1) 2918, 2845, 1705,
1456, 1364, 1116. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.61 (3H, s), 0.9 (3H, t),
1.0 (3H, s), 1.1 (3H, s), 1.7 (1H, q, J = 4.5, 3.9 Hz), 1.7 (1H, t, J = 3.2, Hz), 1.9
(2H, m), 2.2 (1H, dtq, J = 13.4, 3,2, 1.7 Hz), 5.5 (1H, d, J = 5.6 Hz), 2.5 (2H, m),
5.5 (1H, d, J = 5.6 Hz), 5.8 (1H, d, J = 5.7 Hz). 13C NMR (CDCl3, 75 MHz) δ
(ppm): 14.1 (C-24), 15.3 (C-20), 19.7 (C-6), 20.5 (C-2), 22.3 (C-23), 22.4 (C-11),
24.8 (C-17), 26.2 (C-22), 28.6 (C-18), 33.0 (C-12), 37.9 (C-10), 38.0 (C-7), 38.3
(C-3), 39.3(C-1), 39.9 (C-21), 43.2 (C-13), 49.1 (C-8), 49.9 (C-4), 52.2 (C-9),
58.1 (C-5), 60.9 (C-14), 134.6 (C-16), 136.5(C-15), 216.1 (C-19). HRMS (ESI):
[M+H]+ calcd for C24H38O: 343.2995, found: 343.2994.
N-ethyl-ent-16-hydroxy-beyeran-19-carboxamide (14): To solution of 5 (120
mg, 0.2 mmol) in dry THF (3.0 mL) was added an excess of LiAlH4 (1.3 mL, 1.0
M, 2.6 mmol) and was refluxed for 6 h. The reaction mixture was quenched with
NaOH 2.5M (2mL) and extrated with EtOAc (2 x 40 mL). The organic phase
were washed with brine (2 x 30 mL) and dried with Na2SO4, filtered and
evaporated under reduced pressure. The residue was puritied by flash column
chromatography on silica gel (EtOAc) obtaining amide 14 (80.3 mg in 66.5 %
yield). Physical State: white solid (mp 216-218 °C); IR (cm-1): 3267, 2934, 2842,
1734, 1445. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.8 (3H, s), 0.9 (3H, s), 1.1
(3H, t), 1.4 (3H, s), 1.8 (2H, q, J = 2.6 Hz), 1.9 (1H, q, J = 2.5 Hz), 2.1(1H, d, J
= 13.2, 2.8 Hz), 3.2 (2H, m,), 3.8 (dd, J = 10.8, 4.7 Hz). 13C NMR (CDCl3, 75
MHz) δ (ppm): 12.8 (C-20), 14.4 (C-22), 18.9 (C-6), 20.0 (C-2), 22.0 (C-11), 24.1
(C-17), 29.0 (C-18), 33.6 (C-21), 33.6 (C-12), 37.5 (C-7), 37.9 (C-10), 40.2 (C-
15), 41.6 (C-4), 41.9 (C-13), 42.0 (C-3), 42.1 (C-1), 43.4 (C-8), 55.1 (C-14), 56.0
(C-9), 57.8 (C-5), 79.6 (C-16), 178.0 (C-19). HRMS (ESI): [M+H]+ calcd for
C22H37NO2: 348.2897, found: 348.2892.
ent-Beyeran-16,19-diol (15). 2a (63 mg, 0.2 mmol) was dissolved in dry THF
(5 mL) then LiAlH4 (41.4 mg, 1.1 mmol) was added an excess. The reaction
mixture was refluxed for 5 h. Then the mixture was filtered through celite and
evapored under reduced pressure. The residue was purified by flash column
chromatography on silica gel (hexane/EtOAc, 10:1) obtaining 45.3 mg in 81%
yield. Physical State: white solid (mp 150-152 °C); IR (cm-1) 3284, 2927, 2837,
1442, 1331, 1060, 1025; 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.93 (3H, s), 0.95
(3H, s), 1.0 (3H, s), 1.9 (2H, ddt, J = 16.5, 11.8, 2,.7 Hz), 3.5 (1H, m), 3.8 (1H,
d, J = 11.0 Hz), 3.9 (1H, dd, J = 10.7, 4.7 Hz). 13C NMR (CDCl3, 75 MHz) δ
(ppm): 15.5 (C-20), 18.0 (C-6), 20.3 (C-2, C-11), 25.0 (C-17), 27.1 (C-18), 33,7
(C-12), 35.5 (C-7), 37.6 (C-10), 38.5 (C-4), 39.6 (C-3), 39.9 (C-1), 42.0 (C-13),
43.0 (C-15), 55.5 (C-14), 56.7 (C-9), 57.0 (C-5), 65.5 (C-19), 170.9 (C-16).
HRMS (ESI): [M+NH4]+ calcd for C20H34O2: 324.2897, found: 324.2893.
ent-Beyer-15-en-19-mesylate (20):
3 (50mg, 0.17 mmol) dissolved in
dicloromethane (4 mL) was added triethylamine 0.1mL and methanesulfonyl
chloride (0.045 mL, 0,58 mmol) The reaction mixture was stirred for 3 h. Solution
HCl 1N (20 mL) was added to the reaction mixture and extracted with CH2Cl2 (2
× 15 mL). The combined organic layer was washed with brine solution (20 mL)
and dried over Na2SO4, filtered and evaporated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
(Dicloromethane) obtaining a white solid 20 (51,3 mg, 77% yield). Physical
State: white solid (mp: 80-82 °C); IR (cm-1) 2922, 1690, 2924, 1448, 1255; 1H
NMR (CDCl3, 300 MHz) δ (ppm): 0.7 (3H, s), 1.0 (3H, s), 1.0 (3H, s), 1.5 (1H,
m), 1.7 (1H, d, J = 2.1 Hz), 1.7 (1H, d, J = 3.0 Hz), 3.0 (3H, s), 4.0 (1H, m), 4.4
(1H, , J = 9.4 Hz), 5.5 (1H, dd, J = 5.7, 0.9 Hz), 5.6 (1H, d, J = 5.6 Hz). 13C NMR
(CDCl3, 75 MHz) δ (ppm): δ (ppm): 15.8 (C-20), 18.0 (C-6), 19.9 (C-2), 20.3 (C-
11), 24.8 (C-17), 27.1 (C-18), 33.0 (C-12), 35.5 (C-7), 36.5 (C-21), 37.2 (C-10),
21.6 (C-27), 24.8 (C-17), 27.0 (C-18), 33.1 (C-12), 35.5 (C-7), 37.0 (C-4), 37.2
(C-10), 37.3 (C-3), 38.6(C-1), 43.4 (C- 13), 48.6 (C-8), 52.7 (C-9), 56.5 (C-5),
60.9 (C-14), 72.5 (C-19), 134.7 (C-16), 136.7 (C-15). HRMS (ESI): [M+NH4]+
calcd for C21H34O3S: 384.2566, found: 384.2567.
ent-16-Oxime-17-beyeran-19-ol (16): The ethyl ester -oxime of 2a (168 mg,
0.46 mmol) was dissolved in dry THF (8mL) and was added an excess of LiAlH4
(0.4 mL, 1.0 M). The reaction mixture was refluxed for 8 h. Then the reaction
mixture was quenched with NaOH 2.0 M (2.0 mL) and was dissolved in EtOAc,
(30 mL) and washed with brine solution (2 x 30 mL). The combined organic layer
dried over Na2SO4, filtered and evaporated under reduced pressure. The
6
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202001424
European Journal of Organic Chemistry
FULL PAPER
dd, J = 8.3, 6.8 Hz) 7.5 (1H, m), 8.0 (2H, m). 13C NMR (CDCl3, 75 MHz) δ (ppm):
15.7 (C-20), 18.3 (C-6), 20.2 (C-2), 20.2 (C-11), 24.3 (C-17), 27.2(C-18), 33.2
(C-12), 36.2(C-7), 37.2 (C-10), 37.6 (C-3), 37.8 (C-4), 39.2 (C-1), 43.5 (C-13),
49.0 (C-8), 52.9 (C-9), 56.9 (C-5), 59.2 (C-21), 61.0 (C-14), 67.7 (C-19), 128.3
(C-24,26), 129.5 (C-23,27), 130.6 (C-25), 132.5 (C-22), 134.9 (C-16), 136.5 (C-
15), 166.8(C-21). HRMS (ESI): [M+NH4]+ calcd for C27H36O2: 410.3053, found:
410.3055
ent-Beyer-15-en-19-tosylate (21):
3 (50mg, 0.17 mmol) dissolved in
dicloromethane (6 mL) was added p-toluenesulfonyl chloride (49.5 mg,
0.25mmol), triethylamine (0.2 mL) and DMAP (cat). The reaction mixture was
stirred overnight to room temperature. Water (15 mL) was added to the reaction
mixture and extracted with CH2Cl2 (2 × 20 mL). The combined organic layer was
washed with brine solution (2 x 20 mL), dried over Na2SO4, filtered and
evaporated under reduced pressure. The residue was purified by flash column
chromatography on silica gel (Dicloromethane) obtaining a white solid 21 (60,2
mg, 79% yield). Physical State: white solid (mp: 114-116 °C); IR (cm-1) 2925,
1455, 1356, 1173; 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.59 (3H, s), 0.93 (3H,
s), 1.0 (3H, s), 2.5 (3H, s), 3.8 (1H, m), 4.2 (1H, d, J = 9.3 Hz), 5.5 (1H, dd, J =
5.6, 0,9 Hz) 5.6 (1H, d, J = 5.6 Hz), 7.4 (2H, m), 7.8 (2H, m).) 13C NMR (CDCl3,
75 MHz) δ (ppm): 15.6 (C-20), 17.7 (C-6), 19.9 (C-2), 20.2 (C-11), 21.6 (C-27),
24.8 (C-17), 27.0 (C-18), 33.1 (C-12), 35.5(C-7), 37.0 (C-4), 37.2 (C-10), 37.3
(C-3), 38.5 (C-1), 43.4 (C-13), 48.8 (C-8), 52.7 (C-9), 56.4 (C-5), 60.9 (C-14),
73.4 (C-19), 134.7 (C-16), 136.7 (C-15), 127.9 (C-23,.C-25), 129.7 (C-22,.C-26),
134.7 (C-16), 136.6 (C-15), 144. (C-21), 162.4 (C-24). HRMS (ESI): [M+NH4]+
calcd for C28H37O3S: 460.2879, found: 460.2870.
ent-Beyer-15-en-19-azide (26): ent-beyer-15-en-19-ol 3 (100 mg, 0.35 mmol),
triphenylphosphine (273 mg, 1.0 mmol) and Diisopropyl azodicarboxylate
(DIAD; 0.2 mL, 1.2 mmol) was dissolved in dry THF (5 mL). The reaction mixture
was stirred for 15 minutes then diphenylphosphoryl azide (DPPA, 0.2 mL, 1.2
mmol) was added to the reaction mixture and stirred for 24 h at 40 °C. THF was
evaporated and the residue was dissolved in ethyl acetate (20 mL) and washed
with NaHCO3 solution (2 x 15 mL) and dried over Na2SO4, filtered and
evaporated under reduced pressure. The residue was purified by flash column
chromatography on silica gel (hexane/EtOAc, 7:1) yielding 26 (41.2 mg, 40 %
yield). Physical State: yellow oil; 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.8 (3H,
s), 1.0 (3H, s), 1.0 (3H, s), 1.4 (2H, m), 1.8 (1H, dq, J = 13.7, 2.7 Hz), 3.2 (1H,
m), 3.6 (1H, d, J = 12.0 Hz), 5.5 (1H, d, J = 5.8 Hz), 5.7 (1H, d, J = 5.8 Hz). 13C
NMR (CDCl3, 75 MHz) δ (ppm): 15.7 (C-20), 18.1 (C-6), 20.0 (C-2), 20.3 (C-
11), 24.9 (C-17), 28.1 (C-18), 25.4 (C-21), 33.1 (C-12), 36.2 (C-7), 37.2 (C-10),
37.4 (C-3), 38.3 (C-4), 38.9 (C-1), 43.6 (C-13), 48.9 (C-8), 52.9 (C-9), 56.3 (C-
19), 56.6 (C-5), 61.0 (C-14), 134.9 (C-16), 136.7(C-15).
ent-Beyer-15-en-19-acetate (22): 3 (30 mg, 0.10 mmol) dissolved in piridine
(0.4mL) was added acetic anhydride (0.2 mL). The reaction mixture was stirred
to room temperature for 3 h. water (20 mL) was added to the reaction mixture
and extracted with CH2Cl2 (2 × 20 mL). The combined organic layer was washed
with HCl 10 % (3 x 15 mL) and dried over Na2SO4, filtered and evaporated under
reduced pressure. The residue was purified by flash column chromatography
on silica gel (hexane/EtOAc, 20:1) yielding 22 (30.8 mg, 90% yield). Physical
State: white solid (mp: 67-69 °C); IR (cm-1) 2921, 2847, 1733, 1453, 1237, 1029;
1H NMR (CDCl3, 300 MHz) δ (ppm): 0.7 (3H, s), 0.9 (3H, s), 1.2 (3H, s), 1.4 (1H,
dd, J = 6.6, 3,8 Hz) 3.8 (1H, d, J = 10.9 Hz) 4.2 (1H, d, J = 11.0 Hz), 5.4 (1H, d,
J = 5.7 Hz), 5.6 (1H, d, J = 5.6 Hz). 13C NMR (CDCl3, 75 MHz) δ (ppm): 15.7 (C-
20), 18.2 (C-6), 20.2 (C-2), 21.0 (C-11), 24.9 (C-17), 27.5(C-18), 29.8 (C-22),
33.2 (C-12), 36.1 (C-7), 36.9 (C-10), 37.8 (C-4), 37.1 (C-3), 39.0 (C-1), 43.6 (C-
13), 50.0 (C-8), 52.8 (C-9), 56.7 (C-5), 61.0 (C-14), 67.1 (C-19), 135.0 (C-16),
136.5 (C-15), 171.4 (C-21). HRMS (ESI): [M+H]+ calcd for C22H34O2: 331.2631,
found: 331.2629
ent-Beyer-15-en-19-amine (27): Azide 26 (400 mg, 1.26 mmol) and
tryphenylphosphine (429.3 mg, 1.6 mmol) was dissolved in THF/H2O (8:0.6 mL)
and stirred for 24 h at room temperature. The mixture was concentrated, and
the residue was purified by flash column chromatography on silica gel
(CH2Cl2/MeOH, 12:1) obtaining 27 (217.7 mg, 60 % yield). Physical State: pale
yellow solid (mp: 283-285 °C); IR (cm-1) 3018, 2924, 2844, 1601, 1528, 1456m;
1H NMR (CDCl3, 300 MHz) δ (ppm): 0.8 (3H, s), 0.9 (3H, s), 1.0 (3H, s), 1.4 (1H,
d, J = 3.4 Hz), 1.5 (1H, q, J = 2.1 Hz), 1.6 (1H, dt, J = 14.1, 4.0 Hz), 1.8 (1H, m),
2.4 (1H, dt, J = 13.0, 2,3 Hz), 2.7 (1H, d, J =12.9 Hz), 5.5 (1H, d, J = 5,6 Hz),
5.8 (1H, d, J = 5.7 Hz). 13C NMR (CDCl3, 75 MHz) δ (ppm): 15.8 (C-20), 18.5
(C-6), 20.3 (C-2), 20.4 (C-11), 25.0 (C-17), 28.4 (C-18), 33.3 (C-12), 37.2 (C-7),
37.4 (C-10), 37.7 (C-4), 37.8 (C-3), 39.5 (C-1), 43.6 (C-19), 49.1 (C-13), 53.1
(C-8), 54.4 (C-9), 57.0 (C-5), 57.5 (C-14), 61.1, (), 135.2 (C-16), 136.4 (C-15).
HRMS (ESI): [M+H]+ calcd for C20H33N: 288.2685, found: 288.2687.
ent-Beyer-15-en-19-methyl ether (23): To a solution of 3 (60 mg, 0.21 mmol)
in dry THF (5 mL) and inert atmosphere was added an excess of NaH (49.3mg,
2.0mmol). After stirred for 40 minutes, iodomethane was added (110µL, 1.8
mmol) and was refluxed for 4 h. Solution of NaHCO3 5% was added to the
reaction mixture and extracted with CH2Cl2 (2 × 15 mL). The combined organic
layers were washed with brine, dried over Na2SO4, filtered and concentrated.
The residue was purified by flash column chromatography on silica gel
(hexane/EtOAc, 20:1) to afford 23 (54.6 mg, 87% yield). Physical State:
colorless oil; IR (cm-1) 2921, 1449, 1367, 1108, 1043. 1H NMR (CDCl3, 300
MHz) δ (ppm): 0.7 (3H, s), 1.0 (3H, s), 1.0 (3H, s), 1.4 (1H, m), 1.6 (2H, t, J =
2.4, Hz), 1.8 (1H, m) 3.2 (1H, dd, J = 9.1, 1.7 Hz) 3.3 (3H, s), 3.5 (1H, m), 5.5
(1H, dd, J = 5.7, 1.9 Hz), 5.7 (1H, d, J = 5.8, 2.0 Hz). 13C NMR (CDCl3, 75 MHz)
δ (ppm): 15.7 (C-20), 18.3 (C-6), 20.2 (C-2), 20.2 (C-11), 24.3 (C-17), 27.2(C-
18), 33.2 (C-12), 36.2(C-7), 37.2 (C-10), 37.6 (C-3), 37.8 (C-4), 39.2 (C-1), 43.5
(C-13), 49.0 (C-8), 52.9 (C-9), 56.9 (C-5), 59.2 (C-21), 61.1 (C-14), 76.0 (C-19),
135.1 (C-16), 136.4 (C-15). HRMS (ESI): [M+CH3OH+H]+ calcd for C21H34O:
335.2944, found: 335.2980
ent-Beyer-15-en-19-acetamide (28): Amine 27 (70 mg, 0.24 mmol) and
excees of acetic anhydride (32.5µL, 1.4 mmol) was dissolved in pyridine (3 mL).
DMAP (cat, 0.01mmol) were added to the reaction mixture and stirred for 3 h at
room temperature. Water (30 mL) was added to the mixture of reaction and
extracted with EtOAc, (2 x 30 mL). The combined organic layer was washed
with HCl 10 % solution (2 x 20 mL) and dried over Na2SO4, filtered and
evaporated under reduced pressure. The residue was purified by flash column
chromatography on silica gel (hexane/EtOAc, 2:1) yielding 28 (57.6 mg, 80 %).
Physical State: white solid (mp: 189-191 °C); IR (cm-1) 3288, 2923, 2841, 1644,
1292; 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.8 (3H, s), 0.9 (3H, s), 1.0 (3H, s),
2.0 (3H, s), 3.0 (1H, ddd, J = 13.6, 5.7, 1,4 Hz), 3.7 (1H, dd, J =13.6, 6.9 Hz),
5.5 (1H, d, J = 5,7 Hz), 5.7 (1H, d, J = 5.7 Hz). 13C NMR (CDCl3, 75 MHz) δ
(ppm): 15.8 (C-20), 18.3 (C-6), 20.1 (C-2), 20.3 (C-11), 23.5 (C-22), 24.9 (C-17),
27.9 (C-18), 33.2 (C-12), 36.6 (C-7), 37.3 (C-4,10), 37.5 (C-3), 39.1 (C-1), 42.1
(C-19), 43,6 (C-13), 49.0 (C-8), 53.0 (C-9), 56.9 (C-5), 61.1 (C-14), 135.0 (C-
16), 136.5 (C-15), 170.2 (C-21). HRMS (ESI): [M+H]+ calcd for C22H35NO:
330.2791, found: 330.2787.
ent-Beyer-15-en-19-benzyl ether (24): 3 (50.0 mg, 0.17 mmol) in dry THF (3
mL) was added NaH (31.2 mg, 0.8 mmol). the resultant solution was refluxed
for 30 min. and Benzyl Bromide (0.084 mL, 0.12 mmol) was added and the
resulting mixture was refluxed for 3 h. The reaction mixture was quenched with
water (10 mL), extracted with CH2Cl2 (2 × 15 mL). The combined organic layer
was washed with brine solution (15 mL), dried over Na2SO4, filtered and
evaporated under reduced pressure. The residue was purified by flash column
chromatography on silica gel (hexane/EtOAc, 20:0.5) yielding Benzyl ether 24
(59.5 mg, 96 % yield). Physical State: yellow oil. IR (cm-1) 2920, 2845, 1451,
1345. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.7 (3H, s), 1.0 (3H, s), 1.1 (3H, s),
1.6 (2H, dd, J = 5.0, 2.6 Hz), 1.9 (1H, dq, J = 13.3, 2.9 Hz), 3.3 (1H, d, J = 9.0
Hz) 3.6 (1H, d, J = 8.9 Hz), 4.5 (2H, m), 5.5 (1H, d, J = 5.7 Hz), 5.7 (1H, d, J =
5.7 Hz), 7.5 (1H, m), 8.0 (2H, m). 13C NMR (CDCl3, 75 MHz) δ (ppm): 15.7 (C-
20), 18.4 (C-6), 20.3 (C-2), 20.3 (C-11), 25.0 (C-17), 28.1 (C-18), 33.2 (C-12),
36.4 (C-7), 37.2 (C-10), 37.7 (C-4), 37.6 (C-3), 39.2 (C-1), 43.6 (C-13), 49.0 (C-
8), 52.9 (C-9), 56.9 (C-5), 61.0 (C-14), 73.2 (C-19), 128.3 (C-24,26), 128.4 (C-
23,27), 135.2 (C-16), 136.4 (C-15), 139.2 (C-22). HRMS (ESI): [M+H]+ calcd for
C27H38O: 379.2995, found: 379.2995
N-(Maleic acid) ent-Beyer-15-en-19-amide (29): Amine 27 (80 mg, 0.27
mmol), maleic anhydride (107.3 mg, 0.28 mmol), DMAP (0.01mmol) was
dissolved in pyridine (4 mL) was stirred at room temperature for 3.5 h. The
reaction mixture was dissolved in dichloromethane (30 mL) and was washed
with HCl 1.0 M (2 x 20 mL) and brine solution (2 x 15mL) and dried over Na2SO4,
filtered and evaporated under reduced pressure. The residue was purified by
flash column chromatography on silica gel (CH2Cl2/MeOH, 12:0.5) to give the
amide 29 (59.0 mg, 55 %). Physical State: white solid (mp: 215-217 °C); IR (cm-
1) 3245, 2928, 2845, 1716, 1642, 1299; 1H NMR (DMSO-d6, 300 MHz) δ (ppm):
0.7 (3H, s), 0.8 (3H, s), 1.0 (3H, s), 2.9 (1H, dd, J = 13.4, 5.8 Hz) 5.5 (1H, d, J
= 5,6 Hz), 5.7 (1H, d, J = 5.6 Hz), 6.2 (1H, d, J =12.7 Hz), 6.3 (1H, d, J =12.8
Hz) 13C NMR (DMSO-d6, 75 MHz) δ (ppm): 15.9 (C-20), 18.3 (C-6),19,8 (C-2),
20.2 (C-11),), 25.2 (C-17), 28.1 (C-18), 33.1 (C-12), 36.3 (C-7), 37.3 (C10), 37.5
(C-3), 37.9 (C-4), 38.9 (C-1), 41.9 (C-19), 43,7 (C-13), 49.0 (C-8), 52.7 (C-9),
56.8 (C-5), 61.1 (C-14), 131.6 (C-22), 134.4 (C-23), 135.5 (C-16), 136.5 (C-15),
166.3 (C-21), 166.6 (C-24). HRMS (ESI): [M+H]+ calcd for C24H35NO3:
386.2689, found: 386.2686.
ent-Beyer-15-en-19-benzoate (25): 3 (41.1 mg, 0.10 mmol) was dissolved in
dicloromethane (5 mL) and was added Benzoyl chloride (0.023 mL, 0,19 mmol).
Then triethylamine (0.1 mL, 0.6 mmol) and the reaction mixture was stirred for
2 h. Water (10 mL) was added to the reaction mixture and extracted with CH2Cl2
(3 × 15 mL). The combined organic layer was washed with brine solution (2x15
mL) and dried over Na2SO4, filtered and evaporated under reduced pressure.
The residue was purified by flash column chromatography on silica gel
(hexane/EtOAc, 20:1 yielding Benzoyl ester 25 (34.5 mg, 82% yield). Physical
State: pale yellow solid (mp: 99-101 °C); IR (cm-1) 2920, 1711, 1445, 1285; 1H
NMR (CDCl3, 300 MHz) δ (ppm): 0.7 (3H, s), 0.9 (3H, s), 1.0 (3H, s), 1.1 (1H, q,
J = 3.8 Hz), 1.4 (1H, m), 1.4 (1H, d, J = 2.3 Hz), 4.0 (1H, dd, J = 11.1, 1.3 Hz)
4.5 (1H, d, J = 11.0 Hz), 5.4 (1H, d, J = 5.6 Hz), 5.6 (1H, d, J = 5.6 Hz), 7.4 (2H,
N-(Allyl) ent-Beyer-15-en-19-amide (30): 27 (50 mg, 0.17 mmol), acrylic acid
(24 µL, 0.35 mmol), TEA (36 µL, 0.27 mmol), DMAP (0.01mmol) and DCC (51
mg, 0.24 mmol) were dissolved in CH2Cl2 (8 mL) and stirred overnight at room
temperature. The reaction mixture was dissolved in dichloromethane (30 mL),
washed with HCl 10% solution (2 x 15 mL) and water (2 x 15 mL) and dried over
Na2SO4, filtered and evaporated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (hexane/EtOAc, 4:1)
yielding 30 (38.3 mg, 64.4 %,). Physical State: white solid (mp: 164-166 °C); IR
(cm-1) 3298, 2921, 2845, 1658, 1627, 1558, 1244; 1H NMR (CDCl3, 300 MHz) δ
(ppm): 0.8 (3H, s), 0.9 (3H, s), 1.0 (3H, s), 1.7 (2H, dt, J = 10.8, 2.9 Hz), 3.1 (1H,
7
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202001424
European Journal of Organic Chemistry
FULL PAPER
dd, J = 13.6, 5.6 Hz), 3.8 (3H, m), 5.5 (1H, d, J = 5,4 Hz), 5.7 (1H, m), 6.1 (1H,
ddd, J =16.5, 10.0, 2.4 Hz), 6.3 (1H, dq, J =17.0, 1.6 Hz) 13C NMR (DMSO-d6,
75 MHz) δ (ppm): 15.8 (C-20), 18.3 (C-6), 20,1 (C-2), 20.2 (C-11),), 25.0 (C-17),
28.0 (C-18), 33.2 (C-12), 36.7 (C-7), 37.2 (C10), 37.5 (C-3), 37.6 (C4), 39.1 (C-
1), 42.0 (C-19), 43,6 (C-13), 49.0 (C-8), 53.0 (C-9), 57,0 (C-5), 61.1 (C-14),
131.1 (C-22), 126.3 (C-23), 135.1 (C-16), 136.6 (C-15), 165.8 (C-21). HRMS
(ESI): [M+H]+ calcd for C23H35NO: 342.2791, found: 342.2798.
(C-18), 33.3 (C-12), 37.8 (C-10), 38.6 (C-7), 38.9 (C-3), 40.1 (C-1), 40.8 (C-4),
43.5 (C-13), 49.2 (C-8), 53.5 (C-9), 59.2 (C-5), 60.9 (C-14), 76.4 (C-19), 126.5
(C-24), 126.9 (C-23,25), 127.1(C-22,26), 134.9 (C-16), 136.5 (C-15), 143.7 (C-
21). HRMS (ESI): [M+NH4]+ calcd for C26H36O: 382.3104, found 382.3101
19-Phenyl-ent-Beyeran-19-ol (37): Alcohol 36 (20 mg, 0.07 mmol) was
dissolved in methanol and trated with an excess of Pd/C (3.0 mg, 10% mol Pd,
0.01 mmol). Solution was stirred under a hydrogen atmosphere at room
temperature for overnight. The reaction mixture was filtered through a celite path
and the solvent was evaporated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (hexane/EtOAc, 10:1)
yielding 37 (16.3 mg, 82% yield). Physical State: white solid (mp: 141-143 °C);
IR (cm-1): 3406, 2862, 1471, 1365. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.9 (3H,
s), 1.2 (3H, s), 1.3 (3H, s), 1.9 (1H, ddd, J = 14.5, 11.4, 2.9 Hz), 2.1 (1H, d, J =
11.8 Hz), 5.3 (1H, s), 7.30 (1H, s), 7.31 (1H, s), 7.33 (2H, t, J = 1.6 Hz). 13C
NMR (CDCl3, 75 MHz) δ (ppm): 14.8 (C-20), 18.1 (C-6), 20.0 (C-2), 20.6 (C-16),
20.7 (C-15), 23.1 (C-11), 23.2 (C-17), 24.1 (C-18), 32.9 (C-12), 38.8 (C-10),
40.1 (C-7), 40.4 (C-3), 40.1 (C-1), 40.6 (C-4), 41.0 (C-13), 50.0 (C-14), 54.0 (C-
9), 59.2 (C-5), 76.8 (C-19), 127.1 (C-24), 127.5 (C-23,25), 127.7 (C-22,26),
143.6 (C-21). HRMS (ESI): [M+CH3CN+H]+ calcd for C26H38O: 408.3260, found
408.3251
ent-Beyer-15-en-19-carbaldehyde (31): 3 (400 mg, 1.4 mmol) was dissolved
in Dichloromethane (10 mL) and was added PCC (450 mg, 2.1 mmol). The
reaction mixture was stirred for 3.5 h. then the mixture was filtered through celite
and evapored under reduced pressure. The residue was purified by flash
column chromatography on silica gel (hexane/EtOAc, 12:1) yielding aldehyde
31 (268 mg, 72 % yield). Physical State: colorless oil; IR (cm-1) 2926, 1718,
1452, 1260. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.6 (3H, s), 1.0 (3H, s), 1.2
(3H, s), 1.7 (2H, d, J = 6.4 Hz), 1.9 (1H, d, J = 12.7 Hz), 2.1 (2H, dd, J = 18.7,
11.2 Hz), 5.5 (1H, d, J = 5.3 Hz), 5.5 (1H, d, J = 5.31 Hz), 9.8 (1H, s). 13C NMR
(CDCl3, 75 MHz) δ (ppm): 14.5 (C-20), 18.4 (C-6), 19.2 (C-2), 20.5 (C-11), 24.3
(C-17), 29.7 (C-18), 32.2 (C-12), 34.1 (C-7), 37.4 (C-3), 37.5 (C-10), 38.6 (C-1),
43.6 (C-13), 48.3 (C-8), 48.8 (C-4), 51.6 (C-9), 56.7 (C-5), 60.9 (C-14), 134.5
(C-16), 136.8 (C-15), 206.1 (C-19).
19-Ethyl-ent-Beyer-15-en-19-ol (32): The procedure for preparing 32 was
exactly the same as for preparing 36 from compound 31 (40 mg, 0.1 mmol),
yielding 32 (16 mg, 36 %). Physical State: yellow oil; IR (cm-1) 3390, 2847, 1733,
1453, 1237, 1029. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.86 (3H, s), 0.9 (3H, s),
0.9 (3H, s), 1.0 (3H, s), 1.4 (1H, m) 1.6 (2H, dt, J = 0.9, Hz), 1.7 (1H, m), 4.0
(1H, ddd, J = 10,4, 6.3, 2.2 Hz), 5.6 (1H, d, J = 5.6 Hz), 5.7 (1H, d, J = 5.6 Hz.
13C NMR (CDCl3, 75 MHz) δ (ppm): 11.5 (C-22), 15.7 (C-20), 18.1 (C-6), 20.2
(C-2), 22.9 (C-11), 23.1 (C-17), 24.9 (C-18), 25.4 (C-21), 33.2 (C-12), 37.8 (C-
10), 38.5 (C-7), 39.0 (C-3), 40.1 (C-1), 40.6 (C-4), 43.5 (C-13), 49.2 (C-8), 53.5
(C-9), 59.3 (C-5), 61.0 (C-14), 76.3 (C-19), 134.9 (C-16), 136.4(C-15). HRMS
(ESI): [M+NH4]+ calcd for C22H36O: 334.3104, found: 334.3100.
N-Hexyl-ent-beyer-15-en-19-amine (38): Aldehyde 31 (50.0 mg, 0.17 mmol)
was dissolved in MeOH (4.0 mL) then hexylamine (0.34mmol, 2.0 eq) was
added the resulting mixture was stirred for 30 minutes, and an excess of sodium
cyanoborohydride (32.8 mg, 0.5 mmol: 3.0 eq) the mixture was sttired for 24 h.
Metanol was concentered after the residue was purified by flash column
chromatography on silica gel (CH2Cl2/MeOH, 20:1) yielding amine 38 (36 mg,
65 % yield). Physical State: white solid (mp: 168-170 °C); IR (cm-1) 2922, 2847,
2337, 1450, 1121; 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.7 (3H, s), 0.9 (3H, m),
1.0 (3H, s), 1.2 (3H, s), 1.6 (2H, d, J = 5.8 Hz), 1.7 (1H, d, J = 2.5 Hz), 1.9 (1H,
d, J = 14.8 Hz), 2.8 (1H, d, J = 12,3 Hz), 3.0 (1H, dt, J = 7.1, 4.0 Hz), 3.2 (1H,
d, J = 12,4 Hz), 5.5 (1H, d, J = 5,6 Hz), 5.8 (1H, d, J = 5.7 Hz). 13C NMR (CDCl3,
75 MHz) δ (ppm): 14.0 (C-20), 15.7 (C-26), 17.8 (C-6), 19.8 (C-2), 20.2 (C-11),
22.5 (C-25), 25.2 (C-24), 26.3 (C-23), 24.9 (C-17), 27.4 (C-18), 31.1 (C-22) 33.2
(C-12), 36.0 (C-7), 36.2 (C-10), 37.0 (C-3), 37.2 (C-4), 38.4 (C-1), 43.6 (C-13),
48.8 (C-19), 49.8 (C-8), 51.6 (C-21), 52.3 (C-9), 52.7 (C-5), 60.9 (C-14), 134.5
(C-16), 137.0 (C-15). HRMS (ESI): [M+H]+ calcd for C26H45N: 372.3624, found
372.3629
19-Propyl ent-Beyer-15-en-19-ol (33): The procedure for preparing 33 was
exactly the same as for preparing 36 from compound 31 (70 mg, 0.24 mmol),
yielding 33 (36.4 mg, 45.4 %). Physical State: white solid (mp: 91-93 °C); IR
(cm-1) 3485, 2921, 2844, 1725, 1455, 1342, 1272 cm-1. 1H NMR (CDCl3, 300
MHz) δ (ppm): 0.88 (3H, s), 0.9 (3H, s), 0.9 (3H, s), 1.0 (3H, s), 1.5 (2H, m), 1.8
(1H, ddd, J = 9.6, 4.3, 2.2 Hz), 4.1 (1H, dd, J = 10,1, 2.0 Hz), 5.5 (1H, d, J = 5.6
Hz), 5.7 (1H, d, J = 5.6 Hz. 13C NMR (CDCl3, 75 MHz) δ (ppm): 14.1 (C-23),
15.7 (C-20), 18.0 (C-6), 19.8 (C-22), 20.1 (C-2), 22.9 (C-11), 23.0 (C-17), 24.9
(C-18), 33.2 (C-12), 34.9 (C-21), 37.8 (C-10), 38.5 (C-7), 39.0 (C-3), 40.1 (C-1),
40.4 (C-4), 43.5 (C-13), 49.2 (C-8), 53.5 (C-9), 59.2 (C-5), 60.9 (C-14), 74.1 (C-
19), 134.8 (C-16), 136.4(C-15). HRMS (ESI): [M+NH4]+ calcd for C23H38O:
348.3260, found: 348.3263.
N-Ethyl-ent-beyer-15-en-19-amine (39): The procedure for preparing 39 was
exactly the same as for preparing 38 from compound 31 (54.0 mg, 0.18 mmol),
yielding 39 (28 mg, 48 %). Physical State: white solid (mp: 200-202 °C); IR (cm-
1) 2922, 2840, 2343, 1698, 1579, 1438, 1126; 1H NMR (CDCl3, 300 MHz) δ
(ppm): 0.7 (3H, s), 0.9 (3H, m), 1.2 (3H, t, J = 7,3 Hz), 1.5 (2H, m), 1.7 (1H, d, J
= 12.8 Hz), 1.8 (2H, m), 2.7(1H, d, J = 12,5 Hz), 3.0 (4H, m), 5.5 (1H, d, J = 5,6
Hz), 5.7 (1H, d, J = 5.6 Hz). 13C NMR (DMSO, 75 MHz) δ (ppm): 11.3 (C-20),
15.6 (C-22), 17.8 (C-6), 19.6 (C-2), 20.2 (C-11), 25.1 (C-17), 27.3 (C-18), 33.0
(C-12), 35.8 (C-7), 35.9 (C-4), 37.1 (C-3), 38.6 (C-1), 43.6 (C-13), 49.0 (C-8),
50.3 (C-21), 52.3 (C-9), 55.2 (C-19), 57.2 (C-5), 61.0 (C-14), 135.2 (C-16), 136.7
(C-15). HRMS (ESI): [M+H]+ calcd for C22H37N: 316.2998, found 316.2994
19-Butyl-ent-Beyer-15-en-19-ol (34): The procedure for preparing 34 was
exactly the same as for preparing 36 from compound 31 (40 mg, 0.1 mmol),
obtaining 34 (26 mg; 47% yield). Physical State: white solid (mp: 92-94 °C); IR
(cm-1) 3480, 2917, 2845, 1726, 1454, 1365; 1H NMR (CDCl3, 300 MHz) δ (ppm):
4.1 (1H, m), 5.5 (1H, d, J = 5.7Hz), 5.7 (1H, d, J = 5.7 Hz). 13C NMR (CDCl3, 75
MHz) δ (ppm): 14.1 (C-24), 15.5 (C-20), 18.0 (C-6), 20.1 (C-2), 22.7 (C-23), 22.9
(C-11), 23.1 (C-17), 24.9 (C-18), 29.0 (C-22), 32.7 (C-21), 33.3 (C-12), 37.8 (C-
10), 38.5 (C-7), 39.1 (C-3), 39.3(C-1), 40.8 (C-4), 43.5 (C-13), 49.1 (C-8), 53.5
(C-9), 59.3 (C-5), 60.9 (C-14), 74.3 (C-19), 134.6 (C-16), 136.5(C-15). HRMS
(ESI): [M+NH4]+ calcd for C24H40O: 362.3417, found 362.3428
N-Bencyl-ent-beyer-15-en-19-amine (40): The procedure for preparing 40
was exactly the same as for preparing 38 from compound 31 (60.0 mg, 0.21
mmol) to afford 40 (59.3 mg, 75%) Physical State: yellow oil; IR (cm-1) 2919,
2843, 2343, 1452, 1122; 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.6 (3H, s), 1.0
(3H, m), 1.1 (3H, s), 1.5 (1H, dd, J = 8.3, 2.5 Hz), 1.7 (1H, d, J = 9.7, 2.7 Hz)
1.8 (1H, m), 2.6 (1H, d, J = 12,2 Hz), 3.0 (1H, d, J = 12,2 Hz), 4.0 (1H, d, J =
13,4 Hz), 3.2 (1H, m), 5.5 (1H, d, J = 5,6 Hz), 5.6 (1H, d, J = 5.7 Hz), 7.5 (5H,
m), 13C NMR (CDCl3, 75 MHz) δ (ppm):15.5 (C-20), 17.7 (C-6), 19.8 (C-2), 20.1
(C-11), 24.9 (C-17), 27.5 (C-18), 33.1 (C-12), 36.1 (C-7) 36.5 (C-10), 35.9 (C-
4), 37.1 (C-3), 38.6 (C-1), 43.6 (C-13), 49.0 (C-8), 50.3 (C-21), 52.3 (C-9), 55.2
(C-19), 57.2 (C-5), 61.0 (C-14), 135.2 (C-16), 136.7 (C-15). HRMS (ESI): [M+H]+
calcd for C27H39N: 378.3155, found 378.3151
19-Allyl-ent-beyer-15-en-19-ol (35): The procedure for preparing 35 was the
same as for preparing 36 from compound 31 (40 mg, 0.1 mmol), yielding 35 (13
mg, 30 %). Physical State: white solid (mp: 109-111°C); IR (cm-1)3612, 2917,
2845, 1726, 1454, 1365. 1H NMR (CDCl3, 300 MHz) δ (ppm): 0.9 (6H, s), 1.0
(3H, s), 1.4 (1H, d, J = 1.1, Hz), 1.7 (1H, td, J = 3.3, 1.5 Hz), 1.8 (1H, dt, J = 3.8,
1.9 Hz), 2.0 (1H, m), 2.4 (1H, ddq, J = 13.7, 5.5, 1.8 Hz), 4.1 (1H, ddd, J = 10,5,
4.0, 2.4 Hz), 5.1 (1H, dt, J = 8,6, 2.0 Hz), 5.5 (1H, d, J = 5.7 Hz), 5.7 (1H, d, J =
5.7 Hz), 5.8 (1H, dddd, J = 16,5, 11.0, 8.7, 5.6 Hz), 13C NMR (CDCl3, 75 MHz)
δ (ppm): 14.1 (C-23), 15.7 (C-20), 18.0 (C-6), 19.8 (C-22), 20.1 (C-2), 22.9 (C-
11), 23.0 (C-17), 24.9 (C-18), 33.2 (C-12), 34.9 (C-21), 37.8 (C-10), 38.5 (C-7),
39.0 (C-3), 40.1 (C-1), 40.4 (C-4), 43.5 (C-13), 49.2 (C-8), 53.5 (C-9), 59.2 (C-
5), 60.9 (C-14), 74.1 (C-19), 134.8 (C-16), 136.4(C-15). HRMS (ESI): [M+NH4]+
calcd for C22H34O: 332.2947, found 332.2901
Steviol (41): Stevioside 1 (1.0 g, 1.24 mmol) was dissolved in water (15 mL)
and treated with NaIO4 (2.0 g, 9.4 mmol) the mixture was stirred overnight. Then
KOH (0.5 g, 8.9 mmol) was added to the reaction mixture. The suspenssion was
immersed in a hot oil-bath to 110 °C for 1h. Water (15 mL) was added and
reaction was treated with AcOH (0.5 mL) and extracted with ethyl acetate (40
mL) dried over Na2SO4 filtered and evaporated under reduced pressure. The
residue was purified by flash column chromatography on silica gel
(hexane/EtOAc, 1:1) yielding Steviol 41 (32.2 mg, 8 % yield). Physical State:
white solid(mp: 199-201 °C); IR (cm-1) 3476, 2945, 1698, 1460, 1243; 1H NMR
(CDCl3, 300 MHz) δ (ppm): 1.0 (3H, s), 1.2 (3H, s), 1.0 (3H, s), 2.1 (2H, dt, J =
6.1, 2.5 Hz), 2.2 (1H, q, J = 3.3 Hz), 4.8 (1H, d, J = 2.1 Hz), 5.0 (1H, t, J = 2.6
Hz). 13C NMR (CDCl3, 75 MHz) δ (ppm): 15.4 (C-20), 18.9 (C-6), 20.3 (C-2),
21.7 (C-11), 28.1 (C-18), 37.6 (C-12), 39.2 (C-10), 39.4 (C-7), 40.4 (C-3), 41.2
(C-1), 41.7 (C-4), 43.5 (C-8), 46.8 (C-15), 47.3 (C-14), 53.8 (C-9), 56.8(C-5),
80.4 (C-13), 103.1 (C-17), 155.5 (C-16), 183.8 (C-19). HRMS (ESI) calcd for
C20H30O3 [M+H]+ 319.2267, found 319.2254
19-Phenyl-ent-beyer-15-en-19-ol (36): Aldehyde 31 (40.0 mg, 0.10 mmol)
was dissolved in dry THF (3.0 mL) and stirred under an argon atmosphere at
room temperature for 5 minutes. The mixture was cooled to -78 °C, and an
excess the phenylmagnesium bromide solution (2.0 M, THF, 0.2 mL) was added
dropwise: The reaction mixture was then quenched by the addition of aqueous
1N HCl l and extracted with EtOAc (2 x 20 mL). The organic layers were washed
successively with brine solution, dried with Na2SO4, filtered and concentered in
vacuo. The residue was purified by flash column chromatography on silica gel
(hexane/EtOAc, 10:1) yielding 36 (22 mg, 44 % yield). Physical State: white
solid (mp: 151-153 °C); IR (cm-1) 3390, 2849, 1726, 1452, 1273; 1H NMR (CDCl3,
300 MHz) δ (ppm): 0.9 (3H, s), 1.0 (3H, s), 1.2 (3H, s), 1.5 (1H, dd, J = 9.6, 3.2
Hz), 1.8 (2H, m), 1.9 (1H, m) 5.4 (1H, s), 5.5 (1H, d, J = 5,7 Hz), 5.8 (1H, d, J =
5.6 Hz), 7.2 (2H, m), 7.3 (1H, s), 7.3 (2H, d, J = 1.6 Hz). 13C NMR (CDCl3, 75
MHz) δ (ppm): 15.3 (C-20), 18.5 (C-6), 20.4 (C-2), 22.9 (C-11), 23.4 (C-17), 24.8
ent-Kaur-16-en-13,19-diol (42): Steviol 41 (30 mg, 0.1 mmol) was dissolved in
dry THF (8 mL) and was added an excess of LiAlH4 (17.8 mg, 0.5 mmol). The
reaction mixture was refluxed for 3 h. Then the reaction was quenched with
KOH solution 10% (10 mL) and extracted with EtOAc (2 x 15 mL) dried over
8
This article is protected by copyright. All rights reserved.

10.1002/ejoc.202001424
European Journal of Organic Chemistry
FULL PAPER
Na2SO4 filtered and evaporated under reduced pressure. The residue was
purified by flash column chromatography on silica gel (hexane/EtOAc, 4:6)
obtaining Stevidiol 42 (25.3 mg 88% yield). Physical State: white solid (mp: 217-
219 °C); IR (cm-1): 3303, 2977, 2928, 1694,1476, 1014. 1H NMR (CDCl3, 300
MHz) δ (ppm): 1.0 (3H, s), 1.2 (3H, s), 1.0 (3H, s), 1.6 (2H, dd, J = 4.3, 2.0 Hz),
1.8 (2H, dd, J = 12.9, 3.8 Hz), 1.9 (1H, d, J = 10.7 Hz), 4.8 (1H, d, J = 2.1 Hz),
5.0 (1H, t, J = 2.6 Hz). 13C NMR (CDCl3, 75 MHz) δ (ppm): 18.7 (C-20), 19.0 (C-
6), 20.7 (C-2), 20.9 (C-11), 28.5 (C-18), 36.2 (C-12), 39.3 (C-7), 39.5 (C-3), 39.6
(C-10), 41.2 (C-1), 42.0 (C-4), 42.5 (C-8), 47.1 (C-15), 48.3 (C-14), 55.4 (C-9),
57.3 (C-5), 63.6 (C-19), 79.7 (C-13), 103.4 (C-17), 157.3 (C-16). HRMS (ESI)
calcd for C20H34O2 [M+H]+ 305.2475, found 305.2472
[18] D. J. Finney, in Statistical Method in Biological Assay (Mathematics in
Medicine series), Oxford Univ. Pr., 1979
[19] S. A. Pulido, D. L. Muñoz, A. M. Restrepo, C. V. Mesa, J. F. Alzate, I. D.
Velez, S. M. Robledo, Acta Trop. 2012, 122, 36-45.
Acknowledgements.
Jilmar Murillo in memoriam, doctoral student, Universidad de
Antioquia.
This work was sponsored by Colciencias (Grant 111565843059).
Keywords: Cutaneous leishmaniasis • ent-kaurenes • ent-
beyerenes • antiparasites • cytotoxicity.
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10.1002/ejoc.202001424
European Journal of Organic Chemistry
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Synthesis of ent-beyerene and ent-kaurene derivatives of the sweetener Stevia is described, including the cytotoxicity tests and in vitro
activity against Leishmania braziliensis, the most predominant form of the disease in America. Twenty-one of the 39 synthesized
compounds showed high leishmanicidal activity, even at concentrations below 10 mM
Grupo de Química Orgánica de Productos Naturales, Instituto de Química. J. A Murillo, F. Echeverri, W. Quiñones, F. Torres, L. Isaza,
G. A. Escobar*.
PECET, Facultad de Medicina. S. M. Robledo, T. Pineda
Medicinal and Natural Products Chemistry, The University of Iowa. H. Olivo
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@gustavo37378861 (G. A. Escobar)
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