Synthesis and biological activity of cyclopropyl Δ7-dafachronic acids as DAF-12 receptor ligands

Article abstract of DOI:10.1039/d1ob00912e

The four cyclopropyl stereoisomers of Δ7-dafachronic acids were prepared from the bile acid hyodeoxycholic acid and employed as chemical tools to exploit the importance of the orientation and spatial disposition of the carboxyl tail and the C25-methyl gro

Full text of DOI:10.1039/d1ob00912e

Organic &
Biomolecular Chemistry
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Synthesis and biological activity of cyclopropyl
Δ7-dafachronic acids as DAF-12 receptor ligands†
Cite this: Org. Biomol. Chem., 2021,
19, 5403
c
Valentina Mancino,a,b Giada Ceccarelli,a Andrea Carotti, a Laura Goracci,
Roccaldo Sardella,a Daniela Passeri,b Roberto Pellicciarib and Antimo Gioiello
*
a
The four cyclopropyl stereoisomers of Δ7-dafachronic acids were prepared from the bile acid hyodeoxy-
cholic acid and employed as chemical tools to exploit the importance of the orientation and spatial dispo-
sition of the carboxyl tail and the C25-methyl group for the binding at the DAF-12 receptor. The synthesis
route was based on (a) Walden inversion and stereoselective PtO2-hydrogenation to convert the
L-shaped 5β-cholanoid scaffold into the planar 5α-sterol intermediate; (b) two-carbon homologation of
the side chain by Wittig and cyclopropanation reaction; and (c) formation of the 3-keto group and Δ7
double bond. The synthesized isomers were isolated and tested for their activity as DAF-12 ligands by
AlphaScreen assays. Results showed a significant loss of potency and efficacy for all the four stereoi-
somers when compared to the parent endogenous ligand. Computational analysis has evidenced the
configurational and conformational arrangement of both the carboxylic and the C25-methyl group of
dafachronic acids as key structural determinants for DAF-12 binding and activation.
Received 10th May 2021,
Accepted 17th May 2021
DOI: 10.1039/d1ob00912e
rsc.li/obc
are characterized by a planar steroidal body, a double bond at
the C4 or C7 position, a C-3-keto group, a seven-carbon side
Introduction
Dafachronic acids (DAs, 1–4) (Fig. 1A) are C27 acidic steroids chain with a methyl group at the C25 position and a carboxylic
that serve as hormonal ligands for the nuclear hormone recep-
tor DAF-12 (‘DAuer larva formation-12’), a transcription factor
regulating the life cycle fate of nematodes.1 Acting as a mole-
cular switch, the liganded DAF-12 promotes larval reproductive
development, whereas the unliganded receptor directs towards
a long-lived, alternate third larval stage, also called the dauer
diapause, in which the nematode exhibits more stress resis-
tance, an extended larval survival and, interestingly, an increased
life span.2 Remarkably, DAs also activate parasitic DAF-12 ortho-
logues inducing the recovery of the third stage of infective
larvae (iL3) in parasitic nematodes, such as S. stercoralis, and
hookworms.3,4 These important findings have suggested poten-
tial applications of DAF-12 modulators as therapeutic agents for
the treatment of parasitosis as disseminated strongyloidiasis.5
Over the last years, diverse routes for the synthesis of
endogenous DAF-12 ligands 1–4 6,7 have been developed facili-
tating their use as chemical tools to explore the druggability of
the receptor especially in agriculture and veterinary. DAs (1–4)
aDepartment of Pharmaceutical Sciences, University of Perugia, Via del Liceo 1,
06123 Perugia, Italy. E-mail: antimo.gioiello@unipg.it
bTES Pharma S.r.l., Corso Vannucci 47, 06121 Perugia, Italy
cDepartment of Chemistry, Biology and Biotechnology, University of Perugia,
Via dell’Elce di Sotto 8, 06123 Perugia, Italy
Fig. 1 (A) Structure of dafachronic acids (1–4). (B) Structure–activity
relationships of dafachronic acid derivatives as DAF-12 ligands: key
structural determinants and rank order of potency.
†Electronic supplementary information (ESI) available: Copy of NMR spectra
and HPLC analysis of final products. See DOI: 10.1039/d1ob00912e
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tail (Fig. 1A). Efforts in the structural modifications of the DA group and an unsaturated double bond at C-7 or C-4 are
scaffold have been useful to define the structure–activity required for an efficient DAF-12 activation. As mentioned, the
relationship (SAR) of steroidal DAF-12 modulators with the C25(S) diastereoisomers of both Δ4 and Δ7 DAs (1 and 3) are
scope to enable the design and discovery of more effective com- more active than the corresponding R-isomers (2 and 4), indi-
pounds especially in terms of metabolic stability (Fig. 1B).8–12
cating that the configuration of the methyl group affects either
In particular, it was shown that (a) 25(S)-diasteroisomers ligand binding or complex conformation.8 Moreover, it was
are more potent DAF-12 agonists than the corresponding (R)- shown that the lack of the C25-methyl decreased ligand
epimers;8 (b) 5α-steroids (trans A/B-ring junction) are better affinity resulting in a less potent steroid.10b
ligands than the corresponding 5β-analogs;8 (c) the shift of the
Based on these findings, we sought to synthesize 24,25-
double bond to the C5 position and the replacement of the methylen-Δ7-dafachronic acids (5–8) as DAF-12 ligands. The
C20-methyl with a carbonyl group determines a significant frequent appearance of the cyclopropyl ring in bioactive com-
loss of potency;9,10a (d) the side chain shortening has resulted in pounds and drugs is due to the influence that this moiety
partial agonist compounds;10a (e) the introduction of a double plays on the properties of the compounds containing it.13
bond at the C22 position, the insertion of a C24-difluomethylated Indeed, the cyclopropyl ring addresses multiple roadblocks
group, or the replacement of the carboxylic moiety with a hydroxyl that can occur during medicinal chemistry such as enhancing
group yielded derivatives with an antagonistic profile;10,11 (f) sub- potency, reducing off-target effects, increasing metabolic stabi-
stitutions at the C4 position are not allowed.12 It is worth noting lity and permeability, decreasing plasma clearance, altering
that none of the DA derivatives was more potent than the natural pKa, and contributing to an entropically more favorable
ligands 1–4, with the Δ7-(S)-DA (1) being the most active stereoi- binding to the receptor. Moreover, the insertion of a cyclopro-
somer in both in vitro and in vivo appraisals.
pane ring at the α-position of a functional group responsible
Herein, we report the synthesis of 24,25-cyclopropyl DA of key interactions with the receptor ligand binding domain
derivatives 5–8 (Fig. 2) with the aim to evaluate the effect of (LBD) has been an extensively used strategy to obtain con-
the structural constraint and property of the cyclopropyl strained analogues and determine the relative bioactive
moiety in the activation of the DAF-12 receptor. Cyclopropyl conformation.14–16 In our case, the presence of the cyclopropyl
analogues can indeed bind selectively to the target receptor as ring at the C24–C25 position will confer a greater rigidity to
the result of a special arrangement and dedicated interactions, the DA side chain terminus, allowing to get insights into the
superimposed on the bioactive conformation. We also show importance of the configurational and conformational aspects
results from biological screening experiments against the of the carboxyl and C25-methyl group in the interaction with
DAF-12 of S. Stercolaris (ssDAF-12) and provide computational crucial residues of the DAF-12 LBD. This is due to the distinc-
modeling analysis to rationalize the observed DAF-12 activity.
tive geometric, steric and electronic features of the cyclopropyl-
ic nucleus. Moreover, it can be expected to achieve metaboli-
cally stable derivatives upon replacement of the C25 methyl
group with the cyclopropane ring. Indeed, proton abstraction
from a cyclopropane ring would be more difficult from a
methyl group that is needed for oxidative metabolism to occur.
Results and discussion
Design
Since the discovery of DAs 1–4, several semisynthetic DA
Synthesis
derivatives have been reported,8–12 showing that a C-3 keto
In defining the best synthetic approach for the preparation of
the four cyclopropyl diastereoisomers 5–8, the knowledge
acquired with the development of the divergent synthesis of
DAs 1–4 was applied.7 The synthesis pathway started from the
natural bile acid hyodeoxycholic acid (HDCA, 9) and was based
on A/B-ring junction inversion, shift of the OH group from
C6α- to C7β-position, side chain homologation, cyclopropana-
tion reaction and generation of the Δ7 double bond, and C3-
OH oxidation (Fig. 3).
The first stage of the synthesis was focused on modifi-
cations at the HDCA steroid nucleus (Scheme 1). Thus, the
methyl ester of HDCA was treated with tosyl chloride (TsCl)
in freshly distilled pyridine at room temperature to give
the 3α,6α-ditosylated derivative in 94% yield. The methyl
3α,6α-ditosyloxy-5β-cholan-24-oate was refluxed in glacial
AcOH in the presence of potassium acetate (AcOK) to provide
the methyl 3β-acetoxychol-5-en-24-oate through the elimin-
ation of the C6-hydroxy group and the concurrent C3 Walden
inversion.17 Intermediate 11 was formed together with the
Fig. 2 Structure of the four isomers of 24,25-methylen-Δ7-dafachronic
acids (5–8).
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PtO2 to give the 5α-derivative 13 in excellent yield (96%). The
reduction was performed also using Pd/C in THF, but the reac-
tion proceeded considerably slowly. The subsequent stereo-
selective reduction of the C7 carbonyl group with L-selectride®
in dry THF at −78 °C, provided the 7α-hydroxy intermediate,
that was hydrolyzed under alkaline conditions (NaOH/MeOH)
furnishing the acid 15 in 72% yield.
A suspension of 15 and catalytic p-TSA in dioxane at room
temperature was reacted with 3,4-dihydro-2H-pirane to obtain
the 3β,7α-protected acid 16 (>98%) to be converted into the
corresponding aldehyde. As attempts for the direct reduction
were not successful (data not shown), we were forced to first
reduce the acid to alcohol using LiAlH4 in freshly distilled THF
at room temperature (91% yield) and then to oxidize the
alcohol by Swern reagent (90% yield). The following Wittig
reaction with methyltriphenylphoshonium bromide in reflux-
ing THF using t-BuOK as a base afforded the alkene 19 in 92%
yield after chromatography.
Fig. 3 Retrosynthetic approach to cyclopropyl dafachronic acids 5–8.
PG = protecting group.
The cyclopropanation on the C24–C25 double bond was rea-
lized by slow addition of ethyl diazoacetate (EDA) to a solution
of the alkene in dry CH2Cl2 at room temperature using dirho-
dium(II) tetraacetate (Rh2(OAc)4) as the catalyst.18 As expected,
the reaction furnished a mixture composed by the four cyclo-
propyl diastereoisomers with an overall yield of 92%. Finally,
acidic (HCl/MeOH) and alkaline (NaOH/MeOH) hydrolysis pro-
vided isomers 20a and 20b in 6 : 4 ratio as determined by
reverse phase HPLC analysis (Fig. 1S, ESI†). Crude purification
by silica gel flash-chromatography allowed to isolate the two
couples of isomers [Rf1 (20a), Rf2 (20b)], obtained in 45% and
30% isolated yield, respectively. At this stage, the stereo-
chemistry of the two couple of isomers was hypothesized by
NMR analysis, in analogy to previous works.16,19 In cyclopropyl
ursodeoxycholic acid (CUDCA), C-NMR analysis showed that
signals related to the C24 carboxyl group, the C25 methylene
and the C20 were more shielded for cisoid compounds than
their respective transoid analogues (Table 1). The 13C-NMR
spectra of the two isomeric mixtures 20a and 20b revealed a
similar tendency allowing us to speculate that compounds
with the higher TLC retention factor (Rf) were the cisoids,
while the couple of stereoisomers with a lower Rf were the
transoid ones.
Scheme 1 Synthesis of 3β,7α-dihydroxy-24,25-methylen-24-bishomo-
5α-cholan-26-oic acid (20a,b) from hyodeoxycholic acid (9). Reagents
and conditions: (a) p-TSA, MeOH, u.s.; (b) TsCl, dry pyridine; 94% from
9; (c) AcOK, AcOH, reflux, 62%; (d) CrO3, 3,5-dimethylpyrazole, CH2Cl2,
−30 °C, then r.t., 65%; (e) H2, PtO2, 1.5 atm, AcOEt, 96%; (f) L-selectride,
dry THF, −78 °C, then r.t.; (g) NaOH, MeOH, 72% from 13; (h) 3,4-DHP,
p-TSA, dioxane, >98%; (i) LiAlH4, dry THF, 0 °C, then, r.t., 91%; ( j)
(COCl)2, DMSO, Et3N, dry CH2Cl2. −50 °C, 90%; (k) CH3PPh3Br, t-BuOK,
dry THF, r.t., then reflux, 92%; (l) EDA, Rh(OAc)2, dry CH2Cl2; (m) HCl,
MeOH; (n) NaOH, MeOH, 75% from 19.
In the last part of the synthesis, the two couples of isomers
20a and 20b were elaborated separately using the same experi-
mental protocol (Scheme 2). Therefore, the corresponding
methyl chol-3,5-dien-24-oate, whose formation was favored by methyl ester of 20a,b was selectively protected at the C3 posi-
the generation of a conjugated dienic system and by the trans- tion by treating with Ac2O in presence of NaHCO3 in reflux-
diaxial elimination of a water molecule from the C5–C6 posi- ing THF, to afford the desired compounds 21a,b in nearly
tion. The reaction showed a variable yield (21–62%) according quantitative yield (90%). These were treated with SOCl2 in
to the batch size and concentration (data not shown). In par- dry pyridine at room temperature to obtain the methyl-
ticular, the yield resulted generally higher on small scale (up 3β-acetoxy-24,25-methylen-Δ7-DA derivatives 22a and 22b in
to 0.5 g) and in diluted conditions (0.2–0.3 M).
65% and 60% yield, respectively, after purification by flash
Intermediate 11 was next subjected to regioselective allylic chromatography on silica gel. Finally, the alkaline hydrolysis
oxidation by treatment with CrO3 and 3,5-dimethylpyrazole in (NaOH/MeOH) and the treatment of the corresponding acids
dry CH2Cl2 at room temperature to furnish 12 in 65% yield with Jones reagent in acetone furnished the desired cisoid
after flash chromatography. The C5–C6 double bond was 5–6 and transoid products 7–8 as mixtures of isomers, in
reduced by stereoselective hydrogenation in the presence of high yields.
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Table 1 Chemical shifts related to C20, C24 and C25 of 22,23-cyclo-
propyl ursodeoxycholic acids compared with chemical shift of C23, C26
e C27 of intermediates 20a,b
Table 2 DAF-12 activities of 25(S)-Δ7-DA (1) and cyclopropyl con-
strained analogs 5–8 a
DAF-12 activity
Compound
EC50 a (µM)
Efficacyb (%)
25(S)-Δ7-DA (1)
Cisoid (S,R) (5)
Cisoid (R,S) (6)
Transoid (R,R) (7)
Transoid (S,S) (8)
0.011 0.005
2.4 0.6
8.0 1.3
4.0 0.8
11.4 1.5
100
50
41
6
7
a Data represent EC50 mean values SD of at least three independent
experiments. b % of efficacy was calculated at Emax of each curve con-
sidering the Emax of 1 as 100% value.
CUDCA
C20
Intermediates 20a,b
C24
C25
C23
C26
C27
Cisoid
34.8
31.8
39.7
39.4
175.8
176.1
177.9
177.3
16.3
12.0
17.5
12.7
23.04
23.18
24.41
24.61
176.75
13.71
13.98
16.38
15.83
Unexpectedly, results disclosed a significant loss of activity for
all the stereoisomers with derivative 5 being the most potent
with an EC50 of 2.4 μM.
Transoid
178.13
Furthermore, none of the derivatives reached the efficacy of
1. In particular, cisoid compounds 5–6 were partial agonists
while the corresponding transoid cyclopropyl analogs 7 and 8
failed to activate the receptor. Compound 7 was also tested in
antagonist mode using 24-hydroxy-4-cholen-3-one early indi-
cated as micromolar DAF-12 antagonist.10 Data showed that
compound 7 was able to displace the coactivator recruited by
the agonist 1 suggesting an antagonist effect on the DAF-12
receptor. In particular, the dose response experiment using
derivative 7 resulted an IC50 value of 25 μM while in the same
experiment the IC50 value of 24-hydroxy-4-cholen-3-one was
3 μM.
In order to rationalize compound potency, molecular
docking studies were carried out to evaluate the binding mode
of 5–8 in the ssDAF-12 LBD (Fig. 4). In all cases, the steroid
nucleus maintained the same arrangement within the receptor
pocket, while the side chain showed a different arrangement.
Indeed, although the carboxyl still formed H-bonds with the
R599 and T613 residues, the different folding of the carbon
chain caused a variation in the length and distance of these
interactions.
Scheme 2 Synthesis of 24,25-metilen-Δ7-DA (5–8) from intermediates
20a,b. Reagents and conditions: (a) p-TSA, MeOH, u.s.; (b) Ac2O,
NaHCO3, THF, 91% from 20a,b; (c) SOCl2, pyridine, 65% (22a) 60% (22b);
(d) NaOH, MeOH, quantitative yield. (e) (i) Jones regent, acetone, 0 °C to
r.t., 85%. (ii) HPLC purification.
The binding modes analysis highlighted that derivative 8
was the only one of the series that lost the H-bond interaction
with T613. However, the docking score of 8 did not reflect this
difference showing a value (−11.59) similar to the ones
recorded for the other isomers (−11.38, −11.39 and −11.27 for
5, 6 and 7, respectively). Trying to understand the causes for
the loss of activity observed for the cyclopropyl 5–8 with
respect to 1, a conformational analysis was performed to ident-
ify the global minima energy of each investigated compound.
This value was instrumental to understand the energy spent by
the ligand to assume the putative bioactive conformation
inside the DAF-12 binding site. Interestingly, the bioactive con-
formation of 1 corresponded to its minima energy confor-
mation, while all the cyclopropyl constrained DAs resulted
In spite of their diastereomeric nature, compounds 5–8
failed to be separated on a conventional octadecylsilica-based
achiral stationary phase, although it should in principle be
conceptually possible. Therefore, compounds 5–8 were isolated
using research-type non commercial Cinchona alkaloyd-based
anion exchanger type chiral stationary phase.20 The chemical
integrity of the four isolated diastereoisomers was checked via
HPLC, and the relative stereochemical assignment was deter-
mined as we previously reported.20
In vitro DAF-12 activity and molecular modeling
Pure cyclopropyl isomers 5–8 were tested along with 25(S)-Δ7- with a less favorable energy profile. Indeed, compound 8 is
DA (1) as reference control for their ability to bind and activate 12.51 kJ mol−1 far from its energy minima, while the rest of
DAF-12 receptor using AlphaScreen assays (Table 2). the ligands gave slightly better results (7.82, 10.34 and
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Fig. 4 (A) Binding mode of Δ7-(S)-DA (1) (yellow ball and sticks)
observed in docking analysis using the 3GYU pdb code. (B–E) Docking
results of 5–8 in the ssDAF-12 LBD: 24(S),25(R)-methylen-Δ7-dafachro-
nic acid (5) (B); 24(R),25(S)-methylen-Δ7-dafacronic acid (6) (C); 24(R),25
(R)-methylen-dafachronic acid (7) (D); 24(S),25(S)-methylen-Δ7-dafacro-
nic acid (8) (E).
Fig. 5 Visualization of the CRY GRID MIF calculated in 3GYU pdb code
at −1 kcal mol−1 and the docked poses from Fig. 3. (A) Δ7-(R)-DA (2); (B)
Δ7-(S)-DA (1); (C) 24(S),25(R)-methylen-Δ7-dafachronic acid (5); (D) 24
(R),25(S)-methylen-Δ7-dafacronic acid (6); (E) 24(R),25(R)-methylen-
dafachronic acid (7); (F) 24(S),25(S)-methylen-Δ7-dafacronic acid (8).
9.66 kJ mol−1 for 5, 6 and 7, respectively) but still quite far
from the most favored conformational energy value. It is worth
noting that the trend of the conformational energy value calcu-
lated reflects the experimental DAF-12 activity observed. In
conclusion, in silico analyses highlighted that the cyclopropyl
constraint still allowed to keep the H-bond interactions with
Q637, R599 and T613, but all of them spend a high confor-
mational energy cost that may explain the observed drop in
activity.
A further analysis was performed to investigate the possible
role of the methyl group in 1 and 2 interacting with ssDAF-12
(Fig. 5). We hypothesized that its replacement with the cyclo-
propyl group could be also partially responsible of the
decreased activity. To this aim, the hydrophobic regions in the
protein cavities were evaluated in terms of GRID Molecular
Interaction Fields (MIFs)21 using the CRY probe, which simu-
lates hydrophobic interactions, including entropic energy
terms.22 The CRY MIFs were visualized in the docking results
(Fig. 5C–F). In the past, the effect of the replacement of a
hydrogen atom with a methyl group in protein binding was
deeply studied by performing literature analysis in more than
2000 cases.23 As a result, Leung and co-workers noticed that
an activity boost of a factor of ≥10 occurred with an 8% fre-
quency, while a 100-fold boost occurred as a 1 in 200 event.
Therefore, in our study a cooperative effect including the con-
formational constraint and the lack of the methyl group might
be responsible for the lower activity.
Conclusions
(Fig. 5). This study highlighted a hydrophobic region gener- The high incidence of nematode parasitism in developing
ated by V615, L454 and I561, which is almost overlapped with countries and the associated problems make the availability of
the area including R599 and T613 residues, devoted to estab- adequate anthelmintic treatments necessary. Unfortunately,
lish the H-bonds with the carboxyl moiety in the tested the available treatments are not fully effective because of the
ligands. According to our docking results, both stereoisomers onset of resistance mechanisms and the high criticality of dis-
1 and 2 are able to orient the methyl group into the CRY MIFs seminated strongyloidiasis. The implication of the DAF-12
(Fig. 5A and B). On the contrary, the introduction of the cyclo- receptor in the regulation of parasitic life cycles and infective
propyl moiety in 5–8 hampers this hydrophobic interaction stages brought to light the opportunity to develop new
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approaches for the treatment of parasitic diseases by using equipped with a CBM-20A communication bus module, two
DAF-12 ligands. LC-20AD dual piston pumps, a SPD-M20A photodiode array
The scope of this work was to get further insights into the detector and a Rheodyne 7725i injector with a 20 μL stainless
role of the DA side chain terminal, and in particular, of the steel loop. A Lux Amylose-2 RP18 column (Phenomenex®,
C26-carboxyl and C25-methyl group in the activation of the Torrance, CA, USA) (250 mm × 4.6 mm I.D.) was used as the
DAF-12 receptor. To this aim, we have synthesized the four analytical column. The column temperature was controlled
stereoisomers of DA 5–8 characterized by the presence of the through a Grace heather/chiller thermostat. Purity of syn-
cyclopropyl ring at the α-position of the carboxylic acid. While thesized compounds (>95%) was assessed by HPLC-HRMS.20
this modification imposes a certain rigidity influencing com- Spectroscopic characterization of intermediates 12 and 15 are
pound binding at the receptor, the presence of the cyclopro- consistent with previously reported spectral data.24,25
pane can provide metabolically stable DA derivatives and
improve physico-chemical properties as water solubility.
Synthesis
The synthesis of the four stereoisomers 5–8 was realized
from the bile acid hyodeoxycholic acid. Pure compounds 5–8
Synthesis of Δ7-dafachronic acids (5–8)
were prepared in 19 steps and evaluated for their ability to
Methyl 3β-acetoxy-chol-5-en-24-oate (11). Intermediate 11 was
modulate the ssDAF-12 receptor, the most important parasitic prepared from 40 g HDCA (9) in refluxing AcOH following our
receptor orthologue. Our results indicated that constraints at previous reported protocol.17 Isolated yield from HDCA (9):
1
the side chain are detrimental for the potency of DAs likely 56%. Rf (petroleum ether/AcOEt 98 : 2): 0.30. H-NMR (CDCl3,
due to the high conformational energy cost spent by the 200 MHz) δ: 0.66 (3H, s, 18-CH3), 0.90 (3H, d, J = 6.4 Hz,
ligand to assume the putative bioactive conformation within 21-CH3), 0.99 (3H, s, 19-CH3), 2.01 (3H, s, OCOCH3), 3.64 (3H,
the ssDAF-12 LBD. In addition, the lack of a methyl group s, COOCH3), 4.57–4.61 (1H, m, 3-CH), 5.35 (1H, s, 6-CH).
pointing towards the hydrophobic region defined by V615, 13C-NMR (CDCl3, 100.6 MHz) δ: 11.81, 18.26, 19.27, 20.95,
L545 and I561 could also play a role in making the binding 21.42, 24.20, 27.70, 28.06, 30.96 (2×), 31.78 (2×), 35.32, 36.51,
less effective. Taken together, we can summarize that although 36.92, 38.04, 39.61, 42.30, 49.90, 51.48, 55.68, 56.57, 73.94,
the cyclopropyl-constrained DAs 5–8 are less active compared 122.57, 139.57, 170.58, 174.78.
with the endogenous agonist 1, we demonstrated the funda-
Methyl 3α-acetoxy-7-keto-chol-5-en-24-oate (12). To a suspen-
mental role of the DA side chain in the activation of the recep- sion of CrO3 (87.5 g, 0.88 mol) in dry CH2Cl2 (340 mL) at
tor demonstrating the importance of the configurational and −40 °C, dry 3,5-dimethylpyrazole was added (84.0 g, 0.88 mol)
conformational asset of both the carboxyl terminal and methyl and the mixture was stirred at −30 °C for 30 minutes.
group at the C25 position. Our study paves the way for the Intermediate 11 (20.9 g, 48.6 mmol) was added and the
design of novel side chain-modified DA derivatives and fur- mixture was allowed to warm to room temperature and reacted
nishes a synthesis route to prepare useful tools to explore for 2 d. The reaction mixture was filtered through a Celite pad,
specific binding to DAF-12 receptor subtypes, especially to washing the filter with CH2Cl2, and evaporated to dryness. The
those lacking crystal structures.
crude was purified by flash chromatography using petroleum
ether/AcOEt as eluent to furnish the desired product 12 as a
white solid (14.1 g, 31.8 mmol, 65%). Rf (petroleum ether/
AcOEt 85 : 15): 0.25. H-NMR (CDCl3, 400 MHz) δ: 0.68 (3H, s,
18-CH3), 1.86 (3H, d, J = 6.3 Hz, 21-CH3), 1.20 (3H, s, 19-CH3),
2.05 (3H, s, OCOCH3), 2.20–2.25 (1H, m, 8-CH), 3.66 (3H, s,
1
Experimental
General methods
All the reagents were of analytical grade. All reactions were COOCH3), 4.67–4.74 (1H, m, 3-CH), 5.70 (1H, s, 6-CH).
monitored by TLC, performed on aluminum backed silica 13C-NMR (CDCl3, 100.6 MHz) δ: 11.94, 17.21, 18.42, 21.10,
plates 60-F254 (Merck). Spots on TLC were visualized by using 21.22, 26.21, 27.30, 28.35, 30.98 (2×), 35.22, 35.95, 37.69, 38.26,
UV lamp (254 nm) and by staining and warming with phos- 38.56, 43.12, 45.33, 49.70, 49.87, 51.46, 54.41, 72.15, 126.65,
phomolybdate reagent (5% solution in EtOH). The purifi- 163.84, 170.26, 174.63, 201.77.
cations by flash chromatography were performed using silica
Methyl 3β-acetoxy-7-keto-5α-cholan-24-oate (13). To a solution
gel F60 (Merck) (0.040–0.063 mm) or by Combi-flash using pre- of 12 (13.4 g, 30.1 mmol) in AcOEt (280 mL), PtO2 was added
packed columns. 1H NMR spectra were recorded at 200 or (0.68 g, 3.0 mmol) and the resulting mixture was hydrogenated
400 MHz, 13C NMR spectra were recorded at 100.6 MHz using at 1.5 atm at room temperature for 16 h. The reaction product
the solvents indicated below. Chemical shifts are reported in 13 was obtained by filtration through a Celite pad and evapor-
ppm (parts per million). The abbreviations used are as follows: ation of the solvent (12.8 g, 28.8 mmol, 96%). Rf (petroleum
s, singlet; d, doublet; dd, double doublet; ddd, double double ether/AcOEt 8 : 2): 0.31. 1H-NMR (CDCl3, 400 MHz) δ: 0.64 (3H,
doublet, dddd, double double double doublet, t, triplet, dt, s, 18-CH3), 0.91 (3H, d, J = 6.3 Hz, 21-CH3), 1.10 (3H, s,
double triplet, qt, quartet triplet, bs, broad signal. Melting 19-CH3), 2.02 (3H, s, OCOCH3), 2.15–2.25 (1H, m, 8-CH), 2.34
points were determined with an electrothermal apparatus and (2H, t, J = 6.3 Hz, 6-CH2), 3.65 (3H, s, COOCH3), 4.60–4.70 (1H,
are uncorrected. The preparative and analytical HPLC m, 3-CH). 13C-NMR (CDCl3, 100.6 MHz) δ: 11.67, 12.03, 18.33,
measurements were made on a Shimadzu LC-20A Prominence 21.30, 21.73, 24.90, 27.05, 28.21, 30.94, 31.01, 33.78, 35.18,
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35.78, 35.90, 38.61, 42.53, 45.81, 46.43, 48.81, 49.88, 51.46, (20 mL) and brine (20 mL), dried over anhydrous Na2SO4 and
54.66, 54.89, 72.72, 170.46, 174.64, 211.41.
Methyl 3β-acetoxy-7α-hydroxy-5α-cholan-24-oate (14). To
evaporated to dryness, providing the desired product 16 as a
yellow oil (2.15 g, 3.83 mmol, quantitative). Rf (petroleum
a
solution of the 7-keto derivative 13 (12.8 g, 28.8 mmol) in ether/AcOEt 8 : 2): 0.12. 1H-NMR (CDCl3, 400 MHz) δ: 0.62 (3H,
freshly distilled THF (200 mL) at −78 °C, L-selectride® (43 mL, s, 18-CH3), 0.78–0.79 (3H, m, 19-CH3), 0.95 (3H, d, J = 6.2 Hz,
43.0 mmol) was added dropwise in 30 min and the solution 21-CH3), 2.20–2.27 (2H, m, 23-CH2), 2.30–2.40 (2H, m, 23-CH2),
thus obtained was stirred at −78 °C for 50 min. Then, NaHCO3 3.39–3.57 (2H, m, OCH2-THP), 3.58–3.65 (1H, m, 3-CH), 3.70
saturated solution (130 mL), 30% H2O2 (215 mL) and AcOEt (1H, bs, 7-CH), 3.85–3.93 (2H, m, OCH2-THP), 4.57–4.60 (1H,
(280 mL) were sequentially added dropwise. The mixture was m, OCHO-THP), 4.69–4.71 (1H, m, OCHO-THP).
stirred for 30 min at room temperature and the aqueous
3β,7α-Ditetrahydropyranyloxy-5α-cholan-24-ol (17). To a sus-
phase was extracted with AcOEt (2 × 300 mL). The combined pension of LiAlH4 (1.74 g, 7.66 mmol) in dry THF (20 mL) at
organic layers were washed with brine (200 mL), dried over 0 °C, a solution of the acid 16 (2.15 g, 3.83 mmol) in dry THF
anhydrous Na2SO4 and evaporated to dryness, to give the (30 mL) was added dropwise. The solution obtained was
desired product 14 (14.1 g, 31.5 mmol) as a white solid, that stirred at room temperature for 18 hours, then AcOEt (15 mL)
was used for the next step without further purifications. and MeOH (800 mL) were cautiously sequentially added drop-
Rf (petroleum ether/AcOEt 7 : 3): 0.26. 1H-NMR (CDCl3, wise and the mixture was poured into H2O (150 mL) and
400 MHz) δ: 0.64 (3H, s 18-CH3), 0.80 (3H, s, 19-CH3), 0.90 extracted with AcOEt (5 × 60 mL). The organic phase was
(3H, d, J = 6.4 Hz, 21-CH3), 2.00 (3H, s, OCOCH3), 2.15–2.25 washed with brine (150 mL), dried over anhydrous Na2SO4 and
(1H, m, 23-CH2), 2.30–2.40 (1H, m, 23-CH2), 3.65 (3H, s, evaporated under reduced pressure to obtain the desired
COOCH3), 3.81 (1H, bs, 7-CH), 4.66–4.72 (1H, m, 3-CH). alcohol derivative 17 as a yellowish oil (1.91 g, 3.50 mmol,
13C-NMR (CDCl3, 100.6 MHz) δ: 11.08, 11.77, 18.18, 20.87, yield: 91%), used for the next step without further purifi-
21.38, 23.53, 27.32, 28.01, 30.95 (2×), 33.52, 35.30, 35.48, cations. Rf (petroleum ether/AcOEt 8 : 2): 0.15. 1H-NMR
36.30, 36.42, 36.84, 39.35, 39.45, 42.60, 45.61, 50.42, 51.43, (CDCl3, 400 MHz) δ: 0.59 (3H, s, 18-CH3), 0.75–0.76 (3H, m,
55.71, 67.68, 73.52, 170.53, 174.67.
19-CH3), 0.89 (3H, d, J = 4.8 Hz), 3.45–3.51 (2H, m, OCH2-THP),
3β,7α-Dihydroxy-5α-cholan-24-oic acid (15). A solution of ester 3.54–3.55 (1H, m, 3-CH), 3.67 (1H, bs, 7-CH), 3.82–3.88 (2H, m,
14 (14.1 g, 31.5 mmol) in 5% NaOH in MeOH (125 mL) was OCH2-THP), 4.54–4.56 (1H, m, OCHO-THP), 4.68–4.70 (1H, m,
stirred at room temperature overnight. The solvent was evapor- OCHO-THP).
ated under reduced pressure, the resulted residue was dis-
3β,7α-Ditetrahydropyranyloxy-5α-cholan-24-al (18). To a solu-
solved in H2O (200 mL) and washed with Et2O (3 × 200 mL). tion of (COCl)2 (0.40 mL, 4.55 mmol) in CH2Cl2 (5 mL) at
The aqueous phase was acidified to pH = 4 with 3 N HCl and −50 °C, DMSO (0.57 mL, 8.75 mmol) was added dropwise in
extracted with Et2O (2 × 200 mL). The combined organic layers 10 min. The mixture was then stirred for 10 min at −50 °C,
were washed with brine (200 mL), dried over anhydrous prior adding dropwise a solution of the alcohol 17 (1.91 g,
Na2SO4 and evaporated under reduced pressure to obtain 15 as 3.50 mmol) in CH2Cl2 (10 mL). The mixture was stirred for 2 h
a white solid (8.9 g, 22.8 mmol, 72% from 13). Rf (CHCl3/ at −50 °C. Et3N (2.43 mL, 17.49 mmol) was added and the
MeOH 95 : 5): 0.27. 1H-NMR (CDCl3, 400 MHz) δ: 0.54 (3H, reaction was allowed to warm to room temperature in 4 h. The
s 18-CH3), 0.69 (3H, s, 19-CH3), 0.81 (3H, d, J = 6.4 Hz, 21-CH3), mixture was poured into 2 N aqueous KOH (20 mL) and
2.00–2.10 (1H, m, 23-CH2), 2.13–2.17 (1H, m, 23-CH2), extracted with CH2Cl2 (3 × 20 mL). The combined organic
3.48–3.43 (1H, m, 3-CH), 3.68 (1H, bs, 7-CH), 3.80–3.90 (2H, s, layers were washed with H2O (50 mL) and brine (50 mL), dried
3,7-OH). 13C-NMR (CDCl3, 100.6 MHz) δ: 14.97, 15.59, 22.01, over anhydrous Na2SO4 and evaporated to dryness, to provide
24.82, 27.35, 34.76, 34.83, 34.88, 39.25, 39.36, 40.25, 40.60, 1.72 g (3.16 mmol, 90%) of 3β,7α-ditetrahydropyranyloxy-
40.86, 41.13, 43.33, 43.39, 46.47, 49.60, 54.29, 59.67, 71.55, 5α-cholan-24-al (18) that was used for the next step without
74.64, 180.98.
further purifications. Rf (petroleum ether/AcOEt 8 : 2): 0.39.
3β,7α-Ditetrahydropyranyloxy-5α-cholan-24-oic acid (16). To a 1H-NMR (CDCl3, 400 MHz) δ: 0.63 (3H, s, 18-CH3), 0.79–0.80
suspension of the acid 15 (1.5 g, 3.83 mmol) and p-toluensul- (3H, m, 19-CH3), 0.91 (3H, d, J = 6.3, 21-CH3), 2.30–2.38 (2H,
fonic acid (0.11 g, 0.57 mmol) in 1,4-dioxane (40 mL), 3,4- m, 23-CH2), 2.41–2.48 (2H, m, 23-CH2), 3.46–3.50 (2H, m,
dihydro-2H-pyran (3.5 mL, 38.3 mmol) was added dropwise in OCH2-THP), 3.57–3.67 (1H, m, 3-CH), 3.71 (1H, bs, 7-CH),
10 min and the mixture was stirred at room temperature over- 3.85–3.93 (2H, m, OCH2-THP), 4.57–4.85 (1H, m, OCHO-THP),
night. The mixture was poured into H2O (80 mL) and extracted 4.71–4.74 (1H, m, OCHO-THP), 9.76 (1H, s, CHO).
with AcOEt (5 × 50 mL). The organic phase was evaporated to
dryness. The thick yellow oil was dissolved in MeOH (25 mL),
3β,7α-Ditetrahydropyranyloxy-24-homo-5α-chol-24-ene (19). To
suspension of methyltriphenylphosphonium bromide
a
treated with 10% aqueous NaOH (10 mL) and refluxed for 2 h. (4.11 g, 4.31 mmol) in freshly distilled THF (30 mL), 1 M
The solvent was evaporated under reduced pressure and the t-BuOK solution in THF (10.47 mL, 10.47 mmol) was added
resulting residue was dissolved in H2O (50 mL) and extracted and the yellow mixture obtained was stirred overnight. A solu-
with Et2O (3 × 50 mL). The aqueous phase was acidified to tion of the aldehyde 18 (1.9 g, 3.46 mmol) in dry THF (30 mL)
pH = 5 with 1 N HCl and quickly extracted with AcOEt (3 × was added to the suspension, which turned to orange, and the
50 mL). The combined organic layers were washed with H2O mixture obtained was refluxed for 3 h. The mixture was
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allowed to cool to room temperature, diluted with n-hexane reduced pressure, the residue was dissolved in H2O (50 mL)
(100 mL) and H2O/MeOH 1 : 1 (150 mL), and extracted with and extracted with CHCl3 (2 × 50 mL). The combined organic
EtOAc (3 × 100 mL). The combined organic layers were washed layers were washed with saturated NaHCO3 (50 mL), H2O
with brine (100 mL), dried over anhydrous Na2SO4 and evapor- (50 mL) and brine (50 mL), dried over anhydrous Na2SO4 and
ated to dryness. The crude was purified by silica gel chromato- evaporated to dryness, to provide the desired methyl ester as a
graphy using petroleum ether/EtOAc (from only petroleum light yellow solid. To a solution of the ester in dry THF (8 mL),
ether to petroleum ether/EtOAc 6 : 4) as eluent. Intermediate NaHCO3 (360 mg, 4.32 mmol) and Ac2O (0.41 mL, 4.32 mmol)
19 was isolated as a white solid (1.73 g, 3.18 mmol, 92%). Rf were added and the mixture was refluxed overnight under mag-
1
(petroleum ether/AcOEt 8 : 2): 0.48. H-NMR (CDCl3, 400 MHz) netic stirring. The mixture was concentrated in vacuo, the
δ: 0.61 (3H, s, 18-CH3), 0.78–0.79 (3H, m, 19-CH3), 0.90 (3H, d, residue was dissolved in H2O (30 mL) and acidified to pH = 4
J = 6.4, 21-CH3), 3.46–3.48 (2H, m, OCH2-THP), 3.58–3.66 (1H, with 3 N HCl and extracted with CH2Cl2 (3 × 30 mL). The com-
m, 3-CH), 3.70 (1H, bs, 7-CH), 3.83–3.92 (2H, m, OCH2-THP), bined organic layers were washed with H2O (30 mL) and brine
4.55–4.58 (1H, m, OCHO-THP), 4.70–4.72 (1H, m, OCHO-THP), (30 mL), dried over anhydrous Na2SO4 and evaporated under
4.95 (1H, d, J = 1.32 Hz, 25-CH), 4.97 (1H, d, J = 1.33 Hz, reduced pressure, obtaining the desired product 21a as a yel-
25-CH), 5.73–5.83 (1H, m, 24-CH).
lowish solid (106 mg, 0.22 mmol, yield 91%). Rf (petroleum
Methyl 3β,7α-dihydroxy-24,25-methylen-24-bishomo-5α-cholan- ether/EtOAc 8 : 2): 0.21. 1H-NMR (CDCl3, 200 MHz) δ: 0.62–0.63
26-oate (20a,b). To a suspension of the intermediate 19 (1.5 g, (3H, m, 18-CH3), 0.80 (3H, s, 19-CH3), 0.87 (3H, d, J = 6.13,
2.75 mmol) and Rh2(OAc)4 (49 mg, 0.11 mmol) in dry CH2Cl2 21-CH3), 2.01 (3H, s, OCOCH3), 3.65 (3H, s, COOCH3), 3.80
(40 mL), ethyl diazoacetate (EDA) (1.68 mL, 15.99 mmol) in dry (1H, bs, 7-CH), 4.60–4.70 (1H, m, 3-CH). 13C-NMR (CDCl3,
CH2Cl2 (20 mL) was added using a syringe pump, with a rate 100.6 MHz) δ: 11.08, 11.73, 13.35, 17.81, 18.06, 18.71, 20.56,
of 1.5 mL min−1. The mixture was reacted for 20 h, filtered 21.40, 22.26, 22.48, 23.56, 27.31, 28.08, 33.51, 35.47, 35.60,
over a Celite pad and evaporated to dryness. The crude was 35.80, 36.22, 36.40, 36.83, 39.38, 42.54, 45.61, 50.41, 51.50,
treated for 3 h with a 5% HCl solution in MeOH (30 mL). The 55.83, 67.74, 73.54, 170.57, 173.48.
solvent was removed under reduced pressure, then H2O
Methyl
3β-acetoxy-7α-hydroxy-24,25-methylen-24-bishomo-
(100 mL) was added and the residue was extracted with CHCl3 5α-cholan-26-oate (21b). Compound 21b was obtained follow-
(3 × 70 mL). The combined organic layers were washed with ing the same procedure described for 21a, starting from the
H2O (100 mL) and brine (100 mL), dried over anhydrous acid 20b (137 mg, 0.31 mmol) in 90% yield (138 mg,
Na2SO4 and evaporated to dryness. The crude was stirred with 0.28 mmol). Rf (petroleum ether/EtOAc 8 : 2): 0.19. 1H-NMR
a 5% NaOH solution in refluxing MeOH (15 mL) overnight. (CDCl3, 400 MHz) δ: 0.64–0.65 (3H, m, 18-CH3), 0.81 (3H, s,
The reaction mixture was concentrated in vacuo, diluted with 19-CH3), 0.88 (3H, d, J = 6.4, 21-CH3), 2.01 (3H, s, OCOCH3),
H2O (100 mL) and washed with Et2O (50 mL). The aqueous 3.65 (3H, s, COOCH3), 3.82 (1H, bs, 7-CH), 4.65–4.75 (1H, m,
phase was acidified to pH = 4 with 3 N HCl and extracted with 3-CH). 13C-NMR (CDCl3, 100.6 MHz) δ: 11.11, 11.78, 15.39,
EtOAc (5 × 100 mL). The combined organic layers were washed 15.87, 18.56, 19.83, 20.23, 20.91, 21.40, 23.39, 23.58, 27.36,
with H2O (100 mL) and brine (100 mL), dried over Na2SO4 and 28.12, 29.52, 33.56, 35.16, 35.34, 35.52, 36.38, 36.90, 39.44,
evaporated to dryness. The crude isomeric mixture was puri- 42.61, 45.69, 50.49, 51.56, 55.88, 67.78, 73.55, 170.55, 175.01.
fied by silica gel flash chromatography to obtain pure 20a
Methyl 3β-acetoxy-24,25-methylen-24-bishomo-5α-chol-7-en-26-
(400 mg) and 20b (267 mg). 20a: Rf (CH2Cl2/MeOH 9 : 1): 0.32. oate (22a). To a solution of 21a (106 mg, 0.22 mmol) in freshly
1H-NMR (CDCl3, 400 MHz) δ: 0.66 (3H, s, 18-CH3), 0.81 (3H, s, distilled pyridine (4 mL) cooled at 0 °C, dry SOCl2 (0.05 mL,
19-CH3), 0.90 (3H, d, J = 6.4, 21-CH3), 3.61–3.67 (1H, m, 3-CH), 0.66 mmol) was added, and the mixture was stirred at room
3.83 (1H, bs, 7-CH). 13C-NMR (CDCl3, 100.6 MHz) δ: 11.65, temperature for 1 h. The mixture was poured into H2O (15 mL)
12.23, 13.71, 13.98, 18.98, 19.35, 22.13, 23.04, 23.18, 24.72, and extracted with CH2Cl2 (3 × 20 mL). The organic phase was
28.06, 31.29, 35.53 (2×), 36.27, 36.71, 37.04, 37.59, 38.56, 39.53, sequentially washed with a saturated solution of NaHCO3
42.63, 42.62, 45.84, 49.63, 49.84, 50.50, 55.85, 67.97, 71.11, (20 mL), 3 N HCl (2 × 20 mL) and brine (20 mL), dried over
176.75. 20b: Rf (CH2Cl2/MeOH 9 : 1): 0.30. 1H-NMR (CDCl3, anhydrous Na2SO4 and evaporated to dryness, to obtain a
400 MHz) δ: 0.70 (3H, s, 18-CH3), 0.84 (3H, s, 19-CH3), 0.95 thick yellow oil. The crude was purified by flash chromato-
(3H, d, J = 6.4, 21-CH3), 3.51–3.57 (1H, m, 3-CH), 3.77 (1H, bs, graphy eluting with petroleum ether/AcOEt (from only pet-
7-CH). 13C-NMR (MeOD + DMSO, 100.6 MHz) δ: 11.81, 12.41, roleum ether to petroleum ether/EtOAc 7 : 3) to yield 22a as a
15.83, 16.38, 19.29, 20.83, 21.29, 22.19, 24.51, 29.34, 30.64, pure white solid (66 mg, 0.14 mmol, 65%). Rf (petroleum
32.26, 36.55, 36.72, 38.07, 38.26, 38.74, 40.18, 40.39, 41.01, ether/EtOAc 8 : 2): 0.23. 1H-NMR (CDCl3, 400 MHz) δ: 0.51–0.52
43.70, 47.09, 48.43, 49.70, 51.73, 57.51, 68.48, 71.82, 178.13.
(3H, m, 18-CH3), 0.80 (3H, s, 19-CH3), 0.90 (3H, d, J = 6.3 Hz,
Methyl 3β-acetoxy-7α-hydroxy-24,25-methylen-24-bishomo- 21-CH3), 2.02 (3H, s, OCOCH3), 3.67 (3H, s, COOCH3),
5α-cholan-26-oate (21a). To a solution of the acid 20a (107 mg, 4.66–4.72 (1H, m, 3-CH), 5.14 (1H, bs, 7-CH). 13C-NMR (CDCl3,
0.25 mmol) in MeOH (20 mL), para-toluenesulfonic acid 100.6 MHz) δ: 11.78, 12.88, 13.45, 17.84, 18.10, 18.86, 21.41
(20 mg, 0.10 mmol) was added and the solution was treated (2×), 22.38, 22.90, 23.71, 27.45, 27.80, 29.49, 33.77, 34.15,
with ultrasound at room temperature for 90 min, then heated 35.85, 36.79, 39.40, 40.01, 43.31, 49.21, 51.47, 54.92, 55.87,
to reflux for 39 hours. The solvent was evaporated under 73.43, 117.27, 139.46, 170.63, 173.47.
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Methyl 3β-acetoxy-24,25-methylen-24-bishomo-5α-chol-7-en-26- C27H40O3 [M − H]−: calcd 412.29762, found: 412.29385. 7–8:
oate (22b). Compound 22b was obtained following the same 26 mg (0.06 mmol). Rf: 0.41 (CHCl3/MeOH, 95 : 5). 1H-NMR
procedure described for 22a, starting from the alcohol 21b (CDCl3, 400 MHz) δ: 0.56 (3H, s, 18-CH3), 0.91 (3H, d, J = 6.51,
(138 mg, 0.28 mmol) in 60% yield. Rf (petroleum ether/EtOAc 21-CH3), 1.02 (3H, s, 19-CH3), 5.19 (1H, bs, 7-CH). 13C-NMR
8 : 2): 0.20. 1H-NMR (CDCl3, 400 MHz) δ: 0.51–0.52 (3H, m, (CDCl3, 100.6 MHz) δ: 11.85, 12.42, 16.62, 18.74, 19.86, 21.65,
18-CH3), 0.80 (3H, s, 19-CH3), 0.89 (3H, d, J = 6.4 Hz, 21-CH3), 22.89, 24.43, 27.87, 29.67, 30.03, 34.36, 35.11, 35.70, 35.78,
2.02 (3H, s, OCOCH3), 3.65 (3H, s, COOCH3), 4.66–4.71 (1H, m, 38.07, 38.73, 39.38, 42.82, 43.35, 44.23, 48.84, 54.90, 55.85,
3-CH), 5.14 (1H, bs, 7-CH). 13C-NMR (CDCl3, 100.6 MHz) δ: 117.00, 139.48, 180.59, 212.02. HRMS (ESI): C27H40O3
11.79, 12.89, 15.60, 18.74, 19.81, 20.21, 21.41 (2×), 22.87, 23.39, [M − H]−: calcd 412.29762, found: 412.29378. The two couples
27.45, 27.85, 29.57, 33.76, 34.15, 35.12, 35.68, 36.79, 39.44, of isomers were separated by using research-type Cinchona
40.00, 43.32, 49.20, 51.54, 54.92, 55.89, 73.42, 117.35, 139.37, alkaloyd-based anion exchanger type chiral stationary phase
170.62, 175.00.
following our previous reported protocol.20
3β-Hydroxy-24,25-methylen-24-bishomo-5α-chol-7-en-26-oic acid
(23a). The methyl ester 22a (39 mg, 0.08 mmol) was dissolved
in 2.5% NaOH solution in MeOH (1.5 mL) and the mixture was
refluxed for 5 h under magnetic stirring. The solvent was
removed under vacuum, the residue was dissolved in H2O
(20 mL), acidified to pH = 3 with 3 N HCl and extracted with
EtOAc (3 × 20 mL). The combined organic layers were washed
with H2O (20 mL) and brine (20 mL), dried over Na2SO4 and
evaporated to dryness, to furnish 23a as a white solid (35 mg,
0.08 mmol, quantitative). Rf (CHCl3/MeOH 95 : 5): 0.47.
1H-NMR (CDCl3, 400 MHz) δ: 0.49–0.50 (3H, m, 18-CH3), 0.76
(3H, s, 19-CH3), 0.87–0.89 (3H, m, 21-CH3), 3.52–3.60 (1H, m,
3-CH), 5.12 (1H, bs, 7-CH). 13C-NMR (CDCl3, 100.6 MHz) δ:
11.73, 12.94, 13.81, 14.07, 17.85, 18.78, 20.95, 21.46, 22.95,
23.67, 27.80, 29.59, 31.09, 34.11, 35.66, 35.93, 37.08, 37.54,
39.47, 40.17, 43.29, 54.92, 55.86, 60.44, 70.83, 117.38, 139.46,
177.11.
AlphaScreen assays
Activation of the DAF-12 receptor was determined using
AlphaScreen technology. Briefly, assays were conducted in
white, low volume, 384-well ProxyPlate using a final volume of
10 μL containing 10 nM glutathione transferase-tagged
ssDAF-12-LBD protein and 30 nM biotinylated Src-1 peptide.
The stimulation was carried out with different DA concen-
trations for 30 min at 25 °C. Luminescence was read in an
EnVision 2103 microplate analyzer (PerkinElmer, USA) after
incubation with the detection mix (acceptor and donor beads)
for 4 h at 25 °C in the dark. Dose–response curves were per-
formed in triplicate. To test the antagonism effect, the GST-
ssDAF-12 and the biotinylated Rsc-1 were incubate with a fixed
concentration of 1 at 0.3 μM and a dose response of compound
5 per 30 min at 25 °C.
3β-Hydroxy-24,25-methylen-24-bishomo-5α-chol-7-en-26-oic acid
(23b). Compound 23b was obtained following the same pro-
Computational modelling
cedure described for 23a, starting from the methyl ester 22b in The Maestro graphical user interface and the LigPrep module
90% yield (35 mg, 0.08 mmol). Rf (CHCl3/MeOH 95 : 5): 0.36. (Schrödinger, LLC, New York, NY, 2020) were used to build
1H-NMR (CDCl3, 400 MHz) δ: 0.47 (3H, s, 18-CH3), 0.73 (3H, s, and assign a protomeric state to the investigated compounds.
19-CH3), 0.84–0.86 (3H, m, 21-CH3), 3.48–3.54 (1H, m, 3-CH), The PDB code 3GYU4 was used as source for the ssDAF-12 LBD
5.10 (1H, bs, 7-CH). 13C-NMR (CDCl3, 100.6 MHz) δ: 11.69, structure in complex with 1. The complex was treated using
12.88, 15.65, 16.07, 18.66, 19.77, 20.11, 21.42, 22.82, 23.73, the default settings of the Protein Preparation Wizard protocol
27.81, 29.59, 30.95, 34.08, 35.07, 35.70, 37.02, 37.44, 39.45, of the Schrödinger Suite. Next, the Glide SP docking program
40.13, 43.27, 54.90, 55.85, 70.66, 117.42, 139.38, 177.75.
was run to obtain the best ranked putative binding poses. The
3-Keto-24,25-methylen-24-bishomo-5α-chol-7-en-26-oic
acid grid box was centered on the co-crystallized ligand. The confor-
(5–8). To a suspension of 23a (40 mg, 0.09 mmol 23a) or 23b, mational energy of the co-crystallized pose of compound 1 and
(35 mg, 0.08 mg 23b) in acetone (6 mL) at 0 °C, Jones reagent of the putative bioactive conformation of 5–8 obtained by the
was added dropwise untill the colour of the mixture turned docking run was estimated using the Current Energy task of
from green to red (4 drops). The mixture was stirred at room the Macromodel software, using the OPLS3e as force field in
temperature for 2 h, then MeOH (2 mL) and H2O (20 mL) were water solvent, while leaving all the other settings at default
added. The product was extracted with EtOAc (3 × 20 mL). The value.26 In order to calculate the global minima energy of com-
combined organic layers were washed with H2O (20 mL) and pounds 1 and 5–8 a Conformational Search analysis has been
brine (20 mL), dried over anhydrous Na2SO4 and evaporated performed with the Macromodel software using the default
under reduced pressure, providing target products 5–8. 5–6: settings, the OPLS3e force field and the water solvent. The
35 mg (0.08 mmol). Rf: 0.42 (CHCl3/MeOH, 95 : 5). 1H-NMR GRID 2021 graphical user interface (version 2021.1, Molecular
(CDCl3, 400 MHz) δ: 0.55–0.56 (3H, m, 18-CH3), 0.94 (3H, d, J = Discovery Ltd, UK)21 was used to calculate the hydrophobic
6.3, 21-CH3), 1.01 (3H, s, 19-CH3), 5.17 (1H, bs, 7-CH). molecular interaction field generated by the CRY probe in the
13C-NMR (CDCl3, 100.6 MHz) δ: 11.85, 12.42, 14.42, 18.74, DAF-12 protein (pdb code: 3GYU). The MIF was evaluated at
19.86, 21.66, 22.88, 24.42, 27.87, 29.67, 30.03, 34.36, 35.70, −1.00 kcal mol−1. Afterwards, the best docking poses for com-
35.79, 38.08, 38.73, 39.38, 42.82, 43.33, 44.20, 48.80, 54.87, pounds 1 and 5–8 previously obtained were uploaded into the
55.87, 117.04, 139.40, 179.20 (×2), 212.01. HRMS (ESI): GRID session.
This journal is © The Royal Society of Chemistry 2021
Org. Biomol. Chem., 2021, 19, 5403–5412 | 5411

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Paper
Organic & Biomolecular Chemistry
10 (a) M. V. Dansey, L. D. Alvarez, G. Samaja, D. S. Escudero,
A. S. Veleiro, A. Pecci, O. A. Castro and G. Burton, Biochem.
J., 2015, 465, 175–184; (b) G. A. Samaja, O. Castro,
L. D. Alvarez, M. V. Dansey, D. S. Escudero, A. S. Veleiro,
A. Pecci and G. Burton, Bioorg. Med. Chem. Lett., 2013, 23,
2893–2896.
Conflicts of interest
R.P. and A.G. are cofounders of TES Pharma. R.P. is President
of TES Pharma.
11 C. R. Rodriguez, M. C. del Fueyo, V. J. Santillánc,
M. V. Dansey, A. S. Veleiro, O. A. Castro and G. Burton,
Steroids, 2019, 151, 108469.
Acknowledgements
TES Pharma (Perugia, Italy) is gratefully acknowledged for the
financial support. We also thank Tiziana Benicchi (TES 12 R. Martin, E. V. Entchev, T. V. Kurzchaliab and
Pharma) for the technical support in AlphaScreen assays.
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