Phorbol Diesters and 12-Deoxy-16-hydroxyphorbol 13,16-Diesters Induce TGFα Release and Adult Mouse Neurogenesis

Article abstract of DOI:10.1021/acs.jmedchem.1c00156

A small library of phorbol 12,13-diesters bearing low lipophilicity ester chains was prepared as potential neurogenic agents in the adult brain. They were also used in a targeted UHPLC-HRMS screening of the latex of Euphorbia resinifera. Two new 12-deoxy-

Full text of DOI:10.1021/acs.jmedchem.1c00156

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Article
Phorbol Diesters and 12-Deoxy-16-hydroxyphorbol 13,16-Diesters
Induce TGFα Release and Adult Mouse Neurogenesis
̃
Abdellah Ezzanad, Ricardo Gómez-Oliva, Felipe Escobar-Montano, Mónica Díez-Salguero,
Noelia Geribaldi-Doldan, Samuel Dominguez-Garcia, José Manuel Botubol-Ares, Carolina de los Reyes,
Rosa Durán-Patrón, Pedro Nunez-Abades, Antonio J. Macías-Sánchez, Carmen Castro,
and Rosario Hernández-Galán*
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ABSTRACT: A small library of phorbol 12,13-diesters bearing
low lipophilicity ester chains was prepared as potential neurogenic
agents in the adult brain. They were also used in a targeted
UHPLC−HRMS screening of the latex of Euphorbia resinifera.
Two new 12-deoxy-16-hydroxyphorbol 13,16-diesters were iso-
lated, and their structures were deduced using two-dimensional
NMR spectroscopy and NOE experiments. The ability of natural
and synthetic compounds to stimulate transforming growth factor
alpha (TFGα) release, to increase neural progenitor cell
proliferation, and to stimulate neurogenesis was evaluated. All
compounds that facilitated TGFα release promoted neural
progenitor cell proliferation. The presence of two acyloxy moieties
on the tigliane skeleton led to higher levels of activity, which
decreased when a free hydroxyl group was at C-12. Remarkably, the compound bearing isobutyryloxy groups was the most potent on
the TGFα assay and at inducing neural progenitor cell proliferation in vitro, also leading to enhanced neurogenesis in vivo when
administered intranasally to mice.
formation, Figure S1) are capable of inducing adult neural
progenitor cell (NPC) proliferation via PKC activation and of
stimulating neurogenesis in the adult brain neurogenic niche of
the SVZ and DG. Particularly, it was found that the 12-
deoxyphorbol isolated from Euphorbia resinifera, DPB, showed
the highest capacity to stimulate neurogenesis in vivo.9
Furthermore, the long-term intranasal administration of DPB
exerted not only a potent effect in stimulating neurogenesis in
both the SVZ and DG, but the treatment of mice with this
compound also improved their capacity to perform memory
and learning tasks.10 The comparison of the activity of all 12-
deoxyphorbols tested with DPB9 pointed at the role of the C-
13 acyl chains as a key element in defining the potency of these
compounds at activating PKC and promoting neurogenesis in
vivo. Additionally, unlike PMA, other 12-deoxyphorbols 13-
acyl esters previously described9 did not show tumorigenic
INTRODUCTION
■
Brain damage caused by neurological and vascular disorders or
by traumatic injuries is tightly associated with an irreversible
neuronal loss, which may lead to cognitive impairment, motor
dysfunction, and even alterations of the personality.1−3 Despite
the physiological capacity of the adult brain to generate new
neurons from neural stem cells (NSC) in the subventricular
zone (SVZ)4 and the dentate gyrus (DG) of the hippo-
campus,5 neuronal replacement in damaged brain regions
rarely occurs, complicating functional recovery. An association
of alterations in neurogenesis with cognitive impairment has
been described in mouse models of different metabolic and
neurological disorders,6,7 and it is generally accepted that
increased neurogenesis leads to improve cognitive perform-
ance.8 Thus, this field demands the discovery of new drugs that
regenerate damaged brain regions, facilitating functional
recovery. Searching for molecules that potentiate the capacity
of NSC to generate new neurons in the adult brain is a crucial
step in this pathway.
Received: January 27, 2021
Published: May 4, 2021
Our group has previously shown that several phorbol
derivatives such as 12-deoxyphorbol 13-acetate (Prostratin),
phorbol 12-myristate-13-acetate (PMA), 12-deoxyphorbol 13-
phenylacetate (DPP), 12-deoxyphorbol 13-isobutyrate (DPB),
and 12-deoxyphorbol 13-angelate (DPA) (Supporting In-
© 2021 The Authors. Published by
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https://doi.org/10.1021/acs.jmedchem.1c00156
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a
Scheme 1. Synthesis of Monoacyl and Diacylphorbols
a
Reagents and conditions: (a) Ac2O/C5H5N, rt, 7 h for 2a (99% yield); isobutyric acid, DMAP, EDCI, dichloromethane for 3a, 5a, and 9a (99, 99,
and 82%, respectively); phenylacetic acid, DMPA, EDCI, dichloromethane for 4a, 6a, and 7a (82, 67, and 73% yield, respectively); tiglic acid,
DMAP, EDCI, dichloromethane for 8a (47% yield). (b) HClO4, rt 30 min for 2, 3, 4, 5, and 6 (99, 87, 90, 84, and 86%, respectively); HClO4, rt 15
min for 7, 8, and 9 (75, 79, and 52% yield, respectively).
activity, suggesting 12-deoxyphorbols 13-acyl esters as good
candidates to produce potent pharmacological drugs that
promote neurogenesis and facilitate functional repair of
damaged brain tissue.
on the phorbol skeleton. Consequently, it is logical to consider
that compounds with two ester functional groups, like PMA,
but bearing less lipophilic chains, like those found in 12-
deoxyphorbol esters such as the ones mentioned above, would
present a better activity profile.
A review of the literature shows that not only phorbol esters
at C-12 and C-13 have been described but also derivatives with
no substituents at C-12, but with ester groups at C-13 and C-
16 (12-deoxy-16-hydroxyphorbol 13,16-diesters).14,15 Both
groups of compounds would be related to 12-deoxyphorbols
like those described above (Figure S1) but with an extra
acyloxy chain either at C-12 or C-16.
Phorbol diesters bearing different chains at C-12 and C-13
could conceivably be prepared starting from a commercially
available mixture of phorbols coming from Croton oil, an oil
obtained from the seeds of Croton tiglium.16 On the other
hand, as no synthetic procedures have been described for the
preparation of 12-deoxy-16-hydroxyphorbol 13,16-diesters,
they would have to be obtained from natural sources. For
instance, the presence of 12-deoxy-16-hydroxyphorbol deriv-
atives in the latex of E. resinifera17−19 is known.
With this in mind, we have prepared a small library of
phorbols bearing low-lipophilicity ester chains through
chemical transformations on C-20-protected phorbol obtained
from Croton oil. Based on our previous results9 and with the
aim to evaluate the effect of the presence of an extra acyloxy
moiety on the tigliane skeleton, we decided to prepare 12,13-
diester-bearing acetate, phenylacetate, isobutyrate, and meth-
ylbutenoate chains (like prostratin, DPP, DPB, and DPA,
respectively). To analyze the effect of an extra free hydroxyl
group, and as DPB has resulted to be the most potent
compound when tested as a neural precursor promoter,9
phorbol 13-isobutyrate was also prepared. On the other hand,
to study the impact of the location of the chain on the tigliane
skeleton on the capability of induction of the NPC
proliferation, mixed 12,13-diesters carrying a combination of
these chains were prepared, and both groups of compounds
were used to identify possible 12-deoxy-16-hydroxyphorbol
Regarding the mechanism of action of these phorbol
derivatives, it has been reported that conventional PKC
activation facilitates the secretion of neurotrophic factors that
induce NPC proliferation such as the transforming growth
factor alpha (TGFα). This neurotrophic factor stimulates the
proliferation of undifferentiated NPC by binding and activating
the epidermal growth factor receptor (EGFR). PKC-mediated
phosphorylation of membrane-bound pro-TGFα facilitates the
ADAM17-mediated shedding of the soluble TGFα ligand.11−13
Furthermore, it has been recently described that DPB elicits
TGFα release mimicking the effect of TGFα in cultures of
NPC.10
In this paper, we describe the preparation of selected
phorbol 12,13-diesters (2−9) from commercial Croton oil,
their use for identification of 12-deoxy-16-hydroxyphorbol
13,16-diesters through a screening-assisted ultrahigh perform-
ance liquid chromatography (UHPLC)−high-resolution mass
spectroscopy (HRMS) from the latex of E. resinifera and also
the isolation and characterization of two new 12-deoxy-16-
hydroxyphorbol 13,16-diesters (10−11). Furthermore, we
report the evaluation of these compounds as inducers of
TGFα release, promoters of NPC expansion in the absence of
epidermal growth factor, and as neurogenesis-stimulating small
molecules. We also show structure−activity studies that can
help the development of this class of compounds as effective
neurogenic agents.
RESULTS AND DISCUSSION
■
Among the 12-deoxyphorbol esters tested previously,9,10 those
with a phenylacetate or isobutyrate chain on C-13 (DPP and
DPB, respectively) presented good activity at the lower range
of tested concentrations. Therefore, it is reasonable to conceive
that the nature of the biological activity of this class of
compounds depends on the number and type of ester chains
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13,16-diesters in the latex of E. resinifera through a targeted
UHPLC−HRMS method. The exchange of chains between C-
12 and C-13 could tell us if the activity was associated with the
location of each chain in a specific position or simply with their
presence in that area of the molecule. Finally, a comparison of
the effect of diesters in 12,13 with those of 13,16-diesters
would inform us about the influence of the relative position of
the ester group on the tigliane skeleton.
Synthesis of Phorbol Diesters. A set of phorbol esters
was prepared by selective transformations at C-12, C-13, and
C-20 positions on the tigliane skeleton.
Reactivity of hydroxyl groups in phorbol (1) toward
esterification proceeds in the order C-20 > C-13 > C-12 due
to steric effects.20 Therefore, to selectively acylate the hydroxyl
groups on C12 and/or C13 of phorbol, the primary allylic
alcohol at C20 must be first protected.
UHPLC−HRMS-Assisted Identification, Isolation, and
Structural Elucidation of 12-Deoxyphorbol13,16-die-
sters from E. resinifera. 12-Deoxy-16-hydroxyphorbol 13,16-
diesters and phorbol 12,13-diesters with the same ester
moieties are isomeric and, therefore, are expected to show
equivalent molecular ions in mass spectrometry.
Consequently, using the chemical formula of compounds 2−
9 as leads, extracted ion chromatograms (XIC) for
pseudomolecular ions [M + Na]+, in electrospray ionization
(ESI) positive mode and [M + HCOOH−H]− in ESI negative
mode, were obtained by the UHPLC−HRMS analysis of the
fractions obtained from the column chromatography of the
latex of E. resinifera, as described in the Experimental Section.
XIC for m/z 575.2621 (calculated mass for C32H40O8Na,
corresponding to [M + Na]+ for both compounds 7 and 9) in
ESI positive mode and m/z 597.2700 (calculated mass for
C33H41O10, corresponding to [M + HCOOH−H]− for both
compounds 7 and 9) in ESI negative mode provided
chromatographic peaks with different retention times to
those of compound 9, under equivalent elution conditions
(Supporting Information, Figures S36 and S37). Therefore, the
E. resinifera fraction where the above mentioned compounds
were detected, isomeric to 7 and 9, was subject to a careful
isolation procedure, as detailed in the Experimental Section,
yielding compounds 10 and 11 (Figure 1). A comparison of
Croton oil was used as a source of phorbol (1), and a
modified protocol reported by Bertolini et al.20 was used to
isolate phorbol 20-trityl ether (1a) (Supporting Information,
Scheme S1). Once the hydroxyl group at C-20 was protected,
we proceeded to functionalize C-12 and C-13 following a
modification of the previously reported strategy.20
A hydrolysis reaction of the trityl ether allows the recovery
of the hydroxyl group at C-20.
A careful chromatographic purification of precursor 1a must
be carried out in order to avoid the co-occurrence of the
epimer at C-4 ((4S)-phorbol derivatives). Epimerization at C-4
can take place through a retro-aldol-mediated rearrangement in
a basic medium.21,22 Due to the conformational change
associated with the epimerization at C-4, (4S)-phorbol
derivatives are not PKC activators.20
20-Trityl phorbol esters containing identical chains at C-12
and C-13 (2a−4a) were obtained following the procedure
shown in Scheme 1. While ester 2a was obtained by treatment
of 1a with acetic anhydride in pyridine, esters 3a and 4a were
prepared by treatment with either isobutyric or phenyl acetic
acid, N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide
(EDCI) hydrochloride, and N,N-dimethylamino pyridine
(DMAP) (Scheme 1). All attempts to obtain phorbol 12,13-
diangelate were unsuccessful.
As mentioned above, the C-13 hydroxyl group in phorbol
derivatives is more reactive toward esterification than the one
at C-12.20 Therefore, C-13 monoacyl esters 5a and 6a could be
prepared in a similar fashion to 3a and 4a, modifying slightly
the reaction conditions (Scheme 1). In order to achieve
selective acylation on C-13, temperature control and close
monitoring of the reaction using thin layer chromatography
(TLC) are required.
Once selective conditions for the preparation of 13-
acylphorbols were found, it was possible to prepare 12,13-
diacylphorbols with different chains at C-12 and C-13.
Consequently, 20-tritylphorbol 12-phenylacetate-13-isobuty-
rate (7a), 20-tritylphorbol 12-tigliate-13-isobutyrate (8a), and
20-tritylphorbol 12-isobutyrate-13-phenylacetate (9a)
(Scheme 1) were prepared as they would incorporate at C-
12 ester groups already present in bioactive 12-deoxyphor-
bols.9 Treatment of 5a with phenylacetic acid or tiglic acid
yielded 7a and 8a. Diester 9a was prepared, starting from 6a by
treatment with isobutyric acid (Scheme 1). Finally, hydrolysis
of trityl group at C-20 was achieved by treatment of
corresponding diacylphorbol derivatives (2a−9a) with
HClO4 (0.01 M) at room temperature for 30 min to produce
compounds 2−9.
Figure 1. Structures of 10−11.
the HRMS of the isomeric compounds 9, 10, and 11, in ESI
positive mode, and with a data-independent acquisition (MSE),
allowing the fragmentation of all ions within full m/z range,23
shows similarities and some differences on both sets of spectra.
MSE of these compounds shows a relatively abundant
fragmentation corresponding to [M-PhAcOH-(CH3)2CCO2H
+ Na]+ (calculated m/z 351.1572).
Further fragmentations derived from this ion showed
differences in compound 9, on the one hand, and both
compounds 10 and 11, on the other, under equivalent MSE
conditions (Figure 2 and Supporting Information, Schemes S2,
S3, and S4). On the one hand, for compound 9, a further loss
of water (one and two molecules) and CO leads to ions m/z
311.1647, m/z 293.1541, and m/z 265.1592 (calculated).
These are considered diagnostic fragments, associated with
different mass spectrometry strategies to identify phorbol
esters in complex mixtures.24,25 On the other, for compound
10, additional ions at m/z 323.1623, m/z 275.1436, and m/z
247.1487 (calculated) were observed, which were not apparent
in the MSE of compound 9. As detailed in the proposed
fragmentation map for compound 10 (Figure 2 and Supporting
Information, Scheme S3), while the ion at m/z 323.1623 could
be explained by further loss of CO from the ion at m/z
351.1572, ions at m/z 275.1436 and m/z 247.1487 could be
explained by further loss of water and CO, respectively, from
m/z 293.1541 ([M + H-2H2O-PhAcOH-(CH3)2CCO2H]+).
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Figure 2. Comparison of MSE spectra (Data-Independent Acquisition)20 for (A) compound 9, (B) compound 10, and (C) compound 11 (data
acquired in positive ionization with a ramp tramp collision energy of the high-energy function set at 60−120 eV).
Interestingly, the latter fragmentation was the most abundant
one observed in the ESI positive-ion mass spectra of
compound 10 due to in-source fragmentations. Similar
fragmentation patterns were observed for compound 11
(Figure 2 and Supporting Information, Scheme S4).
Nuclear Overhauser effect spectroscopy (NOESY) correlations
observed for H-16α, H-16β/H-14, and H3-17/H-8, H3-17/H-
11 in all compounds (Supporting Information) confirmed that
H2-16 is α-oriented, as H-14, and H3-17 is β-oriented, as H-8
and H-11. Accordingly, compounds 10 and 11 were assigned
as 12-deoxy-16-hydroxyphorbol 16-isobutyrate-13-phenyl ac-
etate (DPPI) and 12-deoxy-16-hydroxyphorbol 13-phenyl-
acetate-16-tigliate (DPPT), respectively. Both are new
compounds and are described here for the first time.
1
Comparison of the H and 13C NMR spectra, as well as
heteronuclear multiple−bond correlation (HMBC) coupling
patterns of 10−11 with those of 7−9, indicates that the
substituents were now located on C-13 and C-16 (Table 1).
The 13C NMR spectra of compounds 10 and 11 showed the
presence of only three methyl groups and three methylenes,
indicating that the substituent on C-12 on phorbol 12,13-
diesters must be now located on one of the methyls of the gem-
dimethyl group on C-15.1H NMR data for 10, and 11 showed
the presence of phenylacetate (3.63, s, 7.31−7.23, m for 10
and 3.62, s, 7.32−7.19, m for 11) and either an isobutyrate
(2.53, sp; 1.15, d; 1.14, d for 10) or a tigliate ester (6.84, q;
1.82, s; 1.81 d for 11), respectively. The main difference
Effect of Phorbol Diesters and 12-Deoxy-16-hydrox-
yphorbol 13,16-Diesters on TGFα Release. A previous
report shows that 12-deoxyphorbol DPB stimulated neuro-
genesis and improved memory in mice by facilitating TGFα
release and promoting the proliferation of NSC.10 In order to
analyze whether the compounds isolated and synthesized had
the ability to facilitate the release of TGFα, we built a fusion
protein in which TGFα was expressed flanked by green
fluorescent protein (GFP) and mCherry in the C-terminal and
N-terminal domains, respectively (Figure 3B). This construct
was transfected into HEK293T cells and the ratio mCherry/
GFP in the cell membrane was quantified by using life imaging
upon addition of the different compounds. By using this TGFα
probe, cells expressing TGFα will show a 1:1 ratio of mCherry
to GFP. However, if the shedding of TGFα occurs, cells will
lose a percentage of mCherry fluorescence proportional to the
amount of TGFα being released to the extracellular medium,
whereas the GFP fluorescence will remain attached to the
membrane. Thus, in the absence of shedding, the m-Cherry/
GFP ratio remains unaltered, whereas the shedding and release
of soluble TGFα should lower this ratio. As shown in Figure
3B,C, in the absence of compound (Ø; see Supporting
Information, Movie 1) or in the presence of 0.5 μM phorbol
(1; see Supporting Information, Movie 2), the mCherry/GFP
ratio remains unaltered; however, the addition of compound 3
(see Supporting Information, Movie 3) and an identical
1
between the H NMR spectra of the natural compounds 10
and 11 and those of the synthetic compounds was the presence
of two doublets (δH 4.05 and 3.85 ppm in 10 and 4.14 and
3.84 ppm in 11), which correlated to a methylene group (δC
70.2 and 70.5 ppm, respectively) in the gHSQC experiments.
These methylene groups were assigned to C-16 because of
their correlation with the carbon signals assigned to C-17 (δC
11.7 ppm in 10 and 11.8 ppm in 11) in the gHMBC. The long-
range couplings of the protons H2-16 with the carbonyl
carbons at δC 178.6 ppm in 10 and 169.5 ppm in 11 in the
gHMBC spectra demonstrated that isobutyril and tiglyl groups
are located at C-16. Consequently, phenylacetyl groups could
only be located on the C-13 quaternary carbon. Furthermore,
the presence of a methylene group at C-12 was confirmed by
the correlations observed in the gHMBC experiments between
the methyl groups at C-11 (δH 0.84 and 0.89 ppm,
respectively) and the C-12 carbons in each compound.
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the effect of compounds 10 (see Supporting Information,
Movie 6) and 11 on TGFα release was similar to that of
compounds 2 and 8. All these results agree with the reported
effect of DPB, stimulating the release of TGFα, in a classical
PKC-dependent manner.10
Table 1. Summary of NMR Data for DPPI (10) and DPPT
(11)
DPPI (10)
DPPT (11)
a
b
c
d
pos
δH (J in Hz)
δC
δH (J in Hz)
7.53, m
δC
1
2
3
4
5
7.53, m
160.8
135.0
210.5
74.7
160.8
134.6
210.5
74.7
Effect of Phorbol Diesters and 12-Deoxy-16-hydrox-
yphorbol 13,16-diesters on NPC Proliferation In Vitro.
In order to analyze whether the capacity of these compounds
to release TGFα correlated with their proliferative activity on
NPC, we tested the effect of phorbol (1) (as a negative
control), compound 5 (no activity on the TGFα assay),
compound 2 (mild effect), and compound 3 (most potent on
the TGFα assay). In addition, we chose compound 10 as the
representative of 12-deoxy-16-hydroxyphorbol 13,16-diesters.
We cultured NPCs isolated from the SVZ and cultured them
under floating conditions in which the NPCs proliferate and
form neurospheres. Cells were left in the absence and presence
of increasing concentrations of the different compounds for 48
h. The size of the neurospheres formed was quantified
afterward.
We observed no effect in cultures treated with either
phorbol (1) or compound 5 (these two had shown no effect
on TGFα release) (Figure 4A,B). However, all compounds
that facilitated TGFα release, independently of their potency in
the previous assay (compound 3 showed a stronger effect
compared with 2 and 10), were able to promote NPC
proliferation in vitro.
This indicated that the amount of TGFα produced by
compounds 3, 2, and 10 is higher than that required for these
cells to proliferate. Interestingly, differences among these
compounds are mainly based on the dose−response curve.
Thus, compounds 3 and 10 showed different dose−response
curves with respect to compound 2 (Figure 4A,B). The first
two showed a bell-shaped dose−response curve, whereas the
latter showed a linear dose response curve, within the
concentration range explored. Maximum effect with 3 and 10
was obtained with nanomolar concentrations, whereas no
effect of compound 2 was observed unless a concentration of 5
μM was used. These results indicated that all compounds with
a capacity to facilitate TGFα release were able to promote the
proliferation of NPC in vitro. However, differences were found
in the doses required to exert its proliferative effect, with
compounds 3 and 10 being more potent than 2 and also more
potent than DPB.10
The bell-shaped curve observed for compounds 3 and 10
may be explained by their mechanism of action. As shown in
Figure 3C, these compounds stimulate the release of the EGFR
ligand TGFα. Previous reports have elucidated that the PKCα-
mediated release of TGFα induced by DPB activates the
receptor and its downstream signaling cascades, thus inducing
proliferation10 activating the receptor. However, another role
for PKCα is mediated by its capacity to phosphorylate EGFR
causing its feedback inhibition.26 Thus, it is likely that higher
doses of compounds 3 and 10 result in the potent inhibition of
EGFR and therefore in a reduction of proliferation and
neurosphere size.
2.50, d (19.0)
2.42, d (19.0)
38.6
2.51, d (19.0)
2.42, d (19.0)
38.6
6
7
8
9
10
11
142.4
129.7
39.3
77.6
57.2
37.4
142.4
129.6
39.3
77.7
57.2
37.4
5.56, bd (5.8)
3.08, t (5.8)
5.58, bd (5.7)
3.10, t (5.7)
3.16 d, (2.9)
2.02,
dt (11.0, 6.7)
3.16, d (2.7)
2.03, m
12β 1.57,
dd (11.0, 14.8)
12α 2.13,
dd (6.7, 14.8)
33.0
1.57, dd (11.0, 15.0)
2.14, dd (7.0, 15.0)
33.1
13
14
15
16
65.1
31.5
28.3
70.2
65.2
31.5
28.3
70.5
1.12, d (5.8)
1.16, d (5.7)
4.05, d (11.4)
3.85, d (11.4)
1.13, s
0.84, d (6.7)
1.73,
dd (2.9, 1.4)
4.14, d (11.4)
3.84, d (11,4)
1.14, s
0.89, d (6,6)
1.74, d (2.7, 0.7)
17
18
19
11.7
18.9
10.2
11.8
18.9
10.2
20
3.93, d (12,9)
3.87, d (12,9)
68.2
3.94, d (12,9)
3.84, d (12,9)
68.3
1′
2′
3′
4′
5′
178.6
35.2
19.4
31.6
169.5
129.7
12.2
138.8
14.4
2.53, sp (7.0)
1.15, d (7.0)
1.14, d (7.0)
1.82, s
6.84, q (6.8)
1.81, d superimposed with
H3-4″
1″
2″
3″
4″
5″
6″
175.23
42.3
134.9
130.4
129.7
128.3
175.1
42.4
134.9
129.6
130.4
128.2
3.63, s
3.62, s
7.26−7.23, m
7.31−7.29, m
7.26−7.23, m
7.24−7.19, m
7.32−7.28, m
7.24−7.19, m
a
b
1H NMR (600 MHz, CD3OD). 13C NMR (151 MHz, CD3OD).
c
d
1H NMR (500 MHz, CD3OD). 13C NMR (126 MHz, CD3OD).
concentration of 4, 7, and 9 significantly reduced this ratio by
25%, whereas other compounds, such as 2 (see Supporting
Information, Movie 4) and 8, exerted a moderate effect on this
ratio (15% reduction) and compound 5 (see Supporting
Information, Movie 5) barely reduced this ratio.
These results indicated that, except for phorbol (1) and
compound 5, the synthetic and naturally occurring compounds
tested were able to facilitate TGFα release. Therefore, for these
compounds to exert their effect, the hydroxyl groups at C-12
and C-13 or at C-13 and C-16 need to be esterified. Higher
activities were observed when phenylacetate and isobutyrate
esters were present, with the most active compound (3)
bearing two isobutyrate chains. The analysis of TGFα release
for compounds isolated from E. resinifera, where position C-12
was unsubstituted and positions C-13 and C-16 were acylated,
was consistent with this hypothesis. As shown in Figure 3B,C,
Effect of Intranasal Administration of Compounds 2
and 3 on Adult Mouse Neurogenesis in the SVZ. In order
to elucidate whether compounds with the high activity in vitro
were able to stimulate neurogenesis in vivo in the adult mouse
brain, compounds 2 (moderate effect in TGFα and neuro-
sphere assays) and 3 (effect at lower concentrations in the
neurosphere assay) were administered intranasally during 3
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Figure 3. Phorbol diesters and 12-deoxy-16-hydroxyphorbol 13,16-diesters can induce mCherry-TGFα-GFP ectodomain shedding in vitro. (A)
Images of mCherry-TGFα-GFP expressing, serum-starved HEK293T cells stimulated with diluent, with 1, 2, 3, 5, or 10 during 180 min. mCherry/
GFP ratio images at the indicated time points are shown in the intensity-modulated display mode. The color ranging goes from red to blue to
represent mCherry/GFP ratios. The upper and lower limits of the ratio range are shown on the right. Scale bar = 20 μm. (B) Scheme of mCherry-
TGFα-GFP construct and its action mechanism. (C) Quantitative analysis of the microscopic images obtained from the time-lapse assays of
HEK293T cells expressing mCherry-TGFα-GFP protein fusion. mCherry/GFP ratios were normalized to the average mCherry/GFP ratio
measured before stimulation with phorbol diesters and 12-deoxy-16-hydroxyphorbol 13,16-diesters. The mean normalized mCherry/GFP ratios are
shown, n = 40. All compounds were tested at 0.5 μM. Ø: none, 1: phorbol (P), 2: phorbol 12,13-diacetate, 3: phorbol 12,13-diisobutyrate, 4:
phorbol 12,13-diphenylacetate, 5: phorbol 13-isobutyrate, 7: phorbol 13-isobutyrate-12-phenylacetate, 8: phorbol 13-isobutyrate-12-tigliate, 9:
phorbol 12-isobutyrate-13-phenylacetate, 10: DPPI, and 11: DPPT.
consecutive days. During the 3 days, the mice received
injections of the thymidine analogue BrdU to label cells that
proliferated during the duration of the treatment. Thereafter,
the mice were sacrificed, their brains were sectioned, and
immunohistochemistry was performed in the sections contain-
ing the SVZ to detect BrdU and the marker of newly generated
neuronal progenitors doublecortin (DCX). As shown in Figure
5, and agreeing with the reported effects of DPB,10 both
compounds increased the number of proliferating cells (BrdU
+) and the number of proliferative neuronal progenitors (BrdU
+/DCX+). However, only compound 3 increased the total
DCX cells (DCX burden). Interestingly, the effect of
compound 3 on neurogenesis was observed when the
concentration of the solution administered daily (12 μL/
day) was 10 nM, whereas no effect was observed when the
concentration was 100 nM (10× higher) (Supporting
Information, Figure S38) or 5 μM (data not shown). Thus,
for compound 3, a daily dose of 12 μL of 10 nM solution had
to be used in order to observe an in vivo effect. These findings
are not surprising, considering the dose−response effect
observed in vitro in which this compound showed a bell-
shaped dose−response curve with the maximum activity at 50
nM and a decline when higher doses of the compound were
used. Although the actual concentrations used in vitro and in
vivo should not be compared because the concentration of this
compound in the SVZ or DG after the 3-day treatment period
is not known, it is reasonable to suggest that the in vitro dose
response curve and the in vivo effect on neurogenesis agree.
The reason for the lack of activity at high doses of compound 3
can be explained by its mechanism of action. As explained
above, the reported effect of DPB on adult hippocampal
neurogenesis was mediated by its capacity to activate PKCα10
inducing TGFα release and EGFR activation. Thus, it is
reasonable to suggest that the mechanism of action of
compound 3 is similar and results in the activation of EGFR
as well. In light of the results shown herein, it is evident that
low concentrations of 3 are enough to release the amount of
TGFα required to saturate the EFGR receptor and initiated
the EGFR-associated signaling pathways that promote
proliferation. In this context, the lack of effect at high doses
might be explained if elevated doses of the compound over-
activate PKCα and directly phosphorylate EGFR, mediating
the feedback inhibition of the receptor.26
Physicochemical Properties and SAR Analysis. To
investigate the influence of an additional ester group, we
compared the previous results obtained with 12-deoxyphor-
bols9,10 with those described herein. Thus, in comparison to
prostratin, the introduction of an additional acetyloxy moiety
leads to an increased proliferative effect in compound 2
(phorbol 12,13-diacetate). A similar effect was observed when
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Figure 4. Effect of phorbol diesters and 12-deoxy-16-hydroxyphorbol 13,16-diesters on NPC proliferation in vitro. (A) Representative phase-
contrast microscopy images of neurospheres cultured for 72 h in the presence of 1, 2, 3, 5, and 10. Scale bar = 100 μm. (B) Graph shows the effect
of the different treatments at different concentrations on the neurosphere area expressed as the percentage of control (only bFGF treatment). Data
shown are the mean
SEM of nine independent measurements. Statistical analysis: ***p < 0.0001 in two-tailed unpaired Student’s t-test
comparing bFGF with bFGF + treatment. 1: phorbol, 2: phorbol 12,13-diacetate, 3: phorbol 12,13-diisobutyrate, 5: phorbol 13-isobutyrate, and
10: DPPI.
comparing DPB with phorbol 12,13-diisobutyrate (3) and
DPP with 12-deoxy-16-hydroxyphorbol 16-isobutyrate-13-
phenyl acetate (10).
differences were observed when the group located at C-13 is
an isobutyrate (compare compounds 3, 7, and 8, Figure 3C).
Table S1 (Supporting Information) summarizes physico-
chemical parameters of the tested compounds, including
molecular lipophilicity (log P), bulkiness (MW), H-bond
features (HBA, HBD, and topological polar surface area
(TPSA)), and molar refractivity. All of them possess a greater
number of H-bond acceptors (HBA = 6−8) than H-bond
donors (HBD = 3−5); only those with HBD > 3 (compounds
1 and 5) proved to show no activity. On the other hand, the
previously reported and active 12-deoxyphorbol esters9,10
possess HBD = 3 (Supporting Information, Table S2), which
is consistent with the analysis shown above, suggesting that the
number of HBD seem to be essential for activity.
Two additional factors directly influence the effect of the
compound described in this work on NPC expansion: the
nature and location of side chains on the tigliane skeleton.
Comparing compounds with the same substituent located in
different positions (9 vs 10 or 7 vs 9), it is observed that
changing a substituent from C-12 to C-16 produces a
significant reduction in the proliferative effect; however, no
difference is observed when substituents are exchanged
between C-12 and C-13. Remarkably, while the modification
of the chain at C-12 in compounds bearing a phenylacetyl
group at C-13 does not produce changes in the activity
(compare compounds 4 and 9, Figure 3C), significant
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Figure 5. Effect of the intranasal administration of phorbol diesters on BrdU+ and DCX + cells in the SVZ in vivo. Intranasal administration of 2 (5
μM), 3 (10 nM), or only the vehicle was performed during 3 days to healthy 2 month-old adult mice. All mice were intraperitoneally injected with
BrdU (100 mg/kg) on the last day of treatment as described in methods. (A) Representative confocal microscopy images of the SVZ of adult mice
treated with 2, 3, or only the vehicle. Slides were processed for the immunohistochemical detection of the proliferation marker BrdU and DCX.
Scale bar = 50 μm. (B) Graph shows the number of proliferating cells marked with BrdU per mm,3 the percentage of DCX burden, and the number
of BrdU + cells per mm3 that co-expressed DCX marker in the SVZ. Data shown are the mean SEM; n = 6 animals per group. Statistical analysis:
*p < 0.05 by Student’s t-test comparing with the control group. 2: phorbol 12,13-diacetate and 3: phorbol 12,13-diisobutyrate.
Docking Analysis of Phorbol Derivatives with
PKCδC1B (PDB: 1PTR) and PKCβC1B (PDB: 3PFQ). PKC
activation is delicately controlled by several factors, involving
its conformational change and membrane translocation.
Phorbol esters show a higher affinity for the C-1 domain
than the natural substrate (diacylglycerol, DAG), which, in
part, explains their ability to promote PKC activation. Phorbol
esters bind to the hydrophilic cleft of C-1 through hydrogen
bonding interactions, which may result in a hydrophobic
capping of the C-1 domain, depending on the nature of the
substituents at C-12 and C-13 in the phorbol ester.27 The
phorbol ester/PKC complex can now eventually translocate to
the membrane, which has been described as part of the
activation process.
A right balance in the lipophilicity of the substituents of the
phorbol esters would translate into a balanced activation of
PKC.28 The crystal structure (PDB ID: 1PTR)27 of PKCδC1B
complexed with phorbol 13-acetate describes the binding
pocket as a V-shape, surrounded by two long β-sheets. The
PKCδC1B binds to phorbol 13-acetate through three residues:
Gly-253, Thr-242, and Leu-251. Such residues target phorbol
13-acetate in specific atoms, where they made three hydrogen
bonds with the hydroxyl group in position C-20 and another
two with both oxygens in positions C-3 and C-4.29 Trans-
formation of any groups located at these positions would affect
the ligand-binding affinity. On the other hand, phorbol esters’
ability to bind PKC isoforms depends on the structural
characteristics of the C-1 domains in each one.29 Previous
results have shown that the capacity to promote proliferation
of NPC is related to their ability to activate classical PKC.10,30
Therefore, to further understand the structure−activity
relationship, selected phorbol esters (1, 3, and 7), 12-
deoxyphorbol esters (DPB and DPA), and 12-deoxy-16-
hydroxyphorbol esters (10 and 11) were subjected to in silico
molecular docking with C-1 domain models of a classical PKC.
Previously, due to the availability of the crystal structure of the
PKCδC1B/phorbol 13-acetate complex, we investigated
possible interactions of the acyl groups at C-13, C-12/C-13,
and C-13/C-16 with residues on the walls of the receptor
groove PKCδ. Then, we extended our analysis such that we
were able to identify key determinants of the interaction of
phorbol esters with the C1 domain of PKCβ, involved in the
promotion of NPC proliferation.10,30 The subdomain C1 of
PKCβ was determined from the full-length proteins PKCβII
(PDB ID: 3PFQ).31 The activator-binding residues of PKCβ
were identified by comparison with the activator-binding
residues of PKCδ-C1B (sequence homology was 62%, Figure
6).
Despite differences in some residues, alignment of the crystal
structures of δC1B (blue) and βC1B (gray) has shown a
remarkable similarity in the activation of binding residues,
including GLY-253 δC1B/GLY-124 βC1B, LEU-251 δC1B/
LEU-122 βC1B, and THR-242 δC1B/THR-113 βC1B). The
differences between δC1B and βC1B were in the following
residues Met-239 δC1B → Ser-110 βC1B, Trp-252 δC1B →
Tyr-123 βC1B, Val-255 δC1B → Ile-126 βC1B, and Lys-256
δC1B → His-127 βC1B.
Identical hydrogen bond interactions to those previously
described for phorbol 13-acetate29 were observed in all
compounds except in phorbol (1), which showed an extra
interaction between the hydroxyl group in C-13 and Ser110 of
PKC β (equivalent to Met239 of PKCδ) (Supporting
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Figure 6. (A) Structural and sequence comparison of the zinc finger of PKCδC1B (blue) and PKCβC1B (gray). In silico interaction with phorbol
13-acetate is included for comparison with the crystal structure (PDB ID: 1PTR) of PKCδC1B complexed with phorbol 13-acetate. (B) Sequence
alignment of PKCδC1B and PKCβII-C1B (amino acids (AA) in red are the AA around the ligand-binding site, and the AA marked in yellow are
the AA that form hydrogen bonds with phorbol esters).
Information, Figures S39 and S40). No differences were
observed in the way in which the C-12, C-13-diester and C-13,
C-16-diester were located inside the pocket, confirming that
hydrogen bond interactions between oxygenated functions on
A and B rings (C3O, C4OH, and C20OH and Gly-124, Thr-
113, and Leu-122 of PKCβ, equivalent to Gly-253, Thr-242,
and Leu-251 of PKCδ) are the factors that determine how
these compounds bind the C-1 domain of PKC.
Experimentally, neurosphere assays have shown that 12-
deoxyphorbols (DPP and DPB)9 are weaker promoters of
NPC proliferation than DPPI (10) and phorbol ester 3 (Figure
4), confirming that the presence of an extra ester function
(either at C-12 or at C-16) favors an activity increase. On the
other hand, TGFα assays, which we have shown to correlate
with the promotion of NPC proliferation, show that phorbol
12,13-diesters 3 and 7 are more potent than 12-deoxy-16-
hydroxyphorbol 13,16-diesters 10 and 11, the difference
between them being the disposition inside (10 and 11) or
outside (3, 7) of the C-1-domain receptor gap of the extra
ester function, either at C-12 or at C-16, respectively
(Supporting Information, Figure S40). This indicates that
the additional side chains present on these compounds are key
factors for activity, which would be consistent with an increase
of the interaction of the phorbol ester/PKC complex with the
membrane and a better modulation of the PKC activity.
CONCLUSIONS
■
In summary, we describe the preparation of a small library of
phorbol 12,13-diesters, which were also used in a targeted
UHPLC−HRMS screening of the latex of E. resinifera. This
approach led to the identification and isolation of two new 12-
deoxy-16-hydroxyphorbol 13,16-diesters. The ability of these
compounds to induce TFGα, to increase NPC proliferation,
and to stimulate neurogenesis was evaluated. All compounds
that facilitated TGFα release were able to promote NPC
proliferation when evaluated in vitro.
The presence of an extra acyloxy moiety at the C or D rings
of the tigliane skeleton leads to an increased level of activity
compared with equivalent 12-deoxyphorbol 13-esters, which
decreases if a free hydroxyl group is at C-12. While the number
of hydrogen bond donors appears to be a determinant factor
for activity, this is modulated by additional side-chain
interactions.
Remarkably, compound 3, bearing isobutyryloxy groups at
C-12 and C-13, was the most potent compound on the TGFα
assay and in the in vitro NPC proliferation assay, also leading
to neurogenesis in vivo when administered intranasally to mice.
Considering the effect of similar compounds like DPB at
improving episodic memory in mice,10 these results point out
at phorbol diesters, like those described here, as relatively
simple-to-manufacture pharmacological agents in the develop-
ment of treatments for disorders associated with a reduction in
neurogenesis and memory impairment such as neurodegener-
ative diseases.
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the resulting mixture was added EDCI (1.13 mmol). After stirring for
18 h, the reaction mixture was evaporated, and then Et2O (5 mL) was
added. The crude was sequentially washed with H2O (5 mL), twice
with saturated sodium bicarbonate solution (5 mL), and H2O (2 mL),
dried over anhydrous sodium sulfate, filtered, and evaporated under
reduced pressure to afford compounds 3a and 4a.
EXPERIMENTAL SECTION
■
General. Silica gel (Merck, grade 60, particle size 0.04−0.063 mm,
230−240 mesh ASTM) was used for column chromatography. TLC
was performed on Merck Kiesegel 60 F254, 0.25 mm thick. Optical
rotations were determined with a Jasco P-2000 digital polarimeter.
Infrared spectra were recorded on a PerkinElmer Spectrum BX FT-IR
Phorbol 12,13-Diisobutyrate-20-trityl (3a) (99%). White amor-
spectrophotometer and reported as wavenumber (cm−1). H and 13C
1
phous powder; [α]D21 + 50.9 (c 2.0, CH3OH). IR (KBr) υmax: 3408,
́
NMR measurements were recorded on Agilent 400, 500, and 600
MHz NMR spectrometers. Chemical shifts are expressed in ppm (δ)
referenced to the solvent used, and J values are reported in Hz. The
multiplicity of the signals (s, singlet; d, doublet; dd, doublet of
doublets; t, triplet; td, triplet of doublets; q, quadruplet; qt,
quintuplet; hept, heptuplet; and m, multiplet), the coupling constants
(J), and the specific characterization of each compound are shown.
Spectra were assigned using appropriate COSY, DEPT, HSQC,
HMBC, and NOESY sequences. HRMS was performed in a Waters
Xevo G2-S QTof mass spectrometer in the positive-ion or negative-
ion ESI mode. HPLC purification was performed with a Merck-
Hitachi LaChrom apparatus equipped with an UV−vis detector (L
4250), a differential refractometer detector (RI-7490), and an Elite
LaChrom-Hitachi apparatus equipped with a differential refractom-
eter detector (RI-2490). LiChroCART LiChrospher Si 60 (5 μm, 250
mm × 4 mm) and LiChroCART LiChrospher Si 60 (10 μm, 250 mm
× 10 mm) columns were used for isolation experiments. The purity of
the tested compounds was determined by HPLC−UV with a Hitachi
Primaide or by HPLC−RI with a Hitachi Chromaster HPLC systems.
The purity of all tested compounds is >95%.
UHPLC−HRMS Conditions. The analyses were run on a
separation system, ACQUITY UPLC H-Class system, with a binary
solvent system and an automatic sample manager that was equipped
with an UPLC BEH C18 (2.1 mm × 100 mm, 1.7 mm) column,
running at a temperature of 55 °C. The mobile phases consisted of
eluent A (0.1% formic acid in water, v/v) and eluent B (methanol).
These phases were delivered at a flow rate of 0.3 mL/min by using a
linear gradient program as follows: 0−0.5 min, 25% A; 0.5−5 min,
25−0% A; 5−7 min, 0% A; 7−8 min, 0−25% A; and 8−10 min, 25%
A. The injection volume of all samples was 2 mL.
3059, 2975, 2934, 1735, 1714, 1469, 1369, 1194, 1156, 1076, 980,
and 758 cm−1. 1H NMR (CD3OD, 500 MHz) δ: 7.56 (m, 1H), 7.41−
7.38 (m, 6H), 7.30−7.26 (m, 6H), 7.24−7.20 (m, 3H), 5.56 (d, J =
5.8 Hz, 1H), 5.42 (d, J = 10.1 Hz, 1H), 3.57 (d, J = 11.1 Hz, 1H),
3.49 (d, J = 11.1 Hz, 1H), 3.19 (m, 2H), 2.61−2.52 (m, 2H), 2.50 (br
s, 2H), 2.23 (dq, J = 10.1, 6.6 Hz, 1H), 1.75 (dd, J = 2.7, 1.4 Hz, 3H),
1.28 (s, 3H), 1.23 (s, 3H), 1.19−1.15 (m, 12H), 1.08 (d, J = 5.1 Hz,
1H), and 1.38 (d, J = 6.6 Hz, 3H). 13C NMR (CD3OD, 125 MHz) δ:
210.2, 180.8, 178.5, 160.4, 145.5 (3C), 140.7, 134.4, 130.8, 129.8
(6C), 128.8 (6C), 128.1 (3C), 88.2, 79.5, 78.1, 74.9, 69.9, 66.8, 57.4,
44.3, 40.4, 38.9, 37.0, 35.5, 27.3, 24.2, 19.5, 19.2, 19.0, 18.9, 17.4,
14.7, and 10.2. HRMS (ESI): m/z calcd for C47H54O8Na [M + Na]+,
769.3716; found, 769.3712.
Phorbol 12,13-Diphenylacetate-20-trityl (4a) (82%). Spectro-
scopic data of compound 4a were identical to those described in the
literature.32
General Procedure for Monoacylation at C-13. Preparation of
Compounds 5a and 6a. A mixture of phorbol-20-trityl ether (1a)
(0.13 mmol), the corresponding isobutyric acid or phenylacetic acid
(0.4 mmol), and DMAP (0.03 mmol) in dry CH2Cl2 (2 mL) was
stirred for 10 min. To the resulting mixture was added EDCI (0.4
mmol). After stirring for 2 h, the reaction mixture was evaporated and
then Et2O (5 mL) was added. The crude was sequentially washed
with H2O (5 mL), twice with saturated sodium bicarbonate solution
(5 mL), and H2O (2 mL), dried over anhydrous sodium sulfate,
filtered, and evaporated under reduced pressure to afford compounds
5a and 6a.
Phorbol 13-Isobutyrate-20-trityl (5a) (99%). White amorphous
powder; [α]2D1 + 38,08 (c 4.7, CH3OH). IR (KBr) υmax: 3411, 3059,
́
2977, 2927, 2877, 1710, 1448, 1324, 1204, 1159, 1048, 992, 758, and
706 cm−1. 1H NMR (CD3OD, 500 MHz) δ: 7.58 (m, 1H), 7.41−7.38
(m, 6H), 7.29−7.25 (m, 6H), 7.23−7.19 (m, 3H), 5.54 (d, J = 5.9 Hz,
1H), 3.87 (d, J = 9.7 Hz, 1H), 3.56 (d, J = 11.0 Hz, 1H), 3.49 (d, J =
11.0 Hz, 1H), 3.15 (m, 2H), 2.63 (hept, J = 7.0 Hz, 1H), 2.49 (s,
2H), 2.04 (dq, J = 9.7, 6.71H), 1.76 (dd, J = 3.0, 1.3 Hz, 3H), 1.24 (s,
6H), 1.19 (d, J = 7.0 Hz, 6H), 1.07 (d, J = 6.5 Hz, 3H), and 0.98 (d, J
= 5.3 Hz, 1H). 13C NMR (CD3OD, 125 MHz) δ: 210.5, 181.6, 160.9,
145.5 (3C), 140.3, 134.2, 131.3, 129.8 (6C), 128.8 (6C), 128.1 (3C),
88.1, 79.4, 77.5, 74.9, 70.0, 69.0, 57.7, 46.2, 40.4, 39.0, 36.6, 35.5,
27.1, 24.3, 19.2, 19.1, 17.4, 15.4, and 10.3. HRMS (ESI): m/z calcd
for C43H48O7Na [M + Na]+, 699.3298; found, 699.3297.
Phorbol 13-Phenylacetate-20-trityl (6a) (67%). Spectroscopic
data of compound 6a were identical to those described in the
literature.33
The UHPLC system was coupled with a quadrupole time-of-flight
tandem mass spectrometer (Xevo-G2-S QTOF; Waters, Manchester,
U.K.) equipped with an ESI source. The operating parameters in ESI
and the data-independent acquisition mode (MSE)23 were set as
follows in positive and negative modes: sample cone voltage of 30 V;
source temperature of 120 °C; cone gas flow of 10 L/h; and
desolvation gas flow of 850 L/h. In the positive mode, capillary
voltage and desolvation temperature were set at 0.7 kV and 400 °C,
respectively. In the negative mode, capillary voltage and desolvation
temperature were set at 3.0 kV and 450 °C, respectively. In the MSE
mode (positive mode), the trap collision energy of the low-energy
function was set at 6 eV, while the ramp trap collision energy of the
high-energy function was set at 60−120 eV.
To ensure mass accuracy and reproducibility, the mass
spectrometer was calibrated over a range of 100−1200 Da using
sodium formiate solution. Leucine-enkephalin (m/z 556.2771 in
positive-ion mode) was used as the external reference of LockSpray
infused at a constant flow of 20 mL/min. Data acquisition was
performed by MassLynx 4.1 software (Waters, Manchester, U.K.) in
positive and negative-ion modes.
Synthesis of Phorbol Esters. Preparation of Phorbol 12,13-
Diacetate-20-trityl (2a). Pyridine (2 drops) was added to a solution
of phorbol-20-trityl (1a) (60.0 mg, 0.1 mmol) in acetic anhydride (2
mL) and stirred at room temperature overnight. Then, cyclohexane
was added (5 mL), and the solvent was evaporated under reduced
pressure. This procedure was repeated three times to give
quantitatively phorbol-12,13-diacetate-20-trityl (2a) (67.6 mg, 99%).
Spectroscopic data of compound 2a were identical to those described
in the literature.20
General Procedure for the Preparation of 12,13-Diacylphorbols
3a and 4a. A mixture of phorbol-20-trityl (1a) (0.13 mmol), the
corresponding isobutyric acid or phenylacetic acid (1.13 mmol), and
DMAP (0.13 mmol) in dry CH2Cl2 (2 mL) was stirred for 10 min. To
General Procedure for the Preparation of 12,13-Diacylphorbols
7a−9a. Phorbols 7a and 8a were prepared from 5a and phorbol 9a
from 6a following the procedure described above for the synthesis of
3a and 4a.
Phorbol 12-Phenylacetate-13-isobutyrate-20-trityl (7a) (73%).
White amorphous powder; [α]2D1 26.3 (c 3.1, CH3OH). IR (KBr)
υmax: 3330, 3059, 3022, 2972, 2917, 2872, 1706, 1447, 1348, 1251,
́
1
1157, 1049, 980, and 702 cm−1. H NMR (CD3OD, 500 MHz) δ:
7.53 (m, 1H), 7.38−7.17 (m, 20H), 5.52 (d, J = 5.8 Hz, 1H), 5.40 (d,
J = 10.5 Hz, 1H), 3.68−3.61 (m, 2H), 3.54 (d, J = 11.2 Hz, 1H), 3.47
(d, J = 11.2 Hz, 1H), 3.16 (t, J = 2.5 Hz, 1H), 3.12 (t, J = 5.8 Hz, 1H),
2.52 (m, 1H), 2.48 (br s, 2H), 2.18 (dq, J = 10.5, 6.5 Hz,1H), 1.73
(dd, J = 2.4, 1.5 Hz, 3H), 1.12 (d, J = 7.0 Hz, 6H), 1.08 (s, 3H), 1.05
(s, 3H), 1.00 (d, J = 5.1 Hz, 1H), and 0.84 (d, J = 6.5 Hz, 3H). 13C
NMR (CD3OD, 125 MHz) δ: 210.2, 180.7, 173.3, 160.3, 145.5 (3C),
140.6, 135.8, 134.4, 130.7, 130.2 (2C), 129.8 (6C), 129.6 (2C), 128.8
(6C), 128, 1 (4C), 88.1, 79.4, 78.7, 74.8, 69.9, 66.6, 57.3, 44.1, 42.5,
40.3, 38.8, 37.0, 35.5, 27.3, 24.0, 19.0, 18.9, 17.0, 14.7, and 10.3.
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HRMS (ESI): m/z calcd for C51H54O8Na [M + Na]+, 817.3716;
found, 817.3730.
67.9, 67.3, 57.3, 44.2, 42.5, 42.2, 39.9, 38.4, 37.1, 27.5, 23.8, 17.0,
14.7, and 10.2. Purity HPLC: 98.1%. HRMS (ESI): m/z calcd for
C36H40O8Na [M + Na]+, 623.2621; found, 623.2616.
Phorbol 12-Tigliate-13-isobutyrate-20-trityl (8a) (47%). White
amorphous powder; [α]2D1 + 17.5 (c 1.25, CH3OH). IR (KBr) υmax
:
Phorbol 13-Isobutyrate (5) (84%). White amorphous solid; [α]D21
+
́
3402, 2978, 2950, 1712, 1448, 1361, 1254, 1219, 1156, 1082, 770,
and 705 cm−1. 1H NMR (CD3OD, 400 MHz) δ: 7.56 (m, 1H), 7.41−
7.37 (m, 6H), 7.30−7.21 (m, 9H), 6.85 (dq, J = 7.2, 1.4 Hz, 1H), 5.56
(d, J = 6.1 Hz, 1H), 5.48 (d, J = 10.4 Hz, 1H), 3.58 (d, J = 11.1 Hz,
1H), 3.50 (d, J = 11.1 Hz, 1H), 3.20 (m, 2H), 2.57 (hept, J = 7.0 Hz,
1H), 2.51 (s, 2H), 2.27 (dq, J = 10.4, 6.4 Hz, 1H), 1.82 (m, 6H), 1.75
(dd, J = 2.6, 1.4 Hz, 3H), 1.31 (s, 3H), 1.23 (s, 3H), 1.15 (d, J = 7.0
Hz, 6H), 1.08 (d, J = 5.1 Hz, 1H), and 0.87 (d, J = 6.4 Hz, 3H). 13C
NMR (CD3OD, 100 MHz) δ: 210.0, 180.7, 169.1, 160.2, 145.3 (3C),
140.5, 138.8, 134.2, 130.6, 129.6 (6C), 129.4, 128.6 (6C), 127.9
(3C), 87.9, 79.3, 78.0, 74.7, 69.7, 66.6, 57.1, 44.3, 40.2, 38.7, 36.9,
35.2, 27.1, 23.9, 18.8, 17.3, 14.5, 14.2, 12.1, and 10.0. HRMS (ESI):
m/z calcd for C48H54O8Na [M + Na]+, 781.3716; found, 781.3712.
Phorbol 12-Isobutyrate-13-phenylacetate-20-trityl (9a) (82%).
White amorphous powder; [α]2D1 + 28.5 (c 1.5, CH3OH). IR (KBr)
48.4 (c 2.0, CHCl3); IR (KBr) υmax: 3414, 2924, 2854, 1638, 1491,
́
1329, 1291, 1159, 1015, and 792 cm−1; 1H NMR (CD3OD, 400
MHz) δ: 7.57 (m, 1H), 5.60 (d, J = 5.7, 1H), 3.95 (d, J = 13.0 Hz,
1H), 3.91 (d, J = 13.0 Hz, 1H), 3.86 (d, J = 10.2 Hz, 1H), 3.26 (t, J =
5.7 Hz, 1H), 3.12 (t, J = 2.8 Hz, 1H), 2.62 (p, J = 7.0 Hz, 1H), 2.52
(d, J = 19.0 Hz, 1H), 2.44 (d, J = 19.0 Hz, 1H), 2.02 (dq, J = 10.2, 6.5
Hz, 1H), 1.74 (dd, J = 2.8, 1.4 Hz, 3H), 1.23 (s, 3H), 1.22 (s, 3H),
1.18 (d, J = 7.0 Hz, 3H), 1.06 (d, J = 6.5 Hz, 3H), and 1.02 (d, J = 5.7
Hz, 1H); 13C NMR (CD3OD, 100 MHz) δ: 210.5, 181.6, 161.0,
142.5, 134.4, 129.9, 79.7, 77.6, 74.8, 69.0, 68.1, 57.7, 46.2, 40.1, 38.6,
36.8, 35.5, 27.2, 24.3, 19.2, 19.1, 17.5, 15.5, and 10.2. Purity HPLC:
96.2%. HRMS (ESI): m/z calcd for C24H34O7Na [M + Na]+,
457.2202; found, 457.2200.
Phorbol 13-Phenylacetate (6) (86%). White amorphous solid;
[α]2D1 + 4.4 (c 1.4, CHCl3). IR (KBr) υmax: 3417, 2921, 2848, 1710,
́
1461, 1125, 1078, 1022, and 792 cm−1. 1H NMR (CD3OD, 500
MHz) δ: 7.55 (m, 1H), 7.35−7.21 (m, 5H), 5.55 (d, J = 5.6 Hz, 1H),
3.94−3.88 (m, 3H), 3.71 (s, 2H), 3.23 (t, J = 5.6 Hz, 1H), 3.10 (t, J =
2.4 Hz, 1H), 2.50 (d, J = 19.0 Hz, 1H), 2.43 (d, J = 19.0 Hz, 1H),
2.01 (dq, J = 10.5, 6.3 Hz, 1H), 1.73 (dd, J = 2.9, 1.4 Hz, 3H), 1.18 (s,
3H), 1.05 (s, 3H), 1.04 (d, J = 6.5 Hz, 3H), and 0.95 (d, J = 5.5 Hz,
1H). 13C NMR (CD3OD, 125 MHz) δ: 210.5, 176.5, 161.0, 142.5,
135.0, 134.4, 130.5 (2C), 129.8, 129.6 (2C), 128.3, 79.7, 77.5, 74.7,
69.7, 68.1, 57.7, 46.2, 42.2, 40.0, 38.6, 36.7, 27.3, 24.0, 17.4, 15.4, and
10.2. Purity HPLC: 96.8%. HRMS (ESI): m/z calcd for C28H34O7Na
[M + Na]+, 505.2202; found, 505.2209.
υmax: 3414, 2976, 2924, 2878, 1715, 1626, 1469, 1269, 1218, 1154,
́
759, and 705 cm−1. 1H NMR (CD3OD, 500 MHz) δ: 7.54 (m, 1H),
7.39−7.19 (m, 20H), 7.30−7.21 (m, 9H), 5.521 (d, J = 6.3 Hz, 1H),
5.47 (d, J = 10.5 Hz, 1H), 3.67 (s, 2H), 3.56 (d, J = 11.1 Hz, 1H),
3.48 (d, J = 11.1 Hz, 1H), 3.15 (m, 2H), 2.56 (hept, J = 7.0 Hz, 1H),
2.51−2.46 (s, 2H), 2.21 (dq, J = 10.5, 6.5 Hz, 1H), 1.74 (dd, J = 3.0,
1.4 Hz, 3H), 1.24 (s, 3H), 1.16 (d, J = 7.0 Hz, 3H), 1.15 (d, J = 7.0
Hz, 3H), 1.05 (s, 3H), 1.02 (d, J = 5.1 Hz, 1H), and 0.88 (d, J = 6.5
Hz, 3H). 13C NMR (CD3OD, 125 MHz) δ: 210.2, 178.6, 175.7,
160.3, 145.5 (3C), 140.7, 134.9, 134.4, 130.7 (2C), 130.6, 129.8
(6C), 129.5 (2C), 128.8 (6C), 128.1, 128.1 (3C), 88.1, 79.5, 78.1,
74.8, 69.8, 67.4, 57.4, 44.3, 42.1, 40.3, 38.8, 36.9, 35.4, 27.5, 24.0,
19.4, 19.2, 17.4, 14.7, and 10.2. HRMS (ESI): m/z calcd for
C51H54O8Na [M + Na]+, 817.3716; found, 817.3707.
Phorbol 12-Phenylacetate-13-isobutyrate (7) (75%). White
amorphous solid; [α]2D1 + 10.0 (c 5.1, CHCl3). IR (KBr) υmax
:
́
3402, 2980, 2926, 2880, 1713, 1497, 1391, 1251, 1161, 988, and 757
cm−1. 1H NMR (CD3OD, 500 MHz) δ: 7.52 (m, 1H), 7.35−7.23 (m,
5H), 5.58 (d, J = 5.5 Hz, 1H), 5.39 (d, J = 10.5 Hz, 1H), 3.94 (d, J =
13.0 Hz, 1H), 3.89 (d, J = 13.0 Hz, 1H), 3.66 (s, 2H), 3.24 (t, J = 5.5
Hz, 1H), 3.14 (t, J = 2.4 Hz, 1H), 2.53 (m, 1H), 2.50−2.47 (m, 2H),
2.17 (dq, J = 10.5, 4.0 Hz, 1H), 1.73 (dd, J = 2.3, 1.6 Hz, 3H), 1.13
(d, J = 1.9 Hz, 3H), 1.12 (d, J = 1.9 Hz, 3H), 1.08 (s, 3H), 1.04 (s,
3H), 1.03 (d, J = 5.1 Hz, 1H), and 0.84 (d, J = 6.6 Hz, 3H). 13C NMR
(CD3OD, 125 MHz) δ: 208.8, 179.3, 171.9, 159.1, 141.4, 134.4,
133.1, 128.8 (2C), 128.2 (2C), 127.8, 126.7, 78.4, 77.4, 73.3, 66.5,
65.3, 55.8, 42.7, 41.1, 38.6, 37.0, 35.8, 34.1, 25.9, 22.6, 17.6, 17.5,
15.7, 13.3, and 8.8. Purity HPLC: 99.0%. HRMS (ESI): m/z calcd for
C32H30O8Na [M + Na]+, 575.2621; found, 575.2639.
General Procedure for the Deprotection of Trityl Ether.
Preparation of Phorbols 2−9. HClO4 (0.01 M solution in methanol,
1 mL, 0.01 mmol) was added to a solution of the corresponding
phorbols 2a−9a (0.05 mmol) in methanol (1 mL). Once the reaction
was completed (30 min.), sodium acetate was added until pH = 7.
The solvent was evaporated under reduced pressure to give a crude
product which was purified by silica gel column chromatography.
Elution with petroleum ether/ethyl acetate (50:50) yielded the
corresponding compounds 2−9.
Phorbol 12,13-Diacetate (2) (99%). Spectroscopic data of
compound 2 were identical to those described in the literature.34
Purity HPLC: 99.8%.
Phorbol 12,13-Diisobutyrate (3) (87%). White amorphous
powder; [α]2D1 + 19.1 (c 2.2, CHCl3). IR (KBr) υmax: 3405, 2975,
Phorbol 12-Tigliate-13-isobutyrate (8) (79%). Spectroscopic data
of compound 8 were identical to those described in the literature.35,36
Purity HPLC: 99.5%.
́
2926, 2878, 1712, 1469, 1389, 1275, 1160, 1080, 978, and 754 cm−1.
1H NMR (CD3OD, 500 MHz) δ: 7.54 (m, 1H), 5.61 (d, J = 4.7 Hz,
1H), 5.41 (d, J = 10.3 Hz, 1H), 3.96 (d, J = 13.9 Hz, 1H), 3.91 (d, J =
13.9 Hz, 1H), 3.30 (m, 1H), 3.16 (t, J = 2.7 Hz, 1H), 2.63−2.53 (m,
4H), 2.22 (dq, J = 10.5, 6.3 Hz, 1H), 1.73 (dd, J = 2.7, 1.4 Hz, 3H),
1.27 (s, 3H), 1.21 (s, 3H), 1.19−1.14 (m, 12H), 1.10 (d, J = 5.3 Hz,
1H), and 0.88 (d, J = 6.5 Hz, 3H). 13C NMR (CD3OD, 125 MHz) δ:
210.3, 180.7, 178.5, 160.5, 142.9, 134.6, 129.3, 79.8, 78.1, 74.7, 68.0,
66.8, 57.3, 44.3, 40.0, 38.5, 37.3, 35.5 (2C), 27.3, 24.2, 19.4, 19.2,
19.0, 18.9, 17.5, 14.8, and 10.2. Purity HPLC: 96.6%. HRMS (ESI):
m/z calcd for C28H39O8 [M − H]−, 503.2645; found, 503.2641.
Phorbol 12,13-Diphenylacetate (4) (90%). White amorphous
Phorbol 12-Isobutyrate-13-phenylacetate (9) (52%). White
amorphous solid; [α]2D1 + 12.8 (c 0.4, CHCl3). IR (KBr) υmax
:
́
3430, 2924, 2854, 1710, 1457, 1376, and 787 cm−1. 1H NMR
(CD3OD, 400 MHz) δ: 7.53 (br s, 1H), 7.32−7.22 (m, 5H), 5.58 (d,
J = 5.7 Hz, 1H), 5.47 (d, J = 10.4 Hz, 1H), 3.94 (d, J = 13.2 Hz, 1H),
3.89 (d, J = 13.2 Hz, 1H), 3.67 (s, 2H), 3.26 (t, J = 5.7 Hz, 1H), 3.14
(d, J = 3.0 Hz, 1H), 2.57 (hept, J = 7.0 Hz, 1H), 2.50 (d, J = 19.0 Hz,
1H), 2.46 (d, J = 19.0 Hz, 1H), 2.21 (dq, J = 10.4, 6.4 Hz, 1H), 1.73
(dd, J = 3.0, 1.3 Hz, 3H), 1.23 (s, 3H), 1.15 (d, J = 7.0 Hz, 3H), 1.05
(d, J = 5.7 Hz, 1H), 1.03 (s, 3H), and 0.88 (d, J = 6.4 Hz, 3H). 13C
NMR (CD3OD, 100 MHz) δ: 210.3, 178.7, 175.7, 160.5, 142.9,
134.9, 134.6, 130.7 (2C), 129.5 (2C), 129.2, 128.2, 79.8, 78.2, 74.7,
67.9, 67.4, 57.3, 44.3, 42.1, 40.0, 38.4, 37.1, 35.4, 27.5, 24.0, 19.4,
19.2, 17.4, 14.8, and 10.2. Purity HPLC: 99.4%. HRMS (ESI): m/z
calcd for C32H39O8 [M − H]−, 551.2645; found, 551.2631.
powder; [α]2D1 + 24.2 (c 7.0, CHCl3). IR (KBr) νmax: 3415, 2924,
́
1
2854, 1710, 1628, 1455, 1330, 1076, 998, and 751 cm−1. H NMR
(CD3OD, 400 MHz) δ: 7.51 (m, 1H), 7.34−7.21 (m, 10H), 5.54 (d, J
= 5.6 Hz, 1H), 5.45 (d, J = 10.4 Hz, 1H), 3.92 (d, J = 13.1 Hz, 1H),
3.89 (d, J = 13.1 Hz, 1H), 3.65 (s, 2H), 3.64 (s, 2H), 3.20 (t, J = 5.6
Hz, 1H), 3.11 (t, J = 2.6 Hz, 1H), 2.49 (d, J = 19.0 Hz, 1H), 2.44 (d, J
= 19.0 Hz, 1H), 2.14 (dq, J = 10.4, 6.6 Hz, 1H), 1.72 (dd, J = 2.6, 1.4
Hz, 3H), 0.98 (d, J = 5.6 Hz, 1H), 0.96 (s, 3H), 0.88 (s, 3H), and
0.84 (d, J = 6.6 Hz, 3H). 13C NMR (CD3OD, 100 MHz) δ: 210.3,
175.7, 173.5, 160.5, 142.8, 135.8, 134.8, 134.6, 130.7 (2C), 130.3
(2C), 129.6 (2C), 129.5 (2C), 129.2, 128.2, 128.1, 79.8, 78.9, 74.7,
Plant Material. Latex from E. resinifera was collected in November
2016 in Demnate, Beni Mellal-Khenifera Province (Marruecos) and
was obtained by making repeated cuts along the branches of plants
and collecting the white milky exudates. A vouch specimen has been
kept at the herbarium of the chemistry laboratory of Natural
Substances, Mohamed V University, Rabat (Morocco).
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Extraction and Isolation. Air-dried sample of E. resinifera latex
(200 g) was macerated in EtOAc (200 mL, 3 times). The resulting
solution was filtered first by filter paper strips and then through a
short bed of TLC-grade silica gel (30 g). The yellowish extract,
obtained after solvent removal, was magnetically stirred with
acetonitrile in order to remove triterpene compounds (white
precipitate). Acetonitrile fraction (yellow organic extract) was
concentrated and subjected to purification by vacuum liquid
chromatography over silica gel column using hexane/EtOAc gradient
and CH3OH to yield six fractions (Fr1: 10%, Fr2-Fr4: 20, 30, and
50%, respectively, Fr5: 100% EtOAc, and Fr6: CH3OH). The
phorbol-containing fraction (Fr4) was purified by open-column
chromatography using hexane/EtOAc (30:70−0:100, v/v) to obtain
four subfractions which were analyzed by UHPLC−HRMS, taking
into account the results of mass spectroscopy of our synthetic phorbol
12,13-diesters. A compound isomeric to phorbol 12-phenylacetate-13-
isobutyrate (7) was detected in subfraction FR4-4 (58.1 mg), which
was further chromatographed through analytic HPLC using Si-60
column and EtOAc/hexane (40% of EtOAc) as the solvent system.
Finally, two compounds not described previously, 12-deoxy-16-
hydroxyphorbol 13-phenylacetate-16-isobutyrate (DPPI, 10, 1.0 mg,
0.0005%, 46.18 min) and 12-deoxy-16-hydroxyphorbol 13-phenyl-
acetate-16-tigliate (DPPT, 11, 1.4 mg, 0.0007%, 51.1 min), were
isolated.
factors EGF and bFGF were used as indicated in the figure legends
(see Supporting Information, Methods).
Neurosphere Assay. The effects of compounds 1, 2, 3, 5, and 11
were tested using neurosphere assays in order to evaluate NPC
proliferation. This assay analyzes the size of the neurospheres formed
after a period of time in the presence of increasing concentrations of
the compounds added at the time of seeding. All experiments were
performed in triplicates and a minimum of five independent times (n
= 5). The numbers and sizes of the neurospheres were measured as
previously described30 using ImageJ41 (see Supporting Information,
Methods).
Intranasal Administration of Compounds 2 and 3.
Compounds were delivered intranasally as previously described.42−44
Treatments were administered manually while the animal was placed
standing with an extended neck.44 A total of 12 μL of each solution
was administered. 3 μL was delivered each time alternating both nasal
cavities using a micropipette. Thereafter, the mouse was kept in such a
position for 10 additional seconds to ensure complete inhalation of
the fluid. Based on the results obtained in vitro, compound 3 was used
at a concentration of 10 and 100 nM in saline, whereas compound 2
was used at 5 mM; saline was used as the vehicle. In all experiments,
the mice were coded, and treatment (vehicle or compound) was
assigned randomly to the code numbers and applied. Blind
quantifications were performed to avoid subjective biases.10
Brain Processing and Immunohistochemistry. At the end of
the treatment, brains were perfused with paraformaldehyde (PFA)
and sliced using a cryotome into 30 μm sections. Immunohistochem-
istry was performed as previously described.30,41,45 Antibodies used
were rat monoclonal anti-BrdU (1:500, Abcam, Cambridge, UK) and
rabbit monoclonal anti-DCX (1:500, Abcam, Cambridge, UK). The
secondary antibodies used were donkey anti-rat IgG labeled with
AlexaFluor 488 (1:1000) and donkey anti-rabbit IgG labeled with
Alexa-Fluor 594 (1:1000) from Invitrogen (Carlsbad, CA).
Quantification of Neurogenesis in Brain Sections. Cells
positive for BrdU and DCX in the SVZ were estimated as
described.40,45 Positive cells were quantified throughout the lateral
and laterodorsal walls of the lateral ventricles in every fifth section; 6−
8 sections per brain were analyzed by fluorescence microscopy at 20×
magnification. Mice were coded depending on the treatment, and
quantification of cells in brain slices was done in blinded analysis.
Cells in the SVZ of both brain hemispheres have been quantified
unless otherwise indicated.
12-Deoxy-16-hydroxyphorbol 13-Phenylacetate-16-isobutyrate
(10). White amorphous powder; [α]2D4 + 9.61 (c 0.08, CH3OH); IR
1
(KBr) υmax: 3416, 1712, 1633, and 1100 cm−1; for H and 13C NMR
́
(CD3OD) data, see Table 1. Purity HPLC: 99.0%. HRESIMS m/z:
575.2621 [M + Na]+, calcd for C32H40O8Na, 575.2621.
12-Deoxy-16-hydroxyphorbol 13-Phenylacetate-16-tigliate (11).
White amorphous powder; [α]2D4 + 18.52 (c 0.14, CH3OH); IR (KBr)
υmax: 3416, 2923, 1705, and 1269 cm−1; for 1H and 13C NMR
́
(CD3OD) data, see Table 1. Purity HPLC: 98.2%. HRESIMS m/z:
587.2662 [M + Na]+, calcd for C33H40O8Na, 587.2621.
Animals. SVZ-derived NPCs were isolated from 7-day postnatal
CD1 mice and used for in vitro experiments. For in vivo experiments,
2-month-old male CD1 mice were used. The animals were housed
under controlled conditions of temperature (21−23 °C) and light
(LD 12:12) with free access to food (AO4 standard maintenance diet,
́
SAFE, Epinay-sur-Orge, France) and water. Care and handling of the
animals were performed according to the Guidelines of the European
Union Council (2010/63/EU) and the Spanish regulations (65/2012
and RD53/2013) for the use of laboratory animals. All studies
involving animals are reported in accordance with the ARRIVE
guidelines for reporting experiments involving animals.37,38 The
protocols used have been authorized by the Ethics Committee of the
Molecular Docking. The crystal structure (PDB: 1PTR)27 of
PKC Delta C1 domain complexed with phorbol 13-acetate (P13-Ac)
and the full-length proteins PKCβII (PDB ID: 3PFQ)31 were used to
perform in silico analysis. To generate three-dimensional (3D) ligands,
two-dimensional structures drawn by ChemDraw Ultra were super-
imposed on the crystal structure of P13-Ac using Forge 10.5.0
(Cresset BioMolecular Discovery LTD). All 3D ligands were verified
using NOESY NMR data. Molecular docking analysis was carried out
using AutoDockTools-1.5.6 (The Scripps Research Institute, La Jolla,
CA, USA). The target protein regions were limited with a grid box of
40 × 40 × 40 Å that was centered (x = 10.879, y = 25.984, and z =
25.53) on the binding site where P 13Ac was removed from the
crystallized enzyme. The binding pocket residues of PKC-β were
identified by comparison with the activator-binding residues of PKCδ-
C1B. The residues selected were limited with grid box of 40 × 40 ×
40 Å centered at (x = −59.148, y = 16.647, and z = −5.326). The
Lamarckian genetic algorithm was used to perform the automated
molecular dockings with the preset parameters. The total number of
genetic algorithm runs and iterations was set to 100 and 1.0 × 107,
respectively. The lowest binding energy conformation was selected for
UCSF Chimera analysis. 3D protein−ligand interactions were
visualized in Discovery Studio Visualizer.
́
“Consejeria de Agricultura, Pesca y Desarrollo sostenible” of the Junta
́
de Andalucia”, Spain, with the approval numbers 30/03/2016/038
and 04/03/2020/033.
HEK293T Culture, Cloning, and Transfection. HEK293T cells
were obtained from ATCC (Manassas, VA, USA), cultured,
transfected, and left overnight.30 Thereafter, the cells were left in
serum-free FluoroBrite DMEM (Thermo Fisher Scientific) for 30 min
and used in time-lapse experiments as previously described.39
Cloning of Human TGFα cDNA Fused to GFP and m-Cherry.
Full-length cDNA encoding the mCherry-TGFα-GFP construct
containing the human TGFα (NCBI reference sequence:
NM_003236.4) containing mCherry cDNA between nucleotides
126 and 127 of TGFα, and the enhanced green fluorescent protein
(EGFP) in frame was designed and synthesized by GeneCust
(Boynes, France) as previously described.10
Time-Lapse Experiments and Fluorescence Analysis of
mCherry-Fused TGFα or Neuregulin in the Culture Medium
of HEK293T. HEK293T cells were plated in μ−dishes (35 mm high,
Ibidi, Munich, Germany). The cells were treated with different
compounds, and images were taken during 3 h as described in the
figure legends.
ASSOCIATED CONTENT
■
sı
* Supporting Information
SVZ-Derived NPC Isolation and Culture. SVZ-derived cells
were isolated following the same procedure described in Rabaneda et
al.40 For in vitro experiments in proliferation conditions, growth
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00156.
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Article
Instituto de Investigación e Innovación Biomédica de Cádiz
(INIBICA), 11009 Cádiz, Spain
mCherry/GFP ratio remains unaltered in the absence of
the compound (AVI)
José Manuel Botubol-Ares − Departamento de Química
Orgánica, Facultad de Ciencias, Universidad de Cádiz,
Puerto Real 11510, Cádiz, Spain; Instituto de Investigación e
Innovación Biomédica de Cádiz (INIBICA), 11009 Cádiz,
Spain; orcid.org/0000-0002-2312-612X
Carolina de los Reyes − Departamento de Química Orgánica,
Facultad de Ciencias, Universidad de Cádiz, Puerto Real
11510, Cádiz, Spain
Rosa Durán-Patrón − Departamento de Química Orgánica,
Facultad de Ciencias, Universidad de Cádiz, Puerto Real
11510, Cádiz, Spain; Instituto de Investigación e Innovación
Biomédica de Cádiz (INIBICA), 11009 Cádiz, Spain;
orcid.org/0000-0002-5307-4164
mCherry/GFP ratio remains unaltered in the presence
of 0.5 μM phorbol (AVI)
mCherry/GFP ratio on the addition of compound 3
(AVI)
mCherry/GFP ratio on the addition of compound 2
(AVI)
mCherry/GFP ratio on the addition of compound 5
(AVI)
Effect of addition of compound 10 on TGFα release
(AVI)
Docking of DPPI with PKCβ (PDB)
Docking of DPPT with PKCβ (PDB)
Docking of DPA with PKCβ (PDB)
Pedro Nunez-Abades − Departamento de Fisiología, Facultad
de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain
Antonio J. Macías-Sánchez − Departamento de Química
Orgánica, Facultad de Ciencias and Instituto de Investigación
en Biomoléculas (INBIO), Universidad de Cádiz, Puerto
Real 11510, Cádiz, Spain; Instituto de Investigación e
Innovación Biomédica de Cádiz (INIBICA), 11009 Cádiz,
Spain; orcid.org/0000-0001-6002-4977
Docking of 7 with PKCβ (PDB)
Docking of 3 with PKCβ (PDB)
Docking of Phorbol with PKCβ (PDB)
Docking of DPB with PKCβ (PDB)
Molecular formula strings of tested compounds (CSV)
Structure of active phorbol derivatives; isolation of
phorbol 20-trityl ether; NMR spectra of compounds 3−
11; HPLC chromatograms for compounds 2−11;
UPLC-HRMS-assisted identification of 12-deoxyphorbol
13,16-diesters from E. resinifera; proposed fragmentation
route for selected ion on MSE spectrum for compounds
9−11; effect of intranasal administration of compound
3; physicochemical properties; docking analysis of
phorbol derivatives; molecular docking of DPPT (11),
DPPI (10), 7 and 3 and Phorbol (1), DPA and DPB on
PKCδC1B; and molecular docking of DPPI (10) and
DPPT (11), 7 and 3 and Phorbol (1), DPBand DPA on
PKCδC1B (PDF)
́
Carmen Castro − Area de Fisiología, Facultad de Medicina,
Universidad de Cádiz, 11002 Cádiz, Spain; Instituto de
Investigación e Innovación Biomédica de Cádiz (INIBICA),
11009 Cádiz, Spain
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00156
Author Contributions
The manuscript was written through the contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
AUTHOR INFORMATION
Corresponding Author
́
Ministerio de Ciencia, Innovacion y Universidades (grant nos
■
RTI2018−099908-B-C21, and RTI2018−099908-B-C22 MI-
́ ́
CINN/FEDER) and Consejeria de Economia, Conocimiento,
Rosario Hernández-Galán − Departamento de Química
Orgánica, Facultad de Ciencias and Instituto de Investigación
en Biomoléculas (INBIO), Universidad de Cádiz, Puerto
Real 11510, Cádiz, Spain; Instituto de Investigación e
Innovación Biomédica de Cádiz (INIBICA), 11009 Cádiz,
Spain; orcid.org/0000-0003-1887-4796;
́
Empresas y Universidades Junta de Andalucia (grant no
FEDER-ANDALUCIA 2018−00106647).
Notes
́
The authors declare no competing financial interest.
Email: rosario.hernandez@uca.es
ACKNOWLEDGMENTS
■
This work was supported by the Spanish Ministerio de Ciencia,
Innovación y Universidades (grant nos RTI2018−099908-B-
C21 and RTI2018−099908-B-C22 MICINN/FEDER granted
to CC and RHG, respectively) and Consejería de Economía,
Conocimiento, Empresas y Universidades Junta de Andalucía
Authors
Abdellah Ezzanad − Departamento de Química Orgánica,
Facultad de Ciencias, Universidad de Cádiz, Puerto Real
11510, Cádiz, Spain
́
Ricardo Gómez-Oliva − Area de Fisiología, Facultad de
́
(grant no FEDER-ANDALUCIA sol2018−00106647-tra). We
Medicina, Universidad de Cádiz, 11002 Cádiz, Spain;
Instituto de Investigación e Innovación Biomédica de Cádiz
(INIBICA), 11009 Cádiz, Spain
thank the “Servicio de experimentación y producción animal
(SEPA) de la Universidad de Cádiz” as well as the “Servicios
Centrales de apoyo a la investigación en Ciencias de la Salud
(SCICS) de la Universidad de Cádiz” and “Servicios centrales
de Ciencia y tecnología (SC-ICYT) de la Universidad de
Cádiz”.
Felipe Escobar-Montano − Departamento de Química
̃
Orgánica, Facultad de Ciencias, Universidad de Cádiz,
Puerto Real 11510, Cádiz, Spain
́
Mónica Díez-Salguero − Area de Fisiología, Facultad de
Medicina, Universidad de Cádiz, 11002 Cádiz, Spain
Noelia Geribaldi-Doldan − Departamento de Anatomía y
Embriología Humanas, Universidad de Cádiz, 11002 Cádiz,
Spain
Samuel Dominguez-Garcia − Area de Fisiología, Facultad de
Medicina, Universidad de Cádiz, 11002 Cádiz, Spain;
ABBREVIATIONS
■
COSY, correlation spectroscopy.; DMAP, dimethylamino
pyridine.; DPA, 12-deoxyphorbol 13-angelate; DPB, 12-
deoxyphorbol 13-isobutyrate; DPP, 12-deoxyphorbol 13-
phenylacetate; DPPI, 12-deoxy-16-hydroxy-13-phenylacetyl
́
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