ACS Medicinal Chemistry Letters
Letter
NMR spectra and optical rotation data. Thus, the structure of
5 was elucidated as 3,5,17-triacetoxy-15-hydroxy-14-oxola-
thyra-6Z,12E-diene and was given the trivial name euphlathyr-
inoid E.
The known compounds, Euphorbia Factor L8 (6),23
Euphorbia Factor L11 (7),24 Euphorbia Factor L9 (8),25 5,15-
diacetoxy-3-benzoyloxy-7-hydroxy-14-oxolathyra-6(17),12E-
diene (9),22 15,17-di-O-acetyl-3-O-benzoyl-5,17-dihydroxy-
isolathyol (10),21 15-O-acety-3-O-nicotinoy-jolkinol-5β,6β-
oxide (11),26 Euphorbia Factor L31 (12),27 Euphorbia Factor
L30 (13),27 Euphorbia Factor L3 (14),25 Euphorbia Factor L2
(15),23 and Euphorbia Factor L1 (16),23 were identified by
comparison of their spectroscopic data with those in the
literatures.
Figure 3. RT-qPCR analysis was used to detect the expressions of the
PXR downstream genes (A) CYP3A4, (B) CYP2B6, and (C) MDR1
in HepaRG cells after treatment of RIF (10 μM) and compounds 8,
26, and 30 (10 μM). Values were relative to house-keeping gene β-
ACTIN. Data were presented as mean SD (n = 3). *P < 0.05, **P
< 0.01, ***P < 0.001, ****P < 0.0001, versus the vehicle.
To clarify the SARs of lathyrane diterpenoids related to the
hPXR, we used the major components, 14 and 15, as the
starting materials for the design of various derivatives (Scheme
1). Briefly, the structural modifications were mainly deployed
on the Δ6(17) terminal double bond, α,β-unsaturated ketone,
cyclopropane ring as well as substituents on C-3, C-5, C-7, and
C-15. First, alkaline hydrolysis of 15 and 14 afforded 17 and
27, respectively, to increase their hydrophilicity. Then, the
acylation of the free hydroxyls in 17 with acetic anhydride or 2-
furoyl/2-thiophenecarbonyl/benzoyl chloride yielded corre-
sponding esters 18−22. The partial palladium catalyzed
hydrogenation of 14 at Δ6(17) or Δ12 afforded 30 or 29,
respectively. The complete hydrogenation of 15 via an excess
of palladium generated 26. The oxidation of 15 with meta-
chorobenzoic acid (m-CPBA) gave the epoxide derivatives 24
and 25. Reduction of 14 and 15 with the sodium borohydride
generated 31 [possessing a rare CH(12)−O−C(15) linkage]
and 23, respectively. Finally, the treatment of 14 with sodium
borohydride followed with dilute hydrochloric acid afforded
the cyclopropane-opening products 32, 33, and 34.
The cytotoxicity of 1−34 was initially performed on
HEK293T cells to exclude the cytotoxic compounds 3, 12,
13, and 28 (Figure S2.5). Then, the remaining compounds
were subjected to the hPXR agonistic screening by using a
dual-luciferase reporter gene system constructed in HEK293T
cells via transient transfection with reporter plasmids.17
Rifampicin (RIF), a classical hPXR agonist, was used as the
positive drug. The results showed most of these lathyrane
diterpenoids exhibited potent hPXR agonistic activity at the
concentration of 10 μM (Figure 2A). Among them, 8, 26, and
30 could significantly enhance the hPXR reporter gene activity
by 6.9, 3.4, and 4.9 fold, respectively, being more active than
that of RIF (activation fold = 2.9). Then, the dose−response
assays of 8, 26, 30, and RIF were performed. As shown in
Figure 2B, all of these compounds could dose-dependently
enhance the hPXR reporter gene activity.
endowed with different agonistic activity on hPXR. In the 3-O-
acyls series, the activities were ranked as 3-O-cinnamoyl (4) ≈
3-O-benzoyl (14) > 3-O-nicotinoyl (6). In the C-7 substitutes
series, different moiety contributed to the activity was ranked
as 7-acyloxy > 7-hydroxyl > 7-alkyl (8 and 15 > 9 > 14; 18, 20,
and 21 > 17 > 27), indicating the presence of acyloxy moieties
at C-7 were beneficial to activity. Remarkably, 7-O-nicotinoyl
significantly increased the activity as compared to 7-O-benzoyl
(6.93-fold in 8 vs 2.03-fold in 15). The acylation of OH-15 was
detrimental to activity, as shown by 7 vs 15. In addition, the
presence of 5-O-benzoyl led to a dramatic decrease of the
activity, as shown by 18, 20, and 21 vs 22, suggesting the
unfavorableness of large substituent at C-5. In α,β-unsaturated
ketone group, the hydrogenation or epoxidation of Δ12
generally had little influence on the activity as shown by 29
vs 14 and 25 vs 15, whereas the reduction of 14-carbonyl
decreased the activity (23 vs 7). The hydrogenation of Δ6(17)
dramatically increased the activity as shown by 30 vs 14 and 26
vs 15, whereas epoxidation or migration had little influence
(24 vs 25; 16 vs 14; 10 vs 14). In addition, the cyclopropane
ring-opening seemed indifferent to the activity, as shown by
32, 33, and 34 vs 14. The above-mentioned SARs information
is summarized in Figure 4.
To confirm the hPXR activation effects of 8, 26, and 30, we
further evaluated their regulation on hPXR downstream key
genes that are responsible for BAs metabolism and transport.
HepaRG cells were incubated with 8, 26, and 30, and the
mRNA expressions of CYP3A4, CYP2B6, and MDR1 were
measured by RT-qPCR. The results indicated that 8
remarkably increased the expressions of CYP3A4, CYP2B6,
and MDR1, suggesting that 8 may activate hPXR to promote
BA detoxification (Figure 3).
In general, the hydrolysis products showed a dramatic
decrease in the activity, as shown by 14 vs 27 and 15 vs 17,
indicating that proper lipophilicity is necessary for activity. On
this basis, the different substituents on C-3, C-5, C-7, and C-15
Figure 4. SARs of lathyrane diterpenoids on hPXR agonistic activity.
↑, increased activity; ↓, decreased activity; →, little influence on
activity.
To further explore the potential molecular recognition
mechanism between these lathyrane diterpenoids and hPXR,
we simulated the binding modes of 8 and RIF with hPXR,
respectively, by docking these agonists into the hPXR ligand-
binding domain (LBD, PDB ID: 1SKX) using MOE2014.0901.
As shown in Figure 5, 8 was docked well into the LBD of
1163
ACS Med. Chem. Lett. 2021, 12, 1159−1165