New estrogen receptor antagonists. 3,20-Dihydroxy-19-norpregna-1,3,5(10)-trienes: Synthesis, molecular modeling, and biological evaluation

Accepted Manuscript New estrogen receptor antagonists. 3,20-Dihydroxy-19-norpregna-1,3,5(10)-trienes: Synthesis, molecular modeling, and biological evaluation Yury V. Kuznetsov, Inna S. Levina, Alexander M. Scherbakov, Olga E. Andreeva, Irina V. Fedyushkina, Andrey S. Dmitrenok, Alexander S. Shashkov, Igor V. Zavarzin PII:

S0223-5234(17)30945-5

DOI:

10.1016/j.ejmech.2017.11.042

Reference:

EJMECH 9917

To appear in:

European Journal of Medicinal Chemistry

Received Date: 19 July 2017 Revised Date:

30 October 2017

Accepted Date: 17 November 2017

Please cite this article as: Y.V. Kuznetsov, I.S. Levina, A.M. Scherbakov, O.E. Andreeva, I.V. Fedyushkina, A.S. Dmitrenok, A.S. Shashkov, I.V. Zavarzin, New estrogen receptor antagonists. 3,20Dihydroxy-19-norpregna-1,3,5(10)-trienes: Synthesis, molecular modeling, and biological evaluation, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.11.042. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

NEW

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ESTROGEN RECEPTOR ANTAGONISTS.

3,20-DIHYDROXY-19-NORPREGNA-1,3,5(10)-

TRIENES: SYNTHESIS, MOLECULAR MODELING, AND BIOLOGICAL EVALUATION

Yury V. Kuznetsov a*, Inna S. Levina a, Alexander M. Scherbakov b, Olga E. Andreeva b, Irina V. Fedyushkina c, Andrey S. Dmitrenok a, Alexander S. Shashkov a, Igor V. Zavarzin a

N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky

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a

prospect 47, Moscow 119991, Russia b

Institute of Carcinogenesis, N.N. Blokhin Cancer Research Centre, Kashirskoye sh. 24,

Moscow 115478, Russia

Orekhovich Institute of Biomedical Chemistry, Pogodinskaya ul. 10, Moscow, 119121 Russia

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c

* Corresponding author. N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky prospect 47, Moscow, 119991 Russia. Tel./Fax: +74991372944/+74991355328. E-mail address:

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[email protected]

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Graphical abstract

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ACCEPTED MANUSCRIPT NEW

ESTROGEN RECEPTOR ANTAGONISTS. 3,20-DIHYDROXY-19-NORPREGNA-1,3,5(10)TRIENES: SYNTHESIS, MOLECULAR MODELING, AND BIOLOGICAL EVALUATION

Yury V. Kuznetsov a, Inna S. Levina a, Alexander M. Scherbakov b, Olga E. Andreeva b, Irina V. Fedyushkina c, Andrey S. Dmitrenok a, Alexander S. Shashkov a, Igor V. Zavarzin a

a

N. D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Leninsky

b

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prospect 47, Moscow, 119991 Russia

Institute of Carcinogenesis, N.N. Blokhin Cancer Research Centre, Kashirskoye sh. 24,

Moscow 115478, Russia c

Orekhovich Institute of Biomedical Chemistry, Pogodinskaya ul. 10, Moscow, 119121 Russia

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* Corresponding author. N. D. Zelinsky Institute of Organic Chemistry, RAS, Leninsky prospect 47, Moscow, 119991 Russia. Tel./Fax: +74991372944/+74991355328. E-mail address:

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[email protected]

ABSTRACT

New estrogen receptor α (ERα) antagonists – 3,20-dihydroxy-19-norpregna-1,3,5(10)-

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trienes containing an additional carbocyclic ring D' at the 16α,17α positions – were synthesized. The effects of the new compounds on the MCF-7 breast cancer cells and ERα activation were investigated. All the steroids studied were synthesized starting from estrone methyl ether. The

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scaffold of compounds containing the six-membered ring D' was constructed via the Diels–Alder reaction of butadiene with 3-methoxy-19-norpregna-1,3,5(10),16-tetraen-20-one 5. The

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hydrogenation of primary 16α,17α-cyclohexeno-adduct 7 followed by 3-demethylation (by HBr– AcOH) and reduction of 20-oxo group (by LiAlH4) or in one step by DIBAH gave target monoand dihydroxy steroids 9 – 11. The Corey–Chaykovsky reaction of the same 3-methoxy-19norpregna-1,3,5(10),16-tetraen-20-one 5 gave 16α,17α-methylene-substituted compound. The reaction of the latter with DIBAH immediately yielded 3,20-dihydroxy-16α,17α-methyleno-19norpregna-1,3,5(10)-triene

13.

The

same

procedures

using

3-methoxy-19-norpregna-

1,3,5(10),16-tetraen-20-one 5 produced corresponding 3,20-dihydroxy-16,17-19-norpregna1,3,5(10)-triene 16 and 3,20-dihydroxy-19-norpregna-1,3,5(10),16-tetraene 17. All compounds

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were fully characterized by 1D and 2D NMR, HRMS, and X-ray diffraction. The molecular docking showed that the target compounds can bind to ER, their binding mode being similar to that of natural estradiol. 16α,17α-Methylene- or unsubstituted compounds exhibit the highest cytotoxicity against MCF-7 cells, being simultaneously relatively weak ERα inhibitors. 3,20-

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Dihydroxy steroids containing the six-membered ring D' proved to be the most effective ERα inhibitors. These compounds displayed moderate cytotoxicity comparable of that of tamoxifen and showed no toxic effect on MCF-10A normal, nontumorigenic epithelial cells. The new ER

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antagonists were found to be good candidates for further testing as agents for the treatment and prevention of ERα-positive breast cancers.

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Keywords: antiestrogens, estrogen receptor α, cytotoxicity, 19-norpregnatriene, cycloaddition, NMR assignment, breast cancer. 1. Introduction

The development of effective drugs for the treatment of hormone-dependent cancer is emerging as an important field of research in medicinal chemistry. For instance, the design of new steroid hormone antagonists, in particular, estrogens that play a significant role in the

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development of breast cancer, has received extensive attention. Estrogens, with 17β-estradiol I (E2) being the predominant one, exert their biological functions via estrogen receptors (ER), thereby playing an essential role in the female

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reproductive system, and also control other important aspects of human physiology. Some effects of estrogens are beneficial (protective effects against injuries in the cardiovascular and central nervous systems, positive effects on bone health), while proliferative effects of estrogens

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in some target organ tissues – targeted tissues – can be pathological (uncontrolled proliferation). Breast cancer is one of the most common types of cancer and the most frequent cause of cancer death in women worldwide [1]. The majority of breast malignant tumors are hormone-dependent, and the growth of (ER+)

cancer cells depends on estrogens [2]. Hence, the major strategies for hormonal therapy are to block the estrogens signaling pathways. The mechanisms of action of hormonal drugs are based on blocking the biosynthesis of estrogens by aromatase inhibitors [3] or by steroid sulfatase (STS) inhibitors [4], and, since estrogens exert most of their functions via estrogen receptors (ER), - on inhibition of estrogen

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receptor by selective ER modulators (SERMs) [5], or degradation of estrogen receptor by selective ER down-regulators (SERDs) [6, 7]. However, the efficacy of known steroidal and nonsteroidal antiestrogens is often limited by drug resistance, insufficient tissue selectivity, and/or unwanted side effects [8]. Therefore, the development of new antitumor agents for antiestrogen therapy is a challenge. Previously, we described a new class of synthetic analogues (called progestins) of the

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natural hormone progesterone containing the additional six-membered ring D' fused to the ring D of the steroid scaffold (so-called pregna-D'-pentaranes) II. These synthetic progestins are promising for practical application because they have high progestogenic activity in vivo and in vitro and can inhibit the growth of cancer cells [9, 10].

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The molecular docking of pregna-D'-pentaranes to the ligand-binding domain (LBD) of progesterone receptor (PR), the main molecular target of progestins in cells, showed that, despite

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a larger volume of these molecules compared to progesterone, they are well-accommodated in the ligand-binding pocket of the receptor, the additional six-membered ring D' fitting well in the α-region of the cavity that is present in this pocket near the ring D, thus increasing the hydrophobic binding without distortion of the overall conformation of the ligand molecule [10]. It is well-known that ER LBD has a substantially larger volume than the endogenous ligand and comprises several subpockets, on of which is at the 16α–17α region of the ring D of the

could bind to ER.

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ligand [11, 12]. Therefore, we supposed that D'-pentaranes containing the aromatic ring A III

Based on the foregoing, we performed the synthesis and molecular modeling of 3,20dihydroxy-19-norpregna-1,3,5(10)-triene derivatives, which are steroids containing two hydroxyl

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groups at both ends of the hydrophobic steroid nucleus characteristic of estrogens, and investigated the effects of these compounds on breast cancer cells and estrogen receptor activity. O

OH

AC C

OH

HO

17 16 H

H HO

O

I

D' (CH2)n

II

III: n = 0, 1, 4

2. Results and discussion 2.1

Chemical synthesis

Key intermediate 5 was synthesized in several steps from estrone methyl ether 1 (Scheme 1). The reaction of the latter with trimethylsilyl cyanide in the presence of anhydrous zinc iodide [13] gave silyl cyanohydrin 2 resulting from the attack on the less hindered α-side of the steroid molecule by isomeric 17β-carbonitrile that is present as a minor impurity. The hydrolysis of silyl 4

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cyanohydrin 2 in aqueous hydrochloric acid in ethanol produced cyanohydrin 3 [14], which was subjected to dehydration in the POCl3–pyridine system giving conjugated nitrile 4. There are contradictory data on the conditions and yields for this dehydration reaction performed by procedures described in the literature [13-18]. Thus, these procedures give unstable results. We supposed that this reaction involves two steps – the formation of chlorophosphonic acid esters of steroid cyanohydrin followed by their cleavage. We found that the completeness of the cleavage

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of intermediate chlorophosphonic acid esters, which can be achieved by refluxing the reaction mixture for a long period of time, is crucial for high yields of target product 4. An alternative approach [13] via the direct conversion of silyl cyanohydrin 2 using tetrabutylammonium, cesium, and potassium fluorides afforded conjugated nitrile 4 in low yield. The reaction of

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conjugated nitrile 4 with methylmagnesium iodide [15, 16] in a toluene–diethyl ether mixture at 65°C followed by the decomposition of the resulting imine magnesium salt with acetic acid and

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hydrolysis of the imine under reflux with 10% HCl produced conjugated ketone 5. In order to obtain the latter in high yield, the temperature should be strictly maintained and the reaction mixture should be thoroughly stirred during the decomposition of imine magnesium salt; otherwise, the reaction gives considerable amounts of by-products. We isolated one of these byproducts and characterized it as “dimer” 6. The formation of this dimer is apparently attributed to the reaction of the intermediate imine magnesium salt with products of its further hydrolysis

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followed by the cyclization to the dihydropyridine derivative and aromatization of the latter (see the Supplementary). When determining the structure of product 6, we also made the complete

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assignment of the NMR spectra of compounds 4 and 5. b

OTMS

O

OH

CN

c

AC C

a

O

O

d

O

O

2

1

NC CN

4

3

O

e

N

+ O

O

O

5

6

Scheme 1. Synthesis of key intermediate 5 and dimerization product 6. Reagents and conditions: (a) TMSCN, ZnI2, CH2Cl2, reflux, 90%; (b) POCl3, KF, pyridine, reflux, 16 h, 48%; (c) HCl (aq.), EtOH, reflux, 85%; (d) POCl3, pyridine, reflux, 12 h, 82%; (e) MeMgI, diethyl ether, toluene, 65°C, 6 h, AcOH, H2O, 5°C, HCl (aq), reflux 2 h, 75% (two steps).

5

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The Lewis acid (AlCl3) catalyzed Diels–Alder reaction [19] of conjugated ketone 5 with butadiene produced pentacyclic steroid 7 containing the additional 16α,17α-cyclohexane ring in high yield. This steroid was hydrogenated under atmospheric pressure in the presence of 10% palladium on activated carbon as the catalyst (Scheme 2). Hydrogenated adduct 8 was demethylated with a mixture of 48% hydrobromic acid and sodium iodide in glacial acetic acid to prepare 3-hydroxysteroid 9 in high yield. The addition of an equimolar amount of sodium

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iodide as a source of a highly reactive nucleophile to a standard demethylation system allowed us to substantially reduce the reaction time.

3,20-Diols 10a and 10b were synthesized by reducing the 20-keto group in hydroxysteroid 9 with lithium aluminum hydride in THF. In this case, we isolated only enantiomer 10a having

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20(R) configuration. The heating of 3-methoxy-20-ketosteroid 8 with a toluene solution of diisobutylaluminum hydride (DIBAH) under reflux allowed the synthesis of a diastereomeric

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mixture of 3,20-diols by one-pot reduction–dementylation. Individual isomers 10a and 10b were isolated from the mixture by preparative reversed-phase HPLC (RP-HPLC). The absolute configuration of the new chiral center С20 was established by NMR spectroscopy. For this purpose, the 1H and

13

C chemical shift assignments (Tables 1a and 1b) and NOE experiments

were performed. The 2D NOESY spectra of 10a and 10b were not clear enough due to consequences of the chemical shifts of protons of CH3-21 methyl with H22 and H25 protons. We

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performed ge-1D NOESY experiments to reveal the differences in NMR for these isomers. Irradiation of the hydroxyl proton at C20 in ge-1D NOESY experiments provided responses for H20, CH3-21, H12 (δН1.81) for 10a (R-isomer), while responses for H20, CH3-21, H16 (δН 2.24), H22 (δН 1.73), H25 (δН 1.89) were observed on irradiation of the hydroxyl proton of 10b

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(S-isomer) (Fig. 1). Besides, compound 10a was studied by X-ray diffraction (Fig. 2). The configuration at C20 was assigned as R.

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3-Methoxy-20-hydroxysteroid 11 was synthesized as a mixture of 20(R) and 20(S) isomers

by reduction of 20-ketosteroid 8 with lithium aluminum hydride or DIBAH at room temperature. A comparative study of the 20-keto group reduction by these reagents showed that the ratio of the 20(R) and 20(S) isomers is 4.3 : 1 for LAH and 2.3 : 1 for DIBAH (NMR data).

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O

O

O

5

e

a

b

HO

c

H

d

H HO

O

O

H

H

7

HO

8

10a: 20(R)

9 d

10b: 20(S)

HO

H O

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11: 20(R,S)

H

SC

Scheme 2. Synthesis of pentacyclic steroids containing the additional 16α,17αcyclohexane ring and functional hydroxyl groups at positions 3 and 20. Reagents and conditions: (a) butadiene, AlCl3, CH2Cl2, RT, 68%; (b) H2, 10% Pd/C, dioxane, 93%; (c) HBr (48%), AcOH, NaI, reflux, 3.5 h, 77%; (d) LiAlH4, THF, RT, 48%; (e) DIBAH, toluene, reflux, 6 h.

H

H

12

H

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20 H

21

H

21 H

H

H

H O

B-ring

25

B-ring

22 H

B-ring

H

16

H

H

12

22 H

O

20

H

H

H

16 25

B-ring

R

S

AC C

EP

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Fig. 1. Structure assignment for compounds 10a and 10b using ge-1D NOESY. Most significant interactions and fragments of the steroid scaffold are shown for clarity.

Fig. 2. General view of one independent molecules of compound 10a in the crystal structure shown with displacement ellipsoids (p=50%).

To evaluate the size effect of the additional ring D' of the steroids on the biological properties, we synthesized 3,20-dihydroxysteroids containing the additional cyclopropane ring D'

7

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annulated at positions 16α,17α (Scheme 3) and steroids 16 and 17 that lack the additional ring D' (Scheme 4). Compound 12 was synthesized by the Corey–Chaykovsky reaction of conjugated ketone 5 with trimethylsulfoxonium iodide. The corresponding 3,20-dihydroxysteroid 13 was synthesized in one step from compound 12 by heating the latter under reflux with DIBAH. The cyclopropane moiety, which can be decomposed during demethylation by strong acids, remains intact under

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these conditions [20, 21]. Steroid 13 was isolated as a mixture of 20(R) and 20(S) isomers. Nevertheless, the structure assignment for both isomers was carried out using 2D 1H/1H COSY, TOCSY, NOESY and 1H/13C HSQC, HMBC techniques. The configurations of asymmetric centers were established by the careful analysis of the NOESY spectra. The distance between

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CH3-21 and H16 for the 20(R) and 20(S) isomers should be different. The hydrogen H16β is the nearest neighbor of CH3-21 in the R isomer, while this methyl is far apart from H16β in the S

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isomer (Fig. 3). The NOESY spectrum of a mixture of these isomers (see the Supplementary) shows a strong CH3-21 (δН 0.84)/H16β (δН 0.96) cross-peak, but no cross-peaks are observed between the corresponding signals CH3-21 (δН 1.09)/H16β (δН 1.23), which is evidence that the former pair of signals is from the R isomer, whereas the latter one is from the S isomer.

O

a

b

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5

HO

H

H

O

HO

12

13: 20(R,S)

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Scheme 3. Synthesis of 3,20-dihydroxy-16α,17α-cyclopropano-19-norpregna1,3,5(10)-triene 13. Reagents and conditions: (a) Me3SOI, NaH, DMSO/THF, RT, 24 h, 59%; (b) DIBAH, toluene, reflux, 6 h, 32%. H H H

O

H

20

H

+

H

H

H

21

H

20

21

O H

H

H

B-ring

B-ring

16

16

B-ring

B-ring

R

S

Fig. 3. Structure assignment for the 20(R) and 20(S) isomers of compound 13 using NOESY. Most significant interactions and fragments of the steroid scaffold are shown for clarity.

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The hydrogenation of conjugated ketone 5 gave saturated ketone 14. The demethylation of the latter with HBr/NaI/AcOH followed by the reduction of 3-hydroxy-20-ketosteroid 15 that formed with lithium aluminum hydride produced 3,20-dihydroxysteroid 16 which was isolated as 20(R) isomer after column chromatography and recrystallization. In the NOESY spectrum of 16, the CH3-21 protons (δН 1.01) show (apart from trivial CH3-21, H-20, and OH-20 mutual signals) a strong cross-peak with H16β (δН 1.63), a weak cross-peak with H16α (δН 1.14), and a

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medium cross-peak with H17 (δН 1.30), while only a cross-peak with H17 appears for the hydroxyl proton at C20 (δН 4.09). This assignment was confirmed by X-ray analysis (Fig. 4).

O

b a HO

O

14

SC

O

5

HO

d HO

17: 20(R,S)

c

HO

16: 20(R)

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15

HO

AC C

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Scheme 4. Synthesis of 3,20-dihydroxy-19-norpregna-1,3,5(10)-tri- and 1,3,5(10),16tetraenes. Reagents and conditions: (a) H2, 10% Pd/C, dioxane, 91%; (b) HBr (48%), AcOH, NaI, reflux, 3.5 h, 53%; (c) LiAlH4, THF, RT, 40%; (d) DIBAH, toluene, reflux, 6 h, 47%.

Fig. 4. General view of one of the two independent molecules in the crystal structure of compound 16 shown with displacement ellipsoids for non-hydrogen atoms (p=50%). A comparative study of the reduction of 16,17-unsubstituted 3-methoxy ketone 14 with lithium aluminum hydride and DIBAL demonstrated that in this case (cf. compound 11), the stereoselectivity of the reduction of the 20-keto group is somewhat higher, while the difference in the ratio of the 20(R)/(S) isomers obtained using these two reducing agents is smaller (4.6 : 1 and 3.9 : 1 for LAH and DIBAL, respectively). Thus, the initially lower content and the complex 9

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isolation procedure for the product were considered to explain the depletion of the 20(S) isomer of compound 16. 3,20-Dihydroxysteroid 17 containing a double bond at position 16 was synthesized by reduction–demethylation of conjugated ketone 5 with DIBAL. Since an almost equimolar mixture of the 20(R) and 20(S) isomers of 17 was obtained, the configuration at C20 was assigned by comparing the intensities of the cross-peaks in the NOESY spectrum (see the

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Supplementary) corresponding to interactions of the side chain protons and the nearest protons of the rigid steroid scaffold. The intensities of the significant cross-peaks H20/CH3-18, OH-20/H16, and CH3-21/H16 are comparable for both isomers, whereas the intensities of the CH3-21/CH3-18 cross-peaks differ clearly between the isomers. The stronger peak (δН 1.22/ δН 0.81) should

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correspond to the R configuration because the distance between the 18- and 21-methyl protons in this configuration is shorter than that in the S configuration (Fig. 5).

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As opposed to the above-described examples, the formation of an equimolar mixture of the 20(R) and 20(S) isomers is apparently attributed to the fact that the 20-keto group that is reduced is far apart from the stereocenter at C13, resulting in lower asymmetric induction. The 1H and 13C NMR signal assignments of compounds 10a, 10b, 13, 16, and 17 are summarized in Tables 1a,b, respectively.

TE D

+

H

H

H

18

H

O

H

21 H

H

C-ring

H

16

H

H

H

H

18

H

H H

21

O H

C-ring

H

16

C-ring

H

EP

C-ring

R

AC C

S

Fig. 5. Structure assignment for the 20(R) and 20(S) isomers of compound 17 using NOESY. Most significant interactions and fragments of the steroid scaffold are shown for clarity. 1

Table 1a. and 17. Compound Atom n. 1 2 3 4 5 6 7 8

10a R

H NMR (DMSO-d6, 600 MHz) spectra of compounds 10a, 10b, 13, 16, 10b S

6.99 6.48 6.42 2.69 1.77; 1.27 1.76;1.25 1.26

13 R

S 6.99 6.48 6.42 2.70 1.72;1.23 1.36

10

16 R 7.03 6.51 6.42 2.70 1.75; 1.25 1.25

17 R

S 7.02 6.50 6.44 2.72 1.82; 1.33 1.45

2.14; 1.27 1.50 -

1.61 1.81; 1.60 2.02

1.71 1.56; 1.30 2.24 -

0.86

0.75

3.81 3.85 1.15 1.10 1.28 1.73; 1.25 1.45; 1.14 1.71; 1.45 1.47 1.42; 1.31 1.59; 1.31 1.89; 1.42 8.93 4.10 4.12 13

2.14 2.27; 1.43 1.93; 1.60 1.88; 1.50 1.52 1.47 2.08; 1.86 5.50 5.52 0.81 0.84

3.51 1.01 8.92 4.10

4.25 1.22

4.20 1.20

3.95

4.44

4.49

C NMR (DMSO-d6, 150 MHz) spectra of compounds 10a, 10b, 13, 16, 10b S

125.8 112.6 154.8 114.9 137.1 29.2

71.1 22.2 23.8 20.2 29.5 33.1

R

S

125.6 112.6 154.9 114.9 137.0 29.0

27.6 38.7 43.1 130.6 25.8 31.7 47.3 48.6 32.3 40.1 49.2 14.1

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27.7 38.9 43.2 130.7 26.1 32.5 49.2 48.2 32.9 35.4 49.1 14.4

13

74.1 20.2 24.6 23.3 20.6 29.8

16 R 125.9 112.6 154.8 114.8 137.0 29.2 27.5 38.5 43.4 130.6 26.3 39.4 42.3 54.5 25.3 23.8 57.9 12.0

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10a R

AC C 2.2

2.15; 1.28 2.12; 1.27 1.16 1.60; 1.12 1.63; 1.14 1.30 0.70

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2.10; 1.29 1.81; 1.52

2.20; 1.30 2.00; 1.36 1.77; 1.36 1.01 1.03 1.53; 1.33 1.53; 1.28 0.96 1.23 0.91 0.87 4.15 4.07 0.84 1.09 0.68; 0.40 0.54; 0.44 8.94 4.02 4.08

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-

Table 1b. and 17. Compound Atom n. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

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2.01

27.6 37.1 43.8 130.7

27.5 37.0 43.7 130.5

TE D

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 3-OH 20-OH

26.1

35.3 42.1 46.1 26.0 19.5 41.0 16.9

35.1 41.0 46.7 25.7 18.1 39.9 17.1 62.5 23.2 4.6

65.4 20.6 6.4 -

68.3 21.7 -

17 R

S 125.6 112.6 154.9 114.9 137.1 29.0 27.4 37.0 43.8 130.6 26.1 34.7 45.9

56.6

56.2 30.1

121.2 159.5 16.4

121.7 160.1 16.5

63.6 23.7

63.4 23.6 -

Molecular modeling

To investigate the possible binding mode and ligand–receptor interactions, we performed molecular docking of the compounds into the ligand binding site of the nuclear receptor ERα (PDB: 1QKU) followed by energy calculations by the MM-PBSA method in order to determine the individual characteristics of these interactions. 11

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The basic requirements for high-affinity ligand binding to ERα are well known due to an extensive structure–activity relationship studies and X-ray crystallographic data for ligand–ER LBD complexes [22-24]. According to the X-ray structure of the estradiol complex with ER LBD [22], two appropriately spaced hydroxyl groups are located at both ends of the nearly planar hydrophobic scaffold in such a way that the phenolic hydroxyl forms hydrogen bonds with the amino acid residues glutamate (Glu353) and arginine (Arg394) and also with a

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conserved water molecule, while another hydroxyl (17β-OH) is recognized by a single histidine (His524). All other contacts in the complex are hydrophobic [22].

The docking results showed that all the compounds under study (8-11, 13, 16, and 17) can bind at the ligand-binding pocket (LBP) of estrogen receptor α (ER-α).

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In particular, as can be seen in Fig. 7, compound 10a interacts with similar protein residues of LBD, like natural estradiol in the X-ray structure of E2 in complex with ERα LBD [22], and

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can be well-accommodated in the ligand-binding pocket despite the larger volume of steroid 10a compared to estradiol. The additional ring D', which enhances hydrophobic binding, fits well in α-region of the cavity near the ring D.

The analysis of the binding of the compounds under study demonstrates that all of them can bind to ER LBD with similar energies that are comparable to or slightly higher than the

AC C

EP

TE D

value estimated for the estradiol molecule (Table 2).

Fig. 7. Interactions (hydrogen bonds) calculated for compound 10a and the key ligandbinding pocket residues of human estrogen receptor α (left) and the arrangement of ligand 10a in the ligand-binding pocket (right) (calculated based on the crystal structure of the ERα LBD–E2 complex, PDB:1QKU). The surfaces are colored by lipophilicity, ranging from brown (most lipophilic) to blue (least lipophilic).

Table 2.

Estimated binding energies of compounds 8–11, 13, and 15–17 to the human estrogen receptor alpha ligand-binding domain.

Compound

∆E (GBTOT), 12

STD

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2.3

Biological evaluation

2.3.1

Сytotoxic activity

kkal/mol -66.09 -74.22 -75.72 -79.14 -81.53 -76.09 -82.08 -69.87 -66.38 -67.72 -71.15 -66.12 -67.63 -70.43

SC

RI PT

±2.91 ±1.60 ±2.62 ±1.71 ±2.12 ±2.09 ±2.70 ±3.50 ±3.54 ±3.08 ±2.53 ±2.03 ±2.20 ±2.81

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(configuration of C20) E2 8 9 10a (R) 10b (S) 11 (R) 11 (S) 13 (R) 13 (S) 15 16 (R) 16 (S) 17 (R) 17 (S)

Compounds 8-11, 13, and 15-17 were tested in vitro for cytotoxic activity against the estrogen receptor-positive MCF-7 breast cancer cell line. Cytotoxicity was evaluated by the

TE D

MTT test based on the accumulation of the MTT reagent (3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide) by living cells. Compounds 13 and 16 displayed the highest activity. They exhibited the cytotoxic effect at submicromolar level. Three steroids – 10a, 10b, and 17 – showed activity comparable to that of the SERM tamoxifen. Only weak effects in

EP

MCF-7 cells were detected for compound 9, while 8, 11, and 15 exhibited no activity in MCF-7 breast cancer cells. To elucidate possible toxicity of prepared compounds against normal

AC C

epithelium MCF-10A non-tumorigenic epithelial cells were tested. The MCF10A human mammary epithelial cell line is a widely used in vitro model for studying normal breast cell function and transformation. As can be seen in Table 3, compounds 13 and 16 displayed high toxicity towards MCF-

10A cells, while 10b has an insignificant effect. The other compounds, as well as the reference drug tamoxifen, exhibited no activity (no toxic effect) against nontumorigenic cells. Table 3.

Compound 8

The IC50 (half-maximal inhibitory concentration) of compounds. The cells were grown for 72 h and then the inhibitory activity of the compounds was evaluated by the MTT test. IC50, µM MCF-7 breast cancer cells MCF-10A normal cells NA NA 13

ACCEPTED MANUSCRIPT

2.3.2

ERα-mediated luciferase reporter gene assay

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15.8 ± 1.4 NA 9 6.8 ± 0.7 NA 10a 4.6 ± 0.5 23.0 ± 2.5 10b NA NA 11* 0.21 ± 0.03 3.1 ± 0.4 13* NA NA 15 0.15 ± 0.03 5.9 ± 0.5 16 4.6 ± 0.4 19.1 ± 1.8 17* tamoxifen 5.3 ± 0.6 NA NA – does not reach 50% growth inhibition at concentrations lower than 25 µM * tested as a mixture of 20(R,S) epimers

SC

The ERα transcriptional activity was evaluated using the ERα luciferase reporter gene assay. The ability of the compounds to inhibit ERα activity was analyzed in MCF-7 cells after

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ERα luciferase reporter plasmid transfection. The β-galactosidase plasmid construct was used to normalize transfection efficiency. Compounds 8, 11, 15, and 17 displayed no antiestrogenic activity, while steroids 9, 10b, 13, and 16 partially inhibited ERα (Fig. 8). Compound 10a exhibited the highest ERα antagonist potency. The treatment with this steroid resulted in strong down-regulation of ERα activity. The incubation with 5 µM 10a led to ca. 70% inhibition of ERα activity in MCF-7 cells, demonstrating the efficacy of 10a being comparable to that of the

TE D

SERM drug tamoxifen.

As can be seen from the results of the ERα transcriptional activity assay (see Fig. 8), only five compounds (9, 10a, 10b, 13, and 16) of the nine tested compounds have pronounced ER antagonist activity.

EP

In both biological assays, compounds containing the hydrophobic steroid scaffold with the aromatic ring A and the 3- and 20-hydroxyl groups at both ends exhibited the highest

AC C

activity. It should be noted that the spatial arrangement of the 20-hydroxyl group has apparently a weaker influence on the effects of the compounds (cf. 10a and 10b). Meanwhile, the phenolic hydroxyl is essential for the manifestation of inhibitory activity. Thus, the steroid containing the 20-hydroxyl and the 3-OMe group instead of the second hydroxyl (11) does not exhibit these properties, whereas the steroid containing 20-keto and 3-OH groups (9) exerts some activity in both assays. The introduction of the additional six-membered ring D' at 16α,17α positions into the steroid containing 3-phenol and 20-hydroxyl groups proved to be an effective modification of the steroid scaffold for the design of agents exhibiting antiestrogenic activity. It should be emphasized that both the compounds containing the 16α,17α-methylene substituent and the compounds containing the ring D devoid of substituents retain this activity to a lesser extent.

14

ACCEPTED MANUSCRIPT 140 120

80 60 40 20 0 8

9

10a

10b

11

13

15

16

17

ta mo

SC

cont

RI PT

ERE-Luc, rel.units

100

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Fig. 8. Estrogen receptor α activity in MCF-7 cells. MCF-7 human breast cancer cells were transfected with the ERE-TK-LUC plasmid containing the luciferase reporter gene under control of the estrogen responsive element (ERE) and cotransfected with the β-galactosidase plasmid. The medium was removed 24 h after the transfection. The synthesized steroids, the SERM drug tamoxifen at 5 µM concentration, or the vehicle control (cont) were added to phenol-free DMEM supplemented with 5% DCC serum (Hyclone) and 10 nM 17β-estradiol. The luciferase and β-galactosidase activities were determined after 24 h as described in Section 4.4.2 using the ERα luciferase reporter gene assay. 3. Conclusion

TE D

A series of 3,20-dihydroxy-19-norpregna-1,3,5(10)-triene derivatives – steroids containing the additional carbocyclic ring D' and two hydroxyl groups at both ends of the hydrophobic steroid nucleus characteristic of estrogens – were synthesized. Docking studies showed that these

EP

compounds can bind to ERα, their binding mode is similar to that of natural estradiol. Most of these compounds exhibit cytotoxic activity against breast cancer cells and inhibit ER transcriptional activity in the ERα luciferase reporter gene assay. The 16α,17α-methylene-

AC C

substituted or unsubstituted compounds display the highest cytotoxicity, being simultaneously relatively weak ERα inhibitors. 3,20-Dihydroxysteroids containing the six-membered ring D' proved to be the most effective estrogen receptor α inhibitors. These compounds exhibit moderate cytotoxicity comparable to that of tamoxifen, while having no toxic effect on normal (nontumorigenic) cells. The results of this study showed that the new ER antagonists are good candidates for further testing as agents for the treatment and prevention of ERα-positive breast cancers. 4. Materials and methods 4.1

General

15

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All reagents were purchased from Acros Organics and were of reagent or analytical grade. Solvents were purified according to standard procedures [25]. The analytical TLC was carried out on silica gel 60 F254 plates (Merck) using dichloromethane, a 40 : 1 toluene–acetone mixture, or a 50 : 1 dichloromethane–acetone mixture as eluents. The spots were visualized by

RI PT

UV light or by spraying with aqueous KMnO4. The preparative separation was carried out by column chromatography on silica gel 60 (0.063–0.100mm) (Merck) at a compound–sorbent ratio of 1 : 40. Preparative RP-HPLC was performed on a Sunfire C18 (19 mm x 250 mm) preparative

SC

column with elution at a flow rate of 20 ml/min using MeCN/water (70 : 30) and UV detection at 280 nm. The melting points were determined on a Boetius micro-melting point apparatus

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(Germany). All yields are given for purified compounds.

One- and two-dimensional NMR spectra were recorded on Bruker AM300 (300.13 MHz for 1H and 75.5 MHz for

13

C) and Bruker AV-6 (600.13 MHz for 1H and 150.9 MHz for

13

C)

spectrometers using standard Bruker software. Chemical shifts are given in δ (ppm) relative to the residual solvent peaks: δH 7.27 and δC 77.0 for CHCl3; δH 2.50 and δC 39.5 for DMSO-d5.

TE D

The mixing time for TOCSY and NOESY experiments were 100 ms and 600 ms, respectively, spin-lock time for ROESY experiments was 150 ms. The 1H/13C and 1H/15N HMBC experiments

EP

were optimized for the spin-spin coupling constant of 8 Hz. High-resolution mass spectra (HRMS) were measured on a Bruker micrOTOF II instrument using electrospray ionization

AC C

(ESI) in positive ion mode (interface capillary voltage 4500 V); the mass range from m/z 50 to m/z 3000 Da; external or internal calibration was performed using Electrospray Calibrant Solution (Fluka). Solutions in acetonitrile or methanol were injected with a syringe (flow rate 3 µL/min). Nitrogen was applied as a dry gas; interface temperature was set at 180 °C. X-ray diffraction data were collected on a Bruker APEX DUO diffractometer. Frames were integrated using the Bruker SAINT software package with a narrow-frame algorithm. A semiempirical absorption correction was applied with the TWINABS or SADABS programs based on intensities of equivalent reflections. The structures were solved by direct methods and refined by

16

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the full-matrix least-squares technique against F2 with anisotropic displacement parameters. The hydrogen atoms of hydroxyl groups were located from difference Fourier maps; all other hydrogen atoms were positioned geometrically. All calculations were performed with the SHELX software package. Atomic coordinates, bond lengths and angles, and thermal parameters

RI PT

were deposited at the Cambridge Crystallographic Data Center (CCDC), the reference numbers are given in the relevant sections of the Experimental section. These data can be obtained, free of charge,

via

http://www.ccdc.cam.ac.uk/data_request/cif,

or

by

e-mailing

SC

[email protected], or by contacting the Cambridge Crystallographic Data Centre, 12 Union Road, Cambridge CB2 1EZ, UK; fax: +44(0)1223-336033. Chemical synthesis

4.2.1

3-Methoxy-17β-trimethylsiloxyestra-1,3,5(10)-triene-17α-carbonitrile 2

M AN U

4.2

Estrone 3-methyl ether (1, 30.1 g, 106 mmol) was taken in dry dichloromethane (250 ml). To this solution, anhydrous zinc iodide (1.00 g, 3 mmol) and trimethylsilyl cyanide (20 ml, 14.9

TE D

g, 150 mmol) were added, and the mixture was refluxed with stirring for 2 h until the starting material disappeared on TLC. Then the reaction mixture was concentrated in vacuo, and the solid residue was dissolved in a boiling mixture of toluene and hexane (1 : 2). The hot solution

EP

was filtered through a silica gel pad (5 g) and allowed to cool in a refrigerator to 8°C. The precipitate was collected, washed with cold hexane, and dried to obtain 3-methoxy-17β-

AC C

trimethylsiloxyestra-1,3,5(10)-triene-17α-carbonitrile 2. Yield: 90%, m.p. 135–136˚C, 1H NMR (CDCl3, 300 MHz): δ 0.26 (s, 9H, Si(CH3)3), 0.85

(s, 3H, 18-CH3), 1.35–2.58 (m, 13H, rest of the 5 x CH2 and 3 x CH of steroid ring), 2.87 (bs, 2H, 6-CH2), 3.79 (s, 3H, OCH3), 6.65 (s, 1H, 4-CH), 6.74 (d, 1H, 2-CH, J = 8.4 Hz), 7.22 (d, 1H, 1-CH, J = 8.4 Hz);

13

C NMR (CDCl3, 75 MHz): δ 1.3, 12.3, 23.1, 26.3, 27.2, 29.8, 33.3,

38.1, 39.4, 43.4, 48.2, 48.4, 55.3, 111.6, 113.9, 122.4, 126.4, 132.2, 137.9, 157.6; HRMS: m/z 406.2175 [M + Na]+ (calcd for C23H33NNaO2Si, 406.2173). 4.2.2

3-Methoxy-17β-hydroxyestra-1,3,5(10)-triene-17α-carbonitrile 3 17

ACCEPTED MANUSCRIPT

Estrone 3-methyl ether (1, 70.2 g, 250 mmol) was taken in dry dichloromethane (600 ml). To this solution, anhydrous zinc iodide (2.4 g, 7.5 mmol) and trimethylsilyl cyanide (43 ml, 31.7 g, 320 mmol) were added. The mixture was refluxed with stirring for 2 h until the starting material disappeared on TLC and then concentrated in vacuo. The solid residue was dissolved in

RI PT

a mixture of ethanol (600 ml), water (90 ml), and conc. hydrochloric acid (5 ml) and refluxed for 1 h until the starting material disappeared on TLC. Then distilled water (350 ml) pre-warmed to 80˚C was added, and the mixture was allowed to cool to ambient temperature. The precipitate

SC

was collected, washed with distilled water, and dried to obtain 3-methoxy-17β-hydroxyestra1,3,5(10)-triene-17α-carbonitrile 3.

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Yield: 85%, m.p. 158–161˚C with decomposition (cf. lit. m.p. 158.5˚C [26]), 1H NMR (DMSO-d6, 300 MHz): δ 0.79 (s, 3H, 18-CH3), 1.22–2.44 (m, 13H, rest of the 5 x CH2 and 3 x CH of steroid ring), 2.77 (bs(m), 2H, 6-CH2), 3.69 (s, 3H, OCH3), 6.56 (bs, 1H, OH), 6.61 (s, 1H, 4-CH), 6.68 (d, 1H, 2-CH, J = 8.3 Hz), 7.17 (d, 1H, 1-CH, J = 8.3 Hz); 13C NMR (CDCl3, 75 MHz): δ 11.9, 122.3, 25.8, 26.7, 29.1, 33.2, 36.6, 38.7, 43.0, 46.8, 49.4, 54.8, 79.8, 111.5,

312.1958).

3-Methoxyestra-1,3,5(10),16-tetraene-17-carbonitrile 4

EP

4.2.3

TE D

113.4, 123.0, 126.2, 131.6, 137.3, 157.1; HRMS: m/z 312.1963 [M + H]+ (calcd for C20H26NO2,

a) Starting from 3-methoxy-17β-trimethylsiloxyestra-1,3,5(10)-triene-17α-carbonitrile 2.

AC C

Cyanohydrin silyl ether (2, 10 g, 26 mmol) and potassium fluoride (2.1 g, 36 mmol) were taken in dry pyridine (20 ml) and then POCl3 (5 ml, 8.23 g, 54 mmol) was added dropwise with stirring. The reaction mixture was incubated for 30 min, refluxed with stirring for 16 h, cooled to ca. 50˚C, poured into cold water acidified with conc. hydrochloric acid to pH 1-2, and extracted with CHCl3 (4 x 100 ml). The organic layer was washed with water and brine, dried over anhydrous Na2SO4, filtered through a silica gel pad (10 g), and concentrated in vacuo. The resulting yellow oil was recrystallized from a mixture of toluene and hexane (3 : 1) to obtain 3methoxyestra-1,3,5(10),16-tetraene-17-carbonitrile 4.

18

ACCEPTED MANUSCRIPT

Yield: 48%, m.p. 174–175˚C (cf. lit. m.p. 168–170˚C [15]), 1H NMR (CDCl3, 300 MHz): δ 0.97 (s, 3H, 18-CH3), 1.22–2.53 (m, 11H, rest of the 4 x CH2 and 3 x CH of steroid ring), 2.91 (m, 2H, 6-CH2), 3.79 (s, 3H, OCH3), 6.66 (bs, 1H, 4-CH), 6.68 (bs, 1H, 16-CH), 6.74 (d, 1H, 2CH, J = 8.3 Hz), 7.22 (d, 1H, 1-CH, J = 8.3 Hz); 13C NMR (CDCl3, 75 MHz): δ 16.3, 26.1, 27.6,

RI PT

29.5, 32.6, 34.0, 37.1, 44.1, 48.4, 55.2, 111.5, 113.9, 115.9, 126.0, 127.5, 132.0, 137.5, 147.3, 157.6; HRMS: m/z 311.2122 [M + NH4]+ (calcd for C20H27N2O, 311.2118). b)

Starting

from

3-methoxy-17β-hydroxyestra-1,3,5(10)-triene-17α-carbonitrile

3.

SC

Cyanohydrin (3, 18.4 g, 59 mmol) was taken in dry pyridine (50 ml) and then POCl3 (11 ml, 18.1 g, 118 mmol) was added dropwise with stirring. The reaction mixture was incubated for 30 min

M AN U

and refluxed with stirring for 12 h until the starting and intermediate compounds disappeared on TLC. The completion of the reaction was confirmed by the easy and clear layer separation as a result of the mixing of a sample of the reaction mixture with CHCl3 and water acidified with HCl. Then the reaction mixture was cooled to ca. 50˚C, poured cold water acidified with conc. hydrochloric acid to pH 1-2, and extracted with CHCl3 (3 x 100 ml). The organic layer was

TE D

washed with water and brine, dried over anhydrous Na2SO4, and concentrated in vacuo to obtain a dark brown tarry mass, which was dissolved in a boiling mixture of toluene and hexane (150

EP

ml and 60 ml). The hot solution was filtered through a silica gel pad (20 g) and allowed to cool to room temperature and then in a refrigerator to 8°C. The precipitate that formed was collected,

AC C

washed with hexane, and dried to obtain 3-methoxyestra-1,3,5(10),16-tetraene-17-carbonitrile 4 in 82% yield. 4.2.4

3-Methoxy-19-norpregna-1,3,5(10),16-tetraen-20-one 5

3-Methoxyestra-1,3,5(10),16-tetraene-17-carbonitrile (4, 10.0 g, 34.1 mmol) was taken in

a mixture of toluene (160 ml) and dry diethyl ether (100 ml) under argon. To this solution, a 3.0 M methylmagnesium iodide solution in diethyl ether (25 ml, 75 mmol) was added. The reaction mixture was incubated with stirring at 60–65˚C for ca. 5 h until the starting nitrile disappeared on TLC. Then the reaction mixture was cooled to 5˚C, and glacial acetic acid (50 ml) was added

19

ACCEPTED MANUSCRIPT

dropwise with cooling and vigorous stirring in such a way as to maintain the internal temperature below 15˚C. Then glacial acetic acid (40 ml) and water (10 ml) were added to the reaction mixture, which was refluxed for 1 h. Next, water (10 ml) and conc. HCl (3 ml) were added to the boiling reaction mixture and the mixture was refluxed for 1 h. After cooling, the organic layer

RI PT

was separated and washed with water and brine. All aqueous layers were pooled and extracted with CHCl3 (3 x 30 ml). The organic fractions were worked-up in the same way, pooled, dried with anhydrous Na2SO4, and concentrated in vacuo to obtain a solid yellow residue, which was

SC

dissolved in a boiling mixture of toluene and hexane (3 : 1). The hot solution was filtered through a silica gel pad (5 g) and allowed to cool in a refrigerator to 8˚C. The crystalline

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precipitate that formed was separated, washed with hexane, and dried to obtain 3-methoxy-19norpregna-1,3,5(10),16-tetraen-20-one 5.

Yield: 75%, m.p. 194–195˚C (cf. lit. m.p. 192–194˚C [15]), 1H NMR (CDCl3, 300 MHz): δ 0.93 (s, 3H, 18-CH3), 1.22–2.53 (m, 11H, rest of the 4 x CH2 and 3 x CH of steroid ring), 2.30 (s, 3H, 21-CH3), 2.91 (m, 2H, 6-CH2), 3.79 (s, 3H, OCH3), 6.65 (bs, 1H, 4-CH), 6.71 (d, 1H, 2-CH,

TE D

J = 8.9 Hz), 6.75 (bs, 1H, 16-CH), 7.22 (d, 1H, 1-CH, J = 8.9 Hz); 13C NMR (CDCl3, 75 MHz): δ 15.9, 26.4, 27.1, 27.7, 29.6, 31.9, 34.7, 36.9, 44.2, 46.4, 55.2, 55.5, 111.3, 113.8, 126.1, 126.1,

311.2006).

“Dimer” 6

AC C

4.2.5

EP

132.7, 137.7, 144.3, 155.5, 157.4, 196.8; HRMS: m/z 311.2015 [M + H]+ (calcd for C21H27O2,

3-Methoxyestra-1,3,5(10),16-tetraene-17-carbonitrile (4, 7.47 g, 25.5 mmol) was taken in

toluene (120 ml) under argon. To this solution, a 3.0 M methylmagnesium iodide solution in diethyl ether (14 ml, 42 mmol) was added. The reaction mixture was incubated with stirring at ca. 65˚C for ca. 5 h until the starting nitrile disappeared on TLC and then cooled to 5˚C followed by the addition of glacial acetic acid (10 ml), which was accompanied by an increase in the internal temperature to ca. 35˚C. Then the reaction mixture was cooled to ambient temperature, water (25 ml) and conc. hydrochloric acid (1 ml) were added, and the mixture was refluxed for 1

20

ACCEPTED MANUSCRIPT

h. After cooling, the organic layer was separated and washed with water and brine. All aqueous layers were pooled and extracted with CHCl3 (3 x 30 ml). The organic fractions were worked-up in the same way, pooled, dried with anhydrous Na2SO4, and concentrated in vacuo to obtain a yellow viscous residue , which was chromatographed on silica gel using, sequentially, a mixture

RI PT

of dichloromethane and hexane (2 : 1) and a mixture of dichloromethane, hexane, and acetone (4 : 1 : 1). Then the relevant fractions were pooled and concentrated. Ketone 5 and “dimer” 6 were obtained in yields of 60% and 5%, respectively. .

SC

“Dimer”: m.p. 266–269˚C (toluene), 1H NMR (CDCl3, 300 MHz): δ 1.06 (s, 3H, 18-CH3), 1.16 (s, 3H, 18’-CH3), 2.37 (s, 3H, 21-CH3), 1.43–2.52 (m, 24H, rest of the 7 x CH2 and 6 x CH

M AN U

of both steroid rings), 2.72 (t, 1H, 15-CH), 2.93 (m, 5H, 6-CH2, 6’-CH2, 15-CH), 3.81 (s, 6H, 2 x 3-OCH3), 6.42 (bs, 1H, 16’-CH), 6.68 (bs, 2H, 4-CH, 4’-CH), 6.75 (m, 2H, 2-CH, 2’-CH), 6.95 (s, 1H, 20-CH), 7.24 (d, 2H, 1-CH, 1’-CH, J = 8.1 Hz); HRMS: m/z 600.3819 [M + H]+ (calcd for C42H50NO2, 600.3836); a sample for X-ray diffraction was additionally recrystallized from toluene, CCDC ID: 1495043.

3-Methoxy-16α,17α-cyclohex-3’,4’-eno-19-norpregna-1,3,5(10)-trien-20-one 7

TE D

4.2.6

3-Methoxy-19-norpregna-1,3,5(10),16-tetraen-20-one (5, 13.5 g, 44 mmol) and anhydrous

EP

aluminum chloride (0.94 g, 7 mmol) were taken in dry dichloromethane (180 ml) under argon. Dry butadiene (6.4 g, 119 mmol) was slowly purged with stirring at ambient temperature through

AC C

the resulting dark beet-red solution. The mixture was further stirred for 18 h in a sealed roundbottom flask until the starting material disappeared on TLC (toluene– acetone). Then the reaction was quenched with methanol (5 ml) and saturated NaHCO3 (10 ml), after which the solution turned pale yellow. The organic layer was separated, washed with water, dried over anhydrous Na2SO4, and concentrated in vacuo. The oily residue was recrystallized from hexane to obtain 3methoxy-16α,17α-cyclohex-3’,4’-eno-19-norpregna-1,3,5(10)-trien-20-one 7. Yield: 68%, m.p. 135–137˚C (cf. lit. m.p. 136–137.5˚C [19]), 1H NMR (CDCl3, 300 MHz): δ 0.76 (s, 3H, 18-CH3), 1.30–2.48 (m, 15H, rest of the 6 x CH2 and 3 x CH of steroid and

21

ACCEPTED MANUSCRIPT

additional rings), 2.15 (s, 3H, 21-CH3), 2.86 (m, 2H, 6-CH2), 3.14 (m, 1H, 16-CH), 3.79 (s, 3H, OCH3), 5.83 (m, 2H, 3’,4’-CH=CH-), 6.64 (s, 1H, 4-CH), 6.72 (d, 1H, 2-CH, J = 8.1 Hz), 7.21 (d, 1H, 1-CH, J = 8.1 Hz); 13C NMR (CDCl3, 75 MHz): δ 16.9, 26.3, 27.1, 27.8, 28.5, 29.8, 29.9, 32,8, 33.8, 34.3, 38.7, 43.5, 46.3, 50.0, 55.2, 66.6, 111.5, 113.8, 126.0, 126.1, 129.4, 132.4,

4.2.7

RI PT

157.5, 211.1; HRMS: m/z 387.2283 [M + Na]+ (calcd for C25H32NaO2, 387.2295). 3-Methoxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-trien-20-one 8

3-Methoxy-16α,17α-cyclohex-3’,4’-eno-19-norpregna-1,3,5(10)-trien-20-one (7, 9.2 g, 25

SC

mmol) and 10% Pd/C (0.45 g) as the catalyst were taken in dioxane (150 ml). The mixture was hydrogenated at atmospheric pressure until the starting material disappeared on TLC

M AN U

(visualization by spraying with aqueous KMnO4). Then the catalyst was filtered off, the solvent was evaporated in vacuo, and the residue was recrystallized from a hexane–toluene mixture (5 : 1) to obtain 3-methoxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-trien-20-one 8. Yield: 93%, m.p. 138–139˚C (cf. lit. m.p. 139.5–142˚C (methanol) [27]), 1H NMR (CDCl3, 300 MHz): δ 0.73 (s, 3H, 18-CH3), 0.81–2.41 (m, 19H, rest of the 8 x CH2 and 3 x CH of steroid

TE D

and additional rings), 2.17 (s, 3H, 21-CH3), 2.87 (m, 2H, 6-CH2), 3.02 (m, 1H, 16-CH), 3.79 (s, 3H, OCH3), 6.64 (s, 1H, 4-CH), 6.72 (dd, 1H, 2-CH, J1 = 8.1 Hz, J2 = 2.3 Hz), 7.19 (d, 1H, 113

C NMR (CDCl3, 75 MHz): δ 16.1, 21.2, 22.5, 26.4, 27.2, 27.3, 27.7, 28.0,

EP

CH, J = 8.8 Hz);

29.8, 29.9, 32.3, 34.2, 38.9, 43.9, 47.3, 49.5, 55.3, 64.4, 111.6, 113.9, 126.1, 132.6, 138.0, 157.6,

AC C

212.5; HRMS: m/z 389.2438 [M + Na]+ (calcd for C25H34NaO2, 389.2451). 4.2.8

3-Hydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-trien-20-one 9.

A mixture of 3-methoxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-trien-20-one (8,

0.37 g, 1.0 mmol), sodium iodide (0.15 g, 1 mmol), glacial acetic acid (5 ml), and conc. hydrobromic acid (3 ml) was refluxed for 3.5 h and then poured into cold water. The precipitate that formed was filtered off, dried, and recrystallized from aqueous methanol to obtain 3hydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-trien-20-one 9.

22

ACCEPTED MANUSCRIPT

Yield: 77%, m.p. 239–240˚C, 1H NMR (DMSO- d6, 300 MHz): δ 0.62 (s, 3H, 18-CH3), 0.71–2.33 (m, 19H, rest of the 8 x CH2 and 3 x CH of steroid and additional rings), 2.09 (s, 3H, 21-CH3), 2.70 (bs(m), 2H, 6-CH2), 2.87 (m, 1H, 16-CH), 6.43 (s, 1H, 4-CH), 6.50 (d, 1H, 2-CH, J = 7.7 Hz), 7.01 (d, 1H, 1-CH, J = 7.7 Hz); 8.97 (bs, 1H, 3-OH);

13

C NMR (DMSO- d6, 75

RI PT

MHz): δ 15.9, 20.9, 22.2, 26.2, 26.6, 27.6, 27.7, 27.9, 29.4, 29.6, 31.8, 33.8, 38.7, 43.4, 47.0, 48.8, 63.8, 113.0, 115.2, 126.0, 130.5, 137.3, 155.2, 211.7; HRMS: m/z 353.2469 [M + H]+ (calcd for C24H33O2, 353.2475).

20(R)-3,20-Dihydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-triene 10a

SC

4.2.9

3-Hydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-trien-20-one (9, 0.25 g, 0.7

M AN U

mmol) was taken in anhydrous tetrahydrofuran (15 ml). To this solution, LiAlH4 (0.07 g, 1.8 mmol) was carefully added. The reaction mixture was stirred for 48 h. Then methanol (5 ml), saturated NH4Cl (10 ml), and conc. hydrochloric acid (2 ml) were sequentially added dropwise. The organic layer was separated, and the aqueous layer was extracted with tetrahydrofuran (2 x 10 ml) and chloroform (2 x 10 ml). Each organic fraction was washed separately with saturated

TE D

NH4Cl. Then all extracts were pooled, dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting oily mass was purified by column chromatography (silica gel, dichloromethane–

EP

acetone, 20 : 1) followed by recrystallization from aqueous ethanol to obtain 20(R)-3,20dihydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-triene 10a.

AC C

Yield: 48%, m.p. 196–197˚C, 1H NMR (DMSO-d6, 300 MHz): δ 0.86 (s, 3H, 18-CH3), 1.05–2.15 (m, 20H, rest of the 8 x CH2 and 4 x CH of steroid and additional rings), 1.14 (d, 3H, 21-CH3, J = 5.8 Hz), 2.70 (m, 2H, 6-CH2), 3.82 (m, 1H, 20-CH), 4.12 (d, 1H, 20-OH), 6.42 (s, 1H, 4-CH), 6.48 (d, 1H, 2-CH, J = 8.1 Hz), 6.99 (d, 1H, 1-CH, J = 8.1 Hz), 8.95 (bs, 1H, 3-OH); 13

C NMR, see the Table 1b in text; HRMS: m/z 355.2639 [M + H]+ (calcd for C24H35O2,

355.2632); a sample for X-ray diffraction was obtained from aqueous methanol, CCDC ID: 1495042. 4.2.10

20(S)-3,20-Dihydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-triene 10b

23

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3-Methoxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-trien-20-one (8, 0.37 g, 1.0 mmol) was taken in anhydrous toluene (15 ml). The system was flushed with argon, and a 1.2 M DIBAH solution in toluene (5 ml, 6 mmol) was added to the reaction mixture under argon. Then the mixture was refluxed with stirring for 6 h, after which methanol (2 ml), water (10 ml), and

RI PT

conc. hydrochloric acid (2 ml) were sequentially added with vigorous stirring. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (4 x 10 ml). The pooled organic fractions were washed with water and saturated NH4Cl, dried over anhydrous Na2SO4,

SC

and concentrated in vacuo. The resulting oily mass was purified by preparative HPLC to obtain 20(R)-3,20-dihydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-triene 10a and 20(S)-3,20-

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dihydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-triene 10b as amorphous solids. After recrystallization from aqueous ethanol, these products were obtained in yields of 44% and 14%, respectively.

20(S)-3,20-Dihydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-triene

10b:

14%,

m.p. 203–204˚C (at 136˚C the phase transition was observed for the initially formed crystals), 1H

TE D

NMR (DMSO-d6, 300 MHz): δ 0.75 (s, 3H, 18-CH3), 1.07–2.29 (m, 20H, rest of the 8 x CH2 and 4 x CH of steroid and additional rings), 1.10 (d, 3H, 21-CH3, J = 5.5 Hz), 2.69 (m, 2H, 6-CH2),

EP

3.85 (m, 1H, 20-CH), 4.13 (d, 1H, 20-OH), 6.42 (bs, 1H, 4-CH, J = 2.2 Hz), 6.48 (dd, 1H, 2-CH, J1 = 8.8 Hz, J2 = 2.2 Hz), 6.99 (d, 1H, 1-CH, J = 8.1 Hz), 8.96 (bs, 1H, 3-OH); 13C NMR, see

AC C

the Table 1b in text; HRMS: m/z 377.2552 [M + Na]+ (calcd for C24H34NaO2, 377.2451). 4.2.11

20(R,S)-3-Methoxy-20-hydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-

triene 11

3-Methoxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-trien-20-one (8, 0.37 g, 1.0

mmol) was taken in anhydrous tetrahydrofuran (15 ml). To this solution, LiAlH4 (0.08 g, 2.1 mmol) was carefully added, and then the reaction mixture was stirred for 48 h, after which methanol (5 ml), saturated NH4Cl (10 ml), and conc. hydrochloric acid were sequentially added dropwise. The organic layer was separated, and the aqueous layer was extracted with

24

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tetrahydrofuran (2 x 10 ml) and chloroform (2 x 10 ml). Each organic fraction was washed separately with saturated NH4Cl. Then all extracts were pooled, dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting oily mass was recrystallized from a 1 : 1 toluene– hexane mixture to obtain 20(R,S)-3-methoxy-20-hydroxy-16α,17α-cyclohexano-19-norpregna-

RI PT

1,3,5(10)-triene 11. Yield: 62%, m.p. 145–147˚C, 1H NMR (CDCl3, 300 MHz): δ 0.85 + 0.99 (s, 3H, 18-CH3, R+S), 1.16–2.43 (m, 21H, rest of the 8 x CH2 and 4 x CH of steroid and additional rings and 20-

SC

OH), 1.29 + 1.33 (d, 3H, 21-CH3, J = 6.61 Hz, R+S), 2.89 (m, 2H, 6-CH2), 3.81 (s, 3H, OCH3), 4.08 (q, 1H, 20-CH, J1 = 6.6 Hz, J2 = 5.9 Hz), 6.67 (s, 1H, 4-CH), 6.48 (dd, 1H, 2-CH, J1 = 8.1

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Hz, J2 = 2.2), 7.22 (d, 1H, 1-CH, J = 8.1 Hz); 13C NMR (CDCl3, 75 MHz): δ (14.4), 14.9, 20.5 (20.1), (20.8), 22.2, 24.2, 24.3, (24.8), (26.2), 26.5, 28.1, 29.9, 30.0, (30.8), (32.2), (33.0), 33.2, 33.5, 36.2, (38.9), 39.1, (40.7), 43.7, (48.1), 48.8, (49.2), 49.6, (49.8), 49.9, 55.2, 73.5, (76.9), 111.4, 113.8, (126.2), 126.3, (132.9), 133.0, 138.1, 157.4; HRMS: m/z 369.2779 [M + H]+ (calcd for C25H37O2, 369.2788).

Comparative study of the 20-keto group reduction of 3-methoxy-16α,17α-

TE D

4.2.12

cyclohexano-19-norpregna-1,3,5(10)-trien-20-one 8 and 3-methoxy-19-norpregna-1,3,5(10)-

EP

trien-20-one 14 with lithium aluminum hydride and DIBAH. a) LAH: Ketone (1 mmol) was taken in anhydrous THF (10 ml). To this solution, LiAlH4

AC C

(0.05 g, 1.3 mmol) was carefully added, and the reaction mixture was stirred for 4 h, after which methanol (5 ml), saturated NH4Cl (10 ml), and conc. hydrochloric acid (1 ml) were sequentially added dropwise with vigorous stirring. The organic layer was separated, and the aqueous layer was extracted with ethyl acetate (4 x 10 ml) and chloroform (3 x 10 ml). The organic fractions were washed with saturated NH4Cl, pooled, dried over anhydrous Na2SO4, and concentrated in vacuo. The crude product was analyzed by 1H NMR (CDCl3, 300 MHz) comparing the intensities of major and minor picks at δН 0.98 and δН 0.84 in the case of compound 8, and at δН 0.81 and δН 0.72 in the case of compound 14.

25

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b) DIBAH: Ketone (1 mmol) was taken in anhydrous toluene (10 ml). The system was flushed with argon, and a 1.2 M DIBAH solution in toluene (2 ml, 2.4 mmol) was added to the reaction mixture under argon. Then the mixture was stirred for 4 h, after which methanol (2 ml), water (10 ml), ethyl acetate (10 ml), and conc. hydrochloric acid (1 ml) were sequentially added

4.2.13

RI PT

dropwise with vigorous stirring. Further processing was the same as in the example (a) above. 3-Methoxy-16α,17α-cyclopropano-19-norpregna-1,3,5(10)-trien-20-one 12

Trimethylsulfoxonium iodide (1.55 g, 7.0 mmol) and sodium hydride (60 % suspension in

SC

oil, 0.4 g, 10.0 mmol) were taken in a mixture of anhydrous DMSO (30 ml) and THF (10 ml), and the solution was stirred until hydrogen evolution ceased. Then a solution of 3-methoxy-19-

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norpregna-1,3,5(10),16-tetraen-20-one (5, 1.55 g, 5 mmol) in anhydrous THF (50 ml) was added. The reaction mixture was stirred for 24 h at room temperature until the starting material disappeared on TLC, poured into water (400 ml), and extracted with chloroform (3 x 50 ml). The organic layers were pooled, washed with water and brine, dried over anhydrous Na2SO4, and concentrated in vacuo. The resulting sticky solid was recrystallized from methanol with an

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addition of a small amount of THF to obtain 3-methoxy-16α,17α-cyclopropano-19-norpregna1,3,5(10)-trien-20-one 12.

EP

Yield: 59%, m.p. 138–140˚C (cf. lit. m.p. 137–139˚C (methanol) [28], 1H NMR (CDCl3, 300 MHz): δ 0.91 (m, 1H, 22α-CH), 1.01 (s, 3H, 18-CH3), 1.04 (m, 1H, 22β-CH), 1.22–2.47 (m,

AC C

12H, rest of the 4 x CH2 and 4 x CH of steroid ring), 2.00 (s, 3H, 21-CH3), 2.88 (m, 2H, 6-CH2), 3.80 (s, 3H, OCH3), 6.65 (s, 1H, 4-CH), 6.73 (d, 1H, 2-CH, J1 = 8.1 Hz), 7.21 (d, 1H, 1-CH, J = 8.1 Hz); 13C NMR (CDCl3, 75 MHz): δ 15.0, 17.2, 23.9, 25.7, 26.4, 27.1, 27.8, 29.7, 34.6, 37.0, 41.3, 44.3, 46.6, 47.7, 55.3, 111.5, 113.9, 126.2, 132.7, 137.8, 157.6, 208.0; HRMS: m/z 347.1985 [M + Na]+ (calcd for C22H28NaO2, 347.1982). 4.2.14

20(R,S)-3,20-Dihydroxy-16α,17α-cyclopropano-19-norpregna-1,3,5(10)-triene 13

20(R,S)-3,20-Dihydroxy-16α,17α-cyclopropano-19-norpregna-1,3,5(10)-triene

13

was

prepared from 3-methoxy-16α,17α-cyclopropano-19-norpregna-1,3,5(10)-trien-20-one (1, 0.32

26

ACCEPTED MANUSCRIPT

g, 1.0 mmol) and a 1.2 M DIBAH solution in toluene (4 ml, 4.8 mmol) as described for 20(S)3,20-dihydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-triene 10b. Yield: 32%, m.p. 210–217˚C*, 1H NMR (DMSO-d6, 300 MHz): δ 0.43 (m, 1H, 22-CH, R+S), 0.53 + 0.68 (m, 1H, 22-CH, R+S), 0.83 + 1.08 (d, 3H, 21-CH3, R+S, J = 6.6 Hz, J = 5.5

RI PT

Hz), 0.87 + 0.91 (s, 3H, 18-CH3, R+S), 0.90 – 2.27 (m, 12H, rest of the 4 x CH2 and 4 x CH of steroid ring), 2.69 (m, 2H, 6-CH2), 4.07 (m, 2H, 20-CH and 20-OH), 6.42 (s, 1H, 4-CH), 6.49 (d, 1H, 2-CH, J = 8.7 Hz), 6.99 (d, 1H, 1-CH, J = 8.8 Hz), 8.99 (bs, 1H, 3-OH); 13C NMR, see the

4.2.15

SC

Table 1b in text; HRMS: m/z 335.1978 [M + Na]+ (calcd for C21H28NaO2, 335.1982). 3-Methoxy-19-norpregna-1,3,5(10)-trien-20-one 14

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3-Methoxy-19-norpregna-1,3,5(10),16-tetraen-20-one (5, 0.71 g, 2.3 mmol) and 10% Pd/C (0.15 g) as the catalyst were taken in dioxane (30 ml) and hydrogenated at atmospheric pressure until the starting material disappeared on TLC (visualization by spraying with aqueous KMnO4). Then the catalyst was filtered off, the solvent was evaporated in vacuo, and the residue was

trien-20-one 14.

TE D

recrystallized from a 2 : 1 hexane–toluene mixture to obtain 3-methoxy-19-norpregna-1,3,5(10)-

Yield: 91%, m.p. 135˚C (cf. lit. m.p. 134–136˚C [29]), 1H NMR (CDCl3, 300 MHz): δ 0.68

EP

(s, 3H, 18-CH3), 1.25–2.45 (m, 13H, rest of the 5 x CH2 and 3 x CH of steroid ring), 2.18 (s, 3H, 21-CH3), 2.64 (m, 1H, 17-CH), 2.87 (m, 2H, 6-CH2), 3.80 (s, 3H, OCH3), 6.66 (s, 1H, 4-CH),

AC C

6.74 (dd, 1H, 2-CH, J1 = 8.1 Hz, J2 = 2.3 Hz), 7.23 (d, 1H, 1-CH, J = 8.8 Hz); 13C NMR (CDCl3, 75 MHz): δ 13.5, 23.0, 24.2, 26.7, 27.8, 29.9, 31.5, 38.8, 39.1, 43.7, 44.5, 55.2, 55.8, 63.9, 111.6, 113.9, 126.3, 132.5, 138.0, 157.6, 209.4; HRMS: m/z 335.1969 [M + Na]+ (calcd for C21H28NaO2, 335.1982). 4.2.16

3-Hydroxy-19-norpregna-1,3,5(10)-trien-20-one 15

A mixture of 3-methoxy-19-norpregna-1,3,5(10)-trien-20-one (14, 0.66 g, 2.2 mmol), sodium iodide (0.3 g, 2.0 mmol), glacial acetic acid (10 ml), and conc. hydrobromic acid (5 ml) was refluxed for 3.5 h and then poured into cold water. The precipitate that formed was filtered

27

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off, dried, dissolved in boiling toluene, and filtered hot through silica gel (0.5 g). After cooling, the precipitate that formed was filtered off and recrystallized from methanol to obtain 3-hydroxy19-norpregna-1,3,5(10)-trien-20-one 15 as yellowish prisms. Yield: 53%, m.p. 247–248˚C (cf. lit. m.p. 244–247˚C [30]), 1H NMR (DMSO- d6, 300

RI PT

MHz): δ 0.57 (s, 3H, 18-CH3), 1.15–2.36 (m, 13H, rest of the 5 x CH2 and 3 x CH of steroid ring), 2.10 (s, 3H, 21-CH3), 2.65 (m, 1H, 17-CH), 2.72 (m, 2H, 6-CH2), 6.46 (s, 1H, 4-CH), 6.53 (d, 1H, 2-CH, J = 8.1 Hz), 7.06 (d, 1H, 1-CH, J = 8.1 Hz), 8.96 (bs, 1H, 3-OH);

13

C NMR

(DMSO- d6, 75 MHz): δ 13.1, 22.3, 23.6, 26.3, 27.3, 29.1, 31.1, 38.1, 38.4, 43.1, 43.6, 54.8,

SC

62.7, 112.7, 114.9, 125.9, 130.1, 137.0, 154.9, 208.5; HRMS: m/z 299.2013 [M + H]+ (calcd for

4.2.17

M AN U

C20H27O2, 299.2006).

20(R)-3,20-Dihydroxy-19-norpregna-1,3,5(10)-triene 16

20(R)-3,20-Dihydroxy-19-norpregna-1,3,5(10)-triene 16 was prepared from 3-hydroxy-19norpregna-1,3,5(10)-trien-20-one (15, 0.25 g, 0.93 mmol) and LiAlH4 (0.08 g, 2.1 mmol) as described for 20(R)-3,20-dihydroxy-16α,17α-cyclohexano-19-norpregna-1,3,5(10)-triene 10a.

TE D

Yield: 40%, m.p. 229–230˚C (cf. lit. m.p. 232–233˚C [31]), 1H NMR (DMSO- d6, 300 MHz): δ 0.71 (s, 3H, 18-CH3), 1.02 (d, 3H, 21-CH3, J = 6.6 Hz), 1.05–2.30 (m, 14H, rest of the

EP

5 x CH2 and 4 x CH of steroid ring), 2.70 (m, 2H, 6-CH2), 3.52 (m, 1H, 20-CH), 4.10 (d, 1H, 20OH, J = 5.5 Hz), 6.43 (s, 1H, 4-CH), 6.49 (dd, 1H, 2-CH, J1 = 8.8 Hz, J2 = 2.2 Hz), 7.03 (d, 1H, 13

C NMR, see the Table 1b in text; HRMS: m/z

AC C

1-CH, J = 8.8 Hz), 8.93 (bs, 1H, 3-OH);

301.2166 [M + H]+ (calcd for C20H29O2, 301.2162); a sample for X-ray diffraction was obtained from aqueous methanol, CCDC ID: 1495041. 4.2.18

20(R,S)-3,20-Dihydroxy-19-norpregna-1,3,5(10),16-tetraene 17

20(R,S)-3,20-Dihydroxy-19-norpregna-1,3,5(10),16-tetraene 17 was prepared from 3methoxy-19-norpregna-1,3,5(10),16-tetraen-20-one (5, 0.37 g, 1.2 mmol) and a 1.2 M DIBAH solution in toluene (4 ml, 4.8 mmol) as described for 20(S)-3,20-dihydroxy-16α,17αcyclohexano-19-norpregna-1,3,5(10)-triene 10b but without a chromatographic step. The sticky

28

ACCEPTED MANUSCRIPT

solid that was obtained after the usual work-up was purified by recrystallization from aqueous methanol to obtain 20(R,S)-3,20-dihydroxy-19-norpregna-1,3,5(10),16-tetraene 17. Yield: 47%, m.p. 195–198˚C, 1H NMR (DMSO-d6, 300 MHz): δ 0.81 + 0.84 (s, 3H, 18CH3, R+S), 1.20–2.33 (m, 11H, rest of the 4 x CH2 and 3 x CH of steroid ring), 1.20 + 1.22 (d,

RI PT

3H, 21-CH3, S+R, J = 6.6 Hz), 2.73 (m, 2H, 6-CH2), 4.22 (m, 1H, 20-CH), 4.44 + 4.49 (d, 1H, 20-OH, R+S, J = 5.3 Hz), 5.50 + 5.52 (bs, 1H, 16-CH, R+S), 6.44 (d, 1H, 4-CH, J = 2.2 Hz), 6.50 (dd, 1H, 2-CH, J1 = 8.1 Hz, J2 = 2.2 Hz), 7.02 (d, 1H, 1-CH, J = 8.8 Hz), 8.96 (bs, 1H, 313

C NMR, see the Table 1b in text; HRMS: m/z 321.1822 [M + Na]+ (calcd for

SC

OH);

C20H28NaO2, 321.1825). Molecular modeling

M AN U

4.3

The three-dimensional structure of the human ER ligand-binding domain (LBD) was taken from the Protein Data Bank (PDB: 1QKU). The structures of compounds 8-11, 13, and 15-were generated using the SYBYL8.1 software suite [32]. The structures of the compounds and the protein molecule were optimized in vacuum using the Powell energy minimization algorithm with the Tripos force field [33]. The partial atomic charges were calculated by the Gasteiger–

TE D

Hückel method. Interactions of the steroids with the ER LBD binding site were modeled using the molecular docking program DOCK 6.5 [34]. The modeling of the ER complex with the natural ligand E2 was used as the control. No more than three candidates (most probable

EP

complexes) were selected using the Dock scoring function. The further optimization of virtual complexes was performed using the Amber 9.0 package [35] with the general AMBER99 force field (GAFF) as described previously [36]. The optimization included the explicit solvation of

AC C

the complex in aqueous environment (TIP3P model) and productive dynamics at 300 K for 10 ps with periodic boundary conditions (NTP ensemble). This computational molecular dynamics experiment was used for the structure optimization and for the assessment of the LBD–ligand binding energies by means of MM-PBSA [35]. The averaging was performed over 10 observations saved at regular time intervals during simulation. The solvation component was calculated as a mean value estimated by the Poisson–Boltzmann method. The final structure was selected based on the minimum value of the complex Gibbs energy calculated by the MM-PBSA method taking into account electrostatic interactions, changes in van der Waals interactions, and solvation effects. 4.4

Biology 29

4.4.1

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Cytotoxicity evaluation

Cell cultures and assessment of inhibitory activity. The ERα-positive hormone dependent MCF-7 human breast cancer cells were obtained from the ATCC collection (USA). The MCF-7 cells were cultured in standard DMEM medium (PanEco, Russia) supplemented with 10% FCS and 0.1 mg/ml sodium pyruvate (Santa Cruz) at 37°C, 5% CO2 and 80-85% humidity. The MCF10A normal breast cells were purchased from ATCC and were cultured in DMEM/F12 (PanEco,

RI PT

Russia) supplemented with 7% horse serum, 20 ng/ml EGF, 0.5 µg/ml hydrocortisone, and 10 µg/ml insulin at 37°C, 5% CO2 and 80-85% humidity. The growth inhibitory activity of the compounds was assessed by the MTT test based on the accumulation of the MTT reagent (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (Applichem, USA) by living cells

SC

[37]. Briefly, the cells were seeded with a density of 104 cells per well in 24-well plates (Corning, USA) in 900 µL of the medium. The tested compounds were dissolved in DMSO

M AN U

(Applichem, USA) at a concentration of 10 mM before experiments and then were diluted in the medium to the required concentrations. The antiestrogen tamoxifen (Sigma-Aldrich, USA) was used as the reference drug. Compounds at different concentrations in 100 µL of the appropriate medium were added 24 h after the seeding, and the cells were grown for 72 h. After incubation with the compounds, the medium was removed, the MTT reagent that was dissolved in the medium was added to the final concentration of 0.2 mg/ml to each well, and the incubation was

TE D

performed for 3 h. Then the cell supernatants were removed and purple MTT formazan crystals were dissolved in 100% DMSO (350 µL per well). Culture plates were gently shaken, and the absorbance was measured at 571 nm on a MultiScan reader (ThermoFisher, USA). The viability of the cells was expressed as percentage of control. Dose-response curves were analyzed by

EP

regression analysis using sigmoid curves (Log(concentration) vs normalized absorbance). In this study, the half-maximal inhibitory concentration (IC50) values were determined using GraphPad

AC C

Prism (USA). 4.4.2

Luciferase reporter gene assay for ERα

Measurements of estrogen receptor α activity. To determine the transcriptional activity of

estrogen receptor α (ERα), MCF-7 cells were transfected with the plasmids containing the luciferase reporter gene controlled by the promoter with estrogen responsive elements [38]. The ERE-TK-LUC reporter plasmid used in this work was kindly provided by Dr. George Reid [39]. The transfection was carried out for 24 h at 37 °C using Metafectene (Biontex Laboratories GmbH) in phenol red-free DMEM supplemented with 10% dextran-coated charcoal-treated (DCC) serum (steroid-free conditions). To control the efficiency and potential toxicity of the transfection, the cells were co-transfected with the β-galactosidase plasmid. The tested 30

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compounds were added to phenol red-free DMEM supplemented with 5% DCC serum and 10 nM 17β-estradiol. The luciferase activity was measured according to a standard protocol (Promega) using an Infinite M200 Pro microplate reader (Tecan). The β-galactosidase activity was assessed at 405 nm using a MultiScan FC reader (ThermoFisher, USA). The luciferase activity was expressed in arbitrary units evaluated as the ratio of the luciferase to galactosidase activity.

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Acknowledgments

This work was supported by Russian Science Foundation (grant 14-50-00126). The biology experiments of the research were supported by Russian Science Foundation (grant 14-15-00362). The plasmid ERE-TK-LUC was kindly provided by Dr. George Reid. We would like to thank

SC

Dr. Olga Susova for helpful consultations on MCF-10A cell culture.

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Appendix A. Supplementary data

Supplementary data associated with this article can be found in the online version of the article. These data include characterization data for most of the compounds described in this article: NMR signal assignments for compounds 4 and 5, structure determination of “dimer” 6 including principal X-ray diffraction data and its probable formation mechanism, relevant 1D

17. References

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and 2D NMR spectra of 3,20-dihydroxy steroids, and 1H and 13C NMR spectra of compounds 2–

[1] R. L. Siegel, K. D. Miller, A. Jemal. Cancer Statistics, 2016. CA Cancer J. Clin. 2016, 66, 7-30. DOI: 10.3322/caac.21332.

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[2] F. Lumachi, D. A. Santeufemia, S. M. M. Basso. Current medical treatment of estrogen receptor-positive breast cancer. World J. Biol. Chem. 2015, 6, 231-239. DOI: 10.4331/wjbc.v6.i3.231.

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[3] I. Ahmad, Shagufta. Recent developments in steroidal and nonsteroidal aromatase inhibitors for the chemoprevention of estrogen-dependent breast cancer, Eur. J. Med. Chem. 2015, 102, 375-386. DOI: 10.1016/j.ejmech.2015.08.010. [4] С. Ouellet, R. Maltais, É. Ouellet, X. Barbeau, P. Lagüe, D. Poirier. Discovery of a sulfamate-based steroid sulfatase inhibitor with intrinsic selective estrogen receptor modulator properties. Eur. J. Med. Chem. 2016, 119, 169-182. DOI: 10.1016/j.ejmech.2016.04.044. [5] D. P. McDonnell, S. E. Wardell. The molecular mechanisms underlying the pharmacological actions of ER modulators: implications for new drug discovery in breast cancer. Cur. Opin. Pharmacol. 2010, 10, 620-628. [6] D.P. McDonnell, S.E. Wardell, J.D. Norris. Oral selective estrogen receptor downregulators (SERDs), a breakthrough endocrine therapy for breast cancer. J. Med. Chem. 2015, 58, 4883-4887. DOI: 10.1021/acs.jmedchem.5b00760. [7] J.F.R. Robertson. Fulvestrant (Faslodex®) — how to make a good drug better. Oncologist. 2007, 12, 774-784. DOI:10.1634/theoncologist.12-7-774. 31

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ERalpha on responsive promoters is an integral feature of estrogen signaling. Mol. Cell. 2003, 11, 695-707. DOI: 10.1016/S1097-2765(03)00090-X.

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Highlights

1) Series of 3,20-dihydroxy-19-norpregna-1,3,5(10)-trienes were synthesized. 2) Studied compounds can bind to ERα and their binding mode is similar to that of estradiol. 3) Synthesized 3,20-dihydroxy-19-norpregna-1,3,5(10)-trienes were cytotoxic against MCF-7.

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4) Additional ring D′ is responsible for ERα antagonist activity of the compounds synthesized.

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ACCEPTED MANUSCRIPT Highlights 1) Series of 3,20-dihydroxy-19-norpregna-1,3,5(10)-trienes were synthesized. 2) Studied compounds can bind to ERα and their binding mode is similar to that of estradiol. 3) Synthesized 3,20-dihydroxy-19-norpregna-1,3,5(10)-trienes were cytotoxic against MCF-7.

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4) Additional ring D′ is responsible for ERα antagonist activity of the compounds synthesized.

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