Chemical synthesis, characterisation and biological evaluation of lactonic-estradiol derivatives as...

Accepted Manuscript

Title: Chemical synthesis, characterisation and biologicalevaluation of lactonic-estradiol derivatives as inhibitors of17�-hydroxysteroid dehydrogenase type 1

Author: Siham Farhane Michelle-Audrey Fournier DonaldPoirier

PII: S0960-0760(13)00067-8DOI: http://dx.doi.org/doi:10.1016/j.jsbmb.2013.05.002Reference: SBMB 3976

To appear in: Journal of Steroid Biochemistry & Molecular Biology

Received date: 21-12-2012Revised date: 28-4-2013Accepted date: 1-5-2013

Please cite this article as: S. Farhane, M.-A. Fournier, D. Poirier, Chemical synthesis,characterisation and biological evaluation of lactonic-estradiol derivatives as inhibitorsof 17�-hydroxysteroid dehydrogenase type 1, Journal of Steroid Biochemistry andMolecular Biology (2013), http://dx.doi.org/10.1016/j.jsbmb.2013.05.002

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http://dx.doi.org/doi:10.1016/j.jsbmb.2013.05.002

http://dx.doi.org/10.1016/j.jsbmb.2013.05.002

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J. Steroid Biochem. Mol. Biol. (revised version)

Special Issue: Challenges in the synthesis and biological testing of steroid derivatives as inhibitors

Chemical synthesis, characterisation and biological evaluation of lactonic-estradiol derivatives

as inhibitors of 17β-hydroxysteroid dehydrogenase type 1

Siham Farhane, Michelle-Audrey Fournier and Donald Poirier*

Laboratory of Medicinal Chemistry, CHU de Québec (CHUL) – Research Center and Laval

University, Québec (Québec), G1V 4G2, Canada

Corresponding author:

Dr. Donald Poirier

Laboratory of Medicinal Chemistry

CHU de Québec (CHUL) - Research Center

2705 Laurier Boulevard

Québec (Québec), G1V 4G2, Canada

Tel: (418) 654-2296; Fax: (418) 654-2761

E-mail: [email protected]

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HighlightsLactone- and lactol-estradiol derivatives were synthesized and characterized.Lactone E-ring was diversified by adding a hydroxymethyl, a methylcarboxylate, a carboxy or an allyl group.A chemical approach was developed to introduce a chemical group on hindered beta-steroid face. Lactone and lactol derivatives inhibited 17β-HSD1 (34-60%) similarly as the natural substrate estrone (53%).

Abstract

To control estradiol (E2) formation, we are interested in synthesizing inhibitors of 17β-

hydroxyteroid dehydrogenase type 1 (17-HSD1). Since the results of docking experiments have

shown that E2-lactone derivatives substituted in position 19 or 20 (E-ring) could generate

interactions with the active site of the enzyme, we carried out their chemical synthesis. After having

prepared the 16,17--lactone-E2 in four steps starting from estrone (E1), we introduced the

molecular diversity by adding a hydroxymethyl, a methylcarboxylate, a carboxy or an allyl group.

The allyl derivative was used as a key intermediate to generate a hydroxyethyl side chain in α or β

position. Two lactols were also obtained from two hydroxyalkyl lactones. Enzymatic assays

revealed that lactone and lactol derivatives weakly inhibited 17β-HSD1 in homogenized HEK-293

cells overexpressing 17β-HSD1 (34-60% at 1 μM) and in intact T-47D cells expressing 17β-HSD1

(10-40% at 10 μM).

Keywords: 17-Hydroxysteroid dehydrogenase, Enzyme, Inhibitor, Steroid, Synthesis.

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Introduction

Estrogens are well known to contribute to the development and progression of estrogen-

dependent diseases such as breast cancer [1]. Intratumoural production of estrogens thus plays a

crucial role in the proliferation of breast cancer cells, especially in postmenopausal women. The

blockade of this pathway may inhibit the growth of breast tumours and thus can represent an

effective treatment for hormone-dependent breast cancer (HDBC). The biologically active estrogen

estradiol (E2) is formed by the reduction of the carbonyl of estrone (E1). The major enzyme

catalysing this reaction is 17-hydroxysteroid dehydrogenase type 1 (17-HSD1), although 17-

HSD7 and 17-HSD12 can also catalyse the transformation of E1 to E2 [2]. Several studies have

reported increased levels of E2 that drive the proliferation of cancer cells and tumours via its action

on the estrogen receptor (ER) [3,4]. Other studies have also indicated that patients with high 17-

HSD1 expression level in tumours have significantly shortened disease-free and overall survival [5-

7]. Taken together, these results suggest that compounds inhibiting the activity of 17-HSD1 may

be of therapeutic benefit in the treatment of HDBC in postmenopausal patients [8,9]. This

therapeutic approach is also supported by the success of inhibition of other enzymes involved in the

steroidogenesis, namely aromatase [10-13] and, more recently, steroid sulfatase [14-17].

The 17-HSD1, which has a preferentially reductive activity using NADPH or NADH as

cofactor [18,20], is expressed in many steroidogenic tissues, including breast tissue. Much

crystallographic information has been obtained for the enzyme in its native form [21] and for the

enzyme that has been complexed with E2 and NADP [22], E2 alone [23], equilin and NADP [24],

the inhibitor EM-1745 [25] or the inhibitor E2B (CC-156) [26]. Such structural information can be

helpful for designing potential inhibitors of 17-HSD1. But although inhibitors of 17-HSD1 have

been reported by several groups [27-31], none of these inhibitors have reached clinical use.

As a part of our ongoing program to synthesize inhibitors of 17-HSD1 for the treatment of

HDBC, we used an E-ring γ-lactone-E2 as a convenient scaffold. Our interest in lactone compounds

as inhibitors of 17β-HSD1 arose from 1) the fact that the addition of a substituted five-member

lactone ring is more likely to eliminate or reduce the estrogenicity associated with an E2 nucleus

[32] and 2) from the results of the preliminary docking studies, using the three-dimensional structure

of EM-1745 [25], an E2/adenosine hybrid inhibitor [33,34], complexed with 17-HSD1. In fact, we

visually analyzed the best docked conformations for positioning of the ligand as well as for

identification of specific interactions, such as hydrogen bonding, which are well known to play a

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crucial role in the binding of a ligand to a protein. The ligand positioning criteria that we used is that

the steroid moiety should adopt the same orientation as the E1 substrate in the active site – a

tendency to adopt a backwards or upside-down position is indicative that the introduced molecular

diversity does not work in synergy with the E1 or E2 scaffold to increase affinity; in other words, to

properly satisfy the new functional group, the molecule must adopt a position that is not optimal for

its steroid moiety, thereby decreasing the final affinity and thus the inhibitory potency. From our

docking experiments, we identified a series of lactone and lactol derivatives that potentially interact

with 17β-HSD1 by producing hydrogen bonding (Table 1). As examples, compounds 7 and 9 were

expected to generate key hydrogen bonding interactions similar to those of the natural substrate E1,

as well as additional hydrogen bonding (Figure 1).

The results of our docking experiments having suggested that lactone and lactol derivatives

(scheme 1) might generate favourable interactions with the active site of 17β-HSD1, we herein

describe their chemical synthesis as well as their biological evaluation as inhibitors.

2. Experimental

2.1. General methods

Reagents were obtained from Sigma-Aldrich Canada Co. (Oakville, ON, Canada). Usual

solvents were obtained from Fisher Scientific and VWR (Montreal, QC, Canada) and were used as

received. Anhydrous solvents were purchased from Aldrich and VWR in SureSeal bottles, which

were conserved under positive argon pressure. All anhydrous reactions were performed in oven-

dried glassware under positive argon pressure. Thin-layer chromatography (TLC) was performed on

0.25-mm silica gel 60 F254 plates (Whatman, Maidstone, England), and compounds were visualized

by exposure to UV light (254 nM) and/or with a solution of ammonium heptamolybdate tetrahydrate

(with heating). Flash chromatography was performed on Silicycle 60 (Québec, QC, Canada) 230-

400 mesh silica gel. 1H and 13C NMR spectra were recorded with a Bruker AVANCE 400

spectrometer (Billerica, MA, USA). The chemical shifts () are expressed in ppm and referenced to

chloroform (7.26 and 77.00 ppm), acetone (2.06 and 206.00 ppm) or methanol (3.30 and 49.0 ppm)

for 1H and 13C, respectively. Low-resolution mass spectra (LRMS) were recorded with an LCQ

Finnigan apparatus (San Jose, CA, USA) equipped with an atmospheric pressure chemical ionisation

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(APCI) source on positive or negative mode. The names of steroid derivatives were generated using

ACD/Labs (Chemist’version) software (Toronto, ON, Canada). However, for NMR assignment and

purpose of discussion, we used the classic numbering of estrane nucleus (C1-C18) and we used

C19-C23 for additional carbons of the extra E-ring and substituent. Partial numbering was also

reported in Schemes 2 and 3.

2.2. Chemical synthesis

2.2.1. Synthesis of alcohol 2

To a solution of lactone 1 [35] (50 mg, 0.126 mmol) in dry tetrahydrofuran (THF) (3.2 mL)

at -78°C was added a solution of sodium bis-(trimethylsilyl) amide (30 µL, 0.14 mmol). After 10

min, the mixture was stirred for 30 min at 0°C, then cooled again at -78°C and ethylformate (33 µL,

0.82 mmol) was added. The reaction mixture was stirred for 10 min at -78°C and then for 6 h at 0°C.

Water was added to quench the reaction and the crude product was extracted with EtOAc. The

organic phase was washed with brine, dried over MgSO4, and evaporated under reduced pressure.

The crude product was dissolved in methanol (5 mL), the mixture was stirred for 15 min at 0°C,

then NaBH4 (12 mg, 0.29 mmol) was added to a cooled (0°C) solution. After the mixture was stirred

for 3-4 h at 0°C, the reaction was quenched by adding water and the extraction was performed with

CH2Cl2. The organic phase was dried over Na2SO4 and evaporated under reduced pressure. The

crude product was purified by flash chromatography (hexanes/EtOAc, 97:3) to give 2.

4bS,6aS,9aR,10aS,10bR)-2-{[tert-butyl(dimethyl)silyl]oxy}-9-(hydroxymethyl)-6a-methyl-

4b,5,6,6a,6b,9,9a,10,10a,10b,11,12-dodecahydro-8H-naphtho[2’,1’:4,5]indeno[1,2-b]furan-8-one

(2). 60% yield. 1H NMR (CDCl3): 0.19 (s, Si(CH3)2), 0.78 (s, 18-CH3), 0.97 (s, SiC(CH3)3), 1.20-

2.35 (unassigned CH and CH2), 2.57 (m, 19-CH), 2.81 (m, 16-CH and 6-CH2), 3.78 and 3.92 (2m,

CH2-OH), 4.42 (d, J = 10.0 Hz, 17-CH), 6.56 (d, J = 2.2 Hz, 4-CH), 6.62 (dd, J1 = 8.4 Hz, J2 = 2.4

Hz, 2-CH), 7.11 (d, J = 8.5 Hz, 1-CH). 13C NMR (CDCl3): -4.4 (2x), 12.7, 18.2, 25.7 (3x), 26.1,

27.5, 29.4, 32.6, 37.4, 38.1, 38.6, 43.6, 43.9, 50.4, 51.4, 62.4, 90.8, 117.3, 120.0, 126.1, 132.3,

137.5, 153.5, 180.0. LRMS: calcd for C27H41O4Si (M+H)+ 457.3, found 457.1.

2.2.2. Synthesis of ester 4

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A solution of diisopropylamine (0.12 mL, 0.87 mmol) in dry THF (2.8 mL) was stirred under argon

at 0°C and a solution of n-butyllithium in hexanes (0.51 mL, 0.87 mmol) was added dropwise. After

45 min, the resulting lithium diisopropylamide (LDA) solution was cooled at -78°C and the lactone

1 [35] (100 mg, 0.23 mmol), dissolved in dry THF (2 mL), was added dropwise. The mixture was

allowed to stir for 1 h at 0°C, then cooled again at -78°C and the methylchloroformate (75 µL, 0.96

mmol) was added dropwise. The reaction mixture was stirred for 30 min at -78°C and then for 4 h at

0°C. Water was added to quench the reaction and the crude product was extracted with EtOAc. The


Purification was done by chromatography (hexanes/EtOAc, 98:2) to give 4.

Methyl(4bS,6aS,9aR,10aS,10bR)-2-{[tert-butyl(dimethyl)silyl]oxy}-6a-methyl-8-oxo-

5,6,6a,6b,8,9,9a,10,10a,10b,11,12-dodecahydro-4bH-naphtho[2’,1’:4,5]indeno[1,2-b]furan-9-

carboxylate (4). 83% yield. 1H NMR (CDCl3): 0.19 (s, Si(CH3)2), 0.76 (s, 18-CH3), 0.98 (s,

SiC(CH3)3), 1.25-2.40 (unassigned CH and CH2), 2.81 (m, 6-CH2), 3.32 (m, 16-CH), 3.39 (d, J =

8.0 Hz, 19-CH), 3.82 (s, CO2CH3), 4.52 (d, J = 9.8 Hz, 17-CH), 6.55 (d, J = 2.3 Hz, 4-CH), 6.62

(dd, J1 = 8.4 Hz, J2 = 2.5 Hz, 2-CH), 7.11 (d, J = 8.5 Hz, 1-CH). 13C NMR (CDCl3): -4.4 (2x), 12.6,

18.2, 25.7 (3x), 26.1, 27.5, 29.4, 32.6, 37.2, 38.0, 40.0, 43.6, 44.0, 51.1, 53.2, 54.3, 90.7, 117.3,

120.0, 126.1, 132.2, 137.4, 153.5, 168.7, 172.9. LRMS: calcd for C28H41O5Si (M+H)+ 485.3, found

485.1.

2.2.3. Synthesis of 3 and 5

The alcohol 2 or ester 4 was dissolved in a methanolic solution of HCl (2%, v/v) and the

mixture was stirred at room temperature for 3 h. Water was added, the methanol evaporated under

reduced pressure and the residue extracted with EtOAc. The organic phase was washed with a

saturated NaCl solution, dried over MgSO4, evaporated under vacuum and purified by flash

chromatography (hexanes/EtOAc) to give 3 or 5.

4bS,6aS,9aR,10aS,10bR)-2-hydroxy-9(hydroxymethyl)-6a-methyl-


(3). 88% yield. 1H NMR (C2D6O): 0.76 (s, 18-CH3), 1.20-2.40 (unassigned CH and CH2), 2.55 (m,

19-CH), 2.78 (m, 6-CH2), 3.02 (m, 16-CH), 3.80 (m, CH2-OH), 4.37 (d, J = 10.1 Hz, 17-CH),

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6.53 (d, J = 2.3 Hz, 4-CH), 6.62 (dd, J1 = 8.4 Hz, J2 = 2.5 Hz, 2-CH), 7.11 (d, J = 8.5 Hz, 1-CH).

LRMS: calcd for C21H27O4 (M+H)+ 343.2, found 343.0.

Methyl(4bS,6aS,9aR,10aS,10bR)-2-hydroxy-6a-methyl-8-oxo-5,6,6a,6b,8,9,9a,10,10a,10b,11,12-

dodecahydro-4bH-naphtho[2’,1’:4,5]indeno[1,2-b]furan-9-carboxylate (5). 76% yield. 1H NMR

(CDCl3): 0.77 (s, 18-CH3), 1.30-2.35 (unassigned CH and CH2), 2.77 (m, 6-CH2), 3.31 (m, 16-

CH), 3.60 (d, J = 7.9 Hz, 19-CH), 3.75 (s, CO2CH3), 4.56 (d, J = 10.1 Hz, 17-CH), 6.53 (d, J = 2.3

Hz, 4-CH), 6.63 (dd, J1 = 8.5 Hz, J2 = 2.4 Hz, 2-CH), 7.11 (d, J = 8.5 Hz, 1-CH), 7.99 (OH). 13C

NMR (C2D6O): 12.7, 26.9, 28.1, 30.0, 32.6, 37.8, 39.0, 41.0, 44.3, 44.5, 51.3, 52.9, 54.4, 91.0,

113.5, 115.8, 126.9, 131.3, 138.0, 155.9, 169.8, ~173.2. LRMS: calcd for C22H27O5 (M+H)+ 371.2,

found 371.1.

2.2.4. Synthesis of acid 7

Ester 4 (57 mg, 0.117 mmol) was dissolved in methanol (3 mL) and few drops of CH2Cl2. A

10% aqueous solution of NaOH (0.11 mL) was added and the resulting mixture was refluxed for 7 h.

Water was then added and the solution washed with CH2Cl2. The aqueous phase was acidified to pH

10 with a 10% aqueous solution of HCl and extraction was performed with EtOAc. The organic

phase was washed with brine, dried over MgSO4 and evaporated to dryness under reduced pressure.

The crude product 6 was dissolved in a methanolic solution of HCl (2%, v/v) and the mixture was

stirred at room temperature for 3 h. Water was added, the methanol evaporated under reduced

pressure and the residue extracted with EtOAc. The organic phase was washed with brine, dried

over MgSO4, and purified by flash chromatography (hexanes/EtOAc) to give 7.

(4bS,6aS,9aR,10aS,10bR)-2-hydroxy-6a-methyl-8-oxo-5,6,6a,6b,8,9,9a,10,10a,10b,11,12-

dodecahydro-4bH-naphtho[2’,1’:4,5]indeno[1,2-b]furan-9-carboxylic acid (7). 55% yield. 1H NMR

(C2D6O): 0.77 (s, 18-CH3), 1.25-2.40 (unassigned CH and CH2), 2.79 (m, 6-CH2), 3.31 (m, 16-

CH), 3.54 (d, J = 7.8 Hz, 19-CH), 4.55 (d, J = 10.1 Hz, 17-CH), 6.54 (d, J = 2.4 Hz, 4-CH), 6.61

(dd, J1 = 8.5 Hz, J2 = 2.4 Hz, 2-CH), 7.10 (d, J = 8.4 Hz, 1-CH). 13C NMR (C2D6O): 12.6, 26.8,

28.0, 29.9, 32.5, 37.7, 38.9, 40.9, 44.2, 44.4, 51.3, ~ 54.0, 90.9, 113.3, 115.6, 126.8, 131.2, 138.0,

155.7, 169.9, 173.9. LRMS: calcd for C21H23O5 (M-H)- 355.2, found 355.5.

2.2.5. Synthesis of lactol 9

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To a solution of 4 (0.083 mmol) in dry THF (1.7 mL) at 0°C under argon atmosphere was added

LiAlH4 (0.166 mmol). The mixture was stirred 16 h at 0°C and quenched by addition of water and

10% aqueous NaOH. The crude compound was extracted with EtOAc, washed with brine and dried

over MgSO4. The crude product 8 was dissolved in a methanolic solution of HCl (2%, v/v) and the

mixture was stirred at room temperature for 3 h. Water was added, the methanol evaporated under

reduced pressure and the residue extracted with EtOAc. The organic phase was washed with brine,

dried over MgSO4 and purified by flash chromatography (hexanes/EtOAc) to give 9 as a mixture of

two lactols in proportions 30 : 70. Data in italic characters correspond to the minor C20-isomer.

(4bS,6aS,6bS,8S,9S,9aR,10aS,10bR)-9-(hydroxymethyl)-6a-methyl-

5,6,6a,6b,8,9,9a,10,10a,10b,11,12-dodecahydro-4bH-naphtho[2’,1’:4,5]indeno[1,2-b]furan-2,8-diol

(9). 54% yield. 1H NMR (CD3OD): 0.80 and 0.93 (2s, 18-CH3), 1.10-2.20 (unassigned CH and

CH2), 2.31 (m, 19-CH), 2.57 (m, 16-CH), 2.80 (m, 6-CH2), 3.64 and 3.72 (2m, CH2OH), 3.94 and

4.26 (2d, J = 9.8 Hz, 17-CH), 5.20 and 5.57 (2d, J = 4.4 Hz, CHOH), 6.49 (d, J = 2.1 Hz, 4-CH),

6.55 (dd, J1 = 2.3 Hz, J2 = 8.5 Hz, 2-CH), 7.08 (d, J = 8.5 Hz, 1-CH). 13C NMR (CD3OD): 14.0

(14.7), 27.8, 28.9, 30.7, 32.4, 39.3, 40.1, 43.9, 44.0, 45.1, 55.1, 55.4, 62.4 (62.6), 93.9 (91.7), 103.6

(105.7), 113.8, 116.0, 127.2, 132.2, 138.7, 155.9. LRMS: calcd for C21H27O4 (M-H)- 343.2, found

343.3.

2.2.6. Synthesis of 13

A solution of diisopropylamine (0.17 mL, 1.17 mmol) in dry THF (1.15 mL) was stirred

under argon at 0°C and a solution of n-butyllithium in hexanes (0.67 mL, 1.17 mmol) was added

dropwise. After 45 min, the resulting lithium diisopropylamide (LDA) solution was cooled at -78°C

and the lactone 1 (100 mg, 0.23 mmol), dissolved in dry THF (1 mL), was added dropwise. The

mixture was allowed to stir for 1 h at 0°C, then cooled again at -78°C and allyl bromide (30 µL, 0.35

mmol) was added dropwise. The reaction mixture was stirred overnight from -78°C to room

temperature. Water was added to quench the reaction and the crude product was extracted with

EtOAc. The organic phase was washed with brine, dried over MgSO4, and evaporated under reduced

pressure. Purification was done by chromatography (hexanes/EtOAc, 98:2) to give 13.

(4bS,6aS,9aR,10aS,10bR)-9-allyl-2-{[tert-butyl(dimethyl)silyl]oxy}-6a-methyl-


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2.35 (unassigned CH and CH2), 2.45 (m, 19-CH), 2.60 (m, 16-CH and 1H of 21-CH2), 2.81 (m, 6-

CH2), 4.36 (d, J = 10.0 Hz, 17-CH), 5.14 (m, CH=CH2), 5.76 (m, CH=CH2), 6.55 (d, J = 2.4 Hz, 4-

CH), 6.61 (dd, J1 = 8.4 Hz, J2 = 2.4 Hz, 2-CH), 7.11 (d, J = 8.5 Hz, 1-CH). 13C NMR (CDCl3): -4.4

(2x), 12.6, 18.2, 25.7 (3x), 26.2, 27.6, 29.5, 33.4, 36.3, 37.4, 38.1, 41.6, 43.7, 44.0, 47.8, 51.4, 90.1,

117.3, 117.8, 120.0, 126.1, 132.4, 134.7, 137.5, 153.5, 180.0. LRMS: calcd for C29H43SiO3 (M+H)+

467.3, found 467.4.

2.2.7. Inversion of C19-configuration (synthesis of 14)

A solution of diisopropylamine (0.31 mL, 2.2 mmol) in dry THF (39 mL) was stirred under

argon at 0°C and a solution of n-butyllithium (1.1 mL, 2.2 mmol) was added dropwise. After 30

min, the solution was cooled at -78°C and the -allyl derivative 13 (200 mg, 0.44 mmol), dissolved

in dry THF (2 mL), was added dropwise to the LDA solution. The mixture was stirred for 1 h at 0°C

then cooled again at -78°C and dry methanol (0.93 mL) was slowly added. The reaction mixture was

stirred at -78°C for 1 h. Water was added and the crude product was extracted with EtOAc. The


The crude product was purified by flash chromatography (hexanes/EtOAc) to afford 14.

(4bS,6aS,6bS,9S,9aR,10aS,10bR)-2-{[tert-butyl(dimethyl)silyl]oxy}-6a-methyl-9-(prop-2-en-1-yl)-

4b,5,6,6a,6b,9,9a,10,10a,10b,11,12-dodecahydro-8H-naphtho[2',1':4,5]indeno[1,2-b]furan-8-one


2.25 (unassigned CH and CH2), 2.71 (m, 1H of 21-CH2), 2.82 (m, 6-CH2 and 19-CH), 3.00 (m, 16-

CH), 4.46 (d, J = 9.6 Hz, 17-CH), 5.10 (d, J = 11.2 Hz, 1H of CH=CH2), 5.14 (d, J = 18.5 Hz, 1H

of CH=CH2), 5.89 (m, CH=CH2), 6.55 (d, J = 2.3 Hz, 4-CH), 6.62 (dd, J1 = 8.4 Hz, J2 = 2.5 Hz, 2-

CH), 7.11 (d, J = 8.5 Hz, 1-CH). 13C NMR (CDCl3): -4.4 (2x), 13.2, 18.1, 25.7 (3x), 26.3, 26.8, 27.8,

29.5, 31.5, 37.7, 37.8, 39.1, 40.5, 43.6, 44.4, 51.0, 89.6, 116.3, 117.3, 120.0, 126.1, 132.4, 135.9,

137.5, 153.5, 180.0.

2.2.8. Synthesis of and -allyl derivatives 15 and 16

According to the general procedure for hydrolysis of silylated ether in acid conditions, 15

and 16 were obtained from 13 and 14, respectively.

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(4bS,6aS,9aR,10aS,10bR)-9-allyl-2-hydroxy-6a-methyl-4b,5,6,6a,6b,9,9a,10,10a,10b,11,12-

dodecahydro-8H-naphtho[2’,1’:4,5]indeno[1,2-b]furan-8-one (15). 97% yield. 1H NMR (CDCl3):

0.76 (s, 18-CH3), 1.20-2.35 (unassigned CH and CH2), 2.45 (m, 19-CH), 2.65 (m, 16-CH and 1H

of 21-CH2), 2.81 (m, 6-CH2), 4.36 (d, J = 10.0 Hz, 17-CH), 4.90 (s, OH), 5.13 (m, CH=CH2), 5.76

(m, CH=CH2), 6.57 (d, J = 2.7 Hz, 4-CH), 6.64 (dd, J1 = 2.7 Hz, J2 = 8.4 Hz, 2-CH), 7.13 (d, J =

8.4 Hz, 1-CH). 13C NMR (CDCl3): 12.6, 26.3, 27.5, 29.4, 33.4, 36.3, 37.4, 38.1, 41.6, 43.6, 44.0,

47.8, 51.4, 90.1, 112.8, 115.2, 117.8, 126.5, 132.1, 134.7, 137.9, 153.4, 180.0. LRMS: calcd for

C23H29O3 (M+H)+ 353.2, found 353.3.

(4bS,6aS,6bS,9S,9aR,10aS,10bR)-2-hydroxy-6a-methyl-9-(prop-2-en-1-yl)-


(16). 93% yield. 1H NMR (CDCl3): 0.70 (s, 18-CH3), 1.20-2.25 (unassigned CH and CH2), 2.68 (m,

1H of 21-CH2), 2.82 (m, 6-CH2 and 19-CH), 3.00 (m, 16-CH), 4.47 (d, J = 9.6 Hz, 17-CH), 5.03

(s, OH), 5.11 (m, CH=CH2), 5.89 (m, CH=CH2), 6.57 (d, J = 2.5 Hz, 4-CH), 6.64 (dd, J1 = 2.6 Hz,

J2 = 8.4 Hz, 2-CH), 7.13 (d, J = 8.4 Hz, 1-CH). 13C NMR (CDCl3): 13.2, 26.4, 26.8, 27.7, 29.4,

31.5, 37.7 (2x), 39.1, 40.5, 43.5, 44.4, 50.9, 89.7, 112.8, 115.2, 116.4, 126.5, 132.0, 135.8, 137.9,

153.6, 179.8. LRMS: calcd for C23H29O3 (M+H)+ 353.2, found 353.2.

2.2.9. Synthesis of 17 (acylation of 15)

To a solution of 15 (0.446 mmol) in dry pyridine (2 mL) at 0°C were added acetyl chloride

(4.46 mmol) and dimethylaminopyridine (DMAP) (0.039 mmol), and the resulting mixture was

stirred for 1 h at 25°C. EtOAc was then added and the organic phase was washed successively with

an aqueous saturated solution of NH4Cl, an aqueous 1 M solution of CuSO4, and H2O. The organic

phase was dried over MgSO4 and evaporated to dryness. Purification by flash chromatography with

hexanes/EtOAc as eluent gave 17.

(4bS,6aS,6bS,9S,9aR,10aS,10bR)-6a-methyl-8-oxo-9-(prop-2-en-1-yl)-5,6,6a,6b,8,9,9a,10,10a,10b,

11,12-dodecahydro-4bH-naphtho[2',1':4,5]indeno[1,2-b]furan-2-yl acetate (17). 92% yield. 1H NMR

(CDCl3): 0.76 (s, 18-CH3), 1.20-2.15 (unassigned CH and CH2), 2.28 (s, COCH3), 2.45 (m, 19-CH),

2.62 (m, 16-CH and 1H of 21-CH2), 2.82 (m, 6-CH2), 4.36 (d, J = 10.0 Hz, 17-CH), 5.14 (m,

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CH=CH2), 5.76 (m, CH=CH2), 6.79 (d, J = 2.3 Hz, 4-CH), 6.85 (dd, J1 = 2.4 Hz, J2 = 8.2 Hz, 2-

CH), 7.27 (d, J = 7.3 Hz, 1-CH). 13C NMR (CDCl3): 12.6, 21.1, 26.1, 27.3, 29.4, 33.4, 36.3, 37.4,

37.8, 41.6, 43.8, 43.9, 47.7, 51.4, 90.0, 117.8, 118.7, 121.5, 126.4, 134.7, 137.4, 137.9, 148.5, 169.8,

180.0. LRMS: calcd for C25H31O4 (M+H)+ 395.2, found 395.3.

2.2.10. Synthesis of 21 (ozonolysis of 17)

Allyl compound 17 (0.221 mmol) was dissolved in dry CH2Cl2 (2 mL) and stirred at room

temperature for 10 min, then ozone was bubbled in the cold (-78°C) mixture. When the solution kept

a light blue colour (nearly 3-5 min), indicating the excess ozone, the gas flow was stopped, and

nitrogen was bubbled to remove excess ozone. The solution was then diluted with dry methanol (2

mL), and NaBH4 (0.331 mmol) was added at -78°C. Stirring was continued for 2 h while allowing

the mixture to slowly warm to room temperature. The crude product 19 was treated with 4 mL of a

solution of 0.5 N NaOH in THF (1:1, v/v) for 30 min at 25°C. The reaction was neutralized with a

10% aqueous solution of HCl, and the crude product was extracted with EtOAc. The combined

organic layer was washed with brine, dried over MgSO4, and the solvent was evaporated under

reduced pressure. Purification by flash chromatography (hexanes/EtOAc, 2:8) gave 21.

(4bS,6aS,9aR,10aS,10bR)-2-hydroxy-9-(2-hydroxyethyl)-6a-methyl-4b,5,6,6a,6b,9,9a,10,10a,10b,

11,12-dodecahydro-8H-naphtho[2’,1’:4,5]indeno[1,2-b]furan-8-one (21). 55% yield. 1H NMR

(CDCl3): 0.78 (s, 18-CH3), 1.20-2.35 (unassigned CH and CH2), 2.57 (m, 16-CH and 19-CH), 2.83

(m, 6-CH2), 3.78 and 3.88 (2m, CH2OH), 4.41 (d, J = 9.7 Hz, 17-CH), 4.62 (s, OH), 6.56 (d, J =

2.6 Hz, 4-CH), 6.63 (dd, J1 = 2.7 Hz, J2 = 8.4 Hz, 2-CH), 7.14 (d, J = 8.5 Hz, 1-CH). 13C NMR

(CDCl3): 12.7, 27.0, 28.2, 33.6, 36.1, 38.0, 39.1, 43.1, 44.3, 44.4, 45.4, 51.8, 59.9, 60.1, 90.3, 113.4,

115.7, 126.9, 131.4, 138.1, 155.9, 180.2. LRMS: calcd for C22H27O4 (M-H)- 355.2, found 355.5.

2.2.11. Synthesis of lactol 22 (reduction of carbonyl function)

According to the general procedure for reduction of carbonyl function with LiAlH4 and the

appropriate procedure for the cleavage of the protective group, the lactol 22 was obtained from 19

and purified by flash chromatography (hexanes/EtOAc). Lactol 22 was obtained as a pure isomer.

(4bS,6aS,8R,9aR,10aS,10bR)-9-(2-hydroxyethyl)-6a-methyl-5,6,6a,6b,8,9,9a,10,10a,10b,11,12-

dodecahydro-4bH-naphtho[2’,1’:4,5]indeno[1,2-b]furan-2,8-diol (22). 56% yield. 1H NMR

(CDCl3): 0.70 (s, 18-CH3), 1.10-2.30 (unassigned CH and CH2), 2.40 and 2.52 (2m, 16-CH and

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19-CH), 2.81 (m, 6-CH2), 3.82 and 3.90 (2m, CH2OH), 4.34 (d, J = 9.6 Hz, 17-CH), 5.85 (d, J =

5.0 Hz, CHOH), 6.55 (d, J = 2.2 Hz, 4-CH), 6.63 (dd, J1 = 2.2 Hz, J2 = 8.4 Hz, 2-CH), 7.14 (d, J =

8.4 Hz, 1-CH). 13C NMR (CDCl3): 13.1, 26.5, 27.5, 29.5, 33.0, 34.1, 37.5, 38.5, 43.7, 44.0, 47.2,

50.8, 51.9, 66.4, 95.6, 112.7, 113.1, 115.2, 126.5, 132.6, 138.1, 153.3. LRMS: calcd for C22H29O3

(M-H2O+H)+ 341.2, found 341.1.

2.2.12. Synthesis of 24 (ozonolysis of 23)

Allyl compound 16 (60 mg, 0.17 mmol) was dissolved in dry CH2Cl2 (2 mL) at 0°C, DMAP

(31 mg, 0.25 mmol) and trifluoroacetic anhydride (0.028 mL, 0.20 mmol) were added and the

solution stirred for 30 min at 0°C. Water was added to quench the reaction and the crude product

was extracted with EtOAc. The organic phase was washed with brine, dried over MgSO4, and

evaporated under reduced pressure. The crude product 23 was submitted to ozonolysis reaction

according to the procedure described above for synthesis of 21. Purification by flash

chromatography (hexanes/EtOAc, 2:8) gave 24.

(4bS,6aS,6bS,9S,9aR,10aS,10bR)-2-hydroxy-9-(2-hydroxyethyl)-6a-methyl-


(24). 53% yield. 1H NMR (CD3OD): 0.70 (s, 18-CH3), 1.25-2.40 (unassigned CH and CH2), 2.80

(m, 6-CH2), 2.94 (m, 19-CH), 3.08 (m, 16-CH), 3.68 and 3.76 (2m, CH2OH), 4.53 (d, J = 9.5 Hz,

17-CH), 4.65 (s, OH), 6.50 (d, J = 2.4 Hz, 4-CH), 6.57 (dd, J1 = 2.6 Hz, J2 = 8.4 Hz, 2-CH), 7.09

(d, J = 8.5 Hz, 1-CH). 13C NMR (CD3OD): 13.8, 27.6, 27.7, 28.9, 30.5, 31.5, 38.5, 38.8, 39.3, 40.2,

44.8, 45.5, 51.9, 61.0, 91.4, 113.8, 116.1, 127.2, 132.1, 138.6, 155.9, 182.8. LRMS: calcd for

C22H29O4 (M+H)+ 357.2, found 357.1.

2.3. Inhibition of 17-HSD1 in homogenized cells

The HEK-293 cells overexpressing human 17-HSD1 (transfected cells) were homogenized

and the homogenate was kept at -80°C in phosphate buffer pH 7.5 (50 mM KH2PO4, 1 mM ethylene

diamine tetraacetic acid (EDTA) and 20% glycerol) until used for testing [36,37]. During the

enzymatic assay, the homogenized cells were incubated at 37°C with agitation for 2 h in presence of

60 nM [14C]-estrone (American Radiolabeled Chemicals Inc., St. Louis, MO, USA), 0.1 mM NADH

(reduced nicotinamide adenine dinucleotide), the inhibitor at a concentration of 1 μM and phosphate

buffer to adjust the final volume to 1 mL. At the end of the incubation period, the reaction is stopped

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with 1 mL of diethyl ether and the steroids are extracted. This is followed by centrifugation for 5

min at 3000 rpm to separate the two phases. The aqueous phase was frozen using a bath of ethanol

and dry ice and the organic phase containing the steroids was placed in a tube to be evaporated. The

residues are dissolved in CH2Cl2 and then deposited by small dot on a sheet of silica gel (TLC, 10

cm x 20 cm x 0.2 mm, EMD Chemicals Inc., Gibbstown, NJ, USA). The plate is eluted with a

mixture of toluene: acetone (4:1). Thereafter, the steroids (E1 and E2) were detected and quantified

by a Storm 860 device to determine the percentage of transformation and percentages of inhibition.

The percentage of transformation and the percentage of inhibition were calculated as follow: %

transformation = 100 x [14C]-E2 ⁄ ([14C]-E1 + [14C]-E2) and % of inhibition = 100 x (%

transformation without inhibitor - % transformation with inhibitor) ⁄ % transformation without

inhibitor.

2.4. Inhibition of 17-HSD1 in intact T-47D cells

T-47D cells were seeded in a 24-well plate (3000 cells/well) in 990 µL of medium

supplemented with insulin (50 ng⁄mL) and 5% dextran-coated charcoal-treated FBS, which was used

rather than untreated 10% FBS, to remove the remaining steroid hormones. Stock solutions of each

compound to be tested were previously prepared in ethanol and diluted with culture medium to

achieve appropriate concentrations prior to use. After 24 h of incubation, 5 µL of the diluted

solution were added to the cells to obtain a final concentration of 0.1, 1 or 10 µM. The final

concentration of ethanol in the well was adjusted to 0.1%. Additionally, 5 µL of a solution of [14C]-

estrone (American Radiolabeled Chemicals, Inc.) was added to obtain a final concentration of 60

nM. Cells were incubated for 24 h and each inhibitor was assessed in triplicate. After incubation, the

culture medium was removed and labeled steroids (E1 and E2) were extracted with 1 mL of diethyl

ether and quantified as reported above.

2.5. Docking studies

Docking studies were carried out using the AutoDock 4.0 docking software and its graphical

interface MGL Tools. The 17β-HSD 3D coordinates used for docking were downloaded from the

Brookhaven Protein DataBank (pdb id:1i5r). Prior to the calculation of the energy grids, the protein

coordinate file was cleared of water and substrates using a text editor. A 125 x 125 x 125 sized grid

with 0.3A spacing centered on the enzyme active site was calculated using the AutoGrid component

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of the AutoDock suite. 3D coordinate files for inhibitors were generated in pdb format using the

ChemOffice suite, energy minimized and their partial charges were calculated using the

antechamber software. Docking was performed using the Lamarkian-Genetic algorithm available in

AutoDock with a rigid enzyme model. Default settings were used, except for population size, which

was set to 300 and maximum evaluations, which was set to 5000000. Fifty (50) runs were performed

for each ligand. Results were analysed using a clustering graph of binding energy at 5.0 rmsd.

Docking was considered complete if only one major cluster per ligand positioning could be

observed. The best conformations and interactions were visualized and analysed using the molecular

viewing software Pymol.

3. Results and Discussion

3.1. Chemical synthesis

The synthesis of lactone derivatives was reported in Schemes 2-4. After having synthesized

the 16β,17--lactone-E2 (intermediate compound 1) in four steps from commercially available E1

as previously reported [35], we introduced molecular diversity by using a direct alkylation.

Depending on the type of functional group, alcohol or ester, two synthetic procedures were applied

to obtain the lactone derivatives 2 and 4 (Scheme 2A). The first procedure consisted in an alkylation

of 1 with sodium bis-(trimethylsilyl) amide (NaHMDS) and ethyl formate followed by reduction

using NaBH4 that afforded alcohol 2 in 60% yield for two steps. By NOESY experiment, the

CH2OH group was found to be on the steroidal -face because a correlation was observed between

18-CH3 and 19-CH suggesting a -H orientation. Hydrolysis of the protective TBDMS group under

acid conditions next provided the final alcohol 3 in a yield of 88%. The second procedure was used

to prepare the ester derivative 4, the precursor of compounds 5-9. Alkylation of 1 with lithium

diisopropylamide (LDA) and methyl chloroformate afforded the ester 4 in a yield of 83%. Similarly

as for the compound 2, the NOESY experiment confirmed the -CH2OH orientation by a correlation

between 18-CH3 and 19-CH. Carboxylic acid derivative 6 and lactol derivative 8 were respectively

obtained from 4 by a hydrolysis (aqueous NaOH) and a reduction (LiAlH4). Ester 5, acid 7 and

lactol 9 were finally obtained by an acid hydrolysis of the TBDMS protective group.

During the synthesis of lactone derivatives 2, 4, 6 and 8 we observed only the -epimer, with

no trace of the. However, preliminary docking results predicted a better affinity for the -epimer.

We therefore decided to explore epimerization of compounds 2, 4 and 6 as a strategy for the

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synthesis of the -epimer (compounds 10-12). For this purpose, we used LDA as base under

different conditions. The isomerisation reaction never yielded any -epimer for all compounds

tested (Scheme 2B). Since the ligands tested carried a carbonyl in position 20, we hypothesized that

1) the lithium enolate generated by the treatment with LDA is chelated in a very stable 6-member

ring (Scheme 2C) and 2) the proton in of the carbonyl is very acidic, easily deprotonated, thus

leading to the formation of the thermodynamically favoured -H isomer over the -H compound.

To test these hypotheses, we synthesized the lactone derivative 13, which does not carry an

oxygen atom at position 21 and thus with a less acidic proton at position 19 (Scheme 3). For the

purpose of synthesizing 13 and 14, and next 15 and 16, we had to optimize two key reactions: 1) the

allylation in alpha position of the lactone carbonyl, and 2) the isomerisation of the allyl group. In

order to obtain the -allyl derivative 13 in a good yield, several bases were used such as LDA,

NaHMDS, KHMDS and NaH. Optimization of the yield was also attempted with the strongest base

KDA (LDA/t-BuOK), which is more efficient for a rapid deprotonation of weakly acidic

compounds, but it was not possible to increase the yield of 13 over that obtained with LDA. The

best yield of 13 (40%) was obtained with 5 equivalents of LDA in THF with a reaction temperature

ranging from -78°C to 0°C for the enolisation step and -78°C to room temperature for the allylation

step. However, the isomer with -allyl stereochemistry (compound 14) was not observed during the

preparation of 13. Several assays were also carried out in order to optimize the isomerisation of the

allyl group ( to ). For the enolisation step of this reaction, four bases (LDA, NaHMDS, KHMDS

and KDA) were used to study the effect of number of equivalent (1.5, 2, 3 and 5), temperature (-78,

-22 and 0°C) and counter ion. For the protonation step, we always used methanol at low temperature

(-100 or -78°C). The isomerisation reaction was never completed, but the -isomer 14 was formed

for the first time in 30% yield using LDA (5 eq) at 0°C (1 h) for the enolisation step and methanol at

-78°C for the protonation step. Despite several attempts with other reaction conditions, we were not

able to increase this yield. For both allylation and isomerisation reactions, we observed that the best

counter ion of the base was lithium, since it is the smallest one. The epimerization was the crucial

step and, consequently, the -isomer was only obtained in 30% yield. An explanation of the result

could be that the -product is formed, but unstable in the reaction conditions, and goes back partly

into the -isomer that is the most thermodynamically stable (E -isomer = 16.57 kcal/mol and E -

isomer = 18.43 kcal/mol, as calculated by CS Chem 3D software). The stereochemistries of

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compounds 13 and 15 as well as 14 and 16 were confirmed by NMR (NOESY) experiments after

the full assignment of 1H- and 13C-NMR signals was carried out for these two isomers using COSY,

APT, HSQC and HMBC experiments (Figures 1 and 2).

Having obtained both α- and β-allyl epimers, we proceeded with the oxidative cleavage by

ozonolysis of the double bond followed by a reduction with NaBH4. Ozonolysis of 3-TBDMS-allyl

derivatives 13 and 14 did not yield the expected corresponding aldehydes. A modification of the

TBDMS protecting group in position 3 was needed to avoid an oxidation in A and B steroid rings

leading to degradation compounds. In fact, an activating group at C3 favours an oxidation of ring A.

The presence of a less activating protecting group such as acetate (Ac) allowed the success of the

ozonolysis of 17 and 18 to give 19 and 20 (Scheme 4). The acetate group of 19 (the -isomer) was

easily hydrolyzed to give the final alcohol 21 whereas the lactol 22 was obtained from 19 by a

reduction and the cleavage of protecting group using LiAlH4. However, since the -isomer is easily

epimerized into the -isomer in the presence of a base, hydrolysis of the acetate group of 20 yielded

the -epimer 21 exclusively and not 23. The choice of a protecting group such as trifluoroacetate

(CF3CO2) (compound 19), easily cleaved in non basic conditions, was needed in order to maintain

the -stereochemistry of the CH2CH2OH group. This change of protecting group also allowed

synthesis to proceed with one less step, since the reduction conditions also cleaved the protecting

group, affording the final -epimer 24 without the need for an additional step. Characteristic proton

signals (H16, H17, H19 and CH3-18) were identified using 1D- and 2D-NMR experiments (APT,

COSY, HSQC, HMBC and NOESY). The -CH2CH2OH stereochemistry of 19 was confirmed by a

NOESY experiment showing a correlation between H19 and CH3-18 whereas the -CH2CH2OH

stereochemistry of 20 was confirmed by the presence of a correlation between H16 and H19.

3.2. Inhibitory activity on 17β-HSD1

New synthesized compounds were first tested in homogenized HEK-293 cells

overexpressing 17β-HSD1 (Table 1). We determined their ability to inhibit the transformation of

tritiated E1 to tritiated E2 at a concentration of 1 µM. All compounds tested inhibited 17β-HSD1 in

a range between 34 and 60%, values that are close to the inhibition produced by untritiated E1 used

as inhibitor (53%). Focusing on the alcohol derivatives, such as the hydroxymethyl compound 3, it

is clear that adding an OH group (lactol 9) or adding a methylene group (hydroxyethyl compound

21) did not increase the inhibitory activity. Moreover, the stereochemistry α or β of the hydroxyethyl

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group did not influence the enzyme inhibition (45 and 38% for 21 and 24, respectively), but the

lactol 22 is a slightly better inhibitor than compound 21 (56 and 25%, respectively). These lactone

and lactol derivatives are clearly less potent than CC-156 [38], which produced 88% of inhibition.

We next evaluated the lactone and lactol derivatives using another source of 17β-HSD1. In fact, we

determined their ability to inhibit the transformation of tritiated E1 to tritiated E2 in T-47D cells, a

breast cancer cell line that is well-known to express endogenous 17β-HSD1. We obtained

disappointing results, since the compounds that we tested only weakly inhibited (10-40%) the

formation of E2 at the highest concentration of 10 μM. As a point of comparison, the reference

inhibitor CC-156 strongly inhibited the 17β-HSD1 at the three concentrations used (60, 92 and 97%

of inhibition at 0.1, 1 and 10 μM, respectively).

4. Conclusion

The chemical synthesis and characterisation of nine new lactone and lactol derivatives was

performed from the intermediate lactone 1. Since the direct alkylation of this lactone generated only

the α-isomer at position 19 (compounds 3, 5, 7, 9, 15, 21 and 22), a strategy was developed to obtain

the β-isomeric compounds 16 and 24. These compounds were selected from a series of docking

experiments that identified their ability to generate potential hydrogen bonding interactions with

17β-HSD1. Unfortunately, these compounds did not effectively inhibit the transformation of E1 to

E2 in two different assays (homogenized and intact cells expressing 17β-HSD1). It is now clear that

the selection of chemical groups that we introduced on the extra E-ring of E2-γ-lactone did not

generate the expected interactions. Consequently, they only weakly inhibited 17β-HSD1 compared

to the known inhibitor CC-156. The use of E2-16β,17β-γ-lactone as a core for the development of

17β-HSD1 inhibitors still remains an attractive option, since this lactone did not inhibit 17β-HSD2

[35], an oxidative 17β-HSD that should not be inhibited by an inhibitor of reductive 17β-HSD1. The

methodology that we developed for the synthesis of hindered β-isomers, as well as for α-isomers,

can now be used for the preparation of new E2-γ-lactone derivatives as selective inhibitors of 17β-

HSD1.

Acknowledgments

We thank the Canadian Institutes of Health Research (CIHR) and the Canadian Breast

Cancer Research Alliance (CBCRA) for an operating grant. We also thank Diane Fournier for

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advice regarding docking experiments and Liviu Ciobanu, Richard Labrecque and Jean-Yves

Sanceau for helpful suggestions. Careful reading of the manuscript by Micheline Harvey was greatly

appreciated.

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Table 1. Lactone and lactol derivatives targeted as inhibitors of 17β-HSD1

CompoundIDa

X R Interactionb Homogenized HEK-293 cells17β-HSD1

Inhibition (%)c

Intact T-47D cells17β-HSD1

Inhibition (%)d

3 O α-CH2OH 3-OH/His22117β-O/Ser142

34 0 / 2 / 20

5 O α-COOCH3 --- 57 0 / 3 / 187 O α-COOH 3-OH/His221

COOH/Gly14160 0 / 2 / 10

9 OH α-CH2OH 3-OH/His22117β-O/Ser142

OH/Tyr155

35 0 / 3 / 30

15 O α-CH2CH=CH2 --- 50 0 / 10 / 2816 O β-CH2CH=CH2 --- 36 0 / 7 / 4021 O α-CH2CH2OH 3-OH/His221

OH/Tyr155 & Lys159

45 (25)e 0 / 4 / 12

22 OH α-CH2CH2OH 3-OH/His22117β-O/Ser142

OH/Val188

--- (56)e ---

24 O β-CH2CH2OH OH/Asp152 & Leu95

38 0 / 2 / 10

E1 Enzyme substrate 3-OH/His22117-O/Gly186 &

Cys185

53 12 / 78 / 97

CC-156 Enzyme inhibitor 3-OH/His22117β-OH/Ser142

& Cys185CONH2/Asn152

88 (90)e 60 / 92 / 97

a See Scheme 1 for the chemical structures and carbon numbering.b Hydrogen bonding interactions identified from docking experiments.c For the transformation of E1 (60 nM) to E2 by 17β-HSD1 overexpressed in HEK-293 cells (homogenized cells). Compounds were tested at 1 μM.d For the transformation of E1 (60 nM) to E2 by 17β-HSD1 in T-47D cells (intact cells).Compounds were tested at 0.1, 1 and 10 μM, respectively.e These data were obtained in the same experimental conditions but from another protocol.

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Legends of Schemes and Figures

Scheme 1. Retrosynthetic analysis for the synthesis of final compounds 3, 5, 7, 9, 15, 16, 21, 22 and 24 (E2-derivatives with an additional E-ring) as potential inhibitors of 17β-HSD1.

Scheme 2. A) Synthesis of alcohol 3, ester 5, acid 7 and lactol 9 from lactone 1. B) Epimerisation of 19α-compounds 2, 4 and 6 to 19β-compounds 10, 11 and 12. C) Hypothetical 6-member ring formed in presence of LDA. Reagents and conditions: a) 1. NaHMDS, HCOOEt, THF; 2. NaBH4, MeOH, 0°C; b) MeOH, HCl 2%; c) LDA, methyl chloroformate, THF; d) NaOH 10%, MeOH; e) LiAlH4, THF, 0°C; f) 1. LDA, THF; 2. MeOH, -78°C.

Scheme 3. Allylation in position of the lactone (synthesis of 15 and 16). Reagents and conditions: a) LDA, allyl bromide, THF; b) 1. LDA, THF; 2. MeOH, -78°C; c) MeOH, HCl 2%.

Scheme 4. Synthesis of 21, 22 and 24. Reagents and conditions: a) Ac2O, DMAP, CH2Cl2, 0°C; b) O3, -78°C, CH2Cl2; c) NaBH4, CH2Cl2/MeOH: 1/1, 0°C; d) NaOH, THF, 0°C; e) (CF3CO)2O, DMAP, CH2Cl2, 0°C; f) LiAlH4, THF, 0°C.

Figure 1. Results of docking experiments showing key hydrogen bonds between 17β-HSD1 and lactone 7 (A) or lactol 9 (B).

Figure 2. Characterisation of and allyl derivatives 15 and 16 by H-NMR spectroscopy. Characteristic protons H16, H17, H19 and H21 were identified using NMR (1H, APT, COSY, HSQC, HMBC and NOESY) experiments.

Figure 3. Characterisation of the stereochemistry of 16 using a NOESY experiment. A characteristic correlation was observed between H16 and H19. These protons were previously identified by COSY, HSQC and HMBC experiments.

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HO

O

HO

OX

TBDMSO

O O

R

Estrone(StartingMaterial)

1(Intermediate

Lactone)

3 (X = O; R = -CH2OH(X = O; R =-COOMe)

7 (X = O; R = -COOH)

9 (X = H/OH; R -CH2OH)

15 (X = O; R = -CH2CH=CH2)

16 (X = O; R =-CH2CH=CH2)

21 (X = O; R = -(CH2)2OH)

22 (X = H/OH; R = -(CH2)2OH)

24 (X = O; R = -(CH2)2OH)

H H

H

A B

C DE

H

H

Scheme 1. Retrosynthetic analysis for the synthesis of final compounds 3, 5, 7, 9, 15, 16, 21, 22 and 24 (E2-derivatives with an additional E-ring) as potential inhibitors of 17β-HSD1.

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RO

O O

OH

a

RO

O O

COOMe

TBDMSO

O O

R

RO

O O

COOH

TBDMSO

O O

R

RO

O OH

OH

1

d

e4 (R = TBDMS)

5 (R = H)

b

b

f

c

2 (R = TBDMS)

3 (R = H)

6 (R = TBDMS)

7 (R = H)b

8 (R = TBDMS)

9 (R = H)b

X

A

B

TBDMSO

O O

2 (R = CH2OH)4 (R = COOMe)6 (R = COOH)

O

X = H2 or OR = H or Me

6-member ring

O

LiO

XR

C

10 (R = CH2OH)11 (R = COOMe)12 (R = COOH)

19

20

1716

18

19 19

H

H

H

H

H

Scheme 2. A) Synthesis of alcohol 3, ester 5, acid 7 and lactol 9 from lactone 1. B) Epimerisation of 19α-compounds 2, 4 and 6 to 19β-compounds 10, 11 and 12. C) Hypothetical 6-member ring formed in presence of LDA. Reagents and conditions: a) 1. NaHMDS, HCOOEt, THF; 2. NaBH4, MeOH, 0°C; b) MeOH, HCl 2%; c) LDA, methyl chloroformate, THF; d) NaOH 10%, MeOH; e) LiAlH4, THF, 0°C; f) 1. LDA, THF; 2. MeOH, -78°C.

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O O

TBDMSOTBDMSO

O O

O O

HO

O O

HO

a

c

16 (isomer )15 (isomer )

1

O O

TBDMSO

b

c

13 14

18

19

20

21

2223

Scheme 3. Allylation in position of the lactone (synthesis of 15 and 16). Reagents and conditions: a) LDA, allyl bromide, THF; b) 1. LDA, THF; 2. MeOH, -78°C; c) MeOH, HCl 2%.

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HO

O O

AcO

O O

RO

O O

OH

a

HO

O O

AcO

O O

AcO

O O

OH

b, c

a

20

1516

b, c

1817

21 (R = H)

19 (R = Ac)

d

CF3COO

O O

23

e

b, c

HO

O O

OH

24

d

HO

O OH

OH

22

f, d

dX

Scheme 4. Synthesis of 21, 22 and 24. Reagents and conditions: a) Ac2O, DMAP, CH2Cl2, 0°C; b) O3, -78°C, CH2Cl2; c) NaBH4, CH2Cl2/MeOH: 1/1, 0°C; d) NaOH, THF, 0°C; e) (CF3CO)2O, DMAP, CH2Cl2, 0°C; f) LiAlH4, THF, 0°C.

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Figure 1. Results of docking experiments showing key hydrogen bonds between 17β-HSD1 and lactone 7 (A) or lactol 9 (B).

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Figure 2. Characterisation of and allyl derivatives 15 and 16 by H-NMR spectroscopy. Characteristic protons H16, H17, H19 and H21 were identified using NMR (1H, APT, COSY, HSQC, HMBC and NOESY) experiments.

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Figure 3. Characterisation of the stereochemistry of 16 using a NOESY experiment. A characteristic correlation was observed between H16 and H19. These protons were previously identified by COSY, HSQC and HMBC experiments.

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Chemical synthesis, characterisation and biological evaluation of lactonic-estradiol derivatives as...

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