Synthesis, Evaluation, and Structure-Activity Relationship Study of Lanosterol Derivatives to Reverse Mutant-Crystallin-Induced Protein Aggregation

Article abstract of DOI:10.1021/acs.jmedchem.8b00705

We describe here the development of potent synthetic analogues of the naturally occurring triterpenoid lanosterol to reverse protein aggregation in cataracts. Lanosterol showed superiority to other scaffolds in terms of efficacy and generality in previous studies. Various modified lanosterol derivatives were synthesized via modification of the side chain, ring A, ring B, and ring C. Evaluation of these synthetic analogues draws a clear picture for SAR. In particular, hydroxylation of the 25-position in the side chain profoundly improved the potency, and 2-fluorination further enhanced the biological activity. This work also revealed that synthetic lanosterol analogues could reverse multiple types of mutant-crystallin aggregates in cell models with excellent potency and efficacy. Notably, lanosterol analogues have no cytotoxicity but can improve the viability of the HLE-B3 cell line. Furthermore, representative compound 6 successfully redissolved the aggregated crystallin proteins from the amyloid-like fibrils in a concentration-dependent manner.

Full text of DOI:10.1021/acs.jmedchem.8b00705

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Article
Synthesis, Evaluation, and Structure-Activity
Relationship Study of Lanosterol Derivatives to Reverse
Mutant Crystallins Induced Protein Aggregation
Xinglin Yang, Xiangjun Chen, Zimo Yang, Yi-Bo Xi, Liguo Wang, Yue Wu, Yong-Bin Yan, and Yu Rao
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 28 Aug 2018
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Synthesis, Evaluation, and Structure-Activity
Relationship Study of Lanosterol Derivatives to
Reverse Mutant Crystallins Induced Protein
Aggregation
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Xinglin Yang1‡, Xiang-Jun Chen2,3‡, Zimo Yang1‡, Yi-Bo Xi2,4, Liguo Wang1, Yue Wu1, Yong-Bin Yan2*,
Yu Rao1*
1 MOE Key Laboratory of Protein Sciences, School of Pharmaceutical Sciences, MOE Key
Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University,
Beijing 100084, P.R. China.
2 State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University,
Beijing 100084, China
3 Eye Center of the Second Affiliated Hospital, Institutes of Translational Medicine, Zhejiang
University School of Medicine, Hangzhou 310058, China.
4 Tsinghua-Peking Joint Center for Life Sciences, Tsinghua University, Beijing 100084, China
‡ These authors contributed equally.
Abstract
We describe here the development of potent synthetic analogues of naturally occurring
triterpenoid lanosterol to reverse protein aggregation in cataracts. Lanosterol showed superiority to
other scaffolds in terms of efficacy and generality in previous studies. Various modified lanosterol
derivatives were synthesized via modification of the side chain, ring A, ring B and ring C.
Evaluation of these synthetic analogues draws a clear picture for SAR. In particular, hydroxylation
of the 25-position in the side chain profoundly improved the potency, and 2-fluorination further
enhanced the biological activity. This work also revealed that synthetic lanosterol analogues could
reverse multiple types of mutant crystalline aggregates in cell models with excellent potency and
efficacy. Notably, lanosterol analogues have no cytotoxicity but can improve the viability of the
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HLE-B3 cell line. Furthermore, representative compound 6 successfully redissolved the
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aggregated crystallin proteins from the amyloid-like fibrils in a concentration-dependent manner.
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Introduction
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Cataracts, which are a clouding of the lens in the eyes, lead to degradation of visual acuity and
even blindness. According to a recent published systematic analysis, cataracts are the leading
cause of blindness worldwide, especially in developing countries1. Every year millions of patients
undergo cataract surgery, which involves removing the opacified lens and replacing it with an
artificial lens implant. While surgical treatment of cataract is effective, the risk of complications,
such as posterior capsular opacification and retinal detachment is inevitable2. In addition,
receiving cataract surgery is limited to those with accessible facilities, doctors, and funds in the
developing world where cataract incidence is increasing3. Lenses are an avascular tissue, and
maturation of lens fiber cells involves coordinated organelle degradation and high soluble
crystallin protein expression. These anatomic characteristics contribute to lens transparency.
Crystallin proteins, including α, β and γ crystallins, comprise 90% of the proteins in the mature
lens fiber cells. Normally, these highly concentrated proteins can remain soluble throughout life,
which is helped by α-crystallin, an ATP-independent chaperone. In cataractous lenses, aggregated
lens proteins with high molecular weight are found, which are insoluble and account for light
scattering4, 5. Mutations in crystallin proteins can cause protein misfolding and induce the
formation of aggregates, which is typical pathogenesis and clinical characteristic of cataracts6. So,
if a compound can dissolve various mutant crystallin aggregates, it may be developed as the
anti-cataract agent in future. Other factors7-9, such as oxidative damage, dysregulation of Ca2+
concentration and an increased glucose level in the lens, can also lead to different kinds of
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cataracts. Based on the etiology of cataracts, different nonsurgical strategies10-12 were developed,
such as the inhibition of aldose reductase and the usage of antioxidants. However, currently, there
are no widespread, efficient drugs to prevent or treat cataracts. Because cataracts are viewed as a
protein aggregation disease, compounds that can inhibit or even reverse protein aggregation are
promising drug candidates for cataract treatment. The screening assays was analysis of
compounds’ capability of dissolving various mutant crystallin aggregates.
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In 2015, it was reported that lanosterol, an amphipathic molecule enriched in the lens, could
reverse protein aggregation in cataracts13. Shortly afterwards, another study found that
25-hydroxycholesterol (C29) could serve as pharmacological chaperone for a-crystallin to
partially restore transparency in cataract models14. Remarkably, these two works provide a
brand-new strategy for the prevention and treatment of cataracts. However, from a perspective of
drug-likeness properties, both lanosterol and 25-hydroxycholesterol have their respective
limitations as potential candidates. For example, in terms of the efficacy against α-crystallin,
25-hydroxycholesterol is better than lanosterol. Additionally, it was found that lanosterol could
reduce intracellular aggregation by various cataract-causing mutant crystallin proteins, including α,
β and γ-crystallin. In contrast, 25-hydroxycholesterol is only effective towards α-crystallin. In
addition to its relatively low efficacy (EC50 = 1.4 µM), lanosterol is an endogenous tetracyclic
triterpenoid that can be transformed to different steroids with important bioactivities15. The
uncontrolled transformation in vivo may decrease the half-time in vivo. Meanwhile,
25-hydroxycholesterol is also an endogenous oxysterol. It was reported that
25-hydroxycholesterol is an LXR agonist that will activate SREBP-1C in the liver leading to
hepatic steatosis and hypertriglyceridemia16.
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Taken together, these facts imply that lanosterol may represent a suitable compound for further
SAR study and we envisioned that an optimization of lanosterol may yield good lead candidates,
which can remain active to all three subtypes of crystalline with improved efficacy to address the
abovementioned limitations. Herein, we reported that through a comprehensive modification of
lanosterol and biological evaluation of diverse synthetic analogues, a few new synthetic lanosterol
analogues could reduce intracellular aggregation by various cataract-causing mutant crystalline
proteins (α, β and γ) with excellent potency and efficacy. More importantly, the aggregated
crystallin proteins from the amyloid-like fibrils can be readily redissolved by representative
compound 6.
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Results and discussion
Figure 1. A. Chemical structures of lanosterol and 25-hydroxycholesterol and their efficacy to inhibit
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α-crystallinR120G induced aggregation in the cell model. Aggregation inhibition (%) by 0.4 μM of compounds.
bAggregation inhibition (%) by 4 μM of compounds.. B, C. Activity analysis of lanosterol and C29, lanosterol
could reduce intracellular aggregation by various cataract-causing mutant crystallin proteins, while C29 is only
effective towards α-crystallin.
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Figure 2. Chemical structure of lanosterol derivatives and their efficacy to inhibit α-crystallinR120G induced
aggregation in the cell model. aAggregation inhibition (%) by 0.4 μM of compounds. bAggregation inhibition (%)
by 4 μM of compounds.
Structure of lanosterol derivatives
As shown in figure 2, a highly diverse derivative of lanosterol was designed and synthesized,
which included three major patterns of modification, namely, 1) side chain modification, 2) ring A
modification, and 3) ring B and ring C modification. At the beginning of our investigation, the
side chain was systematically modified. Different kinds of functional groups were introduced to
the side chain, including alkyl (1), epoxide (2), aldehyde (3), hydroxyl oxime (4), amine (5),
hydroxyl (6) and carbonyl (9) groups. In addition, glycoside (11) with improved aqueous
solubility was also synthesized. Evaluation of these substituents, which can serve as both the
hydrogen bond acceptor and donor, will offer a quite clear picture of the side chain requirement
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for high efficiency. In parallel, the ring A of lanosterol was also modified diversely. 3-OH was
acetylated (12), methylated (13), oxidized (14) or converted to corresponding hydroxyl oxime (15),
methoxy oxime (16) and amine (17) groups. Due to the high efficacy of 25-hydroxylanosterol
(figure 2), this compound was selected for further modification at rings A, B, and C. Same with
ring A modification of compound 12-17, the modification of compound 6 afforded compounds
18-23. Ring A of compound 6 also underwent further chemical diversification. Hydroxyl (24),
lactam (25) and carboxylic acid (26, 27) moieties were introduced into ring A. Additionally, ring A
is further opened via hydroxylation of lactone (32), affording compound 33. The double bond
between ring B and ring C can be smoothly transformed to epoxy (28), dienes (29), and hydroxyl
(30). Carbonyl group was introduced to position 7 and 11, affording compound 31. Compounds
1-3, 5-8, 10, 12-18, 20, and 31 were prepared according to the literature1. Synthesis of other
derivatives is illustrated in schemes 1-3.
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Scheme 1. Preparation of Compounds 4, 6, 9, 10, 26 and 27
The side chain double bond of compound 38 was selectively oxidized to epoxide (39) with
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m-CPBA as the oxidant under mild conditions. The epoxide analogue (39) was further converted
to the aldehyde analogue (40) when treated with HIO4 and subsequently reacted with
hydroxylamine to give oxime analogue (4). Compound 39 can be readily reduced to afford
compound 6 using LiAlH4 and hydrolyzed to compound 41 using H3PO2, respectively. The
Rh-catalyzed O-H insertion reaction using ethyl 2-diazoacetate as a reaction partner and
subsequent hydrolysis can transform compound 6 into compound 27. Compound 6 reacted with
succinic anhydride to provide compound 26. The secondary alcohol in the side chain of compound
41 was selectively oxidized to ketone (9) using DMP as an oxidant. Later, through condensation
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with hydroxylamine hydrochloride, compound 9 was smoothly converted into oxime analogue 10.
Scheme 2. Preparation of Compounds 11 and 21-25
As demonstrated in scheme 2, oxime analogues 21 and 22 were readily obtained when compound
20 reacted with hydroxylamine and methoxyamine, respectively. Oxime 21 was transformed into
the 3-NH2 analogue 23 and lactam analogue 25 via reduction and Beckman rearrangement. The
Pd-catalyzed C-H activation strategy was used to prepare compound 24. The two angular methyl
groups were oxidized using O-methyl oxime as the directing group and PhI(OAc)2 as the oxidant22.
Subsequent hydrolysis gave the desired product 24. Benzoyl-protected glycosyl
dichloroacetamide23 was employed as the donor for the glycosylation of compound 20. Then, the
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ketone group of compound 43 was reduced using NaBH4 and the benzoyl protecting group was
removed using NaOMe to afford compound 11.
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Scheme 3. Preparation of Compounds 19, 28-30, 32 and 33
As shown in scheme 3, the double bond of compound 6 was oxidized to epoxide 28 with m-CPBA
as the oxidant24. Diene analogue 29 was obtained by treatment of epoxide (28) with 40% HF
solution. The epoxide of compound 28 was reduced to a hydroxyl group with lithium as the
reductant, affording compound 3025. The 3-OH of compound 28 was oxidized to a carbonyl group
(46), which was further transformed into a lactone via Baeyer–Villiger oxidation. Subsequently,
ring A was opened by hydrolysis of the lactone under basic conditions, affording the ring
A-opening derivative 33.
Biological evaluation of synthetic lanosterol analogues and SAR discussion
We used mutant crystallin cell lines as screening system and the cells were transfected with
plasmids containing the mutated crystallin genes. After transfection for 16 h, the mutant
crystallins were overexpressed and formed the aggregates (figure S1), then the compounds were
added into cell culture medium. After incubated for 6~8 h, we used the fluorescence microscopy
to analyze compounds’ capability of dissolving various mutant crystallin aggregates13.The
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inhibition of αB R120G crystallin aggregation with 0.4 µM and 4 µM compounds was calculated
based on DMSO-treated cells. The results are summarized in figure 1 and 2. It was found that side
chain modification greatly changed the activity of lanosterol. 2H-Lanosterol (1) showed nearly no
activity at a concentration of 4 µM in our assay system. The transformation of the double bond to
epoxide (2) slightly decreased the activity, which demonstrated that the double bond of the side
chain was not essential for good efficacy. The activity of the aldehyde analogue (3) shows a
similar activity with lanosterol. The oxime derivative (4), which can serve as not only a hydrogen
bond donor but also a hydrogen bond acceptor, demonstrated increased activity with respect to
lanosterol. Interestingly, introduction of the diethylamine group (5), which may serve as a
hydrogen bond acceptor decreased the activity greatly. Notably, the introduction of the hydroxyl
group (6) to the side chain significantly increases the activity, which is consistent with the positive
activity of C29. 25-Hydroxylanosterol (6), with lanosterol skeleton and bearing the same side
chain as C29, shows excellent activity to various mutant crystallin proteins. While cholesterol
shows no activity to mutant crystallin proteins, and C29 bearing cholesterol skeleton, only shows
activity to mutant α-crystallin proteins (Figure 1 and 2). These results imply that the lanosterol
skeleton has priority to cholesterol to be modified as possible therapeutic treatments for cataracts.
Derivatives with a short or long primary alcohol side chain (7, 8) show a loss of potency. When
other functional groups, such as ketone and second hydroxyl, were introduced to the 24-position
of 25-hydroxylanosterol, the activity of the corresponding analogues (9, 10) was generally reduced
in different degrees. Compounds bearing the glucose motif (11) exhibited moderate activity.
As illustrated in figure 2, ring A was also modified, and the corresponding derivatives were tested
in the screening system. 3-OH was acetylated and the activity of the corresponding analogue 12
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slightly increased. Methylation (13) and oxidation (14) of 3-OH in lanosterol decreased the
activity. Compounds 15 and 16 bearing hydroxyl oxime and methoxy oxime showed obvious
activity improvements. Moderate activity was observed when the hydroxyl group was replaced
with an amine group (17). Introduction of the oxime group in the 3-position improved the activity
compared to that of lanosterol. However, compounds 21 and 22 with modifications of both 25-OH
and 3-oxime showed a decreased activity compared to compound 6. This result implied that there
was no synergistic effect between the side chain and the ring skeleton modification of lanosterol.
Similar with the ring A modification of lanosterol, several analogues of 25-hydroxylanosterol
(18-23) were less potent compared to 25-hydroxylanosterol. Then, derivatives of compound 6 with
ring A modification were further tested in this assay. Oxidation of two angular methyl groups leads
to a total activity loss, which suggested that two angular methyl groups were essential for
favorable activity. The activity of 7-membered lactam derivatives 25 remained partial. Compound
26 with the succinic acid motif in the 3-position exhibited a similar activity to the acetylated
derivative 18. However, compound 27 with the acetic acid motif showed nearly no activity. These
results suggested that esterified derivatives (18, 26) but not alkylated derivatives (19, 27) could
remain partly active.
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It was found that the modification of the lanosterol skeleton body, ring B and ring C, generally
decreased the activity of the corresponding derivatives to different degrees. The transformation of
the double bond in compound 6 to the diene group makes rings B and C of compound 29 more flat,
which results in a slight shift of ring A and 3-OH observed from the computer calculation
(Maestro 11). The slight perturbation of conformation undoubtedly accounts for the activity loss.
Compound 29, with an epoxy group at the 8,9-position, had a reduced efficacy with respect to 6.
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Meanwhile, installation of a hydroxyl group at the 8-position dramatically decreased the activity
of compound 30. In addition, introduction of the carbonyl groups at positions 7 and 11 led to an
activity loss as well. It was observed that lactone derivative 32 showed less potency than
compound 28. Opening ring A afforded compound 33, which had a low efficacy. These results
imply that the hydrophobic property and unique conformation of lanosterol skeleton are
indispensable for the high efficacy to inhibit protein aggregation.
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Based on the above testing results, compound 6 demonstrated the best activity among these
synthetic analogues. SAR showed that the introduction of 25-hydroxyl greatly improved the
potency. The conformation and hydrophobicity of ring B and ring C was essential for high potency.
Although some modifications of 3-OH maintained the activity of the lanosterol derivatives,
compound 6 with 3-OH demonstrated the superior activity over other analogues. As shown in
scheme 4, the 2-position of compound 6 was further modified with F and OH functional groups.
The silyl enol ether intermediate (44) was oxidized using selectfluor and m-CPBA, affording
2-fluorinated and 2-hydroxylated ketone derivatives. These two intermediates underwent
sequential reduction and deprotection to furnish compounds 35, 36, and 37, respectively. Then,
compounds 6, 20, 35, 36, and 37 were tested to evaluate their activity regarding the effectivity on
the aggregates of B-R120G. As shown in table 1 and figure 3, to our delight, it was found that
2-fluorination of compound 6 improved the potency further (compound 35, EC50 = 18 nM),
which is possibly due to the special hydrophobic/hydrophilic balance of the fluorine atom26.
Besides, introduction of fluorine might reduce the metabolism rate of corresponding compound in
vivo15. To our delight, the CLogP value of compound 35 is also lower than that of lanosterol
(CLogP of lanosterol is 11.077, CLogP of compound 35 is 8.713). Lower CLogP value means
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better solubility which is important for drug formulation development. 3-Epi-35 (36) showed a
similar activity to compound 35. However, introduction of the OH group in the 2-position (37)
decreased the activity. All these compounds show a good dose-dependent activity.
25-Hydroxyllanosterol (6, EC50 = 57 nM) was approximately 25 times more potent than
lanosterol (1, EC50 = 1420 nM), which implied that the 25-hydroxyl group played a crucial role in
reversing protein aggregation. Compound 20, with oxidation of the 3-hydroxyl group, shows a
2-fold lower potency than compound 6, which suggested that the 3-hydroxyl group may serve as a
hydrogen bond acceptor rather than a donor. Compound 29, with a flatter conformation, showed a
slightly decreased potency.
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Scheme 4. Preparation of Compounds 35-37
Table 1. The efficacy of lanosterol analogues to inhibit α-crystallinR120G induced aggregation in the cell
model
Aggregation inhibition
Aggregation inhibition
Compound
number
Compound
number
(%)（αB R120G）
(%)（αB R120G）
0.4 μM
4 μM
0.4 μM
4 μM
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52.5±0.6
56.7±1.7
60.3±0.8
62.1±1.4
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56.1±1.9
33.4±1.7
60.6±2.6
51.1±0.9
35
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Figure 3. The activity evaluation of lanosterol derivatives and reported compounds that could inhibit or postpone
cataracts. A, C. EC 50 assay of lanosterol derivative B. The activity assay of the reported compounds that could inhibit
or postpone cataracts.
Meanwhile, several reported compounds27-30, which could inhibit or postpone cataracts were
tested in the B R120G HeLa cell lines, including Bendazac lysine (an aldose reductase inhibitor),
pirenoxine sodium (a compound that can reduce selenite or calcium ions), glutathione and
ascorbate (important antioxidant in the eye). As illustrated in figure 3B, no obvious effect could be
observed with these compounds, which verifies the unique biological mechanism of our new
synthetic compounds.
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Figure 4. Activity evaluation of lanosterol derivatives towards different mutant crystallins. A, In HeLa cell lines,
the effectivities of lanosterol analogues was general to the mutated crystallins B, In HLE-B3 cell lines, the
lanosterol analogues also show significant activities, and the effectivities were also general to the mutated
crystallins.
Furthermore, these potent lanosterol derivatives and negative control 4H were evaluated on
aggresomes caused by other crystallin mutations, including αA Y118D, βB2 V187E, γC G129C
and γD W43R31. As shown in figure 4A and 4B, these potent derivatives exhibited general
efficiency towards multiple types of mutant crystallins in both HeLa and HLE-B3 cell lines.
Meanwhile, compound 34, as a negative control, showed no activity towards all the crystallins.
These results demonstrated that lanosterol was a unique scaffold that can inhibit multiple types of
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mutant crystallin induced aggregation. It was worth noting that the efficacy of lanosterol
derivatives remain consistent among different kinds of mutant crystalline. For instance, compound
35, the most potent compound in the αB R120G-containing cell line, is also the most potent
compound in other mutant crystallin cell lines, which suggested that different mutant-crystallin
aggregation was inhibited by lanosterol analogues via a same mechanism.
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Figure 5. Compound 6 redissolved preformed amyloid-like fibrils of crystallin proteins. A. Effect of lanosterol and
compound 6 on the redissolution of crystallin aggregates by ThT fluorescence (n = 3). Left, a-crystallin mutants;
right, β/γ-crystallin mutants. B. EC50 assay of compound 6 on the redissolution of crystallin aggregates by ThT
fluorescence. C. Negatively stained TEM photographs of aggregates of seven crystallin mutant proteins treated by
a liposome vehicle, compounds 6 and 34 in liposomes.
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To investigate whether lanosterol derivatives have a direct effect on the dissolution of aggregated
proteins, the aggregates of seven mutant crystallins were obtained by heating crystallins in the
presence of 1 M guanidine chloride as described before13. Under this condition, all crystallin
proteins formed amyloid-like fibrils as revealed by the enhancement of thioflavin T (ThT)
fluorescence and the fibrillary structures under negatively stained transmission electron
microscopy (TEM). Compound 6, with good efficacy towards different mutant crystallins in the
cell model, was chosen for investigation of the dissolution effect in vitro with compound 34 as a
negative control. Dipalmitoyl phosphatidylcholine was used to increase the solubility of lanosterol
derivatives. As shown in figure 5A, compound 6, but not 34, could efficiently decrease the ThT
fluorescence of all the seven mutant crystallin aggregates. Consistent with the cell model assay,
compound 6 also showed a better efficacy than lanosterol. Then, the EC50 was tested based on
thioflavin T (ThT) fluorescence of αA Y118D and αB R120G. As shown in figure 5B, a good dose
activity relationship was observed and the EC50 of compound 6 is 35.6 nM for αA Y118D, 3116
nM for αB R120G. In addition, it was observed that all the fibrillar structures treated with
compound 6, but not 34, were redissolved in the negatively stained TEM photographs (figure 5C).
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Figure 6. Toxicity evaluation of lanosterol derivatives towards different mutant crystallins in cells. A. HLE-B3 cell
lines and B. HeLa cell lines
The toxicity of new prepared lanosterol derivatives and C29 were tested in HeLa or HLE-B3 cell
lines with peGFP-N1 and multiple types of mutant crystallins. It was observed that lanosterol
analogues could improve the cell viability towards both HeLa and HLE-B3 cell lines with
peGFP-N1. However, a slight decrease in cell viability was observed for C29. These results
demonstrated that lanosterol derivatives are promising compounds for cataract treatment and the
toxicity of C29 may be due to multiple functions of endogenous oxysterol. Mutant crystallin
proteins usually cause cytotoxicity. Both lanosterol derivatives and C29 can inhibit the
aggregation of mutant a-crystallins. Consistently, these compounds could improve the cell
viability of both HeLa and HLE-B3 cell lines, with -crystallins obviously being even at a
concentration of 40 nM. For cells with beta and gamma mutant crystallins, lanosterol derivatives
could inhibit the corresponding protein aggregation; consequently, they can also obviously
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improve the cell viability. In contrast, C29 is not efficient towards β and γ crystallins, which also
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Figure 7. Summary of the structure−activity relationships of lanosterol to reverse mutant crystallins induced
protein aggregation in HeLa cell.
To explore the structural features of lanosterol that are responsible for reversing mutant
crystallin-induced protein aggregation and improve potency, we synthesized and evaluated 37
synthetic lanosterol analogues. As shown in figure 7, the SAR summary was depicted. In general,
modification of structures in green could improve the potency significantly. For example,
introduction of 25-hydroxyl and 2-fluorine greatly improved the potency (35 EC50 = 18 nM vs
lanosterol EC50 = 1.42 µM). In addition, structures in blue can be modified; however,
modification may lead to decrease or loss of activity. Compound 20 with 3-carbonyl groups
maintained the activity but had low potency (20 EC50 = 106 nM vs 6 EC50 = 57 nM). Compound
30 with 8-hydroxyl lost the activity. Oxidation of gem-dimethyl in red also led to a loss of activity.
Conclusion
In summary, using mutant crystallin cell line as a screening system, we demonstrated lanosterol
derivatives as efficient compounds to reverse different kinds of protein aggregation in cataracts
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with good potency and efficacy (6, EC50 = 57 nM, 35, EC50 = 18 nM). These potent compounds
could not only inhibit the mutant crystalline aggregation in the cell model but also redissolve the
mutant crystalline fiber in vitro. In addition, it was found that lanosterol analogues could also
improve cell viability. Extensive modification of lanosterol provides a detailed SAR study, which
lays a solid foundation for the development of drug candidates to treat cataracts and probes to
elucidate the biological mechanism. A preliminary SAR is summarized based on our results.
Further biological evaluation of the new synthetic potent molecule in different kinds of cataract
animal models and detailed mechanism study is ongoing in our laboratory.
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Experimental Section
Materials
All commercial materials (Alfa Aesar, Aladdin, J&K Chemical LTD.) were used without further
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purification. All solvents were analytical grade. The H-NMR and 13C-NMR spectra were
recorded on a Bruker 400 MHz spectrometer in CDCl3 using TMS or solvent peak as a standard.
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The representative signals are shown for H-NMR32. All 13C-NMR spectra were recorded with
complete proton decoupling. Low-resolution mass spectral analyses were performed with a Waters
AQUITY UPLCTM/MS. High-resolution mass spectral analyses were performed with a Bruker
Impact HD/QTOF. Analytical TLC was performed on Yantai Chemical Industry Research Institute
silica gel 60 F254 plates and flash column chromatography was performed on Qingdao Haiyang
Chemical Co. Ltd silica gel 60 (200-300 mesh). The rotavapor was BUCHI’s Rotavapor R-3.
High-purity chemical compounds, such as lanosterol, cholesterol, dimethyl sulfoxide (DMSO),
paraformaldehyde and triton X-100 were purchased from Sigma. Compound 29 (5-cholesten-3β,
25-diol) reported by Leah N. Makley et al. was from Aladdin. Hoechst 33342 was obtained from
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Invitrogen. The p62/SQSTM1 antibody was from Proteintech. Plasmid Maxipre kit was from
Vigorous Biotechnology. The transfection reagent LipofectamineTM 2000 and other cell culture
materials were purchased from Invitrogen. Cell Counting Kit-8 was from Dojindo laboratories. All
other chemicals were products of analytical grade. The purity of tested compounds, determined by
HPLC, was >95%.
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(R,E)-4-((3S,5R,10S,13R,14R,17R)-3-hydroxy-4,4,10,13,14-pentamethyl-2,3,4,5,6,7,10,11,12,1
3,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanal oxime (4). To a
50 ml round-bottom flask, were added 50 mg of compound 40 (0.11 mmol, 1.0 eq) and 10% KOH
in EtOH, the resulting solution was stirred at 90 °C for 2 h. Then the mixture was washed with
water and extracted with DCM. The organic layer was concentrated by evaporation to obtain crude
intermediated which was used without further purification. To a 50 ml round-bottom flask, were
added compound (1.0 eq) prepared above, 23.6 mg of NH2OH.HCl (0.33 mmol, 3.0 eq), 36 mg of
NaOAc (0.44 mmol, 4.0 eq), 5 ml of EtOH. After stirred at 60 °C for 3 h, the resulting solution
was washed with saturated NaHCO3 solution and extracted with EtOAc. The organic layer was
purified by 200-300 mesh silica gel flash column chromatography (Hexane:EtOAc = 5:1),
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yielding 41 mg pure product (I.Y. = 89%). H-NMR (400 MHz, CDCl3, representative signals) δ
(ppm) 7.40 (t, 1H, J = 6 Hz), 3.24 (dd, 1H, J = 11.6 Hz, J = 4.4 Hz), 0.97 (d, 6H, 29-H3, 19-H),
0.92 (m, 3H, 21-H3), 0.86 (s, 3H, 30-H3), 0.79 (s, 3H, 28-H3), 0.67 (s, 3H, 18-H3); 13C-NMR
(100 MHz, CDCl3) δ (ppm)153.1, 134.6, 134.5, 79.2, 50.6, 50.5, 50.4, 50.0, 44.7, 39.1, 37.2, 36.5,
36.3, 35.8, 33.1, 32.6, 31.1, 31.0, 29.9, 28.4, 28.3, 28.1, 28.0, 26.7, 24.4, 21.2, 19.3, 18.54, 18.48,
18.4, 15.9, 15.6. LC-MS: calculated for C27H46NO2 [M+H]+: 416.35, found 416.65.
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(10S,13R,14R,17R)-17-((R)-6-hydroxy-6-methyl-5-oxoheptan-2-yl)-4,4,10,13,14-pentamethyl
-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl
acetate. (9) To a solution of 50 mg compound 41 (0.1 mmol, 1.0 eq) in DCM at room temperature
was added 50 mg of DMP (0.12 mmol, 1.2 eq). After stirred at room temperature for 5 h, the
resulting solution was washed with saturated NaHCO3 solution. The organic layer was dried over
anhydrous Na2SO4 and the solvent was removed by evaporation, then the mixture was purified by
200-300 mesh silica gel flash column chromatography (Hexane:EtOAc= 10:1), yielding 43 mg
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pure product (I.Y. = 85%). H-NMR(400 MHz, CDCl3, representative signals) δ (ppm) 3.84 (s,
1H), 3.23 (d, 1H, J = 8.0 Hz), 2.57-2.48 (m, 2H), 1.38 (s, 6H, 26-H3, 27-H3), 0.98 (d, 6H, 29-H3,
19-H3), 0.90 (m, 6H, 21-H3, 30-H3), 0.80 (s, 3H, 28-H3), 0.69 (s, 3H, 18-H3); 13C-NMR (100
MHz, CDCl3) δ (ppm) 215.1, 134.6, 134.5, 79.1, 76.4, 50.60, 50.57, 50.0, 44.7, 39.1, 37.2, 36.3,
35.8, 32.8, 32.1, 31.2, 31.0, 30.4, 29.9, 29.5, 28.3, 28.1, 26.72, 26.67, 24.4, 22.8, 21.2, 19.3, 18.6,
18.4, 15.9, 15.6, 14.3. MALDI-TOF-MS: calculated for C30H51O3Na [M+Na]+: 481.31, found
481.380.
(6R)-2-hydroxy-6-((10S,13R,14R,17R)-3-hydroxy-4,4,10,13,14-pentamethyl-2,3,4,5,6,7,10,11,12,
13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methylheptan-3-one
(9).
The compound prepared above was dissolved in the solution of KOH in EtOH (10%). After stirred
at 90 °C for 2 h, the mixture was concentrated by evaporation, a moderate amount of water was
added, and the suspension was extracted with DCM. The organic layer was purified by 200-300
mesh silica gel flash column chromatography (Hexane:EtOAc = 5:1), yielding compound 9 as
white powder (I.Y. = 90%). 1H-NMR(400 MHz, CDCl3, representative signals) δ (ppm) 3.25-3.22
(m, 1H), 2.57-2.48 (m, 2H), 1.00 (s, 3H), 0.98 (s, 3H), 0.91-0.88 (m, 10H), 0.81 (s, 3H), 0.69 (s,
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3H); 13C-NMR (100 MHz, CDCl3) δ (ppm) 215.1, 134.6, 134.5, 79.1, 76.4, 50.60, 50.57, 50.0,
44.7, 39.1, 37.2, 36.3, 35.8, 32.8, 32.1, 31.2, 31.0, 30.4, 29.9, 29.5, 28.3, 28.1, 26.72, 26.67, 24.4,
22.8, 21.2, 19.3, 18.6, 18.4, 15.9, 15.6, 14.3. MALDI-TOF-MS: calculated for C30H51O3Na
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[M+Na]+:
481.31,
found
481.380.
(2R,3S,5R,6S)-2-(((R)-6-((3S,5R,10S,13R,14R,17R)-3-hydroxy-4,4,10,13,14-pentamethyl-2,3,
4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methy
lheptan-2-yl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol (11) To a solution of
100mg compound 43 (0.1 mmol, 1.0 eq) in 5ml methanol at room temperature was added 10 mg
of NaBH4 (0.25 mmol, 2.5 eq). After stirred at room temperature for 30 min, the solvent was
removed by evaporation, then the mixture was washed with saturated NaHCO3 solution. The
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organic later was concentrated to obtain 95 mg of crude product. H-NMR(400 MHz, CDCl3,
representative signals) δ (ppm) 8.00 (d, J = 7.7 Hz, 2H), 7.94 (d, J = 7.7 Hz, 2H), 7.90 (d, J = 7.7
Hz, 2H), 7.82 (d, J = 7.7 Hz, 2H), 7.55-7.50 (m, 3H), 7.44-7.33 (m, 10H), 7.30-7.26 (m, 2H), 5.92
(t, J = 9.7 Hz, 1H), 5.58 (t, J = 9.7 Hz, 1H), 5.50 (t, J = 8.4 Hz, 1H), 5.00 (d, J = 7.9 Hz, 2H),
4.60-4.56 (m, 1H), 4.50-4.49 (m, 1H), 4.17-4.15 (m, 1H), 3.24-3.22 (m, 1H), 0.64 (s, 3H);
13C-NMR (100 MHz, CDCl3) δ (ppm) 166.21, 165.9, 165.4, 165.0, 134.49, 133.52, 133.28, 133.18,
129.93, 129.87, 129.79, 129.73, 129.55, 129.00, 128.92, 128.52, 128.44, 128.41, 128.38, 95.91,
79.06, 78.92, 73.25, 72.14, 72.10, 70.32, 63.81, 50.61, 50.50, 49.86, 44.51, 42.84, 38.99, 37.10,
36.69, 36.50, 35.70, 31.05, 30.91, 28.28, 28.07, 27.95, 27.19, 26.59, 25.31, 24.37, 21.09, 20.70,
19.25, 18.64, 18.35, 15.85, 15.54.
To a solution of 58 mg compound (0.06 mmol, 1.0 eq) prepared above in DCM/methanol : 1 ml/3
ml) was added 6 mg of NaOMe (0.11 mmol, 1.9 eq). After stirred at room temperature for 3 h,
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the solvent was concentrated by evaporation, then washed with saturated NaHCO3 solution and
extracted with DCM. The organic layer was dried over anhydrous Na2SO4 and purified by 200-300
mesh silica gel flash column chromatography (Hexane:EtOAc = 5:1), yielding 28 mg of compound
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11 (I.Y. = 80%). H-NMR(400 MHz, CD3OD:CDCl3 = 2:1, representative signals) δ (ppm) 4.43 (d,
1H, J = 7.7 Hz), 3.79-3.77 (m, 1H), 3.70-3.66 (m, 1H), 3.38-3.35 (m, 2H), 3.24-3.23 (m, 1H),
3.18-3.14 (m, 2H), 1.24 (s, 6H, 26-H3, 27-H3), 0.98 (s, 6H, 29-H3, 19-H3), 0.89 (m, 6H, 21-H3,
30-H3), 0.80 (s, 3H, 28-H3), 0.69 (s, 3H, 18-H3); 13C-NMR (100 MHz, CD3OD/CDCl3 = 2/1) δ
(ppm) 135.05, 134.91, 97.67, 79.10, 79.00, 77.22, 76.32, 74.25, 70.94, 62.41, 51.16, 51.06, 50.32,
49.63, 48.36, 45.00, 42.90, 39.34, 37.51, 37.35, 37.07, 36.24, 31.55, 31.32, 30.14, 28.74, 28.24,
27.82, 27.01, 26.72, 26.37, 24.55, 21.49, 19.44, 19.00, 18.78, 16.09, 15.80. MALDI-TOF-MS:
calculated for C36H63O7Na [M+Na]+: 629.44, found 629.429.
(3S,5R,10S,13R,14R,17R)-3-methoxy-4,4,10,13,14-pentamethyl-17-((R)-6-methylhept-5-en-2-
yl)-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene (13)
To a 50 ml round-bottom flask were added 90 mg of lanosterol (0.2 mmol, 1.0 eq) and 10 ml dry
THF, followed by 24.3 mg of NaH (1.0 mmol, 5.0 eq), 62 ul of MeI (1.0 mmol, 5.0 eq). After
stirred at room temperature for 12 h, the reacting solution was quenched by water, and the
extracted with DCM. The organic layer was purified by 200-300 mesh silica gel flash column
chromatography (Hexane:EtOAc = 30:1), yielding 90 mg of compound 13 (I.Y. = 90%). 1H-NMR
(400 MHz, CDCl3, representative signals) δ (ppm) 5.10 (t, J = 6.2 Hz, 3H), 3.58 (s, 3H), 2.67 (dd,
1H, J = 11.6 Hz, J = 4.4 Hz), 0.98 (s, 6H, 29-H3, 19-H3), 0.90 (m, 6H, 21-H3, 30-H3), 0.79 (s, 3H,
28-H3), 0.68 (s, 3H, 18-H3); 13C-NMR (100 MHz, CDCl3) δ (ppm) 134.7, 134.5, 131.1, 125.4,
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88.8, 57.7, 51.1, 50.6, 50.0, 44.7, 39.0, 37.2, 36.5, 36.4, 35.7, 31.2, 31.0, 28.4, 28.1, 26.6, 25.9,
25.1, 24.4, 22.8, 21.2, 19.3, 18.8, 18.3, 17.8, 16.3, 15.9.
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(5R,10S,13R,14R,17R)-4,4,10,13,14-pentamethyl-17-((R)-6-methylhept-5-en-2-yl)-1,2,4,5,6,7,
10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthren-3-one O-methyl oxime
(16). Refer to the synthetic method of compound 22. 1H-NMR (400 MHz, CDCl3, representative
signals) δ (ppm) 5.09 (t, 1H, J = 6.8 Hz), 3.81 (s, 3H), 3.03-3.00 (m, 1H), 1.15 (s, 3H, 28-H), 1.06
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(d, 6H, 29-H3, 19-H3), 0.91 (d, 3H, 21-H3), 0.85 (s, 3H, 30-H3), 0.69 (s, 3H, 18-H3); C-NMR
(100 MHz, CDCl3) δ (ppm) 166.2, 134.9, 133.9, 131.0, 125.4, 61.14, 51.61, 50.55, 50.02, 44.62,
40.18, 37.21, 36.50, 36.41, 35.83, 31.15, 31.01, 28.32, 27.22, 26.56, 25.87, 25.08, 24.38, 23.37,
21.21, 19.13, 18.83, 18.79, 18.25, 17.77, 15.96. MS (ESI): calculated for C31H52NO [M+H]+:
454.40, found 454.41.
(R)-6-((3S,5R,10S,13R,14R,17R)-3-methoxy-4,4,10,13,14-pentamethyl-2,3,4,5,6,7,10,11,12,13,
14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methylheptan-2-ol (19).
To a 50 ml round-bottom flask were added 90 mg of compound 6 (0.2 mmol, 1.0 eq) and 10 ml
dry THF, followed by 24.3 mg of NaH (1.0 mmol, 5.0 eq), 62 ul of MeI (1.0 mmol, 5.0 eq). After
stirred at room temperature for 12 h, the reacting solution was quenched by water, and the
extracted with DCM. The organic layer was purified by 200-300 mesh silica gel flash column
chromatography (Hexane:EtOAc = 30:1), yielding 30 mg of compound 19 (I.Y. = 30%). 1H-NMR
(400 MHz, CDCl3, representative signals) δ (ppm) 3.36 (s, 3H), 2.66 (dd, 1H, J = 11.6 Hz, J = 4.0
Hz), 2.57-2.48 (m, 2H), 1.20 (s, 6H, 26-H3, 27-H3), 0.97 (d, 6H, 29-H3, 19-H3), 0.89 (m, 6H,
21-H3, 30-H3), 0.78 (s, 3H, 28-H3), 0.68 (s, 3H, 18-H3); 13C-NMR (100 MHz, CDCl3) δ (ppm)
134.7, 134.5, 88.8, 77.5, 77.2, 76.8, 74.8, 57.7, 51.1, 50.7, 50.0, 49.2, 44.6, 40.5, 39.0, 37.2, 37.0,
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36.6, 35.6, 31.2, 31.0, 28.4, 28.1, 26.6, 25.2, 24.4, 22.8, 21.2, 20.7, 19.3, 18.9, 18.3, 16.3, 15.9.
MS (ESI): calculated for C31H54O2Na [M+Na]+: 481.40, found 481.40.
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(5R,10S,13R,14R,17R)-17-((R)-6-hydroxy-6-methylheptan-2-yl)-4,4,10,13,14-pentamethyl-1,
2,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthren-3-one oxime
(21). To a 50 ml round-bottom flask, were added 100 mg of compound 20 (0.23 mmol, 1.0 eq), 47
mg of NH2OH.HCl (0.68 mmol, 3.0 eq), 55.8 mg of dry NaOAc (0.68 mmol, 3.0 eq), 10 ml EtOH.
After stirred at 60 °C for 3 h, the resulting solution was concentrated by evaporation. The mixture
was purified by 200-300 mesh silica gel flash column chromatography (Hexane:EtOAc = 10:1),
yielding 95 mg of compound 21 (I.Y. = 90%). 1H-NMR (400 MHz, CDCl3, representative signals)
δ (ppm) 9.11(s, br, 1H), 3.14-3.10(m, 1H), 0.88 (m, 6H, 21-H3, 30-H3), 0.68 (s, 3H, 18-H3);
13C-NMR (100 MHz, CDCl3) δ (ppm) 167.2, 134.9, 133.9, 71.3, 51.5, 50.6, 50.0, 44.6, 44.5, 40.5,
37.3, 36.8, 36.6, 35.8, 31.1, 31.0, 29.5, 29.3, 28.3, 27.0, 26.5, 24.4, 23.2, 21.3, 21.2, 19.1, 18.9,
18.8, 17.7, 15.9. MS (ESI): calculated for C30H52NO2 [M+H]+: 458.40, found 458.40.
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(5R,10S,13R,14R,17R)-17-((R)-6-hydroxy-6-methylheptan-2-yl)-4,4,10,13,14-pentamethyl-1,
2,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthren-3-one
O-methyl oxime (22). To a 50 ml round-bottom flask, were added 100 mg of compound 20 (0.23
.
mmol, 1.0 eq), 56.4 mg of MeONH2 HCl (0.68 mmol, 3.0 eq), 55.8 mg of dry NaOAc (0.68 mmol,
3.0 eq), 10 ml EtOH. After stirred at 60 °C for 3 h, the resulting solution was concentrated by
evaporation. The mixture was purified by 200-300 mesh silica gel flash column chromatography
1
(Hexane:EtOAc = 15:1), yielding 99 mg of compound 22 (I.Y. = 92%). H-NMR (400 MHz,
CDCl3, representative signals) δ (ppm) 3.81 (s, 3H), 3.00 (d, 1H, J = 14.4 Hz,), 0.89 (m, 6H,
21-H3, 30-H3), 0.69 (s, 3H, 18-H3); 13C-NMR (100 MHz, CDCl3) δ (ppm) 166.2, 134.9, 134.0,
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77.5, 77.2, 76.8, 71.3, 61.2, 51.6, 50.7, 50.0, 44.62, 44.56, 40.2, 37.2, 36.9, 36.6, 35.8, 31.1, 31.0,
29.5, 29.4, 28.4, 27.2, 26.5, 24.4, 23.4, 21.3, 21.2, 19.1, 18.8, 18.3, 16.0. LC-MS: calculated for
C31H54O2 [M+H]+: 472.41, found 472.99.
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(R)-6-((3S,5R,10S,13R,14R,17R)-3-amino-4,4,10,13,14-pentamethyl-2,3,4,5,6,7,10,11,12,13,14
,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-methylheptan-2-ol (23). To
a solution of 50 mg compound 21 (0.1 mmol, 1.0 eq) in THF was added 8.3 mg of LiAlH4 (0.2
mmol, 2.0 eq). After stirred at room temperature for 4 h, the reacting system was quenched with
water and then the mixture was extracted with DCM. The organic layer was purified by 200-300
mesh silica gel flash column chromatography (DCM:MeOH = 10:1), yielding 35 mg of compound
23 (I.Y. = 80%). 1H-NMR (400 MHz, CDCl3, representative signals) δ (ppm) 2.68 (s, 1H), 2.43 (d,
13
1H, J = 10.8 Hz), 0.67 (s, 3H, 18-H3); C-NMR (100 MHz, CDCl3) δ (ppm) 134.6, 134.2, 70.1,
68.4, 59.7, 51.1, 50.9, 50.5, 49.7, 44.3, 43.9, 38.3, 37.04, 36.97, 36.9, 36.7, 36.4, 35.8, 35.6, 31.0,
30.9, 30.5, 27.9, 27.7, 27.0, 26.3, 25.4, 25.2, 23.3, 20.69, 20.65, 18.4, 18.13, 18.07. LC-MS:
calculated for C30H54NO [M+H]+: 444.41, found 444.96.
(5R,10S,14R,17R)-17-((R)-6-hydroxy-6-methylheptan-2-yl)-4,4-bis(hydroxymethyl)-10,14-di
methyl-1,2,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-3H-cyclopenta[a]phenanthren-3-o
ne O-methyl oxime (24).
To a 10ml round bottom flask were added compound 22 (0.2 mmol, 94 mg), Pd(OAc)2 (0.1
o
equiv.), PhI(OAc)2 (3.0 equiv.) and AcOH/Ac2O (2 mL/2 mL). The reaction was stirred at 80 C
for 12 h and monitored by TLC. Water was added to dilute the reaction mixture and NaHCO3 was
added to neutralize the reaction mixture. Then reaction mixture was extracted by DCM for three
times, the organic layer dried over anhydrous Na2SO4 and concentrated on rotavapor under
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reduced pressure. Finally, the residue was purified by silica gel column chromatography to give
the compound 42 (51 mg, 45%). Then compound 42 was added to a solution of DCM/MeOH (1
mL/3 mL) containing MeONa (2.0 eq), the reaction system was stirred at room temperature for 12
h. Then reaction mixture was extracted by DCM for three times, the organic layer dried over
anhydrous Na2SO4 and concentrated on rotavapor under reduced pressure. Finally, the residue was
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purified by silica gel column chromatography to give the compound 24 (35 mg, 80%). H-NMR
(400 MHz, CDCl3, representative signals) δ (ppm) 4.08-4.02 (m, 2H), 3.90-3.77 (m, 6H), 3.51 (t,
1H, J = 6.4 Hz), 3.00 (dd, 1H, J = 16 Hz, J = 2.4 Hz), 2.26-2.18 (m, 1H), 1.20 (s, 6H, 26-H3,
27-H3), 1.07 (s, 3H, 19-H3), 0.89 (m, 6H, 21-H3, 30-H3), 0.68 (s, 3H, 18-H3); 13C-NMR (100
MHz, CDCl3) δ (ppm) 164.5, 135.2, 133.5, 71.2, 69.4, 65.4, 61.9, 50.6, 50.0, 49.0, 46.8, 44.53,
44.51, 37.0, 36.8, 36.6, 34.9, 31.0, 30.9, 29.5, 29.3, 28.3, 26.4, 24.4, 21.3, 21.2, 19.3, 19.1, 18.8,
15.9. MS (ESI): calculated for C30H51NO4Na [M+Na]+: 526.39, found 526.38.
(5aS,7aR,8R,10aR,12aR)-8-((R)-6-hydroxy-6-methylheptan-2-yl)-1,1,5a,7a,10a-pentamethyl-
1,4,5,5a,6,7,7a,8,9,10,10a,11,12,12a-tetradecahydrocyclopenta[5,6]naphtho[2,1-c]azepin-3(2
H)-one (25). To a round-bottom flask were added 50 mg of compound 21 (0.1 mmol, 1.0 eq) and
TFA/DCM : 1 ml/1 ml. After stirred at room temperature for 2 h, the resulting solution was
washed with saturated NaHCO3 and extracted with DCM. The organic layer was concentrated and
purified by 200-300 mesh silica gel flash column chromatography (Hexane:EtOAc = 30:1),
yielding 18 mg of compound 25 (I.Y. = 40%). 1H-NMR (400 MHz, CDCl3, representative signals)
δ (ppm) 8.38 (s, br, 1H), 3.13-3.07 (m, 1H), 2.19-2.10 (m, 1H), 1.14 (s, 3H, 28-H3), 1.06 (s, 6H,
29-H3, 19-H3), 0.88-0.84 (m, 6H, 21-H3, 30-H3), 0.69 (s, 3H, 18-H3); 13C-NMR (100 MHz,
CDCl3) δ (ppm) 167.3, 135.0, 134.0, 89.5, 51.5, 50.6, 50.0, 44.6, 40.9, 40.5, 37.3, 36.43, 36.38,
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35.8, 31.1, 31.0, 28.3, 27.1, 26.5, 25.8, 25.7, 24.4, 23.2, 21.2, 20.6, 19.1, 18.9, 18.7, 17.7, 16.0.
LC-MS: calculated for C30H52O2 [M+H]+: 458.39, found 458.74.
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4-(((3S,5R,10S,13R,14R,17R)-17-((R)-6-hydroxy-6-methylheptan-2-yl)-4,4,10,13,14-pentamet
hyl-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)ox
y)-4-oxobutanoic acid (26). To the solution of 40 mg compound 6 (0.1 mmol, 1.0 eq) in 2 ml
pyridine were added 12 mg of succinic anhydride (0.11 mmol, 1.1 eq) and 11mg of DMAP (0.1
mmol, 1.0 eq). After stirred at 80 °C for 3 h, the resulting solution was washed with 10% HCl and
extracted with EtOAc. The organic layer was concentrated and purified by 200-300 mesh silica
gel flash column chromatography (DCM:MeOH = 20:1), yielding 44 mg of compound 26 (I.Y. =
80%). 1H-NMR (400 MHz, CDCl3, representative signals) δ (ppm) 4.52 (dd, J = 11.6 Hz, J = 4.8
Hz, 1H), 2.69-2.63(m, 4H), 1.20 (s, 6H, 26-H3, 27-H3), 0.99 (s, 3H, 28-H3), 0.90-0.86 (m, 12H,
21-H3, 30-H3, 29-H3, 19-H3), 0.68 (s, 3H, 18-H3); 13C-NMR (100 MHz, CDCl3) δ (ppm) 172.0,
170.7, 134.6, 134.3, 81.6, 71.4, 60.6, 50.6, 49.9, 44.6, 44.5, 38.0, 37.0, 36.8, 36.6, 35.3, 31.1, 30.9,
29.5, 29.4, 29.3, 29.1, 28.5, 28.4, 28.0, 26.5, 24.4, 24.2, 21.3. 21.2, 21.1, 19.3, 18.8, 18.2, 16.7.
MALDI-TOF-MS: calculated for C34H56O5Na [M+Na]+: 567.40, found 567.441.
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2-(((3S,5R,10S,13R,14R,17R)-17-((R)-6-hydroxy-6-methylheptan-2-yl)-4,4,10,13,14-pentamet
hyl-2,3,4,5,6,7,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl)ox
y)acetic acid (27). To a sealed tube were added 224 mg of compound 6 (0.5 mmol, 1.0 eq), 8 mg
of Rh2(OAc)4 (5%), ethyl 2-diazoacetate, 10 ml DCM. After stirred at 40 °C for 12 h, the resulting
solution was concentrated by evaporation and purified by 200-300 mesh silica gel flash column
chromatography (Hexane:EtOAc = 30:1). The obtained product was dissolved in MeOH/H2O : 2
ml/1 ml, followed by the addition of 345.5 mg K2CO3 (2.5 mmol, 5.0 eq). After stirred at 80°C for
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5 h, the mixture was concentrated by evaporation and the extracted by DCM. The organic layer
was purified by 200-300 mesh silica gel flash column chromatography (DCM:MeOH = 50:1),
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yielding 62.8 mg of compound 27 (I.Y. = 25%). H-NMR (400 MHz, CDCl3, representative
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signals) δ (ppm) 4.07 (q, 2H), 2.90(d, 1H, J = 10 Hz), 1.15 (s, 6H, 26-H3, 27-H3), 0.97 (d, 6H,
29-H3, 19-H3), 0.83 (m, 9H, 21-H3, 30-H3, 28-H3), 0.66 (s, 3H, 18-H3); 13C-NMR (100 MHz,
CDCl3) δ (ppm) 134.76, 134.71, 88.87, 71.15, 51.15, 50.88, 50.10, 44.78, 44.51, 39.27, 37.23,
37.11, 36.81, 35.74, 31.31, 31.09, 28.94, 28.77, 28.51, 28.19, 26.74, 24.42, 23.52, 21.46, 21.32,
19.34, 18.84, 18.41, 16.35, 15.94.
(5S,8S,9S,10S,13R,14R,17R)-17-((R)-6-hydroxy-6-methylheptan-2-yl)-4,4,10,13,14-pentamet
hyltetradecahydro-11H-8,9-epoxycyclopenta[a]phenanthren-3-ol (28). To a solution of 50 mg
6 (0.11 mmol, 1.0 eq) in DCM (30 ml) was added 22.4 mg of m-CPBA (0.13 mmol, 1.2 eq) and 10
mg of NaHCO3 (0.12 mmol, 1.1 eq) at 0 ˚C. After stirred at room temperature for 12 h, the
resulting solution was washed with saturated NaHCO3. The organic layer was dried over
anhydrous Na2SO4 and the solvent was removed by evaporation, then the mixture was purified by
200-300 mesh silica gel flash column chromatography (Hexane:EtOAc = 5:1), yielding 43 mg of
compound 28 (I.Y. = 85%). ;1H-NMR(400 MHz, CDCl3, representative signals) δ (ppm)
3.21-3.19(m, 1H), 1.23 (s, 6H, 26-H3, 27-H3), 1.13 (s, 3H), 0.96 (s, 3H), 0.88 (d, J = 6.3 Hz, 3H,
21-H3), 0.79 (s, 3H, 30-H3), 0.75 (s, 3H, 28-H3); 13C-NMR (100 MHz, CDCl3) δ (ppm) 78.6,
71.2, 70.8, 68.3, 49.0, 48.5, 44.5, 43.7, 41.9, 38.6, 38.0, 36.7, 36.4, 33.0, 29.42, 29.36, 28.6, 28.4,
27.3, 27.0, 23.6, 21.6, 21.2, 20.1, 19.1, 17.1, 16.6, 16.4, 15.2. LC-MS: calculated for C30H52O3
[M-H]-:459.39, found 459.97.
(3S,5R,10S,13R,14R,17R)-17-((R)-6-hydroxy-6-methylheptan-2-yl)-4,4,10,13,14-pentamethyl
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