Structural Reassignment of rel-(3′ Z,3 R,6 R,7 R,3a′ R,6′ R)-3,8-Dihydrodiligustilide and the Activity of Diligustilide and 3,8-Dihydro- A nd 3,8,7′,7a′-Tetrahydrodiligustilides as Progestins

Article abstract of DOI:10.1021/acs.orglett.9b02762

Several phthalides were semisynthesized, including a 3,8-dihydrodiligustilide with progesterone-like activity, previously isolated from Ligusticum chuanxiong, the structure of which was earlier assigned to a semisynthetic product with nonidentical spectroscopic constants. The structure of this natural phthalide was reassigned with a proposal of its absolute configuration. Phthalides acted as progestins in cell viability assays, immunofluorescence microscopy, and docking analysis. Therefore, the structures for natural and semisynthetic phthalides with potential use in hormone-related therapies were reassigned.

Full text of DOI:10.1021/acs.orglett.9b02762

Letter
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Structural Reassignment of rel-(3′Z,3R,6R,7R,3a′R,6′R)‑3,8-
Dihydrodiligustilide and the Activity of Diligustilide and 3,8-
Dihydro- and 3,8,7′,7a′-Tetrahydrodiligustilides as Progestins
†
‡
́
Luis Avila, Ericka K. P. Almeida-Aguirre, Carlos A. Mendez-Cuesta, Ruben A. Toscano,
́ ́ ́
Jose
Marco A. Cerbon
†
́
Cervantes, and Guillermo Delgado*
Instituto de Química, Universidad Nacional Autonoma de Mexico, Ciudad Universitaria, Coyoacan 04510, Ciudad de Mexico,
́ ́ ́ ́
Mexico
*
S Supporting Information
ABSTRACT: Several phthalides were semisynthesized, includ-
ing a 3,8-dihydrodiligustilide with progesterone-like activity,
previously isolated from Ligusticum chuanxiong, the structure of
which was earlier assigned to a semisynthetic product with
nonidentical spectroscopic constants. The structure of this
natural phthalide was reassigned with a proposal of its absolute
configuration. Phthalides acted as progestins in cell viability
assays, immunofluorescence microscopy, and docking analysis.
Therefore, the structures for natural and semisynthetic
phthalides with potential use in hormone-related therapies
were reassigned.
rogestins are substances that act as progesterone receptor
P
(PR) agonists and can induce the natural activity of
1
progesterone. These compounds are used in hormone
2
−4
5,6
replacement therapy
and contraception
and for the
treatment of infertility, although there are risks associated
with these uses, such as an increased breast cancer incidence,
4
,7
depending upon the progestin. Therefore, identifying new
progestins, with fewer side effects, that could be used for
therapeutic purposes is warranted.
Phthalides are a group of compounds isolated from several
8
natural sources, such as plants, fungi, and lichens, and are
secondary metabolites of some plants belonging to the
9
Apiaceae family. The latter are widely used in traditional
8
−10
medicines in Asia and North America.
For example,
Ligusticum porteri is employed in folk medicine in Northern
Mexico and Southern USA for headaches, colds, stomach
11,12
disorders, diabetes, etc.
The bioactivity of phthalides
correlates in several cases with the traditional uses of the
1
3
plants; e.g., compound 1 has antinociceptive activity,
14
phthalides 1, 2, rac-3, and rac-4 have sedative effects, and
15
rac-3 is a gastroprotector (see Figure 1).
Chuanxiong rhizome (L. chuanxiong) is recommended for
menstrual disorders in traditional Chinese medicine, and this
use correlates with the isolation of progestins rac-5 and,
specifically, 6, which activate the PR according to a gene
Figure 1. Phthalides from Apiaceae plants.
Herein, we report the semisynthesis of both C3 epimers of
3,8-dihydrodiligustilide from diligustilide (rac-3), the structural
correction and absolute configuration of the bioactive Diels−
Alder adduct of L. chuanxiong (reported as 6), as well as the
1
6,17
reporter assay.
However, structure 6 had been previously
18
assigned to one of the hydrogenation products of rac-3.
Therefore, two sets of different physical data were allocated to
the same structure, and one was incorrectly assigned. Analysis
of the spectroscopic data allowed us to hypothesize that we
were observing epimers at C3 of 3,8-dihydrodiligustilide.
Received: August 6, 2019
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.9b02762
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
evaluation of these compounds as progestins. Theoretical
calculations were also performed to explore the interaction
between phthalides and PR.
In the first stage, the hydrogenation of the natural product
rac-3 (reisolated from L. porteri) was achieved, yielding 3,8-
dihydro- and 3,8,7′,7a′-tetrahydro-derivatives, rac-6 and rac-7,
respectively (Scheme 1), which were characterized through
spectroscopic data of compound rac-8 were consistent with
those reported for the L. chuanxiong metabolite (minor
1
3
corrections in C NMR chemical shifts were performed for
C4 and C8, Table 1). Therefore, the correct structure for this
potent natural progestin is one of the enantiomers of rac-8.
The fact that the progestogenic 3,8-dihydrodiligustilide
isolated from L. chuanxiong was found as an enantiomerically
pure compound could be explained through a Diels−Alder
reaction between (Z)-ligustilide (1) as a diene and one
enantiomer of 3,8-dihydroligustilide as a dienophile. Thus, the
absolute configuration of this substance could be deduced as
follows: considering the characterization of the racemic
epimers at C3 of 3,8-dihydrodiligustilides and that only the
a
Scheme 1. Derivatization of Diligustilide (rac-3)
3
S-enantiomer of 3,8-dihydroligustilide has been characterized
as a natural product (senkyunolide A, 9), then the biosynthetic
Diels−Alder reaction for producing the enantiomerically pure
16
adduct (the metabolite of L. chuanxiong) should proceed via
a face-differentiated supra,supra approach between the
C3a′si,C6′si face of diene 1 and the C6si,C7re face of 9
(
(
endo-β approach of the dienophile to the diene) to produce
3′Z,3S,6R,7R,3a′R,6′R)-3,8-dihydrodiligustilide (ent-8 in
Scheme 2).
Thus, ent-8 (not 6) is the structure of the phthalide isolated
from L. chuanxiong. We then verified its activity (and that of
the other phthalides) as progestin.
The T47D cancer cell line viability was determined through
MTT assays. Given that it is a measure of the mitochondrial
metabolic activity, the assay provides information on cell
proliferation. The results showed that rac-6, rac-7, and rac-8
increased cell proliferation after 24 h of treatment, depending
on the concentration (see Figure 2). Of note, rac-6 and rac-7
had a similar effect to that of progesterone (P4). In contrast,
the effect of rac-3 was not significantly different than the
control. The observed effect could be attributed to the
interaction of phthalides with PR because this observation is
consistent with the fact that progestins up-regulate the
a
Reagents: (a) H /Pd−C (rac-6: 66%, rac-7: 12%); (b) DBU/THF
2
(MW or reflux, rac-8: 18%).
19−22
proliferation of T47D cells.
spectroscopic methods. H and 13C NMR assignments of the
obtained compounds (Table 1) were coincident with the
semisynthetic products obtained previously in our group.
The orientation of the hydrogen attached to C3 of rac-6 was
determined by the NOE existing between this hydrogen (δH
1
The translocation of PR from the cytoplasm to the nucleus,
due to the nuclear translocation signal depending on the
hormone, and its retention there, may be a consequence of
exposure to progestins, which induces PR phosphorylation,
18
23,24
particularly at residue Ser 294.
It was observed through
4
.56) and the one bonded to C7 (δ 3.18), indicating that
immunofluorescence microscopy (IMF) that after 20 min of
H
2
94
both atoms should be oriented toward the same side of the
molecule. This observation was confirmed by X-ray crystallog-
raphy, which showed that the butyl substituent pointed to the
inner side of the molecule, and the calculated distance between
hydrogens bound to C3 and C7 is 4.780 Å. The structure of
the tetrahydroderivative (rac-7) was also confirmed by X-ray
crystallography (see the Supporting Information), being
consistent with the former observation. Consequently, the
metabolite from L. chuanxiong could not be 6.
treatment, phosphorylated PR (phospho -PR) is distributed
mainly throughout the cytoplasm for the control. The same
occurs for rac-3 and rac-8 (shown in the Supporting
2
94
Information). On the other hand, phospho -PR was in
perinuclear or nuclear localization after treatment with
compounds rac-6 and rac-7 (see Figure 3). This result is
evidence that this phthalide acts as a progestin, i.e., it is an
agonist of PR, capable of inducing its phosphorylation, which
in turn allows for the translocation of this nuclear receptor
from the cytoplasm to the nucleus after 20 min, where it
remains and activates signaling pathways concerning PR
function.
A substantial difference in the spectroscopic constants was
1
the H NMR chemical shift of the hydrogen bound to C3,
which is δ 4.56 for the semisynthetic product (rac-6) and
δH 4.80 for the natural product isolated from L. chuanxiong.
We envisaged the possibility of epimerizing rac-6 to confirm
that the natural product corresponded to rac-8. This task was
attempted by reacting rac-6 with DBU in refluxing THF for 24
h and for 30 min upon microwave irradiation. By increasing
the temperature, or the reaction length, a retro-Diels−Alder
reaction was observed. As a result, a separable 1:4 mixture of
18
H
16
Furthermore, molecular docking of tested phthalides (both
enantiomers of rac-3, rac-6 and rac-8) on PR supported the
agonist activity of these compounds toward this protein.
Docking calculations between the studied phthalides and PR
(using three different crystallized structures of this protein,
2
5
26
27
4APU, 3ZR7, and 1ZUC ), performed in both AutoDock
28
29
4.2 and AutoDock VINA, showed that all of the tested
phthalides interact with the protein near the ligand binding
1
rac-8 and rac-6 was obtained ( H NMR control). The
B
DOI: 10.1021/acs.orglett.9b02762
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
C
DOI: 10.1021/acs.orglett.9b02762
Org. Lett. XXXX, XXX, XXX−XXX

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Letter
Scheme 2. Regio- and Stereo-Differentiated Biosynthetic
Diels−Alder Reaction (1 + 9) for Producing ent-8
basis of these results, all proven natural and semisynthetic
phthalides could be PR agonists.
Table 2. Calculated Affinity Energies for Interaction
between Phthalides and Different Crystallized Structures of
PR
−
ΔG (kcal/mol)
4
APU
3ZR7
1ZUC
AD4
compd
AD4
VINA
AD4
VINA
VINA
3
6
7
8
ent-3
ent-6
ent-7
ent-8
P4
10.44
10.99
10.61
11.10
10.71
10.97
10.79
11.04
11.14
4.9
4.7
5.1
4.8
4.9
4.7
4.6
4.7
5.3
11.24
10.76
10.98
11.33
10.96
10.88
10.87
10.77
22.48
6.9
6.2
5.8
6.5
6.5
6.2
6.7
7.1
7.6
10.56
10.05
9.88
11.67
9.81
10.43
9.46
10.45
24.27
4.6
4.7
3.9
7.4
5.0
3.7
3.7
4.0
10
As mentioned above, these compounds were located near
the LBD, consistent with the experimental results of the
activity of phthalides as progestins. Of note, the calculated
affinity energies were similar among the tested compounds.
For example, in the case of structure 4APU using AD4, the
energies for all of the compounds are in the range of −10.44
and −11.14 kcal/mol, like P4. With VINA, a similar behavior
was observed, and the range was between −4.7 and −5.3 kcal/
mol, very close to, and similar to, P4. In particular, we found
that compound 6 showed an interaction between the sulfur
(
from residue Met-756) and carbonyl oxygen from the ligand,
as shown in Figure 4 (S−O attractive interactions, though
Figure 2. T47D cell proliferation assays with tested compounds at 24
h. Proliferation increased upon treatment with rac-6, rac-7, and
progesterone at 40, 75, 100, and 150 μM. Results are presented as the
means ± SD (n = 3); ***p < 0.001, **p < 0.002, *p < 0.033
compared with the control.
Figure 4. (A−C) Docking position of 6 with PR. (D) 2D interaction
diagram between 6 and PR.
31
counterintuitive, are common). The correct interaction of
ligands with this residue is relevant to increase agonistic
26
activity, which correlates with the in vitro results for rac-6.
Figure 3. Immunofluorescence microscopy showing subcellular
localization of PR in T47D cells labeled with phospho2 PR (red)
and nucleus stained in blue with 4′,6-diamidino-2-phenylindole
Hence, we demonstrated that, despite the misassignment of
the structure of the compound from L. chuanxiong (ent-8), it
actually acts as a progestin, as do other hydrodiligustilides.
Furthermore, this class of compounds could be a family of
phytoprogestins useful in therapy, different from the natural
94
(
DAPI) for: (a) untreated cells, with PR located in the cytoplasm,
and (b) cells after treatment with test rac-6, pPR is notably located
inside the nucleus. Scale bars: 100 μm.
32,33
flavonoids displaying this activity.
The impact of any side
effects of these phthalides and its optimization remain
unstudied.
In summary, we prepared two bioactive compounds,
correcting the originally reported structure for the natural
25,30
domain (LBD), similar to progesterone and other agonists.
The respective docking scores are shown in Table 2. On the
D
DOI: 10.1021/acs.orglett.9b02762
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
progestin isolated from L. chuanxiong and proposing its
absolute configuration (formula (3′Z,3S,6R,7R,3a′R,6′R)-8).
These compounds increased cell viability in hormonodepend-
ent T47D cancer cells and led to the phosphorylation of PR
and its translocation into the nucleus, according to
experimental and computational studies. These results are
evidence of the progestin-like activity of dimeric phthalides,
such as dihydro- and tetrahydrodiligustilides, which may be
considered as possible candidates for hormone replacement
therapies.
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AUTHOR INFORMATION
Corresponding Author
■
*
(15) Velazquez-Moyado, J. A.; Martínez-Gonzalez, A.; Linares, E.;
́ ́
E-mail: delgado@unam.mx.
ORCID
Bye, R.; Mata, R.; Navarrete, A. Gastroprotective Effect of
Diligustilide Isolated from Roots of Ligusticum porteri Coulter &
Rose (Apiaceae) on Ethanol-Induced Lesions in Rats. J. Ethno-
pharmacol. 2015, 174, 403−409.
Guillermo Delgado: 0000-0002-1394-6300
Present Addresses
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Progestins from Rhizomes of Ligusticum chuanxiong. Phytochemistry
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†(
E.K.P.A.-A., M.A.C.C.) Department of Biology, Facultad de
́
Quimica, Universidad Nacional Auton
Universitaria, Coyoacan 04510, Ciudad de Mex
C.A.M.-C.) Universidad Autonoma Metropolitana, Unidad
Xochimilco, Calzada del Hueso 1100, Ciudad de Mexico
4960, Mexico.
́
oma de Mex
́
ico, Ciudad
2
́
́
ico, Mexico.
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Medica for Chinese Medicine; Carl-Hermann Hempen, T. F., Ed.;
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b978-0-443-10094-9.00015-7.
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o, R. G.; Toscano, R. A.; Bye, R.;
Notes
The authors declare no competing financial interest.
(
ACKNOWLEDGMENTS
This work was taken in part from the PhD thesis of J.L.A. The
authors thank Universidad Nacional Auton
(
■
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(20) Migliaccio, A.; Piccolo, D.; Castoria, G.; Di Domenico, M.;
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Ciencias Quimicas, and Consejo Nacional de Ciencia y
Tecnologia (Scholarship 249731 awarded to J.L.A.). The
authors also thank Maria Isabel Chavez, Angeles Pen
Instituto de Quimica de la UNAM), and Beatriz Quiroz
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