Preparation of antiproliferative terpene-alkaloid hybrids by free radical-mediated modification of ent-kauranic derivatives

Article abstract of DOI:10.3390/molecules26154549

A convenient strategy for molecular editing of available ent-kauranic natural scaffolds has been developed based on radical mediated C–C bond formation. Iodine atom transfer radical addition (ATRA) followed by rapid ionic elimination and radical azidoalkylation were investigated. Both reactions involve radical addition to the exo-methylenic double bond of the parent substrate. Easy transformations of the obtained adducts lead to extended diterpenes of broad structural diversity and artificial diterpene-alkaloid hybrids possessing lactam and pyrrolidine pharmacophores. The cytotoxicity of selected diterpenic derivatives was examined by in vitro testing on several tumor cell lines. The terpene-alkaloid hybrids containing N-heterocycles with unprecedented spiro-junction have shown relevant cytotoxicity and promising selectivity indexes. These results represent a solid basis for following research on the synthesis of such derivatives based on available natural product templates.

Full text of DOI:10.3390/molecules26154549

molecules
Article
Preparation of Antiproliferative Terpene-Alkaloid Hybrids
by Free Radical-Mediated Modification of
ent-Kauranic Derivatives
Elena Pruteanu 1,2 , Vladilena Gîrbu 1 , Nicon Ungur 1 , Leentje Persoons , Dirk Daelemans ,
3
3
2
,
1,
Philippe Renaud * and Veaceslav Kulcitki *
,
1
2
3
,
Institute of Chemistry (MECC), Str. Academiei, 3, MD-2028 Chisinău, Moldova;
elena.pruteanu@unibe.ch (E.P.); vgirbu1@gmail.com (V.G.); nicon.ungur@gmail.com (N.U.)
Department of Chemistry, Biochemistry and Pharmaceutical Sciences, University of Bern, Freiestrasse 3,
3012 Bern, Switzerland
KU Leuven, Department of Microbiology, Immunology and Transplantation, Laboratory of Virology
and Chemotherapy, Rega Institute for Medical Research, Herestraat 49, 3000 Leuven, Belgium;
leentje.persoons@rega.kuleuven.be (L.P.); dirk.daelemans@kuleuven.be (D.D.)
*
Correspondence: philippe.renaud@unibe.ch (P.R.); kulcitki@yahoo.com (V.K.)
Abstract: A convenient strategy for molecular editing of available ent-kauranic natural scaffolds
has been developed based on radical mediated C–C bond formation. Iodine atom transfer radical
addition (ATRA) followed by rapid ionic elimination and radical azidoalkylation were investigated.
Both reactions involve radical addition to the exo-methylenic double bond of the parent substrate.
Easy transformations of the obtained adducts lead to extended diterpenes of broad structural diver-
sity and artificial diterpene-alkaloid hybrids possessing lactam and pyrrolidine pharmacophores.
The cytotoxicity of selected diterpenic derivatives was examined by in vitro testing on several tumor
cell lines. The terpene-alkaloid hybrids containing N-heterocycles with unprecedented spiro-junction
have shown relevant cytotoxicity and promising selectivity indexes. These results represent a solid
basis for following research on the synthesis of such derivatives based on available natural prod-
uct templates.
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Citation: Pruteanu, E.; Gîrbu, V.;
Ungur, N.; Persoons, L.; Daelemans,
D.; Renaud, P.; Kulcit,ki, V.
Preparation of Antiproliferative
Terpene-Alkaloid Hybrids by Free
Radical-Mediated Modification of
ent-Kauranic Derivatives. Molecules
2
021, 26, 4549. https://doi.org/
1
0.3390/molecules26154549
Keywords: diterpene; ent-kaurane; alkaloids; radical chemistry; ATRA; lactam; lactone; spiro com-
pound; azide; pyrrolidine; cytotoxicity
Academic Editor: Maria José U.
Ferreira
Received: 21 June 2021
Accepted: 19 July 2021
Published: 28 July 2021
1
. Introduction
The kauranic family of diterpenes is widespread in diverse plant sources and some
representatives are readily available for large scale processing. The best known examples
are ent-kaurenoic acid and steviol (Figure 1), which can be conveniently isolated from
industrial crop derived products or residues. Iso-steviol can also be included in this
group, since it easily forms upon acidic hydrolysis of steviol glycosides found in the
Stevia Rebaudiana plant and is broadly used as an alternative non-caloric sweetener [ ].
These compounds alone possess relevant biological activities [ ], and numerous SAR
studies have been reported based on their structural modifications [ ]. In particular,
chemical modification of steviol and especially iso-steviol has been well explored and
1
2
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
3
2
1
2–4
5–7
2
3
delivered diverse compound libraries, basing on chemical reactivity of both carboxylic
groups, as well as D-cycle functionalities [6,7].
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
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distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
ent-kaurenoic acid 1 has also been extensively involved in SAR studies and chemical
modification was reported frequently at the level of the carboxylic functional group. The re-
16
activity of the exo-methylenic
addition processes or trivial oxidative transformations and only scarce data involve deeper
skeletal modifications based on this functionality [ ]. The exo-methylene moiety of acid
1 represents an additional opportunity for increasing molecular complexity based upon
∆ -double bond was mostly addressed via the heteroatom
8,9
4
.0/).
Molecules 2021, 26, 4549. https://doi.org/10.3390/molecules26154549
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 4549
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free radical processes. Free radical transformations are mild and versatile molecular edit-
ing tools, which have gained increasing popularity within the natural product chemistry
community due to their tremendous potential for convenient late-stage functionalization
of natural product scaffolds. Remote C–H functionalization is one example [10–12]. As a
logical consequence, medicinal chemistry research can fully benefit from the structural
diversity generated by free radical transformations of available natural product templates.
Figure 1. Structures of ent-kaurenoic acid 1, steviol 2 and iso-steviol 3.
In this context, we turned our attention to two mild free radical processes that proved
efficient for the functionalization of olefinic bonds in other compound families: the atom
transfer radical addition (ATRA) and the azidoalkylation reaction. ATRA are highly attrac-
tive transformations originating from the work of Kharasch, who discovered the addition
of activated alkyl halides to alkenes [13–15]. This pioneering work was further refined into
a valuable synthetic procedure [16] with a positive impact in diverse areas of chemistry [17].
Briefly, ATRA involves a starting alkyl halide with a weak C–X bond, which upon ho-
molytic cleavage generates a carbon-centered radical prone to interaction with the olefinic
moiety of the substrate in an addition process (Scheme 1, top). The resulting free radical
abstracts the X-atom from the alkyl halide and the reaction chain perpetuates. Iodine-ATRA
reactions leading to tertiary iodides are often followed by a rapid elimination of HI and
conversion to alkenes [18
process but involves an extra azidating agent, usually a sulfonyl azide (Scheme 1, bot-
tom) [20 22]. This reaction has been applied for the synthesis of several alkaloids [22 24
,19]. The azidoalkylation reaction is closely related to the ATRA
–
– ]
and its stereochemistry has been investigated in detail [25]. The formal result of both
transformations is a carbo-functionalization of the alkene, including the simultaneous
formation of both C–C and C–heteroatom bonds.
Such transformations are quite efficient and can be run under very mild reaction con-
ditions, compatible with other functional groups present in the substrate. Therefore, it rep-
resents an ideal tool for molecular editing within SAR studies of complex molecules like
ent-kauranes. It is worth mentioning that the exo-methylenic double bond in ent-kauranes
is regarded as one of the crucial elements in the interaction with molecular targets [5] and
the structural modifications of the diterpenic skeleton at the level of C (16)–C (17) carbon
atoms can result in new properties of the corresponding derivatives. In this respect, C (17)
alkylated ent-kauranes have been sporadically isolated from natural sources. A recent
example relates to helikaurolides A (
prenylated at C (17) with a densely functionalized sesquiterpenic unit [26]. Biogenetically,
both and have been hypothesized based upon ent-kaurenic acid alkylation with the
4) and B (5) (Figure 2)—unprecedented ent-kauranes,
4
5
1
sesquiterpenic unit. Therefore, radical C (17) alkylations represent an exact mimic of this
biogenetical mechanism. Having this in mind, we report in the current paper the results of
the synthesis and cytotoxic activity of new ent-kauranes based on radical additions to the
exo-methylenic double bond of ent-kaurenoic acid 1 and its derivatives.

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Scheme 1. Mild transformation of alkenes via ATRA (-elimination) and azidoalkylation: useful tools
El
for molecular editing (R = electrophilic C-residue).
Figure 2. Structures of helikaurolides A (4) and B (5).
2
2
2
. Results and Discussions
.1. Radical Addition to ent-Kaurenoates
.1.1. Iodine-ATRA Reactions
Carboiodination is a process that formally adds an alkyl group and an iodine atom
to the olefinic moieties. It is the simplest version of ATRA, and we started our work with
carboiodination of methyl ent-kaurenoate . It was prepared upon methylation of acid
6
1
with diazomethane. Ethyl iodoacetate was chosen as a readily available alkylating agent.
The reaction was performed in refluxing benzene under inert atmosphere in the presence
of dilauroyl peroxide (DLP) as an initiator (Scheme 2, Method A) [27].
DLP was gradually added to the reaction mixture in portions of 0.05 equivalents every
2
h during 24 h of reflux. Under these conditions, the conversion was sluggish and the
starting ester was recovered unchanged, along with minor amounts of esters (15%).
Their formation can be explained by the instability of the initially formed tertiary iodide,
which eliminates HI to provide and . A hydrogen atom transfer, possibly involving
6
7–9
7
8

Molecules 2021, 26, 4549
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DLP, can explain the formation of saturated
9. The separation of these compounds was
difficult and only the endocyclic isomer
satisfactory spectral data.
7 was isolated in a sufficiently pure form to provide
Scheme 2. Iodine -ATRA of ethyl iodoacetate to methyl-ent-kaurenoate 6.
In order to increase the conversion rate, we performed the reaction under the action
of a hexabutylditin–di-tert-butyl hyponitrite (DTBHN) system, which showed good results
in similar transformations [28]. The reaction resulted in the almost complete consumption
of the starting material over 2 h at reflux and a mixture of esters
8% yield (Scheme 2, Method B, 2:1:1). The formation of the tertiary iodide was not
observed. Such eliminations of tertiary iodides has been described in the literature [18].
7–9 was produced with an
8
7/8/9
2.1.2. Azidoalkylation
At this stage, it became clear that an efficient regiocontrol over HI elimination repre-
sents quite a challenging task for substrates with exo-methylenic double bonds. Therefore,
we turned our attention to the azidoalkylation reaction, a transformation of special value
from a SAR perspective due to the broad opportunities offered by the azide group for fur-
ther transformation [29] such as 1,3-dipolar cycloaddition (click reaction) [30] and reduction
to primary amines.
Azidoalkylation of acid
1 was performed under standard conditions using ethyl
iodoacetate (radical precursor) and phenylsulfonyl azide (azidating agent) in the presence
of hexabutylditin (chain transfer reagent) and DTBHN as an initiator [20]. The reaction
proceeded well and the resulting azide 10 was isolated in 70% yield (Scheme 3, Method A).
The free carboxyl group remained intact. Unfortunately, the purification of the product
was difficult and total removal of residual tin contaminants was not possible. In order to
avoid this problem, we investigated azidoalkylation of the corresponding methyl ester
6
under the same conditions. The azide 11 was isolated with an 83% yield without noticeable
contamination by tin residues. To avoid the use of toxic hexabutylditin, the reaction was
attempted using triethylborane as a mediator [31
,
32]. The compatibility of triethylborane
34]. We de-
with the free carboxylic functional group of was expected to be an issue [33
1
,
cided to circumvent this problem by using a two-fold excess of Et B and aqueous workup
3
to hydrolyze acyloxyboron derivatives. The addition products 10 and 11 were both isolated
in satisfactory 60% yields (Scheme 3, Method B).
Scheme 3. Azidoalkylation of ent-kaurenoic acid 1 and methyl-ent-kaurenoate 6.
To prepare the terpene-alkaloid hybrids, the product
γ-azidoester 11 was converted
to N-containg heterocycles. The hydrogenation of azide 11 went smoothly under 1 atmo-

Molecules 2021, 26, 4549
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sphere of hydrogen pressure and Pd/C as a catalyst. The initially formed primary amine
spontaneously formed the lactam 12 during the reduction (Scheme 4). Further reduction of
the lactam 12 with NaBH /I furnished the pyrrolidine 13 [35]. The spiro-cyclic system
4
2
in both 12 and 13 represents a novel ent-kauranic-alkaloid hybrid that turned out to be a
relevant pharmacophore, as revealed by cytotoxicity testing experiments described below.
The relative stereochemistry of lactam 12 has been determined by single crystal X-ray
crystallography (Scheme 4) [36]. These data confirmed indirectly the relative stereochem-
istry of starting azide 11, showing that the addition of azide radicals occurred in a highly
stereoselective way from the less hindered convex face of the tetracyclic ent-kauranic frame-
work [25]. Furthermore, the ethyl ester moiety of azide 11 could be selectively hydrolyzed
to provide the azido acid 14 in nearly quantitative yield.
Scheme 4. Conversion of the azidoalkylated product 11 into lactam 12, pyrrolidine 13 and azido acid
1
4. X-ray crystal structure of lactam 12 (ellipsoids drawn at 50% probability, CCDC 2023783).
We also observed that the nature of the iodide represents an important factor affecting
azidoalkylation of acid
1 and ester 6. Our attempt to use iodoacetonitrile instead of ethyl
iodoacetate did not result in any azidoalkylated product under Et B initiation. The major
3
reaction products are the unsaturated nitriles 15
–17 (Scheme 5), similar in structure to esters
7
and . This result is presumably due to the formation of an unstable iodide intermediate
8
in the case of the nitrile, while the ester radical adduct reacts directly with the sulfonyl azide
without the formation of a transient iodide. Indeed, iodoacetonitrile is a better iodine atom
donor than the corresponding ester by about two orders of magnitude [37]. Trace amounts
of an azide obtained from the azidoalkylation of acid
1 have been tentatively identified
based on the characteristic 13C NMR peak at 72.25 ppm, corresponding to the C-N carbon
3
atom. The isolation of this minor product turned out to be impossible using standard
chromatography techniques.
Scheme 5. Azidoalkylation attempt of methyl-ent-kaurenoate 6 with iodoacetonitrile.

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2
.2. Radical Addition to 15α-Hydroxy-ent-Kaurenoates
The modification of methyl 15 -hydroxy-ent-kaurenoate 20 was investigated next. The hy-
droxylated ent-kauranic acid derivative 18 comes together with ent-kaurenoic acid in the
α
1
sunflower vegetal mass as an angelic acid ester 19. The latter can be easily isolated and
hydrolyzed to provide pure 18 (Scheme 6). Methylation of 18 with diazomethane furnished
quantitatively the methyl ester 20 used for the radical mediated modification experiments.
Scheme 6. Preparation of methyl 15α-hydroxy-ent-kaurenoate 20.
2.2.1. Iodine ATRA Reactions
Upon heating the ester 20 with ethyl iodoacetate in benzene under DLP initiation,
a mixture of two major unsaturated compounds E-21 and 22 was obtained (Scheme 6).
In line with similar ATRA experiments with substrates and , the hypothetically formed
1
6
tertiary iodide is unstable, and rapid HI elimination leads to the unsaturated products [18].
Due to the presence of the alcohol moiety in 20, lactonization of the Z-alkene resulting from
the ATRA-elimination process occurred spontaneously under these reaction conditions
leading to the unsaturated lactone 22. The E-alkene cannot cyclize and the ester E-21 is
formed. Chromatographic separation of E-21 from lactone 22 proved to be difficult due to
their similar retention factors. Acetylation of the mixture of 21 and 22 with acetic anhydride
resulted in the formation of acetate 23 which could be easily separated by chromatography
and provided satisfactory analytical data for full characterization (Scheme 7). The structure
of lactone 22 was obtained by single crystal X-ray crystallography (Scheme 7) [36]. The basic
challenge at this stage was the demonstration of the E-configuration of the exocyclic double
bond in acetate 23 and indirectly in alcohol 21. The convincing proof came from the
experiments on azidoalkylation of the allylic alcohol 20 (see below).
Scheme 7. Addition of ethyl iodoacetate to 20. X-ray crystal structure of lactone 22·H O (ellipsoids
2
drawn at 50% probability, CCDC 2023785).

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2
.2.2. Azidoalkylation
The azidoalkylation process was investigated under similar conditions that brought
about optimal conversion of methyl ent-kaurenoate to the azide 11. Upon treatment of 20
6
with ethyl iodoacetate in the presence of hexabutylditin–DTBHN and phenylsulfonyl azide,
an optimal 90% conversion of starting material was achieved after 5 h of heating at reflux
in benzene (Scheme 8). However, no azide was detected but the already known ester E-21
and lactone 22 were obtained. When the same reaction was run at room temperature under
triethylborane initiation, again no azide was formed, and a mixture of unsaturated esters E-
21 and Z-21 was obtained (Scheme 8). Interestingly, the lower reaction temperature impedes
the lactonization of Z-21 and its isolation was possible. The absence of the azidoalkylated
product is presumably a consequence of the increased steric hindrance and decreased
nucleophilicity of the hydroxylated radical adduct relative to the non-hydroxylated one,
leading to 11.
Scheme 8. Iodine-ATRA between methyl 15α-hydroxy-ent-kaurenoate 20 and ethyl iodoacetate.
Having accessed all four compounds E-21, Z-21
,
22, and 23 of sufficient purity, we were
1
able to draw conclusions on their stereochemistry based on the H NMR spectra, in partic-
ular upon examining the splitting character of C (18) methylenic protons adjacent to the
carboxylic group in these compounds (Figure 3).
1
Figure 3. H NMR spectra sections of lactone 21 (A), ester 24 (B), ester 22 (C), and acetate 23 (D).
First, these methylenic protons in 22 resonate at 3.02 ppm (Figure 3A) and show a
relevant geminal coupling, which is due to the rigid cyclic structure and strong influence
of the adjacent oxygenated moieties. The vicinal coupling with the vinylic proton is in
1
accordance with dihedral angles observed in X-ray pictures. The H NMR spectrum of Z-21
shows a similar splitting profile of the methylenic group at C (18) (Figure 3B, 3.11–3.32 ppm)

Molecules 2021, 26, 4549
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to that observed in lactone 22, demonstrating the proximity of this group to the hydroxyl at
C (15) position, leading to a larger chemical shift difference between two geminal protons
at C (18). On the other hand, both C (18) methylenic protons in ester E-21 and acetate 23
are almost equivalent due to the conformational mobility of the lateral chain and impeded
interaction with the oxygenated functional group. Their NMR peaks represent doublets,
resonating at 3.08 ppm (Figure 3C) and 2.97 ppm (Figure 3D), respectively, and having an
identical coupling constant of 7.3 Hz with vinylic protons from C (17).
Interestingly, an attempt to run the azidoalkylation process with iodoacetic acid
instead of its ethyl ester led to the azide 24 and lactone 22 (Scheme 9). The azidoalkylation
was also achieved with iodoacetonitrile as the radical precursor. Performing the reaction
with triethylborane as radical initiator was compatible with the free carboxyl group in the
starting ent-kaurane 18 and resulted in product 25, opening up more opportunities for
further functionalization.
Scheme 9. Azidoalkylation of methyl 15-
α-hydroxy-ent-kaurenoate 20 and 15-α-hydroxy-ent-
kaurenoic acid 18 with iodoacetic acid and iodoacetonitrile.
Further transformations of azido-acid 24 included its methylation with diazomethane
to ester 26, which could be converted into lactam 27 during reduction of the azide under
hydrogenation conditions (Scheme 10).
Scheme 10. Conversion of the azidoalkylated acid 24 into ester 26 and lactam 27.
2.3. Cytotoxicity Testing
The described processes resulted in a small library of new compounds with modified
ent-kaurane skeletons, and their cytotoxic activity has been investigated. Proliferation
assays have been performed in seven different tumor cell lines as shown in Table 1: Capan-1
(pancreatic adenocarcinoma), HCT-116 (colorectal carcinoma), NCI-H460 (lung carcinoma),
DND-41 (acute lymphoblastic leukemia), HL-60 (acute myeloid leukemia), K-562 (chronic
myeloid leukemia), and Z-138 (non-Hodgkin lymphoma). A parallel cytotoxicity assay was
performed on non-cancerous retina cells (hTERT RPE-1) in order to estimate the selectivity
of the investigated compounds towards tumor cells. The inhibition of cell proliferation was
calculated as IC50 by interpolation based on the semi-log dose response. Docetaxel and stau-
rosporine have been included as positive controls. The data in Table 1 show that a significant

Molecules 2021, 26, 4549
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portion of the investigated compounds demonstrate relevant antitumoral activity. In partic-
ular, compounds 11–13, 22, 26, and 27 were active with IC50 values below 1 µM concentra-
tions. We were also pleased to observe a selective activity for some compounds, based on
much higher IC50 for normal non-cancerous cells. The calculated selectivity indices (SI),
i.e., the ratio of the IC50 for normal cells to the IC50 for the corresponding tumor cells [38],
are higher than the threshold value of 3 for several samples (see table in the supplementary
material for details). The highest selectivity indices were obtained for compounds 11
, 12,
13,
26, and 27, with IC50 values for retina (hTERT RPE-1) cells up to 43-fold higher than
those for cancerous cells. These data are indicative of a potential therapeutic index for
these compounds, which can be explored in subsequent in vivo toxicity tests.
Table 1. Antiproliferative activity of ent-kauranic derivatives. DT–docetaxel; SP–staurosporine.
IC50
Compound Conc. Unit
hTERT
RPE-1
Capan-1
HCT-116
NCI-H460
DND-41
HL-60
K-562
Z-138
7
µM
µM
µM
µM
µM
µM
µM
µM
µM
µM
µM
nM
nM
29.2 ± 1.2
1.8 ± 0.4
7.6 ± 0.5
32.2 ± 2.0
26.2 ± 0.9
38.2 ± 5.1
1.4 ± 0.1
31.7 ± 3.1
33.6 ± 4.5
25.9 ± 0.3
27.7 ± 4.3
18.7 ± 4.8
1.0
37.1 ± 3.9
1.7 ± 0.5
1.2 ± 0.7
11.5 ± 1.0
44.5 ± 8.3
18.1 ± 3.4
1.3 ± 0.6
33.2 ± 7.6
6.4 ± 1.5
8.3 ± 1.1
3.7 ± 1.7
4.2 ± 1.8
6.2 ± 1.8
30.7 ± 16.4
1.0 ± 0.4
0.8 ± 0.2
11.6 ± 0.5
5.8 ± 0.1
19.0 ± 5.7
0.5 ± 0.2
18.8 ± 8.0
34.4 ± 0.5
9.9 ± 2.7
4.0 ± 3.7
2.2 ± 0.8
1.5
12.0 ± 1.5
1.3 ± 0.1
1.4 ± 0.1
1.0 ± 0.1
31.1 ± 1.6
15.4 ± 6.0
1.4 ± 0.3
41.8 ± 5.3
34.0 ± 2.9
0.6 ± 0.2
1.9 ± 0.2
5.5 ± 1.3
2.2 ± 0.8
36.5 ± 0.7
1.4 ± 0.3
21.4 ± 10.1
6.1 ± 1.1
44.5 ± 4.8
7.8 ± 1.0
2.3 ± 0.3
38.9 ± 4.1
36.4 ± 9.8
6.6 ± 1.3
8.3 ± 2.0
4.7 ± 1.2
8.6 ± 1.5
19.6 ± 7.7
3.0 ± 1.4
38.3 ± 17.8
8.3 ± 1.3
46.2 ± 9.1
33.6 ± 2.6
3.7 ± 1.3
49.3 ± 5.0
>100
41.1 ± 15.9
38.0 ± 11.8
4.3 ± 1.6
9.1 ± 1.6
24.7 ± 11.8
8.8 ± 2.2
36.4 ± 11.5
3.2 ± 1.0
41.0
58.4 ± 2.5
2.5 ± 0.1
76.3 ± 1.4
>100
36.9 ± 0.2
56.5 ± 2.8
5.2 ± 1.2
27.9 ± 3.2
27.6 ± 15.5
0.5 ± 0.2
52.0 ± 33.4
9.5 ± 0.9
22.7
22.1 ± 11.4
2.4 ± 1.1
25.2 ± 0.3
52.0 ± 17.4
21.6 ± 5.4
39.8 ± 10.9
3.7 ± 0.7
6.7 ± 4.4
1
1
2
3
4
6
2
4
5
6
7
1
1
1
1
2
2
2
2
2
DT
SP
From a structural topology point of view, the most active compounds show some
relevant similarities (Figure 4). First of all, most of the active compounds include nitrogen
containing functional groups. The second structural convergence relates to the cyclic
backbone, which includes an extra cycle to the natural ent-kauranic framework. Exceptions
are azides 11 and 26. Finally, three of the four most relevant cytotoxic compounds with high
selectivity towards tumor cells are heterocycles 12, 13, and 27 with a spirocyclic fragment.
To the best of our knowledge, such derivatives of ent-kauranic compounds have never been
reported in SAR studies. The activity levels of these compounds is much higher than that
of the reference drug Cisplatin (IC50 16.5 for HCT-116 and 5.33 for NCI-H460) and in the
same range as the currently marketed drug midostaurine, with IC50 0.237 for HCT-116 and
0
.43 for NCI-H460 [39]. In fact, compounds 12 and 27 share the same
both natural staurosporine and synthetic midostaurine. Although this pharmacophore is
becoming increasingly more studied [40], little is known about the properties of -spiro-1-
γ-lactam unit with
γ
lactams, which are extremely rare in the literature. The same relates to their mechanism
of interaction with cellular targets [41]. Following in vivo toxicity studies can reveal the
therapeutic potential of the reported new ent-kauranic derivatives.

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Figure 4. Relevant cytotoxic activity parameters for selected compounds.
3. Materials and Methods
3.1. Chemistry General Section
IR spectra were recorded on a Perkin–Elmer (Beaconsfield, UK) Spectrum 100 FT-
IR spectrophotometer using the universal ATR sampling accessory. NMR spectra were
recorded in CDCl on a Bruker (Rheinstetten, Germany) Avance III spectrometer (400.13
3
and 100.61 MHz). Chemical shifts (
from tetramethylsilane Si (CH ) (
δ
) are reported in parts per million (ppm) downfield
1
δ
4
= 0.00 for H NMR spectra) using residual solvent signal
3
1
13
1
as an internal standard:
δ singlet 7.26 ( H), triplet 77.0 ( C) for CDCl or δ singlet 4.87 ( H),
3
13
septet 49.0 ( C) for CD OD. Multiplicities are given as s (singlet), d (doublet), t (triplet),
3
q (quadruplet), m (multiplet), and bs (broad signal). Coupling constant (J) is reported in Hz.
1
In case of mix of products, selected values for H NMR were reported (as specified in the
description). In 13C NMR spectra, the peak positions are reported to one decimal unless the
difference in chemical shift between two signals (for example for regio-/diastereoisomers)
is small and required two decimals. Optical rotations were measured in CHCl or MeOH
3
on a Jasco DIP 370 polarimeter, using a 5-cm cell. GC-MS experiments were performed on
an Agilent 7890A (Wilmington, DE, USA) chromatograph equipped with a quadrupole MS
detector MSD 5975C and an HP-5ms capillary column (30 m/0.25 mm/0.25
TLC was performed on Fluka Silica Alu foils. Usual workup included dilution with brine
and extraction with Et O. The organic phases have been dried over Na SO , filtered and the
µm). Analytical
2
2
4
solvent evaporated under reduced pressure to provide the crude reaction product. Column
chromatography (CC) was performed with Fluka Kieselgel 60 silica gel. All solvents were
of reagent grade and other reagents have been used as received from suppliers. DLP was
added in aliquot portions every 2 h to the solution of the corresponding olefin and iodide.
Addition of Et B solutions (commercial) to the reaction mixtures was performed via a
3
syringe with the needle immersed into the reaction mixture to avoid direct contact of
Et B drops with air. ATRA reactions have been performed with ethyl iodoacetate unless
3
otherwise specified. Benzene and THF have been dried according to standard procedures
and distilled before use.
3.2. Crystal-Structure Determination
The crystals were mounted in air at ambient conditions. All measurements were made
on an Oxford Diffraction SuperNova area-detector diffractometer [42] using mirror optics
monochromated Mo K radiation ( = 0.71073 Å) and Al filtered [43]. Data reduction was
α
λ
performed using the CrysAlisPro [42] program. The intensities were corrected for Lorentz
and polarization effects, and an absorption correction based on the multi-scan method
using SCALE3 ABSPACK in CrysAlisPro [42] was applied. Data collection, refinement

Molecules 2021, 26, 4549
11 of 19
parameters and other specific experimental details are given in the Supplementary Materi-
als. The structure was solved by direct methods using SHELXT [44], which revealed the
positions of all non-hydrogen atoms of the title compound. The non-hydrogen atoms were
refined anisotropically. All H-atoms were placed in geometrically calculated positions and
refined using a riding model where each H-atom was assigned a fixed isotropic displace-
ment parameter with a value equal to 1.2Ueq of its parent atom. Refinement of the structure
2
was carried out on F using full-matrix least-squares procedures, which minimized the
2
2 2
o c
function Σw (F –F ) . The weighting scheme was based on counting statistics and in-
cluded a factor to downweight the intense reflections. All calculations were performed
using the SHELXL-2014/7 [45] program in OLEX2 [46].
3.3. Cytotoxicity Testing
3.3.1. Cell Culture and Reference Compounds
All cell lines (HL-60, K-562, Z-138, Capan-1, HCT-116, NCI-H460 and hTERT RPE-1)
were acquired from the American Type Culture Collection (ATCC, Manassas, VA, USA),
except for the DND-41 cell line, which was purchased from the Deutsche Sammlung von
Mikroorganismen und Zellkulturen (DSMZ Leibniz-Institut, Braunschweig, Germany).
All cell lines were cultured as recommended by the suppliers. Culture media were pur-
chased from Gibco Life Technologies, USA, and supplemented with 10% fetal bovine
serum (HyClone, GE Healthcare Life Sciences, Logan, UT, USA). Reference inhibitor com-
pounds staurosporine and docetaxel were obtained from Selleckchem (Munich, Germany).
All stock solutions were prepared in DMSO.
3.3.2. Cell Proliferation Assays
Adherent cell lines Capan-1, HCT-116 and NCI-H460 cells were seeded at a density be-
tween 500 and 1500 cells per well in 384-well clear-bottomed tissue culture plates (Greiner).
After overnight incubation, cells were treated with the test compounds at seven different
−
3
concentrations ranging from 100 to 6.4
×
10
µM. Suspension cell lines DND-41, HL-60,
K-562, and Z-138 were seeded at densities ranging from 2500 to 5500 cells per well in
3
84-well clear-bottomed tissue culture plates containing the test compounds at the same
◦
seven concentration points. The plates were incubated at 37 C and monitored for 72 h in
an IncuCyte device (Essen BioScience Inc., Ann Arbor, MI, USA) for real-time imaging.
®
Images were taken every 3 h, with one field imaged per well under 10
×
magnification.
Cell growth was then quantified based on the percent cellular confluence as analysed by the
®
IncuCyte image analysis software and used to calculate IC values by linear interpolation.
50
All tests have been performed in duplicate and the average values have been calculated.
The corresponding mean values and deviations are provided in Table 1.
3
.4. Experimental Section
DLP-mediated ATRA to methyl ent-kaurenoate
Methyl ent-kaurenoate (50 mg, 0.16 mmol) in dry C H (5 mL) was treated with ethyl
iodoacetate (28 L, 0.24 mmol) and DLP (3 mg, 0.008 mmol). The reaction mixture was
heated on reflux under N for 24 h. The mixture was purified by CC (hexane/EtOAc 98:2)
6
6
6
µ
2
and gave unchanged starting material (40 mg, 80%) and a mixture of compounds
(10 mg, 15%).
7, 8 and
9
Ester 7
Pale yellow viscous oil. [α]20
−1
=
−
45.8 (c = 8.13, CHCl ). IR (
ν
, cm ): 3804, 3736, 3674,
D
3
1
3
608, 2927, 1728, 1445, 1231, 1156. H NMR (400 MHz, CDCl )
δ
H: 5.07 (s, 1H, CH-15), 4.13
3
(
q, J = 7.1 Hz, 2H, -CO2CH2CH ), 3.63 (s, 3H, -CO CH ), 2.44–2.49 (m, 2H, -CH2-COOEt),
3 2 3
1
(
4
.25 (t, J = 7.1 Hz, 3H, -CO CH2CH3), 1.16 (s, 3H, CH -20), 0.83 (s, 3H, CH -22), 2.38–0.93
2 3 3
m, 21H). 13C NMR (100 MHz, CDCl3)
7.8, 43.9, 43.8, 40.8, 39.6, 39.5, 38.2, 32.4, 29.7, 28.7, 25.4, 25.3, 20.8, 19.2, 18.9, 15.2, 14.2.
δC: 178.0, 173.4, 145.1, 134.4, 60.2, 56.9, 51.0, 49.0,
+
GCMS m/z calculated for [C H O ] : 402.28; found: 402.3.
25
38
4

Molecules 2021, 26, 4549
12 of 19
Ester 8
Mixture of 2 diastereomers. IR (v, cm 1): 3674, 2931, 1728, 1449, 1234, 1171, 1150.
H NMR (400 MHz, CDCl ) selected values, H: 4.15–4.09 (m, 2H, -CO2CH2CH ), 3.64 (s,
−
1
δ
3
3
3
2
H, -CO CH ), 1.25 (m, 3H, -CO CH2CH3), 1.17 (m, 3H, CH -20), 0.82 (m, 3H, CH -
2 3 2 3 3
13
2).
C NMR (100 MHz, CDCl ) selected values, δC: 178.04/178.03, 172.57/172.41,
3
1
49.98/148.94, 110.39/109.94, 60.36, 57.12/57.07, 55.17/55.10, 51.03, 49.32/46.21, 43.84,
4
3.59/43.48, 41.41/41.19, 40.80, 39.63/39.47, 39.41/39.27, 38.12, 34.90/34.68, 33.06/30.83,
2
8.73, 21.96, 19.16, 18.76/18.45, 15.45/15.41, 14.21/14.18. GCMS m/z calculated for
C H O : 402.28; found: 402.3.
25
38
4
Ester 9
Colorless oil. IR (
400 MHz, CDCl3) H: 4.12 (q, J = 7.9 Hz, 2H, -CO2CH2CH ), 3.63 (s, 3H, -CO CH ),
.86–2.60 (m, 1H), 2.29 (m, 2H, CH -17), 1.25 (t, J = 7.1 Hz, 3H, -CO CH CH ), 1.16 (s, 3H,
, cm 1): 3750, 2921, 1729, 1506, 1448, 1370, 1191, 1042. H NMR
−
1
ν
(
δ
3
2
3
2
2
2
2
3
13
CH -20), 0.81 (s, 3H, CH -22), 2.18–0.79 (m, 23H). C NMR (100 MHz, CDCl ) δ : 178.10,
3
3
3
C
1
3
74.03, 60.17, 57.15, 56.61, 51.05, 46.74, 44.41, 43.89, 42.22, 40.87, 40.51, 40.13, 39.48, 38.49,
8.21, 34.09, 28.77, 26.66, 25.88, 22.24, 19.19, 19.14, 15.42, 14.28. GCMS m/z calculated for
C H O : 404.29; found: 404.3.
25
40
4
Bu Sn -mediated ATRA to methyl ent-kaurenoate
6
2
Methyl ent-kaurenoate
6
(50 mg, 0.158 mmol) in dry C H (3 mL) was treated under
6
6
N with ethyl iodoacetate (37.4
2
µ
L, 0.316 mmol), Bu Sn (120
µL, 0.237 mmol), and DTBHN
6
2
(
0.8 mg, 0.005 mmol). The reaction mixture was refluxed for 2 h. The crude product
was purified by CC (hexane/EtOAc 98:2) and gave a 2:1:1 mixture of compounds
and (61 mg, 88% total yield). Separation of this mixture on a silver nitrate impregnated
silica afforded a pure sample of ester (45% yield), along with esters and (22% and
1% yield, respectively).
7, 8,
9
7
8
9
2
Bu Sn -mediated ATRA to ent-kaurenoic acid
6
2
ent-Kaurenoic acid
N with ethyl iodoacetate (51.5
1
(87 mg, 0.29 mmol) in dry benzene (2 mL) was treated under
L, 0.44 mmol), PhSO N (159 mg, 0.87 mmol), Bu Sn
µ
2
2
3
6
2
(
222 µL, 0.44 mmol), and DTBHN (1.5 mg, 0.087 mmol). The reaction mixture was refluxed
for 2 h and after this time the crude product was purified by CC (pentane/EtOAc 90:10)
giving compound 10, slightly contaminated with tin compounds (NMR). This impurified
product was methylated with an ethereal solution of diazomethane and purified by CC
(
hexane/EtOAc 98:2) to afford azide 11 (90 mg, 70%) as a colorless oil.
Azide 11
Colorless viscous oil. [α]2
0
D
−1
=
−
79.5 (c = 0.84, CHCl ). IR (
ν
, cm ): 2944, 2873, 2096,
727, 1502. H NMR (400 MHz, CDCl ) δH: 4.12 (q, J = 7.2 Hz, 2H, -CO2CH2CH ), 3.62 (s,
3
3
1
1
3
1
δ
3
4
3
H, -CO CH ), 2.42 (t, J = 7.9 Hz, 2H, -CH2-COOEt), 1.25 (t, J = 7.2 Hz, 3H, -CO CH2CH3),
2
3
2
13
.14 (s, 3H, CH -20), 0.80 (s, 3H, CH -22), 2.06–0.75 (m, 23H). C NMR (100 MHz, CDCl3)
3 3
C: 177.9, 173.4, 73.1, 60.5, 56.7, 55.8, 51.5, 51.1, 44.7, 44.3, 43.7, 41.4, 40.6, 39.4, 38.0, 37.7,
0.3, 29.7, 28.7, 26.1, 22.0, 19.0, 18.5, 15.3, 14.2. GCMS m/z calculated for [C H N O ]:
45.28; found: 402.3 (M-HN3).
25
39
3
4
Bu Sn -mediated ATRA to methyl ent-kaurenoate
6
2
Methyl ent-kaurenoate
N with ethyl iodoacetate (64
6
(100 mg, 0.36 mmol) in dry benzene (2 mL) was treated under
L, 0.54 mmol), PhSO N (200 mg, 1.08 mmol), Bu Sn
µ
2
2
3
6
2
(272 µL, 0.54 mmol), and DTBHN (2 mg, 0.01 mmol). The reaction mixture was refluxed
for 2 h. The crude product was purified by column chromatography (hexane/EtOAc 98:2)
and gave the azide 11 (133 mg, 83%).
Et B-mediated ATRA to ent-kaurenoic acid
3

Molecules 2021, 26, 4549
13 of 19
ent-Kaurenoic acid
with ethyl iodoacetate (35
.584 mmol). The reaction was stirred at room temperature overnight then diluted with
1
(44 mg, 0.146 mmol) in dry EtOH (2 mL) was treated under N2
µ
L, 0.292 mmol), PhSO N (80 mg, 0.438 mmol), Et B (0.6 mL,
2 3 3
0
brine and worked up. The crude product was purified by CC (hexane/EtOAc 94:6) giving
pure compound 10 (39 mg, 60%).
Azide 10
Pale yellow viscous oil. [α]2
0
=
−
69.2 (c = 1.9, CHCl ). IR (
ν
, cm ): 2935, 2900, 2872,
(ppm) 4.14 (q, J = 7.1 Hz, 2H,
CO2CH2CH ), 2.43 (t, J = 8.1 Hz, 2H, -CH2-COOEt), 1.26 (t, J = 7.1 Hz, 3H, -CO CH2CH3),
−1
D
3
1
2
-
848, 2626, 2098, 1734, 1693. H NMR (400 MHz, CDCl ) δ
3
3
2
13
1
.22 (s, 3H, CH -20), 0.93 (s, 3H, CH -22), 2.15–0.73 (m, 23H). C NMR (100 MHz, CDCl3)
3 3
δ
(ppm) 184.9, 173.4, 73.1, 60.6, 56.7, 55.8, 51.5, 44.7, 44.3, 43.7, 41.4, 40.5, 39.6, 37.7, 37.6,
30.3, 29.7, 28.9, 26.1, 21.8, 18.9, 18.5, 15.4, 14.2.
Et B-mediated ATRA to methyl ent-kaurenoate
3
Methyl ent-kaurenoate
N with ethyl iodoacetate (39
6
(50 mg, 0.16 mmol) in dry EtOH (2 mL) was treated under
L, 0.33 mmol), PhSO N (87 mg, 0.47 mmol) and Et B
µ
2
2
3
3
(
0.47 mL). The reaction mixture was stirred for 2 h at room temperature. The crude product
was purified by column chromatography (pentane/EtOAc 98:2) and gave the azide 11
43 mg, 60%).
Lactam 12
A solution of azide 11 (53 mg, 0.12 mmol) and 10% Pd/C (10% w/w) in dry EtOAc
2 mL) was stirred for 48 h at room temperature under H (1 atm). The catalyst was filtered
(
(
2
off, the solvent was removed under reduced pressure, and the crude purified by column
chromatography (CH Cl /MeOH 90:10) giving lactam 12 (42 mg, 95%) as a white powder.
2
2
◦
20
D
White powder. M.p. 150–152 C, from CH Cl . [α]
=
−
77.4 (c = 4.50, CHCl3).
H: 6.68 (s, 1H,
NH), 3.63 (s, 3H, -CO CH ), 2.30 (m, 2H, CH -17), 2.05 (d, J = 11.9 Hz, 2H, CH -18), 1.15
2
2
−
1
1
IR (
-
(
1
ν
, cm ): 3676, 3209, 2937, 2298, 1712, 1683. H NMR (400 MHz, CDCl )
δ
3
2
3
2
2
13
s, 3H, CH -20), 0.81 (s, 3H, CH -22), 2.36–0.79 (m, 21H). C NMR (100 MHz, CDCl3)
δ
C:
77.9, 176.8, 67.2, 57.6, 56.9, 55.6, 51.1, 46.6, 44.9, 43.8, 41.8, 40.7, 39.4, 38.9, 38.1, 31.1, 30.0,
8.7, 27.1, 22.0, 19.1, 18.8, 15.4.
Pyrrolidine 13
To a solution of lactam 12 (50 mg, 0.13 mmol) in THF (4 mL) were added NaBH4
20 mg, 0.53 mmol) and I (102 mg, 0.4 mmol). The reaction mixture was refluxed for 8 h,
3
3
2
(
2
◦
then cooled at 0 C and excess hydride quenched with 3N HCl solution, then neutralized
and worked up as usual. The crude product was purified by CC (CH Cl /MeOH 90:10)
2
2
giving compound 13 (40 mg, 85%) as a colorless oil.
20
−1
Colorless oil. [α]
722, 1599. H NMR (400 MHz, CDCl )
=
−
73.6 (c = 2.74, CHCl ). IR (
ν
, cm ): 3424, 2940, 2871, 2744, 2483,
D
3
1
1
δ
(ppm) 8.46 (s, 1H, NH) 3.63 (s, 3H, -CO CH ), 3.43
3
2
3
(
(
bs, 2H, CH -19), 2.65 (s, 1H, CH-13), 1.15 (s, 3H, CH -20), 0.81 (s, 3H, CH -22), 2.09–0.78
2
3
3
m, 24H). 13C NMR (100 MHz, CDCl3)
δ
(ppm) 177.9, 76.8, 56.7, 55.7, 52.4, 51.1, 45.7, 43.8,
43.3, 42.7, 40.7, 40.3, 39.5, 38.5, 38.1, 33.0, 28.6, 26.9, 22.7, 22.1, 19.0, 18.6, 15.3.
Azide 14
Azide 11 (50 mg, 0.11 mmol) was dissolved in EtOH and treated with KOH (31.4 mg,
0.56 mmol) on reflux for 2 h. Then, the reaction mixture was diluted with brine and extracted
with Et O and ethereal extract washed with 10% sol. H SO , sat. sol. NaHCO , brine and
2
2
4
3
dried over Na SO . The crude product was purified by CC (petroleum ether/EtOAc 90:10),
2
4
providing the azide 14 as colorless oil (45 mg, 98%).
Colorless oil. [α]2
0
=
−
85.6 (c = 4.50, CHCl ). IR (
ν
, cm ): 3855, 3668, 2942, 2098,
H: 3.64 (s, 3H,
−1
D
3
1
1
724, 1715, 1446, 1236, 1154, 1066, 1051, 774. H NMR (400 MHz, CDCl )
δ
3
-
2
CO CH ), 2.51 (t, J = 7.97 Hz, 2H, CH -17,), 1.16 (s, 3H, CH -20), 0.82 (s, 3H, CH -22),
2 3 2 3 3
1
3
.16–0.99 (m, 23H). C NMR (100 MHz, CDCl3)
δC: 178.6, 178.0, 72.9, 56.7, 55.8, 51.5, 51.2,
44.8, 44.3, 43.7, 41.4, 40.6, 39.4, 38.0, 37.7, 29.9, 29.5, 28.7, 26.2, 22.0, 19.0, 18.5, 15.3.

Molecules 2021, 26, 4549
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Et B-mediated ATRA of iodoacetonitrile to ent-kaurenoic acid.
3
ent-Kaurenoic acid
N with iodoacetonitrile (91
1
(190.0 mg, 0.63 mmol) in 3.5 mL EtOH was treated under
L, 1.25 mmol), PhSO N (345.0 mg, 1.88 mmol) and Et B
µ
2
2
3
3
(
(
∆
1.9 mL, 1.88 mmol) at room temperature overnight. The crude product was purified by FC
1
5
petroleum ether/EtOAc 90:10) and provided the isomeric mixture 15 (95 mg, 42%, ∆ :
16
= 45:55, NMR data).
Mixture of nitriles 15
−
1
IR (
CDCl ) selected values,
ν
, cm 1): 3671, 2923, 2854, 2666, 1696, 1469, 1445, 1413. H NMR (400 MHz,
δ
H
: 5.23 (s, CH-15), 5.18–5.14 (m, CH-17), 2.98 (s, 2xCH -18), 2.15
3
2
1
5
13
(
m, CH -17 ∆ ), 1.24, 1.23 (s, CH -20), 0.93, 0.95 (s, CH -22); C NMR (100 MHz, CDCl3)
2
3
3
16
selected values,
δ (major ∆ ): 184.04, 153.35, 118.38, 105.82, 56.99, 54.94, 45.94, 44.02,
C
1
5
4
1
2
1.20, 40.67, 39.67, 39.30, 37.75, 32.87, 28.92, 21.79, 19.05, 18.41, 17.04, 15.59; (minor
84.26, 142.85, 136.04, 119.73, 56.72, 49.22, 47.42, 43.76, 43.40, 40.66, 39.85, 39.26, 37.79, 28.94,
5.96, 25.32, 20.67, 19.05, 18.95, 15.56, 15.38.
∆ ):
Et B-mediated ATRA of iodoacetonitrile to methyl ent-kaurenoate
3
Methyl ent-kaurenoate
N with iodoacetonitrile (82
6
µ
(180.0 mg, 0.56 mmol) in 3.5 mL EtOH was treated under
L, 1.13 mmol), PySO N (313.0 mg, 1.7 mmol), and Et B
2
2
3
3
(
1.7 mL, 1.7 mmol) at room temperature overnight. The crude product was purified by
CC (petroleum ether/EtOAc 95:5) and provided the mixture of unsaturated derivatives 16
and 17 (117 mg, 58%, 16 17 = 3:1, NMR data). This mixture was submitted to a repetitive
/
separation on a silver nitrate impregnated silica column and individual nitriles slightly
contaminated with a second isomer were obtained as a colorless oils.
Nitrile 16
Colorless oil. [α]2
0
D
=
−
48.8 (c = 4.6, CHCl ). IR (
ν
, cm ): 2932, 2848, 1723, 1639, 1468,
446, 1232, 1158. H NMR (400 MHz, CDCl ) δ : 5.22 (s, 1H, CH-15), 3.63 (s, 3H, -CO CH ),
3 H
−1
3
1
1
2
3
2
.49–2.38 (m, 2H, CH -18), 1.15 (s, 3H, CH -20), 0.99 (s, 3H, CH -22), 2.19–0.78 (m, 21H).
C NMR (100 MHz, CDCl3) δC: 178.0, 142.8, 136.0, 119.8, 56.7, 51.1, 49.2, 47.4, 43.8, 43.8,
2
3
3
1
3
43.4, 40.7, 39.6, 39.3, 38.1, 28.7, 25.9, 25.3, 20.7, 19.1, 18.9, 15.5, 15.2.
Nitrile 17
Colorless oil. [α]2
0
D
−1
=
−
100.8 (c = 3.4, CHCl ). IR (
ν
, cm ): 2931, 2855, 1723, 1461, 1447,
150. H NMR (400 MHz, CDCl ) δ : 5.17–5.13 (m, 1H, CH-17), 3.64 (s, 3H, -CO CH ), 3.04
3 H
3
1
1
2
3
(
2
m, 2H, CH -18), 2.84 (bs, 1H), 2.21–1.96 (m, 1H), 1.17 (s, 3H, CH -20), 0.83 (s, 3H, CH -22),
2
3
3
1
3
.06–0.79 (m, 19H). C NMR (100 MHz, CDCl3)
δC: 178.1, 152.5, 118.9, 106.5, 57.2, 55.1,
51.3, 49.3, 44.0, 43.8, 41.2, 40.9, 39.7, 39.6, 38.2, 30.8, 29.7, 28.9, 22.1, 19.3, 18.9, 17.2, 15.7.
DLP-mediated ATRA to methyl 15α-hydroxy-ent-kaurenoate
Methyl 15 -hydroxy-ent-kaurenoate 20 (56 mg, 0.17 mmol) in 3.7 mL benzene was
treated under N with ethyl iodoacetate (60 L, 0.507 mmol) and DLP (3.4 mg, 0.009 mmol).
The reaction was refluxed for 24 h and after this time the crude product was purified by FC
pentane/EtOAc 95:5), giving a mixture of lactone 22 and ester E-21 (25 mg, 22/E-21 = 5:2
α
µ
2
(
ratio, NMR data). This mixture was acetylated with acetic anhydride (0.2 mL, 2.6 mmol)
and DMAP (0.5 mol%) in pyridine (5 mL) at room temperature for 48 h. The crude
mixture was extracted with Et O, neutralized with 10% aqueous H SO and washed with
2
2
4
brine. The organic layer was dried, and the solvent was removed under reduced pressure.
The crude was purified by column chromatography (pentane/EtOAc 97:3) to afford pure
lactone 22 (16 mg, 25%) and acetate 23 (8 mg, 10%).
Lactone 22
◦
20
White solid. M.p. 180–182 C, from CH Cl . [α] = +11.4 (c = 2.83, CHCl ). IR (ν,
2
2
D
3
cm 1): 3054, 2986, 2933, 2865, 1740, 1722. H NMR (400 MHz, CDCl )
−
1
δH: 5.81 (dt, J = 5.4,
3
2
.3 Hz, 1H, CH-17), 4.48 (d, J = 1.66 Hz, 1H, CH-15), 3.64 (s, 3H, -CO CH ), 3.03 (m, 2H,
2
3

Molecules 2021, 26, 4549
15 of 19
CH -18), 2.82 (bs, 1H, CH-13), 1.19 (s, 3H, CH -20), 0.86 (s, 3H, CH -22), 2.20–0.84 (m, 18H).
2
3
3
13
C NMR (100 MHz, CDCl3) δC: 177.9, 172.6, 148.9, 115.3, 88.0, 56.8, 54.6, 51.1, 46.1, 43.8,
4
0.8, 40.7, 39.6, 38.4, 38.0, 34.3, 33.1, 30.0, 28.7, 20.7, 19.0, 18.9, 15.7. GCMS m/z calculated
+
for C H O : 359.28; found 328.2 (M -CO ).
23
32
4
2
Ester 23
Colorless oil. [a]20
−1
1
=
−
69.8 (c = 2.36, CHCl ). IR (
ν
, cm ): 2984, 2930, 2859, 1727. H
D
3
NMR (400 MHz, CDCl3)
δH: 5.62 (t, J = 7.4 Hz, 1H, CH-17), 5.44 (s, 1H, CH-15), 4.12 (q,
J = 7.8, 7.3 Hz, 2H, -CO2CH2CH ), 3.63 (s, 3H, -CO CH ), 2.97 (d, J = 7.1 Hz, 2H, CH -18),
3
2
3
2
2
.79 (bs, 1H, CH-13), 2.07 (s, 3H, -O (CO)CH ), 1.25 (t, J = 7.9 Hz, 3H, -CO CH CH ), 1.15
3 2 2 3
13
(
s, 3H, CH -20), 0.81 (s, 3H, CH -22), 2.20–0.80 (m, 18H). C NMR (100 MHz, CDCl3)
δ
C:
77.9, 171.5, 170.7, 148.7, 117.3, 80.7, 60.6, 56.6, 52.5, 51.1, 47.8, 43.8, 42.3, 40.6, 39.7, 37.9,
7.0, 34.7, 34.3, 33.1, 28.6, 20.9, 20.8, 19.1, 18.4, 15.5, 14.2.
3
3
1
3
Bu Sn -mediated ATRA to methyl 15α-hydroxy-ent-kaurenoate
6
2
Methyl 15
α
-hydroxy-ent-kaurenoate 20 (50 mg, 0.15 mmol) in 3.2 mL benzene was
L, 0.3 mmol), PhSO N (83 mg, 0.45 mmol),
treated under N with ethyl iodoacetate (36
µ
2
2
3
Bu Sn (115 µL, 0.23 mmol) and DTBHN (0.9 mg, 0.005 mmol). The reaction mixture was
6
2
refluxed for 5 h. The crude product was purified by FC (pentane/EtOAc 95:5) and gave a
mixture of compounds 22 and E-21 (43 mg, 22/E-21 = 5:2, NMR data), along with unreacted
starting material (5 mg, 10%).
Et B-mediated ATRA to methyl 15α-hydroxy-ent-kaurenoate
3
Methyl 15
α
-hydroxy-ent-kaurenoate 20 (50 mg, 0.15 mmol) in 3.5 mL EtOH was treated
L, 0.3 mmol), PhSO N (83 mg, 0.45 mmol) and Et B
under N with ethyl iodoacetate (36
µ
2
2
3
3
(
0.45 mL, 0.45 mmol) at room temperature overnight. The crude product was purified by
column chromatography (pentane/EtOAc 95:5) and afforded unreacted starting material
(7 mg, 14%), ester Z-21 (17 mg, 27%, contaminated with cca. 20% E-21), and pure ester
2
0
E-21 (15 mg, 24%).
Ester E-21
Colorless oil. [α]20
=
−
89.8 (c = 4.80, CHCl ). IR (
ν
, cm ): 3484, 2933, 2871, 1724, 1463,
H: 5.80 (t, J = 7.3 Hz, 1H,
−1
D
3
1
1
447, 1370, 1329, 1233, 1191, 1153. H NMR (400 MHz, CDCl )
δ
3
CH-17), 4.13 (q, J = 7.1 Hz, 2H, -CO CH CH ), 3.77 (s, 1H, CH-15), 3.64 (s, 3H, -CO CH ),
2
2
3
2
3
3
.09 (d, J = 7.5 Hz, 2H, CH2-COOEt), 1.25 (t, J = 7.1 Hz, 3H, -CO CH2CH3), 1.17 (s, 3H,
2
13
CH -20), 0.83 (s, 3H, CH -22) 2.85–0.72 (m, 18H). C NMR (100 MHz, CDCl3)
δ
C: 178.11,
71.90, 153.54, 116.42, 83.18, 60.69, 56.96, 52.89, 51.16, 47.33, 43.82, 40.75, 39.60, 38.02, 36.09,
5.14, 35.09, 34.65, 30.56, 28.72, 21.03, 19.13, 18.66, 15.64, 14.19.
3
3
1
3
Ester Z-21
, cm 1): 3484, 2932, 2872, 1725, 1464, 1448, 1372, 1312, 1233, 1191,
155. H NMR (400 MHz, CDCl ) selected values, H: 5.50 (t, J = 7.8 Hz, 1H, CH-17), 4.14
q, J = 7.1 Hz, 2H, -CO2CH2CH ), 3.95 (s, 1H, CH-15), 3.64 (s, 3H, -CO CH ), 3.11-3.32 (m,
−
Colorless oil. IR (
ν
1
1
(
δ
3
3
2
3
2
H, CH2-COOEt), 1.25 (t, J = 7.1 Hz, 3H, -CO CH2CH3), 1.18 (s, 3H, CH -20), 0.82 (s, 3H,
2
3
13
CH -22). C NMR (100 MHz, CDCl ) selected values,
δC: 178.03, 172.79, 154.79, 115.62,
3
3
8
2
0.40, 60.96, 57.06, 53.11, 51.05, 47.79, 43.84, 42.13, 40.80, 39.61, 38.06, 36.00, 35.12, 33.18,
8.69, 21.07, 19.15, 18.27, 15.57, 14.13.
Bu Sn -mediated ATRA of iodoacetic acid to methyl 15α-hydroxy-ent-kaurenoate
6
2
Methyl 15α-hydroxy-ent-kaurenoate 20 (64.0 mg, 0.192 mmol) in 2.0 mL benzene was
treated under N with iodoacetic acid (71.4 mg, 0.38 mmol), PhSO N (105.0 mg, 0.57 mmol),
2
2
3
Bu Sn (145 µL, 0.28 mmol), and DTBHN (1.0 mg, 0.006 mmol). The reaction mixture was
6
2
refluxed for 5 h. The crude product was purified by CC (petroleum ether/EtOAc 70:30) to
provide lactone 22 (23 mg, 28%) and azide 24 as a colorless oil (91 mg, 42%, contaminated
with PhSO N ).
2
3

Molecules 2021, 26, 4549
16 of 19
Azide 24
Colorless oil. IR (
400 MHz, D COD)
, cm 1): 3675, 2988, 2955, 2928, 2902, 2108, 1724, 1553. H NMR
δ : 3.63 (s, 3H, -CO CH ), 3.52 (s, 1H, CH-15), 2.46 (t, 2H, CH -17),
H
−
1
ν
(
3
2
3
2
13
1
δ
2
.17 (s, 3H, CH -20), 0.84 (s, 3H, CH -22), 2.29–0.83 (m, 21H). C NMR (100 MHz, D COD)
C: 179.6 (2xC (O)2), 88.3, 76.0, 58.1, 56.3, 51.6, 48.5, 45.0, 43.0, 41.8, 40.8, 39.1, 37.1, 36.7,
9.1, 29.0, 28.0, 26.7, 22.3, 20.1, 19.6, 16.0.
3
3
3
Et B-mediated ATRA of iodoacetonitrile to 15α-hydroxy-ent-kaurenoic acid
3
1
5
α
-Hydroxy-ent-kaurenoic acid 18 (70 mg, 0.22 mmol) in 2.0 mL EtOH was treated
under N with iodoacetonitrile (32 L, 0.44 mmol), PhSO N (120 mg, 0.66 mmol), and Et B
µ
2
2
3
3
(
0.88 mL, 0.88 mmol). The reaction mixture was purified by FC (CH Cl /MeOH 95:5) and
2 2
provided compound 25 (48 mg, 54%) as a colorless oil (contaminated with PhSO N ).
2
3
Nitrile 25
−
Colorless oil. IR (
ν
, cm 1): 3836, 3673, 2988, 2973, 2902, 2653, 1698, 1404, 1394, 1381,
1
1
(
(
242. H NMR (400 MHz, D COD) : 5.29 (s, 1H, OH), 2.71–2.81 (m, 2H, CH -18), 2.54
δ
H
3
2
1
3
s, 1H, CH-15), 1.24 (s, 3H, CH -20), 0.94 (s, 3H, CH -22), 2.42–0.77 (m, 21H). C NMR
3 3
100 MHz, D COD)
δ
H: 183.9, 119.4, 85.5, 83.2, 56.4, 55.4, 53.4, 50.3, 47.5, 43.6, 40.5, 39.8,
3
38.8, 37.9, 37.6, 36.1, 28.8, 26.4, 20.8, 19.1, 18.8, 15.5.
Azidoester 26
To a stirred solution of azidoacid 24 (45 mg, 0.10 mmol) in approximately 5 mL of
◦
Et O at 0 C, an etheric solution of CH N was added dropwise until the yellow color
2
2
2
persisted (0.2–0.25 mmol). The mixture was stirred for 30 min and concentrated to give the
crude product, which was purified by column chromatography (pentane/EtOAc 95:5) to
give azidoester 26 (41 mg, 99%) as a colorless oil.
Colorless oil. [α]2
0
=
−
55.8 (c = 2.13, CHCl ). IR (
ν
, cm ): 3737, 3526 (w), 2940, 2872,
: 3.69 (s, 3H, C-19 (-CO CH )),
−1
D
3
1
2
3
3
1
3
853, 2299, 2107, 1725, 1445. H NMR (400 MHz, CDCl )
δ
H
3
2
3
.63 (s, 3H, C-21, -CO CH )), 3.40 (s, 1H, CH-15), 2.56 (t, J = 7.9 Hz, 2H, CH -18), 1.16 (s,
2
3
2
13
H, CH -20), 0.81 (s, 3H, CH -22), 2.16–0.78 (m, 21H). C NMR (100 MHz, CDCl3)
δ
C:
77.84, 173.61, 85.22, 75.44, 56.71, 54.72, 51.71, 51.06, 47.55, 43.78, 41.87, 40.59, 39.59, 37.98,
6.05, 35.66, 29.41, 28.63, 28.41, 25.78, 21.03, 18.99, 18.19, 15.34.
3
3
Lactam 27
A solution of azidoester 26 (53 mg, 0.12 mmol) and 10% Pd/C (10% w/w) in dry
EtOAc (2 mL) was stirred for 64 h at room temperature under H (1 atm). The catalyst was
2
filtered off, the solvent was removed under reduced pressure, and the crude purified by
FC (CH Cl /MeOH 85:15), giving lactam 27 (16 mg, 35%) as a yellowish oil.
2
2
2
0
−1
Yellowish oil. [α]
685, 1552. H NMR (400 MHz, CDCl )
=
−
90.8 (c = 3.0, CHCl ). IR (
ν
, cm ): 3351, 2933, 2871, 2322, 1722,
D
3
1
1
1
δ
: 6.73 (s, 1H, NH), 3.63 (s, 3H, -CO CH ), 3.49 (s,
3
H
2
3
H, CH-15), 2.44–2.27 (m, 2H), 1.16 (s, 3H, CH -20), 0.81 (s, 3H, CH -22), 2.14–0.79 (m, 21H).
C NMR (100 MHz, CDCl3) δC: 179.1, 178.0, 88.2, 70.8, 56.8, 54.4, 51.1, 47.4, 44.9, 43.7, 40.7,
3
3
1
3
38.0, 39.6, 36.6, 35.4, 30.6, 29.3, 28.6, 26.3, 21.2, 19.1, 19.0, 15.5.
4. Conclusions
Modifications of ent-kaurenoic acid derivatives at the exo-methylenic double bond of
the tetracyclic diterpenic framework based on mild radical mediated C–C bond forming
reactions such as the iodine ATRA and the azidoalkylation reaction have been performed.
Although this reaction has been demonstrated previously on other substrates, their applica-
tion in complex natural compounds is not well documented and far from trivial due to the
potential side reactions such as intramolecular hydrogen atom transfers. Both steric and
electronic factors strongly influence the outcome of the free radical sequences, providing
unprecedented avenues for expanding structural diversity of natural products.
The azidoalkylation reaction proved to be extremely useful in the context of increasing
structural complexity of the parent natural scaffold, including simultaneous functional-

Molecules 2021, 26, 4549
17 of 19
ization with different heteroatomic functional groups. The advantage of the tertiary azide
moiety was demonstrated by subsequent reductions, leading to spiro-fused lactam and
pyrrolidine fragments. The reported structural modifications led to a series of new com-
pounds with relevant biological activity, which was demonstrated by in vitro cytotoxicity
tests on several tumor cell lines. The hybrid terpene-nitrogen containing heterocycles with
unprecedented spiro-junction have shown relevant cytotoxicity and promising selectivity
indexes. These results represent a solid basis for following research on the synthesis of
such derivatives based on available natural product templates.
Supplementary Materials: The following are available online. X-ray crystal structure report for
lactam 12 (CCDC 2023783), X-ray crystal structure report for lactone 22 (CCDC 2023785), IR and
NMR spectra of compounds 11, 12, 13, 16, 22, 25, 26, 27, IC , and SI data for investigated ent-
50
kauranic derivatives.
Author Contributions: Conceptualization, supervision and funding acquisition, P.R. and V.K.; prepa-
ration of substrates and chemical synthesis, N.U., E.P. and V.G.; evaluation of cytotoxicity, L.P.
and D.D.; writing—original draft preparation, V.K.; writing—review and editing, P.R. All authors
have read and agreed to the published version of the manuscript.
Funding: This research was funded by the SCOPES program of the Swiss National Science Founda-
tion, project IZ73Z0_152346/1; the Swiss National Science Foundation, projects 200020_172621 and
2
00020_201092; and the National Agency for Research and Development (ANCD) of the Republic of
Moldova, grant number 20.80009.8007.03.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: Data of the compounds are available on request from the correspond-
ing authors.
Acknowledgments: The X-ray crystal structure determination service unit of the Department of
Chemistry, Biochemistry, and Pharmaceutical Sciences of the University of Bern (P. Macchi and
J. Hauser) is acknowledged for measuring, solving, refining, and summarizing the structures of
compounds 12 and 22.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available on request from the corresponding authors.
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