Targeting RORs nuclear receptors by novel synthetic steroidal inverse agonists for autoimmune disorders

Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry

journal homepage: w ww.elsevier.com/locate/bmc

Targeting RORs nuclear receptors by novel synthetic steroidal inverse

agonists for autoimmune disorders

Matteo Dal Prà ^a, Davide Carta ^a, Gyorgy Szabadkai ^b, Matteo Suman ^b, Yahima Frión-Herrera ^a,

Nicola Paccagnella ^a, Giulia Castellani ^a, Sara De Martin ^a, Maria Grazia Ferlin ^a,

⇑

^aDepartment of Pharmaceutical and Pharmacological Sciences, University of Padova, 35131 Padova, Italy

^bDepartment of Biomedical Sciences, University of Padova, 35131 Padova, Italy

a r t i c l e i n f o

a b s t r a c t

Article history:

Designing novel inverse agonists of NR ROR

c

t still represents a challenge for the pharmaceutical commu-

Received 5 September 2017

Revised 1 February 2018

Accepted 13 February 2018

Available online xxxx

nity to develop therapeutics for treating immune diseases. By exploring the structure of NRs natural

ligands, the representative arotenoid ligands and RORs speciﬁc ligands share some chemical homologies

which can be exploited to design a novel molecular structure characterized by a polycyclic core bearing a

polar head and a hydrophobic tail. Compound MG 2778 (8), a cyclopenta[a]phenantrene derivative, was

identiﬁed as lead compound which was chemically modiﬁed at position 2 in order to obtain a small

library for preliminary SARs. Cell viability and estrogenic activity of compounds 7, 8, 19a, 30, 31 and

32 were evaluated to attest selectivity. The selected 7, 8, 19a and 31 compounds were assayed in a

Keywords:

NR RORct

Inverse agonists

Gal4 UAS-Luc co-transfection

Autoimmune disorders

Gal4 UAS-Luc co-transfection system in order to determine their ability to modulate RORct activity in

a cellular environment. They were evaluated as inverse agonists taken ursolic acid as reference com-

pound. The potency of compounds was lower than that of ursolic acid, but their efﬁcacy was similar.

Compound 19a was the most active, signiﬁcantly reducing RORct activity at low micromolar

concentrations.

1. Introduction

receptor-related orphan receptors subfamily, which is constituted

by ROR

a

, RORb and ROR

c

or RORc. ROR

c

t is a splice variant of

t is selec-

Nuclear receptors (NRs) form a family of transcription factors

that are composed of modular protein structures with DNA- and

ligand-binding domains (DBDs and LBDs). The DBDs confer gene

target site speciﬁcity, whereas LBDs serve as control switches for

NR function. In each case the overall fold of the LBD is conserved

and the ligand is bound entirely within the protein, completing

the core as the protein refolds around it.¹It was shown that despite

the chemical diversity of the natural nuclear receptor ligands, their

volumes are highly conserved.²

For many NRs, both endogenous and synthetic small molecule

ligands bind to small pockets within the LBDs, resulting in confor-

mational changes that regulate transcriptional activity. This prop-

erty of NRs has proven to be a rich source as targets for developing

of therapeutics for a myriad of human diseases, ranging from

inﬂammatory diseases and cancer to endocrine and metabolic

diseases.³

ROR and is encoded by a single gene called RORc. RORc

c

tively expressed in thymocytes (T cells) and appears to drive the

activation and differentiation of CD4+ and CD8+ cells into IL17-

producing T helper cells (T_H17) and cytotoxic T cells (Tc17). T_H17

and Tc17 are effector cells that promote inﬂammation, adaptive

immunity, and autoimmunity by producing IL17 and other inﬂam-

matory cytokines such as IL21. Both synthetic and putative

endogenous agonists of ROR

basal activity of ROR t enhancing T_H17 cell proliferation. Among

the various transcriptional regulators ROR is a uniquely tractable

drug target for manipulating T_H17 cell development and function

in the context of autoimmune diseases.⁴The ROR

t LBD is an ideal

ct have been shown to increase the

c

domain to target via small molecules. Small molecules targeting

RORs come in at least two types: inverse agonists, which block

ROR-dependent transcriptional activity; and agonists, which

enhance the transactivation of RORs.⁵

The retinoic acid nuclear receptors subfamily includes RAR

a,

Since the discovery of the ﬁrst small molecule T0901317^6,8,9

RARb and RAR

c

and it is evolutionarily closed to the retinoic acid

(Table 1), many ROR

activity have been disclosed in the literature.^7,10Using the

T0901317 scaffold as a lead compound, a series of synthetic ROR

inverse agonists have been developed, including SR1001, SR1555,

ct ligands with agonistic and inverse agonistic

c

⇑

Corresponding author.

E-mail address: mariagrazia.ferlin@unipd.it (M.G. Ferlin).

https://doi.org/10.1016/j.bmc.2018.02.018

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M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

Table 1

Structure of RORs ligands.⁷

Name

Structure

Receptor preferences

Ligand type

Ref.

8,9

T0901317

RORa

, ROR

c

, LXRa

, LXRb, PXR, FXR, other

RORs: inverse agonist

LXRS, PXR, FXR: agonist

SR1001

SR1078

ROR

a

c

Inverse agonist

11

12

ROR

a

c

Agonist

SR3335

SR2211

ROR

a

c

Inverse agonist

6

ROR

12

TMP778

TMP920

ROR

c

Inverse agonist

13

ROR

GSK805

ROR

c

Inverse agonist

Agonist

13

14

Cholesterol

RORa

Cholesterol sulfate

ROR

a

c

Agonist

14

15

Digoxin

ROR

Inverse agonist

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M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

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Table 1 (continued)

Name

Structure

Receptor preferences

Ligand type

Ref.

16

Ursolic acid

ROR

c

Inverse agonist

7

a

-Hydroxycholesterol

ROR

a

c

Inverse agonist

17

7b-Hydroxycholesterol

ROR

a

c

7-Ketocholesterol

ROR

a

c

Inverse agonist

17

18

20a

-hydroxycholesterol

ROR

c

Agonist

22R-Hdroxycholesterol

Agonist

18

25-Hydroxycholesterol

24S-hydroxycholesterol

24,25-Epoxy-cholesterol

ROR

c

Agonist

18

19

ROR

a

c

Inverse agonist

ROR

c

24R-Hydroxycholesterol

Inverse agonist

19

and SR2211.^6–11Some structurally complex natural products, such

as digoxin and ursolic acid have also been reported to be ROR

inverse agonists.^15,16Dan Littman’s group, who discovered the cru-

cial role for ROR t in T_H17 cells, identiﬁed the cardiac glycoside

T cell lineages, and it was efﬁcient in a mouse EAE model. Digoxin

was also identiﬁed in a random screening campaign, as an inhibitor

of mouse and human T_H17 cell differentiation, and the crystal

c

structure of the LBD of RORct in complex with digoxin at 2.2 Å res-

digoxin as a speciﬁc inhibitor for ROR

c

t transcriptional activity

olution has been solved. (Fig. 2).^15,17

using a chemical library screening.¹⁵They conﬁrmed that digoxin

inhibited murine T_H17 cell differentiation without affecting other

Ursolic acid, another natural product, was also found in a com-

pound library screening as an inhibitor of RORc

t.¹⁶Importantly,

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M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

both digoxin and ursolic acid have cholesterol-like chemical struc-

tures, which might account for their similar action on the NR.

Recently, a team at Genentech identiﬁed N-isobutyl-N-((5-(4-

(methylsulfonyl)phenyl)thiophen-2-yl)methyl)-1-phenylmethane-

sulfonamide as a ROR

ing campaign.²⁰Although the development of ROR

agonists has shown signiﬁcant promise,^21,22the development of

new ROR t selective modulators with therapeutic potential still

ct inverse agonist via a biochemical screen-

ct inverse

c

remains an urgent need.

Wang et al.²³ﬁrst reported that the natural products 7

a-

hydroxycholesterol¹⁷and 24S-hydroxycholesterol¹⁹were inverse

agonists (i.e. functional antagonists) of RORa and RORc that sup-

pressed transcriptional activities in hepatocytes. Oxysterols are

well known natural ligands for the related NR including the liver

X receptor (LXR), therefore their interaction with the LBDs of RORs

was not surprising.²⁴Most small molecule inhibitors and drugs are

based on cyclic systems, which leads to a stiffening of the mole-

cule, resulting in enhanced target afﬁnity due to less entropy loss

upon binding. The structural homology of NRs suggested to evalu-

ate ligands for other class of receptors as possible cognate com-

pounds that opportunely modiﬁed could switch their target

classes becoming speciﬁc RARs/RORs agonists or inverse agonists.

Fig. 2. Digoxin binding mode in the RORc

t ligand binding domain.¹⁵

1.1. Designing a lead compound

Very recently, the authors were involved in expanding their

research in the ﬁeld of inﬂammatory and auto-immune diseases,

by modulating the activity of NRs. Looking through the NRs super-

family and the chemical variety of the ligands scaffolds (polyenes,

polycyclic compounds, aromatic or aliphatic rings, eicosanoids, far-

nesoids, oxysterols, and tryptamine) (Fig. 1), it could seem very

by a cyclopenta[a]phenantrene scaffold. The design of a novel

ROR inverse agonist lead candidate was rationalized by means

of a structure-based approach founded on hybridization of chemi-

cal structures, which mix the features of ROR natural ligands

(cholesterol-like derivatives, digoxin, ursolic acid) with the fea-

tures of representative arotenoids (Fig. 3).²⁴This choice was made

because RARs and RORs receptors are evolutionarily closed and

shared sequence homology.⁶

c

unlikely that a novel ROR

be designed. However, the authors decide to explore the possibility

to target ROR receptor with a novel lead candidate, characterized

c inverse agonist lead candidate could

c

Fig. 1. The natural ligands of nuclear receptors²

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M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

5

Fig. 3. Arotenoids selective ligands.

The envisaged novel RORs inverse agonist may result then con-

ceptually deﬁned by the following attributes:

compounds (7, 8, 19a, 31) were assayed in a Gal4 UAS-Luc co-

transfection system in order to determine their ability to modulate

RORct activity in a cellular environment. They were evaluated as

1) A central polycyclic fused structure is present in other natu-

ral ligands of different classes of NRs assuring a suitable

molecular volume to fulﬁll the LBD of the ROR receptors.

The conservation of volumes among the natural ligands of

nuclear receptors is likely to be a useful criterion in the

design of high-afﬁnity analogs.²It serves as a linker and sup-

porting structure for the other fundamental chemical func-

tions necessary for delivering the biological activity of the

compound.

inverse agonists taken ursolic acid as reference compound.²⁶

Results from the synthetic work and preliminary biological evalu-

ation are reported.

2. Results and discussion

2.1. Docking simulations of MG 2778 (8) in RORct LBD

We further employed molecular modelling analysis to simulate

MG 2778 (8) binding in the ROR t binding pocket. We selected the

crystal structure of ROR

2) An aromatic ring as usually represented in arotenoids²⁴

3) A large lipophilic scaffold (cyclic, polycyclic or poly methy-

lated scaffold) mimicking the cyclic RA function or other

bulky substituents connected to the polycyclic linker

4) A polar terminus corresponding to or mimicking the RA and

ursolic acid acidic function (COOH or any of the known

bioisosters or derivatives)

c

t in complex with one of the best-known

inverse agonists, digoxin (PDB code 3B0W).²⁷Computer docking

simulation of compound 8 was performed using Maestro 10.5

Glide software SP precision.

Fig. 5 shows the binding mode of the most favoured pose of

compound 8 in the presumptive binding site in comparison with

digoxin. We found that compound 8 could be readily accommodate

5) A hydroxylic function, as represented in arotenoids, choles-

terol-like ligands and ursolic acid

in the pocket. Moreover, RORct shows a binding pocket mostly

characterized by hydrophobic residues (Leu-287, Leu-292, Trp-

317, Cys-320, Ala-321, Ala-327, Val-361, Met-365, Ala-368, Val-

376, Phe-377, Phe-378, Phe-388, Leu-396) which suggests a bind-

ing interaction mode mainly characterized by hydrophobic interac-

tions. No direct interaction between compound 8 and the residues

responsible for digoxin binding was found.¹⁵However, even if the

molecular volume of compound 8 is smaller than that of digoxin, it

is possible that the bulky substituent in position 2 of the cyclo-

penta[a]phenanthrene core (which occupies the position of the

ﬁrst sugar ring in digoxin) might be sufﬁcient to disturb the polar

The molecular structure of a lead compound might be the tetra-

cycle MG 2778 as shown in Fig. 4:

The early objective was to develop an efﬁcient synthetic path

for obtaining the proposed compound as described in Fig. 4 (MG

2778). MG 2778 is a cyclopenta[a]phenantrene derivative bearing

an adamanthyl group at 2 position. This large group in position 2

was placed also because it was found to be effective in reducing

hormonal effects of estrone and estradiol analogs in non-feminiz-

ing neuroprotective agents and so preventing estrogen receptor

binding.²⁵It also has an

a-b-unsaturated carboxylic group at 16

interactions observed in the agonist-bound RORct LBD, involving

and a phenolic hydroxyl at position 3. Next, with the aim to obtain

preliminary SARs, a small series of analogs modiﬁed at position 2 of

the polycyclic nucleus with groups other than adamanthyl but

maintaining the lipophilic and bulky features was planned, since

a suitable substitution at this position is considered signiﬁcant

for giving selectivity. To synthesize 2-substituted analogs we

adopted methods such as Friedel-Crafts alkylation, acylation and

Suzuki-Miyaura cross-coupling reactions on aromatic ring. Six

compounds (7, 8, 19a, 30–32) were tested for cytotoxicity and

estrogen receptor activity. The selected four non-cytotoxic

His-479, Tyr-502 and Phe-506 which would be important to stabi-

lize the active conformation of helix H12.^15,17

2.2. Chemistry

The synthetic work has been organized into four schemes that

describe the optimized synthetic pathways as a result of trials to

improve yields and purity of reaction products. The schemes report

the routes carrying to ﬁnal compounds for the synthesis of which

the pre-formed polycyclic scaffold 3-hydroxyestra-1,3,5(10)-tri-

ene-17-one (estrone) was selected as starting material. In all cases,

the early protection of phenolic OH was necessary to prevent unre-

solvable mixtures formation along the pathway. Schemes 1 and 2

describe two alternative routes to obtain compound 8 (named

MG 2778) by performing the same reactions in a different order.

For this purpose, intermediate 1 was obtained from the starting

commercial estrone by alkylating with CH₃I in the presence of Bu₄-

NI and NaOH 10% in CH₂Cl₂at 70 °C (99% yield).²⁸As previously

reported,²⁹compound 1 was submitted to a Friedel-Crafts reaction

conducted with adamanthanol, BF₃Et₂O in hexane for 4 h. The

reaction proved to be highly region-speciﬁc yielding only the 2-

adamanthyl substituted compound 2 (95%yield). The following

16-C methoxycarbonylation reaction³⁰was carried out with

Fig. 4. Lead structure of MG 2778. The molecule can be divided into three parts: an

acidic head, a cyclopenta[a]phenantrene backbone, and a lipophilic tail. The activity

was investigated after structural modiﬁcation of lipophilic group.

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M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

Fig. 5. Comparison of the crystallographic structure of digoxin (in orange) in complex with RORct ligand binding domain (Protein Data Bank code 3B0W) and the

energetically most favourable pose of compound 8 (in green) obtained by molecular docking simulation. Hydrophobic residues are shown in white. Hydrogen atoms are

omitted.

Scheme 1. Synthesis of compound 8. Reagents and conditions: a) CH₃I, Bu₄N+I^ꢀ, CH₂Cl₂, NaOH 10%, ref., 3 h, 99%; b) 1-adamantanol, BF₃Et₂O, hexane, 4 h, 95%; c) C₃H₆O₃,

NaH, ref., 3 h, 93%; d) NaBH₄, THF/CH₃OH 9:1, 1 h, 90%; e) MsCl, Et₃N, anhydrous CH₂Cl₂; f) DBU, C₆H₆, ref., 6 h, 60%; g) NaOH, MeOH, CH₂Cl₂, 96 h, 90%; h) NaSCH₃, NMP, ref.,

9 h, 56%.

dimethyl carbonate, NaH at reﬂuxing (85 °C) for 3 h yielding com-

pound 3 (yield 93%). In order to form the 16–17 double bond, at

ﬁrst the 17-carbonyl group was reduced to secondary alcohol 4

2-adamanthyl substitution the procedure resulted difﬁcult. Thus,

the adamanthyl moiety was inserted at the end of the pathway.

Henceforward, compound

1 was transformed into the 16-

methoxycarbonylated derivative 9³⁰(93%) that was reduced to

the 17-hydroxylic derivative 10³¹(60%). Then, the last was mesy-

lated to compound 11 and this reacting with DBU produced the

precursor compound 12³²(84%) showing the 16–17 double bond.

At this point, the introduction of the adamanthyl group again pro-

duced only compound 6 but unfortunately with low yields (12%).²⁹

Evidently, the presence of the 16–17 double bond provoked the

formation of byproducts in the F-C reaction. Following, compound

13 gave the described acid 8 by reacting with NaSCH₃and NMP at

reﬂux.³⁴Accordingly, by comparing the two synthetic pathways

(Schemes 1 And 2), it was concluded that by the pathway in

Scheme 2 the scope to facilitate the synthetic work was achieved,

but despite the laborious work up, the pathway in Scheme 1 was

undoubtedly the more advantageous because of the higher yields.

Next, in view of the synthesis of various 2-substituted analogs

of 8, the synthetic work has proceeded with an assessment of the

reactivity of 3-methoxylated estrone 1 towards the Friedel-Crafts

31

by a chemo-selective reaction with NaBH₄in a mixture of THF/

CH₃OH 9:1 for 1 h at room temperature (yield 90%). The obtained

32

alcohol 4 was mesylated with MsCl in anhydrous CH₂Cl₂giving

the intermediate ester 17-methylsulfonate 5, which by treatment

with DBU in benzene³²for 6 h at 60 °C and after Flash Chromatog-

raphy puriﬁcation, furnished the precursor intermediate 6 (60%

yield).

The last step to produce the designed compound 8 was

attempted with various hydrolytic methods and most of them

failed. Among all, the treatment with, MeOH, NaOH 2 M, in

33

CH₂Cl₂for 96 h gave the acid derivative 7 by 95% yield and only

the method involving the use of NaSCH₃in NMP at reﬂuxing for

9 h³⁴was successful in giving the desired compound 8 with a yield

of 56%.

In Scheme 2, the route to compound 8 was set up in an attempt

to improve the work up of reaction mixtures. Indeed, through the

previous reactions Scheme 1, with compounds bearing the

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tion progress by TLC. After work-up of the reaction mixture, the

raw material was puriﬁed by Flash Chromatography giving three

compounds, identiﬁed as 19a, b and c. Unfortunately, the desired

compound 19a was present in lesser amount (25%), 19b (37,5%)

and 19c (37%). The different reactivity of benzoyl compound 18

in comparison with compound 6 (Schemes 1 and 2) towards

NaSCH₃has not been understood. In this case, the F-C acylation

reaction of 3-methoxy-estrone, as for some reported alkylation³⁵

other than with adamanthanol, was proved not to be a regio-speci-

ﬁc reaction. Therefore, it is possible to conclude that the lack of

region speciﬁcity of F-C acylation towards position 2 together with

the low yields of compound 19a might represent a drawback for

the future synthesis of novel 2-substituted analogs.

It is known that the Suzuki-Miyaura cross-coupling reaction³⁶is

a robust method to obtain a variety of aromatic derivatives because

of the large amount of commercially available boronic acids, there-

fore it was planned to study also the possibility to obtain novel 2-

substituted analogs by this kind of cross-coupling. In Scheme 4, the

synthesis of three novel 2-substituted compounds by this method

is reported.

Scheme 2. An alternative pathway for the synthesis of compound 8. Reagents and

conditions: a) C₃H₆O₃, NaH, ref., 3 h, 95%; b) NaBH₄, THF/CH₃OH 9:1, 1 h, 60%; c)

MsCl, Et₃N, anhydrous CH₂Cl₂, 84%; d) DBU, C₆H₆, ref., 6 h, 99%; e) NaOH, MeOH,

CH₂Cl₂, 96 h, 90%; f) 1-adamanthanol, BF₃Et₂O, hexane, 4 h, 12%; g) NaSCH₃, NMP,

ref., 5 h, 32%.

Preliminary results suggested an optimal pathway where the

starting estrone was protected as benzyl ether, easily removable

later in the path, giving compound 20 (BzCl, Bu₄NI) (99%)²⁸that

was then transformed into the 16-methoxycarbonylated derivative

21, as before (81%).³⁰This was ﬁrst selectively reduced with NaBH₄

to the corresponding alcohol 22 (68%).³¹After mesylation of 17-

hydroxy (23) and the next treatment with DBU, compound 24

was obtained (54%). In previous experiments it was seen that as

for 3-methoxy compound 12, also the 2-benzyloxy derivative 24

resulted not to be a suitable intermediate for iodination step. Thus,

compound 22 was catalytically reduced (Pd/C 10%, H₂)³⁷producing

the 2,17-dihydroxylic derivative 25 (93%) that was submitted to

the successful iodination to compound 26 with NIS, (CF₃SO₃)₃In

in CH₃CN for 8 h.³⁸Bromination had previously been carried out

on 3-methoxy-estrone 1 (Scheme 1) but it was slightly region-

selective (data not shown) and mainly with the 2-Br-derivatives

the cross-coupling did not take place later in the synthesis. The

iodination of compound 25 with NIS yielded the desired 2-iodi-

nated product 26 (51%) and a little amount of 4-iodinated and

2,4-diiodinated as deduced from ¹H NMR spectrum of the reaction

mixture. Therefore, iodinating with NIS and (CF₃SO₃)₃In proved to

be more region-selective compared with the other methods carried

out (data not shown). It is worth to underline that the chromato-

graphic puriﬁcation of 26 in presence of other two iodinated com-

pounds was only feasible when the two phenolic and alcoholic

hydroxyls were free. Unfortunately, for compound 26 16–17 dou-

ble bond formation was no longer possible. Preliminarily, the S-

M cross-coupling reaction of compound 26 was accomplished with

three boronic acids of different hindrance and following two differ-

ent methods: conventional synthesis³⁸and MW added organic

synthesis.³⁹The ﬁrst one provided only complex mixtures, while

the second one was found to be successful due to the following

advantages: shorter reaction times, higher yields, less by-products

and thus easier to process mixtures. After ﬂash chromatography

puriﬁcation, compounds 27–29 were obtained in good yields

26%, 33%, 42%, respectively.

Scheme 3. Synthesis of compound 19. Reagents and conditions: a) benzoyl

chloride, AlCl₃, anhydrous DCM, 3 h, 91%; b) C₃H₆O₃, NaH, rif., 3 h, 31%; c) NaBH₄,

THF/CH₃OH 9:1, 0.5 h, 94%; d) MsCl, Et₃N, anhydrous CH₂Cl₂; e) DBU, C₆H₆, rif., 5 h,

21%; f) NaSCH₃, DMF, 1 h., 63%.

(F-C) acylation and the Suzuky-Miyaura (S-M) cross-coupling reac-

tion. For this purpose, following the above useful pathway and car-

rying out the same kind of reactions as in Scheme 1, Scheme 3

describes the synthesis of 2-benzoyl-compound 14. The 3-meth-

oxy-estrone 1 was submitted to the F-C reaction with benzoyl

chloride in the presence of AlCl₃in CH₂Cl₂at 0 °C for 3 h.³⁵In this

case, a mixture of three compounds was obtained that were sepa-

rated by Flash Chromatography. As expected, due to the more elec-

tron-rich position 2, the 2-benzoyl-3-methoxy-derivative 14a was

retrieved in greater amount (58%), the 4-benzoyl-methoxy isomer

14b (31%) and in lesser amount the 2-benzoyl-3-hydroxy deriva-

tive 14c (2%). The last formed due to the demethylating property

of reaction conditions. Compound 14a was then transformed into

the 16-methoxycarbonylated derivative 15 (33%)³⁰before being

selectively reduced to the 17-hydroxylic compound 16 by NaBH₄

(97%).³¹This compound was ﬁrst mesylated (17, 17-OSO₂CH₃)³²

and thereafter by treatment with DBU, compound 18 (17-H)³²

showing the 16–17 double bond, was obtained (21%). Finally, com-

pound 18 was reacted with NaSCH₃in DMF³⁴for 1 h when at this

time the starting compound disappeared on monitoring the reac-

Finally, the three methyl esters 27–29 were transformed into

the corresponding acids by treatment with MeOH-NaOH 10% giv-

ing the compounds 30 (99%), 31 (98%) and 32 (97%).⁴⁰For all the

synthesized compounds, complete characterization was carried

out by mono-dimensional ¹H- ¹³C- and bi-dimensional HSQC,

HMBC and COSY NMR experiments.

Furthermore, it is noted that the synthesis described in

Scheme 4, despite the successful S-M cross-coupling on the iodi-

nated 26, presents a strong restriction due to the impracticality

to obtain the designed compounds with 16–17 double bond.

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M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

Scheme 4. Synthesis of compounds 30–32. Reagents and conditions: a) BzCl, Bu₄N⁺I^ꢀ, CH₂Cl₂, NaOH 10%, rif., 3 h, 99%; b) C₃H₆O₃, NaH, rif., 3 h, 31%; c) NaBH₄, THF/CH₃OH

9:1, 1 h, 68%; d) Pd/C, H₂, EtOAc, r.t., 8 h, 93%; e) NIS, (CF₃SO₃)₃In, CH₃CN, 8 h, 51%; f) 1. C₁₂H₁₁BO₂, Pd(PPh₃)₄, K₂CO₃, C₄H₈O₂, MW (160 °C), 30 min, 33%; 2. Pd(PPh₃)₄, K₂CO₃,

C₄H₈O₂, MW (160 °C), 30 min, 42%; 3. C₆H₇BO₂, Pd(PPh₃)₄, K₂CO₃, C₄H₈O₂, MW (160 °C), 30 min, 26%; g) MeOH, NaOH 10%, rif, 1 h, 99%.

Indeed, iodination reaction with NIS didn’t work with compounds

12 and 24 and additionally the chromatographic puriﬁcation of the

2-iodinated derivative was achievable only with the di-hydroxylic

compound 26 that however was not suitable for the removal of 17-

alcoholic OH by the method reported before.

formed on HepG2 cells. As shown in Fig. 6, compound 19a was

found to be toxic at the highest concentrations tested (25 M, p

< 0.01 vs vehicle; 50 M, p < 0.001 vs vehicle), whereas compounds

30 and 32 caused a signiﬁcant decrease of cell viability even at

lower concentrations. No cytotoxic effects were observed on after

incubation of HepG2 cells with compounds 7, 8 and 31 (Table 2).

l

2.3. Biology

2.3.2. Estrogenic activity of the synthetic compounds 7, 8, 19a and 30–

32

2.3.1. Effect of compounds 7, 8, 19a, 30–32 on cell viability

In order to verify whether the synthetic ROR

had any effect on cell growth and survival, MTT assay was per-

c

t inverse agonists

Estrogenic activity of the novel steroidal compounds 7, 8, 19a

and 30–32 was evaluated because of the molecular structure being

Fig. 6. Cell viability assay on HepG2 cells treated with the synthetic compounds 7, 8, 19a and 30–32, reported as percentage of viable cells with respect to control treated

with medium. Results are mean SEM. ^*p < 0.05, ^**p < 0.01 and ^***p < 0.001 vs vehicle, one-way ANOVA followed by Dunnett post hoc test. Three independent experiments

were performed in quadruplicate.

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M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

9

Table 2

Structure of compounds tested for cytotoxic and estrogenic activity.

Compound

Structure

Formula

C₃₀H₃₈O₃

MW

7

446.62

8

C₂₉H₃₆O₃

432.27

402.18

19a

C₂₆H₂₆O₄

30

C₃₁H₃₂O₄

468.59

31

32

C₃₁H₃₀O₅

482.57

392.49

C₂₅H₂₈O₄

derived from estrone, a known estrogenic agent. Real time PCR

analysis was performed on RNA extracts from an estrogen-recep-

tors (ERs) expressing cell line (MCF-7). Cells were treated with

the compounds in order to test whether the expression of GREB1

and CXCL12, two target genes for ERs, was altered. Fig. 7 shows

that the expression of both GREB1 and CXCL12 was increased by

compound 8 (p < 0.05 and p < 0.001 for GREB and CXCL12 mRNA

expression vs vehicle, respectively), 30 (p < 0.001 for GREB and

CXCL12 mRNA expression vs vehicle), 31 (p < 0.001 for GREB and

CXCL12 mRNA expression vs vehicle) and 32 (p < 0.001 and p <

0.05 for GREB and CXCL12 mRNA expression vs vehicle, respec-

tively), while neither compound 7 nor 19a display any estrogenic

activity.

fected with the plasmids UAS-Luc and NanoGlo do not express

the protein containing the ROR t LBD. Densitometric analysis con-

t is not expressed in lanes 2 and 3 (Data not

c

ﬁrmed that ROR

shown).

c

Fig. 9 shows the ability of the tested compounds to decrease

activity, as luminescence lessening, at various concentrations. After

2

l

M treatment only compound 19a displayed a slight but signiﬁ-

cant activity, at 5 M both compounds 8 and 19a decreased activ-

ity in a signiﬁcant amount, at 10 M a dramatic decrease in ROR

activity could be observed after addition for all the tested com-

pounds (p < 0.001), and ﬁnally, at 20 M all compounds showed

l

c

l

an inhibitory effect comparable to that of ursolic acid. Fig. 9 shows

that compounds 7, 8, 19a and 31 displayed a concentration-depen-

dent activity. Extrapolated IC50 values were similar for compounds

19a and 31 (4,4 mM and 4,7 mM, respectively), and increased for

compounds 7 and 8 (6,8 and 6,5 mM, respectively). The most rele-

2.3.3. Evaluation of inverse-agonist activity of compounds 7, 8, 19a

and 31 on RORct

Based on the MTT assay results, where compounds 7, 8 and 31

did not display any cytotoxic activity, and compound 19a was

cytotoxic only at the highest concentrations (25–50 mM), com-

pounds 7, 8, 19a and 31 were selected for evaluating their ability

vant outcome of the in vitro ROR

selected compounds was that compound 19a signiﬁcantly reduced

ROR t activity at low concentrations (2–5 mM, p < 0.05 vs vehicle).

ct inhibitory activity by the

c

to modulate the in vitro ROR

c

t activity in a cellular environment

2.3.4. Effect of compounds 7, 8, 19a and 31 on cell cycle distribution

In order to complete the characterization of the selected syn-

thetic compounds, we analyzed their effect on cell cycle distribu-

tion both in HepG2 and HEK-293 cells. Fig. 10 shows the effect of

compounds 7, 8, 19a and 31 on both cell viability of HepG2 and

by means of a Gal UAS-Luc cotransfection system taken ursolic acid

as reference compound. Since the absence of in vitro cytotoxicity at

low concentrations is a promising feature for candidate drugs

designed for lifetime lasting diseases such as autoimmune dis-

eases, no further in vitro characterization of 30 and 32 was per-

formed. To ascertain whether HEK-293 cells had been

successfully transfected with the plasmids, ROR

sion was evaluated by means of Western Blot analysis. As shown

in Fig. 8, the cells transfected with all three plasmids (ROR

Gal4, UAS-Luc, NanoGlo) express ROR t, whereas the cells trans-

HEK-293 either transfected or not with RORct-Gal4 plasmid, and

cell cycle distribution. After conﬁrming the absence of cytotoxicity

of the selected synthetic compounds on both cell lines, we also

demonstrated that cell cycle distribution was not affected even

ct protein expres-

c

-

after incubation with the highest concentrations (10 and 20

tested previously (see Section 2.3.3).

lM)

c

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10

M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

Fig. 7. Gene expression of GREB1 (A) and CXCL12 (B) in MCF-7 cells treated with the synthetic compounds 7, 8, 19a and 30–32. All the compounds were tested at 2 mM

concentration. Results are mean SEM. ^*p < 0.05 and ^***p < 0.001 vs vehicle, one-way ANOVA followed by Dunnett post hoc test. Three independent experiments were

performed in triplicate.

3. Conclusion

55 kDa

Looking the NRs natural ligands structure through, representa-

tive arotenoids ligands and RORs ligands, by means of a structure-

37 kDa

based approach founded on hybridization of chemical structures, a

lead compound 8 (MG 2778) was identiﬁed, synthesized and

chemically modiﬁed in order to obtain a small series of novel ster-

oidal compounds acting as ROR

tions of compounds 8 and 19a into ROR

digoxin showed a potential binding afﬁnity.

The four non-cytotoxic compounds 7, 8, 19a and 31 were tested

by means of a Gal UAS-Luc co-transfection system taken ursolic

c

inverse agonists. Docking simula-

c

t LBD in complex with

Fig. 8. Western blot analysis of RORct (58 kDa) protein in whole protein extracts of

HEK-293 cells transfected with Gal4-RORg LBD plasmid, UAS-luc and NanoLuc

reporter plasmid (+) or not-transfected cells (ꢀ). GAPDH (43 KDa) was used as

loading control.

acid as reference compound, resulting to act as RORct inverse ago-

nists in a dose dependent manner. Considering these preliminary

biological results, we can propose that using the tetracycle scaffold

is an appropriate approach for the further design of RORct inverse

2.4. Docking study of compound 19a

agonists. Regarding the bound groups at 2, 3 and 16 positions, we

can deduce that a bulky alkyl or aryl group in the 2 position is nec-

essary in order to reduce estrogenic activity, although low estro-

genic activity is maintained in presence of the free 3-phenolic

OH as for compound 8 with respect to compound 7 (3-OCH₃). How-

ever, no estrogenic activity was observed for compound 19a having

the free 3-OH. In this case, we suggest the existence of a H-bond,

between the carbonyl of the ﬂexible benzoylic group and the phe-

nolic OH. Probably, this event could hamper the interaction of the

OH itself at the ER, however, at the docking simulation of 19a in

Compound 19a was docked using the crystal structure of RORct

in complex with digoxin (PDB code 3B0W).²⁷Computer docking

simulation of 19a was performed using Maestro 10.5 Glide soft-

ware SP precision. The most favoured pose of 19a (Fig. 11) in the

presumptive binding site is similar to the one found for 8 (Fig. 5).

Compound 19a could be readily accommodated in the pocket,

but also in this case, no signiﬁcant interactions with residues

responsible for digoxin binding were found.¹⁵Again, we can sug-

gest the possibility that the substituent in position 2 (benzoyl

group in this case), could perturb the interactions necessary for

RORc

t activity.^15,17

RORct LBD (Fig. 10) we didn’t see it. The polar terminus (16-COOH)

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M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

11

Fig. 9. Evaluation of inverse-agonist activity of compounds 7, 8, 19a and 31 on ROR

vs vehicle, one-way ANOVA followed by Dunnett post hoc test. Three independent experiments were performed in triplicate.

c

t (A) and dose-dependent efﬁcacy (B). Results are mean Â SEM. ^*p < 0.05 and ^***p < 0.001

is essential for activity while the 16–17 double bond not as noted

for compound 31 that was active as well as compounds showing

the double bond at that position. The potency of our compounds

is lower than that of ursolic acid, the strongest known RORct

inverse agonist, but their efﬁcacy is similar. In particular, com-

pound 19a was the most active, causing a signiﬁcant reduction of

RORct activity at low micromolar concentrations. From the above

considerations, we can conclude that 19a may represent a good

candidate for further in vitro and in vivo characterization and

ﬁeld from tetramethylsilane as internal reference. Coupling con-

stants are given in hertz. In the case of multiplets, chemical

shifts were measured starting from the approximate centre. Inte-

grals were satisfactorily in line with those expected based on com-

pound structure. Mass spectra were obtained on a Mat 112 Varian

Mat Bremen (70 eV) mass spectrometer and Applied Biosystems

Mariner System 5220 LC/MS (nozzle potential 140 eV). Column

ﬂash chromatography was performed on Merck silica gel (250–

400 mesh ASTM); chemical reactions were monitored by analytical

thin-layer chromatography (TLC) on Merck silica gel 60F-254 glass

plates. Microwave assisted reactions were performed on a CEM

Discover^Òmonomode reactor with a built-in infrared sensor

assisted-temperature monitoring and automatic power control;

all reactions were performed in closed devices under pressure con-

trol. Solutions were concentrated on a rotary evaporator under

reduced pressure. The purity of new tested compounds was

checked by HPLC using the instrument HPLC VARIAN ProStar

may serve as a useful tool for developing RORct inverse agonists.

4. Experimental section

Melting points were determined on a Buchi M-560 capillary

melting point apparatus and are uncorrected. ¹H NMR spectra were

determined on Bruker 300 and 400 MHz spectrometers, with the

solvents indicated; chemical shifts are reported in d (ppm) down-

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12

M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

Fig.. 10. Cell viability of HepG2 (left) and HEK-293 (right) cells after incubation with compounds 7, 8, 19a and 31 at 10 and 20

20 M. The results are expressed as mean SEM. Three independent experiments were performed in duplicate.

lM. Below, cell cycle distribution analysis at

l

Fig. 11. Comparison of the crystallographic structure of digoxin (in orange) in complex with ROR

ct ligand binding domain (Protein Data Bank code 3B0W) and the

energetically most favourable pose of 19a (in green) obtained by molecular docking simulation. Hydrophobic residues are shown in white. Hydrogen atoms are omitted.

model 210, with detector DAD VARIAN ProStar 335. The analysis

was performed with a ﬂow of 1 mL/min, a C-8 column of dimen-

sions 250 mm ꢁ 4.6 mm, a particle size of 5 mm, and a loop of

10 mL. The detector was set at 254 nm. The mobile phase consisted

of phase A (Milli-Q H₂O, 18.0 MU, TFA 0.05%) and phase B (95%

MeCN, 5% phase A). Gradient elution was performed as reported:

0 min, % B ¼ 10; 20 min, % B ¼ 90; 25 min, % B ¼ 90; 26 min, % B

¼ 10; 31 min, % B ¼ 10.

Starting materials were purchased from Sigma-Aldrich and Alfa

Aesar, and solvents were from Carlo Erba, Fluka and Lab-Scan.

DMSO was obtained anhydrous by distillation under vacuum and

stored on molecular sieves.

Dulbecco’s modiﬁed Eagle’s medium (DMEM), was obtained

from Sigma-Aldrich Italy (Milan, Italy). Dulbecco’s modiﬁed Eagle’s

medium (DMEM), was obtained from Sigma-Aldrich Italy (Milan,

Italy). Foetal bovine serum (FBS), glutamine and penicillin-strepto-

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M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

13

mycin (pen-strep) solutions were obtained from Gibco (Life Tech-

nologies Italia, Monza, Italy).

solvent was removed under vacuum. The obtained residue was

treated with water to obtain a yellowish solid. The solid was ﬁl-

trated and dried overnight under vacuum to yield 1.55 g of yellow

powder. Yield 95%; R_f= 0.50 (cyclohexane/ethyl acetate, 8:2); mp

= 253 °C; ¹H NMR (400 MHz, CDCl₃): d 0.93 (s, 3H), 1.47 (m, 1H),

1.53 (m, 1H), 1.54 (m, 1H), 1.56 (m, 1H), 1.64 (m, 1H), 1.65 (m,

1H), 1.76 (6H), 1.97 (m, 1H), 2.01 (m, 1H), 2.06 (m, 1H), 2.11

(3H), 2.20–2.08 (6H), 2.30 (m, 1H), 2.47 (m, 1H), 2.53 (m, 2H),

2.92 (m, 2H), 3.83 (s, 3H), 6.63 (s, 1H), 7.18 ppm (s, 1H); ¹³C

NMR (101 MHz, CDCl₃): d 13.90, 21.61, 26.04, 26.63, 29.17, 29.30,

31.60, 35.92, 36.94, 37.16, 38.55, 40.79, 44.36, 48.08, 50.42,

55.03, 112.09, 123.68, 131.03, 134.72, 136.07, 156.87, 221.12

ppm. HRMS (ESI-MS, 140 eV): m/z [M+H⁺] calculated for

C₂₉H₃₉O⁺₂, 419.2950; found, 419.2932.

4.1. Synthesis

4.1.1. General procedure for the synthesis of of-protected estrone deri-

vatives 1 and 20

As a typical procedure, the synthesis of the-3-methoxy-estrone

derivative is described in detail. A mixture of commercial estrone

(1.00 g, 3.70 mmol) and tetrabutylammonium iodide (0.068 g,

0.185 mmol) was suspended in CH₂Cl₂(18 mL). Methyl iodide

(0.875 mL, 14.06 mmol) and a 10% NaOH solution (18 mL) were

added. The mixture was reﬂuxed at 70 °C for 3 h. The reaction

was monitored by TLC analysis (eluent chloroform/methanol

95:5). At the end of the reaction, the two phases were clearly trans-

parent and were separated. The aqueous phase was extracted with

CH₂Cl₂(30 mLꢁ3) and the combined organic phases were washed

with brine, dried over sodium sulphate, ﬁltered and evaporated

under vacuum to give a white solid product (1.045 g).

4.1.3. (8R,9S,13S,14S)-7,8,9,11,12,13,15,16-Octahydro-2-benzoyl-3-

methoxy-13-methyl-6H-cyclopenta[a]phenanthre ne-17(14H)-one (14)

In a dried round-bottomed ﬂask, a suspension of anhydrous

AlCl₃(1.260 g, 9.453 mmol) in 15 mL of anhydrous CH₂Cl₂was pre-

pared. The mixture was cooled to 0 °C and benzoyl chloride (0.880

mL, 7.574 mmol) was added dropwise. The mixture turned pink

and was stirred for 1 h at room temperature. After this period,

the mixture was cooled again at 0 °C and then a solution of com-

pound 1 (1.077 g, 3.787 mmol) in anhydrous CH₂Cl₂(10 mL) was

added dropwise. The reaction mixture turned yellow immediately

and it was kept at 0 °C for all the duration. The progression of the

reaction was monitored by TLC analysis (hexane/ethyl acetate 6:4).

At the end of the reaction, the suspension was poured into an ice/

water mixture and it was acidiﬁed with concentrated HCl. The two

phases were separated: the aqueous phase was extracted with

CH₂Cl₂and the resulting organic phase was washed with saturated

sodium bicarbonate solution, brine and dried over sodium sul-

phate. The mixture was ﬁltered, and the solvent evaporated under

vacuum to yield 1.344 g of white solid. Yield 58% 14a, 31% 14b, 2%;

14c; R_f= 0.49 (hexane/ethyl acetate, 6:4); mp = 229 °C; ¹H NMR

(300 MHz, CDCl₃): d 0.91 (s, 3H), 1.45 (m, 1H), 1.49 (m, 1H), 1.50

(m, 1H), 1.55 (m, 1H), 1.59 (m, 1H), 1.60 (m, 1H), 1.92 (m, 1H),

2.05 (m, 1H), 2.05 (m, 1H), 2.06 (m, 1H), 2.25 (m, 1H), 2.32 (m,

1H), 2.51 (m, 1H), 2.97 (m, 2H), 3.68 (s, 3H), 6.70 (s,1H), 7.30 (s,

1H), 7.39–7.46 (m, 2H); 7.50–7.57 (m, 1H), 7.81 ppm (dd, J = 8.3,

1.3 Hz, 2H); ¹³C NMR (400 MHz, CDCl₃): d 13.83, 21.56, 25.77,

26.40, 29.94, 31.45, 35.81, 38.26, 43.80, 47.94, 50.35, 55.64,

111.79, 126.47, 127.07, 128.08, 129.77, 131.98, 132.64, 138.20,

140.97, 155.54, 196.47, 220.60 ppm. HRMS (ESI-MS, 140 eV): m/z

[M+H⁺] calculated for C₂₆H₂₉O⁺₃, 389.2117; found, 389.2212.

4.1.1.1. (8R,9S,13S,14S)-7,8,9,11,12,13,15,16-Octahydro-3-methoxy-

13-methyl-6H-cyclopenta[a]phenanthren-17(14H)-one

(1). Yield

99%; R_f= 0.88 (chloroform/methanol, 95:5); mp = 177–178 °C; ¹H

NMR (300 MHz, CDCl₃): d 0.84 (s, 3H), 1.49 (m, 1H), 1.50 (m, 1H),

1.52 (m, 1H), 1.53 (m, 1H), 1.61 (m, 1H), 1.65 (m, 1H), 1.97 (m,

1H), 2.05 (m, 1H), 2.09 (m, 1H), 2.27 (m, 1H), 2.42 (m, 1H), 2.51

(m, 1H), 2.53 (m, 1H), 2.93 (m, 2H), 3.80 (s, 3H), 6.67 (d, J = 2.73

Hz, 1H), 6.75 (dd, J = 8.61 Hz, J = 2.73 Hz, 1H), 7.23 ppm (d, J =

8.61 Hz, 1H); ¹³C NMR (75 MHz, CDCl₃): d 13.86, 21.60, 25.94,

26.57, 29.68, 31.60, 35.88, 38.39, 43.99, 48.03, 50.43, 55.22,

111.59, 113.89, 126.35, 132.03, 137.77, 157.91, 220.94 ppm. HRMS

(ESI-MS, 140 eV): m/z [M+H⁺] calculated for C₁₉H₂₅O⁺₂, 285.1855;

found, 285.1865.

4.1.1.2. (8R,9S,13S,14S)-3-(Benzyloxy)-7,8,9,11,12,13,15,16-octahydro-

13-methyl-6H-cyclopenta[a]phenanthren-17(14H)-one (20). Com-

pound 20 was prepared as for compound 1 by reacting estrone

(2.50 g, 9.25 mmol), tetrabutylammonium iodide (0.178 g, 0.462

mmol), benzyl bromide (4.18 mL, 35.14 mmol) in a mixture of CH₂-

Cl₂/10% NaOH solution (45 mL each). After the workup, the obtained

residue was washed with hexane to remove excess benzyl bromide

yielding 3.301 g of yellow solid. Yield 99%; R_f= 0.38 (n-hexane/ethyl

acetate, 8:2); mp = 128–129 °C; ¹H NMR (400 MHz, CDCl₃): 0.91

ppm (s, 3H), 1.50 (m, 1H), 1.52 (m, 1H), 1.53 (m, 1H), 1.56 (m,

1H), 1.62 (m, 1H), 1.65 (m, 1H), 1.99 (m, 1H), d 2.04 (m, 1H), 2.09

(m, 1H), 2.17 (m, 1H), 2.29 (m, 1H), 2.43 (m, 1H), 2.51 (m, 1H),

2.88 (m, 2H), 5.04 (s, 2H), 6.73 (d, J = 2.7 Hz, 1H), 6.79 (dd, J = 8.6,

2.8 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.34–7.29 (m, 1H), 7.40–7.35

(m, 2H), 7.45–7.41 ppm (m, 2H); ¹³C NMR (101 MHz, CDCl₃): d

13.89, 21.62, 25.95, 26.58, 29.69, 31.63, 35.90, 38.40, 44.04, 48.04,

50.47, 70.00, 112.42, 114.94, 126.37, 127.45, 127.88, 128.57,

132.36, 137.29, 137.82, 156.90, 220.94 ppm. HRMS (ESI-MS, 140

eV): m/z [M+H⁺] calculated for C₂₅H₂₉O⁺₂, 361.2168; found,

361.2149.

4.1.4. General procedure for the synthesis of derivatives (3,9,15,21)

As a typical procedure, the synthesis of the methyl 2-adaman-

tyl-3-methoxy-16-carboxylate estrone derivative 3 is described

in detail. Compound 2 (0.640 g, 1.53 mmol) was suspended in

dimethyl carbonate (11.2 mL, 132.91 mmol) and NaH (0.320 g,

13.33 mmol) was added. A catalytic amount of CH₃OH was added.

The mixture was reﬂuxed for 3 h at controlled temperature (85 °C).

The reaction was monitored by TLC analysis (eluent cyclohexane/

ethyl acetate, 8:2). At the end of the reaction, the mixture was

cooled at room temperature and quenched with CH₃OH (1 mL).

The mixture was acidiﬁed with glacial acetic acid and poured into

water (150–200 mL). The suspension was stirred and once the pre-

cipitate was formed, ﬁltrated to obtain a yellow precipitate that

was dried overnight under vacuum to yield 0.511 g of yellow

powder.

4.1.2. (8R,9S,13S,14S)-7,8,9,11,12,13,15,16-Octahydro-2-adamantyl-

3-methoxy-13-methyl-6H-cyclopenta[a]phenanthre ne-17(14H)-one

(2)

Into a two-necked 100 mL round-bottomed ﬂask, compound 1

(1.08 g, 3.80 mmol) and 1-adamantanol (0.70 g, 4.60 mmol) were

placed and stirred for 15 min in hexane at 0 °C. Under N₂atmo-

sphere, BF₃Et₂O (1.6 mL, 12.74 mmol) was added dropwise with

a syringe. The mixture was stirred at room temperature for 4 h.

The reaction was monitored by TLC analysis (eluent cyclohexane/

ethyl acetate, 8:2). At the end of the reaction, the mixture was

transferred to a single-necked round-bottomed ﬂask and the

4.1.4.1. (8R,9S,13S,14S)-methyl-7,8,9,11,12,13,14,15,16,17-Decahy-

dro-2-adamantyl-3-methoxy-13-methyl-17-oxo-6H-cyclopenta[a]

phenanthrene-16-carboxylate (3). Yield 93%; R_f= 0.30 (cyclohex-

ane/ethyl acetate, 8:2); mp = 180–181 °C; ¹H NMR (300 MHz,

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M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

CDCl₃): d 0.98 (s, 3H), 1.37 (m, 1H), 1.51 (m, 1H), 1.52 (m, 1H), 1.57

(m, 1H), 1.63 (m, 1H), 1.65 (m, 1H), 1.76 (6H), 1.85 (m, 1H), 1.99

(m, 1H), 2.03 (m, 1H), 2.11 (3H), 2.20–2.08 (6H), 2.29 (m, 1H),

2.44 (m, 1H), 2.88 (m, 2H), 3.21 (dd, J = 9.9, 8.5 Hz, 1H), 3.76–

3.79 (s, 3H), 3.80 (s, 3H), 6.61 (s, 1H), 7.14 ppm (s, 1H); ¹³C NMR

(75 MHz, CDCl₃): d 13.31, 25.90, 26.40, 29.17, 29.30, 32.50, 36.90,

36.94, 37.16, 38.40, 40.79, 44.80, 47.87, 50.42, 52.50, 54.10,

55.03, 112.09, 124.20, 131.03, 134.72, 136.07, 156.87, 169.87,

212.05 ppm. HRMS (ESI-MS, 140 eV): m/z [M+H⁺] calculated for

(ESI-MS, 140 eV): m/z [M+H⁺] calculated for C₂₇H₃₁O⁺₄, 419.2222;

found, 419.2237.

4.1.5. General procedure for the synthesis of derivatives (4,10,16,22)

As a typical procedure, the synthesis of methyl 17-hydroxy-2-

adamantyl-3-methoxy-13-methyl-16-carboxylate 4 is described

in detail. Compound 3 (1711 g, 3.59 mmol) was suspended in a

mixture THF/CH₃OH 9:1 (20 mL). The mixture was cooled and stir-

red for 15 min at 0 °C, then NaBH₄(0.156 g, 4.12 mmol) was added

carefully in portions. The temperature was maintained at 0 °C and

the reaction was monitored by TLC analysis (eluent cyclohexane/

ethyl acetate, 2:1). The reaction was completed in 0.5 h. The mix-

ture was acidiﬁed with HCl 2 N solution and extracted with ethyl

acetate. The combined organic phases were washed with brine,

dried over sodium sulphate, ﬁltered and evaporated to dryness to

yield 1.725 g of spongy solid. The crude product was puriﬁed by

silica gel ﬂash column chromatography (hexane/ethyl acetate) to

give 1.53 g of white solid.

C

₃₁H₄₁O⁺₄, 477.3005; found, 477.3015.

4.1.4.2. (8R,9S,13S,14S)-methyl7,8,9,11,12,13,14,15,16,17-decahydro-

3-methoxy-13-methyl-17-oxo-6H-cyclopenta[a]phenanthrene-16-

carboxylate (9). Compound 9 was prepared as for compound 3 by

reacting compound 1 (1.045 g; 3.674 mmol) with dimethyl carbon-

ate (26.9 mL, 319 mmol) and NaH (0.768 g, 32.01 mmol). Yield

93%; R_f= 0.33 (hexane/ethyl acetate, 8:2); ¹H NMR (300 MHz,

CDCl₃): d 0.98 (s, 3H), 1.37 (m, 1H), 1.51 (m, 1H), 1.52 (m, 1H),

1.57 (m, 1H), 1.63 (m, 1H), 1.65 (m, 1H), 1.85 (m, 1H), 1.99 (m,

1H), 2.03 (m, 1H), 2.29 (m, 1H), 2.44 (m, 1H), 2.88 (m, 2H), 3.21

(dd, J = 9.9, 8.5 Hz, 1H), 3.76–3.79 (s, 3H), 3.80 (s, 3H), 6.67

(d, J = 2.73 Hz, 1H), 6.75 (dd, J = 8.61 Hz, 2.73 Hz, 1H), 7.23 ppm

(d, J = 8.61 Hz, 1H).

4.1.5.1. (8R,9S,13S,14S)-Methyl 7,8,9,11,12,13,14,15,16,17-decahydro-

17-hydroxy-2-adamantyl-3-methoxy-13-methyl-6H-cyclopenta[a]

phenanthrene-16-carboxylate (4). Yield 90%; R_f= 0.57 (hexane/

ethyl acetate, 2:1); mp = 210–211 °C; ¹H NMR (300 MHz, CDCl₃):

d 0.83 (s, 3H), 1.18 (m, 1H), 1.32 (m, 1H), 1.35 (m, 1H), 1.45 (m,

1H), 1.55 (m, 1H), 1.68 (m, 1H), 1.76 (6H), 1.91 (m, 1H), 1.80 (m,

1H), 2.02 (m, 1H), 2.02 (3H), 2.10 (6H), 2.32 (m, 1H), 2.35 (m,

1H), 2.83 (m, 2H), 3.14 (dd, J = 18.7, 9.1 Hz, 1H), 3.72 (s, 3H), 3.80

(s, 3H), 3.89 (d, J = 10.0 Hz, 1H), 6.59 (s, 1H), 7.16 ppm (s, 1H);

¹³C NMR (300 MHz, CDCl₃): d 11.67, 26.71, 27.25, 27.74, 29.48,

29.71, 37.24, 37.49, 37.62, 38.72, 41.11, 44.44, 44.57, 44.69,

48.96, 52.04, 55.10, 82.16, 112.39, 124.03, 131.70, 135.07, 136.25,

156.87, 175.94 ppm. HRMS (ESI-MS, 140 eV): m/z [M+H⁺] calcu-

lated for C₃₁H₄₃O⁺₄, 479.3161; found, 479.3149.

4.1.4.3. (8R,9S,13S,14S)-Methyl-7,8,9,11,12,13,14,15,16,17-decahydro-

2-benzoyl-3-methoxy-13-methyl-17-oxo-6H-cyclopenta[a]phenan-

threne-16-carboxylate (15). Compound 15 was prepared as for

compound 3 by reacting compound 14 (1.259 g; 3.241 mmol)

with dimethyl carbonate (23.7 mL, 281.5 mmol) and NaH

(0.677 g, 28.23 mmol). An amount of 1.290 g of a crude product

was obtained, and this was puriﬁed by silica gel ﬂash column

chromatography (hexane/ethyl acetate) to give 0.430 g of com-

pound 15. Yield 33%; R_f= 0.35 (hexane/ethyl acetate, 2:1); mp

= 114 °C; ¹H NMR (400 MHz, CDCl₃): d 0.99–0.96 (m, 3H), 1.40

(m, 1H), 1.41 (m, 1H), 1.42 (m, 1H), 1.45 (m, 1H), 1.52 (m, 1H),

1.90 (m, 1H), 2.07 (m, 1H), 2.07 (m, 1H), 2.24 (m, 1H), 2.26 (m,

1H), 2.27 (m, 1H), 2.91 (m, 2H), 3.22 (dd, J = 9.9, 8.4 Hz, 1H),

3.68 (s, 3H), 3.77 (s, 3H), 6.70 (s,1H), 7.29 (s, 1H), 7.39–7.46

(m, 2H); 7.50–7.57 (m, 1H), 7.81 ppm (dd, J = 8.3, 1.3 Hz, 2H);

¹³C NMR (101 MHz, CDCl₃): d 13.27/14.34, 25.70, 26.31, 26.39,

30.05, 31.57/31.77, 37.82, 43.78, 47.82, 48.89, 52.61, 54.01,

55.62, 111.75, 126.49, 127.05, 128.09, 129.77, 131.75, 132.69,

138.14, 140.91, 155.54/155.56, 169.80/170.32, 196.48, 211.79

ppm. HRMS (ESI-MS, 140 eV): m/z [M+H⁺] calculated for

4.1.5.2. (8R,9S,13S,14S)-Methyl 7,8,9,11,12,13,14,15,16,17-decahydro-

17-hydroxy-3-methoxy-13-methyl-6H-cyclopenta[a]phenanthrene-

16-carboxylate (10). Compound 10 was prepared as for compound

4 by reacting compound 9 (1.195, 3.49 mmol) with NaBH₄(0.151 g,

4.005 mmol) for 0.5 h, to give 1.166 g of spongy solid. The crude

product was puriﬁed by silica gel ﬂash column chromatography

(hexane/ethyl acetate) to give 0.717 g of white solid. Yield 60%;

R_f= 0.53 (hexane/ethyl acetate, 2:1); ¹H NMR (300 MHz, CDCl₃):

d 0.83 (s, 3H), 1.18 (m, 1H), 1.32 (m, 1H), 1.35 (m, 1H), 1.45 (m,

1H), 1.57 (m, 1H), 1.63 (m, 1H), 1.68 (m, 1H), 1.85 (m, 1H), 1.99

(m, 1H), 2.02 (m, 1H), 2.32 (m, 1H), 2.35 (m, 1H), 2.83 (m, 2H),

3.14 (dd, J = 18.7, 9.1 Hz, 1H), 3.72 (s, 3H), 3.80 (s, 3H), 3.88

(d, J = 10.0 Hz, 1H), 6.67 (d, J = 2.73 Hz, 1H), 6.75 (dd, J = 8.61 Hz,

2.73 Hz, 1H), 7.23 ppm (d, J = 8.61 Hz, 1H).

C

₂₈H₃₁O⁺₅, 447.2171; found, 447.2157.

4.1.4.4. (8R,9S,13S,14S)-Methyl 3-(benzyloxy)-7,8,9,11,12,13,14,15,

16,17-decahydro-13-methyl-17-oxo-6H-cyclopenta[a]phenanthrene-

16-carboxylate (21). Compound 21 was prepared as for compound

3 by reacting compound 20 (3.301 g; 9.157 mmol) with dimethyl

carbonate (67.03 mL, 795.46 mmol) and NaH (1.914 g, 79.78

mmol) to yield 3.104 g of yellow powder. Yield 81%; R_f= 0.27

(hexane/ethyl acetate, 8:2); mp = 155 °C; ¹H NMR (300 MHz,

CDCl₃): d 0.98 ppm (s, 3H), 1.45 (m, 1H), 1.46 (m, 1H), 1.48 (m,

1H), 1.51 (m, 1H), 1.62 (m, 1H), 1.63 (m, 1H), 1.98 (m, 1H), 2.03

(m, 1H), 2.07 (m, 1H), 2.25 (m, 1H), 2.40 (m, 1H), 2.89 (m, 2H),

3.21 (dd, J = 9.9, 8.5 Hz, 1H), 3.76 (s, 3H), 5.04 (s, 2H), 6.74 (d, J

= 2.6 Hz, 1H), 6.79 (dd, J = 8.6, 2.7 Hz, 1H), 7.20 (d, J = 8.5 Hz,

1H), 7.32 (t, J = 7.1 Hz, 1H), 7.38 (t, J = 7.3 Hz, 2H), 7.43 ppm

(d, J = 6.9 Hz, 2H); ¹³C NMR (75 MHz, CDCl₃): d 13.29, 25.78,

26.54, 29.56, 31.94, 36.90, 37.94, 43.99, 47.95, 48.94, 52.57,

54.07, 69.97, 112.47, 114.92, 126.32, 127.42, 127.87, 128.55,

132.00, 137.22, 137.67, 156.94, 169.85, 212.90 ppm. HRMS

4.1.5.3. (8R,9S,13S,14S)-Methyl 2-benzoyl-7,8,9,11,12,13,14,15,16,

17-decahydro-17-hydroxy-3-methoxy-13-methyl-6H-cyclopenta[a]

phenanthrene-16-carboxylate (16). Compound 16 was prepared as

for compound 4 by reacting compound 15 (0.564 g, 1.263 mmol)

with NaBH₄(0.055 g, 1.449 mmol) for 0.25 h, to give 0.553 g of

spongy solid. Yield 97%; R_f= 0.54 (hexane/ethyl acetate, 1:1); mp

= 250 °C; ¹H NMR (400 MHz, CDCl₃): d 0.84 (s, 3H), 1.23 (m, 1H),

1.31(m, 1H), 1.31 (m, 1H), 1.47 (m, 1H), 1.70 (m, 1H), 1.87 (m, 1H),

1.87 (m, 1H), 1.93 (m, 1H), 2.01 (m,1H), 2.17 (m, 1H), 2.98 (m, 2H),

3.14 (dd, J = 18.8, 9.1 Hz, 1H), 3.67 (s, 3H), 3.72 (s, 3H), 3.87 (d, J =

10.0 Hz, 1H), 6.68 (s,1H), 7.30 (s, 1H), 7.42 (t, J = 7.5 Hz, 2H), 7.54

(t, J = 7.3 Hz, 1H), 7.83–7.77 ppm (m, 2H); ¹³C NMR (101 MHz,

CDCl₃): d 11.28, 27.19, 27.44, 29.67/29.25, 30.01, 37.16/37.07,

38.14, 43.75, 44.05, 44.38, 48.65, 51.86, 55.64, 81.71, 111.76,

126.41, 127.16, 128.11/128.07, 129.79, 132.33, 132.59, 138.27,

Please cite this article in press as: Dal Prà M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.02.018

M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

15

141.02, 155.47, 175.41, 196.53 ppm. HRMS (ESI-MS, 140 eV): m/z [M

mmol). The obtained crude product 11 (0.659 g, 1.560 mmol) was

treated with DBU (0.466 mL, 3.120 mmol) and after the work-up,

it was puriﬁed by silica gel ﬂash column chromatography (hex-

ane/ethyl acetate) to give 0.523 g of white solid. Yield 84%; R_f=

0.83 (hexane/ethyl acetate, 2:1); ¹H NMR (300 MHz, CDCl₃): d

0.88 (s, 3H), 1.50 (m, 1H), 1.63 (m, 1H), 1.65 (m, 1H), 1.70 (m,

1H), 1.91 (m, 1H), 1.97 (m, 1H), 2.10 (m, 1H), 2.27 (m, 1H), 2.37

(m, 1H), 2.42 (m, 1H), 2.58 (m, 1H), 2.86 (m, 2H), 3.76 (s, 3H),

3.81 (s, 3H), 6.67 (d, 1H), 6.75 (d, J = 1.7 Hz, 1H), 7.23 ppm (d, 1H).

+H⁺] calculated for C₂₈H₃₃O⁺₅, 449.2328; found, 449.2548.

4.1.5.4. (8R,9S,13S,14S)-Methyl 3-(benzyloxy)-7,8,9,11,12,13,14,15,

16,17-decahydro-17-hydroxy-13-methyl-6H-cyclopenta[a]phenan-

threne-16-carboxylate (22). Compound 22 was prepared as for

compound 4 by reacting compound 21 (3.104 g, 7.42 mmol) with

NaBH₄(0.322 g, 8.52 mmol) for 0.5 h, to give 3.586 g of orange

solid. The crude product was puriﬁed by silica gel ﬂash column

chromatography (cyclohexane/ethyl acetate) to give 2.44 g of

white solid. Yield 68%; R_f= 0.45 (cyclohexane/ethyl acetate,

2:1); mp = 186 °C; ¹H NMR (300 MHz, CDCl₃): d 0.84 ppm (s,

3H), 1.38 (m, 1H), 1.41 (m, 1H), 1.55 (m, 1H), 1.56 (m, 1H),

1.77 (m, 1H), 1.90 (m, 1H), 2.05 (m, 1H), 2.12 (m, 1H), 2.26 (m,

1H), 2.36 (m, 1H), 2.89 (m, 2H), 3.13 (m, 1H), 3.73 (s, 3H), 3.88

(d, 1H), 5.03 (s, 2H), 6.72 (d, J = 2.7 Hz, 1H), 6.78 (dd, J = 8.4,

2.6 Hz, 1H), 7.20 (d, J = 8.5 Hz, 1H), 7.31 (dd, J = 8.5, 5.9 Hz,

1H), 7.37 (dd, J = 8.1, 6.5 Hz, 2H), 7.43 ppm (d, J = 6.9 Hz, 2H);

¹³C NMR (75 MHz, CDCl₃): d 11.29, 26.24, 27.32, 29.33, 29.69,

37.16, 38.19, 43.90, 44.04, 48.62, 51.88, 69.93, 81.76, 112.29,

114.79, 126.35, 127.43, 127.84, 128.53, 132.64, 137.25, 137.81,

156.74, 175.57 ppm. HRMS (ESI-MS, 140 eV): m/z [M+H⁺] calcu-

lated for C₂₇H₃₃O⁺₄, 421.2379; found, 421.2364.

4.1.6.3. (8S,9S,13S,14S)-Methyl 7,8,9,11,12,13,14,15-octahydro-2-

benzoyl-3-methoxy-13-methyl-6H-cyclopenta[a]phenanthrene-16-

carboxylate (18). Compound 18 was prepared as for compound 6

by reacting compound 16 (0.656 g, 1.462 mmol) with triethy-

lamine (0.289 mL, 2.077 mmol) and methanesulfonyl chloride

(0.119 mL, 1.546 mmol). The obtained crude product 17 (0.495 g,

0.940 mmol) was treated with DBU (0.317 mL, 2.126 mmol) and

after the work-up, it was puriﬁed by silica gel ﬂash column chro-

matography (hexane/ethyl acetate) to give 0.104 g of white solid.

Yield 21%; R_f= 0.66 (hexane/ethyl acetate, 2:1); mp = 107 °C; ¹H

NMR (300 MHz, CDCl₃): d 0.91 (s, 3H), 1.54 (m, 1H), 1.61 (m, 1H),

1.68 (m, 1H), 1.72 (m, 1H), 1.74 (m, 1H), 1.90 (m,1H), 2.04 (m,

1H), 2.35 (m, 1H), 2.38 (m,1H), 2.39 (m, 1H), 2.60 (m, 1H), 2.89

(m, 2H), 3.68 (s, 3H), 3.71 (s, 3H), 6.65 (s, 1H), 6.85 (d, J = 1.8 Hz,

1H), 7.22 (s, 1H), 7.44 (t, J = 7.4 Hz, 2H), 7.58–7.50 (m, 1H), 7.84–

7.77 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): d 15.95, 26.04, 27.65,

29.99, 31.23, 34.78, 37.10, 43.95, 46.99, 51.54, 54.88, 55.60,

111.57, 126.47, 126.79, 128.09, 129.83, 132.52, 132.59, 132.60,

134.72, 141.27, 154.60, 155.43, 166.51, 196.63 ppm. HRMS (ESI-

MS, 140 eV): m/z [M+H⁺] calculated for C₂₈H₃₁O⁺₄, 431.2222; found,

419.2473.

4.1.6. General procedure for the synthesis of derivatives (6,12,18)

As a typical procedure, the synthesis of methyl 2-adamantyl-3-

methoxy-16-carboxylate derivative 6 is described in detail. In a

double-necked round bottomed ﬂask compound

4 (0.659 g,

1.377 mmol) was dissolved in anhydrous CH₂Cl₂. Under a N₂atmo-

sphere, triethylamine (0.273 mL, 1.956 mmol) was added dropwise

to the solution and then methanesulfonyl chloride (0.112 mL,

1.456 mmol) was poured into the mixture. The obtained solution

was stirred overnight. The mixture was then washed with water,

saturated NaHCO₃solution, brine, ﬁltered and evaporated under

vacuum to yield a yellow solid (5). The obtained residue (0.741 g,

1.331 mmol) was then dissolved in benzene (20 mL), and DBU

(0.397 mL, 2.662 mmol) was added. Under a N₂atmosphere, the

reaction mixture was reﬂuxed for 5 h. The progress of the reaction

was monitored by TLC analysis (hexane/ethyl acetate 2:1). Even

though the reaction was not completed, the mixture was cooled

and washed with equivalent volumes of 5% HCl solution, brine

and saturated NaHCO₃solution. The organic phase was evaporated

to dryness under vacuum and the obtained crude product was

puriﬁed by silica gel ﬂash column chromatography (hexane/ethyl

acetate) to give 0.179 g of white solid correspondent to the desired

product (6) and 0.287 g of starting material.

4.1.7. (8S,9S,13S,14S)-7,8,9,11,12,13,14,15-Octahydro-2-adamantyl-

3-methoxy-13-methyl-6H-cyclopenta[a]phenanthrene-16-carboxylic

acid (7)

In a round bottomed ﬂask, compound 6 (0.166 g, 0.360 mmol)

was dissolved in a mixture of CH₂Cl₂/CH₃OH (9:1), and then 2 mL

of 3 M methanolic NaOH solution were added. The mixture was

stirred at room temperature for 96 h. The progression of the reac-

tion was monitored by TLC analysis (hexane/ethyl acetate 2:1). At

the end of the reaction, 1 M HCl solution was added and the

organic phase was extracted with CHCl₃. The combined organic

phases were washed with 1 M HCl solution, brine and dried over

sodium sulphate. After ﬁltration, the organic phase was evaporated

to dryness to yield 0.153 g of white solid. Yield 95%; R_f= 0.49 (hex-

ane/ethyl acetate, 2:1); mp = over 300 °C; ¹H NMR (300 MHz,

CDCl₃): d 0.89 (s, 3H), 1.46 (m, 1H), 1.58 (m, 1H), 1.65 (m, 1H),

1.72 (m, 1H), 1.77 (6H), 1.81 (m, 1H), 1.95 (m, 1H), 2.03 (m, 1H),

2.05 (3H), 2.09–2.06 (6H), 2.27 (m, 1H), 2.36 (m, 1H), 2.37 (m,

1H), 2.56 (m, 1H), 2.88 (m, 2H), 3.80 (s, 3H), 6.61 (s, 1H), 7.05 (d,

J = 1.7 Hz, 1H), 7.14 (s, 1H); ¹³C NMR (75 MHz, CDCl₃): d 16.38,

26.65, 28.13, 29.46, 29.59, 31.19, 35.20, 37.22, 37.47, 37.72,

41.09, 44.85, 47.79, 55.27, 55.35, 112.43, 123.62, 131.77, 134.74,

135.15, 136.19, 157.03, 157.88, 170.75 ppm. HRMS (ESI-MS, 140

eV): m/z [M+H⁺] calculated for C₃₀H₃₉O⁺₃, 447.2899; found,

447.2878. RP-C8 HPLC: t_R= 19.80 min, 98.9% (A%).

4.1.6.1. (8S,9S,13S,14S)-Methyl 7,8,9,11,12,13,14,15-octahydro-2-

adamantyl-3-methoxy-13-methyl-6H-cyclopenta [a] phenanthrene-

16-carboxylate (6). Yield 60%; R_f= 0.80 (hexane/ethyl acetate,

2:1); mp = 179 °C; ¹H NMR (300 MHz, CDCl₃): d 0.88 (s, 3H), 1.50

(m, 1H), 1.63 (m, 1H), 1.65 (m, 1H), 1.70 (m, 1H), 1.77 (6H), 1.91

(m, 1H), 1.97 (m, 1H), 2.05 (3H), 2.09–2.06 (6H), 2.10 (m, 1H),

2.27 (m, 1H), 2.37 (m, 1H), 2.42 (m, 1H), 2.58 (m, 1H), 2.86 (m,

2H), 3.76 (s, 3H), 3.81 (s, 3H), 6.62 (s, 1H), 6.92 (d, J = 1.7 Hz, 1H),

7.14 (s, 1H); ¹³C NMR (75 MHz, CDCl₃): d 16.48, 26.68, 28.13,

29.48, 29.55, 31.51, 35.38, 37.24, 37.49, 37.73, 41.11, 44.90,

47.50, 51.73, 55.30, 55.34, 112.44, 123.60, 131.85, 135.14, 135.26,

136.16, 155.12, 157.02, 166.78 ppm. HRMS (ESI-MS, 140 eV): m/z

[M+H⁺] calculated for C₃₁H₄₁O⁺₃, 461.3056; found, 461.3067.

4.1.8. (8S,9S,13S,14S)-7,8,9,11,12,13,14,15-Octahydro-2-adamantyl-

3-hydroxy-13-methyl-6H-cyclopenta[a]phenanthrene-16-carboxylic

acid (8)

Compound 7 (0.103 g, 0.217 mmol) was dissolved in 5 mL of

NMP and treated with a suspension of NaSCH₃(0.092 g, 1.32

mmol) in 5 mL of NMP. The mixture was reﬂuxed for 5 h and mon-

itored by TLC analysis. Once the starting material spot disappeared

on TLC, a mixture of water and ice was added, and then 1 M HCl

solution until pH = 1. The mixture was extracted with ethyl acet-

4.1.6.2. (8S,9S,13S,14S)-Methyl 7,8,9,11,12,13,14,15-octahydro-3-

methoxy-13-methyl-6H-cyclopenta[a]phenanthrene-16-carboxylate

(12). Compound 12 was prepared as for compound 6 by reacting

compound 10 (0.660 g, 1.916 mmol) with triethylamine (0.379

mL, 2.722 mmol) and methanesulfonyl chloride (0.119 mL, 2.026

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16

M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

ate, washed with water, brine and dried over sodium sulphate. The

solvent was evaporated under vacuum and the black residue

obtained was dissolved with diluted NH₃solution. The solution

was acidiﬁed again with 1 M HCl until pH = 1 to obtain a subtle

precipitate. The suspension was centrifugated and the supernatant

discarded. The obtained powder was dried to yield 0,057 g of ﬁnal

product. Yield 56%; R_f= 0.49 (hexane/ethyl acetate, 2:1); mp = over

300 °C; ¹H NMR (400 MHz, CDCl₃): d 0.89 (s, 3H), 1.46 (m, 1H), 1.72

(m,1H), 1.75 (m, 1H), 1.76 (m, 1H), 1.77 (6H), 1.93 (m, 1H), 1.97 (m,

1H), 2.06 (m, 1H), 2.07 (3H), 2.11 (6H), 2.30 (m, 1H), 2.31 (m, 1H),

2.42 (m, 1H), 2.66 (m, 1H), 2.88 (m, 2H), 6.39 (s, 1H), 7.03 (d, J = 1.7

Hz, 1H), 7.12 ppm (s, 1H). ¹³C NMR (101 MHz, CDCl₃): d 16.98,

26.16, 28.91, 29.46, 29.59, 32.06, 34.85, 36.45, 37.47, 37.65,

40.07, 44.52, 47.39, 55.15, 116.3, 123.74, 131.74, 133.69, 134.19,

134.73, 151.14, 157.60, 168.99 ppm. HRMS (ESI-MS, 140 eV): m/z

[M+H⁺] calculated for C₂₉H₃₇O⁺₃, 433.2743; found, 433.2761. RP-

C8 HPLC: t_R= 16.59 min, 99.1% (A%).

1H), 2.26 (m, 1H), 2.71 (m, 2H), 3.04 (q, J = 8.7 Hz, 1H), 3.60 (s,

3H), 3.77 (s, 1H), 5.00 (dd, J = 8.8, 5.4 Hz, 1H), 6.43 (d, J = 2.6 Hz,

1H), 6.50 (dd, J = 8.4, 2.5 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 8.99

ppm (s, 1H); ¹³C NMR (75 MHz, DMSO d₆): d 12.11, 26.62, 27.46,

28.59, 29.63, 37.08, 38.71, 43.88, 44.53, 46.82, 48.94, 51.72,

80.89, 113.32, 115.38, 126.64, 130.85, 137.71, 155.37, 175.70

ppm. HRMS (ESI-MS, 140 eV): m/z [M+H⁺] calculated for

C

₂₀H₂₇O⁺₄, 331.1909; found, 331.1901.

4.1.11. (8R,9S,13S,14S)-Methyl 7,8,9,11,12,13,14,15,16,17-decahydro-

3,17-dihydroxy-2-iodo-13-methyl-6H-cyclopenta[a]phenanthrene-

16-carboxylate (26)

Compound 25 (1.550 g, 4.69 mmol), N-iodosuccinimide (1.161

g, 5.160 mmol), Indium (III) triﬂuoromethanesulfonate (0.264 g,

0.47 mmol) were mixed together and dissolved in acetonitrile.

The mixture was stirred overnight in the dark (wrapped in foil)

at room temperature. The progression of the reaction was moni-

tored by TLC analysis (cyclohexane/ethyl acetate 1:1). At the end

of the reaction water was added and the organic phase was

extracted with ethyl acetate. The combined organic phases were

washed with brine and dried over sodium sulphate. After ﬁltration,

the solvent was evaporated under vacuum to yield 2.183 g of yel-

low product. The product was puriﬁed by silica gel column chro-

matography (d = 3 cm, l = 35 cm, 230–400 mesh, eluent

cyclohexane/ ethyl acetate 1:1) to yield 0.639 g of white product.

Yield 30%; R_f= 0.66 (cyclohexane/ethyl acetate, 1:1); mp = 179 °C

¹H NMR (300 MHz, CDCl₃): d 0.82 (s, 3H), 1.16 (m, 1H), 1.31 (m,

1H), 1.32 (m, 1H), 1.48 (m, 1H), 1.51 (m, 1H), 1.76 (m, 1H), 1.88

(m, 1H), 2.05 (m, 1H), 2.09 (m, 1H), 2.16 (m, 1H), 2.24 (m, 1H),

2.78 (m, 2H), 3.13 (dd, J = 18.7, 9.2 Hz, 1H), 3.72 (s, 3H), 3.88 (d,

J = 10.1 Hz, 1H), 5.74 (s br, 1H), 6.68 (s, 1H), 7.51 ppm (s, 1H); ¹³C

NMR (75 MHz, CDCl₃): d 11.33, 26.28, 27.10, 29.23, 29.28, 37.02,

37.94, 43.52, 44.15, 45.99, 48.53, 51.99, 81.68, 82.23, 115.00,

134.75, 135.20, 138.94, 152.83, 175.63 ppm. HRMS (ESI-MS, 140

eV): m/z [M+H⁺] calculated for C₂₀H₂₆IO⁺₄, 457.0876; found,

457.0853.

4.1.9. (8S,9S,13S,14S)-7,8,9,11,12,13,14,15-Octahydro-2-benzoyl-3-

hydroxy-13-methyl-6H-cyclopenta[a]phenanthrene-16-carboxylic acid

(19)

Compound 18 (0.124 g, 0.289 mmol) was dissolved in 5 mL

DMF and treated with NaSCH₃(0.123 g, 1.759 mmol). The mixture

was reﬂuxed for 1 h and monitored by TLC analysis (hexane/ethyl

acetate 1:1). Once the starting material spot disappeared on TLC,

DMF was evaporated under vacuum and the residue was acidiﬁed

with 1 M HCl. The mixture was extracted with ethyl acetate,

washed with water, brine and dried over sodium sulphate. The sol-

vent was evaporated under vacuum to give 0.074 g of a spongy yel-

low solid. The crude product was puriﬁed by RP-C18 ﬂash column

chromatography (tetrahydrofuran/water 8:2) to give a solid corre-

spondent to the products: 25% 19a, 37,5% 19b and 37% 19c as

approximately evaluated by ¹H NMR. The mixture was further sep-

arated by a ﬂash column chromatography (Ethyl acetate/hexane

8:2) yielding the desired compound 0.0185 g. Overall yield 16%;

mp = over 300 °C; ¹H NMR (300 MHz, CDCl₃): d 0.87 (s, 3H), 1.56

(m, 1H), 1.66 (m, 1H), 1.68 (m, 1H), 1.70 (m, 1H), 1.71 (m, 1H),

1.91 (m, 1H), 2.02 (m, 1H), 2.31 (m, 1H), 2.31 (m, 1H), 2.36 (m,

1H), 2.58 (m, 1H), 2.91 (m, 2H), 6.69 (s, 1H), 6.90 (d, J = 1.7 Hz,

1H), 7.28 (s, 1H), 7.42 (t, J = 7.4 Hz, 2H), 7.57–7.50 (m, 1H), 7.83–

7.78 (m, 2H); ¹³C NMR (75 MHz, CDCl₃): d 16.09, 26.11, 27.56,

29.98, 31.16, 34.81, 37.12, 43.99, 47.10, 55.62, 111.77, 126.27,

126.78, 128.07, 129.79, 132.52, 132.61, 132.61, 134.88, 141.17,

154.67, 155.45, 166.40, 196.66 ppm. HRMS (ESI-MS, 140 eV): m/z

[M+H⁺] calculated for C₂₆H₂₇O⁺₄, 403.1909; found, 403.1889. RP-

C8 HPLC: t_R= 17.75 min, 98.7% (A%).

4.1.12. General procedure for the synthesis of derivatives (27, 28, 29)

As a typical procedure, the synthesis of (8R,9S,13S,14S)-methyl

7,8,9,11,12,13,14,15,16,17-decahydro-3,17-dihydroxy-13-methyl-

2-(4-byphenyl)-6H-cyclopenta[a]phenanthrene-16-carboxylate 27

is described in detail. Compound 26 (0.200 g, 0.438 mmol) was dis-

solved in dioxane (2 mL) and then biphenyl boronic acid (0.174 g,

0.880 mmol), potassium carbonate (0.243 g, 1.760 mmol) and tet-

rakis(triphenylphosphine)palladium (0) (0.050 g, 0.045 mmol)

were added. The mixture was microwave irradiated at 160 °C

(power set point 250 W, ramp time 60 s, hold time 30 min). The

reaction progression was monitored by TLC analysis (hexane/ethyl

acetate 1:1). At the end of the reaction, the mixture was diluted

with water (10 mL) and extracted with ethyl acetate. The com-

bined organic phases were dried over sodium sulphate, ﬁltered

and the solvent removed under vacuum. The crude product was

puriﬁed by silica gel ﬂash-column chromatography (hexane/ethyl

acetate) to give 0.081 g of compound 27.

4.1.10. (8R,9S,13S,14S)-Methyl 7,8,9,11,12,13,14,15,16,17-decahydro-

3,17-dihydroxy-13-methyl-6H-cyclopenta[a]phenanthrene-16-carbo-

xylate (25)

Into a double-necked round bottomed ﬂask, previously dried in

oven, about 0.300 g of Pd/C 10% and approximately 40 mL of ethyl

acetate were placed. After connecting the ﬂask to an elastomer bal-

loon containing hydrogen gas, the mixture was stirred at room

temperature for 1 h to saturate the suspension of Pd/C with hydro-

gen. Then, compound 22 (2.121 g, 5.04 mmol) in 20 mL of ethyl

acetate was added dropwise to the suspension, and the mixture

was stirred under hydrogen at atmospheric pressure and heated

by means of an oil bath at 50 °C for 8 h, monitoring the progression

of the reaction by TLC analysis (cyclohexane/ethyl acetate 2:1). At

the end of the reaction the mixture was ﬁltered, and the solution

was concentrated to dryness on a rotavapor to give 1.550 g of

white solid. Yield 93%; R_f= 0.20 (cyclohexane/ethyl acetate, 2:1);

mp = 125 °C ¹H NMR (300 MHz, DMSO d₆): d 0.76 (s, 3H), 1.14

(m, 1H), 1.21 (m, 1H), 1.24 (m, 1H), 1.25 (m, 1H), 1.32 (m, 1H),

1.41 (m, 1H), 1.81 (m, 1H), 1.82 (m, 1H), 1.83 (m, 1H), 2.11 (m,

4.1.12.1. (8R,9S,13S,14S)-Methyl 7,8,9,11,12,13,14,15,16,17-decahy-

dro-3,17-dihydroxy-13-methyl-2-(4-byphenyl)-6H-cyclopenta[a]phe-

nanthrene-16-carboxylate (27). Yield 33%; R_f= 0.54 (hexane/ethyl

acetate, 1:1); mp = 232 °C; ¹H NMR (400 MHz, MeOD-d₄): d 0.94

(s, 3H), 1.46 (m, 1H), 1.65 (m, 1H), 1.69 (m, 1H), 1.79 (m, 1H),

1.81 (m, 1H), 1.85 (m, 1H), 1.90 (m, 1H), 1.91 (m, 1H), 1.93 (m,

1H), 2.03 (m, 1H), 2.65 (m, 1H), 2.74 (m, 1H), 2.76 (m, 1H), 2.95

(m, 1H), 3.70 (s, 3H), 3.82 (d, J = 10.5 Hz, 1H), 6.75 (s, 1H), 7.39

(s, 1H), 7.41 (m, AA’BB’, 2H), 7.45 (m, AA’BB’, 2H), 7.62 (m, 1H),

7.68 (m, 2H), 7.65 (m, 2H) ppm. ¹³C NMR (101 MHz, MeOD-d4):

d 11.64, 24.70, 26.41, 29.32, 34.27, 36.48, 39.02, 42.54, 44.97,

Please cite this article in press as: Dal Prà M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.02.018

M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

17

47.91, 53.39, 55.64, 84.23, 114.35, 127.01, 127.28, 127.50, 127.61,

127.67, 128.78, 129.03, 137.41, 138.56, 139.34, 140.25, 142.65,

158.70, 174.60 ppm. HRMS (ESI-MS, 140 eV): m/z [M+H⁺] calcu-

lated for C₃₂H₃₅O⁺₄, 483.2535; found, 483.2547.

centrifugated and the supernatant discarded. The obtained powder

was dried to yield 0,069 g of ﬁnal product.

4.1.13.1. (8R,9S,13S,14S)-7,8,9,11,12,13,14,15,16,17-Decahydro-3,

17-dihydroxy-13-methyl-2-(4-byphenyl)-6H-cyclopenta[a]phenan-

threne-16-carboxylic acid (30). Yield 98%; mp = over 300 °C; ¹H

NMR (400 MHz, MeOD-d₄): d 0.94 ppm (s, 3H), 1.46 (m, 1H), 1.65

(m, 1H), 1.69 (m, 1H), 1.79 (m, 1H), 1.81 (m, 1H), 1.85 (m, 1H),

1.90 (m, 1H), 1.91 (m, 1H), 1.93 (m, 1H), 2.03 (m, 1H), 2.65 (m,

1H), 2.74 (m, 1H), 2.76 (m, 1H), 2.95 (m, 1H), 3.82 (d, 1H), 6.77

(s, 1H), 7.39 (s, 1H), 7.41 (m, AA’BB’, 2H), 7.45 (m, AA⁰BB⁰, 2H),

7.64 (m, 1H), 7.68 (m, 2H), 7.69 ppm (m, 2H); ¹³C NMR (101

MHz, MeOD-d₄): d 11.12, 25.94, 26.17, 29.23, 34.16, 36.72, 38.98,

42.57, 44.89, 47.95, 53.47, 79.66, 114.12, 127.07, 127.34, 127.54,

127.69, 127.82, 128.87, 128.92, 137.42, 138.43, 139.24, 140.32,

142.68, 158.76, 177.55 ppm. HRMS (ESI-MS, 140 eV): m/z [M+H⁺]

calculated for C₃₁H₃₃O⁺₄, 469.2379; found, 469.2354. RP-C8 HPLC:

t_R= 18.89 min, 99.23% (A%).

4.1.12.2. (8R,9S,13S,14S)-Methyl 7,8,9,11,12,13,14,15,16,17-decahy-

dro-3,17-dihydroxy-13-methyl-2-(4-dibenzofuranyl)-6H-cyclopenta[a]

phenanthrene-16-carboxylate (28). Compound 28 was prepared as

for compound 27 by reacting compound 26 (0.308 g, 0.675 mmol)

with 4-(dibenzofuranyl)-boronic acid (0.287 g, 1.356 mmol),

potassium carbonate (0.375 g, 2.710 mmol) and tetrakis(triph-

enylphosphine)palladium (0) (0.078 g, 0.068 mmol). The obtained

crude product was puriﬁed by silica gel ﬂash column chromatogra-

phy (hexane/ethyl acetate) to give 0.129 g of white solid. Yield

42%; R_f= 0.53 (hexane/ethyl acetate, 1:1); mp = 215 °C; ¹H NMR

(400 MHz, MeOD-d₄): d 0.80 (s, 3H), 1.14 (m, 1H), 1.15 (m, 1H),

1.24 (m, 1H), 1.31 (m, 1H), 1.42 (m, 1H), 1.43 (m, 1H), 1.52 (m,

1H), 1.72 (m, 1H), 1.94 (m, 1H), 2.07 (m, 1H), 2.24 (m, 1H), 2.82

(m, 2H), 3.16 (q, J = 6.82 Hz, 1H), 3.70 (s, 3H), 3.91 (d, J = 5.07 Hz,

1H), 7.13 (d, J = 9.04 Hz, 1H), 7.59 (d, J = 2.24 Hz, 1H), 7.65

(dd, J = 9.12, 1.94 Hz, 1H), 7.88 (m, J = 7.54, 1.12 Hz, 1H), 7.98 (m,

J = 7.45, 0.98 Hz, 1H), 8.08 (d, J = 8.94 Hz, 1H), 8.15 (d, J = 9.14 Hz,

1H), 8.22 (s, 1H), 8.55 ppm (s, 1H). ¹³C NMR (101 MHz, MeOD-

d4): d 10.45, 27.02, 27.76, 29.53, 30.01, 32.31, 38.45, 44.12,

45.02, 47.54, 47.35, 55.72, 82.01, 113.06, 124.23, 125.08, 125.10,

126.11, 126.40, 127.21, 128.05, 128.86, 130.09, 131.20, 134.89,

138.55, 139.71, 142.89, 149.81, 154.89, 159.11, 169.81 ppm. HRMS

(ESI-MS, 140 eV): m/z [M+H⁺] calculated for C₃₂H₃₃O⁺₅, 497.2328;

found, 497.2341.

4.1.13.2. (8R,9S,13S,14S)-7,8,9,11,12,13,14,15,16,17-Decahydro-3,17-

dihydroxy-13-methyl-2-(4-dibenzofuranyl)-6H-cyclopenta[a]phenan-

threne-16-carboxylic acid (31). Compound 31 was prepared as for

compound 30. Yield 97%; mp = over 300 °C; ¹H NMR (400 MHz,

DMSO d₆): d 0.81 (s, 3H), 1.14 (m, 1H), 1.15 (m, 1H), 1.24 (m,

1H), 1.31 (m, 1H), 1.42 (m, 1H), 1.43 (m, 1H), 1.52 (m, 1H), 1.72

(m, 1H), 1.94 (m, 1H), 2.07 (m, 1H), 2.24 (m, 1H), 2.82 (m, 2H),

3.16 (q, J = 6.82 Hz, 1H), 3.91 (d, J = 5.07 Hz, 1H), 7.13 (d, J = 9.04

Hz, 1H), 7.59 (d, J = 2.24 Hz, 1H), 7.65 (dd, J = 9.12, 1.94 Hz, 1H),

7.89 (m, J = 7.54, 1.12 Hz, 1H), 7.98 (m, J = 7.45, 0.98 Hz, 1H), 8.08

(d, J = 8.94 Hz, 1H), 8.15 (d, J = 9.14 Hz, 1H), 8.22 (s, 1H), 8.59

ppm (s, 1H); ¹³C NMR (101 MHz, DMSO d₆): d 10.94, 26.98, 27.88,

29.72, 30.04, 32.24, 38.74, 44.17, 45.09, 47.53, 47.88, 82.04,

113.12, 124.12, 125.06, 125.07, 126.15, 126.34, 127.93, 128.01,

128.92, 130.14, 131.24, 134.93, 138.56, 139.72, 142.82, 149.82,

154.83, 159.14, 169.87 ppm. HRMS (ESI-MS, 140 eV): m/z [M+H⁺]

calculated for C₃₁H₃₁O⁺₅, 483.2171; found, 483.2161. RP-C8 HPLC:

t_R= 14.32 min, 98.8% (A%).

4.1.12.3. (8R,9S,13S,14S)-Methyl 7,8,9,11,12,13,14,15,16,17-decahy-

dro-3,17-dihydroxy-13-methyl-2-phenyl-6H-cyclopenta[a]phenan-

threne-16-carboxylate (29). Compound 29 was prepared as for

compound 27 by reacting compound 26 (0.131 g, 0.287 mmol)

with phenyl boronic acid (0.070 g, 0.577 mmol), potassium carbon-

ate (0.158 g, 1.150 mmol) and tetrakis(triphenylphosphine)palla-

dium (0) (0.033 g, 0.029 mmol). The obtained crude product was

puriﬁed by silica gel ﬂash column chromatography (hexane/ethyl

acetate) to give 0.081 g of white solid. Yield 26%; R_f= 0.47 (hex-

ane/ethyl acetate, 1:1); mp = 240 °C; ¹H NMR (400 MHz, MeOD-

d₄): d 0.89 (s, 3H), 1.22 (m, 1H), 1.34 (m, 1H), 1.37 (m, 1H), 1.54

(m, 1H), 1.55 (m, 1H), 1.91 (m, 1H), 1.94 (m, 1H), 1.97 (m, 1H),

2.23 (m, 1H), 2.36 (m, 1H), 2.41 (m, 1H), 2.85 (m, 2H), 3.18

(q, J = 8.8 Hz, 1H), 3.71 (s, 3H), 3.96 (d, J = 10.5 Hz, 1H), 6.62 (s,

1H), 7.14 (s, 1H), 7.29–7.27 (m, 1H), 7.41–7.38 (m, 2H), 7.54–

7.51 ppm (m, 2H); ¹³C NMR (101 MHz, MeOD-d₄): d 10.74, 26.17,

27.23, 28.30, 28.94, 37.36, 38.51, 43.91, 46.43, 48.30, 48.85,

50.78, 82.90, 115.51, 125.89, 126.08, 127.17, 127.65, 129.12,

131.38, 136.76, 139.31, 151.47, 176.20 ppm. ¹³C NMR (101 MHz,

MeOD-d4): d 10.74, 26.17, 27.23, 28.30, 28.94, 37.36, 38.51,

43.91, 46.43, 48.30, 48.85, 50.78, 82.90, 115.51, 125.89,

126.08, 127.17, 127.65, 129.12, 131.38, 136.76, 139.31, 151.47,

176.20 ppm. HRMS (ESI-MS, 140 eV): m/z [M+H⁺] calculated for

4.1.13.3. (8R,9S,13S,14S)-7,8,9,11,12,13,14,15,16,17-Decahydro-3,17-

dihydroxy-13-methyl-2-phenyl-6H-cyclopenta[a]phenanthrene-16-

carboxylic acid (32). Compound 32 was prepared as for compound

30. Yield 90%; mp = over 300 °C; ¹H NMR (400 MHz, MeOD-d₄): d

0.94 (s, 3H), 1.46 (m, 1H), 1.65 (m, 1H), 1.69 (m, 1H), 1.79 (m,

1H), 1.81 (m, 1H), 1.85 (m, 1H), 1.90 (m, 1H), 1.91 (m, 1H), 1.93

(m, 1H), 2.03 (m, 1H), 2.65 (m, 1H), 2.74 (m, 1H), 2.76 (m, 1H),

2.95 (m, 1H), 3.82 (d, 1H), 6.88 (s, 1H), 7.38 (s, 1H), 7.40 (m, 1H),

7.42 (m, 2H), 7.45 ppm (m, 2H); ¹³C NMR (101 MHz, MeOD-d4):d

11.48, 26.12, 26.17, 29.42, 34.16, 36.72, 39.65, 42.57, 44.12,

47.93, 53.47, 79.66, 113.01, 127.69, 127.81, 128.87, 128.92,

129.04, 137.42, 138.76, 139.24, 158.76, 177.52 ppm. HRMS (ESI-

MS, 140 eV): m/z [M+H⁺] calculated for C₂₅H₂₉O⁺₄, 393.2066; found,

393.2973. RP-C8 HPLC: t_R= 11.81 min, 99.1% (A%).

4.2. Biology

C

₂₆H₃₁O⁺₄, 407.2222; found, 407.2234.

4.2.1. Cell viability assay

4.1.13. General procedure for the synthesis of derivatives (30, 31, 32)

As typical procedure, the synthesis of (8R,9S,13S,14S)-

Cell viability was determined by the 3-(4,5-dimethyl-thiazole-

2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, St

Louis, MO, USA) assay, as previously described.⁴¹Brieﬂy, HepG2

cells were cultured in DMEM supplemented with 1% glutamine,

pen-strep and 10% FBS; cells were seeded in 96-multiwells culture

plates at a concentration of (5000 cells/well) and treated with com-

a

7,8,9,11,12,13,14,15,16,17-decahydro-3,17-dihydroxy-13-methyl-

2-(4-byphenyl)-6H-cyclopenta[a]phenanthrene-16-carboxylic acid

30 is described in detail. Compound 27 was dissolved in 8 mL of

methanol and then 4 mL of 10% NaOH solution were added. The

mixture was heated to reﬂux for 1 h and monitored by TLC analysis

(hexane/ ethyl acetate, 2:1). As the starting reagent spot disap-

peared, the solvent was reduced with rotavapor and the mixture

acidiﬁed with concentrated HCl until pH = 1. The suspension was

pounds 7, 8, 19a, 30, 31 and 32 (6.25, 12.5, 25, 50 lM) for 24 h. The

formazan absorbance was measured at 570 nm, using a Multilabel

Plate Reader VICTOR^TMX3 (Wallac Instruments, Turku, Finland).

Three independent experiments were performed in quadruplicate.

Please cite this article in press as: Dal Prà M., et al. Bioorg. Med. Chem. (2018), https://doi.org/10.1016/j.bmc.2018.02.018

18

M. Dal Prà et al. / Bioorganic & Medicinal Chemistry xxx (2018) xxx–xxx

Table 3

Primer sequences used in this study, NCBI reference sequences and amplicon sizes (base pairs).

Gene

Forward primer

Reverse primer

RefSeq

Size(bp)

GREB-1

CXCL12

GAPDH

5⁰-gtt-ctg-aag-cta-gac-acg-ga-3⁰

5⁰-tac-aga-tgc-cca-tgc-cga-tt-3⁰

5⁰-aca-tca-aga-agg-tgg-tga-agc-a-3⁰

5⁰-ttg-agc-aatcgg-tcc-acc-aa-3⁰

5⁰-gaa-tcc-act-tta-gct-tcg-gg-3⁰

5⁰-gtc-aaa-ggt-gga-gga-gtg-ggt-3⁰

NM_014668.3

NM_000609.6

NM_001289746.1

185

157

119

4.2.2. Cell cycle distribution analysis

measured using Nano-glo dual-luciferase reporter assay system

(Promega) following manufacturer instructions with a Perkin

Elmer en-vision system. All the assays were performed in tripli-

cate, and the standard errors were calculated accordingly.

Cell cycle distribution analysis was evaluated by ﬂow cytometry

(Epics XL, Beckmann Coulter) with CXP software, according to an

already described method.⁴²Cells (200000 per well), 24 h after

seeding into 6 well-plates, were treated with compounds 7, 8,

19a, 31 at 20 mM for 24 h. The cells were washed with PBS and

ﬁxed with ethanol 70%. After 15 min of incubation, cells were

resuspended with RNase A (0,1 mg/mL) and 25 mL of propidium

iodide (1 mg/mL) for 15 min at room temperature. The results of

the different experiments were analyzed with CXP software. Three

independent experiments were performed in duplicate.

4.2.5. Western blot analysis

Protein expression levels of RORct in transient transfected

HEK293 cells was evaluated by Western blot analysis, as already

described.⁴⁶Brieﬂy, 20 mg per lane of proteins were subjected to

sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-

PAGE) on 10% polyacrylamide gels (100 mV for 15⁰and 150 mV

for 90⁰) and transferred to a 0.45 mm nitrocellulose membrane

(Bio-Rad Laboratories S.r.l., Segrate, Milan, Italy) at 250 mA for

90 min in the presence of 25 mM Tris – 192 mM glycine. Mouse

4.2.3. Evaluation of the estrogenic activity by qRT-PCR

The estrogenic activity of the compounds 7, 8, 19a, 30, 31 and

32 was evaluated in the human breast adenocarcinoma cell line

MCF-7 which highly expresses estrogen receptor (ER).⁴³MCF-7

cells were cultured in high glucose DMEM without phenol red sup-

plemented with 1% glutamine, pen-strep and 10% FBS, and seeded

in 6-well culture plates at a concentration of (25,0000 cells/well).

Samples were treated with compounds 7, 8, 19a, 30, 31 and 32

monoclonal anti-RORc (Millipore, Billerica, MA, USA) and rabbit

polyclonal anti-GAPDH (Santa Cruz Biotechnology, Dallas, TX,

USA) primary antibodies (both diluted 1:500) were used to detect

RORct and GAPDH (used as a loading control). Signal intensity of

immunoreactive bands was analyzed by the Quantity One software

(Bio-Rad Laboratories S.r.l.).

(2

lM) for 24 h. Estrone (2 lM) was used as positive control. At

4.2.6. Statistical analysis

the end of the incubation period, MCF-7 were scraped away from

cell culture dishes and total RNA was extracted and puriﬁed by

means of the SV Total RNA Isolation System (Promega Corporation,

Madison, WI), as already described.⁴⁴Integrity and quantity of RNA

were evaluated by an RNA 6000 Nano assay in an Agilent BioAnal-

yser (Agilent Technologies Inc., Palo Alto, CA, USA). The relative

expression of GREB-1 and CXCL12, two genes which increase their

transcription after the activation of ER⁴⁵was determined by real-

time PCR (Eco^TMIllumina, Real-Time PCR system, San Diego, CA,

USA) using One Step SYBR PrimeScript RT-PCR Kit (Takara Bio,

Inc., Otsu, Shiga, Japan). PCR ampliﬁcations were tested for linear-

ity and efﬁciency using standard curves obtained with serial dilu-

tion of cDNA; the speciﬁcity of ampliﬁcation and absence of dimers

were conﬁrmed by melt-curve analysis. All genes were normalized

to Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The pri-

mers used in this study are listed in Table 3. Expression levels of

GREB-1 and CXCL12 genes were calculated by the DDCt method

using the Eco^TMSoftware v4.0.7.0. Modiﬁcations of mRNA levels

were expressed as fold variation compared with that of untreated

cells. Three independent experiment were performed in triplicate.

Comparison of the experimental data obtained from control cell

cultures and those treated with the synthetic compounds was

made by one-way analysis of variance (ANOVA). In the case of sig-

niﬁcant differences (a = 0.05), the analysis of variance was fol-

lowed by the Dunnett post hoc test. P < 0.05 was considered

statistically signiﬁcant. If not otherwise stated, data are presented

as mean standard deviation.

4.3. Molecular docking simulations

The 3D structure of orphan nuclear receptor RORct in complex

with the inverse agonists, digoxin, was retrieved from the Protein

Data Bank (www.rcsb.org, PDB code 3B0W).

Prior to docking simulation, protein structure was processed

with Maestro 10.5 Protein Preparation tool using OPLS-2005 force

ﬁeld. Maestro 10.5⁴⁷Receptor Grid tool was used for the docking

site identiﬁcation, indicating bound digoxin as the grid centre

and a length of 10 Å. Molecular structures of compounds 8 and

19a used for virtual docking were designed using the Builder tool

of MOE 2015.10⁴⁸and prepared for the docking simulation with

Maestro 10.5⁴⁷Ligand Preparation tool using OPLS-2005 force

ﬁeld. The docking simulations were performed with Maestro

10.5⁴⁷Glide software SP precision, using ﬂexible ligand sampling

and performing post-docking minimization.

4.2.4. Cell transient transfection assays

HEK293 cells were maintained in DMEM supplemented with

10% FBS, 1% penicillin and streptomycin. Cells were plated at a con-

centration of 30,000/well in 96-well plates, according to an already

described method.²⁶HEK293 cells were transiently transfected in

OPTIMEM (Gibco) medium using lipofectamine 2000 (Invitrogen)

following manufacturer instructions. Each plate was cotransfected

with 0,2 mg of Gal4-RORg LBD plasmid (Gal4-driven reporter

assays), 0.1 mg UAS-luciferase expression plasmid (both plasmids

were kindly furnished by prof. Grifﬁn) and 0.01 mg of NanoLuc

reporter plasmid (Promega, Italy). 6 h after transfection medium

was replaced with DMEM supplemented with 1% FBS. The follow-

ing day compounds 7, 8, 19a and 32 were added at different con-

centrations (2, 5, 10, 20 mM). Ursolic acid was used as a positive

control. 48 h after transfection luminescence emission was

A. Supplementary data

Supplementary data associated with this article can be found, in

the online version, at https://doi.org/10.1016/j.bmc.2018.02.018.

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Targeting RORs nuclear receptors by novel synthetic steroidal inverse agonists for autoimmune disorders

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